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Biomaterials 34 (2013) 3667e3677

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Biomaterials

journal homepage: www.elsevier .com/locate/biomater ia ls

Galactosylated trimethyl chitosanecysteine nanoparticles loaded with Map4k4siRNA 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

Article history:Received 8 January 2013Accepted 24 January 2013Available online 15 February 2013

Keywords:Chitosan derivate nanoparticlesGalactosylated modificationsiRNAOral administrationUlcerative colitisTargeted gene delivery

* Corresponding author. Tel.: þ86 21 6564 3797; faE-mail address: chyin@fudan.edu.cn (C. Yin).

0142-9612/$ e see front matter � 2013 Elsevier Ltd.http://dx.doi.org/10.1016/j.biomaterials.2013.01.079

a b s t r a c t

Galactosylated trimethyl chitosanecysteine (GTC) nanoparticles (NPs) were developed for oral deliveryof a mitogen-activated protein kinase kinase kinase kinase 4 (Map4k4) siRNA (siMap4k4) to the activatedmacrophages for treatment of dextran sulfate sodium (DSS)-induced ulcerative colitis (UC). siRNA loadedGTC 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 theirphysicochemical characteristics. GTC/TPP NPs with smaller particle size and lower zeta potential pos-sessed superior structural stability in gastrointestinal environment compared to GTC/HA NPs. Cellularuptake of GTC/TPP NPs in activated macrophages was significantly enhanced compared to trimethylchitosan-cysteine (TC)/TPP NPs owing to galactose receptor-mediated endocytosis. The in vitro andin vivo gene knockdown measurement showed that siMap4k4 loaded GTC/TPP NPs effectively inhibitedTNF-a production, which remarkably outperformed siMap4k4 loaded TC/TPP NPs. Compared to TC/TPPNPs, GTC/TPP NPs more efficiently promoted the distribution of siRNA in ulcerative colon following oraladministration. Daily oral administration of GTC/TPP NPs containing siMap4k4 significantly improvedDSS-induced body weight loss, colon length shortening, and increase of myeloperoxidase activity. Thisstudy would provide an effective approach for oral siRNA delivery in the treatment of inflammatorybowel diseases.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Ulcerative colitis (UC) is a chronic inflammatory bowel diseasefor which existing treatments are largely limited by low effective-ness and severe systemic side effects [1]. Tumor necrosis factoralpha (TNF-a) as a proinflammatory cytokine plays a central role inthe onset and progression of UC [2]. Intervention of the inflam-matory responses with TNF-a monoclonal antibodies has becomeamajor successful immunotherapy in the clinic [3], which however,shows limitations including severe side effects caused by theantibody and high cost [4]. Small interfering RNA (siRNA)-mediatedknockdown of functional protein at the messenger RNA (mRNA)level offers an alternative therapeutic approach to various inflam-matory diseases owing to its high specificity and efficiency [5,6]. Asan important protein kinase of the mammalian STE20/MAP4Kfamily [7], mitogen-activated protein kinase kinase kinase kinase 4(Map4k4) has been demonstrated to be a key upstreammediator of

x: þ86 21 5552 2771.

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TNF-a action [6]. Therefore, it was hypothesized that gene knock-down targeting Map4k4 for suppressing TNF-a production wouldprovide a promising siRNA-based therapeutic strategy for thetreatment of UC.

To achieve RNAi for clinical applications, effective carriers areessential for delivering intact siRNA to the cytoplasm of target cells,based on the specific pathophysiology of each disease. In inflam-matory bowel diseases, such as UC, TNF-a secreted bymacrophagesis a major contributor to the development of bowel inflammation[2], therefore RNAi therapeutics targeting macrophage Map4k4present a potential approach for the treatment of UC. Macrophagegalactose-type lectin (MGL) is expressed at high levels by activatedmacrophages under inflammatory conditions [8,9]. Zuo et al. [10]demonstrated the targeted delivery ability of the galactosylatedlow molecular weight chitosan (CS)/antisense oligonucleotidecomplex into activated macrophages and its therapeutic action inexperimental colitis by intracolonic administration. For the man-agement of UC as chronic inflammation, high dose frequency ofnucleic acid drugs would be required to achieve continuouslycurative effect [1]. Considering patient compliance, oral delivery ofsiRNA will be an optimal alternative [11]. However, the harsh

Abbreviations

CS chitosanDSS dextran sulfate sodiumFAM-NC siRNA FAM-labeled negative control small interfering

RNAGT galactosylated trimethyl chitosanGTC galactosylated trimethyl chitosanecysteineGTC/HA NPs hyaluronic acid-crosslinked GTC nanoparticlesGTC/TPP NPs tripolyphosphate-crosslinked GTC nanoparticlesLA lactobionic acidLPS lipopolysaccharideMap4k4 mitogen-activated protein kinase kinase kinase kinase 4

