diverse applications of nanomedicine

Diverse Applications of NanomedicineBeatriz Pelaz Philipps Universitaumlt MarburgChristoph Alexiou University Hospital ErlangenRamon A Alvarez-Puebla Universitat Rovira I VirgiliFrauke Alves University Medical Center GoumlttingenAnne M Andrews California NanoSystems InstituteSumaira Ashraf Philipps Universitaumlt MarburgLajos P Balogh AA Nanomedicine amp Nanotechnology Consultants NorthAndover Massachusetts 01845 United StatesLaura Ballerini International School for Advanced Studies (SISSAISAS)Alessandra Bestetti University of MelbourneCornelia Brendel Philipps Universitaumlt Marburg

Only first 10 authors above see publication for full author list

Journal Title ACS NanoVolume Volume 11 Number 3Publisher American Chemical Society | 2017-03-14 Pages 2313-2381Type of Work Article | Post-print After Peer ReviewPublisher DOI 101021acsnano6b06040Permanent URL httpspidemoryeduark25593rz88m

Final published version httpdxdoiorg101021acsnano6b06040

Copyright informationcopy 2017 American Chemical Society

Accessed April 9 2022 310 PM EDT

Diverse Applications of NanomedicineBeatriz Pelazdagger Christoph AlexiouDagger Ramon A Alvarez-Pueblasect∥ Frauke Alvespara

Anne M AndrewsΦ Sumaira Ashrafdagger Lajos P Balogh Laura Ballerini Alessandra Bestetti

Cornelia Brendel Susanna Bosi Monica Carril Warren C W Chan$ Chunying Cheninfin

Xiaodong Chenotimes Xiaoyuan Chenint Zhen Cheng⧧ Daxiang Cuiℏ Jianzhong Duforall Christian Dullinpara

Alberto Escuderodaggerƛ Neus Feliupart Mingyuan Gao∮ Michael Georgeprod Yury Gogotsi

Arnold Grunwelleroplus Zhongwei Guamp Naomi J Halascurren Norbert Hampp Roland K Hartmannoplus

Mark C Hersam Patrick Hunziker⟠loz Ji Jian⧫ Xingyu Jianginfin Philipp Jungebluth

Pranav Kadhiresan$ Kazunori Kataoka⊡ Ali Khademhosseinidaggerdagger Jindrich KopecekDaggerDagger

Nicholas A Kotovsectsect Harald F Krugperpperp Dong Soo Leeparapara Claus-Michael Lehr

Kam W Leong Xing-Jie Lianginfin Mei Ling Limpart Luis M Liz-Marzan Xiaowei Ma

Paolo Macchiarini Huan Meng Helmuth Mohwald Paul Mulvaney

Andre E Nel Shuming Nie Peter Nordlandercurren Teruo Okano Jose Oliveira$$

Tai Hyun Parkinfininfinotimesotimes Reginald M Pennerint int Maurizio Prato Victor Puntes∥⧧⧧ΦΦ

Vincent M Rotelloℏℏ Amila Samarakoon$ Raymond E Schaakforallforall Youqing Shenƛƛ

Sebastian Sjoqvistpart Andre G Skirtachpartpart Mahmoud G Solimandagger Molly M Stevens∮ ∮

Hsing-Wen Sungoplusoplus Ben Zhong Tangprodprod Rainer TietzeDagger Buddhisha N Udugama$

J Scott VanEppssectsect Tanja Weilampamp Paul S Weisscurrencurren Itamar Willner Yuzhou Wu

Lily Yang$$ Zhao Yuedagger Qian Zhangdagger Qiang Zhang⟠⟠ Xian-En Zhanglozloz Yuliang Zhaoinfin

Xin Zhou⧫⧫ and Wolfgang J Parakdagger

daggerFachbereich Physik Fachbereich Medizin oplusFachbereich Pharmazie and Department of Chemistry Philipps UniversitatMarburg 35037 Marburg GermanyDaggerENT-Department Section of Experimental Oncology amp Nanomedicine (SEON) Else Kroner-Fresenius-Stiftung-Professorship forNanomedicine University Hospital Erlangen 91054 Erlangen GermanysectDepartment of Physical Chemistry Universitat Rovira I Virgili 43007 Tarragona Spain∥ICREA Pg Lluiacutes Companys 23 08010 Barcelona SpainDepartment of Haematology and Medical Oncology paraDepartment of Diagnostic and Interventional Radiology University MedicalCenter Gottingen 37075 Gottingen GermanyDepartment of Molecular Biology of Neuronal Signals Max-Planck-Institute for Experimental Medicine 37075 GottingenGermany

California NanoSystems Institute Department of Chemistry and Biochemistry and ΦDepartment of Psychiatry and SemelInstitute for Neuroscience and Human Behavior Division of NanoMedicine and Center for the Environmental Impact ofNanotechnology and currencurrenDepartment of Materials Science and Engineering University of California Los Angeles Los AngelesCalifornia 90095 United StatesAA Nanomedicine amp Nanotechnology Consultants North Andover Massachusetts 01845 United StatesInternational School for Advanced Studies (SISSAISAS) 34136 Trieste ItalySchool of Chemistry amp Bio21 Institute University of Melbourne Parkville Victoria 3010 AustraliaDepartment of Chemical and Pharmaceutical Sciences University of Trieste 34127 Trieste ItalyCIC biomaGUNE Paseo de Miramon 182 20014 Donostia - San Sebastian SpainIkerbasque Basque Foundation for Science 48013 Bilbao Spain$Institute of Biomaterials and Biomedical Engineering University of Toronto Toronto Ontario M5S 3G9 CanadainfinCAS Center for Excellence in Nanoscience and CAS Key Laboratory for Biomedical Effects of Nanomaterials and NanosafetyNational Center for Nanoscience and Technology of China Beijing 100190 China

Received September 7 2016Published March 14 2017

Nano

Focus

wwwacsnanoorg

copy 2017 American Chemical Society 2313 DOI 101021acsnano6b06040ACS Nano 2017 11 2313minus2381

This is an open access article published under an ACS AuthorChoice License which permitscopying and redistribution of the article or any adaptations for non-commercial purposes

otimesSchool of Materials Science and Engineering Nanyang Technological University Singapore 639798int Laboratory of Molecular Imaging and Nanomedicine National Institute of Biomedical Imaging and Bioengineering NationalInstitutes of Health Bethesda Maryland 20892 United States

⧧Molecular Imaging Program at Stanford and Bio-X Program Canary Center at Stanford for Cancer Early Detection StanfordUniversity Stanford California 94305 United States

ℏInstitute of Nano Biomedicine and Engineering Department of Instrument Science and Engineering School of ElectronicInformation and Electronical Engineering National Center for Translational Medicine Shanghai Jiao Tong University 200240Shanghai China

forallDepartment of Polymeric Materials School of Materials Science and Engineering Tongji University Shanghai ChinaƛInstituto de Ciencia de Materiales de Sevilla CSIC Universidad de Sevilla 41092 Seville SpainpartDepartment of Clinical Science Intervention and Technology (CLINTEC) Karolinska Institutet 141 86 Stockholm Sweden∮ Institute of Chemistry Chinese Academy of Sciences 100190 Beijing ChinaprodNanion 80636 Munchen GermanyampCollege of Polymer Science and Engineering Sichuan University 610000 Chengdu ChinaDepartment of Materials Science and Engineering and AJ Drexel Nanomaterials Institute Drexel University PhiladelphiaPennsylvania 19104 United States

currenDepartments of Physics and Astronomy Rice University Houston Texas 77005 United StatesDepartments of Materials Science and Engineering Chemistry and Medicine Northwestern University Evanston Illinois 60208United States

⟠University Hospital 4056 Basel SwitzerlandlozCLINAM European Foundation for Clinical Nanomedicine 4058 Basel Switzerland⧫Department of Polymer Science and Engineering and ƛƛCenter for Bionanoengineering and Department of Chemical and BiologicalEngineering Zhejiang University 310027 Hangzhou ChinaThoraxklinik Heidelberg Universitatsklinikum Heidelberg 69120 Heidelberg Germany⊡The University of Tokyo Bunkyo Tokyo 113-8654 JapandaggerdaggerHavard University Cambridge Massachusetts 02139 United StatesDaggerDaggerBiomedical Polymers Laboratory University of Utah Salt Lake City Utah 84112 United StatessectsectEmergency Medicine University of Michigan Ann Arbor Michigan 48019 United StatesperpperpEMPA Federal Institute for Materials Science and Technology CH-9014 St Gallen SwitzerlandparaparaDepartment of Molecular Medicine and Biopharmaceutical Sciences and infininfinSchool of Chemical and Biological Engineering SeoulNational University Seoul South KoreaDepartment of Pharmacy Saarland University 66123 Saarbrucken GermanyHIPS - Helmhotz Institute for Pharmaceutical Research Saarland Helmholtz-Center for Infection Research 66123 Saarbrucken GermanyDepartment of Biomedical Engineering Columbia University New York City New York 10027 United StatesLaboratory of Controllable Nanopharmaceuticals Chinese Academy of Sciences (CAS) 100190 Beijing ChinaBiomedical Research Networking Center in Bioengineering Biomaterials and Nanomedicine Ciber-BBN 20014 Donostia - San

Sebastian SpainLaboratory of Bioengineering Regenerative Medicine (BioReM) Kazan Federal University 420008 Kazan RussiaDepartment of Interfaces Max-Planck Institute of Colloids and Interfaces 14476 Potsdam GermanyEmory University Atlanta Georgia 30322 United StatesTokyo Womenrsquos Medical University Tokyo 162-8666 Japan$$SMALL Wiley-VCH Weinheim GermanotimesotimesAdvanced Institutes of Convergence Technology Suwon South Koreaint intDepartment of Chemistry University of California Irvine California 92697 United States⧧⧧Institut Catala de Nanotecnologia UAB 08193 Barcelona SpainΦΦVall drsquoHebron University Hospital Institute of Research 08035 Barcelona SpainℏℏDepartment of Chemistry University of Massachusetts Amherst Massachusetts 01003 United StatesforallforallDepartment of Chemistry The Pennsylvania State University University Park Pennsylvania 16802 United StatespartpartDepartment of Molecular Biotechnology University of Ghent B-9000 Ghent Belgium∮ ∮Department of Materials Department of Bioengineering Institute for Biomedical Engineering Imperial College London London

SW7 2AZ United KingdomprodprodHong Kong Branch of Chinese National Engineering Research Center for Tissue Restoration and Reconstruction Hong Kong China

ACS Nano Nano Focus

DOI 101021acsnano6b06040ACS Nano 2017 11 2313minus2381

2314

oplusoplusDepartment of Chemical Engineering and Institute of Biomedical Engineering National Tsing Hua University Hsinchu CityTaiwan ROC 300

ampampInstitut fur Organische Chemie Universitat Ulm 89081 Ulm GermanyMax-Planck-Institute for Polymer Research 55128 Mainz GermanyInstitute of Chemistry The Center for Nanoscience and Nanotechnology The Hebrew University of Jerusalem Jerusalem 91904

IsraelSchool of Chemistry and Chemical Engineering Huazhong University of Science and Technology 430074 Wuhan China⟠⟠School of Pharmaceutical Science Peking University 100191 Beijing ChinalozlozNational Laboratory of Biomacromolecules CAS Center for Excellence in Biomacromolecules Institute of Biophysics Chinese

Academy of Sciences 15 Datun Road Chaoyang District Beijing 100101 China⧫⧫Key Laboratory of Magnetic Resonance in Biological Systems State Key Laboratory of Magnetic Resonance and Atomic and

Molecular Physics National Center for Magnetic Resonance in Wuhan Wuhan Institute of Physics and Mathematics ChineseAcademy of Sciences Wuhan 430071 China

ABSTRACT The design and use of materials in the nanoscale size rangefor addressing medical and health-related issues continues to receiveincreasing interest Research in nanomedicine spans a multitude of areasincluding drug delivery vaccine development antibacterial diagnosis andimaging tools wearable devices implants high-throughput screeningplatforms etc using biological nonbiological biomimetic or hybridmaterials Many of these developments are starting to be translated intoviable clinical products Here we provide an overview of recentdevelopments in nanomedicine and highlight the current challenges andupcoming opportunities for the field and translation to the clinic

Medicine is the science engineering and practice ofdiagnosing treating curing monitoring predictingand preventing diseases Most people associate

nanomedicine with pharmaceutical formulations where soft orhard particles of nanometer dimensions are injected into humansfor diagnosis and treatment However this field covers a broaderrange of research and development Nanomedicine differs fromother types of medicine in that it involves the development andapplication of materials and technologies with nanometer lengthscales to function in all the ways described below1minus5

Properties of nanoscale objects are transitional betweenmolecular and bulk regimes Nanoscale properties exist for allmaterials regardless of whether they are found in nature or aresynthetic However only synthetic objects are typically con-sidered part of ldquonanoscience and engineeringrdquo whereas the studyof biological nanoscale structures is often thought as part ofcharacterization without considering biological propertiesBecause of the transitional nature of ldquonanoscalerdquo materials it isdifficult to limit a materialrsquos reach and define its borders bystrict definitions and solid numbers (eg larger than atoms orsmall molecules but smaller than 100 nm) More importantlynanoscale particles can demonstrate new properties that can beexploited for the design of new therapeutic effects and diagnosticsNanomedicine is an interdisciplinary field where nanoscience

nanoengineering and nanotechnology interact with the lifesciences Given the broad scope of nanomedicine we expectit eventually to involve all aspects of medicine Moreovernanomedicine like medicine can enter the clinics and can be partof conventional clinical practice assuming all aspects oftranslation are satisfied including safety regulatory and ethicalrequirements It is expected that nanomedicine will lead to thedevelopment of better devices drugs and other applications forearly diagnoses or treatment of a wide range of diseases with high

specificity efficacy and personalization with the objective beingto enhance patientsrsquo quality of life In this Nano Focus we donot attempt to define nanomedicine but rather to provide anoverview of recent achievements materials and technologiesbelonging to nanomedicineNanoparticles (NPs) are key components of nanomedicine

and currently a large variety of nanoparticle types exist How-ever no standardized nomenclature exists in the literaturetherefore terms such as engineered nanomaterials nonbiologicalcomplex drugs (NBCDs) nanomedicalsnanomedicines etc areused freely Many nanomaterials can replicate some functionsof globular biological macromolecules6 Examples are lipidmicelles7 different polymeric nanostructures8 protein con-structs9 ribonucleic acid (RNA) NPs (RNPs)10 carbon dots(C-dots)11 nanodiamonds (NDs)12 carbon nanotubes(CNTs)13 graphene14 as well as inorganic materials such asmesoporous silica NPs (MSNP) superparamagnetic iron oxideNPs (SPIONs)15 quantum dots (QDs)16 plasmonic NPs17 goldnanoclusters (GNCS)18 upconverting NPs (UCNPs)19 etcMany of these nanoscale materials have unique size- and shape-dependent optical electronic and magnetic properties and theseproperties are dependent upon methods to synthesize to purifyand to characterize them20minus23 Many researchers note that smallchanges in size and shape can significantly affect the properties ofthe NPs Precision syntheses are therefore necessary to producesamples with tightly focused distributions in order to achieve thetargeted functions specifically and to correlate observed functionswith specific NP characteristics Detailed characterization of NPsamples that are used in a medical application is also criticalbecause onemust know and understandwhat is being injected intothe body A sample of NPs may be heterogeneous with distinctsubpopulations after synthesis2425 Microscopic imaging isconventionally used but this technique may be insufficient

ACS Nano Nano Focus


2315

because it is limited to a small number of NPs that may or may notbe representative of the whole sample Thus microscopic imagingmay not provide sufficient information about surface functional-ization composition and other property-determining featuresOther techniques that are starting to become part of thecharacterization scheme of NPs prior to use in humans aredynamic light scattering transmission electron microscopy gelelectrophoresis and ζ-potential analysis However there are nostandardized characterization requirements of NPs26 prior to usein humans and thismust be a focus for nanomedicine applicationsThe main reason is that the biodistribution and interaction of NPswith proteins is strongly size- and surface-dependent and thus in aheterogeneous sample many NPs will distribute differently andmay exhibit undesired effects or even toxicity In addition tocharacterization there is also a need to develop new and improvedmethods of NP separation and purification to produce optimalsamples for nanomedical applications and for studying NPbehavior inside the body2728 (which is important to designoptimal NP formulations for medical use)Despite the need to standardize characterization methods

NPs are expected to improve the detection and diagnosis ofdiseases First smart NPs can be designed to provide contrast atthe zone of interest and report information about the localenvironment after administration into the body This informa-tion can aid in imaging the anatomical fine structures of organsand labeling tissues with certain markers and enables local read-out of the concentrations of molecules of interest which helps toanalyze diseases directly inside the human body Second NPs arekey components of many high-throughput diagnostics machinesthat can analyze extracted samples (such as blood tissue etc)outside of the body for rapidly detecting biological makers andmolecular alterations The ability to analyze multiple biomarkerssimultaneously may improve diagnostic precision Moreovermultifunctional or theranostic NPs that can simultaneouslydiagnose treat and even monitor therapeutic efficacy are beingengineered29

Nanoparticles are also being developed for the treatment ofdisease NPs are used as delivery vehicles for pharmaceuticalagents30 as bioactive materials or as important components inimplants31 In the case of delivery NP-based carrier systems havea unique ability to cross biological barriers Thus NPs can entertumors via their localized leaky vasculature and are retained dueto poor lymphatic drainage in the tumor microenvironment32

This passive targeting is called the enhanced permeation andretention (EPR) effect33 There is an ongoing debate in theliterature about the effectiveness of active ie ligandreceptor-mediated targeting versus passive targeting but any carrier hasto be delivered to the designated site before it can bind to cellsurface receptors or be retained by other effects34

In addition to using nanomedicine to diagnose and to treatdiseases it is also important to establish NPsrsquo efficacy and safetyin biological systems After the NP has functioned as designedafter administration into the body what happens to the carrierparticle when the drug has been delivered or the tissue imagedElimination of the particles can potentially occur via renal orhepatobiliary clearance If they do not get cleared the long-termfate of the NPs is not clear These particles may degrade and getcleared renally because they are small enough to transportthrough the kidneyrsquos filtration slits35minus37 or they may accumulatein different organs and interact with off-target cells The in vivofate of NPs can potentially be a dynamic process and thus thereis a need to understand nanobiokinetics (nanopharmacokinetics

and pharmacodynamics) which may relate to unique andinteresting toxicological responses of NPsNanomedicine is not limited to colloidal materials and

technologies to evaluate them for in vivo applications Nano-medicine developments go beyond the ldquomagic particulate bulletrdquoconcept38 Nanomedicine could involve the design of newscaffolds and surfaces for engineering sensors or implantablesystems and electronics to aid in the regeneration of tissues(ie regenerative medicine) Many of these concepts are still atthe early stages of development but some have already reachedclinical practiceThis Nano Focus article is organized into four subsections

that are focused on specific applications of materials or systemswith nanoscale properties (a) in vivo diagnostics (b) in vitrodiagnostics (c) in vivo therapeutics and (d) implantablenanomaterials

IN VIVO DIAGNOSIS (ldquoSMART IMAGINGrdquo)A key focus in nanomedicine involves the use of nanomaterials ascontrast agents for anatomical and functional imaging Usingnanomaterials as contrast agents enables visualization of struc-tures inside the human body and helps clinicians to delineatehealthy from diseased tissues and to recommend proper treat-ment Nanoparticles can be engineered with different contrastproperties The most common modalities are computedtomography (CT) magnetic resonance imaging (MRI) imagingof radioactivity such as positron emission tomography (PET)or single photon emission computed tomography (SPECT)fluorescence imaging and photoacoustic imaging For all thesetechniques material development is crucial because the NPs arecontrast agents that enable visualization of biological tissuesFor this application NPs can be engineered to localize in specifictissues and potentially produce high contrast

Computed Tomography X-ray-based imaging enableshigh-resolution anatomical and in the case of CT also three-dimensional (3D) imaging of mostly skeletal tissues at unlimiteddepth in human applications Computed tomography imaging isthe work-horse in clinical diagnostics due to the simplicity ofthe technique the comparable low demands on infrastructurethe rapid image generating and the low costs for a standardexaminationIn recent years around 250 out of every 1000 people in the

United States underwent CT imaging39 Classical X-ray imagingharnesses the tissue-specific attenuation of X-ray energy togenerate contrast in the recorded radiographs Therefore bonesgenerate more contrast than soft tissue because of the largerrelative electron density of bone In order to boost the lowcontrast of soft tissue contrast agents with elements such asiodine and barium characterized by a high-electron density aretypically applied to visualize blood vessels in gastrointestinal(GI) tract tumors and other soft tissues In contrast to typicalfunctional imaging techniques such as PET and SPECT in CTthe photonsX-rays are produced outside the body and onlymodulated by the tissues through which they travel Thus thelarge photon flux enables a high signal-to-noise ratio leading CTto outperform other 3D imaging techniques in terms of spatial

A key focus in nanomedicine involvesthe use of nanomaterials as contrastagents for anatomical and functionalimaging

ACS Nano Nano Focus


2316

resolution However the weak interaction between tissue and theincident X-ray beam results in comparably limited sensitivity ofCT which might explain why specifically targeting contraststrategies are virtually non-existentThis poor sensitivity has encouraged nanomedicine research-

ers to develop nanoparticles as contrast agents for X-ray imagingGold nanoparticles (AuNPs) are central to this developmentDue to its high atomic number and electron density (79 and1932 gcm3 respectively versus the typical X-ray contrast agentiodine with values of 53 and 49 gcm3 respectively) AuNPshave higher attenuation coefficients and can be used as contrastagents for X-ray imaging CT and micro-CT40 The AuNPs aretypically coated with targeting molecules such as folic acid so thatthey can highlight distinct tissue structures Gold nanoclusters(NCs) which are smaller structures than AuNPs are also beingdeveloped as CT contrast agents1840 Folic-acid-conjugatedAuNCs with silica shells were demonstrated to exhibit goodbiocompatibility and could actively target the folic acid receptor(+) in vitro of MGC-803 cells and in vivo gastric cancer tissueswith 5 mm diameter in nude mice models The researchersshowed that the use of NCs exhibited excellent contrast forCT imaging In addition to CT imaging these NCs can also beused for molecular imaging since red fluorescence emissionwas observed4142 These NCs also penetrated the tumor and

were retained but their small size enabled them to be clearedrenally4142 Thus these NPs are promising candidates for futureclinical useBesides gold NPs composed of other materials with a high

atomic number are also suited for CT One example is NaGdF4-based UCNPs Besides providing contrast for CT these NPs canbe imaged optically (see Figure 1) In addition to their abilityto provide contrast for CT potential toxicity of NPs plays animportant role toward translation to clinics (vide inf ra)In parallel to the development of new CT contrast agents

there has also been progress in instrumentation relating to thepreparation and detection of the agents Novel X-ray sourcessuch as synchrotrons provide tremendously more flux and ahigher degree of coherence and therefore enable the exploitationof the wave nature of X-rays for imaging purposes These novelX-ray sources have led to new applications like phase contrastCT diffraction enhanced imaging and holotomography toname a few44minus46 For example phase contrast CT provides10minus200 times more contrast in soft tissue applications thanclassical absorption-based CT4748 This gain in contrast directlytranslates into an increase in sensitivity By coupling thisincreased sensitivity with the use of target-specific contrastagents submicrometer-scale 3D spatial resolution can beachieved This technique can be applied to gather structural

Figure 1 (I) In vitro CT images of (a) lanthanide-doped NaGdF4 upconversion ldquonanoclustersrdquo (lt5 nm) suspended in aqueous solution (b) CTattenuation plot (given in Hounsfield units HU) of NaGdF4 NPs in dependence of the concentration of each sample from 02 to 10 mgmL tofurther investigate the CT contrast effect (II) Images of a control group before injection of NPs (c) photograph of a nude mouse loaded withgastric cancerMGC-803 cells (d) X-ray image and (eminusg) CT images of nudemice from the control group (III) (hminusk)CT images and (IV) (lminusn)MRI images of mice after intravenous injection with NaGdF4 UCNPs making use of passive targeting (EPR effect) The pulse sequenceelectromagnetic conversion (EC) = 11417 kHz repetition time (TR time) = 2000 echo time (TE time) = 656Ef (echo frequency)Parameters of transverse plane the pulse sequence EC = 11417 kHz TR time = 2000 TE time = 438Ef It took about 6 h to acquire oneimage The relaxivity value of NaGdF4UCNPs at 15 T is about 45mMsminus1 Adapted with permission from ref 43 Copyright 2015 Royal Society ofChemistry

ACS Nano Nano Focus


2317

information indicative of asthma within the lung of a mouse49

It can also be applied in the visualization of macrophages loadedwith barium sulfate after intracheal instillation49 In a study byDullin et al the researchers achieved 9 μm resolution whichenabled the visualization of macrophage function within thelocal 3D environment46 The spatial resolution could be furtherimproved to 400 nm by using holotomography (see Figure 2)50

TheNP-based CT imaging technologies can potentially changethe way we perform CT-based clinical diagnosis51 Currently softtissue imaging via CT requires a large dose of iodine- or barium-containing contrast agents to overcome poor sensitivity How-ever the high dose may not be well tolerated by patientsFurthermore it is difficult to design traditional contrastagents to target specifically and to bind to cellular biomarkersor accumulate in tissues of interest such as sites of inflammation orprimary tumors and metastasis Combining targeted AuNPswith the recent development of energy-resolved detectors52 thediscrimination of a multitude of different materials in a singleCT scan should be possible in the near future Therefore thechallenge facing the field is to design and to engineer smallNP probes of high-atomic-number materials like iodine gold orbarium conjugated to targeting moieties that specifically labelcertain cell types in vivo similar to probes used in optical imagingMoreover since novel CT techniques do not focus on X-rayattenuation but on phase shift or scattering as sources of contrastother types of NPs like hollow spheres53 or NPs with X-rayscattering properties will become increasingly important Despitethe negative effects of the applied X-ray dose CT is the mostapplied technique in clinical diagnosis (eg sim50 of imaging-based diagnostics performed in Germany in 2013 were by CT)54

Improved detectors and the implementation of novel algorithmsespecially newly developed reconstruction techniques havealready dramatically lowered X-ray doses55 Techniques likelimited angle reconstruction and zoom tomography will furtheraid in this development5056

Magnetic Resonance Imaging Magnetic resonanceimaging is widely used for in vivo applications due to its safetyspatial resolution soft tissue contrast clinical relevance andability to record anatomical and functional information aboutsoft tissues and organs Notably MRI-responsive contrastagents provide physiological information that complementsroutine anatomical images Since the technology is based on theinteraction of nuclei with surrounding molecules in a magneticfield MRI has no need for ionizing radiation and possessesunlimited depth of penetration and unparalleled soft tissuecontrast57 However MRI has relatively poor sensitivity incomparison to nuclear and optical modalities thus leading tolonger acquisition time and use of large amounts of contrastagents Although the introduction of higher magnetic fields(higher than 47 T) could increase the signal-to-noise ratiothereby permitting higher resolution and faster scanning thesafety of high static magnetic field strengths on human health isof great concern5859 The development of hyperpolarized MRI(ie polarization of the nuclear magnetic moments far beyondthermal equilibrium conditions)60 has improved the sensitivity ofthe desired nuclei and enables quantitative in vivo imaging andreal-time metabolic profiling using stable isotope precursors60

Current contrast agents such as paramagnetic agents or SPIONsplay important roles in enhancing contrast in T1 or T2 imageswhich provide higher MRI sensitivity and accuracy for imagingliving subjects These agents accelerate the rate of T1 and T2relaxation thereby enhancing local contrast61 Paramagneticagents (eg gadolinium-based agents) principally acceleratelongitudinal T1 recovery generating positive contrast whilesuperparamagnetic agents (eg iron oxide-based agents such asSPIONs) primarily increase the rate of dephasing or transverseT2 decay resulting in negative contrast effects61 However bothtypes of agents have been associated with toxic effects whichneeds to be taken into account for their use on humans and inveterinary medicine (vide inf ra)6263

Conventional T1 contrast agents such as paramagnetic com-plexes (based on Gd3+ Mn2+ or Fe3+) are small moleculesthat leave the vascular system within minutes and are renallycleared The short circulation time makes it difficult toacquire a high-resolution image of desired sites BiocompatibleNP-based T1 contrast agents have been developed because theyhave a number of advantages over conventional T1 contrast

6465

Researchers can tailor the size shape and composition circula-tion time target cells and tissues and optical and physicalproperties to meet the biological requirements for optimizingimaging For example Gd(III)minusnanodiamond conjugatesenable contrast enhancement with a much smaller amount ofGd compared to other agents used66 For example melanin hasbeen intensively studied as a target for melanoma imaging andwas recently a major focus for designing a multimodal imagingnanoplatform Melanin NPs with diameters of 45 nm notonly retain their unique optical properties but also have naturalbinding capability with various metal ions After chelation withFe3+ ions and subsequent conjugation with cyclic arginine-glycine-aspartic acid (RGD) molecules melanin NPs serve as T1contrast agents for targeted MRI of U87MG glioblastoma6768

In another example a noncytotoxic asymmetrical cancer-targeting polymer vesicle based on R-poly(L-glutamic acid)-block-poly(ε-caprolactone) [R is folic acid or diethylenetriami-nepentacetatic acid (DTPA)] as shown in Figure 3 was reportedas a T1 MRI contrast agent with enhanced sensitivity and it alsoserved as a delivery vehicle for cancer chemotherapy69 Suchasymmetrical vesicles have a cancer-targeting outer corona

Figure 2 Three-dimensional localization of labeled macrophagesin a 500 μm thick lung section of a healthy mouse scanned byholotomography Three orthogonally oriented slices are showntogether with automatically labeled barium clusters (green)representing macrophages loaded with barium sulfate and alveolarwalls in a small region of interest (ROI yellow) A part of a bloodvessel has been marked semi automatically (purple) Adapted withpermission from ref 50 Copyright 2015 Nature Publishing Group

ACS Nano Nano Focus


2318

coupled with a Gd(III)-chelating and drug-loading-enhancinginner corona Liu and colleagues demonstrated that such vesiclesexhibited an extremely high T1 relaxivity (4239 mMminus1 sminus1 8-foldbetter than that of DTPA-Gd) and anticancer drug loadingefficiency (526 for doxorubicin hydrochloride DOXmiddotHCl)69

Moreover the DOX-loaded vesicles exhibited 2-fold betterantitumor activity than free DOX69

As T2 contrast agents SPIONs establish a substantial locallyperturbed dipolar field to shorten proton relaxation of thesurrounding tissues significantly The magnetism of NPs undernormal magnetic field strengths is dependent on the magneto-crystalline anisotropy and the size of the NPs In comparison totheir spherical counterparts for example iron oxide NPs with anoctapod shape exhibit an ultrahigh transverse relaxivity value anddramatically increase the sensitivity of MRI for early stage cancerdetection largely due to their effective radius and the local fieldinhomogeneity of the magnetic core70 Because of the negativecontrast effect and magnetic susceptibility artifacts it is still amajor challenge to distinguish the region of signals induced byiron oxide NPs from low-level background magnetic resonance(MR) signals originating from adjacent tissues such as bonevasculature calcification fat hemorrhage blood clots etcAs a combination of paramagnetic and SPIONs eg in coreshell structures71 dual-mode T1T2 contrast agents havebeen developed for dual T1 and T2 mode MRI because theyhelp validate reconstruction and visualization of the data inan accurate and reliable way However interference inevitablyoccurs when both T1 and T2 contrast agents are integrated intoa single nanostructure in close proximity The magnetic fieldinduced by the T2 contrast material perturbs the relaxa-tion process of paramagnetic T1 contrast materials leading toundesirable quenching of the T1 signal Coreshell structurescan efficiently reduce their magnetic coupling by introducinga separating layer between the superparamagnetic core andparamagnetic shell Such magnetically decoupled dual-modeT1T2 contrast agents not only provide superior MR contrasteffects in both modes but also enable self-confirmation ofimages and essentially function as an ldquoAND logic gaterdquo to reducesusceptibility artifacts from the raw images to enhance accuracyof the MRI7273 In contrast to coreshell structures dumbbellhybrid nanostructures combine T1 and T2 contrast agentstogether in a nanocomposite via a bridge NP which spatiallyseparates two contrast agents at a certain distance to reducetheir magnetic coupling74 Unlike coreshell structures whereT2 contrast agents are sequestered within a supporting matrixdumbbell nanostructures allow both T1 and T2 contrast agents tobe exposed to their immediate environment without compro-mising their magnetic properties Moreover both surfaces of

T1 and T2 contrast agents are available for subsequent surfacecoating for targeting molecular imagingBesides being ldquomererdquo contrast agents for MRI7576 activatable

or ldquosmartrdquoNPs can respond to a change in tumor microenviron-ment to instigate the therapeutic and diagnostic mechanism7778

