Delivery systems for siRNA drug development in cancer therapy

Cong-fei Xu,Jun Wang

School of Life Sciences and Hefei National Laboratory for Physical Sciences at the Microscale,University of Science and Technology of China,Hefei,Anhui 230027,China

Review

Delivery systems for siRNA drug development in cancer therapy

Cong-fei Xu,Jun Wang＊

School of Life Sciences and Hefei National Laboratory for Physical Sciences at the Microscale,University of Science and Technology of China,Hefei,Anhui 230027,China

ARTICLEINFO

Article history:

Received 17 April 2014

Received in revised form

17 July 2014

Accepted 20 August 2014

Available online 28 August 2014

RNA interference Cancer therapy Delivery systems siRNA

Since the discovery of the Nobel prize-winning mechanism of RNA interference(RNAi)ten years ago,it has become a promising drug target for the treatment of multiple diseases, including cancer.There have already been some successful applications of siRNA drugs in the treatment of age-related macular degeneration and respiratory syncytial virus infection.However,signif i cant barriers still exist on the road to clinical applications of siRNA drugs,including poor cellular uptake,instability under physiological conditions,off-target effects and possible immunogenicity.The successful application of siRNA for cancer therapy requires the development of clinically suitable,safe and effective drug delivery systems.Herein,we review the design criteria for siRNA delivery systems and potential siRNA drug delivery systems for cancer therapy,including chemical modif i cations,lipidbased nanovectors,polymer-mediated delivery systems,conjugate delivery systems,and others.

©2015 Shenyang Pharmaceutical University.Production and hosting by Elsevier B.V.All rights reserved.

1.Introduction

RNA interference(RNAi)was f i rst discovered in plants,but it was not widely noted in animals until Fire and Mello demonstrated that double-stranded RNA(dsRNA)can cause greater suppression of gene expression than single-stranded RNA(ssRNA)in Caenorhabditis elegans[1].Due to the excellent gene silencing potential of RNAi,it has attracted broad attention in terms of how to harness the capabilities of RNAi. In 2001,Tuschl et al.f i rst transferred dsRNA into mammalian cells and solved the interferon effect of dsRNA transfection in these cells,which broadened the therapeutic use of Rania[2]. In 2010,Davis et al.reported the f i rst targeted siRNA delivery nanoparticle in humans via systemic injection,which provided a reference and a solid foundation for siRNA clinical use [3].In recent years,RNAi has become more and more important in gene silencing and drug development because of its high specif i city,signif i cant effect,minor side effects and ease of synthesis.

Naturally,RNAi is an important defense mechanism by which eukaryotic cells can degrade exogenous genes,like viruses.When dsRNA enters the cell,it is f i rst cleaved into short double stranded fragments of～20 nucleotide siRNAs bythe enzyme Dicer.Then,each double stranded siRNA is split into the passenger strand and the guide strand.After that,the guide strand is incorporated into the RNA-induced silencing complex(RISC),while the passenger strand is degraded.In the RISC,the guide strand of siRNA pairs with a complementary sequence in a messenger RNA molecule and induces cleavage by Argonaute,which causes post-transcriptional gene silencing.There are three strategies for RNAi:short hairpin RNA(shRNA),endogenous microRNA(miRNA)and small interfering RNA(siRNA).siRNA is more suitable for drug use because it does not require genome integration and can be easily synthesized.Since rational design of siRNA can specif i callyinhibitendogenousandheterologousgene expression,itcanmodulateanydisease-relatedgene expression.For example,most cancer is caused by oncogene overexpression or gene mutation,so it may be possible to cure cancer by disease-related gene suppression via rational siRNA design.Owing to its great potential in biological research and drug development,RNAi was awarded the Nobel Prize for medicine in 2006.Since then,billions of dollars have been invested in the therapeutic application of RNAi in humans.At least 22 RNAi-based drugs have entered clinical trials(Table 1).

Among these clinical trials,most siRNAs were administered by local delivery,typically via the intravitreal or intranasal routes.However,local delivery may not be appropriate for all diseases.Under some circumstances,systemic drug administration by intravenous(i.v.)injection is needed,and delivery systems will be necessary to administer the siRNA payload.For example,PRO-040201(ApoB-SNALP)administrated by i.v.injection was developed by Tekmira with a stable nucleicacid lipid particle(SNALP)system.It was developed for the treatment of hypercholesterolemia by targeting ApoB, which is produced by hepatocytes.In July 2009,Tekmira initiated a Phase I clinical trial for PRO-040201.Seventeen subjects receiveda single dose at one of seven different dosing levels and six subjects receiveda placebo.The results revealed that ApoB siRNA was delivered into hepatocytes eff i ciently and resulted in a signif i cant reduction of LDL and triglycerides in blood.However,Tekmira terminated the clinical trial in January 2010 because one of the two subjects treated with the highest dose experienced f l u-like symptoms consistent with stimulation of the immune system caused by the ApoB siRNA payload[4].Calando Pharmaceuticals(Pasadena,California, USA)has developed an siRNA therapeutic(CALAA-01),which is a cyclodextrin-based polymeric nanoparticle containing the M2 subunit of ribonucleotide reductase(RRM2)targeted siRNA.CALAA-01 was modif i ed with the human transferrin (TF)protein and polyethylene glycol(PEG)to improve its stability[3].Unfortunately,its phase I clinical trial has been terminated in 2013 according to U.S.Food and Drug Administration(FDA).In addition to the abovementioned siRNA drugs,many more are in the developmental pipeline.

