Biomaterials 35 (2014) 4589e4600

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Oral delivery of shRNA and siRNA via multifunctional polymeric nanoparticles for synergistic cancer therapy Lu Han, Cui Tang, Chunhua Yin* State Key Laboratory of Genetic Engineering, Department of Pharmaceutical Sciences, School of Life Sciences, Fudan University, Shanghai 200433, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 3 February 2014 Accepted 15 February 2014 Available online 6 March 2014

Galactose modified trimethyl chitosan-cysteine (GTC) conjugates with various galactose grafting densities were developed for oral delivery of Survivin shRNA-expression pDNA (iSur-pDNA) and vascular endothelial growth factor (VEGF) siRNA (siVEGF) in the synergistic and targeted treatment of hepatoma. iSur-pDNA and siVEGF loaded GTC nanoparticles (NPs) were prepared via electrostatic complexation and showed desirable stability in physiological fluids and improved intestinal permeation compared to naked genes. Galactose grafting density of GTC NPs significantly affected their in vitro and in vivo antitumor activities. GTC NPs with moderate galactose grafting density, termed GTC2 NPs, were superior in facilitating cellular uptake, promoting nuclear distribution, and silencing target genes, leading to notable inhibition of cell growth. In tumor-bearing mice, orally delivered GTC2 NPs could effectively accumulate in the tumor tissues and silence the expression of Survivin and VEGF, evoking increased apoptosis, inhibited angiogenesis, and thus the most efficient tumor regression. Moreover, compared with single gene delivery, co-delivery of iSur-pDNA and siVEGF showed synergistic effects on inhibiting in vitro cell proliferation and in vivo tumor growth. This study could serve as an effective approach for synergistic cancer therapy via oral gene delivery, and highlighted the importance of ligand grafting density in the rational design of targeted nanocarriers. Ó 2014 Elsevier Ltd. All rights reserved.

Keywords: Polymeric nanoparticles Survivin shRNA VEGF siRNA Oral delivery Cancer therapy

1. Introduction Gene silencing by short hairpin RNA (shRNA) or small interfering RNA (siRNA), denoted RNA interference (RNAi), has emerged as an attractive modality in cancer therapy [1]. RNAi strategy aiming at single-gene silencing is extensively applied, nevertheless hardly obtained satisfactory therapeutic efficacy due to the multiple gene mutations in the onset and progression of cancer [2]. To address this problem, cancer therapies involving multiple-gene silencing have been attempted in recent years, which mainly focus on the combined use of different shRNA or different siRNA [3,4]. It is well known that shRNA and siRNA down-regulate gene expression via different mechanisms [5]. shRNA, which is endogenously and stably generated after nucleic delivery of shRNAincorporating plasmid DNA (pDNA), offers long-lasting silencing of target genes. However, the passage of pDNA through nuclear envelope is restricted, which leads to the delayed RNAi effect [5]. Comparatively, siRNA directly interacts with messenger RNA

* Corresponding author. Tel.: þ86 21 6564 3797; fax: þ86 21 5552 2771. E-mail address: [email protected] (C. Yin). http://dx.doi.org/10.1016/j.biomaterials.2014.02.027 0142-9612/Ó 2014 Elsevier Ltd. All rights reserved.

(mRNA) in the cytosol and initiates prompt RNAi effect subsequently, while its long-term efficacy is strongly compromised by the dilution following cell proliferation and enzymatic degradation in the cytosol [5]. Consequently, simultaneous application of shRNA and siRNA for cancer therapy is anticipated to synergistically integrate the advantages of each mechanism for timely and stable gene knockdown to enhance the antitumor efficacy, wherein siRNA and shRNA respectively trigger early and late gene silencing. Unfortunately, to the best of our knowledge, few efforts have been made in this area so far. In despite of the great therapeutic potential of shRNA and siRNA, naked genes are known to be poorly internalized by cancer cells owing to their negative charges and hydrophilic nature [6,7]. Worse still, the rapid degradation by nucleases seriously limits their in vivo application, especially for oral delivery [8,9]. The harsh biological milieu of enzyme-rich gastrointestinal (GI) tract and poor permeation across intestinal epithelium make efficient oral delivery of genes a much more difficult task than the other administration routes [10]. However, due to its advantages in convenience, patient compliance, and cost-effectiveness, oral route is always considered the holy grail for gene delivery and attracts extensive attention [10]. Several oral delivery systems for genes based on nanoparticles

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Abbreviations ANOVA CLSM DMEM EDAC FBS FITC GAPDH GI GT GTC HRP LA mRNA MTT NHS

analysis of variance confocal laser scanning microscopy Dulbecco’s modified Eagle’s medium 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride fetal bovine serum fluorescein isothiocyanate glyceraldehyde-3-phosphate dehydrogenase gastrointestinal galactose modified trimethyl chitosan galactose modified trimethyl chitosan-cysteine horseradish peroxidase lactobionic acid messenger RNA methyl tetrazolium N-hydroxysuccinimide

(NPs) have demonstrated impressive anti-inflammation efficacies through targeting the macrophages [11,12]. However, few antitumor studies involving oral gene delivery are available so far. Our recent work demonstrated galactose modified trimethyl chitosan-cysteine (GTC) conjugate as a potent siRNA delivery vector with notable therapeutic efficacy following intratumoral injections [13]. In the present study, considering the benefits of trimethyl and cysteine groups in promoting mucoadhesion and opening tight junctions [14], GTC conjugate was exploited for a more challenging and meaningful application, i.e. co-delivery of antitumor shRNA and siRNA via oral administration. Moreover, GTC conjugates with various galactosylation degrees were developed to delineate the effects of ligand grafting density on the entire co-delivery behaviors to optimize the antitumor efficacy. Survivin shRNA-expression pDNA (iSur-pDNA) and vascular endothelial growth factor (VEGF) siRNA (siVEGF) were chosen as the therapeutic genes with the functions of apoptosis induction and angiogenesis inhibition, respectively [15,16]. iSur-pDNA and siVEGF were encapsulated into GTC NPs via electrostatic complexation. Their stability in physiological fluids and permeation across intestinal epithelia were investigated. In vitro cellular uptake, intracellular distribution, gene silence, and cell growth inhibition were determined in human hepatoma BEL-7402 cells. In vivo distribution and antitumor efficacy of GTC NPs were evaluated in tumor-bearing nude mice following oral administration. In vitro and in vivo safety assessments of GTC NPs were also conducted.

