best practices in cancer nanotechnology_perspective from nci nanotechnology alliance

Review

Best Practices in Cancer Nanotechnology: Perspective fromNCI Nanotechnology Alliance

William C. Zamboni1, Vladimir Torchilin2, Anil K. Patri4, Jeff Hrkach3, Stephen Stern4, Robert Lee6,Andre Nel7, Nicholas J. Panaro4, and Piotr Grodzinski5

AbstractHistorically, treatment of patients with cancer using chemotherapeutic agents has been associated with

debilitating and systemic toxicities, poor bioavailability, and unfavorable pharmacokinetics. Nanotech-

nology-based drug delivery systems, on the other hand, can specifically target cancer cells while avoiding

their healthy neighbors, avoid rapid clearance from the body, and be administered without toxic solvents.

They hold immense potential in addressing all of these issues, which has hampered further development of

chemotherapeutics. Furthermore, such drug delivery systems will lead to cancer therapeutic modalities that

are not only less toxic to the patient but also significantly more efficacious. In addition to established

therapeutic modes of action, nanomaterials are opening up entirely newmodalities of cancer therapy, such

as photodynamic and hyperthermia treatments. Furthermore, nanoparticle carriers are also capable of

addressing several drug delivery problems that could not be effectively solved in the past and include

overcoming formulation issues, multidrug-resistance phenomenon, and penetrating cellular barriers that

may limit device accessibility to intended targets, such as the blood–brain barrier. The challenges in

optimizing design of nanoparticles tailored to specific tumor indications still remain; however, it is clear that

nanoscale devices carry a significant promise towardnewways of diagnosing and treating cancer. This review

focuses on future prospects of using nanotechnology in cancer applications and discusses practices and

methodologies used in the development and translation of nanotechnology-based therapeutics.ClinCancer

Res; 18(12); 3229–41. �2012 AACR.

Defining Oncology Applications forNanotechnology ConstructsMoving a prospective new drug to the clinic is an

arduous task. Despite decades of experience and well-defined best practices for evaluating small molecules asdrug candidates, only 1 of every 5,000 to 10,000 prospec-tive formulations reaches U.S. Food and Drug Adminis-tration (FDA) approval, and only 5% of oncology drugsentering phase I clinical trials are approved (1).

Recently, chemists, pharmaceutical scientists, biologists,biomedical engineers, andoncologists have turned tonano-technology in their quest for innovation and improve-ment of the success rate in drug development. Nanotech-nology is defined by the National Nanotechnology Initia-tive (http://www.nano.gov) as research and technologydevelopment at the atomic, molecular, or macromolecularscale leading to the controlled creation anduseof structures,devices, and systems with a length scale of approximately100 nm (Fig. 1). Examples of nanoparticle platforms areincluded in Figs. 1 and 2. The multifunctional constructsbased on novel nanomaterials can be delivered directly tothe tumor site and eradicate cancer cells selectively. Anappropriate nanoconstruct design allows for improved drugefficacy at lower doses as comparedwith the small-moleculedrug treatment, as well as a wider therapeutic window andlower side effects. In addition to established therapeuticmodes of action, nanomaterials are opening up entirelynew modalities of cancer therapy, such as photodynamicand hyperthermia treatments. Furthermore, nanoparticlecarriers are also capable of addressing several drug deliveryproblems, which could not be effectively solved in the pastand include overcoming multidrug-resistance phenome-non and penetrating cellular barriers that may limit deviceaccessibility to intended targets, such as the blood–brainbarrier, among others. Polyethylene glycol (PEG)ylated-

Authors' Affiliations: 1UNC Eshelman School of Pharmacy, UNC Line-berger Comprehensive Cancer Center, Carolina Center for CancerNanotechnology Excellence, UNC Institute for Pharmacogenomics andIndividualized Therapy, The University of North Carolina at Chapel Hill,Chapel Hill, North Carolina; 2Northeastern University, Bouvé College ofHealth Sciences, Department of Pharmaceutical Sciences, Boston; and3BIND Biosciences, Cambridge; 4Nanotechnology CharacterizationLaboratory, Advanced Technology Program, SAIC-Frederick Inc.,NCI-Frederick, Frederick; and 5NCI Nanotechnology Alliance, NationalCancer Institute, Bethesda, Maryland; 6Particle Sciences, Inc., Bethle-hem, Pennsylvania; and 7Division of NanoMedicine, Department ofMedicine, David Geffen School of Medicine at UCLA, Los Angeles,California

Corresponding Author: William C Zamboni, Genetic Medicine Building,Room 1013, CB# 7361, 120 Mason Farm Road, Chapel Hill, NC 27599.Phone: 919-843-6665; Fax: 919-966-5863; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-11-2938

�2012 American Association for Cancer Research.

ClinicalCancer

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liposomal doxorubicin (Doxil; Janssen and Caelyx; Scher-ing-Plough), liposomal daunorubicin (DaunoXome; GalenLtd.), liposomal cytarabine (DepoCyt; Celgene), and pacli-taxel albumin-bound particles (Abraxane; Abraxis Bios-

ciences) are the only members of this relatively new classof agents that are approved in the United States (2–7).

The challenges in optimizing design of nanoparticlestailored to specific tumor indications still remain; however,it is clear that nanoscale devices carry significant promisetoward new ways of diagnosing and treating cancer. Thisreview focuses on future prospects of using nanotechno-logy in cancer applications and discusses practices andmethodologies used in the development and translationof nanotechnology-based therapeutics.

