# Nanoparticles Escaping RES and Endosome: Challenges for siRNA Delivery for Cancer Therapy

Authors: Shutao Guo; Leaf  Huang
Journal: Journal of Nanomaterials
 (2011)

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## Abstract

Small interfering RNAs (siRNAs) technology has emerged as a promising potential treatment for viral, genetic diseases and cancers. Despite the powerful therapeutic potential of siRNA, there are challenges for developing efficient and specific delivery systems for systemic administration. There are extracellular and intracellular barriers for nanoparticle-mediated delivery. First, nanoparticles are rapidly cleared from the circulation by the reticuloendothelial system (RES). Second, following their cellular uptake, nanoparticles are trapped in endosomes/lysosomes, where siRNA would be degraded by enzymes. In this review, we describe strategies for grafting a polyethylene glycol (PEG) brush to the nanoparticles for evading RES, such that they may effectively accumulate in the tumor by the enhanced permeability and retention (EPR) effect. PEG has to shed from the nanoparticles to allow close interaction with the tumor cells. Current strategies for facilitating endosome escape, such as ion pair formation, “proton sponge effect”, destabilizing endosome membrane, and hydrophobic modification of the vector, are discussed.


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## Body

## 1. Introduction

RNA interference was firstly discovered by Fire et al. in 1998 [1], and this technology has emerged as a powerful tool for analyzing gene function and inhibiting gene expression in cell culture and in animal models. Small interfering RNA (siRNA), resulting from the cleavage of longer double stranded RNA precursors by endonuclease dicer, could enter the RNA-induced silencing complex, where the complementary mRNA is degraded and as a result the expression of the corresponding protein is reduced [2, 3]. However, systemic delivery of naked siRNA is limited by rapid blood clearance, RNA degradation, and poor cellular penetration due to the large molecular weight and negative charge of siRNA.The success in the application of siRNA for cancer therapy is highly dependent on the development of vectors which are nontoxic and can selectively and efficiently deliver siRNA into the specific tissue in vivo [4, 5]. Gene therapy vectors can be generally divided into two categories: viral vectors and nonviral vectors. Viral vectors are highly efficient, but the drawbacks of high cost, safety concerns and immunity significantly limit their application. To over the limitations of viral vectors, nonviral vectors have been widely developed as alternatives. The majority of nonviral vectors are based on synthetic polymers and lipids. To date, these synthetic vectors are still relatively less efficient than viral vectors. It is a result of many barriers, extracellular as well as intracellular, encountered between the site of administration and the nucleus or cytoplasm of the target cells for DNA or siRNA delivery [5–8]. Extracellular barriers include condensing nucleic acid into stable complexes which would not be deassembled in the solution and blood, maintaining stability and circulation of nanoparticles in the blood stream, penetrating the tissue, and specific binding to the target cells of interest. Following internalization, gene delivery vectors are challenged by intracellular barriers, including endosome entrapment and nucleic acid unpacking from vectors. Among these barriers, escaping the reticuloendothelial system (RES) uptake and endosome will be focused in this review.