The most common triggers for activatable NPs are stimulisuch as pH temperature redox reactions79 metabolites ionsproteases ultrasound and light80 The tumor microenvironmentregulates tumor progression and the spread of cancer in the bodyResponsive agents capable of reporting diagnostically relevantphysicochemical properties of the microenvironment in whichthe contrast agent distributes have gained tremendous attentionVarious nanocarriers have been tested in combination with amyriad of imaging contrast agents payload drugs and targetingmoieties leading to the formulation of theranostic NPs capableof delivering therapy concomitant with diagnosis In the case ofMRI different ldquosmartrdquo NP probes have been demonstratedThe design of pH-responsive probes is of great interest since it isan important physiological parameter and pH dysregulationcan be a cancer marker Gao and colleagues reported a smartpH-activatable 19F-probe for detecting specific and narrow pHtransitions ranging from 55 to 74 in biological systems81 Theydeveloped micelles composed of fluorinated polymers withtertiary amines having different pKa values The protonation ofsuch amines at pH below their pKa results in micelle disassemblyand 19F-MRInuclear magnetic resonance spectroscopy (NMR)signal activation They were able to determine the pH throughthe identification of the corresponding activated 19F-reporter Inanother related example hypoxia in the tumor microenviron-ment results in lactic acid production and hence acidicconditions Zhou and colleagues fabricated pH-triggered NPsfor 19F-MRI and fluorescence imaging (MRI-FI) of cancercells82 19F was attached to AuNPs by acid-labile hydrazonelinkers Fluorescence imaging revealed highly selective uptake ofthese NPs by lung cancer cells The ldquotrickrdquo to these NPs involvesselective release of 19F-MRI contrast agent after the NPs havereached the tumor environment In the acidic intracellularcompartments of cancer cells the 19F detached from the AuNPsand appreciably enhances the intracellular 19F-MRI signal asshown in Figure 4Temperature differences between tissues is a common feature

in pathological conditions especially in tumors Thus a secondclass of ldquosmartrdquoNP probes for MRI uses hypersensitive detectionof temperature changes in different tissues83 Langereis andcolleagues84 reported a combined temperature-sensitive lip-osomal 1H chemical exchange saturation transfer (CEST)85

and 19F magnetic resonance (MR) contrast agent as a potentialcarrier system for MRI-guided drug delivery The liposomescontain both a chemical shift agent as well as a highly fluorinatedcompound Upon reaching the melting temperature of theagentsrsquo lipid membrane the lipo-CEST contrast enhancementvanishes due to the release of the chemical shift agentSimultaneously the 19F-MRI probe is freed from the influenceof the paramagnetic shift agent causing an appearance of a19F-MRI signal The combined CEST and 19F-MR temperature-sensitive liposomal carrier provides CEST-based contrastenhancement which can be switched on and off to localize theliposomes before release while the 19F-MR signal can potentiallyquantify local drug release of drugs with MRIRedox reactions play crucial roles in biological processes and

abnormal redox reactions are implicated in various conditionsincluding liver damage and human immunodeficiency virus(HIV) A third class of ldquosmartrdquo NP probes for MRI uses redox

Figure 3 Illustration of multifunctional asymmetrical polymervesicles for ultrasensitive T1 MRI and effective cancer targeteddrug delivery Adapted from ref 69 Copyright 2015 AmericanChemical Society

ACS Nano Nano Focus


2319

reactions in the tumor microenvironment for cleavage of atomsfrom the NP structure Kikuchi and co-workers designed redoxactivatable NPs to image reducing environments86 Gd3+

complexes were attached to the surface of 19F containing NPsby disulfide linkers Vicinity of the Gd3+ to 19F reduces thetransveral relaxation time T2 of the fluorine compounds by theparamagnetic relaxation enhancement effect which attenuatesthe 19F NMRMRI signal (see Figure 5) When the disulfide

bonds enabling attachment of Gd3+ are reduced the Gd3+

complexes are cleaved from the NP surface Then the T2NMRMRI signal of the encapsulated 19F compounds increasesindicating the presence of the NPs in redox active environmentImaging Radiolabels There has been rapid progress in

the development of NP-based radiolabels87 Chelators such as14710-tetraazacyclododecane-14710-tetraacetic acid (DOTA)and 147-triazacyclononane-147-trisacetic acid are commonly

used to label diagnostic positron emitters eg 68Ga or 64Cu ortherapeutic beta-emitters eg 177Lu or 90Y The sequential useof different poly(ethylene glycol) (PEG) -coated emitters eg68Ga and 177Lu enable multiplexed labeling as well as improvedcirculation time For this purpose micelle encapsulation wasintroduced for combining a mixture of amphiphiles comprisingchelators bearing the radiolabels ligands for targeting and PEGfor surface passivation with originally hydrophobic NPs88 Thesame method could successfully be applied for different types ofNPs such as SPIONs plasmonic NPs and coreshell UCNPsSometimes the NPs can be labeled postsynthetically without theuse of chelators for radiometals89 The resulting radiolabeled NPsenable researchers to trace their biodistribution when adminis-tered systemically and to study fundamental NP in vivo process(eg determining the contribution of active versus passive tumortargeting in mouse models)90

In fact radiolabels are particularly suited for quantitativebiodistribution studies γ-emission is barely absorbed by tissueand radioactivity is independent of the probersquos local environ-ment ie there are no substantial quenching effects such as in thecase of fluorophores which often exhibit pH-dependentemission Using radiolabels produces excellent signals and there-fore a low dose can yield sufficient image quality to make adiagnostic interpretation One additional advantage is the abilityfor multiplexed read-out of radiolabels with different γ-emissionenergies in parallel This has been recently used for recording thebiodistribution of several compounds of NPs independently37

Most NPs are hybrid structures composed of their functional part(ie contrast for imaging) and an organic surface coating thatprovides colloidal stability and targeting capability as well as thecorona of adsorbed proteins9192 By adding radiolabels ofdifferent energies to different parts of the NPs the NPs coulddisintegrate after in vivo administration ie the organic surfacecapping may come off the functional particle core37 Such studiesin the future will enable imaging of the different NP componentsto elucidate the fate of the different parts of the NP This will beimportant for understanding the biodegradability of all differentparts of a NP and their elimination This is decisive for guidingthe design of NPs for use in in vivo delivery applications How-ever when different labeling methods and radioisotopes are usedto label NPs to study their fate it is crucial to ensure that theradioisotopes are incorporated onto the NPs with little impact ontheir original biological behavior

Fluorescence Imaging Fluorescence is a useful imagingmodality because the emission of the probes after excitation canbe visualized by the naked eye or at higher resolution with opticalmicroscopy There is an abundance of available fluorophores thatcan be tailored to specific applications Many traditional organicfluorophores suffer from aggregation-caused quenching (ACQ)thereby limiting design schemes where specific interactionspromote fluorophore localization This has forced researchers touse dilute solutions (often at the nanomole level) of fluoro-phores Given the minuscule amount of fluorophore they can beeasily photobleached which imposes a limit on the achievablecontrast Contrary to ACQ aggregation-induced emission (AIE)is where increased aggregation yields a stronger fluorescentsignal93 This makes AIE fluorogens (AIEgens) good candidatesfor bioimaging applications94 The accumulation of AIEgens atregions of interest results in the formation of nanoaggregates(AIE dots) which intensifies the fluorescence signal The AIEdots are resistant to photobleaching enabling high-quality bio-imaging over a wide time window They are suitable for long-term tracking of theranostic processes such as tumor-metastasis

Figure 4 (A) Synthesis of AuNPs capped with folic acid and 19Fcontrast agent Folic acid with a high tumor affinity due to theoverexpression of its receptors on the cancer cells was conjugatedonto the surface of the AuNPs 19F contrast agent was covalentlyconjugated onto the AuNPs via acid-labile hydrazone linkers(B) pH-triggered release of the 19F contrast agent from the AuNPsto the cytosol via the selective removal of the pH-labile cap in theacidic intracellular compartments of cancer cells Adapted withpermission from ref 82 Copyright 2014 Royal Society of Chemistry

Figure 5 Design of redox activatable NPs Adapted with permissionfrom ref 86 Copyright 2015 Wiley-VCH Verlag GmbH amp Co

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2320

monitoring drug deliveryrelease and stem-cell therapy95

Organic AIE dots enjoy almost all advantages of inorganicQDs9697 yet are free of the disadvantages of the latter (egcytotoxicity9899) In addition AIE molecules and nanoaggre-gates can be decorated by functional groups with bindingspecificity to particular receptors that enables active target-ing949697 Integration of an RGD unit with the AIEgen structurefor example has generated integrin-specific fluorescence turn-onbioprobes for tumor cell targeting image-guided drug deliveryin situ monitoring of therapeutic effect etc100101

Two decades ago fluorescent semiconductor QDs wereintroduced for the fluorescent labeling of biological moleculesand cells102103 Due to their narrow emission bands QDs aresuited for multiplexed read-out as detailed below104105

Semiconductor QDs are also bright and show reducedphotobleaching which makes them suitable for long-termimaging at the single-molecule level106minus108 However semi-conductor QDs can be cytotoxic toward cells due to the releaseof metal ions9899 and many researchers take this factinto consideration in designing QDs for their long-termimaging studies109minus111 For example CdSe QDs can be coatedwith ZnS polymer shells or SiO2 layers to suppress thedissolution of possibly toxic semiconductor core materials99

Details about the mechanism for potential toxicity are still underdiscussionRare earth (RE)-based nanophosphors which consist of a

host inorganic matrix doped with luminescent lanthanide (Ln)cations constitute another type of luminescent materials Theirmain advantages are their low toxicity high photostability highthermal and chemical stability high luminescence quantum yieldand sharp emission bands112 However they normally show lowluminescence intensity which is caused by the low absorptionof the parity-forbidden Ln3+ 4fminus4f transitions113 The opticalproperties of such NPs can be further modified by dopingas shown recently in the case of Eu3+ Bi3+ codoped REVO4(RE = Y Gd) NPs Intense red luminescence was achieved byusing indirect excitation of the Eu3+ cations via the vanadateanions The incorporation of Bi3+ into the REVO4 structureresulted in a shift of the original absorption band correspondingto the vanadate toward longer wavelengths yielding nano-phosphors excitable by near-ultraviolet and visible light114

Carbon dots are another class of fluorescent NPs Carbon dotsare zero-dimensional carbon nanomaterials and possess theadvantageous characteristics of QDs over organic fluorophoressuch as high photostability tunable emission and large two-photon excitation cross sections11Moreover C-dot fluorescencedoes not blink They are water-soluble can be functionalized canbe produced economically and have excellent cell permeabilityand biocompatibility making them attractive for intracellularsensing11115116 Also C-dots without heavy metal content areenvironmentally friendly and can be much safer for biologicalapplications than traditional Cd-containing QDs11115116 In arecent study C-dots were subjected to a systematic safety evalua-tion involving acute toxicity subacute toxicity and genotoxicityexperiments (including a mouse bone marrow micronuclear testand a Salmonella typhimurium mutagenicity test)117 Carbon dotdosages of 51 mgkg weight body showed no significant toxiceffects ie no abnormality or lesions and no genotoxicity in theorgans of the animalsNanodiamonds another group of carbon NPs are receiving

growing interest for drug delivery and other biomedicalapplications because they have good biocompatibility118 Intra-venously administered ND complexes at high dosages didnot change serum indicators of liver and systemic toxicity119

Nanodiamond powder produced by detonation (5minus6 nm seeFigure 6) or other methods (20minus50 nm) are readily available incommercial quantities at moderate cost and are among the mostpromising carbon nanomaterials for drug delivery imaging andtheranostic applications Fluorescence of NDs due to nitrogen-vacancy (NV) centers (see Figure 6) that are composed of asubstitutional nitrogen and a next neighbor vacancy enablesbiomedical imaging120121 Fluorescent NPs can also be producedby linking or adsorbing various fluorophores onto NDs Forexample bright blue fluorescent NDs were produced via covalentlinking of octadecylamine to carboxylic groups on theND surface(ND-ODA)122 In biomedical imaging fluorescent NDs com-bine the advantages of semiconductor QDs (small NP size highphotostability and bright multicolor fluorescence) with biocom-patibility lack of toxicity and tunable surface chemistry Thereforethey are promising for in vivo imaging Since the sizes of NDparticles can be smaller than 5 nm NPs they can be removed byrenal excretion without leaving any toxic residue in the body123124

Figure 6 (A) Schematic model of a 5 nm ND with a fluorescent NV center and a variety of surface terminations after oxidative purification isshown The diamond core is covered by a layer of surface functional groups that stabilize the NP by terminating its dangling bonds The surfacecan also be stabilized by the conversion of sp3 carbon to sp2 carbon Adapted with permission from ref 118 Copyright 2012 Nature PublishingGroup (B) Plot showing the strength of binding (KL) versus the monolayer capacity (Amax) based on a Langmuir model for DOX polymyxin Btetracycline and vancomycin adsorbed on detonation-produced ND from two sources (ZH and ND) with different surface chemistries theas-received surface like in the right picture carboxylated (minusCOOH) and aminated (minusNH2) surfaces Adapted with permission fromrefs 125 and 126 Copyright 2016 Elsevier and copyright 2013 American Chemical Society respectively

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Besides using fluorescent NPs as contrast labels for imagingldquosmartrdquo fluorophores that change their fluorescence signaldepending on the local environment can be designed Byintegrating pH-sensitive fluorophores on the surfaces of NPsinformation about their local environment can be gained forexample whether they are located extracellularly in neutralpH or intracellularly inside acidic endosomeslysosomes127128

By combining analyte-sensitive fluorophores with NPs theconcentrations of different analytes in the vicinity of the NPs canbe determined129130

Fluorescent NPs based on AIE offer another concept in thisdirection The AIE process has been utilized to develop light-upbiosensors Cell apoptosis for example has been followed inreal time by making use of the AIE process (see Figure 7)131

Tetraphenylethene (TPE) an archetypical AIEgen has beenfunctionalized by a short oligopeptide (DEVDK) throughclick chemistry affording DEVDK-TPE a peptideminusAIEgenadduct DEVDK-TPE is soluble in aqueous buffers and henceemits no fluorescence (ie no residual emission) It remainsnonfluorescent in intracellular media after cells internalize itDuring apoptosis caspase enzyme cleaves the DEVD segmentThe residual segment (K-TPE) is not hydrophilic enough tobe dissolved in aqueous media and thus forms intracellularnanoaggregates The aggregate formation turns on the fluo-rescence of TPE which is gradually intensified with the progress ofapoptosis This enables real-time and in situ monitoring of thebiological process of programmed cell death131

For cellular and in vivo cancer imaging Nie Gao and theirco-workers have developed a class of activatable NPs based onthe use of copolymer materials with ionizable tertiary aminegroups and covalently conjugated fluorescence dyes132133 Theself-assembled NP structures undergo a dramatic and sharptransition within a narrow range of pH (often less than 02 pHunits) This pH-induced transition leads to rapid and completedissociation of the nanomicelles As a result the covalently linkeddyes change from a self-quenched ldquooffrdquo state to a highly emissivebright ldquoonrdquo state This supersensitive and nonlinear responseto external pH enables targeting acidic organelles in cancer cellsas well as the acidic microenvironment in solid tumors Thisfeature is important in addressing the tumor heterogeneityproblem which is a major challenge for various imaging andtherapeutic approaches based onmolecular or receptor targetingBy targeting the common ldquohallmarksrdquo of tumors (ie theacidic habitat or microenvironment and the growth of newblood vessels or angiogenesis) this work has opened exciting

opportunities in detecting and potentially treating a broad rangeof human solid tumors This approach leads to improveddetection sensitivity because each NP contains multiple dyemolecules which are turned on (restored to fluorescence) in anall-or-none fashion leading to amplified fluorescence signals thatare many times brighter than those of single dye molecules

LABORATORY-BASED DIAGNOSIS(ldquoHIGH-THROUGHPUT SCREENINGrdquo)Nanoparticles can also be used for detection of moleculescells and tissues outside the human body In this diagnosticapplication the function of the NP is to identify unique bio-logical molecules in biological fluids that are associatedwith the health of the patient The NPs act as transducersand are coated with ligands to enable the biorecognition ofunique biological molecules in the fluid in the in vitro sensingapplications For example AuNPs have been modified withligands that specifically bind to a complementary proteinThe presence of these proteins induces the cross-linking of theNPs (ie agglutination) This controlled agglomeration canbe observed colometrically by the change of color of the NPsolution134135 These concepts have been later refined forexample in rapid colorimetric DNA sensing136137 This AuNP-based diagnostic technology has advanced to testing of patientsamples and is now used in the clinic138 Nanotechnologypresents an opportunity to improve the overall diagnostic processby lowering the limit of detection thus enabling high throughputand multiplexed detections of biological targets with highsensitivity

Screening Based on Fluorescence Read-Out Quantumdots are frequently used as fluorescence labels in proteins ornucleic acid assays One example in this direction is a QD-basedfluorescence polarization assay for screening of antigen surfaceepitopes139 In this example a method for quickly screeningand identifying dominant B cell epitopes was developed usinghepatitis B virus (HBV) surface antigen as a target Eleven aminoacid fragments from the HBV surface antigen were synthesizedby 9-fluorenylmethoxy carbonyl solid-phase peptide synthesisstrategy and then CdTe QDs were used to label the N-terminals

Figure 7 Diagrammatic illustration of the real-time monitoring of cell apoptosis process by DEVDK-TPE an AIE-active fluorescent bioprobeAdapted from ref 131 Copyright 2012 American Chemical Society

Nanoparticles can also be used formolecular detection of molecules cellsand tissues outside the human body

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of all peptides After optimizing the factors for this fluorescencepolarization (FP) immunoassay the antigenicities of syntheticpeptides were determined by analyzing the recognition andcombination of peptides and standard antibody samples Theresults of the FP assays confirmed that 10 of the 11 syntheticpeptides had distinct antigenicities In order to screen dominantantigenic peptides the FP assays were carried out to investigatethe antibodies against the 10 synthetic peptides of the HBVsurface antigen in 159 samples of anti-HBV surface antigen-positive antiserum The results showed that 3 of the 10 antigenicpeptides might be immunodominant because the antibodiesagainst them existed more widely among the samples and theirantibody titers were higher than those of other peptides Usingthree dominant antigenic peptides 293 serum samples weredetected for HBV infection by the FP assays The results showedthat the antibody-positive ratio was 519 and the sensitivityand specificity were 843 and 982 respectively ThisQD-based FP assay is a simple rapid and convenient methodfor determining immunodominant antigenic peptides and haspotential in applications such as epitope mapping vaccinedesigning or clinical disease diagnosisAnother application of QDs is multiplexed detection as

described by Nie and co-workers Quantum dots have beenincorporated into microbeads to generate barcodes whereunique optical emission for each bead can be generated bydifferent amounts and sizes of QDs140 Fluorescent QDs are idealoptical coders because they have narrow fluorescence linewidths size- and shape-tunable emission photostability and allfluorescence emission can be excited with a single wavelengthThus barcodes can be designed with high coding precisioncost-effective instrumentation can be used by simplifying theoptical components and excitation source for read out andfinally it has been suggested that over 1 million unique barcodescan be generated using different emitting QDs and intensitiesThere are several methods to prepare QD barcodes (a) theldquoswellingrdquo technique where polystyrene beads are mixed in asolvent to enable beads to increase in volume which allows thediffusion of hydrophobic QDs into the outermost layer140

(b) layer-by-layer (LbL) assembly of different emitting QDsonto the surface of the beads141 (c) incorporation of QDs insidea silica NP with a hydrophobic core142143 and (d) microfluidicflow focusing to incorporate QDs inside the microbeads144minus146

Each method of barcode preparation has a different fluorescencesignal stability Some formulations tend to expose QDs to smallions in the buffer which leads to high read-out variability147

Hence careful selection of preparation method is requiredfor reproducibility in the biological assay This optimization isimportant for the use of barcodes in diagnosing patient samplesas outlined in the following examplesTo make use of barcodes for biological applications the sur-

face of the barcodes typically needs to be coated with bio-recognition molecules (eg oligonucleotides or antibodies)Panels of different uniquely emitting barcodes can be designedfor detecting ldquosetsrdquo of diseases For example a mixture of fiveunique emitting barcodes with three distinct target proteins hasbeen designed for diagnosing patients with HIV hepatitis B andhepatitis C The two other barcodes were used as positive andnegative controls These five barcodes can be mixed with apatientrsquos plasma and secondary probes If the patient presentsthe antigen that recognizes the antibody on the barcode surfaceand the secondary probe molecular assembly forms sandwichcomplexes of barcodeminuspatient antigenminussecondary probesThe overall optical signal of the barcode for positive detection

changes upon the assembly process and can be distinguishedby using flow cytometry148 or a smartphone camera149 Ademonstration of the use of QD barcodes for diagnosing patientsamples was reported by Ming et al who found that barcodescould be used to detect the genetic targets of patients infected withHIV and hepatitis B149 Kim et al described the clinical sensitivityand specificity for QD barcode diagnostic analysis of hepatitis B tobe greater than 90150 This is a promising step for the clinicaltranslation of this technology The QD barcode is an example of anumber of emerging nanotechnology-based diagnostic deviceswhere multiplexing occurs by the design of the barcoding system(eg Raman-based barcodes graphic barcodes)151152

Novel approaches in instrumentation have also been developedto enable automation of barcode-based diagnostic assays Micro-fluidic chips are an ideal platform for high-throughputmultiplexedread-out This approach has been demonstrated by couplingmicrofluidics with traditional Western blots (WBs) in proteinidentification153 Parallel microfluidic channels are designed toincorporate the internal molecular weight marker loadingcontrol and antibody titration in one protocol Compared tothe conventional WB that detects only one protein the micro-fluidicWB (μWB) can analyze at least 10 proteins simultaneouslyfrom a single sample while requiring only about 1of the amountof antibody used in conventional WB For nucleic acid analysesmicrocapillary and loop-mediated isothermal amplification areincorporated (cLAMP) to achieve straightforward robustmultiplexed and point-of-care testing (see Figure 8) ThecLAMP uses capillaries (glass or plastic) to introduce and tohouse samplesreagents segments of water droplets to preventcontamination pocket warmers to provide heat and a hand-heldflashlight for visual read-out of the fluorescence signal It enablesthe simultaneous detection of two regions of the HIV genomefrom multiple plasma samples As few nucleic acid detectionmethods can be wholly independent of external power supply andequipment the cLAMP holds great promise for point-of-careapplications in resource-poor settings154 To enhance thethroughput for detection and to integrate the analysis of proteinsand nucleic acids into one protocol barcode-based bioassays thatare capable of encoding and decoding are introduced A singlebarcoded microchip can carry out tens of individual proteinnucleic acid assays (encode) and immediately yield all assayresults by a portable barcode reader or a smartphone (decode)The applicability of barcoded microchips has been demonstratedby HIV immunoassays for simultaneous detection of three targets(anti-gp41 antibody anti-gp120 antibody and anti-gp36 anti-body) from six human serum samples However the barcode-based assay can also be applied for the simultaneous detection ofpathogen-specific oligonucleotides by a single chip containingboth positive and negative controls155

Another multiplex detection technology that is gaining interestis ldquochemical noserdquo sensors156 Array-based ldquochemical noserdquosensors have been designed where individual spots are coatedwith chemical agents to recognize specificmolecules in a complexanalyte mixture These systems use selective recognition ofanalytes a complementary approach to traditional biomarker-based strategies Nanoparticle-based chemical noses have beenused for sensing of sera157 cell-surface based discriminationof bacteria158 and genotyping of cancer cells159 These systemsused separate recognition elements to generate the selectivity-based pattern required for analyte identification Recentlyan alternative multiplexing strategy was used to create a high-throughput multichannel sensor that was able to determine themechanism of chemotherapeutics within minutes160 instead of

ACS Nano Nano Focus


2323

the weeks required for traditional biochemical approaches(see Figure 9) The multiplexed platform complexed a singleAuNP with three different fluorescent proteins (FPs) to detectdrug-induced physicochemical changes on cell surfacesThis result demonstrates the ability of nanomaterials to senseminute changes in cell surfaces rapidly enabling high-throughputscreening These systems are promising for a wide variety ofapplications including diagnostics for wound biofilms161

While in the above example the fluorophores were merelyused as passive labels they can also be used for active sensing inintracellular assays The functionalization of luminescent NPsand their incorporation into cells provide versatile means toimage cells However the design of functional NPs that quanti-tatively respond to specific cellular biomarkers or intracellularconditions remains challenging Several recent reviews haveaddressed advances in the application of luminescent NPs forintracellular sensing162minus164 Quantum dots have been widelyapplied as luminescent transducers for the development ofoptical biosensors165166 Quantum dots can be functionalizedwith moieties that alter their fluorescence properties in responseto particular cellular conditions or cellular biomarkers Thisapproach can be used to develop intracellular sensors As anexample coreshell CdSeZnS QDs were functionalized with adopamine-conjugated peptide monolayer to act as pH sensors(see Figure 10A) Under aerobic conditions the dopamineligands are oxidized to quinone units which quench the transferof electrons The quenching efficiency of the QDs is dependenton the redox potential of quinone units which in turn are

pH-dependent167 By altering the pH the quenching efficiency ofthe QDs can be tuned167 The ability of the QDs to respond tointracellular pH changes was demonstrated experimentally byMedintz et al167 By subjecting COS-1 cells loaded with QDs toextracellular nystatin which induces pH changes in the cells theintracellular pH changes could be quantified using an in vitro-derived calibration curve (see Figure 10B)Similarly cytochrome c functionalized CdSeZnS QDs can

be used for detection of the reactive oxygen species (ROS)superoxide radical (O2

bullminus) The O2bullminus-mediated reduction of

cytochrome c enhances the fluorescence of QDs The abilityof the QDs to respond to intracellular changes in O2

bullminus wasdemonstrated experimentally by Li et al168 By subjecting HeLaand HL-7702 cells to phorbol myristate-induced generation ofO2

bullminus the resulting fluorescence could provide a quantitativemeasure of the time-dependent concentration of O2

bullminus in thecells168

Quantum dots can also be applied for the detection ofdihydronicotinamide adenine dinucleotide (NADH)169 CdSeZnS QDs were functionalized with a monolayer of Nile Blue(NB+) which quenches the fluorescence via an energy transfermechanism (see Figure 11A) Freeman et al demonstratedthat NADH a cofactor generated via the metabolic Krebscycle induced reduction of NB+ to the colorless NBH reducedstate which resulted in the recovery of QD fluorescence (seeFigure 11B) The metabolism of HeLa cells can be inhibitedby anticancer drugs eg taxol which substantially lowersthe intracellular NADH concentration to yield low fluorescence

Figure 8 Illustration of μWB and multiplexed cLAMP carried out in a variety of forms by means of microcapillaries (A) Proteins are transferredfrom a polyacrylamide gel to a polyvinylidene fluoride (PVDF) membrane by electroblotting (B) μWB chip is assembled by incorporating apolydimethylsiloxane (PDMS) microfluidic network with the blotted PVDF membrane Panels (A) and (B) adapted from ref 153 Copyright2010 American Chemical Society (C) Microfluidic channels are oriented perpendicular to the protein bands on the membrane Antibodies forspecific proteins are introduced in parallel microfluidic channels Adapted from ref 154 Copyright 2014 American Chemical Society

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2324

Figure 9 Multichannel AuNP fluorescent protein sensor generating different responses for drug-induced phenotypes Clustering of themechanisms was performed using linear discriminant analysis enabling identification of new drugs as ldquoknownrdquo or ldquonovelrdquo Adapted from ref 160Copyright 2015 Nature Publishing Group

Figure 10 (A) Dopamine-functionalized QDs for the photoluminescence (PL) sensing of pH via the pH-dependent oxidation of dopamine to thequinone derivative and the accompanying electron-transfer quenching of theQDs (B)Differential interference contrast (DIC) and fluorescenceconfocal imaging of pH-changes in COS-1 cell subjected to internalized pH-responsive dopamine-functionalized QDs (fluorescence at 550 nm)and co-incorporated red fluorescent Fluorophorex (FLX) 20 nm nanospheres acting as an internal strand emitting at 680 nm The calibrationcurve corresponding to the intracellular fluorescence changes derived from the confocal fluorescence images is displayed at the bottom Adaptedwith permission from ref 167 Copyright 2010 Nature Publishing Group

ACS Nano Nano Focus


2325

(see Figure 11C) This latter example highlights the potentialuse of intracellular QDs biosensors for drug screeningapplications169

The potential toxicity associated with the intracellular applica-tion of QDs can be avoided by using alternatives such as C-dotsFigure 12 illustrates the application of fluorescein-modified

Figure 11 (A) Schematic analysis of NADH using NB+-functionalized CdSeZnS QDs (B) Calibration curve corresponding to the fluorescencechanges of the CdSeZnS QDs upon analyzing different concentrations of NADH (C) Time-dependent fluorescence changes observed upon theglucose (50 mM)-stimulated activation of the metabolism in HeLa cancer cells loaded with NB+-functionalized CdSeZnS QDs in (1) untreatedHeLa cells (2) Taxol-treated HeLa cells Inset Time-dependent confocal microscopy images of a HeLa cell (without Taxol treatment) upontriggering the metabolism with glucose 50 mM Adapted with permission from ref 169 Copyright 2008 John Wiley amp Sons Inc

Figure 12 (A) Schematic pH-stimulated fluorescence changes of fluorescein isothiocyanate (FITC)-functionalized C-dots (B) Fluorescencespectra of the FITC-modified C-dots at different pH values (C) Confocal microscopy images (fluorescence FL and bright-field BF) followingthe spatial pH changes in L929 cells loaded with the FITC-modified C-dots Adapted with permission from ref 170 Copyright 2013 IOPPublishing

ACS Nano Nano Focus


2326

C-dots as optical transducers in the fluorescence resonanceenergy transfer (FRET)-based detection of intracellular pH170

Carbon dots serve as the donor in the FRET pairing withfluorescein to provide a ratiometric detection of pH within aregion of pH = 40 to pH = 80 (Figure 12B) Similar experimentshave been performed with QDs modified with a pH-sensitivefluorophore171 demonstrating that the concept of active pHsensing can be carried out with a variety of different NPmaterialsDu et al170 demonstrated the use of fluorescein-functionalizedC-dots in monitoring temporal and spatial intracellular pHchanges in L929 cells using fluorescence spectroscopy (seeFigure 12C)In addition to the sensor mentioned above several other

intracellular fluorescent C-dot-based pH sensors have alsobeen reported172173 The quantification of intracellular pH isparticularly interesting since abnormal pH values are oftenassociated with certain diseases such as cancer or Alzheimerrsquosdisease174175 Moreover functionalized C-dots have beenmodified for intracellular sensing of metal ions176177 Forexample quinoline-functionalized C-dots were applied to senseZn2+ ions in the intracellular environment (see Figure 13)177

Organic and inorganic polymer NPs have been implementedas functional units for intracellular sensing applications Forexample silica NPs have been widely used to transport fluoro-phores for the intracellular sensing of oxygen levels178 pH179 ormetal ions180 In these systems the fluorophore embeddedwithin the SiO2 NPs responds selectively to the intracellularanalyte thus providing an optical output in response to stimuliOften the combination of two fluorophores where only onefluorophore responds to the respective analyte are integratedwith the NPs to enable the ratiometric detection of theanalyte For example the pH-sensitive FITC and pH-insensitiveRu(bpy)3

