enhancing endosomal escape for nanoparticle mediated sirna delivery

Nanoscale

FEATURE ARTICLE

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Enhancing endos

DfHUwSdUWDowt

based nanoparticles. He will becFudan University in China.

Department of Chemistry, Fudan University

China. E-mail: [email protected]

Cite this: Nanoscale, 2014, 6, 6415

Received 2nd January 2014Accepted 10th April 2014

DOI: 10.1039/c4nr00018h

www.rsc.org/nanoscale

This journal is © The Royal Society of C

omal escape for nanoparticlemediated siRNA delivery

Da Ma*

Gene therapy with siRNA is a promising biotechnology to treat cancer and other diseases. To realize siRNA-

based gene therapy, a safe and efficient delivery method is essential. Nanoparticle mediated siRNA delivery

is of great importance to overcome biological barriers for systemic delivery in vivo. Based on recent

discoveries, endosomal escape is a critical biological barrier to be overcome for siRNA delivery. This

feature article focuses on endosomal escape strategies used for nanoparticle mediated siRNA delivery,

including cationic polymers, pH sensitive polymers, calcium phosphate, and cell penetrating peptides.

Work has been done to develop different endosomal escape strategies based on nanoparticle types,

administration routes, and target organ/cell types. Also, enhancement of endosomal escape has been

considered along with other aspects of siRNA delivery to ensure target specific accumulation, high cell

uptake, and low toxicity. By enhancing endosomal escape and overcoming other biological barriers,

great progress has been achieved in nanoparticle mediated siRNA delivery.

1. Introduction to siRNA delivery

Since its discovery by Fire et al. in 1998,1 RNA interference(RNAi) has emerged as a promising technology to treat cancerand other diseases by halting the production of targetproteins.2,3 Small interfering RNA (siRNA) is a synthetic double-stranded RNA (dsRNA) with approximately 21 base pairs,4 whichis capable of entering the RNA-induced silencing complex(RISC), interfering with and inhibiting the expression of specicgenes.5 Since the size of siRNA is much smaller compared to fullsize RNA, siRNA can be chemically synthesized, which

a Ma received his B.S. degreerom Peking University in 2005.e later graduated from theniversity of Maryland in 2010ith a Ph.D. degree in chemistry.ince 2010, he has been a post-octoral research associate at theniversity of North Carolina.orking with Professor JosepheSimone, he is currently devel-ping siRNA delivery technologyith PRINT® (Particle Replica-ion in Non-wetting Templates)ome a principle investigator at

, 220 Handan Road, Shanghai, 200433,

hemistry 2014

signicantly lowers its production cost. The high target speci-city of RNAi and relatively low synthetic cost of siRNA givesiRNA-based gene therapy great potential for a variety ofapplications.6

As a large and negatively charged biological molecule, nakedsiRNA is unstable in the blood stream, is unable to penetratecell membranes, and can be immunogenic.7 A safe and efficientdelivery method is crucial to realize the broad potential ofsiRNA-based therapeutics. Both viral and non-viral vectors canbe used to deliver siRNA.8 Non-viral vectors, especially nano-particles, are less expensive to produce and carry a lower risk ofprovoking an immune response compared to viral vectors. As aresult, nanoparticles are of great interest to deliver siRNA.9–14

Nanoparticle mediated siRNA delivery is an intensively investi-gated research eld with approximately 1000 research paperspublished in the past three years alone.

Currently, siRNA delivery, especially systemic delivery in vivo,remains a difficult task.15 The difficulty of siRNA delivery isrooted in several biological barriers that present challengeswhen siRNA is delivered via systemic administration. First,nanomaterials for siRNA delivery need to form a stable complexwith the cargo to protect it from degradation during circulationin the blood stream. Next, siRNA loaded nanoparticles need toevade fast clearance from the blood and avoid an immuneresponse, which generally is realized by the surface modica-tion with poly(ethylene glycol) (PEG) to protect and stabilizenanoparticles. Furthermore, a sufficient amount of carriersneeds to accumulate in the target tissue and be taken up bytarget cells. To achieve this, proper surface characteristics andtargeting groups are essential to enable accumulation in targettissues and uptake by target cells. Since the RNAi machinery is

Nanoscale, 2014, 6, 6415–6425 | 6415

http://dx.doi.org/10.1039/C4NR00018H

http://pubs.rsc.org/en/journals/journal/NR

http://pubs.rsc.org/en/journals/journal/NR?issueid=NR006012

Fig. 1 Gateways for endocytic entry. “Other” pathways representclathrin- and caveolae-independent pathways. Adapted with permis-sion from ref. 21. Copyright American Chemical Society 2012.