## 2. Strategies for Nanoparticles Escaping RES Uptake

The applications of siRNA in vitro and in vivo are hampered by their high molecular weight, negative charge, low stability, and rapid blood clearance [9–11]. Approaches to overcome these drawbacks have relied on nonviral siRNA carriers based on cationic polymers or lipids. Cationic vectors suffer from fast blood clearance by the RES. Nanoparticle size and surface charge are the two major properties strongly influencing the clearance [12, 13]. Nanoparticle with the size of 100–200 nm would readily accumulate and retain in the tumor interstitium because of the enhanced permeability and retention (EPR) effect [14–17], which is further facilitated by the lack of a draining lymphatic system in tumor tissues. The success of stealth nanoparticles for tumor therapy is highly dependent on reduced RES uptake and prolonged circulation time in the blood.To prolong the circulation time, coating by polyethylene glycol (PEG), or PEGylation, is the most effective method to reduce protein adsorption in vivo and thereby helps to avoid the RES system [18–22]. Although there are successful attempts to develop alternative polymers to PEG, such as poloxamer [23, 24], polyvinyl alcohol [25, 26], poly(amino acid)s [27], and polysaccharide [28–31], PEG is still the most widely used material. PEG-lipid (such as PEG-DSPE) is usually inserted into liposomes to form a hydrated layer on the liposome surface. There are two kinds of conformation for PEG covering the surface of nanoparticles. For PEG-DSPE-stabilized liposomes, PEG is ready to take mushroom conformation at low degree of PEGylation and will shift to brush conformation as the content of PEG-DSPE is increased to certain levels. The brush mode is the ideal configuration for protecting nanoparticles from serum absorption. However, due to the detergent-like property of PEG-lipid, it is difficult to form stable liposomes with high content of PEG-lipid. To tackle this problem, Li and Huang [32, 33] developed PEGylated liposome-polycation-DNA (LPD) nanoparticles by inserting PEG-DSPE after formation of LPD nanoparticles. Negative and nanosized cores were formed by condensing DNA with protamine, which then bind cationic lipids to form DNA/protamine-encapsulated liposomes. The DNA/protamine cores played an important role to stabilize liposomes by electrostatic interaction. Therefore, LPD nanoparticles were still stable, even if 10.6% (by molar) of PEG-lipid was incorporated into the LPD nanoparticles [32, 33]. Stealth LPD nanoparticles were characterized by lower liver uptake through evasion of RES uptake and efficient delivery of siRNA to tumors [34, 35]. After 4 hours of i.v. injection, RES uptake of LPD nanoparticles was as low as 5–15% of the injected dose, and 30% of the dose accumulated in the tumor. It was hypothesized that the brushed PEG shed overtime, resulting in reducing the PEG content in the nanoparticles and further blood clearance [33, 36–38]. Pharmacokinetics (PK) data showing a rapid distribution phase and not much prolonged circulation time of the injected particles is consistent with the hypothesis. PEG shedding, which is not well understood and requires further study, is important for endosome escape and cargo discharge after the nanoparticle enters into the cell by endocytosis. This aspect will be discussed in Section 3.To overcome the PEG dilemma, several strategies are designed. The first strategy is to modify the nanoparticles with tumor-specific ligand to enhance intracellular uptake. Among the ligands, iron-saturated transferrin (Tf) has been widely investigated to target tumor cells overexpressing Tf-receptors [39–43]. Ogris et al. [40] demonstrated that Tf-bearing PEG-polyethyleneimine (PEI) could selectively deliver plasmid into tumor, leading to 100-fold higher gene expression in tumor cells than that of other tissues. Bartlett et al. [10] prepared siRNA-containing, Tf-targeted nanoparticles. Although Tf could not enhance the accumulation of nanoparticles in the tumor in mice, it facilitated cell uptake of nanoparticles, thereby improved gene knockdown efficiency. Very recently, they performed the first gene inhibition experiment by administration of Tf-targeted, siRNA-containing cyclodextrin containing polycation (CDP) nanoparticles in humans [42] and showed that siRNA successfully silenced the target gene in both mRNA and protein levels. In addition, several other ligands, such as RGD [44, 45], CNGRC [46], anisamide [35, 45, 47], and folate [48, 49], have also been applied to target specific tumor delivery in vivo.Ligand-mediated nanoparticles are usually internalized into tumor cells by receptor-mediated endocytosis and finally taken up into endosomal/lysosomal vesicles. Therefore, the second strategy is to incorporate sheddable PEG to facilitate drug escape from endosomal/lysosomal vesicles. Exploiting the microenvironment of tumor and acidic nature of endosome, PEG-lipids and polyplexes with pH-sensitive degradable spacers are used to prepare PEG sheddable liposomes and cationic polymer vectors. Usually, pH-sensitive degradable bonds are orthoester [50, 51], hydrazone [52, 53], vinyl ethers [54], or acetals [55], and this has been reviewed elsewhere [56]. Enzyme cleavable PEG has also been developed [57, 58]. In addition to use sheddable PEG, we will discuss other methods to facilitate disruption of the endosomal membrane in the next section.In addition to the PEG dilemma, another problem of PEGylation, that is, accelerated blood clearance (ABC), has been reported [59]. Although PEGylation could significantly prolong the circulation time of nanoparticles in the blood, repeated i.v. injections of the PEGylated nanoparticles resulted in the lost of the long-circulating characteristics [59–61]. The mechanism of ABC developed by Ishida et al. is that first injected dose of PEGylated liposomes activates the splenic synthesis of anti-PEG IgM, resulting in the opsonization of the second dose of PEGylated liposomes and uptake by the liver Kupffer cells [62–64].To overcome the ABC problem, alternative hydrophilic polymers (such as poly(N-vinyl-2-pyrrolidone) (PVP) and poly(hydroxyethyl-l-asparagine) (PHEA)) [65, 66] and cleavable PEG-lipid derivatives [67] are used. PVP-coated nanoparticles showed improved blood circulation and no ABC phenomenon [65]. For in vivo siRNA delivery, Kiwada group [68] demonstrated that conventional PEG-coated siRNA-lipoplex (PSCL) also caused anti-PEG IgM production, which is lower than that of PEG-coated naked cationic liposomes (PCL). A similar PEGylated lipid nanocarrier termed PEGylated wrapsome (PEG-WS) was further developed for siRNA delivery [69, 70]. Anti-PEG IgM production of the new PEG-WS formulation is less than that of PSCL and significantly dependant on the sequence of siRNA [70]. It shows that anti-PEG IgM production induced by potent immune stimulatory siRNA is much higher, and 2′-O-methyl (2′-OMe) uridine modification can significantly reduce anti-PEG IgM production by inhibiting cytokine induction.


## 3. Strategies for Promoting Nanoparticles Escape from Endosome

Having solved the problems with RES uptake, another challenge for siRNA delivery is to have the cargo escape from endosomes to reach cytoplasm. Here several strategies designed to enhance endosomal escape are described (Table1).Table 1
Examples of endosomal escape compounds for systemic siRNA delivery.
CategoryCompoundsMechanism of endosomal escapeCompatible with RES evasionReferencesPolymersPolyethylenimine PEIProton sponge effectUnlikely[71–73]Poly(amidoamine)s (PAAs)Proton sponge effectUnknown[74]Charge-reversal copolymersProton sponge effectUnknown[75–79]Poly(propylacrylic acid) (PPAA) and its derivativesDestabilizing membraneUnknown[80–87]Amphiphilic cationic copolymersHydrophobic interaction and Proton sponge effectLikely[88–99]pH-sensitive degradable polymersIncreasing endosomal osmotic pressureUnknown[100–103]LipidsCationic lipidsForming ion pairs with anionic phospholipids within the endosomal membraneYes[104–107]Zwitterionic lipidsForming ion pairs with anionic phospholipids within the endosomal membraneLikely[108]Lipid coated nanoparticlesLipid coated calcium phosphate (LCP)Forming ion pairs and increasing endosomal osmotic pressureYes[109]Liposome-polycation-DNA (LPD)Forming ion pairs and Proton sponge effectYes[32, 34, 35, 110, 111]Inorganic nanoparticlesLayered double hydroxide (LDH)Increasing endosomal osmotic pressureUnknown[112]Calcium phosphate (CaP)Increasing endosomal osmotic pressureUnlikely[113–116]Carbonate apatite (CAp)Increasing endosomal osmotic pressureUnlikely[117]Magnesium and manganous phosphate nanoparticlesIncreasing endosomal osmotic pressureUnlikely[118, 119]Calcium carbonateIncreasing endosomal osmotic pressureUnlikely[120]PeptidesKALADestabilizing membraneUnknown[121, 122]GALADestabilizing membraneUnknown[123–126]Hemagglutinin proteinDestabilizing membraneUnknown[127]


