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Biomaterials 33 (2012) 3316e3323

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Biomaterials

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Simultaneous gene transduction and silencing using stimuli-responsive viral/nonviral chimeric nanoparticles

Soo Kyung Cho a, Young Jik Kwon a,b,c,d,*

aDepartment of Chemical Engineering and Materials Science, University of California, Irvine, CA 92697, United StatesbDepartment of Pharmaceutical Sciences, University of California, Irvine, CA 92697, United StatescDepartment of Biomedical Engineering, University of California, Irvine, CA 92697, United StatesdDepartment of Molecular Biology and Biochemistry, University of California, Irvine, CA 92697, United States

a r t i c l e i n f o

Article history:Received 29 December 2011Accepted 9 January 2012Available online 24 January 2012

Keywords:Adeno-associated virusCombined viral/nonviral chimericnanoparticleGene transferSimultaneous expression and silencingsiRNAStimuli-responsive delivery

* Corresponding author. Department of PharmaceCalifornia, 132 Sprague Hall, Irvine, CA 92697-3958, U8714; fax: þ1 949 824 4023.

E-mail address: kwonyj@uci.edu (Y.J. Kwon).

0142-9612/$ e see front matter � 2012 Elsevier Ltd.doi:10.1016/j.biomaterials.2012.01.027

a b s t r a c t

Despite viral vectors’ predominant use in clinical trials, due to higher gene delivery efficiency thannonviral counterparts, intrinsic immunogenicity and limited tunability for multi-modal effects are majorconcerns for their usage in gene therapy. An adeno-associated viral (AAV) particle was shielded withacid-degradable, siRNA-encapsulating polyketal (PK) shell, resulting in coreeshell viral/nonviral chimericnanoparticles (ChNPs). The AAV core of a ChNP is protected from immune responses by the PK shellwhich also facilitates the intracellular trafficking of the AAV core and efficiently releases the encapsulatedsiRNA into the cytoplasm. ChNPs led to significantly enhanced gene transduction, compared tounmodified free AAVs, and simultaneous silencing of a target gene, while avoiding inactivation byrecognition from the immune system. Furthermore, conjugation of sialic acid (SA) on the surface ofChNPs enabled receptor-mediated targeted gene delivery to CD22-expressing cells. The ChNPs developedin this study combine the advantages of both viral and nonviral vectors and are a promising platform fortargeted co-delivery of DNA and siRNA in inducing synergistic therapeutic effects by simultaneousexpression and silencing of multiple genes.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Development of efficient and safe delivery methods remainsa pivotal challenge in gene therapy. Recombinant viral vectors aresuperior to nonviral vectors in delivering genes, especially in vivo.However, viral vectors also offer considerable limitations, such asimmune responses (particularly upon repeated administrations)[1e3], difficulties in large-scale production [4e7], limited size ofgenes that can be packaged, narrow cell tropisms [8], and lack ofsurface modalities for molecular (synthetic) modifications withoutaltering viral stability and infectivity [9e12]. Many studies haveattempted to overcome the drawbacks of viral vectors. Immuno-suppression is often required prior to viral gene delivery butincreases the chance of opportunistic infection [13]. Geneticallymodifying the viral capsid and envelope [14,15], conjugatingvarious functional moieties (e.g., targeting molecules) [16,17], and

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electrostatically or covalently incorporating lipids or polymers[18e22] are often accompanied by compromised infectivity [23] orretained/new immunogenicity upon repeated administrations[24e28]. Nonviral vectors using synthetic materials (e.g., cationiclipids and polymers), on the other hand, are easy to manufacture atlarge scales, can deliver larger payloads, is readily tunable fordesirable structure/performance, and exhibits low immunogenicity[29]. However, poor transfection, particularly in vivo, has been thefundamental challenge in employing nonviral vectors for genetherapy [30,31].

Abnormalities in biological homeostases are often a result of animbalance in molecular signaling caused by multiple inconsis-tencies at the genetic level (or inconsistencies in gene expression).For example, cancer cells simultaneously upregulate bcl-2 genewhile suppressing pro-apoptotic p53 genes to enhance survival[32]. Therefore, disrupting this imbalance in gene expression byantagonizing overly expressed gene prior to introducing a newtherapeutic gene (transgene) is a logical approach to maximizingthe effects of gene therapy (synergistic gene therapy by simulta-neous gene expression and silencing). Synergistic gene therapyrequires the co-delivery of a transgene and antagonist nucleic acids(e.g., siRNA) against an overly expressed gene using a single carrier

S.K. Cho, Y.J. Kwon / Biomaterials 33 (2012) 3316e3323 3317

platform, in order to minimize discrepancies in pharmacokineticsand biodistribution in vivo. More importantly, in order to beappropriately processed and effective, transgene and siRNA mustbe transported to the nucleus and cytoplasm, respectively. Selec-tively controlled, ordered, and targeted delivery of therapeuticnucleic acids to intracellular targets will also maximize therapeuticefficacy, while reducing undesired side effects [33,34].

