• <tr id="yyy80"></tr>
  • <sup id="yyy80"></sup>
  • <tfoot id="yyy80"><noscript id="yyy80"></noscript></tfoot>
  • 99热精品在线国产_美女午夜性视频免费_国产精品国产高清国产av_av欧美777_自拍偷自拍亚洲精品老妇_亚洲熟女精品中文字幕_www日本黄色视频网_国产精品野战在线观看 ?

    Delivery systems for siRNA drug development in cancer therapy

    2015-05-15 08:29:18CongfeiXuJunWang

    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.

    REFERENCES

    [1]Fire A,Xu SQ,Montgomery MK,et al.Potent and specif i c genetic interference by double-stranded RNA in Caenorhabditis elegans.Nature 1998;391:806-811.

    [2]Elbashir SM,Harborth J,Lendeckel W,et al.Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells.Nature 2001;411:494-498.

    [3]Davis ME,Zuckerman JE,Choi CH,et al.Evidence of RNAi in humans from systemically administered siRNA via targeted nanoparticles.Nature 2010;464:1067-1070.

    [4]Vaishnaw AK,Gollob J,Gamba-Vitalo C,et al.A status report on RNAi therapeutics.Silence 2010;1:14.

    [5]Ohkuma S,Poole B.Fluorescence probe measurement of the intralysosomal pH in living cells and the perturbation of pH by various agents.Proc Natl Acad Sci 1978;75:3327-3331.

    [6]Dominska M,Dykxhoorn DM.Breaking down the barriers: siRNA delivery and endosome escape.J Cell Sci 2010;123:1183-1189.

    [7]Alexis F,Pridgen E,Molnar LK,et al.Factors affecting the clearance and biodistribution of polymeric nanoparticles. Mol Pharm 2008;5:505-515.

    [8]Layzer JM,McCaffrey AP,Tanner AK,et al.In vivo activity of nuclease-resistant siRNAs.RNA 2004;10:766-771.

    [9]Van de Water FM,Boerman OC,Wouterse AC,et al. Intravenously administered short interfering RNA accumulates in the kidney and selectively suppresses gene function in renal proximal tubules.Drug Metab Dispos 2006;34:1393-1397.

    [10]Mosser DM,Edwards JP.Exploring the full spectrum of macrophage activation.Nat Rev Immunol 2008;8:958-969.

    [11]Larson SD,Jackson LN,Chen LA,et al.Effectiveness of siRNA uptake in target tissues by various delivery methods.Surgery 2007;142:262-269.

    [12]Jackson AL,Linsley PS.Recognizing and avoiding siRNA offtarget effects for target identif i cation and therapeutic application.Nat Rev Drug Discov 2010;9:57-67.

    [13]Schultz N,Marenstein DR,De Angelis DA,et al.Off-target effects dominate a large-scale RNAi screen for modulators of the TGF-pathway and reveal microRNA regulation of TGFBR2.Silence 2011;2:1-20.

    [14]Hornung V,Guenthner-Biller M,Bourquin C,et al.Sequencespecif i c potent induction of IFN-α by short interfering RNA in plasmacytoid dendritic cells through TLR7.Nat Med 2005;11:263-270.

    [15]Judge AD,Sood V,Shaw JR,et al.Sequence-dependent stimulation of the mammalian innate immune response by synthetic siRNA.Nat Biotechnol 2005;23:457-462.

    [16]Karikˊo K,Bhuyan P,Capodici J,et al.Small interfering RNAs mediate sequence-independent gene suppression and induce immune activation by signaling through toll-like receptor 3.J Immunol 2004;172:6545-6549.

    [17]Marques JT,Williams BR.Activation of the mammalian immune system by siRNAs.Nat Biotechnol 2005;23:1399-1405.

    [18]Heil F,Hemmi H,Hochrein H,et al.Species-specif i c recognition of single-stranded RNA via toll-like receptor 7 and 8.Science 2004;303:1526-1529.

    [19]Iwasaki A,Medzhitov R.Toll-like receptor control of the adaptive immune responses.Nat Immunol 2004;5:987-995.

    [20]Whitehead KA,Langer R,Anderson DG.Knocking down barriers:advances in siRNA delivery.Nat Rev Drug Discov 2009;8:129-138.

    [21]Deleavey GF,Damha MJ.Designing chemically modif i ed oligonucleotides for targeted gene silencing.Chem Biol 2012;19:937-954.

    [22]Wang AZ,Langer R,Farokhzad OC.Nanoparticle delivery of cancer drugs.Annu Rev Med 2012;63:185-198.

    [23]Malek A,Merkel O,Fink L,et al.In vivo pharmacokinetics, tissue distribution and underlying mechanisms of various PEI(-PEG)/siRNA complexes.Toxicol Appl Toxicol 2009;236:97-108.

    [24]Wolfrum C,Shi S,Jayaprakash KN,et al.Mechanisms and optimization of in vivo delivery of lipophilic siRNAs.Nat Biotechnol 2007;25:1149-1157.

    [25]Akinc A,Querbes W,De S,et al.Targeted delivery of RNAi therapeutics with endogenous and exogenous ligand-based mechanisms.Mol Ther 2010;18:1357-1364.