Design Trends for a Successful In Vivo CarrierGeneral concepts

The therapeutic effects of many anticancer drugs and theoutcome of anticancer therapies could be significantlyimproved if (i) delivery of the drug occurs specifically totumors (cancer cells) or preferably inside specific organellesin cells and (ii) reduction of drug toxic side effects isachieved. In the case of poorly soluble drug candidates,the solubility and/or bioavailability problem could also beovercome. Various pharmaceutical nanocarriers (e.g., lipo-somes, polymeric micelles, and polymeric nanoparticles)have been used in preparing novel dosage formulationswith good bioavailability and specific drug delivery to tu-mors. Zeta potential, size, cationic surface charge, and solu-bility are factors that affect the biocompatibility of thesenanocarriers (Fig. 3). These factors influence the cytotoxicity

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Figure 1. Definition of nanotechnology and examples of nanotechnology platforms used in drug development. This figure was obtained with permission fromthe Society for Leukocyte Biology (Fig. 3 of ref. 97).

Figure 2. Collage of nanomedical particles and devices developed bymembers of the NCI Alliance for Nanotechnology in Cancer. This figurewas obtained with permission from IEEE (Fig. 1 of ref. 98; photo courtesyof the NCI Alliance for Nanotechnology in Cancer, NanotechnologyImage Library).

Zamboni et al.

Clin Cancer Res; 18(12) June 15, 2012 Clinical Cancer Research3230




(surface reactivity), clearance process (renal or biliary),mononuclear phagocyte system (MPS)/reticuloendothelialsystem (RES) recognition, and enhanced permeability andretention (EPR) effect.Multifunctional nanomedicines that are being developed

can combine not only different biologic properties (e.g.,increased circulation time in blood, ability to accumulatein tumors via the EPR effect, and stimulus sensitivity), butalso carry a combination of several drugs and diagnosticlabels and/or markers (8). Targeted nanosystems that havea capability of specifically targeting cell surfaces or intra-cellular components and, as such, contribute to enhancedaccumulation of the drug in tumor are also emerging (9).Currently, lipid-based nanomedicines (such as liposomes,lipid-core polymeric micelles, solid lipid nanoparticles)have gained increased interest owing to their good biologiccompatibility, easy control over their composition andproperties, and suitability for scale-up to large-scale pro-duction at reasonable costs (10).

Active targeting with ligands and antibodiesNearly all cancer nanomedicines use some aspect of

targeting. Most rely solely upon "passive" targeting, alsoknown as the EPR effect, which allows for the extravasationof nanoparticles from the circulation via abnormal fenes-trations in tumor vasculature.

"Active" targeting of nanomedicines provides the addi-tional targeting mechanism of receptor-mediated bindingof nanoparticles to surface receptors expressed on tumorcells or blood vessels such as anb3-integrins (11), folic acid(12), and prostate-specific membrane antigen, also knownas PSMA (13). Research has been conducted on severaltargeting ligands, including antibodies, aptamers, peptides,and small molecules. Studying transferrin-targeted nano-particles, Choi and colleagues at the California Institutefor Technology showed that actively targeted nano-particles deliver a higher payload to cancer cells than theirpassively targeted counterparts (14).

A successful, actively targeted nanomedicine requiresa delicate balance of ligand content and surface exposurethat minimizes immunologic recognition and clearanceto provide sufficient nanoparticle circulation time to reachthe target cells, while achieving appropriate binding affi-nity to the surface receptors expressed on tumor cells orblood vessels. The presence ofmultiple targeting ligands pernanoparticle yields a binding affinity stronger than for theligand alone, thus enhancing the ligand-receptor–bindinginteraction for the nanoparticles.

Gu and colleagues, at MIT and Harvard University,showed the importance of this balance with their apta-mer-functionalized, actively targeted poly(lactic-co-glycolicacid)–PEG (PLGA-PEG) nanoparticles in which 5%

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Figure 3. Nanoparticle biocompatibility trends. The zeta potential, size, and solubility affect the cytotoxicity (surface reactivity), clearance process (renal orbiliary), MPS/RES recognition, and EPR effect. This figure was obtained with permission from John Wiley and Sons (Fig. 3 of ref. 99).

Cancer Nanotechnology: Perspective from NCI

www.aacrjournals.org Clin Cancer Res; 18(12) June 15, 2012 3231




aptamer surface coverage was ideal for optimal tumoraccumulation (13). These fundamental findings havebeen advanced by BIND Biosciences, replacing the aptamerwith a small-molecule–targeting ligand, better suited forpharmaceutical development, with their PSMA-targeteddocetaxel (BIND-014) in a phase I clinical study for arange of solid tumor cancers (ClinicalTrials.gov identifierNCT01300533). The efficacy of BIND-014 PSMA-targeteddocetaxel nanoparticles in a PSMA-expressing humanLNCaP prostate cancer xenograft mouse model is present-ed in Fig. 4.

