Acid-Degradable Cationic Dextran Particles for the Delivery of SiRNA DEXTRANA E ESPERMINA

Published: May 03, 2011

r 2011 American Chemical Society 1056 dx.doi.org/10.1021/bc100542r | Bioconjugate Chem. 2011, 22, 10561065

ARTICLE

pubs.acs.org/bc

Acid-Degradable Cationic Dextran Particles for the Delivery of siRNATherapeuticsJessica L. Cohen, Stephanie Schubert, Peter R. Wich, Lina Cui, Joel A. Cohen, Justin L. Mynar,and Jean M. J. Frechet*

College of Chemistry, University of California, Berkeley, California 94720-1460, United States

INTRODUCTION

RNA interference (RNAi) has drawn much attention in theeld of medicine due to its potential for treating chronic diseasesand genetic disorders by harnessing the endogenous RNAipathway.13 RNAi is a biological mechanism wherein double-stranded RNAs can be used to reduce expression of targetproteins.4,5 Once the RNA is present in the cytoplasm of the cell,it is shortened and processed by the RNase III enzyme, Dicer,6 andincorporated into a protein complex called the RNA-inducedsilencing complex (RISC).7 One of the two strands of the short,double-stranded RNA is cleaved, and the activated RISC (whichcontains the guide strand of the RNA) binds to a complementarysequence of mRNA and results in its degradation.8 The activatedRISC is capable of multiple rounds of mRNA cleavage, whichpropagates gene silencing.9 Due to its potential to silence genes ina sequence-specic manner, RNAi holds promise for treatingmany diseases that may not otherwise be accessed with currenttherapeutic technology.1

Various approaches have been developed that allow forexploitation of the RNAi process, principally through the useof exogenous synthetic small interfering RNA (siRNA), double-stranded RNAs that are typically 1923 base pairs in length.Synthetic siRNA can be designed to target nearly any gene in thebody, and is therefore attractive for a variety of medical applica-tions. Previous reports have demonstrated that synthetic siRNAsare capable of knocking down targets in several diseases in vivo,including hepatitis B virus, human papillomavirus, and ovariancancer.10 Despite great therapeutic potential, the clinical applica-tion of siRNA is limited by delivery problems. siRNA does notcross cellular membranes eciently due to its relatively large size,negative charge, and hydrophilicity. In addition, siRNA is un-stable under in vivo conditions due to rapid degradation by serumnucleases.11 Thus, the widespread use of RNAi therapeutics fordisease prevention and treatment requires the development of

clinically suitable, safe, and eective delivery vehicles.10 In orderto induce eective RNAi, these vehicles must overcome a varietyof extracellular and intracellular obstacles; i.e., they shouldprovide protection against nuclease activity and facilitate inter-nalization and intracellular tracking of the siRNA.12 Eventhough signicant advances have been made in the eld, thedevelopment of vehicles that can eciently deliver RNAi ther-apeutics both in vitro and in vivo remains a major challenge.

Both viral and non-viral carriers have been developed for thedelivery of siRNA.1216 Although viral vectors are very ecient,they can cause immunogenic and inammatory responses,17,18

which raise concerns about their safety as delivery vectors. Non-viral vectors provide opportunities for improved safety, greaterexibility, and more facile manufacturing; however, most of theexisting carriers suer from low delivery eciencies. The mostcommon non-viral vectors involve complexes formed betweencationic lipids19,20 or polymers21,22 and siRNA through electro-static interactions between the negative phosphates along thenucleic acid backbone and the positive charges displayed on thevector. In addition to low transfection eciencies, these systemsalso suer from high toxicity due to their polycationic nature andlimited stability in vivo due to non-specic interactions with serumproteins. The limitations associated with current delivery vehiclesmotivate the development of novel systems for siRNA deliverythat may be able to overcome these obstacles.

Among many alternatives to cationic polymers and lipidscommonly used to form polyplexes/lipoplexes with geneticmaterial,10,23,24 particles made from biodegradable and non-toxicmaterials such as slow-hydrolyzing poly(lactic-co-glycolic acid)(PLGA),25,26 fast-degradingpolyesters (such asDEAPA-PVA-PLGA),27

Received: December 2, 2010Revised: April 10, 2011

ABSTRACT: We report a new acid-sensitive, biocompatible,and biodegradable microparticulate delivery system, sperminemodied acetalated-dextran (Spermine-Ac-DEX), which can beused to eciently encapsulate siRNA. These particles demon-strated ecient gene knockdown inHeLa-luc cells with minimaltoxicity. This knockdown was comparable to that obtainedusing Lipofectamine, a commercially available transfectionreagent generally limited to in vitro use due to its high toxicity.

1057 dx.doi.org/10.1021/bc100542r |Bioconjugate Chem. 2011, 22, 10561065

Bioconjugate Chemistry ARTICLE

and acid-sensitive poly(orthoesters) (POEs)28 have beenexplored as in vivo gene delivery vectors. Another system basedon acid-sensitive polyketals has also recently shown promise fordelivery of siRNA both in vitro and in vivo.29 These priorexamples of particulate systems for siRNA and DNA deliverytypically employ small quantities of cationic polymers25,30 (i.e.,poly(-amino ester), PBAE, or polyethyleneimine, PEI), lipidssuch as DOTAP,29 or small molecules such as spermine26

blended with the carrier polymer to enhance loading of DNA/RNA and delivery eciency. Formed by standard emulsiontechniques, these particles combine physical entrapment of theirpayload with electrostatic complexation of genetic material whileretaining biocompatible degradation mechanisms. However,some limitations of these systems relate to the synthetic ex-ibility, biocompatibility of the degradation products, and thepaucity of chemical methods for the modication of the particlesurface. For example, despite the promising transfection resultsobtained from PLGA microspheres, they still suer from slowrelease rates31 and the formation of DNA-damaging acidicby-products.32,33

Dextran, a homopolysaccharide of glucose, appears to be wellpoised for use as a polymeric carrier due to its biodegradability,wide availability, and ease of modication.34 In addition, dextranalready has a history of human use in clinical applications forplasma volume expansion and plasma substitution. The potentialapplication of dextran for siRNA delivery has recently beendemonstrated.3537 Previously, we described the developmentof a modular and tunable particle system based on acetal-modied dextran (Ac-DEX).38,39 We have shown that Ac-DEXparticles prepared by standard emulsion techniqueseitherwater in oil (w/o) or water in oil in water (w/o/w)have highencapsulation eciencies for both hydrophobic smallmolecules40 and high molecular weight hydrophilic cargoes,38,39

such as proteins, and that the release rate of the encapsulatedcargo was tunable.39 These acid-degradable Ac-DEX particleswere capable of delivering protein antigens to macrophages anddendritic cells. Besides their use as a successful vaccine carrier,microparticles prepared from Ac-DEX blended with a cationicpolymer proved eective at delivering plasmid DNA to bothphagocytic and non-phagocytic cells.41 Although siRNA andplasmid can be applied to achieve similar functional outcomes,successful plasmid delivery carriers cannot necessarily achieveecient siRNA delivery due to the major intrinsic structuraldierences and dierent location of action for siRNA andplasmid.4244 For example, reports from other groups haveshown the diculty of encapsulating the highly charged, hydro-philic, and rigid siRNA in particles by w/o/w emulsion, pre-sumably due to leakage into the outer water phase.45