MGL macrophage galactose-type lectinmRNA messenger RNANC siRNAnegative control small interfering RNAsiRNA small interfering RNAsiMap4k4Map4k4 siRNAScr scrambled siMap4k4TMC N-trimethyl chitosanTC TMC-cysteine conjugatesTC/HA NPs hyaluronic acid-crosslinked TC nanoparticlesTC/TPP NPs tripolyphosphate-crosslinked TC nanoparticlesTNF-a tumor necrosis factor alphaUC ulcerative colitis

J. Zhang et al. / Biomaterials 34 (2013) 3667e36773668

environment of the gastrointestinal tract including high ionicstrength, dramatic pH alteration, and ubiquitous digestive enzymewould potentially damage siRNA integrity, thus leading to theinefficacy of RNAi after oral delivery [12].

CS as a carrier for oral delivery of siRNA has many advantagesincluding its strong nucleic acid affinity, bioadhesion, and bio-degradability [5]. However, its insolubility at neutral and alkalineenvironment limits its application in oral delivery of nucleic acid[13]. To improve the solubility of CS over a wide pH range, trime-thylationmodification of CS has been introduced [14]. Furthermore,thiolated modification of CS with thiol-bearing compounds canenhance its bioadhesion capacity through covalent bonding withmucin glycoproteins [15]. In the present investigation, galactosy-lated trimethyl chitosanecysteine (GTC) was developed as anactivated macrophages-targeting carrier for oral siRNA admin-istration. The siRNA loaded GTC nanoparticles (NPs) were preparedthrough ionic gelation with tripolyphosphate (TPP) or hyaluronicacid (HA), and characterized in terms of particle size, zeta potential,and siRNA integrity in physiological environment. In vitro assess-ment of cell binding, cellular uptake, cytotoxicity, and geneknockdown efficiency of GTC NPs were carried out in lip-opolysaccharide (LPS)-activated Raw 264.7 cells. Biodistributionand in vivo RNAi efficiency of orally delivered siRNA loaded GTC NPswere determined in mice suffering from dextran sulfate sodium(DSS)-induced UC.

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

2. Materials and methods

2.1. Materials

CS (deacetylation degree of 85% and molecular weight (Mw) of 200 kDa) wasobtained 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 Exper-imental Reagent Co., Ltd. (Shanghai, China). LPS (obtained from Escherichia coli) waspurchased from Sigma (St. Louis, MO, USA). Lipofectamine 2000 was obtained fromInvitrogen (Carlsbad, USA). DSS was from MP Biomedicals (Mw ¼ 36e50 kDa, Ill-kirch, France). All other reagents were of analytic grade.

Map4k4 siRNA (siMap4k4), scrambled siMap4k4 (Scr), and negative controlsiRNA (NC siRNA) duplexes were supplied by GenePharma (Shanghai, China) anddissolved in DEPC-treated water before use. siMap4k4 contained the sequences ofsense 50-GACCAACUCUGGCUUGUUA-30 and antisense 50-UAACAAGCCAGA-GUUGGUC-30 . The Scr contained the scrambled sequences of sense 50-CAGUCGC-GUUUGCGACUGG-30 and antisense 50-CCAGUCGCAAACGCGACUG-30 . The NC siRNAcontained the sequences of sense 50-UUCUCCGAACGUGUCACGUTT-30 and antisense50-ACGUGACACGUUCGGAGAATT-30 . FAM-labeled NC siRNA (FAM-NC siRNA) wasused 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, and36B4 were synthesized by Shanghai Sangon Biotech Co., Ltd. (Shanghai, China) andthe sequences are as follows: CCCTCACACTCAGATCATCTTCT (TNF-a forward);GCTACGACGTGGGCTACAG (TNF-a reverse); CATCTCCAGGGAAATCCTCAGG (Map4k4forward); TTCTGTAGTCGTAAGTGGCGTCTG (Map4k4 reverse); TCCAGGCTTTGGG-CATCAC (36B4 forward); CTTTATCAGCTGCACATCACTCAGA (36B4 reverse).

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 SlaccasExperimental Animals Co., Ltd. Animal experiments were performed according tothe Guiding Principles for the Care and Use of Experiment Animals in Fudan Uni-versity. The study protocol was reviewed and approved by the Institutional AnimalCare and Use Committee, Fudan University.