NPs nanoparticles Papp apparent permeability coefficient pDNA plasmid DNA FITC-pDNA FITC labeled pDNA iSur-pDNA Survivin shRNA-expression pDNA iVEGF-pDNA VEGF shRNA-expression pDNA RLU relative light unit RNAi RNA interference SEM scanning electron microscopy shRNA short hairpin RNA siRNA small interfering RNA Scr scrambled control siRNA TAMRA-siRNA TAMRA labeled siRNA siSurvivin Survivin siRNA TEER transepithelial electrical resistance TIR tumor inhibition ratio VEGF vascular endothelial growth factor siVEGF VEGF siRNA.

lymphoma (Raji B) cells were obtained from the American Type Culture Collection (Rockville, MD, USA). All cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS). Hepatoma H-22 ascites were obtained from Shanghai Institute of Materia Medica (Shanghai, China). Female Balb/c nude mice (18e22 g) and female Kunming mice (18e22 g) were obtained from Slaccas Experimental Animal Co., Ltd. (Shanghai, China) and kept under standard housing conditions. Animal experiments were performed according to the Guiding Principles for the Care and Use of Experiment Animals in Fudan University. The study protocol was reviewed and approved by the Institutional Animal Care and Use Committee, Fudan University. 2.2. Preparation and characterization of GTC conjugate GTC conjugate was synthesized through sequential conjugation of chitosan with CH3I, LA, and cysteine as described in Supplementary Information [13]. GTC conjugates and galactose modified trimethyl chitosan (GT) prepared at the trimethyl chitosan/LA ratios of 2:1, 1:1, and 1:2 (w/w) were denoted as GTC1 (GT1), GTC2 (GT2), and GTC3 (GT3), respectively. 1H NMR spectra were measured for the calculation of trimethyl and galactose densities [17,18], and the amounts of immobilized sulphydryl were determined using Ellman’s reagent [19]. Trimethyl chitosancysteine (TC) conjugate was synthesized accordingly as a comparison. 2.3. Preparation and characterization of GTC NPs GTC conjugates, iSur-pDNA, and siVEGF were dissolved in DEPC-treated water at 2.0, 0.2, and 0.2 mg/mL, respectively. iSur-pDNA was directly mixed with siVEGF at 5:1 (w/w) and the mixture was added into GTC solution at the GTC/iSur-pDNA ratio of 10:1 (w/w). The resultant GTC NPs were incubated for 30 min at 37  C before use. Particle size and zeta potential of GTC NPs were determined with Zetasizer Nano (Malvern, UK) without further dilution. Morphology was observed with scanning electron microscopy (SEM, Vega TS5136, Tescan, Czech). 2.4. Stability of NPs

2. Materials and methods 2.1. Materials, cell culture, and animals Chitosan (deacetylation degree of 85% and molecular weight (Mw) of 200 kDa) was purchased from Golden-Shell Biochemical Co., Ltd. (Zhejiang, China). Lactobionic acid (LA), L-cysteine hydrochloride, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC), N-hydroxysuccinimide (NHS), fluorescein isothiocyanate (FITC), and Hoechst 33258 were from Sigma (St. Louis, MO, USA). Lipofectamine 2000 and LipoRNAiMAX were obtained from Invitrogen (Carlsbad, USA). pGL3-control vector without shRNA cassette, iSur-pDNA (target sequence of GAATTAACCCTTGGTGAAT), and iVEGF-pDNA (target sequence of GGCCAGCACAUAGGAGAGA) were amplified in Escherichia coli and purified using TIANGEN Plasmid Maxprep Kit (Beijing, China). siVEGF, its scrambled control siRNA (Scr), TAMRA labeled siRNA (TAMRA-siRNA), and Survivin siRNA (siSur) were supplied by GenePharma Co., Ltd. (Shanghai, China). The primers for Real-Time PCR were purchased from Sangon Biotech Co., Ltd. (Shanghai, China). The sequences of siRNA and primers were listed in Supplementary Information Table S1. All other reagents were of analytic grade. BEL-7402 cells were provided by Chinese Academy of Sciences (Shanghai, China). Human colon adenocarcinoma (Caco-2) cells and human Burkitt’s

2.4.1. Structural stability against dilution, ionic strength, pH alteration, and serum treatment NPs suspensions were diluted with phosphate buffered solution (PBS, 0.2 M, pH 7.4) by 250 folds to simulate the massive dilution and ionic challenge in physiological conditions. To mimic pH alteration in the GI tract, the pH of NPs suspension was modulated to 1.2 using 1 M HCl solution and then back to 7.4 using 1 M NaOH solution. To evaluate the stability against serum, NPs were treated with equal volume of DMEM containing 10% FBS and incubated for 30 min at 37  C. Structural stability of NPs was monitored in terms of particle size and zeta potential. 2.4.2. Stability against enzymatic degradation The serum, gastric fluids, intestinal fluids, and intestinal homogenate fluids of mice were prepared as described previously [20]. NPs containing 1 mg of iSur-pDNA and 200 ng of siVEGF were incubated with equal volume of serum, gastric fluids, intestinal fluids, intestinal homogenate fluids, DNase I solution (20 mg/mL), and RNase A solution (40 mg/mL) at 37  C for 2 h. The samples were then heated at 80  C for 5 min to terminate enzymatic activity and heparin (5 mg/mL) was added to dissociate iSur-pDNA and siVEGF from NPs. The integrity of iSur-pDNA and siVEGF was examined on 1% (w/v) agarose gel at 100 V for 1 h and 4% (w/v) agarose gel electrophoresis at 56 V for 1 h, respectively.

L. Han et al. / Biomaterials 35 (2014) 4589e4600 2.5. Permeation across in vitro Caco-2 monolayers iSur-pDNA was covalently labeled with FITC as previously reported [21]. FITC labeled pDNA (FITC-pDNA) and TAMRA-siRNA were allowed to form NPs as described in Section 2.3. Caco-2 mono-cultured and co-cultured monolayers were established as normal intestinal epithelia model and M cell model, respectively [22]. Briefly, Caco-2 cells were seeded at 5  104 cells/well on MillicellÒ (pore size of 0.4 mm, surface area of 0.6 cm2, Merck Millipore, Germany) and further cultured for 21 days to form mono-cultured monolayers. As for co-cultured monolayers, Raji B cells were seeded on the basolateral side at 5  104 cells/well after Caco-2 cells were cultured on MillicellÒ for 16 days, and co-cultured for another 7 days. Mono-cultured and co-cultured monolayers were allowed to equilibrate with 500 mL of transport buffer (0.5% BSA in Hank’s balanced salt solution) at 37  C for 30 min. NPs were subsequently added to the apical side at 2 mg pDNA and 400 ng siRNA/well. At predetermined time interval, transepithelial electrical resistance (TEER) was measured and an aliquot of 50 mL was withdrawn from the basolateral side to quantify transported FITC-pDNA and TAMRA-siRNA by fluorimetry (FITC: lex ¼ 488 nm, lem ¼ 519 nm; TAMRA: lex ¼ 560 nm, lem ¼ 585 nm). An equal volume of fresh Hank’s balanced salt solution was added to the basolateral side to keep a constant volume. The apparent permeability coefficient (Papp) for FITC-pDNA and TAMRA-siRNA was calculated according to the following equation:  Papp ¼

dQ dT



AC

where dQ/dt represents the permeation rate of FITC-pDNA or TAMRA-siRNA (mg/s), C is the initial concentration of FITC-pDNA or TAMRA-siRNA in the apical medium (mg/ mL), and A is the area of monolayers (cm2).

