Nanoscale View Article Online
FEATURE ARTICLE
Published on 15 April 2014. Downloaded by Université Laval on 16/06/2014 12:47:56.
Cite this: Nanoscale, 2014, 6, 6415
View Journal | View Issue
Enhancing endosomal escape for nanoparticle mediated siRNA delivery Da Ma* Gene therapy with siRNA is a promising biotechnology to treat cancer and other diseases. To realize siRNAbased 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,
Received 2nd January 2014 Accepted 10th April 2014
administration routes, and target organ/cell types. Also, enhancement of endosomal escape has been
DOI: 10.1039/c4nr00018h
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,
www.rsc.org/nanoscale
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 cancer and other diseases by halting the production of target proteins.2,3 Small interfering RNA (siRNA) is a synthetic doublestranded RNA (dsRNA) with approximately 21 base pairs,4 which is capable of entering the RNA-induced silencing complex (RISC), interfering with and inhibiting the expression of specic genes.5 Since the size of siRNA is much smaller compared to full size RNA, siRNA can be chemically synthesized, which
Department of Chemistry, Fudan University, 220 Handan Road, Shanghai, 200433, China. E-mail: [email protected]
Da Ma received his B.S. degree from Peking University in 2005. He later graduated from the University of Maryland in 2010 with a Ph.D. degree in chemistry. Since 2010, he has been a postdoctoral research associate at the University of North Carolina. Working with Professor Joseph DeSimone, he is currently developing siRNA delivery technology with PRINT® (Particle Replication in Non-wetting Templates) based nanoparticles. He will become a principle investigator at Fudan University in China.
This journal is © The Royal Society of Chemistry 2014
signicantly lowers its production cost. The high target specicity of RNAi and relatively low synthetic cost of siRNA give siRNA-based gene therapy great potential for a variety of applications.6 As a large and negatively charged biological molecule, naked siRNA is unstable in the blood stream, is unable to penetrate cell membranes, and can be immunogenic.7 A safe and efficient delivery method is crucial to realize the broad potential of siRNA-based therapeutics. Both viral and non-viral vectors can be used to deliver siRNA.8 Non-viral vectors, especially nanoparticles, are less expensive to produce and carry a lower risk of provoking an immune response compared to viral vectors. As a result, nanoparticles are of great interest to deliver siRNA.9–14 Nanoparticle mediated siRNA delivery is an intensively investigated research eld with approximately 1000 research papers published in the past three years alone. Currently, siRNA delivery, especially systemic delivery in vivo, remains a difficult task.15 The difficulty of siRNA delivery is rooted in several biological barriers that present challenges when siRNA is delivered via systemic administration. First, nanomaterials for siRNA delivery need to form a stable complex with the cargo to protect it from degradation during circulation in the blood stream. Next, siRNA loaded nanoparticles need to evade fast clearance from the blood and avoid an immune response, which generally is realized by the surface modication with poly(ethylene glycol) (PEG) to protect and stabilize nanoparticles. Furthermore, a sufficient amount of carriers needs to accumulate in the target tissue and be taken up by target cells. To achieve this, proper surface characteristics and targeting groups are essential to enable accumulation in target tissues and uptake by target cells. Since the RNAi machinery is
Nanoscale, 2014, 6, 6415–6425 | 6415
View Article Online
Published on 15 April 2014. Downloaded by Université Laval on 16/06/2014 12:47:56.
Nanoscale
housed in the cytoplasm, successful delivery of siRNA relies on the ability of nanoparticles to enter the cell, reach the cytoplasm, and then release the cargo. In most cases, nanoparticles are internalized through an endocytosis and endo-lysosomal pathway. Therefore, endosomal escape is of particular importance for the delivery of siRNA.8 Recent mechanistic investigations on nanoparticle intracellular trafficking indicate that insufficient endosomal escape could signicantly limit siRNA delivery efficiency.9,16 Entrapment in the hostile endo-lysosomal vesicles and degradation by lysosomal enzymes in an acidic environment could be a dead end for siRNA delivery. To achieve RNAi, siRNA containing nanoparticles need to escape from the endosome within a short period of time to avoid the fate of being degraded or recycled. In systemic delivery, a nanoparticle design must be able to achieve multiple functions of elongated blood circulation time, improved stability, and reduced toxicity in addition to enhanced endosomal escape.16–19 Therefore, designing multifunctional siRNA delivery systems with efficient endosomal escape is a great challenge. This feature article highlights the challenges of and solutions for endosomal escape in nanoparticle mediated siRNA delivery. The discussion focuses on recent progress regarding this topic. The goal is to show that enhanced endosomal escape can be achieved by chemical composition control, surface property modication, and other creative nanoparticle design approaches. The author hopes that this article will raise awareness of the importance of addressing endosomal escape when designing nanoparticles for siRNA delivery. Endosomal escape enhancing strategies are summarized in this article, which can hopefully assist fellow researchers to design their own siRNA delivery systems.
2. Endosomal escape: the critical challenge in siRNA delivery As mentioned above, a few biological barriers have to be overcome, when siRNA is delivered in vivo via systemic administration. Among them, endosomal escape is a key biological barrier in siRNA delivery. Nanoparticles are typically taken up via endocytosis. Depending on nanoparticle properties (size, shape, surface properties, etc.) and cell types, endocytosis of nanoparticles may occur via different pathways.20 Generally, endocytosis can be divided into two broad categories: phagocytosis and pinocytosis. While phagocytosis mostly occurs with specialized phagocytes, such as macrophages and dendritic cells, pinocytosis is present in all types of cells. Based on the proteins involved, pinocytosis occurs either via clathrin-mediated pathways or clathrin-independent pathways. Clathrin-independent pathways can be further divided into caveolae-mediated endocytosis, clathrin- and caveolae-independent pathways, and macropinocytosis. The endocytic entry pathways are summarized in Fig. 1.21 Another classication of endocytosis is based on material interaction with the cellular membrane (receptormediated, adsorptive, uid phase).
6416 | Nanoscale, 2014, 6, 6415–6425
Feature Article
Gateways for endocytic entry. “Other” pathways represent clathrin- and caveolae-independent pathways. Adapted with permission from ref. 21. Copyright American Chemical Society 2012.
Fig. 1
Endocytosis of nanoparticles is a complex process. For most nanoparticles, more than one pathway could be used to achieve cellular entry. Among these endocytosis pathways, clathrinmediated endocytosis is generally considered to be the most common route of cellular entry, which goes through the endolysosomal pathway. Some endocytosis pathways, such as some cases of caveolae-mediated endocytosis and macropinocytosis, may bypass lysosomes.20 In these cases, an active endosomal escape mechanism is unnecessary. Nevertheless, this feature article will focus on endosomal escape strategies for the more common route of endocytosis via the endo-lysosomal pathway. Facilitating endosomal escape has long been the focus of gene delivery research. In the “classical” endo-lysosomal pathway, nanoparticles start intracellular trafficking with early endosome vesicles, which become progressively acidic as they mature into late endosomes.22–24 By accumulating protons in the vesicle, the proton pump vacuolar ATPase generates acidication until the pH drops to pH 5–6. With the fusion of the late endosomes with the lysosomes (pH 4–5), the content would be degraded by enzymes if it does not escape the endosome. Cationic nanoparticles with a strong buffering capacity in the pH range from 5 to 7 have displayed the ability to escape the endosome potentially through the so-called “proton sponge” effect. To escape from the endosome in a timely fashion is essential to achieve efficient siRNA delivery. Two recent reports studying nanoparticle intracellular trafficking give more details about the endosomal escape mechanism, which indicate some “hidden” pathways that might compromise siRNA delivery efficiency.9,16 In the rst report, Gilleron et al. investigated the intracellular trafficking of siRNA containing lipid nanoparticles (LNPs), which were labelled by either uorescent dyes or gold nanoparticles.16 Quantitative uorescence imaging and electron microscopy were used to analyse nanoparticle trafficking. The LNPs were found to enter cells through both macropinocytosis and clathrin-mediated endocytosis. The key discovery was that escape of siRNA from endosomes into the cytosol occurs at low efficiency (1–2%) and only during a limited period of time when the LNPs reside in a specic compartment sharing early and late endosome characteristics. This discovery further stressed the importance of quick and efficient endosomal escape in order to realize high siRNA delivery efficiency.
This journal is © The Royal Society of Chemistry 2014
View Article Online
Published on 15 April 2014. Downloaded by Université Laval on 16/06/2014 12:47:56.
Feature Article
Nanoscale
Different types of nanoparticles used for gene delivery. Adapted with permission from ref. 27. Copyright Wiley-VCH 2008.
Fig. 3
Schematic illustration of LNP intracellular trafficking pathways summarized in Gilleron et al. and Sahay et al. reports. Adapted with permission from ref. 25. Copyright Nature Publishing Group 2013. Fig. 2
The second study was carried out by Sahay et al.9 Researchers used high-throughput confocal microscopy to screen a library of small-molecule inhibitors and identify critical signalling pathways that regulated the cellular uptake and intracellular trafcking of siRNA in HeLa cells. LNPs were also used in their investigation. Results showed that LNPs were internalized by macropinocytosis and trafficked directly into endosomes. Surprisingly, it was discovered that siRNA dissociated from the LNPs was exocytosed to the extracellular milieu. The amount of siRNA lost in this manner was calculated to be approximately 70% of the dose taken up by cells. This discovery indicates that nanoparticle endocytic recycling is limiting the efficiency of siRNA delivery. Intracellular trafficking pathways of both reports are summarized in Fig. 2.25 Although these two investigations were based on LNPs, the observations may also apply to other siRNA delivery platforms. As both reports show, it is essential to design siRNA loaded nanoparticles that are capable of escaping from the endosome efficiently. Otherwise, nanoparticles will either be degraded or recycled, which severely limits siRNA delivery efficiency.26
3. Endosomal escape enhancement for different nanoparticle types Various types of nanoparticles have been used for gene delivery. As depicted in Fig. 3, these nanoparticles include lipid-based nanoparticles, polymer-based nanoparticles, gold nanoparticles, mesoporous silica nanoparticles, carbon nanotubes, and
This journal is © The Royal Society of Chemistry 2014
nanoparticle assemblies.27 Depending on the type of nanoparticles, different strategies to enhance endosomal escape are used. The general method is to improve pH buffering capacity and increase the “proton sponge” effect. With this “proton sponge” mechanism, the buffering capacity prevents acidication of the endosomes by acting as “proton sponge”, which leads to an increase in the proton inux followed by an enhanced accumulation of counter anions and osmotic swelling. There had only been indirect evidence supporting this pH buffering mechanism, until a recent investigation reported direct visualization of this “proton sponge” mechanism with confocal microscopy.28 In this section, discussion focuses on recent progress in siRNA delivery with lipid nanoparticles, polyplex nanoparticles, polymer nanospheres, and inorganic nanoparticles. Cationic lipid-based nanoparticles are the most widely used non-viral gene delivery vectors.29 Currently, they are also the type of nanoparticles that holds the greatest promise to achieve clinical breakthroughs.30 Cationic lipids can self-assemble into nanoparticles, and encapsulate negatively charged siRNA. Further modication of these nanoparticles gives stabilizing and targeting capabilities when delivered in vivo via systemic administration.15,31–34 To design optimized delivery systems for siRNA therapeutics, Anderson and co-workers pioneered the use of robotic methods to systematically screen lipids.10,35,36 Cationic and pH sensitive lipids, which have a high pH buffering capacity, are used to enhance endosomal escape via the “proton sponge” effect. Systematic studies have been carried out to investigate how to use pH sensitive lipids to achieve the optimal endosomal escape effect.37,38 A combination of lipidbased nanoparticles with special endosomal escape strategies, such as calcium phosphate and cell penetrating peptides, has led to successful nanosystems for systemic delivery.39
Nanoscale, 2014, 6, 6415–6425 | 6417
View Article Online
Published on 15 April 2014. Downloaded by Université Laval on 16/06/2014 12:47:56.
Nanoscale
Polyplex nanoparticles are generally based on electrostatic complexation of cationic polymers and anionic nucleic acid.40–48 Cationic polymers, such as polyethyleneimine (PEI), poly-Llysine (PLL), chitosan, and synthetic dendrimers, are used to form polyplexes. The cationic characteristics of polymers enable the formation of stable complexes, which contributes to cationic nanoparticle mediated cell uptake and improves endosomal escape. One major advantage of polyplex nanoparticles is the innite number of polymers, which researchers can design and synthesize to incorporate multiple functions, such as protection with PEG and targeting toward certain cell types. The cationic feature of polymers contributes to endosomal escape typically by increasing the pH buffering capacity. More importantly, pH sensitive groups can be introduced into polymer structures for enhanced endosomal escape and reduced toxicity.49–54 In addition, it is possible to prepare hybrid materials of polymers and peptides to realize improved endosomal escape. Polymer nanospheres are another type of polymer based nanoparticles. Poly-L-lactic acid (PLA) and poly-lactic-co-glycolic acid (PLGA) can encapsulate siRNA via non-ionic interaction to form polymer nanospheres.55 Approved by the FDA for pharmaceutical use, PLA and PLGA are well known for their biocompatibility. As neutral nanoparticles without modication, PLA and PLGA based nanospheres need to be optimized to improve cellular uptake and endosomal escape by introducing a coating of cationic lipids or polymers.56,57 PLA or PLGA containing hybrid nanoparticles have been developed. Desai et al. developed a PLGA and cationic lipid hybrid nanoparticle to deliver siRNA and capsaicin via topical administration to inhibit skin inammation 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 calcium phosphate nanoparticles.39,75–77 Inorganic nanoparticles are generally highly stable in maintaining their size, shape and composition. Nevertheless, since inorganic nanoparticles lack charge, they are unable to electrostatically interact with either anionic nucleic acids or the negatively charged cell membrane. Successful transfection with inorganic nanoparticles requires surface coating or modication with cationic polymers to improve cellular uptake and endosomal escape. Cationic polymers, such as PEI, PLL or specically designed polymers, contribute to the improved cell uptake and endosomal escape capability. Among these inorganic nanoparticles, calcium phosphate nanoparticles have a unique advantage. With a tendency to quickly dissolve under acidic conditions like that inside endosomes and lysosomes, calcium phosphate nanoparticles can greatly enhance endosomal escape with minimum toxicity.78 In summary, different nanoparticles have their unique strategies to enhance endosomal escape. The common strategy is to take advantage of the acidic environment inside the endosome and enhance endosomal escape with the “proton sponge” effect to destabilize the endosome membrane.
