Nanocarriers: a tool to overcome biological barriers in siRNA delivery

1. Introduction

2. Difficulties in siRNA delivery

3. Conjugation of siRNA with

nanosystems

4. Nanosystems for siRNA delivery

5. siRNA delivery systems in

clinical trials

6. Off-target effects in siRNA

delivery

7. Conclusions

8. Expert opinion

Review

Nanocarriers: a tool to overcomebiological barriers in siRNAdeliveryAvnesh Kumari, Vineet Kumar & Sudesh Kumar Yadav†

Council of Scientific and Industrial Research (CSIR), Institute of Himalayan Bioresource

Technology, Biotechnology Division, Nanobiology Lab, Palampur, India

Introduction: siRNA has poor in vivo stability and has a plasma half life of only

a few minutes after intravenous administration. These problems can be over-

come by conjugating/encapsulating siRNA with various nanosystems. Surface

modifications of such nanosystems can further improve the cellular uptake

of siRNA-nanosystems. In this review, the authors have highlighted the prob-

lems encountered in siRNA delivery, conjugation strategies and nanosystems

for siRNA delivery and for improving their in vivo delivery performance.

Areas covered: The authors briefly cover various problems encoun-

tered in siRNA delivery and discuss nanocarriers for overcoming these

biological barriers.

Expert opinion: siRNA binding and unpacking are important factors for opti-

mizing the interactions between siRNA and nanosystems. Several crucial con-

jugation parameters, such as the conjugation site of siRNA, and the nature of

molecules to be conjugated (charge, molecular weight and hydrophobicity),

should be carefully considered for maximising delivery efficiency of siRNA

conjugated nanosystems.

Keywords: conjugation, encapsulation, nanocarriers, siRNA

Expert Opin. Biol. Ther. (2011) 11(10):1327-1339

1. Introduction

RNA interference (RNAi) is a new strategy for clinical trials in siRNA therapy.Double stranded RNA of 21 -- 24 nucleotides known as siRNA are known to medi-ates RNAi and effectively silenced target genes [1,2]. In the cytosol, siRNA bindswith specific protein complex known as the RNA induced silencing complex(RISC). This complex mediates the unwinding of siRNA duplex that binds to thetarget mRNA. Publications related to RNAi increased exponentially from 5 in1998 to more than 2400 in 2008 [3]. Remarkable progress has been made on differ-ent aspects of the RNAi technology. Attempts are now being strenuously made touse siRNA for therapeutic applications [3]. Unfortunately, applications of RNAiare limited in clinical developments due to lack of efficient and specific delivery sys-tems for therapeutic siRNA [4]. siRNA has poor in vivo stability and a plasma half-life of only a few minutes after intravenous administration [5]. In order to achieveeffective RNAi activity of clinical significance, suitable delivery systems are requiredto protect siRNA from degradation and specifically deliver siRNA to target cells [6].

Success of siRNA therapy is highly dependent on the delivery systems. The crea-tion and design of nanosystems for the delivery of nucleic acids such as siRNAs haverecently gained interest because of their possible clinical applications [7,8]. Nanosys-tems escaped into endolysosomes within 10 min of incubation with cells andentered the cytoplasm [9]. Surface charge reversal of nanosystems in acidic pH ofendolysosomes is responsible for endolysosomal escape [9]. Nanosystems can alsobe used as efficient delivery vehicles for intracellular targeting. Nanosystems having

10.1517/14712598.2011.587801 © 2011 Informa UK, Ltd. ISSN 1471-2598 1327All rights reserved: reproduction in whole or in part not permitted

Exp

ert O

pin.

Bio

l. T

her.

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Rye

rson

Uni

vers

ity o

n 03

/18/

13Fo

r pe

rson

al u

se o

nly.

the ability to change their surface charges from anionic tocationic can escape the endosomal compartment, whereasnegatively charged nanosystems remain mostly in theendosomal compartment [10].siRNA has been evaluated as a potential therapeutic agent

for a variety of nucleic acid based diseases such as HIV [11],hepatitis C [12] and cancer [13]. The use of nanosystems forsiRNA delivery can significantly improve the efficacy of can-cer gene therapy mediated by RNAi [14]. Nanosystems forsiRNA delivery not only allowed attainment of tumoricidaleffect through increasing the level of siRNA expression, butalso by exerting a longer inhibitory effect on cancer cells [15].Nanosystems coupled to ligands or receptors can be used fortargeted delivery. Targeted delivery results in higher bioavail-ability of siRNA at its site of action and reduces sideeffects [15]. Targeted delivery can be achieved by either activetargeting or passive targeting [16]. Active targeting is achievedby conjugating siRNA to tissue- or cell-specific ligands [17].While passive targeting is achieved by coupling siRNA tonanosystems that passively reach the target organ. RNAitechnology has become an excellent strategy for cancer genetherapy. In 2008, the first, to our knowledge, targeted deliveryof siRNA in humans was accomplished. A Phase I trial usingthe experimental therapeutic CALAA-01 was initiated andthe first patient was treated during May, 2008. Under thisprotocol, targeted nanoparticles (NPs) were administeredintravenously for the treatment of solid tumors [18].A plethora of materials that can form complexes with

siRNA have previously been reviewed [19]. Most studies havefocussed on cationic lipids and polymers, which form randomself aggregates with nucleic acids that are often larger than100 nm. These systems are generally heterogenous and arepoorly defined particle collectives [20]. These cationic materialsform stable NPs with anionic siRNA via charge complexationand provide protection to siRNA from degradation. These

siRNA delivery systems are either toxic or inefficient for spe-cific siRNA delivery. Use of non-viral vectors [21] andpolymer-siRNA conjugates have also been reviewed earlier [22].Non-viral vectors and polymers form looser complexes withsiRNA than with plasmid DNA. Such incomplete encapsula-tion of the nucleic acid leads to the exposure of siRNA to enzy-matic or physical degradation prior to delivery to the targetedcells [23]. However, nanoencapsulation of siRNA in nanosys-tems can improve the siRNA delivery, transfection efficiencyand cellular uptake [24].

In this article we have reviewed various problems encoun-tered in siRNA delivery, conjugation of siRNA with nanosys-tems for improved therapeutic efficacy and nanosystems forsiRNA delivery for improving their in vivo performance.

2. Difficulties in siRNA delivery

There is huge scope in using siRNA as therapeutic agents, butthe full potential of siRNA cannot be attained until bettermethodologies for delivery to cells and tissues will be devel-oped [25]. The interaction with cell membranes is one impor-tant criteria for these delivery devices. Multiple pathways ofsiRNA entry into mammalian cells have been documentedbased on the nature and size of the payload [8]. The pathwayof cellular entry will determine if the respective siRNA isunloaded at its site of action and if it is effective or not [26].Therefore, delivery of siRNA-nanosystems to specific intra-cellular targets for specific biological function small, mono-disperse nanosystems with a defined zeta potential andsurface chemistry are recommended [26]. Although the fieldof siRNA therapeutics has made significant progress, the effec-tive delivery of siRNA still remains a challenge. AdministeredsiRNA come across many biological barriers. Nuclease activityin plasma membrane and other tissues is one of the firstbiological barriers encountered by administered siRNA [27].

The covalent binding of PEG to the nanosystems cangreatly reduce undesired effects such as immune response,unspecific interactions and degradation [28]. Renal clearanceis another barrier encountered by the administered siRNA.The biphasic plasma half-lives of administered siRNA are inthe range of few minutes to several hours. They get accumu-lated in the kidney and liver. PEG association with siRNA-nanosystems improved extracellular stability and circulationhalf-life. However, one disadvantage with PEG association isthat it decreased the transfection efficiency. Reticuloendothe-lial system (RES) clearance is one more hurdle in siRNAdelivery. Phagocytic cells of the RES, particularly the abun-dant Kupffer cells in the liver and macrophages, also detectand phagocytose siRNA as well as nanosystems and therefore,may be used to enhance their delivery [29]. These organs accu-mulate high concentrations of siRNA following systemicadministration. Strategies to avoid uptake and clearance byRES following systemic administration include the use ofnanosystems, targeting ligands and conjugation with PEG.To further increase circulation time, the particles can be

Article highlights.