2+fluorophores were incorporated into SiO2 NPs The

labeled NPs were then applied for sensing the drug-inducedlysosomal pH changes in murine macrophages stimulated bychloroquine and for the dexamethasone-induced acidification inapoptotic HeLa cells181 A sense-and-treat SiO2 NPs systemwas demonstrated by the immobilization of Atto 647 dye and aDOX-functionalized polymer on the SiO2 NP The func-tionalized NPs were incorporated in Hep-G2 cells and thepH-induced release of the DOX in endosomal and lysosomaldomains of the cells was probed by the two fluorophores whilereleasing the anticancer DOX drugOrganic polymer NPs exhibit several advantages for trans-

porting intracellular sensing moieties Many different nontoxicpolymer matrices are available and their hydrophilichydro-phobic properties can be tailored for optimal cell permeationFurthermore the versatile methods available to modify polymerNPs enable the functionalization of recognition ligandsfluorophores and cell-targeting ligands and functionalitiesand minimize nonspecific adsorption Figure 14 illustrates two

general strategies to assemble polymer-based NPs for intra-cellular sensing applications One type of sensing NPs involvesthe incorporation of a fluorophore into the NP matrices where itserves as both binding ligand and optical transducer panel IThe second type of intracellular optical sensors involves themodification of the NPs with two functional elements (i) arecognition ligand that binds the analyte and (ii) a transducerthat responds optically to the recognition event panel II Thistwo-element NP composite is known as an opt(r)ode in analogyto electrochemical sensing electrodes In this approach electro-chemical reactions stimulated by the recognition complex orintracellular environmental changes eg pH changes induced bythe sensing events trigger the optical transducer in the NPsFluorescent sensors for the intracellular detection of spe-

cific ions such as Cu2+ Mg2+ and Zn2+ were developed182minus184

using ion-responsive fluorophores embedded in polymer NPs(Figure 14 panel I) An interesting optical sensor that follows theprinciple shown in Figure 14 panel I and probes intracellularlevels of H2O2 is depicted in Figure 15 Polyacrylonitrile (BPAN)NPs modified with the Schiff base ligands of aminopyridineboronate ester (50 nm diameter) were used as the sensor NPs(Figure 15A) In the presence of H2O2 oxidative cleavage ofthe boronate ester units occurred leading to the formation of thehydroxy-pyridine-substituted polymer nanoparticles whichexhibited a characteristic fluorescence band at λ = 400 nmThe intensity of the fluorescence band was correlated with theconcentration of H2O2 (Figure 15B) and other ROS species didnot interfere with it185

In yet another example researchers developed bifunctionalpolymer NPs that acted as optrodes to probe intracellular levelsof H2O2 according to the principle shown in Figure 14panel II186 They modified PEG hydrogel nanospheres(250minus350 nm) with horseradish peroxidase (HRP) and theAmplex Red (10-acetyl-37-dihydroxy phenoxazine) transducer(Figure 16A) In the presence of stress-induced and intracellularformation of H2O2 the HRP-catalyzed oxidation of AmplexRed proceeds yielding the fluorescent resorufin transducer186

The hydrogel NPs were introduced into macrophages andthese responded to exogenous H2O2 (100 μM) or endogenousperoxide stimulated by lipopolysaccharides (1 μgmiddotmLminus1)

Figure 13 Carbon-dot-based sensor for the intracellular sensing of Zn2+ ions Adapted from ref 177 Copyright 2014 Royal Society of Chemistry

Figure 14 Chemically modified polymer NPs for intracellularsensing (I) NPs function as optically responsive ligands (II)Particles functionalized with an analyte recognition unit and anoptical-transducing element The recognition event activates theoptical transducer

ACS Nano Nano Focus


2327

Similarly a nanosensor capable of monitoring intracellularglucose levels is depicted in Figure 16B) Polyacrylamide NPswere implemented as a carrying matrix for glucose oxidase GOxand the Ru(II)-tris-bipyridine Ru(bpy)3

2+ transducer While theRu(bpy)3

2+fluorescence is quenched by intracellular O2 levels

the O2-driven GOx-catalyzed oxidation of glucose depletesthe O2 levels resulting in the triggered-on fluorescence of theRu(bpy)3

2+ luminescent probe As the degree of O2 depletion bythe biocatalytic process is controlled by the concentration ofglucose the resulting fluorescence of the transducer provided aquantitative measure for the concentration of glucose187 Theuse of composite polymer NPs carrying enzymes and opticaltransducers as bifunctional sensing elements is particularlyattractive since the products generated by many enzyme-drivenprocesses may activate optical (fluorescent) transducers The useof such intracellular nanosensors should be implemented withcaution however since intracellular environmental conditionseg changes in pH might alter the enzyme activities thusperturbing the intracellular nanosensor performancesScreening Based on Surface Plasmon Resonance

Plasmonic NPs are also designed for optical read-out188minus192 inparticular in the form of colorimetric responses The aggregationof AuNPs shifts the absorption maximum to longer wavelengths

Aggregation can be induced by the selective binding of analytemolecules to the functionalized surfaces of AuNPs193 The mostpopular example in this research direction is DNA detectionas developed by Mirkin and co-workers136194195 There are nowmany similar assays For example Stevens and co-workers usedAuNPs to detect the enzyme phospholipase A2 at a concentrationof 700 pM190 A more complex scheme was used by Pompa andco-workers to detect cancer-related point mutations in theKirsten rat sarcoma viral oncogene homologue (KRAS) gene189

In this case AuNP aggregates form when the target genehybridizes with the complementary single-stranded captureprobes functionalized to AuNPs Subsequently the AuNPminustarget-gene complex undergoes a secondary binding eventwith DNA-functionalized magnetic Fe3O4 microparticles Inaddition to nucleic acid detection AuNPs functionalized withcapture antibodies and patterned onto dielectric surfaces canbe used to fabricate a protein biosensor192 The characteristicsurface plasmon resonance (SPR) of such AuNPs is shiftedby the binding of target proteins and this spectral shift canbe detected using conventional SPR imaging spectrometersTamiya and co-workers demonstrated a 300-channel versionof this device capable of label-free detection of targets downto 100 pgmL192

Figure 15 (A) Chemically modified boronate ester-functionalized BPAN NPs for intracellular fluorescence probing H2O2 (B) Fluorescencespectra changes of the modified polymer NPs upon interaction with increasing amounts of H2O2 0 20 40 60 and 80 μM Adapted from ref 185Copyright 2012 American Chemical Society

Figure 16 Bifunctional recognitiondye polymer NPs for sensing (A) Sensing of H2O2 via the HRP-driven oxidation of Amplex-Red to thefluorescent Resorufin product (B) Sensing of glucose by the depletion of O2 by the GOx-mediated oxidation of glucose using Ru(bpy)3

2+ asauxiliary fluorescent probe

ACS Nano Nano Focus


2328

Taking advantage of the high cross-section absorbance ofplasmonic NPs their SPR can also be used in plasmonic-driventhermal sensing Qin et al presented an improved lateral flowimmunoassay using AuNPs which improved the analyticalsensitivity of the method 32-fold achieving similar sensitivity toan enzyme-linked immunosorbent assay (ELISA)196 Further-more Polo et al reported a SPR-based detection of carcino-embryonic antigen (CEA) with a sensitivity 3 orders ofmagnitude better than standard ELISAs using anisotropicAuNPs197

One of the emerging techniques related to plasmon resonancethat demonstrated high sensitivity and versatility is the chiro-plasmonic method developed by Kotov and Xu198minus200 Thistechnique is based on the giant polarization rotation character-istic of nanoscale assemblies highly polarizable metallic nano-particles The chiroptical effects in these structures are severalorders of magnitude higher than in small organic molecules dueto high polarizability of the inorganic nanomaterials and largerdimension Notably the physics of chiroplasmonic detection

differs from that of red-blue plasmon coupling assays orscreening with Raman scattering based (see below) Polarizationrotation in NP assemblies is primarily based on interactionsof the electromagnetic field with asymmetric nanostructuresrather than on the formation of so-called lsquohot spotsrsquo or plas-mon coupling201 This difference is essential for biomedicaldiagnostics because it enables detection of long strands ofDNA and large proteins For instance translation-inspiring sen-sitivity was obtained for prostate specific antigen using thismethod202 Most recently this method in combination withUCNPs also enabled dual detection of one of the most promisingdiagnostic targets micro RNAs (miRNA) at the levels sufficientfor its application in cancer diagnostics203

Screening based on surface-enhanced Raman scatter-ing Surface-enhanced Raman scattering (SERS) is anotherclass of NP-based molecular detection204205 Surface-enhancedRaman scattering is an ultrasensitive molecular spectroscopytechnique that benefits from the electromagnetic fields generatedupon excitation of plasmons in nanomaterials Despite the

Figure 17 (A) Scheme showing prionmutation and prion ultradetection in human blood Surface-enhanced Raman spectra (SERS) of (a) naturaland (b) spiked human blood (c) natural and (d) spiked human plasma (e) spiked human plasma after spectral subtraction of the matrix (humanplasma) (f) the scrambled prion Adapted with permission from ref 205 Copyright 2011 National Academy of Sciences (B) (a) Optical spectraand SERS (mapped at 1548 cmminus1 as marked with the arrow below) images of 3T3 cells in the presence of capsules The SERS spectra (bottom)show the signals for the colored circles in the image (top) (b) Optical images and intracellular NO formation over time (obtained through theI1583(I1583 + I1548) relation) for three different samples upon NO induction with hydrogen peroxide (H2O2) A control sample without thepresence of H2O2 is also shown for comparison Representative normalized SERS spectra obtained at different times are shown The SERSdashed (blue) and dotted (red) spectra represent the reference vibrational pattern for aminobezenethiol and hydroxybenzenethiol respectivelyAdapted with permission from ref 212 Copyright 2013 John Wiley amp Sons Inc (C) (a) Outline of the c-Fosc-Jun dimerization on the metalsurface and the resulting deformation of the Raman label structure (b) Details of the 1000minus1100 cmminus1 spectral regions of the SERS of themolecular spring (benzenethiol) interfacing the NP and the protein c-Fos(c) Spectral shift of the benzenethiol band at ca 1075 cmminus1 as afunction of c-Jun concentration (logarithmic scale) in HEPES buffer Adapted from ref 215 Copyright 2013 American Chemical Society (D) (a)In vivo cancer targeting and SERS detection by using ScFv-antibody-conjugated AuNPs that recognize the tumor biomarker epidermal growthfactor receptor (EGFR) Top Photographs showing a laser beam focusing on the tumor site or on the anatomic location of liver Bottom SERSspectra obtained from the tumor and the liver locations by using (a) targeted and (b) nontargeted NPs Two nude mice bearing human head andneck squamous cell carcinoma (Tu686) xenograft tumors (3 mm diameter) received 90 mL of ScFv EGFR-conjugated SERS tags or PEGylatedSERS tags (460 pM) The NPs were administered via tail vein single injection SERS spectra were taken 5 h postinjection In vivo SERS spectrawere obtained from the tumor site (red) and the liver site (blue) with 2 s signal integration and at 785 nm excitation The spectra werebackground-subtracted and shifted for better visualization The Raman reporter molecule was malachite green with distinct spectral signaturesas labeled Adapted with permission from ref 216 Copyright 2008 Nature Publishing Group

ACS Nano Nano Focus


2329

impressive detection limits of SERS down to the single moleculelevel206 its application in diagnosis has been restricted untilrecently by the inherent complexity of biological samples Thusdirect SERS detection of disease markers have typically beencarried out either in lab samples where the marker was diluted ina controlled solution or in biological samples where the markeris characterized by an extraordinary affinity for the plasmonicsurfaces such as the use of SERS detection for Creutzfeldt-Jakobprionic diseases (see Figure 17A)205 The use of chemoreceptorsintermediary molecular species with selective affinity for themolecular target (ie the disease marker)207 has notablyincreased the applicability of SERS as an efficient diagnostictool Surface-enhanced Raman scattering has therefore beenapplied to ex vivo detection of drugs208 proteins209 metabolitesindicative of disease210 or even toxic ions211 but also to in vitromonitoring of relevant small inorganic molecules such as nitricoxide212 or pH levels213 inside living cells (see Figure 17B)In this approach SERS detection is achieved indirectly as thespectral changes for the chemoreceptor are monitored beforeand after reaction with the target analyte rather than directlydetecting the analyte Ideally the chemoreceptor needs to show arecognizable spectral change either structural or electronicallyupon conjugation with its target Although this property canbe found easily in small aromatic molecules such as aromaticthiols and amines porphyrins and dyes macromoleculessuch as peptides and proteins usually do not present spectrallydiscernible differences In such cases however the use ofmolecular springs as an interface between the metallic surfaceand the protein chemoreceptor can be employed to monitor theinteractions with the analytical target This strategy has beendemonstrated successfully in the quantification of the oncogenicprotein c-JUN in cell lysates by using the transcription factorFOS214 coupled with mercaptobenzoic acid as the molecularspring (see Figure 17C)215 An alternative approach to the use ofSERS for diagnostics and bioimaging relies on the highly intensesignals that can be obtained from plasmonic NPs labeled withmolecules featuring large SERS cross sections This strategypioneered by Shuming Nie has been demonstrated extensivelyfor bioimaging of tumors in mice (see Figure 17D) and for use asa contrast agent for the multiplex labeling in tissue prepara-tions217 The use of different Raman labels in combination withdifferent antibodies can further expand the multiplexingcapability of these systems Moreover a variety of membranereceptors can be targeted specifically to obtain pertinent spatialinformation218 Surface-enhanced Raman scattering NP tagshave also been used for multiplexed detection of circulatingtumor cells and cardiovascular protein biomarkers in bloodsamples219 Nie and co-workers developed a sandwich-type assayinvolving magnetic capturing beads and SERS tags for measuringa panel of four cardiac protein biomarkers (sVCAM-1 suPARHSP70 and CRP) The magnetic beads enable easy purifica-tion while the SERS tags enable simultaneous quantificationDue to multiplexing high sensitivity and the large dynamicrange of SERS multiple biomarkers can be assessed over a wideconcentration range in a single tube Since this system removesmany purification and enrichment steps SERS-based assays canprovide better positive and negative predictive values in high-riskpatients In the context of coronary arterial disease a majorclinical problem is the prediction of sudden cardiac events such asplaque rupture and myocardial infarction Hence it is importantto develop assays to distinguish high-risk populations for plaquerupture that require immediate intervention and treatment

Screening Based on Electronic Read-Out Nano-wires220minus229 are enabling a novel class of biosensors that arecapable of detecting disease markers in blood saliva and urineand able to convert the detection events into electronic signalsSemiconductor nanowire field-effect transistors (FETs) makeuse of nanowires that are sensitive to surface-binding events230

This sensitivity is derived from the proximity of the bindingsites to the charge carriers within the nanowire231minus233 Nano-engineered FETs differ considerably from conventional micro-lithography-based FETs for solution-based detection234minus236 Forexample nanoengineering enables miniaturization and alsoenables the detector to be close to the target Silicon nanowirescan detect proteins at 10 fM concentrations in low ionic strengthbuffers however the detection of analyte molecules by nanowireFETs in high ionic strength (gtmM) solutions including allphysiological solutions of interest has posed problems In suchsolutions the Debye length is reduced to 02minus20 nm and thescreening of analyte charge by the electrolytes either reducessensitivity or more often prevents detection of the analytealtogether221237 Andrews Weiss and Yang showed thataptamer-functionalized FETs can operate in full ionic strengthbuffer presumably because of the proximity of the chargedbackbone to the gated semiconductor and thus show promise forin vivomeasurements238239 A recent study demonstrated230 thatbackfilling antibody-functionalized silicon nanowires with aPEG layer extends the Debye length enabling the detectionof prostate-specified antigen at a concentration of 10 nM in100 mM phosphate buffer230 Nanowires composed of con-ductive polymers (eg polyaniline240minus243) have also beenadapted for electrical biosensing using either FET-basedtransduction244245 or chemoresistive transduction228229 Inthese systems the binding sites are either antibodies or virusparticles displaying peptide motifs both of which are capable ofrecognizing and binding analyte molecules Conveniently eitherof these two ldquoreceptorsrdquo can be entrained in the polymer from thepolymerization solution during nanowire fabrication In contrastto nanowire FETs transduction of analyte binding to polymernanowires is not completely understood Carbon nanotubeshave been also used as transducers for biosensing246 All thesebiosensors are ldquolabel-freerdquocapable of providing a signal cor-related to the concentration of analyte by immersion in a solutionwithout added reagents or process steps Thus these devices areideally suited for use in point-of-care disease diagnosticsAn electrically responsive NP sensing scheme requires

electrical contact One approach described by Gadopadhyayet al240 involves the incorporation of AuNPs into polyanilinefilms on electrode surfaces240 In this case AuNPs facilitate theattachment of single-stranded DNA (ssDNA) capture strands tothe nanowire using a thiol-based immobilization schemeBinding of target DNA to the ssDNA capture strands impedesthe redox responsiveness of polyaniline and reduces peak cur-rents by providing an insulating layer to the electrode surface240

Gadopadhyay et al demonstrated that target nucleotideconcentrations down to 10minus18 M can be detected by monitoringchanges in peak currents Similarly QDs have been used astransducer in electrochemistry based detection schemes247minus254

Biological molecules can also act as transducers that directlyconvert binding events into electrical signals This action can bein the form of ion channels which open in response to bindingevents and subsequently result in a signal cascade255 Olfactoryreceptors for example can be used for the development ofsensitive and selective odor-sensing biosensors256minus258 Thesebiosensors can screen numerous volatile compounds generated

ACS Nano Nano Focus


2330

by the human body such as exhaled breath and body odors todetect diseases and health conditions259260 Olfactory receptorshave also been combined with various transducers such as quartzcrystal microbalance261minus263 SPR sensors264265 microelectro-des266267 NPs268 graphene269270 and nanotubes271minus277 togenerate different types of read-outs For example a biosensorwith an electrical read-out was constructed by immobilizingolfactory receptors on nanotubes271272 Similarly a nanovesicle-based olfactory biosensor where nanovesicles with surfaceolfactory receptors were immobilized on nanotubes wassuccessfully used for cancer diagnosis263276277 In addition toolfactory receptors others such as hormone receptors268 tastereceptors278279 and peptides280 can also efficiently be used inbiosensors that can detect components of saliva tears bloodurine cerebral spinal fluid mucous and even tissues281282

Similarly whole cells can be used as transducers wherebyelectric currents of electrically excitable cells are recorded283

Primary cells284 and stem cells285 from patients have becomeinteresting targets that can be used as potential tools for per-sonalized medicine In addition to whole cells specific ionchannels can also be used as transducers Ion channels areinvolved in electrical signal conduction (such as in the nervousor cardiac systems) as well as in other disease areas such asoncology286 cystic fibrosis287 diabetes288 and infection289 Thegold standard for monitoring ion channel currents is the patchclamp technique290 which provides highly accurate functionalrecordings from single or multiple ion channels in cellsHowever the low throughput and the need for highly trainedoperators exclude the classical patch clamp technique frommanyscreening applications This situation changed with the develop-ment of the planar patch clamp technique291292 The latestgenerations of planar patch clamp devices like the SyncroPatch384PE (2013 by Nanion Technologies) enable recordings of upto 768 cells at a time and can be integrated into high-throughputscreening cascades of the pharmaceutical industry Nano-technology has had a leading role in shaping these technologiesby providing nanopores responsible for sealing the cell andthe detector surface Such nanopores can be used not only forelectrophysiological measurements of cells but also havebeen used in the context of DNA detection293 In most caseselectrophysiological detection is used for monitoring over-expressing ion channels which has implications in primary drugtargeting or for eliciting dangerous side effects One prominentexample is the cardiac herG channel that plays an important rolein heart arrythmia and QT-prolongation284285294minus296

At the tissue and organ levels indicators like electro-physiological signals have been widely employed for evaluationof organ function However conventional electrodes areconfronted with the mismatch between rigidplanar electrodesand softcurvilinear tissues By designing gold nanobelts withsinusoidal structures on flexible tripod substrates one is able tocreate stretchable biointegrated electrodes for direct recordingof electrophysiological signals of rat cerebral cortex withdesirable sensitivity and stability297 The transfer of thin flexibleelectronics onto polymers also enables conformal placement andmeasurements298 Given the abundance of material can-didates and nanofabrication techniques more novel and smarttools capable of monitoring function and quantity would emergeand boost the development of nanomedicine and its transitioninto practical applicationsBiomechanical AssaysNanoenabled tools can be designed

for diagnostics and therapies based on the mechanical propertiesof cells299 At the subcellular and cellular levels many biophysical

properties have recently emerged as indicators of cell physiologyand pathology as complementary or regulatory alternatives fordisease development Cell migration for example can be tracedby removing the trail of adherent cells left on a substrate coatedwith AuNPs300301 or with QDs110 The morphology of the trailscorrelates with the metastatic potential of the cells302 The forcethat adherent cells exert on a substrate can also be measured Forexample cell-traction-force microscopy based on the deforma-tion of the polymer substrates on which cells are grown is able tospatiotemporally map cell traction force as precise as a nano-newton303minus305 It can also be used to uncover previously hiddendetails of drugminuscell interactions and mechanical contributionsduring cell migration303minus305 For example previously retardedcell migration upon NP uptake was attributed to cytoskeletondisruption However the uptake of the NPs actually transformscells from the motile phenotype into an adhesive phenotype asrevealed by the increased cell traction force and altered patternsof cell traction force306 More importantly ultrasensitive celltraction force microscopy could be competitive to traditional cellbiology methods such as the 3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazolium bromide assays307 WB and flow cytometrysince early changes can be readily observed under cell-traction-force microscopy

IN VIVO TREATMENT (ldquoPARTICLE-BASED DELIVERYrdquo)Nanoparticles have been designed to treat many diseases but themost prominent disease focus has been in cancer The literatureis replete with large sets of in vivo data obtained from animals andsome NP formulations for cancer treatment have already beenapproved by regulatory agencies and have reached hospitalsshowing much reduced adverse effects compared to baredrugs308 However their therapeutic efficacies are most oftennot or only marginally improved309310 Thus there are ongoingefforts to develop systems with high therapeutic efficacies311

The key in such developments is to improve the design of thenanodelivery system by precisely controlling the functionsproperties of the materials

Nanoparticles as Anticancer Drugs The cancer drugdelivery process to a solid tumor consists of five critical stepstermed the CAPIR cascade circulation in blood accumulationand penetration into the tumor cellular internalization andintracellular drug release Thus the overall therapeutic efficiencyof a nanomedicine is determined by its efficiency in each step(see Figure 18)312 A nanomedicine efficiently accomplishing thewhole CAPIR cascade should have a high therapeutic indexCorrespondingly such a nanomedicine should have 2R2SPproperties the abbreviation of ldquodrug retention vs release (2R)rdquoldquosurface stealthy vs sticky (2S)rdquo and ldquotumor penetration (P)rdquo313

The 2R indicates the required properties of the nanomedicine interms of the loaded drug ie it must tightly retain the drugwithout burst release during the transport in blood compart-ments and tumor tissues while efficiently releasing the drugat the intracellular target to exert its pharmaceutical actionSimilarly the 2S gives the needed properties in terms of the NPsurface which should be stealthy while in blood circulation for

Nanoparticles have been designed totreat many diseases but the mostprominent disease focus has been incancer

ACS Nano Nano Focus


2331

passive tumor accumulation whereas it should become stickyonce nearby the tumor cells to interact with them for efficientcellular uptake Besides the 2R2S properties the nanomedicineshould be able to penetrate in tumor tissues to reach remotetumor cells from blood vessels Only if a nanomedicine possesses2R2SP properties will it be able to accomplish the CAPIRcascade successfully and to deliver active drugs at the righttime and place to provide an overall high therapeutic efficacyand favorable prognosis312313 Therefore the current focuson developing nanomedicines of high therapeutic index lieson tailoring the basic physiochemical properties of NPsie ldquosize surface properties and stability (3S nanoproperties)rdquoto achieve 2R2SP properties to accomplish the CAPIR cascadeefficientlyA demonstration of such a 2R2SP nanomedicine is a

dendrimer coreminuslipid shell nanoassembly mimicking a ldquocluster-bombrdquo which shows enhanced efficacy as demonstrated by Sunet al312 The nanocomplex is able to cruise to a tumor shedldquobombletsrdquo to penetrate into the tumor and then ldquofirerdquo at tumorcells to accomplish the CAPIR cascade A sixth-generationnontoxic degradable polyaminoester dendrimer with a diameterof 5 nmwas chosen as the ldquobombletrdquo because of its ability to carryanticancer drugs and more importantly its pH-dependent2-(NN-diethylamino)ethyl termini312314315 The lipid shell wascomposed of fusogenic DOPE phospholipids PEGylated DSPE-PEG lipids and cholesterol in order to keep the nanoassemblystable and to provide a stealth surface in the blood compartmentThe 45 nm NPs contained sim27 5 nm dendrimers surrounded bya PEGylated lipid layer The NPs could circulate in blood andaccumulate in the tumor via the EPR effect Once in the tumorthe fusion of the lipid layer with the cell membrane released thedendrimers intracellularly or extracellularly The nearly neutraldendrimers were shown to penetrate into tumor tissues easilydue to their size where the extracellular pH is acidic (pHsim6minus7)The dendrimers were then protonated and became positivelycharged efficiently triggering fast cellular uptake and releasingthe drug inside the cells The dendrimerminuslipid nanoassemblyefficiently accomplished the CAPIR cascade and thus exertedstrong anticancer activity in DOX-resistant tumor comparedwith typical PEGminusPCL micelles312

Nanoparticulate Delivery Vehicles A vast number of classesof nanoparticulate delivery vehicles have been reported316minus351

As demonstrated by Huynh et al317 drug-loaded deliveryvehicles are attractive because they can be passively or activelytargeted to cancer tissues to improve anticancer drug selectivityand thereby reduce severe side effects Liposomes316 are perhapsone of the most advanced delivery vehicles concerning clinicaltranslation with several formulations approved by the US Foodand Drug Administration (FDA)316 Liposome-based systemsencapsulating drugs are already used in some cancer therapies(eg Myocet Daunoxome Doxil) However liposomes havesome significant drawbacks they have a low capacity toencapsulate lipophilic drugs they are manufactured throughprocesses involving organic solvents they are often leaky and areunstable in biological fluids and aqueous solutions317

Polymeric micelles embedded with drugs can also reduceundesirable side effects Micelle formation involves the self-assembly of amphiphilic molecules in which upon separationof hydrophilic and hydrophobic moieties hollow capsules areformed In water the interior of those micelles forms thehydrophobic container whereas the outside imposes the hydro-philic interface Encapsulation of anticancer drugs withinmicellescan reduce toxicity and improve circulation318352353 Forexample nephrotoxicity is significantly reduced by loadingcisplatin into polymeric micelles318 Shielding the cisplatin-loaded core with PEG improves longevity in blood circulation byreducing acute kidney accumulation of polymeric micelles In thisregard the secondary structure of the micelle core also plays acritical role in stabilizing polymeric micelles in diluted conditionsin the blood For example since the core-forming segment in thisformulation is poly(L-glutamic acid) (PLG) cisplatin binding tothe side chain of the PLG segment induces α-helix formationRegular assembly of cisplatin-bound helical bundles in the coreis key to keeping the micellar structure intact during bloodcirculation319 Five different formulations of polymeric micellardrugs based on poly(ethylene glycol)-poly(amino acid) blockcopolymers developed by Kataoka and colleagues are alreadyin clinical translation in Asia and North America320 Specificallya polymeric micellar drug loaded with paclitaxel is close tocompleting its Phase III clinical trial and is expected to be up forapproval soon Polymeric micelles thus provide a mature plat-form with numerous advantages to improve patient complianceappreciably including outpatient treatment without hospital-izationMicelles can also be jointly fabricated with polymers as well as

lipids Microfluidic chips can be used for the controlled pro-duction of these micelles for the delivery of therapeuticagents321322 By adopting a microfluidic platform (see Figure 19)NPs of varying water content and rigidity but with the samechemical composition size and surface properties can besynthesized It is important to be able to vary the properties ofthese micelles efficiently since cellular uptake can be entirelydependent on these characteristics For example rigid or ldquohardrdquoNPs with less interfacial water between the polymeric core andlipid shell can enter cells more easily than those that are flexible(ldquosoftrdquo) In contrast bigger particles have poor accumulation incells128323 In this regard a hollow-structured rigid nanovesicle(RNV) was fabricated to entrap various hydrophilic reagentseffectively by multistage microfluidic chips in one step withoutcomplicated synthesis extensive use of emulsifiers or stabilizersor laborious purification procedures The RNVs contain a hollowwater core a rigid poly(lactic-co-glycolic acid) (PLGA) shell andan outermost lipid layer The entrapment efficiency of

Figure 18 (A) Five-step CAPIR cascade in targeted cancer drugdelivery Reprinted with permission from ref 312 Copyright 2014John Wiley amp Sons Inc (B) Needed properties of a nanomedicinecapable of accomplishing the cascade

ACS Nano Nano Focus


2332

hydrophilic reagents such as calcein rhodamine B and smallinterfering ribonucleic acid (siRNA) inside the hollow water coreof RNVs was approximately 9042

Unlike micelles polymer capsules provide a delivery platformfor encapsulating hydrophilic drugs Polymer capsules can beformed with entirely hydrophilic polymers for example theLbL approach can form these capsules by templating parti-cles324325354 While such capsules are often on the micrometerscale smaller polymer NPs can be obtained from polymercapsules by thermal shrinkage327 In this approach hollowcapsules ldquofilledrdquo with a desired medicine with an even poly-electrolyte layer number are subjected to elevated temperatures(above the temperature of the glass transition temperature of thepolyelectrolyte complex ie sim40 degC) At temperatures above60 degC polymeric NPs are formedIn contrast to the aforementioned NPs where lab-made

synthesis is required exosomes are naturally occurring nano-sized-vesicles endogenously secreted by cells328 They areinvolved in intercellular and tissue-level communication by trans-ferring biological material (ie microRNAs [miRNAs] siRNA)among cells Exosomes exhibit great potential as nanocarriers fora wide range of therapies including inflammatory diseases andcancer329 as well as for diagnosis330

Nanocarriers are not always in the form of vesicles there arenumerous other types of delivery vehicles including water-solu-ble polymers dendrimers and even polysaccharides331334340341

Water-soluble polymers in a random coil conformation can beefficient carriers of anticancer drugs324331 Recently backbonedegradable N-(2-hydroxypropyl)methacrylamide (HPMA)copolymerminusdrug conjugates synthesized by reversible additionminusfragmentation chain transfer copolymerization demonstratedhigh efficacy in treating solid tumors These conjugates containa degradable oligopeptide sequence (GFLG) in the backbone anddrug in the backbone and at the termini This design permits theuse of high molecular weight long-circulating polymer carrierswithout impairing their biocompatibility (the carrier will degradeinto fragments that will be eliminated from the body)355

Consequently as a result of maintaining the concentrationgradient (blood to tumor) for an extended period of time theEPR effect is considerably more effective the drug can accu-mulate in solid tumor to a higher extent Conjugates with

epirubicin332 DOX333 and a combination of conjugates withgemcitabine (GEM) and paclitaxel8 were highly effective in thetreatment of experimental ovarian cancer using this platformDendrimers334 are another form of polymeric NPs which have

the advantage of being nearly monodisperse in contrast topolymerization products while having a defined structure andcomposition334335 Remarkably dendrimers with adequatehydrophilicity unlike liposomes and polymer micelles do nothave a critical micelle concentration336 Therefore dendrimersshould not ldquoin principlerdquo fragment upon dilution337 Addition-ally the number of functional groups grows exponentially asthe number of generations increases These features makedendrimers a powerful platform for drug delivery Drugs can beloaded into the dendrimers in different ways such as byencapsulation or by covalent binding of the drug Drug encap-sulation can be realized in unimolecular micelles or insupramolecular assemblies338 This approach enables encapsu-lation of the most widely used chemotherapeutic agentseg DOX methotrexate paclitaxel and campthothecin335 Thecovalent binding of drugs is traditionally used to overcomethe encapsulation problems and the drugs are linked to thedendrimer surface using groups that will be cleaved followingcellular uptake (eg cis-aconityl or acyl hydrazine groups estergroups or disulfide groups)335 All these characteristics distin-guish these materials as potential carriers for different therapeuticapplications339

Another class of carrier vehicles is based on polysaccharidessuch as chitosan which are promising carriers for oral delivery oftherapeutic proteins356 The oral route is considered to be themost convenient and comfortable means of drug administrationfor patients357 Chitosan is a biocompatible polysaccharidederived from the shells of crustaceans It is a mucoadhesive agentand a permeation enhancer that is able to mediate the opening oftight junctions between epithelial cells reversibly358 A NP carriersystem formed by self-assembly of positively charged chitosanand negatively charged poly(γ-glutamic acid) in an aqueousmilieu has been developed for delivering protein drugsorally340341 The as-prepared chitosan NPs are pH-responsivein the intestines because the pKa of chitosan is approximately 65The working mechanism for this oral delivery system is that themucoadhesive chitosan NPs adhere to and infiltrate the mucuslayer on the intestinal surface The infiltrated NPs can transientlyopen the tight junctions between epithelial cells while becomingunstable and disintegrated owing to their pH-responsivenessand releasing the loaded protein The released protein thenpermeates the paracellular pathway via opened tight junctionsultimately absorbing into the systemic circulation342 Howeverthe enhanced absorption of therapeutic proteins as delivered bychitosan NPs occurs mainly in the duodenum most likelybecause chitosan is insoluble in a neutralbasic pH environmentin the jejunum and ileum343 Additionally these chitosan NPs arenot able to protect the released protein from degradation by theproteases present in the mucus layer One strategy of inhibitingintestinal protease activities is to remove essential metal ionssuch as Ca2+ from the enzyme structure using chelating agentssuch as ethylene glycol tetraacetic acid (EGTA) Furthermorethe use of chelating agents for removing extracellular Ca2+ candisrupt intercellular junctions and increase paracellular perme-ability According to reported experiments EGTA-conjugatedchitosan NPs can significantly reduce enzymatic degradation bythe specific removal of extracellular Ca2+ from the intestinallumen This enhanced stability causes intracellular internal-ization of junctional components and thereby increases the