6418 | Nanoscale, 2014, 6, 6415–6425
Feature Article
4. Endosomal escape enhancing methods Most endosomal escape strategies are based on the acidic endosome micro-environment and “proton sponge” effect, although external stimulations, such as photochemical internalization (PCI), are also used.79 In this section, several endosomal escape enhancing strategies will be introduced, which include the use of cationic polymers, pH sensitive polymers, calcium phosphate, and cell penetrating peptides. This section will also address how to design multifunctional systems to enhance endosomal escape and overcome other biological barriers in siRNA delivery. 4.1
Cationic polymers
Cationic polymers are used to complex with siRNA to form polyplexes or to coat inorganic and other nanoparticles. PEI, PLL and other polyammonium polymers are generally used based on the “proton sponge” effect. As an alternative to polyammonium polymers, Ornelas-Megiatto et al. reported the use of polyphosphonium polymers as an efficient and non-toxic transfection agent.80 In many cases, polymers are specically designed and synthesized to facilitate multiple functions including endosomal escape, protection and targeting. A cationic polymer coating is necessary for many inorganic nanoparticle based siRNA carriers to enhance endosomal escape and cell uptake. Xia et al. developed PEI coated mesoporous silica nanoparticles to deliver siRNA and DNA.71 There are several reports of PEI capped gold nanoparticles for siRNA delivery.59–61,72 Other than commercially available polyammonium polymers, Lee et al. synthesized and tested biodegradable poly(b-amino ester) type polymers to improve the transfection efficiency of gold nanoparticles.62 In their study, a library of polymers was screened to nd the best formulation to enhance gold nanoparticle transfection efficiency. Kozielski et al. developed a bioreducible poly(b-amino ester), which could deliver siRNA and release the cargo in an intracellular environment.81 In their design, the cleavable disulde bonds in the polymer structure ensured the degradation of the polymer and the release of cargo in the reducing intracellular environment. There are many reports of multi-functional cationic polymers. A triblock poly(amido amine)–poly(ethylene glycol)–polyL-lysine (PAMAM-PEG-PLL) nanocarrier was developed by Patil et al.82 This triblock copolymer had a combination of improved endosomal escape (PAMAM), protection (PEG) and siRNA condensation (PLL), which could be easily tuned to achieve the optimal transfection efficacy. Yu et al. reported an amphotericin B (AmB)-loaded, poly(2-(dimethylamino)ethyl methacrylate)block-poly(2-(diisopropylamino)ethyl methacrylate) (PDMA-bPDPA) micelleplex siRNA delivery system.83 PDMA-b-PDPA is a cationic polymer, which can complex with siRNA and improve its transfection capability. AmB is a hydrophobic antifungal drug, known to increase membrane permeability at sublethal concentrations by the formation of transmembrane pores. Their study conrmed that AmB was released from the micelleplexes in the early endosome to assist endosomal escape. In
This journal is © The Royal Society of Chemistry 2014
View Article Online
Published on 15 April 2014. Downloaded by Université Laval on 16/06/2014 12:47:56.
Feature Article
this dual pH-responsive system, the combination of membrane poration by AmB and endosome swelling by polycations contributed to efficient endosomal escape. Shrestha et al. designed and synthesized a polymer with primary amines and histamines, which can be assembled into cationic shell crosslinked knedel-like nanoparticles (cSCKs) (Scheme 1).50 Amphiphilic block copolymers were synthesized, followed by selective crosslinking throughout the hydrophilic shell layer to form cSCKs. To reduce toxicity, histamine groups were introduced into the structure of the copolymer. By increasing the ratio of histamines to primary amines, siRNAbinding affinity was decreased and the cytotoxicity was reduced. By controlling the ratio of primary amines and histamines, siRNA-binding affinity, cytotoxicity, immunogenicity, and transfection efficiency could be controlled. Endosomal escape was facilitated by having these two species of low and high pKas. With this tuning capability, while maintaining adequate endosomal escape, toxicity of these cationic nanoparticles was reduced and their biocompatibility was increased. Peptide/polymer hybrid materials, which combine the advantages of precise structural control of peptides and low synthetic cost of polymers, are interesting gene delivery vectors. Meyer et al. developed a polycation with a pH responsive peptide and PEG. While PEG was used to protect the cargo, the sequence of the peptide could be tuned to have the optimal transfection efficacy.84 Zeng et al. reported a multifunctional dendronized peptide polymer platform for siRNA delivery.85 The ratio of cationic modules, hydrophilic groups, and hydrophobic groups in this peptide/polymer hybrid material could be adjusted in order to achieve high siRNA delivery efficiency and excellent biocompatibility. In addition, the polymer was reducing environment degradable in its design. Polymer degradation in the intracellular environment not only triggered the release of cargo, but also further reduced the possible long-term toxicity. In addition, polymeric nanoparticles can be coated with cationic lipids to improve cellular uptake and endosomal escape. Yang et al. developed a polymeric nanoparticle assembled from mPEG-PLA, PLA, cationic lipid, and siRNA in a single step.34 The resulting hybrid nanoparticles exhibited excellent stability in serum and showed signicantly improved biocompatibility compared to that of pure cationic lipid nanoparticles. 4.2
pH sensitive polymers
While cationic polymers are capable of enhancing endosomal escape, they may lead to toxicity in the physiological
Nanoscale
environment. By using pH sensitive polymers, the nanoparticle toxicity can be reduced during circulation. Aerwards, pH sensitive polymers will be further protonated or degraded to expose the membrane disruptive inner core to assist endosomal escape. Polymers can be modied with pH sensitive groups. The ratio of pH sensitive groups to other functional groups can be adjusted and balanced to achieve the optimal overall transfection efficiency. While cationic polymers in the previous section were discussed as having certain pH sensitive characteristics, this section will give a detailed discussion of the application of pH sensitive polymers in siRNA delivery. Regular amine groups tend to change their protonation status as pH changes. Nevertheless, imidazole and other pH sensitive groups are more widely used. The use of imidazole and other pH-sensitive groups can decrease the amount of positive surface charge in the physiological environment to reduce toxicity and make nanoparticles “stealthy” during circulation. They can also give nanoparticles an enhanced pH buffering capacity. Davis was one of the earliest researchers to use imidazole modied polymers for siRNA delivery. He and co-workers reported an imidazole modied linear, cyclodextrin-containing polycation nanomaterial for gene delivery and established the endosomal escape enhancing effect of imidazole groups.86 They later applied this discovery to a nanoparticle design and succeeded in achieving the rst targeted delivery of siRNA in humans.3 Lin et al. introduced imidazole into a polymer-based liposomal dual-shell siRNA delivery carrier.52 Malamas et al. designed and evaluated an imidazole based pH-sensitive amphiphilic cationic lipid for siRNA delivery.37 Gu et al. developed an imidazole containing polymer nanocarrier system to achieve endosomal escape and timed release of siRNA.87 In their study, an inuenza virus-inspired block copolymer was prepared. The imidazole containing polymer chains were capable of fusing with the endosome membrane to assist the escape. In addition, polyhistidine peptides were used as a pH sensitive domain. Benns et al. used a pH sensitive poly(L-histidine)-gra-poly(L-lysine) comb shaped polymer as a gene delivery vector.88 In addition to cationic pH sensitive polymers, anionic pH sensitive polymers, especially propylacrylic acid containing copolymers, have been used to enhance endosomal escape. Propylacrylic acid is pH sensitive with a membrane disruptive capability under endosomal conditions. Convertine et al. developed a novel propylacrylic acid containing endosomolytic diblock copolymer for siRNA delivery.89 This copolymer was
Scheme 1 Self-assembly of polymers with various amounts of primary amines and histamines into micelles followed by selective crosslinking in the hydrophilic shell regions. Adapted with permission from ref. 50. Copyright Elsevier 2012.
This journal is © The Royal Society of Chemistry 2014
Nanoscale, 2014, 6, 6415–6425 | 6419
View Article Online
Published on 15 April 2014. Downloaded by Université Laval on 16/06/2014 12:47:56.
Nanoscale
composed of a positively-charged block of dimethylaminoethyl methacrylate (DMAEMA) to mediate siRNA complexation, and a second endosomal-releasing block composed of DMAEMA and propylacrylic acid (PAA). Nanoparticles assembled with this copolymer had low cytotoxicity and enhanced endosomal escape. To make nanoparticles even more “stealthy”, chargeconversion polymers were developed in one early report to assemble a ternary polyplex nanoparticle, which could dramatically change its surface charge in response to pH change.90 Guo et al. reported charge-conversion polymer coated gold nanoparticles to deliver siRNA.91,92 As depicted in Scheme 2, this gold nanoparticle based ternary complex was composed of siRNA, polycations, and a charge-conversion polymer. This complex had a negative x-potential in the physiological environment to avoid blood clearance and reduce toxicity. Once it entered the acidic endosome, pH change would trigger the charge conversion. This charge conversion resulted in a positive surface charge of nanoparticles, which assisted endosomal escape to transport siRNA into the cytoplasm. The overall effect of this design was to realize two functions: prolonged circulation time and enhanced endosomal escape. A supramolecular endosome destabilizing strategy was reported by Tamura et al.93 In their study, N,N-dimethylaminoethyl (DMAE) modied cyclodextrin polyrotaxanes were incorporated into the polyplex system. Polyrotaxanes were constructed with acid labile sulfanylpropionyl ester linkers. Aer entering the acidic endosome, sulfanylpropionyl ester linkers would be cleaved to remove stoppers on both ends. Therefore, DMAE modied cyclodextrin would be released, which destabilized the endosome membrane and resulted in enhanced endosomal escape. While similar design strategies have been applied to enable drug release from mesoporous silica nanoparticles, their study was the rst example of supramolecular endosomal escape enhancement. Plasmalogens, acid labile lipids, have been used to deliver gene therapeutics and other drugs.94 Plasmalogens contain acid labile vinyl ether. The degradation of vinyl ether and plasmalogen liposomes in an acidic environment would trigger the release of cargo and disruption of the membrane. Based on the same principle, acid labile “encrypted polymers” were developed to achieve reduced toxicity and improved endosomal escape. Murthy et al. developed pH sensitive polymeric carriers capable of delivering oligonucleotides.95 As shown in Fig. 4, the backbone of this polymer was covalently conjugated to a PEG
Feature Article
Fig. 4 Chemical structures of the “encrypted polymers”. The aciddegradable linker is a para-amino benzaldehyde-acetal. Adapted with permission from ref. 95. Copyright Elsevier 2003.
“mask” via an acetal linker. The toxicity of this carrier was reduced as a result of the PEG layer. Aer internalization, the PEG layer was removed as the acetal linker was cleaved inside the acidic endosome. The exposed hydrophobic, membrane disruptive backbone would assist endosomal escape to improve the efficiency of gene delivery. The combination of pH sensitive nanomaterials with other stimuli responsiveness could help achieve improved transfection. Enzymes are an important stimulus in addition to pH change. Matrix metalloproteinase (MMP), an enzyme overexpressed by some cell types including certain types of cancer cells, was used for this purpose. Li et al. developed a pH responsive, smart polymeric siRNA delivery system with an MMP dependent proximity-activated targeting system.96 This delivery system had a PEG corona during circulation. Once reaching MMP rich target cells, cancer cells in their study, the PEG layer would be removed to expose the dimethylaminoethyl methacrylate containing cationic layer, which leads to cell uptake. Membrane disruptive propylacrylic acid in the inner core of pH sensitive polymers was designed to enhance endosomal escape. This design also realized the targeting effect toward cancer tissues, since nanoparticles would only be taken up by cancer cells, where high MMP concentrations would ensure the removal of PEG corona. 4.3
Scheme 2 Enhanced intracellular payload release inside endosomes by using pH dependent charge-conversion polymers on nanoparticles. Adapted with permission from ref. 91. Copyright American Chemical Society 2010.
6420 | Nanoscale, 2014, 6, 6415–6425
Calcium phosphate
Inorganic nanomaterials that quickly dissolve in acidic environments are of great interest to enhance endosomal escape. Being non-toxic and able to form nanoparticles, calcium phosphate (CaP) is the most important pH sensitive inorganic nanomaterial.76,97 A series of CaP nanoparticle transfection systems have been developed. The most successful is the lipid coated CaP nanoparticle (Liposome/Calcium/Phosphate or LCP).39,77 With a reverse water-in-oil micro-emulsion method, LCPs were prepared with calcium phosphate as the inner core and lipid DOTAP as the outer layer (Fig. 5). The inner core of
This journal is © The Royal Society of Chemistry 2014
View Article Online
Feature Article
Nanoscale
demonstrated the use of two or more endosomal escape enhancing strategies to improve siRNA transfection efficacy.
Published on 15 April 2014. Downloaded by Université Laval on 16/06/2014 12:47:56.
4.4
Fig. 5 The formation process of liposome/calcium/phosphate (LCP) nanoparticles. Adapted with permission from ref. 77. Copyright Elsevier 2010.