### 3.1. Ion-Pair Formation

The mechanism whereby cationic lipids destabilize endosome membrane to facilitate the endosomal escape of nucleic acid such as plasmid DNA or siRNA is originally proposed by Xu and Szoka [105]. Cationic lipids form ion pairs with anionic lipids within endosome membrane to destabilize the endosome membrane. Because the cross-sectional area of the combined headgroup in ion pair is less than that of the sum of individual headgroup areas in isolation, a “cylindrical” shape of individual charged lipids is transformed to a “cone” shape of ion pair which further promote the formation of the inverted hexagonal (HII) phase as proposed by Cullis et al. [128, 129].The exposure of the positive charge in liposomes to endosomal membranes is a prerequisite for electrostatic interaction between cationic lipids and anionic lipids. PEGylation of liposomes for systemic drug delivery therefore inhibits the formation of ion pairs. As mentioned in Section2, the shedding character of LPD nanoparticles is helpful to overcome the PEG dilemma [32–35]. After PEG comes off the LPD nanoparticles, cationic lipids are exposed to anionic lipids. In this novel formulation, the brushed but sheddable PEG not only grants LPD nanoparticles evasion of the RES for the initial period of time such that LPD nanoparticles can penetrate into the tumor by EPR effect but also facilitates the endosomal escape of the cargo siRNA. Due to successful RES evasion and endosomal escape, LPD nanoparticle is a very promising carrier for systemic delivery of siRNA.Obata et al. [108] synthesized zwitterionic lipids with amino acid-modified head group. The fusogenic potential of these liposomes with endosome-mimicking anionic membrane is pH-responsive, and it increases as pH decreases. The zeta potential of these liposomes in physiological conditions is negative and switches to positive as pH declines, which means that these lipoplexes may have prolonged circulation time in the blood. The properties of these liposomes provided great promise for drug delivery in vivo but no such report has been seen yet.Heyes et al. [130] synthesized 1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA) lipid with two double bonds per alkyl chain, which has strong fusogenicity and high gene transfection efficiency. Recently, Semple et al. [131] developed DLinDMA derivatives by optimizing the headgroup and linker moiety to formulate stable nucleic acid-lipid particles (SNALPs). They introduced an acid degradable ketalized linker. SNALP formulation containing DLin-KC2-DMA lipid presented excellent in vivo silencing activity in liver in rodents and nonhuman primates. This novel formulation could evade RES uptake and circulate in the blood for longer time due to its low pKa under neutral pH, because the amine head groups remain unprotonated and the zeta potential of nanoparticles was nearly neural or negative at pH 7.4. After the nanoparticles were internalized into intracellular endosomes, the amine groups became protonated to form cationic lipids, which was necessary to escape endosome by ion-pair mechanism. As known, it is difficult for liposome formulations to avoid drug leakage in the blood on one hand and achieve rapid drug release in target tissues on the other [132–137]. In this novel SNALP formulation, the hydrolysis of ketal bond might increase the content of neutral lipid in liposomes, which likely triggers siRNA release due to reorganization of the lipid domains [138, 139] and is key for its excellent performance at in vivo siRNA delivery. Therefore, this formulation may be advantageous to release cargo efficiently from both endosome and liposomes.


