Galactosylated trimethyl chitosan–cysteine nanoparticles loaded with Map4k4 siRNA for targeting activated macrophages

Biomaterials 34 (2013) 3667e3677

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Galactosylated trimethyl chitosanecysteine nanoparticles loaded with Map4k4 siRNA for targeting activated macrophages Jing Zhang, 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 8 January 2013 Accepted 24 January 2013 Available online 15 February 2013

Galactosylated trimethyl chitosanecysteine (GTC) nanoparticles (NPs) were developed for oral delivery of a mitogen-activated protein kinase kinase kinase kinase 4 (Map4k4) siRNA (siMap4k4) to the activated macrophages for treatment of dextran sulfate sodium (DSS)-induced ulcerative colitis (UC). siRNA loaded GTC NPs were prepared based on ionic gelation of GTC with anionic crosslinkers (tripolyphosphate (TPP) or hyaluronic acid (HA)). The types of crosslinkers involved in GTC NPs significantly affected their physicochemical characteristics. GTC/TPP NPs with smaller particle size and lower zeta potential possessed superior structural stability in gastrointestinal environment compared to GTC/HA NPs. Cellular uptake of GTC/TPP NPs in activated macrophages was significantly enhanced compared to trimethyl chitosan-cysteine (TC)/TPP NPs owing to galactose receptor-mediated endocytosis. The in vitro and in vivo gene knockdown measurement showed that siMap4k4 loaded GTC/TPP NPs effectively inhibited TNF-a production, which remarkably outperformed siMap4k4 loaded TC/TPP NPs. Compared to TC/TPP NPs, GTC/TPP NPs more efficiently promoted the distribution of siRNA in ulcerative colon following oral administration. Daily oral administration of GTC/TPP NPs containing siMap4k4 significantly improved DSS-induced body weight loss, colon length shortening, and increase of myeloperoxidase activity. This study would provide an effective approach for oral siRNA delivery in the treatment of inflammatory bowel diseases. Ó 2013 Elsevier Ltd. All rights reserved.

Keywords: Chitosan derivate nanoparticles Galactosylated modification siRNA Oral administration Ulcerative colitis Targeted gene delivery

1. Introduction Ulcerative colitis (UC) is a chronic inflammatory bowel disease for which existing treatments are largely limited by low effectiveness and severe systemic side effects [1]. Tumor necrosis factor alpha (TNF-a) as a proinflammatory cytokine plays a central role in the onset and progression of UC [2]. Intervention of the inflammatory responses with TNF-a monoclonal antibodies has become a major successful immunotherapy in the clinic [3], which however, shows limitations including severe side effects caused by the antibody and high cost [4]. Small interfering RNA (siRNA)-mediated knockdown of functional protein at the messenger RNA (mRNA) level offers an alternative therapeutic approach to various inflammatory diseases owing to its high specificity and efficiency [5,6]. As an important protein kinase of the mammalian STE20/MAP4K family [7], mitogen-activated protein kinase kinase kinase kinase 4 (Map4k4) has been demonstrated to be a key upstream mediator of

* Corresponding author. Tel.: þ86 21 6564 3797; fax: þ86 21 5552 2771. E-mail address: [email protected] (C. Yin). 0142-9612/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biomaterials.2013.01.079

TNF-a action [6]. Therefore, it was hypothesized that gene knockdown targeting Map4k4 for suppressing TNF-a production would provide a promising siRNA-based therapeutic strategy for the treatment of UC. To achieve RNAi for clinical applications, effective carriers are essential for delivering intact siRNA to the cytoplasm of target cells, based on the specific pathophysiology of each disease. In inflammatory bowel diseases, such as UC, TNF-a secreted by macrophages is a major contributor to the development of bowel inflammation [2], therefore RNAi therapeutics targeting macrophage Map4k4 present a potential approach for the treatment of UC. Macrophage galactose-type lectin (MGL) is expressed at high levels by activated macrophages under inflammatory conditions [8,9]. Zuo et al. [10] demonstrated the targeted delivery ability of the galactosylated low molecular weight chitosan (CS)/antisense oligonucleotide complex into activated macrophages and its therapeutic action in experimental colitis by intracolonic administration. For the management of UC as chronic inflammation, high dose frequency of nucleic acid drugs would be required to achieve continuously curative effect [1]. Considering patient compliance, oral delivery of siRNA will be an optimal alternative [11]. However, the harsh

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Abbreviations CS chitosan DSS dextran sulfate sodium FAM-NC siRNA FAM-labeled negative control small interfering RNA GT galactosylated trimethyl chitosan GTC galactosylated trimethyl chitosanecysteine GTC/HA NPs hyaluronic acid-crosslinked GTC nanoparticles GTC/TPP NPs tripolyphosphate-crosslinked GTC nanoparticles LA lactobionic acid LPS lipopolysaccharide Map4k4 mitogen-activated protein kinase kinase kinase kinase 4

environment of the gastrointestinal tract including high ionic strength, dramatic pH alteration, and ubiquitous digestive enzyme would potentially damage siRNA integrity, thus leading to the inefficacy of RNAi after oral delivery [12]. CS as a carrier for oral delivery of siRNA has many advantages including its strong nucleic acid affinity, bioadhesion, and biodegradability [5]. However, its insolubility at neutral and alkaline environment limits its application in oral delivery of nucleic acid [13]. To improve the solubility of CS over a wide pH range, trimethylation modification of CS has been introduced [14]. Furthermore, thiolated modification of CS with thiol-bearing compounds can enhance its bioadhesion capacity through covalent bonding with mucin glycoproteins [15]. In the present investigation, galactosylated trimethyl chitosanecysteine (GTC) was developed as an activated macrophages-targeting carrier for oral siRNA administration. The siRNA loaded GTC nanoparticles (NPs) were prepared through ionic gelation with tripolyphosphate (TPP) or hyaluronic acid (HA), and characterized in terms of particle size, zeta potential, and siRNA integrity in physiological environment. In vitro assessment of cell binding, cellular uptake, cytotoxicity, and gene knockdown efficiency of GTC NPs were carried out in lipopolysaccharide (LPS)-activated Raw 264.7 cells. Biodistribution and in vivo RNAi efficiency of orally delivered siRNA loaded GTC NPs were determined in mice suffering from dextran sulfate sodium (DSS)-induced UC.

