Báo cáo khoa học: Efficient and targeted delivery of siRNA in vivo pdf

Moreover, the first evidence Keywords administration routes; barriers in siRNA delivery; chemically modified RNA; in vivo disease models; nanoparticles; nonviral carriers; nucleic acid th

Efficient and targeted delivery of siRNA in vivo

Min Suk Shim1and Young Jik Kwon1,2,3

1 Department of Chemical Engineering and Materials Science, University of California, Irvine, CA, USA

2 Department of Pharmaceutical Sciences, University of California, Irvine, CA, USA

3 Department of Biomedical Engineering, University of California, Irvine, CA, USA

Introduction

RNA interference (RNAi) is a highly conserved

biologi-cal process among yeasts, worms, insects, plants and

humans [1] A single strand of exogenously introduced

double-stranded small interfering RNA (siRNA; 20–30

nucleotides) guides an RNA-inducing silencing protein

complex to degrade the mRNA with the matching

sequence; thus, translation into the target proteins is

silenced [2–4] RNAi has been of great interest not only

as a powerful research tool to suppress the expression of

a target gene, but also as an emerging therapeutic

strat-egy to silence disease genes [5] Theoretically, siRNA

can interfere with the translation of almost any mRNA,

as long as the mRNA has a distinctive sequence, whereas the targets of traditional drugs are limited by types of cellular receptors and enzymes [6]

Cancer, viral infections, autoimmune diseases and neurodegenerative diseases have been explored as promising disease targets of RNAi [7,8] Recent pro-gress in clinical trials using siRNA to cure age-related macular degeneration (bevasiranib; Opko Health, Inc., Miami, FL, USA; phase III) and respiratory syncytial virus infection (ALN-RSV01; Alnylam, Cambridge,

MA, USA; phase II) have demonstrated the therapeu-tic potential of RNAi [9] Moreover, the first evidence

Keywords

administration routes; barriers in siRNA

delivery; chemically modified RNA; in vivo

disease models; nanoparticles; nonviral

carriers; nucleic acid therapeutics; RNA

interference; targeted delivery in vivo;

viral vectors

Correspondence

Y J Kwon, Department of Pharmaceutical

Sciences, 916 Engineering Tower,

University of California, Irvine, CA 92697,

USA

Fax: +1 949 824 2541

Tel: +1 949 824 8714

E-mail: kwonyj@uci.edu

(Received 7 July 2010, accepted

26 August 2010)

doi:10.1111/j.1742-4658.2010.07904.x

RNA interference (RNAi) has been regarded as a revolutionary tool for manipulating target biological processes as well as an emerging and prom-ising therapeutic strategy In contrast to the tangible and obvious effective-ness of RNAi in vitro, silencing target gene expression in vivo using small interfering RNA (siRNA) has been a very challenging task due to multiscale barriers, including rapid excretion, low stability in blood serum, nonspecific accumulation in tissues, poor cellular uptake and inefficient intracellular release This minireview introduces major challenges in achiev-ing efficient siRNA delivery in vivo and discusses recent advances in over-coming them using chemically modified siRNA, viral siRNA vectors and nonviral siRNA carriers Enhanced specificity and efficiency of RNAi

in vivo via selective accumulations in desired tissues, specific binding to target cells and facilitated intracellular trafficking are also commonly attempted utilizing targeting moieties, cell-penetrating peptides, fusogenic peptides and stimuli-responsive polymers Overall, the crucial roles of the interdisciplinary approaches to optimizing RNAi in vivo, by efficiently and specifically delivering siRNA to target tissues and cells, are highlighted

Abbreviations

ApoB, apolipoprotein B; CPP, cell-penetrating peptide; FA, folic acid; GFP, green fluorescent protein; HER-2, human epidermal growth factor 2; i.p., intraperitoneal; i.t., intratumoral; i.v., intravenous; 9R, nonamer arginine residues; RGD, Arg-Gly-Asp peptide; RNAi,

RNA interference; siRNA, small interfering RNA; VEGF, vascular endothelial growth factor.

of targeted in vivo gene silencing for human cancer

therapy via systemic delivery of siRNA using

transfer-rin-tagged, cyclodextrin-based polymeric nanoparticles

(CALAA-01; Calando Pharmaceuticals, Pasadena, CA,

USA; phase I) has been recently announced [10]