We now describe the preparation and preliminary evaluationof a new polymeric platformspermine-modied Ac-DEX(spermine-Ac-DEX)for the delivery of siRNA. The newsystem combines facile synthesis and biocompatibility with theadditional benet of controlled payload release sensitive tophysiologically relevant acidic conditions. Acid-sensitive systemshave particularly desirable characteristics, as cargo release can betriggered in response to endosomal acidication upon cellularuptake. Acid-catalyzed hydrolysis of spermine-Ac-DEX generatesspermine-modied dextran, which may be able to be furthermetabolized by enzymes in vivo.4652

The ability of spermine-Ac-DEX particles to overcome avariety of cellular obstacles and function as an ecient deliveryvehicle for siRNA can be rationalized by its tailor-made design

(Figure 1). We hypothesize that particulate formulation shouldprovide protection of the encapsulated siRNA against chemicaland enzymatic degradation. The cationic characteristics of sper-mine-Ac-DEX can facilitate the encapsulation of siRNA insideparticles, and it may also favorably contribute to cellular uptakeby enhancing interaction of the particles with negatively chargedcell membranes. Once inside cells, hydrolysis of the polymer inthe acidic endolysosomal compartment can allow the siRNA tobe released from the particles. Endosomal escape may beachieved via the proton sponge eect of the amine moieties,as well as increased endosomal osmotic pressure by degradationof the spermine-Ac-DEX material. Overall, we speculate that it isthe combination of protection and endolysosomal release thatwould be themost likely contributors to the successful delivery ofsiRNA using a spermine-Ac-DEX carrier.

EXPERIMENTAL PROCEDURES

General Materials and Methods. All chemicals were fromSigma-Aldrich (St. Louis, MO) unless otherwise noted. Theantiluciferase siRNA (sense strand: 50-CUU ACG CUG AGUACU UCG A dTdT-30) was obtained from Dharmacon(Lafayette, CO) and Silencer Negative Control #1 siRNA waspurchased from Ambion (Austin, TX). Phosphate buffered saline(PBS, pH 7.4) was from Invitrogen (Carlsbad, CA). Reactionsrequiring anhydrous conditions were performed in flame-driedvessels and under a positive pressure of dry nitrogen. Water (dd-H2O) for buffers and particle washing steps was purified to aresistance of 18 M using a NANOpure purification system(Barnstead, USA). When used in the presence of acetal contain-ing materials, dd-H2O was rendered basic (pH 8) by the additionof triethylamine (TEA) (approximately 0.01%). 1HNMR spectrawere recorded at 400 or 500 MHz on a Bruker spectrometer.Elemental analyses were performed at the UC Berkeley MassSpectrometry Facility. Fluorescence measurements were ob-tained using a Spectra Max Gemini XS plate-reading fluorimeter

Figure 1. Particles are expected to eciently transfect HeLa-luc cellsdue to their ability to overcome several obstacles to gene delivery. Ac-DEX particles should protect siRNA from degradation. (1) The particlesare endocytosed by HeLa cells, and the vesicle is acidied upon fusion ofthe endosome with the lysosome. (2) Spermine Ac-DEX particlesdegrade in the acidic environment of the endolysosome and (3) thesiRNA is released into the cytoplasm.



(Molecular Devices, USA), usage courtesy of Prof. CarolynBertozzi. Absorbance measurements were obtained using aSpectra Max 190 microplate reader (Molecular Devices, USA),usage courtesy of Prof. Carolyn Bertozzi. Luminescence measure-ments were obtained using a GloMax 96 microplate luminometer(Promega, USA), usage courtesy of Prof. Eva Harris.Synthesis of Spermine-Ac-DEX. Partial Oxidation of Dex-

tran. Dextran (5.0 g, 30.9 mmol, Mw 911 000 g/mol, fromLeuconostoc mesenteroides) was dissolved in 20 mL water. Afteradding sodium periodate (1.1 g, 51.4 mmol), the solution wasstirred for 5 h at rt. The product was purified by dialysis of thesolution against distilled water using a regenerated cellulosemembrane with a MWCO of 3500 g/mol. The water waschanged 5 times and the sample was lyophilized to obtain awhite powder (4.2 g, 8.4 mol aldehyde functions/100 molanhydroglucose unit, AGU). The degree of oxidation wasdetermined colorimetrically (UV absorption at 562 nm) usinga microplate reductometric bicinchoninic acid assay (Micro BCAProtein Assay Kit, Pierce, USA) according to the manufacturersprotocol and glucose monohydrate for calibration.

Synthesis of Partially Oxidized Acetalated Dextran. Acetala-tion of partially oxidized dextran was performed in a similarmanner as described previously.38 Briefly, 3.0 g partially oxidizeddextran (18.5 mmol, 8.4 mol aldehyde functions/100 mol AGU)was modified with 2-methoxypropene (10.6 mL, 111 mmol)yielding partially oxidized acetalated dextran (4.3 g) containing100 mol acyclic and 72.5 mol cyclic acetals/100 mol AGU. Thedegree of functionalization was determined by 1H NMR spec-troscopy in DCl/D2O according to the method described byBroaders et al.39 1H NMR (400 MHz, CDCl3): 1.40 (s, br,acetal), 3.25 (br, acetal), 3.45, 3.504.10, 4.90, 5.10 (br, dextran).

Synthesis of Spermine-Ac-DEX. Partially oxidized Ac-DEX(2.0 g, 12.3 mmol) was stirred with spermine (4.0 g, 19.8 mmol)in 10 mLDMSO at 50 C for 22 h. The reduction was performedfor 18 h at room temperature by adding NaBH4 (2.0 g,52.9 mmol) to the DMSO solution. The spermine modifieddextran was precipitated in dd-H2O (40 mL). The product wasisolated by centrifugation at 4000g for 5 min, and the resultingpellet was washed thoroughly with dd-H2O (5 40 mL, pH 8)by resuspension followed by centrifugation and removal of thesupernatant. Residual water was removed by lyophilization,yielding spermine functionalized acetalated dextran, spermine-Ac-DEX, (1.6 g) as a white powder containing 6.6 mol spermine/100 mol AGU. The degree of functionalization was determinedby elemental analysis using the nitrogen content. Anal.(spermine-Ac-DEX) C, found 55.79; H, found 8.29; N, found,1.24. 1H NMR (400 MHz, CDCl3): 1.40 (s, br, acetal), 1.60,1.80, 2.60, 2.65, 2.75 (br, spermine), 3.25 (br, acetal), 3.45,3.504.10, 4.90, 5.10 (br, dextran).Synthesis of PBAE. PBAE, a white solid, was synthesized by

the Michael-type addition of 4,40-trimethylenedipiperidine to 1,4-butanediol diacrylate inTHF as the solvent according to themethoddescribed by Lynn et al. (GPC: Mn = 42.6 kDa, PDI = 2.78).

