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Biomaterials 33 (2012) 270e282

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Biomaterials

journal homepage: www.elsevier .com/locate/biomateria ls

EGFR-specific PEGylated immunoliposomes for active siRNA deliveryin hepatocellular carcinoma

Jie Gaoa,c,1, Yongsheng Yua,1, Yingying Zhangb,1, Jinjing Songa, Huaiwen Chena,c,Wei Lia,c, Weizhu Qiana,c, Li Dengb, Geng Koua,c, Jianming Chenb,**, Yajun Guoa,c,d,*a International Joint Cancer Institute, the Second Military Medical University, 800 Xiang Yin Road, Shanghai 200433, PR ChinabDepartment of Pharmaceutical Science, College of Pharmacy, the Second Military Medical University, 325 Guo He Road, Shanghai 200433, PR ChinacNational Engineering Research Center for Antibody Medicine & Shanghai Key Laboratory of Cell Engineering and Antibody, 399 Libing Road, Shanghai 201203, PR Chinad PLA General Hospital Cancer Center, PLA Graduate School of Medicine, Beijing 100853, PR China

a r t i c l e i n f o

Article history:Received 27 August 2011Accepted 14 September 2011Available online 2 October 2011

Keywords:AntibodyImmunoliposomessiRNA deliveryEGFRHCC

* Corresponding author. International Joint CancerMedical University, Shanghai, China. Tel./fax: þ86 21** Corresponding author. Tel./fax: þ86 21 81871291.

E-mail addresses: yjcjm@163.com (J. Chen), yjguo1 Jie Gao, Yongsheng Yu and Yingying Zhang contr

0142-9612/$ e see front matter � 2011 Elsevier Ltd.doi:10.1016/j.biomaterials.2011.09.035

a b s t r a c t

The development of immunoliposomes for systemic siRNA (small interfering RNA) delivery is highlydesired. We reported previously the development of targeted LPD (liposomeepolycationeDNA complex)conjugated with anti-EGFR (epidermal growth factor receptor) Fab0 (TLPD-FCC) for siRNA delivery, whichshowed superior gene silencing activity in EGFR-overexpressing breast cancers. However, TLPD-FCC didnot achieve satisfactory gene silencing activity in EGFR-overexpressing hepatocellular carcinoma (HCC).In this study, some modifications including increased antibody conjugation efficiency and reducedPEGylation degree were made to TLPD-FCC to increase gene silencing activity in HCC. The resultantoptimized liposomes denoted as TLPD-FP75 efficiently bound and delivered to EGFR-overexpressing HCC,resulting in enhanced gene silencing activity compared to untargeted LPD (NTLPD-FP75). Tissue distri-bution in vivo revealed that the accumulation of TLPD-FP75 was higher than NTLPD-FP75 in orthotopicHCC model of mice. The promoted uptake of TLPD-FP75 in HCC cells was confirmed by confocalmicroscopy. To investigate the in vivo gene silencing activity, we administered TLPD-FP75 by intravenousinjections into mice bearing orthotopic HCC. The results showed TLPD-FP75 potently suppressed lucif-erase expression, while little silencing was observed in NTLPD-FP75. TLPD-FP75 was demonstrated topossess potent gene silencing activity in HCC and will potentially increase the feasibility of HCC genetherapy.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction

Hepatocellular carcinoma (HCC) is one of the most commoncancers worldwide, with more than 500,000 new cases a year [1]. Amajority of these cases originates from developing countries andoccursmore frequently inmen thanwomen. In theUnited States, theincidence of HCC has increased by more than 90% in the past threedecades [2]. Despite great advances have been achieved in HCCdiagnosis and therapy, themorality rate remains high, especially forHCC in the advanced stage when the disease is usually diagnosed.

Gene therapy is the insertion, alteration, or removal of geneswith the cells and biological tissues to treat diseases, and it has

Institute, the Second Military81870801.

@smmu.edu.cn (Y. Guo).ibuted equally to this paper.

All rights reserved.

emerged as a potential treatment for HCC. Classical HCC genetherapy strategies consist of restoration of tumor suppressor genes,genetic prodrug-activating therapy, genetic immunotherapy andoncolytic viruses [3]. These gene therapy strategies have beenproven to be beneficial in animal models of HCC, and some of themeven have reached the early phases of clinical development [3]. Oneof the most famous therapeutic agents, Gendicine, the adenoviralvector carrying the wild-type human p53 gene, has been used intreating head and neck squamous carcinoma in combination withchemotherapy [4]. In spite of their efficacy in HCC therapy, theapplication of these classical gene therapy strategies was severelylimited by the following issues. Firstly, viral vectors are mostly usedin these classical gene therapy strategies. Although viral vectors areeffective in achieving high efficiency for both gene delivery andexpression, the limitations associated with them are safety,potential immunogenicity and high cost [5]. Secondly, viral vectorsusually only deliver a single specific gene associated with inhibitionof cancer progression. However, HCC often has multiple genetic

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alterations and signal pathways, which make the disease verycomplex [6]. The effectiveness of targeting a single specific genetends to be compensated by activation of other signal pathways.Finally, the gene delivery of viral vectors was non-specific. It isimpossible to modify the viral vectors with a target-specific ligandwhich could achieve active targeting and specific receptor-mediated endocytosis [7].

Gene therapy using non-viral vectors represents a preferredapproach because of their low immunogenicity, high biocompati-bility, low cost and convenient surface modification [8]. Althoughthe transfection efficiency of non-viral vectors is still lower thanthat of their viral counterparts, surface modification by target-specific ligands could significantly improve the transfection effi-ciency of non-viral vectors. siRNA (small interfering RNA) is 21e25nucleotide, double-stranded RNA and could be used to mediatetranscript degradation for specific gene silencing. Non-viral vectorsdelivering siRNA represent a potent and specific method in tumorgene therapy [9]. However, the efficient siRNA delivery to targettissues following systemic administration is the most importanthurdle for widespread use of non-viral vectors delivering siRNAin vivo [5,10]. Currently, lipid-based delivery system, one kind ofnon-viral vectors, has been successfully applied in delivering siRNAin vivo [11]. Zimmermann et al. developed a stable nucleic acid lipidparticle (SNALP) for delivering siRNA against apoB to the liver ofmonkeys [12]. The apoB protein, low-density lipoprotein, andcholesterol level was significantly reduced for up to 11 days. Huanget al. developed a well-established LPD (liposomeepoly-cati-oneDNA complex) and it was shown to deliver siRNA efficiently totumor cells overexpressing sigma receptor [13,14]. We have earlierobtained optimized targeted LPD conjugated with tumor-specificantibody (TLPD) conjugated with anti-EGFR (epidermal growthfactor receptor) Fab0 by conventional conjugation (TLPD-FCC),which possessed a small size around 150 nm and superior in vitrostability. The results showed that TLPD-FCC possessed superiorgene silencing activity in EGFR-overexpressing MDA-MB-231breast cancers both in vitro and in vivo [15].