2.Advantages of siRNA and barriers to siRNA in cancer therapeutics

Compared to chemotherapeutic anti-cancer drugs,there are a lotof advantages of siRNAdrug.Duetothe specialmechanism of siRNA,it has four advantages as a potential cancer therapeutic strategy.The fi rst is its high degree of safety.siRNA acts on the post-translational stage of gene expression,so it does not interact with DNA and thereby avoids the mutation and teratogenicity risks of gene therapy.The second advantage of siRNA is its high ef fi cacy.In a single cancer cell,siRNA can cause dramatic suppression of gene expression with just several copies.Compared to other small molecule drugs or antibody-based drugs,the greatest advantages of siRNA are the unrestricted choice of targets and speci fi city determined by the principle of complementary base pairing.This strategy also bene fi ts from rapid developments in molecular biology and whole-genome sequencing.In addition,comprehensive nucleotidesequencedatabaseshavebeenestablished, including human genomic databases,cDNA databases and disease gene databases,which have laid a solid foundation for siRNA drug development.The basic strategy of an siRNA drug is to treat cancer by silencing the speci fi c cancer-promoting gene with rationally designed siRNA.Of course,it is also possible to design effective siRNA drug targeting for any disease gene according to the mRNA sequence.

However,several barriers still exist on the road to siRNA clinical use for cancer therapy(Fig.1).Firstly,siRNA is unstable under physiological conditions.When siRNA traf fi cs through the blood,it is easily digested by nucleases in the serum.The intracellular traf fi cking of siRNA delivered by different reagents generally begins in early endosomes.These early endosomes subsequently fuse with sorting endosomes, which in turn transfer their contents into late endosomes.The endosomal compartments of cell are signi fi cantly acidic(pH 5.0～6.2),while the cytosol or intracellular space is neutral (pH≈7.4)[5].Endosome is then relocated to the lysosomes, which are further acidi fi ed(pH≈4.5)and contain various nucleases that promote the degradation of siRNA[6].The ideal administration route of siRNA is systemic injection,so that siRNA can reach cancer cells more ef fi ciently.After injection into the blood,siRNA is easily enzymatically degraded by endogenous nucleases, fi ltered by the kidney,taken up by phagocytes and aggregated with serum proteins[7].One of the fi rst biological barriers encountered by administered siRNA is the nuclease activity in plasma and tissues.The major nuclease in plasma is a 3′exonuclease;however,cleavage of internucleotide bonds can also take place.The reported halflife for unmodi fi ed siRNA in serum ranges from several minutes to 1 h[8].In addition,the kidney plays a key role in siRNA clearance;severalstudiesin animalshavereportedthat the biodistribution of siRNA shows the highest uptake in the kidney[9].In addition to circulating nuclease degradation and renal clearance,a major barrier to in vivo delivery of siRNA is uptake by the reticuloendothelial system(RES).The RES is composed of phagocytic cells,including circulating monocytes and tissue macrophages,the physiological function of which is to clear foreign pathogens and to remove cellular debris and apoptotic cells[10].Tissue macrophages are most abundant in the liver(where they are called Kupffer cells)and the spleen,tissues that also receive high blood fl ow and exhibit a fenestrated vasculature.Thus,it is not surprising that these organs accumulate high concentrations of siRNA following systemic administration.siRNA uptake after standard i.v.tail vein injection or intraperitoneal(i.p.)injectionhas been noted in the liver,spleen,kidney and bone marrow at 4 h,but the overall signal was weak[11].

Secondly,free siRNA,which is a type of anionic and hydrophilic double-stranded small RNA,is not readily taken up by cells.Moreover,the hydrophilicity and negative charge of siRNA molecules prevents them from readily crossing biological membranes.Therefore,siRNA needs to be packaged in vesicles in order to enter cells.

The third barrier is the off-target effects of siRNA,which lead to unanticipated phenotypes that complicate the interpretation of the therapeutic benef i ts of siRNA,including siRNA-inducedsequence-dependentregulationofunintended transcripts through partial sequence complementarity to their 3′UTRs,as well as widespread effects on miRNA processing and function through saturation of the endogenous RNAi machinery by exogenous siRNA[12].The scale of off-target effects was found to be remarkable during the identif i cation of novel components of signal transduction pathways by RNAi screens[13].All siRNA hits,whatever their intended direct target,reduced the mRNA levels of two known upstream pathway components,TGF-β receptor 1 and 2 (TGFBR1 and TGFBR2),via miRNA-like off-target effects[13]. Transfection of small RNAs can globally perturb gene regulationby endogenousmiRNA.Targetsof endogenousmiRNAare expressed at signif i cantly higher levels after specif i c siRNA transfection,consistent with the impaired effectiveness of endogenous miRNA repression,which results in unexpected changes in gene expression.