53

Preparation of Particles. Particles Encapsulating siRNA. Ac-DEX particles containing siRNA were prepared using a doubleemulsion water/oil/water (w/o/w) evaporation method similarto that described previously.38,39 Stock siRNA solutions (2.66,5.32, or 10.64 mg/mL depending on the desired initial siRNAfeed) were prepared in nuclease-free distilled water. Spermine-Ac-DEX (25 mg) was dissolved in ice-cold CH2Cl2 (0.5 mL).The stock siRNA solution (25 L) was added and the mixturewas sonicated for 30 s on ice using a probe sonicator (Branson

Sonifier 450) with a 1/2 in. flat tip, an output setting of 5,and a duty cycle of 80%. This primary emulsion was then addedto an aqueous solution of poly(vinyl alcohol) (PVA, Mw =13 00023 000 g/mol, 8789% hydrolyzed) (1 mL, 3% w/w inPBS) and sonicated for an additional 30 s on ice using the samesettings. The resulting double emulsion was immediately pouredinto a second PVA solution (5 mL, 0.3% w/w in PBS) and stirredfor 3 h at rt allowing the organic solvent to evaporate. The particleswere isolated by centrifugation (10 250 rpm, 30 min) and washedwith PBS (25 mL) and dd-H2O (2 25 mL, pH 8) by vortexingand sonication followed by centrifugation and removal of thesupernatant. The washed particles were resuspended in dd-H2O(2 mL, pH 8) and lyophilized to yield a white, fluffy solid. Yieldswere typically between 50% and 85% per batch based on startingpolymer and siRNA mass (12.521 mg of particles).

Ac-DEX/PBAE Particles. Particles consisting of Ac-DEX andPBAE were made in the same manner as described above, exceptthat unmodified Ac-DEX and 10 wt % PBAEwere used instead ofspermine-Ac-DEX.

Empty Particles. Particles that did not contain siRNA weremade in the same manner described above, except that theaqueous buffer in the primary emulsion consisted of dd-H2O(25 L) and no siRNA.Quantification of Encapsulated siRNA. Particles containing

siRNA were suspended at a concentration of 10 mg/mL in a0.3 M acetate buffer (pH 5) and incubated at 37 C under gentleagitation for 3 d using a Thermomixer R heating block(Eppendorf). After the particles had been fully degraded, aliquotswere taken and analyzed for siRNA content using the Quant-iTPicogreen dsDNA assay (Molecular Probes) according to themanufacturers instructions. Empty Ac-DEX particles were de-graded in a similar fashion and used to determine backgroundfluorescence. For this experiment, all solutions included heparinat 10 mg/mL to disrupt electrostatic interactions between blendpolymers and siRNA to enable quantification. The results werecompared to a standard curve and the mass of siRNA encapsu-lated was calculated. Fluorescence was measured using a Spec-traMax Gemini XS microplate reader (Molecular Devices,Sunnyvale, CA, ex. 480 nm, em. 520 nm).Zeta-Potential Analysis. Zeta-potentials of particles were

measured using a Nano ZS ZetaSizer (Malvern Instruments,UK) at 25 C after suspending particles in HEPES buffer (5 mM,pH 7.4) at 0.1 mg/mL. Data shown represent the average zeta-potential( standard deviation of distributions of five sequentialmeasurements.Characterization of Particle Size by Scanning Electron

Microscopy (SEM). Particles were suspended in dd-H2O (pH 8)at a concentration of 0.3 mg/mL and the resulting dispersionswere dripped onto silicon wafers. After 15 min, the water waswicked away using tissue paper and the samples were furtherdried under a stream of N2 gas. The particles were then sputter-coated with a 2 nm layer of a palladium/gold alloy and imagedusing a scanning electron microscope (S5000, Hitachi). Theparticle diameter and size distribution of the particles weredetermined by measuring 100 particles and analyzing the datausing Excel. These micrographs were also used to assess particlemorphology.pH-Dependent Degradation of Ac-DEX Particles. Empty

Ac-DEX particles were suspended in triplicate at a concentrationof 1 mg/mL in either a 0.3 M acetate buffer (pH 5.0) or PBS(pH 7.4) and incubated at 37 C under gentle agitation using aThermomixer R heating block (Eppendorf). At various time



points, 50 L aliquots were removed, centrifuged at 14 000g for4 min to pellet out insoluble materials, and the supernatant wasstored at 20 C. The collected supernatant samples wereanalyzed for the presence of reducing polysaccharides using amicroplate reductometric bicinchoninic acid based assay (UVabsorption at 562 nm) according to the manufacturers protocol(Micro BCA Protein Assay Kit, Pierce, USA). The curve wasmade by applying a Boltzmann fit.pH-Dependent Release of siRNA from Ac-DEX Particles.

This experiment was performed essentially in the same manneras above except siRNA-loaded particles were used instead ofempty particles. The quantity of siRNA in the supernatantsamples was determined by using the Quant-iT PicogreendsDNA assay (Molecular Probes) according to the manufac-turers instructions. The amount of siRNA in each sample wascalculated by fitting the emission to a calibration curve using theQuant-iT Picogreen dsDNA assay (Molecular Probes). For thisexperiment, all solutions included heparin at 12.5 mg/mL todisrupt electrostatic interactions between blend polymers andsiRNA to enable quantification. The curve was made by applyinga Boltzmann fit.Cell Lines and Culture. HeLa cell lines stably expressing

firefly luciferase (HeLa-luc) were maintained in DulbeccosModified Eagles Medium (DMEM) supplemented with 10%(v/v) fetal bovine serum (FBS), 1% GlutaMAX, and 500 g/mLZeocin (all from Invitrogen except the serum, which was fromHyclone (Logan, UT)). Cell incubations were performed in awater-jacketed 37 C/5% CO2 incubator.