Design trends for nanoparticles intended for deliveryof special agents or distinct indications: highly toxicagents

Because many anticancer drugs are highly toxic, it is verydesirable to develop nanomedicines that limit their toxicityat tumor sites with a minimal toxicity toward normaltissues. Ideally, drugs should be developed that possesstoxicity only against diseased ormutated cells. For example,some inhibitors of poly (ADP-ribose) are highly toxic onlyagainst cell lines exhibiting certain cancer-associated muta-tions and do not affect cells without such mutations (15).However, the development of such drugs with mechanism-based selectivity is in its infancy. Approaches that are moredeveloped are associated with the specific targeting ofdrug-loaded nanoparticles to tumor cells or providingcontrolled or on-demand drug delivery. The paradox oftargeted delivery is that even with effective targeting, themajority of the administered dose ends up in normaltissues throughout the body, resulting in pronounced non-specific off-target toxicity. If targeting efficiency could be

increased such that as little as 3% of the injected dosereaches the tumor site, this would allow for the dramaticdecrease in the total administered dose, thus sharply dimin-ishing the toxic effect to normal tissues and still providingmore drug in the tumor than after "traditional" adminis-tration. Thus, the high cytotoxicity of many anticancerdrugs can be predominantly localized only in the tumor.On-demand drug delivery by mesoporous silica nanopar-ticles functionalized with a pH-sensitive nanovalve thatonly opens in acidifying intracellular endosomal compart-ments is an example of a novel type of nanocarrier capableof controlled drug release (16).

Pharmacologic Characterization and PropertiesPharmacologic nomenclature

The terms used to describe the pharmacokinetic disposi-tion of carrier-mediated drugs are "encapsulated" or "con-jugated" (drug within or covalently bound to the carrier),"released" (the active drug released from the carrier), and"sum total" (encapsulated or conjugated drug plus releas-ed drug; refs. 17, 18). The released drug has also been calledthe legacy drug, regular drug, or warhead (17–19). Thereleased drug consists of a protein-bound or free drug. Thepharmacokinetic disposition of these nanoparticle agentsis dependent upon the carrier and not the parent druguntil the drug is released from the carrier (20). The drugthat remains encapsulated in nanoparticles or linked to aconjugate or polymer is an inactive prodrug and, thus, thedrug must be released from the carrier to be active (17, 21).Whether the drug needs to be released outside of the cellin the tumor extracellular fluid (ECF) or within the cell

© 2012 American Association for Cancer Research

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600Figure 4. Efficacy of BIND-014PSMA-targeted docetaxelnanoparticles in PSMA-expressing human LNCaPprostate cancer xenograftmouse model. Passivelytargeted docetaxelnanoparticles (PTNP, green)decrease tumor growth ratecompared with conventionaldocetaxel (DTXL, red). BIND-014 (blue) is identical to PTNP inevery way except for PSMA-targeting ligand on the surface.The additional active PSMAbinding by BIND-014 results intumor shrinkage of nearly 50%,a vast improvement over DTXL.Micewere treated 4 timeswith 5mg/kg of DTXL, PTNP, or BIND-014 at 4-day intervals.

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depends on the formulation of the carrier and the mecha-nism of release (17, 19, 22). After the drug is released fromthe carrier, the pharmacokinetic disposition of the drugwill be the same as after administration of the noncarrierform of the drug (17, 18). In certain formulations, theremay be unencapsulated drug along with the encapsulateddrug; separation and quantitation of each of these specieswould help explain complex pharmacokinetics. Many for-mulations use a combination of drugs for cancer. In suchcases, the encapsulation and/or conjugation efficiencies andrelease profiles of individual drugsmay be different depend-ing on the hydrophobicity and/or hydrophilicity of the drugand the formulation platform. For synergy, the appropriateconcentration of drugs reaching the desired site of action,suchas the tumor, is critical for efficacy.As such, carryingoutappropriate pharmacokinetic studies to understand theindividual release profiles and fine tuning the nanomaterialplatform for enhanced efficacy are critically important.Thus, the pharmacology and pharmacokinetics of theseagents are complex, and detailed studies must be done toevaluate the disposition of the encapsulated or conjugatedform of the drug and the released active drug and metabo-lites in plasma, tumors, and tissues (21).

Systemic, tissue, and tumor disposition ofnanoparticlesNanoparticles can alter both the tissue distribution and

the rate of clearance of the drug by making the drug take onthe pharmacokinetic characteristics of the carrier (23–25).Pharmacokinetic parameters of the nanoparticles dependon the physiochemical characteristics of the nanoparticle,such as size, surface charge, shape, nature and density ofcoating, composition, stability, membrane lipid packing(in case of liposomal particles), steric stabilization, deform-ability, dose, and route of administration (23). The primarysites of accumulation of nanoparticles are the tumor, liver,and spleen compared with nonnanoparticle formulations(23, 24, 26–30). The development of PEGylated nanoparti-cles was based on the discovery that incorporation of PEGonto the surface of nanoparticles yields preparations withsuperior prolonged plasma exposures and tumor deliverycompared with non-PEGylated nanoparticles (23, 26, 27).The clearance of nanoparticles has been proposed to

occur by uptake of the carrier by the RES, which is alsocalled the MPS (Fig. 5; refs. 23, 27, 31). The MPS uptake ofcationic or hydrophobic nanoparticles results in their rapidremoval from the blood and accumulation in tissuesinvolved in the MPS, such as the liver and spleen. Uptakeby the MPS may result in irreversible sequestering of theencapsulated drug in the MPS, in which it can be degraded.In addition, the uptake of the nanoparticles by the MPSmay result in acute impairment of the MPS and toxicity.The presence of the negatively charged coating on theoutside of the nanoparticles does not prevent uptake bythe MPS, but simply reduces the rate of uptake (Fig. 5;refs. 25–27, 32). The exact mechanism by which stericstabilization of nanoparticles decreases the rate of uptakeby the MPS is unclear (23, 30, 32).