A gene carrier combining both viral and nonviral vectors intoa single nanoparticle platform (chimeric nanoparticles [ChNPs]) forco-delivery of a transgene and siRNA was developed in this study.This carrier incorporates the advantages of viral vectors’ hightransduction efficiency in its core as well as nonviral vectors’controlled cytosolic release of siRNA and low immunogenicity in itspolymeric shell, toward achieving efficient, controlled, and targetedco-delivery of a transgene (i.e., GFP) and siRNA (i.e., anti-luciferasesiRNA, LucsiRNA). The ChNPs developed in this study is a promisingtool in obtaining synergistic therapeutic effects via simultaneousexpression and silencing of multiple genes.

2. Materials and methods

2.1. Materials

GFP-encoding AAVs (GFPAAV; serotype 2) and luciferase-encoding AAVs (LucAAV;serotype 2) were purchased from Vector Biolabs (Philadelphia, PA) and ViroVek(Hayward, CA), respectively. Anti-AAV polyclonal antibodies were purchased fromIMGENEX (San Diego, CA). siRNA against firefly luciferase (LucsiRNA) and Dye547-labeled siRNA (Dye547-siRNA) were purchased from Ambion (Foster City, CA).Acid-degradable amino ketal methacrylamide monomer and acid-degradable ketalbismethacrylamide cross-linker were synthesized as previously reported [35e37],with slight modifications. Non-degradable cationic monomer and cross-linker,which contain an additional ethoxy group instead of ketal linkage, were alsosynthesized as recently reported [35]. N-Hydroxysuccinimide (NHS) and N,N-dii-sopropylethylamine (DIPEA) were purchased from Acros Organics (Thermo FisherScientific, Pittsburg, PA), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide(EDC) was purchased from Advanced ChemTech (Louisville, KY). Sialic acid waspurchased from Nacalai USA (San Diego, CA).

Eosin-5-isothiocyanate, Alexa Fluor 488 carboxylic acid succinimidyl ester, andQuant-iT PicoGreen nucleic acid assay kit were purchased from Invitrogen (Carlsbad,CA). PD10 size-exclusion column (MWCO 5 kDa) was purchased from GE Healthcare(Pittsburgh, PA) and Amicon Ultra Centrifugal filters (MWCO 10 kDa) werepurchased from Millipore (Billerica, MA). QuickTiter AAV quantitation kit waspurchased from Cell Biolabs (San Diego, CA). 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from SigmaeAldrich (St.Louis, MO) and nucleus-staining dye DRAQ5 was purchased from BioStatus (Lei-cestershire, UK). Forward (50-CTTCTTCAAGTCCGCCATGC-30) and reverse (50-GTCGCCCTCGAACTTCA-30) primers for GFP gene were purchased from Invitrogen(Carlsbad, CA). TaqMan Fast Universal PCR Mater Mix was purchased from AppliedBiosystems (Carlsbad, CA). Steady-Glo� luciferase assay solution (Promega, Madison,WI) and BCA protein assay kit (Pierce, Rockford, IL) were used to quantify Lucexpression and total protein amounts, respectively.

Human diffuse large B cell lymphoma (DLBCL)-Val cells expressing Luc (DLBCL-Val/Luc) were gifted from David Fruman (UC Irvine) and cultured in Iscove’sModified Dulbecco’s Medium (IMDM) (MediaTech, Herndon, VA) supplementedwith 10% fetal bovine serum (FBS) (Hyclone, Logan, UT) and 1% antibiotics (100units/mL penicillin and 100 mg/mL streptomycin) (MediaTech). Human B lympho-blast SUP-B15 cells also gifted from David Fruman (UC Irvine) were cultured inIMDM supplemented with 20% heat-inactivated FBS (Hyclone, Logan, UT), 5 mM

HEPES, 55 mM 2-mercaptoethanol, and 1% antibiotics (100 units/mL penicillin and100 mg/mL streptomycin).