2.3. Preparation of GTC conjugates

GTC was synthesized through a three-step route. Firstly, CS was reacted withmethyl iodide (CH3I) in methyl-2-pyrrolidone (NMP)/NaOH solution for 120 min at65 �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 describedby Park et al. [16]. Briefly, LA (0.25 mmol) was dissolved in 3 mL of N,N,N0 ,N0-tet-ramethylethylenediamine (TEMED)/HCl buffer solution (10 mM, pH 4.7), into which1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC)/N-hydrox-ysuccinimide (NHS) (molar ratio ¼ 1:1) were added at a final concentration of300 mM. The reaction was allowed for 2 h under stirring at room temperature, fol-lowed 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 purifiedvia ultrafiltration (MWCO 10 kDa). Finally, GT was reacted with cysteine at pH 5.0and 1:2 (w/w) for 5 h at room temperature as mediated by EDC/NHS (80 mM), andthe resultant GTC was dialyzed against pH 5.0 HCl solution and lyophilized. Theamount of immobilized sulfhydryl was determined with Ellman’s reagent [17]. Thetrimethylation degree of TMC and galactosylated modification degree of GTC werecalculated by 1H NMR (AVANCE DMX 500, Bruker, Germany) [14,18]. As compari-sons, TMC-cysteine conjugates (TC) were prepared as previously described [17].

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-crosslinkedGTC 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 mix-ture 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 withHA solution (1 mg/mL) at 1:10 (w/w). The GTC solution (6 mg/mL) was addeddropwise into the mixture under stirring at the GTC/HA weight ratio of 5:1, 7:1, and9:1, 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 wereprepared 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 ZetasizerNano (Malvern, Worcestershire, UK). The association of siRNA with the NPs wasmonitored with gel retardation assay on 4% agarose gel electrophoresis stained withethidium bromide (0.5 mg/mL), and the electrophoresis was performed at 56 V for1 h. Morphology of GTC/TPP(10) NPs and GTC/HA(9) NPs was observed with scan-ning electron microscopy (SEM, Vega TS5136, Tescan, Czechoslovakia).

J. Zhang et al. / Biomaterials 34 (2013) 3667e3677 3669

Stability of GTC/TPP(10) NPs and GTC/HA(9) NPs against ionic strength and pHalteration was evaluated in terms of particle size and zeta potential. Ionic strengthwas adjusted to 0.2 M using NaCl solution. To mimic the pH alteration in the gas-trointestinal tract, the pH of GTC/TPP(10) NPs and GTC/HA(9) NPs suspensions wasadjusted to 1.2 using 1 M HCl solution, and then back to pH 7.4 using 1 M NaOHsolution.

2.5. siRNA integrity in physiological fluids

Blood was collected from the orbital sinus of male C57BL/6 mice and cen-trifugated at 12,000 rpm and 4 �C for 4min. The supernatant was collected as serum.Ten milliliters of phosphate buffered saline (PBS, pH 7.4) was used to rinse the in-testinal lumen. The obtained solution was centrifugated at 12,000 rpm and 4 �C for20 min, and the supernatant was collected as intestinal fluids. Serum and intestinalfluids were stored at �20 �C before use.

To assess the stability of encapsulated siRNA in the cell culture media and in-testinal lumen, GTC/TPP(10) NPs, TC/TPP(10) NPs, and naked siRNA solution con-taining 400 ng of NC siRNA were mixed with equal volume of serum and intestinalfluids 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. siRNAintegrity was subsequently evaluated on 2% agarose gel electrophoresis. Naked NCsiRNA 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. Cellpellets were washed three times with PBS and resuspended in isotonic buffer so-lution (glucose 44.4 g/L, KCl 0.2 g/L, Na2HPO4$12H2O 2.9 g/L, and KH2PO4 0.2 g/L) ata concentration of 0.5e1 � 106 cells/mL. GTC/TPP(10) NPs and TC/TPP(10) NPscontaining 0.7 mg of siMap4k4was mixed with 1 mL of Raw 264.7 cell suspension inthe presence of 10 ng/mL LPS, and was incubated at 37 �C for 2 h. After the solutionwas 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 withisotonic 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 andincubated for 24 h. The culture medium was replaced by the fresh serum-freeDMEM. GTC/TPP(10) NPs and TC/TPP(10) NPs containing 400 ng of FAM-NC siRNAwere 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 microplatereader (Thermofisher, USA) (lex¼ 480 nm, lem¼ 520 nm) and protein content by theLowry method. Naked FAM-NC siRNA served as a control. Uptake was expressed asthe 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 andgalactose at a final concentration of 100 and 200 mM were added 1 h prior to NPsapplication, respectively, and incubated with the cells throughout the uptakemeasurement. Results were expressed as the percentage uptake of the controlwhere cells were incubated with NPs in the absence of LA and galactose at 37 �Cfor 4 h.