Numerous studies reported that EGFR overexpression occursfrequently in HCC, and in some cases has been correlated withpoor prognosis, suggesting that EGFR is a potential target for HCCtherapy [6,16]. We hypothesized that TLPD-FCC can be constructedto provide efficient intracellular siRNA delivery in EGFR-overexpressing HCC. However, our preliminary studies revealedthat although TLPD-FCC achieved a noticeable success in siRNAdelivery, the release of siRNA into the cytoplasm is variabledepending on the cells and still needs improvement. As for HCC,TLPD-FCC could selectively bind to and be internalized in EGFR-overexpressing HCC but did not achieve satisfactory genesilencing activity. Our previous research and other groups’ studieshave showed that endosomal escape is a major barrier for efficacyof siRNA-mediated gene silencing, and PEGylation representsa major barrier for endosomal escape [11,17,18]. Therefore, wespeculated that the high degree of surface PEGylation may lead topoor gene silencing activity of TLPD-FCC in EGFR-overexpressingHCC. In contrast, reduction of PEGylation degree may enhancethe gene silencing activity of TLPD-FCC in HCC. However, PEGy-lation degree should not be reduced significantly, as the PEGcoating increases blood circulation time, consequently leavingtime for the nano-scale liposomes to reach tumors owing to theenhanced permeability and retention (EPR) effect [5,11]. Moreover,the plasma membrane is a significant barrier for siRNA uptake, andeffective siRNA delivery approaches need to overcome this limi-tation by facilitating cellular uptake. Many studies showed thattarget-specific ligand like antibody can induce active targeting andsignificantly promotes siRNA cellular uptake. Noteworthily, Fenget al. reported that the cytotoxic effect enhanced with the increase

of the targeted nanoparticles surface density of the antibody,indicating that the quantity of conjugated antibody plays animportant role in therapeutic effect of targeted nanoparticles [19].Our results showed that the anti-EGFR antibody was surelyconjugated on TLPD-FCC, but the quantity was small anda considerable amount of unconjugated antibody still existed [15].If the quantity of conjugated anti-EGFR antibody can be raised,a better gene silencing activity of TLPD-FCC might be achieved inHCC.

To obtain an effective non-viral vector for siRNA delivery in HCCgene therapy, we investigated the effect of PEGylation and antibodyconjugation on the essential physicochemical properties and tar-geting efficiency of TLPD, which was previously obtained and well-characterized. Some modifications including increased antibodyconjugation efficiency and reduced PEGylation degree were madeto TLPD-FCC to increase gene silencing activity in HCC. We evalu-ated parameters that affect the gene silencing activity to find anoptimal formulation, and subsequently characterized the lipo-somes for physical stability, in vitro and in vivo targeting ability andgene silencing activity.

2. Materials and methods

2.1. Materials

1,2-Dioleoyl-3-trimethylammonium-propane (chloride salt) (DOTAP), choles-terol (Chol), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[mal-eimide(polyethylene glycol)-2000] (ammonium salt) (DSPE-PEG-Mal), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt) (DSPE-mPEG) and 1,2-dioleoyl-sn-glycero-3-phosphoe-thanolamine-N-carboxyfluorescein (CFPE) were purchased from Avanti Polar Lipids(Alabaster, AL). Deoxyribonucleic acid solution from calf thymus (calf thymus DNA,sonicated DNA for hybridization), protamine sulfate salt from salmon (Grade X,amorphous powder) and G418 were obtained from SigmaeAldrich (St. Louis, MO).All organic reagents are of analytical grade and purchased from Sinopharm(Shanghai, China). Human-mouse chimeric anti-EGFR mAb was kindly provided byNational Engineering Research Center for Antibody Medicine (Shanghai, China) andFab0 of anti-EGFR mAb (anti-EGFR Fab0) was prepared as we described previously[20]. FITC labeled goat anti-human IgG (H þ L) was purchased from Zymed (SanFrancisco, CA). 2-Iminothiolane (Traut’s Reagent) was obtained from Pierce (OudBeijerland, NL). FAM-labeled negative control (NC) siRNA (FAM-siRNA), anti-luciferase siRNA, anti-RhoA siRNA and NC siRNA were synthesized by Gene-Pharma Co., Ltd (Shanghai, China). The sequence of siRNA and primers were listed inSupplementary Table 1. Cy5-labeled NC siRNA (Cy5-siRNA) was synthesized byRibobio Co., Ltd (Guangzhou, China). Dulbecco’s modified Eagle’s medium (DMEM)and fetal bovine serum (FBS) were purchased from Invitrogen (Carlsbad, CA, USA).

2.2. Cell lines

The human HCC cell lines SMMC-7721, LM3 and Hep3B were purchased fromATCC (American Type Culture Collection, VA). The SMMC-7721, LM3 and Hep3B cellswere stably transfected with luciferase gene using pcDNA3.1 vector. All the cellsused in this study were stably transfected with luciferase gene. The cells weremaintained in DMEM supplemented with 10%FBS, 25 mM HEPES buffer, 0.4 mg/mlG418,100 U/ml penicillin and 100 mg/ml streptomycin in a humidified atmosphere of5% CO2 at 37 �C. Supplementary Fig. 1 shows that SMMC-7721 cells overexpressedEGFR, LM3 cells expressed EGFR moderately, while Hep3B cells expressed EGFRlowly.

2.3. Liposome preparation

Liposomeswere prepared as described previously with slight modifications [15].Briefly, cationic liposomes composed of DOTAP and Chol (1:1 molar ratio, 10 mM)were prepared by the thin film hydrationmethod. For the preparation of fluorescentliposomes, 0.1% (molar ratio) CFPEwas added in the lipid film. To prepare naked LPD,124 ml cationic liposomes, 15 ml protamine (2 mg/ml) and 11 ml DEPC water weremixed as solution A and kept at room temperature (RT) for 10 min. Meanwhile, 90 mlsiRNA (24 mg, 20 mM), 2.4 ml calf thymus DNA (24 mg, 10 mg/ml) and 57.6 ml DEPCwater were also mixed as solution B and kept at RT for 10 min. The solution A and Bwere mixed quickly to form naked LPD and incubated at RT for 15 min. Forconventional conjugation, naked LPD was mixed with 37.8, 28.35 or 18.9 ml micellesolution of DSPE-PEG-Mal (10 mg/ml) to form LPD at 50 �C for 10 min. 600 mgthiolated anti-EGFR Fab0 was mixed with LPD and the mixture was incubated at16 �C overnight to form targeted LPD conjugated with anti-EGFR antibody (TLPD).