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housed in the cytoplasm, successful delivery of siRNA relies onthe ability of nanoparticles to enter the cell, reach the cyto-plasm, and then release the cargo. In most cases, nanoparticlesare internalized through an endocytosis and endo-lysosomalpathway. Therefore, endosomal escape is of particular impor-tance for the delivery of siRNA.8

Recent mechanistic investigations on nanoparticle intracel-lular trafficking indicate that insufficient endosomal escapecould signicantly limit siRNA delivery efficiency.9,16 Entrap-ment in the hostile endo-lysosomal vesicles and degradation bylysosomal enzymes in an acidic environment could be a deadend for siRNA delivery. To achieve RNAi, siRNA containingnanoparticles need to escape from the endosome within a shortperiod of time to avoid the fate of being degraded or recycled. Insystemic delivery, a nanoparticle design must be able to achievemultiple functions of elongated blood circulation time,improved stability, and reduced toxicity in addition toenhanced endosomal escape.16–19 Therefore, designing multi-functional siRNA delivery systems with efficient endosomalescape is a great challenge.

This feature article highlights the challenges of and solu-tions for endosomal escape in nanoparticle mediated siRNAdelivery. The discussion focuses on recent progress regardingthis topic. The goal is to show that enhanced endosomal escapecan be achieved by chemical composition control, surfaceproperty modication, and other creative nanoparticle designapproaches. The author hopes that this article will raiseawareness of the importance of addressing endosomal escapewhen designing nanoparticles for siRNA delivery. Endosomalescape enhancing strategies are summarized in this article,which can hopefully assist fellow researchers to design theirown siRNA delivery systems.

2. Endosomal escape: the criticalchallenge in siRNA delivery

As mentioned above, a few biological barriers have to be over-come, when siRNA is delivered in vivo via systemic adminis-tration. Among them, endosomal escape is a key biologicalbarrier in siRNA delivery.

Nanoparticles are typically taken up via endocytosis.Depending on nanoparticle properties (size, shape, surfaceproperties, etc.) and cell types, endocytosis of nanoparticles mayoccur via different pathways.20 Generally, endocytosis can bedivided into two broad categories: phagocytosis and pinocy-tosis. While phagocytosis mostly occurs with specializedphagocytes, such as macrophages and dendritic cells, pinocy-tosis is present in all types of cells. Based on the proteinsinvolved, pinocytosis occurs either via clathrin-mediated path-ways or clathrin-independent pathways. Clathrin-independentpathways can be further divided into caveolae-mediated endo-cytosis, clathrin- and caveolae-independent pathways, andmacropinocytosis. The endocytic entry pathways are summa-rized in Fig. 1.21 Another classication of endocytosis is basedon material interaction with the cellular membrane (receptor-mediated, adsorptive, uid phase).

6416 | Nanoscale, 2014, 6, 6415–6425

Endocytosis of nanoparticles is a complex process. For mostnanoparticles, more than one pathway could be used to achievecellular entry. Among these endocytosis pathways, clathrin-mediated endocytosis is generally considered to be the mostcommon route of cellular entry, which goes through the endo-lysosomal pathway. Some endocytosis pathways, such as somecases of caveolae-mediated endocytosis and macropinocytosis,may bypass lysosomes.20 In these cases, an active endosomalescape mechanism is unnecessary. Nevertheless, this featurearticle will focus on endosomal escape strategies for the morecommon route of endocytosis via the endo-lysosomal pathway.

Facilitating endosomal escape has long been the focus ofgene delivery research. In the “classical” endo-lysosomalpathway, nanoparticles start intracellular trafficking with earlyendosome vesicles, which become progressively acidic as theymature into late endosomes.22–24 By accumulating protons inthe vesicle, the proton pump vacuolar ATPase generates acidi-cation until the pH drops to pH 5–6. With the fusion of the lateendosomes with the lysosomes (pH 4–5), the content would bedegraded by enzymes if it does not escape the endosome.Cationic nanoparticles with a strong buffering capacity in thepH range from 5 to 7 have displayed the ability to escape theendosome potentially through the so-called “proton sponge”effect. To escape from the endosome in a timely fashion isessential to achieve efficient siRNA delivery. Two recent reportsstudying nanoparticle intracellular trafficking give more detailsabout the endosomal escape mechanism, which indicate some“hidden” pathways that might compromise siRNA deliveryefficiency.9,16

In the rst report, Gilleron et al. investigated the intracellulartrafficking of siRNA containing lipid nanoparticles (LNPs),which were labelled by either uorescent dyes or gold nano-particles.16 Quantitative uorescence imaging and electronmicroscopy were used to analyse nanoparticle trafficking. TheLNPs were found to enter cells through both macropinocytosisand clathrin-mediated endocytosis. The key discovery was thatescape of siRNA from endosomes into the cytosol occurs at lowefficiency (1–2%) and only during a limited period of time whenthe LNPs reside in a specic compartment sharing early and lateendosome characteristics. This discovery further stressed theimportance of quick and efficient endosomal escape in order torealize high siRNA delivery efficiency.