. Reticuloendothelial system clearance, renal clearanceand endosomal trapping are the major barriersencountered in siRNA delivery.

. Conjugation of various cleavable and non-cleavablefunctional and non-functional molecules to siRNA couldnot only improve their pharmacokinetic behaviours andcellular uptake efficiencies, but also promotecell specificities.

. The suitable nanoencapsulation of siRNA onbiodegradable and biocompatible nanocarriers eliminatesthe possibilities of off-target effects, degradation andstructural and functional deformities.

. Nanocarriers efficiently deliver siRNA to tumor cells andother normal tissues with leaky vasculature, whileconjugates of siRNA-nanocarriers with targeting ligandshave advantages in targeting siRNA to specific cellsor tissues.

This box summarizes key points contained in the article.

Nanocarriers: a tool to overcome biological barriers in siRNA delivery

1328 Expert Opin. Biol. Ther. (2011) 11(10)

Exp

ert O

pin.

Bio

l. T

her.

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Rye

rson

Uni

vers

ity o

n 03

/18/

13Fo

r pe

rson

al u

se o

nly.

coated with molecules that provide them with a hydrophilicprotective layer, such as PEG and poly-vinyl pyrrolidone(PVP) [29]. This static layer prevents macromolecules to inter-act with the particle, even at low surface coverage for particles,to release their load at a specific site.

The accumulation of siRNA-loaded nanosystems at the tar-get site is important instead of their circulation and retentionin the circulatory system of the body. This can be ensured bypassive or active targeting. In passive targeting, NPs are accu-mulated at tumor sites because of the leaky vasculature charac-teristic of tumor tissue [29]. While in active targeting the NPscarry siRNA molecules on the target surface that are able tointeract with the surrounding tissue [30]. siRNA reach theirtherapeutic targets by receptor-mediated endocytosis [9].Receptor binding and internalisation facilitate the entry ofsiRNA into cells. Initial uptake is followed by intracellulartrafficking into a variety of low pH endosomes [9]. Endosomaltrapping is another barrier for siRNA delivery. siRNA mustexit from the endosomes to reach its target in cytoplasm ornucleus. Grafted aminoacid groups on polymer backboneshave strong proton absorbing capabilities inside endosomesand lysosomes, leading to rapid osmotic swelling and siRNArelease [31]. Delivered siRNA entered the nucleus or cytoplasmof cells and affected gene expression.

3. Conjugation of siRNA with nanosystems

siRNA is a negatively charged molecule and hence cannotenter cells spontaneously [32]. Therefore some kind of formu-lation is necessary to bring siRNA to the cytoplasm of targetcells. siRNA can be conjugated to NPs through electrostaticinteractions [31] or covalent attachment. Various nanosizedpolyelectrolytes are known to form complexes with negativelycharged siRNA by ionic interactions. One of the majordrawbacks of electrostatically attached siRNA is that they arereleased when they come in contact with serum proteins.Another strategy for attaching siRNA is conjugation. siRNAis a hybridized product of sense and antisense complementarystrands. After cellular uptake, an antisense strand of siRNA isincorporated into the RISC to initiate the RNAi mecha-nism [33]. The conjugate is usually formed through a covalentattachment of the targeting molecule to the sense strand ofthe siRNA, so as not to disrupt silencing activity. Most ofthe conjugates employ cleavable linkages that include acid-labile and reducible bonds between siRNA and the conjuga-tion partner. Such conjugations facilitate the release of intactsiRNA inside cells. The acid-labile and disulfide linkagesget cleaved in the acidic endosome compartments and thereductive atmosphere of cytoplasm, respectively [33]. Thiol-containing siRNA can be coupled to NPs by using sulfosucci-nimidyl 6-(3¢-[2-pyridyldithio]-propionamido)hexanoate(sulfo-LC-SPDP) [34] and sulfosuccinimidyl 4-(N-maleimido-methyl) cyclohexane-1-carboxylate (sulfo-SMCC) cross-link-ers [35,36]. The choice of cross-linker however, may affect theability of the siRNA cargo to interact with RISC. The interior

reducing environment of the cell led to cleavage of thedisulfide bond generated by sulfo-LC-SPDP and allowed thesiRNA delivery. On the other hand, stable thioether linkageof sulfo-SMCC is unaffected by reducing conditions leavingsiRNA intact [35].

siRNA can be conjugated to thiol protected gold NPs(AuNPs) for targeted delivery [37]. siRNA has also been immo-bilized onto the surface of magnetic NPs. For this, primaryamine groups on the iron oxide NPs were activated with a het-erofunctional cross-linker, m-maleimidobenzoyl-N-hydroxy-succinimide ester (MBS) [36], and allowed to conjugate withthiol group of the antisense strand of siRNA [38].

siRNA can also be conjugated to nanosystems by avi-din--biotin interactions (Figure 1). Biotinylated siRNA canreact with avidin-labelled NPs to form siRNA--nanoparticleconjugates [39]. Owing to the very high affinity of avidin bind-ing of biotin (KD = 10--15 M, dissociation t½ = 89 days), thereis rapid and high affinity capture of the biotinylated ligand bythe avidin. The avidin--biotin linkage between the therapeuticand the targeting antibody has been demonstrated to bestable in the blood of rats and mice [40]. siRNAs can be bioti-nylated on either the 3¢-or 5¢-end of the sense strand of theduplex and conjugated to the targeting antibody withoutloss of RNAi activity. In such an example, exposure of cellsto streptavidin (SA)-bound 3¢-biotinyl-siRNA and humaninsulin receptor (HIRMAb/SA conjugate)-bound mAb,caused > 90% reduction in luciferase gene expression [39].

siRNA-containing thiol groups can be easily coupled tomaleimide groups through thioether bonds. This strategyhas been used for conjugating maleimide-containing PEGpeptide conjugates with 1-iminobis[N-(oleicylcysteinylhis-tinyl-1-aminoethyl)propionamide (EHCO)/siRNA NPs [6].Single-walled carbon nanotubes (SWNT) have been surfacefunctionalised with PEG-maleimide through Van der Waalsforces. The amine or maleimide terminal on the PEG immo-bilized on SWNT can then be used to conjugate with a widerange of biological molecules [34].

Conjugation of various cleavable and non-cleavablefunctional and non-functional molecules to siRNA couldnot only improve their pharmacokinetic behaviours and cellu-lar uptake efficiencies, but also promote cell specificities [33].Several crucial conjugation parameters, such as the conjuga-tion site of siRNA, cleavability, the length of spacer and thenature of molecules to be conjugated (charge, molecularweight and hydrophobicity), should be carefully consideredfor maximising delivery efficiency of siRNA conjugatednanosystems [33].

4. Nanosystems for siRNA delivery

Most siRNA delivery systems have been developed based onthe concept for the delivery of plasmid DNA, using cationiclipids [41] or polymers [42]. These cationic materials form sta-ble NPs with anionic siRNA via charge complexation andprovide protection of siRNA from degradation. These

Kumari, Kumar & Yadav

Expert Opin. Biol. Ther. (2011) 11(10) 1329

Exp

ert O

pin.

Bio

l. T

her.

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Rye

rson

Uni

vers

ity o

n 03

/18/

13Fo

r pe

rson

al u

se o

nly.

charged moieties of cationic molecules form polyplexes withthe siRNA and the resulting aggregates can be taken up intothe cells. In the cytosol, these aggregates released siRNA.However, these carriers can have notable drawbacks withrespect to toxicity and difficulties in specific cell targeting [33],thereby giving rise to a need for alternative delivery methods.These systems are generally heterogenous and have poorlydefined particle distribution [20].The creation and design of nanosystems (Table 1) for the

delivery of nucleic acids such as siRNAs have recently gainedinterest because of their possible clinical applications [7].siRNA binding and unpacking are important factors for opti-mizing the interactions between siRNA and nanosystems.Nanosystems should comprise a nucleic acid containing coreenabling them to perform intracellular siRNA delivery andprocessing. Also, a ‘stealth biocompatibility layer’ should beincorporated in nanosystems to ensure biological stabilityreflected in a sufficient circulation time, evasion of the RESand resistance against particle aggregation. Finally the nano-systems should contain a targeting layer to enhance particleuptake in the target cells.