MGL macrophage galactose-type lectin mRNA messenger RNA NC siRNAnegative control small interfering RNA siRNA small interfering RNA siMap4k4Map4k4 siRNA Scr scrambled siMap4k4 TMC N-trimethyl chitosan TC TMC-cysteine conjugates TC/HA NPs hyaluronic acid-crosslinked TC nanoparticles TC/TPP NPs tripolyphosphate-crosslinked TC nanoparticles tumor necrosis factor alpha TNF-a UC ulcerative colitis

2.2. Cell line and animals Raw 264.7 cells were obtained from the American Type Culture Collection (Rockville, MD, USA) and cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, NY, USA) containing 10% fetal calf serum (FCS). Male C57BL/6 mice (6 weeks, 20  2 g) were obtained from Shanghai Slaccas Experimental Animals Co., Ltd. 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.

2.3. Preparation of GTC conjugates GTC was synthesized through a three-step route. Firstly, CS was reacted with methyl iodide (CH3I) in methyl-2-pyrrolidone (NMP)/NaOH solution for 120 min at 65  C to obtain N-trimethyl chitosan (TMC) with trimethylation degree of about 30% as previously reported [14]. Secondly, LA was covalently bound on TMC as described by Park et al. [16]. Briefly, LA (0.25 mmol) was dissolved in 3 mL of N,N,N0 ,N0 -tetramethylethylenediamine (TEMED)/HCl buffer solution (10 mM, pH 4.7), into which 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC)/N-hydroxysuccinimide (NHS) (molar ratio ¼ 1:1) were added at a final concentration of 300 mM. The reaction was allowed for 2 h under stirring at room temperature, followed by the addition of 12 mL of TMC solution (42 mM, dissolved in TEMED/HCl) and a further reaction for 72 h. The obtained galactosylated TMC (GT) was purified via ultrafiltration (MWCO 10 kDa). Finally, GT was reacted with cysteine at pH 5.0 and 1:2 (w/w) for 5 h at room temperature as mediated by EDC/NHS (80 mM), and the resultant GTC was dialyzed against pH 5.0 HCl solution and lyophilized. The amount of immobilized sulfhydryl was determined with Ellman’s reagent [17]. The trimethylation degree of TMC and galactosylated modification degree of GTC were calculated by 1H NMR (AVANCE DMX 500, Bruker, Germany) [14,18]. As comparisons, TMC-cysteine conjugates (TC) were prepared as previously described [17].

2. Materials and methods 2.1. Materials CS (deacetylation degree of 85% and molecular weight (Mw) of 200 kDa) was obtained from Golden-shell Biochemical Co., Ltd. (Zhejiang, China). Lactobionic acid (LA) and cysteine were purchased from Shanghai Yuanju Biochemical Co., Ltd. (Shanghai, China). TPP and HA (Mw 20 kDa) were obtained from Shanghai Experimental Reagent Co., Ltd. (Shanghai, China). LPS (obtained from Escherichia coli) was purchased from Sigma (St. Louis, MO, USA). Lipofectamine 2000 was obtained from Invitrogen (Carlsbad, USA). DSS was from MP Biomedicals (Mw ¼ 36e50 kDa, Illkirch, France). All other reagents were of analytic grade. Map4k4 siRNA (siMap4k4), scrambled siMap4k4 (Scr), and negative control siRNA (NC siRNA) duplexes were supplied by GenePharma (Shanghai, China) and dissolved in DEPC-treated water before use. siMap4k4 contained the sequences of sense 50 -GACCAACUCUGGCUUGUUA-30 and antisense 50 -UAACAAGCCAGAGUUGGUC-30 . The Scr contained the scrambled sequences of sense 50 -CAGUCGCGUUUGCGACUGG-30 and antisense 50 -CCAGUCGCAAACGCGACUG-30 . The NC siRNA contained the sequences of sense 50 -UUCUCCGAACGUGUCACGUTT-30 and antisense 50 -ACGUGACACGUUCGGAGAATT-30 . FAM-labeled NC siRNA (FAM-NC siRNA) was used for in vitro siRNA quantification. TAMRA-labeled NC siRNA (TAMRA-NC siRNA) was used for in vivo siRNA quantification. The primers for mouse TNF-a, Map4k4, and 36B4 were synthesized by Shanghai Sangon Biotech Co., Ltd. (Shanghai, China) and the sequences are as follows: CCCTCACACTCAGATCATCTTCT (TNF-a forward); GCTACGACGTGGGCTACAG (TNF-a reverse); CATCTCCAGGGAAATCCTCAGG (Map4k4 forward); TTCTGTAGTCGTAAGTGGCGTCTG (Map4k4 reverse); TCCAGGCTTTGGGCATCAC (36B4 forward); CTTTATCAGCTGCACATCACTCAGA (36B4 reverse).

2.4. Preparation and characterization of GTC NPs GTC NPs were prepared based on the ionic gelation of GTC with TPP or HA. GTC, HA, TPP, and siRNA were dissolved in DEPC-treated water. As for TPP-crosslinked GTC NPs (GTC/TPP NPs), siRNA (0.2 mg/mL) was mixed with TPP solution (1 mg/ mL) at 1:20 (w/w). The GTC solution (4 mg/mL) was added dropwise into the mixture under stirring at the GTC/TPP weight ratio of 10:1, 12.5:1, and 15:1, respectively. As for HA-crosslinked GTC NPs (GTC/HA NPs), siRNA (0.2 mg/mL) was mixed with HA solution (1 mg/mL) at 1:10 (w/w). The GTC solution (6 mg/mL) was added dropwise into the mixture under stirring at the GTC/HA weight ratio of 5:1, 7:1, and 9:1, respectively. The resultant NPs were incubated at 37  C for 30 min before use, and they were named as “GTC/crosslinker(m)1 NPs”, wherein m was the GTC/ crosslinker weight ratio. As comparisons, TPP- and HA-crosslinked TC NPs were prepared with the same methods for GTC NPs, named as “TC/crosslinker(m) NPs” wherein m was the TC/crosslinker weight ratio. The particle size and zeta potential of NPs were determined using Zetasizer Nano (Malvern, Worcestershire, UK). The association of siRNA with the NPs was monitored with gel retardation assay on 4% agarose gel electrophoresis stained with ethidium bromide (0.5 mg/mL), and the electrophoresis was performed at 56 V for 1 h. Morphology of GTC/TPP(10) NPs and GTC/HA(9) NPs was observed with scanning electron microscopy (SEM, Vega TS5136, Tescan, Czechoslovakia).