2.8. Cytotoxicity

Raw 264.7 cells were seeded on 96-well plates at a density of 1 � 104 cells perwell, 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 methyltetrazolium (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 andincubated for 24 h. The culture medium was replaced by the fresh serum-freeDMEM. GTC/TPP(10) NPs and TC/TPP(10) NPs loaded with siMap4k4 were addedat 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 andnegative controls, respectively. After a 4-h incubation, the culture medium wasdiscarded and a further culture in serum-containing medium was allowed for 20 hbefore LPS (10 ng/mL) stimulation for 5 h. The supernatant of culture medium wascollected for the quantification of the extracellular TNF-a production by ELISA. Inaddition, RNA was isolated from the transfected Raw 264.7 cells according to theTrizol reagent protocol (Invitrogen, USA), and cDNAwas synthesized from 500 ng ofthe total RNA using PrimeScript�RT reagent kit (Takara Biotechnology Co. Ltd.,China) according to themanufacturer’s instructions. Synthesized cDNA, forward andreverse primers, and the SYBR Premix Ex Taq� (Takara Biotechnology Co. Ltd.,China) were run on the ABI PRISM 7900HT Real-Time PCR system (Applied

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 overa period of 8 days. C57BL/6 mice suffering from DSS-induced UC were given a gav-age 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 ofmice and plasma was isolated via centrifugation. Mice were sacrificed and heart,liver, spleen, lung, kidney, small intestine, and colon were taken, weighed, andhomogenized with RIPA lysis buffer. The homogenate was centrifugated at3000 rpm for 15 min, and the amount of TAMRA-NC siRNA in the supernatant aswell as plasmawas 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 siRNAdelivered.

2.11. In vivo RNAi against UC via gavage

UC was induced by replacing the drinking water of mice with 3% (w/v) DSS overaperiodof 8days. Normal controlmice receivedwater.Micewere givenadailygavageof 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-treatedgroup) at the dose of 250 mg siRNA/kg body weight for 6 consecutive days (days 0e5), or PBS (colitis control group). The dose adopted in this investigation (250 mgsiRNA/kg/day) was comparable to that inWilson et al.’s study (230 mg siRNA/kg/day)[19], which was relatively low among anti-inflammation studies involving TNF-aknockdown [19,20]. Bodyweightwasmonitored daily. Micewere sacrificed on day 7,colonic segment of each mouse was gathered and homogenized with cold PBS (pH7.4). The homogenate was centrifuged at 12,000 rpm and 4 �C for 20 min, and thesupernatant was collected for TNF-a quantification by ELISA. For the evaluation ofMap4k4 and TNF-a mRNA level in colonic tissues, colonic segments were cut intosmall pieces, washed with saline, immersed in RNAlater solution (Qiagen, USA) for24 h, and homogenized in liquid nitrogen. RNA in the cell lysate was extracted withTrizol reagent and intracellular Map4k4 and TNF-a mRNA levels in colonic tissueswere thereaftermonitoredby realtime-PCR.Neutrophil infiltration into the colonwasevaluated by measuring myeloperoxidase (MPO) activity in colonic tissues. Fur-thermore, 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 wasperformed by Student’s unpaired t test between two groups or One-Way Analysis ofVariance (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 trime-thylation, galactosylation, and thiolation of chitosan as shown inFig.1A. After CS was allowed to react with CH3I to achieve TMCwitha trimethylation degree of 34% as determined by 1H NMR (Fig. 1B)[14], galactosylation was performed to yield GT. The grafting effi-ciency of galactose residues on GT was calculated by comparing thepeak area of eCHe on LA (4.5 ppm) to that of eNHeOCeCH3 onchitosan (2.0 ppm) [18]. The chemical composition of the galactosegroup on GT was determined to be 24% (Fig. 1B). Thiolation of GTwith cysteine was achieved via amide bond formation between e

NH2 on GT and eCOOH on cysteine, with free sulfhydryl content of102.6 � 2.5 mmol/g and disulfide content of 166.0 � 18.0 mmol/g asquantified using Ellman’s reagent.

3.2. Preparation and characterization of GTC NPs

GTC NPs were prepared through ionic crosslinking of cationicGTC with anionic crosslinkers TPP or HA and simultaneousencapsulation of siRNA. As summarized in Table 1, GTC NPs hadparticle sizes ranging from 140 nm to 160 nm with polydispersityindex (PDI) of 0.100e0.250 and zeta potentials ranging from 20 mVto 42mV. The GTC/HA NPs possessed larger particle size and higher

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J. Zhang et al. / Biomaterials 34 (2013) 3667e36773670

Table 1Particle size and zeta potential of GTC/TPP NPs and GTC/HA NPs containing NC siRNAwith different weight ratios of GTC/crosslinker. Data were presented as mean � SD(n ¼ 3).

