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Effects of mannose density on in vitro and in vivo cellular uptake and RNAi efficiency of polymeric nanoparticles
Biomaterials 52 (2015) 229e239
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Effects of mannose density on in vitro and in vivo cellular uptake and RNAi efficiency of polymeric nanoparticles Shuang Chu, 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 19 September 2014 Received in revised form 5 February 2015 Accepted 6 February 2015 Available online
To evaluate the effects of mannose density on in vitro and in vivo cellular uptake and RNA interference (RNAi) efficiency of polymeric nanoparticles (NPs) in macrophages, mannose-modified trimethyl chitosan-cysteine (MTC) conjugates with mannose densities of 4%, 13%, and 21% (MTC-4, MTC-13, and MTC-21) were synthesized. Tumor necrosis factor-alpha (TNF-a) siRNA loaded MTC NPs with particle sizes of ~150 nm exhibited desired structural stability and effectively protected siRNA from enzymatic degradation. Generally, cellular uptake and RNAi efficiency were affected by mannose density. As expected, MTC-21 NPs presented the maximum in vitro uptake and RNAi efficacy in Raw 264.7 cells among all NPs tested. However, MTC-4 NPs exhibited the optimal in vivo uptake by peritoneal exudate cell macrophages (PECs). In the inflammation model of acute hepatic injury, orally delivered MTC-4 and MTC-13 NPs worked better in silencing TNF-a expression and alleviating liver damage than MTC-21 NPs. As for the ulcerative colitis model, MTC-4 NPs outperformed MTC-13 and MTC-21 NPs with respect to TNF-a knockdown and therapeutic efficacy following oral administration. These results highlighted the importance of ligand density in cellular uptake and RNAi efficiency, which could serve as a guideline in the rational design of targeted nanocarriers for anti-inflammation therapy. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Gene delivery Polymeric nanoparticles Mannose density Cellular uptake Gene silencing
1. Introduction Oral delivery of small interfering RNA (siRNA) offers an appealing strategy for the treatment of various inflammatory diseases [1,2]. However, its further application in vivo would be severely restricted by the low selectivity and poor internalization [3]. To address this challenge, numerous cationic polymeric vectors bearing targeting ligands have been under investigation, intending for the delivery of siRNA to macrophages which could aggravate inflammatory responses by overexpressing pro-inflammatory cytokines [4,5]. Owing to mannose receptors (MR) excessively expressed on the surface of macrophages [6], mannose functionalized polymeric carriers have been extensively applied in siRNA-mediated anti-inflammation therapy and impressive progress has been made [7,8]. Nevertheless, to maximize the therapeutic efficacy of siRNA and reduce the risk for adverse effects, rational optimization of carriers is eagerly needed. One of the key aspects in the design of actively targeted delivery systems is ligand density [9]. It has been demonstrated to exert
* Corresponding author: Tel.: þ86 21 6564 3556; fax: þ86 21 5552 2771. E-mail address: [email protected] (C. Tang). http://dx.doi.org/10.1016/j.biomaterials.2015.02.044 0142-9612/© 2015 Elsevier Ltd. All rights reserved.
significant implications on the cellular uptake of targeted carriers, such as liposomes [10], polymeric nanoparticles (NPs) [11], and gold NPs [12]. In general, a critical minimal threshold in ligand density is needed to initiate the ligand-receptor recognition [13]. Higher ligand densities often promote the cellular internalization of NPs, which have been affirmed in wheat germ agglutinin modified PEG-PLA NPs [14] and super-para-magnetic iron oxide NPs labeled with HER2/neu targeting antibodies [15]. However, in some cases, actively targeted NPs coverage of highly dense ligands are unfavorable for cellular uptake [11]. In addition, ligand density can affect the in vitro gene transfer and in vivo antitumor activity, wherein carriers with the highest ligand density do not necessarily result in the most desirable efficacies [16,17]. Collectively, in view of different cell lines and disease models applied, there exists an optimum ligand density for biological behaviors of targeted vectors. To our knowledge, the effects of ligand density on the in vivo uptake and anti-inflammation efficacy of siRNA-loaded targeted delivery systems have not yet been investigated and little is known about their optimal ligand density. Mannose-modified trimethyl chitosan-cysteine (MTC) conjugates were prepared in our previous study as an oral delivery vector for tumor necrosis factor-alpha (TNF-a) siRNA, which exhibited remarkable anti-inflammation efficacy [7]. In this study, to
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elucidate the effects exerted by ligand density on in vitro and in vivo cellular uptake and RNA interference (RNAi) efficiency, MTC conjugates with various mannose densities were synthesized by adjusting the feed mass ratio of mannopyranosylphenylisothiocyanate (MPITC) to trimethyl chitosan (TMC). MTC NPs containing siRNA were developed via ionic gelation and their physicochemical properties, siRNA protection capability, in vitro uptake in Raw 264.7 cells, and in vivo uptake in peritoneal exudate cell macrophages (PECs) following oral administration were investigated. Gene silencing efficacy of MTC NPs encapsulating TNF-a siRNA was determined in in vitro Raw 264.7 cells and in vivo mouse models of acute hepatic injury and ulcerative colitis (UC). 2. Materials and methods 2.1. Materials, cell lines, and animals Chitosan (deacetylation degree of 85% and molecular weight of 200 kDa) was obtained from Golden-shell Biochemical Co., Ltd. (Zhejiang, China). Methyl iodide, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), and L-cysteine hydrochloride were purchased from Yuanju Biochemical Co., Ltd. (Shanghai, China). Tripolyphosphate (TPP) was bought from Experimental Reagent Co., Ltd. (Shanghai, China). MPITC, lipopolysaccharide (LPS), and D-galactosamine (D-GalN) were supplied by Sigma (St. Louis, MO, USA). Dextran sulfate sodium (DSS, molecular weight of 36e50 kDa) was obtained from MP Biomedicals (Illkirch, France). All other reagents were of analytical grade. 20 -O-methyl modified TNF-a-specific siRNA duplex (TNF-a siRNA) and FAMtagged siRNA (FAM-siRNA) were purchased from GenePharma (Shanghai, China) and dissolved in DEPC-treated water before use. TNF-a siRNA contained the sequences of sense 50 -GUCUCAGCCUCUUCUCAUUCCUGCT-30 and antisense 50 AGCAGGAAmUGmAGmAAmGAmGGmCUmGAmGAmCmAmU-30 , wherein “m” referred to a 20 -O-methyl base. Raw 264.7 cells were purchased from the American Type Culture Collection (Rockville, DS, USA) and maintained in Dulbecco's Modified Eagle Medium (DMEM, Gibco, NY, USA) supplemented with 10% fetal calf serum (FCS). PECs were harvested by sacrificing male C57BL/6 mice, intraperitoneally injecting DMEM with 10% FCS, aspirating the abdominal fluids, and centrifugating at 1500 rpm for 5 min. The isolated PECs were cultured in DMEM containing 10% FCS. Male C57BL/6 mice (6 weeks) were provided by Slaccas Experimental Animals Co., Ltd. (Shanghai, China). The study protocols were reviewed and approved by the Institutional Animal Care and Use Committee, Fudan University.
2.2. Synthesis and characterization of MTC conjugates MTC conjugates with different mannose densities were synthesized through adjusting the feed mass ratio of MPITC to TMC. MTC conjugate was synthesized through a three-step route as previously described [7]. Firstly, TMC was synthesized through reaction of chitosan with methyl iodide in methyl-2-pyrrolidone/NaOH solution for 2 h at 65 C and its quaternization degree was evaluated by 1H NMR (Bruker, Germany) [18]. Secondly, TMC (1%, w/v) was reacted with MPITC in dimethyl sulfoxide (0.5%, w/v) at determined feed mass ratio (20:1, 5:1, and 2.5:1, w/w) for 24 h at pH 9.0 and room temperature to obtain mannosylated TMC (MT) with various mannose densities. Thirdly, L-cysteine hydrochloride was conjugated to MT at a feed mass ratio of 1:2 (w/w) and pH 5.0 for 5 h at room temperature by the catalysis of EDC/NHS (200 mM). The resultant MTC conjugates were purified via ultrafiltration (MWCO 10 kDa) and dialysis against HCl solution (pH 5.0) for 72 h. The mannose density of MTC conjugate was characterized by 1H NMR and the amount of immobilized sulphydryl was determined by Ellman's reagent [19].