This journal is © The Royal Society of Chemistry 2014


Fig. 2 Schematic illustration of LNP intracellular trafficking pathwayssummarized in Gilleron et al. and Sahay et al. reports. Adapted withpermission from ref. 25. Copyright Nature Publishing Group 2013.

Fig. 3 Different types of nanoparticles used for gene delivery. Adaptedwith permission from ref. 27. Copyright Wiley-VCH 2008.

Feature Article Nanoscale

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The second study was carried out by Sahay et al.9 Researchersused high-throughput confocal microscopy to screen a library ofsmall-molecule inhibitors and identify critical signalling path-ways that regulated the cellular uptake and intracellular traf-cking of siRNA in HeLa cells. LNPs were also used in theirinvestigation. Results showed that LNPs were internalized bymacropinocytosis and trafficked directly into endosomes.Surprisingly, it was discovered that siRNA dissociated from theLNPs was exocytosed to the extracellular milieu. The amount ofsiRNA lost in this manner was calculated to be approximately70% of the dose taken up by cells. This discovery indicates thatnanoparticle endocytic recycling is limiting the efficiency ofsiRNA delivery.

Intracellular trafficking pathways of both reports aresummarized in Fig. 2.25 Although these two investigations werebased on LNPs, the observations may also apply to other siRNAdelivery platforms. As both reports show, it is essential to designsiRNA loaded nanoparticles that are capable of escaping fromthe endosome efficiently. Otherwise, nanoparticles will eitherbe degraded or recycled, which severely limits siRNA deliveryefficiency.26

3. Endosomal escape enhancementfor different nanoparticle types

Various types of nanoparticles have been used for gene delivery.As depicted in Fig. 3, these nanoparticles include lipid-basednanoparticles, polymer-based nanoparticles, gold nanoparticles,mesoporous silica nanoparticles, carbon nanotubes, and


nanoparticle assemblies.27 Depending on the type of nano-particles, different strategies to enhance endosomal escape areused. The general method is to improve pH buffering capacityand increase the “proton sponge” effect. With this “protonsponge” mechanism, the buffering capacity prevents acidica-tion of the endosomes by acting as “proton sponge”, which leadsto an increase in the proton inux followed by an enhancedaccumulation of counter anions andosmotic swelling. There hadonly been indirect evidence supporting this pH buffering mech-anism, until a recent investigation reported direct visualizationof this “proton sponge”mechanism with confocal microscopy.28

In this section, discussion focuses on recent progress in siRNAdelivery with lipid nanoparticles, polyplex nanoparticles, poly-mer nanospheres, and inorganic nanoparticles.

Cationic lipid-based nanoparticles are the most widely usednon-viral gene delivery vectors.29 Currently, they are also thetype of nanoparticles that holds the greatest promise to achieveclinical breakthroughs.30 Cationic lipids can self-assemble intonanoparticles, and encapsulate negatively charged siRNA.Further modication of these nanoparticles gives stabilizingand targeting capabilities when delivered in vivo via systemicadministration.15,31–34 To design optimized delivery systems forsiRNA therapeutics, Anderson and co-workers pioneered theuse of robotic methods to systematically screen lipids.10,35,36

Cationic and pH sensitive lipids, which have a high pH buff-ering capacity, are used to enhance endosomal escape via the“proton sponge” effect. Systematic studies have been carriedout to investigate how to use pH sensitive lipids to achieve theoptimal endosomal escape effect.37,38 A combination of lipid-based nanoparticles with special endosomal escape strategies,such as calcium phosphate and cell penetrating peptides, hasled to successful nanosystems for systemic delivery.39

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Polyplex nanoparticles are generally based on electrostaticcomplexation of cationic polymers and anionic nucleic acid.40–48

Cationic polymers, such as polyethyleneimine (PEI), poly-L-lysine (PLL), chitosan, and synthetic dendrimers, are used toform polyplexes. The cationic characteristics of polymers enablethe formation of stable complexes, which contributes tocationic nanoparticle mediated cell uptake and improvesendosomal escape. One major advantage of polyplex nano-particles is the innite number of polymers, which researcherscan design and synthesize to incorporate multiple functions,such as protection with PEG and targeting toward certain celltypes. The cationic feature of polymers contributes to endo-somal escape typically by increasing the pH buffering capacity.More importantly, pH sensitive groups can be introduced intopolymer structures for enhanced endosomal escape andreduced toxicity.49–54 In addition, it is possible to prepare hybridmaterials of polymers and peptides to realize improved endo-somal escape.