Samplea Particle size(nm)

Polydispersityindex

Zeta potential(mV)

GTC/TPP(10) NPs 147.6 � 5.5 0.131 � 0.013 21.4 � 2.4GTC/TPP(12.5) NPs 144.6 � 7.1 0.194 � 0.027 24.7 � 2.4GTC/TPP(15) NPs 147.2 � 7.8 0.249 � 0.004 26.2 � 2.0GTC/HA(5) NPs 158.5 � 1.7 0.128 � 0.023 23.8 � 2.1GTC/HA(7) NPs 154.3 � 2.2 0.126 � 0.015 34.2 � 0.3GTC/HA(9) NPs 153.3 � 2.0 0.102 � 0.038 41.6 � 1.3

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

J. Zhang et al. / Biomaterials 34 (2013) 3667e3677 3671

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, themigration of siRNA loaded into GTC/TPP(10) NPs, GTC/TPP(12.5)NPs, GTC/TPP(15) NPs, and GTC/HA(9) NPs was completely retar-ded, which suggested their high binding affinity for siRNA. Fur-thermore, the morphology of GTC/TPP(10) NPs and GTC/HA(9) NPswas observed by SEM (Fig. 2B), which exhibited spherical/sub-spherical structures. In addition, the particle size, zeta potential,and siRNA association ability of TC NPs were similar with those ofGTC NPs (Table S1 and Fig. S1).

Fig. 2. (A) Agarose gel electrophoresis of naked siRNA as well as GTC/TPP NPs and GTC/HA Nof GTC/TPP(10) NPs and GTC/HA(9) NPs. Bars ¼ 200 nm.

Under high ionic strength condition (0.2 M), the particle size ofGTC/TPP(10) NPs and GTC/HA(9) NPs was 144.0 � 13.6 nm and792.4 � 15.8 nm, respectively, and their zeta potentials were20.9 � 1.6 mV and 15.5 � 0.4 mV, respectively. Compared with theinitial values shown in Table 1, it was found that elevating the ionicstrength exerted inappreciable effects on the particle size and zetapotential of GTC/TPP(10) NPs, however, a 5-fold increment in par-ticle size and 26.1 mV decrease in zeta potential of GTC/HA(9) NPswere noted, indicating that involvement of HA introduced struc-tural instability to NPs under high ionic strength. To simulate thepH alterations that orally delivered NPs would encounter in thegastrointestinal tract, the pH of NPs suspensions was adjusted to 1.2and back to 7.4. Following pH alteration, the particle size and zetapotential of GTC/TPP(10) NPs were 193.8 � 27.1 nm and13.5 � 1.3 mV, respectively, and those of GTC/HA(9) NPs were641.3 � 27.3 nm and 19.8 � 0.9 mV, respectively. Compared to GTC/HA(9) NPs, GTC/TPP(10) NPs demonstrated better structural sta-bility after oral administration.

3.3. siRNA integrity in physiological fluids

As illustrated in Fig. 3, naked siRNA treated with serum andintestinal fluids was completely degraded, whereas siRNA encap-sulated into GTC/TPP(10) NPs and TC/TPP(10) NPs was preserved asindicated by the migrating bands on the gel.

Ps containing NC siRNA with different weight ratios of GTC/crosslinker; (B) SEM images

Fig. 3. Stability of naked NC siRNA and NPs containing NC siRNA after incubation withserum 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.

J. Zhang et al. / Biomaterials 34 (2013) 3667e36773672

3.4. Cell binding and cellular uptake

Binding of NPs to cell surface could be detected by the change ofzeta potential of the cell. Surface negative charges of Raw 264.7cells 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 cellbinding affinity for Raw 264.7 cells.

Compared to the naked FAM-NC siRNA, FAM-NC siRNA encap-sulated into NPs significantly facilitated the uptake by Raw 264.7cell (Fig. 4B). Uptake amount of FAM-NC siRNA loaded into GTC/TPP(10) NPs showed a 5.8- and 1.6-fold increment in comparisonwith naked FAM-NC siRNA and TC/TPP(10) NPs, respectively.

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Fig. 4. Cell binding and cellular uptake of TC/TPP(10) NPs and GTC/TPP(10) NPs. (A) Cell bindNPs containing FAM-NC siRNA by LPS-activated Raw 264.7 cells. Naked FAM-NC siRNA solusiRNA in LPS-activated Raw 264.7 cells following treatment with LA or galactose (Gal). Indicasignificant differences observed from the values of blank (###P < 0.001).

Furthermore, the addition of LAwhich possessed galactose residuesor galactose substantially reduced uptake of FAM-NC siRNAencapsulated into GTC/TPP(10) NPs by 50.4% and 56.4%, respec-tively (Fig. 4C), implying that the enhanced uptake of GTC/TPP(10)NPs was attributed to the involvement of galactose-receptorrecognition.