2.2. Synthesis and characterization of ChNPs

GFPAAV particles (7.0 � 1011 genome copies [GC]) in 2.5 mL of 10 mM sodiumbicarbonate buffer (pH 8.0) were mixed with 3 mg of eosin-5-isothiocyanate dis-solved in 30 mL DMSO and stirred at room temperature for 2 h. Un-conjugated eosinwas purified using a PD 10 size-exclusion column equilibrated with 10 mM HEPESbuffer (pH 7.4). The eosin-conjugated GFPAAV (2.0 � 1011 GC in 1 mL of 10 mM HEPESbuffer) was transferred to a glass vial where 10 mg of ascorbic acid in 50 mL de-ionized water was added and stirred on ice. Meanwhile, acid-degradable aminoketal methacrylamide monomer (5 mg in 50 mL de-ionized water) was mixed with3 mg of LucsiRNA in 50 mL of de-ionized water. The mixture was incubated withoutstirring for 30 min at room temperature prior. Immediately after the mixture ofmonomer and siRNA was added to the solution of eosin-conjugated GFPAAV andascorbic acid, polymerization was initiated by irradiating with halogen light at 700

lux with constant stirring. At 10 min of photopolymerization, the mixture of 5 mg ofamino ketal methacrylamide monomer in 50 mL of de-ionized water and 5 mg ofketal bismethacrylamide cross-linker in 50 mL of 10mMHEPES buffer (i.e., siRNA-freemixture of monomer and cross-linker) was added and further polymerized foradditional 5 min. The resulting polymerization solutionwas added into a centrifugalfilter (MWCO 10 kDa) and centrifuged at 3500 rpm (2100 g) at 4 �C for 30 min toremove un-reacted monomer, cross-linker, and ascorbic acid. The harvested nano-particles were resuspended in 3mL of de-ionized water, centrifuged again under thesame conditions, and finally dispersed in 1 mL of nuclease-free water. Two types ofcontrol ChNPswere also synthesized using the samemethods. By polymerizing non-degradable monomer and cross-linker, ND-ChNPs containing a GFPAAV core andnon-degradable, LucsiRNA-encapsulating shell were synthesized. siRNA witha scrambled sequence was also encapsulated in acid-degradable polyketal (PK) shellof Sc-ChNPs with a GFPAAV core.

The size and the surface charge of the resulting nanoparticles in 1 mL ofnuclease-free water were measured using a Malvern Zetasizer Nano ZS (MalvernInstruments, Westborough, MA) at 25 �C. An aliquot (10 mL) of a ChNP-containingsolution was dropped on a carbon-coated grid (Ted Pella, Redding, CA), and thegrid was stained with uranyl acetate and dried overnight at room temperature. Themorphology and the size of the ChNPs were then examined under a Philips CM20transmission electron microscope (Philips Electronic Instruments, Mahwah, NJ) at80 kV.

2.3. Quantification and gel electrophoresis of siRNA and AAV DNA

ChNP solution (50 mL in nuclease-free water) was mixed with 25 mL of 100 mM

acetate buffer, achieving a final pH of 5.0, and incubated at 37 �C for 12 h with slightagitation. After acid-hydrolysis, the released siRNA amount was quantified byQuant-iT PicoGreen nucleic acid assay kit, according to the manufacturer’s protocol.After further incubation with RNase A at a concentration of 20 mg/mL at 37 �C for20 min, AAV particles were lysed by proteinase K at a concentration of 1 mg/mL for20 min at room temperature. AAV DNA extracted using a Wako DNA Extractor SP kit(Wako Chemicals, Richmond, VA) was quantified by using a QuickTiter� AAVQuantitation Kit.

The siRNA and AAV DNA released from ChNPs were visualized by agarose gelelectrophoresis. Because of substantially less AAV DNA than siRNA released fromChNPs, 3 mL of the solutions containing unhydrolyzed, acid-hydrolyzed, and acid-hydrolyzed and virus-lysed ChNPs were mixed with 1 mL of 5 mM forward-primer,1 mL of 5 mM reverse-primer, and 5 mL of nuclease-free water. 7 mL of TaqMan FastUniversal PCR Mater Mix was added and released AAV DNA was amplified usinga Taqman 7900HT Fast Real-Time PCR System (Applied Biosystems, Carlsbad, CA) for30 cycles.10 mL of resulting solutionwasmixedwith 10 mL of an unamplified solutionto enrich siRNA. After 20 mL of each sample was loaded in a 1% agarose gel containing1 mg/mL of ethidium bromide in TBE buffer, the gel was electrophoresed at 110 V for30 min, and the nucleic acids bands were visualized on a UV transilluminator (AlphaInnotech, Santa Clara, CA).