The development of effective chemotherapeutic agentsfor the treatment of solid tumors depends, in part, on theability of those agents to achieve cytotoxic drug exposurewithin the tumor via the EPR effect (33, 34). In addition,studies suggest that the cells of the MPSmay also play a rolein the tumor disposition of liposomal agents and in thesensitivity of the tumors to liposomal agents (35–37). Oncein the tumor, the nonligand-targeted PEGylated nanopar-ticles are localized in the ECF surrounding the tumor cell,but do not enter the cell (38, 39). Thus, for the nano-particles to deliver the active form of the anticancer agent,the drug must be released from the nanoparticle into theECF and then diffuse into the cell, or be taken up into thecell directly and then released (21). As a result, the abilityof the nanoparticle to carry the anticancer agent to thetumor and release it into the ECF are equally importantfactors in determining the antitumor effect of nanoparticleanticancer agents. In general, the kinetics of this local releaseare unknown, as it is difficult to differentiate between thenanoparticle-encapsulated and released forms of the drugin solid tissue, although with the development of micro-dialysis, this is becoming easier (21).

Factors affecting pharmacokinetic andpharmacodynamic variability in patients

There is significant interpatient variability in the phar-macokinetic disposition of nanoparticle and liposomalencapsulated agents in patients (17, 37, 40–42). It seemsthat the pharmacokinetic variability of the carrier formu-lation of a drug is several-fold higher compared with thenonnanoparticle formulationof thedrug (17, 41, 42). Thus,there is a need to identify factors associated with the signif-icant pharmacokinetic variability. Most of the studies eval-uating the factors that affect the pharmacokinetic variabilityof nanoparticle agents in patients have involved liposomalagents. The factors associatedwith altered pharmacokineticsof PEGylated liposomal agents are age, body composition,gender, presence of tumors in the liver, and changes inand the function of monocytes in blood (Fig. 5). There isa 2- to 3-fold lower clearance of PEGylated liposomal doxo-rubicin (Doxil) and PEGylated liposomal CKD-602(S-CKD602) in patients �60 years of age compared withpatients <60 years of age (43). Results also suggest thatmonocytes in blood engulf S-CKD602, which causes therelease of CKD-602 from the liposome and toxicity to themonocytes, and that the effects are more prominent inpatients <60 years old (44). Patients with a lean bodycomposition have an increased plasma exposure of encap-sulated drug after administration of S-CKD602 (P ¼0.02; Fig. 4). It has also been reported that women have alower clearance of encapsulated drug after administrationof PEGylated liposomal agents compared with men. Popu-lation pharmacokinetic studies have reported that patientswith refractory solid tumorswho have primary ormetastatictumors in the liver have a higher clearance of S-CKD602compared with patients who do not have tumors in theirliver (45). In theory, the presence of tumors in the liver mayinduce the MPS cells in the liver and, thus, increase the






sequestering of the liposome in the liver, which would leadto an increase in systemic clearance of encapsulated drug(45).

Gabizon and colleagues reported that the clearance ofDoxil decreased by approximately 25% to 50% from cycle 1to 3 (Fig. 5; ref. 46). In addition, La-Beck and colleaguesreported that this reduction in clearance of Doxil fromcycle 1 to cycle 3 was associated with a reduction in pre-cycle monocyte count (47). These studies suggest that thereis a reduction in the clearance of liposomes over time, and,thus, dose reductions may be needed in subsequent cyclesto minimize the risk of toxicity (46). Interestingly, repeatdose studies of PEGylated liposomal doxorubicin in miceand rats did not report accumulation of drug in plasma,suggesting that these preclinical models may not accuratelyreflect the disposition of PEGylated liposomal agents

after repeated dosing (48, 49). Thus, there is also a needto develop better preclinical animal models for pharma-cology and toxicology studies of liposomal and nano-particle agents.

Future studies need to evaluate the mechanism of clear-ance of nanoparticle agents and identify the factors associat-ed with pharmacokinetic and pharmacodynamic variabilityof nanoparticle anticancer agents in patients and specificallyin tumors (30, 40, 50–53). Future studies also need to deve-lop phenotypic probes that can be used to predict this vari-ability and individualize therapy with nanoparticle agents.

Translation of nanotherapeutics: perception andreality

The successful translation of anticancer nanomedicinesfrom bench to bedside requires significant efforts that

© 2012 American Association for Cancer Research

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Figure 5. Summary of the clearance of nanoparticle agents via theMPS.Most of the studies evaluating factors affecting nanoparticle agents have been done inpatients receiving PEGylated and non-PEGylated liposomal agents, and, thus, these carrier systems are depicted. However, in theory, these factorsmay alsoaffect other nanocarrier systems, but need to be evaluated in future studies. Nanoparticle agents are primarily cleared via the monocytes, macrophages,and dendritic cells of theMPS that are located in the liver, spleen, and blood. In addition, theMPS cells in the lung and bonemarrow also seem to be involved.The tumor delivery of nanoparticle agents is determined by the EPR effect and potentially MPS in tumors. The factors affecting the pharmacokinetics(PK) and pharmacodynamics (PD) of nanoparticle agents in patients and animal models included age, gender, body composition, tumors in the liver, the doseand regimen, other drugs, type of cancer, and prior therapy. PBMC, peripheral blood mononuclear cell.