### 3.2. “Proton Sponge Effect”

Successful escape of siRNA carriers from endosome and release of the payload into cytoplasm is necessary to improve the efficiency of gene silencing. Due to the acid nature of endosomal/lysosomal vesicles, pH-buffering agents are widely exploited to promote cargo release. Under acidic condition, various macromolecules with low pKa amine group have been shown to exhibit “proton sponge effect”, such as PEI and its derivates [74, 94, 140, 141]. When the complexes formed by these compounds and nucleic acid are internalized into the cell, these compounds are capable of buffering the endosomal vesicle, leading to endosomal swelling and lysis, thus releasing the nucleic acids into the cytoplasm.Charge-reversal copolymers could shift their charge nature between positive and negative in a pH-dependent fashion [76–79, 142]. Charge conversion can occur in acidic intracellular organelles such as endosome or lysosome (pH = 5~6), and then these copolymers facilitate the endosomal escape of nucleic acids by enhancing the capacity of “proton sponge”. Pittella et al. [79] synthesized a hybrid nanocarrier system composed of calcium phosphate (CaP), a block copolymer PEG, and a charge-conversional polymer (CCP) to deliver siRNA. Confocal laser scanning microscopic observation confirmed that CCP was helpful for endosomal escape of siRNA with the nanoparticles. This hybrid nanocarrier system achieved significant knockdown of vascular endothelial growth factor (VEGF) in PanC-1 cells with low cytotoxicity. Guo et al. prepared charge-reversal functionalized gold nanoparticles (CRFGNs) with cis-aconitic anhydride-functionalized poly(allylamine) (PAH-Cit) [75]. The charge reversion of PAH-Cit was confirmed by polyacrylamide gel electrophoresis and confocal laser scanning microscopy. In vitro quantification of lamin A/C protein expression by western blot indicated that the knockdown efficiency of siRNA delivered by CRFGN was dose-dependent in Hela cell line. At 10:1 Au : siRNA ratio, siRNA delivered by CRFGN achieved the highest (80.0%) knockdown efficiency, which is better than Lipofectamine 2000 which inhibited about 66.0%. CRFGN achieved similar results in DNA transfection experiments.In fact, layered double hydroxide (LDH) [112], calcium phosphate [116, 124], and some other inorganic nanoparticles [118] also belong to materials which are able to deplete protons in acid environment. Because these nanoparticles could be degraded in acidic buffer, they are also able to release cargo from endosomal vesicles. For example, siRNA-loaded LDH nanoparticles [112] were dissolved due to the low pH in the endosome, which facilitated cargo escape from endosomes into the cytoplasm and significant downregulation of protein expression in HEK293T cells. Carbonate apatite nanoparticles [117] were developed to facilitate siRNA escape from the endosomes. It was remarkable that carbonate apatite nanoparticles could efficiently silence reporter genes at a low dose and were more efficient than Lipofectamine. Calcium phosphate has been widely used in biomaterials due to excellent biocompatibility. In vitro gene transfection efficiency of calcium phosphate nanoparticles with hydroxyapatite phase structure was found to be higher than that of the commercial transfecting reagent Polyfect [116]. Nevertheless, these particles are not stable and readily form large aggregates; therefore their in vivo applications are limited. To synthesize stable siRNA-loaded calcium phosphate, Kataoka et al. [113–115] utilized PEG-polycarboxylate block copolymer (such as PEG-b-poly(aspartic acid) and PEG-b-poly(methacrylic acid)) to achieve stable and size-controlled CaP nanoparticles, which showed good stability in serum and significant gene knockdown in vitro. Incorporation of PEG-SS-siRNA instead of PEG-polycarboxylate for stabilization achieved high PEG density in CaP nanoparticles, which could be potentially useful for the systemic delivery of siRNA. The PEG-stabilized CaP nanoparticles were observed to escape from endosome/lysosome exhibited RNAi efficacy for in vitro gene silencing. To improve the endosomal escape of CaP nanoparticles, a diblock copolymer composed of PEG and a charge-reversal polymer was incorporated [79] and resulting hybrid nanoparticles showed significant VEGF knockdown in PanC-1 cells because of the rapid endosomal escape. However, there is no in vivo study of PEG-stabilized CaP nanoparticles for siRNA delivery.Recently, Li et al. [109] prepared siRNA-encapsulated calcium phosphate by reverse water-in-oil microemulsion technique and then coated calcium phosphate nanoparticles with lipid to obtain a novel siRNA formulation of lipid-coated calcium phosphate (LCP) nanoparticle for siRNA systemic delivery. As mentioned before, calcium phosphate is acid-sensitive, and its degradation in endosome/lysosome increases osmotic pressure of endosome, leading to release of siRNA into the cytoplasm. In addition, lipid component is well known to be helpful for nanoparticles uptake and cargo release from endosome. Antiluciferse (luc) siRNA-loaded LCP nanoparticles showed significant gene silencing of luciferase both in vitro and in vivo with negligible immunotoxicity. The in vivo results suggested that LCP nanoparticles offer significant promise for siRNA delivery in clinical trails.


### 3.3. Membrane-Destabilizing Macromolecules

To date, there is no kind of synthetic vector which has comparable efficiency as viral vectors. Hemagglutinin protein, which is pH-sensitive and membrane-destabilizing, helps viral vectors to disrupt the endosome efficiently and enter the cytoplasm [127]. The escape mechanism of hemagglutinin and other fusion proteins is that they shift from an ionized and hydrophilic conformation to a hydrophobic and membrane-active conformation as the environment changes from neutral to acidic, resulting in destabilization of the endosomal membrane and its leakage subsequently. Therefore, incorporation of membrane-destabilizing peptides is another effective strategy to utilize the low pH environment of endosomes/lysosomes.To mimic the function of viral hemagglutinin protein, many peptides [123, 143–145] and polymers [80, 146–149] with similar properties were synthesized to enhance gene and siRNA delivery. Among the synthetic peptides, the GALA peptide  (WEAALAEALAEALAEHLAEALAEALEALAA) [123] was the most studied. Harashima group has developed a kind of multifunctional envelope type nanodevice (MEND) combining PEGylation, targeting ligand, and GALA. It was demonstrated that GALA facilitated endosomal release of siRNA, resulting in efficient knockdown [124–126]. In an in vivo gene silencing study, different MENDs with antiluciferase siRNA were directly injected into tumors of T1080-luc-tumor-bearing mice, and compared to unmodified MEND, GALA-modified MEND exhibited greater gene silencing in tumor tissues. On the other hand, poly(propylacrylic acid) (PPAA) was the most efficient and most studied membrane-destabilizing synthetic polymers, and its membrane-destabilizing capacity could be enhanced by copolymerization with hydrophobic monomers [80–84]. PPAA was demonstrated to enhance the transfection efficiency of cationic lipid/pDNA complexes both in vitro and in vivo [81, 85]. PPAA is negatively charged at physiological condition and therefore cannot condense siRNA directly. Stayton et al. synthesized a kind of diblock polymer composed of poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) to condense siRNA and a second endosomal-releasing block composed of DMAEMA and propylacrylic acid (PAA) in roughly equimolar ratios, together with butyl methacylate (BMA). These carriers became sharply lytic at endosomal pH range. In HeLa cells, a copolymer with the most hydrophobic second block (highest BMA content) exhibited the best knockdown effect [86]. When the length of endosomolytic block and its hydrophobic content were increased, this type of copolymer could self-assemble into spherical micelle. The cationic micelle presented much higher knockdown efficiency at low siRNA dose as compared to nonmicelle formulations. These results also indicated that hydrophobic property should contribute significantly to enhancing siRNA knockdown efficiency [87]. Similarly, Lin et al. [150] synthesized a comb-like polymers constructed by a copolymer of pH-sensitive EAA monomers and hydrophobic BMA or hexyl methacrylate (HMA) as the backbone and a second copolymer of hydrophobic HMA and cationic trimethylaminoethylmethacrylate (TMAEMA) at a 50/50 molar feed ratio as grafts. The grafts were linked to the backbone by the acid-labile hydrazone bond. These comb-like polymers could deliver anti-GAPDH siRNA molecules and successfully silenced GAPDH expression at both the mRNA and protein levels in MCF-7 cells.