4.1 NanogelsNanogels possess a high degree of porosity, permitting highload capacity and can also be selectively surface functionalisedto enable tumor-specific targeting [15]. Core/shell hydrogelNPs (nanogels) with surface localized peptides that specificallytarget ovarian carcinoma cell lines possessing high expressionlevels of the ephrin A2 receptor (Eph2A) or have been devel-oped for siRNA delivery [15]. These nanogels were alsodemonstrated to be highly effective in the non-covalentencapsulation of siRNA and their delivery to targeted cell inserum-containing medium. Nanogel-mediated delivery ofsiRNA has also been documented to knockdown the EGFR.Nanogels have been reported to provide excellent protection

of siRNA during endosomal uptake and endosomal escapeduring targeted delivery [15].

4.2 NanoparticlesIn order to deliver siRNA-loaded NPs to specific intracellulardestinations to elicit a distinctive biological effect, it is opti-mal to apply small, monodisperse NPs with a defined zetapotential and surface chemistry [26]. Current focus is onadjustment of the size and surface properties of NPs for effi-cient transport of highly charged macromolecules such assiRNA [26]. AuNPs have been used to deliver plasmid DNAand siRNA into cells. AuNPs as a core of nanocomplex iseasy to synthesise and is highly biocompatible to cells and tis-sues [43,44]. Mirkin and coworkers [37] have documented theattachment of siRNA molecules to the surface of AuNPs viaa thiol group. The polyvalent siRNA/nanoparticle conjugateshave been reported to show a six time greater half life andprolonged gene knockdown compared with free RNAduplexes [37]. This has demonstrated the general use of AuNPsas a delivery vehicle. However, in some cases AuNPs seemedto aggregate upon assembly with nucleic acids. Two oppo-sitely charged polyelectrolytes have been coated on the surfaceof AuNPs in a layer by layer manner to prevent the self assem-bly of nucleic acids and aggregation of NPs [26]. Poly-b-aminoesters-siRNA--AuNPs has been used to knockdown afirefly luciferase expression in a dose dependent manner [45].

Solid lipid NPs (SLN) have been prepared by modified sol-vent emulsification method for use in siRNA delivery. siRNAwas conjugated with PEG via a disulfide linkage and the resul-tant siRNA--PEG conjugate was anchored on the cationic SLNvia charge interactions. siRNA--PEG/SLN complex showedhigher gene silencing efficiency, serum stability and reducedtoxicity [46]. siRNA targeting estrogen receptor-a (ERa) hasbeen encapsulated in polyisobutylcyanoacrylate (PIBCA) andPEG-co-poly(e-caprolactone-co-dodecyl-malate) (PEG-PLGA)nanocapsules. The size of such siRNA nanocapsules wasbetween 100 and 200 nm [47] documenting the suitability forcellular delivery. However, encapsulation yield with PIBCAnanocapsules was low and no release of trapped siRNA wasobserved for PEG-PLGA nanocapsules [47]. siRNA-loadednanocapsules resulted in persistent loss of the ER-a inMCF-7 human breast cancer cells. While intravenous injectionof these nanocapsules into estradiol-stimulated MCF-7 cellxenografts led to a significant decrease in tumor growth [47].

The NPs of EHCO have also been used to deliver siRNA [6]

(Table 1). EHCO-siRNA NPs enhanced the cellular uptake ofsiRNA, possibly due to nonspecific internalization throughcharge interaction of the positively charged NPs with thenegatively charged cell surface. While PEGylation ofEHCO--siRNA NPs has limited the nonspecific chargeinteraction and resulted in reduced cellular uptake of mPEG-EHCO--siRNA NPs [6]. The incorporation of tumor-specific peptides, RGD and bombesin, into the NPs via PEGspacer facilitated receptor-mediated endocytosis alsoincreased the cellular uptake of RGD-PEG-EHCO-siRNA

Nanoparticle

Streptavidin

Biotin

siRNA

Tetraethylene glycollinker

Figure 1. Schematic representation of antibody-siRNA

conjugate: biotinylated siRNA is coupled to

streptavidin-conjugated nanoparticles.



Exp

ert O

pin.

Bio

l. T

her.

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Rye

rson

Uni

vers

ity o

n 03

/18/

13Fo

r pe

rson

al u

se o

nly.

Table

1.NanosystemsforsiRNA

delivery.

TypeofNP

Size(nm)

Modeofattach

mentofsiRNA

Celllines

Advantages

Demerits

Ref.

Cationic

solid

lipid

nanoparticles

117

siRNAanchoredonthe

surface

ofsolid

lipid

nanoparticlesvia

electrostaticinteractions

PC3andMDAMB435

siRNA-PEG/SLN

complexes

exhibitedcomparable

gene

silencingefficiency

tosiRNA-PEG/PEIcomplexes

while

showingvery

low

cytotoxicity

Rapidly

clearedbyRES

[45]

LPD

nanoparticles

--Electrostaticinteractions

B16F10melanomacells

Hightumortargetingand

low

RESuptake

Rapidly

clearedbyRES

[21]

Cyclodextrincontaining

polymernanoparticles

(CDP)

100

Electrostaticinteractions

Cancercells

Luciferase

geneinhibition

wassignificantlygreaterfor

thetransferrin-targeted

nanoparticles

--[76]

(1-aminoethyl)iminobis

[N-(oleicylcysteinylhistinyl-

1-aminoethyl)propionamide]

(EHCO)

--Electrostaticinteractions

U87cells

PEG

modifiedEHCO/siRNA

nanoparticleswere

able

tofacilitate

endosomalescape

ofthesiRNA

delivery

systems

Show

aggregation

whencoupledto

siRNA

[6]

Liposomes

--Encapsulatedinsidethe

core

viaelectrosatic

interactions

--Encapsulatesalm

ost

50%

of

thesiRNA

inthecore

ofthe

PEGylatedliposomes

Rapidly

clearedbyRES

[32]

Liposomes

--Encapsulation

K562andLA

MA-84cells

Thetransferrin-liposomes

encapsulatingsiRNApromote

sequence-specificdown-regulation

oftheBCR--A

BLmRNA

Rapidly

clearedbyRES

[61]

Dendrimers

2.5

--5

Encapsulationinside

thecore

viaelectrostatic

interactions

A2780cells

Targetingofthedendrimer

specifically

totheplasm

amembraneofcancercells

Toxicatconcentration

of200nM

[63]

Dendriworm

s--

Noncovalentattachment

Humanglioblastomacells

Reduce

protein

levelsofEGFR

inhumanglioblastomacells

by

70--80%

,2.5-fold

more

efficiently

thancommercialcationiclipids

--[64]

Quantum

dots

17

Electrostaticinteractions

MDA-M

B-231

Proton-spongelayerform

edby

covalentgraftingoftertiary

amine

groupsontheQD

surface

leadsto

efficientsiRNA

release

from

intracellularvesicles

Toxicdueto

heavy

metalslikecadmium

[31]

Carbonnanotubes

--Bioconjugation

Tcells

andprimary

cells

Silencingeffect,knownto

block

HIV

viralentryandreduce

infection,is

superiorto

thatobservedwith

conventionalliposome-based

nonviraldelivery

agents

Showscytotoxicity

[59]

BCR--A

BL:

breakpointclusterregion--A

belsonleukemia

oncogenefusion;ER:estrogenreceptor;LPD:lipid

polycationDNA;MDR:multidrugresistant;PEI:polyethylenim

ine;QD:quantum

dot;RES:reticuloendothelial

system;SLN

:solid

lipid

nanoparticle.



Exp

ert O

pin.