Figure 19 Scheme of a two-stage microfluidic platform forassembling polymerminuslipid hybrid NPs with varying amounts ofwater (a) Nanoparticles composed of the lipid shell and PLGA coreare produced by injecting the PLGA solution in the first stage andlipidminusPEG solution in the second stage (mode 1 PminusL NPs)(b) Nanoparticles composed of the lipid shell interfacial water layerand PLGA core are produced by injecting the lipidminusPEG solution inthe first stage and the PLGA solution in the second stage (mode 2PminusWminusL NPs) Adapted with permission from ref 322 Copyright2015 John Wiley amp Sons Inc

ACS Nano Nano Focus


2333

absorption of protein throughout the small intestine344 A recentlarge animal study performed with beagle dogs revealed thatEGTA-conjugated chitosan NPs could successfully deliverproteins into the plasma345 Although chitosan-based NPs arepromising for oral delivery of therapeutic proteins furtherpreclinical studies are required to test their efficacy and safetyProteins can themselves form the basis of another class of drug

carriers346 The abundant plasma protein serum albumin hasbeen successfully converted into a drug transporter and waslaunched to the market in 2005 for delivering the anticancer drugpaclitaxel (Abraxane)347 Serum albumin is a nontoxic non-antigenic and biodegradable material In fact albumins areexcellent charge-reversible materials Albumins have character-istic isoelectric points due to the existence of both carboxylic acidand amino groups When the environmental pH is higher thanthe pI albumins have more negative charge on their surfaceotherwise albumins are more positively charged In addition thepI of albumins can be adjusted by changing the ratio between thecarboxylic acid and amino groups through chemical modifica-tions By tuning charge albuminNPs have been successfully usedto deliver negatively charged nucleic acids as biomolecules forgene therapy359 Recent approaches broadened the applicabilityof this successful platform and there is further development ofnovel features that aims to advance intelligent drug deliveryBoronic-acid-rich albumin NPs have been equipped with tar-geting peptides cRGD on the surface and DOX encapsulatedinside the NPs348 The boronic acid groups enhance the recog-nition of NPs to cancer cells since the boronic acids reversiblyinteract with overexpressed sialic acid residues Encouraged bythe success of albumin NPs other endogenous proteins are beinginvestigated such as milk casein349 or the iron-transport pro-tein ferritin350 Proteins in their denatured form reproduciblyenable the attachment of PEG chains as well as drug moleculesto improve circulation times and solubility In this waypolypeptideminusPEG copolymers of human serum albumin havebeen obtained that can form small micelles These micelles wereable to transport and to release multiple copies of the drug DOXin leukemia cells via a two-step release mechanism351

Similar to protein carriers ribonucleic acid (RNA) nano-technology has emerged as a potential platform for drug deliveryapplications This use is largely due to the ability to designstructures with high thermodynamic stability and favorable anddistinctive in vivo attributes360 RNA can act both as a deliveryvehicle as well as an active therapeutic agent As an example Qiuet al combined the bacteriophage phi29 DNA packaging motorpRNA three-way junction (3WJ) with folic acid a near-infrared(NIR) marker and BRCAA1 (breast cancer associated antigen 1AF208045) siRNA (see Figure 20)43 These RNA NPs comprisefunctionality for targeting imaging delivery gene silencing andregression of gastric cancer In vitro assays revealed that the RNANPs specifically bind to gastric cancerMGC803 cells The siRNAincorporated in the RNA NPs thereby significantly silencesthe BRCAA1 gene The apoptosis of gastric cancer cells wasobserved as a result of silencing the antiapoptosis factor BCl-2and up-regulation of the proapoptosis factor Rb and Baxexpression Animal trials using a gastric tumor-bearing nude micemodel confirmed that these RNA NPs could be used to imagegastric cancer in vivo while showing little accumulation in crucialorgans and tissues several hours postsystemic injection Thegrowth of the gastric tumor noticeably decreased during thecourse of treatment No damage to important organs by the RNANPs was detected All results show that RNA nanotechnologycan overcome some conventional cancer therapeutic limitations

and open new opportunities for specific delivery of therapeuticssuch as siRNA miRNA andor chemotherapeutic drugs tostomach cancer In recent work siRNA attached to glucose-modified AuNPs (AuNPPEGglucosesiRNA) were appliedin vitro to a luciferase-CMT167 adenocarcinoma cancer cell lineand in vivo to the lungs of B6 albino mice361 The siRNA-bearingNPs induced the expression of pro-apoptotic proteins such asFasCD95 and caspases 3 and 9 in CMT167 adenocarcinomacells in a dose-dependent manner independent of the inflam-matory response Moreover in vivo pulmonary delivered siRNA-bearing NPs were capable of targeting c-Myc gene expression(a crucial regulator of cell proliferation and apoptosis) via in vivoRNA interference (RNAi) in tumor tissue This led to an sim80reduction in tumor size without associated inflammation Over-all RNA NPs can improve therapeutic efficacy while reducingtoxicity and side effects and therefore provide an avenue for usein clinical tumor therapy in the near futureNanodiamonds are among the most promising carbon nano-

materials for drug delivery as well as imaging and theranosticapplications Nanodiamonds that are sim5 nm have a largeaccessible surface and tailorable surface chemistry combinedwith unique optical mechanical and thermal properties118

In drug delivery the rational surface modification of NDs allowsfor enhanced adsorption and chemical binding of the drugs forsustained or triggered drug release Both the amount of drugadsorbed and the strength of adsorption can be controlled bythe surface chemistry By attaching drugs to the surface of NDsand by achieving controlled release it is possible to renew thepotency of anticancer chemotherapeutics125 Similarly the devel-opment of NDs for targeted delivery of antibiotics and otherdrugs may also restore the drug efficiency and reduce systemictoxicity Currently NDs are being investigated for the deliveryand sustained release of anticancer chemotherapeutics119363

Figure 20 Global structure of therapeutic RNA NPs with BRCAA1siRNA (A) Design of the RNA NPs The left image shows the NPstructure as used in animal trials On the right an extended structureimaged by atomic forcemicroscopy (AFM) is shown (B) AFM imageof extended 3WJ RNA NPs The RNA complex as shown in (A) onthe left is estimated to be around 10 nm in size Due to convolution ofthe tip size (sim10 nm in diameter) in AFM images features smallerthan the size of the tip cannot be resolved To characterize thestructure of the RNA constructs the 3WJ NPs were extended by39minus60 base pairs (in red color A on the right) which is within thepersistence length of dsRNA and will not affect the 3WJ folding asdescribed before by Shu et al362 to generate the AFM image asshown Adapted with permission from ref 360 Copyright 2015Nature Publication Group

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nucleic acids364 and insulin365 Nanodiamonds have also beendemonstrated for covalent binding of proteins sometimesenhancing their activity366

Similarly inorganic NPs are frequently used as delivery vehi-cles Among the inorganic nanocarriers mesoporous silica nano-particles (MSNPs) have emerged as a multifunctional platformthat enables the development of custom-designed treatmentmodalities for disease-specific applications One example is theuse of MSNPs to address cancer-specific challenges such as thedysplastic stroma of pancreatic ductal adenocarcinoma (PDAC)The stroma serves as a treatment barrier due to inaccessibletumor blood vessels as well as the expression of cytidinedeaminase which is responsible for rapid degradation of the firstline chemotherapeutic agent GEM It is possible to use a varietyof MSNP designs to address the stromal barrier includingdrug delivery to decrease the stromal abundance or negotiatingvascular access by interfering with the action of pericytes thatclosely adhere to vascular fenestrations in the stromal bloodvessels (see Figure 21A)367minus370 In one formulation a MSNPcarrier was developed to deliver a small-molecule inhibitor of theTGF-β receptor kinase which is responsible for providing thecellular signal for the adherence of pericytes to endothelialcells371 This was accomplished by placing a polycationicpolymer on the NP surface to interact with the small moleculeinhibitor via hydrogen bonding Under acidic conditions thesmall molecule inhibitor was rapidly released in the tumorstroma When used as a first-wave carrier this NP constructopened vascular fenestrations within 1minus2 h thereby making itpossible for a second-wave carrier (eg a liposome) to deliverGEM to the tumor site (see Figure 21B)371 Another formulationincluded the development of a dual delivery carrier where a lipid-bilayer-coated MSNP (LB-MSNP) was used to codeliver asynergistic GEMpaclitaxel combination to the site of orthotopichuman tumor implants in the pancreas of a murine model372

This dual-delivery MSNP carrier was sim10times more potent thanAbraxane which recently received FDA approval in combinationwith GEM Not only did the ratiometric-designed carriersuppress the tumor stroma but it also decreased the expressionof cytidine deaminase enabling increased uptake of activatedGEM at the cancer site (see Figure 21C)372 The LB-MSNPcould also be used with considerable efficacy to deliver irinotecanto the pancreatic cancer site This result can be achieved using aremote loading method in which the drug is imported for highloading efficiency across the lipid bilayer through the use ofa proton gradient373 This carrier was more effective than aliposomal equivalent that was recently approved by the FDA butit also improved safety because of its more stable drug retentionTogether these examples demonstrate the use of multifunctionalMSNPs for the treatment of pancreatic cancerNaturally there are many more types of NPs used for delivery

applications such as CNTs graphene and also the alreadymentioned inorganic NPs such as SPIONs plasmonic NPs etcHowever due to their sheer number it is impossible to describethem all in this Nano FocusTargeting Targeting of NPs refers to delivery to specific

tissues and cells in vivo Nanoparticles can be designed to bestealthy to escape immune clearance and avoid nonspecific celluptake but should also be capable of being sticky to target tissuesand interacting with andor be taken up by desired cellsNanoparticles can passively or actively accumulate in the desiredtissue via transport through leaky vessels and unique intraorganpressures or adhere to specific biological structures in thetargeted tissue via molecule recognition to surface-bound

ligands374 Recently researchers designed smarter NPs that canturn from stealthy to sticky Switching between these twoproperties may enhance the biodistribution and targeting of NPsto diseased tissuesResearchers have found inspiration in nature by using

principles of viruses transport proteins and their interactionswith cell membranes to achieve ldquostealthy and stickyrdquo propertiesMimicking envelope-type viruses different envelope-type375376

or shell-sheddable nanocarriers377378 have been explored toenhance the distribution of NPs in target tissue Polymers withstrong antifouling properties such as PEG or zwitterionicpolymers were tethered onto NPs by cleavable anchor groupsthat are responsive to different stimuli eg pH redox andproteolytic enzymes The NPs with the stealthy shell hadprolonged circulation times and enhanced accumulation in solid

Figure 21 (A) Schematic to show the barriers and challenges that areresponsible for failed chemotherapy in PDAC This includesabundant dysplastic stroma which serves as a physical and biologicalbarrier interference in vascular access and the presence of a highlocal concentration of deaminase activity which leads to in activationof GEM Trichrome staining of human PDAC is also shown bluestaining is collagen deposition (B) Two-wave approach for PDACtreatment PEIPEG-MSNP binds to the TGFβ inhibitor LY364947The complexation is highly stable in the physiological conditions butcan be disrupted in the acidic stromal environment NIR-labeledparticles retention was increased 10-fold prior to injecting the TGFβicarrier into mice with BxPC3 xenografts (circle) with significantlydecreased pericyte coverage on endothelial cells in tumor bloodvessel Reprinted from ref 371 Copyright 2013 American ChemicalSociety (C) Schematic describing GEM trapping in MSNP poreswhich are sealed off by the LB that contains a subtoxic dose ofpaclitaxel (PTX) Ratiometric codelivery of GEMPTX by LB-MSNPinhibits PANC-1 orthotopic tumor growth through increaseddelivery of GEM at the tumor site GEMPTX loaded LB-MSNPleads to CDA inhibition in parallel with increased oxidative stressAdapted from ref 372 Copyright 2015 American Chemical Society

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tumors due to the EPR effect ie passive targeting The uniquestimuli within the targeting tissue enable the NP to release theshell thus converting from stealthy into sticky In a similardirection zwitterionic surfaces containing mixed positive andnegative charges have received great attention due to theirunique stealthy properties and their similarity to proteins Manyproteins behave stealthily to most cells and they can have longcirculation times in vivo However they can alternate betweenldquostealthy and stickyrdquo under even a slight stimulus such as a minorchange in pH379 Although the detailed mechanism of thischange has not been elucidated and might indeed be complexthe zwitterionic properties of amino acids and the mixed-chargestructures of proteins might contribute to this feature By exploit-ing pH changes in different structures (eg in tumor tissues65minus68 in endosomes 55minus68 and in lysosomes 40minus50)zwitterionic polymer-based NPs capable of altering their chargedensities in different tissues can be prepared This responsive-ness involves introducing a surface moiety sensitive to tumorextracellular acidity (pH sim68)379 as the anionic part of thezwitterionic polymer (see Figure 22)Nanoparticles can also be covered by neoglycoconjugates or

sugar-like functional groups and other functionalities suchas thiol groups to convert the NPs from stealthy to stickyThough typically uncharged such glycan-based ligands provideextremely high stability in biofluids while minimizing non-specific cell internalization Whereas much research has beencarried out with nanometer-size AuNPs381 these moieties haveonly recently been shown to work for larger AuNPs with variousmorphologies including nanorods375382 Interestingly eventhough the stealthiness was shown to be superior to that ofPEG ligands specific protein-binding was retained enabling theAuNPs to target cell types that overexpress certain proteinreceptors The NPs thus exhibit prolonged circulation times dueto the pronounced antifouling properties of the zwitterionicpolymer However when the NPs are in an acidic tumorenvironment surface charges change from negative to positiveleading to enhanced cellular uptake and accumulation of the NPswithin tumor tissue Compared with the NPs that switch theircharge states by cleavage of the responsive covalent bondscharge conversion of weakly electrolytic groups at different pHproceeds rapidly and better mimics the changes in charge densityof amino acid segments within proteins Surface engineering of

inorganic NPs viamix-charged ligands can enhance NP retentionin tumors by modulating the aggregation of NPs380383384 Themixed-charge modified AuNPs present not only a transition fromstealthy to sticky under the acidic extracellular pH of solidtumors but also a significant aggregation of NPs with muchhigher tumor accumulation retention and cellular internal-ization Aggregation of AuNPs can also shift the surface plasmonband to the near-infrared wavelengths for photothermaltherapy385 Moreover recent studies on 3D spheroids of livercells made in inverted colloidal crystals demonstrated thattargeted nanotubes display unusually fast passive diffusion in thetissues386 Such an effect originates from the effective two-dimensional (2D) translation of nanotubes over the surface ofthe cells in the tissues when the attractive forces betweennanotubes are balanced with electrostatic repulsion This findingsuggests that nanoscale design of drug delivery vehicles can resultin deep and effective penetration into the tumorsIn addition to tailoring the surface properties of NPs opti-

mizing the sizes of NPs is also particularly important for tumoraccumulation penetration and finally treatment efficacy387

particularly in targeting stroma-rich tumors388 By using micellesranging from 20 to 300 nm with the same chemical structure andphysical properties Wang et al found that the blood circulationtime and tumor accumulation of micelles increased with anincrease in their diameters with optimal diameters ranging from100 to 160 nm However higher tumor accumulation of the largemicelles (100 nm) did not result in significantly improvedtherapeutic efficacy because larger micelles had poorer tumorpenetration than did smaller micelles (30 nm) An optimal sizethat balances drug accumulation and penetration in tumors iscritical for improving the therapeutic efficacy of nanoparticulateformulations387 The size effect is more pronounced in pancreatictumors which usually have thicker stroma and hypovascularcharacteristics that limit NP penetration In the same directionKataoka and co-workers discovered that tuning the size ofpolymeric micelles in the range below 50 nm significantlyimproved the penetrability into stroma-rich pancreatic tumorand consequently improved antitumor efficacy367 In addition30 nm polymeric micelles loaded with cisplatin have proceededonto Phase III clinical trials for the treatment of pancreatic cancerpatients by extending survival times in Phase III compared tostandard treatment using GEMApart from passive targeting as described above there are also

exciting recent developments in actively targeting NPs As one ofthe early studies in designing integrin-targeted nanomedicinesRGD-modified stealth liposomes achieved higher intracellularlevels of DOX389 In recent years more novel ligands againstvarious tumor targets have been employed by making use ofdifferent targeting motifs including alpha-conotoxin ImIPHSCNK lanreotide octreotide and chlorotoxin for alpha 7nAChR integrin α5β1 somatostatin receptors and chloridechannels390minus394 However receptor redistribution induced byligand binding decreases receptor affinity for ligands A specialdimeric RGDwith proper distance between two RGDmotifs wasused to circumvent this problem395 On the other hand anelectrically neutral but hydrophobic penetration peptide wasintroduced to modify NPs against different breast cancer cellphenotypes irrespective of their receptor expression displayinglasting accumulation and better inhibition of the tumor396 Stillthere are ongoing discussions about the benefits of active versuspassive targeting34397

Besides ligand-mediated active targeting active physicaltargeting has also been demonstrated Magnetic focusing can

Figure 22 Interchange between ldquostealthy and stickyrdquo for zwitterionicpolymer micelles (left) and the mix-charged AuNPs (right) Adaptedwith permission from ref 379 and 380 Copyright 2012 and 2014 JohnWiley amp Sons Inc

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2336

enrich iron oxide NPs in the tumor region Materials mostcommonly used for magnetic drug delivery contain metal ormetal oxide NPs such as SPIONs Superparamagnetic iron oxideNPs consist of an iron oxide core often coated with organicmaterials such as fatty acids polysaccharides or polymers toimprove colloidal stability and to prevent separation into NPsand carrier medium398399 For some applications a permanentmagnet is placed in the targeted region prior to theadministration of magnetic NPs This has been done for ferricsteel implants which were placed in the subarachnoid space in anin vitro human spine model prior to NP administration into thespine400 Similarly dilute microferrimagnetic wires implantedwithin blood vessels in combination with an externally appliedmagnetic field can provide specific enrichment of simultaneouslyadministered ferromagnetic NPs401 The challenge of NPenrichment in deeper body regions was addressed by He andco-workers who deployed Halbach-like magnet arrays402 Nacevet al also developed a sophisticated approach based on fastmagnetic pulses on ferromagnetic rods that led to reversing thesign of the potential energy term in Earnshawrsquos theorem Thisprocess enabled a quasi-static stable trap between magnetsresulting in the concentration of ferromagnetic rods on a centraltarget403 These concepts are still far away from clinics Magneticdrug targeting (MDT) based on intra-arterial administration ofdrug-loaded SPIONs is closer to application in patients Thistechnology involves enriching SPIONS via a strong externalmagnetic field A proof-of-principle preclinical study wasconducted to demonstrate successful application of SPIONsfor cancer treatment404 Simultaneous injection of Mitoxan-toneminusSPIONs into the tumor-supplying vessel in rabbits and theapplication of a strong external magnetic field over a VX2squamous cell led to complete tumor remissions without sideeffects By applying only 5minus10 of the conventional chemo-therapeutic dose complete tumor remissions were achievedThe distribution profile after MDT displayed 572 of the totalrecovery of administered drug with 663 of the particleslocalized in the tumor region with magnetic targeting comparedto less than 1 of drug and NPs reaching the tumor regionduring conventional intravenous application Magnetic drugtargeting enables better enrichment of drug formulations and

consequently enables more specific treatment The majority ofmagnetic material is based on magnetic NPs either encapsulatedor coated by different inorganic coreshell coatings405 or byvarious polymer-basedmatrices which are from natural406minus408 orartificial origin409410 To translate these magnetic nanoparticlesfor clinical applications the development of methods for large-scale synthesis is required A promising approach to synthesizingfatty acid-coated SPIONs shielded with albumin and function-alized with adsorptive mitoxantrone was developed with easyscale-up synthesis pathways in order to obtain antitumor-effective magnetic NPs411 Nanoparticles derived from thisprocess have proven to be stable under appropriate storageconditions412

Delivery of Macromolecular Biopharmaceuticalsacross Biological Barriers Nanomedicines are also beingdeveloped for diagnosing and treating infectious neurodegener-ative or cardiovascular diseases413 Anticancer drugs are typicallysmall organic molecules however the number and impact ofmacromolecular biopharmaceuticals (ie polypeptides andpolynucleotides) are increasing enormously Their size andcomplexity impose significant challenges to their use as safe andeffective medicines including fast elimination and poor metabolicstability Furthermore this class of compounds notoriouslysuffers from limited permeability across biological barriers suchas cellular membranes or epithelial tissues (see Figure 23)414 Onthe other hand their medical use offers not only the ability toameliorate symptoms but also the opportunity to cure or toprevent diseases either by directly correcting hormonal disorders(eg insulin in diabetes) or by correcting inhibiting or repro-gramming genetically disordered cells The delivery of macro-molecular biopharmaceuticals preferentially via noninvasive(ldquoneedle freerdquo) approaches is therefore another importantfuture application of nanomedicines This approach takesadvantage of the fact that NPs can both stabilize and protectthe therapeutic cargo while translocating across epithelial tissuebarriers like those of the gastrointestinal (GI) tract skin orlungs415

Similar to the EPR effect which occurs at the endothelium oftumor blood vessels NPs can also accumulate in inflamedmucosal areas This phenomenon was demonstrated recently by

Figure 23 Cellular and noncellular barriers of the lung after landing on lung lining fluids (1) the drug (or eventually entire NPs) must cross thepulmonary epithelium (2) in order to reach the underlying tissue or the systemic circulation respectively Besides inhalationnanopharmaceuticals must also overcome effective clearance processes (3) provided by either mucus in the central lung (bronchi) or bymacrophages in the peripheral lung (alveoli) Reprinted with permission from ref 414 Copyright 2014 Elsevier

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2337

confocal laser endoscopy in colitis patients416 In several colitismouse models oral administration of enteric-coated NPs thatencapsulated an anti-inflammatory compound showed signifi-cant alleviation of symptoms and inflammatory markerscompared to the same dose of free drug and nanocarrierswithout enteric coating At the same time adverse effects elicitedby drugs can be reduced through targeted delivery which reducesthe dosing frequency as well as adverse side effects417418

Nanoparticles can also accumulate in hair follicles and skinfolds after transport through the stratum corneum (SC) Thisphenomenon depends on the NP size and shape As a medicalapplication it offers the potential to reach the perifollicularantigen-presenting cells without impairing the SC barrierTransfollicular delivery of ovalbumin using polymeric NPs wasrecently shown to induce a significant cellular and humoralimmune response without compromising the SC barrier by anypretreatment when combined with innovative adjuvants419minus421

Lastly pulmonary delivery of inhalation nanopharmaceuticalsis an area of emerging interest for the treatment of both systemicand pulmonary diseases Market approval of several inhalativeformulations demonstrates the preference of the public for lessinvasive means of drug delivery However the lung featuresseveral biological barriers that normally protect our bodiesagainst potentially noxious substances which need to beovercome for effective delivery422

Transport of biomolecular pharmaceuticals across barrierssuch as biofilms has also been a topic of focus For examplepatients suffering from cystic fibrosis a genetic disorder typicallyget infections by opportunistic bacteria most notablyPseudomonas aeruginosa These Gram-negative bacteria protectthemselves from attacks from the immune system by formingbiofilms Interfering with their so-called quorum sensingcommunication systems by novel types of anti-infectives mayprevent biofilm formation therefore combatting the bacteriawhile minimizing the development of resistance Using solid lipidNPs (lt100 nm) researchers observed about a 7-fold increase inpotency to reduce the virulence factor pyocyanin compared tothe free drug423 Nanoparticles can also be used to addressrespiratory problems with implications for bacterial or viralinfections Aerosolized chitosan-coated PLGA nanoparticleshave been used for in vivo genome editing by nuclease-encodingRNA of a lethally deficient surface protein B As opposed to viralvectors which caused lethal adverse effects in clinical trials thenanotechnology-based nonviral carriers can be prepared by well-controlled chemical processes and do not bear the typical risks ofviral vectors424

Stimuli-Responsive Release Controlled-release systemscan potentially reduce or alter toxicity by selectively controllingthe rate of drug release and directly killing diseased cellsResearchers have thus far developed many different controlled-release methods30 For instance transporter polymers containingpH-sensitive groups are protonated or deprotonated in thelocal microenvironment This responsiveness again leads to thedisruption of the hydrophilicminushydrophobic balance of thenanocarriers triggering drug release Alternatively cleavage ofchemical bonds in response to pH can degrade the nanocarrierand release drug molecules Acid-sensitive polypeptide nanogelshave been reported for efficient DOX loading and release DualpH-sensitive polymerminusdrug conjugates that reversed theirsurface charges from negative to positive at tumor extracellularpH (sim68) promoted cell internalization and DOX drug releaseat pH 50 (endolysosomal pH) by cleaving acid-labile hydrazinebonds425

Besides pH redox potential has recently emerged as aubiquitous natural stimulus for intracellular drug and genedelivery because of the large concentration gradient ofglutathione (GSH) in healthy (sim2minus10 mM) and tumor tissues(sim10minus25 mM) Glutathione-responsive nanocarriers have beendesigned containing disulfide bonds426427 either in the hydro-phobic block of amphiphilic copolymers428 at the interconnec-tion of two polymer blocks429 or as cross-linkers430 The redoxpotential differences of disulfide and diselenide bonds431432

could even facilitate a dual reduction-sensitive polyplex forprogrammed gene transfection by sequentially deshielding thereductive conditions of tumor tissues and consequentlydegrading in response In addition thioether bridges of higherchemical stability have been introduced offering better handlingand storage compared to disulfide bridges433 Cleavage of thethioethers is only triggered by high GSH concentrations above10 mM which maximizes the local specificity of redox triggereddrug release in tumor cellsAnother powerful stimulus to release NPs are enzymes which

can be in overabundance under pathological conditions such ascancer or inflammation Elevated local concentrations of matrixmetalloproteinases (MMPs) phospholipases or glycosidaseshave been exploited to release drug molecules from drugtransporters containing the respective enzyme substrates Forinstance MMP-sensitive nanocarriers have been designed byconjugating DOX to silica NPs via a MMP-sensitive peptide375

The delivery of the nanocarrier to the tumor exposed the carrierto the presence of MMPs in the tumor which degrades thepeptide linker and causes rapid release of the drug DOX from thesilica NPTumor cells are characteristically heterogeneous in many

aspects434 As a result nanocarriers responding to a single type ofsignal would release the drug only in the fraction of tumor cellsoverexpressing that particular signal but not in other tumor cellsthereby decreasing the overall therapeutic efficacy of the drugformulation435 For instance cancer cells may be under reducingconditions as a result of their elevated intracellular GSH levelswhich may be several-fold higher than those of normal cells436

Also many tumor cells are reported to overproduce ROS egperhaps more than 1 order of magnitude higher than healthycells437 and thus increase oxidative stress438 These heteroge-neous cells may exist in different tumors but may also coexist indifferent regions in one tumor Tumor cells at different stagesmay also have different GSHROS levels435436438minus440

To compensate for this observed tumor heterogeneity Wanget al proposed an amphiphilic SN38-prodrug formed nano-capsule that could respond to both intracellular GSH and ROS torelease the carried drug (see Figure 24)441 The thioether linkerof the prodrug was designed to undergo thiolysis in the presenceof GSH or fast hydrolysis due to ROS oxidation of the linkergiving rise to high in vitro cytotoxicity and in vivo anticancertherapeutic activity The nanocapsules had a SN38 loadingcontent of 35 wt and were able to target the tumor passively viathe EPR effect making them ideal for translational nano-medicine441

The development of intracellular stimuli-responsive drug-loaded NPs has recently attracted substantial research efforts442

Specifically the intracellular release of drugs by biomarkertriggers or intracellular environmental conditions holds greatpromise as a means to localize the drug in disease-carrying cellsConsequently this decreases the dose of drugs requiredfor treatment and reduces harmful effects on normal cellsThe recognition and catalytic properties encoded in nucleic acids

ACS Nano Nano Focus


2338

have been implemented to developmesoporous SiO2NPs for theintracellular controlled release of anticancer drugs Figure 25Aexemplifies the controlled release of the anticancer drugcamptothecin (CPT) from mesoporous SiO2 NPs (100 nmdiameter) using intracellular triggers443 The systemmakes use ofthe fact that adenosine triphosphate (ATP) is overexpressed incancer cells due to their enhanced metabolism and the fact thatspecific enzymes such as the exonuclease EndoGI are producedin certain cancer cells Accordingly the mesoporous SiO2 NPswere loaded with the CPT anticancer drug and the pores werecapped with bulky hairpin nucleic acid units The hairpinstructures included programmed sequence-specific aptamerdomains which recognize the ATP biomarker The interactionof intracellularly generated ATP with functionalized NPsresulted in the reconfiguration of the hairpin units into ATPminusaptamer complexes that were recognized by the secondaryintracellular cancer cell biomarker EndoGI The biocatalyticdigestion of the duplex domains of the ATPminusaptamer complexesresulted in the regeneration of the ATP biomarker and theEndoGI-stimulated unlocking of the capped pores leading to therelease of the CPT drug Figure 25B shows the cytotoxic effectsof the stimuli-responsive CPT-loaded mesoporous SiO2 NPs onMDA-MB-231 breast cancer cells panel I and on normalepithelial MCF-10a breast cells panel II After a time interval of48 h the cell viability of the MDA-MB-231 cells decreased by65 whereas the viability of normal cells only decreased by 20The effective cell-death of the cancer cells was attributed to theATP-EndoGI-driven release of CPT in cancer cellsMesoporous SiO2 NPs were also loaded with anticancer drugs

such as DOX and capped with various other stimuli-responsivenucleic acids responding to substances or environmental

Figure 24 SN38 prodrug formed nanocapsule responsive to tumor GSHROS heterogeneity releasing the parent drug SN38 via thiolysis in thepresence of GSH or via enhanced hydrolysis due to ROS oxidation of the linker This process thereby gives rise to high in vitro cytotoxicity andin vivo anticancer therapeutic activity Reprinted with permission from ref 441 Copyright 2013 John Wiley amp Sons Inc

Figure 25 ATP-EndoGI sense-and-treat system involvingCPT-loaded mesoporous SiO2 NPs (A) Reconfiguring the DNAcapping units via the formation of ATPminusaptamer complexesfollowed by the Endo GI digestion of these complexes which leadsto the recycling of the ATP biomarker and the release of CPT(B) Cytotoxicity of the ATPEndoGI-responsive CPT-loaded SiO2NPs toward (I) MDA-MB 231 cells (II) MCF-10a cells Entries(a) correspond to treatment of the cells with unloaded NPs and(b) correspond to CPT-loaded SiO2 NPs Gray bars correspond tocell viability after 24 h and black bars represent the cell viabilitiesafter 48 h Adapted from ref 443 Copyright 2013 AmericanChemical Society

ACS Nano Nano Focus


2339

conditions existing in cancer cells For example the in vitrorelease of DOX from mesoporous NPs was triggered by themiR-21 biomarker present in cancer cells444 or by releasing thei-motif locking units under the acidic conditions available incancer cells445 Similarly Zhang et al reported the cleavageof DNA-capped DOX-loaded mesoporous SiO2 NPs by theATP-promoted activation of a catalyticMg2+-dependentDNAzymethat was associated with the capping units446 The intracellularrelease of DOX from DNA-capped mesoporous nanoparticlesusing photogenerated ROS species has also been demonstrated447

In order to realize the potential of these stimuli-responsive NPsfully toxicity must be evaluated in cells and animalsAlthough numerous internal stimuli have been investigated

it remains a challenge to receive a sharp response to subtle changesin environmental conditions As a result many exogenous stimuli-responsive drug nanocarriers have been designed to respond toexternal stimuli such as temperature changes magnetic or electricfields and light (ie electromagnetic waves) to control the spatiallocation of drug release448449 Particularlymagnetically responsivetransporters have been developed to target tumor tissue underthe influence of an external magnetic field450 This concept hasdemonstrated great potential in cancer therapy and increasedtherapeutic efficacy at lower doses with fewer toxic effectsIn addition magnetic resonance imaging combines diagnosticsand therapy in the same system as we will emphasize in thetheranostics section Light is another attractive stimulus for theconstruction of stimuli-responsive drug nanocarriers since thisapproach enables remote and spatiotemporal drug release bytuning the wavelength energy and site of irradiation Theadvantages of phototriggered drug release are obvious howeveraccessibility of the body for light is limited451 Skin treatment isthe most obvious application of this technology but there arenumerous other methods used for drug release on or through theskin by epitopical methods Interestingly Sykes and co-workersshowed that QDs and AuNPs can accumulate and transit throughthe skin after tail vein injection Their results suggest that skinmay be a depot for light-activated NP drug release452