Lastly,siRNA is not as safe as expected.High levels of siRNA have been known to result in the activation of innate immune responses and the production of cytokines in vitro and in vivo[14,15].Mammalian immune cells express a subfamily of pattern-recognition receptors called Toll-like receptors(TLRs)that recognize pathogen-associated molecular patterns,including unmethylated CpG DNA and viral dsRNA [12].Several TLRs are involved in the recognition of siRNA, including TLR3,TLR7 and TLR8[16,17].TLR3 is the receptor for dsRNA,and cultured human embryonic kidney HEK-293 cells overexpressing TLR3 are capable of recognizing siRNA.siRNA has been shown to activate TLR3 signaling in a sequenceindependent manner[16].TLR7 and TLR8 were initially shown to mediate the recognition of RNA viruses and small synthetic antiviral compounds referred to as imidazoquinolines[18].It has been shown that TLR7 is absolutely required for the induction of cytokines using the appropriate knockout mice in murine immune cells in response to siRNA[14,15]. siRNA can be recognized by human plasmacytoid dendritic cells(pDCs)through TLR7 and by human monocytes,likely via TLR8[19].TLR7and TLR8mediatetherecognitionof siRNA in a sequence-dependent manner,and RNA sequences,including UG dinucleotides and the 5′-UGU-3′motif,are preferentially recognized[18].Thus,the sequence issue of siRNA-mediated immune stimulation requires further investigation.

In consideration of these barriers to realizing the broad potential of siRNA-based therapeutics,safe and effective siRNA delivery methods are desired.Therefore,chemical modif i cations and/or delivery methods are required to bring siRNA to its site of action without adverse effects.A broad diversity of materials is under exploration to address the challenges of in vivo delivery,including polymers,lipids, peptides,antibodies,aptamers,and small molecules.Successful systems have been developed by rational design or discovered using high-throughput screens.

3.The design criteria of an siRNA delivery system for cancer therapy

To apply siRNA into cancer therapy,the delivery barriers of siRNA in vivo are the predominant problems to be solved.According to the barriers of siRNA encountered in cancer therapy,there are several criteria for siRNA delivery system. As siRNA molecules are too large(～13 kDa)and too negatively charged to diffuse across cancer cell membranes alone,the issue of effective and non-toxic delivery is a key challenge and serves as the most signif i cant barrier between siRNA technology and its therapeutic application[20].To administer siRNA systemically and allow it to cross physiological barriers to reach itssite of action,deliverysystemsmustbeengineered to(I)provide serum stability,(II)allow immune evasion,(III) mitigate interactions with serum proteins and non-cancer cells,(IV)resist renal clearance,(V)enhance vascular permeability to reach cancer tissues,(VI)permit cell entry and endosome escape to enter the RNAi machinery[7,20]and(VII) have low toxicity.

Firstly,siRNA should be injected into blood for cancer therapy.As soon as naked RNA molecules are administered to the blood,the innate immunesystem is stimulated and serum nucleases immediately degrade the RNA.A common strategy to avoid these problems is to modify the siRNA backbone through chemical elements.The most frequently used strategies of chemical modif i cation are incorporation of 2′-O-methyl and 2′-deoxy-2′-f l uoro groups,locked or unlocked nucleic acids,or phosphorothioate linkages[21].Special design of siRNA sequence and structure can also avoid recognition by the innate immune system.Although chemical modif i cations can solve some problems of siRNA delivery, nanoparticles that encapsulate siRNA are better at protecting it from degradation and immune recognition[22].So,not only modif i cations of the siRNA chemical structure are needed,but additional delivery materials are also necessary to surmount other barriers in the body.

There are many components in the blood that will interact with siRNA delivery in various ways.High positive charges on the surface of nanoparticles can cause unfavorable aggregation with erythrocytes[23],but this kind of interaction between nanoparticles and serum proteins can also aid uptake by cancer cells[24,25].For example,many liposomal delivery systems,as well as siRNA conjugated to lipophilic molecules, interact with serum lipoproteins and subsequently gain entry into hepatocytes that take up those lipoproteins[24].However,serum opsonin proteins can also be adsorbed on the surface of delivery nanoparticles,and tag them for uptake by the mononuclearphagocytesystem(MPS)[7,26].Themain pathway by which nanoparticles are cleared from the blood is opsonization and subsequent uptake by the MPS,which prevents them from reaching their targets.The most commonly used and best characterized strategy to minimize interaction betweendeliverynanoparticlesandserumproteinsis shielding the nanoparticle surface with polyethylene glycol (PEG)[7,27].Rational PEGylation of delivery nanoparticles can prolong blood circulation time by minimizing non-specif i c interactionsofnanoparticleswithserumproteins,the innate immune system and other non-target tissues.PEG forms a barrier around nanoparticles that provides steric stabilization and protection from the physiological surroundings[28].The length of the PEG chain can have a signif i cantinf l uenceonitsstabilizationandprotective properties,and chain length is typically optimized for each individual delivery system.