1 The nomenclature of m in “GTC/crosslinker(m) NPs” and “TC/crosslinker(m) NPs” referred to the polymer/crosslinker weight ratio.

J. Zhang et al. / Biomaterials 34 (2013) 3667e3677 Stability of GTC/TPP(10) NPs and GTC/HA(9) NPs against ionic strength and pH alteration was evaluated in terms of particle size and zeta potential. Ionic strength was adjusted to 0.2 M using NaCl solution. To mimic the pH alteration in the gastrointestinal tract, the pH of GTC/TPP(10) NPs and GTC/HA(9) NPs suspensions was adjusted to 1.2 using 1 M HCl solution, and then back to pH 7.4 using 1 M NaOH solution. 2.5. siRNA integrity in physiological fluids Blood was collected from the orbital sinus of male C57BL/6 mice and centrifugated at 12,000 rpm and 4  C for 4 min. The supernatant was collected as serum. Ten milliliters of phosphate buffered saline (PBS, pH 7.4) was used to rinse the intestinal lumen. The obtained solution was centrifugated at 12,000 rpm and 4  C for 20 min, and the supernatant was collected as intestinal fluids. Serum and intestinal fluids were stored at 20  C before use. To assess the stability of encapsulated siRNA in the cell culture media and intestinal lumen, GTC/TPP(10) NPs, TC/TPP(10) NPs, and naked siRNA solution containing 400 ng of NC siRNA were mixed with equal volume of serum and intestinal fluids at 37  C for 6 h, respectively. The mixtures were heated at 80  C for 5 min, followed by the addition of heparin sodium (40 mg/mL) to dissociate siRNA. siRNA integrity was subsequently evaluated on 2% agarose gel electrophoresis. Naked NC siRNA incubated with DEPC-treated water served as a negative control. 2.6. Cell binding Raw 264.7 cells were collected by centrifugation at 3000 rpm for 5 min. Cell pellets were washed three times with PBS and resuspended in isotonic buffer solution (glucose 44.4 g/L, KCl 0.2 g/L, Na2HPO4$12H2O 2.9 g/L, and KH2PO4 0.2 g/L) at a concentration of 0.5e1  106 cells/mL. GTC/TPP(10) NPs and TC/TPP(10) NPs containing 0.7 mg of siMap4k4 was mixed with 1 mL of Raw 264.7 cell suspension in the presence of 10 ng/mL LPS, and was incubated at 37  C for 2 h. After the solution was centrifugated at 3000 rpm for 5 min, cell pellets were resuspended with 0.2 M PBS (pH 7.4), which was subjected to zeta potential analysis. Cells incubated with isotonic buffer solution instead of NPs served as blank controls. 2.7. Cellular uptake Raw 264.7 cells were seeded on 24-well plates at 5  104 cells per well and incubated for 24 h. The culture medium was replaced by the fresh serum-free DMEM. GTC/TPP(10) NPs and TC/TPP(10) NPs containing 400 ng of FAM-NC siRNA were added in the presence of 10 ng/mL LPS. Following incubation at 37  C for 4 h, the cells were washed three times with PBS and lysed with 0.5% SDS (w/v, pH 8.0). The cell lysate was quantified for FAM-NC siRNA by a VARioSKAN Flash microplate reader (Thermofisher, USA) (lex ¼ 480 nm, lem ¼ 520 nm) and protein content by the Lowry method. Naked FAM-NC siRNA served as a control. Uptake was expressed as the amount of FAM-NC siRNA associated with 1 mg of cellular protein. To evaluate the effect of free LA and galactose on the cellular uptake, LA and galactose at a final concentration of 100 and 200 mM were added 1 h prior to NPs application, respectively, and incubated with the cells throughout the uptake measurement. Results were expressed as the percentage uptake of the control where cells were incubated with NPs in the absence of LA and galactose at 37  C for 4 h. 2.8. Cytotoxicity Raw 264.7 cells were seeded on 96-well plates at a density of 1  104 cells per well, and were cultured for 24 h. Then the cells were incubated with GTC/TPP(10) NPs and TC/TPP(10) NPs containing siMap4k4 at the siMap4k4 concentrations of 0.1, 0.2, 0.5, 1, and 2 mg/mL for 6 h in the presence of 10 ng/mL LPS, followed by methyl tetrazolium (MTT) assay. The untreated cells served as 100% cell viability. 2.9. In vitro TNF-a knockdown Raw 264.7 cells were seeded on 24-well plates at 4  104 cells per well and incubated for 24 h. The culture medium was replaced by the fresh serum-free DMEM. GTC/TPP(10) NPs and TC/TPP(10) NPs loaded with siMap4k4 were added at 400 ng siMap4k4 per well in the presence of 10 ng/mL LPS. Lipofectamine 2000/ siMap4k4 complexes and GTC/TPP(10) NPs loaded with Scr served as positive and negative controls, respectively. After a 4-h incubation, the culture medium was discarded and a further culture in serum-containing medium was allowed for 20 h before LPS (10 ng/mL) stimulation for 5 h. The supernatant of culture medium was collected for the quantification of the extracellular TNF-a production by ELISA. In addition, RNA was isolated from the transfected Raw 264.7 cells according to the Trizol reagent protocol (Invitrogen, USA), and cDNA was synthesized from 500 ng of the total RNA using PrimeScriptÒRT reagent kit (Takara Biotechnology Co. Ltd., China) according to the manufacturer’s instructions. Synthesized cDNA, forward and reverse primers, and the SYBR Premix Ex TaqÔ (Takara Biotechnology Co. Ltd., China) were run on the ABI PRISM 7900HT Real-Time PCR system (Applied