Despite quite efficient and reliable gene silencing

in vitro, only limited RNAi has been achieved in vivo

because of rapid enzymatic degradation in

combina-tion with poor cellular uptake of siRNA [11]

There-fore, novel delivery systems, which enable prolonged

circulation of siRNA with resistance against enzymatic

degradation, high accessibility to target cells via

clini-cally feasible administration routes and optimized

cytosolic release of siRNA after efficient cellular

uptake, are indispensably required [12] In this

mini-review, major factors in determining overall RNAi

efficiency in vivo are introduced Moreover, up-to-date

progress in achieving efficient and targeted siRNA

delivery in vivo, particularly by overcoming multiscale

hurdles using novel siRNA carriers, is discussed

Challenges in RNAi in vivo

Design and in vivo delivery of siRNA

There are multiple key considerations in order to

achieve efficient RNAi in vivo by delivering exogenous

siRNA siRNA has to be designed to target

hybridiza-tion-accessible regions within the target mRNA while

avoiding unintended (off-target) effects [13–15], which

is extensively reviewed in this series by Walton et al

[16] In addition, siRNA can also induce adverse

effects such as immune responses, as discussed by

Samuel-Abraham & Leonard [17] siRNA may induce

interferon responses either through the

double-stranded RNA-activated protein kinase PKR [18] or

toll-like receptor 3 [19] Therefore, a combination of

computer algorithms and experimental validation

should be employed to determine the optimized siRNA

sequences that are complementary to target mRNA

while inducing minimal immune responses [20]

Naked siRNA is relatively unstable in blood in its

native form and is rapidly cleared from the body (i.e

short half-lives in vivo) via degradion by ribonucleases,

rapid renal excretion and nonspecific uptake by the

reticuloendothelial system [21] The phosphorothioate

backbone, or various 2¢ positions in the sugar moiety

of siRNA, is conventionally modified to enhance its

stability and activity against nuclease degradation

[22,23], without affecting gene silencing activity [24]

siRNA is an anionic macromolecule and does not

readily enter cells by passive diffusion mechanisms An

appropriate siRNA delivery system enhances cellular

uptake, protects its payload from enzymatic digestion and immune recognition, and improves the pharmacoki-netics by avoiding excretion via the reticuloendothelial system and renal filtration (i.e prolonged half-life

in vivo) [25–27] In addition, targeted delivery systems localize siRNA in the desired tissue, resulting in a reduction in the amount of siRNA required for efficient gene silencing in vivo, as well as minimized side effects Therefore, the development of effective

in vivo delivery systems is pivotal in overcoming the challenges in achieving efficient and targeted siRNA delivery in vivo Major hurdles in siRNA delivery

in vivo and various approaches to overcoming them are illustrated in Fig 1

Local versus systemic delivery The types of target tissues and cells dictate the optimum administration routes of local versus systemic delivery For example, siRNA can be directly applied to the eye, skin or muscle via local delivery, whereas sys-temic siRNA delivery is the only way to reach meta-static and hematological cancer cells Local delivery offers several advantages over systemic delivery, such as low effective doses, simple formulation (e.g no targeting moieties), low risk of inducing systemic side effects and facilitated site-specific delivery [28] Therefore, if applicable, local delivery is likely to be a more cost-efficient strategy for siRNA delivery in vivo than systemic administration For example, initial clini-cal trials for RNAi-based treatment of age-related mac-ular degeneration have exclusively used local injections

of siRNA directly into the eye [10] Other promising local routes include intranasal siRNA administration for pulmonary delivery [10,29–31] and direct injection into the central nervous system [10,32,33]

Alternatively, systemic delivery via intravenous (i.v.), intraperitoneal (i.p.) or oral administration is widely applicable when the target sites are not locally confined or not readily accessible Metastatic tumors are especially amenable for systemic delivery compared with local administration For example, human bcl-2 oncogene-targeting siRNA, which was complexed with cationic liposomes and i.v injected, effectively inhib-ited tumor growth in a mouse liver metastasis model [34] Another study showed that siRNA encapsulated

in a lipid vesicle was able to impart efficient and per-sistent antiviral activity after being injected into a hepatitis B virus mouse model [35] However, impor-tantly, systemic siRNA delivery imposes several additional barriers in comparison with local delivery siRNA should remain in its active form during circula-tion and be able to reach target tissues after passing

through multiple barrier organs (e.g liver, kidney and

lymphoid organs)