After systemic administration,there are many ways by which siRNA leaves the bloodstream,including through the liver,spleen,kidney and lung.However,kidney clearance is the most common pathway.The kidney is composed of many glomeruli,which work as a natural f i ltration barrier that allows water and small molecules to pass into nascent urine while larger molecules are retained in the circulation[29].The pore size of the glomerular f i ltration barrier is roughly 8 nm [30],and excretion through the kidney typically occurs for molecules less than 50 kDa in size[31];the molecular weight of naked siRNA is about 13 kDa[20].Therefore,siRNA passes through glomeruli and f l ow into the urine.By complexing siRNA with synthetic materials,the size of the delivery nanoparticle can be increased to avoid glomerular f i ltration through the kidneys and reserve the siRNA for alternative organ targets[31].Many delivery systems are designed to be larger than 20 nm[32].However,20 nm is a strict limit as dynamic polyconjugates(DPCs;10 nm)[33]and triantennary N-acetylgalactosamine(GalNAc)conjugates are both highly effective delivery systems.

Based on the enhanced permeability and retention effect (EPR effect),which means that nanoparticles ranging in size from tens to hundreds of nanometers are passively accumulated in tumors to a greater extent than in normal tissue, mainly because newly formed tumor vessels are usually abnormal in form and architecture,many nanosized drug delivery systems have been developed including micelles or vesicles,dendrimers,liposomes and inorganic hybrid particles for cancer therapy.

Most siRNA delivery systems undergo cellular internalization through endocytosis.Various delivery systems aim to improve the rate of cellular uptake by incorporating targeting ligands that bind specif i cally to receptors on target cells to induce receptor-mediated endocytosis[34].Adsorption of serum proteins on the nanoparticle surface may hinder this ligand-receptor interaction[35].Other systems use cellpenetrating peptides that can induce cell uptake through endocytosis or non-endocytic mechanisms[36].Endocytosed materials are taken up into membrane-bound endocytic vesicles,which fuse with early endosomes and become increasingly acidic as they mature into late endosomes.Some delivery systems incorporate materials that are designed to respond to a low pH environment by becoming membranedisruptive in order to trigger the release of siRNA from endosomes into the cytoplasm[33,37].Still,the exact endosomal release mechanism of many siRNA delivery systems is poorly understood.

Additionally,low toxicity is the most important part of siRNA delivery systems.If siRNA delivery provokes unacceptabletoxicityoneither acellularorsystemiclevel,eventhe most eff i cacious siRNA delivery system will be rendered useless.Viral vectors,which were among the f i rst vehicles to be studied for siRNA delivery,can induce unacceptable levels of toxicity through the activation of immune responses[38]. Therefore,synthetic lipids and polymers have been developed to offer alternatives to viral vectors for nucleic acid delivery applications,and are carefully formulated to avoid stimulation of the immune system[15].Clearance of larger molecular mass materials typically requires them to be biodegradable. The use of biodegradable,high molecular mass polycationsand polymers containing linkages that can be cleaved inside the cell can help reduce cytotoxicity[39].

4.Potential siRNA drug delivery systems for cancer therapy

Although many strategies that can deliver siRNA into the cytoplasm of cancer cells have been reported,most of them can only satisfy in vitro applications.The majority of siRNA drugs in clinical trials are directly administered to pathologybearing regions to avoid the complexity of systemic delivery. They can be divided into nine classes according to their targets,including eye diseases,pachyonychia congenita,viral diseases,asthma,hypercholesterolemia,acute kidney injury, thyroxine amyloidosis,and cancer[40].However,the excellent therapeutic potential of siRNA for cancer therapyremains uncovered.It is necessary to introduce systemic routes of siRNA delivery to treat most cancers.

As mentioned above,the design criteria of an in vivo,systemic siRNA delivery system should include biocompatibility, biodegradability,and non-immunogenicity.Additionally,the system should protect siRNA from serum nucleases and deliver it into target cells eff i ciently.Finally,the delivery systemshouldprovidesiRNAan endosome escapeability to enter the RNAi machinery and activate RNAi pathways[41,42].The currently developed siRNA delivery systems for cancer therapy can be divided into four categories:chemical modif i cations,lipid-based nanovectors,polymer-mediated delivery systems,conjugate delivery systems,and others(exosomes, RNAi-microsponges,oligonucleotide nanoparticles).

4.1.Chemical modif i cations of anti-cancer siRNA

Although chemical modi fi cations do not provide a carrier for siRNA,they show great potential and are necessary in cancer therapeutic siRNA delivery systems.With rational chemical modi fi cations,siRNA can acquire advantages such as serum stability,immune escape ability,and RNAi machinery access [8,43,44].

Chemical modi fi cations can be introduced at the 5′or 3′-terminus,backbone,sugar or nucleobase of siRNA.The most common modi fi cation site of siRNA is the 2′position of the ribose ring,which has been proven to enhance siRNA stability by preventing degradation by endonucleases.The two modifi cation strategies,i.e.2′-O-methyl and 2′-deoxy-2′- fl uoro,are quite well-understood and commercialized,and have been shown to enhance the serum stability of siRNA and increase its in vivo potential.Some other approaches also exist,such as replacement of the phosphodiester(PO4)group with phosphothioate(PS)at the 3′-end of RNA backbone,or the combination of 4′-thiolation with 2′-O-alkyl modi fi cation[44,45].