J. Gao et al. / Biomaterials 33 (2012) 270e282272

For post-insertion, 600 mg thiolated anti-EGFR Fab0 was first incubated with 37.8,28.35 or 18.9 ml micelle solution of DSPE-PEG-Mal (10 mg/ml) at 16 �C overnight.Then naked LPD was mixed with the antibody-conjugated micelles to form TLPD at50 �C for 10 min.

Nontargeted LPD (NTLPD) was prepared in the same way as TLPD except thatDSPE-PEG-Mal was replaced by DSPE-mPEG. All the reagents and equipment used inpreparing liposomes were sterile. The prepared naked LPD, TLPD and NTLPD werestored at 4 �C. The following designations are used: TLPD-FC (TLPD conjugated withanti-EGFR Fab0 by conventional conjugation), TLPD-FP (TLPD conjugated with anti-EGFR Fab0 by post-insertion). For convenience, TLPD-FC with 37.8, 28.35 or 18.9 mlDSPE-PEG-Mal micelle was denoted as TLPD-FC100, TLPD-FC75 and TLPD-FC50respectively. TLPD-FP with 37.8, 28.35 or 18.9 ml DSPE-PEG-Mal micelle was deno-ted as TLPD-FP100, TLPD-FP75 and TLPD-FP50 respectively. Similarly, their respec-tive controls are NTLPD-FC100, NTLPD-FC75, NTLPD-FC50, NTLPD-FP100, NTLPD-FP75 and NTLPD-FP50.

2.4. Characterization of liposomes

2.4.1. Liposome size and zeta potentialThe liposomes were dispersed in deionized water. Average size and zeta

potential were analyzed using Zeta sizer Nano S (Malvern instruments, UK).

2.4.2. siRNA encapsulation efficiency (EE)The siRNA EE was determined by ultra-filtrating the liposomes entrapping FAM-

siRNA using Amicon� Ultra-4 centrifugal filter devices (100,000 NMWL, Millipore,Massachusetts). After thoroughly ultra filtration with DEPC water, unencapsulatedFAM-siRNA was collected and quantified using a calibration line obtained withstandard lead FAM-siRNA solutions. The fluorescence of FAM-siRNAwas determinedwith the spectrofluorometer (Synergy� 4, Biotek, Wi, USA) using excitation andemissionwavelengths of 495 and 525 nm, respectively. The siRNA EE was calculatedusing the formula: (Mi � MU)/Mi � 100%. MU and Mi were defined as the mass ofunencapsulated siRNA and initially added siRNA, respectively.

Fig. 1. The size (A) and zeta potential (B) of TLPD-FC and TLPD-FP, which wereprepared by conventional conjugation or post-insertion strategies. The detailedimplications of TLPD-FC100, TLPD-FC75, TLPD-FC50, TLPD-FP100, TLPD-FP75 andTLPD-FP50 were seen in Section 2.3. Their particle size and zeta potential wereanalyzed using Zeta sizer Nano S. Data are expressed as mean � SD (n ¼ 3).

2.4.3. Gel retardation assayThe siRNA binding efficiency of the liposomes was evaluated by gel retardation

assay. Briefly, after destroyed by Triton X-100, the liposomes were loaded intoindividual wells of agarose gel, electrophoresed and visualized with Goldenview�dye staining. Meanwhile, untreated liposomes were used as a negative control.

2.4.4. siRNA serum stabilitySerum stability of siRNA in its aqueous solution and liposome formulations was

evaluated using agarose gel electrophoresis. Samples of siRNA either in aqueoussolution or liposomes were mixed with FBS in a 1:1 volume ratio to give 50% serumconcentration and incubated at 37 �C. At different times, aliquots containing 0.25 mgsiRNA of each sample (the liposomes were first destroyed by Triton X-100) wereloaded onto a gel and electrophoresis was performed to visualize intact siRNA.

2.5. Determination of conjugated antibody on the surface of liposomes

The presentation and integrity of anti-EGFR Fab0 conjugated to the liposomeswere confirmed by SDS-PAGE. The gels were run under non-reducing conditions ata constant voltage of 160 V in a Tris/glycine/SDS buffer. The gels were stained withcoomassie brilliant blue to detect protein, destained and dried. The antibodyconjugation efficiency was determined by scanning densitometry.

2.6. Cell viability

The non-specific toxicity of the liposomes against the HCC cells was measuredby Cell Counting Kit-8 (Dojindo laboratories, Kumamoto, Japan) according to themanufacturer’s protocol. Briefly, the cells were seeded into 96-well plates(3 � 104 cells per well) and incubated overnight. Then the cells were incubated withthe liposomes for 24 h (0.2 mg or 0.4 mg siRNA per well, the final siRNA concen-tration was 100 or 200 nM) until the fresh culture medium was changed. 48 h later,the cell proliferation was evaluated by adding 10 ml of the CCK-8 solution to eachwell of the plate. After incubation for 2 h at 37 �C, the absorbance was measured at450 nm/630 nm using BIO-TEK ELx800 Universal Microplate Reader (Bio-Tek,

Fig. 2. Determination of anti-EGFR Fab0 on the surface of the liposomes. (A) Thepresentation and integrity of anti-EGFR Fab0 after conjugated to the liposomes surfacewere detected by SDS-PAGE. Lane 1: protein marker; lane 2: free anti-EGFR Fab0; lanes3e5: TLPD-FC100, TLPD-FC75, TLPD-FC50; lanes 6e8: TLPD-FP100, TLPD-FP75, TLPD-FP50. The upper arrow indicated the conjugated anti-EGFR Fab0 to the liposomes,which had a slightly larger molecular weight than free Fab0 (indicated by the lowerarrow). (B) The antibody conjugation efficiency was determined by scanningdensitometry.

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Winooski, VT). The cell viability was calculated using the formula: [(AE � AB)/(AC � AB)] � 100%. AE, AC and AB were defined as the absorbance of experimentalsamples, untreated samples and blank controls, respectively.

2.7. In vitro cellular uptake

For in vitro cellular uptake analysis, SMMC-7721, LM3 or Hep3B cells wereseeded in 48-well plates at a density of 6 � 104 cells per well overnight. Cells weretreated with the liposomes at a concentration of 250 nM or 500 nM FAM-siRNA for24 h. For transfection efficiency analysis at different time points, SMMC-7721 cellswere chosen and treated with the liposomes at a concentration of 250 nM FAM-siRNA for 1, 2, 4, 8, 12 or 24 h. After then, the cells were trypsinized, washed andthe mean fluorescence intensity was analyzed by flow cytometry (FCM). FCM wasperformed using a FACScan flow cytometer (Becton Dickinson, San Jose, CA).