    [26]Romberg B,Hennink WE,Storm G.Sheddable coatings for long-circulating nanoparticles.Pharm Res 2008;25:55-71.

    [27]Bazile D,Prud'homme C,Bassoullet MT,et al.Stealth Me. PEG-PLA nanoparticles avoid uptake by the mononuclear phagocytes system.J Pharm Sci 1995;84:493-498.

    [28]Martina MS,Nicolas V,Wilhelm C,et al.The in vitro kinetics of the interactions between PEG-ylated magnetic-f l uidloaded liposomes and macrophages.Biomaterials 2007;28:4143-4153.

    [29]Jarad G,Miner JH.Update on the glomerular f i ltration barrier. Curr Opin Nephrol Hypertens 2009;18:226.

    [31]Rappaport J,Hanss B,Kopp JB,et al.Transport of phosphorothioate oligonucleotides in kidney:implications for molecular therapy.Kidney Int 1995;47:1462-1469.

    [32]Lee H,Lytton-Jean AKR,Chen Y,et al.Molecularly selfassembled nucleic acid nanoparticles for targeted in vivo siRNA delivery.Nat Nanotechnol 2012;7:389-393.

    [33]Rozema DB,Lewis DL,Wakef i eld DH,et al.Dynamic PolyConjugates for targeted in vivo delivery of siRNA to hepatocytes.Proc Natl Acad Sci 2007;104:12982-12987.

    [34]Yu B,Zhao X,Lee LJ,et al.Targeted delivery systems for oligonucleotide therapeutics.AAPS J 2009;11:195-203.

    [35]Salvati A,Pitek AS,Monopoli MP,et al.Transferrinfunctionalized nanoparticles lose their targeting capabilities when a biomolecule corona adsorbs on the surface.Nat Nanotechnol 2013;8:137-143.

    [36]Bolhassani A.Potential eff i cacy of cell-penetrating peptides for nucleic acid and drug delivery in cancer.Biochim Biophys Acta-Rev Cancer 2011;1816:232-246.

    [37]Shim MS,Kwon YJ.Stimuli-responsive polymers and nanomaterials for gene delivery and imaging applications. Adv Drug Deliv Rev 2012;64:1046-1059.

    [38]Barquinero J,Eixarch H,Perez-Melgosa M.Retroviral vectors:new applications for an old tool.Gene Ther 2004;11:S3-9.

    [39]Vandenbroucke RE,De Geest BG,Bonnˊe S,et al.Prolonged gene silencing in hepatoma cells and primary hepatocytes after small interfering RNA delivery with biodegradable poly (β-amino esters).J Gene Med 2008;10:783-794.

    [40]Kanasty R,Dorkin JR,Vegas A,et al.Delivery materials for siRNA therapeutics.Nat Mater 2013;12:967-977.

    [41]Juliano R,Alam MR,Dixit V,et al.Mechanisms and strategies for effective delivery of antisense and siRNA oligonucleotides.Nucleic Acids Res 2008;36:4158-4171.

    [42]Aigner A.Nonviral in vivo delivery of therapeutic small interfering RNAs.Curr Opin Mol Ther 2007;9:345-352.

    [43]Braasch DA,Jensen S,Liu Y,et al.RNA interference in mammalian cells by chemically-modif i ed RNA.Biochemistry 2003;42:7967-7975.

    [44]Chiu YL,Rana TM.siRNA function in RNAi:a chemical modif i cation analysis.RNA 2003;9:1034-1048.

    [45]Dande P,Prakash TP,Siouf iN,et al.Improving RNA interference in mammalian cells by 4′-thio-modif i ed small interfering RNA(siRNA):effect on siRNA activity and nuclease stability when used in combination with 2′-O-alkyl modif i cations.J Med Chem 2006;49:1624-1634.

    [46]Hall AH,Wan J,Shaughnessy EE,et al.RNA interference using boranophosphate siRNAs:structure-activity relationships.Nucleic Acids Res 2004;32:5991-6000.

    [47]Santel A,Aleku M,Keil O,et al.A novel siRNA-lipoplex technology for RNA interference in the mouse vascular endothelium.Gene Ther 2006;1:1222-1234.

    [48]Hayes ME,Drummond DC,Hong K,et al.Assembly of nucleic acid-lipid nanoparticles from aqueous-organic monophases.Biochim Biophys Acta Biomembr 2006;1758:429-442.

    [49]Khoury M,Louis-Plence P,Escriou V,et al.Eff i cient new cationic liposome formulation for systemic delivery of small interfering RNA silencing tumor necrosis factor α in experimental arthritis.Arthritis Rheum 2006;54:1867-1877.

    [50]Zuhorn IS,Oberle V,Visser WH,et al.Phase behavior of cationic amphiphiles and their mixtures with helper lipid inf l uences lipoplex shape,DNA translocation,and transfection eff i ciency.Biophys J 2002;83:2096-2108.

    [51]Landen CN,Chavez-Reyes A,Bucana C,et al.Therapeutic EphA2 gene targeting in vivo using neutral liposomal small interfering RNA delivery.Cancer Res 2005;65:6910-6918.

    [52]Wu SY,McMillan NA.Lipidic systems for in vivo siRNA delivery.AAPS J 2009;11:639-652.