CaP plays two roles: (1) to form solid inorganic nanoparticles and (2) to assist endosomal escape. Inside the endosome, CaP would dissolve to disassemble the nanoparticles. This dissolving process would also increase the osmotic pressure and cause endosome swelling. Their design combined the advantages of lipid nanoparticles for a prolonged circulation time and CaP nanoparticles for an enhanced endosomal escape. LCP carriers were able to efficiently deliver siRNA to a xenogra tumor model by intravenous administration. LCP achieved 70% and 50% of luciferase silencing for the tumor cells in culture and those grown in a xenogra model, respectively. LCP achieved signicantly higher gene silencing than LPD (lipid nanoparticles without a CaP core), which proved the important role of CaP for an enhanced endosomal escape. Later an asymmetric lipid bilayer coated CaP nanoparticle system was developed with a small size of 25–30 nm. This new design had an improved transfection effect both in vitro and in vivo. Pittella et al. combined calcium phosphate nanoparticles and pH conversion polymers to enhance endosomal escape and reduce positive surface charge.98,99 They successfully synthesized sub-100 nm CaP nanoparticles, which could incorporate siRNA and a PEG modied anionic polymer. Inside the acidic endosome, the anionic polymer would be converted to a cationic polymer to destabilize the endosome membrane. Meanwhile, CaP would dissolve to generate the “proton sponge” effect to further enhance endosomal escape. Their study
This journal is © The Royal Society of Chemistry 2014
Cell penetrating peptides
Based on the outstanding transfection efficiency of viral vectors, cell penetrating peptides are designed and incorporated into non-viral vectors. Peptides are highly tunable in structure with 20 natural amino acids to choose from and an innite number of sequences. Many cell penetrating peptides are derived from bacterial or viral proteins, such as HIV-Tat based fusogenic peptides. Typical physicochemical features of cell penetrating peptides include a high positive charge (a large number of arginines or lysines) and amphiphilicity (capability to strongly interact with a lipid membrane). As mentioned above, nanoparticles can be taken up via more than one pathway. The precise internalization mechanism of cell penetrating peptides remains controversial. Generally, it is believed that cell penetrating peptides are internalized either by fusing with lipid cell membranes following a vacuole-based endocytosis or creating pores on the cell membrane.100,101 Akita et al. developed lipid enveloped-type nanoparticles modied with cell penetrating peptides, which could deliver siRNA to dendritic cells.102 Work has been done to optimize peptide sequences with the highest cell penetrating efficiency. Research by van Asbeck et al. evaluated molecular parameters of siRNA-cell penetrating peptide nanocomplexes for efficient cellular delivery.103 They discovered that the most active cell penetrating peptide displayed high serum resistance but also high sensitivity to decomplexation by polyanionic macromolecules. Karagiannis et al. used siRNA loaded lipid-like nanoparticles decorated with different cell penetrating peptides and evaluated both in vitro and in vivo efficacy.104 They could correlate the transfection efficiency with the physical and chemical properties of peptides. Similar work was done by Asai et al. to develop cell penetrating peptide conjugated lipid nanoparticles for siRNA delivery.105 On the other hand, Ren et al. focused on tumor cell targeting and internalization with cell penetrating peptides.106 They designed and screened a library of tandem tumor-targeting and cell-penetrating peptides, which were capable of condensing siRNA into stable nanocomplexes (Fig. 6). Through physicochemical and biological characterization, they identied a group of nanocomplexes that were capable of cell type specic siRNA delivery. These nanocomplexes were taken up by cells via endocytosis. Cell
Schematic representation of the tumor penetrating nanocomplex, with siRNA (blue), a cyclic tumor-penetrating domain (LyP-, green) and various cell-penetrating peptide domains (purple). Adapted with permission from ref. 106. Copyright American Chemical Society 2012.
Fig. 6
Nanoscale, 2014, 6, 6415–6425 | 6421
View Article Online
Nanoscale
penetrating peptides triggered endosomal escape, followed by the release of cargo, which resulted in gene silencing in a receptor-specic fashion. It is difficult to deliver siRNA to tumor cells in a target specic manner. This strategy has great potential for siRNA delivery to cancer cells.
Published on 15 April 2014. Downloaded by Université Laval on 16/06/2014 12:47:56.
4.5 Enhancing endosomal escape and overcoming other biological barriers Systemic siRNA delivery is a challenging task, which requires a sophisticated design to overcome multiple biological barriers. Endosomal escape is only one of the biological barriers researchers have to overcome while designing nanocarriers. In the previous sections, several examples of multi-functional nanomaterials were discussed. Nevertheless, it would be interesting and intriguing to design a nanosystem, which can be precisely tuned to overcome multiple biological barriers in siRNA delivery. To achieve this, Nelson et al. developed a copolymer as a model system to study how to enhance the efficiency of systemic siRNA delivery by incorporating multiple functions.17 They designed a PEG-(DMAEMA-co-BMA) diblock polymer. By varying the relative ratio of DMAEMA and BMA (butyl methacrylate), they formulated this polymer for optimal siRNA delivery. The cationic and hydrophobic contents were balanced to enhance siRNA packaging and endosomal escape. The PEG corona and optimized hydrophobic content increased the nanoparticle stability in the presence of human serum. Compared to the standard polymer, the polymer with an optimized hydrophobic content 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 the liver and other organs and signicantly improved gene silencing in vivo. Their study is of great signicance as a good example of enhancing multiple functions by tuning the chemical composition.
5.
Conclusion and perspective
This feature article discusses recent advances in endosomal escape enhancement strategies for nanoparticle mediated siRNA delivery. Mechanistic studies of nanoparticle intracellular trafficking indicate the importance of efficient endosomal escape. Based on different nanoparticle types, different endosomal escape strategies are deployed to tackle the challenge. For siRNA delivery, it is oen a difficult task to translate a nanoparticle design that works in vitro into animal studies due to multiple biological barriers. The solution is to design nanoparticles with multiple functions to overcome multiple biological barriers. For a rational multi-functional nanoparticle design, two major challenges exist. The rst challenge is how to manage conicting functions. For example, elevated circulation time and enhanced endosomal escape have opposite requirements for nanoparticle characteristics. Examples to solve this challenge include the use of charge-conversion polymers and enzyme proximity activation. The second challenge is how to have a simple and general design. Complex delivery systems
6422 | Nanoscale, 2014, 6, 6415–6425
Feature Article
designed to achieve multiple functions could lead to poor reproducibility and high production costs. Simple and costeffective nanoparticle systems should be creatively designed. One good example is the liposome/calcium phosphate system, which was inexpensive to prepare and achieved success both in vitro and in vivo. Endosomal escape, a critical biological barrier in siRNA delivery, should be overcome with the criteria mentioned above. When designing nanoparticles for siRNA delivery, researchers should constantly keep in mind the necessity of maintaining a high endosomal escape efficiency. It is also important for future endosomal escape enhancement design strategies to be considered along with other aspects of siRNA delivery. Chemical tuning capability is highly desirable to nd the optimal formulation to realize the highest efficacy and the lowest toxicity. As the endeavour to achieve safe and efficient siRNA delivery continues, we expect to see more innovative designs and the ultimate clinical success of siRNA-based gene therapy.
Acknowledgements The author wants to thank Professor Joseph DeSimone for his guidance and support, and Crista Farrell for proofreading.
Notes and references 1 A. Fire, S. Xu, M. K. Montgomery, S. A. Kostas, S. E. Driver and C. C. Mello, Nature, 1998, 391, 806–811. 2 F. Leuschner, P. Dutta, R. Gorbatov, T. I. Novobrantseva, J. S. Donahoe, G. Courties, K. M. Lee, J. I. Kim, J. F. Markmann, B. Marinelli, P. Panizzi, W. W. Lee, Y. Iwamoto, S. Milstein, H. Epstein-Barash, W. Cantley, J. Wong, V. Cortez-Retamozo, A. Newton, K. Love, P. Libby, M. J. Pittet, F. K. Swirski, V. Koteliansky, R. Langer, R. Weissleder, D. G. Anderson and M. Nahrendorf, Nat. Biotechnol., 2011, 29, 1005–1010. 3 M. E. Davis, J. E. Zuckerman, C. H. J. Choi, D. Seligson, A. Tolcher, C. A. Alabi, Y. Yen, J. D. Heidel and A. Ribas, Nature, 2010, 464, 1067–1070. 4 S. M. Elbashir, J. Harborth, W. Lendeckel, A. Yalcin, K. Weber and T. Tuschl, Nature, 2001, 411, 494–498. 5 R. Kanasty, J. R. Dorkin, A. Vegas and D. Anderson, Nat. Mater., 2013, 12, 967–977. 6 J.-P. Behr, Acc. Chem. Res., 2012, 45, 980–984. 7 K. A. Whitehead, R. Langer and D. G. Anderson, Nat. Rev. Drug Discovery, 2009, 8, 129–138. 8 E. Wagner, Acc. Chem. Res., 2012, 45, 1005–1013. 9 G. Sahay, W. Querbes, C. Alabi, A. Eltoukhy, S. Sarkar, C. Zurenko, E. Karagiannis, K. Love, D. Chen, R. Zoncu, Y. Buganim, A. Schroeder, R. Langer and D. G. Anderson, Nat. Biotechnol., 2013, 31, 653–658. 10 C. A. Alabi, K. T. Love, G. Sahay, H. Yin, K. M. Luly, R. Langer and D. G. Anderson, Proc. Natl. Acad. Sci. U. S. A., 2013, 110, 12881–12886. 11 S. J. Tan, P. Kiatwuthinon, Y. H. Roh, J. S. Kahn and D. Luo, Small, 2011, 7, 841–856.
This journal is © The Royal Society of Chemistry 2014
View Article Online
Published on 15 April 2014. Downloaded by Université Laval on 16/06/2014 12:47:56.
Feature Article
12 H. M. Aliabadi, B. Landry, C. Sun, T. Tang and H. Uluda˘ g, Biomaterials, 2012, 33, 2546–2569. 13 P. Kesharwani, V. Gajbhiye and N. K. Jain, Biomaterials, 2012, 33, 7138–7150. 14 H. Tian, J. Chen and X. Chen, Small, 2013, 9, 2034–2044. 15 K. Buyens, S. C. De Smedt, K. Braeckmans, J. Demeester, L. Peeters, L. A. van Grunsven, X. de Mollerat du Jeu, R. Sawant, V. Torchilin, K. Farkasova, M. Ogris and N. N. Sanders, J. Controlled Release, 2012, 158, 362–370. 16 J. Gilleron, W. Querbes, A. Zeigerer, A. Borodovsky, G. Marsico, U. Schubert, K. Manygoats, S. Seifert, C. Andree, M. St¨ oter, H. Epstein-Barash, L. Zhang, V. Koteliansky, K. Fitzgerald, E. Fava, M. Bickle, Y. Kalaidzidis, A. Akinc, M. Maier and M. Zerial, Nat. Biotechnol., 2013, 31, 638–646. 17 C. E. Nelson, J. R. Kintzing, A. Hanna, J. M. Shannon, M. K. Gupta and C. L. Duvall, ACS Nano, 2013, 7, 8870–8880. 18 Y. J. Kwon, Acc. Chem. Res., 2012, 45, 1077–1088. 19 M. Dominska and D. M. Dykxhoorn, J. Cell Sci., 2010, 123, 1183–1189. 20 G. Sahay, D. Y. Alakhova and A. V. Kabanov, J. Controlled Release, 2010, 145, 182–195. 21 R. Duncan and S. C. W. Richardson, Mol. Pharm., 2012, 9, 2380–2402. 22 J. Nguyen and F. C. Szoka, Acc. Chem. Res., 2012, 45, 1153– 1162. 23 J. Haensler and F. C. Szoka, Bioconjugate Chem., 1993, 4, 372–379. 24 N. D. Sonawane, F. C. Szoka and A. S. Verkman, J. Biol. Chem., 2003, 278, 44826–44831. 25 Y. Wang and L. Huang, Nat. Biotechnol., 2013, 31, 611– 612. 26 There is a recent report of microRNA/siRNA loaded PLGA nanoparticles that are capable of staying inside endosome for a long period of time without being degraded or exocytosed: J. Devalliere, W. G. Chang, J. W. Andrejecsk, P. Abrahimi, C. J. Cheng, D. Jane-Wit, W. M. Saltzman and J. S. Pober, FASEB J., 2014, 28, 908–922. 27 V. Sokolova and M. Epple, Angew. Chem., Int. Ed., 2008, 47, 1382–1395. 28 Z. ur Rehman, D. Hoekstra and I. S. Zuhorn, ACS Nano, 2013, 7, 3767–3777. 29 W. Li and F. C. Szoka, Pharm. Res., 2007, 24, 438–449. 30 J. C. Burnett, J. J. Rossi and K. Tiemann, Biotechnol. J., 2011, 6, 1130–1146. 31 D. J. Gary, H. Lee, R. Sharma, J.-S. Lee, Y. Kim, Z. Y. Cui, D. Jia, V. D. Bowman, P. R. Chipman, L. Wan, Y. Zou, G. Mao, K. Park, B.-S. Herbert, S. F. Konieczny and Y.-Y. Won, ACS Nano, 2011, 5, 3493–3505. 32 T. Lobovkina, G. B. Jacobson, E. Gonzalez-Gonzalez, R. P. Hickerson, D. Leake, R. L. Kaspar, C. H. Contag and R. N. Zare, ACS Nano, 2011, 5, 9977–9983. 33 Z. U. Rehman, I. S. Zuhorn and D. Hoekstra, J. Controlled Release, 2013, 166, 46–56. 34 X.-Z. Yang, S. Dou, Y.-C. Wang, H.-Y. Long, M.-H. Xiong, C.-Q. Mao, Y.-D. Yao and J. Wang, ACS Nano, 2012, 6, 4955–4965.