In Vitro siRNA Transfection Assay. HeLa-luc cells wereseeded (15 000 cells/well) into each well of a 96-well clear tissueculture plate (Costar, Corning, NY) and allowed to attachovernight in growth medium. Growth medium was composedof DMEM (with phenol red), 10% FBS, and 1% GlutaMAX.Particle samples (encapsulating either luciferase siRNA or con-trol siRNA) were prepared in culture medium (without anti-biotics) by alternately vortexing and sonicating in a Branson 2510water bath for 20 s to generate homogeneous suspensions. Thesamples were then serially diluted in medium to give theindicated particle concentrations or equivalent siRNA doses.Existing medium was replaced with 100 L of each particledilution (2.1935 pmol siRNA) in quadruple wells. The cellswere allowed to grow for an additional 48 h before being analyzedfor gene expression. Lipofectamine 2000 (Invitrogen, Carlsbad,CA) was used as a positive control for siRNA delivery and wasprepared according to the manufacturers instructions. Com-plexes containing equivalent doses of siRNA to particles wereprepared bymixing Lipofectamine 2000 and siRNA (3:5:1 ratio).As negative controls, both equivalent doses of free siRNA inmedium and medium alone were used.After 48 h, the cells were washed with PBS (containing Mg2

and Ca2, 3 100 L), Glo Lysis Buer (120 L, Promega,Madison, WI) was added to each well, and the plate was vortexedat rt for 20min. Samples fromeachwell (100L)were transferredto the wells of a white 96-well tissue culture plate (Corning,Lowell, MA). Steady-Glo luciferase assay reagent (Promega) wasreconstituted according to the manufacturers instructions andinjected into each well in series (100 L/well) using a GloMax 96microplate luminometer (Promega). After a 10 s post-injectiondelay, each well was read with a 2 s integration time.Total Protein Assay. Cells treated identically and in parallel

with transfection assays were tested on a second 96-well plate.After washing, the cells were lysed with M-PER Mammalian

Protein Extraction Reagent (50 L/well, Pierce, Rockford, IL)by incubating for 10 min at rt. PBS (50 L/well) was then added,and the plate was briefly vortexed. Samples from each well(50 L) were transferred to a black 96-well plate (Corning) alreadycontaining PBS (100 L/well). A solution of 3 mg/mL fluor-escamine in acetone (50 L) was added to each well and mixedwell using a multichannel pipet. After 5 min, fluorescence wasmeasured using a SpectraMaxGemini XS reader (ex. 400 nm, em.460 nm). Protein concentrations were determined using bovineserum albumin as a standard. Relative light units (RLU) from theluminometer were normalized to the total mass of cellularprotein. The resulting data (RLU/mg of protein) are given as amean ( standard deviation of four independent measurements.Percentage knockdown was calculated by comparison of treatedcells to untreated cells. The data was compared to the knock-down of cells treated with particles loaded with control siRNA orcontrol siRNA/Lipofectamine complexes.Viability Assay. Cells treated identically and in parallel with

transfection assays were tested on a third 96-well plate. A 3.0 mg/mL solution of MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphen-yl-2H-tetrazolium bromide) in medium (40 L) was addeddirectly to each well, and the plate was incubated for an additional30 min. The medium was then replaced with DMSO (200 L/well), 100 L of which was transferred to another clear-bottom96-well assay plate (Pro-Bind, Falcon) containing 100 LDMSOand 25 L of glycine buffer (0.1M glycine, 0.1MNaCl, pH 10.5)per well. The absorbances at 570 nm were measured using aSpectraMax 190 reader (Molecular Devices). Cell viability wasnormalized to the absorbance measured from untreated cells.Data are represented as a mean ( standard deviation of fourmeasurements.

RESULTS AND DISCUSSION

Synthesis of Spermine-Modified Ac-DEX. Ac-DEX hasseveral characteristics that make it well suited for the deliveryof bioactive cargoes. Although this system has been used tosuccessfully deliver protein antigens, adjuvants, and small mole-cule agents, the neutrally charged polymer cannot efficientlyencapsulate siRNA. Taking advantage of the blend approachdescribed above, we have recently reported the preparation ofparticles encapsulating plasmid DNA by formulating Ac-DEXwith small amounts of the cationic polymer PBAE.41 Theseparticles were able to efficiently transfect both phagocytic andnon-phagocytic cells in vitro. Inspired by this work, we tried toprepare Ac-DEX particles encapsulating siRNA by blending witheither small polyamines or cationic polymers. While high loadingand efficient delivery of plasmid DNA was achieved by blendingAc-DEX with PBAE, attempts to prepare siRNA-loaded particlesby the same method only afforded particles with low loading andlow encapsulation efficiency.Domb and co-workers have reported the synthesis of various

oligoamine polysaccharide conjugates for use in gene delivery.54

They found that, of 300 dierent polycations prepared, only a fewwere active in transfecting cells. Dextran-spermine displayedespecially high transfection eciency, which they attributed tounique complexation properties between DNA and the graftedspermine moieties.54 Dextran-spermine, and derivatives thereof,have shown high transfection of plasmid DNA both in vitro andin vivo.55,56 Combining this pioneering work with the uniquecharacteristics of our Ac-DEX, we have modied Ac-DEX withspermine for use in siRNA delivery.



Spermine-Ac-DEX was prepared by using reductive aminationchemistry for the conjugation of spermine to Ac-DEX(Scheme 1). To increase the number of available aldehydefunctionalization sites beyond the reducing chain ends of thepolysaccharide, dextran was rst lightly oxidized with sodiumperiodate, thus increasing signicantly its aldehyde content57 asevaluated by a reductometric bicinchoninic acid assay (BCAassay). Using this method, we settled on a loading of 8.4 aldehydefunctions per 100 anhydroglucose units (AGU). Reaction of theremaining hydroxyl groups with 2-methoxypropene aordedpartially oxidized Ac-DEX, an acid-sensitive, hydrophobic poly-mer that can be easily processed into microparticles. Finally,amine modication was performed between spermine andaldehyde-containing-Ac-DEX using sodium borohydride as thereducing agent. The amount of spermine conjugated to thedextran, calculated from the nitrogen content (%N) as deter-mined by elemental analysis, averaged 6.6 spermine units per100 AGU.Preparation and Characterization of Spermine-Ac-DEX

Particles. Spermine-Ac-DEX particles encapsulating siRNAwere prepared via a standard double-emulsion technique(Figure 2). Particles prepared from unmodified Ac-DEX andAc-DEX blended with 10 wt % PBAE, a formulation previouslyused for plasmid DNA, were also made for comparison purposes.The particles were visualized by scanning electron microscopy(SEM) to determine the average particle size and morphology.All particles were found to be spherical in shape with diameters inthe range of 180 to 230 nm in the dry state, irrespective of particleformulation. The surface charge of the particles was determinedby zeta-potential measurements. Unmodified Ac-DEX particles

have a slightly negative zeta-potential due to the encapsulatedsiRNA. Blending the polymer with PBAE made the surfacecharge less negative owing to the cationic nature of the polymer,while the surface charge of spermine-Ac-DEX particles waspositive. As expected, the surface charge decreased with increas-ing loadings of siRNA but remained positive for all particleformulations. Various particle characteristics are known toinfluence particle behavior in vivo, including particle size, shape,and surface charge.58 When administered intravenously, posi-tively charged particles are susceptible to opsonization bynegatively charged serum proteins and clearance by cells of thereticuloendothelial system (RES). While a positive surfacecharge might be disadvantageous for delivery into the blood-stream, pulmonary delivery represents an attractive alternativedelivery strategy for which there is precedent of success utilizingpositively charged particles.59,60 Indeed, we envision utilizingaerosolization for the delivery of our particles to the lungs.The siRNA loading and loading eciencies were determined

by degrading a sample of particles under acidic conditions andanalyzing the siRNA concentration using a Picogreen dsDNAassay (Table 1). The siRNA loading eciency for unmodiedAc-DEX particles was low (5%), as expected based on pre-vious reports with other particle systems, such as PLGA.26