Quantitative determination of siRNA uptaken by the HCC cells was performedusing a microplate reader. Briefly, SMMC-7721 cells were seeded in 48-well plates ata density of 6� 104 cells per well overnight. Cells were treated with the liposomes ata concentration of 250 nM FAM-siRNA for 1, 2, 4, 8, 12 or 24 h. After then, cells werewashed three times with PBS followed by incubation with 300 ml lysis buffer (5%Triton X-100 in PBS) at RT for 0.5 h. The quantity of FAM-siRNA in the cell lysissolution was quantified using a calibration line obtained with standard lead FAM-siRNA solutions. The fluorescence of FAM-siRNA was determined with the spec-trofluorometer (Synergy� 4, Biotek, Wi, USA) using excitation and emission wave-lengths of 495 and 525 nm, respectively. The siRNA uptake percentage was

Fig. 3. In vitro gene silencing of NTLPD and TLPD of different PEGylation degree. SMMC-7721D) (the final siRNA concentration was 250 nM or 500 nM). After 48 h, luciferase activity ofpercent luminescence intensity compared to the untreated control. Data are expressed as m

calculated from the following formula: QU/QI � 100%. QU and QI were defined as themass of uptaken siRNA and initially added siRNA, respectively.

2.8. Confocal microscopic study

SMMC-7721 cells (2.5 � 105 cells per well) were seeded overnight on coverslipsin a 12-well tissue culture plate and treated with CFPE labeled liposomes entrappingCy5-siRNA for 2 h (1.6 mg siRNA per well, the final siRNA concentrationwas 100 nM).After washing with PBST (PBS containing 0.1% Tween-20), the cells were fixed with4% paraformaldehyde (PFA). Cells were then incubated with 40 ,6-diamidino-2-phenylindole dihydrochloride (DAPI; Sigma, Fluka Chemie, Buchs, Israel) fornuclear staining and mounted under glass coverslips. The immunofluorescence ofthe cells was visualized with Leica TCS SP5 Confocal Microscope.

2.9. In vitro gene silencing

The in vitro gene silencing assay was taken in SMMC-7721, LM3 and Hep3B cellsin 48-well plates. Briefly, HCC cells were seeded at a density of 6 � 104 cells per wellovernight. Cells were treated with liposomes entrapping NC or anti-luciferase siRNA(the final siRNA concentration was 250 nM or 500 nM) for 24 h until fresh culturemedium was changed. 48 h later, the luciferase activity of the cells was measured.Briefly, the cells werewashed and incubatedwith lysis buffer at RT for 20min. Five mlcell lysate was mixed with 25 ml substrate (Luciferase Assay System, Promega Co.,Madison, WI) and the luminescence was measured by GloMax� 96 Microplate

cells were treated with the liposomes entrapping anti-luciferase siRNA (A, B) or NC (C,the cells was analyzed, normalized with the protein concentration and expressed asean � SD (n ¼ 3).

J. Gao et al. / Biomaterials 33 (2012) 270e282274

Luminometer (Promega). The protein concentrations of the samples were deter-mined by using a Micro BCA protein assay kit (Beyotime Biotechnology. Haimen,China). Luciferase activity of a sample was normalized with the protein concentra-tion and expressed as percent luminescence intensity compared to the untreatedcontrol.

For in vitro gene silencing analysis at different time points, SMMC-7721 cellswere chosen and treated with the liposomes entrapping NC or anti-luciferase siRNA(the final siRNA concentration was 250 nM) for 24 h until fresh culture mediumwaschanged. 24, 48, 72, 96 h later, the luciferase activity of the cells was measured asdescribed above.

2.10. Quantitative reverse transcription polymerase chain reaction

Total RNAwas extracted from the cells with Trizol reagent (Invitrogen, Carlsbad,CA) and the first-strand complementary DNA (cDNA) was reversely transcribed fromRNA using the Reverse Transcription System kit (Promega, Madison, WI). Real-timepolymerase chain reaction (real-time PCR) was performed using an Applied Bio-systems 7500 Sequence Detection system.

2.11. Effect of RhoA silencing on cell migration

SMMC-7721 cell migration in response to RhoA silencing was evaluated usingthe modified Boyden chamber (6.5 mm transwells) with polycarbonate membranes(8.0-mm pore size) (Costar Corp., Cambridge, Mass) as described before [18]. Briefly,SMMC-7721 cells (1.25 � 105 cells/well) were seeded overnight in a 24-well tissueculture plate and transfected with the liposomes entrapping 4 mg anti-RhoA or NCsiRNA (the final siRNA concentration was 500 nM) for 24 h until fresh culturemedium was changed. 48 h later, total RNA was extracted from the cells with Trizolreagent and the RhoA silencing was demonstrated by real-time PCR. In addition,1 � 105 SMMC-7721 cells after transfection were trypsinized and seeded into theupper 8.0-mm pore size membrane inserts in the 12-well tissue culture plates.Culture medium containing 10%FCS was placed in the bottomwells. After 48 h, cellsthat did not migrate were removed from the top side of the inserts with cottonswabs. Cells that had migrated to the underside of the inserts were stained with 1%crystal violet solution and counted in five random fields at �200 magnification.

2.12. Animal studies

All animals were purchased from the Shanghai Experimental Animal Center ofChinese Academic of Sciences (Shanghai, China). Mice were placed in a pathogen-

Fig. 4. Gel retardation assay (A) and siRNA EE (B). (A) Gel retardation assay. TLPD-FP75(lanes 1, 2), NTLPD-FP75 (lanes 3, 4), Naked LPD (lanes 5, 6). The latter and formerlanes indicated samples destroyed by Triton X-100 or untreated, respectively.Untreated siRNA was run in lane 7. Each lane contains 0.3 mg siRNA. The siRNA wasvisualized with Goldenview� dye staining. The arrows indicated the free siRNA. (B)The siRNA EE determined by ultra filtration method. The siRNA EE was calculated usingthe formula: (MI � MU)/MI � 100%. MU and MI were defined as the mass of unen-capsulated siRNA and initially added siRNA, respectively. Data are expressed asmean � SD (n ¼ 3).

free environment and allowed to acclimate for a week before being used instudies. All procedures were performed in accordance with guideline of theCommittee on Animals of the Second Military Medical University, Shanghai, China.

2.13. Tissue distribution

Balb/c nude mice (Male, 4e6 weeks, w20 g) were inoculated s.c. on the rightback with 5 � 106 SMMC-7721 cells. Once tumors reached about 300e500 mm3,tumors were excised and cut into small pieces (about 1e2mm3). One piece was thenimplanted into the left liver lobe of each mouse using a trocar. After 2 weeks, thesuccessfully establishment of orthotopic HCC model was demonstrated by tumorluminescent images. Briefly, mice were given an intraperitoneal injection of luciferin(Promega) at a dose of 150 mg/kg. The successfully establishment of orthotopic HCCmodel was demonstrated by IVIS� Lumina II Imaging System (Xenogen), which wastaken to capture the visible light photograph and luminescent image.