Bio

l. T

her.

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Rye

rson

Uni

vers

ity o

n 03

/18/

13Fo

r pe

rson

al u

se o

nly.

Table

1.NanosystemsforsiRNA

delivery

(continued).

TypeofNP

Size(nm)

Modeofattach

mentofsiRNA

Celllines

Advantages

Demerits

Ref.

PEG-e-caprolactone-m

alic

acid

100--200

Encapsulation

MCF-7cell

Astronganddurable

inhibition

ofERatranslationwasobtainedin

BC

cells

aswellasin

xenografts

--[46]

PNIPMAm

nanogels

100

Encapsulation

HeyorBG-1

cells

TheencapsulatedsiRNA

istransportedinto

thecell

interior,where

itisavailable

forgenesilencing

--[15]

Mesoporoussilica

nanoparticles(M

SNP)

306

Adsorption

RAW

264.7,BEAS-2B,

PANC-1,andBxPC3

Noncovalentattachmentof

PEIpolymers

tothesurface

notonly

increasesMSNP

cellularuptakebutalso

generatesacationicsurface

towhichDNAandsiRNA

constructscould

beattached

--[77]

Gold

nanoparticles

13

Conjugation

Hela

cells

RNA--g

old

nanoparticle

conjugates,

whichcanbe

usedto

effectively

regulate

genes

Show

aggregation

[36]

Polymericmicelles

303.8

Electrostaticinteraction

Hu-7

cells

Polymericmicellescanbe

usedforthecodelivery

of

doxorubicin

andsiRNA

Show

cytotoxicity

inpresence

ofserum

proteins

[70]

Multifunctionalnanocarrier

100--140

Electrostaticinteraction

MDRlungcancercells

Multifucntionalnanoacrriers

canbeusedforovercoming

MDRin

cancercells

--[66]

BCR--A

BL:

breakpointclusterregion--A

belsonleukemia

oncogenefusion;ER:estrogenreceptor;LPD:lipid

polycationDNA;MDR:multidrugresistant;PEI:polyethylenim

ine;QD:quantum

dot;RES:reticuloendothelial

system;SLN

:solid

lipid

nanoparticle.



Exp

ert O

pin.

Bio

l. T

her.

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Rye

rson

Uni

vers

ity o

n 03

/18/

13Fo

r pe

rson

al u

se o

nly.

or BN-PEG-EHCO-siRNA NPs significantly comparedwith mPEG-EHCO-siRNA NPs. EHCO-siRNA NPs andPEG-modified EHCO-siRNA NPs were able to facilitateendosomal escape of the siRNA delivery systems. Systemicadministration of a therapeutic siRNA with the peptide-tar-geted delivery systems resulted in significantly improvedtumor growth inhibition compared with a nontargeted deliv-ery system or free siRNA via intravenous injection in nudemice bearing human glioma U87 xenografts [6].

Surface-modified lipid polycation DNA (LPD) NPs havebeen used to deliver siRNA to solid and metastatic tumors [48].The major components of this delivery system are cationic lip-ids and protamine (a cationic polypeptide), which facilitate theinteraction with negatively charged siRNA [48]. Surface stericstability was introduced by PEGylation to prevent the aggrega-tion of the resulting complex with serum components [48].Ligands were attached to the distal end of the PEG chain toincrease their cellular bioavailability. Cationic lipid is necessaryfor endosome lysis and intracellular release of siRNA. Themechanism of the endosome membrane destabilization couldbe due to the formation of an ion pair complexes betweenthe cationic lipid in the NPs and the negatively chargedanionic lipids in the endosome membrane [49]. Anisamide tar-geted PEGylated LPD formulation showed significant increasein cellular uptake via a specific receptor-mediated pathway.The function of anisamide was the specificity to tumor uptakeas nontargeted LPD NPs accumulated in the tumor as effi-ciently as the targeted particles via enhanced permeabilityretention effect (EPR) [50]. The surface modification of LPDNPs has been found to enhance the permeability and retentioneffect in the tumour [48]. This targeted formulation has alsobeen observed for strong gene-silencing effect. Surface-modified LPD has delivered siRNA predominantly to thetumor, a major uptake organ. It provides an advantage ofhigh tumor targeting and low RES uptake [21].

4.3 Quantum dotsQuantum dots (QDs) consist of a semiconductor core sur-rounded by organic ligands. Recently, attention has beenfocussed on using quantum dots for the delivery of siRNA(Figure 2). QDs are photostable CdSe/ZnS fluorescentnanocrystals that exhibit tunable emission properties for awide range of color possibilities [51]. Combining QDs withsiRNA for RNAi tracking requires neither chemical labelingof siRNA, which is costly and can potentially deter complex-ing with RISC, nor the expression of reporter plasmids. QDsare also brighter than most conventional fluorescent dyes [52].Therefore, they provide easier in vivo detection than greenfluorescent protein (GFP) in the autofluorescence back-ground [52]. Furthermore, QDs are far less susceptible to pho-tobleaching, fluorescing more than 20 times longer thanconventional fluorescent dyes under continuous mercurylamp exposure [53].

For siRNA delivery, the surface of QDs have been modifiedwith different ligands to make them suitable for attaching

siRNA. Quantum dots of poly(maleic anhydride-alt-1-decene) (QD-PMAL) modified with dimethylaminopropylamine has been used for the delivery of siRNA [31].QD-PMAL is efficient in siRNA intracellular delivery forboth serum-free and complete media. In contrast, lipofect-amine only works well in serum-free media. Clustered tertiaryamines mounted on a polymer backbone have strong protonabsorbing capabilities inside endosomes (pH 5 -- 6) and lyso-somes (pH 4 -- 5), leading to rapid osmotic swelling andsiRNA release. The ratio of carboxylic and tertiary aminegroups provides a tunable parameter for optimizing theRNA silencing efficiency and its cellular toxicity [31]. Withless tertiary amines, the affinity between QDs and siRNAwas weak, whereas with more amines the cytotoxicty ofQDs was increased. Tertiary amines provide a positive chargefor electrostatic binding of siRNA as well as protection fromenzymatic degradation. The coexistence of carboxylic andamino groups in a zwitterion form is an important structuralfeature. Such formation weakens siRNA binding to the carrierQDs and thus facilitates siRNA unpackaging [31]. At low pHcarboxylic and amine groups are protonated, while at high pHthey are deprotonated. Therefore, the zwitterionic surfacebehaves like a buffer system that can quickly neutralize excessprotons in the endosome. The osmotic pressure buildingalong this proton buffering process will eventually rupturethe endosomes, releasing siRNA [54].

QDs coated with primary amines were reported to bindsiRNA more strongly than the tertiary amine. But these arelargely ineffective for gene silencing. Sonawane et al. [55]

have also shown that QDs coated with a proton sponge layer(PEG-modified PEI) are able to penetrate cells and deliversiRNA through escape from intracellular organelles. Specifictargeting can be developed in QD--siRNA by use of peptides,antibodies and aptamers. Generally, QDs are attached withsiRNA through disulfide crosslinkers rather than electrostaticinteractions (Figure 2). Tumor bearing peptide (f3), targetingcell surface nucleolin has been attached to the surface of QDsfor increasing cellular uptake. Delivery of these f3-siRNA-QDs to enhanced GFP (EGFP) transfected Hela cells hascaused a significant knockdown of EGFP signal [35]. Further-more, QDs-siRNA formulation produced with the disulfidebond (using sulfo-LC-SPDP) has led to greater EGFP knock-down. The level of knockdown attained with disulfide-linked QDs-siRNA, however, was less than observed whenan equal concentration of free siRNA was delivered withlipofectamine [35]. Note the proton-sponge effect, which canbe precisely controlled by partially converting the carboxylicacid groups into tertiary amines. When both are linked tothe surface of nanometer-sized particles, these two functionalgroups provide steric and electrostatic interactions. Suchinteractions are highly responsive to the acidic organellesand are also well suited for siRNA binding and cellular entry.Use of QDs systems has been found to increase gene silencingactivity by 10-fold -- 20-fold and reduce the cellular toxicityby 5-fold -- 6-fold in MDA-MB-231 cells compared to



Exp

ert O

pin.