Blend particles formulated from Ac-DEX and a cationic polymerpoly(-amino ester) (PBAE) also showed low encapsulationeciency for siRNA (7%), much lower than the plasmidencapsulation achieved with a similar particle formulation. Thislower encapsulation eciency for siRNA as compared to plasmidDNA may be due to the fact that siRNA has a lower molar massthan plasmid DNA (approximately 250 less) and, thus, may be

Scheme 1. Schematic Illustration of the Synthesis of Spermine-Modied Ac-DEX

Figure 2. Preparation of spermine-modied Ac-DEX particles encapsulating siRNA showing a representative scanning electron micrograph of particles(scale bar = 500 nm) (center) and a cartoon illustration of siRNA-loaded particles (right).



more able to diuse through the condensing polymer during theemulsion process, resulting in reduced loading eciency.61

Improved loading eciencies (7598%) were obtained forparticles prepared from spermine-Ac-DEX. This loading com-pares favorably with other particle formulations, such as PLGAblended with spermidine for which 56% of the siRNA wasencapsulated.26 In contrast to previously reported particulatedelivery systems, however, particles prepared using spermine-Ac-DEX did not require blending of a cationic material with the

polymer due to the ability of the polymer to electrostaticallyinteract with siRNA.

Table 1. Characterization of Particle Formulations

particle formulation (siRNA, feed)a diameterb,c [nm] zeta-potentialb [mV] siRNA loadingb,d [g mg1] loading eciencyb (%)

Ac-DEX (Luc, 5) 230( 80 5.5( 0.42 0.26 5Ac-DEX/10% PBAE (Luc, 5) N.D. 3.4( 0.44 0.34 7Spermine Ac-DEX (Luc, 10) 178( 76 9.2( 0.69 7.95 76e

Spermine Ac-DEX (Luc, 5) 229( 59 16.2( 0.58 4.70 91Spermine Ac-DEX (Luc, 2.5) 185( 65 19.7( 0.63 2.28 95f

Spermine Ac-DEX (Control, 5) 195( 45 16.1( 0.71 5.11 98a Luc antiluciferase siRNA, Control Silencer Negative Control #1 siRNA, Feed amount of siRNA used in particle preparation, g of siRNA permg of polymer. bCharacterized as described in the main text. c Some particle aggregation was observed upon resuspension following lyophilization. Themean particle diameter determined by light scattering for Spermine Ac-DEX (Luc, 5) and Spermine Ac-DEX (Control, 5) was 2.71 ( 1.54 m and6.45( 2.47 m, respectively (Horiba Partica LA-950, Horiba Scientic). d siRNA loading ing of siRNA permg of particles (100% eciency 5.0g ofsiRNA per mg of particles). A loading of 5 g of siRNA per mg of particles corresponds to an N/P ratio of approximately 100. e 100% eciency 10 gof siRNA per mg of particles. f 100% eciency 2.5 g of siRNA per mg of particles. N.D. = not determined.

Figure 3. (a) Degradation of particles when incubated at pH 7.4 (blackcircles) and pH 5 (white circles), as determined by analysis of releasedsoluble dextran. (b) Release of siRNA from spermine Ac-DEX particleswhen incubated at pH 7.4 (black circles) or pH 5 (white circles). Datarepresent the mean ( standard deviation of triplicate measurements. Figure 4. Optimized siRNA loading in particles and its eect on

luciferase knockdown. (a) siRNA loading in particles compared withthe feed amount of siRNAused in particle synthesis. Dashed diagonal linerepresents 100% loading eciency. (b) Knockdown of luciferase activitywith varying siRNA loadings. HeLa-luc cells were treated with particledoses equivalent to 17.2 pmol siRNA/well. Results were compared tountreated cells and the percentage knockdown of luciferase expressionwas calculated. Results (columns) are combined with results from aconcurrently performed cytotoxicity assay (closed circles and line).Knockdown correlates with siRNA concentration, not particle loading.



A signicant limitation of other commonly used particledelivery systems is their lack of ability to tune the release rateof the encapsulated cargo. Ac-DEX particles are designed to berelatively stable under physiological conditions (pH 7.4) butdegrade under the mildly acidic conditions typically found in thelysosome (pH 5.05.5), thus allowing for the selective release ofthe therapeutic agent once inside the cell. We have shown thatthe rate of microparticle degradation can be easily varied bycontrolling the type of acetals (i.e., cyclic vs acyclic) formed onthe dextran.39 To determine the degradation rate of spermine-Ac-DEX particles, empty particles were incubated at 37 C ateither pH 5.0 or pH 7.4 and monitored for the release of solubledextran (Figure 3a). No soluble dextran was detected after 48 hfor the particles incubated at pH 7.4. In contrast, particlesincubated in pH 5.0 buer showed continuous release of dextranin the rst 24 h at 37 C, with a degradation half-life of 8 h. Wealso monitored the release of siRNA from the particles at pH 5.0and pH 7.4 to determine if the release occurred in a pH-controlled manner (Figure 3b). Little or no release was observedat pH 7.4 after 48 h, while continuous release of siRNA wasobserved over 24 h at pH 5.0.To study siRNA loading, three dierently loaded batches of

spermine-Ac-DEX particles encapsulating luciferase siRNA wereprepared. The feed values for these particles were 2.5, 5.0, and10 g of siRNA per mg of polymer, respectively. Following prepara-tion, the siRNA loading of the particles was quantied as describedabove (Figure 4a). Loadings of up to 8.0 g of siRNA per mg ofparticle were achieved and the loading eciency was above 75%for all particle batches. Ecient loading of siRNA into spermine-Ac-DEX particles is important because of the high cost of siRNA.

In VitroAnalysis of siRNADelivery toHeLa Cells.To test thetransfection efficiency of spermine-Ac-DEX particles in vitro, wechose to knockdown a model reporter protein, firefly luciferase.HeLa cells that stably express firefly luciferase (HeLa-luc cells)were used as a model cell line. A more effective delivery systemwill lead to more cytosolic siRNA and luciferase mRNA cleavage,and thus a lower expression of luciferase protein. Firefly luciferasecatalyzes the mono-oxygenation of luciferin, and during this

process, a photon of light is produced. Thus, reduced expressionof luciferase will result in the generation of fewer photons whenthe cells are incubated with the enzyme substrate.We were interested in determining if particle loading or

concentration inuences the delivery eciency of siRNA-loadedparticles. Dierentially loaded particles were prepared andHeLa-luc cells were incubated with particle doses equivalent to17.2 pmol of siRNA per well. All of the particles could reduceluciferase expression of the cells (approximately 5560% com-pared to untreated cells) with low toxicity (greater than 85%viability) (Figure 4b). The luciferase expression was comparablefor all three batches of particles indicating that the particleloading and concentration within the tested range have aminimalimpact on the overall performance, and the knockdown resultedfrom successful delivery of siRNA.To determine if the knockdown was dose-dependent, HeLa-