Extracellular and intracellular barriers in siRNA

delivery in vivo

Regardless of administration routes, the final

desti-nation of siRNA is the cytoplasm of the target cell,

where it incorporates into RNA-inducing silencing

protein complex and encounters target mRNAs First,

siRNA that survives in the plasma and is transported

close to a target tissue must extravasate through the

tight vascular endothelial junctions It has been

reported that microvascular transport of

macromoel-cules > 5 nm in diameter is significantly inhibited in

normal tissues [36] However, transport of

macromole-cules across the tumor endothelium is more efficient

than that of normal endothelium because of its leaky

and discontinuous vascular structures with poor

lym-phatic drainage Thus, tumor endothelium allows the penetration of high molecular mass macromolecules (> 40 kDa), which is also referred to as ‘enhanced permeation and retention effect’ [37] siRNA, in its native form or formulated in a delivery carrier, must then diffuse through the extracellular matrix, a dense network of fibrous protein and carbohydrates surrounding a cell [38], before accessing target cells siRNA or its complex adheres preferably to target cells via receptor-mediated specific binding, followed by cellular uptake Even after it is internalized by a cell, siRNA should be released from the endosome, while avoiding entrapment and degradation [39,40] Because the condition in the endosome⁄ lysosome is mildly acidic, facilitated cytosolic release of siRNA using acid-responsive delivery carriers has been a popular strategy to overcome this intracellular hurdle [41,42] Fusogenic peptides which undergo acid-triggered con-formational changes have also been shown to

acceler-Fig 1 Interdisciplinary approaches to achieving efficient and targeted RNAi in vivo by overcoming multiscale barriers in systemic siRNA delivery Detailed design parameters of an ideal siRNA carrier are depicted in Fig 2.

ate endosomal escape of nucleic acids [43,44] Finally,

siRNA delivered by a carrier should be decomplexed

in the cytoplasm [45] A broad range of novel materials

that provide enhanced siRNA release have been

devel-oped (e.g disulfide-based cationic polymers) [46]

Fig 1 shows extracellular and intracellular barriers in

siRNA delivery with various approaches to

overcom-ing them

Chemically modified siRNA for

enhanced RNAi in vivo

Various molecular positions in siRNA have been

chem-ically replaced or modified, mainly to resist enzymatic

hydrolysis For example, phosphodiester (PO4) linkages

were replaced with phosphothioate (PS) at the 3¢-end,

and introducing O-methyl (2¢-O-Me), fluoro (2¢-F)

group or methoxyethyl (2¢-O-MOE) group greatly

pro-longed half-lives in plasma and enhanced RNAi

effi-ciency in cultured cells [47–51] In addition, effieffi-ciency

enhancer molecules were conjugated to either the 5¢- or

3¢-end of the sense strand, without affecting the activity

of the antisense strand [52] There are some potential

risks that chemically modifying siRNA may

compro-mise RNAi efficiency For example,

boranophospho-nate modification at the central position of the

antisense strand of siRNA showed improved resistance

to nuclease degradation, but simultaneously reduced

RNAi activity [53] In addition, non-natural molecules

produced upon the degradation of a chemically

modi-fied siRNA may generate metabolites that might be

unsafe or trigger unwanted effects To date, cholesterol

and aptamers are the most promising siRNA conjugates

that have demonstrated efficient RNAi in vivo

Cholesterol–siRNA conjugates

Improved pharmacokinetic and cellular uptake

proper-ties of cholesterol–siRNA conjugates silenced

apolipo-protein B (ApoB) in mice via i.v administration [22]

By contrast, ApoB siRNA unconjugated with

choles-terol was unable to induce mRNA interference and

was rapidly cleared The mechanisms of improved

dis-tribution and cellular uptake of siRNA through

cho-lesterol conjugation were demonstrated in a recent

study; cholesterol–siRNA conjugates seem to

incorpo-rate into circulating lipoprotein particles (i.e improved

distribution in vivo) and are efficiently internalized by

hepatocytes via receptor-mediated processes (i.e

effi-cient cellular uptake) [54] Prebinding of cholesterol–

siRNA conjugates to lipoparticles dramatically

improved silencing efficacy in mice, and lipoparticle

types affected cholesterol–siRNA conjugate

distribu-tion in various tissues [54] Using a transgenic mouse model for Huntington’s disease, it was also demon-strated that a single intrastriatal injection of choles-terol–siRNA conjugates silenced a mutant Huntingtin gene, attenuating neuronal pathology as well as delaying the abnormal behavioral phenotype [55]