Polymer nanospheres are another type of polymer basednanoparticles. Poly-L-lactic acid (PLA) and poly-lactic-co-gly-colic acid (PLGA) can encapsulate siRNA via non-ionicinteraction to form polymer nanospheres.55 Approved bythe FDA for pharmaceutical use, PLA and PLGA are wellknown for their biocompatibility. As neutral nanoparticleswithout modication, PLA and PLGA based nanospheresneed to be optimized to improve cellular uptake and endo-somal escape by introducing a coating of cationic lipids orpolymers.56,57 PLA or PLGA containing hybrid nanoparticleshave been developed. Desai et al. developed a PLGA andcationic lipid hybrid nanoparticle to deliver siRNA andcapsaicin via topical administration to inhibit skin inam-mation in vivo.58

Inorganic nanoparticles include gold nanoparticles,59–62

carbon nanotubes,63–67 mesoporous silica nanoparticles,68–71

iron oxide nanoparticles,72 quantum dots,73,74 and calciumphosphate nanoparticles.39,75–77 Inorganic nanoparticles aregenerally highly stable in maintaining their size, shape andcomposition. Nevertheless, since inorganic nanoparticles lackcharge, they are unable to electrostatically interact with eitheranionic nucleic acids or the negatively charged cell membrane.Successful transfection with inorganic nanoparticles requiressurface coating or modication with cationic polymers toimprove cellular uptake and endosomal escape. Cationic poly-mers, such as PEI, PLL or specically designed polymers,contribute to the improved cell uptake and endosomal escapecapability. Among these inorganic nanoparticles, calciumphosphate nanoparticles have a unique advantage. With atendency to quickly dissolve under acidic conditions like thatinside endosomes and lysosomes, calcium phosphate nano-particles can greatly enhance endosomal escape with minimumtoxicity.78

In summary, different nanoparticles have their uniquestrategies to enhance endosomal escape. The commonstrategy is to take advantage of the acidic environment insidethe endosome and enhance endosomal escape with the“proton sponge” effect to destabilize the endosomemembrane.

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4. Endosomal escape enhancingmethods

Most endosomal escape strategies are based on the acidicendosome micro-environment and “proton sponge” effect,although external stimulations, such as photochemical inter-nalization (PCI), are also used.79 In this section, several endo-somal escape enhancing strategies will be introduced, whichinclude the use of cationic polymers, pH sensitive polymers,calcium phosphate, and cell penetrating peptides. This sectionwill also address how to design multifunctional systems toenhance endosomal escape and overcome other biologicalbarriers in siRNA delivery.

4.1 Cationic polymers

Cationic polymers are used to complex with siRNA to formpolyplexes or to coat inorganic and other nanoparticles. PEI,PLL and other polyammonium polymers are generally usedbased on the “proton sponge” effect. As an alternative to poly-ammonium polymers, Ornelas-Megiatto et al. reported the useof polyphosphonium polymers as an efficient and non-toxictransfection agent.80 In many cases, polymers are specicallydesigned and synthesized to facilitate multiple functionsincluding endosomal escape, protection and targeting.

A cationic polymer coating is necessary for many inorganicnanoparticle based siRNA carriers to enhance endosomalescape and cell uptake. Xia et al. developed PEI coated meso-porous silica nanoparticles to deliver siRNA and DNA.71 Thereare several reports of PEI capped gold nanoparticles for siRNAdelivery.59–61,72 Other than commercially available poly-ammonium polymers, Lee et al. synthesized and tested biode-gradable poly(b-amino ester) type polymers to improve thetransfection efficiency of gold nanoparticles.62 In their study, alibrary of polymers was screened to nd the best formulation toenhance gold nanoparticle transfection efficiency. Kozielskiet al. developed a bioreducible poly(b-amino ester), which coulddeliver siRNA and release the cargo in an intracellular envi-ronment.81 In their design, the cleavable disulde bonds in thepolymer structure ensured the degradation of the polymer andthe release of cargo in the reducing intracellular environment.