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Biosystems, USA) for the evaluation of intracellular Map4k4 and TNF-a mRNA level. The ribosomal mRNA 36B4 was used as the internal loading control. 2.10. Biodistribution UC was induced by replacing the drinking water of mice with 3% (w/v) DSS over a period of 8 days. C57BL/6 mice suffering from DSS-induced UC were given a gavage of GTC/TPP(10) NPs or TC/TPP(10) NPs containing 5 mg of TAMRA-NC siRNA. At 2, 6, and 12 h post oral administration, blood was collected from the orbital sinus of mice and plasma was isolated via centrifugation. Mice were sacrificed and heart, liver, spleen, lung, kidney, small intestine, and colon were taken, weighed, and homogenized with RIPA lysis buffer. The homogenate was centrifugated at 3000 rpm for 15 min, and the amount of TAMRA-NC siRNA in the supernatant as well as plasma was quantified by a microplate reader (lex ¼ 544 nm, lem ¼ 576 nm). The results were expressed as percentage of total amount of TAMRA-NC siRNA delivered. 2.11. In vivo RNAi against UC via gavage UC was induced by replacing the drinking water of mice with 3% (w/v) DSS over a period of 8 days. Normal control mice received water. Mice were given a daily gavage of Scr loaded GTC/TPP(10) NPs (Scr-treated group), siMap4k4 loaded TC/TPP(10) NPs (TC NPs-treated group), and siMap4k4 loaded GTC/TPP(10) NPs (GTC NPs-treated group) at the dose of 250 mg siRNA/kg body weight for 6 consecutive days (days 0e 5), or PBS (colitis control group). The dose adopted in this investigation (250 mg siRNA/kg/day) was comparable to that in Wilson et al.’s study (230 mg siRNA/kg/day) [19], which was relatively low among anti-inflammation studies involving TNF-a knockdown [19,20]. Body weight was monitored daily. Mice were sacrificed on day 7, colonic segment of each mouse was gathered and homogenized with cold PBS (pH 7.4). The homogenate was centrifuged at 12,000 rpm and 4  C for 20 min, and the supernatant was collected for TNF-a quantification by ELISA. For the evaluation of Map4k4 and TNF-a mRNA level in colonic tissues, colonic segments were cut into small pieces, washed with saline, immersed in RNAlater solution (Qiagen, USA) for 24 h, and homogenized in liquid nitrogen. RNA in the cell lysate was extracted with Trizol reagent and intracellular Map4k4 and TNF-a mRNA levels in colonic tissues were thereafter monitored by realtime-PCR. Neutrophil infiltration into the colon was evaluated by measuring myeloperoxidase (MPO) activity in colonic tissues. Furthermore, colonic tissue was harvested, fixed in paraffin (4%, w/v), cross-sectioned, and stained with haematoxylin/eosin (H&E) for the histological examination. 2.12. Statistical analysis All experimental data were expressed as mean  SD. Statistical analysis was performed by Student’s unpaired t test between two groups or One-Way Analysis of Variance (ANOVA) followed by Tukey’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 of GTC conjugates GTC conjugates were synthesized through sequential trimethylation, galactosylation, and thiolation of chitosan as shown in Fig. 1A. After CS was allowed to react with CH3I to achieve TMC with a trimethylation degree of 34% as determined by 1H NMR (Fig. 1B) [14], galactosylation was performed to yield GT. The grafting efficiency of galactose residues on GT was calculated by comparing the peak area of eCHe on LA (4.5 ppm) to that of eNHeOCeCH3 on chitosan (2.0 ppm) [18]. The chemical composition of the galactose group on GT was determined to be 24% (Fig. 1B). Thiolation of GT with cysteine was achieved via amide bond formation between e NH2 on GT and eCOOH on cysteine, with free sulfhydryl content of 102.6  2.5 mmol/g and disulfide content of 166.0  18.0 mmol/g as quantified using Ellman’s reagent. 3.2. Preparation and characterization of GTC NPs GTC NPs were prepared through ionic crosslinking of cationic GTC with anionic crosslinkers TPP or HA and simultaneous encapsulation of siRNA. As summarized in Table 1, GTC NPs had particle sizes ranging from 140 nm to 160 nm with polydispersity index (PDI) of 0.100e0.250 and zeta potentials ranging from 20 mV to 42 mV. The GTC/HA NPs possessed larger particle size and higher

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J. Zhang et al. / Biomaterials 34 (2013) 3667e3677 Table 1 Particle size and zeta potential of GTC/TPP NPs and GTC/HA NPs containing NC siRNA with different weight ratios of GTC/crosslinker. Data were presented as mean  SD (n ¼ 3). Samplea

Particle size (nm)

GTC/TPP(10) NPs GTC/TPP(12.5) NPs GTC/TPP(15) NPs GTC/HA(5) NPs GTC/HA(7) NPs GTC/HA(9) NPs

147.6 144.6 147.2 158.5 154.3 153.3

a

     

5.5 7.1 7.8 1.7 2.2 2.0

Polydispersity index 0.131 0.194 0.249 0.128 0.126 0.102

     

0.013 0.027 0.004 0.023 0.015 0.038

Zeta potential (mV) 21.4 24.7 26.2 23.8 34.2 41.6

     

2.4 2.4 2.0 2.1 0.3 1.3

Values in parentheses represented the weight ratios of GTC/crosslinker.

zeta potential compared to GTC/TPP NPs. As depicted in Fig. 2A, compared to naked siRNA, GTC/HA(5) NPs, and GTC/HA(7) NPs, the migration of siRNA loaded into GTC/TPP(10) NPs, GTC/TPP(12.5) NPs, GTC/TPP(15) NPs, and GTC/HA(9) NPs was completely retarded, which suggested their high binding affinity for siRNA. Furthermore, the morphology of GTC/TPP(10) NPs and GTC/HA(9) NPs was observed by SEM (Fig. 2B), which exhibited spherical/subspherical structures. In addition, the particle size, zeta potential, and siRNA association ability of TC NPs were similar with those of GTC NPs (Table S1 and Fig. S1).