Bio

l. T

her.

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Rye

rson

Uni

vers

ity o

n 03

/18/

13Fo

r pe

rson

al u

se o

nly.

various other siRNA delivery agents (lipofectamine, JetPEI,and TransIT) [31].

4.4 Carbon nanotubesThe unusual electrical and mechanical properties of carbonnanotubes (CNTs) make them innovative nanosystems withinthe emerging platforms for therapeutic and diagnostic pur-poses [56,57]. However, native CNTs have poor solubility andbiocompatibility. Hence, functionalization of CNTs withconjugating molecules such as peptides, nucleic acids andpolymers is required to facilitate their use in siRNA delivery.The potential of SWNTs for the cellular delivery of DNAplasmids and siRNA has recently been investigated. The func-tionalized SWNTs with lipids and natural-amino-acid-baseddendrimers (tetra-oleoyl lysine dendrimer tubules; TOT)have been used to complex siRNA [58]. The TOT--siRNAcomplex has been found quite effective in gene silencing [58].Multiwalled carbon nanotubes (MWNTs) have beenfunctionalised with dendrons and used for efficient cyto-plasmic delivery of siRNA [59]. siRNA linked to phospho-lipid functionalized SWNTs via cleavable disulfide linkagehave shown highly efficient siRNA delivery and a more

potent RNAi functionality than a widely used transfectionagent, lipofectamine [34]. A SWNT-based siRNA deliverysystem has been found useful for transfecting human T cellsand primary cells, which are resistant to delivery byconventional cationic liposome-based transfection agents [60].Surface-functionalization-dependent cell uptake of SWNTswas also observed. Compared with SWNTs coated withlong PEG (5.4 kDa) groups, shorter PEG (2 kDa)-coatedSWNTs with more exposed hydrophobic surface have beenreported for higher cellular uptake. This was due to incom-plete coverage of nanotube sidewalls that facilitate the bindingand association by hydrophobic-hydrophobic interactionswith cell membrane. Cell binding of SWNTs is the first stepfor cellular entry via endocytosis. Balanced chemical function-alization schemes that impart sufficient aqueous solubilityand biocompatibility to nanotubes could be important forintracellular delivery of siRNA by CNTs [61].

4.5 Lipid nanoformulationsLipid nanoparticles are based on lipoplex formulations. Size,lamellarity and surface charge of such NPs can be controlledby the variation of formulation conditions. Typically, lipid

QD core

Nucleus

Interaction with RISC

Endosome escape

siRNA

Ligand

PEG

mRNA degradation

Internalization

QD core

QD core

Figure 2. Quantum dots (QDs) are suitable vehicles for carrying siRNA into live cells in vitro and in vivo. Conjugation of

ligands (along with the siRNA cargo) to the QD surface allows targeted internalization in tumor cells. Once internalized, these

particles must escape the endolysosomal pathway and reach the cytoplasm to interact with the RNA-induced silencing

complex (RISC), which leads to degradation of mRNA homologous to the siRNA sequence.



Exp

ert O

pin.

Bio

l. T

her.

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Rye

rson

Uni

vers

ity o

n 03

/18/

13Fo

r pe

rson

al u

se o

nly.

nanoparticles consist of cationic lipid, a PEGylated lipid,cholesterol, neutral helper lipids and the siRNA. The lipid com-ponent is responsible for both cell association and endosomalescape. PEGylated lipid is required to stabilise the particle toimprove circulation half-life and systemic exposure [62]. Distri-bution of lipid nanoparticles can be controlled by varyingformulation parameters. Lipid nanoparticles undergo acationic lipid-mediated mechanism of endosomal escape. Bycombination of the optimal headgroups pKa and optimalphase-transition biophysical properties, cationic lipids can besynthesized which undergo endosomal escape. This propertymakes lipid nanoparticles unique from other nanocarriers andthe most advanced siRNA delivery system which has enteredinto clinical trials [62]. Liposome is also made up of an aqueouscompartment enclosed in a phospholipid and is capable ofentrapping a hydrophilic molecule. The lipid component ofthe liposome bilayer is often a cationic and/or a fusogenic lipid,cholesterol and PEG-lipid [63]. The use of liposomes to deliversiRNA allows protection from nuclease degradation, confer sus-tained plasma concentration and provide targeted delivery [63].PEG-liposomes have been used for encapsulating siRNA. ThesiRNA encapsulation yield of liposome has been found to bedependent on the concentration of the encapsulation buffer.At 20 mM of citrate buffer, a 92.17% encapsulation yield wasachieved, whereas at 300 mM drastic reduction of the encapsu-lation yield to 7.72% was observed [62]. The encapsulation effi-ciency of PEGylated liposomes was found to be 50% [32].Transferrin ligand (Trf) was coupled to the surface of liposomesfor targeted siRNA delivery to Trf receptor. The Trf-liposomesencapsulating siRNA has been documented to promotesequence-specific down-regulation of the breakpoint clusterregon--Abelson murine leukemia gene fusion (BCR-ABL)mRNA [63].

4.6 DendrimersDendrimers are a type of regular and highly branched, mono-disperse, spherical nanomaterial with dense peripheral groupsthat can be functionalized with drugs, targeting moieties andother biologically active components [64]. These highly effi-cient delivery systems have been explored to a lesser extentfor siRNA delivery [65]. Quaternary poly(amidoamine) (QPA-MAM-NHAc) dendrimers had neutral outer surfaces withinternal positive charges. Therefore, siRNA could be inter-nalized at least partially, inside the dendrimer. Since siRNAis smaller than DNA, it can easily penetrate between den-drimer branches. Occassionaly, siRNA is partially accommo-dated inside the dendrimer branches and the remainingpart of the siRNA is internalized in another molecule ofdendrimer [65]. The surface-modified and internally quater-nized QPAMAM-NHAc dendrimer has been reported toshow an excellent cell permeability. This has resulted in a sub-stantial accumulation of siRNA in cells within 80 min. Thecellular uptake of siRNA was significantly improved by theuse of dendrimer conjugated with targeting ligand luteinizinghormone-releasing hormone (LHRH) [65].

Dendriworms have also been used for efficient siRNAdelivery. Dendriworms consist of PAMAM dendrimer andcross-linked iron oxide nanoworms, which are labeledwith near-infrared fluorochromes-reduced dendrons. Nano-worms were conjugated to siRNA linked dendrimers usingthe heterobifuctional linker N-succinimidyl 3-(2- pyridyldi-thio)-propionate, which links amine groups on the nano-particle with the central thiols on reduced dendrons. A keyfeature of the dendriworms is that dendrons are conjugatedvia a reducible linker (SPDP) to the nanoparticle surface.This may allow removal of dendrons from the NPs surfacein the reducing intracellular environment, resulting inimproved siRNA delivery and diffusion inside cells [66]

(Table 1).

4.7 Multifunctional nanocarriersMultifunctional nanocarriers can encapsulate siRNA, over-come endosomal uptake and enter into cytoplasm and a tar-geting ligand can be attached to them for targeted delivery.Multifunctional nanocarriers can be used for the treatmentof multidrug resistance by codelivery of anticancer drug andsuppressors of cellular resistance [67]. This strategy has beenused for the dual delivery of doxorubicin and siRNA forinducing cell death and suppression of cellular resistancein multidrug resistant cells [67]. Combinatorial delivery ofsiRNA with the anticancer drug cisplatin has significantlyimproved the antitumor effects and imparted cell-targetedselectivity with the specificity of siRNA [68]. In anotherstudy, multifunctional nanocarriers have also been used forthe codelivery of siRNA and doxorubicin to overcome drugresistance in cancer. This study highlighted the potential ofmultifunctional nanocarriers for siRNA delivery [69].