This journal is © The Royal Society of Chemistry 2014
Nanoscale
35 E. D. Karagiannis, C. A. Alabi and D. G. Anderson, ACS Nano, 2012, 8484–8487. 36 Y. Zhang, L. Arrington, D. Boardman, J. Davis, Y. Xu, K. DiFelice, S. Stirdivant, W. Wang, B. Budzik, J. Bawiec, J. Deng, G. Beutner, D. Seifried, M. Stanton, M. Gindy and A. Leone, J. Controlled Release, 2014, 174, 7–14. 37 A. S. Malamas, M. Gujrati, C. M. Kummitha, R. Xu and Z.-R. Lu, J. Controlled Release, 2013, 171, 296–307. 38 Y. Sato, H. Hatakeyama, Y. Sakurai, M. Hyodo, H. Akita and H. Harashima, J. Controlled Release, 2012, 163, 267– 276. 39 J. Li, Y. Yang and L. Huang, J. Controlled Release, 2012, 158, 108–114. 40 M. Yan, M. Liang, J. Wen, Y. Liu, Y. Lu and I. S. Y. Chen, J. Am. Chem. Soc., 2012, 134, 13542–13545. 41 M. Zheng, G. M. Pavan, M. Neeb, A. K. Schaper, A. Danani, G. Klebe, O. M. Merkel and T. Kissel, ACS Nano, 2012, 6, 9447–9454. 42 H. Lim, J. Noh, Y. Kim, H. Kim, J. Kim, G. Khang and D. Lee, Biomacromolecules, 2013, 14, 240–247. 43 B. J. Hong, A. J. Chipre and S. T. Nguyen, J. Am. Chem. Soc., 2013, 135, 17655–17658. 44 Z. J. Deng, S. W. Morton, E. Ben-Akiva, E. C. Dreaden, K. E. Shopsowitz and P. T. Hammond, ACS Nano, 2013, 7, 9571–9584. 45 J. Kloeckner, S. Boeckle, D. Persson, W. Roedl, M. Ogris, K. Berg and E. Wagner, J. Controlled Release, 2006, 116, 115–122. 46 S. S. Dunn, S. Tian, S. Blake, J. Wang, A. L. Galloway, A. Murphy, P. D. Pohlhaus, J. P. Rolland, M. E. Napier and J. M. DeSimone, J. Am. Chem. Soc., 2012, 134, 7423– 7430. 47 P. Posocco, X. Liu, E. Laurini, D. Marson, C. Chen, C. Liu, M. Fermeglia, P. Rocchi, S. Pricl and L. Peng, Mol. Pharm., 2013, 10, 3262–3273. 48 M. L. Patil, M. Zhang, O. Taratula, O. B. Garbuzenko, H. He and T. Minko, Biomacromolecules, 2009, 10, 258–266. 49 K. Raemdonck, B. Naeye, K. Buyens, R. E. Vandenbroucke, A. Høgset, J. Demeester and S. C. De Smedt, Adv. Funct. Mater., 2009, 19, 1406–1415. 50 R. Shrestha, M. Elsabahy, S. Florez-Malaver, S. Samarajeewa and K. L. Wooley, Biomaterials, 2012, 33, 8557–8568. 51 C. Dohmen, D. Edinger, T. Fr¨ ohlich, L. Schreiner, U. L¨ achelt, C. Troiber, J. R¨ adler, P. Hadwiger, H.-P. Vornlocher and E. Wagner, ACS Nano, 2012, 6, 5198– 5208. 52 S.-Y. Lin, W.-Y. Zhao, H.-C. Tsai, W.-H. Hsu, C.-L. Lo and G.-H. Hsiue, Biomacromolecules, 2012, 13, 664–675. 53 X. Yang, J. Du, S. Dou, C. Mao, H. Long and J. Wang, ACS Nano, 2012, 771–781. 54 T. Kim, T. Rothmund, T. Kissel and S. W. Kim, J. Controlled Release, 2011, 152, 110–119. 55 A. Alshamsan, A. Haddadi, S. Hamdy, J. Samuel, A. O. S. El-kadi and H. Uludag, Mol. Pharm., 2010, 7, 1643–1654. 56 S. H. Lee, H. Mok, Y. Lee and T. G. Park, J. Controlled Release, 2011, 152, 152–158.
Nanoscale, 2014, 6, 6415–6425 | 6423
View Article Online
Published on 15 April 2014. Downloaded by Université Laval on 16/06/2014 12:47:56.
Nanoscale
57 W. Hasan, K. Chu, A. Gullapalli, S. S. Dunn, E. M. Enlow, J. C. Lu, S. Tian, M. E. Napier, P. D. Pohlhaus, J. P. Rolland and J. M. DeSimone, Nano Lett., 2012, 287–292. 58 P. R. Desai, S. Marepally, A. R. Patel, C. Voshavar, A. Chaudhuri and M. Singh, J. Controlled Release, 2013, 170, 51–63. 59 S. T. Kim, A. Chompoosor, Y.-C. Yeh, S. S. Agasti, D. J. Solell and V. M. Rotello, Small, 2012, 8, 3253–3256. 60 M.-Y. Lee, S.-J. Park, K. Park, K. S. Kim, H. Lee and S. K. Hahn, ACS Nano, 2011, 5, 6138–6147. 61 W.-J. Song, J.-Z. Du, T.-M. Sun, P.-Z. Zhang and J. Wang, Small, 2010, 6, 239–246. 62 J. Lee, J. J. Green, K. T. Love, J. Sunshine, R. Langer and D. G. Anderson, Nano Lett., 2009, 2402–2406. 63 K. S. Siu, D. Chen, X. Zheng, X. Zhang, N. Johnston, Y. Liu, K. Yuan, J. Koropatnick, E. R. Gillies and W.-P. Min, Biomaterials, 2014, 35, 3435–3442. 64 X. Jiang, G. Wang, R. Liu, Y. Wang, Y. Wang, X. Qiu and X. Gao, Nanoscale, 2013, 5, 7256–7264. 65 S. Foillard, G. Zuber and E. Doris, Nanoscale, 2011, 3, 1461– 1464. 66 P. Singh, C. Samor`ı, F. M. Toma, C. Bussy, A. Nunes, K. T. Al-Jamal, C. M´ enard-Moyon, M. Prato, K. Kostarelos and A. Bianco, J. Mater. Chem., 2011, 21, 4850–4700. 67 N. W. S. Kam, Z. Liu and H. Dai, J. Am. Chem. Soc., 2005, 127, 12492–12493. 68 J. Shen, R. Xu, J. Mai, H.-C. Kim, X. Guo, G. Qin, Y. Yang, J. Wolfram, C. Mu, X. Xia, J. Gu, X. Liu, Z.-W. Mao, M. Ferrari and H. Shen, ACS Nano, 2013, 9867–9880. 69 D. Lin, Q. Cheng, Q. Jiang, Y. Huang, Z. Yang, S. Han, Y. Zhao, S. Guo, Z. Liang and A. Dong, Nanoscale, 2013, 5, 4291–4301. 70 S. B. Hartono, W. Gu, F. Kleitz, J. Liu, L. He, A. P. J. Middelberg, C. Yu, G. Q. M. Lu and S. Z. Qiao, ACS Nano, 2012, 6, 2104–2117. 71 T. Xia, M. Kovochich, M. Liong, H. Meng, S. Kabehie, S. George, J. I. Zink and A. E. Nel, ACS Nano, 2009, 3, 3273–3286. 72 S. Jiang, A. A. Eltoukhy, K. T. Love, R. Langer and D. G. Anderson, Nano Lett., 2013, 13, 1059–1064. 73 L. Qi and X. Gao, ACS Nano, 2008, 2, 1403–1410. 74 M. V Yezhelyev, L. Qi, R. M. O'Regan, S. Nie and X. Gao, J. Am. Chem. Soc., 2008, 130, 9006–9012. 75 T. Devarasu, R. Saad, A. Ouadi, B. Frisch, E. Robinet, P. Laquerri` ere, J.-C. Voegel, T. Baumert, J. Ogier and F. Meyer, J. Mater. Chem. B, 2013, 1, 4692–4700. 76 M. Zhang, A. Ishii, N. Nishiyama, S. Matsumoto, T. Ishii, Y. Yamasaki and K. Kataoka, Adv. Mater., 2009, 21, 3520– 3525. 77 J. Li, Y.-C. Chen, Y.-C. Tseng, S. Mozumdar and L. Huang, J. Controlled Release, 2010, 142, 416–421. 78 S. Bisht, G. Bhakta, S. Mitra and A. Maitra, Int. J. Pharm., 2005, 288, 157–168. 79 Y. Matsushita-Ishiodori and T. Ohtsuki, Acc. Chem. Res., 2012, 45, 1039–1047. 80 C. Ornelas-Megiatto, P. R. Wich and J. M. J. Fr´ echet, J. Am. Chem. Soc., 2012, 134, 1902–1905.
6424 | Nanoscale, 2014, 6, 6415–6425
Feature Article
81 K. L. Kozielski, S. Y. Tzeng and J. J. Green, Chem. Commun., 2013, 49, 5319–5321. 82 M. L. Patil, M. Zhang and T. Minko, ACS Nano, 2011, 5, 1877–1887. 83 H. Yu, Y. Zou, Y. Wang, X. Huang, G. Huang and B. D. Sumer, ACS Nano, 2011, 9246–9255. 84 M. Meyer, A. Philipp, R. Oskuee, C. Schmidt and E. Wagner, J. Am. Chem. Soc., 2008, 130, 3272–3273. 85 H. Zeng, H. C. Little, T. N. Tiambeng, G. A. Williams and Z. Guan, J. Am. Chem. Soc., 2013, 4962–4965. 86 S. Mishra, J. D. Heidel, P. Webster and M. E. Davis, J. Controlled Release, 2006, 116, 179–191. 87 W. Gu, Z. Jia, N. P. Truong, I. Prasadam, Y. Xiao and M. J. Monteiro, Biomacromolecules, 2013, 14, 3386–3389. 88 J. M. Benns, J. S. Choi, R. I. Mahato, J. S. Park and S. W. Kim, Bioconjugate Chem., 2000, 11, 637–645. 89 A. J. Convertine, D. S. W. Benoit, C. L. Duvall, A. S. Hoffman and P. S. Stayton, J. Controlled Release, 2009, 133, 221–229. 90 Y. Lee, K. Miyata, M. Oba, T. Ishii, S. Fukushima, M. Han, H. Koyama, N. Nishiyama and K. Kataoka, Angew. Chem., Int. Ed., 2008, 47, 5163–5166. 91 S. Guo, Y. Huang, Q. Jiang, Y. Sun, L. Deng, Z. Liang, Q. Du, J. Xing, Y. Zhao, P. C. Wang, A. Dong and X. Liang, ACS Nano, 2010, 4, 5505–5511. 92 L. Han, J. Zhao, X. Zhang, W. Cao, X. Hu, G. Zou and X. Duan, ACS Nano, 2012, 7340–7351. 93 A. Tamura and N. Yui, J. Mater. Chem. B, 2013, 1, 3535. 94 J. A. Boomer and D. H. Thompson, Chem. Phys. Lipids, 1999, 99, 145–153. 95 N. Murthy, J. Campbell, N. Fausto, A. S. Hoffman and P. S. Stayton, J. Controlled Release, 2003, 89, 365–374. 96 H. Li, S. S. Yu, M. Miteva, C. E. Nelson, T. Werfel, T. D. Giorgio and C. L. Duvall, Adv. Funct. Mater., 2013, 23, 3040–3052. 97 V. Sokolova, A. Kovtun, O. Prymak, W. Meyer-Zaika, E. A. Kubareva, E. A. Romanova, T. S. Oretskaya, R. Heumann and M. Epple, J. Mater. Chem., 2007, 17, 721–727. 98 F. Pittella, K. Miyata, Y. Maeda, T. Suma, S. Watanabe, Q. Chen, R. J. Christie, K. Osada, N. Nishiyama and K. Kataoka, J. Controlled Release, 2012, 161, 868–874. 99 F. Pittella, M. Zhang, Y. Lee, H. J. Kim, T. Tockary, K. Osada, T. Ishii, K. Miyata, N. Nishiyama and K. Kataoka, Biomaterials, 2011, 32, 3106–3114. 100 L. Crombez, A. Charnet, M. C. Morris, G. Aldrian-Herrada, F. Heitz and G. Divita, Biochem. Soc. Trans., 2007, 35, 44– 46. 101 I. Nakase, H. Akita, K. Kogure, A. Gr¨ aslund, U. Langel, H. Harashima and S. Futaki, Acc. Chem. Res., 2012, 45, 1132–1139. 102 H. Akita, K. Kogure, R. Moriguchi, Y. Nakamura, T. Higashi, T. Nakamura, S. Serada, M. Fujimoto, T. Naka, S. Futaki and H. Harashima, J. Controlled Release, 2010, 143, 311– 317. 103 A. H. van Asbeck, A. Beyerle, H. McNeill, P. H. M. BoveeGeurts, S. Lindberg, W. P. R. Verdurmen, M. H¨ allbrink,
This journal is © The Royal Society of Chemistry 2014
View Article Online
Feature Article
105 T. Asai, T. Tsuzuku, S. Takahashi, A. Okamoto, T. Dewa, M. Nango, K. Hyodo, H. Ishihara, H. Kikuchi and N. Oku, Biochem. Biophys. Res. Commun., 2014, 444, 599–604. 106 Y. Ren, S. Hauert, J. H. Lo and S. N. Bhatia, ACS Nano, 2012, 8620–8631.
Published on 15 April 2014. Downloaded by Université Laval on 16/06/2014 12:47:56.
U. Langel, O. Heidenreich and R. Brock, ACS Nano, 2013, 7, 3797–3807. 104 E. D. Karagiannis, A. M. Urbanska, G. Sahay, J. M. Pelet, S. Jhunjhunwala, R. Langer and D. G. Anderson, ACS Nano, 2013, 7, 8616–8626.