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Under high ionic strength condition (0.2 M), the particle size of GTC/TPP(10) NPs and GTC/HA(9) NPs was 144.0  13.6 nm and 792.4  15.8 nm, respectively, and their zeta potentials were 20.9  1.6 mV and 15.5  0.4 mV, respectively. Compared with the initial values shown in Table 1, it was found that elevating the ionic strength exerted inappreciable effects on the particle size and zeta potential of GTC/TPP(10) NPs, however, a 5-fold increment in particle size and 26.1 mV decrease in zeta potential of GTC/HA(9) NPs were noted, indicating that involvement of HA introduced structural instability to NPs under high ionic strength. To simulate the pH alterations that orally delivered NPs would encounter in the gastrointestinal tract, the pH of NPs suspensions was adjusted to 1.2 and back to 7.4. Following pH alteration, the particle size and zeta potential of GTC/TPP(10) NPs were 193.8  27.1 nm and 13.5  1.3 mV, respectively, and those of GTC/HA(9) NPs were 641.3  27.3 nm and 19.8  0.9 mV, respectively. Compared to GTC/ HA(9) NPs, GTC/TPP(10) NPs demonstrated better structural stability after oral administration. 3.3. siRNA integrity in physiological fluids As illustrated in Fig. 3, naked siRNA treated with serum and intestinal fluids was completely degraded, whereas siRNA encapsulated into GTC/TPP(10) NPs and TC/TPP(10) NPs was preserved as indicated by the migrating bands on the gel.

Fig. 2. (A) Agarose gel electrophoresis of naked siRNA as well as GTC/TPP NPs and GTC/HA NPs containing NC siRNA with different weight ratios of GTC/crosslinker; (B) SEM images of GTC/TPP(10) NPs and GTC/HA(9) NPs. Bars ¼ 200 nm.

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Furthermore, the addition of LA which possessed galactose residues or galactose substantially reduced uptake of FAM-NC siRNA encapsulated into GTC/TPP(10) NPs by 50.4% and 56.4%, respectively (Fig. 4C), implying that the enhanced uptake of GTC/TPP(10) NPs was attributed to the involvement of galactose-receptor recognition.

3.6. In vitro TNF-a knockdown

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The efficiency of in vitro gene knockdown induced by NPs containing siMap4k4 could be assessed by the change of correlative protein and mRNA production. TNF-a production in Raw 264.7 cells was measured by ELISA. As depicted in Fig. 5A, GTC/TPP(10) NPs containing siMap4k4 induced a significant decrease in TNFa secretion from LPS-stimulated Raw 264.7 cells compared to TC/ TPP(10) NPs containing siMap4k4 and Lipofectamine 2000/ siMap4k4 complexes. GTC/TPP(10) NPs containing Scr had no TNFa inhibition effect, indicating that the inhibition was sequence-

N

Binding of NPs to cell surface could be detected by the change of zeta potential of the cell. Surface negative charges of Raw 264.7 cells could be counteracted if bound by positively charged NPs, verifying the NPs binding onto cell membranes. As shown in Fig. 4A, GTC/TPP(10) NPs outperformed TC/TPP(10) NPs in the aspect of cell binding affinity for Raw 264.7 cells. Compared to the naked FAM-NC siRNA, FAM-NC siRNA encapsulated into NPs significantly facilitated the uptake by Raw 264.7 cell (Fig. 4B). Uptake amount of FAM-NC siRNA loaded into GTC/ TPP(10) NPs showed a 5.8- and 1.6-fold increment in comparison with naked FAM-NC siRNA and TC/TPP(10) NPs, respectively.

As shown in Fig. S2, cell viability was not affected by the addition of NPs or naked siMap4k4. Thus, TC/TPP(10) NPs and GTC/ TPP(10) NPs containing siMap4k4 were confirmed non-toxic at the test concentrations.

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3.4. Cell binding and cellular uptake

3.5. Cytotoxicity

(1

Fig. 3. Stability of naked NC siRNA and NPs containing NC siRNA after incubation with serum and intestinal fluids at 37 BC for 6 h evaluated by agarose gel electrophoresis. Lane 1: naked NC siRNA without any treatment; Lane 2: naked NC siRNA; Lane 3: GTC/ TPP(10) NPs; Lane 4: TC/TPP(10) NPs.

Fig. 4. Cell binding and cellular uptake of TC/TPP(10) NPs and GTC/TPP(10) NPs. (A) Cell binding of NPs containing siMap4k4 in LPS-activated Raw 264.7 cells; (B) cellular uptake of NPs containing FAM-NC siRNA by LPS-activated Raw 264.7 cells. Naked FAM-NC siRNA solution served as a control. (C) Uptake inhibition of GTC/TPP(10) NPs containing FAM-NC siRNA in LPS-activated Raw 264.7 cells following treatment with LA or galactose (Gal). Indicated values were mean  SD (n ¼ 3). *P < 0.05, **P < 0.01, and ***P < 0.001. #Statistically significant differences observed from the values of blank (###P < 0.001).