For tissue distribution study, mice were randomly assigned to treatment groups:3 mice per group � 3 groups. Liposomes entrapping Cy5-siRNA were injected i.v. asa single dose (1.2 mg siRNA/kg) via tail vein. The mice were anaesthetized byinhalation. At different time points after injection, the in vivo images were observedwith IVIS imaging system (excitation 640 nm) and recorded by a built-in CCDcamera. After 24 h, the mice were killed, and the excised tumors and major organswere also imaged.

For in vivo uptake study, tumors were collected from sacrificed mice, immedi-ately placed in OCT medium, and rapidly frozen in liquid nitrogen. Frozen sections

Fig. 5. In vitro cellular uptake analysis at different time points. SMMC-7721 cells werechosen and treated with TLPD-FP75 and NTLPD-FP75 at a concentration of 250 nMFAM-siRNA for 1, 2, 4, 8, 12 or 24 h. After then, the cells were trypsinized, washed andthe mean fluorescence intensity was analyzed by FCM (A). (B) The quantitativedetermination of siRNA uptaken by the HCC cells was performed using a microplatereader. The siRNA uptake percentage was calculated from the following formula: QU/QI � 100%. QU and QI were defined as the mass of uptaken siRNA and initially addedsiRNA, respectively. Data are expressed as mean � SD (n ¼ 3).

Fig. 6. In vitro cellular uptake analysis in three HCC cell lines. SMMC-7721, LM3 orHep3B cells were treated with the liposomes at a concentration of 250 nM or 500 nM

FAM-siRNA for 24 h. After then, the cells were trypsinized, washed and the meanfluorescence intensity was analyzed by FCM. Data are expressed as mean � SD (n ¼ 3).

J. Gao et al. / Biomaterials 33 (2012) 270e282 275

were cut at 10 mm sections and fixedwith acetone at�20 �C. Afterwashing with PBS,sections were counterstained with DAPI and visualized with Leica TCS SP5 ConfocalMicroscope.

2.14. In vivo gene silencing

After the orthotopic HCC model in mice was successfully established asdescribed above, mice were randomly assigned to treatment groups: 3 mice pergroup � 4 groups. Mice were injected via the tail vein with different formulations(1.2 mg siRNA/kg, one injection per day for 4 days). One day after the final injection,the tumor luminescent images were taken again. Then the mice were euthanizedand the tumors were excised. The tumors were weighed and homogenized in lysisbuffer (1 ml lysis buffer per 100 mg) followed by centrifugation at 12,000 g for10 min. The luciferase activity of the samples was determined as described inSection 2.9.

2.15. Statistical analysis

Direct comparison between two groups was conducted by Student’s unpaired ttest. P value of <0.05 was considered statistically significant.

3. Results

3.1. Liposome size and zeta potential

We investigated the correlation of the conjugation strategytypes and PEGylation degree with the size and zeta potentials ofTLPD. A certain amount (600 mg) of Fab0 was conjugated to LPD byconventional conjugation and post-insertion strategies. As shownin Fig. 1A, the size of TLPD varied markedly. Accompanied withdecreased PEGylation degree, the size of TLPD-FC and TLPD-FPincreased rapidly. Specifically, TLPD-FP50 had a size larger than1000 nm. It has to be noted that the general size of TLPD-FC100,TLPD-FP100 and TLPD-FP75 possessed appropriate size of aboutw200 nm. The liposomes of such a small size may be desired forlong circulation time and high cellular uptake efficiency.

The zeta potential of the liposomes was also investigated. Thezeta potential of naked LPD was 49.4 � 4.1 mV (mean � SD, n ¼ 4).After PEGylation and antibody conjugation, the zeta potential ofliposomes decreased rapidly, to a value of no more than 15 mV(Fig. 1B).

3.2. Determination of conjugated antibody on the surface ofliposomes

The presentation and integrity of anti-EGFR Fab0 on TLPD wereconfirmed by SDS-PAGE (Fig. 2A). Compared with the native form(Lane 2), anti-EGFR Fab0 conjugated to TLPD was intact and showeda slightly larger molecular weight (Lanes 3e8). The resultsdemonstrated that the anti-EGFR Fab0 was conjugated to TLPD andhad a slower swimming motility in the gels. No conjugated anti-body was detected in naked LPD and NTLPD (data not shown). Asdetermined by scanning densitometry (Fig. 2B), the quantity ofconjugated anti-EGFR Fab0 of TLPD-FP was larger than TLPD-FC(38%, 38%, 29%, 81%, 80% and 81% of total anti-EGFR Fab0 wasconjugated to TLPD-FC100, TLPD-FC75, TLPD-FC50, TLPD-FP100,TLPD-FP75 and TLPD-FP50 respectively).

3.3. In vitro gene silencing of liposomes

We examined the luciferase gene silencing activity of TLPD-FC(TLPD-FC100 and TLPD-FC75) and TLPD-FP (TLPD-FP100, TLPD-FP75 and TLPD-FP50) in SMMC-7721 cells. TLPD-FC50 was notincluded in this experiment, owing to its large size (>1000 nm) andinstability. As shown in Fig. 3A, accompanied with reduced PEGy-lation degree, the gene silencing activity of TLPD-FC and TLPD-FPincreased, indicating that PEGylation always reduce gene trans-fection efficiency [5,18]. It was notable to see that, at the same

PEGylation degree, the gene silencing activity of TLPD-FP washigher than that of TLPD-FC. Among all the TLPD, TLPD-FP50showed that best gene silencing activity. In contrast, NTLPD-FCand NTLPD-FP did not show any gene silencing activity (Fig. 3B).Considering PEGylation plays an important role in prolonging theblood circulation time of liposomes and PEGylation degree shouldnot be reduced significantly, TLPD-FP75 was selected and used inthe subsequent experiments.

3.4. Characterization of liposomes

3.4.1. Gel retardation assay and siRNA EEGel retardation assay was a commonly used method to examine

the siRNA binding efficiency of liposomes. As shown in Fig. 4A, nosiRNA migration was observed for naked LPD, NTLPD-FP75 andTLPD-FP75. However, after destructed by Triton X-100, the siRNAwas released from the liposomes, indicating that naked LPD,NTLPD-FP75 and TLPD-FP75 all had powerful binding affinity tosiRNA. Furthermore, the ultra-filtrating method was adopted toprecisely calculate the siRNA EE of liposomes. As shown in Fig. 4B,the siRNA EE of all liposomes was about 90%, suggesting thatPEGylation and antibody conjugation have little adverse impact onsiRNA EE for NTLPD-FP75 and TLPD-FP75.