2.3. Preparation and characterization of MTC NPs MTC conjugate, TPP, and siRNA were dissolved in DEPC-treated water at the concentration of 1, 1, and 0.2 mg/mL, respectively. TPP solution was added into siRNA solution at the TPP/siRNA ratio of 17:1 (w/w). The mixture was then added into the MTC conjugate solution at the TPP/MTC ratio of 1:8 (w/w), and incubated at 37 C for 30 min to form MTC NPs. The hydrodynamic particle sizes and zeta potentials of MTC NPs were monitored by dynamic light scattering (DLS) with a zetasizer Nano-ZS (Malvern, Worcestershire, UK). The morphology of MTC NPs was visualized with scanning electron microscopy (SEM) (Vega TS5136, Tescan, Czech). The cytotoxicity of MTC NPs was evaluated in Raw 264.7 cells. Cells were seeded in 96-well plates at 1 104 cells/well and cultured at 37 C for 24 h. Then they were treated with MTC NPs at a final siRNA concentration of 0.05, 0.1, 0.2, 0.5, and 1 mg/mL for 6 h, followed by methyl tetrazolium (MTT) assay. The untreated cells were regarded as 100% cell viability.
2.4. Structural stability and siRNA protection of MTC NPs Heparin displacement was employed to estimate the structural stability of MTC NPs. Heparin sodium was added into MTC NPs containing 200 ng of siRNA at a final concentration of 0.04, 0.07, 0.10, and 0.13 mg/mL, respectively. The mixture was incubated at room temperature for 30 min to dissociate MTC NPs and then it was evaluated on a 4% (w/v) agarose gel at 56 V for 1 h. MTC NPs (0.2 mL) were incubated with equal volume of RNase A (20 mg/mL) for 2 h or physiological fluids for determined time at 37 C (2 h for intestinal fluids and 4 h for mouse serum, intestinal homogenates, and peritoneal fluids). Then the mixture was treated at 80 C for 5 min to inactivate nuclease and heparin sodium was added to dissociate MTC NPs. The integrity of siRNA was determined via 4% (w/v) agarose gel electrophoresis at 56 V for 1 h. 2.5. siRNA encapsulation and release The encapsulation of MTC conjugates toward siRNA was evaluated by gel retardation assay. MTC NPs were loaded in a 4% agarose gel stained with ethidium bromide and the electrophoresis was performed at 56 V for 1 h. MTC NPs containing FAM-siRNA were suspended in 1 mL of 0.2 M phosphate buffered solution (PBS, pH 7.4) and incubated at 37 C and 100 rpm. At predetermined time, the suspension was ultrafiltrated (MWCO 30 kDa) at 3000 rpm for 30 min and 60 mL of the filtrate was withdrawn to quantify siRNA content by fluorimetry (lex ¼ 480 nm, lem ¼ 520 nm). Then MTC NPs were resuspended in 1 mL of 0.2 M PBS for further incubation. 2.6. In vitro cellular uptake Raw 264.7 cells and PECs were seeded in 24-well plates at 5 104 and 5 105 cells/well, respectively, and cultured at 37 C for 24 and 2 h, respectively. The culture media were replaced by 1 mL of fresh DMEM, and MTC NPs containing FAM-siRNA were added and incubated with cells for 4 h at a final siRNA concentration of 0.4 mg/mL. The cells were washed with PBS and lysed with 0.5% SDS (w/v, pH 8.0). The content of FAM-siRNA and protein in the cell lysate were quantified by spectrofluorimetry (lex ¼ 480 nm, lem ¼ 520 nm) and the Lowry method, respectively. Cellular uptake was represented as the amount of FAM-siRNA normalized with per mg of total cellular protein. To confirm MR-mediated internalization, the cells were incubated with free mannose which could bind MR at a final concentration of 1.2 mM for 30 min prior to MTC NPs treatment and throughout the uptake process. To directly visualize the cellular uptake, Raw 264.7 cells treated with MTC NPs containing FAM-siRNA for 4 h were washed with PBS, fixed with paraformaldehyde, and stained with Hoechst 33258 (10 mg/mL). The cells were observed with confocal laser scanning microscopy (CLSM, Zeiss, Germany). The detailed uptake mechanisms were explored as previously described [7]. Raw 264.7 cells were incubated with endocytic inhibitors genistein (200 mg/mL), methylb-cyclodextrin (Me-b-CD, 50 mM), wortmannin (50 nM), amiloride (500 mM), and chlorpromazine (10 mg/mL) for 30 min prior to MTC NPs application and throughout the uptake process. Results were denoted as the relative percentage of uptake compared to the control without inhibitor pretreatment. 2.7. In vivo cellular uptake Male C57BL/6 mice were orally administered with FAM-siRNA loaded MTC NPs at the siRNA dose of 100 mg/kg. After 6 h, PECs were isolated, seeded in 24-well plates, and cultured for another 2 h. The uptake level was measured as described above. 2.8. In vitro RNAi Raw 264.7 cells were seeded in 24-well plates at 4 104 cell/well and cultured at 37 C for 24 h. The culture media were replaced by the fresh DMEM. Then MTC NPs containing TNF-a siRNA were added and incubated with the cells at a final siRNA concentration of 0.4 mg/mL for 24 h, after which LPS (10 ng/mL) was incubated for another 5 h. The culture media were collected for the determination of extracellular TNF-a level by ELISA kit (R&D, USA). Cells were collected for the isolation of RNA with Trizol reagent (Sigma, USA). cDNA was synthesized from 500 ng RNA using PrimeScript® RT reagent kit (Takara Biotechnology Co., Ltd., China) for the determination of intracellular TNF-a mRNA level via Real-Time PCR. The ribosomal mRNA 36B4 was used as an internal loading control. 2.9. In vivo RNAi 2.9.1. In vivo RNAi against acute hepatic injury Male C57BL/6 mice were orally administered with PBS or various TNF-a siRNA loaded MTC NPs at a siRNA dose of 30 mg/kg. After 24 h, LPS (12.5 mg/kg) and D-GalN (1.25 g/kg) were intraperitoneally injected to induce acute hepatic injury. After 2 h, blood was collected and centrifugated at 12,000 rpm for 4 min to isolate the serum for monitoring the TNF-a level by ELISA kit (R&D, USA). Mice were sacrificed and liver, spleen, and lung were collected and immersed in the RNAlater solution. Then they were homogenized in liquid nitrogen and RNA was extracted with Trizol
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reagent (Sigma, USA). The intracellular TNF-a mRNA level in the liver, spleen, and lung was determined by Real-Time PCR. Male C57BL/6 mice were orally delivered with various TNF-a siRNA loaded MTC NPs at a siRNA dose of 30 mg/kg or PBS, and 24 h post administration, LPS (12.5 mg/kg) and D-GalN (1.25 g/kg) were intraperitoneally injected. After 6 h, liver was harvested, fixed with 4% paraformaldehyde (w/v), and stained with haematoxylin/eosin (H&E) for histological analyses.
As depicted in Fig. S2, MTC NPs exerted inappreciable cytotoxicity on Raw 264.7 cells even at the siRNA concentration of 1 mg/mL which was 2.5-fold higher than the dose applied in the in vitro transfection.
2.9.2. In vivo RNAi against UC Male C57BL/6 mice bearing UC were obtained by DSS solution (3%, w/v) as drinking water over a period of 8 days (day 0e7). Healthy control mice were treated with normal water. Mice were orally administered each day with PBS or various TNF-a siRNA loaded MTC NPs at a siRNA dose of 30 mg/kg for 5 consecutive days (day 0e4). Body weight was daily monitored. On day 7, mice were sacrificed and colons were collected. The colonic segments were homogenized with cold PBS (pH 7.4), followed by centrifuging at 12,000 rpm and 4 C for 20 min. The supernatant was collected to quantify the TNF-a level by ELISA kit (R&D, USA). Colonic segments were immersed in the RNAlater solution and then homogenized in liquid nitrogen. RNA was extracted with Trizol reagent (Sigma, USA) and intracellular TNF-a mRNA level in the colons was monitored by Real-Time PCR. Colon sections were fixed with 4% paraformaldehyde (w/v) and stained with H&E for histological analyses.
To evaluate the effects of mannose density on the structural stability of MTC NPs, heparin sodium was adopted as the competitive polyanion to dissociate siRNA from MTC NPs. As shown in Fig. 2B, the lowest concentration of heparin sodium to completely dissociate siRNA was 0.1 mg/mL for MTC-4 and MTC-13 NPs and 0.13 mg/mL for MTC-21 NPs, suggesting the higher structural stability of MTC-21 NPs against the attack of ionic molecules in physiological fluids. As displayed in Fig. 2C, naked siRNA was largely degraded after incubation with RNase A and various physiological fluids as evidenced by the invisible bands. However, all MTC NPs could effectively protect the encapsulated siRNA from degradation by nuclease and physiological fluids as indicated by the obviously brighter bands compared with naked siRNA.