Nanoscale
This journal is © The Royal Society of Chemistry 2014
Nanoscale, 2014, 6, 6415–6425 | 6425
FEATURE ARTICLE
Published on 15 April 2014. Downloaded by Université Laval on 16/06/2014 12:47:56.
Cite this: Nanoscale, 2014, 6, 6415
View Journal | View Issue
Enhancing endosomal escape for nanoparticle mediated siRNA delivery Da Ma* Gene therapy with siRNA is a promising biotechnology to treat cancer and other diseases. To realize siRNAbased 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,
Received 2nd January 2014 Accepted 10th April 2014
administration routes, and target organ/cell types. Also, enhancement of endosomal escape has been
DOI: 10.1039/c4nr00018h
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,
www.rsc.org/nanoscale
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 cancer and other diseases by halting the production of target proteins.2,3 Small interfering RNA (siRNA) is a synthetic doublestranded RNA (dsRNA) with approximately 21 base pairs,4 which is capable of entering the RNA-induced silencing complex (RISC), interfering with and inhibiting the expression of specic genes.5 Since the size of siRNA is much smaller compared to full size RNA, siRNA can be chemically synthesized, which
Department of Chemistry, Fudan University, 220 Handan Road, Shanghai, 200433, China. E-mail: [email protected]
Da Ma received his B.S. degree from Peking University in 2005. He later graduated from the University of Maryland in 2010 with a Ph.D. degree in chemistry. Since 2010, he has been a postdoctoral research associate at the University of North Carolina. Working with Professor Joseph DeSimone, he is currently developing siRNA delivery technology with PRINT® (Particle Replication in Non-wetting Templates) based nanoparticles. He will become a principle investigator at Fudan University in China.
This journal is © The Royal Society of Chemistry 2014
signicantly lowers its production cost. The high target specicity of RNAi and relatively low synthetic cost of siRNA give siRNA-based gene therapy great potential for a variety of applications.6 As a large and negatively charged biological molecule, naked siRNA is unstable in the blood stream, is unable to penetrate cell membranes, and can be immunogenic.7 A safe and efficient delivery method is crucial to realize the broad potential of siRNA-based therapeutics. Both viral and non-viral vectors can be used to deliver siRNA.8 Non-viral vectors, especially nanoparticles, are less expensive to produce and carry a lower risk of provoking an immune response compared to viral vectors. As a result, nanoparticles are of great interest to deliver siRNA.9–14 Nanoparticle mediated siRNA delivery is an intensively investigated research eld with approximately 1000 research papers published in the past three years alone. Currently, siRNA delivery, especially systemic delivery in vivo, remains a difficult task.15 The difficulty of siRNA delivery is rooted in several biological barriers that present challenges when siRNA is delivered via systemic administration. First, nanomaterials for siRNA delivery need to form a stable complex with the cargo to protect it from degradation during circulation in the blood stream. Next, siRNA loaded nanoparticles need to evade fast clearance from the blood and avoid an immune response, which generally is realized by the surface modication with poly(ethylene glycol) (PEG) to protect and stabilize nanoparticles. Furthermore, a sufficient amount of carriers needs to accumulate in the target tissue and be taken up by target cells. To achieve this, proper surface characteristics and targeting groups are essential to enable accumulation in target tissues and uptake by target cells. Since the RNAi machinery is
Nanoscale, 2014, 6, 6415–6425 | 6415
View Article Online
Published on 15 April 2014. Downloaded by Université Laval on 16/06/2014 12:47:56.
Nanoscale
housed in the cytoplasm, successful delivery of siRNA relies on the ability of nanoparticles to enter the cell, reach the cytoplasm, and then release the cargo. In most cases, nanoparticles are internalized through an endocytosis and endo-lysosomal pathway. Therefore, endosomal escape is of particular importance for the delivery of siRNA.8 Recent mechanistic investigations on nanoparticle intracellular trafficking indicate that insufficient endosomal escape could signicantly limit siRNA delivery efficiency.9,16 Entrapment in the hostile endo-lysosomal vesicles and degradation by lysosomal enzymes in an acidic environment could be a dead end for siRNA delivery. To achieve RNAi, siRNA containing nanoparticles need to escape from the endosome within a short period of time to avoid the fate of being degraded or recycled. In systemic delivery, a nanoparticle design must be able to achieve multiple functions of elongated blood circulation time, improved stability, and reduced toxicity in addition to enhanced endosomal escape.16–19 Therefore, designing multifunctional siRNA delivery systems with efficient endosomal escape is a great challenge. This feature article highlights the challenges of and solutions for endosomal escape in nanoparticle mediated siRNA delivery. The discussion focuses on recent progress regarding this topic. The goal is to show that enhanced endosomal escape can be achieved by chemical composition control, surface property modication, and other creative nanoparticle design approaches. The author hopes that this article will raise awareness of the importance of addressing endosomal escape when designing nanoparticles for siRNA delivery. Endosomal escape enhancing strategies are summarized in this article, which can hopefully assist fellow researchers to design their own siRNA delivery systems.
2. Endosomal escape: the critical challenge in siRNA delivery As mentioned above, a few biological barriers have to be overcome, when siRNA is delivered in vivo via systemic administration. Among them, endosomal escape is a key biological barrier in siRNA delivery. Nanoparticles are typically taken up via endocytosis. Depending on nanoparticle properties (size, shape, surface properties, etc.) and cell types, endocytosis of nanoparticles may occur via different pathways.20 Generally, endocytosis can be divided into two broad categories: phagocytosis and pinocytosis. While phagocytosis mostly occurs with specialized phagocytes, such as macrophages and dendritic cells, pinocytosis is present in all types of cells. Based on the proteins involved, pinocytosis occurs either via clathrin-mediated pathways or clathrin-independent pathways. Clathrin-independent pathways can be further divided into caveolae-mediated endocytosis, clathrin- and caveolae-independent pathways, and macropinocytosis. The endocytic entry pathways are summarized in Fig. 1.21 Another classication of endocytosis is based on material interaction with the cellular membrane (receptormediated, adsorptive, uid phase).
6416 | Nanoscale, 2014, 6, 6415–6425
Feature Article
Gateways for endocytic entry. “Other” pathways represent clathrin- and caveolae-independent pathways. Adapted with permission from ref. 21. Copyright American Chemical Society 2012.
Fig. 1
Endocytosis of nanoparticles is a complex process. For most nanoparticles, more than one pathway could be used to achieve cellular entry. Among these endocytosis pathways, clathrinmediated endocytosis is generally considered to be the most common route of cellular entry, which goes through the endolysosomal pathway. Some endocytosis pathways, such as some cases of caveolae-mediated endocytosis and macropinocytosis, may bypass lysosomes.20 In these cases, an active endosomal escape mechanism is unnecessary. Nevertheless, this feature article will focus on endosomal escape strategies for the more common route of endocytosis via the endo-lysosomal pathway. Facilitating endosomal escape has long been the focus of gene delivery research. In the “classical” endo-lysosomal pathway, nanoparticles start intracellular trafficking with early endosome vesicles, which become progressively acidic as they mature into late endosomes.22–24 By accumulating protons in the vesicle, the proton pump vacuolar ATPase generates acidication until the pH drops to pH 5–6. With the fusion of the late endosomes with the lysosomes (pH 4–5), the content would be degraded by enzymes if it does not escape the endosome. Cationic nanoparticles with a strong buffering capacity in the pH range from 5 to 7 have displayed the ability to escape the endosome potentially through the so-called “proton sponge” effect. To escape from the endosome in a timely fashion is essential to achieve efficient siRNA delivery. Two recent reports studying nanoparticle intracellular trafficking give more details about the endosomal escape mechanism, which indicate some “hidden” pathways that might compromise siRNA delivery efficiency.9,16 In the rst report, Gilleron et al. investigated the intracellular trafficking of siRNA containing lipid nanoparticles (LNPs), which were labelled by either uorescent dyes or gold nanoparticles.16 Quantitative uorescence imaging and electron microscopy were used to analyse nanoparticle trafficking. The LNPs were found to enter cells through both macropinocytosis and clathrin-mediated endocytosis. The key discovery was that escape of siRNA from endosomes into the cytosol occurs at low efficiency (1–2%) and only during a limited period of time when the LNPs reside in a specic compartment sharing early and late endosome characteristics. This discovery further stressed the importance of quick and efficient endosomal escape in order to realize high siRNA delivery efficiency.
This journal is © The Royal Society of Chemistry 2014
View Article Online
Published on 15 April 2014. Downloaded by Université Laval on 16/06/2014 12:47:56.
Feature Article
Nanoscale
Different types of nanoparticles used for gene delivery. Adapted with permission from ref. 27. Copyright Wiley-VCH 2008.
Fig. 3
Schematic illustration of LNP intracellular trafficking pathways summarized in Gilleron et al. and Sahay et al. reports. Adapted with permission from ref. 25. Copyright Nature Publishing Group 2013. Fig. 2
The second study was carried out by Sahay et al.9 Researchers used high-throughput confocal microscopy to screen a library of small-molecule inhibitors and identify critical signalling pathways that regulated the cellular uptake and intracellular trafcking of siRNA in HeLa cells. LNPs were also used in their investigation. Results showed that LNPs were internalized by macropinocytosis and trafficked directly into endosomes. Surprisingly, it was discovered that siRNA dissociated from the LNPs was exocytosed to the extracellular milieu. The amount of siRNA lost in this manner was calculated to be approximately 70% of the dose taken up by cells. This discovery indicates that nanoparticle endocytic recycling is limiting the efficiency of siRNA delivery. Intracellular trafficking pathways of both reports are summarized in Fig. 2.25 Although these two investigations were based on LNPs, the observations may also apply to other siRNA delivery platforms. As both reports show, it is essential to design siRNA loaded nanoparticles that are capable of escaping from the endosome efficiently. Otherwise, nanoparticles will either be degraded or recycled, which severely limits siRNA delivery efficiency.26
3. Endosomal escape enhancement for different nanoparticle types Various types of nanoparticles have been used for gene delivery. As depicted in Fig. 3, these nanoparticles include lipid-based nanoparticles, polymer-based nanoparticles, gold nanoparticles, mesoporous silica nanoparticles, carbon nanotubes, and
This journal is © The Royal Society of Chemistry 2014
nanoparticle assemblies.27 Depending on the type of nanoparticles, different strategies to enhance endosomal escape are used. The general method is to improve pH buffering capacity and increase the “proton sponge” effect. With this “proton sponge” mechanism, the buffering capacity prevents acidication of the endosomes by acting as “proton sponge”, which leads to an increase in the proton inux followed by an enhanced accumulation of counter anions and osmotic swelling. There had only been indirect evidence supporting this pH buffering mechanism, until a recent investigation reported direct visualization of this “proton sponge” mechanism with confocal microscopy.28 In this section, discussion focuses on recent progress in siRNA delivery with lipid nanoparticles, polyplex nanoparticles, polymer nanospheres, and inorganic nanoparticles. Cationic lipid-based nanoparticles are the most widely used non-viral gene delivery vectors.29 Currently, they are also the type of nanoparticles that holds the greatest promise to achieve clinical breakthroughs.30 Cationic lipids can self-assemble into nanoparticles, and encapsulate negatively charged siRNA. Further modication of these nanoparticles gives stabilizing and targeting capabilities when delivered in vivo via systemic administration.15,31–34 To design optimized delivery systems for siRNA therapeutics, Anderson and co-workers pioneered the use of robotic methods to systematically screen lipids.10,35,36 Cationic and pH sensitive lipids, which have a high pH buffering capacity, are used to enhance endosomal escape via the “proton sponge” effect. Systematic studies have been carried out to investigate how to use pH sensitive lipids to achieve the optimal endosomal escape effect.37,38 A combination of lipidbased nanoparticles with special endosomal escape strategies, such as calcium phosphate and cell penetrating peptides, has led to successful nanosystems for systemic delivery.39
Nanoscale, 2014, 6, 6415–6425 | 6417
View Article Online
Published on 15 April 2014. Downloaded by Université Laval on 16/06/2014 12:47:56.
Nanoscale
Polyplex nanoparticles are generally based on electrostatic complexation of cationic polymers and anionic nucleic acid.40–48 Cationic polymers, such as polyethyleneimine (PEI), poly-Llysine (PLL), chitosan, and synthetic dendrimers, are used to form polyplexes. The cationic characteristics of polymers enable the formation of stable complexes, which contributes to cationic nanoparticle mediated cell uptake and improves endosomal escape. One major advantage of polyplex nanoparticles is the innite number of polymers, which researchers can design and synthesize to incorporate multiple functions, such as protection with PEG and targeting toward certain cell types. The cationic feature of polymers contributes to endosomal escape typically by increasing the pH buffering capacity. More importantly, pH sensitive groups can be introduced into polymer structures for enhanced endosomal escape and reduced toxicity.49–54 In addition, it is possible to prepare hybrid materials of polymers and peptides to realize improved endosomal escape. Polymer nanospheres are another type of polymer based nanoparticles. Poly-L-lactic acid (PLA) and poly-lactic-co-glycolic acid (PLGA) can encapsulate siRNA via non-ionic interaction to form polymer nanospheres.55 Approved by the FDA for pharmaceutical use, PLA and PLGA are well known for their biocompatibility. As neutral nanoparticles without modication, PLA and PLGA based nanospheres need to be optimized to improve cellular uptake and endosomal escape by introducing a coating of cationic lipids or polymers.56,57 PLA or PLGA containing hybrid nanoparticles have been developed. Desai et al. developed a PLGA and cationic lipid hybrid nanoparticle to deliver siRNA and capsaicin via topical administration to inhibit skin inammation 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 calcium phosphate nanoparticles.39,75–77 Inorganic nanoparticles are generally highly stable in maintaining their size, shape and composition. Nevertheless, since inorganic nanoparticles lack charge, they are unable to electrostatically interact with either anionic nucleic acids or the negatively charged cell membrane. Successful transfection with inorganic nanoparticles requires surface coating or modication with cationic polymers to improve cellular uptake and endosomal escape. Cationic polymers, such as PEI, PLL or specically designed polymers, contribute to the improved cell uptake and endosomal escape capability. Among these inorganic nanoparticles, calcium phosphate nanoparticles have a unique advantage. With a tendency to quickly dissolve under acidic conditions like that inside endosomes and lysosomes, calcium phosphate nanoparticles can greatly enhance endosomal escape with minimum toxicity.78 In summary, different nanoparticles have their unique strategies to enhance endosomal escape. The common strategy is to take advantage of the acidic environment inside the endosome and enhance endosomal escape with the “proton sponge” effect to destabilize the endosome membrane.