4.8 Polymeric micellesPolymeric micelles are formed from amphiphilic diblockcopolymers that self-assemble to form micelles with hydro-phobic cores and cationic coronas. These micelles can electro-statically form a complex with siRNA and their hydrophobiccore can be used for the loading of chemotherapeuticdrugs. Polymeric micelles made up of diblock copolymers ofdimethylaminoethyl methacrylate and butyl methacrylatehave been used for the dual delivery of siRNA and doxorubi-cin. Co-delivery of siRNA and doxorubicin has resulted inless potent cell killing and caspase upregulation whencompared with independent delivery of siRNA and doxo-rubicin [70]. Polymeric micelles made up of stearic-acid-grafted polyethyleneimine have been used for siRNA anddoxorubicin delivery. siRNA delivered by this nanocarrierhas demonstrated higher cellular uptake [71]. Interestingly,polymeric micelles composed of poly(N,N-dimethylami-noethyl methacrylate), negatively charged propylacrylic acidresidues and hydrophobic butyl methacrylate residues showedhigh serum stability and mediated endosomal escape ofsiRNA in a pH-dependent fashion [72]. Also, GFP--siRNAwith a phosphothioethanol formulated into micelles with



Exp

ert O

pin.

Bio

l. T

her.

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Rye

rson

Uni

vers

ity o

n 03

/18/

13Fo

r pe

rson

al u

se o

nly.

polyethylene glycol--phosphothioethanol has downregulatedthe GFP production more efficiently than free siRNA [73].

5. siRNA delivery systems in clinical trials

Lipid nanoparticles represent the superior macromolecularsiRNA delivery systems. The most advanced of these isthe cyclodextrin-containing polycation system [62]. Cationiccyclodextrins undergo association with negatively-chargedsiRNA. Cyclodextrin groups can be capped with admantaneshaving PEG groups, some of which contain targetingligands. This technology was licensed to Calando. Thiscompany developed a product which is into Phase I studies.Alnylam is studying the delivery of two siRNA genes (kid-ney-specific cadherin (KSP) and VEGF) formulated in stablenucleic acid lipid particle designed by Tekmira. Tekmirahas also used stable nucleic acid based lipid particle in aPhase I trial to study the safety of apolipoprotein B (ApoB)siRNA [62].

6. Off-target effects in siRNA delivery

The most vital effect while using RNAi pathway for thera-peutic application is the recognition and binding of siRNAin the cytosol to the correct mRNA for efficient gene silenc-ing. Besides the barriers related to stability and bioavailabil-ity several classes of off-target effects have been reported forsiRNA. Unmethylated CpG motifs have been found to pro-voke the immune system [74]. Other off-target effects resultfrom guanine quartet sequences, which bind to many cellu-lar proteins. It is possible that siRNA can bind to unin-tended mRNA through partial mismatch. This problemcan be overcome by using chemical modification of thesiRNA guide strand, which conserves their ability to bindwith their targets. Chemical modification of the siRNAguide strand can be used to increase specificity to targetmRNA as well as to reduce off-target effects [75]. A secondapproach could be the use of nanocarriers to reduce off-target effects in siRNA delivery. Association of siRNA withnanocarriers protects siRNA from degradation and modifiesits tissue and cellular distribution to enable it to reachthe target.

7. Conclusions

Development of an efficient delivery system is one of the mostchallenging hurdles to turn siRNAs into clinically acceptabletherapeutic drugs. RES, renal clearance, and endosomal trap-ping are the major barriers encountered in siRNA delivery.Conjugation of siRNAs to different nanosystems couldimprove their pharmacokinetic behaviours, cellular uptakeefficiencies and cell specificities. The use of nanosystemsincreases cellular uptake and transfection efficiency, and alsoreduces the toxicity associated with cationic materials. Surfacemodification of nanosystems with PEG will improve the

cellular uptake, protect siRNA from degradation and facilitateentry into target cells. Nanocarriers are able to efficientlydeliver siRNA to tumor cells and other normal tissues withleaky vasculature. Furthermore, the conjugates of siRNA-nanocarriers with targeting ligands have advantages intargeting siRNA to specific cells or tissues.

8. Expert opinion

Development of an efficient delivery system is one of the mostchallenging hurdles to turn siRNAs into clinically acceptabletherapeutic drugs. RES, renal clearance and endosomaltrapping and off-target effects are the major barriers encoun-tered in siRNA delivery. siRNA delivered to the wrong cellsmay have undesirable consequences. Nanosystems can signif-icantly reduce the off-target effects associated with siRNAdelivery. Conjugation of siRNA to different nanosystemscould improve their pharmacokinetic behaviours, cellularuptake efficiencies and cell specificities. Interestingly, the useof nanosystems has reduced the toxicity associated with cat-ionic materials. Another benefit of using ligand-targetednaocarriers with siRNA is the potential to develop dual levelsof specificity, selective tissue localisation by the nanoparticlesand selective therapeutic activity by siRNA. Surface modifica-tion of nanosystems with PEG has been found useful forfurther improvement in the cellular uptake and protectionof siRNA from degradation and facilitates entry into targetcells. The field of siRNA therapeutics has achieved lineargrowth along with nanotechnology. The suitable nanoencap-sulation of siRNA on biodegradable and biocompatible nano-carriers eliminates the possibilities of degradation, structuraland functional deformities and metabolism before reachingthe target sites. Overcoming biological barriers through theuse siRNA as therapeutic agents by advances in ligand-targeted nanocarriers pave the way towards the developmentof better targeted therapeutics.

siRNA therapy can be focussed against cancer using a diver-sity of strategies, including the inhibition of overexpressedoncogenes, promoting apoptosis, regulating the cell cycle,antiangiogenesis and enhancing the efficacy of chemotherapyand radiotherapy. The conjugation of nanocarriers withsiRNA offers substantial prospects for a revolution in thediagnosis and treatment of diseases such as cancer. Nanocar-riers efficiently deliver siRNA to tumor cells and other normaltissues with leaky vasculature, while conjugates of siRNA-nanocarriers with targeting ligands have advantages in target-ing siRNA to specific cells or tissues. Co-delivery of anticancerdrug and siRNA with nanocarriers can overcome multidrugresistance in cancer cells. Through careful combination ofanticancer drug and siRNA, it is possible to induce cell deathand suppress cellular resistance in multidrug-resistant cells.Although there is still a long way to go before we eradicatecancers, we believe that delivery of siRNA by nanocarrierswill eventually serve as an essential tool in cancer therapyand enter into the clinical field in the near future.



Exp

ert O

pin.

Bio

l. T

her.

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Rye

rson

Uni

vers

ity o

n 03

/18/

13Fo

r pe

rson

al u

se o

nly.

Among different nanosystems, lipid nanoparticles are themost advanced systems for siRNA delivery. By strategicallydesigning lipid nanoparticles, properties can be optimisedfor better siRNA delivery. Due to this flexibility, their usehas entered into clinical trials. Three leading companies,Tekmira, Calando and Alnylam, are actively engaged in devel-oping lipid-nanoparticle-based delivery vehicles for siRNAdelivery. The combination of nanosystems with siRNA offerssubstantial potential for a revolution in the diagnosis andtreatment of diseases.

Though nanosystems have proven excellent for siRNAdelivery, the possibility of associated risks of toxicity shouldnot be ignored. For example, quantum dots use heavy metalslike cadmium which are toxic for in vivo applications. There-fore, toxicity evaluations of such nanosystems for siRNAdelivery are recommended.

Acknowledgements

The Director, CSIR-Institute of Himalayan BioresourceTechnology (IHBT) is duly acknowledged for providingnecessary facility and guidance.

Declaration of interest

The Council of Scientific and Industrial Research (CSIR)and Department of Biotechnology (DBT), of theGovenment of India are duly acknowledged for financialsupport in the lab. Avnesh Kumari thanks the Depart-ment of Science & Technology Women ScientistAward (DST-WOSA) of the Government of Indiascheme for financial assistance to conduct research inour laboratory.

BibliographyPapers of special note have been highlighted as

either of interest (�) or of considerable interest(��) to readers.