### 3.4. Hydrophobic Modifications of Cationic Polymers

For gene delivery, it has been widely reported that hydrophobic modification of cationic polymers could improve the interaction between vectors, resulting in more efficient escape by lysis of the endosomes and therefore higher gene transfection efficiency [151–155]. Recently, hydrophobic-modified or amphiphilic cationic copolymers are also used as vectors to deliver siRNA [89–91, 94–98, 156]. Kim et al. [88, 89] synthesized water soluble lipopolymer (WSLP) by conjugating cholesterol with short PEI (1.8 kDa) or oligoarginine. WSLP/siRNA complexes showed successful VEGF knockdown in vitro and significant repression of tumor growth in vivo via intratumoral injection. Similarly, branched PEI (25 kDa) derivatives, such as PEI modified by oleic acid and stearic acid [90], tyrosine [92] or PEI grafted with hydrophobic poly(γ-benzyl L-glutamate) segment [93], and low molecular weight PEI (800 Da) modified by Michael addition with alkyl acrylates [91], could condense siRNA into more stable nanoparticles and present better siRNA silencing effect than PEI and were comparable to some of the commercially available transfection agents. These branched PEI derivatives also have low cytotoxicity.Mao et al. [96, 97] reported self-assembled nanoparticles of monomethoxy poly(ethylene glycol)-block-poly(caprolactone)-block poly(2-aminoethyl ethylene phosphate) (PPEEA) (mPEG-b-PCL-b-PPEEA). Micellar nanoparticles have several advantages over hydrophilic cationic coplolymers. For example, micellar nanoparticles could load nucleic acids and hydrophobic anticancer drugs simultaneously. Anticeramidase siRNA-loaded nanoparticles showed significant gene knockdown activities toward the endogenous acid ceramidase gene in vitro and significant inhibition of tumor growth in a BT474 xenograft murine modelviatail vein injection. Evaluation of immunotoxicity indicated that this delivery system did not induce immune response. Xiong et al. [95] synthesized a type of biodegradable amphiphilic poly(ethylene oxide)-block-polycaprolactone (PEO-b-PCL) with grafted polyamines for siRNA delivery. These polyamine-grafted PEO-b-PCL micelles could efficiently deliver MDR-1-targeted siRNA to silence P-gp expression in vitro and showed effective endosomal escape after cellular uptake.Additionally, amphiphilic copolymers have been explored in an effort to deliver nucleic acids and hydrophobic drug paclitaxel into cancer cells [98, 99]. Zhu et al. [98] synthesized PDMAEMA-PCL-PDMAEMA triblock copolymers by RAFT polymerization method. Polymeric micelles/anti-GFP siRNA complexes showed more efficient knockdown than PDMAEMA (20 kDa) and PEI (25 kDa) in MDA-MB-435-GFP cells, and codelivery of VEGF siRNA and paclitaxel with polymeric micelles/siRNA complexes achieved synergistic effects in inhibiting tumor cell growth in vitro.


### 3.5. pH-Sensitive Degradable Vectors

In addition to the ability to escape from endosomes, the ideal siRNA delivery vectors should be able to release siRNA into the cytoplasm. Among the pH-sensitive bonds, acetal bond has been widely used to construct intelligent macromolecules or lipid vectors for drug delivery [100–103, 131, 157], due to its fast degradation in endosomal environment. Materials containing acetal bonds are supposed to be degraded in endosomes which precedes the release of their cargo. Hydrolysis of acetal bond will consume hydrogen, which also helps cargo escape from endosome by increasing endosomal osmotic pressure.Shim and Kwon [101, 157, 158] developed acid-degradable ketalized branched PEI and linear PEI. These derived PEIs showed significant gene transfection efficiency and siRNA knockdown effect when compositions of the polymers were optimized. Some of them could selectively release siRNA from the endosome into cytoplasm with reduced cytotoxicity. To achieve efficient dissociate of carrier with nucleic acids, they synthesized PEG-conjugated acid-degradable poly(ketalized serine) [100] and degradable polyspermine by Michael addition [103].