    [53]S?rensen DR,Leirdal M,Sioud M.Gene silencing by systemic delivery of synthetic siRNAs in adult mice.J Mol Biol 2003;327:761-766.

    [54]Bao Y,Jin Y,Chivukula P,et al.Effect of PEGylation on biodistribution and gene silencing of siRNA/lipid nanoparticle complexes.Pharm Res 2013;30:342-351.

    [55]Lin SY,Zhao WY,Tsai HC,et al.Sterically polymer-based liposomal complexes with dual-shell structure for enhancing the siRNA delivery.Biomacromolecules 2012;13:664-675.

    [56]Aleku M,Schulz P,Keil O,et al.Atu027,a liposomal small interfering RNA formulation targeting protein kinase N3, inhibits cancer progression.Cancer Res 2008;68:9788-9798.

    [57]Strumberg D,Schultheis B,Traugott U,et al.Phase I clinical development of Atu027,a siRNA formulation targeting PKN3 in patients with advanced solid tumors.Int J Clin Pharmacol Ther 2012;50:76.

    [58]Morrissey DV,Lockridge JA,Shaw L,et al.Potent and persistent in vivo anti-HBV activity of chemically modif i ed siRNAs.Nat Biotechnol 2005;23:1002-1007.

    [59]Judge AD,Robbins M,Tavakoli I,et al.Conf i rming the RNAimediated mechanism of action of siRNA-based cancer therapeutics in mice.J Clin Invest 2009;119:661.

    [60]Shen H,Sun T,Ferrari M.Nanovector delivery of siRNA for cancer therapy.Cancer Gene Ther 2012;19:367-373.

    [61]Akinc A,Zumbuehl A,Goldberg M,et al.A combinatorial library of lipid-like materials for delivery of RNAi therapeutics.Nat Biotechnol 2008;26:561-569.

    [62]Sahoo SK,Labhasetwar V.Nanotech approaches to drug delivery and imaging.Drug Discov Today 2003;8:1112-1120.

    [63]Wang X,Wang Y,Chen ZG,et al.Advances of cancer therapy by nanotechnology.Cancer Res Treat 2009;41:1-11.

    [64]Davis ME.The f i rst targeted delivery of siRNA in humans via a self-assembling,cyclodextrin polymer-basednanoparticle:from concept to clinic.Mol Pharm 2009;6:659-668.

    [65]Gonzalez H,Hwang SJ,Davis ME.New class of polymers for the delivery of macromolecular therapeutics.Bioconjug Chem 1999;10:1068-1074.

    [66]Bellocq NC,Pun SH,Jensen GS,et al.Transferrincontaining,cyclodextrin polymer-based particles for tumor-targeted gene delivery.Bioconjug Chem 2003;14:1122-1132.

    [67]Bartlett DW,Davis ME.Insights into the kinetics of siRNA-mediated gene silencing from live-cell and live-animal bioluminescent imaging.Nucleic Acids Res 2006;34:322-333.

    [68]Bartlett DW,Su H,Hildebrandt IJ,et al.Impact of tumorspecif i c targeting on the biodistribution and eff i cacy of siRNA nanoparticles measured by multimodality in vivo imaging. Proc Natl Acad Sci 2007;104:15549-15554.

    [69]Urban-Klein B,Werth S,Abuharbeid S,et al.RNAi-mediated gene-targeting through systemic application of polyethylenimine(PEI)-complexed siRNA in vivo.Gene Ther 2005;12:461-466.

    [70]Philipp A,Zhao X,Tarcha P,et al.Hydrophobically modif i ed oligoethylenimines as highly eff i cient transfection agents for siRNA delivery.Bioconjug Chem 2009;20:2055-2061.

    [71]Grayson AC,Doody AM,Putnam D.Biophysical and structural characterization of polyethylenimine-mediated siRNA delivery in vitro.Pharm Res 2006;23:1868-1876.

    [72]Navarro G,Sawant RR,Essex S,et al. Phospholipid-polyethylenimine conjugate-based micellelike nanoparticles for siRNA delivery.Drug Deliv Transl Res 2011;1:25-33.

    [73]Navarro G,Sawant RR,Biswas S,et al.P-glycoprotein silencing with siRNA delivered by DOPE-modif i ed PEI overcomes doxorubicin resistance in breast cancer cells. Nanomedicine 2012;7:65-78.

    [74]Sun TM,Du JZ,Yan LF,et al.Self-assembled biodegradable micellar nanoparticles of amphiphilic and cationic block copolymer for siRNA delivery.Biomaterials 2008;29:4348-4355.

    [75]Mao CQ,Du JZ,Sun TM,et al.A biodegradable amphiphilic and cationic triblock copolymer for the delivery of siRNA targeting the acid ceramidase gene for cancer therapy. Biomaterials 2011;32:3124-3133.

    [76]Liu XQ,Xiong MH,Shu XT,et al.Therapeutic delivery of siRNA silencing HIF-1 alpha with micellar nanoparticles inhibits hypoxic tumor growth.Mol Pharm 2012;9:2863-2874.