Photochemical drug release has also been studied in the lungsPractically all organs accessible by endoscopic tools may betreated this way453 For the combination of light and interven-tional therapy fixed-point drug release can be realized by takingadvantage of optical fibers Near-infrared laser-triggered drugnanocarriers are particularly promising due to deeper tissuepenetration lower scattering properties and minimal harmimposed on tissues Thus NIR-responsive systems are promisingplatforms for clinical applications454minus456 Several studies havedemonstrated that uponNIR irradiation457 AuNPs incorporatedin the wall of the polymer-based delivery container can convertlight into heat through a series of photophysical processesThe heat can directly destroy cancer cells or facilitate disruptionof thermoresponsive NPs resulting in the release of encapsulateddrugs458459 Controlling this process by minimizing heataccumulation can lead to nondestructive opening of capsulesand intracellular delivery of medicine460minus462 However eventhough these strategies have proven successful in severalexamples deep tissue penetration to reach less accessible targetsis still challenging and the accuracy is often low due tocomplicated tissue and bone environments inside the bodyThus the future success of this strategy is not only dependent onmaterial design but also on the development of advancedinstruments to position the therapeutic stimulus accuratelyIn ophthalmology there is a need to release drugs for the

treatment of secondary cataracts years after implantation of an

artificial intraocular lens In this case since the cornea is a goodultraviolet light absorber the use of ultraviolet light is notappropriate for treatment Photochemical drug release frompolymers by two-photon absorption can potentially overcomethese problems In addition to low molecular weight drugs NPscan similarly be released by two-photon absorption463 Examplesof such materials have been developed and successfully tested inthe lab and in animal models Thermoresponsive drug nano-carriers have been intensively investigated as drug delivery systemsThese transporters reveal high stability at body temperature(sim37 degC) but rapidly release their drug cargos within locallyheated tissue (sim40minus42 degC)464 This approach is particularlyattractive in combination with AuNP-assisted NIR plasmonicphotothermal therapy

Alternative Delivery Strategies An alternative deliverystrategy involves combining multiple antitumor drugs inone carrier465466 The poor specificity of most drugs and thecomplexity of tumors (eg multiple targets) contribute greatly tothe poor prognosis of chemotherapy Passively or activelytargeted nanopreparations usually enhance the selectivity ofanticancer drugs while the combination of therapies against morethan one single tumor target improves the therapeutic out-come467 Co-delivery of chemotherapeutic drugs and nucleicacids has shown promising results in overcoming multidrugresistance Simultaneous delivery of small-molecule drugs andnucleic acids is challenging due to the pronounced differences intheir physicochemical properties which may greatly affect theirbiodistribution and pharmacokinetics The preparation ofdelivery systems that enable the delivery of structurally differingdrugs with comparable pharmacokinetics is attractive to inhibittumor growth efficiently For example Zhang et al demonstratedthat the combined use of octreotide-modified paclitaxel micellesand salinomycin micelles eradicated both breast cancer andcancer stem cells468 Zhou et al have shown that treatment ofprostate cancer with a combination of two nanoconjugates onetargeting the hedgehog-signaling pathway of stem cells (HPMAcopolymer-cyclopamine conjugate) and the other differen-tiated cells (HPMA copolymer-docetaxel conjugate) improvedsurvival rates469 Dai et al reported a strategy combining themTOR inhibitor rapamycin and DOX-loaded octapeptideliposomes to combat triple-negative breast cancer470 Fenget al studied the synergistic inhibition of breast cancer bycodelivering VEGF siRNA and paclitaxel via vapreotide-modifiedcoreshell NPs471 Fan et al found that the reduction of tumorinterstitial fluid pressure by liposomal imatinib improved theantitumor effect of liposomal DOX472 Finally Wang et aldemonstrated the sequential killing of tumor vasculature andtumor cells via integrin-targeted polymeric micelles loaded withboth combretastatin A4 and DOX473

In addition to intravenous delivery nanomedicines can bedelivered locally Most chemotherapeutics are delivered systemi-cally where high doses result in severe side effects and only alimited amount of drug reaches the tumor site Intratumor orperitumor delivery is thought to improve these issues but thechallenge is in simultaneously achieving high drug loadingsustained and stable drug release and long-term local drugretention One platform achieved these requirements byincorporating micelles or nanocrystals of a hydrophobic anti-tumor drug such as docetaxel and paclitaxel into a thermo-sensitive injectable hydrogel474475

Tumor metastasis accounts for 90 of cancer-associateddeaths and is almost inaccessible by chemotherapy surgicaloperation or radiotherapy A nanomedicine modified with a

ACS Nano Nano Focus


2340

peptide-targeting metastasis can facilitate DOX in inhibitingmetastatic tumor growth and prevent the initiation andprogression of tumor metastasis476 Its targeting to highlymetastatic tumors was demonstrated in a double tumor-bearingmodel with both metastatic and nonmetastatic tumors in thesame mouse477 Lymph-targeting peptides can enhance nano-particle delivery to a highly metastatic tumor and itslymphatics478 Chlorotoxin a venom peptide previously usedfor brain targeting was found to inhibit metastatic tumor growthandmetastasis479 After intravenous injection CPTNPs accumu-lated in the lungs a major metastatic site for many types ofcancers which led to greater antimetastatic efficacy480 Finally amacrophage-based biomimetic delivery system inhibited meta-stasis due to its tumor-homing properties481

Regulating Cells with RNA Targeting protein-codingmessenger RNAs (mRNAs) and regulating noncoding RNAsby delivering therapeutic RNA effectors or modified derivativeshas become an important research topic in nanomedicine Inprinciple there are four different strategies to deliver nucleic acideffector molecules (i) through viral vectors that encode RNAeffectors such as short hairpin (shRNAs) usually adeno-associatedviruses or lentiviruses482minus484 (ii) via NPs based on liposomes(lipoplexes) polycations or polyamines (polyplexes)485486

(iii) as conjugates with hydrophobic molecules (eg cholesterolor tocopherol)487488 N-acetylgalactosamine489 or aptamers490

and (iv) by gymnotic (ldquonakedrdquo) delivery of chemically modifiedRNA which means functional delivery into cells without anyfurther formulation491 There are various possibilities to formnanoplexes with nucleic acid effectors For example chitosansdendrimers polyethylenimine atelocollagen or cyclodextrin canbe used to produce polymeric NPs for delivery Cationic orneutral liposomes as well as zwitterionic polymer micelles areavailable options among liposome-based NPs Several ofthese nanoplexes are now in clinical studies illustrating thatRNA-based therapeutics are considered promising for futurehealthcare Functionalization of nanoplex delivery sytems opensmany perspectives for a more specific delivery of NPs to theirtarget cellsWe will focus our discussion on relatively small nucleic acid

effectors usually not exceeding 50 nucleotides (nt U1 adaptorssiRNA duplexes) and as short as 8 nt (tiny locked nucleic acids[LNAs]) Such antisense oligonucleotide (ASO) effectors areroutinely designed with strategic nucleotide modifications toimprove various features such as stability in biological fluidscellular uptake target RNA affinity and specificity or tominimize toxicity nonspecific immune stimulation and off-target effects492 At present the most effective and widely usedchemical modifications are phosphorothioates ribose 2prime-OHmodifications (eg 2prime-O-methyl 2prime-fluoro or 2prime-O-methoxyethylmodifications) and LNA residues with their ribose moietylocked in the 3prime-endo conformation by a methylene bridge con-necting the 2prime-oxygen and the 4prime-carbon atom (see Figure 26A)Locked nucleic acid modifications make ASOs nuclease-resistantand increase duplex stability with target RNAs more than anyother known nucleotide modification without compromisingtarget specificity as long as immune stimulatory sequences areavoided493494 Gapmers are ASOs carrying an internal stretch ofat least 6 DNA oligonucleotides for the recruitment ofendogenous RNase H activities that cleave the target RNAin the RNADNA hybrid region The 5prime- and 3prime-terminalnucleotides of gapmers preferentially modified with LNAresidues (termed LNA gapmers)495 to optimize target affinityand to confer nuclease resistance (see Figure 26B) Such LNA

Figure 26 (A) Chemical nucleoside modifications frequently used fornucleic acid effectors In many cases phosphorothioate (PS)modifications (one of the two nonbridging oxygens of thephosphodiester replaced with sulfur) are incorporated to improve thepharmacokinetic properties and cellular uptake (B) LNA gapmerscomposed of terminal LNA residues and at least 6 central DNAnucleotides enable cleavage of RNA target strands as part of RNADNAhybrids by endogenous RNase H activities (C) In the U1 adaptorapproach a U1 small nuclear ribonucleoprotein (snRNP) (an abundantspliceosomal RNP) is guided to the 3prime-terminal exon of a targetpre-mRNA via a dual antisense oligonucleotide that simultaneouslyanneals to the 5prime-end of U1 RNA This blocks polyadenylation via aninhibitory interaction between poly(A) polymerase (PAP) and theU1minus70K protein As a result the pre-mRNA is still cleaved but notpolyadenylated which induces its degradation Efficient down-regulation by ldquoU1 interference (U1i)rdquowas observed with target domainsof 12minus15 nt (with up to 15 LNA residues) and 10minus13 nt in the U1 RNAbinding domain (good efficiency observed with eight 2prime-O-methyl andfive LNA residues) A PS backbonewas also found to be compatible withU1i499500 U1A U1C U1minus70k and the Sm ring proteins are integralcomponents of the U1 snRNP PAP poly(A) polymerase CPSFcleavage and polyadenylation specificity factor (including theendonucleolytic subunit 73)CA cleavage site Adaptedwith permissionfrom ref 500 Copyright 2009 John Wiley amp Sons Inc

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gapmers are applied to knocking down mRNAs and longnoncoding RNAs (lncRNAs) which have emerged like miRNAs(see below) as an abundant class of regulators of gene expres-sion496 Aberrant expression of lncRNAs is often causativelylinked to pathological processes497498

The classical RNAi effectors are siRNAs usually delivered inthe siRNA duplex format (19 bp plus 2 nt overhangs at the3prime-ends see Figure 27) although other formats have beenreported often combined with strategic chemical modifica-tions492 siRNAs can be delivered indirectly by using viral vectors(eg lentiviral vectors) that encode one or a few shRNAs (seeFigure 27) Such approaches have been explored for anti-HIVtherapy484501 as they permit controlled expression of shRNAsand increase the duration of the RNAi effectThe miRNAs representing the endogenous RNAi effector

branch are interesting targets because their aberrant expression(down- or up-regulation) is also causatively linked to manypathological processes such as tumorigenesis Here two differenttherapeutic strategies have emerged (i) delivery of so-calledmiRNA mimics (aiming at restoring the normal levels of amiRNA) and (ii) ASOs that sequester overexpressed maturesingle-stranded miRNAs called anti-miRNAs as the overarchingterm Classical anti-miRs are complementary to 15 or more

nucleotides of the miRNA single strand and are modified with2prime-O-methyl groups and PS internucleotide bonds502 RecentlyMiravirsen (Roche Innovation Center Copenhagen AS) a15-meric miR-122-specific anti-miR with a PS-modified back-bone and a mixture of LNA and DNA residues503minus505 suc-cessfully passed a phase 2 clinical trial after significantly reducinghepatitis C virus replication in liver cells503minus505 As a peculiarityof miRNAs the essential base-pairing interaction with targetmRNAs is confined to the so-called seed region comprising the5prime-terminal 6minus8 nt of the miRNA In fact there are families ofmiRNAs whose members share the same seed sequence and arelikely to interact with the same target mRNAs For the sake of astrong therapeutic effect in many cases it appears reasonableto inactivate all miRNA members of the same family This inacti-vation is achieved by a class of anti-miRs termed ldquotiny LNAsrdquowhich are uniformly PS- and LNA-modified 8-mers directedagainst the miRNA seed region Tiny LNAs specific to miR-122have been successfully delivered without any formulation to thelivers of primates and nonprimates where they down-regulatedcholesterol levels506 In addition 14-meric fully LNA-modifiedanti-miRs termed antiseeds carrying a natural PO backbone andcovering the seed region plus the central part of the targetmiRNA were shown to exert a remarkably persistent inhibition

Figure 27 Exogenous RNA effectors utilizing the RNAi machinery of mammalian cells (siRNA duplexes shRNAs miRNAmimics) or interferingwith the function of endogenous miRNAs (antimiRs) siRNA duplexes 19 bp long mimicking the processing products of the RNase Dicer areincorporated into the RNA-induced silencing complex (RISC) One of the two siRNA strands (termed the guide strand) is selected by RISC toguide the complex to complementary mRNAs sequences that are cleaved by the endonuclease Ago2 followed by mRNA decay This catalyticprocess is highly efficient as one siRNA-programmed RISC can cleave many complementary mRNA molecules In the case of small shRNAexpression vectors a single ormultiple shRNAs are usually transcribed fromRNApolymerase III promoters after theDNA vector has reached thenucleus alternatively miRNA polycistrons can be transcribed from RNA polymerase II (Pol II) promoters The miRNA primary transcripts(termed pri-miRNAs) vector-encoded shRNAs or polycistronic miRNA transcripts are processed by Drosha and DGCR8 and are exported asprecursor intermediates (pre-miRNAs or shRNAs) from the nucleus by the export factor exportin 5 (Exp 5) The final maturation to shortduplexes is catalyzed by the RNase Dicer in the cytoplasm siRNA and miRNA pathways differ after guide strand selection by RISC whereassiRNAs are fully complementary to the target mRNA and induce its cleavage miRNA strands guide the RISC primarily to the 3prime-UTR of theirtarget mRNAs where they form only partial base pairing interactions The prevailing biological miRNA effects are inhibition of the5prime-cap-dependent initiation of protein biosynthesis andor induction of mRNA transfer into cytoplasmic ldquoprocessing (P) bodiesrdquo where themRNAs are stored or degraded m7G 7-methyl-guanosine-5prime5prime-triphosphate cap at the 5prime end of mRNAs An poly(A) tail at the 3prime-end ofmRNAs

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effect against miRNA families Such antiseeds can also bedelivered as NPs with a branched low-molecular-weight poly-ethylenimine (PEI LMW-25) whereas tiny LNAs can not507

More efficient formulation of longer antiseeds relative to tinyLNAs may be of advantage for NP-based delivery into tissues notaccessible by gymnotic delivery A PO instead of PS backbonemay reduce spurious interactions with extra- and intracellularproteinsAnother promising ASO strategy is based on U1 adaptors

which are dual antisense molecules that base-pair with the3prime-terminal region of U1 RNA as part of the highly abundant U1snRNPs and with an accessible sequence in the last exon of thetarget mRNA500 This recruitment of the U1 snRNP blockspolyadenylation of the mRNA (but not its cleavage) whichconsequently results in its degradation by exonucleases499

(see Figure 26C)508 It will be interesting to see if the U1adaptor strategy is applicable to pathologically overexpressedlncRNAs that also undergo polyadenylationHere we have described short nucleic acid effectors Their

potential advantages are (i) cost-effective synthesis (ii) modu-lation and control of their properties by introduction of chemicalmodifications and (iii) facile incorporation into many NPplatforms Of course more complex RNA-based therapeuticshave been conceived As an example Qiu et al combined thebacteriophage phi29 DNA packaging motor pRNA 3WJ withfolic acid targeting a NIR marker for imaging and a BRCAA1(breast cancer associated antigen 1 AF208045) siRNA forRNAi43 (see Figure 20)Inorganic NP-Mediated Cell Death Most nanoparticle

formulations for cancer treatment act as delivery vehicles ofcytotoxic agents to induce a cytostatic effect on tumors Anotheremerging application of nanomedicine is the direct use of NPs ashighly efficient and low-toxic drugs without carrying drug

molecules the inorganic NPs themselves are used as thetherapeutic agent For example GdC82(OH)22 with a doselevel as low as 10minus7 molkg exhibits an antineoplastic efficiency intumor-bearing mice509 The GdC82(OH)22 NPs directlypossess a strong capacity to improve immunity510511 andinterfere with tumor metastases in vivo512minus515 with tolerabletoxicity in vivo and in vitro The high antitumor efficiency of theseNPs is not due to direct toxic effects induced on tumor cellsindicating that the anticancer mechanisms are different fromclassical cancer therapeutics Results from recent studies suggesta new concept of ldquocaging cancerrdquowith GdC82(OH)22 NPs versusldquokilling cancerrdquo by classical therapeutics The ldquocaging cancerrdquoconcept515516 refers to rendering the tumor microenvironmentinhospitable for tumor growth by forming an induced fibrouslayer around the surface of a solid tumor while using theGdC82(OH)22 NPs to regulate the diverse components ofthe tumor microenvironment By employing this concept theGdC82(OH)22 NPs were able to prevent tumor resistance totherapeutic drugs513 while reducing ROS production in tumor-bearing mice Furthermore these NPs mitigated damage to themain organs by decreasing all metalloprotease enzyme activitieswhile restoring other parameters such as oxidative stress close tonormal levels517518 These NPs also induced dendritic cells tobecome functionally mature and increased their capacity toactivate allogeneic T cells in tumor-bearing mice510511519

Tumor angiogenesis can also be inhibited by simultaneouslydown-regulating more than 20 angiogenic factors513 Moreinterestingly GdC82(OH)22 NPs were found to be capable ofinhibiting triple-negative breast cancer cells by blocking theepithelial-to-mesenchymal transition resulting in the efficientelimination of cancer stem cells (CSCs) in vitro and in vivo520

In comparison the use of classical anticancer drugs like paclitaxelresulted in a relatively high enrichment of CSC and promoted

Figure 28 (A) Relative catalytic activity of galactosidase in the presence of three different shapes of ZnO NPs The relative catalytic activity ofgalactosidase with each of the ZnONPs was normalized with respect to free enzyme activity (B) Pictorial representation of pyramid-shaped NPsinteracting with the active site of galactosidase (CminusE) LineweaverminusBurk plots of galactosidase activity with various concentrations of ZnO(C) nanopyramids (D) nanoplates and (E) nanospheres Adapted from ref 524 Copyright 2015 American Chemical Society

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tumor sphere formation and abrogation of tumor initiation andmetastasis probably due to its selective toxicity to regulartumor cells only but not to CSCs520 However the nontoxicGdC82(OH)22 NPs selectively target CSCs but not regulartumor cells under hypoxic conditions Such effects have not beenobserved in normal stem cells The direct use of nontoxicnanomedicines with new acting mechanisms like ldquocaging cancerrdquoinstead of killing cancer can be considered a potentiallyrevolutionary shift in cancer therapeutics in clinical applica-tions521 In the future such strategies based on shutting down thetumor infrastructure will gain more importance due to theirreduced side effects Despite the broad medical utility of this NPsystem the use of Gd-based nanomedicine has recently facedcontroversy related to potential toxicity issues522523

In another example Cha et al proposed ZnO NPs as atherapeutic agent since these NPs can be engineered to specificshapes that act like biomimetic enzyme inhibitors524 The ZnONPs were able to inhibit the activity of beta-galactosidasereversibly in a shape-dependent manner (see Figure 28A)That inhibition was not the result of protein denaturation butrather a subtler conformational frustration of the enzyme thatmore closely resembles the mechanism of naturally occurringenzyme inhibitors Furthermore Cha et al demonstrated that theNP-based enzyme inhibition can be characterized using theclassical MichaelisminusMenten and Eadie-Hofstee formalisms(see Figure 28CminusE) As many traditional pharmacological thera-peutics are enzyme inhibitors this work opens the door forthe design of a new class of degradation-resistant inorganicNP enzyme inhibitors that can be particularly suitable forantibiotic resistant bacteria exemplified by methicillin-resistantStaphylococcus aureus (MRSA see below)Another approach used to design NP-based drugs to induce

apoptosis in malignant cells is based on the cross-linkinginduction of the surface noninternalizing receptors Receptorcross-linking involves the biorecognition of high-fidelity naturalbinding motifs with cell receptors or with side chains of water-soluble polymers The general design concept of drug-free macro-molecular therapeutics is shown in Figure 29 An important featureof these designs is the absence of low-molecular-weight cytotoxiccompounds The concept was validated for the treatment of non-Hodgkin lymphoma (NHL) using nanoconjugates that targetCD20 (noninternalizing) receptors on B cells The system iscomposed of two nanoconjugates (a) an anti-CD20 Fabprime frag-ment covalently linked to a biological moiety (oligopeptide oroligonucleotide) and (b) a polyHPMA grafted with multiplecopies of the complementary oligopeptideoligonucleotideExposure of human NHL Raji B cells to anti-CD20 Fabprime-Motif 1decorates the cell surface with peptidesoligonucleotidesFurther exposure of the decorated cells to graft copolymerP-(Motif 2)x results in heterodimerization or hybridization andtriggers the cross-link between CD20 receptors and initiatingapoptosis in vitro and in a NHL animal model in vivo525minus527

Magnetic resonance imaging and flow cytometry analysisindicated that no residual cancer cells were detectable in long-term survivors (125 days)527 Multimodality imaging studiesinvestigating the interaction between cellular membranes andnanoconstructs confirmed that self-assembly plays a criticalrole in this highly specific therapeutic system528529 This newparadigm demonstrated activity in the treatment of cells isolatedfrom patients with chronic lymphocytic leukemia and mantle celllymphoma530531 This interdisciplinary strategy has potentialto overcome rituximab-related resistance and become a newdrug development pipeline for the treatment of NHL and other

B-cell-derived hematological neoplastic diseases and auto-immune disorders532

Killing Cells with Inorganic NPs upon External StimuliInorganic NPs can respond to external stimuli to elicit tumor celldeath In the following section we describe three commonexamples of external stimuli-driven NP therapies includingphotodynamic therapy (PDT) photothermal therapy (PTT)and magnetic hyperthermia533minus535

In its simplest form PDT relies on the interaction between aphotosensitizer (PS) a light source and oxygen A PS is anontoxic compound capable of absorbing light and producingROS upon de-excitation producing ROS Photosensitizerexcitation is usually achieved via one-photon transition betweenits ground state S0 and a singlet excited state S1 At this point themolecule may return to its ground state via emission of a photon(fluorescence) but the excited PS may also generate a tripletstate T1 by intersystem crossing The lifetime of T1 is longer(microseconds) than S1 (nanoseconds) enabling the system toreact in one of two ways called the Type I and Type IImechanisms The Type I mechanism involves electron transferbetween the PS and a substrate yielding free radicals that canthen react with oxygen to form ROS such as superoxide radicalanions In the Type II mechanism singlet oxygen is generated viaan energy transfer between the PS and triplet oxygen536

Desirable characteristics of PSs are (i) high absorptioncoefficient in the spectral region of the excitation light preferablyin the NIR emission range if PSs are to be used in PDT of cancerstates (ii) a triplet state of appropriate energy (ET gt 95 kJ molminus1)to enable efficient energy transfer to ground state oxygen(iii) high quantum yield (QY) for formation of the triplet stateand long triplet state lifetimes (τ gt 1 μs) and (iv) high photo-stability Organic dyes and aromatic hydrocarbons are oneclass of molecules that possess these properties They includedyes such as rose bengal eosin and methylene blue that areeffective PSs because they possess triplet states of appropriateenergy They have high singlet oxygen yields with rose bengal

Figure 29 Schematic of overall design and possible mechanism ofdrug-free macromolecular therapeutics for treatment of NHLAdapted from ref 527 Copyright 2014 American Chemical Society

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reaching a value of Φ = 071537 and strong absorption bands inthe green region of the spectrum Aromatic hydrocarbons suchas napthalenes anthracenes and biphenyls have also beeninvestigated as PSs Studies found that the efficiency of energytransfer to triplet oxygen is greatly affected by the substituents onthe biphenyl ring and by the polarity of the solvent Finallyquinones are good PSs with QYs in the range of 011minus076538but sometimes they lack strong absorption bands at wavelengthsshorter than 600 nm Porphyrins phthalocyanines and relatedtetrapyrroles are another class of molecules that were identifiedas excellent PSs Porphyrins and their analogues are ideal PScandidates because they occur naturally in biological systems andtherefore generally lack cytotoxicity in the absence of lightThese compounds have high QYs and properties that can beeasily tuned by changing the substituents on the macrocycle butthey are susceptible to photobleaching Also worth mentioningare naphthalocyanines which bear a second benzene ring on theprimary phthalocyanine ring This addition is responsible for anattractive red-shift in absorption from 680 to 770 nm Chlorinsderived from the porphyrin structure through saturation of oneor two double bonds also have absorbance spectra red-shiftedinto the therapeutic spectral window The most studied memberof this class is hematoporphyrin which has high singlet oxygenproduction QY of 065 but unfortunately only a weak absorptionband at 630 nm (ε = 3500 Mminus1 cmminus1) Some transition metalcomplexes have been identified as efficient photosensitizershaving singlet oxygen QY comparable to the well-studied organiccompoundsmethylene blue and rose bengal Ru(II) tris-bipyridineand [Ru(bpy)3]

2+ in particular are effective singlet oxygenproducers with a QY of 073 in oxygen-saturated deuteratedmethanol and 022 in water539 Lastly little attention has been paidto semiconductorNPs as PSsHowever singlet oxygen productionand subsequent cytotoxicity from photoexcited semiconductorssuch as TiO2 and ZnO have been reported540

In addition to the aforementioned requirements theapplication of PDT to the treatment of cancer or other patho-logical conditions places other requirements on potential PSsFirst the compound should have low dark toxicity (ie it shouldnot be toxic to the organism in the absence of light) Second itshould selectively accumulate into the affected tissue in order tominimize side effects and maximize cytotoxic efficiency In factthe half-life of singlet oxygen in biological systems is lt004 μstranslating into a radius of action of lt002 μm541 Finally strongabsorbance in the red-NIR portion of the spectrum would beadvantageous for the PS as these wavelengths are able topenetrate the deepest into biological tissue (see Figure 30)The ability to excite a PS in this region would increase ROSproduction efficiency lowering the number of scattered photonsand achieve maximum attainable treatment depthKeeping in mind these requirements PSs for PDT of cancer

have been classified into three generations (i) The firstgeneration consists of hematoporphyrin derivative (HpD) andits analogues Beginning in the 1970s this compound wasthe first tested and authorized for clinical use HpD (stage 1)is obtained from hematoporphyrin and is a mixture of sim10 com-ponents Descending directly from HpD and now one of themost widely used PSs porfimer sodium (Photofrin Sinclair) isHpD stage 2 which is HpD purified from its monomeric fractionvia high performance liquid chromatography Despite being themost studied this group of PSs suffers from major drawbackstheir selectivity is poor and only 01minus30 of injected PS is foundin tumor tissue542 The cutaneous tissue can take up and retainthese compounds for up to 10 weeks after intake thus causing

photosensitivity in the patient Furthermore their absorbance inthe NIR optical window is weak limiting the efficiency of lightuptake (ii) The second generation has been designed with theaim of overcoming previous limitations and includes structurallydistinct compounds with long-wavelength absorption and highsinglet oxygen QYs Part of this generation is hematoporphyrinderivatives with enhanced solubility properties for fasterclearance from the organism and chlorins Meso-tetrakis(m-hydroxyphenyl)chlorin (m-THPCTemoporfin commercial-ized as Foscan Biolitech Pharma) for example causes lessphotosensitization than Photofrin but is 100 times morephotoactive Other members of the group are purpurineswhich have the disadvantage of being highly hydrophobic andphthalocyanines (iii) The third generation focuses on PSs withadditional properties through the use of composite structuresBetter selectivity toward the tumor tissue is often achieved byconjugation of PSs to monoclonal antibodies or other ligandsthat bind specifically to the surface of cancerous cells Also theuse of delivery vehicles like proteins or NPs leads to improvedoverall hydrophilicity and higher PS loads (iv) Emerging PDTsystems target the development of mesoscale carriers which canincorporate large numbers of sensitizer molecules Thesemesoscale particles are surface modified to minimize removalby immunogenic response mechanisms and reduce nonspecificbinding Specific antibodies attached to these larger functionaldelivery particles offer advantages over simpler PSs which havelow absorption cross sections and which do not localizespecifically to tumorsThere are three main mechanisms by which PDT mediates

tumor destruction direct killing vascular damage and activationof an immune response against tumor cells In the first case theROS generated by PDT can kill tumor cells directly Howevercomplete eradication is not always realized because of thenonhomogeneous distribution of the PS within the tumor non-homogeneous penetration of light and the possibility of limitedoxygen availability within the tissue due to the photodynamicprocess itself Vascular damage on the other hand targets thetumor vasculature system and therefore the concentration ofnutrients and oxygen that reach it In this case the precise action

Figure 30 Relative absorbance of body tissue components such aswater hemoglobin (Hb) oxygenated hemoglobin (HbO2) andmelanin The window that will allow the deepest penetration is the600minus1200 nm region in the red-NIR Adapted from ref 547Copyright 2001 Nature Publishing Group

ACS Nano Nano Focus


2345

mechanism is not yet fully understood but a biphasic vascularreaction following PDT has been suggested543 namely a firstimmediate response consisting of vasoconstriction and after 3 ha second long-term response characterized by thrombusformation Both of these effects were correlated with delay orinhibition of tumor growth Depending on the kind of pathologyto be treated the interval between the sensitizer administrationand light exposure might be a deciding factor for one mechanismover the other When a short interval (minutes) is allowedbetween drug and light administration the sensitizer predom-inantly accumulates in the vascular compartment thus favoringindirect tumor killing by vascular damage Conversely a longerinterval (hours) between intravenous drug injection and lightadministration results in localization of the PS within theextravascular compartment of the tumor and subsequent directtumor-killing action Therefore administration of PSs at multipletime intervals before light activation (fractionated drug-dosePDT) is found to be the most effective way to target both tumorblood vessels and tumor cells More recent studies in the late1980s and early 1990s reported infiltration of lymphocytesleukocytes and macrophages into PDT-treated tissue indicatingactivation of the immune response Differences in the natureand intensity of the inflammatory reaction between normal andcancerous tissues could contribute to the selectivity of PDT-inducedtissue damagePhotodynamic therapy is a reliable tool for treatment of a

variety of diseases whose clinical use is common and wellestablished This process involves two major steps theadministration of a PS and the delivery of light The admini-stration of a PS can be topical or intravenous depending on thepathology to be treated and the ease of access to the area skindiseases that allow for topical application will require drugswith different characteristics than will diseases for which anintravenous approach is required Alongwith vascular permeabilityand interstitial diffusion chemical properties like molecular sizeconfiguration charge and the hydrophilic or lipophilic character ofthe compound will all impact transport and retention of the PSConventionally PDT is administered as a single treatment

using the highest PS dose that avoids systemic toxicity and skinphotosensitivity and the highest light fluence rate that avoidstissue heating and photochemical depletion of oxygen Howeverfor reasons discussed above there have been a few studies aboutf ractionated PDT in which the PS administration is spread overtime Preliminary studies have shown improved responses to thiskind of metronomic PDT compared to single doses most likelydue to the simultaneous action of direct tumor cell killing andblood vessel damage Light can be delivered by surface orinterstitial irradiation Surface irradiation includes cases wherethe light source is external to the body (eg treatment of skinlesions) or is placed within and illuminates the inner surface of abody cavity (eg a hollow organ or a surgical resection space)Interstitial treatments are usually carried out by placing an opticalfiber that carries the light directly into the tissue Typically1minus5W of usable power are required in the 630minus850 nm range atirradiances of up to several hundred mWmiddotcmminus2 in order to delivertreatments in tens of minutes For this reason there are threemain classes of clinical PDT light sources lasers light-emittingdiodes (LEDs) and filtered lamps (see Figure 31) Lasers arepowerful and efficient but they are expensive single-wavelengthdevices where a separate unit is required for each photosensitizerIn the past few years LEDs have become a viable technology forPDT particularly for irradiation of easily accessible tissuesurfaces The main advantages over (diode) laser sources are

the low cost and ease of configuring arrays of LEDs into differentirradiation geometries On the other hand the relatively poorelectrical-to-light conversion efficiency (less than 15) remainsan obstacle because the generated heat must be removedFinally a number of lamp systems can be spectrally filtered tomatch any photosensitizer and they allow flexible geometriesTheir drawback is that they are not suitable for endoscopic usebecause they can be efficiently coupled only into optical fiberbundles or liquid light guides (5minus10 mm diameter)544