2.4. Intracellular trafficking of fluorescently labeled ChNPs

Prior to polymerization, eosin-conjugated AAV particles were labeled with AlexaFluor 488. In order to avoid overlapping fluorescence with Alexa Fluor 488, AAVparticles encoding Luc (LucAAV) was used to prepare fluorescently labeled ChNPs.Briefly, LucAAV particles (2 � 1011 GC in 500 mL of 10 mM sodium bicarbonate buffer)were mixed at room temperature with 1 mg of eosin-5-isothiocyanate in 10 mLDMSO. After 3 h, 10 mg of Alexa Fluor 488 carboxylic acid succinimidyl ester in 10 mLwas added and stirred for additional 3 h. Eosin-conjugated, Alexa Fluor 488-labeledLucAAV particles purified using a PD 10 column equilibratedwith 10mMHEPES bufferwere further shelled with Dye547-siRNA-encapsulating PK layer, as describedearlier. Fluorescently labeled AAVs without polymeric shell and ChNPs weredispersed in 250 mL of 10% serum-containing media and incubated with 3 � 104

DLBCL-Val/Luc cells per well in a Falcon 8-well culture slide in 250 mL of media (finalvolume of 500 mL/well). After 6 h-incubation at 37 �C, the cells were spun down andresuspended in fresh media. After additional incubation for 4 and 18 h (total incu-bation times of 10 and 24 h, respectively), the cells were transferred to 1.5 mLeppendorf tubes, spun down, washed twice with PBS, and fixed with 2% formal-dehyde in PBS. Then the fixed cells were washed twice with PBS and their nucleiwere stainedwith 5 mM DRAQ5 solution in PBS. Finally, the stained cells werewashedtwice with PBS and suspended in 50% (v/v) glycerol in PBS to minimize the move-ment of cells during confocal imaging. Using an Olympus IX2 inverted microscopecoupled with a Fluoview 1000 confocal scanning microscopy setup (FV10-ASW)(Olympus America, Inc., Melville, NY), cell images were observedwith a 40� /1.3 NAoil immersion planapochromat objective. Fluorescence of Alexa Fluor 488 wasimaged using a 488 nm excitation light from a multiple argon laser, a 488 nmdichroic mirror, and a 505e605 nm band pass barrier filter. A 559 nm helium-neonlaser, a SMD640 dichroic mirror, and a 575e620 nm band pass barrier filter wereused to obtain the fluorescence of Dye547. Images of DRAQ5-stained nuclei wereacquired using a 635 nm diode laser excitation light, a 635 nm dichroic mirror, anda 655e755 nm band pass barrier filter. Fluorescence images were captured andprocessed using FV10-ASW 1.6 viewer (Olympus America, Inc.). The cells were

S.K. Cho, Y.J. Kwon / Biomaterials 33 (2012) 3316e33233318

scanned in three dimensions as a z-stack of two-dimensional images (1024 � 1024pixels) and an image cutting horizontally through approximately the middle of thecellular height was selected to differentiate intracellular fluorescence from those onthe cellular surface. Each emission light was scanned separately for individualexcitations of the dyes to eliminate fluorescence cross-talk. Numbers of green andred pixels were counted using Image J software in order to quantify intracellulardistributions of AAV particles and siRNA, respectively.

2.5. Gene transduction and silencing

Various amounts of free AAVs and the ChNPs were incubated with DLBCL-Val/Luc cells plated at a density of 2.0 � 104 cells/well in a 24-well plate 24 h prior totransduction. After 6 h of incubation, the cells were washed twice with fresh media,further incubated for 3 days, and washed twice again. The cell-containing samples(100 mL) were mixed with an equal volume of fresh media, and the fluorescence ofthe cells in 100 mL of media was analyzed using a Guava EasyCyte flow cytometer(Guava Technologies, Hayward, CA). The rest of the cells in 100 mL of media weremixed with 100 mL of Steady-Glo� luciferase assay solution and relative Lucexpression was quantified by bioluminescence intensity using a microplate reader(Molecular Devices, Sunnyvale, CA). The measured bioluminescence intensity wasfurther normalized by a protein amount determined by BCA protein assay kit. Inorder to quantify cytotoxicity, the cells (5 � 103 cells/well in a 96-well plate) wereincubated with free AAVs and ChNPs at various concentrations (final volume of 200mL/well). After 24 h of incubation at 37 �C, relative viability of the cells was deter-mined by conventional MTT assay.

In order to assess inhibited transduction by ChNPs by AAV-neutralizing anti-bodies, DLBCL-Val/Luc cells (2.0 � 104 cells/well in a 24-well plate) were incubatedwith anti-AAV antibodies at different concentrations 1 h prior to incubation withfree AAVs and ChNPs. After 3 days of incubation with free AAVs and ChNPs, the cellswere washed twice with PBS and analyzed for GFP expression by flow cytometry.