6418 | Nanoscale, 2014, 6, 6415–6425
Feature Article
4. Endosomal escape enhancing methods Most endosomal escape strategies are based on the acidic endosome micro-environment and “proton sponge” effect, although external stimulations, such as photochemical internalization (PCI), are also used.79 In this section, several endosomal escape enhancing strategies will be introduced, which include the use of cationic polymers, pH sensitive polymers, calcium phosphate, and cell penetrating peptides. This section will also address how to design multifunctional systems to enhance endosomal escape and overcome other biological barriers in siRNA delivery. 4.1
Cationic polymers
Cationic polymers are used to complex with siRNA to form polyplexes or to coat inorganic and other nanoparticles. PEI, PLL and other polyammonium polymers are generally used based on the “proton sponge” effect. As an alternative to polyammonium polymers, Ornelas-Megiatto et al. reported the use of polyphosphonium polymers as an efficient and non-toxic transfection agent.80 In many cases, polymers are specically designed and synthesized to facilitate multiple functions including endosomal escape, protection and targeting. A cationic polymer coating is necessary for many inorganic nanoparticle based siRNA carriers to enhance endosomal escape and cell uptake. Xia et al. developed PEI coated mesoporous silica nanoparticles to deliver siRNA and DNA.71 There are several reports of PEI capped gold nanoparticles for siRNA delivery.59–61,72 Other than commercially available polyammonium polymers, Lee et al. synthesized and tested biodegradable poly(b-amino ester) type polymers to improve the transfection efficiency of gold nanoparticles.62 In their study, a library of polymers was screened to nd the best formulation to enhance gold nanoparticle transfection efficiency. Kozielski et al. developed a bioreducible poly(b-amino ester), which could deliver siRNA and release the cargo in an intracellular environment.81 In their design, the cleavable disulde bonds in the polymer structure ensured the degradation of the polymer and the release of cargo in the reducing intracellular environment. There are many reports of multi-functional cationic polymers. A triblock poly(amido amine)–poly(ethylene glycol)–polyL-lysine (PAMAM-PEG-PLL) nanocarrier was developed by Patil et al.82 This triblock copolymer had a combination of improved endosomal escape (PAMAM), protection (PEG) and siRNA condensation (PLL), which could be easily tuned to achieve the optimal transfection efficacy. Yu et al. reported an amphotericin B (AmB)-loaded, poly(2-(dimethylamino)ethyl methacrylate)block-poly(2-(diisopropylamino)ethyl methacrylate) (PDMA-bPDPA) micelleplex siRNA delivery system.83 PDMA-b-PDPA is a cationic polymer, which can complex with siRNA and improve its transfection capability. AmB is a hydrophobic antifungal drug, known to increase membrane permeability at sublethal concentrations by the formation of transmembrane pores. Their study conrmed that AmB was released from the micelleplexes in the early endosome to assist endosomal escape. In
This journal is © The Royal Society of Chemistry 2014
View Article Online
Published on 15 April 2014. Downloaded by Université Laval on 16/06/2014 12:47:56.
Feature Article
this dual pH-responsive system, the combination of membrane poration by AmB and endosome swelling by polycations contributed to efficient endosomal escape. Shrestha et al. designed and synthesized a polymer with primary amines and histamines, which can be assembled into cationic shell crosslinked knedel-like nanoparticles (cSCKs) (Scheme 1).50 Amphiphilic block copolymers were synthesized, followed by selective crosslinking throughout the hydrophilic shell layer to form cSCKs. To reduce toxicity, histamine groups were introduced into the structure of the copolymer. By increasing the ratio of histamines to primary amines, siRNAbinding affinity was decreased and the cytotoxicity was reduced. By controlling the ratio of primary amines and histamines, siRNA-binding affinity, cytotoxicity, immunogenicity, and transfection efficiency could be controlled. Endosomal escape was facilitated by having these two species of low and high pKas. With this tuning capability, while maintaining adequate endosomal escape, toxicity of these cationic nanoparticles was reduced and their biocompatibility was increased. Peptide/polymer hybrid materials, which combine the advantages of precise structural control of peptides and low synthetic cost of polymers, are interesting gene delivery vectors. Meyer et al. developed a polycation with a pH responsive peptide and PEG. While PEG was used to protect the cargo, the sequence of the peptide could be tuned to have the optimal transfection efficacy.84 Zeng et al. reported a multifunctional dendronized peptide polymer platform for siRNA delivery.85 The ratio of cationic modules, hydrophilic groups, and hydrophobic groups in this peptide/polymer hybrid material could be adjusted in order to achieve high siRNA delivery efficiency and excellent biocompatibility. In addition, the polymer was reducing environment degradable in its design. Polymer degradation in the intracellular environment not only triggered the release of cargo, but also further reduced the possible long-term toxicity. In addition, polymeric nanoparticles can be coated with cationic lipids to improve cellular uptake and endosomal escape. Yang et al. developed a polymeric nanoparticle assembled from mPEG-PLA, PLA, cationic lipid, and siRNA in a single step.34 The resulting hybrid nanoparticles exhibited excellent stability in serum and showed signicantly improved biocompatibility compared to that of pure cationic lipid nanoparticles. 4.2
pH sensitive polymers
While cationic polymers are capable of enhancing endosomal escape, they may lead to toxicity in the physiological
Nanoscale
environment. By using pH sensitive polymers, the nanoparticle toxicity can be reduced during circulation. Aerwards, pH sensitive polymers will be further protonated or degraded to expose the membrane disruptive inner core to assist endosomal escape. Polymers can be modied with pH sensitive groups. The ratio of pH sensitive groups to other functional groups can be adjusted and balanced to achieve the optimal overall transfection efficiency. While cationic polymers in the previous section were discussed as having certain pH sensitive characteristics, this section will give a detailed discussion of the application of pH sensitive polymers in siRNA delivery. Regular amine groups tend to change their protonation status as pH changes. Nevertheless, imidazole and other pH sensitive groups are more widely used. The use of imidazole and other pH-sensitive groups can decrease the amount of positive surface charge in the physiological environment to reduce toxicity and make nanoparticles “stealthy” during circulation. They can also give nanoparticles an enhanced pH buffering capacity. Davis was one of the earliest researchers to use imidazole modied polymers for siRNA delivery. He and co-workers reported an imidazole modied linear, cyclodextrin-containing polycation nanomaterial for gene delivery and established the endosomal escape enhancing effect of imidazole groups.86 They later applied this discovery to a nanoparticle design and succeeded in achieving the rst targeted delivery of siRNA in humans.3 Lin et al. introduced imidazole into a polymer-based liposomal dual-shell siRNA delivery carrier.52 Malamas et al. designed and evaluated an imidazole based pH-sensitive amphiphilic cationic lipid for siRNA delivery.37 Gu et al. developed an imidazole containing polymer nanocarrier system to achieve endosomal escape and timed release of siRNA.87 In their study, an inuenza virus-inspired block copolymer was prepared. The imidazole containing polymer chains were capable of fusing with the endosome membrane to assist the escape. In addition, polyhistidine peptides were used as a pH sensitive domain. Benns et al. used a pH sensitive poly(L-histidine)-gra-poly(L-lysine) comb shaped polymer as a gene delivery vector.88 In addition to cationic pH sensitive polymers, anionic pH sensitive polymers, especially propylacrylic acid containing copolymers, have been used to enhance endosomal escape. Propylacrylic acid is pH sensitive with a membrane disruptive capability under endosomal conditions. Convertine et al. developed a novel propylacrylic acid containing endosomolytic diblock copolymer for siRNA delivery.89 This copolymer was
Scheme 1 Self-assembly of polymers with various amounts of primary amines and histamines into micelles followed by selective crosslinking in the hydrophilic shell regions. Adapted with permission from ref. 50. Copyright Elsevier 2012.
This journal is © The Royal Society of Chemistry 2014
Nanoscale, 2014, 6, 6415–6425 | 6419
View Article Online
Published on 15 April 2014. Downloaded by Université Laval on 16/06/2014 12:47:56.
Nanoscale
composed of a positively-charged block of dimethylaminoethyl methacrylate (DMAEMA) to mediate siRNA complexation, and a second endosomal-releasing block composed of DMAEMA and propylacrylic acid (PAA). Nanoparticles assembled with this copolymer had low cytotoxicity and enhanced endosomal escape. To make nanoparticles even more “stealthy”, chargeconversion polymers were developed in one early report to assemble a ternary polyplex nanoparticle, which could dramatically change its surface charge in response to pH change.90 Guo et al. reported charge-conversion polymer coated gold nanoparticles to deliver siRNA.91,92 As depicted in Scheme 2, this gold nanoparticle based ternary complex was composed of siRNA, polycations, and a charge-conversion polymer. This complex had a negative x-potential in the physiological environment to avoid blood clearance and reduce toxicity. Once it entered the acidic endosome, pH change would trigger the charge conversion. This charge conversion resulted in a positive surface charge of nanoparticles, which assisted endosomal escape to transport siRNA into the cytoplasm. The overall effect of this design was to realize two functions: prolonged circulation time and enhanced endosomal escape. A supramolecular endosome destabilizing strategy was reported by Tamura et al.93 In their study, N,N-dimethylaminoethyl (DMAE) modied cyclodextrin polyrotaxanes were incorporated into the polyplex system. Polyrotaxanes were constructed with acid labile sulfanylpropionyl ester linkers. Aer entering the acidic endosome, sulfanylpropionyl ester linkers would be cleaved to remove stoppers on both ends. Therefore, DMAE modied cyclodextrin would be released, which destabilized the endosome membrane and resulted in enhanced endosomal escape. While similar design strategies have been applied to enable drug release from mesoporous silica nanoparticles, their study was the rst example of supramolecular endosomal escape enhancement. Plasmalogens, acid labile lipids, have been used to deliver gene therapeutics and other drugs.94 Plasmalogens contain acid labile vinyl ether. The degradation of vinyl ether and plasmalogen liposomes in an acidic environment would trigger the release of cargo and disruption of the membrane. Based on the same principle, acid labile “encrypted polymers” were developed to achieve reduced toxicity and improved endosomal escape. Murthy et al. developed pH sensitive polymeric carriers capable of delivering oligonucleotides.95 As shown in Fig. 4, the backbone of this polymer was covalently conjugated to a PEG
Feature Article
Fig. 4 Chemical structures of the “encrypted polymers”. The aciddegradable linker is a para-amino benzaldehyde-acetal. Adapted with permission from ref. 95. Copyright Elsevier 2003.
“mask” via an acetal linker. The toxicity of this carrier was reduced as a result of the PEG layer. Aer internalization, the PEG layer was removed as the acetal linker was cleaved inside the acidic endosome. The exposed hydrophobic, membrane disruptive backbone would assist endosomal escape to improve the efficiency of gene delivery. The combination of pH sensitive nanomaterials with other stimuli responsiveness could help achieve improved transfection. Enzymes are an important stimulus in addition to pH change. Matrix metalloproteinase (MMP), an enzyme overexpressed by some cell types including certain types of cancer cells, was used for this purpose. Li et al. developed a pH responsive, smart polymeric siRNA delivery system with an MMP dependent proximity-activated targeting system.96 This delivery system had a PEG corona during circulation. Once reaching MMP rich target cells, cancer cells in their study, the PEG layer would be removed to expose the dimethylaminoethyl methacrylate containing cationic layer, which leads to cell uptake. Membrane disruptive propylacrylic acid in the inner core of pH sensitive polymers was designed to enhance endosomal escape. This design also realized the targeting effect toward cancer tissues, since nanoparticles would only be taken up by cancer cells, where high MMP concentrations would ensure the removal of PEG corona. 4.3
Scheme 2 Enhanced intracellular payload release inside endosomes by using pH dependent charge-conversion polymers on nanoparticles. Adapted with permission from ref. 91. Copyright American Chemical Society 2010.
6420 | Nanoscale, 2014, 6, 6415–6425
Calcium phosphate
Inorganic nanomaterials that quickly dissolve in acidic environments are of great interest to enhance endosomal escape. Being non-toxic and able to form nanoparticles, calcium phosphate (CaP) is the most important pH sensitive inorganic nanomaterial.76,97 A series of CaP nanoparticle transfection systems have been developed. The most successful is the lipid coated CaP nanoparticle (Liposome/Calcium/Phosphate or LCP).39,77 With a reverse water-in-oil micro-emulsion method, LCPs were prepared with calcium phosphate as the inner core and lipid DOTAP as the outer layer (Fig. 5). The inner core of
This journal is © The Royal Society of Chemistry 2014
View Article Online
Feature Article
Nanoscale
demonstrated the use of two or more endosomal escape enhancing strategies to improve siRNA transfection efficacy.
Published on 15 April 2014. Downloaded by Université Laval on 16/06/2014 12:47:56.
4.4
Fig. 5 The formation process of liposome/calcium/phosphate (LCP) nanoparticles. Adapted with permission from ref. 77. Copyright Elsevier 2010.