2.10. Statistical analysis Data were represented as mean ± SD. Statistical analysis was performed via Student's unpaired t test between two groups or One-Way Analysis of Variance (ANOVA) with Tukey's post-hoc test among more than two groups (SPSS software, version 12.0, SPSS Inc.). A value of P < 0.05 was judged to be significant.
3. Results 3.1. Synthesis and characterization of MTC conjugates MTC conjugates were synthesized through sequential trimethylation, mannosylation, and thiolation of chitosan as previously described [7]. TMC possessed the quaternization degree of 34% as determined by 1H NMR (Fig. S1) [18]. Various amounts of mannose residues were conjugated to TMC by altering the feed mass ratio of MPITC to TMC (Table 1). In the 1H NMR spectra, the peaks at 7.0e7.4 ppm were assigned to the proton on benzene of MPITC, confirming that mannose had been conjugated onto the backbone of TMC (Fig. 1). By comparing peak areas of benzene protons in MPITC (7.0e7.4 ppm) with acetyl protons (2.1 ppm) in chitosan, the mannose densities were calculated to be 4%, 13%, and 21%, respectively. Cysteine was sequentially conjugated onto the MT to form MTC via amidation reaction. Free sulphydryl and disulfide content of MTC conjugates were summarized in Table 1, indicating that about 13% of the amino groups in chitosan were occupied by cysteine for all MTC conjugates. MTC conjugates with mannose densities of 4%, 13%, and 21% were denoted as MTC-4, MTC-13, and MTC-21, respectively. 3.2. Preparation and characterization of MTC NPs MTC NPs encapsulating siRNA were prepared through ionic gelation in the presence of TPP as crosslinkers. As summarized in Table 2, MTC NPs with distinct mannose densities possessed particle sizes ranging from 140 nm to 170 nm (polydispersity index (PDI) < 0.2). Their zeta potentials decreased with the increase of mannose density. SEM observation revealed the well-dispersed and subsphaeroidal morphology of MTC NPs (Fig. 2A).
Table 1 Preparation and characterization of MTC conjugates. Name
MPITC:TMC (w/w)
Mannose density (%)
Free sulphydryl (mmol/g)
Disulfide (mmol/g)
MTC-4 MTC-13 MTC-21
1:20 1:5 1:2.5
4 13 21
100.3 ± 2.5 110.5 ± 2.1 100.2 ± 13.4
280.7 ± 16.5 260.6 ± 7.9 283.6 ± 6.3
3.3. Structural stability and siRNA protection of MTC NPs
3.4. siRNA encapsulation and release As indicated in Fig. 3A, MTC NPs exhibited complete retardation of siRNA in the loading well, demonstrating efficient encapsulation of siRNA in all NPs. In vitro release profiles showed that the accumulative release percentage of siRNA within 8 h decreased with the elevation of mannose density, which was about 93%, 82%, and 66% for MTC-4, MTC-13, and MTC-21 NPs, respectively (Fig. 3B). 3.5. In vitro cellular uptake As for the uptake in Raw 264.7 cells (Fig. 4A), the maximum in vitro uptake amount was found in MTC-21 NPs with the highest mannose density, which was 1.5 and 3.5 folds enhancement compared to that of MTC-4 and MTC-13 NPs, respectively. Preferable uptake was detected in MTC-4 NPs with higher zeta potential in spite of their lower ligand density in comparison with MTC-13 NPs. CLSM images revealed the similar tendency with quantitative analysis in which MTC-4 and MTC-21 NPs exhibited superior cellular uptake compared to MTC-13 NPs (Fig. 4B). For in vitro internalization by PECs, the uptake amounts of MTC-4 NPs were higher than those of MTC-13 and MTC-21 NPs (Fig. S3A). Free mannose was pre-incubated with cells to occupy MR prior to the addition of MTC NPs and a remarkable decrease in the uptake amount of MTC NPs was observed, confirming the MR-mediated endocytosis (Fig. 4C, Fig. S3B). Additionally, the inhibition ratios of internalization in Raw 264.7 cells of MTC-4, MTC-13, and MTC-21 NPs in the presence of free mannose were 17.3%, 44.1%, and 61.5%, respectively, implying that the MR-mediated endocytosis was more involved in the internalization of MTC NPs with higher mannose density (Fig. 4C). However, as for PECs, there was no significant difference in the inhibition ratios among different MTC NPs, suggesting that MR-mediated endocytosis was saturated even in the case of MTC-4 NPs with the lowest ligand density (Fig. S3B). Detailed uptake mechanisms were investigated by utilizing various endocytic inhibitors. The cellular uptake of MTC NPs was remarkably inhibited by genistein and Me-b-CD, proposing a caveolae-mediated endocytosis pathway. The suppressed uptake in the presence of wortmannin and amiloride indicated macropinocytosis participation in the uptake process. Comparatively, chlorpromazine exerted minimal effect on the internalization of MTC NPs, excluding the involvement of clathrin-dependent pathway (Fig. S4). Clathrin-independent endocytosis of all MTC NPs would
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Fig. 1. (A) Synthetic route of MTC conjugates with different mannose densities. (B) 1H NMR spectra of MTC conjugates with different mannose densities. The peaks at 7.0e7.4 and 2.1 ppm were assigned to the protons on benzene of MPITC and acetyl groups of TMC, respectively.
avoid the endosomal/lysosomal entrapment, which was beneficial for their highly efficient cellular uptake and gene silencing.
NPs, which was in agreement with the results of in vitro internalization by PECs (Fig. S3A).
3.6. In vivo cellular uptake
3.7. In vitro RNAi
As depicted in Fig. 4D, orally delivered MTC-4 NPs presented higher in vivo cellular uptake by PECs than MTC-13 and MTC-21
In terms of in vitro RNAi in Raw 264.7 cells against LPS-triggered TNF-a secretion, MTC-21 NPs with the highest mannose density
S. Chu et al. / Biomaterials 52 (2015) 229e239 Table 2 The particle sizes and zeta potentials of MTC NPs. Sample
Particle size (nm)a,b
Zeta potential (mV)a
MTC-4 NPs MTC-13 NPs MTC-21 NPs
166.6 ± 0.7 (0.190 ± 0.015) 148.6 ± 2.4 (0.046 ± 0.025) 143.3 ± 1.1 (0.151 ± 0.029)
35.8 ± 1.7 22.7 ± 1.7 18.7 ± 0.6
a b
Data represented the mean ± SD (n ¼ 3). Values in parentheses represented the polydispersity index.
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presented exceptional silencing efficiency of 72.8%, which outperformed MTC-4 and MTC-13 NPs. In addition, MTC-4 NPs dramatically depleted TNF-a protein production by 62.6%, which was significantly higher than the corresponding value of 51.6% in MTC-13 NPs (Fig. 5A). For the efficacy of silencing TNF-a mRNA expression, the tendency was in accordance with the results in efficiency of suppressing TNF-a protein. MTC-21 NPs exhibited higher depletion ratio of 65.1% than that of MTC-4 and MTC-13 NPs (Fig. 5B).
Fig. 2. Characterization of MTC NPs and their protection of siRNA integrity. (A) SEM images of MTC NPs. Bar represented 200 nm. (B) Agarose gel electrophoresis of MTC NPs at 56 V for 1 h following incubation with various concentrations of heparin sulfate. (C) Agarose gel electrophoresis of MTC NPs at 56 V for 1 h after treatment with physiological fluids or RNase A. Naked siRNA treated with physiological fluids and RNase A served as the positive controls. Naked siRNA without any treatment served as the negative controls.
Fig. 3. Encapsulation and release of siRNA in MTC NPs. (A) Agarose gel electrophoresis of MTC NPs at 56 V for 1 h. (B) In vitro release profiles of FAM-siRNA from MTC NPs in 0.2 M PBS (pH 7.4) at 37 C.