1. Dykxhoorn DM, Novina CD, Sharp PA.

Killing the messenger: short RNAs that

silence gene expression. Nat Rev Mol

Cell Biol 2003;11:457-67

2. Huang C, Li M, Chen C, Yao Q. Small

interfering RNA therapy in cancer:

mechanism, potential targets, and clinical

applications. Expert Opin Ther Targets

2008;12:637-45

3. Cheng K, Mahato RI. siRNA delivery

and targeting. Mol Pharm 2009;6:649-50

4. Jeong JH, Kim SW, Park TG. Molecular

design of functional polymers for gene

therapy. Prog Polym Sci

2007;32:1239-74

5. Behlke MA. Progress towards in vivo

use of siRNAs. Mol Ther

2006;13:644-70

6. Wang XL, Xu R, Wu X, et al. Targeted

systemic delivery of a therapeutic

siRNA with a multifunctional carrier

controls tumor proliferation in mice.

Mol Pharm 2009;6:738-46

7. Aagaard L, Rossi JJ. RNAi therapeutics:

principles, prospects and challenges.

Adv Drug Deliv Rev 2007;59:75-86

8. Conner SD, Schmid SL. Regulated

portals of entry into the cell. Nature

2003;422:37-44.. This paper is of considerable interest

for its description of portals of entry

into the cell.

9. Panyam J, Zhou WZ, Prabha S, et al.

Rapid endo-lysosomal escape of poly

(DL-lactide-co-glycolide) nanoparticles:

implications for drug and gene delivery.

Faseb J 2002;16:1217-26

10. Vasir JK, Reddy MK, Labhasetwar VD.

Nanosystems in drug targeting:

opportunities and challenges.

Current Nanoscience 2005;1:47-64

11. Chakraborty C. Potentiality of small

interfering RNAs (siRNA) as recent

therapeutic targets for gene-silencing.

Curr Drug Targets 2007;8:469-82

12. Wilson JA, Richardson CD. Future

promise of siRNA and other nucleic acid

based therapeutics for the treatment of

chronic HCV. Infect Disord

Drug Targets 2006;6:43-56

13. Storvold GL, Andersen TI, Perou CM,

et al. siRNA: a potential tool for future

breast cancer therapy? Crit Rev Oncog

2006;12:127-50

14. Khaled A, Guo S, Li F, Guo P.

Controllable self-assembly of

nanoparticles for specific delivery of

multiple therapeutic molecules to cancer

cells using RNA nanotechnology.

Nano Lett 2005;5:1797-08

15. Blackburn WH, Dickerson EB,

Smith MH, et al. Peptide-functionalized

nanogels for targeted siRNA delivery.

Bioconjug Chem 2009;20:960-8

16. Sinha R, Kim GJ, Nie S, Shin DM.

Nanotechnology in cancer therapeutics:

bioconjugated nanoparticles for drug

delivery. Mol Cancer Ther

2006;5:1909-17

17. Ikeda Y, Taira K. Ligand-targeted

delivery of therapeutic siRNA.

Pharm Res 2006;23:1631-40

18. Davis ME. The first targeted delivery of

siRNA in humans via a self-assembling,

cyclodextrin polymer-based nanoparticle:

from concept to clinic. Mol Pharm

2009;6:659-68

19. Zhang S, Zhao B, Jiang H, et al.

Cationic lipids and polymers mediated

vectors for delivery of siRNA.

J Control Release 2007;123:1-10

20. Neu M, Fischer D, Kissel T. Recent

advances in rational gene transfer vector

design based on poly(ethylene imine) and

its derivatives. J Gene Med

2005;7:992-1009

21. Gao K, Huang L. Nonviral methods

for siRNA delivery. Mol Pharm

2009;6:651-8. This article is of interest for its

description of non viral methods for

siRNA delivery.

22. Gary DJ, Puri N, Won YY.

Polymer-based siRNA delivery:

perspectives on the fundamental and

phenomenological distinctions from

polymer-based DNA delivery.


23. Dash PR, Read ML, Barrett LB, et al.

Factors affecting blood clearance and

in vivo distribution of polyelectrolyte

complexes for gene delivery. Gene Ther

1999;6:643-50

24. Davis ME, Zuckerman JE, Choi CHJ,

et al. Evidence of RNAi in humans from

systemically administered siRNA via

targeted nanoparticles. Nature

2010;464:1067-70.. This article is of substantial interest for

its description of RNAi in humans



Exp

ert O

pin.

Bio

l. T

her.

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Rye

rson

Uni

vers

ity o

n 03

/18/

13Fo

r pe

rson

al u

se o

nly.

from systemically administered

siRNA via targeted nanoparticles.

25. Vornlocher H-P. Antibody-directed

cell-type-specific delivery of siRNA.

Trends Mol Med 2006;12:1-3

26. Elbakry A, Zaky A, Liebl R, et al.

Layer-by-layer assembled gold

nanoparticles for siRNA delivery.

Nano Lett 2009;9:2059-64

27. Juliano R, Bauman J, Kang H, Ming X.

Biological barriers to therapy with

antisense and siRNA oligonucleotides.

Mol Pharm 2009;6:686-95

28. Kim SH, Jeong JH, Lee SH, et al. Local

and systemic delivery of VEGF

siRNA using polyelectrolyte complex

micelles for effective treatment of cancer.


29. Kumari A, Yadav SK, Yadav SC.

Biodegradable polymeric nanoparticles

based drug delivery systems.

Colloids Surf B Biointerfaces

2010;75:1-18

30. Baigude H, Rana TM. Delivery of

therapeutic RNAi by nanovehicles.

Chembiochem 2009;10:2449-54

31. Yezhelyev MV, Qi L, O’Regan RM,

et al. Proton-sponge coated quantum

dots for siRNA delivery and intracellular

imaging. J Am Chem Soc

2008;130:9006-12

32. Buyens K, Demeester J, De Smedt SS,

Sanders NN. Elucidating the

encapsulation of short interfering

RNA in PEGylated cationic liposomes.

Langmuir 2009;25:4886-91

33. Jeong JH, Mok H, Oh YK, Park TG.

siRNA conjugate delivery systems.


34. Kam NW, Liu Z, Dai H.

Functionalization of carbon nanotubes

via cleavable disulfide bonds for efficient

intracellular delivery of siRNA and

potent gene silencing. J Am Chem Soc

2005;127:12492-3.. This article is of great interest for its

description of efficient delivery of

siRNA by carbon nanotubes.

35. Derfus AM, Chen AA, Min DH, et al.

Targeted quantum dot conjugates for

siRNA delivery. Bioconjug Chem

2007;18:1391-6

36. Hermanson GT. Heterobifunctional

crosslinkers. In: Bioconjugate techniques,

Illinois, USA: Elsevier; 2008

37. Giljohann DA, Seferos DS,

Prigodich AE, et al. Gene regulation with

polyvalent siRNA-nanoparticle

conjugates. J Am Chem Soc

2009;131:2072-3

38. Medarova Z, Pham W, Farrar C, et al.

In vivo imaging of siRNA delivery and

silencing in tumors. Nat Med

2007;13:372-7

39. Xia CF, Zhang Y, Zhang Y, et al.

Intravenous siRNA of brain cancer with

receptor targeting and avidin-biotin

technology. Pharm Res 2007;24:2309-16

40. Wu D, Pardridge WM. Central nervous

system pharmacologic effect in conscious

rats after intravenous injection of a

biotinylated vasoactive intestinal peptide

analog coupled to a blood-brain barrier

drug delivery system. J Pharmacol

Exp Ther 1996;279:77-83

41. Akhtar S, Benter IF. Nonviral delivery of

synthetic siRNAs in vivo. J Clin Invest

2007;117:3623-32

42. Kim SH, Jeong JH, Lee SH, et al.

LHRH receptor-mediated delivery of

siRNA using polyelectrolyte complex

micelles self-assembled from

siRNA-PEG-LHRH conjugate and PEI.