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include thedevelopment of reasonably simple, inexpensive,and scalable protocols to prepare such medicines andcontrol over the biologic behavior and pharmacologicproperties of these preparations. In general, the real use ofnanomedicine as an essential part of personalizedmedicinewill also require the development of multiple regulatoryguidelines and appropriate training and education (54).The creation of an "industrial culture" of making nano-medicines, including their standardized testing and char-acterization, is another challenge. It seems that nanomedi-cines currently under development will target the mostcommon cancers, such as breast and prostate cancer(55). The first generation of anticancer nanomedicines isbased on their "passive" (EPR-mediated) delivery intotumors, and drugs (such as Doxil) and diagnostic agents(such as superparamagnetic iron oxide nanoparticle-basednuclear magnetic resonance contrast agents) are nowapproved and in active clinical trials, respectively (56).Still, despite a clear understanding that effective anticancernanomedicines should specifically recognize the disease(diseased cells), provide an imaging signal from the affectedzone, and effectively deliver drugs in this zone, a clear setof uniform requirements to such preparation is still absent(57). Despite receiving significant attention (58), rationaldesign of such systems has yet to suggest clear guidelinesfor clinical translation. Another important, but unresolvedissue for the translation of nanoparticle-based medicinesis the role of nanoparticle shape and architecture in theirbiologic behavior and therapeutic properties (59).

Animal models for preclinical evaluations ofnanomedicinesAs the translation of nanomedicines into clinical practice

is still in the early stages, the concordance of preclinical andclinical data with regard to pharmacokinetics, toxicology,and efficacy are still unknown. However, the developingparadigms underlying nanoparticle distribution and bio-compatibility offer perspective about what preclinicalmod-els are likely to have the greatest translational utility. Oneimportant determinant of nanoparticle distribution is theMPS, previously referred to as RES, which is responsible forparticle sequestration (60). It seems that the primary MPSorgan(s) of particle sequestration is species dependent,with more common laboratory animals (e.g., dog, rat, andrabbit) having particle distribution primarily to Kupffercells of the liver and splenic macrophages, similar to man(61). In less commonly used preclinical species (e.g., goatand pig), distribution of particles to pulmonary intravas-cular macrophage is primarily observed (61). For example,i.v. injection of nanoparticulate iron oxide resulted in highpulmonary intravascular uptake (>85% of dose) in sheep,calf, pig, goat, and cat, whereas uptake was primarilyobserved in hepatic Kupffer cells (>65% of dose) in mon-key, hyrax, hamster, rabbit, guinea pig, rat, mouse, andchicken (62). This finding would support the use of themore traditional species for toxicologic and pharmacoki-netic evaluation of nanomedicines owing to the similarMPS distribution profile. This correspondence in nanopar-

ticle distribution for common laboratory species andhumans is also supported by a recent allometeric analysisof clearance of aPEGylatedTNF-boundgoldnanoparticle inrats, rabbits, andhumans (63). Across these species, the TNFclearance–brain-weight product was found to scale pro-portional to body weight, as has been found for manymacromolecular therapeutics, and would suggest commonmechanisms of nanomedicine disposition in these species.However, in a recent study Caron and colleagues found thatthe clearance of a series of PEGylated liposomal anticanceragents did not allometrically scale from mice, rats, anddogs to patients (64). Thus, the ability to scale the phar-macokinetic disposition of nanoparticle agents acrossspecies may be nanoparticle andmodel specific. Caron andcolleagues found that the physiologic cofactors that pro-duced the best scaling of clearance across animal modelsand patients were factors associated with the MPS, suchas monocyte count in blood. Thus, new methods of allo-metric scaling of nanoparticle agents and the use of MPScharacteristics and function need to be evaluated anddeveloped.

The issue of selective tissue distribution and accumula-tion of nanoparticles is a concern (65). Accumulation ofnanoparticles in organs of the MPS, or selectively targetedtissues, is a commonoccurrence for nanomedicines. For thisreason, repeat-dose tissue distribution studies may berequired to identify such tissues, which may then be sub-jected to greater scrutiny in subsequent toxicity studies (65).Similarly, nanoparticle biopersistence is also a concern,especially for metals and nonbiodegradable polymers,and may necessitate lengthy toxicology studies to identifypotential chronic toxicities.

In addition to general pharmacokinetic evaluation,assessing tumor distribution and efficacy of oncologynanomedicines in relevant preclinical cancer models isalso crucial. One issue unique to nanomedicine tumordistribution in comparison with small molecules is thedependency upon long systemic circulation and vascularpermeability for uptake into the interstitial space. Studieshave identified nanoparticle properties associated withlong circulation, such as PEGylation and small size,which correlate with increased tumor concentration max-ima and total exposure (area-under-the-time-concentra-tion curve; ref. 66). However, there have been no system-atic studies evaluating the clinical relevance of vascularpermeabilities found in these animal models. Studieshave shown that tumor vascular pore size can be highlyvariable in animal xenografts, ranging from hundreds ofnanometers to microns (67). As nanomedicine tumorpermeability is at least partially dependent upon vascularpore size, it is important that the tumor model vascula-ture resemble the clinical case (68). In addition to his-tologic type, tumor vascular permeability has also beenshown to vary depending on site of tumor implantation,with orthotopic brain tumors, for example, having lowerpermeability than peripherally implanted tumors (69).This finding would suggest a role for the tumor micro-environment in vascular permeability.