RNA aptamer–siRNA conjugates RNA aptamers have been popularly used to selectively deliver siRNA in vivo to target tissues and cells, such

as prostate cancer cells and tumor vascular endothe-lium overexpressing prostate-specific membrane anti-gen [56] A key advantage of aptamer-mediated targeted delivery systems is that RNA aptamers can be facilely obtained by in vitro transcription reaction and, therefore, avoid contamination by cell or bacterial products Promising in vitro and in vivo RNAi was obtained using siRNA that was directly linked with prostate-specific membrane antigen aptamers [57] An aptamer-based delivery system has also been used to suppress HIV infection Anti-gp120 RNA aptamers were covalently conjugated with a strand of siRNA, and the other siRNA strand was subsequently annealed to the aptamer-conjugated strand These aptamer–siRNA conjugates were able to access HIV-infected cells and silence viral replication in vitro [58]

Viral vectors: natural siRNA carriers Various recombinant viral vectors have been shown to

be efficient in obtaining gene silencing for an extended period in a wide range of mammalian cells [59] For example, an adenoviral vector encoding siRNA against pituitary tumor transforming gene 1 significantly inhib-ited the growth of the pituitary tumor transforming gene 1-overexpressing hepatocellular carcinoma cells

in vitro and in vivo [60] It was also demonstrated that the herpes simplex virus type 1-based amplicon vectors suppressed in vivo tumorigenicity of human polyomavi-rus BK-transformed cells (pRPc cells) [61] Recombi-nant lentiviral vectors have also been frequently used to achieve in vivo gene silencing In particular, lentiviral vectors containing the U6 promoter were found to be efficient in green fluorescent protein (GFP) silencing

in vitro, resulting in 80% gene silencing at an average

of one integrated vector genome per target cell genome

In addition, the U6 promoter was shown to be superior

to the H1 promoter in achieving in vivo gene silencing and led to persistent GFP knockdown in the mouse brain for at least 9 months [62] This indicates that lentivirus-mediated RNAi is a promising strategy for long-term gene silencing in vitro and in vivo Other viral

siRNA carriers such as retroviral vectors have not been

intensively explored for their use in vivo [63–65]

Although viral vectors provide excellent tissue-specific

tropism and high RNAi efficiency, safety concerns (e.g

insertion mutagenesis and immunogenicity) and

difficul-ties with large-scale manufacture may limit the use of

viral vectors for siRNA delivery in clinical setting

[66,67] Therefore, synthetic counterparts (nonviral

vectors) have been more and more intensively explored

as safe and effective alternatives that are easy to be

prepared and can deliver large payloads of siRNA

Nonviral carriers: Trojan horses for

efficient, biocompatible and versatile

siRNA delivery in vivo

Delivery of siRNA in its unmodified form has several

advantages over using a chemically modified form

Unmodified siRNA possesses untouched RNAi

capability (maximized RNAi per siRNA molecule) and

does not require potentially inefficient and time⁄

labor-consuming modification processes (cost-effective

preparation) However, its highly anionic nature and

the macromolecular size of siRNA necessitates using

efficient carriers to overcome multiscale barriers

Unlike viral vectors, which deliver siRNA in the form

of a viral genome, nonviral carriers deliver native

siRNA, generate low immunogenicity and offer high

structural and functional tunability An ideally designed

nonviral siRNA carrier with its desirable structural

and functional multicomponents is depicted in Fig 2

Liposomes and lipoplexes

One of the most significant advances in RNAi in vivo

is successful knockdown of ApoB in nonhuman

primates by systemically delivered siRNA in stable

nucleic acid–lipid particles [68] The siRNA–lipid

complexes showed significantly enhanced cellular

inter-nalization and endosomal escape of siRNA ApoB

siRNA-carrying stable nucleic acid–lipid particles

greatly reduced ApoB expression and serum

choles-terol levels in monkeys when a clinically acceptable

single siRNA dose of 2.5 mgÆkg)1 was injected i.v

[68] Importantly, expression of ApoB was silenced for

at least 11 days With addressing the high toxicity

of the currently available liposomes for siRNA

deliv-ery in vitro and in vivo [69,70], cationic cardiolipin

analog-based liposomes carrying c-raf siRNA inhibited

the growth of breast tumor xenografts in mice [71]