3.5. Cytotoxicity

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

3.6. In vitro TNF-a knockdown

The efficiency of in vitro gene knockdown induced by NPs con-taining siMap4k4 could be assessed by the change of correlativeprotein andmRNA production. TNF-a production in Raw 264.7 cellswas measured by ELISA. As depicted in Fig. 5A, GTC/TPP(10) NPscontaining siMap4k4 induced a significant decrease in TNF-a 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 TNF-a inhibition effect, indicating that the inhibition was sequence-

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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 comparedto 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).

J. Zhang et al. / Biomaterials 34 (2013) 3667e3677 3673

specific to TNF-a expression. Fig. 5B showed that Map4k4 and TNF-amRNA levels in LPS-stimulated Raw 264.7 cells. After treatment ofGTC/TPP(10) NPs containing siMap4k4, the expression levels ofMap4k4 and TNF-a mRNA were sharply decreased by 79.9% and78.9%, respectively. The corresponding values of TC/TPP(10) NPscontaining siMap4k4 treatment were about 46.3% and 62.1%,respectively. Neither Scr loaded GTC/TPP(10) NPs nor Lipofect-amine 2000/siMap4k4 complexes showed any gene silencing ef-fects in LPS-stimulated Raw 264.7 cells. These results indicated thatsiMap4k4 loaded GTC/TPP(10) NPs could suppress the expression ofTNF-a in LPS-stimulated Raw 264.7 cells.

3.7. Biodistribution

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

TC/TPP(10) NPs was decreased with time extending. The TAMRA-NC siRNA distribution levels in plasma and liver were remarkablyincreased within 6 h post oral administration. Negligible amount ofTAMRA-NC siRNA was detected in the heart and relatively lowamounts of TAMRA-NC siRNA were delivered to spleen, lung, andkidney. Notably, GTC/TPP(10) NPs promoted the colonic distribu-tion of TAMRA-NC siRNA compared to TC/TPP(10) NPs, which wasevidenced by a 3-fold increment in distribution levels of TAMRA-NC siRNA in the colon. Accordingly, GTC/TPP(10) NPs showedlower distribution percentages in the small intestine, plasma, andliver than TC/TPP(10) NPs. These results demonstrated that GTC/TPP(10) NPs localized orally delivered siRNA to the colon ratherthan entering systemic circulation via intestinal absorption.

3.8. Induction of colitis and oral delivery of siRNA

The levels of TNF-a production as well as Map4k4 and TNF-a mRNA expression in colonic tissues were examined on day 7 inDSS-induced experimental UC. siMap4k4 loaded GTC/TPP(10) NPsand TC/TPP(10) NPs suppressed TNF-a production in colonic tis-sues, while GTC/TPP(10) NPs containing Scr failed to reduce TNF-a production (Fig. 7A). The inhibition ratio of siMap4k4 loaded GTC/TPP(10) NPs in colonic tissues was 77.6%, which was significantlyhigher than that of siMap4k4 loaded TC/TPP(10) NPs (61.1%). Asshown in Fig. 7B, GTC/TPP(10) NPs containing siMap4k4 sig-nificantly decreased Map4k4 and TNF-a mRNA expression incolonic tissues by 92.1% and 69.0%, respectively, whichwas superiorto TC/TPP(10) NPs containing siMap4k4. GTC/TPP(10) NPs contain-ing Scr had no effect on Map4k4 and TNF-a mRNA expression.

MPO is a primary inflammatory mediator in the pathogenesis ofUC, of which the activity was related to the recruitment of leuko-cytes to the inflammation site of colon. As shown in Fig. 7C, thelevel of MPO activity was low in colonic tissues of normal mice, butwas markedly increased in those of mice suffering from colitis. Theelevated MPO activity induced by colitis was significantly inhibitedby siMap4k4 loaded GTC/TPP(10) NPs and TC/TPP(10) NPs via oraladministration.

DSS-induced UC was characterized by sustained weight loss andshortened colon length. As shown in Fig. 7D, no significant bodyweight loss was observed for GTC NPs-treated mice as well asnormal control mice. However, the colitis control mice and Scr-treated mice exhibited sharply drop in body weight. On day 7, thebody weights of TC NPs-treated mice were notably lower thanthose of GTC NPs-treated mice. After 7 days of treatment with DSS,the colon lengths of colitis control mice and Scr-treated mice weresignificantly shorter than those of normal control mice, whiletreatment of siMap4k4 loaded GTC/TPP(10) NPs (GTC NPs-treatedgroup) 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 comparisonwith those of normal control mice (Fig. 8A), such as abnormality ofcrypts, loss of epithelial cells, and marked infiltration of mononu-clear cells, in accordance with the previous report [21]. Scr-treatedmice exhibited similar histopathological characteristics (Fig. 8C).The treatment of siMap4k4 loaded GTC/TPP(10) NPs (GTC NPs-treated group) produced significant histological improvements,including inappreciable damage of epithelial cells and minimalinfiltration of mononuclear cells (Fig. 8E), while TC NPs-treatedgroup exhibited relatively poor therapeutic effects (Fig. 8D).