## 3.1. Ion-Pair Formation

The mechanism whereby cationic lipids destabilize endosome membrane to facilitate the endosomal escape of nucleic acid such as plasmid DNA or siRNA is originally proposed by Xu and Szoka [105]. Cationic lipids form ion pairs with anionic lipids within endosome membrane to destabilize the endosome membrane. Because the cross-sectional area of the combined headgroup in ion pair is less than that of the sum of individual headgroup areas in isolation, a “cylindrical” shape of individual charged lipids is transformed to a “cone” shape of ion pair which further promote the formation of the inverted hexagonal (HII) phase as proposed by Cullis et al. [128, 129].The exposure of the positive charge in liposomes to endosomal membranes is a prerequisite for electrostatic interaction between cationic lipids and anionic lipids. PEGylation of liposomes for systemic drug delivery therefore inhibits the formation of ion pairs. As mentioned in Section2, the shedding character of LPD nanoparticles is helpful to overcome the PEG dilemma [32–35]. After PEG comes off the LPD nanoparticles, cationic lipids are exposed to anionic lipids. In this novel formulation, the brushed but sheddable PEG not only grants LPD nanoparticles evasion of the RES for the initial period of time such that LPD nanoparticles can penetrate into the tumor by EPR effect but also facilitates the endosomal escape of the cargo siRNA. Due to successful RES evasion and endosomal escape, LPD nanoparticle is a very promising carrier for systemic delivery of siRNA.Obata et al. [108] synthesized zwitterionic lipids with amino acid-modified head group. The fusogenic potential of these liposomes with endosome-mimicking anionic membrane is pH-responsive, and it increases as pH decreases. The zeta potential of these liposomes in physiological conditions is negative and switches to positive as pH declines, which means that these lipoplexes may have prolonged circulation time in the blood. The properties of these liposomes provided great promise for drug delivery in vivo but no such report has been seen yet.Heyes et al. [130] synthesized 1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA) lipid with two double bonds per alkyl chain, which has strong fusogenicity and high gene transfection efficiency. Recently, Semple et al. [131] developed DLinDMA derivatives by optimizing the headgroup and linker moiety to formulate stable nucleic acid-lipid particles (SNALPs). They introduced an acid degradable ketalized linker. SNALP formulation containing DLin-KC2-DMA lipid presented excellent in vivo silencing activity in liver in rodents and nonhuman primates. This novel formulation could evade RES uptake and circulate in the blood for longer time due to its low pKa under neutral pH, because the amine head groups remain unprotonated and the zeta potential of nanoparticles was nearly neural or negative at pH 7.4. After the nanoparticles were internalized into intracellular endosomes, the amine groups became protonated to form cationic lipids, which was necessary to escape endosome by ion-pair mechanism. As known, it is difficult for liposome formulations to avoid drug leakage in the blood on one hand and achieve rapid drug release in target tissues on the other [132–137]. In this novel SNALP formulation, the hydrolysis of ketal bond might increase the content of neutral lipid in liposomes, which likely triggers siRNA release due to reorganization of the lipid domains [138, 139] and is key for its excellent performance at in vivo siRNA delivery. Therefore, this formulation may be advantageous to release cargo efficiently from both endosome and liposomes.