CaP plays two roles: (1) to form solid inorganic nanoparticles and (2) to assist endosomal escape. Inside the endosome, CaP would dissolve to disassemble the nanoparticles. This dissolving process would also increase the osmotic pressure and cause endosome swelling. Their design combined the advantages of lipid nanoparticles for a prolonged circulation time and CaP nanoparticles for an enhanced endosomal escape. LCP carriers were able to efficiently deliver siRNA to a xenogra tumor model by intravenous administration. LCP achieved 70% and 50% of luciferase silencing for the tumor cells in culture and those grown in a xenogra model, respectively. LCP achieved signicantly higher gene silencing than LPD (lipid nanoparticles without a CaP core), which proved the important role of CaP for an enhanced endosomal escape. Later an asymmetric lipid bilayer coated CaP nanoparticle system was developed with a small size of 25–30 nm. This new design had an improved transfection effect both in vitro and in vivo. Pittella et al. combined calcium phosphate nanoparticles and pH conversion polymers to enhance endosomal escape and reduce positive surface charge.98,99 They successfully synthesized sub-100 nm CaP nanoparticles, which could incorporate siRNA and a PEG modied anionic polymer. Inside the acidic endosome, the anionic polymer would be converted to a cationic polymer to destabilize the endosome membrane. Meanwhile, CaP would dissolve to generate the “proton sponge” effect to further enhance endosomal escape. Their study
This journal is © The Royal Society of Chemistry 2014
Cell penetrating peptides
Based on the outstanding transfection efficiency of viral vectors, cell penetrating peptides are designed and incorporated into non-viral vectors. Peptides are highly tunable in structure with 20 natural amino acids to choose from and an innite number of sequences. Many cell penetrating peptides are derived from bacterial or viral proteins, such as HIV-Tat based fusogenic peptides. Typical physicochemical features of cell penetrating peptides include a high positive charge (a large number of arginines or lysines) and amphiphilicity (capability to strongly interact with a lipid membrane). As mentioned above, nanoparticles can be taken up via more than one pathway. The precise internalization mechanism of cell penetrating peptides remains controversial. Generally, it is believed that cell penetrating peptides are internalized either by fusing with lipid cell membranes following a vacuole-based endocytosis or creating pores on the cell membrane.100,101 Akita et al. developed lipid enveloped-type nanoparticles modied with cell penetrating peptides, which could deliver siRNA to dendritic cells.102 Work has been done to optimize peptide sequences with the highest cell penetrating efficiency. Research by van Asbeck et al. evaluated molecular parameters of siRNA-cell penetrating peptide nanocomplexes for efficient cellular delivery.103 They discovered that the most active cell penetrating peptide displayed high serum resistance but also high sensitivity to decomplexation by polyanionic macromolecules. Karagiannis et al. used siRNA loaded lipid-like nanoparticles decorated with different cell penetrating peptides and evaluated both in vitro and in vivo efficacy.104 They could correlate the transfection efficiency with the physical and chemical properties of peptides. Similar work was done by Asai et al. to develop cell penetrating peptide conjugated lipid nanoparticles for siRNA delivery.105 On the other hand, Ren et al. focused on tumor cell targeting and internalization with cell penetrating peptides.106 They designed and screened a library of tandem tumor-targeting and cell-penetrating peptides, which were capable of condensing siRNA into stable nanocomplexes (Fig. 6). Through physicochemical and biological characterization, they identied a group of nanocomplexes that were capable of cell type specic siRNA delivery. These nanocomplexes were taken up by cells via endocytosis. Cell
Schematic representation of the tumor penetrating nanocomplex, with siRNA (blue), a cyclic tumor-penetrating domain (LyP-, green) and various cell-penetrating peptide domains (purple). Adapted with permission from ref. 106. Copyright American Chemical Society 2012.
Fig. 6
Nanoscale, 2014, 6, 6415–6425 | 6421
View Article Online
Nanoscale
penetrating peptides triggered endosomal escape, followed by the release of cargo, which resulted in gene silencing in a receptor-specic fashion. It is difficult to deliver siRNA to tumor cells in a target specic manner. This strategy has great potential for siRNA delivery to cancer cells.
Published on 15 April 2014. Downloaded by Université Laval on 16/06/2014 12:47:56.
4.5 Enhancing endosomal escape and overcoming other biological barriers Systemic siRNA delivery is a challenging task, which requires a sophisticated design to overcome multiple biological barriers. Endosomal escape is only one of the biological barriers researchers have to overcome while designing nanocarriers. In the previous sections, several examples of multi-functional nanomaterials were discussed. Nevertheless, it would be interesting and intriguing to design a nanosystem, which can be precisely tuned to overcome multiple biological barriers in siRNA delivery. To achieve this, Nelson et al. developed a copolymer as a model system to study how to enhance the efficiency of systemic siRNA delivery by incorporating multiple functions.17 They designed a PEG-(DMAEMA-co-BMA) diblock polymer. By varying the relative ratio of DMAEMA and BMA (butyl methacrylate), they formulated this polymer for optimal siRNA delivery. The cationic and hydrophobic contents were balanced to enhance siRNA packaging and endosomal escape. The PEG corona and optimized hydrophobic content increased the nanoparticle stability in the presence of human serum. Compared to the standard polymer, the polymer with an optimized hydrophobic content 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 the liver and other organs and signicantly improved gene silencing in vivo. Their study is of great signicance as a good example of enhancing multiple functions by tuning the chemical composition.
5.
Conclusion and perspective
This feature article discusses recent advances in endosomal escape enhancement strategies for nanoparticle mediated siRNA delivery. Mechanistic studies of nanoparticle intracellular trafficking indicate the importance of efficient endosomal escape. Based on different nanoparticle types, different endosomal escape strategies are deployed to tackle the challenge. For siRNA delivery, it is oen a difficult task to translate a nanoparticle design that works in vitro into animal studies due to multiple biological barriers. The solution is to design nanoparticles with multiple functions to overcome multiple biological barriers. For a rational multi-functional nanoparticle design, two major challenges exist. The rst challenge is how to manage conicting functions. For example, elevated circulation time and enhanced endosomal escape have opposite requirements for nanoparticle characteristics. Examples to solve this challenge include the use of charge-conversion polymers and enzyme proximity activation. The second challenge is how to have a simple and general design. Complex delivery systems
6422 | Nanoscale, 2014, 6, 6415–6425
Feature Article
designed to achieve multiple functions could lead to poor reproducibility and high production costs. Simple and costeffective nanoparticle systems should be creatively designed. One good example is the liposome/calcium phosphate system, which was inexpensive to prepare and achieved success both in vitro and in vivo. Endosomal escape, a critical biological barrier in siRNA delivery, should be overcome with the criteria mentioned above. When designing nanoparticles for siRNA delivery, researchers should constantly keep in mind the necessity of maintaining a high endosomal escape efficiency. It is also important for future endosomal escape enhancement design strategies to be considered along with other aspects of siRNA delivery. Chemical tuning capability is highly desirable to nd the optimal formulation to realize the highest efficacy and the lowest toxicity. As the endeavour to achieve safe and efficient siRNA delivery continues, we expect to see more innovative designs and the ultimate clinical success of siRNA-based gene therapy.
Acknowledgements The author wants to thank Professor Joseph DeSimone for his guidance and support, and Crista Farrell for proofreading.
Notes and references 1 A. Fire, S. Xu, M. K. Montgomery, S. A. Kostas, S. E. Driver and C. C. Mello, Nature, 1998, 391, 806–811. 2 F. Leuschner, P. Dutta, R. Gorbatov, T. I. Novobrantseva, J. S. Donahoe, G. Courties, K. M. Lee, J. I. Kim, J. F. Markmann, B. Marinelli, P. Panizzi, W. W. Lee, Y. Iwamoto, S. Milstein, H. Epstein-Barash, W. Cantley, J. Wong, V. Cortez-Retamozo, A. Newton, K. Love, P. Libby, M. J. Pittet, F. K. Swirski, V. Koteliansky, R. Langer, R. Weissleder, D. G. Anderson and M. Nahrendorf, Nat. Biotechnol., 2011, 29, 1005–1010. 3 M. E. Davis, J. E. Zuckerman, C. H. J. Choi, D. Seligson, A. Tolcher, C. A. Alabi, Y. Yen, J. D. Heidel and A. Ribas, Nature, 2010, 464, 1067–1070. 4 S. M. Elbashir, J. Harborth, W. Lendeckel, A. Yalcin, K. Weber and T. Tuschl, Nature, 2001, 411, 494–498. 5 R. Kanasty, J. R. Dorkin, A. Vegas and D. Anderson, Nat. Mater., 2013, 12, 967–977. 6 J.-P. Behr, Acc. Chem. Res., 2012, 45, 980–984. 7 K. A. Whitehead, R. Langer and D. G. Anderson, Nat. Rev. Drug Discovery, 2009, 8, 129–138. 8 E. Wagner, Acc. Chem. Res., 2012, 45, 1005–1013. 9 G. Sahay, W. Querbes, C. Alabi, A. Eltoukhy, S. Sarkar, C. Zurenko, E. Karagiannis, K. Love, D. Chen, R. Zoncu, Y. Buganim, A. Schroeder, R. Langer and D. G. Anderson, Nat. Biotechnol., 2013, 31, 653–658. 10 C. A. Alabi, K. T. Love, G. Sahay, H. Yin, K. M. Luly, R. Langer and D. G. Anderson, Proc. Natl. Acad. Sci. U. S. A., 2013, 110, 12881–12886. 11 S. J. Tan, P. Kiatwuthinon, Y. H. Roh, J. S. Kahn and D. Luo, Small, 2011, 7, 841–856.
This journal is © The Royal Society of Chemistry 2014
View Article Online
Published on 15 April 2014. Downloaded by Université Laval on 16/06/2014 12:47:56.
Feature Article
12 H. M. Aliabadi, B. Landry, C. Sun, T. Tang and H. Uluda˘ g, Biomaterials, 2012, 33, 2546–2569. 13 P. Kesharwani, V. Gajbhiye and N. K. Jain, Biomaterials, 2012, 33, 7138–7150. 14 H. Tian, J. Chen and X. Chen, Small, 2013, 9, 2034–2044. 15 K. Buyens, S. C. De Smedt, K. Braeckmans, J. Demeester, L. Peeters, L. A. van Grunsven, X. de Mollerat du Jeu, R. Sawant, V. Torchilin, K. Farkasova, M. Ogris and N. N. Sanders, J. Controlled Release, 2012, 158, 362–370. 16 J. Gilleron, W. Querbes, A. Zeigerer, A. Borodovsky, G. Marsico, U. Schubert, K. Manygoats, S. Seifert, C. Andree, M. St¨ oter, H. Epstein-Barash, L. Zhang, V. Koteliansky, K. Fitzgerald, E. Fava, M. Bickle, Y. Kalaidzidis, A. Akinc, M. Maier and M. Zerial, Nat. Biotechnol., 2013, 31, 638–646. 17 C. E. Nelson, J. R. Kintzing, A. Hanna, J. M. Shannon, M. K. Gupta and C. L. Duvall, ACS Nano, 2013, 7, 8870–8880. 18 Y. J. Kwon, Acc. Chem. Res., 2012, 45, 1077–1088. 19 M. Dominska and D. M. Dykxhoorn, J. Cell Sci., 2010, 123, 1183–1189. 20 G. Sahay, D. Y. Alakhova and A. V. Kabanov, J. Controlled Release, 2010, 145, 182–195. 21 R. Duncan and S. C. W. Richardson, Mol. Pharm., 2012, 9, 2380–2402. 22 J. Nguyen and F. C. Szoka, Acc. Chem. Res., 2012, 45, 1153– 1162. 23 J. Haensler and F. C. Szoka, Bioconjugate Chem., 1993, 4, 372–379. 24 N. D. Sonawane, F. C. Szoka and A. S. Verkman, J. Biol. Chem., 2003, 278, 44826–44831. 25 Y. Wang and L. Huang, Nat. Biotechnol., 2013, 31, 611– 612. 26 There is a recent report of microRNA/siRNA loaded PLGA nanoparticles that are capable of staying inside endosome for a long period of time without being degraded or exocytosed: J. Devalliere, W. G. Chang, J. W. Andrejecsk, P. Abrahimi, C. J. Cheng, D. Jane-Wit, W. M. Saltzman and J. S. Pober, FASEB J., 2014, 28, 908–922. 27 V. Sokolova and M. Epple, Angew. Chem., Int. Ed., 2008, 47, 1382–1395. 28 Z. ur Rehman, D. Hoekstra and I. S. Zuhorn, ACS Nano, 2013, 7, 3767–3777. 29 W. Li and F. C. Szoka, Pharm. Res., 2007, 24, 438–449. 30 J. C. Burnett, J. J. Rossi and K. Tiemann, Biotechnol. J., 2011, 6, 1130–1146. 31 D. J. Gary, H. Lee, R. Sharma, J.-S. Lee, Y. Kim, Z. Y. Cui, D. Jia, V. D. Bowman, P. R. Chipman, L. Wan, Y. Zou, G. Mao, K. Park, B.-S. Herbert, S. F. Konieczny and Y.-Y. Won, ACS Nano, 2011, 5, 3493–3505. 32 T. Lobovkina, G. B. Jacobson, E. Gonzalez-Gonzalez, R. P. Hickerson, D. Leake, R. L. Kaspar, C. H. Contag and R. N. Zare, ACS Nano, 2011, 5, 9977–9983. 33 Z. U. Rehman, I. S. Zuhorn and D. Hoekstra, J. Controlled Release, 2013, 166, 46–56. 34 X.-Z. Yang, S. Dou, Y.-C. Wang, H.-Y. Long, M.-H. Xiong, C.-Q. Mao, Y.-D. Yao and J. Wang, ACS Nano, 2012, 6, 4955–4965.