2.6. Sialic acid (SA)-conjugation to ChNPs for targeted gene delivery

In order to activate its carboxylic acid group, sialic acid (SA) (19.4 mg in 100 mL of10 mM sodium bicarbonate buffer) was reacted with 24 mg of EDC and 14.5 mg ofNHS for 15 min at room temperature. To ChNPs (2 � 1011 GFPAAV) in 1 mL of 100 mM

sodium bicarbonate buffer, 10 mL of DIPEA was added and the activated SA solutionwas added. After the resulting mixture was mildly agitated overnight at 4 �C, SA-ChNPs were purified twice by centrifugal filtration (MWCO 10 kDa) at 3500 rpm(2100 g) at 4 �C for 30 min.

CD22þ DLBCL-Val/Luc and CD22- SUP-B15 cells were plated at a density of2.0 � 104 cells/well in a 24-well. ChNPs and SA-ChNPs (5 � 109 GC; prepared withAF488-conjugated LucAAV particles and Dye547-siRNA) in 100 mL PBS were added tothe cell-containing wells in a final volume of 500 mL/well. At different periods ofincubation, the cells were spun down and washed twice with PBS. Average fluo-rescence intensity of the cells was determined by flow cytometry and used toquantify a relative amount of internalized ChNPs. Uptake of ChNPs by DLBCL-Val/Luccells was normalized by that by SUP-B15 cells to present receptor-mediated, tar-geted cellular uptake of SA-ChNPs. Transduction of DLBCL-Val/Luc and SUP-B15 cellsby free GFPAAV, GFPAAV/LucsiRNA ChNPs, and SA-conjugated GFPAAV/LucsiRNA ChNPswas determined by measuring GFP expression as described earlier. The cells werealso incubated with ChNPs and SA-ChNPs in the presence of free SA at variousconcentrations. Interfered transduction by free SA was determined by reduced GFPexpression measured by flow cytometry.

3. Results and discussion

3.1. Design and synthesis of ChNPs

Among many recombinant viruses employed in gene therapy,AAV was chosen as the core of ChNPs that deliver a transgene withhigh in vivo transduction efficiency. AAV is one of the smallestviruses, not known for any clear links with human illness, andincapable of replicationwithout a helper virus [38e40]. In addition,AAV integrates its genome into a region ofw2 kbps on the long armof human chromosome 19 [41]. This property, unique among allknown eukaryotic viruses, offers a low risk of mutagenesis [42].Finally, AAV is relatively easy for synthetic conjugation [43] and itssmall size allows high siRNA loading while keeping ChNP withina desirable size range.

Transduction by GFPAAV particles is hypothesized to beenhanced by an acid-degradable polyketal (PK) shell that 1) shieldsthe viral core from immune responses, 2) enhances cytosolicrelease from the mildly acidic endosome upon acid-hydrolysis, and

3) enables facile conjugation of targeting molecules on the surface.Most importantly, siRNA (e.g., LucsiRNA) can be encapsulated in thePK shell via in situ surface-initiated photopolymerizationwith acid-degradable ketal cationic monomer and cross-linker (Fig. 1A).Therefore, GFP gene and Luc siRNA are co-delivered in the viral coreand nonviral acid-degradable polyketal (PK) shell, respectively. Theresulting ChNPs are artificially enveloped AAVs in structure. Theyare synthetically equipped for improved extracellular performance(protection from immune recognition and targeted delivery) andintracellular trafficking (endosomal escape and simultaneousdelivery of viral DNA and siRNA), because of the stimuli-responsiveand easily tunable PK shell (artificial envelope). Surface-initiatedpolymerization by visible light was used in order to prevent thegeneration of bulk AAV- or siRNA-free particles as well as AAVinactivation and siRNA degradation. The PK shell of an internalizedChNP is hydrolyzed in the mildly acidic endosome, which desta-bilizes the endosome by increasing osmotic pressure [44,45], andfinally releases siRNA into the cytoplasm where it silences Lucexpression (Fig. 1B). Simultaneously, the AAV core trafficks to thenucleus where lysogenic disassembly releases the viral DNA andinitiates GFP expression. Therefore, two distinct genetic materials,siRNA and AAV DNA, are selectively delivered to their intracellulartargets by viral/nonviral chimeric gene carriers for simultaneoussilencing and expression of multiple genes. Co-delivery of addi-tional therapeutic molecules with a viral particle has been highlydesired but hampered by poor and cumbersome loading methods[46e52]. The method used in this study, adding siRNA duringsurface-initiated photopolymerization of cationic monomers,enables efficient, convenient, and tunable incorporation in thestimuli-responsive polymeric shell around a viral particle.