Photodynamic therapy design is still a relatively youngfield and substantial improvements could come from betterPSs higher absorption in the optical window larger extinctioncoefficients more efficient singlet oxygen production and betterchemical and physical properties Interesting new directionsthat could overcome some of the intrinsic limitations of currentcompounds are currently being explored (i) In two-photonPDT the PS ground state is able to absorb two photons of NIRlight simultaneously which can be administered in short pulsesthe total energy absorbed is the same as that from one-photonabsorption at λNIR2 and the resulting photophysical andphotochemical processes are the same This two-photonabsorption enables a much wider choice of PS absorbing athigh extinction coefficients in the visible while retaining thepossibility of using light in the biological optical window Alsothe quadratic dependence of the two-photon absorptionprobability can be exploited to give more precise spatialconfinement of the PDT effect by high-numerical-aperturefocusing of the activating laser beam One of the drawbacks ofthis technique is that in order for the photons to be absorbedsimultaneously the light pulse must typically be shorter thanca 100 fs a factor that substantially increases the cost of lightsources (ii) Another concept currently being investigated is theuse of molecular beacons to improve target selectivity Molecularbeacons comprise a targeting moiety a PS and optionally aquencher In their simplest form the targeting group isresponsible for accumulation of a higher number of PSs in thecancer cells and therefore higher selectivity Alternatively atarget-labile linker keeps a PS and a quencher in close proximityso that FRET prevents activation of the PS Upon interactingwith the target the linker is either broken or opened so that thePS and quencher are separated enabling FRET-driven PDTaction to occur (iii) Finally NP technology provides several

Figure 31 Surgeons delivering laser light through an optical fiber forthe treatment of an internal tumor

ACS Nano Nano Focus


2346

different novel approaches to PDT A first approach is the useof NPs as PS carriers a method already applied to many otherimaging contrast agents and therapeutic compounds The advan-tages are that the loading of PSmolecules per NP can be high andthat theNPs can impart new desirable qualities to the compoundsuch as greater hydrophilicity or targeting ability Examplesinclude the incorporation of PS into modified silica NPs or intolipoprotein NPs with antibody targeting capabilities thedecoration of AuNPs with PS or the use of NPs that arethemselves photoactive Another approach is the use of NPminusPSconjugates where the NP serves as the primary light absorberthat through FRET activates the PS An issue with all these NPstrategies besides potential toxicity is the delivery of the NPs orNPminusPS conjugates to the target tissues because the pharma-cokinetics are strongly dependent on the NP propertiesIn contrast to PDT which kills cells by the generation of ROS

hyperthermia kills cells by heat Tumor treatment by heat is oneof the oldest ways to cure cancer The application of pyrogensinduces endogenous hyperthermia where heat is generated by asubstance or agent that reacts with the human immune systemThis was the standard treatment for inoperable tumors until themiddle of the 20th century After administration of variousbacterial toxins (or later on vaccines) and the subsequentfeverish reaction a significantly higher survival rate was observedBecause of the inability to control the induced fever and theinsufficient outcome it is now applied only rarely545 Modernhyperthermia can be performed using plasmonic NPs and lightas well as magnetic NPs and alternating magnetic fields InorganicNPs act as local heat sources triggered by light or alternatingmagnetic fields Hyperthermia can be used as a stand-aloneapproach or in combination with certain chemotherapeuticsOne of the most promising therapeutic strategies in

nanomedicine is that of photothermal cancer therapy546

In this drug-free therapy these NPs absorb energy at awavelength of 700minus1000 nm547 and generate heat They canburn off the tumor when the irradiation occurs after the particleshave accumulated in the tumor Intensity of the incident laserirradiation the nature of the NPs (ie the localization of thesurface plasmon resonance) and the size of NPs are the factorsthat will determine the efficiency of the localized heating It wasshown at an in vitro level that such localized heating can beapplied to kill cancer cells459548minus552 In vivo the highly localizedhyperthermic response induced by resonant laser irradiationresults in tumor remission for subcutaneous tumors with highefficacy (see Figure 32)553 Researchers have demonstrated bothpassive and active uptake of nanoshells a type of heat-generatingNPs into tumors554555 In addition to nanoshells other plas-monic NPs such as gold nanorods and gold nanocages have beenshown to be useful for hyperthermia applications549556

In preparation for transition to clinical trials the toxicity ofnanoshells was investigated extensively No toxic effects werefound in this study557 Recent improvements in nanoshell designcan enhance their clinical utility The addition and integration ofimaging functionalities with the therapeutic function of theseNPs resulted in NIR-enhanced fluorescence and NIR-enhancedimaging of tumors558minus560 Smaller diameter multilayer NPsknown as ldquonanomatryoshkasrdquo which also have NIR-absorptive

responses can enhance NIR fluorescence for emitters encasedbetween their inner and outer gold layers561 These NPs haveshown increased uptake in tumors relative to nanoshells andoutstanding remission rates with active targeting of aggressivetriple negative breast cancer tumors562

Local heat can also be produced by magnetic NPs uponexposure to alternating magnetic fields564minus567 The basicprecondition for effective magnetic heating is that the NPshave adequate magnetization values Iron oxides (magnetitemaghemite) are particularly preferred since they have been usedas contrast agents in radiology for many years eg in thedetection of cancers in the liver and spleen The generation ofheat is based on NP morphology as well as on the energydissipation during the NPs magnetization process568minus570

This means that the magnetization reversal of NPs with corediameters larger than 100 nm is governed by the motion ofmagnetic domain walls In contrast cores that are smaller than100 nm behave like single-domain NPs where no domain wallsexist Even smaller NPs behave as if they are superparamagneticand the magnetization reversal proceeds either by rotation of themagnetic moment inside the core (Neel relaxation) or bymechanical rotation of the whole NP (Brown relaxation) duringexposure to an alternating magnetic field It has been demon-strated that sufficient thermal energies can be obtained with NPswith core sizes between 10 to 50 nm571minus573 Concerning NPsynthesis the current challenge is the stabilization of NPs with a

One of the most promising therapeuticstrategies in nanomedicine is that ofphotothermal cancer therapy

Figure 32 Evaluation of tumor response to PTT by bioluminescenceimaging The bioluminescence signal is generated only in livingcancer cells as a result of luciferase activity (A) Representative miceof each experimental group showing the luciferase activity in thetumor The mice injected with nanomatryoshkas or nanoshells andtreated with laser experienced loss of bioluminescence in the areailluminated by the laser as seen after therapy Mice were euthanizedwhen tumor volume reached 1500 mm3 or if the tumor persisted at60 days after treatment (B) Luciferase activity in the tumor Theluciferase signal was normalized to the signal before treatmentAdapted from ref 563 Copyright 2014 American Chemical Society

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2347

relatively larger core size distribution574 Promising materials inthis context are magnetosomes derived from magnetotacticbacteria which were successfully deployed in a mouse breastcancer model575 To enhance treatment efficacy coappliedchemotherapeutics or drug-carrying magnetic NPs are includedCisplatin in combination with magnetic heating exhibitsadditive cytotoxic effects particularly when it reaches its targetsite of action where it forms DNA adducts after its chloride ionshave been displaced by hydroxyl groups576 A promisingNP formulation with cisplatin payloads was developedby Unterweger et al406 Most magnetic hyperthermia develop-ments for treating diseases are in the preclinical stage with a fewused in the clinicBiodegradation There is increasing awareness of the

potential threats of using exogenous nanomaterials in vivoIn particular the development of increasingly sophisticatednanocarriers further increases the risk of undesired side effectsdiscussed below Therefore it is highly desirable that drugdelivery vehicles exert only minimal effects on the biologicalsystem after their cargo has been delivered where the vehiclesshould ideally be eliminated after delivery to target tissuesConsequently in vivo degradation with well-defined rates andwith the formation of nontoxic and degradable byproductsthat can be excreted becomes increasingly important577578

Currently there are a number of organic NPs that can bedegraded which will be discussed belowWater-soluble polymeric drug carriers are one such class of

compounds that can be designed to be biodegradable For thesecarriers molecular weight and molecular weight distribution areimportant factors in their design impacting both theirtherapeutic efficacy and their biocompatibility The higher themolecular weight of the polymer carrier the higher theaccumulation of polymerminusdrug conjugate in the tumor tissuewith concomitant increase in therapeutic efficacy331579 How-ever the renal clearance threshold limits the molecular weight ofnondegradable polymeric carriers (such as HPMA-based) tobelow 50 kDa580 This issue lowers the retention time of theconjugate in circulation with simultaneous decrease inpharmaceutical efficiency Consequently higher molecularweight drug carriers with nondegradable backbones depositand accumulate in various organs impairing biocompatibilityThese concerns can be addressed by the incorporation ofdegradable polymer backbones such as those seen in backbonedegradable (second generation) HPMA copolymer carriersThese carriers have long circulation times while preservingbiocompatibility and are based on the combination ofcontrolledliving radical polymerization and click chemis-try355581582 The backbone degradable HPMA copolymerminusdrug conjugates provide long-circulating water-soluble ther-apeutics that effectively accumulate in tumor tissue They areeliminated from the body after enzymatic cleavage of theoligopeptide segments in the backbone (producing polymerfragments below the renal threshold) and in side chains(releasing the drug) The synthesis procedures proposed areversatile they provide a platform for the preparation of a largevariety of polymerminusdrug conjugates with tailor-made propertiessuch as predetermined circulation time and compositionControlled degradationnonspecific cargo release from slow

hydrolysis in physiological fluids like the bloodstreamremainsa challenge Therefore design of biodegradable polymersrequires incorporation of fast and efficient polymer degradationat the target site while retaining high stability in circulation and atoff-target sites Polypeptides offer great potential in this aspect

since their stability can be adjusted based on their monomersequence their molecular weight or by attaching stabilizing ordegradable side chains along the backbone The most commonlyused hydrophilic polypeptides have protonated amino sidechains eg poly(lysine) poly(arginine) poly(glutamate) andpoly(aspartate) which ensure excellent water solubility andincrease the interaction with the negatively charged cellmembrane to facilitate cellular uptake Amphiphilic blockcopolymers of two or three homopolypeptides have beenprepared with hydrophobic segments such as poly(leucine)and hydrophilic segments such as poly(lysine) that formpolymersomes and encapsulate hydrophobic drugs Howeverhighly positively charged polypeptides often reveal uncontrolledprotein absorption immediate immune responses opsonizationby macrophages or rapid clearance from the bloodstream byrenal or hepatobiliary excretion To overcome these limitationswhile taking advantage of the aforementioned propertiesinnovative materials in biological systems that have a degradablebut peptidic nature are highly desirablePolymers based on the bodyrsquos own amino acid sarcosine

(N-methylated glycine) have been obtained and are one of thefirst polymers synthesized by living ring-opening polymer-ization583 The polypeptoid polysarcosine offers high watersolubility nonimmunogenicity584minus586 and protein resistance587

due to its special characteristics eg weak hydrogen bondacceptor with the lack of hydrogen donor capabilities588

Polysarcosine is a nonionic and highly water-soluble polymerthat is similar to PEG but potentially offers in vivo degradability assuggested from in vitro studies under physiologically relevantconditions589 The synthesis of block copolymers based onpolysarcosine and polylysine polycysteine polyglutamic acid orderivatives thereof by ring-opening polymerization of thecorresponding α-N-amino acid carboxyanhydrides in a con-trolled fashion offers high end-group integrity precise controlover molecular weight and low dispersities of around 11 Theamphiphilic block copolypeptides are nontoxic up to concen-trations of 3 mgmL and stabilize hydrophilichydrophobicinterfaces in drug formulations and mini emulsion techniques589

The deprotected polymers polysarcosine-block-polylysineenabled the formation of coreshell polyplexes for efficientin vitro transfections590 Degradable polypeptide copolymershave been achieved from natural protein precursors Unlikesynthetic polypeptides which consist of repeating amino acidsequences the polypeptide backbone of proteins provides ahigher diversity of reactive functional groups which enablesfurther chemical modifications Moreover depending on theprecursor protein chosen these polypeptides offer distinctiveand adjustable molecular weights precisely defined sequenceslow cytotoxicity and full degradability They have been appliedfor coating fluorescent NPs as well as for the delivery of cytotoxicdrug moleculesA challenge when using inorganic NPs is the ability of the

animal to degrade and to eliminate them Sub-5 nm NPs such asNDs can be eliminated from the body renally withoutbiodegradation and without leaving toxic residues591 Howeverlarger inorganic NPs are more difficult to clear from the bodyStudies have shown that AuNPs reside in macrophages afteruptake and do not appear to be degraded592 ZnS-CdSe QDshave also been shown to remain stable in mice and rats afterseveral months593594 There is evidence to suggest that someinorganic NPs (eg CdSe QDs and hollow AuNPs) can breakdown by oxidation or other chemical processes This process isslow but there is evidence to show they are excreted through the

ACS Nano Nano Focus


2348

kidneys35595596 The chemistry and design of the inorganic NPscan influence the degradation pattern Although the corecomposition is difficult to degrade for most inorganic NPs theorganic surface coating has been shown to degrade in vivoThis effect was demonstrated by using multiplexed radiolabelsthat tag the inorganic core and surface coating with an organicmolecule This labeling strategy enabled the measurements ofdifferential temporal biodistribution of all different parts of a NPafter in vivo administration It was discovered that inorganic NPsstabilized by an amphiphilic polymer shell via amide bonds loseparts of the organic shell upon enzymatic cleavage in vivo37

Taking the concept further a number of researchers have startedto use organic molecules to assemble inorganic NPs into complexsystems for in vivo medical applications For example DNA canassemble inorganic NPs into a complex coreminussatellite systemthat can target tumors but these NPs can subsequently enterliver macrophages36 The enzymes in these cells degrade theDNA linker and release the inorganic NPs back into thebloodstream to be eliminated through the kidneys Linkermolecules to build nanosystems are not limited to DNA but canalso be polymers that can potentially be responsive to enzymaticdegradation597598 The clinical translation of inorganic NPs maydepend on whether they are cleared from the body as the long-term toxicity of metal-containing NPs remains unclear Thisresearch area will continue to be active from the fundamental(ie understanding the clearance mechanism of inorganic NPs)to the design (ie using assembly techniques to build degradablenanosystems containing inorganic NP components) perspec-tives

LABORATORY-BASED MATERIAL PRODUCTION FORTREATMENT (ldquoIMPLANTS MADE WITHNANOTECHNOLOGYrdquo)While some nanomaterials are designed to deliver a drug or anaction to diseased tissue and programmed to degrade afterwardsresearchers are also synthesizing nanomaterials that can beintroduced into the body with the intention ldquoto stayrdquo Surfacemodeling on the nanoscale can provide new functions andproperties to implants etc599 Incorporation of nanomaterialswith key features for driving tissue engineering is a usefulmeans to achieve unprecedented performance600601 In par-ticular carbon nanostructures (eg single- or multiwall CNTs(SWCNTMWCNT) graphene carbon nanohorns NDsfullerenes etc) can provide mechanical strength and usefulelectronic properties thanks to their chemical structures602

They have been shown to be biocompatible with and to supportthe growth and proliferation of many classes of cells Despite theissues associated with CNT use in medicine603 there is a growingbody of work showcasing their utility in nanomedicine afterappropriate chemical functionalization604 For example CNTaddition to hydrogels are biomaterials that can be designed torespond to diverse stimuli eg for drug release applications605

Some selected examples and applications are detailed belowNano- and Microengineered Implants Nanodiamondminus

polymer composites can be used in tissue engineering andregenerative medicine particularly showing potential forrestoring damaged tissue Excellent mechanical properties ofNDs in combination with fluorescence delivery of drugs andbiologically active molecules and biocompatibility are usedfor reinforcement of implantable polymers to create multifunc-tional tissue engineering scaffolds Octadecylamine-modifiedNDs incorporated into poly L-lactic acid (PLLA) providedfluorescence for monitoring the implant biodegradation (see

Figure 33) While biodegradable polymers such as PLLA are notmechanically robust the incorporation of NDs brings thecomposite properties close to those of human cortical bone Itincreased the compositersquos mechanical strength and strain tofailure and enabled the use of the composite as a bioresorbablebone-growth scaffold for manufacturing bone fixation devices31

In terms of toxicity murine 7F2 osteoblast morphology andproliferation were unaffected when cultured with the ND-PLLAThese composites possess clinically relevant properties whilebeing nontoxic and highly scalable to produce606

Naturally for such implants the interface between theimplanted devices and surrounding cells and tissues is importantSelection of the geometry offers a useful handle in this directionElongated objects such as nanoneedles for example havesuitable properties for the delivery of bioactive agents to cells andtissues This field of nanoneedles for cell-interfacing which is stillemerging is now growing rapidly and shows great promiseA number of interesting nanoneedles have been developedrecently with a multitude of capabilities Some of these capabilitiesinclude transferring biological information to many cells inparallel607 sensing intracellular biological signaling such as thepresence of enzymes608 and measuring intracellular pH at manypoints across individual cells609 The nanoneedles can deliver tothe cell cytosol and yet due to their nanoscale dimensions arenot destructive to the cells These nanoneedles represent a highlypromising technology for intracellular delivery probing andinterfacing Porous silicon-based nanoneedles were developedand utilized to deliver DNA and siRNA to cells in vitro efficientlyand showed transfection efficiencies greater than 90 whilesilencing (of GAPDH expression) was quantified as 80effective610 encouraging for in vivo applications The authorsshowed that they could achieve neo-vascularization in themuscles on the backs of mice by using porous silicon nano-needles to deliver human VEGF165 plasmid DNA610 Thisfinding is of interest when considering the potential toprevascularize sites at which tissue engineered implants mightbe transplanted in vivo It also has the added advantage thatnucleic acids can simply be loaded into the nanoneedles and nocovalent modifications are required The nanoneedles are aplatform technology that could potentially be used to deliver awide range of nucleic acids and other bioactive agents for all typesof tissues

Toward Interfacing Electrically Active Tissues Electricalsignals can be used to control the interactions of nanomaterialswith tissues This strategy to control tissue function may beimportant for many applications such as in the design of neuro-prostheses Below we describe a number of these applications

Figure 33 Bone fixation screw made of a biodegradable polymer(PLLA) reinforced with octadecylamine (ODA)-funtionalizedfluorescent nanodiamond NPs (ND-ODA) that provide reinforce-ment monitoring of biodegradation and eventually drug deliveryAdapted with permission from ref 606 Copyright 2011 Elsevier

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2349

First attempts for electrically interfacing cells were based onmicrostructured devices611minus613 but more recently nanostruc-tured devices such as nanowires have been used614 Two criteriafor building successful devices are ldquowiringrdquo and size whereldquowiringrdquo involves the connection of the recording sites to theoutside615 and size is essential for brain implants to avoid localinflammation616 Neuroprosthetic implants made from LbLbiomimetic nanocomposites investigated by Kotov and coworkersprovide a unique combination of properties that enable manu-facturing of smallest possible brain implants that can be safelyintegrated within brain tissues617 Some of these propertiesinclude high strength conductivity toughness and biocompat-ibility618619 Additionally615minus617 brain implants have been madewith gold nanowires620

A major step forward in this field has emerged by exploring theinteractions of CNTs with neuronal systems Indeed thepresence of CNTs influences the electrical activity of neuronsespecially in terms of synaptic events In the past decade it hasbeen established that CNT-based substrates are able to impactneuronal physiology profoundly from the functional (electrical)point of view With their exceptional properties of small sizeflexibility strength inertness electrical conductivity and ease ofcombination with various biological compounds CNTs are theexcellent candidates for interfacing with damaged neuronaltissues In 2000 it was found that neurons deposited on func-tionalized CNTs were not only surviving but also elongatingtheir neurites in all directions621 Since then the study of thesematerials as functional components of composites for thesupport of the regeneration of neural tissue has been set up bymany research groupsCarbon nanotubes have high electronmobility have an affinity

for neurons and can integrate with cell membranes at bothdendrites and somas As a result of these tight interactions notonly neurons but also entire 3D slices of spinal cord (SC) tissueexhibit desirable behavior622minus625 The most important resultsobtained can be summarized in three points (i) neuronalsignaling is enhanced when brain circuits are cultured inter-faced to CNT films (ii) CNT networks are able to induce theformation of a higher number of new synaptic contacts withrespect to reference substrates and (iii) CNT interfaces promotenerve fibers regrowth potentiating growth cone activity andsynaptic responses in entire SC slices this is an efficient translationfrom single cell to complex tissues such as SC explantsIt has been found that neonatal hippocampal neurons laid

over a film of CNTs increase their spontaneous electricalactivity Thereafter high-resolution electron microscopy eluci-dated the intimate contacts between regrown neuronal processesand CNTs where the boundary line between organic andinorganic matter is ambiguous623625 The most outstandingresults concerning the interface between CNTs and neurons arerelated to the effects on the electrical activity of neuronalnetworks Lovat et al compared the electrical activity ofhippocampal neuronal networks directly grown on a MWCNTmat with that of networks grown on control substrate by meansof the patch-clamp technique622 The frequency of spontaneousevents (postsynaptic currents) in networks cultured on CNTswas strongly increased (approximately 6-fold) compared withcontrols The balance between inhibitory and excitatorycomponents in the neuronal network was not affected622

By means of single-cell electrophysiology techniques electronmicroscopy analysis and theoretical modeling it was hypothe-sized that CNTs can provide a kind of shortcut between theproximal and distal compartments of the neuron624 This

hypothesis supported by the observation that neuronalmembranes establish tight contacts with the CNT substratewas further corroborated by other experiments When cells wereforced to fire trains of action potentials reverberations weredetected following depolarization potentials and this reverber-ation occurred more frequently on CNT-deposited cells than onthose grown on inert glassy supports This type of back-propagatingaction potential represents a regenerative ability that neuronsexhibit in cellular processes such as the tuning of synapticactivity the expression of spike-timing-dependent plasticity therelease of modulatory messengers and the modulation ofsynaptic plasticityAnother interesting observation concerns the impact of CNTs

on the synaptic activity of neuronal networks The probability offinding synaptically connected pairs of neurons is almost doubledin the presence of the CNT substrate The synaptic plasticitywas also affected because cells grown on CNTs demonstratepotentiated short-term synaptic conditions instead of normaldepression in response to afferent presynaptic spike trainsThis activity mimicked the high frequency flow of informationAll this gain of function is entirely attributable to the peculiarfeatures of conductivity and physiochemical properties of CNTsthat affect the network activity and spike propagation624

The ability of CNT substrates to have an effect on centralnervous system tissue has been tested by coculturing embryonicspinal cord and dorsal root ganglia multilayered explants chronicallyinterfaced to a film of purified MWCNTs625 With respect to thecontrols spinal explants cultured on CNTs displayed highernumbers of longer neuronal processes growing in tight contactwith substrates bearing higher numbers of growth cones at theirtips These neuronal processes seemed to be slack on the CNTcarpet that increased their contact surface but were less stiff thanin the control apparently integrating CNTs as their ownsupports or exoskeletons The overall interactions of the spinaltissue with the substrate appear to be intimate and similar to theresults reported for isolated cell cultures When sensory afferentpathways preserved in spinal explants differentiated in vitro wereexogenously activated the resulting synaptic responses recordedfrom single neurons not in direct contact with the CNT layerwere strongly increased This result indicates that the boostingeffects of CNTs at the interface were transferred from the layersof neurons directly exposed to the CNTs to those functionallyconnected yet physically far from the interfaceIn order to improve the biocompatibility of unconstrained

CNTs groups have tried to incorporate CNTs into polymericscaffolds In addition to improved biocompatibility they aim toproduce 3D structures that are able to be colonized by neuronalcells and to foster the communication among them where theCNTs play strengthening and electrically functional roles Tothis end CNTs have been integrated into various biopolymer-based hydrogels as collagen chitosan626 and agarose627 Ingeneral all these substrates are good supports for neuronal cellsable to sustain their growth and their ability to extend neuritesand growth cones without a remarkable toxicity None of thesescaffolds have yet been tested in vivo however Similar resultshave been achieved by CNT composites based on syntheticpolymers mostly polyester polymers such as electrospunfibers of PLGA628 and of poly(L-lactic acid-co-caprolactone)629

Bosi et al have recently produced a nanocomposite systemwhereCNTs are integrated into a polymeric matrix that can bemodeledinto different porosities stiffnesses and shapes This nano-composite consists of a microporous selfminusstanding elastomericscaffold whose skeleton material is made of PDMS while the

ACS Nano Nano Focus


2350

micrometric cavities are generated by dissolving a sugar templateembedded in PDMS630 This 3D structure showed viability as asupporting scaffold allowing neuronal colonization and 3Dsynaptic network reconstruction but was soft enough to matchthe viscoelastic nature of native hippocampal neural tissue631

The PDMS porous scaffold surfaces was then enriched ofMWCNTs endowing the structure with nanofeatures and imple-menting its electrical conductivityBecause of their peculiar electrical features CNTs are useful as

interfaces with other electrically active tissues such as cardiactissue Martinelli et al632 discovered outstanding effects oncardiac cells cultured on CNT substrates Neonatal ratventricular myocytes (NRVM) were able to interact withnonfunctionalized MWNTs deposited on glass coverslips byforming tight contacts with the material (see Figure 34) Cardiac

myocytes modify their viability proliferation growth matura-tion and electrophysiological properties when interacting withCNT scaffolds Two opposing effects on the development appearto take place Cells prolong the proliferative state whichmaintains some cells in an undifferentiated state They acceleratethe maturation of the differentiated cardiac myocytes in terms ofa more negative NRVM resting potential compared to thecontrol so that the cells become more ldquoadult-likerdquo By trans-mission electron microscopy it has been observed that CNTsestablish irregular tight contacts with the membranes in away that is morphologically similar to neurons cultured onMWNTs622 It is not excluded that other modifications indirectlybrought about by MWNTs such as the deposition of theextracellular matrix (ECM) or the cell contact driven cytoskeletaldynamics are ultimately responsible for the positive effectSuch an effect holds promise to solve the issue of arrhythmias

arising from nonconductive scaffolds as exemplified also by achitosan-based hydrogel where the inserted CNTs act asnanobridges that electrically connect cardiomyocytes633 Anoth-er interesting cardiac construct has been produced by Shin andcolleagues634 CNTs were embedded into photo-cross-linkablegelatin methacrylate (Gel-MA) hydrogels resulting in ultrathin2D patches where neonatal rat cardiomyocytes were seededThese cells showed strong spontaneous and stimulatedsynchronous beating In addition a protective effect againstDOX (cardiotoxic) and heptanol (cardio-inhibitor) wasobserved When released from glass substrates the 2D cardiacpatches (centimeter size) formed 3D soft actuators withcontrollable linear contractile pumping and swimmingactuation behaviors For heart tissue engineering CNTs canalso constitute a templating guide for the electrospinning of

hybrid hydrogel nanofibers that form a superb biomimickingscaffold that is capable of matching the orientation index of theleft ventricular tissue thus promoting cardiomyocyte alignmentand synchronous beating635636

The high potential of CNTs in the field of excitable tissueengineering stems from their exceptional nature and the desire todevelop synthetic materials with appropriate nanostructure andcell-recognition properties that are able to regulate cell adhesionsurvival and growth The in vivo application of nanotechnologytools and of CNT-based platforms in particular is largelydirected to basic research applications However the extra-ordinary effects of these materials hint at developing safer devicesto promote tissue reconstruction A major challenge forbiomedical applications of CNTs is to overcome the currentlimitations of CNT degradation time elimination andor safetyof use

Biological Cell-Based Implants Implants used forregenerative medicine can also be of biological origin Cell-based therapies offer great potential For example stem cellsdelivered to injured tissue have the potential to regeneratedefective tissue637638 One general problem with cell-basedtherapies however is that individually delivered cells havelimited retention times in the target tissue In order to improveretention times cells can for example be delivered in alreadyconnected form (so-called cell patches) or they can be seededinto matrices to which they have high binding affinity Treatmentbased on connected cell patches instead of isolated cells thus mayoffer decisive advantages However it is not trivial to cultivate cellpatches as at one point connected cells need to be isolated fromthe culture substrate Cell adhesion typically can be suspendedby enzymatic treatment (eg with trypsin) or by depletion incalcium However while this enables the collection of cells fromculture substrates it also interferes with intracellular contactsNanotechnology has helped create culture substrates that enableswitching on and off adhesive properties to cells For examplesmart surfaces enable switching from hydrophobic to hydrophilicproperties upon changes in temperature In this way whole cellpatches can be detached from the culture substrate and can beused for transplants639 Also plasmonic heating may be a usefultechnique in this direction640 Such advanced culture methodspave the way for future cell and tissue-engineering therapiesCell sheet patch therapies were clinically applied to humanpatients suffering from diseases such as cornea epitheliumdeficient disease recovery from endoscopic submucosaldissension surgery for esophageal epithelial cancer cardiomy-opathy641642 periodontal regeneration643 and osteoarthritis644

While the number of cell layers grown on top of each otherwithin one cell patch is limited as transport of nutrients andoxygen to the cells needs to be warranted645 subsequenttransplantation of several cell patches of up to sim100 μmthickness is possible and vascularization of the transplants hasbeen demonstrated646647 Cells can also be seeded into matricesresembling replicas of cell surfaces648 into decellularizedmatrices649 or even into completely synthetic scaffolds toimprove their biocompatibility and engraftment650651 Therehave been some attempts for translation to the clinic652653 butresults have been and are still heavily scrutinized654minus656 Thebasic requirements such transplants need to fullfill involvevascularization in order to warrant continuous blood supply657

and in case of exposure to the environment (eg air food)antimicrobial coatings Severe skepticism toward the use of thesetechnologies in general is reported658

Figure 34 Left Scanning electron microscopy (SEM) image of aspinal explant peripheral neuronal fiber on a MWCNT substrateNote the tight and intimate contacts (red arrows) between theneurite membrane and the MWCNTs Scale bar 500 nm Reprintedfrom ref 625 Copyright 2012 American Chemical Society RightSEM image of cardiac myocyte deposited on a layer of MWNTAdapted from ref 636 Copyright 2013 American Chemical Society

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2351

Toward Artificial Organs A current need in medicine iswith the construction of whole organs in vitro to address the issueof shortages in organ donation659 Nanotechnology is emerging

as a powerful tool that can help to fulfill this need by assisting inthe engineering of artificial organs for regenerative medicineas well as organs-on-a-chip applications660661 The need fornanoscale architecture is derived from the ability of cells tosense the nanoscale structures in their microenvironment662

In particular many ECM molecules that surround cells formnanofibrous structures that help to organize cellular architectureand to induce directional migration and alignment663 Asoutlined in the case of implants one approach to engineersuch nanoscale structures is through the generation of 3D-fibrousscaffolds663minus665 Techniques like electrospinning and micro-fluidic-based fiber fabrication have emerged as methods toengineer 3D scaffolds for inducing cellular organization andalignment666minus669 Furthermore self-assembling peptides havebeen used to form nanofibrous structures which mimic thecomplex nanoscale architecture of native ECM moleculesUtilization of biomimetic composite coatings made using LbL

assembly on inverted colloidal crystal scaffolds on ceramics670

gels671672 PLGA polymer673 enabled for instance the creationof ex vivo models of bone marrow674 thymus675 and liver676

One of the advantages of the ex vivo organs is that they enablemultifaceted evaluation of the toxicity of NPs in human tissues inthe cellular constructs replicating real organs677

In addition nanomaterials can be integrated into hydrogels todirect cellular behavior For example the integration of nano-scale silicate materials can induce the directed differentiation ofmesenchymal stem cells (MSCs) into osteogenic fates678

Polymeric nano- and microparticles that release growth factorsand cytokines in a controlled manner can be integrated intoscaffolds to regulate the surrounding biology For examplecontrolled release of angiogenic factors such as VEGF andPDGF through polymeric NPs can induce the formation ofblood vessels Other types of nanomaterials such as CNTs andgrapheneNPs can be incorporated into hydrogels to change theirmechanical and electrical properties as described aboveThe resulting gels have been shown to enhance the formationof cardiac and skeletal muscle tissues634679680

Nanoscale tools also provide means to form tissues withcontrolled microarchitectures For example cell-laden hydrogelscan be induced to assemble into particular structures that bettermimic the microarchitecture of tissues in the body through theuse of nanoscale ldquoprogrammable gluesrdquo681 In this approach thevarious sides of microgels of controlled shapes were coated withadhesive long single-stranded DNA where the function of thestrands is to induce programmed assembly It was demonstratedthat the resulting DNA strands form nanoscale objects on thesurface of gels that uniquely recognized their complementarybase-paired strands on other gels to induce assembly Suchstructures could 1 day provide scalable ways of engineering tissuestructures with predefined architecturesThese and many other examples have shown the power of

nanotechnology to direct cellular behavior for inducing tissue

formation It is anticipated that with our increased knowledge ofhow nanoscale objects interact with cells along with our ability toengineer more controllable nanomaterials will lead to a new eraof nanomedicine for regenerative medicine applications