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TC/TPP(10) NPs was decreased with time extending. The TAMRANC siRNA distribution levels in plasma and liver were remarkably increased within 6 h post oral administration. Negligible amount of TAMRA-NC siRNA was detected in the heart and relatively low amounts of TAMRA-NC siRNA were delivered to spleen, lung, and kidney. Notably, GTC/TPP(10) NPs promoted the colonic distribution of TAMRA-NC siRNA compared to TC/TPP(10) NPs, which was evidenced by a 3-fold increment in distribution levels of TAMRANC siRNA in the colon. Accordingly, GTC/TPP(10) NPs showed lower distribution percentages in the small intestine, plasma, and liver than TC/TPP(10) NPs. These results demonstrated that GTC/ TPP(10) NPs localized orally delivered siRNA to the colon rather than entering systemic circulation via intestinal absorption. 3.8. Induction of colitis and oral delivery of siRNA

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Fig. 5. In vitro gene silencing of Scr loaded GTC/TPP(10) NPs (Scr), siMap4k4 loaded TC/ TPP(10) NPs and GTC/TPP(10) NPs, and Lipofectamine 2000/siMap4k4 complexes. (A) Inhibition of TNF-a production and (B) Map4k4 and TNF-a mRNA expression compared to control in LPS-activated Raw 264.7 cells. Raw 264.7 cells with treatment of LPS (10 ng/mL) served as a control. Indicated values were mean  SD (n ¼ 3). *P < 0.05 and **P < 0.01. #Statistically significant differences observed from the values of control (#P < 0.05, ##P < 0.01, and ###P < 0.001).

specific to TNF-a expression. Fig. 5B showed that Map4k4 and TNFa mRNA levels in LPS-stimulated Raw 264.7 cells. After treatment of GTC/TPP(10) NPs containing siMap4k4, the expression levels of Map4k4 and TNF-a mRNA were sharply decreased by 79.9% and 78.9%, respectively. The corresponding values of TC/TPP(10) NPs containing siMap4k4 treatment were about 46.3% and 62.1%, respectively. Neither Scr loaded GTC/TPP(10) NPs nor Lipofectamine 2000/siMap4k4 complexes showed any gene silencing effects in LPS-stimulated Raw 264.7 cells. These results indicated that siMap4k4 loaded GTC/TPP(10) NPs could suppress the expression of TNF-a in LPS-stimulated Raw 264.7 cells.

The levels of TNF-a production as well as Map4k4 and TNFa mRNA expression in colonic tissues were examined on day 7 in DSS-induced experimental UC. siMap4k4 loaded GTC/TPP(10) NPs and TC/TPP(10) NPs suppressed TNF-a production in colonic tissues, while GTC/TPP(10) NPs containing Scr failed to reduce TNFa production (Fig. 7A). The inhibition ratio of siMap4k4 loaded GTC/ TPP(10) NPs in colonic tissues was 77.6%, which was significantly higher than that of siMap4k4 loaded TC/TPP(10) NPs (61.1%). As shown in Fig. 7B, GTC/TPP(10) NPs containing siMap4k4 significantly decreased Map4k4 and TNF-a mRNA expression in colonic tissues by 92.1% and 69.0%, respectively, which was superior to TC/TPP(10) NPs containing siMap4k4. GTC/TPP(10) NPs containing Scr had no effect on Map4k4 and TNF-a mRNA expression. MPO is a primary inflammatory mediator in the pathogenesis of UC, of which the activity was related to the recruitment of leukocytes to the inflammation site of colon. As shown in Fig. 7C, the level of MPO activity was low in colonic tissues of normal mice, but was markedly increased in those of mice suffering from colitis. The elevated MPO activity induced by colitis was significantly inhibited by siMap4k4 loaded GTC/TPP(10) NPs and TC/TPP(10) NPs via oral administration. DSS-induced UC was characterized by sustained weight loss and shortened colon length. As shown in Fig. 7D, no significant body weight loss was observed for GTC NPs-treated mice as well as normal control mice. However, the colitis control mice and Scrtreated mice exhibited sharply drop in body weight. On day 7, the body weights of TC NPs-treated mice were notably lower than those of GTC NPs-treated mice. After 7 days of treatment with DSS, the colon lengths of colitis control mice and Scr-treated mice were significantly shorter than those of normal control mice, while treatment of siMap4k4 loaded GTC/TPP(10) NPs (GTC NPs-treated group) prevented the DSS-induced colon shortening (Fig. S3). H&E-stained colonic sections obtained from colitis control mice (Fig. 8B) revealed various histological characteristics in comparison with those of normal control mice (Fig. 8A), such as abnormality of crypts, loss of epithelial cells, and marked infiltration of mononuclear cells, in accordance with the previous report [21]. Scr-treated mice exhibited similar histopathological characteristics (Fig. 8C). The treatment of siMap4k4 loaded GTC/TPP(10) NPs (GTC NPstreated group) produced significant histological improvements, including inappreciable damage of epithelial cells and minimal infiltration of mononuclear cells (Fig. 8E), while TC NPs-treated group exhibited relatively poor therapeutic effects (Fig. 8D). 4. Discussion

3.7. Biodistribution As shown in Fig. 6, the distribution of TAMRA-NC siRNA in the small intestine and colon of orally delivered GTC/TPP(10) NPs and

The development of clinically available siRNA-based therapeutics has been stymied by two key challenges: 1) siRNA degradation before entry into the targeted cells in vivo [4] and 2) nonspecific

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J. Zhang et al. / Biomaterials 34 (2013) 3667e3677

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Fig. 6. Biodistribution of GTC/TPP(10) NPs and TC/TPP(10) NPs after oral administration in mice suffering from DSS-induced UC. UC was induced by replacing the drinking water of mice with 3% (w/v) DSS over a period of 8 days. Indicated values were mean  SD (n ¼ 4).