    [77]Mao CQ,Xiong MH,Liu Y,et al.Synthetic lethal therapy for KRAS mutant non-small-cell lung carcinoma with nanoparticle-mediated CDK4 siRNA Delivery.Mol Ther 2014;24:964-973.http://dx.doi.org/10.1038/mt.2014.18.

    [78]Singh M,Briones M,Ott G,et al.Cationic microparticles:a potent delivery system for DNA vaccines.Proc Natl Acad Sci 2000;97:811-816.

    [79]Jilek S,Merkle HP,Walter E.DNA-loaded biodegradable microparticles as vaccine delivery systems and their interaction with dendritic cells.Adv Drug Deliv Rev 2005;57:377-390.

    [80]Luu YK,Kim K,Hsiao BS,et al.Development of a nanostructured DNA delivery scaffold via electrospinning of PLGA and PLA-PEG block copolymers.J Control Release 2003;89:341-353.

    [81]Woodrow KA,Cu Y,Booth CJ,et al.Intravaginal gene silencing using biodegradable polymer nanoparticles densely loaded with small-interfering RNA.Nat Mater 2009;8:526-533.

    [82]Yang XZ,Dou S,Sun TM,et al.Systemic delivery of siRNA with cationic lipid assisted PEG-PLA nanoparticles for cancer therapy.J Control Release 2011;156:203-211.

    [83]Shen S,Mao CQ,Yang XZ,et al.Cationic lipid-assisted polymeric nanoparticles-mediated GATA2 siRNA delivery for synthetic lethal therapy of KRAS mutant non-small-cell lung carcinoma.Mol Pharm 2014;11:2612-2622.http://dx.doi.org/ 10.1021/mp400714z.

    [84]Jeong JH,Mok H,Oh YK,et al.siRNA conjugate delivery systems.Bioconjug Chem 2008;20:5-14.

    [85]Soutschek J,Akinc A,Bramlage B,et al.Therapeutic silencing of an endogenous gene by systemic administration of modif i ed siRNAs.Nature 2004;432:173-178.

    [86]Chiu YL,Ali A,Chu C,et al.Visualizing a correlation between siRNA localization,cellular uptake,and RNAi in living cells. Chem Biol 2004;11:1165-1175.

    [87]Moschos SA,Jones SW,Perry MM,et al.Lung delivery studies using siRNA conjugated to TAT(48-60)and penetratin reveal peptide induced reduction in gene expression and induction of innate immunity.Bioconjug Chem 2007;18:1450-1459.

    [88]Cesarone G,Edupuganti OP,Chen CP,et al.Insulin receptor substrate 1 knockdown in human MCF7 ER+ breast cancer cells by nuclease-resistant IRS1 siRNA conjugated to a disulf i de-bridged D-peptide analogue of insulin-like growth factor 1.Bioconjug Chem 2007;18:1831-1840.

    [89]Xia CF,Zhang Y,Zhang Y,et al.Intravenous siRNA of brain cancer with receptor targeting and avidin-biotin technology. Pharm Res 2007;24:2309-2316.

    [90]Chu TC,Twu KY,Ellington AD,et al.Aptamer mediated siRNA delivery.Nucleic Acids Res 2006;34:e73.

    [91]Simpson RJ,Lim JWE,Moritz RL,et al.Exosomes:proteomic insights and diagnostic potential.Expert Rev Proteomics 2009;6:267-283.

    [92]Baj-Krzyworzeka M,Szatanek R,We?glarczyk K,et al. Tumour-derived microvesicles carry several surface determinants and mRNA of tumour cells and transfer some of these determinants to monocytes.Cancer Immunol Immunother 2006;55:808-818.

    [93]Ratajczak J,Miekus K,Kucia M,et al.Embryonic stem cellderived microvesicles reprogram hematopoietic progenitors: evidence for horizontal transfer of mRNA and protein delivery.Leukemia 2006;20:847-856.

    [95]Mittelbrunn M,Gutiˊerrez-Vˊazquez C,Villarroya-Beltri C, et al.Unidirectional transfer of microRNA-loaded exosomes from T cells to antigen-presenting cells.Nat Commun 2011;2:282.

    [96]Montecalvo A,Larregina AT,Shufesky WJ,et al.Mechanism of transfer of functional microRNAs between mouse dendritic cells via exosomes.Blood 2012;119:756-766.

    [97]Alvarez-Erviti L,Seow Y,Yin HF,et al.Delivery of siRNA to the mouse brain by systemic injection of targeted exosomes. Nat Biotechnol 2011;29:341-345.

    [98]El-Andaloussi S,Lee Y,Lakhal-Littleton S,et al.Exosomemediated delivery of siRNA in vitro and in vivo.Nat Protoc 2012;7:2112-2126.

    [99]Goodman RP,Schaap IAT,Tardin CF,et al.Rapid chiral assembly of rigid DNA building blocks for molecular nanofabrication.Science 2005;310:1661-1665.

    *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

    1818-0876/?2015 Shenyang Pharmaceutical University.Production and hosting by Elsevier B.V.All rights reserved.