luc cells were incubated with varying siRNA doses for 48 h andanalyzed for the expression of luciferase. As shown in Figure 5,spermine-Ac-DEX particles containing luciferase-specic siRNAeciently knock down luciferase expression at all siRNA con-centrations tested. Transfection with luciferase siRNA loadedspermine-Ac-DEX particles resulted in signicant gene silencing(up to 60% knockdown compared with untreated cells). Incomparison, treatment of cells with free siRNA did not aectthe luciferase expression, indicating the importance of encapsula-tion of siRNA in particles. The extent of gene silencing dependson the amount of siRNA incubated with the cells (Figure 5).Treating cells with lower particle concentrations, and thus lesssiRNA, resulted in reduced knockdown. The optimal knock-down was obtained with the three highest siRNA dosages tested(8.75, 17.5, and 35 pmol of siRNA). This silencing eect wassequence specic as spermine-Ac-DEX particles loaded with anon-specic siRNA sequence (Silencer Negative control #1siRNA) did not result in obvious reduction of luciferase expres-sion. Importantly, there was no signicant cytotoxicity associatedwith intracellular accumulation of spermine-Ac-DEX particles.MTT assay of cells incubated with particles showed greater than80% cell viability for all particle concentrations tested (Figure 6).

Figure 5. Spermine Ac-DEX particles can eciently deliver siRNA to HeLa-luc cells. In vitro transfection of HeLa-luc cells with siRNA-loaded spermineAc-DEX particles. HeLa-luc cells were treated with particles encapsulating either luciferase siRNA or control siRNA at various concentrations. Relativelight units (RLU) from the luminometer were normalized to the total mass of cellular protein determined from a uorescamine assay. Data represent themean( standard deviation of quadruplicate measurements. Statistical dierence was performed with Students t-test between untreated cells and eachtreatment group: p < 0.05 were marked with *, p < 0.01 with **, and p < 0.001 with ***.



Lipofectamine 2000, a commercially available cationic lipid-based reagent, was used as a positive control in the transfec-tion experiment. This reagent is commonly used for in vitrotransfection,62 but it faces several obstacles for clinicaltranslation.63 Lipofectamine complexes with the luciferase-tar-geting siRNA as well as with the control siRNA were prepared sothat the dose of siRNA would match that used with two of theparticle concentrations tested (35 pmol siRNA per well and8.75 pmol siRNA per well). We observed some non-specic reduc-tion of luciferase expression using Lipofectamine 2000, mostlikely due to slight toxicity of the complexes. Thus, the knock-down was normalized to the data from the cells treated with thecontrol siRNA complexes. The knockdown obtained with thespermine-Ac-DEX particles was comparable to that obtainedwith Lipofectamine 2000 at both siRNA doses tested (Figure 7).Due to their ability to provide ecient siRNA delivery withreduced cytotoxicity (97% cell viability for spermine-Ac-DEXparticles compared to 75% for Lipofectamine), spermine-Ac-DEX

particles represent an attractive delivery system that may oerseveral advantages over previously reported materials for siRNAdelivery.

CONCLUSION

In summary, spermine-Ac-DEX particles represent a noveldelivery vehicle which can eciently deliver siRNA to cancer cellswith minimal toxicity. Spermine-Ac-DEX combines the attractiveproperties of cationic polymers with those of polymeric particlesaording a material that is safe and eective for gene delivery.Spermine modication of Ac-DEX facilitated the preparation ofparticles capable of encapsulating siRNA with high loadingeciency. In vitro evaluation demonstrated that the particles arecapable of knocking down luciferase expression in HeLa-luc cellsin a dose-dependent manner with low cytotoxicity. In comparisonto previously reported gene delivery materials, spermine-Ac-DEXshows attractive new transfection and biodegradability features,and in addition can be easily modied64 (for example, withpeptide targeting ligands). Due to its acid-sensitivity, spermine-Ac-DEX allows for the selective release of siRNA after cellularinternalization, with potential tunability of cargo release rate.Thus, spermine-Ac-DEX expands the potential application of Ac-DEXbased particles and represents a promising vehicle for siRNAdelivery. Future studies will explore the feasibility of using theseparticles to regulate protein expression in vivo.

AUTHOR INFORMATION

Corresponding Author*Prof. JeanM. J. Frechet, 718 LatimerHall, College of Chemistry,University of California, Berkeley, Berkeley, CA 94720-1460.E-mail: [email protected]. Tel: (510) 643-3077. Fax: (510)643-3079.

Present AddressesInstitute of Pharmacy, Friedrich Schiller University of Jena,07743 Jena, Germany.Liquidia Technologies, Inc., Research Triangle Park, NC27709, USA.

ACKNOWLEDGMENT

This project has been funded in part with Federal funds fromthe National Heart, Lung, and Blood Institute, National Insti-tutes of Health, Department of Health and Human Services,under Contract No. HHSN268201000043C, and in part throughthe Frechet various gifts fund for the support of research in newmaterials. We thank Ann Fischer andMichelle Yasukawa for helpwith cell culture, Dr. Peter Friebe and Dr. Eva Harris forassistance with and the use of their luminescence plate reader,and Dr. Chris Contag for kindly providing the HeLa-luc cell line.S. Schubert gratefully acknowledges the Deutsche Forschungs-gemeinschaft (DFG) and P. R. Wich thanks the Alexander vonHumboldt Foundation (AvH) for funding.

REFERENCES

(1) Fougerolles, A., Vornlocher, H.-P., Maraganore, J., and Lieberman,J. (2007) Interfering with disease: A progress report on siRNA-basedtherapeutics. Nat. Rev. Drug Discovery 6, 443453.

(2) Bumcrot, D., Manoharan, M., Koteliansky, V., and Sah, D. W. Y.(2006) RNAi therapeutics: A potential new class of pharmaceuticaldrugs. Nat. Chem. Biol. 2, 711719.

Figure 6. Spermine Ac-DEX particles are nontoxic at concentrations upto 1 mg/mL. MTT assay was used to measure cell viability comparedwith untreated cells. Data represent the mean ( standard deviation ofquadruplicate measurements.

Figure 7. Spermine Ac-DEX particles show comparable activity com-pared with Lipofectamine 2000. Knockdown was normalized to the datafrom the cells treated with the control siRNA particles or complexes.Data represent the mean ( standard deviation.



(3) Kurreck, J. (2009) RNA interference: From basic research totherapeutic applications. Angew. Chem., Int. Ed. 48, 13781398.

(4) Hammond, S. M., Caudy, A. A., and Hannon, G. J. (2001) Post-transcriptional gene silencing by double-stranded RNA. Nat. Rev. Genet.2, 110119.

(5) Tuschl, T. (2001) RNA interference and small interfering RNAs.ChemBioChem 2, 239245.

(6) Bernstein, E., Caudy, A. A., Hammond, S. M., and Hannon, G. J.(2001) Role for a bidentate ribonuclease in the initiation step of RNAinterference. Nature 409, 363366.