3.2. Characterization of ChNPs and differential release of AAV DNAand siRNA

The synthesized ChNPs were 165 nm in diameter witha cationic surface charge (þ28 mV) (Table 1), while free AAVs wereanionic (�11 mV) and 27 nm in diameter, estimating the thicknessof siRNA-encapsulating PK shell to be approximately 70 nm. Thestructure of ChNPs consisting of an AAV core and acid-degradable,siRNA-encapsulating PK shell was confirmed by the differentialreleases of siRNA and AAV DNA at an endosomal pH of 5.0 andunder virus lysis conditions, respectively (Table 2). While a mildlyacidic endosomal pH alone efficiently released siRNA (condition iiiin Table 2), combined treatment of both acid-hydrolysis of PK shelland AAV core lysis was required to release AAV DNA (condition ivin Table 2). Virus lysis alone without PK shell hydrolysis was notable to release AAV DNA from ChNPs (condition ii in Table 2),indicating sequential stimuli-responsive intracellular release ofsiRNA and viral DNA from the ChNPs. The quantification ofreleased AAV DNA and siRNA also indicated a high efficiency ofsiRNA encapsulation (w100%) in PK shell without losing AAVparticles. TEM confirmed the ChNP size measured by DLS anddemonstrated the AAV release upon acid-hydrolysis of PK shell(Fig. 2A). ChNPs exhibited a dense and spherical shape witha relatively uniform size. Upon incubation at an endosomal pH of5.0, AAV particles (yellow arrow) were released from acid-hydrolyzed polymeric debris (blue arrow). The differentialrelease of siRNA and AAV DNA was also demonstrated by gelelectrophoresis (Fig. 2B). The results shown in Fig. 2 and Table 2imply that the AAV core was effectively shielded by PK shell andAAV core is released only after acid-hydrolysis of PK shell in themildly acidic endosome/lysosome. Therefore, ChNPs mimics theintracellular trafficking of some enveloped viruses such as influ-enza virus whose capsid is released from the endosome when itsenvelope is fused with the endosomal membrane [53].

Table 2

Fig. 1. (A) Synthesis of ChNPs with a GFP-encoding AAV core and Luc siRNA-encapsulating PK shell (GFPAAV/LucsiRNA ChNPs). (B) Intracellular trafficking of siRNA and AAV core thatare differentially delivered by a ChNP to the cytoplasm and the nucleus, respectively.

S.K. Cho, Y.J. Kwon / Biomaterials 33 (2012) 3316e3323 3319

3.3. Intracellular release and distribution of siRNA and AAVparticles delivered by ChNPs

ChNPs made of Alexa Fluor 488-labeled AAV core (green) andDye547-labeled siRNA (red) were synthesized and their intracel-lular trafficking in DLBCL-Val/Luc cells was studied using confocallaser scanning fluorescence microscopy (Fig. 3). Free AAVs werefound in the cytoplasm at 10 h of incubation, and further incubationfor another 14 h did not result in noticeable changes in intracellulardistribution (Fig. 3A). On the contrary, a significant portion of freesiRNA (red dots) and AAV core (green dots) were released fromChNPs at 10 h of incubation when there were still a number ofunhydrolyzed ChNPs carrying both siRNA and AAV, represented byoverlapping fluorescence in yellow (Fig. 3A). At 24 h of incubation,complete release of siRNA and AAVs from ChNPs were observed,

Table 1Size and surface charge of free AAVs, ChNPs, ND-ChNPs, Sc-NPs, and SA-ChNPs.Triplicate measurements were averaged.

Size (nm) (PDIa) Zeta-potential (mV)

Free AAVs (GFPAAV) 26.6 (0.405) �10.70 � 7.62ChNPs 165.1 (0.213) þ27.67 � 3.40ND-ChNPs 162.2 (0.211) þ19.50 � 2.11Sc-ChNPs 158.1 (0.205) þ25.7 � 1.80SA-ChNPs 178.4 (0.236) þ0.10 � 0.26

a PDI: Polydispersity index (0e1) (0: Perfectly monodispersed).

which was confirmed by separate green and red dots (almost noyellow dots) (Fig. 3A; zoomed images at the bottom). siRNA (reddot) released from ChNPs were found selectively in the cytoplasm(88%) and a significant portion of AAVs (green dots) released fromChNPs were found inside the nucleus (30%). Interestingly, a signif-icantly higher portion of AAV particles released from ChNPs wereidentified in the nucleus (30%) than those of free AAVs (10%)(Fig. 3B). This result implies that enhanced endosomal escape ofChNPs aided by PK shell improved intracellular trafficking ofreleased AAV core to the nucleus. Weakened fluorescence signalsafter 24 h of incubation may be a result of a decreased fluorescencedensity upon AAV disassembly and siRNA degradation. To demon-strate the crucial roles of acid-degradable PK shell, ChNPswith non-

Confirmed structure of GFPAAV/LucsiRNA ChNPs by differential release of siRNA andAAV DNA under endosomal and virus lysis conditions.a

Condition

i ii iii iv

pH 5.0 e e þ þAAV lysis e þ e þReleased siRNA (mg) 0.47 � 0.036 0.54 � 0.072 3.19 � 0.037 3.18 � 0.15Released AAV DNA

(�1011 GC)0.20 � 0.011 0.23 � 0.019 0.18 � 0.010 2.04 � 0.047

a The starting amounts of AAV particles and siRNA for ChNP synthesis were2 � 1011 GC (genome copies) and 3 mg, respectively.