    久久久国产精品麻豆| 国产片特级美女逼逼视频| 99精国产麻豆久久婷婷| 考比视频在线观看| 99热这里只有是精品在线观看| 亚州av有码| 欧美日韩亚洲高清精品| 一级毛片电影观看| 成年人免费黄色播放视频| 国产精品一区www在线观看| 久久久久久久久大av| 大话2 男鬼变身卡| 91精品一卡2卡3卡4卡| 欧美 日韩 精品 国产| 久久国产亚洲av麻豆专区| 亚洲精品乱久久久久久| 久久精品久久久久久噜噜老黄| 免费观看在线日韩| 国产欧美亚洲国产| 亚洲不卡免费看| 亚洲欧洲日产国产| 久久免费观看电影| 亚洲欧美成人精品一区二区| 午夜老司机福利剧场| 99热网站在线观看| 日韩人妻高清精品专区| 在线精品无人区一区二区三| 亚洲少妇的诱惑av| 一本一本综合久久| 国产成人av激情在线播放 | 日韩,欧美,国产一区二区三区| 亚洲精品乱码久久久v下载方式| 另类精品久久| 秋霞在线观看毛片| 亚洲色图综合在线观看| 黑人猛操日本美女一级片| 最新中文字幕久久久久| 永久网站在线| 国产日韩欧美在线精品| 欧美成人精品欧美一级黄| 秋霞在线观看毛片| 麻豆乱淫一区二区| 日韩强制内射视频| 国产精品成人在线| 91久久精品电影网| 国产有黄有色有爽视频| 久久久久久人妻| 久久久久网色| 午夜91福利影院| 亚洲欧美成人综合另类久久久| 少妇的逼水好多| 久久久精品区二区三区| 久久久久人妻精品一区果冻| 只有这里有精品99| www.av在线官网国产| 97在线人人人人妻| 人妻系列 视频| 曰老女人黄片| 亚洲av欧美aⅴ国产| h视频一区二区三区| 久久久久久人妻| 国产片特级美女逼逼视频| 午夜老司机福利剧场| av免费在线看不卡| 大香蕉久久成人网| 午夜日本视频在线| 天天操日日干夜夜撸| 性高湖久久久久久久久免费观看| 97超碰精品成人国产| 十八禁高潮呻吟视频| 久热久热在线精品观看| 一本一本综合久久| 日本免费在线观看一区| 国产乱人偷精品视频| 99热这里只有是精品在线观看| 九色成人免费人妻av| 精品熟女少妇av免费看| 80岁老熟妇乱子伦牲交| 极品少妇高潮喷水抽搐| 九色亚洲精品在线播放| 免费观看的影片在线观看| 精品卡一卡二卡四卡免费| 亚洲av电影在线观看一区二区三区| 日韩大片免费观看网站| 极品人妻少妇av视频| 欧美日韩在线观看h| 老女人水多毛片| 天堂俺去俺来也www色官网| 婷婷色综合www| 一区二区三区乱码不卡18| 欧美精品一区二区免费开放| 亚洲av二区三区四区| 尾随美女入室| 国产免费又黄又爽又色| 女的被弄到高潮叫床怎么办| 午夜激情福利司机影院| 春色校园在线视频观看| 久久 成人 亚洲| 亚洲精品久久午夜乱码| 2018国产大陆天天弄谢| 最近最新中文字幕免费大全7| 91aial.com中文字幕在线观看| 黄色配什么色好看| 亚洲三级黄色毛片| 嘟嘟电影网在线观看| 欧美成人精品欧美一级黄| 伊人久久国产一区二区| 在线观看人妻少妇| 国产成人精品一,二区| 国产午夜精品一二区理论片| 少妇 在线观看| 最后的刺客免费高清国语| 搡老乐熟女国产| 日韩三级伦理在线观看| 亚洲综合色惰| 亚洲欧美一区二区三区黑人 | 成人手机av| a级毛片在线看网站| 免费观看av网站的网址| 国产精品一区二区三区四区免费观看| 男女无遮挡免费网站观看| 亚洲美女搞黄在线观看| 涩涩av久久男人的天堂| 久久久国产欧美日韩av| 人人妻人人添人人爽欧美一区卜| 久久久国产一区二区| 亚洲精品日韩在线中文字幕| 99re6热这里在线精品视频| 久久久欧美国产精品| 亚洲精品中文字幕在线视频| av国产精品久久久久影院| 99热国产这里只有精品6| 欧美激情国产日韩精品一区| 一区二区日韩欧美中文字幕 | 伦理电影免费视频| 午夜精品国产一区二区电影| 综合色丁香网| 国产成人精品在线电影| 中文字幕久久专区| 一区二区三区乱码不卡18| 日本vs欧美在线观看视频| 日本免费在线观看一区| 精品国产一区二区三区久久久樱花| kizo精华| 少妇被粗大的猛进出69影院 | 国产精品女同一区二区软件| 伦理电影免费视频| av天堂久久9| 青春草亚洲视频在线观看| 91久久精品国产一区二区三区| 久久ye,这里只有精品| 成人18禁高潮啪啪吃奶动态图 | 人妻人人澡人人爽人人| 一级毛片电影观看| 制服诱惑二区| 精品人妻偷拍中文字幕| 