There are many reports of multi-functional cationic poly-mers. A triblock poly(amido amine)–poly(ethylene glycol)–poly-L-lysine (PAMAM-PEG-PLL) nanocarrier was developed by Patilet al.82 This triblock copolymer had a combination of improvedendosomal escape (PAMAM), protection (PEG) and siRNAcondensation (PLL), which could be easily tuned to achieve theoptimal transfection efficacy. Yu et al. reported an amphotericinB (AmB)-loaded, poly(2-(dimethylamino)ethyl methacrylate)-block-poly(2-(diisopropylamino)ethyl methacrylate) (PDMA-b-PDPA) micelleplex siRNA delivery system.83 PDMA-b-PDPA is acationic polymer, which can complex with siRNA and improveits transfection capability. AmB is a hydrophobic antifungaldrug, known to increase membrane permeability at sublethalconcentrations by the formation of transmembrane pores.Their study conrmed that AmB was released from the micel-leplexes in the early endosome to assist endosomal escape. In




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this dual pH-responsive system, the combination of membraneporation by AmB and endosome swelling by polycationscontributed to efficient endosomal escape.

Shrestha et al. designed and synthesized a polymer withprimary amines and histamines, which can be assembled intocationic shell crosslinked knedel-like nanoparticles (cSCKs)(Scheme 1).50 Amphiphilic block copolymers were synthesized,followed by selective crosslinking throughout the hydrophilicshell layer to form cSCKs. To reduce toxicity, histamine groupswere introduced into the structure of the copolymer. Byincreasing the ratio of histamines to primary amines, siRNA-binding affinity was decreased and the cytotoxicity was reduced.By controlling the ratio of primary amines and histamines,siRNA-binding affinity, cytotoxicity, immunogenicity, andtransfection efficiency could be controlled. Endosomal escapewas facilitated by having these two species of low and high pKas.With this tuning capability, while maintaining adequate endo-somal escape, toxicity of these cationic nanoparticles wasreduced and their biocompatibility was increased.

Peptide/polymer hybrid materials, which combine theadvantages of precise structural control of peptides and lowsynthetic cost of polymers, are interesting gene delivery vectors.Meyer et al. developed a polycation with a pH responsive peptideand PEG.While PEG was used to protect the cargo, the sequenceof the peptide could be tuned to have the optimal transfectionefficacy.84 Zeng et al. reported a multifunctional dendronizedpeptide polymer platform for siRNA delivery.85 The ratio ofcationic modules, hydrophilic groups, and hydrophobic groupsin this peptide/polymer hybrid material could be adjusted inorder to achieve high siRNA delivery efficiency and excellentbiocompatibility. In addition, the polymer was reducing envi-ronment degradable in its design. Polymer degradation in theintracellular environment not only triggered the release of cargo,but also further reduced the possible long-term toxicity.

In addition, polymeric nanoparticles can be coated withcationic lipids to improve cellular uptake and endosomalescape. Yang et al. developed a polymeric nanoparticle assem-bled from mPEG-PLA, PLA, cationic lipid, and siRNA in a singlestep.34 The resulting hybrid nanoparticles exhibited excellentstability in serum and showed signicantly improved biocom-patibility compared to that of pure cationic lipid nanoparticles.

4.2 pH sensitive polymers

While cationic polymers are capable of enhancing endosomalescape, they may lead to toxicity in the physiological

Scheme 1 Self-assembly of polymers with various amounts of primary amthe hydrophilic shell regions. Adapted with permission from ref. 50. Cop


environment. By using pH sensitive polymers, the nanoparticletoxicity can be reduced during circulation. Aerwards, pHsensitive polymers will be further protonated or degraded toexpose the membrane disruptive inner core to assist endosomalescape. Polymers can be modied with pH sensitive groups. Theratio of pH sensitive groups to other functional groups can beadjusted and balanced to achieve the optimal overall trans-fection efficiency. While cationic polymers in the previoussection were discussed as having certain pH sensitive charac-teristics, this section will give a detailed discussion of theapplication of pH sensitive polymers in siRNA delivery.

Regular amine groups tend to change their protonationstatus as pH changes. Nevertheless, imidazole and other pHsensitive groups are more widely used. The use of imidazole andother pH-sensitive groups can decrease the amount of positivesurface charge in the physiological environment to reducetoxicity and make nanoparticles “stealthy” during circulation.They can also give nanoparticles an enhanced pH bufferingcapacity. Davis was one of the earliest researchers to use imid-azole modied polymers for siRNA delivery. He and co-workersreported an imidazole modied linear, cyclodextrin-containingpolycation nanomaterial for gene delivery and established theendosomal escape enhancing effect of imidazole groups.86 Theylater applied this discovery to a nanoparticle design and suc-ceeded in achieving the rst targeted delivery of siRNA inhumans.3 Lin et al. introduced imidazole into a polymer-basedliposomal dual-shell siRNA delivery carrier.52 Malamas et al.designed and evaluated an imidazole based pH-sensitiveamphiphilic cationic lipid for siRNA delivery.37 Gu et al. devel-oped an imidazole containing polymer nanocarrier system toachieve endosomal escape and timed release of siRNA.87 In theirstudy, an inuenza virus-inspired block copolymer wasprepared. The imidazole containing polymer chains werecapable of fusing with the endosome membrane to assist theescape. In addition, polyhistidine peptides were used as a pHsensitive domain. Benns et al. used a pH sensitive poly(L-histi-dine)-gra-poly(L-lysine) comb shaped polymer as a genedelivery vector.88