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Fig. 4. The cellular uptake of MTC NPs containing FAM-siRNA in in vitro Raw 264.7 cells and in vivo PECs. (A) In vitro cellular uptake of MTC NPs in Raw 264.7 cells. Indicated values were mean ± SD (n ¼ 4). *P < 0.05, **P < 0.01, ***P < 0.001. (B) CLSM images showing cellular uptake of MTC NPs loaded with FAM-siRNA (green) in Raw 264.7 cells following incubation for 4 h. The nuclei were stained with Hoechst 33258 (blue). Bar represented 10 mm. (C) Uptake inhibition of MTC NPs in Raw 264.7 cells following treatment with free mannose (1.2 mM). Indicated values were mean ± SD (n ¼ 4). (D) In vivo cellular uptake of MTC NPs in PECs following oral administration. Indicated values were mean ± SD (n ¼ 4). *P < 0.05. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
3.8. In vivo RNAi 3.8.1. In vivo RNAi against acute hepatic injury LPS/D-GalN-induced acute hepatic injury mouse model was established to investigate RNAi efficiency of orally-delivered MTC NPs in systemic and acute inflammation disease. MTC NPs with various mannose densities at a siRNA dose of 30 mg/kg remarkably blocked serum TNF-a production. The silencing efficiency of MTC-4 and MTC-13 NPs was 81% and 79%, respectively, which were superior to that of 42% in MTC-21 NPs (Fig. 6A). Accordingly, notable suppression of TNF-a mRNA expression for all NPs was detected in macrophage-enriched organs (liver, spleen, and lung), among which MTC-21 NPs exhibited relatively inferior TNF-a mRNA depletion efficiency of about 43%, 45%, and 51% in liver,
spleen, and lung, respectively (Fig. 6B). Histological sections from MTC-4 and MTC-13 NPs-treated mice showed obvious alleviation of LPS/D-GalN-triggered liver damage including congested central vein as well as swollen and disarranged hepatocytes (Fig. 6C). 3.8.2. In vivo RNAi against UC DSS-induced UC mouse model was employed to investigate RNAi efficiency of orally-delivered MTC NPs in local and chronic inflammation disease. MTC NPs significantly suppressed TNF-a protein and mRNA expression in colonic tissues. MTC-4 NPs suppressed TNF-a production in colonic tissues with the inhibition ratio of 75.8%, which was significantly higher than that of MTC-13 and MTC-21 NPs (Fig. 7A). Similar trend was found in the TNF-a mRNA expression (Fig. 7B). MTC-4 NPs significantly blocked TNF-a
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Fig. 5. In vitro gene silencing of MTC NPs containing TNF-a siRNA in Raw 264.7 cells. Inhibition of TNF-a expression in the protein (A) and mRNA (B) levels compared to the controls in LPS-stimulated Raw 264.7 cells. Cells activated by LPS (10 ng/mL) without NPs treatment served as the controls. Indicated values were mean ± SD (n ¼ 3). *P < 0.05, ***P < 0.001.
mRNA expression in colonic tissues by 79.4%, which were superior to MTC-13 and MTC-21 NPs. UC was characterized by sustained weight loss and shortened colon length. As illustrated in Fig. 7C and D, the body weight and colon length of mice without any treatment dramatically decreased by 19% and 37% after 8-day exposure to DSS, respectively. By contrast, oral administration of MTC NPs effectively alleviated the weight loss within 5% (Fig. 7C) and the colon shortening within 16% (Fig. 7D). MTC-4 NPs exerted the superiority in prevention of colon shortening among all NPs as evidenced by the minimal reduction of 7%. H&E stained colonic sections of UC control mice exhibited various histological damages as compared with those of healthy mice, including depletion of crypt and goblet cells, abnormality or loss of epithelial cells, marked infiltration of mononuclear cells, and irregular mucosal structure (Fig. 8) [20]. Sections from MTC-4 NPstreated mice revealed histological characteristics resembling those of healthy mice, while MTC-13 and MTC-21 NPs-treated mice showed relatively poor therapeutic efficacy. 4. Discussion Due to the non-selectivity and hydrophilic nature of siRNA [3], numerous works had been focused on the rational design of mannose-modified vectors for macrophage-targeting treatment of inflammatory diseases [7,8]. However, little emphasis had been laid on the optimization of ligand density to improve gene silencing efficiency. Therefore, the study herein was aimed at exploring the effects of mannose density of polymeric NPs on cellular uptake and RNAi efficiency both in vitro and in vivo. MTC conjugate-based delivery systems have shown significantly superior cellular uptake and gene silencing to trimethyl chitosancysteine NPs via mannose ligand-mediated macrophage-targeting in our previous work [2,7], thus representing an ideal platform for us to elucidate the correlation between the ligand density and cellular uptake as well as RNAi efficiency of polymeric NPs. On the basis of prior reports that mannose density of about 5% was necessary for sufficient MR-mediated internalization [21,22], we successfully synthesized a series of MTC conjugates with mannose densities of 4%, 13%, and 21% through adjusting the feed mass ratio of MPITC to TMC, alternatively to examine whether a mannose density above 5% would further increase cellular uptake and gene
transfer efficiency. MTC conjugates and siRNA self-assembled into NPs through ionic gelation with anionic TPP. All NPs possessed similar particle sizes and preferable protection of siRNA integrity in physiological fluids. This might probably be attributed to the complete complexation of MTC conjugates towards anionic siRNA, thus preventing the approach of nucleases during delivery to the macrophages. To clarify the effects of mannose density on the structural stability of MTC NPs, polyanion heparin was employed to simulate the attack of anionic molecules to positively-charged NPs in physiological fluids. MTC NPs with higher mannose density displayed stronger resistance to heparin, thereby possessing more stable structure (Fig. 2B). This phenomenon might be explained by the following two reasons. On one hand, substitution of primary amino groups with mannose residues would diminish positively-charged groups, leading to weak electrostatic interaction with polyanion heparin [16]. On the other hand, uncharged and hydrophilic mannose residues with plentiful hydroxyl groups would likely orientate towards the surface of MTC NPs and link with each other, forming a corona structure which was able to resist the approach of competitive molecules [23]. Moreover, the aforementioned dense corona composed of mannose residues might prevent the dissociation of siRNA from MTC NPs via steric hindrance and retain the cargoes inside the core of MTC NPs. Consequently, slower siRNA release rate was noted in MTC NPs with higher mannose density (Fig. 3B). Additionally, the above-mentioned charge shielding and steric hindrance effects might contribute to the phenomenon that zeta potentials decreased with the increase of mannose density, which would further exert an implication on the cellular uptake by macrophages (Table 2). Since mannose conjugation was aimed at promoting the uptake of MTC NPs by macrophages, we went on to investigate the effects of mannose density on the in vitro internalization by Raw 264.7 cells of MTC NPs (Fig. 4AeC). In contrast to MTC-4 and MTC-13 NPs, as expected, MTC-21 NPs bearing higher mannose residues were more readily internalized into Raw 264.7 cells, which was in line with previous results [10,22]. The highest cellular uptake level of MTC-21 NPs was assumed to majorly result from the enhanced MR-mediated internalization, as evidenced by the maximum uptake inhibition in the presence of free mannose (Fig. 4C). Meanwhile, the strongest structural stability and slowest siRNA
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Fig. 6. In vivo gene silencing of MTC NPs containing TNF-a siRNA in acute hepatic injury mouse model at the siRNA dose of 30 mg/kg. (A) TNF-a production in the serum evaluated by ELISA assay. Indicated values were mean ± SD (n ¼ 6). *P < 0.05. (B) TNF-a mRNA levels in mouse liver, spleen, and lung determined by Real-Time PCR. Indicated values were mean ± SD (n ¼ 3). *P < 0.05. Acute hepatic injury mice induced by LPS (12.5 mg/kg) and D-GalN (1.25 g/kg) with the treatment of PBS served as the controls. (C) H&E stained liver sections for histopathological analyses. Bar represented 500 mm.
release in MTC-21 NPs would lead to less premature release, which might also contribute to their preferable uptake amount. Interestingly, the lower mannose density of MTC-4 NPs did not result in decreased internalization compared with MTC-13 NPs. A possible explanation for this phenomenon would be the strong electrostatic interactions between negatively charged cell membrane and MTC-4 NPs with the highest positive charges among all NPs [24]. TNF-a was elicited by LPS-activated macrophages, and served as an important regulator of inflammation [2]. Therefore it was selected as the therapeutic target in this investigation. MTC-21 NPs performed better in attenuating TNF-a production and TNF-a mRNA expression in LPS-stimulated Raw 264.7 cells as compared to MTC-4 and MTC-13 NPs. And MTC-4 NPs mediated superior TNF-a knockdown compared to MTC-13 NPs (Fig. 5). The tendency of in vitro silencing efficiency with respect to mannose density was consistent with in vitro cellular uptake results. Unlike plasmid DNA,
the dissociation of siRNA from cationic polymeric vector was much easier due to its short length and rigid nature, e.g. the majority of siRNA was released within 8 h for all NPs in our study (Fig. 3B). Moreover, disulfide reduction of MTC NPs would occur under the high concentration of intracellular glutathione. Taken together, siRNA would be rapidly dissociated from MTC NPs and interact with target mRNA once entry into the macrophages. Therefore, the in vitro gene silencing efficiencies of MTC NPs might be closely related to their uptake amounts. Due to the greater complexity of physiological surroundings and transport processes following oral administration than in vitro conditions, the correlation between ligand density and in vivo cellular uptake as well as RNAi efficacy could not be accurately predicted by in vitro results. Hence, the in vivo uptake of MTC NPs in PECs was examined after oral gavage to mice. The highest in vivo uptake amounts by PECs were noted in MTC-4 NPs rather than
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Fig. 7. In vivo gene silencing and the therapeutic efficacy of MTC NPs containing TNF-a siRNA in the UC mouse model. (A) Inhibition of TNF-a secretion in the colonic tissues measured by ELISA. Indicated values were mean ± SD (n ¼ 6). *P < 0.05, **P < 0.01. (B) Attenuation of TNF-a mRNA in the colonic tissues detected by Real-Time PCR. Indicated values were mean ± SD (n ¼ 3). *P < 0.05. (C) Changes of body weight over time in mice treated with or without 3% DSS in drinking water. Indicated values were mean ± SD (n ¼ 6). (D) Changes of colon length over time in mice treated with or without 3% DSS in drinking water. Indicated values were mean ± SD (n ¼ 6). *P < 0.05.