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relative VEGF level of cells without NPs treatment. As for iSur-pDNA, cells were collected via trypsinization and washed with 0.2 M PBS (pH 7.4). The relative light unit (RLU) was measured with fluorometer (Promega, USA) using Luciferase Reporter Gene Assay Kit (Beyotime Institute of Biotechnology, China). The total protein was determined with BCA protein assay kit (Beyotime Institute of Biotechnology, China). Transfection efficiency was presented as RLU per milligram of cellular protein. Lipofectamine 2000 and LipoRNAiMAX were used as positive controls for iSurpDNA and siVEGF, respectively. For the determination of Survivin expression, BEL7402 cells were seeded on 6-well plate at 2  105 cells/well and transfected with GTC NPs containing 4 mg of iSur-pDNA and 800 ng of siVEGF for 48 h as described above. The subsequent western blotting measurements were carried out as described in Supplementary Information.

2.10. In vitro cell growth inhibition BEL-7402 cells were seeded on 96-well plates at 1  104 cells/well and incubated at 37  C for 24 h. Cells were transfected with TC NPs, GTC1 NPs, GTC2 NPs, GTC3 NPs, GTC2/iSur-pDNA/Scr NPs (GTC2-D NPs), and GTC2/pGL3/siVEGF NPs (GTC2-R NPs) containing 5 mg of pDNA and 1 mg of siRNA for 24 h, 48 h, and 72 h, followed by MTT assay. Untreated cells served as 100% cell viability. Cells were also treated with GTC2/ iSur-pDNA þ iVEGF-pDNA/Scr NPs (GTC2-D-D NPs, 5 mg of iSur-pDNA and 1 mg of iVEGF-pDNA) and GTC2/pGL-3/siSur þ siVEGF NPs (GTC2-R-R NPs, 5 mg of siSur and 1 mg of siVEGF) to compare the cell growth inhibition of different combinational strategies. The non-specific cytotoxicity of NPs containing 5 mg of pGL3 and 1 mg of Scr was excluded using MTT assay.

2.11. In vivo distribution 2.6. Cellular uptake BEL-7402 cells were seeded on 24-well plates at 1  105 cells/well and incubated for 24 h. Following treatment with NPs containing 2 mg of FITC-pDNA and 400 ng of TAMRA-siRNA for 0.5, 1, 2, 4, and 6 h, cells were washed with 0.2 M PBS (pH 7.4) and lysed with 0.5% (w/v) sodium dodecyl sulfate (SDS, pH 8.0). The cell lysate was quantified for the contents of FITC-pDNA and TAMRA-siRNA by fluorimetry and total protein content by the Lowry method. Results were expressed as the amount of FITC-pDNA or TAMRA-siRNA (mg) per milligram of cellular protein. To elucidate the detailed uptake mechanism, BEL-7402 cells were incubated with sodium azide (10 mM), methyl-b-cyclodextrin (5 mM), chlorpromazine (10 mg/ mL), genistein (200 mg/mL), or wortmannin (50 nM) for 30 min prior to the addition of NPs and throughout the 4-h uptake period. Results were expressed as the relative uptake percentage compared to cells without the inhibitor pre-treatment. BEL-7402 cells were also incubated with these inhibitors at the adopted concentrations for 6 h to evaluate their cytotoxicity with MTT assay. 2.7. Glutathione-responsive release NPs containing 2 mg of FITC-pDNA and 400 ng of TAMRA-siRNA were incubated at 37  C in 1 mL of 0.2 M PBS (pH 7.4) containing 0 mM, 4.5 mM, or 10 mM glutathione. At each time interval, the suspension was centrifugated at 12,000 rpm for 30 min and 400 mL of the supernatant was quantified for the contents of pDNA and siRNA by fluorimetry. The precipitate was resuspended with 400 mL of 0.2 M PBS (pH 7.4) containing 0 mM, 4.5 mM, or 10 mM glutathione before further incubation. 2.8. Intracellular distribution For quantitative analysis, the nuclei of BEL-7402 cells were isolated as described previously [23], after incubation with NPs for 0.5, 1, 2, 4, and 8 h. Briefly, BEL-7402 cells were collected through centrifugation at 1500 rpm for 5 min. The cell pellet was washed with 0.2 M PBS (pH 7.4), resuspended in TM-2 buffer (10 mM TriseHCl, 2 mM MgCl2, 0.5 mM PMSF), and incubated for 5 min in ice. After treatment with 1% (w/v) Triton X-100 for 5 min, cells were centrifugated at 800 rpm for 10 min. The supernatant was collected to determine the contents of pDNA and siRNA in the cytoplasm while the resultant pellet was resuspended in TM-2 buffer to determine their contents in the nuclei. Results were expressed as the percentage associated with the total amount of intracellular pDNA or siRNA. For qualitative observation, BEL-7402 cells were seeded onto 20 mm coverslips in 6-well plates and cultured for 24 h. Following treatment with NPs for 8 h, cells were washed with 0.2 M PBS (pH 7.4), fixed with 4% (w/v) paraformaldehyde, and stained with Hoechst 33258. The coverslips were observed with confocal laser scanning microscopy (CLSM, Zeiss, Germany). 2.9. In vitro gene silencing BEL-7402 cells were seeded on 24-well plates at 5  104 cells/well and cultured at 37  C for 24 h. Then the culture medium was replaced by serum-free DMEM and GTC NPs containing 2 mg of iSur-pDNA and 400 ng of siVEGF were added. After 4-h incubation, the culture medium was replaced by fresh DMEM containing FBS and incubated for another 20, 44, 68, or 92 h. The culture medium was collected for VEGF quantification by ELISA assay (R&D Systems, USA). Results were expressed as the

Hepatic tumors were established by subcutaneous injection of 0.2 mL of hepatoma H-22 ascites into the right axillary space of Kunming mice. Tumor-bearing mice were given a gavage of TC NPs, GTC1e3 NPs, or GT2 NPs containing 20 mg of FITC-pDNA and 4 mg of TAMRA-siRNA once the tumor volume reached 200 mm3. Naked FITC-pDNA and TAMRA-siRNA were orally administered as the controls. At predetermined time interval, the blood was collected and the plasma was isolated via centrifugation. After the sacrifice of mice, major organs including tumor, heart, liver, spleen, lung, kidney, and intestine were excised, weighed, and homogenized with RIPA buffer. Following centrifugation at 3000 rpm for 10 min, FITC-pDNA and TAMRA-siRNA contents in the supernatant and plasma were quantified by fluorimetry and calculated as the percentage of the total amount.