Preliminary studies suggest that some clinical tumorscontain ultrastructural features, such as fenestrations,similar to those found in animal models (70). However,other human cancers, such as certain brain tumors, seemto be devoid of these pores (71). Because the vascularpermeability of human cancers in comparison with animalmodels havenot been thoroughly evaluated, the selectionofanimal models most appropriate for specific cancer typeswith regard to nanoparticle permeability is difficult. Atthis time, the same recommendations for small-moleculeoncology animal model selection would apply to nano-medicines. Researchers in the National Cancer Institute(NCI) Developmental Therapeutics Program, after analyz-ing the clinical and preclinical data sets of several small-molecule chemotherapeutics, found that medicines thatwere successful in multiple xenograft models were morelikely to succeed in the clinic (72). Because histologic corre-lations of treatment success between clinical and preclinicalcancers were not observed in most cases, this would suggestthat xenograft cancer models are not predictive of specificcancer activity, but rather activity in general. Together withthe variability in tumor vascular architecture and nanome-dicine permeability discussed above, thiswould suggest thatevaluation of nanomedicines inmultiplemodels is prudent.Although veterinary cancers and transgenic models maybe more physiologically relevant than xenograft and/orsyngeneic models, they often suffer from low availabilityor prolonged time in generating tumors. For this reason,xenograft and/or syngeneic models are themost commonlyused models. Syngeneic models, with intact immune sys-tems, would conceivably be an improvement over xenograftmodels in athymic nude or severe combined immunode-ficient mice, especially for evaluation of nanomedicines,which are prone to immunologic interactions (see below).Likewise, orthotopically implanted tumors, with relevantmicroenvironments, may also have advantages.

Previous reviews have suggested that the current regula-tory framework for assessing the safety of small molecules,biologics, and devices is considered sufficient for nanome-dicines (65). Although that suggestion seems to be true atthis time, the choice of themost relevant preclinical modelsfor toxicologic evaluation has yet to be identified. Majortoxicologic issues commonly observed with cancer nano-medicines are development of immunologic and hemato-logic complications (73). As an example, cationic dendri-mers have recently been reported to induce disseminatedintravascular coagulation inmice (74). Anaphylactoid reac-tions are a primary concern for translational developmentof iron oxide nanoparticle magnetic resonance contrastagents, which may be related to the polymer coatings usedand has resulted in removal of many from the market (75,76). Additionally, endotoxin contamination and associatedimmunologic complications, such as complement activa-tion and pyrexia, are a common issue for nanomedicines(77). For this reason, the use of immunologically sensitivespecies, such as rabbits, in addition to historic models (e.g.,rodents and dogs), which tend to be less sensitive, iswarranted early in preclinical development to identify these

possible concerns. A meta-analysis comparing study designissues of nanoparticle and small-molecule anticancer agentsin preclinical models and in phase I clinical trials was doneby Morgan and colleagues (78). In this study, the degree ofdose escalation from starting dose to maximum tolerateddose, number of dose levels, and time to complete the phaseI clinical studies were significantly greater for nanoparticleagents compared with small-molecule drugs. These datasuggest that the standard animal models and cross-species–scaling paradigms employed to define the starting dose inthe phase I clinical study for small-molecule agentsmay notbe optimal for nanoparticle agents.

In addition to the suggestions about animal model selec-tion made above, proper study design is very importantfor evaluation of nanomedicines. Whereas the use of intra-peritoneal (i.p.) administration in place of i.v. administra-tion may have little consequence when evaluating preclin-ical efficacy or toxicity of small molecules in rodents, owingto the high tissue permeability of these agents, this is notlikely the case for many nanomedicines. Because of theirsize, nanomedicines do not freely diffuse across tissuebarriers, and for this reason, i.p. administration cannot bethought of as a parenteral route equivalent to intravenousadministration. Indeed, studies have shown substantialdifferences in distribution when comparing i.p. and i.v.routes of administration (79). Thus, it is important to usethe intended clinical dosing route when evaluating nano-medicines. Another issue is the use of proper controls. Therehave been many examples of both nanoparticle-dependenttoxicities and distribution-related shifts in drug toxicities(80, 81). Consequently, it is important to include bothempty (drug-free) nanoparticles and a nonnanoparticle(small molecule) formulation of the drug as controls toidentify toxicities related to the nanoparticle platform andshifts in drug-related toxicity.

Role of Nanotech Characterization LaboratoryTheNanotechnology Characterization Laboratory (NCL)

is part of the Alliance for Nanotechnology in Cancer atNCI’s federally funded research and development center(managed by SAIC Frederick, Inc.) and is a resource forpreclinical development of nanomaterial-based drug deliv-ery and imaging agents that are beyond a proof-of-principlestage of development, with proven biologic efficacy. Thefacility was established to accelerate clinical translation ofpromising nanotechnology-derived formulations. ThisNCI-funded resource, available to academic investigators,industry collaborators, and government laboratories, wasestablished by NCI in collaboration with the FDA and theNational Institute of Standards and Technology. Once aproject is approved through a simple submission andmate-rial transfer agreement process, a large-scale batch isobtained from the collaborator for testing at NCL facilities.The laboratory conducts preclinical assessment throughan established assay cascade that includes thorough phys-icochemical assessment, relevant in vitro studies to investi-gate biocompatibility, and in vivo absorption, distribution,metabolism, excretion, toxicity, efficacy, and imaging

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studies in rodent models as appropriate. The outcomeof the NCL studies is a client report that is provided tothe collaborators to further their concept toward an inves-tigational new drug (IND) submission or an investigationaldevice exemption (82) with the FDA.In addition to the preclinical assessment, NCL is actively

engaged in standard protocol development and referencematerial standards development through collaborations.The NCL also plays an active role in educational andknowledge sharing efforts to advance the nanomedicinefield. For further information, visit http://ncl.cancer.gov.