MTC-13 and MTC-21 NPs (Fig. 4D), which was consistent with the results of in vitro cellular uptake by PECs (Fig. S3A). In contrast to the highly-efficient in vitro internalization by Raw 264.7 cells, the poor in vitro uptake by PECs of MTC-21 NPs was observed, which might be due to their differences in mannose density on the cell membranes. Receptor density has been proven to exert an important influence on ligand-receptor interaction [25]. Compared with Raw 264.7 cells (leukemic macrophages) containing abundant MR, there probably exists limited MR on the cell membranes of PECs (primary macrophages). Such speculation was confirmed by competitive inhibition experiment in the presence of free mannose wherein MR-mediated endocytosis in PECs was saturated in the case of MTC-4 NPs with the lowest ligand density (Fig. S3B). Owing to the saturation of ligand-receptor recognition, increasing mannose density in MTC NPs from 4% to 21% would cause decreased surface charges and thus weaken electrostatic interaction with cell membranes rather than enhanced MR-mediated endocytosis, thereby reducing in vitro and in vivo internalization by PECs of MTC-21 NPs. Moreover, steric hindrance effect stemming from excessive mannose residues on the surface of NPs would presumably be amplified when the binding sites for mannose were limited. For instance, overcrowding could prevent ligand molecules
from obtaining the correct orientation essential for the receptor recognition, while lowering the mannose density enhanced their congruency to MR, thus providing more effective binding and uptake by macrophages. Accordingly, the highest in vivo cellular uptake was found in MTC-4 NPs while further increasing mannose density would exert adverse effects on in vivo internalization of MTC NPs (Fig. 4D), which was consistent with the previous results that the mannose density of about 5% would be sufficient for MR-mediated gene delivery to macrophages [21,22]. Inflammation models of acute liver injury and chronic UC, characterized by excessive secretion of TNF-a from macrophages, are related to discrepant target macrophages (systemic vs. local) and focal tissues (liver vs. colon), which might have distinct demands on the mannose density for highly enhanced therapeutic efficacy. These models were consequently established to evaluate disease-specific effects of mannose density on in vivo RNAi efficiency and anti-inflammatory efficacy mediated by orally delivered MTC NPs (Fig. 6 and 7). Considering the detailed pathogenesis of inflammation models adopted and the relatively slow absorption of siRNA via oral administration, to assure the high possibility for uncovering the effects of mannose density on in vivo RNAi and antiinflammation efficacy, the prophylactic treatment regimens
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Fig. 8. H&E stained colon sections for histopathological analyses. Bar represented 100 mm.
frequently reported, dosing started either before or on day 0, were therefore adopted for their effectiveness in siRNA delivery for attenuating inflammatory responses [1,2,4,5,7]. MTC-4 NPs presented robust anti-inflammation efficacy against both chronic UC and acute hepatic injury, which was likely due to their highest in vivo cellular uptake among all NPs (Fig. 4D). Compared to published vectors involving TNF-a knockdown for intestinal inflammation therapy, MTC-4 NPs demonstrated outstanding in vivo RNAi potency, wherein a total therapeutic dose of 150 mg siRNA/kg was 2e3 orders of magnitude less [26]. These results supported the strategy that optimization of ligand density was essential to achieve highly-performed therapeutic efficacy with minimal siRNA dose. It was intriguing to note that in spite of the unsatisfactory efficacy in UC model, MTC-13 NPs mediated efficient RNAi against acute hepatic injury. This inconsistent therapeutic outcome might be attributed to the distinct requirements for RNAi stemming from different types of inflammation (chronic UC vs. acute hepatic injury). The relatively quick siRNA release from MTC-13 NPs could lead to the rapidly increased intracellular concentration of free siRNA, which was beneficial to initiate prompt RNAi and thus offer timely protection against acute hepatic injury. However, such release behavior of MTC-13 NPs might compromise the long-time silencing efficiency. Therefore, their anti-inflammation efficacy against UC which preferred sustained TNF-a silencing was relatively inefficient. The discrepancy might also be due to the differences in the phenotype and functions of macrophages involved in relevant inflammation (systemic vs. local) and related tissues (liver vs. colon) [27]. As for MTC-21 NPs, poor in vivo RNAi efficiency and therapeutic effect were observed in both inflammation models, which was in accordance with the results of in vivo cellular uptake. The unsatisfactory RNAi performance of MTC-21 NPs supported the presumption that a higher ligand density above a certain optimum value would enhance the “off-target” effect, thus leading to decreased “selective delivery” of encapsulated drugs to target cells and lowered therapeutic efficacy [25].
5. Conclusions Through controlling the feed mass ratio of MPITC and TMC, a series of MTC conjugates with different mannose densities ranging from 4% to 21% were synthesized to underscore the effects of mannose density on cellular uptake and RNAi efficiency of polymeric NPs both in vitro and in vivo. MTC-4 NPs provided a relatively high in vitro and in vivo cellular uptake, resulting in the highly-performed in vitro and in vivo RNAi efficiency as well as antiinflammatory efficacy at a low dose of siRNA. Comparatively, MTC-21 NPs were correlated with supreme in vitro cellular uptake and gene knockdown, but their performance in in vivo RNAi remained unsatisfactory because of poor in vivo cellular uptake. MTC-13 NPs showed excellent TNF-a silencing in acute hepatic injury, whereas exerted disadvantageous influence on therapeutic efficacy of UC. Our data provided the in vivo evidence that polymeric carriers with lower ligand density would be more favorable for the treatment of inflammatory diseases than their counterparts with higher ligand density and the effects of ligand density were dependent on the type of inflammatory diseases. Therefore, the ligand density should be identified as a crucial parameter for screening targeted polymeric vehicles of siRNA for anti-inflammation therapy, and the idea could be further extended to other applications including cancer therapy.
Acknowledgements The authors are thankful for the financial support from the National Natural Science Foundation of China (No. 81072595).
Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.biomaterials.2015.02.044.
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Contents lists available at ScienceDirect
Biomaterials journal homepage: www.elsevier.com/locate/biomaterials
Effects of mannose density on in vitro and in vivo cellular uptake and RNAi efficiency of polymeric nanoparticles Shuang Chu, 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 19 September 2014 Received in revised form 5 February 2015 Accepted 6 February 2015 Available online
To evaluate the effects of mannose density on in vitro and in vivo cellular uptake and RNA interference (RNAi) efficiency of polymeric nanoparticles (NPs) in macrophages, mannose-modified trimethyl chitosan-cysteine (MTC) conjugates with mannose densities of 4%, 13%, and 21% (MTC-4, MTC-13, and MTC-21) were synthesized. Tumor necrosis factor-alpha (TNF-a) siRNA loaded MTC NPs with particle sizes of ~150 nm exhibited desired structural stability and effectively protected siRNA from enzymatic degradation. Generally, cellular uptake and RNAi efficiency were affected by mannose density. As expected, MTC-21 NPs presented the maximum in vitro uptake and RNAi efficacy in Raw 264.7 cells among all NPs tested. However, MTC-4 NPs exhibited the optimal in vivo uptake by peritoneal exudate cell macrophages (PECs). In the inflammation model of acute hepatic injury, orally delivered MTC-4 and MTC-13 NPs worked better in silencing TNF-a expression and alleviating liver damage than MTC-21 NPs. As for the ulcerative colitis model, MTC-4 NPs outperformed MTC-13 and MTC-21 NPs with respect to TNF-a knockdown and therapeutic efficacy following oral administration. These results highlighted the importance of ligand density in cellular uptake and RNAi efficiency, which could serve as a guideline in the rational design of targeted nanocarriers for anti-inflammation therapy. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Gene delivery Polymeric nanoparticles Mannose density Cellular uptake Gene silencing
1. Introduction Oral delivery of small interfering RNA (siRNA) offers an appealing strategy for the treatment of various inflammatory diseases [1,2]. However, its further application in vivo would be severely restricted by the low selectivity and poor internalization [3]. To address this challenge, numerous cationic polymeric vectors bearing targeting ligands have been under investigation, intending for the delivery of siRNA to macrophages which could aggravate inflammatory responses by overexpressing pro-inflammatory cytokines [4,5]. Owing to mannose receptors (MR) excessively expressed on the surface of macrophages [6], mannose functionalized polymeric carriers have been extensively applied in siRNA-mediated anti-inflammation therapy and impressive progress has been made [7,8]. Nevertheless, to maximize the therapeutic efficacy of siRNA and reduce the risk for adverse effects, rational optimization of carriers is eagerly needed. One of the key aspects in the design of actively targeted delivery systems is ligand density [9]. It has been demonstrated to exert
* Corresponding author: Tel.: þ86 21 6564 3556; fax: þ86 21 5552 2771. E-mail address: [email protected] (C. Tang). http://dx.doi.org/10.1016/j.biomaterials.2015.02.044 0142-9612/© 2015 Elsevier Ltd. All rights reserved.