4. Discussion

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

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J. Zhang et al. / Biomaterials 34 (2013) 3667e36773674

interactions of siRNA delivery carriers with non-targeted cells. Theaim of this investigation was therefore to develop GTC-based NPsto resolve these problems. These siRNA-encapsulated GTC NPswere prepared through ionic gelation, and anticipated to havepreferable stability in the gastrointestinal tract and be selectivelyinternalized by activated macrophages after oral administration,thus silencing the endogenous gene involved in the developmentof DSS-induced UC.

Activated macrophages could migrate to the inflammation sitesof 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 pro-duction in activated macrophages holds great promises for thetreatment of UC [19,20]. A previous report had demonstrated thatgalactosylated modification of chitosan remarkably enhanced thedelivery efficiency of antisense oligonucleotide to activated mac-rophages bearing MGL [10]. Thiolated modification of chitosancould enhance its bioadhesion capacity [22], thereby promotingcellular uptake and gene transfection efficiency [23]. TMC with

good solubility under physiological condition could improve thegene transfection efficiency by facilitating cellular internalization[13]. Based on these advantages above-mentioned, we functional-ized TMCwith galactose and thiol to achieve GTC conjugates, whichwere assumed to target to activated macrophages, improve thesilencing efficiency of siRNA, and suppress TNF-a production fol-lowing oral delivery in mice suffering from UC. Through pre-liminary optimization of the reaction parameters including the feedratios of TMC, LA, and cysteine, the pH of reaction medium, andreaction time, GTC conjugates with appropriate amounts ofimmobilized galactose residues and thiol groups were obtained[17,18]. GTC conjugates were synthesized via carbodiimide chem-istry in aqueous solution, so the water-soluble EDC was chosen asa catalyst. To protect the reactive intermediate O-acylurea andprevent the racemization of GT, NHS was employed. Additionally,TEMED/HCl buffer systemwas adopted for GT synthesis to promotethe galactosylation reaction [24]. Thiolation was applied as the laststep of GTC synthesis since its mild reaction condition would notaffect 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 waterwith 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 controlgroup). (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 thevalues of colitis control group (#P < 0.05 and ###P < 0.001).

J. Zhang et al. / Biomaterials 34 (2013) 3667e3677 3675

avoid the possible oxidization of the thiol groups during the 72-hgalactosylation process.

As for oral delivery of siRNA, polymeric NPs are required topossess compact and stable structure to overcomemultiple barriersin the gastrointestinal tract including high ionic strength, dramaticpH alteration, and ubiquitous endogenous enzymes [12]. In thisstudy, GTC NPs were prepared though ionic gelation of cationic GTCconjugates with anionic crosslinkers, which occurred sponta-neously in aqueous solution without sonication or heating, result-ing in the preservation of the biological activity of siRNA [13,25].TPP and HAwere used as anionic crosslinkers in the preparation ofGTC NPs due to their high biocompatibility and low immunoge-nicity [25,26]. Based on the criteria of smaller particle size, lowerPDI, and higher binding efficiency with siRNA of NPs (Table 1 andFig. 2A), GTC/TPP(10) NPs and GTC/HA(9) NPs were chosen for themeasurement of structural stability. Compared to GTC/HA(9) NPs, it

was found that GTC/TPP(10) NPs could maintain their structuralstability in the gastrointestinal tract, as evidenced by their slightchanges in particle size and zeta potential under high ionic strengthand pH alternation. The poor structural stability of GTC/HA(9) NPsmight be ascribed to the reduction in the electrostatic attractionbetween the oppositely charged polyelectrolytes when salts actedas the counter-ions [27]. The desired structural stability of GTC/TPP(10) NPs was also beneficial for protecting siRNA from degra-dation by shielding siRNA away from nucleases (Fig. 3), Consideringtheir preferable structural stabilities and siRNA protection in theintestinal tract, GTC/TPP(10) NPs were used for the subsequentinvestigations.

LPS was well-known to activate macrophages to over expressMGL and secrete TNF-a at an elevated level [10,20], therefore, thepresent investigation adopted LPS-stimulated Raw 264.7 as acti-vated macrophage models. Because of the high affinity between

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: TCNPs-treated group; E: GTC NPs-treated group.