2.12. In vivo tumor growth inhibition Nude mice bearing BEL-7402 tumor were modeling by subcutaneous injection of cell suspension into the right flank region (4  106 cells/mouse). When the tumor grew to approximately 100 mm3, the mice were randomly assigned to one of the following groups (n ¼ 6): saline (control), TC NPs, GTC1 NPs, GTC2 NPs, GTC3 NPs, GTC2-D NPs, and GTC2-R NPs. NPs were given daily via oral gavage for 20 days at the dose of 1 mg pDNA/kg and 200 mg siRNA/kg. Tumor volumes and body weights were recorded every other day. Tumor volumes were calculated as (S2  L)/2, where S and L represented the short and long diameter of tumors, respectively. All mice were sacrificed one day after last administration, and the tumors were excised, weighed, and stored at 80  C. The tumor inhibition ratio (TIR) of NPs was calculated as (1  Wt/Wc)  100%, where Wt and Wc denoted the average tumor weight of the treatment and control group, respectively. The apoptosis and angiogenesis in the tumors were determined by TdT-mediated dUTP nick end labeling (TUNEL) and immunohistochemistry, respectively, and major organs were stained with hematoxylin and eosin for toxicity detection. Detailed methods were presented in Supplementary Information.

2.13. In vivo gene silencing To determine Survivin and VEGF mRNA level, tumors were homogenized in liquid nitrogen and extracted for RNA with TRI-reagent (Invitrogen, USA). The extracted RNA was then reverse-transcribed into cDNA using PrimeScriptÒRT reagent kit (Takara Biotechnology Co., Ltd, China). Primers, cDNA, and SYBR Premix Ex TaqÔ (Takara Biotechnology Co. Ltd., China) were run on ABI PRISM 7900HT RealTime PCR System (Applied Biosystems, USA) and normalized to b-actin mRNA level. Results were expressed as the relative VEGF mRNA level of tumors in the saline group. For the evaluation of Survivin expression, tumors were homogenized in RIPA buffer containing protease inhibitors and then centrifugated at 12,000 rpm for 5 min. Survivin in the supernatant was detected by western blotting. To quantify the VEGF secretion, tumors were homogenized in 0.2 M PBS (pH 7.4) containing protease inhibitors. Following centrifugation at 12,000 rpm for 5 min, the VEGF amount in the supernatant was measured by ELISA assay and the total protein was determined by the Lowry method. The VEGF level was normalized to total protein content. Results were expressed as the relative VEGF level of tumors in the saline group.

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2.14. Statistical analysis Experimental data were expressed as the mean  SD. Statistical analysis was carried out via Student’s unpaired t test between two groups or single factor analysis of variance (ANOVA) with Turkey’s post-hoc test among three or more groups. The differences were judged to be significant at P < 0.05.

3. Results 3.1. Preparation and characterization of GTC conjugate Synthesis of GTC conjugate was successfully carried out through sequential conjugation of CH3I, LA, and cysteine onto the amino groups of chitosan. After reaction with CH3I, trimethyl chitosan with quaternization degree of 27.5% was obtained as determined by 1 H NMR (Supplementary Information Fig. S1) [17]. The galactosylation degrees of GT1, GT2, and GT3 were calculated to be 6.8%, 17.3%, and 25.2%, respectively, by comparing the integrated peak area of eCHe on LA (4.5 ppm) with that of eNHeOCeCH3 on chitosan (2.0 ppm) [18]. About 10% of amino groups of GTC conjugates were occupied by cysteine as determined with Ellman’s reagent [19]. GTC conjugates possessed free sulphydryl content of 100e120 mmol/g and disulfide content of 180e220 mmol/g. 3.2. Preparation and characterization of GTC NPs Based on gel retardation assays as described in Supplementary Information, the GTC/iSur-pDNA/siVEGF weight ratio was optimized to be 50:5:1 for the purpose of maximally encapsulating both genes (Supplementary Information Fig. S2). GTC NPs possessed particle sizes of 130e160 nm and positive charges of 22e 29 mV (Supplementary Information Table S2). Their uniform particle sizes and morphologies with spherical shape were observed in the SEM images (Fig. 1A). 3.3. Structural stability and protection ability of NPs As depicted in Supplementary Information Table S2, all NPs maintained particle size below 250 nm after PBS dilution, pH

changes, and serum treatment, indicating that they might be structurally stable during transit in the GI tract and blood circulation. Additionally, the presence of serum and high ionic strength diminished the positive charges of NPs, which might assure their in vivo safety. Fig. 1BeC showed that naked pDNA and siRNA were completely degraded with no visible bands, while NPs could protect the loaded pDNA and siRNA from digestion by nucleases and physiological fluids as evidenced by the corresponding bands on the gel. 3.4. Permeation across in vitro Caco-2 monolayers As shown in Fig. 2AeB, the TEER of Caco-2 monolayers rapidly decreased upon the addition of NPs and slowly recovered to the initial values after the removal of NPs, suggesting that NPs could reversibly open the tight junctions between cells to facilitate paracellular transport. The permeation of pDNA and siRNA loaded into NPs across both mono-cultured and co-cultured Caco-2 monolayers was remarkably enhanced compared to that of naked pDNA and siRNA (Fig. 2CeD), implying that the NPs had the ability of promoting transcytosis of pDNA and siRNA in normal enterocytes and intestinal M cells. Fig. 2CeD also revealed that the transport levels of pDNA and siRNA in co-cultured monolayers were higher than those in mono-cultured monolayers, demonstrating the importance of M cells in absorption process of NPs. In addition, the transport levels of GTC3 NPs were relatively lower than those of the other tested NPs in mono-cultured and co-cultured Caco-2 monolayers. 3.5. In vitro cellular uptake As demonstrated in Fig. 3AeB, cellular uptake of all NPs was time-dependent in BEL-7402 cells and the uptake levels reached the plateau within 4 h. GTC1 NPs showed similar uptake amounts of pDNA and siRNA to TC NPs. In contrast, the cellular uptake levels of GTC2 NPs and GTC3 NPs were superior to TC NPs, especially for GTC2 NPs which displayed approximately 1.9-fold uptake amount

Fig. 1. (A) SEM images of NPs. Bar represented 500 nm. Integrity of iSur-pDNA (B) and siVEGF (C) loaded into NPs after incubation with various physiological fluids and nuclease solution as evaluated by agarose gel electrophoresis. Naked iSur-pDNA or siVEGF with the same treatment served as the positive control (þ). Naked iSur-pDNA or siVEGF without any treatment served as the negative control (). Lane a: gastric fluids; Lane b: intestinal fluids; Lane c: intestinal homogenate fluids; Lane d: serum; Lane e: nuclease solution (DNase I for iSur-pDNA and RNase A for siVEGF).