significant implications on the cellular uptake of targeted carriers, such as liposomes [10], polymeric nanoparticles (NPs) [11], and gold NPs [12]. In general, a critical minimal threshold in ligand density is needed to initiate the ligand-receptor recognition [13]. Higher ligand densities often promote the cellular internalization of NPs, which have been affirmed in wheat germ agglutinin modified PEG-PLA NPs [14] and super-para-magnetic iron oxide NPs labeled with HER2/neu targeting antibodies [15]. However, in some cases, actively targeted NPs coverage of highly dense ligands are unfavorable for cellular uptake [11]. In addition, ligand density can affect the in vitro gene transfer and in vivo antitumor activity, wherein carriers with the highest ligand density do not necessarily result in the most desirable efficacies [16,17]. Collectively, in view of different cell lines and disease models applied, there exists an optimum ligand density for biological behaviors of targeted vectors. To our knowledge, the effects of ligand density on the in vivo uptake and anti-inflammation efficacy of siRNA-loaded targeted delivery systems have not yet been investigated and little is known about their optimal ligand density. Mannose-modified trimethyl chitosan-cysteine (MTC) conjugates were prepared in our previous study as an oral delivery vector for tumor necrosis factor-alpha (TNF-a) siRNA, which exhibited remarkable anti-inflammation efficacy [7]. In this study, to
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elucidate the effects exerted by ligand density on in vitro and in vivo cellular uptake and RNA interference (RNAi) efficiency, MTC conjugates with various mannose densities were synthesized by adjusting the feed mass ratio of mannopyranosylphenylisothiocyanate (MPITC) to trimethyl chitosan (TMC). MTC NPs containing siRNA were developed via ionic gelation and their physicochemical properties, siRNA protection capability, in vitro uptake in Raw 264.7 cells, and in vivo uptake in peritoneal exudate cell macrophages (PECs) following oral administration were investigated. Gene silencing efficacy of MTC NPs encapsulating TNF-a siRNA was determined in in vitro Raw 264.7 cells and in vivo mouse models of acute hepatic injury and ulcerative colitis (UC). 2. Materials and methods 2.1. Materials, cell lines, and animals Chitosan (deacetylation degree of 85% and molecular weight of 200 kDa) was obtained from Golden-shell Biochemical Co., Ltd. (Zhejiang, China). Methyl iodide, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), and L-cysteine hydrochloride were purchased from Yuanju Biochemical Co., Ltd. (Shanghai, China). Tripolyphosphate (TPP) was bought from Experimental Reagent Co., Ltd. (Shanghai, China). MPITC, lipopolysaccharide (LPS), and D-galactosamine (D-GalN) were supplied by Sigma (St. Louis, MO, USA). Dextran sulfate sodium (DSS, molecular weight of 36e50 kDa) was obtained from MP Biomedicals (Illkirch, France). All other reagents were of analytical grade. 20 -O-methyl modified TNF-a-specific siRNA duplex (TNF-a siRNA) and FAMtagged siRNA (FAM-siRNA) were purchased from GenePharma (Shanghai, China) and dissolved in DEPC-treated water before use. TNF-a siRNA contained the sequences of sense 50 -GUCUCAGCCUCUUCUCAUUCCUGCT-30 and antisense 50 AGCAGGAAmUGmAGmAAmGAmGGmCUmGAmGAmCmAmU-30 , wherein “m” referred to a 20 -O-methyl base. Raw 264.7 cells were purchased from the American Type Culture Collection (Rockville, DS, USA) and maintained in Dulbecco's Modified Eagle Medium (DMEM, Gibco, NY, USA) supplemented with 10% fetal calf serum (FCS). PECs were harvested by sacrificing male C57BL/6 mice, intraperitoneally injecting DMEM with 10% FCS, aspirating the abdominal fluids, and centrifugating at 1500 rpm for 5 min. The isolated PECs were cultured in DMEM containing 10% FCS. Male C57BL/6 mice (6 weeks) were provided by Slaccas Experimental Animals Co., Ltd. (Shanghai, China). The study protocols were reviewed and approved by the Institutional Animal Care and Use Committee, Fudan University.
2.2. Synthesis and characterization of MTC conjugates MTC conjugates with different mannose densities were synthesized through adjusting the feed mass ratio of MPITC to TMC. MTC conjugate was synthesized through a three-step route as previously described [7]. Firstly, TMC was synthesized through reaction of chitosan with methyl iodide in methyl-2-pyrrolidone/NaOH solution for 2 h at 65 C and its quaternization degree was evaluated by 1H NMR (Bruker, Germany) [18]. Secondly, TMC (1%, w/v) was reacted with MPITC in dimethyl sulfoxide (0.5%, w/v) at determined feed mass ratio (20:1, 5:1, and 2.5:1, w/w) for 24 h at pH 9.0 and room temperature to obtain mannosylated TMC (MT) with various mannose densities. Thirdly, L-cysteine hydrochloride was conjugated to MT at a feed mass ratio of 1:2 (w/w) and pH 5.0 for 5 h at room temperature by the catalysis of EDC/NHS (200 mM). The resultant MTC conjugates were purified via ultrafiltration (MWCO 10 kDa) and dialysis against HCl solution (pH 5.0) for 72 h. The mannose density of MTC conjugate was characterized by 1H NMR and the amount of immobilized sulphydryl was determined by Ellman's reagent [19].
2.3. Preparation and characterization of MTC NPs MTC conjugate, TPP, and siRNA were dissolved in DEPC-treated water at the concentration of 1, 1, and 0.2 mg/mL, respectively. TPP solution was added into siRNA solution at the TPP/siRNA ratio of 17:1 (w/w). The mixture was then added into the MTC conjugate solution at the TPP/MTC ratio of 1:8 (w/w), and incubated at 37 C for 30 min to form MTC NPs. The hydrodynamic particle sizes and zeta potentials of MTC NPs were monitored by dynamic light scattering (DLS) with a zetasizer Nano-ZS (Malvern, Worcestershire, UK). The morphology of MTC NPs was visualized with scanning electron microscopy (SEM) (Vega TS5136, Tescan, Czech). The cytotoxicity of MTC NPs was evaluated in Raw 264.7 cells. Cells were seeded in 96-well plates at 1 104 cells/well and cultured at 37 C for 24 h. Then they were treated with MTC NPs at a final siRNA concentration of 0.05, 0.1, 0.2, 0.5, and 1 mg/mL for 6 h, followed by methyl tetrazolium (MTT) assay. The untreated cells were regarded as 100% cell viability.