43. Daniel MC, Astruc D. Gold

nanoparticles: assembly, supramolecular

chemistry, quantum-size-related

properties, and applications toward

biology, catalysis, and nanotechnology.

Chem Rev 2004;104:293-346

44. Shukla R, Bansal V, Chaudhary M, et al.

Biocompatibility of gold nanoparticles

and their endocytotic fate inside

the cellular compartment: a

microscopic overview. Langmuir

2005;21:10644-54

45. Lee JS, Green JJ, Love KT, et al. Gold,

poly(beta-amino ester) nanoparticles for

small interfering RNA delivery.

Nano Lett 2009;9:2402-6

46. Kim HR, Kim IK, Bae KH, et al.

Cationic solid lipid nanoparticles

reconstituted from low density

lipoprotein components for

delivery of siRNA. Mol Pharm

2008;5(4):622-31

47. Bouclier C, Moine L, Hillaireau H, et al.

Physicochemical characteristics and

preliminary in vivo biological evaluation

of nanocapsules loaded with

siRNA targeting estrogen receptor

alpha. Biomacromolecules

2008;9:2881-90

48. Li SD, Huang L. Surface-modified LPD

nanoparticles for tumor targeting. Ann N

Y Acad Sci 2006;1082:1-8

49. Xu Y, Szoka FC Jr. Mechanism of

DNA release from cationic liposome/

DNA complexes used in cell transfection.

Biochemistry 1996;35:5616-23

50. Li SD, Huang L. Targeted delivery of

antisense oligodeoxynucleotide

and small interference RNA into

lung cancer cells. Mol Pharm

2006;3:579-88

51. Akerman ME, Chan WC, Laakkonen P,

et al. Nanocrystal targeting in vivo.

Proc Natl Acad Sci USA

2002;99:12617-21

52. Gao X, Cui Y, Levenson RM, et al. In

vivo cancer targeting and imaging with

semiconductor quantum dots.

Nat Biotechnol 2004;22:969-76

53. Derfus AM, Chan WCW, Bhatia SN.

Intracellular delivery of quantumdots for

live cell labeling and organelle tracking.

Adv Mater 2004;16:961-6

54. Boussif O, Lezoualc’h F, Zanta MA,

et al. A versatile vector for gene and

oligonucleotide transfer into cells in

culture and in vivo: polyethylenimine.

Proc Natl Acad Sci USA

1995;92:7297-301

55. Sonawane ND, Szoka FC Jr,

Verkman AS. Chloride accumulation

and swelling in endosomes enhances

DNA transfer by polyamine-DNA

polyplexes. J Biol Chem

2003;278:44826-31

56. Bianco A, Kostarelos K, Prato M.

Opportunities and challenges of

carbon-based nanomaterials for cancer

therapy. Expert Opin Drug Deliv

2008;5:331-42

57. Peer D, Karp JM, Hong S, et al.

Nanocarriers as an emerging platform for

cancer therapy. Nat Nanotechnol

2007;2:751-60. This article is of importance for its

description of nanocarriers in

cancer therapy.

58. McCarroll J, Baigude H, Yang CS,

Rana TM. Nanotubes functionalized

with lipids and natural amino acid

dendrimers: a new strategy to create

nanomaterials for delivering systemic

RNAi. Bioconjug Chem 2009;21:56-63



Exp

ert O

pin.

Bio

l. T

her.

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Rye

rson

Uni

vers

ity o

n 03

/18/

13Fo

r pe

rson

al u

se o

nly.

59. Herrero MA, Toma FM, Al-Jamal KT,

et al. Synthesis and characterization of a

carbon nanotube-dendron series for

efficient siRNA delivery. J Am

Chem Soc 2009;131:9843-8

60. Liu Z, Winters M, Holodniy M, Dai H.

siRNA delivery into human T cells and

primary cells with carbon-nanotube

transporters. Angew Chem Int Ed Engl

2007;46:2023-7

61. Liu Z, Tabakman S, Welsher K, Dai H.

Carbon Nanotubes in biology and

medicine: In vitro and in vivo detection,

imaging and drug delivery. Nano Res

2009;2:85-120

62. Stanton MG, Colletti SL. Medicinal

chemistry of siRNA delivery.

J Med Chem 2010;53:7887-901. This article is important for its

description of medicinal chemistry

of siRNA.

63. Mendonca LS, Firmino F, Moreira JN,

et al. Transferrin receptor-targeted

liposomes encapsulating anti-BCR-ABL

siRNA or asODN for chronic myeloid

leukemia treatment. Bioconjug Chem

2010;21:157-68

64. Gillies ER, Frechet JM. Dendrimers and

dendritic polymers in drug delivery.

Drug Discov Today 2005;10:35-43

65. Patil ML, Zhang M, Betigeri S, et al.

Surface-modified and internally cationic

polyamidoamine dendrimers for efficient

siRNA delivery. Bioconjug Chem

2008;19:1396-03

66. Agrawal A, Min DH, Singh N, et al.

Functional delivery of siRNA in mice

using dendriworms. ACS Nano

2009;3:2495-504

67. Saad M, Garbuzenko OB, Minko T.

Co-delivery of siRNA and an anticancer

drug for treatment of multidrug-resistant

cancer. Nanomedicine 2008;3:761-76

68. Chen Y, Bathula SR, Li J, Huang L.

Multi-functional nanoparticles delivering

siRNA and doxorubicin overcome drug

resistance in cancer. J Biol Chem

2010;285:22639-50

69. Taratula1 O, Garbuzenko O, Savla R,

et al. Multifunctional nanomedicine

platform for cancer specific delivery of

siRNA by superparamagnetic iron oxide

nanoparticles-dendrimer complexes.

Curr Drug Delivery 2011;8:59-69

70. Benoit DSW, Henry SM, Shubin AD,

et al. pH-Responsive polymeric

siRNA carriers sensitize multidrug

resistant ovarian cancer cells to

doxorubicin via knockdown of Polo-like

Kinase 1. Mol Pharm 2010;7:442-55

71. Huang HY, Kuo WT, Huang YY.

Combining gene and chemo therapy

using multifunctional polymeric micelles.

World Acad Sci Eng Technol

2010;65:1142-5

72. Convertine AJ, Diab C, Prieve M, et al.

pH-Responsive polymeric micelle carriers

for siRNA drugs. Biomacromolecules

2010;11:2904-11

73. Musacchio T, Vaze O, D’Souza G,

Torchilin VP. Effective Stabilization and

delivery of siRNA: reversible

siRNA-phospholipid conjugate in

nanosized mixed polymeric micelles.

Bioconjugate Chem 2010;21:1530-6

74. Fattal E, Barratt G. Nanotechnologies

and controlled release systems for the

delivery of antisense oligonucleotides and

small interfering RNA. Br J Pharmacol

2009;157:179-94

75. Tokatlian T, Segura T.

siRNA applications in nanomedicine.

Nanomed Nanobiotechnol

2010;2:305-15

76. Bartlett DW, Davis ME. Impact of

tumor-specific targeting and dosing

schedule on tumor growth inhibition

after intravenous administration of

siRNA-containing nanoparticles.

Biotechnol Bioeng 2008;99:975-85

77. Xia T, Kovochich M, Liong M, et al.

Polyethyleneimine coating enhances the

cellular uptake of mesoporous silica

nanoparticles and allows safe delivery of

siRNA and DNA constructs. ACS Nano

2009;3:3273-86

AffiliationAvnesh Kumari, Vineet Kumar &

Sudesh Kumar Yadav†

†Author for correspondence

Council of Scientific and Industrial Research

(CSIR), Institute of Himalayan Bioresource

Technology,

Biotechnology Division, Nanobiology Lab,

Palampur-176061 (HP),

India

Tel: +91 9418050116;

E-mail: [email protected];

[email protected]



Exp

ert O

pin.

Bio

l. T

her.

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Rye

rson

Uni

vers

ity o

n 03

/18/

13Fo

r pe

rson

al u

se o

nly.

Documents

Nanocarriers: a tool to overcome biological barriers in siRNA delivery