In addition to cationic pH sensitive polymers, anionic pHsensitive polymers, especially propylacrylic acid containingcopolymers, have been used to enhance endosomal escape.Propylacrylic acid is pH sensitive with a membrane disruptivecapability under endosomal conditions. Convertine et al.developed a novel propylacrylic acid containing endosomolyticdiblock copolymer for siRNA delivery.89 This copolymer was

ines and histamines into micelles followed by selective crosslinking inyright Elsevier 2012.

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Fig. 4 Chemical structures of the “encrypted polymers”. The acid-degradable linker is a para-amino benzaldehyde-acetal. Adapted withpermission from ref. 95. Copyright Elsevier 2003.


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composed of a positively-charged block of dimethylaminoethylmethacrylate (DMAEMA) to mediate siRNA complexation, and asecond endosomal-releasing block composed of DMAEMA andpropylacrylic acid (PAA). Nanoparticles assembled with thiscopolymer had low cytotoxicity and enhanced endosomalescape.

To make nanoparticles even more “stealthy”, charge-conversion polymers were developed in one early report toassemble a ternary polyplex nanoparticle, which coulddramatically change its surface charge in response to pHchange.90 Guo et al. reported charge-conversion polymer coatedgold nanoparticles to deliver siRNA.91,92 As depicted in Scheme2, this gold nanoparticle based ternary complex was composedof siRNA, polycations, and a charge-conversion polymer. Thiscomplex had a negative x-potential in the physiological envi-ronment to avoid blood clearance and reduce toxicity. Once itentered the acidic endosome, pH change would trigger thecharge conversion. This charge conversion resulted in a positivesurface charge of nanoparticles, which assisted endosomalescape to transport siRNA into the cytoplasm. The overall effectof this design was to realize two functions: prolonged circula-tion time and enhanced endosomal escape.

A supramolecular endosome destabilizing strategy wasreported by Tamura et al.93 In their study, N,N-dimethylami-noethyl (DMAE) modied cyclodextrin polyrotaxanes wereincorporated into the polyplex system. Polyrotaxanes wereconstructed with acid labile sulfanylpropionyl ester linkers.Aer entering the acidic endosome, sulfanylpropionyl esterlinkers would be cleaved to remove stoppers on both ends.Therefore, DMAE modied cyclodextrin would be released,which destabilized the endosome membrane and resulted inenhanced endosomal escape. While similar design strategieshave been applied to enable drug release from mesoporoussilica nanoparticles, their study was the rst example ofsupramolecular endosomal escape enhancement.

Plasmalogens, acid labile lipids, have been used to delivergene therapeutics and other drugs.94 Plasmalogens contain acidlabile vinyl ether. The degradation of vinyl ether and plasmal-ogen liposomes in an acidic environment would trigger therelease of cargo and disruption of the membrane. Based on thesame principle, acid labile “encrypted polymers” were devel-oped to achieve reduced toxicity and improved endosomalescape. Murthy et al. developed pH sensitive polymeric carrierscapable of delivering oligonucleotides.95 As shown in Fig. 4, thebackbone of this polymer was covalently conjugated to a PEG

Scheme 2 Enhanced intracellular payload release inside endosomesby using pH dependent charge-conversion polymers on nano-particles. Adapted with permission from ref. 91. Copyright AmericanChemical Society 2010.

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“mask” via an acetal linker. The toxicity of this carrier wasreduced as a result of the PEG layer. Aer internalization, thePEG layer was removed as the acetal linker was cleaved insidethe acidic endosome. The exposed hydrophobic, membranedisruptive backbone would assist endosomal escape to improvethe efficiency of gene delivery.