2.4. Structural stability and siRNA protection of MTC NPs Heparin displacement was employed to estimate the structural stability of MTC NPs. Heparin sodium was added into MTC NPs containing 200 ng of siRNA at a final concentration of 0.04, 0.07, 0.10, and 0.13 mg/mL, respectively. The mixture was incubated at room temperature for 30 min to dissociate MTC NPs and then it was evaluated on a 4% (w/v) agarose gel at 56 V for 1 h. MTC NPs (0.2 mL) were incubated with equal volume of RNase A (20 mg/mL) for 2 h or physiological fluids for determined time at 37 C (2 h for intestinal fluids and 4 h for mouse serum, intestinal homogenates, and peritoneal fluids). Then the mixture was treated at 80 C for 5 min to inactivate nuclease and heparin sodium was added to dissociate MTC NPs. The integrity of siRNA was determined via 4% (w/v) agarose gel electrophoresis at 56 V for 1 h. 2.5. siRNA encapsulation and release The encapsulation of MTC conjugates toward siRNA was evaluated by gel retardation assay. MTC NPs were loaded in a 4% agarose gel stained with ethidium bromide and the electrophoresis was performed at 56 V for 1 h. MTC NPs containing FAM-siRNA were suspended in 1 mL of 0.2 M phosphate buffered solution (PBS, pH 7.4) and incubated at 37 C and 100 rpm. At predetermined time, the suspension was ultrafiltrated (MWCO 30 kDa) at 3000 rpm for 30 min and 60 mL of the filtrate was withdrawn to quantify siRNA content by fluorimetry (lex ¼ 480 nm, lem ¼ 520 nm). Then MTC NPs were resuspended in 1 mL of 0.2 M PBS for further incubation. 2.6. In vitro cellular uptake Raw 264.7 cells and PECs were seeded in 24-well plates at 5 104 and 5 105 cells/well, respectively, and cultured at 37 C for 24 and 2 h, respectively. The culture media were replaced by 1 mL of fresh DMEM, and MTC NPs containing FAM-siRNA were added and incubated with cells for 4 h at a final siRNA concentration of 0.4 mg/mL. The cells were washed with PBS and lysed with 0.5% SDS (w/v, pH 8.0). The content of FAM-siRNA and protein in the cell lysate were quantified by spectrofluorimetry (lex ¼ 480 nm, lem ¼ 520 nm) and the Lowry method, respectively. Cellular uptake was represented as the amount of FAM-siRNA normalized with per mg of total cellular protein. To confirm MR-mediated internalization, the cells were incubated with free mannose which could bind MR at a final concentration of 1.2 mM for 30 min prior to MTC NPs treatment and throughout the uptake process. To directly visualize the cellular uptake, Raw 264.7 cells treated with MTC NPs containing FAM-siRNA for 4 h were washed with PBS, fixed with paraformaldehyde, and stained with Hoechst 33258 (10 mg/mL). The cells were observed with confocal laser scanning microscopy (CLSM, Zeiss, Germany). The detailed uptake mechanisms were explored as previously described [7]. Raw 264.7 cells were incubated with endocytic inhibitors genistein (200 mg/mL), methylb-cyclodextrin (Me-b-CD, 50 mM), wortmannin (50 nM), amiloride (500 mM), and chlorpromazine (10 mg/mL) for 30 min prior to MTC NPs application and throughout the uptake process. Results were denoted as the relative percentage of uptake compared to the control without inhibitor pretreatment. 2.7. In vivo cellular uptake Male C57BL/6 mice were orally administered with FAM-siRNA loaded MTC NPs at the siRNA dose of 100 mg/kg. After 6 h, PECs were isolated, seeded in 24-well plates, and cultured for another 2 h. The uptake level was measured as described above. 2.8. In vitro RNAi Raw 264.7 cells were seeded in 24-well plates at 4 104 cell/well and cultured at 37 C for 24 h. The culture media were replaced by the fresh DMEM. Then MTC NPs containing TNF-a siRNA were added and incubated with the cells at a final siRNA concentration of 0.4 mg/mL for 24 h, after which LPS (10 ng/mL) was incubated for another 5 h. The culture media were collected for the determination of extracellular TNF-a level by ELISA kit (R&D, USA). Cells were collected for the isolation of RNA with Trizol reagent (Sigma, USA). cDNA was synthesized from 500 ng RNA using PrimeScript® RT reagent kit (Takara Biotechnology Co., Ltd., China) for the determination of intracellular TNF-a mRNA level via Real-Time PCR. The ribosomal mRNA 36B4 was used as an internal loading control. 2.9. In vivo RNAi 2.9.1. In vivo RNAi against acute hepatic injury Male C57BL/6 mice were orally administered with PBS or various TNF-a siRNA loaded MTC NPs at a siRNA dose of 30 mg/kg. After 24 h, LPS (12.5 mg/kg) and D-GalN (1.25 g/kg) were intraperitoneally injected to induce acute hepatic injury. After 2 h, blood was collected and centrifugated at 12,000 rpm for 4 min to isolate the serum for monitoring the TNF-a level by ELISA kit (R&D, USA). Mice were sacrificed and liver, spleen, and lung were collected and immersed in the RNAlater solution. Then they were homogenized in liquid nitrogen and RNA was extracted with Trizol
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reagent (Sigma, USA). The intracellular TNF-a mRNA level in the liver, spleen, and lung was determined by Real-Time PCR. Male C57BL/6 mice were orally delivered with various TNF-a siRNA loaded MTC NPs at a siRNA dose of 30 mg/kg or PBS, and 24 h post administration, LPS (12.5 mg/kg) and D-GalN (1.25 g/kg) were intraperitoneally injected. After 6 h, liver was harvested, fixed with 4% paraformaldehyde (w/v), and stained with haematoxylin/eosin (H&E) for histological analyses.
As depicted in Fig. S2, MTC NPs exerted inappreciable cytotoxicity on Raw 264.7 cells even at the siRNA concentration of 1 mg/mL which was 2.5-fold higher than the dose applied in the in vitro transfection.
2.9.2. In vivo RNAi against UC Male C57BL/6 mice bearing UC were obtained by DSS solution (3%, w/v) as drinking water over a period of 8 days (day 0e7). Healthy control mice were treated with normal water. Mice were orally administered each day with PBS or various TNF-a siRNA loaded MTC NPs at a siRNA dose of 30 mg/kg for 5 consecutive days (day 0e4). Body weight was daily monitored. On day 7, mice were sacrificed and colons were collected. The colonic segments were homogenized with cold PBS (pH 7.4), followed by centrifuging at 12,000 rpm and 4 C for 20 min. The supernatant was collected to quantify the TNF-a level by ELISA kit (R&D, USA). Colonic segments were immersed in the RNAlater solution and then homogenized in liquid nitrogen. RNA was extracted with Trizol reagent (Sigma, USA) and intracellular TNF-a mRNA level in the colons was monitored by Real-Time PCR. Colon sections were fixed with 4% paraformaldehyde (w/v) and stained with H&E for histological analyses.
To evaluate the effects of mannose density on the structural stability of MTC NPs, heparin sodium was adopted as the competitive polyanion to dissociate siRNA from MTC NPs. As shown in Fig. 2B, the lowest concentration of heparin sodium to completely dissociate siRNA was 0.1 mg/mL for MTC-4 and MTC-13 NPs and 0.13 mg/mL for MTC-21 NPs, suggesting the higher structural stability of MTC-21 NPs against the attack of ionic molecules in physiological fluids. As displayed in Fig. 2C, naked siRNA was largely degraded after incubation with RNase A and various physiological fluids as evidenced by the invisible bands. However, all MTC NPs could effectively protect the encapsulated siRNA from degradation by nuclease and physiological fluids as indicated by the obviously brighter bands compared with naked siRNA.
2.10. Statistical analysis Data were represented as mean ± SD. Statistical analysis was performed via Student's unpaired t test between two groups or One-Way Analysis of Variance (ANOVA) with Tukey's post-hoc test among more than two groups (SPSS software, version 12.0, SPSS Inc.). A value of P < 0.05 was judged to be significant.
3. Results 3.1. Synthesis and characterization of MTC conjugates MTC conjugates were synthesized through sequential trimethylation, mannosylation, and thiolation of chitosan as previously described [7]. TMC possessed the quaternization degree of 34% as determined by 1H NMR (Fig. S1) [18]. Various amounts of mannose residues were conjugated to TMC by altering the feed mass ratio of MPITC to TMC (Table 1). In the 1H NMR spectra, the peaks at 7.0e7.4 ppm were assigned to the proton on benzene of MPITC, confirming that mannose had been conjugated onto the backbone of TMC (Fig. 1). By comparing peak areas of benzene protons in MPITC (7.0e7.4 ppm) with acetyl protons (2.1 ppm) in chitosan, the mannose densities were calculated to be 4%, 13%, and 21%, respectively. Cysteine was sequentially conjugated onto the MT to form MTC via amidation reaction. Free sulphydryl and disulfide content of MTC conjugates were summarized in Table 1, indicating that about 13% of the amino groups in chitosan were occupied by cysteine for all MTC conjugates. MTC conjugates with mannose densities of 4%, 13%, and 21% were denoted as MTC-4, MTC-13, and MTC-21, respectively. 3.2. Preparation and characterization of MTC NPs MTC NPs encapsulating siRNA were prepared through ionic gelation in the presence of TPP as crosslinkers. As summarized in Table 2, MTC NPs with distinct mannose densities possessed particle sizes ranging from 140 nm to 170 nm (polydispersity index (PDI) < 0.2). Their zeta potentials decreased with the increase of mannose density. SEM observation revealed the well-dispersed and subsphaeroidal morphology of MTC NPs (Fig. 2A).