interactions of siRNA delivery carriers with non-targeted cells. The aim of this investigation was therefore to develop GTC-based NPs to resolve these problems. These siRNA-encapsulated GTC NPs were prepared through ionic gelation, and anticipated to have preferable stability in the gastrointestinal tract and be selectively internalized by activated macrophages after oral administration, thus silencing the endogenous gene involved in the development of DSS-induced UC. Activated macrophages could migrate to the inflammation sites of colon and produce proinflammatory cytokines such as TNF-a, which were believed to be involved in the pathogenesis of UC [2,19]. Therefore, the therapeutic strategy of depleting TNF-a production in activated macrophages holds great promises for the treatment of UC [19,20]. A previous report had demonstrated that galactosylated modification of chitosan remarkably enhanced the delivery efficiency of antisense oligonucleotide to activated macrophages bearing MGL [10]. Thiolated modification of chitosan could enhance its bioadhesion capacity [22], thereby promoting cellular uptake and gene transfection efficiency [23]. TMC with

good solubility under physiological condition could improve the gene transfection efficiency by facilitating cellular internalization [13]. Based on these advantages above-mentioned, we functionalized TMC with galactose and thiol to achieve GTC conjugates, which were assumed to target to activated macrophages, improve the silencing efficiency of siRNA, and suppress TNF-a production following oral delivery in mice suffering from UC. Through preliminary optimization of the reaction parameters including the feed ratios of TMC, LA, and cysteine, the pH of reaction medium, and reaction time, GTC conjugates with appropriate amounts of immobilized galactose residues and thiol groups were obtained [17,18]. GTC conjugates were synthesized via carbodiimide chemistry in aqueous solution, so the water-soluble EDC was chosen as a catalyst. To protect the reactive intermediate O-acylurea and prevent the racemization of GT, NHS was employed. Additionally, TEMED/HCl buffer system was adopted for GT synthesis to promote the galactosylation reaction [24]. Thiolation was applied as the last step of GTC synthesis since its mild reaction condition would not affect the structure of conjugated trimethyl and galactose, and

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Fig. 7. GTC/TPP(10) NPs containing siMap4k4 protected mice against DSS-induced UC by attenuating colonic TNF-a production. UC was induced by replacing the drinking water with a 3% (w/v) DSS. Normal control mice received water. Mice were given a daily gavage of Scr encapsulated GTC/TPP(10) NPs (Scr-treated group), siMap4k4 loaded TC/TPP(10) NPs (TC NPs-treated group), and siMap4k4 loaded GTC/TPP(10) NPs (GTC NPs-treated group) at a siRNA dose of 250 mg/kg for 6 consecutive days (days 0e5), or PBS (colitis control group). (A) On day 7, colonic TNF-a production was measured by ELISA. (n ¼ 8) (B) Map4k4 and TNF-a mRNA levels in mouse colonic tissue were detected by realtime-PCR. (n ¼ 3) (C) Colonic MPO activity after the 8-day treatment period. (n ¼ 8) (D) Body weight change. (n ¼ 8) *P < 0.05, **P < 0.01. #Statistically significant differences observed from the values of colitis control group (#P < 0.05 and ###P < 0.001).

avoid the possible oxidization of the thiol groups during the 72-h galactosylation process. As for oral delivery of siRNA, polymeric NPs are required to possess compact and stable structure to overcome multiple barriers in the gastrointestinal tract including high ionic strength, dramatic pH alteration, and ubiquitous endogenous enzymes [12]. In this study, GTC NPs were prepared though ionic gelation of cationic GTC conjugates with anionic crosslinkers, which occurred spontaneously in aqueous solution without sonication or heating, resulting in the preservation of the biological activity of siRNA [13,25]. TPP and HA were used as anionic crosslinkers in the preparation of GTC NPs due to their high biocompatibility and low immunogenicity [25,26]. Based on the criteria of smaller particle size, lower PDI, and higher binding efficiency with siRNA of NPs (Table 1 and Fig. 2A), GTC/TPP(10) NPs and GTC/HA(9) NPs were chosen for the measurement of structural stability. Compared to GTC/HA(9) NPs, it

was found that GTC/TPP(10) NPs could maintain their structural stability in the gastrointestinal tract, as evidenced by their slight changes in particle size and zeta potential under high ionic strength and pH alternation. The poor structural stability of GTC/HA(9) NPs might be ascribed to the reduction in the electrostatic attraction between the oppositely charged polyelectrolytes when salts acted as the counter-ions [27]. The desired structural stability of GTC/ TPP(10) NPs was also beneficial for protecting siRNA from degradation by shielding siRNA away from nucleases (Fig. 3), Considering their preferable structural stabilities and siRNA protection in the intestinal tract, GTC/TPP(10) NPs were used for the subsequent investigations. LPS was well-known to activate macrophages to over express MGL and secrete TNF-a at an elevated level [10,20], therefore, the present investigation adopted LPS-stimulated Raw 264.7 as activated macrophage models. Because of the high affinity between

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Fig. 8. Histopathological analysis was performed on H&E-stained sections of colons. Bars ¼ 200 mm. A: Normal control group; B: colitis control group; C: Scr-treated group; D: TC NPs-treated group; E: GTC NPs-treated group.

galactose residues on NPs and MGL receptors on macrophages, GTC/TPP(10) NPs exhibited higher cell binding affinity compared to TC/TPP(10) NPs. The higher cellular uptake amount of GTC/TPP(10) NPs in comparison with TC/TPP(10) NPs as well as the uptake inhibition effects following the addition of free LA and galactose collectively indicated that the MGL receptor-mediated endocytosis was involved in the internalization of GTC/TPP(10) NPs. Because galactosylated and thiolated modification reduced the positive charges on polymers, GTC/TPP(10) NPs and TC/TPP(10) NPs caused negligible cytotoxicity (Fig. S2), suggesting that they might not induce cell death during the gene transfection assessment. Aouadi et al. first demonstrated that Map4k4 was a new target for suppression of TNF-a expression in LPS-induced macrophages [6]. As a key upstream mediator of TNF-a action [28], the effective downregulation of Map4k4 mRNA expression would suppress TNFa production. Therefore, Map4k4 was chosen as the target gene in this investigation. Owing to their higher cell binding and cellular uptake, GTC/TPP(10) NPs containing siMap4k4 worked better in suppressing the TNF-a production as well as Map4k4 and TNFa mRNA expression in LPS-activated Raw 264.7 cells as compared to siMap4k4 loaded TC/TPP(10) NPs and Lipofectamine 2000/ siMap4k4 complexes (Fig. 5). Based on the promising in vitro evidence, we further evaluated the in vivo efficacy of orally delivered GTC/TPP(10) NPs in mice suffering from DSS-induced UC. Compared to TC/TPP(10) NPs, GTC/ TPP(10) NPs loaded with siMap4k4 more effectively delivered siRNA to the inflammatory colon (Fig. 6) and protected mice from DSS-induced UC after oral administration, as indicated by reduced colonic MPO activity, inappreciable body weight loss and colon length shortening, and significant histological improvement (Fig. 7, Fig. S3, and Fig. 8). Moreover, to achieve equivalent therapeutic efficacy, GTC/TPP(10) NPs loaded with siMap4k4 were orally administered at a siRNA dose of 250 mg/kg per day for 6 consecutive days, which represented a remarkable reduction in the administration dose used compared to previous studies [10,20,29]. This minimized therapeutic dose of siRNA would further reduce the risk for side effects owing to massive therapeutic exposure to non-