3.4.2. siRNA serum stabilitysiRNA must be stable to digestion by nuclease in serum for

transfection activity in cells [21]. To address the question ofliposomes-siRNA formulations stability and protection from serumdegradation, they were incubated with an equal volume of serumand incubated at 37 �C. As shown in Supplementary Fig. 2, puresiRNA started to degrade after 3 h and was fully degraded after 6 h.In contrast, siRNA in naked LPD, NTLPD-FP75 and TLPD-FP75 star-ted to degrade after 48 h and did not fully degrade even after 96 h.We proved here that encapsulation of siRNA within LPD formula-tion can protect siRNA from serum degradation to a great extent.

3.5. Cell viability

For the concerns of efficient gene delivery, the liposomes shouldpossess good biocompatibility and low cytotoxicity. The viability ofHCC cells was evaluated in the presence of NTLPD-FP75 and TLPD-FP75 (Supplementary Fig. 3). Compared with untreated cells, no

J. Gao et al. / Biomaterials 33 (2012) 270e282276

significant decrease in cellular viability was observed, indicatingthat NTLPD-FP75 and TLPD-FP75 were not toxic at the concentra-tions examined. In contrast, naked LPD showed significant non-specific toxicity against the HCC cells. These results were accor-dance with previous findings showing that PEGylation couldsignificantly reduce non-specific toxicity of liposomes [5]. Thesedata provided preliminary evidence for the safety of our immu-noliposomes for siRNA delivery.

3.6. In vitro cellular uptake

To investigate the siRNA delivery efficiency of the liposomes, thein vitro cellular uptake study was performed in SMMC-7721 cells.

Fig. 7. Confocal microscopic study. SMMC-7721 cells were seeded overnight on coverslips insiRNA for 2 h. After washing with PBST, the cells were fixed with 4% PFA. Cells were theimmunofluorescence of the cells was visualized with Leica TCS SP5 Confocal Microscope. Roand e: Bar represents 100 mm; Rows b, d and f: Bar represents 75 mm. (For interpretation of tthis article.)

As demonstrated by FCM analysis, TLPD-FP75 had significantlyincreased transfection efficiency compared with NTLPD-FP75 atevery time point (Fig. 5A). Notably, the transfection efficiency ofTLPD-FP75 gradually increased, and reaching plateau at 12 h timepoint, while the transfection efficiency of NTLPD-FP75 alwaysincreased during 24 h. Considering FCM analysis can only reflectthe relative value of transfection efficiency, we further employeda microplate reader to determine the actual quantity of FAM-siRNAuptaken. Consistent with the FCM analysis results, TLPD-FP75showed significantly higher siRNA uptake percentage comparedwith NTLPD-FP75 at every time point (Fig. 5B). At 12 h time point,TLPD-FP75 showed a very high siRNA uptake percentage (51.86%),while NTLPD-FP75 only had a very low siRNA uptake percentage

a 12-well tissue culture plate and treated with CFPE labeled liposomes entrapping Cy5-n incubated with DAPI for nuclear staining and mounted under glass coverslips. Thews a and b: untreated; Rows c and d: NTLPD-FP75; Rows e and f: TLPD-FP75; Rows a, che references to colour in this figure legend, the reader is referred to the web version of

Fig. 8. In vitro gene silencing. The gene silencing assay was taken in SMMC-7721 cells(A) or LM3 cells (B) or Hep3B cells (C). After 48 h, the luciferase activity of the cells wasanalyzed, normalized with the protein concentration and expressed as percent lumi-nescence intensity compared to the untreated control. Data are expressed asmean � SD (n ¼ 3).

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(19.16%). Taken together, these data suggested that the in vitrocellular uptake of TLPD-FP75 was significantly higher than that ofNTLPD-FP75.

We chose three HCC cell lines with different EGFR expressionlevels to evaluate the transfection efficiency of TLPD-FP75 andNTLPD-FP75. Fig. 6 shows that TLPD-FP75 had significantlyincreased transfection efficiency in SMMC-7721 cells comparedwith NTLPD-FP75 (1.5 or 3.1 fold higher in 250 nM or 500 nM siRNA).In LM3 cells, the increased transfection efficiency was moderate(1.3 or 1.7 fold higher in 250 nM or 500 nM siRNA). However, theincreased transfection efficiency was the lowest in Hep3B cells (1.17or 1.75 fold higher in 250 nM or 500 nM siRNA). These results firmlydemonstrated that the binding of TLPD-FP75 to EGFR-over-expressing HCC cells occurs through liposome-conjugated anti-EGFR Fab0, and not through non-specific electrostatic interactionsbetween liposomes and cell membranes. Furthermore, anti-EGFRFab0 could significantly enhance the binding affinity of TLPD-FP75in EGFR-overexpressing HCC cells.

3.7. Confocal microscopic study

To confirm the internalization of the liposomes in HCC cells,SMMC-7721 cells were incubated with CFPE labeled liposomesentrapping Cy5-siRNA for 2 h. Fig. 7 shows that SMMC-7721 cellstreated with TLPD-FP75 showed internalization of both the Cy5-siRNA (red fluorescence) and CFPE (green fluorescence). Incontrast, SMMC-7721 cells treated with NTLPD-FP75 showed nosignificant uptake or internalization as shown by the faint greenand red fluorescence. On superimposing the images, the green andred fluorescence co-localized within the cells, suggesting that thesiRNA was co-localized with the fluorescent lipid. These resultsconfirmed that TLPD-FP75 specifically binds to EGFR and wasinternalize into the cancer cells via receptor-mediated endocytosis.

3.8. In vitro gene silencing

TLPD-FP75 and NTLPD-FP75 were investigated for their lucif-erase gene silencing activity in the three HCC cell lines. As shown inFig. 8A, TLPD-FP75 showed significantly higher gene silencingactivity than NTLPD-FP75 at 250 nM and 500 nM anti-luciferasesiRNA concentration in SMMC-7721 cells (P < 0.01; P < 0.01). It isnoteworthy that, in other two HCC cell lines LM3 and Hep3B,treatment with TLPD-FP75 also significantly decreased the lucif-erase expression but NTLPD-FP75 not (Fig. 8B and C). These datasuggested TLPD-FP75 possess significantly enhanced gene silencingactivity compared to NTLPD-FP75, not only in EGFR-overexpressingHCC cells but also in HCC cells moderately or lowly expressingEGFR.

Furthermore, we measured persistence of gene silencinginduced by TLPD-FP75 in vitro. Previous reports showed that thesilencing effects of siRNA can last an average of approximately 66 h[22,23]. The results showed that TLPD-FP75 could inhibit luciferaseexpression for 48 h, while NTLPD-FP75 showed no gene silencingactivity (Supplementary Fig. 4).