Cationic liposomes formulated with

anisamide-conjugated poly(ethylene glycol) effectively penetrated

the lung metastasis of melanoma tumors in mice and

resulted in 70–80% gene silencing after a single i.v injection [72]

Further noticeable progress in siRNA delivery using liposomes is the use of neutral lipids for systemic siRNA delivery in order to address the toxicity of cat-ionic lipids For example, cyclin D1 (CyD1) siRNA was efficiently encapsulated in neutral phospholipid-based liposomes coated with hyaluronan [73] The resulting siRNA-carrying liposomes were stable during circulation in vivo after i.v injection and suppressed leukocyte proliferation and cytokine secretion by type 1 T-helper cells Another neutral dioleoyl phos-phatidylcholine-based delivery system, which targets EphA2 [74] and focal adhesion kinase [75], demon-strated significantly inhibited tumor growth in an orthotropic ovarian cancer model in mice The same type of liposome has also been reported to efficiently silence neuropilin-2 expression and inhibit the growth

of colorectal cancer xenografts in the mouse liver [76]

Polymers and peptides Nucleic acids such as siRNA are easily complexed with synthetic cationic polymers e.g., polyethylenimine

Fig 2 An ideally designed nonviral siRNA carrier for efficient and targeted RNAi in vivo.

(PEI), biodegradable cationic polysaccharide (e.g.

chitosan) and cationic polypeptides [e.g atelocollagen,

poly(l-lysine) and protamine], via attractive

electro-static interactions For example, i.t injection of

siR-NA– atelocollagen complexes silenced luciferase

expression in germ cell tumor xenografted in mice and

inhibited tumor growth [77] In another study, vascular

endothelial growth factor (VEGF)

siRNA–atelocolla-gen complexes significantly suppressed tumor

angio-genesis and growth in a prostate tumor model in mice

[78] Intravenous administration of chitosan–RhoA

siRNA complexes resulted in effective gene silencing in

subcutaneously implanted breast cancer cells in mice

[79] In addition, intranasally administered chitosan–

siRNA complexes efficiently silenced GFP expression

in bronchiole epithelial cells in GFP-transgenic mice

[29] Tumor necrosis factor expression in systemic

mac-rophages was silenced in mice after i.p administration

of chitosan⁄ siRNA complexes, thus downregulating

systemic and local inflammation [80]

Polyethylenimine is one of the most popularly

inves-tigated synthetic cationic polymers for nucleic acid

delivery in vitro and in vivo Polyethylenimine is very

potent in transfection with its uniquely high buffering

capability at an endosomal pH (proton sponge effect)

which releases nucleic acid payloads into the cytoplasm

[39] c-erbB2⁄ neu (HER-2) siRNA was delivered to

subcutaneous tumors via i.p administration of siRNA⁄

polyethylenimine complexes and resulted in a

remark-able reduction of tumor growth [81] Pain receptors for

N-methyl-d-aspartate were effectively knocked-down by

intrathecal delivery of polyethylenimine-conjugated

siRNA in rats [82] Inhibited viral propagation in the

lungs was also observed after deacetylated linear

polyethylenimine⁄ siRNA complexes targeting influenza

nucleoprotein was retro-orbitally administered [83] In

another study, polyethylenimine-conjugated siRNA

against secreted growth factor pleiotrophin reduced

tumor growth and cell proliferation with no toxicity or

abnormal animal behaviors after intracerebral

adminis-tration in an orthotopic glioblastoma mouse model [84]

Overall, polyethylenimine seems to be a promising

nonviral carrier for siRNA delivery in vivo, if its high

toxicity and limited biodegradability are appropriately

addressed

Polypeptides, such as poly(l-lysine) and protamine,

have also commonly been used to deliver siRNA

A sixth generation of dendritic poly(l-lysine) was

employed to systemically deliver siRNA to silence

ApoB expression without hepatotoxicity in hepatocytes

of apolipoprotein E-deficient mice [85] Protamine, a

natural arginine-rich cationic polypeptide, condenses

negatively charged nucleic acids and has been used as

an efficient gene-delivery carrier [86] An in vivo study demonstrated that complexes of siRNA and low molecular mass protamine, which possess membrane-translocating potency, were accumulated in tumors via i.p administration and successfully inhibited the expression of VEGF, thereby suppressing the growth

of hepatocarcinoma tumors in mice [87] In addition,

no noticeable increase in inflammatory cytokines, including interferon-a and interleukin-12, in serum was observed when the low molecular mass protamine⁄ siRNA complexes were administered, indicating negligible immunostimulatory effects