The basic requirement of successful modi fi cations is enhancing siRNA serum stability without negative effects on its gene silencing activity.Indeed,some kinds of modi fi cation can compromise ef fi ciency.For example,boranophosphonate modi fi cation at the center of the antisense strand enhances the resistance of siRNA to nucleases,although it reduces RNAi activity[46].In addition,the metabolites of these modi fi cations should also be addressed as a safety issue.

4.2.Lipid-based vectors for anti-cancer siRNA delivery

Lipofectamine 2000 is a kind of cationic lipid formulation that is widely used for in vitro plasmid DNA or siRNA transfection. Lipofectamine 2000 or the recently developed lipofectamine RNAimax are effective siRNA transfection agents in vitro which can improve the transfection eff i cacy by thousands of times[47].The transfection mechanism of liposomes involves electrostatic interactions between negatively charged nucleic acids and positively charged lipids.When mixed together, they spontaneously form lipoplexes[48-50].

Because the surface charge of all biological membranes is negative,electronegative or neutral liposomes are more biocompatible than cationic liposomes and have superior pharmacokinetics in general.DOPC(1,2-dioleoylsn-glycero-3-phosphatidylcholine)is a kind of neutral lipid which has been used to improve siRNA entrapment eff i ciency.In 2005,Landen et al.developed the oncoprotein EphA2 targeting DOPC-encapsulated siRNA liposome,which was highly effective in reducing EphA2 expression 48 h after administration of a single dose in an orthotopic model of ovarian carcinoma[51]. Currently,the EphA2 targeting DOPC-encapsulated siRNA liposome(siRNA-EphA2-DOPC)is in a Phase I clinical trial initiated by the M.D.Anderson Cancer Center.Since electronegative or neutral liposomes are not easily endocytosed by cells,cationic liposomes are still the best choice.For example, dioleoyl-phosphatidylethanol-amineand1,2-dioleoyl-3-trimethylammonium-propane(DOTAP)iscationiclipids which form cationic liposomes with negatively charged siRNA [52].Sorensen et al.used cationic DOTAP liposomes to deliver siTNF-α,and the lethal reaction to LPS injection in a mouse model of sepsis was suppressed[53].To maintain an overall positive surface charge for adsorption through the cell membrane and to reduce the possible clearance caused by positive charge,the N/P(nitrogen to phosphate)ratio usual ranges from 2 to 3[47].

Coating liposomes with lipid-anchored PEG can reduce particle size[54],prevent aggregation during storage,increase circulatory half-life and reduce uptake by the reticuloendothelial system(RES)such as red blood cells and macrophages [54].But using PEG is not always advantageous,as the steric effect and charge effect of PEG block the interaction between the liposome and the endosomal membrane and prevent the liposome from escaping the endosome.Many studies have been performed to improve the eff i cacy of PEGylated nanoparticles,including rationally designed PEG length and density or incorporation of pH-sensitive bonds linking PEG to the liposome.How to achieve the best outcome with modulation of PEG length and density is still controversial,but pH-sensitive modif i ed PEG with ionic interactions,such as the HEMA-histidine-methacrylic acid modif i ed PEG liposome, has been shown to be effective.At neutral pH,the PEG copolymer has a net negative charge,whereas the liposomal core,which consists of DOPE and cholesterol,has a net positive charge.In the endosome,imidazole and methacrylic acid residues become protonated,and the net charge of the PEG becomes positive,which results in PEG release and positively charged liposomal membrane exposure,after which the liposome can fuse with the endosome and escape successfully [55].Atu027 is a lipoplexed siRNA drug targeting proteinkinase N3,which has been reported for the treatment of lymph node metastases in mouse models of prostate and pancreatic cancer and various mouse models of lung metastasis[56].Silence Therapeutics(London,UK)is performing a Phase I trial of Atu027.Preliminary result revealed that Atu027 was well tolerated up to a dose of 0.18 mg/kg and was not associated with dose-dependent toxicity[57].

The most famous lipid based vectors that used for clinical trials are the SNALPs(stable nucleic acid-lipid particles). SNALPs are a kind of lipid nanoparticles which encapsulate siRNAand deliveritto thetargetcells.SNALPsaremicroscopic particles approximately 120 nm in diameter.They have been used to deliver siRNAs therapeutically to mammals in vivo.In SNALPs,the siRNA is surrounded by a lipid bilayer containing a mixture of cationic and fusogenic lipids,coated with diffusible polyethylene glycol[58].With enhanced permeability and retention due to prolonged circulation time in the blood,SNALPs are highly bioavailable,which leads to the accumulation of SNALPs at the sites of vascular leakage, especially at cancer growth sites.After accumulation,SNALPs are easily endocytosed by cancer cells and deliver siRNA into cells successfully.SNALPs have been used for the treatment of many diseases,including hepatitis B viral infection,dyslipidemia and Ebola(Zaire)[20].Judge et al.have successfully demonstrated a 75%reduction in subcutaneous tumor size with SNALP-siPlk1 treatment[59].Tekmira Pharmaceuticals Corporation(Burnaby,BC,Canada)initiated a Phase I trial of SNALP-encapsulated siRNA targeting Plk1(TKM080301)in adult patients with solid tumors or lymphomas in December 2010.Alnylam Pharmaceuticals(Cambridge,MA,USA)has developedthef i rstdual-targetedsiRNAdrug,SNALP-formulated siRNAs targeting vascular endothelial growth factor(VEGF)and KSP in ALN-VSP02.A Phase I trial for the treatment of advanced solid tumors with liver involvement was initiated in April 2009.Interim data from the initial 28 patients in the f i rst six-dose cohorts demonstrated that ALNVSP02 was generally well tolerated at the highest dose (1.25 mg/kg)[60].