## 3.2. “Proton Sponge Effect”

Successful escape of siRNA carriers from endosome and release of the payload into cytoplasm is necessary to improve the efficiency of gene silencing. Due to the acid nature of endosomal/lysosomal vesicles, pH-buffering agents are widely exploited to promote cargo release. Under acidic condition, various macromolecules with low pKa amine group have been shown to exhibit “proton sponge effect”, such as PEI and its derivates [74, 94, 140, 141]. When the complexes formed by these compounds and nucleic acid are internalized into the cell, these compounds are capable of buffering the endosomal vesicle, leading to endosomal swelling and lysis, thus releasing the nucleic acids into the cytoplasm.Charge-reversal copolymers could shift their charge nature between positive and negative in a pH-dependent fashion [76–79, 142]. Charge conversion can occur in acidic intracellular organelles such as endosome or lysosome (pH = 5~6), and then these copolymers facilitate the endosomal escape of nucleic acids by enhancing the capacity of “proton sponge”. Pittella et al. [79] synthesized a hybrid nanocarrier system composed of calcium phosphate (CaP), a block copolymer PEG, and a charge-conversional polymer (CCP) to deliver siRNA. Confocal laser scanning microscopic observation confirmed that CCP was helpful for endosomal escape of siRNA with the nanoparticles. This hybrid nanocarrier system achieved significant knockdown of vascular endothelial growth factor (VEGF) in PanC-1 cells with low cytotoxicity. Guo et al. prepared charge-reversal functionalized gold nanoparticles (CRFGNs) with cis-aconitic anhydride-functionalized poly(allylamine) (PAH-Cit) [75]. The charge reversion of PAH-Cit was confirmed by polyacrylamide gel electrophoresis and confocal laser scanning microscopy. In vitro quantification of lamin A/C protein expression by western blot indicated that the knockdown efficiency of siRNA delivered by CRFGN was dose-dependent in Hela cell line. At 10:1 Au : siRNA ratio, siRNA delivered by CRFGN achieved the highest (80.0%) knockdown efficiency, which is better than Lipofectamine 2000 which inhibited about 66.0%. CRFGN achieved similar results in DNA transfection experiments.In fact, layered double hydroxide (LDH) [112], calcium phosphate [116, 124], and some other inorganic nanoparticles [118] also belong to materials which are able to deplete protons in acid environment. Because these nanoparticles could be degraded in acidic buffer, they are also able to release cargo from endosomal vesicles. For example, siRNA-loaded LDH nanoparticles [112] were dissolved due to the low pH in the endosome, which facilitated cargo escape from endosomes into the cytoplasm and significant downregulation of protein expression in HEK293T cells. Carbonate apatite nanoparticles [117] were developed to facilitate siRNA escape from the endosomes. It was remarkable that carbonate apatite nanoparticles could efficiently silence reporter genes at a low dose and were more efficient than Lipofectamine. Calcium phosphate has been widely used in biomaterials due to excellent biocompatibility. In vitro gene transfection efficiency of calcium phosphate nanoparticles with hydroxyapatite phase structure was found to be higher than that of the commercial transfecting reagent Polyfect [116]. Nevertheless, these particles are not stable and readily form large aggregates; therefore their in vivo applications are limited. To synthesize stable siRNA-loaded calcium phosphate, Kataoka et al. [113–115] utilized PEG-polycarboxylate block copolymer (such as PEG-b-poly(aspartic acid) and PEG-b-poly(methacrylic acid)) to achieve stable and size-controlled CaP nanoparticles, which showed good stability in serum and significant gene knockdown in vitro. Incorporation of PEG-SS-siRNA instead of PEG-polycarboxylate for stabilization achieved high PEG density in CaP nanoparticles, which could be potentially useful for the systemic delivery of siRNA. The PEG-stabilized CaP nanoparticles were observed to escape from endosome/lysosome exhibited RNAi efficacy for in vitro gene silencing. To improve the endosomal escape of CaP nanoparticles, a diblock copolymer composed of PEG and a charge-reversal polymer was incorporated [79] and resulting hybrid nanoparticles showed significant VEGF knockdown in PanC-1 cells because of the rapid endosomal escape. However, there is no in vivo study of PEG-stabilized CaP nanoparticles for siRNA delivery.Recently, Li et al. [109] prepared siRNA-encapsulated calcium phosphate by reverse water-in-oil microemulsion technique and then coated calcium phosphate nanoparticles with lipid to obtain a novel siRNA formulation of lipid-coated calcium phosphate (LCP) nanoparticle for siRNA systemic delivery. As mentioned before, calcium phosphate is acid-sensitive, and its degradation in endosome/lysosome increases osmotic pressure of endosome, leading to release of siRNA into the cytoplasm. In addition, lipid component is well known to be helpful for nanoparticles uptake and cargo release from endosome. Antiluciferse (luc) siRNA-loaded LCP nanoparticles showed significant gene silencing of luciferase both in vitro and in vivo with negligible immunotoxicity. The in vivo results suggested that LCP nanoparticles offer significant promise for siRNA delivery in clinical trails.


## 3.3. Membrane-Destabilizing Macromolecules

To date, there is no kind of synthetic vector which has comparable efficiency as viral vectors. Hemagglutinin protein, which is pH-sensitive and membrane-destabilizing, helps viral vectors to disrupt the endosome efficiently and enter the cytoplasm [127]. The escape mechanism of hemagglutinin and other fusion proteins is that they shift from an ionized and hydrophilic conformation to a hydrophobic and membrane-active conformation as the environment changes from neutral to acidic, resulting in destabilization of the endosomal membrane and its leakage subsequently. Therefore, incorporation of membrane-destabilizing peptides is another effective strategy to utilize the low pH environment of endosomes/lysosomes.To mimic the function of viral hemagglutinin protein, many peptides [123, 143–145] and polymers [80, 146–149] with similar properties were synthesized to enhance gene and siRNA delivery. Among the synthetic peptides, the GALA peptide  (WEAALAEALAEALAEHLAEALAEALEALAA) [123] was the most studied. Harashima group has developed a kind of multifunctional envelope type nanodevice (MEND) combining PEGylation, targeting ligand, and GALA. It was demonstrated that GALA facilitated endosomal release of siRNA, resulting in efficient knockdown [124–126]. In an in vivo gene silencing study, different MENDs with antiluciferase siRNA were directly injected into tumors of T1080-luc-tumor-bearing mice, and compared to unmodified MEND, GALA-modified MEND exhibited greater gene silencing in tumor tissues. On the other hand, poly(propylacrylic acid) (PPAA) was the most efficient and most studied membrane-destabilizing synthetic polymers, and its membrane-destabilizing capacity could be enhanced by copolymerization with hydrophobic monomers [80–84]. PPAA was demonstrated to enhance the transfection efficiency of cationic lipid/pDNA complexes both in vitro and in vivo [81, 85]. PPAA is negatively charged at physiological condition and therefore cannot condense siRNA directly. Stayton et al. synthesized a kind of diblock polymer composed of poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) to condense siRNA and a second endosomal-releasing block composed of DMAEMA and propylacrylic acid (PAA) in roughly equimolar ratios, together with butyl methacylate (BMA). These carriers became sharply lytic at endosomal pH range. In HeLa cells, a copolymer with the most hydrophobic second block (highest BMA content) exhibited the best knockdown effect [86]. When the length of endosomolytic block and its hydrophobic content were increased, this type of copolymer could self-assemble into spherical micelle. The cationic micelle presented much higher knockdown efficiency at low siRNA dose as compared to nonmicelle formulations. These results also indicated that hydrophobic property should contribute significantly to enhancing siRNA knockdown efficiency [87]. Similarly, Lin et al. [150] synthesized a comb-like polymers constructed by a copolymer of pH-sensitive EAA monomers and hydrophobic BMA or hexyl methacrylate (HMA) as the backbone and a second copolymer of hydrophobic HMA and cationic trimethylaminoethylmethacrylate (TMAEMA) at a 50/50 molar feed ratio as grafts. The grafts were linked to the backbone by the acid-labile hydrazone bond. These comb-like polymers could deliver anti-GAPDH siRNA molecules and successfully silenced GAPDH expression at both the mRNA and protein levels in MCF-7 cells.