target tissues. The following facts contributed to the enhanced accumulation in the colon and improved therapeutic efficacy of orally delivered GTC/TPP(10) NPs containing siMap4k4 at a low dose. First, when mice were receiving DSS to develop UC, a large number of activated macrophages bearing MGL migrated to the colonic mucosa. GTC/TPP(10) NPs with a high affinity to MGL receptors can be primarily internalized by activated macrophages located in the focus of inflammation [10]. Second, the slight changes in particle sizes of GTC/TPP(10) NPs in the gastrointestinal tract could allow them to be accumulated in the inflamed colon tissue [30], and these NPs could strongly adhere to mucus layers based on the disulfide bond formation with mucus glycoprotein [17]. Third, GTC/TPP(10) NPs could preserve the bioactivity of siMap4k4 before internalization into activated macrophages residing in the inflammation sites of colon. Despite the robust in vitro RNAi efficiencies of siMap4k4 loaded Lipofectamine 2000 and adenovirus [28,31,32], the successful in vivo applications of these carriers might be impeded due to the poor in vivo stability of lipid and safety concerns [4], respectively. The b1,3-D-glucan particles (GeRPs) delivered Map4k4 siRNA to the systemic circulation and silenced gene expression in murine macrophages after oral administration [6]. However, the GeRPs were difficult to be produced with uniformity [33]. As comparisons, the high efficiency of targeting to activated macrophages, preferable structural stability and siRNA protection, low cytotoxicity, and simplicity in formulation collectively indicated the potentiality of GTC NPs for oral delivery of Map4k4 siRNA to suppress colonic inflammation. 5. Conclusions Targeted therapeutic strategy using NPs was developed based on the pathophysiologic characteristics of inflammatory bowel diseases. Map4k4 could be selected as a target for the treatment of DSS-induced UC. Orally delivered siMap4k4 loaded GTC/TPP(10) NPs was effective in protecting mice from DSS-induced UC at a relatively low therapeutic dose by attenuating colonic TNF-

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a production. It was expected that GTC/TPP(10) NPs might be a potential carrier for oral delivery of siRNA because of their desired structural stability, enhanced cell binding and cellular uptake in activated macrophages, low cytotoxicity, high transfection efficiency in vitro, and direct delivery to the focus of disease. Acknowledgments This work was funded by grants from National Natural Science Foundation of China (81072595, 81172995, and 51173029). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.biomaterials.2013.01.079. References [1] Laroui H, Wilson DS, Dalmasso G, Salaita K, Murthy N, Sitaraman SV, et al. Nanomedicine in GI. Am J Physiol-Gastr L 2011;300:G371e83. [2] Papadakis KA, Targan SR. Role of cytokines in the pathogenesis of inflammatory bowel disease. Annu Rev Med 2000;51:289e98. [3] D’Haens G. Anti-TNF therapy for Crohn’s disease. Curr Pharm Design 2003;9: 289e94. [4] Singha K, Namgung R, Kim WJ. Polymers in small-interfering RNA delivery. Nucleic Acid Ther 2011;21:133e47. [5] Howard KA, Paludan SR, Behlke MA, Besenbacher F, Deleuran B, Kjems J. Chitosan/siRNA nanoparticle-mediated TNF-a knockdown in peritoneal macrophages for anti-inflammatory treatment in a murine arthritis model. Mol Ther 2009;17:162e8. [6] Aouadi M, Tesz GJ, Nicoloro SM, Wang MX, Chouinard M, Soto E, et al. Orally delivered siRNA targeting macrophage Map4k4 suppresses systemic inflammation. Nature 2009;458:1180e4. [7] Machida N, Umikawa M, Takei K, Sakima N, Myagmar BE, Taira K, et al. Mitogenactivated protein kinase kinase kinase kinase 4 as a putative effector of Rap2 to activate the c-Jun N-terminal kinase. J Biol Chem 2004;279:15711e4. [8] van Vliet SJ, Saeland E, van Kooyk Y. Sweet preferences of MGL: carbohydrate specificity and function. Trends Immunol 2008;29:83e90. [9] Coombs PJ, Taylor ME, Drickamer K. Two categories of mammalian galactosebinding receptors distinguished by glycan array profiling. Glycobiology 2006; 16:1Ce7C. [10] Zuo LS, Huang Z, Dong L, Xu LQ, Zhu Y, Zeng K, et al. Targeting delivery of antiTNF alpha oligonucleotide into activated colonic macrophages protects against experimental colitis. Gut 2010;59:470e9. [11] Morishita M, Peppas NA. Is the oral route possible for peptide and protein drug delivery? Drug Discov Today 2006;11:905e10. [12] O’Neill MJ, Bourre L, Melgar S, O’Driscoll CM. Intestinal delivery of non-viral gene therapeutics: physiological barriers and preclinical models. Drug Discov Today 2011;16:203e18. [13] Dehousse V, Garbacki N, Jaspart S, Castagne D, Piel G, Colige A, et al. Comparison of chitosan/siRNA and trimethylchitosan/siRNA complexes behaviour in vitro. Int J Biol Macromol 2010;46:342e9. [14] Kean T, Roth S, Thanou M. Trimethylated chitosans as non-viral gene delivery vectors: cytotoxicity and transfection efficiency. J Control Release 2005;103: 643e53.

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