Anotherlipid-likedeliverysystemis lipidoidnanoparticles, which are comprised of cholesterol and PEG-modif i ed lipids specif i c for siRNA delivery[60].To improve SNALP-mediated delivery,Akinc et al.developed a new chemical method for the rapid synthesis of a large library of lipidoids and tested their eff i cacy in siRNA delivery[61].One of the most potential lipidoid drugs was the lipidoid-based siRNA formulation 98N12-5,which led to a 75-90%reduction in ApoB or FVII factor expression in hepatocytes in non-human primates and mice[61].

4.3.Polymer-mediated anti-cancer siRNA delivery systems

Polymer-mediated delivery systems,usually called polymeric nanoparticles,are solid,biodegradable,colloidal systems which have been widely studied as drug vesicles[62].According to the material used,polymer-mediated delivery systems can be divided into two categories:water-soluble cationic polymers and polymer nanoparticles.For anticancersiRNAdelivery,water-solublecationicpolymers mainly include cyclodextrin or polyethyleneimine(PEI),while polymer nanoparticles are usually based on polycaprolactone (PCL),poly(D,L-lactide)(PLA)and poly(D,L-lactide-co-glycolide) (PLGA)[63].

Cyclodextrin is the most promising candidate natural polymer for siRNA delivery.It was fi rst introduced for the delivery of plasmid DNA in 1999 and later reoptimized for siRNA delivery.Less than a decade later,cyclodextrin polymer (CDP)-based nanoparticles were moved into clinical trials for siRNA delivery.Cyclodextrin polymer nanoparticle was the fi rst targeted siRNA delivery system which entered clinical trials for cancer treatment[64].Cyclodextrin polymers are polycationic oligomers synthesized by a step-growth polymerization between diamine-bearing cyclodextrin monomers and dimethyl suberimidate,yielding oligomers with amidine functional groups[65].In cyclodextrin polymer-mediated siRNA delivery systems,adamantane-PEG(AD-PEG)and adamantane-PEG-transferrin(AD-PEG-Tf)are usually used to improve delivery ef fi cacy in vivo[66,67].For AD-PEG-Tf, adamantane can stabilize the cyclodextrin core by form a stable inclusion complex.PEG shielding can reduce blood clearance by protecting particles from serum proteins while decreasing cellular uptake and silencing ef fi cacy.Conjugated transferrin is a targeting component which can bind to the transferrin receptor CD71[68].Calando Pharmaceuticals (Pasadena,CA,USA)have developed CALLA-01,which targets the M2 subunit of ribonucleotide reductase(R2)to inhibit tumor growth[3].

Polyethylenimine(PEI)has been used successfully for nucleic acid delivery under both in vitro and in vivo conditions [69-71].However,high molecular weight PEIs provide high transfection ef fi ciency but also have high toxicity,while low molecular weight PEIs are more biocompatible but are much less ef fi cient.Navarro et al.reported a type of micelle-like nanoparticle(MNP),based on the combination of a covalent conjugate between a phospholipid and low molecular weight PEI(1.8 kDa)with PEG-stabilized liposomes as the outer layers [72].The MNP complexes had a size of～200 nm and a neutral surface charge after the addition of a PEG-lipid coating,which protected the loaded siRNA against enzymatic digestion and enhanced the cellular uptake of the siRNA payload.MNPs have been shown to have the capacity for siRNA delivery and gene silencingwith improved biocompatibility properties.The MNP delivery system was further utilized in silencing P-gp to overcome doxorubicin resistance in MCF-7 human breast cancer cells.The presence of P-gp on the surface of resistant cells decreased after treating cells with MNP-loaded siRNA targeting MDR-1,which effectively inhibited the drug ef fl ux activity.The amount of doxorubicin inside MDR-1-treated cells doubled compared control cells,and led to a two-fold decreased in cell viability after drug treatment for different intervals,similar to values in sensitive cells[73].