(7) Rand, T. A., Ginalski, K., Grishin, N. V., and Wang, X. (2004)Biochemical identication of Argonaute 2 as the sole protein requiredfor RNA-induced silencing complex activity. Proc. Natl. Acad. Sci. U.S.A.101, 1438514389.

(8) Ameres, S. L., Martinez, J., and Schroeder, R. (2007) Molecularbasis for target RNA recognition and cleavage by human RISC. Cell130, 101112.

(9) Hutvagner, G., and Zamore, P. D. (2002) A microRNA in amultiple-turnover RNAi enzyme complex. Science 297, 20562060.

(10) Whitehead, K. A., Langer, R., and Anderson, D. G. (2009)Knocking down barriers: Advances in siRNA delivery. Nat. Rev. DrugDiscovery 8, 129138.

(11) Frohlich, T., and Wagner, E. (2010) Peptide- and polymer-based delivery of therapeutic RNA. Soft Matter 6, 226234.

(12) Pack, D. W., Homan, A. S., Pun, S., and Stayton, P. S. (2005)Design and development of polymers for gene delivery. Nat. Rev. DrugDiscovery 4, 581593.

(13) During, M. J. (1997) Adeno-associated virus as a gene deliverysystem. Adv. Drug Delivery Rev. 27, 8394.

(14) Verma, I. M., and Somia, N. (1997) Gene therapy - Promises,problems, and prospects. Nature 389, 239242.

(15) Schaer, D. V., Koerber, J. T., and Lim, K. (2008) Molecularengineering of viral gene delivery vehicles. Annu. Rev. Biomed. Eng.10, 169194.

(16) Mintzer, M. A., and Simanek, E. E. (2009) Nonviral vectors forgene delivery. Chem. Rev. 109, 259302.

(17) Thomas, C. E., Ehrhardt, A., and Kay, M. A. (2003) Progressand problems with the use of viral vectors for gene therapy. Nat. Rev.Genet. 4, 346358.

(18) Zaiss, A. K., and Muruve, D. A. (2008) Immunity to adeno-associated virus vectors in animals and humans: A continued challenge.Gene Ther. 15, 808816.

(19) Malone, R. W., Felgner, P. L., and Verma, I. M. (1989) Cationicliposome-mediated RNA transfection. Proc. Natl. Acad. Sci. U.S.A.86, 60776081.

(20) Zimmerman, T. S., Lee, A. C. H., Akinc, A., Bramlage, B.,Bumcrot, D., Fedoruk, M. N., Harborth, J., Heyes, J. A., Jes, L. B., John,M., Judge, A. D., Lam, K., McClintock, K., Nechev, L. V., Palmer,L. R., Racie, T., Rohl, I., Seiert, S., Shanmugam, S., Sood, V., Soutschek,J., Toudjarska, I., Wheat, A. J., Yaworski, E., Zedalis, W., Koteliansky, V.,Manoharan, M., Vornlocher, H.-P., and MacLachlan, I. (2006)RNAi-mediated gene silencing in non-human primates. Nature 441,111114.

(21) Neu, M., Fischer, D., and Kissel, T. (2005) Recent advances inrational gene transfer vector design based on poly(ethylene imine) andits derivatives. J. Gene Med. 7, 9921009.

(22) Zhang, S., Zhao, B., Jiang, H., Wang, B., and Ma, B. (2007)Cationic lipids and polymers mediated vectors for delivery of siRNA.J. Controlled Release 123, 110.

(23) Lares, M. R., Rossi, J. J., and Ouellet, D. L. (2010) RNAi andsmall interfering RNAs in human disease therapeutic applications.Trends Biotechnol. 28, 570579.

(24) Shim, M. S., and Kwon, Y. J. (2010) Ecient and targeteddelivery of siRNA in vivo. FEBS J. 277, 48144827.

(25) Little, S. R., Lynn, D. M., Ge, Q., Anderson, D. G., Puram, S. V.,Chen, J., Eisen, H. N., and Langer, R. (2004) Poly-B-amino ester-containing microparticles enhance the activity of nonviral geneticvaccines. Proc. Natl. Acad. Sci. U.S.A. 101, 95349539.

(26) Woodrow, K. A., Cu, Y., Booth, C. J., Saucier-Sawyer, J. K.,Wood, M. J., and Saltzman, W. M. (2009) Intravaginal gene silencingusing biodegradable polymer nanoparticles densely loaded with small-interfering RNA. Nat. Mater. 8, 526533.

(27) Nguyen, J., Steele, T. W. J., Merkel, O., Reul, R., and Kissel, T.(2008) Fast degrading polyesters as siRNA nano-carriers for pulmonarygene therapy. J. Controlled Release 132, 243251.

(28) Wang, C., Ge, Q., Ting, D., Nguyen, D., Shen, H.-R., Chen, J.,Eisen, H. N., Heller, J., Langer, R., and Putnam, D. (2004) Molecularlyengineered poly(ortho ester) microspheres for enhanced delivery ofDNA vaccines. Nat. Mater. 3, 190196.

(29) Lee, S., Yang, S. C., Kao, C.-Y., Pierce, R. H., and Murthy, N.(2009) Solid polymeric microparticles enhance the delivery of siRNA tomacrophages in vivo. Nucleic Acids Res. 37, e145.

(30) Little, S. R., Lynn, D. M., Puram, S. V., and Langer, R. (2005)Formulation and characterization of poly (B amino ester) microparticlesfor genetic vaccine delivery. J. Controlled Release 107, 449462.

(31) Pack, D. W. (2004) Timing is everything. Nat. Mater. 3, 133134.

(32) Fu, K., Pack, D. W., Klibanov, A. M., and Langer, R. (2000)Visual evidence of acidic environment within degrading poly(lactic-co-glycolic acid) (PLGA) microspheres. Pharm. Res. 17, 100106.

(33) Walter, E., Moelling, K., Pavlovic, J., and Merkle, H. P. (1999)Microencapsulation of DNA using poly(DL-lactide-co-glycolide):Stability issues and release characteristics. J. Controlled Release 61, 361374.

(34) Heinze, T., Liebert, T., Heublein, B., and Hornig, S. (2006)Functional polymers based on dextran. Adv. Polym. Sci. 205, 199291.

(35) Raemdonck, K., Naeye, B., Buyens, K., Vandenbroucke, R. E.,Hgset, A., Demeester, J., and De Smedt, S. C. (2009) Biodegradabledextran nanogels for RNA interference: Focusing on endosomal escapeand intracellular siRNA delivery. Adv. Funct. Mater. 19, 14061415.

(36) Raemdonck, K., Naeye, B., Hgset, A., Demeester, J., and DeSmedt, S. C. (2010) Prolonged gene silencing by combining siRNAnanogels and photochemical internalization. J. Controlled Release145, 281288.

(37) Nagane, K., Jo, J.-I., and Tabata, Y. (2010) Promoted adipogen-esis of rat mesenchymal stem cells by transfection of small interferingRNA complexed with a cationized dextran. Tissue Eng., Part A 16, 2131.