色94色欧美一区二区| 精品国产一区二区三区久久久樱花| 永久免费av网站大全| 中文欧美无线码| 精品久久久久久电影网| 国产精品免费大片| 26uuu在线亚洲综合色| 在线观看免费视频网站a站| 人人澡人人妻人| 精品久久国产蜜桃| 一级二级三级毛片免费看| 亚洲天堂av无毛| 欧美日韩在线观看h| 亚州av有码| 成人影院久久| 亚洲欧美日韩卡通动漫| 免费观看无遮挡的男女| 2021少妇久久久久久久久久久| 丝袜脚勾引网站| 超碰97精品在线观看| 美女视频免费永久观看网站| 欧美精品国产亚洲| 国产午夜精品久久久久久一区二区三区| 国产午夜精品久久久久久一区二区三区| 狠狠婷婷综合久久久久久88av| 18+在线观看网站| 亚洲欧美成人精品一区二区| 丝袜喷水一区| 国产日韩欧美亚洲二区| 日本欧美视频一区| 久久这里有精品视频免费| 日韩一区二区视频免费看| 欧美+日韩+精品| 亚洲第一区二区三区不卡| 成人手机av| 美女国产视频在线观看| 欧美精品一区二区大全| 亚洲av欧美aⅴ国产| 国产精品国产三级专区第一集| 日本色播在线视频| 国产精品 国内视频| 99国产综合亚洲精品| 99精国产麻豆久久婷婷| 免费观看av网站的网址| 久久久久久久久久久久大奶| 亚洲精品,欧美精品| 国产欧美日韩综合在线一区二区| 国产成人91sexporn| 有码 亚洲区| 久久午夜综合久久蜜桃| 在线观看美女被高潮喷水网站| 国产精品女同一区二区软件| 嫩草影院入口| 久久精品久久久久久久性| 一区二区日韩欧美中文字幕 | 国产日韩一区二区三区精品不卡 | 少妇 在线观看| 久久久久国产精品人妻一区二区| 国产精品久久久久久精品电影小说| 久久久久国产精品人妻一区二区| 亚洲人成网站在线播| 另类亚洲欧美激情| 国产亚洲av片在线观看秒播厂| 久久久国产欧美日韩av| 欧美激情 高清一区二区三区| 九九在线视频观看精品| 蜜桃在线观看..| 欧美人与善性xxx| 寂寞人妻少妇视频99o| 欧美日韩精品成人综合77777| 亚洲伊人久久精品综合| 69精品国产乱码久久久| 精品一区二区免费观看| 久久久精品免费免费高清| 精品熟女少妇av免费看| 啦啦啦在线观看免费高清www| 最近中文字幕高清免费大全6| 大香蕉97超碰在线| 久久久久久久国产电影| 久久这里有精品视频免费| 亚洲国产日韩一区二区| 久久热精品热| 如日韩欧美国产精品一区二区三区 | 少妇高潮的动态图| 久久热精品热| 亚洲精品自拍成人| 日本午夜av视频| 各种免费的搞黄视频| 国产视频首页在线观看| 精品少妇内射三级| 久久久精品区二区三区| 99热6这里只有精品| 亚洲国产成人一精品久久久| h视频一区二区三区| 最近中文字幕2019免费版| 少妇丰满av| 亚洲精品色激情综合| 免费大片18禁| 成年人午夜在线观看视频| 中文字幕免费在线视频6| 在线观看一区二区三区激情| 久久av网站| 特大巨黑吊av在线直播| 大又大粗又爽又黄少妇毛片口| 在线观看人妻少妇| 免费高清在线观看日韩| 黑丝袜美女国产一区| 日韩人妻高清精品专区| 婷婷色麻豆天堂久久| 日本av免费视频播放| 18禁裸乳无遮挡动漫免费视频| 精品一区在线观看国产| 久久午夜福利片| 超碰97精品在线观看| 一级毛片aaaaaa免费看小| 黑丝袜美女国产一区| 如日韩欧美国产精品一区二区三区 | 天堂中文最新版在线下载| 91国产中文字幕| 一区二区av电影网| 色哟哟·www| 一二三四中文在线观看免费高清| 久久人人爽人人片av| 老司机亚洲免费影院| 成人二区视频| 国产亚洲精品第一综合不卡 | 成年av动漫网址| 亚洲精品久久成人aⅴ小说 | 亚洲精品亚洲一区二区| 国产精品秋霞免费鲁丝片| 一级a做视频免费观看| 久久午夜福利片| 美女视频免费永久观看网站| 免费看不卡的av| 日韩一本色道免费dvd| 草草在线视频免费看| av国产精品久久久久影院| 色婷婷久久久亚洲欧美| 亚洲精品国产av蜜桃| 秋霞伦理黄片| 国产伦理片在线播放av一区| 欧美精品一区二区大全| 亚洲av男天堂| 中文字幕精品免费在线观看视频 | 国产亚洲精品久久久com| 女的被弄到高潮叫床怎么办| 久久这里有精品视频免费| 国产精品99久久久久久久久| 久久人妻熟女aⅴ| 久久精品国产自在天天线| 美女主播在线视频| 亚洲av电影在线观看一区二区三区| 熟女人妻精品中文字幕| 