The combination of pH sensitive nanomaterials with otherstimuli responsiveness could help achieve improved trans-fection. Enzymes are an important stimulus in addition to pHchange. Matrix metalloproteinase (MMP), an enzyme overex-pressed by some cell types including certain types of cancercells, was used for this purpose. Li et al. developed a pHresponsive, smart polymeric siRNA delivery system with anMMP dependent proximity-activated targeting system.96 Thisdelivery system had a PEG corona during circulation. Oncereaching MMP rich target cells, cancer cells in their study, thePEG layer would be removed to expose the dimethylaminoethylmethacrylate containing cationic layer, which leads to celluptake. Membrane disruptive propylacrylic acid in the innercore of pH sensitive polymers was designed to enhance endo-somal escape. This design also realized the targeting effecttoward cancer tissues, since nanoparticles would only be takenup by cancer cells, where high MMP concentrations wouldensure the removal of PEG corona.

4.3 Calcium phosphate

Inorganic nanomaterials that quickly dissolve in acidic envi-ronments are of great interest to enhance endosomal escape.Being non-toxic and able to form nanoparticles, calcium phos-phate (CaP) is the most important pH sensitive inorganicnanomaterial.76,97 A series of CaP nanoparticle transfectionsystems have been developed. The most successful is the lipidcoated CaP nanoparticle (Liposome/Calcium/Phosphate orLCP).39,77 With a reverse water-in-oil micro-emulsion method,LCPs were prepared with calcium phosphate as the inner coreand lipid DOTAP as the outer layer (Fig. 5). The inner core of



Fig. 5 The formation process of liposome/calcium/phosphate (LCP)nanoparticles. Adaptedwith permission from ref. 77. Copyright Elsevier2010.

Fig. 6 Schematic representation of the tumor penetrating nano-complex, with siRNA (blue), a cyclic tumor-penetrating domain (LyP-,green) and various cell-penetrating peptide domains (purple). Adaptedwith permission from ref. 106. Copyright American Chemical Society2012.


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CaP plays two roles: (1) to form solid inorganic nanoparticlesand (2) to assist endosomal escape. Inside the endosome, CaPwould dissolve to disassemble the nanoparticles. This dissolv-ing process would also increase the osmotic pressure and causeendosome swelling. Their design combined the advantages oflipid nanoparticles for a prolonged circulation time and CaPnanoparticles for an enhanced endosomal escape. LCP carrierswere able to efficiently deliver siRNA to a xenogra tumor modelby intravenous administration. LCP achieved 70% and 50% ofluciferase silencing for the tumor cells in culture and thosegrown in a xenogra model, respectively. LCP achieved signi-cantly higher gene silencing than LPD (lipid nanoparticleswithout a CaP core), which proved the important role of CaP foran enhanced endosomal escape. Later an asymmetric lipidbilayer coated CaP nanoparticle system was developed with asmall size of 25–30 nm. This new design had an improvedtransfection effect both in vitro and in vivo.

Pittella et al. combined calcium phosphate nanoparticlesand pH conversion polymers to enhance endosomal escape andreduce positive surface charge.98,99 They successfully synthe-sized sub-100 nm CaP nanoparticles, which could incorporatesiRNA and a PEG modied anionic polymer. Inside the acidicendosome, the anionic polymer would be converted to acationic polymer to destabilize the endosome membrane.Meanwhile, CaP would dissolve to generate the “proton sponge”effect to further enhance endosomal escape. Their study


demonstrated the use of two or more endosomal escapeenhancing strategies to improve siRNA transfection efficacy.

4.4 Cell penetrating peptides

Based on the outstanding transfection efficiency of viral vectors,cell penetrating peptides are designed and incorporated intonon-viral vectors. Peptides are highly tunable in structure with20 natural amino acids to choose from and an innite numberof sequences. Many cell penetrating peptides are derived frombacterial or viral proteins, such as HIV-Tat based fusogenicpeptides. Typical physicochemical features of cell penetratingpeptides include a high positive charge (a large number ofarginines or lysines) and amphiphilicity (capability to stronglyinteract with a lipid membrane). As mentioned above, nano-particles can be taken up via more than one pathway. Theprecise internalization mechanism of cell penetrating peptidesremains controversial. Generally, it is believed that cell pene-trating peptides are internalized either by fusing with lipid cellmembranes following a vacuole-based endocytosis or creatingpores on the cell membrane.100,101