Fig. 2. (A) AAV core release from ChNPs upon acid-hydrolysis. Yellow and blue arrows in the TEM image indicate released AAV core and hydrolyzed polymer debris, respectively. (B)Differential release of siRNA and AAV DNA from ChNPs under the same conditions used in Table 1. It should be noted that the size of the bands does not represent relative amountsof AAV DNA and siRNA released from ChNPs because of PCR-amplification of AAV DNA before gel electrophoresis (see Section 2.3). (For interpretation of the references to colour inthis figure legend, the reader is referred to the web version of this article.)

S.K. Cho, Y.J. Kwon / Biomaterials 33 (2012) 3316e33233320

degradable polymethacrylamide polymeric shell (ND-ChNPs) weresynthesized to carry the same labeled siRNA and AAV core. Evenafter 24 h of incubation, almost all of siRNA and AAV cores (99%overlap represented by yellow dots) were found co-localized (i.e.,assembled in the ND-ChNPs) in the cytoplasm (98%) (Fig. 3A and B).It was clearly demonstrated that acid-degradable PK shell of ChNPsnot only releases the encapsulated siRNA upon acid-hydrolysisunder a mildly acidic condition (Fig. 2B) but also facilitates thetransnuclear localization of AAV core (Fig. 3B).

Fig. 3. Intracellular release and distribution of Alexa Fluor 488-labeled AAV particles (green d24 h with free AAVs, ChNPs, and ND-ChNPs. Yellow dots represent co-localized siRNA and Aand DIC images. (B) Quantified intracellular distributions of fluorescently labeled AAV particlcolour in this figure legend, the reader is referred to the web version of this article.)

3.4. Transduction and simultaneous gene silencing by ChNPs

DLBCL-Val/Luc cells were incubated with free AAVs and variousChNPs (Fig. 4). AAVs are known to efficiently transduce muscle,liver, neuron, and skin cells. However, inefficient transduction of Bcells by AAVs often requires high viral titers [54,55]. Overall, bothfree AAVs and ChNPs transduced the DLBCL-Val/Luc cells ina concentration-dependent manner, while ND-ChNPs showedminimal transduction at all concentrations (Fig. 4A). Importantly, at

ots) and Dye547-labeled siRNA (red dots). (A) DCBCL-Val/Luc cells incubated for 10 andAV particles in the ChNPs. Upper: fluorescence images; Lower: Combined fluorescencees and siRNA at 24 h of incubation with the cells. (For interpretation of the references to

Fig. 4. DCBCL-Val/Luc cells incubated with free AAVs, ChNPs, ND-ChNPs, and Sc-ChNPs. (A) Transduction of the cells by free AAVs, ChNPs, and ND-ChNPs, measured by GFPexpression. (B) Relative transduction by free AAVs and ChNPs in the presence of anti-AAV antibodies, compared with transduction efficiency under antibody-free conditions. (C)Silenced Luc expression by the cells after incubated with AAVs, ChNPs, and Sc-ChNPs. (D) Relative viability of the cells after incubated with free AAVs, ChNPs, and ND-ChNPs.

S.K. Cho, Y.J. Kwon / Biomaterials 33 (2012) 3316e3323 3321

a concentration of 2.5 � 109 GC/well, ChNPs showed substantiallyhigher transduction efficiency than free AAVs. This result can beexplained by the finding that the acid-degradable PK shell facili-tates intracellular trafficking of AAV particles (Fig. 3), overcomingthe, otherwise, limited B cell transduction by free AAVs. Ashypothesized, PK shell of ChNPs effectively shielded AAV core fromanti-AAV antibodies (Fig. 4B). In comparison, the transductioncapability of free AAVs was greatly interfered by the antibodies. Lucexpression by the cells was silenced by ChNPs in a concentration-dependent manner, while no meaningful gene silencing wasobserved when the cells were incubated with ND-ChNPs andChNPs encapsulating control siRNA with scrambled sequences (Sc-ChNPs) (Fig. 4C). ChNPs showed cell viability slightly lower than orequivalent to those of free AAVs and ND-ChNPs (Fig. 4D), impli-cating minimal adverse effects by acid-degradable PK shell. Toensure complete dissociation of PK shell from AAV core uponhydrolysis, eosin was conjugated on the AAV surface via ketallinkage before polymerization (Supplementary Information). Theresulting ChNPs polymerized via cleavable eosin did not result inimproved transduction (data not shown), indicating either efficientdissociation of acid-hydrolyzed PK shell upon AAV disassembly or

no noticeable effects of hydrolyzed PK remaining on the AAV coreon transduction efficiency.