久热久热在线精品观看| 精品一品国产午夜福利视频| 我的老师免费观看完整版| 大码成人一级视频| 美女cb高潮喷水在线观看| 久久久久久人妻| 欧美最新免费一区二区三区| 免费观看a级毛片全部| 国产极品粉嫩免费观看在线 | av不卡在线播放| 中文字幕免费在线视频6| 五月开心婷婷网| 一区二区三区精品91| 精品午夜福利在线看| 自拍欧美九色日韩亚洲蝌蚪91| 亚洲,一卡二卡三卡| 一区二区三区免费毛片| 亚洲精品第二区| 亚洲图色成人| 国产毛片在线视频| 日本免费在线观看一区| 一区在线观看完整版| 亚州av有码| 卡戴珊不雅视频在线播放| 欧美丝袜亚洲另类| 国产精品国产av在线观看| 2022亚洲国产成人精品| √禁漫天堂资源中文www| 欧美日韩av久久| 久热久热在线精品观看| 熟女av电影| av播播在线观看一区| 777米奇影视久久| 亚洲综合精品二区| 中文字幕免费在线视频6| 伦理电影大哥的女人| 午夜福利网站1000一区二区三区| 七月丁香在线播放| 亚洲欧洲精品一区二区精品久久久 | 免费黄网站久久成人精品| 国产精品99久久久久久久久| 成人毛片a级毛片在线播放| 中文精品一卡2卡3卡4更新| 亚洲婷婷狠狠爱综合网| 久久精品国产亚洲网站| 亚洲av成人精品一区久久| 边亲边吃奶的免费视频| 搡女人真爽免费视频火全软件| 国产免费一区二区三区四区乱码| 精品久久久噜噜| 亚洲精品乱码久久久久久按摩| 综合色丁香网| 妹子高潮喷水视频| 成人亚洲精品一区在线观看| 色婷婷av一区二区三区视频| 成人国语在线视频| 亚洲国产精品专区欧美| 能在线免费看毛片的网站| 日韩精品免费视频一区二区三区 | 亚洲av在线观看美女高潮| 欧美一级a爱片免费观看看| 少妇高潮的动态图| 日本vs欧美在线观看视频| 亚洲精品日本国产第一区| 国产午夜精品一二区理论片| 满18在线观看网站| 各种免费的搞黄视频| 99久久精品国产国产毛片| 80岁老熟妇乱子伦牲交| 免费观看a级毛片全部| 王馨瑶露胸无遮挡在线观看| 日本欧美视频一区| 熟女电影av网| 国产一区亚洲一区在线观看| 亚洲欧美一区二区三区国产| 成人综合一区亚洲| 国产有黄有色有爽视频| 日日撸夜夜添| 99久久综合免费| 丁香六月天网| 制服丝袜香蕉在线| 国产精品国产三级国产av玫瑰| 狂野欧美激情性bbbbbb| 99国产精品免费福利视频| 精品一区二区免费观看| a 毛片基地| 黄色欧美视频在线观看| 免费观看av网站的网址| 中文精品一卡2卡3卡4更新| 我要看黄色一级片免费的| av在线app专区| 肉色欧美久久久久久久蜜桃| 亚洲精品乱久久久久久| 午夜激情福利司机影院| a级片在线免费高清观看视频| 国产一区二区三区av在线| 高清不卡的av网站| 国产精品麻豆人妻色哟哟久久| 久久免费观看电影| 久久青草综合色| 丝瓜视频免费看黄片| 亚洲av日韩在线播放| 欧美性感艳星| 亚洲图色成人| 欧美精品人与动牲交sv欧美| 一区二区三区四区激情视频| 在线观看人妻少妇| 寂寞人妻少妇视频99o| 欧美日韩精品成人综合77777| 国产精品久久久久成人av| 九九爱精品视频在线观看| 国产免费现黄频在线看| 99热国产这里只有精品6| 亚洲,一卡二卡三卡| 久久久久久人妻| 女人久久www免费人成看片| 自拍欧美九色日韩亚洲蝌蚪91| 热99国产精品久久久久久7| a级片在线免费高清观看视频| 欧美亚洲 丝袜 人妻 在线| 国产综合精华液| 日本91视频免费播放| 蜜臀久久99精品久久宅男| 日日爽夜夜爽网站| 亚洲丝袜综合中文字幕| 久久精品久久久久久噜噜老黄| 久久国内精品自在自线图片| 国产精品不卡视频一区二区| 国产伦精品一区二区三区视频9| a 毛片基地| 99国产精品免费福利视频| 亚洲av男天堂| 在线 av 中文字幕| 大香蕉久久网| 一级a做视频免费观看| 国产色爽女视频免费观看| 三级国产精品欧美在线观看| 国产成人av激情在线播放 | 日韩在线高清观看一区二区三区| 亚洲av成人精品一二三区| 好男人视频免费观看在线| 大又大粗又爽又黄少妇毛片口| 国产黄频视频在线观看| 久久97久久精品| 黄色欧美视频在线观看| 91国产中文字幕| 99九九在线精品视频| 国产午夜精品一二区理论片| 少妇丰满av| 国产精品久久久久久精品电影小说| 在线播放无遮挡| 少妇人妻 视频| 在线免费观看不下载黄p国产| 