3.9. Cell migration after RhoA silencing

The low-molecular weight GTPases of the Ras superfamily,RhoA, is a potential target of anti-cancer therapy and inhibitors thatdirectly affect RhoA activity or expression prevent cancerprogression and metastasis [24]. In this study, we used RhoA asa cancer therapeutic target to demonstrate the potential of TLPD-FP75 in HCC gene therapy. Compared with the untreated cells,SMMC-7721 cells transfected with TLPD-FP75 entrapping anti-RhoA siRNA showed significantly down-regulated RhoA mRNA

expression by 50% (Fig. 9C) and statistically reduced cell migrationcompared with NTLPD-FP75 entrapping anti-RhoA siRNA(P < 0.001, Fig. 9A and B). In contrast, NTLPD-FP75 entrapping anti-RhoA siRNA, TLPD-FP75 or NTLPD-FP75 entrapping NC siRNA had

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no RhoA gene silencing activity (Fig. 9C). These results demon-strated that TLPD-FP75 could specifically silence RhoA expressionand inhibit cell invasion in EGFR-overexpressing SMMC-7721 cells,whereas NTLPD-FP75 had no such effect.

3.10. Tissue distribution

Cy5 fluorophore is characterized by its superior stability andlong-wavelength excitation and emission, so it is very suitable forin vivo imaging [15]. We used IVIS imaging system to visualize thein vivo distribution of the liposomes entrapping Cy5-siRNA inorthotopic HCC model at different time point. Although strongbackground fluorescence existed in untreated mice, TLPD-FP75 andNTLPD-FP75 showed obviously enhanced fluorescence in liver thannegative controls at every time point (Fig. 10A). However, we couldnot discriminatewhether the fluorescence comes from liver or HCC.To clearly observe the fluorescence signals, the tumors and majororgans were excised and collected at 24 h post injection. As shownin Fig. 10B, two tumors from the mice treated with TLPD-FP75showed much stronger signal compared to tumors from othergroups, suggesting that TLPD-FP75 possess enhanced ability toaccumulate in HCC compared with NTLPD-FP75.

Furthermore, we employed confocal microscopy to investigatethe binding and internalizing of TLPD-FP75 and NTLPD-FP75

Fig. 9. Effect of RhoA silencing on cell migration. SMMC-7721 cells were seeded overnight inor NC siRNA (the final siRNA concentration was 500 nM). (A) 48 h after the transfection, SMMBoyden chamber with polycarbonate membranes. Representative images (200�) of migracalculated from 5 random fields. (C) 48 h after the transfection, total RNA was extracted fromas mean � SD (n ¼ 3).

in vivo, in tumor sections from above isolated tumors. As shownin Fig. 10D, TLPD-FP75 accumulated profusely throughout thetumors tissues (including intercellular substance and cytoplasm) ina pattern consistent with receptor-mediated endocytosis, reflectingby a strong and extensive cytosolic delivery of Cy5-siRNA. Incontrast, NTLPD-FP75 showed only minimal binding or uptake,which did not seem to be significantly different than untreatedcontrols, suggesting that it is less efficient in intracellular delivery.

3.11. In vivo gene silencing

The in vivo activity of TLPD-FP75 and NTLPD-FP75was evaluatedby the luciferase gene silencing in SMMC-7721 tumor xenograft.The tumor luminescent images were taken before and after lipo-some treatment. Fig. 11A shows that, in TLPD-FP75 entrapping anti-luciferase siRNA treated group, the tumor luminescence becamemuch weaker after treatment, whereas in other groups, the tumorluminescence did not change or even became stronger after lipo-some treatment. To evaluate the luciferase activity precisely, thetumors were excised, homogenized and the luciferase activity wasdetermined after four consecutive injections. Fig. 11B shows thein vivo gene silencing activity of siRNA in different formulations.The luciferase activity was compared with the untreated control. Itis noteworthy that TLPD-FP75 entrapping anti-luciferase siRNA

a 12-well tissue culture plate and transfected with the liposomes entrapping anti-RhoAC-7721 cells migration in response to RhoA silencing was assessed using the modifiedtion assays of SMMC-7721 cells were shown. (B) The number of cells per field wasthe cells and RhoA expression was demonstrated by real-time PCR. Data are expressed

Fig. 10. Tissue distribution in vivo. Balb/c nude mice bearing orthotopic HCC of SMMC-7721 cells were injected via tail vein as a single dose with the liposomes entrapping Cy5-siRNA (1.2 mg siRNA/kg). (A) At different time points (6 h, 12 h and 24 h) after injection, the in vivo images were taken with IVIS imaging system. (B) Mice were killed 24 h afterinjection. Tumors and various organs (heart, liver, spleen, lung and kidney) were collected and imaged with IVIS imaging system. (C) Gross morphology of orthotopic HCC and liver.The orthotopic HCC was indicated using the black arrows. (D) In vivo uptake study. Frozen sections from tumors were cut at 10 mm sections and fixed with acetone. After then,sections were counterstained with DAPI and visualized with Leica TCS SP5 Confocal Microscope. Bar represents 20 mm.

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Fig. 11. In vivo gene silencing. After the orthotopic HCC model in mice was successfully established, mice were randomly assigned to treatment groups: 3 mice per group � 4 groups.Mice were injected via the tail vein with different formulations (1.2 mg siRNA/kg, one injection per day for 4 days). (A) The tumor luminescent images taken before (upper images)and after injections (below images). After the tumors were excised and homogenized, the luciferase activity of the tissue lysate was analyzed, normalized with the proteinconcentration and expressed as percent luminescence intensity compared to the untreated control (B). (C) Gross morphology of orthotopic HCC and liver. The orthotopic HCC wasindicated using the black arrows.

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showed a significant enhanced in vivo gene silencing activitycompared with NTLPD-FP75 entrapping anti-luciferase siRNA(P < 0.05). Taken together, these data suggested that TLPD-FP75have a significant enhanced gene silencing activity in vivocompared with NTLPD-FP75.

4. Discussion

HCC is one of the most frequent fatal malignancies worldwide. Itis hoped that gene therapy may provide not only effective treat-ment but also a cure for HCC [25]. Gene therapy using non-viralvectors delivering siRNA represents a preferred approach becausethey have many advantages such as safety, lower toxicity, highbiocompatibility and convenient surface modification. We reportedpreviously the development of TLPD-FCC, which possessed supe-rior gene silencing activity in EGFR-overexpressing breast cancersboth in vitro and in vivo [15]. However, in EGFR-overexpressingHCC, TLPD-FCC did not achieve satisfactory gene silencing activity(Fig. 3A), suggesting that the release of siRNA in TLPD-FCC is vari-able depending on the cells and need improvement.