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Fig. 2. The transport of NPs across in vitro Caco-2 cell monolayers. TEER Alteration in Caco-2 cell mono-culture (A) and co-cultures (B) in the presence of NPs. NPs were removed after 4 h and cells were cultured in fresh DMEM for another 20 h. Papp of FITC-pDNA (C) and TAMRA-siRNA (D) across Caco-2 cell mono-culture and co-cultures after treatment with NPs for 4 h. Indicated values were mean  SD (n ¼ 3). **P < 0.01, ***P < 0.001. # Statistical significance from GTC3 NPs in Caco-2 mono-cultured model (##P < 0.01, ###P < 0.001). $ Statistical significance from GTC3 NPs in Caco-2 co-cultured model ($$ P < 0.01, $$$ P < 0.001).

of both pDNA and siRNA in BEL-7402 cells as compared with TC NPs. Detailed uptake mechanism of NPs was explored using endocytosis inhibitors. The non-specific cytotoxicity of endocytosis inhibitors was excluded by MTT assay (Supplementary Information Fig. S3A). As shown in Supplementary Information Fig. S3B, preincubation with sodium azide yielded dramatic depression in the

uptake of NPs, indicating the involvement of an energy-consuming process. Negligible uptake inhibition exerted by wortmannin suggested that macropinocytosis was not involved [24]. As for TC NPs and GTC1 NPs, chlorpromazine and genistein evenly inhibited their cellular uptake, which proposed that clathrin- and caveolindependent pathway played equal roles in their endocytosis (Fig. 3C) [25]. The uptake of GTC2 NPs and GTC3 NPs was notably

Fig. 3. The cellular uptake of NPs in BEL-7402 cells. In vitro cellular uptake of FITC-pDNA (A) and TAMRA-siRNA (B) in BEL-7402 cells after incubation for 0.5, 1, 2, 4, and 6 h. Indicated values were mean  SD (n ¼ 3). *P < 0.05, **P < 0.01, ***P < 0.001. (C) The effect of endocytosis inhibitors chlorpromazine (10 mg/mL) and genistein (200 mg/mL) on cellular uptake of NPs in BEL-7402 cells. Results were expressed as the percentage values of cells without inhibitor treatment. Indicated values were mean  SD (n ¼ 3).

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inhibited by chlorpromazine while less depressed by genistein, implying that stronger galactoseereceptor interaction might tend to promote the entry the GTC NPs into cells through clathrinmediated endocytosis. 3.6. Glutathione-responsive release Fig. 4 revealed that all NPs exhibited glutathione-sensitive release profiles and the release rate of siRNA was significantly higher than that of pDNA. At the glutathione concentration of 0 mM and 4.5 mM, the 2-h accumulative release percentages of pDNA and siRNA were 6.1e13.6% and 42.0e53.9%, respectively. Comparatively, the presence of high concentration of glutathione (10 mM) effectively promoted the disassociation of genes from NPs with 25.8e 44.6% of pDNA and 80.8e85.3% of siRNA being released within 2 h. Additionally, an increased release of pDNA was positively related to the galactose grafting density of GTC conjugates as illustrated in Fig. 4C. Under the glutathione concentration of 10 mM, the 8-h accumulative release percentages of pDNA from TC NPs, GTC1 NPs, GTC2 NPs, and GTC3 NPs were 29.1%, 33.8%, 45.8%, and 56.0%, respectively. 3.7. Intracellular distribution In the quantitative analysis based on fractional centrifugation, no nucleic fluorescence of siRNA was detected, indicating its total distribution in the cytosol, while pDNA showed accumulation in both the nuclei and the cytosol. As shown in Fig. 5A, the relative nucleic fraction of pDNA increased in the order of TC NPs, GTC1 NPs < GTC3 NPs < GTC2 NPs, suggesting that the galactose grafting density of GTC conjugates had important effects on the nucleic transport of pDNA. CLSM images also confirmed that siRNA was completely accumulated in the cytoplasm while pDNA was localized to the nucleic and cytoplasmic region (Fig. 5B). 3.8. In vitro gene silencing Luciferase reporter gene was incorporated into iSur-pDNA to evaluate the tendency of intracellular shRNA expression. Fig. 6A showed that the 48-h luciferase level of TC NPs and GTC1 NPs was slightly lower than that of GTC3 NPs. Maximal transfection efficiency at 48 h was detected in GTC2 NPs, exerting a 1.8-fold higher luciferase level than the commercial transfection reagent Lipofectamine 2000. Moreover, GTC2 NPs demonstrated sustained luciferase expression within 96 h (Fig. 6A and Supplementary Information Fig. S4A). The gene silencing efficiency of NPs

evaluated by western blotting showed the similar tendency with the luciferase assay, in which GTC2 NPs mediated the most efficient knockdown of Survivin (Fig. 6B). In addition, negligible Survivin knockdown was found in the pGL3-control vector, indicating the gene-specific silencing effect of NPs. The VEGF level in cell culture media was determined by ELISA assay. As demonstrated in Fig. 6C, TC NPs, GTC1 NPs, and GTC3 NPs mediated similar VEGF silencing, which decreased to about 50% of control level within 48 h post-administration. GTC2 NPs exhibited superiority in VEGF silencing with the reduction of 70.2%, which was comparable with a commercial transfection reagent LipoRNAiMAX. However, this high silencing efficiency of GTC2 NPs was rapidly weakened with the increase of time, exhibiting only a reduction of 41.7% at 96 h (Supplementary Information Fig. S4B). VEGF secretion was not altered by Scr loaded into LipoRNAiMAX, indicating the sequence-specific silencing of siVEGF. 3.9. In vitro cell growth inhibition The cell viability was not affected by the addition of NPs containing pGL3 and Scr (Supplementary Information Fig. S5), excluding their non-specific cytotoxicity, which might be due to their decreased positive charges in the presence of serum and massive ionic components (Supplementary Information Table S2). Among NPs containing iSur-pDNA and siVEGF, strongest inhibition of cell growth was observed in GTC2 NPs (Fig. 6D), in agreement with the in vitro results of cellular uptake and gene silencing. The inhibition ratio of cell growth of GTC2 NPs was also much higher than that of GTC2-D NPs and GTC2-R NPs which incorporated a single therapeutic gene, which implied the synergistic antitumor effect of interference against two target genes. As compared with GTC2 NPs, GTC2-D-D NPs displayed inferior 24-h effect of suppressing cell growth while unsatisfactory 72-h inhibition of cell growth was observed in GTC2-R-R NPs (Supplementary Information Fig. S6). This might be attributed to the delayed response of shRNA and rapid degradation of siRNA, respectively. The steady and potent efficacy of GTC2 NPs in the entire tested period revealed the benefits of the combination of shRNA with siRNA. 3.10. In vivo distribution Compared with naked genes, NPs following oral administration exhibited significantly higher distribution in the plasma and tumor, while their accumulation in the intestine were decreased with time (Fig. 7), validating that NPs could promote the intestinal

Fig. 4. The release profiles of FITC-pDNA and TAMRA-siRNA from NPs at the glutathione concentration of 0 mM (A), 4.5 mM (B), and 10 mM (C). Indicated values were mean  SD (n ¼ 3).