Scale-up and manufacturing issuesA critical element for successful development and com-

mercialization of any pharmaceutical product is a scalable,reproducible manufacturing process. Besides typical con-siderations and challenges with scale-up and commercialmanufacture, there are additional challenges for nanotech-nology-basedproducts for treating cancer. Someof themostcritical aspects of themanufacture of cancer nanomedicinesinclude sterility (most will be administered intravenously),nanoparticle size and polydispersity, encapsulation effi-ciency, removal of free drug, and drug-release rate. Inaddition, for actively targeted cancer nanomedicines thatemploy receptor-mediated binding of nanoparticles totumors, the amount and appropriate surface exposure oftargeting ligand must be addressed.The most important quality parameter for an injectable

product is sterility. Achieving sterility may be quite difficultbecause terminal heat sterilization could disrupt nanopar-ticle size and polydispersity, negatively affecting traffickingand biodistribution. Also, if nanoparticle size is muchgreater than 100 nm and polydispersity is broad, sterilefiltration may not work. This leaves few options, includinguse of sterile raw materials and aseptic processing, which isvery costly, or gamma irradiation,whichmanynanoparticleproducts also may not withstand.The removal of unencapsulated drug from nanoparticle

drug products is often difficult, yet free or nonencapsulateddrug contamination will compromise both efficacy andsafety. Likewise, if the manufacturing process cannot con-trol the drug-release rate from nanoparticles, performancewill be unreliable and potentially unsafe if there is a rapid orhigh burst of drug released from the carrier. Finally, activelytargeted nanomedicines require a well-controlled processproviding consistent ligand exposure on nanoparticle sur-faces to fully benefit from effective nanoparticle binding toyield higher tumor-drug concentrations and/or cellulartrafficking.

Interaction with Regulatory AgenciesPathway to the clinic: preinvestigationalnewdrug–andinvestigational new drug–related studiesAlthough the FDA and the pharmaceutical industry have

developed standards to assess drug and material biocom-patibility, immune reactivity, purity, and sterility, theuniqueproperties of nanomaterials often hamper the execution ofthese standardized protocols and require special consider-

ation. Thus, although the FDA has criteria for the preclinicaldata that should be presented in an IND for small-moleculedrugs, there is no standardized set of characterizationmeth-ods for engineered nanomaterials. In consideration of thenovel properties andoftenmulticomponent nature of nano-particle-based therapeutics, a rational characterization strat-egy comprises 3 elements, namely physicochemical charac-terization, in vitro assays, and in vivo studies. For the phys-icochemical characterization, reproducible synthesis andcharacterization assays are needed for batch-to-batch con-sistency, which are predictive of in vivo fate. Although thebasic criteria for the chemistry, manufacture, and controlsectionof an INDfiling are the same, themethodologies andinstrumentation should be appropriate to the type of nano-material being assessed. Additional physicochemical prop-erties that need to be considered include particle size, sizedistribution, polydispersity, surface ligand density, surfacearea, surface charge, surface functionality, shape and con-firmation, composition, purity, and stability.

Although the number of properties to assess seems sub-stantial, once a set of characterization assays and tools havebeen identified that are predictive of components thatcause variation in safety, efficacy, and potency profiles, themethodologies canbe standardized toqualify lots for batch-to-batch consistency. Nanomaterial size and surface char-acteristics are critical for predictive biodistribution andtoxicity. For example, most nanomaterials have PEG coat-ing. Differences in polydispersity of the PEG used and thedensity of the ligands on the surface of the nanoparticleswill result in significantly different toxicity profiles. For core-shell nanoparticles, impurities can come from the core andshell reagents that are used. They need to be appropriatelyassessed to see if residual free components are present inthe drug product. If so, they need to be quantified usingappropriate methods. Purity in the nanomedicine sensecan be affected by the presence of residual solvents, boundand free components (such as unchelated gadolinium orfree drug), and finally, homogeneity, inhomogeneity, andheterogeneity in the ligand distribution that will havesignificant biologic impact.

Apart from these parameters, the stability of the formula-tions has to be measured as a function of time, storage,temperature, pH, light (photo stability), diluent, vehicle,lyophilization, and centrifugation with appropriate meth-ods that will be predictive of biologic effects. Thesecharacterization methodologies become much more chal-lenging for multifunctional nanomaterials intended fordrug delivery and imaging. Some of these challenges canbe addressed during the synthesis, purification, and char-acterization steps in a multistep synthetic methodology,which would allow for purification of unreacted compo-nents and have controls in place to assure uniformity frommultiple lots. For self-assembly methodologies, for exam-ple, in the case of liposomes or emulsions, the character-ization for a multicomponent system is significantly morechallenging. Thus, optimization of the methodology itselfwith appropriate controls is critical for its success in trans-lation. In the case of targeted drug delivery systems, such as






those with active targeting ligands that bind to overex-pressed receptors on tumor, a bioassay predictive of in vivobehavior is critical to have as part of the analytical techni-ques to assure activity.