3.5. Targeted gene delivery by SA-ChNPs

DLBCL-Val/Luc cells express CD22 which binds to SA of cellsurface proteins [56,57]. The abundant amine groups on the surfaceof ChNPs’ PK shell were further conjugated with SA using EDC-NHScoupling method, in order to achieve targeted gene delivery toDLBCL-Val/Luc cells. It was confirmed that a significant number ofcationic amines on the ChNP surface were used in SA conjugation(Fig. S1), resulting in nearly neutral SA-ChNPs (Table 1). Human Blymphoblast SUP-B15 cells, which do not express CD22, were alsoincubated with ChNPs with or without SA conjugation. No differ-ence in cellular uptake of ChNPs by DLBCL-Val/Luc and SUP-B15cells was observed, while the uptake of SA-ChNPs by DLBCL-Val/Luc cells dramatically increased, reaching 9-fold higher uptakethan SUP-B15 cells after 60 min of incubation (Fig. 5A). No suchincreased cellular uptake of SA-ChNPs was observed with the SUP-B15 cells. Significantly higher uptake of SA-ChNPs resulted inhigher transduction of DLBCL-Val/Luc than SUP-B15 cells (Fig. 5B).

Fig. 5. (A) Selective uptake of SA-ChNPs by CD22þ DLBCL-Val/Luc cells over CD22- SUP-B15 cells. (B) Relative transduction of DLBCL-Val/Luc cells (CD22þ) over SUP-B15 cells(CD22-) by free AAVs, ChNPs, and SA-ChNPs, represented by the GFP expression levels in DLBCL-Val/Luc cells after normalized by those in SUP-B15 cells. (C) Transduction ofDLBCL-Val/Luc cells by ChNPs and SA-ChNPs in the presence of free SAs, compared with transduction efficiency under SA-free conditions.

S.K. Cho, Y.J. Kwon / Biomaterials 33 (2012) 3316e33233322

Free AAVs and ChNPs showed no clear preferential transduction ofDLBCL-Val/Luc cells to SUP-B15 cells. A reason for lower increase intargeted transduction (i.e., 2.3 fold in Fig. 5B) than selective uptakeof SA-ChNPs (i.e., 9 fold in Fig. 5A) may indicate limited expressionefficiency of AAV DNA or that not all quantified AAV particles werebiologically active. Due to competitive binding to CD22, the trans-duction of DLBCL-Val/Luc cells by SA-ChNPs greatly decreased inthe presence of free SAs in a concentration-dependent manner,while the transduction efficiency of SA-free ChNPs was not signif-icantly affected by free SAs (Fig. 5C). The results shown in Fig. 5clearly demonstrate the high feasibility of tethering the surface ofthe ChNPs for rendering additional properties to ChNPs (e.g., tar-geted delivery as demonstrated in this study) via convenient, effi-cient, and versatile conjugation of desired functional molecules.

4. Conclusion

Two types of nucleic acids (DNA and siRNA) were co-deliveredby ChNPs with an AAV core and acid-degradable, siRNA-encapsu-lating PK shell in order to achieve simultaneous expression andsilencing for synergistic gene therapy with combined advantages ofboth viral and nonviral vectors. The ChNPs were also tethered fortargeted gene delivery by conjugating targeting molecules on thePK shell. The ratio of AAV DNA to siRNA can be easily tuned byvarying siRNA amount encapsulated in the PK shell in order toobtain optimized expression and silencing of target biologicalpathways. The ChNP platform developed in this study can also beeasily applied to co-delivery of nucleic acids and synthetic drugs forcombined gene and chemotherapy (e.g., ChNPs with an AAV coreand PK shell encapsulating anti-cancer drugs). The polymeric shellcan be synthetically programmed to be responsive to other stimuli,such as hypoxia, enzymes, heat, radicals, and light, and the AAVcore can be replaced by other types of viruses, proteins, polymers,and inorganic nanomaterials, depending on specific needs.

Acknowledgement

This work was funded by the Gabrielle’s Angel Foundation forCancer Research (Award # 056) and American Cancer Society (ACS)Institutional Research Grant (IRG-98-279-07).

Appendix. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.biomaterials.2012.01.027

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