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effectively reduce the tumor size with a TIR of 49.2% and 28.8%, respectively, while combination of iSur-pDNA and siVEGF could dramatically improve the TIR to 81.5%. Such synergistic antitumor effect was consistent with the assessments of in vitro cell growth inhibition. Fig. 8D indicated that no loss in body weight was observed in all treated groups compared with the control saline. Survivin and VEGF levels in tumors were measured to further elucidate the different antitumor efficacies of NPs. As shown in Fig. 9, GTC2 NPs outperformed TC NPs, GTC1 NPs, and GTC3 NPs in terms of both Survivin and VEGF knockdown. Interestingly, significant down-regulation of Survivin mRNA occurred in GTC2-R NPs (Fig. 9A), implying that VEGF silencing alone was capable of inhibiting Survivin expression. The silencing efficiency of Survivin in GTC2 NPs was superior to that of GTC2-D NPs, which also suggested that shRNA-mediated Survivin knockdown could be enhanced by the incorporation of siVEGF. Apoptotic, angiogenic, and histologic analysis were performed to clarify in vivo antitumor mechanism of GTC NPs. The presence of apoptotic DNA fragments and decreased vessel density demonstrated that GTC2 NPs effectively induced apoptosis and inhibited angiogenesis in tumors (Supplementary Information Fig. S8AeB). In addition, Supplementary Information Fig. S8C revealed that no histological toxicity in major organs was noted after oral administration of GTC NPs, assuring their safety for in vivo application. 4. Discussion

Fig. 5. The subcellular distribution of NPs in BEL-7402 cells. (A) Nuclear fraction of FITC-pDNA in BEL-7402 cells after incubation for 0.5, 1, 2, 4, and 8 h. Indicated values were mean  SD (n ¼ 3). *P < 0.05, **P < 0.01. (B) CLSM images of BEL-7402 cells showing nucleic transfer of FITC-pDNA (green) and cytoplasmic accumulation of TAMRA-siRNA (red) following incubation for 8 h. The nuclei were stained with Hoechst 33258 (blue). Bar represented 20 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

permeation of their cargos as well as the subsequent trafficking to the tumor tissues. Decreased distribution in the intestine and plasma was observed in GT2 NPs compared with GTC2 NPs, indicating the significance of cysteine groups in mucoadhesion. GTC2 NPs outperformed the other NPs in terms of tumor accumulation, which might be attributed to the efficient internalization by cancer cells. As depicted in Supplementary Information Fig. S7, both pDNA and siRNA displayed higher distribution in the liver than the other major organs (heart, lung, spleen, and kidney). 3.11. In vivo antitumor efficacy As illustrated in Fig. 8AeC, all formulations containing iSurpDNA and siVEGF remarkably inhibited the tumor growth following oral administration compared with the control saline. GTC2 NPs exerted stronger antitumor efficacy than TC NPs, GTC1 NPs, and GTC3 NPs, in accordance with their higher in vitro cellular uptake and gene silencing. Additionally, oral delivery of either iSurpDNA (GTC2-D NPs) or siVEGF (GTC2-R NPs) alone could also

shRNA and siRNA are two distinct RNAi techniques which have both shown great potential in cancer therapy [1,2]. In addition to the synergistic effect of dual-gene silencing, combined therapy based on shRNA and siRNA is anticipated to integrate the rapid response of siRNA and sustained silencing of shRNA, resulting in the timely and stable knockdown of aberrant genes. However, the clinical potential of orally delivered genes has been hampered by the extensive degradation, poor intestinal permeation, and nonspecific cellular uptake [10]. The aim of this investigation was therefore to develop GTC NPs-based systems for the co-delivery of shRNA and siRNA to overcome the aforementioned barriers in cancer therapy via oral administration. Survivin and VEGF were selected as the therapeutic targets in the current study. Survivin, a member of the inhibitors of apoptosis (IAP) family, plays an important role in regulating cell division and suppressing apoptosis, enabling it a potential target gene for cancer therapy [15]. VEGF, the most relevant promoter of tumor angiogenesis, is often up-regulated in cancers [16,26]. Inhibition of VEGF expression has been proved to retard tumor growth in various models [16]. Considering the constitutive synthesis and residence of Survivin in cancer cells [15], down-regulation of substantially expressed Survivin appears to be a long-term task wherein shRNA is preferable to siRNA. Therefore, iSur-pDNA rather than siSur was adopted in the current study to offer sustained silencing of Survivin. Accordingly, VEGF was depleted with siVEGF to pursue the synergistic antitumor efficacy with iSur-pDNA. GTC conjugates were synthesized as multifunctional vectors for oral gene delivery by integrating the benefits of trimethyl, thiol, and galactose groups. Trimethyl modification aimed to improve the condensing capacity for therapeutic genes and the electrostatic adhesion with negatively-charged mucosa [27]. Cysteine conjugation was anticipated to enhance intestinal permeation through forming disulfide bond with mucin glycoprotein and opening the tight junctions, and provide glutathione-sensitive release of cargos loaded into GTC NPs [28]. The introduction of galactose ligands could promote the internalization of GTC NPs in liver cancer cells that overexpressed asialoglycoprotein receptors [18]. The modification degrees of trimethyl (w30%) and cysteine groups (w10%), as

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Fig. 6. In vitro gene silencing and cell growth inhibition of NPs in BEL-7402 cells. (A) The luciferase expression in BEL-7402 cells at 48 h post-NPs administration. Cells without any treatment served as the control. Indicated values were mean  SD (n ¼ 3). ***P < 0.001. (B) The expression of Survivin in BEL-7402 cells at 48 h post-NPs administration evaluated by western blotting. GAPDH was used as an internal control. (C) VEGF secretion from BEL-7402 cells at 48 h post-NPs administration evaluated by ELISA assay. Results were expressed as the percentage values of cells without any treatment. Indicated values were mean  SD (n ¼ 3). **P < 0.01. (D) The inhibition ratio of cell growth at 48 h post-NPs administration compared with cells without any treatment. Indicated values were mean  SD (n ¼ 6). Statistical significance from GTC2 NPs (***P < 0.001).