This journal is © The Royal Society of Chemistry 2014
Nanoscale
35 E. D. Karagiannis, C. A. Alabi and D. G. Anderson, ACS Nano, 2012, 8484–8487. 36 Y. Zhang, L. Arrington, D. Boardman, J. Davis, Y. Xu, K. DiFelice, S. Stirdivant, W. Wang, B. Budzik, J. Bawiec, J. Deng, G. Beutner, D. Seifried, M. Stanton, M. Gindy and A. Leone, J. Controlled Release, 2014, 174, 7–14. 37 A. S. Malamas, M. Gujrati, C. M. Kummitha, R. Xu and Z.-R. Lu, J. Controlled Release, 2013, 171, 296–307. 38 Y. Sato, H. Hatakeyama, Y. Sakurai, M. Hyodo, H. Akita and H. Harashima, J. Controlled Release, 2012, 163, 267– 276. 39 J. Li, Y. Yang and L. Huang, J. Controlled Release, 2012, 158, 108–114. 40 M. Yan, M. Liang, J. Wen, Y. Liu, Y. Lu and I. S. Y. Chen, J. Am. Chem. Soc., 2012, 134, 13542–13545. 41 M. Zheng, G. M. Pavan, M. Neeb, A. K. Schaper, A. Danani, G. Klebe, O. M. Merkel and T. Kissel, ACS Nano, 2012, 6, 9447–9454. 42 H. Lim, J. Noh, Y. Kim, H. Kim, J. Kim, G. Khang and D. Lee, Biomacromolecules, 2013, 14, 240–247. 43 B. J. Hong, A. J. Chipre and S. T. Nguyen, J. Am. Chem. Soc., 2013, 135, 17655–17658. 44 Z. J. Deng, S. W. Morton, E. Ben-Akiva, E. C. Dreaden, K. E. Shopsowitz and P. T. Hammond, ACS Nano, 2013, 7, 9571–9584. 45 J. Kloeckner, S. Boeckle, D. Persson, W. Roedl, M. Ogris, K. Berg and E. Wagner, J. Controlled Release, 2006, 116, 115–122. 46 S. S. Dunn, S. Tian, S. Blake, J. Wang, A. L. Galloway, A. Murphy, P. D. Pohlhaus, J. P. Rolland, M. E. Napier and J. M. DeSimone, J. Am. Chem. Soc., 2012, 134, 7423– 7430. 47 P. Posocco, X. Liu, E. Laurini, D. Marson, C. Chen, C. Liu, M. Fermeglia, P. Rocchi, S. Pricl and L. Peng, Mol. Pharm., 2013, 10, 3262–3273. 48 M. L. Patil, M. Zhang, O. Taratula, O. B. Garbuzenko, H. He and T. Minko, Biomacromolecules, 2009, 10, 258–266. 49 K. Raemdonck, B. Naeye, K. Buyens, R. E. Vandenbroucke, A. Høgset, J. Demeester and S. C. De Smedt, Adv. Funct. Mater., 2009, 19, 1406–1415. 50 R. Shrestha, M. Elsabahy, S. Florez-Malaver, S. Samarajeewa and K. L. Wooley, Biomaterials, 2012, 33, 8557–8568. 51 C. Dohmen, D. Edinger, T. Fr¨ ohlich, L. Schreiner, U. L¨ achelt, C. Troiber, J. R¨ adler, P. Hadwiger, H.-P. Vornlocher and E. Wagner, ACS Nano, 2012, 6, 5198– 5208. 52 S.-Y. Lin, W.-Y. Zhao, H.-C. Tsai, W.-H. Hsu, C.-L. Lo and G.-H. Hsiue, Biomacromolecules, 2012, 13, 664–675. 53 X. Yang, J. Du, S. Dou, C. Mao, H. Long and J. Wang, ACS Nano, 2012, 771–781. 54 T. Kim, T. Rothmund, T. Kissel and S. W. Kim, J. Controlled Release, 2011, 152, 110–119. 55 A. Alshamsan, A. Haddadi, S. Hamdy, J. Samuel, A. O. S. El-kadi and H. Uludag, Mol. Pharm., 2010, 7, 1643–1654. 56 S. H. Lee, H. Mok, Y. Lee and T. G. Park, J. Controlled Release, 2011, 152, 152–158.
Nanoscale, 2014, 6, 6415–6425 | 6423
View Article Online
Published on 15 April 2014. Downloaded by Université Laval on 16/06/2014 12:47:56.
Nanoscale
57 W. Hasan, K. Chu, A. Gullapalli, S. S. Dunn, E. M. Enlow, J. C. Lu, S. Tian, M. E. Napier, P. D. Pohlhaus, J. P. Rolland and J. M. DeSimone, Nano Lett., 2012, 287–292. 58 P. R. Desai, S. Marepally, A. R. Patel, C. Voshavar, A. Chaudhuri and M. Singh, J. Controlled Release, 2013, 170, 51–63. 59 S. T. Kim, A. Chompoosor, Y.-C. Yeh, S. S. Agasti, D. J. Solell and V. M. Rotello, Small, 2012, 8, 3253–3256. 60 M.-Y. Lee, S.-J. Park, K. Park, K. S. Kim, H. Lee and S. K. Hahn, ACS Nano, 2011, 5, 6138–6147. 61 W.-J. Song, J.-Z. Du, T.-M. Sun, P.-Z. Zhang and J. Wang, Small, 2010, 6, 239–246. 62 J. Lee, J. J. Green, K. T. Love, J. Sunshine, R. Langer and D. G. Anderson, Nano Lett., 2009, 2402–2406. 63 K. S. Siu, D. Chen, X. Zheng, X. Zhang, N. Johnston, Y. Liu, K. Yuan, J. Koropatnick, E. R. Gillies and W.-P. Min, Biomaterials, 2014, 35, 3435–3442. 64 X. Jiang, G. Wang, R. Liu, Y. Wang, Y. Wang, X. Qiu and X. Gao, Nanoscale, 2013, 5, 7256–7264. 65 S. Foillard, G. Zuber and E. Doris, Nanoscale, 2011, 3, 1461– 1464. 66 P. Singh, C. Samor`ı, F. M. Toma, C. Bussy, A. Nunes, K. T. Al-Jamal, C. M´ enard-Moyon, M. Prato, K. Kostarelos and A. Bianco, J. Mater. Chem., 2011, 21, 4850–4700. 67 N. W. S. Kam, Z. Liu and H. Dai, J. Am. Chem. Soc., 2005, 127, 12492–12493. 68 J. Shen, R. Xu, J. Mai, H.-C. Kim, X. Guo, G. Qin, Y. Yang, J. Wolfram, C. Mu, X. Xia, J. Gu, X. Liu, Z.-W. Mao, M. Ferrari and H. Shen, ACS Nano, 2013, 9867–9880. 69 D. Lin, Q. Cheng, Q. Jiang, Y. Huang, Z. Yang, S. Han, Y. Zhao, S. Guo, Z. Liang and A. Dong, Nanoscale, 2013, 5, 4291–4301. 70 S. B. Hartono, W. Gu, F. Kleitz, J. Liu, L. He, A. P. J. Middelberg, C. Yu, G. Q. M. Lu and S. Z. Qiao, ACS Nano, 2012, 6, 2104–2117. 71 T. Xia, M. Kovochich, M. Liong, H. Meng, S. Kabehie, S. George, J. I. Zink and A. E. Nel, ACS Nano, 2009, 3, 3273–3286. 72 S. Jiang, A. A. Eltoukhy, K. T. Love, R. Langer and D. G. Anderson, Nano Lett., 2013, 13, 1059–1064. 73 L. Qi and X. Gao, ACS Nano, 2008, 2, 1403–1410. 74 M. V Yezhelyev, L. Qi, R. M. O'Regan, S. Nie and X. Gao, J. Am. Chem. Soc., 2008, 130, 9006–9012. 75 T. Devarasu, R. Saad, A. Ouadi, B. Frisch, E. Robinet, P. Laquerri` ere, J.-C. Voegel, T. Baumert, J. Ogier and F. Meyer, J. Mater. Chem. B, 2013, 1, 4692–4700. 76 M. Zhang, A. Ishii, N. Nishiyama, S. Matsumoto, T. Ishii, Y. Yamasaki and K. Kataoka, Adv. Mater., 2009, 21, 3520– 3525. 77 J. Li, Y.-C. Chen, Y.-C. Tseng, S. Mozumdar and L. Huang, J. Controlled Release, 2010, 142, 416–421. 78 S. Bisht, G. Bhakta, S. Mitra and A. Maitra, Int. J. Pharm., 2005, 288, 157–168. 79 Y. Matsushita-Ishiodori and T. Ohtsuki, Acc. Chem. Res., 2012, 45, 1039–1047. 80 C. Ornelas-Megiatto, P. R. Wich and J. M. J. Fr´ echet, J. Am. Chem. Soc., 2012, 134, 1902–1905.
6424 | Nanoscale, 2014, 6, 6415–6425
Feature Article
81 K. L. Kozielski, S. Y. Tzeng and J. J. Green, Chem. Commun., 2013, 49, 5319–5321. 82 M. L. Patil, M. Zhang and T. Minko, ACS Nano, 2011, 5, 1877–1887. 83 H. Yu, Y. Zou, Y. Wang, X. Huang, G. Huang and B. D. Sumer, ACS Nano, 2011, 9246–9255. 84 M. Meyer, A. Philipp, R. Oskuee, C. Schmidt and E. Wagner, J. Am. Chem. Soc., 2008, 130, 3272–3273. 85 H. Zeng, H. C. Little, T. N. Tiambeng, G. A. Williams and Z. Guan, J. Am. Chem. Soc., 2013, 4962–4965. 86 S. Mishra, J. D. Heidel, P. Webster and M. E. Davis, J. Controlled Release, 2006, 116, 179–191. 87 W. Gu, Z. Jia, N. P. Truong, I. Prasadam, Y. Xiao and M. J. Monteiro, Biomacromolecules, 2013, 14, 3386–3389. 88 J. M. Benns, J. S. Choi, R. I. Mahato, J. S. Park and S. W. Kim, Bioconjugate Chem., 2000, 11, 637–645. 89 A. J. Convertine, D. S. W. Benoit, C. L. Duvall, A. S. Hoffman and P. S. Stayton, J. Controlled Release, 2009, 133, 221–229. 90 Y. Lee, K. Miyata, M. Oba, T. Ishii, S. Fukushima, M. Han, H. Koyama, N. Nishiyama and K. Kataoka, Angew. Chem., Int. Ed., 2008, 47, 5163–5166. 91 S. Guo, Y. Huang, Q. Jiang, Y. Sun, L. Deng, Z. Liang, Q. Du, J. Xing, Y. Zhao, P. C. Wang, A. Dong and X. Liang, ACS Nano, 2010, 4, 5505–5511. 92 L. Han, J. Zhao, X. Zhang, W. Cao, X. Hu, G. Zou and X. Duan, ACS Nano, 2012, 7340–7351. 93 A. Tamura and N. Yui, J. Mater. Chem. B, 2013, 1, 3535. 94 J. A. Boomer and D. H. Thompson, Chem. Phys. Lipids, 1999, 99, 145–153. 95 N. Murthy, J. Campbell, N. Fausto, A. S. Hoffman and P. S. Stayton, J. Controlled Release, 2003, 89, 365–374. 96 H. Li, S. S. Yu, M. Miteva, C. E. Nelson, T. Werfel, T. D. Giorgio and C. L. Duvall, Adv. Funct. Mater., 2013, 23, 3040–3052. 97 V. Sokolova, A. Kovtun, O. Prymak, W. Meyer-Zaika, E. A. Kubareva, E. A. Romanova, T. S. Oretskaya, R. Heumann and M. Epple, J. Mater. Chem., 2007, 17, 721–727. 98 F. Pittella, K. Miyata, Y. Maeda, T. Suma, S. Watanabe, Q. Chen, R. J. Christie, K. Osada, N. Nishiyama and K. Kataoka, J. Controlled Release, 2012, 161, 868–874. 99 F. Pittella, M. Zhang, Y. Lee, H. J. Kim, T. Tockary, K. Osada, T. Ishii, K. Miyata, N. Nishiyama and K. Kataoka, Biomaterials, 2011, 32, 3106–3114. 100 L. Crombez, A. Charnet, M. C. Morris, G. Aldrian-Herrada, F. Heitz and G. Divita, Biochem. Soc. Trans., 2007, 35, 44– 46. 101 I. Nakase, H. Akita, K. Kogure, A. Gr¨ aslund, U. Langel, H. Harashima and S. Futaki, Acc. Chem. Res., 2012, 45, 1132–1139. 102 H. Akita, K. Kogure, R. Moriguchi, Y. Nakamura, T. Higashi, T. Nakamura, S. Serada, M. Fujimoto, T. Naka, S. Futaki and H. Harashima, J. Controlled Release, 2010, 143, 311– 317. 103 A. H. van Asbeck, A. Beyerle, H. McNeill, P. H. M. BoveeGeurts, S. Lindberg, W. P. R. Verdurmen, M. H¨ allbrink,
This journal is © The Royal Society of Chemistry 2014
View Article Online
Feature Article
105 T. Asai, T. Tsuzuku, S. Takahashi, A. Okamoto, T. Dewa, M. Nango, K. Hyodo, H. Ishihara, H. Kikuchi and N. Oku, Biochem. Biophys. Res. Commun., 2014, 444, 599–604. 106 Y. Ren, S. Hauert, J. H. Lo and S. N. Bhatia, ACS Nano, 2012, 8620–8631.
Published on 15 April 2014. Downloaded by Université Laval on 16/06/2014 12:47:56.
U. Langel, O. Heidenreich and R. Brock, ACS Nano, 2013, 7, 3797–3807. 104 E. D. Karagiannis, A. M. Urbanska, G. Sahay, J. M. Pelet, S. Jhunjhunwala, R. Langer and D. G. Anderson, ACS Nano, 2013, 7, 8616–8626.
Nanoscale
This journal is © The Royal Society of Chemistry 2014
Nanoscale, 2014, 6, 6415–6425 | 6425