One of the fundamental concerns in using synthetic polymers for siRNA delivery in vivo is dose-dependent toxicity upon systemic administration For example, polyethylenimine and poly(l-lysine) have been shown

to trigger necrosis and apoptosis in a variety of cell lines [88,89] The toxicity can be ameliorated by conjugation with biocompatible, hydrophilic polymers such as poly(ethylene glycol) or by removing excess (i.e., uncom-plexed) cationic polymers In gneral, natural cationic polymers (e.g chitosan and protamine), which are bio-compatible, biodegradable and nontoxic, are more desir-able in siRNA delivery in vivo than synthetic polymers

Targeted siRNA delivery in vivo

In order to achieve RNAi in vivo via systemic delivery,

it is crucial for siRNA to be efficiently located in desired tissues⁄ cells This requires three important pro-cesses: prolong circulation in the body, high accessibil-ity to target tissues and specific binding to target cells Targeted siRNA delivery maximizes the local concen-tration in the desired tissue (maximized and localized silencing effects) and prevents nonspecific siRNA dis-tribution (minimized unwanted effects in non-target tissues) For example, recent studies have reported can-cer-targeted siRNA delivery using nanoparticles that specifically bind to cancer-specific or cancer-associated antigens and receptors [90,91]

Folate-conjugated siRNA carriers One of the most popular target molecules in cancer-specific gene and drug delivery is the folate receptor [92] Folic acid (FA) is needed for rapid cell growth, and many cancer cells overexpress folate receptors to which FA and monoclonal antibodies specifically bind [93] FA can be easily conjugated onto the surface of liposomal and polymeric siRNA carriers with or with-out a poly(ethylene glycol) spacer [92] For example, FA-conjugated polyethylenimine showed enhanced gene silencing via receptor-mediated endocytosis [94]

Chimeric survivin siRNA incorporated with

bacterio-phage phi29-encoded RNA and when further

conjugated with FA suppressed the growth of

naso-pharyngeal carcinoma in mice, whereas control

FA-free siRNA–phi29-encoded RNA hybrid did not

affect tumor development [95] As described earlier,

RNA aptamer-mediated targeted siRNA delivery by

direct conjugation with siRNA or tethering onto

carriers has been a frequently adapted strategy

Arg–Gly–Asp peptide-conjugated siRNA carriers

Arg–Gly–Asp (RGD) peptide targets tumor

vascula-ture expressing avb3 integrin Poly(ethylene glycol)

ylated poly(ethylenimine) conjugated with RGD

peptides was developed to selectively deliver VEGF

siRNA to tumors [96] In this study, i.v injected

poly-ethylenimine-poly(ethylene glycol)-RGD⁄ siRNA

com-plexes inhibited tumor angiogenesis and the growth of

integrin-expressing murine neuroblastoma tumors in

mice [96] Systemic delivery (i.v injection) and local

delivery of poly(ethylene glycol)-polyethylenimine-RGD

complexing VEGF siRNA also showed a significant

inhibitory effect on virus-induced angiogenesis as well

as the development of herpetic stromal keratitis lesions

[97]

Antibody-conjugated siRNA carriers

Many studies have suggested that antibodies are good

targeting modalities for targeted siRNA delivery

in vivo, when careful selection of target antigen is

made Ideal antigens should be exclusively expressed

or substantially overexpressed on target cells

Exam-ples of antigens that have been used for

cancer-tar-geted drug and gene delivery include HER-2 [98] and

epidermal growth factor receptor [99] For example,

HER-2 siRNA-carrying liposomes decorated with

transferrin receptor-specific antibody fragments (i.e

nanoimmunoliposome) silenced the HER-2 gene in

xenograft tumors in mice, significantly inhibiting

tumor growth [100] An antibody fragment against an

HIV gp160 has also been used for targeted siRNA

delivery in vivo siRNA linked to a

protamine–anti-body fusion protein, called F105-P, showed inhibited

HIV replication in infected primary T cells [101]