well as the Mw of chitosan (200 kDa), were adopted here based on our previous work wherein chitosan with such grafting extents and Mw highly performed in gene delivery including both pDNA and siRNA [14,20,29]. As for the galactose groups, since their proper grafting degree for the co-delivery of pDNA and siRNA was not available in the literatures, GTC conjugates with distinct galactose densities were therefore constructed to maximize the synergistic efficacy of iSur-pDNA and siVEGF. Self-assembled GTC NPs were obtained after simple complexation between GTC conjugates, iSur-pDNA, and siVEGF initiated by electrostatic interactions. Based on the criteria of higher encapsulation efficiency with genes, GTC NPs were prepared at the GTC/ iSur-pDNA/siVEGF weight ratio of 50:5:1 in the following studies. It was worth noting that due to the short and rigid nature of siRNA chain, NPs formed at such a low polymer/siRNA ratio (50:1) normally possessed larger and more loosely packed structure, and in turn, leading to poor silencing effects [30]. Here, a helper polyanion, iSur-pDNA, was incorporated into GTC NPs along with siVEGF, resulting in stronger electrostatic interaction with GTC conjugates and thus more compact structure. Besides, the introduction of iSurpDNA instead of commonly-used crosslinkers provided the opportunity of dual therapeutic delivery in a single carrier.

As for oral delivery of gene drugs, desirable stability is a prerequisite for NPs to overcome multiple barriers existing in the GI tract and blood vessel, including dilution in the massive digestive fluids (0.2 M ionic strength), drastic pH alteration, and prevailing nuclease degradation [31]. All NPs were able to maintain their structural stability during the transit in the GI tract and blood stream, as evidenced by the slight alternation in particle size after the challenge of dilution, high ionic strength, pH changes, and serum treatment (Supplementary Information Table S2). This led to the protection of encapsulated genes via steric hindrance [32], thereby partially preventing iSur-pDNA and siVEGF from enzymatic degradation in the GI tract and bloodstream. The effective permeation across the intestinal epithelium is necessary for robust gene therapies via oral route. In this study, two in vitro models were employed to assess the ability of GTC NPs to permeate across the intestinal epithelium. Polarized mono-cultured Caco-2 monolayer with well-developed microvilli and co-cultured Caco-2 monolayer with Raji B lymphocytes containing a distinctive phenotype of M cells were applied as non-follicle-associated and follicle-associated epithelial models, respectively [22]. The TEER reduction and restoration in both models indicated that GTC NPs were able to reversibly open the intercellular tight junctions to facilitate their

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Fig. 7. In vivo distribution of FTIC-pDNA (A) and TAMRA-siRNA (B) in the plasma, intestine, and tumor after oral administration of NPs in tumor-bearing mice (n ¼ 3).

Fig. 8. The in vivo antitumor efficacy of NPs in BEL-7402 tumor bearing nude mice following oral administration. (A) Tumor growth curves of the mice after treatment with NPs. (B) Photographs of the excised tumors at day 20. (C) Weights of the excised tumors at day 20. (D) The body weight of tumor bearing mice. Indicated values were mean  SD (n ¼ 6). *P < 0.05, **P < 0.01, ***P < 0.001.

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Fig. 9. The expression of Survivin and VEGF in tumor tissues. Survivin mRNA (A) and VEGF mRNA (B) levels in tumor tissues determined by Real-Time PCR. Indicated values were mean  SD (n ¼ 3). *P < 0.05, **P < 0.01. (C) Survivin expression in tumor tissues determined by western blotting. GAPDH served as an internal control. (D) VEGF levels in tumor tissues determined by ELISA assay. Indicated values were mean  SD (n ¼ 6). ***P < 0.001.

paracellular transport. Higher Papp in the co-cultured models demonstrated the more important roles of M cells than normal intestinal enterocytes in the transcellular transport of GTC NPs. The superior permeation ability of GTC NPs might be attributed to the good bioadhesion of chitosan backbone, together with the electrostatic and disulfide interactions with mucin glycoproteins arising from the trimethyl and cysteine groups, respectively [14]. Interestingly, GTC3 NPs with high galactose grafting density induced significant reduction in the intestinal permeation. The insufficient amount of positive charges on their surface (Supplementary Information Table S2) might lead to weaker electrostatic interaction with the intestinal mucosa and the subsequent inferior permeation capacity. Having demonstrating that GTC NPs could facilitate the intestinal permeation of iSur-pDNA and siVEGF, we reasonably moved on to investigate their internalization by cancer cells. Naked genes could not be readily internalized due to their negative charges and hydrophilic property [6]. However, once encapsulation into positively-charged GTC NPs, they could electrostatically adsorb onto the cell membrane and subsequently be internalized. Moreover, galactose ligand could further promote the uptake of GTC NPs in BEL-7402 cells through receptor-mediated endocytosis. The similar uptake amount between TC NPs and GTC1 NPs indicated that a critical grafting density of galactose was needed to initiate the ligand-receptor recognition [33]. The highest uptake level was

noted in GTC2 NPs rather than GTC3 NPs in spite of their lower ligand grafting density. Such result might be explained by the limited amount of receptors existing on the plasma membrane and the inefficient cellular adsorption of GTC3 NPs resulting from less positive charges. In addition, galactose grafting density of GTC NPs was proven to be correlated with their internalization mechanisms. Clathrin-dependent endocytosis tended to take more involvement in the internalization of GTC NPs with increased galactose grafting density, and thus their distinct intracellular trafficking might be seen [25]. These results suggested that not only the cellular uptake but also the subsequent intracellular fate might be significantly affected by the ligand grafting density of NPs, which should be paid much more attention in the rational design of targeted nanocarriers. To achieve effective gene silencing, ideal delivery vehicles should be capable of providing sufficient protection of their gene payloads prior to efficiently releasing them within target cells [34]. In this study, such necessities were fulfilled by the glutathionesensitivity of GTC NPs derived from cysteine functionalities. Aside from electrostatic interaction between positively and negatively charged components, the intra- and inter-molecular disulfide crosslinking of GTC conjugates mainly contributed to the compact structure of GTC NPs. In the extracellular spaces with minimal glutathione (4.5 mM), GTC NPs were structurally stable due to the extensive disulfide crosslinking, showing limited premature release