## 3.4. Hydrophobic Modifications of Cationic Polymers

For gene delivery, it has been widely reported that hydrophobic modification of cationic polymers could improve the interaction between vectors, resulting in more efficient escape by lysis of the endosomes and therefore higher gene transfection efficiency [151–155]. Recently, hydrophobic-modified or amphiphilic cationic copolymers are also used as vectors to deliver siRNA [89–91, 94–98, 156]. Kim et al. [88, 89] synthesized water soluble lipopolymer (WSLP) by conjugating cholesterol with short PEI (1.8 kDa) or oligoarginine. WSLP/siRNA complexes showed successful VEGF knockdown in vitro and significant repression of tumor growth in vivo via intratumoral injection. Similarly, branched PEI (25 kDa) derivatives, such as PEI modified by oleic acid and stearic acid [90], tyrosine [92] or PEI grafted with hydrophobic poly(γ-benzyl L-glutamate) segment [93], and low molecular weight PEI (800 Da) modified by Michael addition with alkyl acrylates [91], could condense siRNA into more stable nanoparticles and present better siRNA silencing effect than PEI and were comparable to some of the commercially available transfection agents. These branched PEI derivatives also have low cytotoxicity.Mao et al. [96, 97] reported self-assembled nanoparticles of monomethoxy poly(ethylene glycol)-block-poly(caprolactone)-block poly(2-aminoethyl ethylene phosphate) (PPEEA) (mPEG-b-PCL-b-PPEEA). Micellar nanoparticles have several advantages over hydrophilic cationic coplolymers. For example, micellar nanoparticles could load nucleic acids and hydrophobic anticancer drugs simultaneously. Anticeramidase siRNA-loaded nanoparticles showed significant gene knockdown activities toward the endogenous acid ceramidase gene in vitro and significant inhibition of tumor growth in a BT474 xenograft murine modelviatail vein injection. Evaluation of immunotoxicity indicated that this delivery system did not induce immune response. Xiong et al. [95] synthesized a type of biodegradable amphiphilic poly(ethylene oxide)-block-polycaprolactone (PEO-b-PCL) with grafted polyamines for siRNA delivery. These polyamine-grafted PEO-b-PCL micelles could efficiently deliver MDR-1-targeted siRNA to silence P-gp expression in vitro and showed effective endosomal escape after cellular uptake.Additionally, amphiphilic copolymers have been explored in an effort to deliver nucleic acids and hydrophobic drug paclitaxel into cancer cells [98, 99]. Zhu et al. [98] synthesized PDMAEMA-PCL-PDMAEMA triblock copolymers by RAFT polymerization method. Polymeric micelles/anti-GFP siRNA complexes showed more efficient knockdown than PDMAEMA (20 kDa) and PEI (25 kDa) in MDA-MB-435-GFP cells, and codelivery of VEGF siRNA and paclitaxel with polymeric micelles/siRNA complexes achieved synergistic effects in inhibiting tumor cell growth in vitro.


## 3.5. pH-Sensitive Degradable Vectors

In addition to the ability to escape from endosomes, the ideal siRNA delivery vectors should be able to release siRNA into the cytoplasm. Among the pH-sensitive bonds, acetal bond has been widely used to construct intelligent macromolecules or lipid vectors for drug delivery [100–103, 131, 157], due to its fast degradation in endosomal environment. Materials containing acetal bonds are supposed to be degraded in endosomes which precedes the release of their cargo. Hydrolysis of acetal bond will consume hydrogen, which also helps cargo escape from endosome by increasing endosomal osmotic pressure.Shim and Kwon [101, 157, 158] developed acid-degradable ketalized branched PEI and linear PEI. These derived PEIs showed significant gene transfection efficiency and siRNA knockdown effect when compositions of the polymers were optimized. Some of them could selectively release siRNA from the endosome into cytoplasm with reduced cytotoxicity. To achieve efficient dissociate of carrier with nucleic acids, they synthesized PEG-conjugated acid-degradable poly(ketalized serine) [100] and degradable polyspermine by Michael addition [103].


## 4. Conclusions

To date, lipid and polymeric nanoparticles have already been widely used for siRNA delivery. For nanoparticles to efficiently deliver siRNA into target tissue and silence target genes, they must overcome two major hurdles: RES uptake and endosome entrapment. Long-circulation property is the prerequisite for nanoparticles to carry most of the siRNA cargo into the target site of interest. Circulation half-life in the blood can be improved by PEGylation or coating a neural and anionic shell. Facilitating endosomal escape is another important strategy in improving drug bioavailability. Incorporation of sheddable-PEG into nanoparticles can help solve the PEG dilemma and promote nanoparticles to escape from endosomes. Tested strategies exploiting ion pair formation, “proton sponge effect”, or adding endosome destabilizing agents are effective in improving endosome escape. However, most of these endosome escape mechanisms are not compatible with RES evasion such that only local administration of the dosage form can be attempted. Some newly developed nanoparticles (LPD and LCP), which are able to escape from the endosome, deassemble, and release siRNA simultaneously, certainly represent a class of improved vectors for siRNA delivery in vivo.



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*Source: 742895-2011-08-23.xml*