Polycaprolactone(PCL)is usually used for polymeric micelle siRNA drug delivery systems.Sun et al.described the production of self-assembled micellar nanoparticles(MNPs)of a triblock copolymer,monomethoxy poly(ethylene glycol)-block-poly(ε-caprolactone)-blockpoly(2-aminoethylethylene phosphate)(PPEEA)(mPEG-b-PCL-b-PPEEA)(Fig.2)[74].In this system,thehydrophilicphosphoesterPPEEA,whichis considered biocompatible and biodegradable,served as the siRNA binding site,and another hydrophilic block PEG surrounded the hydrophobic core to protect the siRNA and nanoparticles from clearance in the circulation.The siRNA-loaded nanoparticles,known as Micelleplex,can be effectively internalized and subsequently release siRNA into cells, resulting in signif i cant gene knockdown activity,which was demonstrated by delivering two siRNAs targeting green f l uorescenceprotein(GFP)that effectively silencedGFP expression in 40-70%of GFP-expressing HEK293 cells[74].mPEG-b-PCL-b-PPEEA has also been used for acid ceramidase(AC),HIF1 and CDK4 siRNA delivery to successfully treat different kinds of cancer in the mouse[75-77].

Poly(D,L-lactide)(PLA)andpoly(D,L-lactide-co-glycolide) (PLGA)have also demonstrated the potential for sustained nucleic acid delivery[78-80].In 2009,Saltzman and coworkers reported that PLGA nanoparticles can be densely loaded with siRNA in the presence of spermidine and,when applied topically to the vaginal mucosa,lead to eff i cient and sustained gene silencing[81].Yang et al.reported a cationic lipid assisted polymeric nanoparticle system with stealthy property for eff i cientsiRNA encapsulation and delivery,which was fabricated with poly(ethylene glycol)-b-poly(D,L-lactide), siRNA and a cationic lipid,using a double emulsion-solvent evaporation technique(Fig.3).Byincorporationofthe cationic lipid,the encapsulation eff i ciency of siRNA into the nanoparticles was greater than 90%.The siRNA loading weight ratio was up to 4.47%,while the diameter of the nanoparticles was around 170-200 nm.The siRNA retained its integrity within the nanoparticles,which were effectively internalized by cancer cells and escaped from the endosome, resulting in signif i cant gene silencing.Systemic delivery of specif i c siRNA by nanoparticles signif i cantly inhibited luciferase expression in an orthotopic murine liver cancer model and suppressed tumor growth in a MDA-MB-435s murine xenograft model,suggesting its therapeutic promise in disease treatment[82].Using the same cationic lipid-assisted polymeric nanoparticle system,Shen et al.delivered GATA2 siRNA to non-small-cell lung carcinoma(NSCLC)harboring oncogenic KRAS mutations and successfully inhibited tumor growth in mouse model[83].

4.4.Conjugate siRNA delivery systems for cancer therapy

Directly conjugation of delivery materials to siRNA has been shown to be a promising system for siRNA delivery.The most common conjugate materials are small drug molecules,aptamers,lipids,peptides,proteins and polymers[84]. This system has a quite obvious advantage for cancer therapeutic clinical use,since the system is simple and welldef i ned.

Lipophile-siRNA conjugates,which were the f i rst conjugate delivery systems to show eff i cacy in vivo,consist of siRNA conjugated to cholesterol[85]and other lipophilic molecules [24].Cholesterol was conjugated to the 3′-terminus of the sense strand of siRNA via a pyrrolidone linkage.Cholesterol not only increased the transfection eff i cacy of siRNA in vitrobut also improved siRNA pharmacokinetic behavior in vivo [85].To further optimize cholesterol-siRNA,high density lipoprotein(HDL)was bound which increased gene silencing eff i cacy by 8-15 fold in vivo[24].

CPPs(cell-penetrating peptides)are another conjugate material used for siRNA transfection eff i cacy improvement.A well-known CPP is the TAT trans-activator protein from human immunodeficiency virus type-1(HIV-1).TAT has been conjugated to the 3′-terminus of the antisense strand of an siRNA using a heterobifunctional cross-linker(HBFC),i.e. sulfosuccinimidyl-4-(p-maleimidophenyl)butyrate[86].The TAT-siRNA conjugate demonstrated a dramatic improvement in the intracellular delivery of siRNA.However,CPP-siRNA conjugates may exhibit cytotoxicity caused by cell membrane perturbation or immunogenicity[87].

A targeted delivery system is always the dream for anticancer drug development.For siRNA targeted delivery,peptides,antibodies and aptamers have been used.For the receptor-ligand mediated delivery of siRNA,the carboxylic acid group of a peptide mimetic of IGF1,D-(Cys-Ser-Lys-Cys), was activated and conjugated to an amine group of the 5′-sensestrandofsiRNA.Thisstrategyresultedin60%expression reduction of IRS1(insulin receptor substrate 1),which was similar to the chol-siRNA conjugate[88].Antibody-mediated targeted drug delivery systems have attracted much attention due to their superior stability and high specif i city.A monoclonal antibody targeting the transferrin receptor at the blood-brain barrier was directly conjugated to siRNA via a biotin-streptavidinlinkage.Theintravenousadministrationof the antibody-siRNA conjugate led to the eff i cient suppression of reporter gene expression in a rat model bearing intracranially transplanted brain tumors[89].Aptamers,such as the prostate-specificmembraneantigen(PSMA)targetedaptamer, are modif i ed oligonucleotides with selective aff i nities toward specif i c proteins.When conjugated to siRNA using streptavidinviastreptavidin-biotininteractions,PSMA-targeted aptamers have successfully facilitated siRNA uptake by PSMA-overexpressing cells without using transfection agents [90].Regarding these promising conjugate delivery systems, the two most advanced conjugate systems,i.e.DPCs and Gal-NAc conjugates,are already in clinical trials[40].