Antibacterial Coatings Biofilm formation and dissem-ination of antibiotic-resistant bacteria by indirect transmissionthrough contaminated surfaces is becoming a major global publichealth threat Steel and aluminum surfaces commonly presentin hospital settings as knobs buttons frames taps etc possesslimited or very low antimicrobial activity A promising approachfor limiting indirect transmission is the realization of stable andeffective antimicrobial surfaces obtained by nanostructuredcoatings Similarly infections associated with medical devices(catheters implants etc) have become a major concernThe adhesion of bacteria to the devicesrsquo surfaces during theinitial 24 h is believed to be a decisive period for implant-associated infections and the design of optimal antiadhesion ofmicrobes in this period is key Multilayer coating by LbL self-assembly provides a robust method to design multifunc-tional dynamic antibacterial surfaces For example a multilayerof (heparinchitosan)10minus(polyvinylpyrrolidonepoly(acrylicacid))10 [(HEPCHI)10minus(PVPPAA)10] provides the propertiesof contact killing of bacteria due to the underlying (HEPCHI)10while the top (PVPPAA)10 multilayer film shows a top-downdegradable capability in the determined period This activityleads to almost no adhesion of bacteria within 24 h The com-bination of the adhesion resistance and the contact-killing prop-erties shows enhanced antibacterial capability through targetingthe ldquodecisive periodrdquo of implantation and may have potential forapplications in medical implants tissue engineering etc682

Surfaces with immobilized antimicrobial peptides present highand broad antimicrobial activity683 Similarly a topic worthexploring involves immobilizing antimicrobial ligands onto NPsto enhance their antimicrobial activity A quaternary ammoniumcationic surfactant didodecyldimethylammonium bromide(DDAB) was immobilized onto silica NPs to develop anti-microbial NPs These NPs revealed much lower minimalinhibitory concentrations against bacteria and fungi than thesoluble surfactant The electrostatic interaction of the DDABwith the NP is strong since no measurable loss of antimicrobialactivity was observed after suspension in aqueous solution for60 days This result means that the antimicrobial activity of theNP does not require the leaching of the surfactant from thesurface of the NPs More interestingly the NP propertiesexpanded to virucidal activity The versatility relative facility inpreparation low cost and large antimicrobial activity of thoseNPs makes them attractive as coatings for biomedical devices684

Suspensions of ZnO NPs are effective against a broadspectrum of microbial organisms685minus687 but there are manymisconceptions about their mechanism(s) of killing bacteria524

Generation of ROS was often considered to be the source ofantibacterial activity688 However according to analysis of themechanism of bacterial inhibition by ZnO NPs of differentshapes524 at least part of the antibacterial activity originates fromenzyme inhibition Anisotropic ZnO and potentially other NPscan mimic biological inhibitors by partially matching theirgeometries with those of the protein inhibitors Such activityoriginates from the similarity of the surface chemistry and size ofwater-soluble NPs and those of many globular proteins6

Applerot et al used ultrasound irradiation to generate ZnONP-coated surfaces that resist bacterial biofilm formation689

Layer-by-layer coatings of ZnO NPs reduce biofilm growth ofMRSA by 95 Silver in bulk and nanomaterial forms has been

A current need in medicine is theconstruction of whole organs in vitro toaddress the issue of shortages in organdonation

ACS Nano Nano Focus


2352

used extensively as medical device coatings but these deviceshave not lived up to the promise of their in vitro data690 Silverrsquosantimicrobial function is based on the concentration of silver ionsand their associated toxicity691692 Unfortunately the therapeu-tic window between efficacy and toxicity for silver is quite narrowwhich has led to disappointing clinical effectiveness of silver-coated medical devices690 ZnO is significantly less expensivethan silver and has selective toxicity for bacteria over mammaliancells693694 ZnO is generally recognized as safe by the FDANanoparticles can be designed with antibacterial coatings to

circulate in the blood to target bacterial infections post-tumorsurgery During chemotherapy some patients may be moresusceptible to bacterial infections695696 This susceptibilitycannot be addressed using antibiotics alone however as someantibiotics may lead to serious side effects for some patientspostsurgery Abuse of antibiotics has caused the emergence ofmore resistant and virulent strains of pathogens As a result insome cases clinicians need to prescribe multiple antibiotics inaddition to anticancer drugs before and after the surgeryParticles can be engineered with antibacterial properties to targetbacteria in vivo specifically which can potentially minimizeresistance For example a polymer vesicle antibacterial drugcarrier has been developed recently The chitosan-based carrieris grafted with an antibacterial polypeptide and demon-strated to kill both Gram-positive and Gram-negative bacteria(see Figure 35) This polymer vesicle has excellent blood

compatibility and low cytotoxicity which may benefit patientsafter tumor surgery697698 The enhanced antibacterial efficacyof polymer vesicles compared with an individual antibacterialpolymer chain results from the high concentrations of localpositive charges in the polymer vesicles

IN VIVO COMBINATION OF DIAGNOSIS ANDTREATMENT (ldquoTHERANOSTICSrdquo)Theranostics involves the combination of both diagnosis andtreatment Image-guided tumor resection is one such applicationthat uses theranostics The diagnostic use of NPs to label cancercells in particular tumor borders and small metastatic regionscan be used to guide the surgeon for subsequent therapeuticsurgical removal of the tumors699 These diagnostic NPs couldalso be loaded with therapeutic agents for treating postsurgeryAnother interesting application of theranostic agents is NPs thatcan deliver therapeutic agents into tumors while tracking NP

drug delivery700 These agents enable noninvasive imagingto provide information on the fate of the drug once they areadministered in vivo Theranostics research is an exciting area ofresearch as many different combinations of diagnostic andtherapeutic strategies can be built into NP systems

Image-Guided Surgery Recently first-in-human pilotstudies have successfully used both passive and active targetingagents for image-guided surgery of human tumors701

The specific tumor types studied include breast lung ovarianand pancreatic cancers Although still preliminary the resultshave helped clarify two important issues (i) most human tumors(but not all) have moderately leaky vasculatures so the EPReffect provides a general means for passive delivery of imagingagents (5minus6 nm albumin-bound indocyanine green) to a broadrange of human tumors (ii) fluorescent dyes such as fluoresceincan be conjugated to targeting ligands such as folic acid forspecific targeting and high-contrast imaging of human tumorsIn the first approach passive fluorescent agents do not involve

targeting ligands but rely simply on the biodistribution of thefree fluorophore Singhal and co-workers conducted a small trialon nontargeted NIR dyes enrolling 5 patients undergoingsurgery for removal of their cancers (3 lung nodules 1 chest wallmass and 1 anterior mediastinal mass)701702 Two surgeonsreached a consensus about the clinical stage and operativeapproach prior to surgery All enrolled patients were thought tohave limited disease be amenable to surgery and have nometastases (ie potentially curable) Patients were injected withindocyanine green (ICG) prior to surgery703 At the time ofsurgery the body cavity was opened and inspected The resultsdemonstrated that NIR imaging can identify tumors from normaltissues provides excellent tissue contrast and facilitates theresection of tumors701 In situations where there is significantperitumoral inflammation NIR imaging with ICG is not helpfulThis suggests that nontargeted NIR dyes that accumulate inhyperpermeable tissues will have significant limitations in thefuture and receptor-specific NIR dyes may be necessary to over-come this problemIn contrast to passive agents active fluorescent agents are

linked to targeting ligands that are designed to recognize specificreceptors at the target tissue Near-infrared-dye-labeled antibod-ies (MW sim 150 kDa) that are currently used in the clinic fortargeted cancer therapy such as VEGF antibody (bevacizumab)EGFR antibody (cetuximab) and HER2 antibody (trastuzu-mab) have been evaluated as targeted optical imaging probes inpreclinical animal tumor models704minus706 Several antibody-basedimaging probes are approved for clinical trials for image-guidedsurgery707 Potential limitations for antibody-based opticalimaging probes include increased background fluorescenceresulting from relatively long blood circulation times as wellas low efficiency in penetrating through tumor tissues704

To address these issues smaller antibody fragments such asaffibodies (MW sim 7minus8 kDa) against HER2 or EGFR werelabeled with NIR dyes for targeted optical imaging Specificityand sensitivity of those affibody optical imaging probes have beendemonstrated in various animal tumor models704708minus710 withseveral groups in the process of conducting clinical trials Forintraoperative imaging of tumor margins a class of proteaseactivatable optical imaging probes can detect tumor edges sincemany proteases are highly expressed in the invasive tumor areasthat are close to the tumor edge711 The first-in-human resultshave been reported from intraoperative tumor-specific fluo-rescence imaging by using fluorescein-conjugated agents totarget the folate receptors in both ovarian cancer and lung

Figure 35 Antibacterial polypeptide-grafted chitosan-based vesiclescapable of delivering anticancer and antiepileptic drugs simulta-neously Reprinted from ref 697 Copyright 2013 AmericanChemical Society

ACS Nano Nano Focus


2353

cancer712713 In patients with lung cancer the results show thattargeted molecular imaging could identify 46 (92) of the50 lung adenocarcinomas and had no false positive uptake in thechest In vivo prior to exposing the tumor molecular imagingcould only locate 7 of the lesions After dissecting the lungparenchyma 39 more nodules could be detected Four noduleswere not fluorescent and immunohistochemistry showedthese nodules did not express FRα (folate receptor alpha)In 46 positive nodules tumor fluorescence was independent ofsize metabolic activity histology and tumor differentiationTumors closer to the pleural surface were more fluorescent thantumors deep in the parenchyma In two cases this strategy wasable to discover tumor nodules that were not located preop-eratively or intraoperatively by standard techniques These pilotclinical trials have demonstrated that targeted intraoperativeimaging may lead to more complete surgical resections andpotentially better stagingFluorescent NPs can be used for similar image-guided

applications as organic fluorophores Targeted NP-based opticalimaging probes have been developed by conjugating peptidesnatural ligands antibodies antibody fragments and smallmolecules to NPs that have optical imaging properties or arelabeled with fluorescence dyes Results of preclinical studies werepromising showing increased specificity and sensitivity in tumorimaging by systemic delivery of targeted NP optical imagingprobes as opposed to nontargeted probes714715 It has beenshown that optical imaging probes can be targeted to highlyexpressed tumor cell receptors and active tumor stromal fibro-blasts and macrophages such as urokinase plasminogen activatorreceptor (uPAR) They are good imaging probes for sensitivedetection of tumor margins716 In many solid tumors activetumor stromal cells with a high level of uPAR expression arefound in the peripheral tumor areas Even at the early stage oftumor development for example in ductal carcinoma in situ ofthe breast small tumor lesions were surrounded by uPARexpressing macrophages and fibroblasts Therefore opticalimaging probes targeting active tumor stromal cells offer theopportunity of intraoperative detection of small tumor lesions(lt1 mm) that lack tumor vessels that may enable EPR effect tooccur or that cannot be delivered to tumor cells717 Howeverclinical translation of targeted NP imaging probes has been achallenging task The preclinical studies required for filing anInvestigational New Drug Application to the FDA for a clinicaltrial are much more extensive compared to those requiredfor developing small-molecule peptide or antibody-basedimaging probes Additionally the cost of production of targetingligand-conjugated imaging NPs in good manufacturing practice(GMP)-grade for human use is higher than those of peptide andantibody-based imaging probesTracing of Drug DeliveryNanoparticles can be engineered

to observe the delivery process directly Theranostic nanocarriersoffer high capacity drug loading as well as the ability to track andimage the in vivo pathway of both the transporter and the drug inthe body Human tumors are highly heterogeneous in theirtumor vascular structures microenvironment and tumor cellsEfficiency of intratumoral drug delivery and distribution variesamong different tumors Drug delivery using theranostic NPsenables noninvasive in vivo imaging of the NP delivery processand may enable assessment of treatment responses at an earlystage As a tool to visualize cellular uptake and cargo releasefluorescent NPspolymers have been widely used Quantumdots AuNPs iron oxide NPs and NDs have emerged as valuabletools for combined therapy and diagnostic imaging due to their

high stability in vivo DNA as well as drug transport and release ofpolycationic QDs and NDs into cells has been visualized byconfocal imaging which in principle offers monitoring of theentire gene delivery pathway inside cells363718

Polymer NPs with intrinsic imaging properties that do notrequire conjugation of imaging groups have recently beendeveloped For instance the weakly fluorescent polyethyleni-mine (PEI) polymer was conjugated to hydrophobic polylactide(PLA) yielding amphiphilic PEI with a high payload capacity forthe antitumor drug paclitaxel After conjugation with PLA theformed NPs exhibited bright and multicolor fluorescence whichcould be tuned to the NIR region Although fluorescence enablesspatially resolved imaging at the cellular and tissue levels it is notapplicable for in vivo imaging deep inside the body The highbackground autofluorescence from the organism the poorpenetration of the fluorescent signal in and out of the animal andthe risk of tissue damage by high intensity excitation light limitthe application of fluorescence-based imaging in vivo Magneticresonance imaging PET and CT imaging are considered moreattractive and promising for clinical applications Nanoparticlescan be designed to containMRI contrast agents (eg Gd-DOTA)or magnetic iron oxide NPs and to carry drugs using polymers toconstruct a theranostic agentVasiljeva and co-workers developed an effective delivery

system for targeting both tumors and their stromal componentsbased on ferrimagnetic nanoclusters719 This multiplexedtargeting strategy is important for imaging and treating tumorsas the tumor microenvironment has many diverse componentsincluding extracellular matrix components and stroma cellsThe universal lipidated magnetic NPs (called ferri-liposomes bythe authors) enhance the MRI contrast properties and areeffectively taken up by tumors and their stromal componentsChemical compounds within the ferri-liposomes are successfullyreleased when administered in vivo and can be visualized at boththe cellular and tissue levels Furthermore this theranostic agentoffers an exciting possibility to deliver and to detect therapeuticagents simultaneously in vivo As such the cathepsin inhibitorJPM-565 was targeted by ferri-liposomes to the peri-tumoralregion of mouse breast cancer which resulted in a significantreduction in tumor sizeAnother class of theranostic NPs uses light for activation One

example incorporates a second-generation PDT drug Pc 4 witha cancer-targeting ligand and iron oxide NPs as a MRI-guidedPDT platform (see Figure 36)720 The delivery of Pc 4 by NPsenhanced the treatment efficacy and reduced the required PDTdrug dose The targeted IO-Pc 4 NPs have potential to serve asboth an MRI agent and PDT drugOther NPmaterials may also be used for simultaneous imaging

and therapy One example involves chlorin e6-conjugated C-dotsas the light-triggered theranostics NP platform This platformuses simultaneous enhanced photosensitizer fluorescence detec-tion (PFD) and PDT by FRET721 Huang et al reported folicacid-conjugated silica-modified gold nanorods showing highlyselective targeting enhanced radiation therapy and PTT effectson MGC803 gastric cancer cells and also strong X-ray

Drug delivery using theranostic NPsenables noninvasive in vivo imaging ofthe NP delivery process and mayenable assessment of treatmentresponses at an early stage

ACS Nano Nano Focus


2354

attenuation for in vivo X-ray and CT imaging722 In a similarstrategy multifunctional PEGylated gold nanostar NPs wereprepared with CD44v6 monoclonal antibodies conjugated as thetargeting ligands723 The prepared NPs had high affinity towardgastric cancer stem cell spheroid colonies Upon NIR lasertreatment with a low power density (790 nm 15Wcm2 5 min)those spheroid colonies could be completely destroyed in vitrowhile being simultaneously observed with X-rayCT imagingand photoacoustic imaging Orthotopic and subcutaneousxenografted nude mice models of human gastric cancer wereestablished Computed tomography imaging observed in situgastric cancer clearly at 4 h postinjection and photoacousticimaging revealed that CD44v6-modified NPs could activelytarget the gastric cancer vascular system at 4 h postinjectionwhile the NPs inhibited tumor growth upon NIR laser irradiationand even extended survivability of the gastric cancer-bearing miceThese NPs exhibited good potential for applications of gastriccancer targeted imaging and PTT treatmentHybrid materials are increasingly attracting attention since

they enable the combination of different properties and functionsin one multipurpose material with high adaptability Phosphate-based inorganicminusorganic hybrid NPs (IOH-NPs) are attractivetools as imaging and therapeutic drugs724 The NPs are generallycomposed of [ZrO]2+[Rfunction(O)PO3]

2minus (Rfunction = functionalorganic group such as drug andor fluorescent dye) and enablemultipurpose and multifunctional combination of a wide rangeof properties such as drug delivery (eg chemotherapeutic agentsor anti-inflammatory drugs) as well as optical and CT-baseddetection or MRI For example the anti-inflammatory poten-tial of [ZrO]2+[BMP]2minus0996[DUT]

2minus0004 was tested in vitro

on an alveolar macrophage cell line where MHS cells weretreated with the endotoxin lipopolysaccharide (LPS 200 ngmL)to provoke an inflammatory response Cells were simultaneouslyincubated either with IOH-NPs (10minus10 to 10minus5 M) or the

control drug dexamethasone (DM 10minus7 M) Incubation ofLPS-stimulated macrophages with increasing amounts of[ZrO]2+[BMP]2minus0996[DUT]

2minus0004 resulted in a dose-dependent

reduction in TNFα IL-6 and NO secretion with an efficacy of10minus5 M IOH-NPs comparable to 10minus7 M of dissolved molecularDM Fluorescence microscopy confirmed these resultsAnother efficient way to implement theranostic delivery

carriers is to design multicompartment nanomedicinevehicles725 This approach is based onmulticompartment capsuleswhich can be designed in the form of concentric pericentricinnercentric and anisotropic or acentric structures726 Themulti-compartments are composed of smaller nanocapsules or NPswhich are often assembled around a bigger nano- or micro-capsule or particle In such an arrangement some multicompart-ments are responsible for diagnostics by for exampleincorporating sensing molecules727 while other subcompart-ments are constructed for therapy and treatment ie foron-demand delivery of biomedicine Among other alternativestructures are capsosomes and polymersomes Capsosomesrepresent delivery carriers formed by adsorbing liposomes andpolymers on a particle template followed by removal of templatecores A number of interesting applications have been demon-strated for capsosomes including controlling the rates ofenzyme-catalyzed reactions and pH- and temperature triggeredrelease728 Polymersomes on the other hand are delivery carriersbased on block copolymers729 The versatility of controlling theirproperties makes them together with polymeric vesicles730 anindispensable class of drug delivery vehiclesTheranostic NPs not only provide signals for imaging the

locations of drug delivery vehicles they also may respond to thelocal environment and thus report delivery by a change inthe read-out signal A first look at delivery in vitro helps todemonstrate this concept When NPs are incorporated into cellsvia endocytotic pathways the NPs experience radical changes intheir ambient environments This involves a change in pH fromneutralslightly alkaline extracellular medium to highly acidicendosomeslysosomes which for example enables reportingcellular internalization by observing pH changes by pH-sensitivefluorophores integrated into the NPs128171 Endosomes andlysosomes are also rich in proteolytic enzymes which act oninternalized NPs Fluorescence markers that change signal uponproteolytic activity enable the detection of intracellular internal-ization731732 These principles have been applied for onlineobservation of the delivery of micelles Here a color changereports successful delivery upon degradation of the micellesThe delivery of embedded drugs can be imaged directly9733734

Murakami and co-workers demonstrated this approach usingmicelles of BODIPY FL-PEG-b-poly(glutamic acid)-BODIPYTR loaded with 12-(diaminocyclohexane)platinum(II) Themicelles were built to emit due to the shell-conjugated dye whilethe core-conjugated dye was quenched The micelle degradationis triggered by changes in pH and concentration of chloride ionsie changes that occur in late endosomes The last approach wastested in vivo using confocal laser scanning microscopy Despiterapid progress in this field theranostic NPs are still at an earlystage of development

Nanoparticle-Labeled StemCells for Targeted Imagingand Therapy Stem cell nanotechnology as a newly emerginginterdisciplinary field refers to the application of nano-technology in stem-cell research and development Hundredsof clinical trials with autologous or allogeneic MSCs are currentlyongoing with efforts being made to ensure the safety and efficacyof these advanced therapy medicinal products Although

Figure 36 Construction of targeted NPs carrying the PDT drug Pc 4Quantitative measures of T2 values show that the cells with Fmp-ironoxide (IO) NPs had lower T2 values as compared to those with onlySPIONs Reprinted from ref 720 Copyright 2014 AmericanChemical Society

ACS Nano Nano Focus


2355

significant advances in the field of stem cells have been and arecontinuously being made several obstacles must be over-come before their therapeutic application can be realizedThese steps include the development of advanced techniques tounderstand and to control functions of microenvironmentsignals as well as novel methods to track and to guide trans-planted stem cells The application of nanomaterials andnanotechnology in stem-cell research and development exhibitsattractive technological prospects to solve current problems ofstem-cell research and development735 Mesenchymal stem cellsare multipotent stem cells that can differentiate into a variety ofcell types including osteoblasts (bone cells) chondrocytes(cartilage cells) and adipocytes (fat cells) In addition MSCspossess immunosuppressive or immunomodulatory propertiesand have the tendency to home to the sites of active tumo-rigenesis Thus MSCs can be considered as a candidate cell typefor cell-based tissue engineering cancer therapeutics andregenerative medicine applicationsOne fascinating and upcoming application is the generation of

induced pluripotent stem cells (iPSC) fromMSCs with potentialin regenerative medicine Combining negatively charged nucleicacids such as transcription factors and siRNA with cationizedNP formulations provided superior reprogramming efficiencycompared to conventional ldquoYamanaka factorsrdquo (see Figure 37)736

The highly tumorigenic c-MYC and KLF4 oncogenes weredispensable in this approach which employed polysaccharides toform self-assembling NPs instead of lentiviral transduction foriPSC generation from MSCsHowever the distribution and final fate of MSCs inside the

human body have still not been clarified for which novel labelingand in vivo tracking technologies are urgently needed Nano-particles are potentially suitable labels However little is knownabout alteration of MSC biology after exposure or uptake of NPsAs an example silica-coated fluorescent supermagnetic NPswere evaluated for their effects on MSCs and then used to labelMSCs The distribution and final sites of the labeled MSCs wereobserved in vivo in nude mice models with gastric cancerMesenchymal stem cells were efficiently labeled with NPsyielding stable fluorescent signals and magnetic properties within14 days The NP-labeled MSCs targeted and could be used toimage in vivo gastric cancer cells after being intravenouslyinjected for 14 days (see Figure 38)737 Nanoparticle-labeledMSCs could significantly inhibit the growth of gastric cancerin vivo based on hyperthermia effects of the NPs and theCCL19CCR7 and CXCL12CXCR4 axis loops may play keyroles in targeting MSCs to gastric cancer in vivo This NP-basedlabeling and tracking technology has potential in applicationssuch as labeling and tracking in implanted cells evaluatingcell therapeutic effects and recognizing andmapping early gastriccancer cellsDendritic cells (DCs) also display promising potential in

cancer immunity However enhancing DCsrsquo immunotherapeuticeffect in cancer-targeted immunotherapy and prevention remainsa challenge Li et al reported an allogenic DC and tumor cellfused vaccine combined with cytokine-induced killing cells(CIKs) for targeted imaging and enhanced immunotherapeuticefficacy of gastric cancer (GC)738 The fused vaccine wasprepared by PEG-mediated fusion between mature DCs andinactive GC MGC803 cells The immunotherapeutic andprophylactic potentials of the fused cells (FCs) were evaluatedin tumor-bearing postsurgery and tumor-free mice modelsThemigration and homing process of NIR-QD-labeled FCs wereinvestigated with a real-time animal-imaging system Resultsshowed that the FCs and FC + CIKs could trigger the tumor-specific cytotoxic T lymphocytes against GC cells They targetthe tumor tissue enhance the prophylactic effects and suppress

Figure 37 Negatively charged plasmid mixture (encoding Oct4Sox2 miR302-367) and the positively charged cationized Pleurotuseryngii polysaccharide (CPEPS) self-assembled into NPs named asCPEPS- OS-miR NPs which were applied to human umbilical cordmesenchymal stem cells for iPSCs generation Adapted from ref 736Copyright 2015 American Chemical Society

Figure 38 Fluorescence and MR imaging of NP-labeled MSCs targeting gastric cancer cells in vivo (A) In vivo fluorescence images show that(right) tumor sites of the mice in the test group had fluorescence signals of NP-labeled MSCs postinjection after 7 and 14 days and (left) tumorsites of the mice in the control group had no fluorescence signal of NPs postinjection after 7 and 14 days (B) Fluorescence images of majororgans show that (left) no signal was detected in the tumor and organs of the control group and (right) obvious fluorescence signals weredetected in the tumor tissues of the test group (C) MR imaging of NP-labeled MSCs targeting gastric cancer cells after 7 and 14 dayspostinjection Adapted with permission from ref 737 Copyright 2012 Ruan et al

ACS Nano Nano Focus


2356

the tumor growth in vivo These allogenic DC and tumor cellfused vaccine can be used for targeted imaging and enhancedimmunotherapeutic efficacy of GC Similarly the FC + CIKstrategy shows potential in clinical applications such as earlytherapy and prevention of tumor metastasis and relapse in nearfuture

REDUCING SIDE EFFECTS WITH SAFE-BY-DESIGNAPPROACHESPotential Risks of Nanomedicines While there are many

applications of nanomedicines there are concerns over theirpotential side effects when they are used in patients Theseconcerns may limit their translation to patients Thus the ques-tions of how to reduce risks and to increase benefits are vital forthe development of safe and effective nanomedicines First we

need to understand how physicochemical properties of nano-carriers and nanomedicine relate to their biological behavior andfate in vivo577739 These physicochemical properties include sizeshape surface chemistry hydrophobicity and hydrophilicitychirality aggregation degradability and catalytic ability All ofthese properties may influence their fate and subsequent bio-logical responses The other issue to take into consideration isthe protein corona When nanocarriers and nanomedicineare injected in the blood they can quickly adsorb blood proteinssuch as albumin fibrinogen transferrin immunoglobulinlipoproteins and so on740minus743 This corona determines thedistribution and transport behavior of such NBCDs744 Alteredbiokinetics which are dependent on the types of polymer usedand on surface chemistry may enhance the transport velocitybut at the same time they may cause the NPs to enrichundesirably in some specific tissues thereby leading to high localconcentrations at these parts of the body745746 These distri-bution and metabolism changes may induce new andunpredictable effects that must be taken into considerationSecond we need to design and to utilize those propertiesrationally to optimize their application for therapeutic efficacyand safety As an example if the NPs are coated with ldquostealthrdquocomponents to keep them in the bloodstream longer the releaseof the drug molecules should be low at this stage in order not totreat the blood cells and endothelium with the drugThe much more complex structure of nanomedicines

compared to simple drug molecules makes it important tocharacterize them carefully This is relevant not only for theunderstanding of their activity and effects but also for theresponsible production of such structures For this purposein Europe the ldquoNanomedicine Characterization Laboratoryrdquo(httpwwweuncleu) was founded and is funded by the EuropeanCouncilA further critical issue and major problem for nanomedicine is

the quick uptake by cells typically in the reticuloendothelialsystem (RES) which results in their high accumulation in theliver and spleen747minus749 Toxicity against these cells and tissuesshould be carefully tested Besides these ldquonormalrdquo distributionkinetics NPs may have novel metabolic and excretion profileswhich arise due to the interactions of NPs with tissue micro-

structure and specific biomicroenvironments750minus752 Small NPsmight be removed from the blood by renal clearance (lt5 nm) orrapid liver uptake (10minus200 nm)753 whereas large NPs arefiltered in the sinusoidal spleen (gt200 nm)754 or are recognizedand cleared by the RES755 Renal clearance studies in mice usingQDs with zwitterionic cysteine demonstrated that the sizethreshold for glomerular filtration of QDs was about 55 nmwhile renal excretion was prevented when the diameter wasabove this value123 Therefore NPs between 20 and 200 nm canremain in the circulation for extended periods of time754 thoughthere is still debate in the literature on this front756

Usually the increased drug uptake by cells and favorable drugrelease profiles can be easily achieved using nanodeliverysystems However toxicity stability and degradation propertiesof such NPs are yet to be well evaluated Moreover toxicitytesting with NPs is not easy as good models for testing are notavailable and most simple in vitro systems have not been suitableso far757 There are significant variabilities in how different organsinteract with NPs and this variability is difficult to capture in vitroIt is well accepted that the transport of NPs across tissue barriersis clearly size-dependent This has been demonstrated for variousuptake pathways and different tissues such as the gastrointestinaltract758 the lung759 the bloodminusbrain barrier760761 or the humanplacenta762763 Logically these tissues have to be tested intenselyin toxicity assays747758minus764

For assessment of the safety and therapeutic efficacy propermethodology and state-of-art techniques are necessary to studybiological behaviors and transformation of nanomaterials andnanomedicine in vivo and in vitro Systemic and quantitativeinformation from proteomics genomics transcriptomics andmetabolomics can capture the changes on metabolism signalingpathways and biological functions as well765766

Safe-by-Design Approaches An important considerationin the development of new nanotherapeutics is the intrinsicsafety of the design platform from conception to implementa-tion Appropriate consideration should be given to materialcompositions that are already considered safe by the FDA andcontain degradable ingredients that do not lead to biopersistanceor bioaccumulation It is also important to consider intrinsicnanomaterial properties that are generally known to be morehazardous for nanomaterial profiling studies including highcationic density high surface reactivity dissolution and sheddingof potentially toxic metal ions (eg gadolinium522523) highaspect ratio etc One approach involves the selection of the initialNP composition and subjecting its components to multi-parametric high-throughput screening This screening wouldenable one to discern possible effects on cells of potential targettissues and could be used to eliminate hazardous in vitro effectssuch as cytotoxicity membrane disruption organellar damage(eg mitochondria and lysosomes) unwanted redox activityleading to oxidative stress biopersistance off target genotoxicityetc Attempts are made to relate any adverse in vitro effects tospecific physicochemical properties which could then beadapted eliminated or redesigned to render a safer in vitroprofile This approach reduces the number of subsequent animalstudies to be performed if the intrinsic material design isintended to be safe However ultimately the designrsquos efficacyand safety need to be demonstrated through the performance ofin vivo studies including making use of well-established safetyscreening procedures as well as screening for specific materialproperties that cannot be assessed in vitro eg immunogenicityof PEG-coated particles

The questions of how to reduce risksand to increase benefits are vital for thedevelopment of safe and effectivenanomedicines

ACS Nano Nano Focus


2357

Carbon nanomaterials including one-dimensional CNTs and2D graphene exhibit unique physical chemical and electricalproperties that make them attractive candidates for electronicsenergy technologies medical diagnostics and drug delivery767

Due to this significant potential for industrial applicationsmethods for safe handling and use of these nanomaterials are ofcritical importance Furthermore emerging biomedical uses ofthese nanomaterials768769 necessitate the development ofstrategies to minimize toxicity For example in a series of studiesinvolving SWCNTs770 MWCNTs771 and graphene772 it wasshown that stable aqueous dispersions of carbon nanomaterialscan be achieved with the amphiphilic block copolymer surfactantPluronic F108773 In contrast to aggregated samples carbonnanomaterials that are effectively dispersed with PluronicF108 further minimize toxicity in vivo770minus774 This samedispersion strategy also minimizes negative environmentaloutcomes of these nanomaterials in aquatic environ-ments775minus778 The positive benefits of Pluronic block copolymersurfactants have also recently been realized for related 2Dnanomaterials779 such as transition metal dichalcogenides(eg MoS2) both in vivo780 and in aquatic environments781

Challenges to Clinical Translation To reach the patientand to contribute to the benefit of society medical nanomaterialsneed to succeed in an industrial technology transfer phase and inclinical translation This translation is a lengthy costly andcomplex process that should be considered early on in thedevelopment process of novel nanotechnology-based thera-pies308 For nanomedicine to be considered for ldquofrom thebenchtop to the bedsiderdquo clinical translation high therapeuticefficacy alone is not sufficient782minus785 The nanomedicine mustsimultaneously meet the basic requirement triangleshightherapeutic efficacy material excipientability and scale-upability313to meet the safety effectiveness and producibilityrequirements for a drug (see Figure 39) Typical roadblocks for

clinical translation of academic research projects into nano-medicine include (i) the failure either to target an unmet clinicalneed or to do so beyond what can be expected with simplerproducts in the near future (ii) reproducibility and character-ization problems of the nanomaterials involved (iii) designcomplexity forbidding economic production (iv) scale-upchallenges (v) lack of efficacy in humans (vi) unexpectedclinical toxicity (vii) lack of a convincing business case (viii) afragile patent situation (ix) a delay in the registration process ofnanomaterials and nanodevices and (x) crowding of research

project topics toward specific disease groups (cancer) whileother important disease classes (eg poverty diseases metabolicdisease vascular diseases) are neglectedLack of efficacy is frequently seen in humans despite success in

animal models of disease because the physiology of humansdiffers from that of small animals in many aspects This is parti-cularly important in cancer where the usual xenograft models ofhuman cancer in mice are often curable by nanotechnologieswhile current clinical success in cancer therapy remains limitedwith differences in tumor vascularization playing a key role786