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of iSur-pDNA and siRNA (Fig. 4B). Different from the extracellular environment, the intracellular space possessed a reducing condition owing to the relatively high concentration of glutathione (10 mM). Therefore, a rapid breakage of the disulfide crosslinking between GTC conjugates occurred following cellular internalization, leading to the disassembly of NPs and the accelerated intracellular release of genes. The release of iSur-pDNA was much slower than that of siVEGF at the intracellular concentration of glutathione, which might be attributed to the stronger interaction between GTC conjugates and polyanionic pDNA compared to the short and rigid siRNA. Consequently, it was reasonable to hypothesize that following entry of GTC NPs into cancer cells, siVEGF would be rapidly disassociated in the cytosol, while the majority of iSurpDNA might stay entrapment into GTC NPs. Intracellular GTC NPs then electrostatically interacted with the negatively-charged microtubules, moved along the cytoskeleton networks with the help of motor protein, and started the slow release of iSur-pDNA at the perinuclear region [35]. This hypothesis was confirmed by the complete cytoplasmic localization of siVEGF and abundant nucleic distribution of iSur-pDNA as observed in CLSM images (Fig. 5B). Such subcellular distribution was favorable for triggering prompt siRNA-mediated silencing and sustained shRNA expression. Quantitative analysis revealed that nucleic distribution of iSur-pDNA was different among GTC NPs with various galactose grafting densities. GTC2 NPs exhibited suitable binding affinity for iSur-pDNA and moderate release rate of iSur-pDNA (Fig. 4C), which enabled iSurpDNA to be encapsulated into GTC2 NPs in the movement along the microtubules, efficiently dissociated from GTC2 NPs at the perinuclear region, and mainly localized in the nuclei. As previously mentioned, cancer cells expressed a high level of Survivin and VEGF for their immortalization [15,16]. Therefore, iSur-pDNA and siVEGF delivered by GTC NPs were expected to specifically silence the dual-gene expression and further inhibit the growth of cancer cells. All tested NPs containing iSur-pDNA and siVEGF successfully reduced the expression of both genes and the growth of cancer cells. Owing to their highest cellular uptake and nucleic accumulation, GTC2 NPs outperformed the others in terms of in vitro gene silencing and cell growth inhibition. In addition, the cell growth inhibition of GTC2 NPs was stronger than those of GTC2-D NPs and GTC2-R NPs, which verified the synergistic effect of dual-gene silencing. More importantly, combinational application of shRNA and siRNA worked better in suppressing cell growth than simultaneous delivery of different shRNA or different siRNA (Fig. 6D). Due to the complicated and multiple steps in shRNAmediated gene silencing, co-delivery of iSur-pDNA and iVEGFpDNA failed to elicit efficient suppression of cell growth at 24 h (Supplementary Information Fig. S6A). As for siRNA, notwithstanding its direct interaction with cytoplasmic mRNA, the dilution by cell division and the vulnerability to nuclease degradation severely compromised the long-term gene silencing effect of codelivery of siSur and siVEGF, leading to inferior suppression of cell growth at 72 h (Supplementary Information Fig. S6B). Fortunately, when shRNA and siRNA were combined, the timely response of siRNA and the duration expression of shRNA would trigger the early and later gene silencing, respectively, overcoming the individual limitation of each approach. Therefore, GTC2 NPs containing iSur-pDNA and siVEGF were capable of inducing steady and potent inhibition of cell growth in the entire tested period (Fig. 6D and Supplementary Information Fig. S6). No cytotoxicity was noted when pGL-3 and Scr was used (Supplementary Information Fig. S5), indicating the in vitro cell growth inhibition was not an artifact of reduced cell viability. The promising in vitro results motivated us to further evaluate the in vivo therapeutic efficacy of GTC NPs via oral administration in tumor bearing mice. Since indistinguishably inferior antitumor

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efficacy arising from the harsh in vivo conditions would cover synergistic effect of dual gene silencing and density-associated effect of galactose modification, oral administration was performed every day to enhance the in vivo antitumor efficacy of GTC NPs, thereby assuring the high possibility to unravel the aforementioned effects. All NPs significantly inhibited the tumor growth due to a combination of desirable stability in the GI tract, elevated intestinal permeation, enhanced tumor accumulation, high cellular uptake, reasonable subcellular distribution, and efficient gene silencing. As expected, the in vivo antitumor capacity and silencing efficacy accorded well with the in vitro results in that GTC2 NPs exhibited the most efficient tumor regression and gene knockdown, due to their suitable binding affinity for therapeutic genes (Fig. 4), the highest distribution in the tumor tissues, and the most efficient uptake in cancer cells (Fig. 3). Interestingly, Survivin mRNA expression in the tumors was down-regulated following single siVEGF treatment (GTC2-R NPs) (Fig. 9A). Moreover, combined use of siVEGF with iSur-pDNA (GTC2 NPs) was capable of improving the silencing effect of Survivin mRNA in the tumors as compared with single iSur-pDNA treatment (GTC2-D NPs) (Fig. 9A). Such synergistic effect in in vivo gene silencing was also reflected by the smaller tumor size in the groups of GTC2 NPs compared with GTC2D NPs and GTC2-R NPs (Fig. 8). Increased apoptosis and decreased angiogenesis in the tumors might be resulted from Survivin and VEGF knockdown, respectively [15,16], which accounted for the remarkable antitumor efficacy of GTC2 NPs. Neither significant loss in body weight nor noticeable histological damage was observed following repeated administration, which suggested that there was no immediate or severe toxicity associated with GTC NPs. These results collectively apprehended the suitability and safety of GTC NPs as oral delivery systems for shRNA and siRNA in synergistic cancer therapy. 5. Conclusions Multifunctional GTC conjugates with different galactosylation degrees were synthesized for co-delivery of shRNA and siRNA via oral route. GTC NPs could surmount the extracellular and intracellular barriers in oral gene delivery by preventing enzymatic degradation in the GI tract, improving the intestinal permeation, promoting tumor accumulation and cellular uptake, and facilitating intracellular release of therapeutic genes. Synergistic antitumor efficacy was achieved by simultaneously incorporating iSur-pDNA as a crosslinking agent and siVEGF into a single carrier (GTC NPs), which provided a promising addition to existing co-delivery formulations and represented a potential strategy for cancer therapy via oral administration. In addition, we also highlighted the significant effects of ligand grafting density on the entire process of oral gene delivery including intestinal permeation, tumor distribution, cellular uptake, and intracellular distribution, which should be taken into serious consideration in the design of active-targeting nanocarriers. Acknowledgments The authors are thankful for the financial support from the National Natural Science Foundation of China (No. 81172995 and No. 81273460), Graduate Innovation Foundation of Fudan University of China (EZH1322302), and Doctoral Science Foundation of Fudan University of China (EZH1322370). Appendix A. Supplementary data Supplementary data related to this article can be found online at http://dx.doi.org/10.1016/j.biomaterials.2014.02.027.

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