J. Zhang et al. / Biomaterials 34 (2013) 3667e36773676

galactose residues on NPs and MGL receptors on macrophages,GTC/TPP(10) NPs exhibited higher cell binding affinity compared toTC/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 in-hibition effects following the addition of free LA and galactosecollectively indicated that the MGL receptor-mediated endocytosiswas involved in the internalization of GTC/TPP(10) NPs. Becausegalactosylated and thiolated modification reduced the positivecharges on polymers, GTC/TPP(10) NPs and TC/TPP(10) NPs causednegligible cytotoxicity (Fig. S2), suggesting that they might notinduce cell death during the gene transfection assessment.

Aouadi et al. first demonstrated that Map4k4 was a new targetfor suppression of TNF-a expression in LPS-induced macrophages[6]. As a key upstream mediator of TNF-a action [28], the effectivedownregulation of Map4k4mRNA expression would suppress TNF-a production. Therefore, Map4k4 was chosen as the target gene inthis investigation. Owing to their higher cell binding and cellularuptake, GTC/TPP(10) NPs containing siMap4k4 worked better insuppressing the TNF-a production as well as Map4k4 and TNF-amRNA expression in LPS-activated Raw 264.7 cells as compared tosiMap4k4 loaded TC/TPP(10) NPs and Lipofectamine 2000/siMap4k4 complexes (Fig. 5).

Based on the promising in vitro evidence, we further evaluatedthe in vivo efficacy of orally delivered GTC/TPP(10) NPs in micesuffering from DSS-induced UC. Compared to TC/TPP(10) NPs, GTC/TPP(10) NPs loaded with siMap4k4 more effectively deliveredsiRNA to the inflammatory colon (Fig. 6) and protected mice fromDSS-induced UC after oral administration, as indicated by reducedcolonic MPO activity, inappreciable body weight loss and colonlength shortening, and significant histological improvement (Fig. 7,Fig. S3, and Fig. 8). Moreover, to achieve equivalent therapeuticefficacy, GTC/TPP(10) NPs loaded with siMap4k4 were orallyadministered at a siRNA dose of 250 mg/kg per day for 6 consecutivedays, which represented a remarkable reduction in the admin-istration dose used compared to previous studies [10,20,29]. Thisminimized therapeutic dose of siRNAwould further reduce the riskfor side effects owing to massive therapeutic exposure to non-

target tissues. The following facts contributed to the enhancedaccumulation in the colon and improved therapeutic efficacy oforally delivered GTC/TPP(10) NPs containing siMap4k4 at a lowdose. First, when mice were receiving DSS to develop UC, a largenumber of activated macrophages bearing MGL migrated to thecolonic mucosa. GTC/TPP(10) NPs with a high affinity to MGL re-ceptors can be primarily internalized by activated macrophageslocated in the focus of inflammation [10]. Second, the slightchanges in particle sizes of GTC/TPP(10) NPs in the gastrointestinaltract could allow them to be accumulated in the inflamed colontissue [30], and these NPs could strongly adhere to mucus layersbased on the disulfide bond formation with mucus glycoprotein[17]. Third, GTC/TPP(10) NPs could preserve the bioactivity ofsiMap4k4 before internalization into activated macrophagesresiding in the inflammation sites of colon.

Despite the robust in vitro RNAi efficiencies of siMap4k4 loadedLipofectamine 2000 and adenovirus [28,31,32], the successfulin vivo applications of these carriers might be impeded due to thepoor in vivo stability of lipid and safety concerns [4], respectively.The b1,3-D-glucan particles (GeRPs) delivered Map4k4 siRNA to thesystemic circulation and silenced gene expression in murine mac-rophages after oral administration [6]. However, the GeRPs weredifficult to be produced with uniformity [33]. As comparisons, thehigh efficiency of targeting to activated macrophages, preferablestructural stability and siRNA protection, low cytotoxicity, andsimplicity in formulation collectively indicated the potentiality ofGTC NPs for oral delivery of Map4k4 siRNA to suppress colonicinflammation.

5. Conclusions

Targeted therapeutic strategy using NPs was developed basedon the pathophysiologic characteristics of inflammatory boweldiseases. Map4k4 could be selected as a target for the treatment ofDSS-induced UC. Orally delivered siMap4k4 loaded GTC/TPP(10)NPs was effective in protecting mice from DSS-induced UC ata relatively low therapeutic dose by attenuating colonic TNF-

J. Zhang et al. / Biomaterials 34 (2013) 3667e3677 3677

a production. It was expected that GTC/TPP(10) NPs might bea potential carrier for oral delivery of siRNA because of their desiredstructural stability, enhanced cell binding and cellular uptake inactivated macrophages, low cytotoxicity, high transfection effi-ciency in vitro, and direct delivery to the focus of disease.

Acknowledgments

This work was funded by grants from National Natural ScienceFoundation 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.

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