4.5.Other possible anti-cancer siRNA delivery systems

Apart from the previously studied siRNA delivery systems describedabove,therearesomenewsiRNAdeliverystrategies, such as exosome-mediated siRNA delivery systems,oligonucleotide nanoparticles and RNAi-microsponges.Although thesedeliveryplatformsarenotvery well-developed,they still show great potential in siRNA delivery.

Exosomes are small vesicles(40～100 nm)released from cells upon the fusion of a multivesicular body(MVB)containing intraluminal vesicles with the plasma membrane [91].They have been shown to be natural carriers of coding and non-coding RNA,including miRNA,with the ability to induce de novo transcriptional and translational changes in target cells[92-96].The ability of exosomes to transfer mRNAandmiRNAbetweencellsandsubsequentlyto mediate changes in gene expression in recipient cells, together with their high abundance in most body f l uids, highlights their potential as delivery vehicles for RNAi.El-Andaloussi et al.were the f i rst to harness this potential and provide the f i rst proof of concept for the biotechnological exploitation of exosomes[97].They specif i cally targeted dendritic cell-derived exosomes to the brain by displaying a rabies virus glycoprotein(RVG)-derived peptide,and then loaded them with siRNA for delivery both in vitro and in vivo (Fig.4).By using this method,they demonstrated specif i c delivery of siRNA to neurons in the brain following systemic delivery in mice,with up to 60%RNA and protein knockdown predominantly in the midbrain,cortex and striatum,and little homing of the exosome cargo to the liver.In addition to eff i cient and specif i c delivery of siRNA,these exosomes produced little or no toxicity or immunogenicity,even after repeated i.v.administration[98].

Oligonucleotide nanoparticles(ONPs)are composed of complementary DNA fragments designed to hybridize into predef i ned three-dimensional structures(Fig.5)[40].A previously described method[99]of constructing DNA tetrahedra was adapted by incorporating single-stranded overhangs on each edge[32].siRNAs were modif i ed by extension of the 3′-sense strands with DNA overhangs that enabled hybridization to the edges of the tetrahedra.By using unique overhang sequences,six siRNA strands could be attached to each particle,each in a specif i ed position.The resulting nanoparticles had a hydrodynamic diameter of about 29 nm[40].Oligonucleotide nanoparticles modif i ed with folate ligands were used to study the minimum number of targeting ligands required for delivery and to probe the optimal arrangement of these ligands.A minimum of three folate ligands was required to achieve signif i cant gene silencing,yet incorporation of more than three ligands did not greatly improve silencing eff iciency.Furthermore,the positioning of the three ligands was critical:ONPs with three ligands arranged to maximize local density(all three ligands arranged around one side or one vertex)showed eff i cient silencing,whereas those with ligands distant from one another had lower silencing activity [32].At a dose of 2.5 mg/kg,folate-ONPs silenced luciferase expressioninthetumorby～60%withoutsignif i cant immunostimulation.

5.Conclusions and future prospects

As one of the most promising drugs for cancer treatment, siRNA has great advantages,such as excellent safety,high eff i cacy,unrestricted choice of targetsand specif i city.To solve the delivery problems of siRNA,many delivery systems have been developed.These highly effective delivery systems are quite different in terms of structure,size and chemistry,but there are still some guidelines regarding the characteristics of optimal delivery systems.Nanoparticulate delivery systems should have a particle size of about 20-200 nm,i.e.be large enough to avoid renal f i ltration but small enough to evade phagocytic clearance.PEG as the shieldingagenthas proven to be valuable in preventing non-specif i c interactions and avoiding immune recognition in the circulation[40].Chemical modif i cations,such as 2′-O-methyl substitutions,are necessary to minimize non-specif i c effects and avoid nuclease digestion.In addition,endogenous or exogenous targeting ligands are also often benef i cial for siRNA uptake by cancer cells.Although a number of reports have demonstrated the great potential of siRNA in cancer treatment,challenges remain in bringing the full potential of siRNA to the clinic,and most siRNA drug delivery systems are still in preclinical studies.In recent years,siRNA drug development has experienced highs and lows.The attitude of big pharmaceutical companies to RNAi drugs has also become over-optimistic.In summary,a good delivery system is the key to siRNA drug development.Once research into siRNA drug delivery systems makes a signif i cant breakthrough,siRNA will occupy a strong position in the drug market,especially the anti-cancer drug market.

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*Corresponding author.Tel./fax:+86(0)551 63600402.

E-mail address:jwang699@ustc.edu.cn(J.Wang).

Peer review under responsibility of Shenyang Pharmaceutical University.

http://dx.doi.org/10.1016/j.ajps.2014.08.011

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