(38) Bachelder, E. M., Beaudette, T. T., Broaders, K. E., Dashe, J.,and Frechet, J. M. J. (2008) Acetal-derivatized dextran: An acid-responsive biodegradable material for therapeutic applications. J. Am.Chem. Soc. 130, 1049410495.

(39) Broaders, K. E., Cohen, J. A., Beaudette, T. T., Bachelder, E. M.,and Frechet, J. M. J. (2009) Acetalated dextran is a chemically andbiologically tunable material for particulate immunotherapy. Proc. Natl.Acad. Sci. U.S.A. 106, 54975502.

(40) Bachelder, E. M., Beaudette, T. T., Broaders, K. E., Frechet,J. M. J., Albrecht, M. T., Mateczun, A. J., Ainslie, K. M., Pesce, J. T., andKeane-Myers, A. M. (2010) In vitro analysis of acetalated dextranmicroparticles as a potent delivery platform for vaccine adjuvants. Mol.Pharmaceutics 7, 826835.

(41) Cohen, J. A., Beaudette, T. T., Cohen, J. L., Broaders, K. E.,Bachelder, E. M., and Frechet, J. M. J. (2010) Acetal-modied dextranmicroparticles with controlled degradation kinetics and surface func-tionality for gene delivery in phagocytic and non-phagocytic cells. Adv.Mater. 22, 35933597.

(42) Rao, D. D., Vorhies, J. S., Senzer, N., and Nemunaitis, J. (2009)siRNA vs. shRNA: Similarities and dierences. Adv. Drug Delivery Rev.61, 746759.

(43) Gary, D. J., Puri, N., and Won, Y.-Y. (2007) Polymer-basedsiRNA delivery: Perspectives on the fundamental and phenomenologi-cal distinctions from polymer-based DNA delivery. J. Controlled Release121, 6473.

(44) Takahashi, Y., Nishikawa, M., and Takakura, Y. (2009) Non-viral vector-mediated RNA interference: Its gene silencing character-istics and important factors to achieve RNAi-based gene therapy. Adv.Drug Delivery Rev. 61, 760766.



(45) Murata, N., Takashima, Y., Toyoshima, K., Yamamoto, M., andOkada, H. (2008) Anti-tumor eects of anti-VEGF siRNA encapsulatedwith PLGA microspheres in mice. J. Controlled Release 126, 246254.

(46) Vercauteren, R., Bruneel, D., Schacht, E., and Duncan, R.(1990) Eect of the chemical modication of dextran on the degradationby Dextranase. J. Bioact. Compat. Polym. 5, 415.

(47) Vercauteren, R., Schacht, E., and Duncan, R. (1992) Eect ofthe chemical modication of dextran on the degradation by rat liverlysosomal enzymes. J. Bioact. Compat. Polym. 7, 346357.

(48) Crepon, B., Jozefonvicz, J., Chytry, V., Rihova, B., and Kopecek,J. (1991) Enzymatic degradation and immunogenic properties ofderivatized dextrans. Biomaterials 12, 550554.

(49) Gray, I. (1953) Metabolism of plasma expanders studied withcarbon-14-labeled dextran. Am. J. Physiol. 174, 462466.

(50) de Belder, A. N. (1996)Medical Applications of Dextran and ItsDerivatives. Polysaccharides in Medicinal Applications (Dumitriu, S., Ed.)pp 505524, Chapter 16, Marcel Dekker, New York.

(51) Rosenfeld, E. L., and Lukomskaya, I. S. (1957) The splitting ofdextran and isomaltose by animal tissues. Clin. Chim. Acta 2, 105114.

(52) Rosenfeld, E. L., and Saienko, A. S. (1964) Metabolism in vivoof clinical dextran. Clin. Chim. Acta 10, 223228.

(53) Lynn, D. M., and Langer, R. (2000) Degradable poly(B-aminoesters): Synthesis, characterization, and self-assembly with plasmidDNA. J. Am. Chem. Soc. 122, 1076110768.

(54) Azzam, T., Eliyahu, H., Shapira, L., Linial, M., Barenholz, Y., andDomb, A. J. (2002) Polysaccharide-oligoamine based conjugates forgene delivery. J. Med. Chem. 45, 18171824.

(55) Azzam, T., Eliyahu, H., Makovitzki, A., Linial, M., and Domb,A. J. (2004) Hydrophobized dextran-spermine conjugate as potentialvector for in vitro gene transfection. J. Controlled Release 96, 309323.

(56) Hosseinkhani, H., Azzam, T., Tabata, Y., and Domb, A. J.(2004) Dextranspermine polycation: An ecient nonviral vector forin vitro and in vivo gene transfection. Gene Ther. 11, 194203.

(57) Bernstein, A., Hurwitz, E., Maron, R., Arnon, R., Sela, M., andWilchek, M. (1978) Higher antitumor ecacy of daunomycin linked todextran. J. Natl. Cancer Inst. 60, 379383.

(58) Petros, R. A., and DeSimone, J. M. (2010) Strategies in thedesign of nanoparticles for therapeutic applications. Nat. Rev. DrugDiscovery 9, 615627.

(59) Gautam, A., Densmore, C. L., Golunski, E., Xu, B., andWaldrep,J. C. (2001) Transgene expression in mouse airway epithelium byaerosol gene therapy with PEI/DNA complexes.Mol. Ther. 3, 551556.

(60) Jiang, H.-L., Xu, C.-X., Kim, Y.-K., Arote, R., Jere, D., Lim,H.-T.,Cho, M.-H., and Cho, C.-S. (2009) The suppression of lung tumorigen-esis by aerosol-delivered folate-chitosan-graft-polyethyleneimine/Akt1shRNA complexes through the Akt signaling pathway. Biomaterials 30,58445852.

(61) Fuller, J. E. (2008) in Chemical Engineering, MassachusettsInstitute of Technology, Cambridge.

(62) Dalby, B., Cates, S., Harris, A., Ohki, E. C., Tilkins, M. L., Price,P. J., and Ciccarone, V. C. (2004) Advanced transfection with Lipofec-tamine 2000 reagent: Primary neurons, siRNA, and high-throughputapplications. Methods 33, 95103.

(63) Kongkaneramit, L., Sarisuta, N., Azad, N., Lu, Y., Iyer, A. K. V.,Wang, L., and Rojanasakul, Y. (2008) Dependence of reactive oxygenspecies and FLICE inhibitory protein on Lipofectamine-induced apop-tosis in human lung epithelial cells. J. Pharmacol. Exp. Ther. 325, 969977.

(64) Beaudette, T. T., Cohen, J. A., Bachelder, E. M., Broaders, K. E.,Cohen, J. L., Engleman, E. G., and Frechet, J. M. J. (2009) Chemose-lective ligation in the functionalization of polysaccharide-based particles.J. Am. Chem. Soc. 131, 1036010361.

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Acid-Degradable Cationic Dextran Particles for the Delivery of SiRNA DEXTRANA E ESPERMINA