Enhancing endosomal escape for nanoparticle mediated siRNA delivery

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  • Nanoscale

    FEATURE ARTICLE

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    View Article OnlineView Journal | View IssueEnhancing endosDfHUwSdUWDowt

    based nanoparticles. He will becFudan University in China.

    Department of Chemistry, Fudan University

    China. E-mail: dama@live.unc.edu

    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 Comal 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, whicha 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 2014signicantly 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.914

    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 isNanoscale, 2014, 6, 64156425 | 6415

    http://dx.doi.org/10.1039/C4NR00018Hhttp://pubs.rsc.org/en/journals/journal/NRhttp://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.

    Nanoscale Feature Article

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    View Article Onlinehoused 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.1619 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, 64156425Endocytosis 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.2224 By accumulating protons inthe vesicle, the proton pump vacuolar ATPase generates acidi-cation until the pH drops to pH 56. With the fusion of the lateendosomes with the lysosomes (pH 45), 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 spongeeffect. 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 somehidden 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 (12%) 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

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

  • 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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    View Article OnlineThe 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.263. 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, andThis journal is The Royal Society of Chemistry 2014nanoparticle 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 spongemechanism 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.15,3134 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 theproton 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.39Nanoscale, 2014, 6, 64156425 | 6417

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

  • Nanoscale Feature Article

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    View Article OnlinePolyplex nanoparticles are generally based on electrostaticcomplexation of cationic polymers and anionic nucleic acid.4048

    Cationic polymers, such as polyethyleneimine (PEI), poly-L-lysine (PLL), chitosan, and...

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