In this study, we continued our previous work of TLPD-FCC todevelop a superior non-viral vector for siRNA delivery to HCC. Wesupposed that high PEGylation and low antibody conjugation

efficiency give rise to poor gene silencing activity of TLPD-FCC inHCC. The emphasis was given to optimization of PEGylation degreeand antibody conjugation in favor of gene silencing activity in HCC.Therefore, a series of TLPD with different PEGylation degree wereprepared and examined for their gene silencing activity. The resultsshowed that the gene silencing activity of TLPD-FP was higher thanthat of TLPD-FC at the same PEGylation degree (Fig. 3A). Pivotalstudies performed by Kirpotin et al. have demonstrated that cellbinding and internalization of anti-HER2 immunoliposomesincreased at higher surface density of conjugated Fab0 [26]. In thecurrent study, SDS-PAGE analysis confirmed that the quantity ofconjugated anti-EGFR Fab0 of TLPD-FP was larger than TLPD-FC(Fig. 2). Hence, the superior gene silencing activity of TLPD-FPmight be partially due to its enhanced antibody conjugation effi-ciency. The reason why the antibody conjugation efficiency ofTLPD-FP is higher than TLPD-FC is still not very clear. In addition toantibody conjugation, PEGylation is another important factor thatcan affect the transfection efficiency of liposomes [18,27]. Gener-ally, PEGylation inhibits the endosomal escape of siRNA and dras-tically reduced transfection efficiency of liposomes [5]. The genesilencing activity of TLPD-FC and TLPD-FP increased, accompaniedwith reduced PEGylation degree of liposomes (Fig. 3A). On theother hand, PEGylation can significantly reduce opsonization and

J. Gao et al. / Biomaterials 33 (2012) 270e282 281

prolong the circulation lifetime of liposomes. Nevertheless, PEGchains should be arranged in the brush mode with >8 mol%PEGylation [28]. This brush configuration ensures that the entiresurface of nanoparticles is covered and provides the nanoparticleswith full protein from opsonization [29]. Among a series of TLPDwith different PEGylation degree, TLPD-FP75 not only showedprominent gene silencing activity, but also retained a high PEGy-lation degree which ensures the long circulation time of liposomes.As a result, TLPD-FP75 was chosen in the subsequent experiments.

We examined the siRNA EE and serum stability of TLPD-FP75.Fig. 4 showed that TLPD-FP75 and NTLPD-FP75 possessed highsiRNA EE of approximately 90% as naked LPD, suggesting that thepost-PEGylation method, as well as the antibody conjugation, havelittle adverse impact on siRNA EE for NTLPD-FP75 and TLPD-FC.Using serum stability assay, we further characterized the in vitrostability of TLPD-FP75. As shown in Supplementary Fig. 2, TLPD-FP75, NTLPD-FP75, as well as naked LPD, could protect siRNAfrom serum degradation to a great extent, suggesting that the post-PEGylation method provide a favorable protection to siRNAdegradation in serum.

To confirm that TLPD-FP75 possesses enhanced specific bindingaffinity in EGFR-overexpressing HCC cells, we performed in vitrotransfection efficiency assay in three HCC cell lines with differentEGFR expression levels. As shown in Fig. 6, in the three HCC celllines, TLPD-FP75 had significantly increased transfection efficiencycompared with NTLPD-FP75, suggesting that anti-EGFR Fab0

conjugation can significantly enhance the binding affinity of TLPD-FP75. This was demonstrated again by a confocal microscopic study(Fig. 7), which showed that TLPD-FP75 specifically bound to andwas internalized in SMMC-7721 cells. High transfection efficiencydoes not always mean superior gene silencing activity, as genesilencing takes effect only when endosomal escape of siRNA takesplace. Fig. 8 showed that TLPD-FP75 possessed significantlyenhanced gene silencing activity compared with NTLPD-FP75,indicating efficient endosomal escape of siRNA occur for TLPD-FP75. Notably, TLPD-FP75 achieved superior gene silencingactivity not only in EGFR-overexpressing HCC cells, but also in HCCcells moderately or lowly expressing EGFR. This result is fairlyimportant as TLPD-FP75 has a wide application prospect in tar-geting HCC expressing EGFR.

In vivo distribution assay showed that TLPD-FP75 possessedenhanced ability to accumulate in EGFR-overexpressing HCCcompared with NTLPD-FP75 (Fig. 10). Furthermore, in vivo uptakestudy showed that TLPD-FP75 accumulated profusely throughoutthe tumors tissues in a pattern consistent with receptor-mediatedendocytosis. However, NTLPD-FP75 showed only minimal bindingor uptake. More importantly, TLPD-FP75 showed significantlyhigher gene silencing activity in vivo than other formulations(Fig. 11). We speculated that the mechanism of enhanced in vivogene silencing activity was mainly due to the significantlyimproved tumor accumulation and uptake of TLPD-FP75. First, longcirculating TLPD-FP75 and NTLPD-FP75 accumulated in tumortissue owing to EPR effect. Then TLPD-FP75 bound to and inter-nalized in tumor cells via ligandereceptor interactions, whileNTLPD-FP75 remained in the extracellular space and underwentnon-specific endocytosis. It should be noted that our preparedTLPD-FP75 possessed a little larger size (w202 nm) and reducedPEGylation degree than our previously prepared TLPD-FCC(w165 nm). Numerous reports showed that the nanoparticles of100e200 nm in size may have a longer circulation time than largenanoparticles (>200 nm) [30e33]. It is interesting to note that ourprepared TLPD-FP75 still showed specific accumulation andexcellent gene silencing activity in EGFR-overexpressing HCC. Wespeculated that the liposomes have a tendency to accumulate inliver, which is called by passive targeting, resulting in abundant

accumulation of liposomes in liver. Therefore, in our orthotopicHCCmodel, it is easy for TLPD-FP75 to reach HCC, although it mightpossess a shorter circulation time due to its a little larger size andreduced PEGylation degree. In conclusion, our prepared TLPD-FP75,demonstrated a potent gene silencing activity in HCC and poten-tially increased the feasibility of RNA interfering therapy of HCC.

5. Conclusion

In this study, we investigated extensively the effect of PEGyla-tion and antibody conjugation on the essential physicochemicalproperties and targeting efficiency of TLPD. As a result, we obtainedTLPD-FP75 which possessed high siRNA EE and superior serumstability. Compared with NTLPD-FP75, TLPD-FP75 showed a signif-icantly enhanced EGFR targeting efficiency and achieved a superiorgene silencing activity both in vitro and in vivo.

Acknowledgments

This work was financially supported by National Natural ScienceFoundation of China, Shanghai Commission of Science & Tech-nology, Ministry of Science and Technology of China (973 & 863program projects), Pudong Commission of Science and Technologyof Shanghai and a special grant fromMinistry of Education of China(Key Laboratory) and Shanghai Commission of Education andNational Special Projects for New Drug Development and InfectiousDiseases. We thank Ms. Yang Yang, Ms. Jing Xu for their technicalassistance.

Appendix. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.biomaterials.2011.09.035.

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