Moreover, i.t or i.v injection of F105-P⁄ siRNA

com-plexes into mice successfully targeted gp160-expressing

B16 melanoma cells A synthetic chimeric peptide,

which consists of nonamer arginine residues (9R)

added to the C-terminus of a rabies virus glycoprotein

peptide (29 amino acids) (RVG-9R), was able to

spe-cifically deliver siRNA to acetylcholine

receptor-expressing neuronal cells after i.v administration [102]

In addition, treating mice with Japanese encephalitis virus siRNA complexed with RVG-9R showed robust protection of the animals from lethal infection

Intracellular siRNA delivery

In many aspects, siRNA delivery is similar to that of delivering other types of nucleic acids such as plasmid DNA, because they share most extracellular and intra-cellular barriers However, several unique challenges in siRNA delivery make achieving efficient RNAi difficult compared with plasmid DNA delivery First, the final target destination of siRNA is the cytoplasm, whereas plasmid DNA must be transported into the nucleus This implies that siRNA should be rapidly released from its carrier upon endosomal escape Second, overall RNAi efficiency is proportional to the number of siRNAs complexed with RNA-inducing silencing protein complex, whereas a successfully delivered single copy of plasmid DNA might be sufficient to express new transgene proteins In other words, the maximum possi-ble number of siRNA needs to be delivered in the cyto-plasm in order to achieve the desired biological effects Third, siRNA acts only once, whereas plasmid DNA can be replicated or even can be incorporated into the host chromosome [103] (short vs permanent effects)

Cell-penetrating peptide-mediated siRNA delivery Cell-penetrating peptides (CPPs), short cationic poly-peptides with a maximum of 30 amino acids, have been extensively used to obtain enhanced intracellular deliv-ery of a wide range of macromolecules [104] CPPs have been shown to bind the anionic cell surface through elec-trostatic interactions and rapidly induce cellular inter-nalization through relatively unclear mechanisms, although recent evidence shows that CPP-mediated internalization might be an endocytosis-mediated pro-cess [105,106] Various CPPs, including TAT and MPG proteins from HIV-1 [107–110], as well as penetratin and polyarginine [111,112], have been employed for intracel-lular delivery of various proteins and nucleic acids Oligoarginine (e.g 9 arginine, 9R), the simplest CPP, conjugated with cholesterol was shown to effi-ciently deliver siRNA to a transplanted tumor in mice [113] It was also reported that HER-2 siRNA com-plexed with short arginine peptide was localized in perinuclear regions of the cytoplasm in vitro, further significantly inhibiting tumor growth of ovarian cancer xenografts [114] Polyamidoamine dendrimer-TAT conjugated with bacterial magnetic nanoparticles was also used to deliver epidermal growth factor receptor

siRNA to human glioblastoma cells in vitro as well as

xenografts [115] Another type of CPP, MPG-8, was

also used to complex cyclin B1 siRNA, and the

result-ing complexes were further decorated with cholesterol

for i.v injection to the mice bearing human prostate

carcinoma and human lung cancer xenografts [116]

The results showed efficient siRNA delivery in vivo at

a low effective dose (0.5 mgÆkg)1), indicated by

inhibited tumor growth

CPP-mediated cellular internalization via endosis requires additional molecules for facilitated cyto-solic release of siRNA For example, it was found that TAT–siRNA conjugates resulted in no gene silencing because they were entrapped in the endosomes even after efficiently entering cells [117] Photostimulating fluorescently labeled TAT efficiently released TAT– siRNA conjugates from the endosome, resulting in enhanced gene silencing efficiency Chloroquine and

Table 1 siRNA delivery systems for RNAi in vivo BCL-2, B-cell lymphoma 2; Cyb1, cyclin B1; CyD1, cyclin D1; DOPC, 1,2-dioleoyl-sn-glyce-ro-3-phosphatidylcholine; DOPE, dioleoyl phosphatidylethanolamine; DOTAP, (N-[1-(2,3-dioleoyloxy)]-N-N-N-trimethyl ammonium propane); DPPE, dipalmitoyl phosphatidylethanolamine; DSPE, distearoyl phosphatidylethanolamine; FAK, focal adhesion kinase; HST-1 ⁄ FGF-4, fibroblast growth factor; i.c.v., intracerebroventricular; i.p., intraperitoneal; i.v., intravenous; MMP-2, matrix metalloproteinase-2; NMDA, N-methyl- D -aspartate; NR2B, NMDA-R2B receptor subunit protein receptors; PAMAM, polyamidoamine dendrimer; PLK-1, polo-like kinase 1; PTTG1, pituitary tumor transforming gene 1; RVG, rabies virus glycoprotein; SNALP, stable nucleic acid-lipid particles; TNF-a, tumor necrosis factor-a; VEGF, vascular endothelial growth factor.