To achieve rational prospective design criteria for the develop-ment of future nanomaterials for particular tasks a strongscientific understanding of nanominusbio interactions will berequired Predictive models of such interactions can be used todesign novel materials in silicowhere their interaction with bloodcomponents biological barriers cell membranes and intra-cellular structures can be predicted using the model This goalcan be achieved in part by utilizing the combined power ofmacromolecule modeling supramolecular modeling cell model-ing pharmacokinetic modeling and computational toxicologyToxicity as discussed in the prior section is a second key

reason for the pharmaceutical industry to abandon a therapy afterit has reached first-in-human and phase 1 or phase 2 clinical trialsExcipientability ie the feasibility of the carrier material(s) tobe finally proven as an excipient used in humans is directlyrelated to the safety of the nanomedicine313 To have goodexcipientability the carrier material itself should be nontoxic andeasy to excrete completely from the body via the liver (into bile)or the kidneys (into urine) or both According to Choi andFrangioni782 safety and clearance (renal or hepatic) should beincluded among the basic criteria for clinical translation offormulationmaterials administered to humans Accumulation ofany foreign material in the body even inert polymers like PVPcan cause health problems787minus789 Additionally in order toexpedite and to increase the probability of successful translationthe carrier materials should have a clear and simple structure withknown degradation products An even better case as mentionedearlier would be for the carrier material to be made of FDA-approved building blocks In addition to concerns surroundingaccumulation complement activation upon intravenous injec-tion by nanomaterials is a frequent and potentially dangerousevent790 The potential for complement activation of novelnanomaterials can be assessed by combining in vitro tests andin vivo models791 but a full mechanistic understanding ofcomplement activation on nanosurfaces is still missing Rationaldesign of nanomaterials that will not react with complement bydesign is highly desirable but not yet realityFor the pharmaceutical industry the lack of clear and

standardized pathways to regulatory approval for nanosystemsrenders development riskier and therefore less attractive unlesshighly convincing first-in-human studies can be demonstrated byacademia Patent protection is also of key importance forindustry but the flooding of the patent field by a myriad ofnanoscience patents in the past decade with content that is oftenoverlapping and insufficiently examined has rendered thebusiness case for nanomaterials more difficult Too manyopen-ended promises of new technologies may paradoxicallyreduce the propensity of managers and regulators to act792 Jointefforts by the scientific community industry and internationalregulatory bodies to find a common ground through annualinterdisciplinary discussion of the challenges of nanomedicinedesign and toxicity and regulation in the CLINAM conferences

Figure 39 Essential requirements for a nanomedicine to betranslational Adapted with permission from ref 313 Copyright2012 Elsevier

ACS Nano Nano Focus


2358

(httpwwwclinamorg) have contributed to streamlining theregulatory aspects of nanomedicine in recent yearsProcess scale-up ability is the feasibility of making large

volumes of NPs with consistent and reproducible quality toestablish GMP313 For instance a nanomedicine of drug-loadedmicelles made of block copolymers must be produced withreproducible NP size NP size distribution and drug-loadingefficiency and content As the nanomedicine structures becomemore complex the number of quality-control parametersdramatically increases793794 which makes it increasingly difficultto produce acceptably consistent formulations A nanomedicinerequiring only simple production processes is more favorablefor the establishment of a GMP process For instance self-assembling prodrugs can spontaneously form nanomedicineswith advantages that include clear and well-characterized molecularstructures fixed drug loading contents 100 loading efficiency noburst-release problems and easy fabrication all of which arefavorable for clinical translation441795796 For nanomedicine aimedat clinical translation it is advisable to look into excipientability andscale-up ability issues early at the bench to expedite the process

CONCLUSIONS AND PROSPECTSNew NPs tuned for nanomedicine applications are emergingespecially in the fields of drug delivery antibiotic resistanceimaging diagnostics and cancer therapies Recent studies haveshown that the use of multiple nanomaterials (ie NDs andproteins) or a single nanoplatform functionalized with severaltherapeutic agents can successfully image and treat tumors withimproved efficacy NPs-based drug delivery has been includedin the rational biomimetic and systematic design of optimaltherapeutic combinationsNanomedicine in cellular preclinical and clinical studies has

led to many important advances both fundamental andtranslational Many of these advances however have been inthe field of cancer diagnosis and treatment This dispropor-tionate focus is expected to be addressed in upcoming years withresearch focuses expanding to other medical challenges such asantibiotic resistance and artificial organs Nanomedicine is poisedto be of benefit in these areas by virtue of the versatility of thenanomaterial platform design be it through multimodaltherapeutic approaches or through highly specialized multi-faceted design for relevant biological applicationsAlthough nanomedicine has raised exciting expectations for

many medical problems scientific challenges have arisen as wellmainly due to the lack of knowledge about the behavior ofnanomaterials inside living organisms However due to the basicresearch focused on these issues we are now closer to solvingthem and to reaching ldquorealrdquo medical solutions based onnanomedicine

AUTHOR INFORMATIONCorresponding AuthorE-mail wolfgangparakphysikuni-marburgdeORCIDBeatriz Pelaz 0000-0002-4626-4576Anne M Andrews 0000-0002-1961-4833Laura Ballerini 0000-0001-8420-0787Monica Carril 0000-0002-1232-8658Warren C W Chan 0000-0001-5435-4785Chunying Chen 0000-0002-6027-0315Xiaodong Chen 0000-0002-3312-1664Xiaoyuan Chen 0000-0002-9622-0870

Jianzhong Du 0000-0003-1889-5669Alberto Escudero 0000-0002-0850-6589Mingyuan Gao 0000-0002-7360-3684Yury Gogotsi 0000-0001-9423-4032Zhongwei Gu 0000-0003-1547-6880Naomi J Halas 0000-0002-8461-8494Norbert Hampp 0000-0003-1614-2698Mark C Hersam 0000-0003-4120-1426Ji Jian 0000-0001-9870-4038Xingyu Jiang 0000-0002-5008-4703Ali Khademhosseini 0000-0002-2692-1524Jindrich Kopecek 0000-0002-4451-6944Nicholas A Kotov 0000-0002-6864-5804Xing-Jie Liang 0000-0002-4793-1705Luis M Liz-Marzan 0000-0002-6647-1353Paul Mulvaney 0000-0002-8007-3247Shuming Nie 0000-0002-7328-1144Tai Hyun Park 0000-0003-4254-0657Reginald M Penner 0000-0003-2831-3028Vincent M Rotello 0000-0002-5184-5439Raymond E Schaak 0000-0002-7468-8181Youqing Shen 0000-0003-1837-7976Molly M Stevens 0000-0002-7335-266XJ Scott VanEpps 0000-0002-5437-3123Tanja Weil 0000-0002-5906-7205Paul S Weiss 0000-0001-5527-6248Itamar Willner 0000-0001-9710-9077Yuliang Zhao 0000-0002-9586-9360Xin Zhou 0000-0002-5580-7907Wolfgang J Parak 0000-0003-1672-6650NotesThe authors declare no competing financial interest

ACKNOWLEDGMENTSThis report is partly based on presentations held at the Sino-German SymposiumNanomaterials for Biomedical ApplicationsOctober 27minus31 2013 Hangzhou China ChinaNanomedicine2015 April 6minus9 2015 Hangzhou China the third InternationalSymposium on Molecular Imaging and Nanomedicine April25minus29 Suzhou China and the 5th Asian Biomaterials Congress(ABMC5) May 6minus9 2015 Taipei Taiwan (ROC) and theMicro- and Nanotechnologies for Medicine Emerging Frontiersand Applications July 27minus31 2015 Cambridge MA UnitedStates This work was supported by the Deutscher AkademischerAustauschdienst (DAAD to Philipps Universitat Marburg andZhejiang University Hangzhou) the Chinesisch DeutschesZentrum fur Wissenschaftsforderung (ldquoCDZrdquo to ZG andWJP) and the Chinese Academy of Science (CAS) Part ofthis work was supported by the National Natural ScienceFoundation (51390481 81227902 81625011) National BasicResearch Program (2014CB931900) of China (to YS) by theEuropean Commission grant Futurenanoneeds (to VP andWJP) by the Spanish Ministerio de Economia y Compet-itividad (CTQ2011-23167 and CTQ2014-59808R to RAAP)the Generalitat of Catalunya (2014-SGR-612 to RAAP) theDeutsche Forschungsgemeinschaft (DFG) (AL5528-1 to RT)the Swiss National Science Foundation (NRP62 to PH) theClaude amp Julianna Foundation (grant to PH) the NationalScience Foundation (NSF) grants CHE-1306928 (to RP) andECS-0601345 CBET 0933384 CBET 0932823 and CBET

ACS Nano Nano Focus


2359

1036672 (to NAK) Canadian Institute of Health Research(grant to WCWC) and Natural Sciences and EngineeringResearch Council of Canada (grant to WCWC) SA and BPacknowledge a fellowship from the Alexander von HumboldtFoundation NF acknowledges the Lars Hiertas MinneFoundation MC acknowledges Ikerbasque for a ResearchFellow position XC acknowledges the Intramural ResearchProgram (IRP) National Institute of Biomedical Imaging andBioengineering (NIBIB) National Institutes of Health (NIH)BZT acknowledges the Innovation and Technology Commis-sion of Hong Kong (ITC-CNERC14SC01) The PancreaticCancer research of AEN and HM was funded by the USNational Cancer Institute NIH grant U01CA198846 AEacknowledges Junta de Andaluciacutea (Spain) for a Talentia PostdocFellowship co-financed by the European Unions SeventhFramework Programme grant agreement no 267226 AGSacknowledges support by BOF (UGent) and FWO (ResearchFoundation Flanders) Part of this work was supported by theNational Natural Science Foundation of China (81625011)

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(354) Sukhorukov G B Rogach A L Zebli B Liedl T Skirtach AG Kohler K Antipov A A Gaponik N Susha A S WinterhalterM Parak W J Nanoengineered Polymer Capsules Tools forDetection Controlled Delivery and Site Specific Manipulation Small2005 1 194minus200(355) Pan H Yang J Kopeckova P Kopecek J BackboneDegradable Multiblock N-(2-Hydroxypropyl)methacrylamide Copoly-mer Conjugates via Reversible AdditionminusFragmentation Chain Trans-fer Polymerization and Thiolminusene Coupling Reaction Biomacromole-cules 2011 12 247minus252(356) Agnihotri S A Mallikarjuna N N Aminabhavi T M RecentAdvances on Chitosan-Based Micro- and Nanoparticles in DrugDelivery J Controlled Release 2004 100 5minus28(357) Pouton C W Lipid Formulations for Oral Administration ofDrugs Non-Emulsifying Self-Emulsifying and ldquoSelf-MicroemulsifyingrdquoDrug Delivery Systems Eur J Pharm Sci 2000 11 S93minus8(358) Yeh T-H Hsu L-W Tseng M T Lee P-L Sonjae K HoY-C Sung H-WMechanism and Consequence of Chitosan-MediatedReversible Epithelial Tight Junction Opening Biomaterials 2011 326164minus6173(359) Nicoligrave E Syga M I Bosetti M Shastri V P Enhanced GeneSilencing through Human Serum Albumin-Mediated Delivery ofPolyethylenimine-siRNA Polyplexes PLoS One 2015 10 e0122581(360) Cui D X Zhang C L Liu B Shu Y Du T Shu D WangK Dai F P Liu Y L Li C Pan F Yang Y M Ni J Li H Brand-Saberi B Guo PX Regression of Gastric Cancer by Systemic Injectionof RNA Nanoparticles Carrying both Ligand and siRNA Sci Rep 20155 10726(361) Conde J Tian F R Hernandez Y Bao C C Baptista P VCui D X Stoeger T de la Fuente J M RNAi-BasedGlyconanoparticles Trigger Apoptotic Pathways for in Vitro and inVivo Enhanced Cancer-Cell Killing Nanoscale 2015 7 9083minus9091(362) Shu D Shu Y Haque F Abdelmawla S Guo P XThermodynamically Stable RNA Three-Way Junction for ConstructingMultifunctional Nanoparticles for Delivery of Therapeutics NatNanotechnol 2011 6 658minus667(363)Wu Y Ermakova A LiuW Pramanik G Vu TM Kurz AMcGuinness L Naydenov B Hafner S Reuter R Wrachtrup JIsoya J Fortsch C Barth H Simmet T Jelezko F Weil TProgrammable Biopolymers for Advancing Biomedical Applications ofFluorescent Nanodiamonds Adv Funct Mater 2015 25 6576minus6585(364) Alhaddad A Adam M P Botsoa J Dantelle G PerruchasS Gacoin T Mansuy C Lavielle S Malvy C Treussart FBertrand J R Nanodiamond as a Vector for siRNA Delivery to EwingSarcoma Cells Small 2011 7 3087minus3095(365) Shimkunas R A Robinson E Lam R Lu S Xu X Y ZhangX Q Huang H J Osawa E Ho D Nanodiamond-InsulinComplexes as pH-Dependent Protein Delivery Vehicles Biomaterials2009 30 5720minus5728(366) Knapinska A M Tokmina-Roszyk D Amar S Tokmina-Roszyk M Mochalin V N Gogotsi Y Cosme P Terentis A CFields G B Solid-Phase Synthesis Characterization and CellularActivities of Collagen-Model Nanodiamond-Peptide ConjugatesBiopolymers 2015 104 186minus195(367) Cabral H Matsumoto Y Mizuno K Chen Q MurakamiM Kimura M Terada Y Kano M R Miyazono K Uesaka MNishiyama N Kataoka K Accumulation of Sub-100 nm PolymericMicelles in Poorly Permeable Tumours Depends on Size NatNanotechnol 2011 6 815minus823(368) Sugahara K N Teesalu T Karmali P P Kotamraju V RAgemy L Greenwald D R Ruoslahti E Coadministration of aTumor-Penetrating Peptide Enhances the Efficacy of Cancer DrugsScience 2010 328 1031minus1035(369) Frese K K Neesse A Cook N Bapiro T E Lolkema M PJodrell D I Tuveson D A Nab-Paclitaxel Potentiates GemcitabineActivity by Reducing Cytidine Deaminase Levels in a Mouse Model ofPancreatic Cancer Cancer Discovery 2012 2 260minus269(370) Liu J Liao S Diop-Frimpong B Chen W Goel SNaxerova K Ancukiewicz M Boucher Y Jain R K Xu L TGF-β

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Blockade Improves the Distribution and Efficacy of Therapeutics inBreast Carcinoma by Normalizing the Tumor Stroma Proc Natl AcadSci U S A 2012 109 16618minus16623(371) Meng H Zhao Y Dong J Xue M Lin Y-S Ji Z Mai WX Zhang H Chang C H Brinker C J Zink J I Nel A E Two-Wave Nanotherapy To Target the Stroma and Optimize GemcitabineDelivery To a Human Pancreatic Cancer Model in Mice ACS Nano2013 7 10048minus10065(372) Meng H Wang M Liu H Liu X Situ A Wu B Ji ZChang C H Nel A E Use of a Lipid-Coated Mesoporous SilicaNanoparticle Platform for Synergistic Gemcitabine and PaclitaxelDelivery to Human Pancreatic Cancer in Mice ACS Nano 2015 93540minus3557(373) Liu X Situ A Kang Y Villabroza K R Liao Y Chang CH Donahue T Nel A E Meng H Irinotecan Delivery by Lipid-Coated Mesoporous Silica Nanoparticles Shows Improved Efficacy andSafety over Liposomes for Pancreatic Cancer ACS Nano 2016 102702minus2715(374) Torchilin V Tumor Delivery of Macromolecular Drugs Basedon the EPR Effect Adv Drug Delivery Rev 2011 63 131minus135(375) Zhang J Yuan Z F Wang Y Chen W H Luo G F ChengS X Zhuo R X Zhang X Z Multifunctional Envelope-TypeMesoporous Silica Nanoparticles for Tumor-Triggered Targeting DrugDelivery J Am Chem Soc 2013 135 5068minus5073(376) Quan C Y Chen J X Wang H Y Li C Chang C ZhangX Z Zhuo R X Core-Shell Nanosized Assemblies Mediated by theAlpha-Beta Cyclodextrin Dimer with a Tumor-Triggered TargetingProperty ACS Nano 2010 4 4211minus4219(377) Zhong Y N Yang W J Sun H L Cheng R Meng F HDeng C Zhong Z Y Ligand-Directed Reduction-Sensitive Shell-Sheddable Biodegradable Micelles Actively Deliver Doxorubicin intothe Nuclei of Target Cancer Cells Biomacromolecules 2013 14 3723minus3730(378) Chen W Zou Y Meng F Cheng R Deng C Feijen JZhong Z Glyco-Nanoparticles with Sheddable Saccharide Shells AUnique and Potent Platform for Hepatoma-Targeting Delivery ofAnticancer Drugs Biomacromolecules 2014 15 900minus907(379) Yuan Y Y Mao C Q Du X J Du J Z Wang F Wang JSurface Charge Switchable Nanoparticles Based on ZwitterionicPolymer for Enhanced Drug Delivery to Tumor Adv Mater 201224 5476minus5480(380) Liu X S Li H Jin Q Ji J Surface Tailoring of Nanoparticlesvia Mixed-Charge Monolayers and Their Biomedical ApplicationsSmall 2014 10 4230minus4242(381) Marradi M Chiodo F Garcia I Penades S Glyconano-particles as Multifunctional and Multimodal Carbohydrate SystemsChem Soc Rev 2013 42 4728minus4745(382) Garcia I Sanchez-Iglesias A Henriksen-Lacey M GrzelczakM Penades S Liz-Marzan L M Glycans as Biofunctional Ligands forGold Nanorods Stability and Targeting in Protein-Rich Media J AmChem Soc 2015 137 3686minus3692(383) Liu X S Chen Y J Li H Huang N Jin Q Ren K F Ji JEnhanced Retention and Cellular Uptake of Nanoparticles in Tumorsby Controlling Their Aggregation Behavior ACS Nano 2013 7 6244minus6257(384) Liu X S Li H Chen Y J Jin Q Ren K F Ji J Mixed-Charge Nanoparticles for Long Circulation Low ReticuloendothelialSystem Clearance and High Tumor Accumulation Adv HealthcareMater 2014 3 1439minus1447(385) Li H Liu X S Huang N Ren K F Jin Q Ji J Mixed-Charge Self-Assembled MonolayersPrime as a Facile Method to Design pH-induced Aggregation of Large Gold Nanoparticles for Near-InfraredPhotothermal Cancer Therapy ACS Appl Mater Interfaces 2014 618930minus18937(386) Wang Y C Bahng J H Che Q T Han J S Kotov N AAnomalously Fast Diffusion of Targeted Carbon Nanotubes in CellularSpheroids ACS Nano 2015 9 8231minus8238(387)Wang J QMaoWW Lock L L Tang J B Sui M H SunW L Cui H G Xu D Shen Y Q The Role of Micelle Size in Tumor

Accumulation Penetration and Treatment ACS Nano 2015 9 7195minus7206(388) Huo S D Ma H L Huang K Y Liu J Wei T Jin S BZhang J C He S T Liang X J Superior Penetration and RetentionBehavior of 50 nmGold Nanoparticles in Tumors Cancer Res 2013 73319minus330(389) Xiong X B Huang Y Lu W L Zhang X Zhang H NagaiT Zhang Q Enhanced Intracellular Delivery and Improved AntitumorEfficacy of Doxorubicin by Sterically Stabilized Liposomes Modifiedwith a Synthetic RGD Mimetic J Controlled Release 2005 107 262minus275(390) Mei D Lin Z Q Fu J J He B Gao W Ma L Dai W BZhang H Wang X Q Wang J C Zhang X Lu W L Zhou D MZhang Q The Use of Alpha-Conotoxin ImI to Actualize the TargetedDelivery of Paclitaxel Micelles to Alpha 7 nAChR-Overexpressing BreastCancer Biomaterials 2015 42 52minus65(391) Dai W B Yang T Y Wang Y G Wang X Q Wang J CZhang X Zhang Q Peptide PHSCNK as an Integrin alpha(5)beta(1)Antagonist Targets Stealth Liposomes to Integrin-OverexpressingMelanoma Nanomedicine 2012 8 1152minus1161(392) Zheng N Dai W DuW Zhang H Lei L Zhang HWangX Wang J Zhang X Gao J Zhang Q A Novel Lanreotide-EncodedMicelle SystemTargets Paclitaxel to the Tumors with Overexpression ofSomatostatin Receptors Mol Pharmaceutics 2012 9 1175minus1188(393) Zhang J L Jin W Wang X Q Wang J C Zhang X AZhang Q A A Novel Octreotide Modified Lipid Vesicle Improved theAnticancer Efficacy of Doxorubicin in Somatostatin Receptor 2 PositiveTumor Models Mol Pharmaceutics 2010 7 1159minus1168(394) Xiang Y Liang L Wang X Wang J Zhang X Zhang QChloride Channel-Mediated Brain Glioma Targeting of Chlorotoxin-Modified Doxorubicine-Loaded Liposomes J Controlled Release 2011152 402minus410(395) Guo Z M He B Jin H W Zhang H R Dai W B ZhangL R Zhang H Wang X Q Wang J C Zhang X Zhang QTargeting Efficiency of RGD-Modified Nanocarriers with DifferentLigand Intervals in Response to Integrin Alpha v Beta 3 ClusteringBiomaterials 2014 35 6106minus6117(396) Cai D Gao W He B Dai W Zhang H Wang X Wang JZhang X Zhang Q Hydrophobic Penetrating Peptide PFVYLI-Modified Stealth Liposomes for Doxorubicin Delivery in Breast CancerTherapy Biomaterials 2014 35 2283minus2294(397) Sykes E A Chen J Zheng G Chan W C W Investigatingthe Impact of Nanoparticle Size on Active and Passive Tumor TargetingEfficiency ACS Nano 2014 8 5696minus5706(398) Laurent S Saei A A Behzadi S Panahifar A Mahmoudi MSuperparamagnetic Iron Oxide Nanoparticles for Delivery of Ther-apeutic Agents Opportunities and Challenges Expert Opin DrugDelivery 2014 11 1449minus1470(399) Alexiou C Arnold W Klein R J Parak F G Hulin PBergemann C Erhardt W Wagenpfeil S Lubbe A S LocoregionalCancer Treatment with Magnetic Drug Targeting Cancer Res 2000 606641minus6648(400) Lueshen E Venugopal I Soni T Alaraj A Linninger AImplant-Assisted Intrathecal Magnetic Drug Targeting to Aid inTherapeutic Nanoparticle Localization for Potential Treatment ofCentral Nervous System Disorders J Biomed Nanotechnol 2015 11253minus261(401) Hournkumnuard K Natenapit M Magnetic Drug Targetingby Ferromagnetic Microwires Implanted within Blood Vessels MedPhys 2013 40 062302(402) He W Ji Y Luo C Xu Z Development of Single-SideMagnet Array for Super Paramagnetic Nano-Particle Targeting Res JAppl Sci Eng Technol 2014 7 3022minus3029(403) Nacev A Weinberg I N Stepanov P Y Kupfer S Mair LO Urdaneta M G Shimoji M Fricke S T Shapiro B DynamicInversion Enables External Magnets To Concentrate FerromagneticRods to a Central Target Nano Lett 2015 15 359minus364(404) Tietze R Lyer S Durr S Struffert T Engelhorn TSchwarz M Eckert E Goen T Vasylyev S Peukert W Wiekhorst

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F Trahms L Dorfler A Alexiou C Efficient Drug-Delivery UsingMagnetic Nanoparticles minus Biodistribution and Therapeutic Effects inTumour Bearing Rabbits Nanomedicine 2013 9 961minus971(405) Karamipour S Sadjadi M S Farhadyar N Fabrication andSpectroscopic Studies of Folic Acid-Conjugated Fe3O4Au Core-Shellfor Targeted Drug Delivery Application Spectrochim Acta Part A 2015148 146minus155(406) Unterweger H Tietze R Janko C Zaloga J Lyer S DurrS Taccardi N Goudouri O M Hoppe A Eberbeck D SchubertD W Boccaccini A R Alexiou C Development and Characterizationof Magnetic Iron Oxide Nanoparticles with a Cisplatin-Bearing PolymerCoating for Targeted Drug Delivery Int J Nanomed 2014 9 3659minus3676(407) Hamarat Sanlier S Yasa M Cihnioglu A O AbdulhayogluM Yilmaz H Ak G Development of Gemcitabine-AdsorbedMagnetic Gelatin Nanoparticles for Targeted Drug Delivery in LungCancer Artif Cells Nanomed Biotechnol 2016 1minus7(408) Huang J Shu Q Wang L Y Wu H Wang A Y Mao HLayer-by-Layer Assembled Milk Protein Coated Magnetic NanoparticleEnabled Oral Drug Delivery with High Stability in Stomach andEnzyme-Responsive Release in Small Intestine Biomaterials 2015 39105minus113(409) Halupka-Bryl M Asai K Thangavel S Bednarowicz MKrzyminiewski R Nagasaki Y Synthesis and in Vitro and in VivoEvaluations of Poly(ethylene Glycol)-Block-poly(4-Vinylbenzyl-phosphonate) Magnetic Nanoparticles Containing Doxorubicin as aPotential Targeted Drug Delivery System Colloids Surf B 2014 118140minus147(410) Wang N Guan Y P Yang L R Jia L W Wei X T Liu HZ Guo C Magnetic Nanoparticles (MNPs) Covalently Coated byPEO-PPO-PEO Block Copolymer for Drug Delivery J Colloid InterfaceSci 2013 395 50minus57(411) Zaloga J Janko C Nowak J Matuszak J Knaup SEberbeck D Tietze R Unterweger H Friedrich R P Duerr SHeimke-Brinck R Baum E Cicha I Dorje F Odenbach S Lyer SLee G Alexiou C Development of a Lauric AcidAlbumin HybridIron Oxide Nanoparticle System with Improved Biocompatibility Int JNanomed 2014 9 4847minus4866(412) Zaloga J Janko C Agarwal R Nowak J Muller RBoccaccini A R Lee G Odenbach S Lyer S Alexiou C DifferentStorage Conditions Influence Biocompatibility and PhysicochemicalProperties of Iron Oxide Nanoparticles Int J Mol Sci 2015 16 9368minus9384(413) Lepeltier E A Nuhn L Lehr C M Zentel R Not Just forTumor Targeting Unmet Medical Needs and Opportunities forNanomedicine Nanomedicine 2015 10 3147minus3166(414) de Souza Carvalho C Daum N Lehr C M CarrierInteractions with the Biological Barriers of the Lung Advanced in VitroModels and Challenges for Pulmonary Drug Delivery Adv DrugDelivery Rev 2014 75 129minus140(415) Mitragotri S Burke P A Langer R Overcoming theChallenges in Administering Biopharmaceuticals Formulation andDelivery Strategies Nat Rev Drug Discovery 2014 13 655minus672(416) Schmidt C Lautenschlaeger C Collnot EM SchumannMBojarski C Schulzke J D Lehr C M Stallmach A Nano- andMicroscaled Particles for Drug Targeting to Inflamed IntestinalMucosa-A First in Vivo Study in Human Patients J Controlled Release 2013 165139minus145(417) Ali H Weigmann B Collnot E M Khan S A WindbergsM Lehr C M Budesonide Loaded PLGA Nanoparticles for Targetingthe Inflamed Intestinal Mucosa-Pharmaceutical Characterization andFluorescence Imaging Pharm Res 2016 33 1085minus1092(418) Ali H Weigmann B Neurath M F Collnot E MWindbergs M Lehr CM Budesonide Loaded Nanoparticles with pH-Sensitive Coating for Improved Mucosal Targeting in Mouse Models ofInflammatory Bowel Diseases J Controlled Release 2014 183 167minus177(419) Mittal A Schulze K Ebensen T Weissmann S Hansen SGuzman C A Lehr C M Inverse Micellar Sugar Glass (IMSG)

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Diverse Applications of NanomedicineBeatriz Pelazdagger Christoph AlexiouDagger Ramon A Alvarez-Pueblasect∥ Frauke Alvespara

Anne M AndrewsΦ Sumaira Ashrafdagger Lajos P Balogh Laura Ballerini Alessandra Bestetti

Cornelia Brendel Susanna Bosi Monica Carril Warren C W Chan$ Chunying Cheninfin

Xiaodong Chenotimes Xiaoyuan Chenint Zhen Cheng⧧ Daxiang Cuiℏ Jianzhong Duforall Christian Dullinpara

Alberto Escuderodaggerƛ Neus Feliupart Mingyuan Gao∮ Michael Georgeprod Yury Gogotsi

Arnold Grunwelleroplus Zhongwei Guamp Naomi J Halascurren Norbert Hampp Roland K Hartmannoplus

Mark C Hersam Patrick Hunziker⟠loz Ji Jian⧫ Xingyu Jianginfin Philipp Jungebluth

Pranav Kadhiresan$ Kazunori Kataoka⊡ Ali Khademhosseinidaggerdagger Jindrich KopecekDaggerDagger

Nicholas A Kotovsectsect Harald F Krugperpperp Dong Soo Leeparapara Claus-Michael Lehr

Kam W Leong Xing-Jie Lianginfin Mei Ling Limpart Luis M Liz-Marzan Xiaowei Ma

Paolo Macchiarini Huan Meng Helmuth Mohwald Paul Mulvaney

Andre E Nel Shuming Nie Peter Nordlandercurren Teruo Okano Jose Oliveira$$

Tai Hyun Parkinfininfinotimesotimes Reginald M Pennerint int Maurizio Prato Victor Puntes∥⧧⧧ΦΦ

Vincent M Rotelloℏℏ Amila Samarakoon$ Raymond E Schaakforallforall Youqing Shenƛƛ

Sebastian Sjoqvistpart Andre G Skirtachpartpart Mahmoud G Solimandagger Molly M Stevens∮ ∮

Hsing-Wen Sungoplusoplus Ben Zhong Tangprodprod Rainer TietzeDagger Buddhisha N Udugama$

J Scott VanEppssectsect Tanja Weilampamp Paul S Weisscurrencurren Itamar Willner Yuzhou Wu

Lily Yang$$ Zhao Yuedagger Qian Zhangdagger Qiang Zhang⟠⟠ Xian-En Zhanglozloz Yuliang Zhaoinfin

Xin Zhou⧫⧫ and Wolfgang J Parakdagger

daggerFachbereich Physik Fachbereich Medizin oplusFachbereich Pharmazie and Department of Chemistry Philipps UniversitatMarburg 35037 Marburg GermanyDaggerENT-Department Section of Experimental Oncology amp Nanomedicine (SEON) Else Kroner-Fresenius-Stiftung-Professorship forNanomedicine University Hospital Erlangen 91054 Erlangen GermanysectDepartment of Physical Chemistry Universitat Rovira I Virgili 43007 Tarragona Spain∥ICREA Pg Lluiacutes Companys 23 08010 Barcelona SpainDepartment of Haematology and Medical Oncology paraDepartment of Diagnostic and Interventional Radiology University MedicalCenter Gottingen 37075 Gottingen GermanyDepartment of Molecular Biology of Neuronal Signals Max-Planck-Institute for Experimental Medicine 37075 GottingenGermany

California NanoSystems Institute Department of Chemistry and Biochemistry and ΦDepartment of Psychiatry and SemelInstitute for Neuroscience and Human Behavior Division of NanoMedicine and Center for the Environmental Impact ofNanotechnology and currencurrenDepartment of Materials Science and Engineering University of California Los Angeles Los AngelesCalifornia 90095 United StatesAA Nanomedicine amp Nanotechnology Consultants North Andover Massachusetts 01845 United StatesInternational School for Advanced Studies (SISSAISAS) 34136 Trieste ItalySchool of Chemistry amp Bio21 Institute University of Melbourne Parkville Victoria 3010 AustraliaDepartment of Chemical and Pharmaceutical Sciences University of Trieste 34127 Trieste ItalyCIC biomaGUNE Paseo de Miramon 182 20014 Donostia - San Sebastian SpainIkerbasque Basque Foundation for Science 48013 Bilbao Spain$Institute of Biomaterials and Biomedical Engineering University of Toronto Toronto Ontario M5S 3G9 CanadainfinCAS Center for Excellence in Nanoscience and CAS Key Laboratory for Biomedical Effects of Nanomaterials and NanosafetyNational Center for Nanoscience and Technology of China Beijing 100190 China

Received September 7 2016Published March 14 2017

Nano

Focus

wwwacsnanoorg

copy 2017 American Chemical Society 2313 DOI 101021acsnano6b06040ACS Nano 2017 11 2313minus2381

This is an open access article published under an ACS AuthorChoice License which permitscopying and redistribution of the article or any adaptations for non-commercial purposes









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