Akita et al. developed lipid enveloped-type nanoparticlesmodied with cell penetrating peptides, which could deliversiRNA to dendritic cells.102 Work has been done to optimizepeptide sequences with the highest cell penetrating efficiency.Research by van Asbeck et al. evaluated molecular parameters ofsiRNA-cell penetrating peptide nanocomplexes for efficientcellular delivery.103 They discovered that the most active cellpenetrating peptide displayed high serum resistance but alsohigh sensitivity to decomplexation by polyanionic macromole-cules. Karagiannis et al. used siRNA loaded lipid-like nano-particles decorated with different cell penetrating peptides andevaluated both in vitro and in vivo efficacy.104 They couldcorrelate the transfection efficiency with the physical andchemical properties of peptides. Similar work was done by Asaiet al. to develop cell penetrating peptide conjugated lipidnanoparticles for siRNA delivery.105 On the other hand, Renet al. focused on tumor cell targeting and internalization withcell penetrating peptides.106 They designed and screened alibrary of tandem tumor-targeting and cell-penetratingpeptides, which were capable of condensing siRNA into stablenanocomplexes (Fig. 6). Through physicochemical and biolog-ical characterization, they identied a group of nanocomplexesthat were capable of cell type specic siRNA delivery. Thesenanocomplexes were taken up by cells via endocytosis. Cell

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penetrating peptides triggered endosomal escape, followed bythe release of cargo, which resulted in gene silencing in areceptor-specic fashion. It is difficult to deliver siRNA to tumorcells in a target specic manner. This strategy has greatpotential for siRNA delivery to cancer cells.

4.5 Enhancing endosomal escape and overcoming otherbiological barriers

Systemic siRNA delivery is a challenging task, which requires asophisticated design to overcome multiple biological barriers.Endosomal escape is only one of the biological barriersresearchers have to overcome while designing nanocarriers. Inthe previous sections, several examples of multi-functionalnanomaterials were discussed. Nevertheless, it would be inter-esting and intriguing to design a nanosystem, which can beprecisely tuned to overcome multiple biological barriers insiRNA delivery.

To achieve this, Nelson et al. developed a copolymer as amodel system to study how to enhance the efficiency of systemicsiRNA delivery by incorporating multiple functions.17 Theydesigned a PEG-(DMAEMA-co-BMA) diblock polymer. By varyingthe relative ratio of DMAEMA and BMA (butyl methacrylate),they formulated this polymer for optimal siRNA delivery. Thecationic and hydrophobic contents were balanced to enhancesiRNA packaging and endosomal escape. The PEG corona andoptimized hydrophobic content increased the nanoparticlestability in the presence of human serum. Compared to thestandard polymer, the polymer with an optimized hydrophobiccontent enhanced the blood circulation half-life by three-fold,due to improved stability and reduced rate of renal clearance.This optimized polymer enhanced siRNA biodistribution to theliver and other organs and signicantly improved genesilencing in vivo. Their study is of great signicance as a goodexample of enhancing multiple functions by tuning the chem-ical composition.

5. Conclusion and perspective

This feature article discusses recent advances in endosomalescape enhancement strategies for nanoparticle mediatedsiRNA delivery. Mechanistic studies of nanoparticle intracel-lular trafficking indicate the importance of efficient endosomalescape. Based on different nanoparticle types, different endo-somal escape strategies are deployed to tackle the challenge.

For siRNA delivery, it is oen a difficult task to translate ananoparticle design that works in vitro into animal studies dueto multiple biological barriers. The solution is to design nano-particles with multiple functions to overcome multiple biolog-ical barriers. For a rational multi-functional nanoparticledesign, two major challenges exist. The rst challenge is how tomanage conicting functions. For example, elevated circulationtime and enhanced endosomal escape have opposite require-ments for nanoparticle characteristics. Examples to solve thischallenge include the use of charge-conversion polymers andenzyme proximity activation. The second challenge is how tohave a simple and general design. Complex delivery systems

6422 | Nanoscale, 2014, 6, 6415–6425

designed to achieve multiple functions could lead to poorreproducibility and high production costs. Simple and cost-effective nanoparticle systems should be creatively designed.One good example is the liposome/calcium phosphate system,which was inexpensive to prepare and achieved success both invitro and in vivo.

Endosomal escape, a critical biological barrier in siRNAdelivery, should be overcome with the criteria mentioned above.When designing nanoparticles for siRNA delivery, researchersshould constantly keep in mind the necessity of maintaining ahigh endosomal escape efficiency. It is also important for futureendosomal escape enhancement design strategies to beconsidered along with other aspects of siRNA delivery. Chem-ical tuning capability is highly desirable to nd the optimalformulation to realize the highest efficacy and the lowesttoxicity. As the endeavour to achieve safe and efficient siRNAdelivery continues, we expect to see more innovative designsand the ultimate clinical success of siRNA-based gene therapy.

Acknowledgements

The author wants to thank Professor Joseph DeSimone for hisguidance and support, and Crista Farrell for proofreading.

Notes and references

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enhancing endosomal escape for nanoparticle mediated sirna delivery

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