DSPE–poly(ethylene glycol)

–DOTAP–cholesterol liposome

RGD–poly(ethylene glycol)–poly(ethylenimine) VEGF Corneal neovascularization Subconjunctival, i.v 97

Oligoarginine (9R) conjugated-water-soluble

lipopolymer (WSLP)

GALA peptide–poly(ethylene glycol)

–MMP-2 cleavable peptide-DOPE

a All the listed in vivo models involved a mouse model except Zimmermann et al [68] and Tan et al [82].

influenza virus-derived hemagglutinin peptide have also

been frequently used to destabilize the endosomal

membrane and enhance the cytosolic release of

CPP-conjugated macromolecules [118–120]

Fusogenic or pH-responsive intracellular delivery

of siRNA

Fusogenic peptides and lipids and pH-responsive

lipo-plexes and polylipo-plexes have been used to ensure

facili-tated siRNA into the cytoplasm from the endosomes

For example, the incorporation of polypeptides derived

from the endodomain of the HIV-1 envelope (HGP) or

influenza virus fusogenic peptide (diINF-7)

signifi-cantly promoted the liposomal fusion with the

endoso-mal membrane, enhancing siRNA escape into the

cytoplasm [40,121] Similarly, equipping lipoplexes

with fusogenic lipids, such as dioleoyl

phosphatidyl-ethanolamine (DOPE), was shown to facilitate the

endosomal release of siRNA payload [122,123]

Stimuli-triggered macromolecule release from the

mildly acidic endosome (e.g pH 5.0–6.0) has been

popularly investigated using a number of novel

acid-responsive polymers [124–126] For example,

poly(eth-ylene glycol) shielding the surface of a highly fusogenic

phosphatidylethanolamine lipid vesicles was cleaved

upon acid hydrolysis of the vinyl ether bond, triggering

fusion with the endosomal membrane [127] A matrix

metalloproteinase-cleavable and pH-sensitive GALA

peptide was also used to link poly(ethylene glycol) and

dioleoyl phosphatidylethanolamine (DOPE) lipid to

obtain enhanced siRNA delivery specifically into cancer

cells [128] Highly efficient siRNA-mediated knockdown

of luciferase expression was achieved in human

fibrosar-coma cells in vitro and xenografted tumors using this

method Acid-degradable ketalized linear

polyethyl-enimine significantly increased gene silencing efficiency

via efficient cytosolic release with high resistance to

serum and low cytotoxicity [129] It was demonstrated

that ketalized linear polyethylenimine⁄ siRNA

poly-plexes were efficiently released into the cytoplasm upon

acid-hydrolysis of ketal branches in the endosomes,

fol-lowed by enhanced siRNA disassembly from ketalized

linear polyethylenimine in the cytoplasm [129]

Conclusion

RNAi is an emerging therapeutic strategy and has

been widely investigated Despite a few promising

clinical trials, effectively delivering siRNA in vivo

remains a pivotal challenge in translating RNAi in the

clinic as a conventional treatment option A number of

delivery systems and strategies have been developed to

overcome multiscale extracellular and intracellular barriers to siRNA delivery in vivo, as summarized in Table 1 Chemically modified siRNA is stable against enzymatic degradation but can be cleared easily, gener-ating potentially hazardous metabolites Viral siRNA delivery raises several safety and preparation concerns such as immune responses and limited large-scale pro-duction Nonviral siRNA carriers are efficient, safe and versatile in tackling the barriers in siRNA circula-tion, permeation into desired tissues, specific binding

to target cells and optimized intracellular trafficking Recent advances clearly indicate that interdisciplinary approaches using biology, chemistry and engineering play crucial roles in achieving efficient and targeted siRNA delivery in vivo

Acknowledgement This work was supported by NSF CAREER Award (DMR-0956091) and a Council on Research Comput-ing and Libraries Research Grant (UC Irvine)

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