人妻制服诱惑在线中文字幕| 九色成人免费人妻av| 男男h啪啪无遮挡| 一级毛片我不卡| 97在线人人人人妻| 国产黄色免费在线视频| 毛片一级片免费看久久久久| av专区在线播放| 欧美日韩视频精品一区| 最近手机中文字幕大全| 国产毛片在线视频| 少妇的逼好多水| 国产在线一区二区三区精| 中文欧美无线码| 亚洲精品日本国产第一区| 亚洲中文av在线| 欧美另类一区| 乱人伦中国视频| 黄片无遮挡物在线观看| 在线 av 中文字幕| 国产白丝娇喘喷水9色精品| 丰满迷人的少妇在线观看| 一个人免费看片子| 久久久久精品久久久久真实原创| 久久精品夜色国产| 全区人妻精品视频| 国产av国产精品国产| 亚洲丝袜综合中文字幕| 亚洲美女视频黄频| 亚洲精品中文字幕在线视频| 精品熟女少妇av免费看| 国产视频首页在线观看| av在线老鸭窝| 欧美日韩成人在线一区二区| 在线观看免费高清a一片| 一级毛片aaaaaa免费看小| 制服诱惑二区| 亚洲精品日本国产第一区| 国产探花极品一区二区| 一本色道久久久久久精品综合| 国产爽快片一区二区三区| 成年美女黄网站色视频大全免费 | 伊人久久国产一区二区| 国产精品一国产av| 日韩 亚洲 欧美在线| 欧美日韩一区二区视频在线观看视频在线| 国产免费视频播放在线视频| 亚洲,欧美,日韩| 97在线视频观看| videos熟女内射| 国产极品粉嫩免费观看在线 | 久久婷婷青草| 成人国语在线视频| 精品99又大又爽又粗少妇毛片| 国产精品成人在线| 亚洲欧美日韩另类电影网站| 一级,二级,三级黄色视频| 国产 精品1| 国产精品一区二区三区四区免费观看| 777米奇影视久久| 久久久久久久久久久丰满| 国产精品一国产av| 国产成人aa在线观看| 99视频精品全部免费 在线| 青青草视频在线视频观看| 久久久精品免费免费高清| 亚洲精品乱久久久久久| 高清毛片免费看| 亚洲色图 男人天堂 中文字幕 | av免费在线看不卡| 中文字幕制服av| 国产精品偷伦视频观看了| 日韩 亚洲 欧美在线| 国产免费一级a男人的天堂| 嫩草影院入口| 岛国毛片在线播放| 中国国产av一级| 免费看不卡的av| 久久久国产一区二区| 新久久久久国产一级毛片| 最黄视频免费看| 99热6这里只有精品| 亚洲精品aⅴ在线观看| 一区二区三区乱码不卡18| 大香蕉久久网| 国产综合精华液| 国产伦理片在线播放av一区| 午夜久久久在线观看| 亚洲少妇的诱惑av| 国产国语露脸激情在线看| 亚洲国产欧美日韩在线播放| 日韩av在线免费看完整版不卡| 免费日韩欧美在线观看| 你懂的网址亚洲精品在线观看| 少妇猛男粗大的猛烈进出视频| 国产探花极品一区二区| 欧美少妇被猛烈插入视频| 国产一区有黄有色的免费视频| 少妇人妻 视频| 国产爽快片一区二区三区| 3wmmmm亚洲av在线观看| 亚洲成人一二三区av| 国产精品成人在线| 天堂俺去俺来也www色官网| 日韩人妻高清精品专区| 曰老女人黄片| 人成视频在线观看免费观看| a级毛片黄视频| 人成视频在线观看免费观看| 男人爽女人下面视频在线观看| 边亲边吃奶的免费视频| 亚洲婷婷狠狠爱综合网| 人人妻人人澡人人爽人人夜夜| 久久97久久精品| 99国产精品免费福利视频| 有码 亚洲区| 日韩欧美一区视频在线观看| 黑人猛操日本美女一级片| 久久久欧美国产精品| 亚洲av电影在线观看一区二区三区| 国产精品国产av在线观看| 在线观看国产h片| 一本一本久久a久久精品综合妖精 国产伦在线观看视频一区 | 久久人人爽av亚洲精品天堂| 考比视频在线观看| 午夜久久久在线观看| 人妻 亚洲 视频| 狂野欧美激情性xxxx在线观看| 99国产精品免费福利视频| 色视频在线一区二区三区| 女性生殖器流出的白浆| 久久国内精品自在自线图片| 成人毛片60女人毛片免费| 国内精品宾馆在线| 日本-黄色视频高清免费观看| 色婷婷久久久亚洲欧美| 久久热精品热| 久久人人爽人人片av| av播播在线观看一区| 丰满饥渴人妻一区二区三| 国产午夜精品久久久久久一区二区三区| 丰满饥渴人妻一区二区三| 日本av免费视频播放| 性色av一级| 看非洲黑人一级黄片| 99久久人妻综合| 日韩大片免费观看网站| 免费少妇av软件| 亚洲一级一片aⅴ在线观看| 国产日韩欧美在线精品| 国产一区亚洲一区在线观看| 美女中出高潮动态图| 精品久久国产蜜桃|