In vitro characterization is principally done to elucidatemechanisms of biologic interaction and toxicity and notstrictly to screen for biocompatibility. Nonetheless, these invitro studies could be very useful to identify areas requiringattention during execution of in vivo animal studies.Although a host of in vitro studies can be carried out withprimary and transformed tissue culture cells to assess fea-tures such as nanoparticle uptake, subcellular localization,intracellular drug delivery, cytotoxic killing, reactive oxygenspecies generation, and proinflammatory effects, biocom-patibility can also be evaluated ex vivo, using blood or bloodcells to discern effects on coagulation, hemolysis, plateletaggregation, complement activation, and phagocytosis.Many in vitro assays can be used to elucidate potentialproblems that might be encountered during the in vivoassessment phase. Although all in vitro assays are not pre-dictive of in vivooutcome, a set of relevant in vitro assaysmaybe used to characterize potential issues with the nanopar-ticle formulation. Because most rodent animal models arenot predictive of human immunotoxicity, the use of humanblood, albeit in an in vitro setting, would point to potentialissues during the clinical phase of development and can beused as a screening tool and tooptimize the formulation. Animportant in vitro analysis is also checking sterility andendotoxin contamination. Preclinical pharmacology andtoxicity studies in animals should be conducted in themostclinically relevant animal model, as discussed above.

Regulatory agencies are becoming increasingly stringentabout characterization of the particle size distribution ofnanotechnology-based products. Because the particle sizedistribution may have a significant influence on the biodis-tribution and biologic efficacy of the formulation, thisparameter is critical to measure and track. This task is nottrivial and needs to be well understood to generate mean-ingful and actionable data. A review of particle size analysisis beyond the scope of this article, and the reader is referredto the literature (83).

Summary and future directionsHistorically, treatment of patients with cancer using

chemotherapeutic agents has been associated with debili-tating and systemic toxicities, poor bioavailability, andunfavorable pharmacokinetics. Nanotechnology-baseddrug delivery systems that can specifically target cancer cellswhile avoiding their healthy neighbors, avoid rapid clear-

ance from the body, and be administered without toxicsolvents hold immense potential in addressing all of theseissues. Such drug delivery systems will lead to cancer ther-apeutic agents that are not only less toxic to the patient, butalso significantly more efficacious.

With Doxil and Abraxane (84, 85) approved by the FDA,and several new agents undergoing clinical trials, confi-dence is growing that nanotechnology-based therapeuticswill become an important addition to currently availabletreatments. It is possible that, initially, these new formula-tions will have limited use because of their incrementalimprovement in performance and high cost. However,emerging research efforts indicate that nanotechnology canaddress uniquely serious cancer problems that do not haveexisting solutions. For example, effective systemic deliveryof siRNA has been shown to date only using nanoparticledelivery vehicles (86). An additional host of examplesincludes reduction or elimination of multidrug resistance(16, 87, 88), broadening of the therapeutic index of existingdrug formulations (89–91), and development of antimeta-static drugs (92–96). Thus, persistent further developmentof nanoparticle drug delivery technologies will continue,and, thus, these approaches will eventually become animportant part of contemporary cancer care.

Disclosure of Potential Conflicts of InterestJ. Hkrach is employed by and acknowledges ownership interest in BIND

Biosciences. R. Lee is a member of the advisory boards of both SavaraPharmaceuticals and NanoScan Imaging. W.C. Zamboni has benefited fromresearch funding and served on the advisory boards of Alza Pharmaceuticals,Mersana Therapeutics, Covidien, Yakult Pharmaceutical Industry, and HanaBioSciences. SciDose, LLC has contributed to the research of W.C. Zamboniand he has served as a consultant for Liquidia Technologies and AzayaTherapeutics.

Authors' ContributionsConception and design: W.C. Zamboni, P. Grodzinski, N.J. Panaro, A.K.PatriDevelopment of methodology:W.C. Zamboni, P. Grodzinski, N.J. PanaroAcquisition of data: W.C. Zamboni, V. Torchlin, A.K. Patri, J. Hrkach,S. Stern, R. Lee, A. Nel, N.J. Panaro, P. GrodzinskiAnalysis and interpretationofdata:W.C.Zamboni, V. Torchlin, A.K. Patri,J. Hrkach, S. Stern, R. Lee, A. Nel, N.J. Panaro, P. GrodzinskiWriting, review, and/or revision of the manuscript: W.C. Zamboni,V. Torchlin, A.K. Patri, J. Hrkach, S. Stern, R. Lee, A. Nel, N.J. Panaro,P. Grodzinski

Grant SupportThis project has been funded in whole or in part with federal funds

from the National Cancer Institute, NIH, under contract no.HHSN261200800001E. The content of this publication does not necessarilyreflect the views or policies of theDepartment ofHealth andHuman Services,nor does mention of trade names, commercial products, or organizationsimply endorsement by the U.S. Government.

Received November 16, 2011; revised February 24, 2012; accepted March11, 2012; published OnlineFirst June 5, 2012.

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2012;18:3229-3241. Published OnlineFirst June 5, 2012.Clin Cancer Res William C. Zamboni, Vladimir Torchilin, Anil K. Patri, et al. Nanotechnology AllianceBest Practices in Cancer Nanotechnology: Perspective from NCI

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