Table 1 Preparation and characterization of MTC conjugates. Name
MPITC:TMC (w/w)
Mannose density (%)
Free sulphydryl (mmol/g)
Disulfide (mmol/g)
MTC-4 MTC-13 MTC-21
1:20 1:5 1:2.5
4 13 21
100.3 ± 2.5 110.5 ± 2.1 100.2 ± 13.4
280.7 ± 16.5 260.6 ± 7.9 283.6 ± 6.3
3.3. Structural stability and siRNA protection of MTC NPs
3.4. siRNA encapsulation and release As indicated in Fig. 3A, MTC NPs exhibited complete retardation of siRNA in the loading well, demonstrating efficient encapsulation of siRNA in all NPs. In vitro release profiles showed that the accumulative release percentage of siRNA within 8 h decreased with the elevation of mannose density, which was about 93%, 82%, and 66% for MTC-4, MTC-13, and MTC-21 NPs, respectively (Fig. 3B). 3.5. In vitro cellular uptake As for the uptake in Raw 264.7 cells (Fig. 4A), the maximum in vitro uptake amount was found in MTC-21 NPs with the highest mannose density, which was 1.5 and 3.5 folds enhancement compared to that of MTC-4 and MTC-13 NPs, respectively. Preferable uptake was detected in MTC-4 NPs with higher zeta potential in spite of their lower ligand density in comparison with MTC-13 NPs. CLSM images revealed the similar tendency with quantitative analysis in which MTC-4 and MTC-21 NPs exhibited superior cellular uptake compared to MTC-13 NPs (Fig. 4B). For in vitro internalization by PECs, the uptake amounts of MTC-4 NPs were higher than those of MTC-13 and MTC-21 NPs (Fig. S3A). Free mannose was pre-incubated with cells to occupy MR prior to the addition of MTC NPs and a remarkable decrease in the uptake amount of MTC NPs was observed, confirming the MR-mediated endocytosis (Fig. 4C, Fig. S3B). Additionally, the inhibition ratios of internalization in Raw 264.7 cells of MTC-4, MTC-13, and MTC-21 NPs in the presence of free mannose were 17.3%, 44.1%, and 61.5%, respectively, implying that the MR-mediated endocytosis was more involved in the internalization of MTC NPs with higher mannose density (Fig. 4C). However, as for PECs, there was no significant difference in the inhibition ratios among different MTC NPs, suggesting that MR-mediated endocytosis was saturated even in the case of MTC-4 NPs with the lowest ligand density (Fig. S3B). Detailed uptake mechanisms were investigated by utilizing various endocytic inhibitors. The cellular uptake of MTC NPs was remarkably inhibited by genistein and Me-b-CD, proposing a caveolae-mediated endocytosis pathway. The suppressed uptake in the presence of wortmannin and amiloride indicated macropinocytosis participation in the uptake process. Comparatively, chlorpromazine exerted minimal effect on the internalization of MTC NPs, excluding the involvement of clathrin-dependent pathway (Fig. S4). Clathrin-independent endocytosis of all MTC NPs would
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Fig. 1. (A) Synthetic route of MTC conjugates with different mannose densities. (B) 1H NMR spectra of MTC conjugates with different mannose densities. The peaks at 7.0e7.4 and 2.1 ppm were assigned to the protons on benzene of MPITC and acetyl groups of TMC, respectively.
avoid the endosomal/lysosomal entrapment, which was beneficial for their highly efficient cellular uptake and gene silencing.
NPs, which was in agreement with the results of in vitro internalization by PECs (Fig. S3A).
3.6. In vivo cellular uptake
3.7. In vitro RNAi
As depicted in Fig. 4D, orally delivered MTC-4 NPs presented higher in vivo cellular uptake by PECs than MTC-13 and MTC-21
In terms of in vitro RNAi in Raw 264.7 cells against LPS-triggered TNF-a secretion, MTC-21 NPs with the highest mannose density
S. Chu et al. / Biomaterials 52 (2015) 229e239 Table 2 The particle sizes and zeta potentials of MTC NPs. Sample
Particle size (nm)a,b
Zeta potential (mV)a
MTC-4 NPs MTC-13 NPs MTC-21 NPs
166.6 ± 0.7 (0.190 ± 0.015) 148.6 ± 2.4 (0.046 ± 0.025) 143.3 ± 1.1 (0.151 ± 0.029)
35.8 ± 1.7 22.7 ± 1.7 18.7 ± 0.6
a b
Data represented the mean ± SD (n ¼ 3). Values in parentheses represented the polydispersity index.
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presented exceptional silencing efficiency of 72.8%, which outperformed MTC-4 and MTC-13 NPs. In addition, MTC-4 NPs dramatically depleted TNF-a protein production by 62.6%, which was significantly higher than the corresponding value of 51.6% in MTC-13 NPs (Fig. 5A). For the efficacy of silencing TNF-a mRNA expression, the tendency was in accordance with the results in efficiency of suppressing TNF-a protein. MTC-21 NPs exhibited higher depletion ratio of 65.1% than that of MTC-4 and MTC-13 NPs (Fig. 5B).
Fig. 2. Characterization of MTC NPs and their protection of siRNA integrity. (A) SEM images of MTC NPs. Bar represented 200 nm. (B) Agarose gel electrophoresis of MTC NPs at 56 V for 1 h following incubation with various concentrations of heparin sulfate. (C) Agarose gel electrophoresis of MTC NPs at 56 V for 1 h after treatment with physiological fluids or RNase A. Naked siRNA treated with physiological fluids and RNase A served as the positive controls. Naked siRNA without any treatment served as the negative controls.
Fig. 3. Encapsulation and release of siRNA in MTC NPs. (A) Agarose gel electrophoresis of MTC NPs at 56 V for 1 h. (B) In vitro release profiles of FAM-siRNA from MTC NPs in 0.2 M PBS (pH 7.4) at 37 C.
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Fig. 4. The cellular uptake of MTC NPs containing FAM-siRNA in in vitro Raw 264.7 cells and in vivo PECs. (A) In vitro cellular uptake of MTC NPs in Raw 264.7 cells. Indicated values were mean ± SD (n ¼ 4). *P < 0.05, **P < 0.01, ***P < 0.001. (B) CLSM images showing cellular uptake of MTC NPs loaded with FAM-siRNA (green) in Raw 264.7 cells following incubation for 4 h. The nuclei were stained with Hoechst 33258 (blue). Bar represented 10 mm. (C) Uptake inhibition of MTC NPs in Raw 264.7 cells following treatment with free mannose (1.2 mM). Indicated values were mean ± SD (n ¼ 4). (D) In vivo cellular uptake of MTC NPs in PECs following oral administration. Indicated values were mean ± SD (n ¼ 4). *P < 0.05. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
3.8. In vivo RNAi 3.8.1. In vivo RNAi against acute hepatic injury LPS/D-GalN-induced acute hepatic injury mouse model was established to investigate RNAi efficiency of orally-delivered MTC NPs in systemic and acute inflammation disease. MTC NPs with various mannose densities at a siRNA dose of 30 mg/kg remarkably blocked serum TNF-a production. The silencing efficiency of MTC-4 and MTC-13 NPs was 81% and 79%, respectively, which were superior to that of 42% in MTC-21 NPs (Fig. 6A). Accordingly, notable suppression of TNF-a mRNA expression for all NPs was detected in macrophage-enriched organs (liver, spleen, and lung), among which MTC-21 NPs exhibited relatively inferior TNF-a mRNA depletion efficiency of about 43%, 45%, and 51% in liver,
spleen, and lung, respectively (Fig. 6B). Histological sections from MTC-4 and MTC-13 NPs-treated mice showed obvious alleviation of LPS/D-GalN-triggered liver damage including congested central vein as well as swollen and disarranged hepatocytes (Fig. 6C). 3.8.2. In vivo RNAi against UC DSS-induced UC mouse model was employed to investigate RNAi efficiency of orally-delivered MTC NPs in local and chronic inflammation disease. MTC NPs significantly suppressed TNF-a protein and mRNA expression in colonic tissues. MTC-4 NPs suppressed TNF-a production in colonic tissues with the inhibition ratio of 75.8%, which was significantly higher than that of MTC-13 and MTC-21 NPs (Fig. 7A). Similar trend was found in the TNF-a mRNA expression (Fig. 7B). MTC-4 NPs significantly blocked TNF-a
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Fig. 5. In vitro gene silencing of MTC NPs containing TNF-a siRNA in Raw 264.7 cells. Inhibition of TNF-a expression in the protein (A) and mRNA (B) levels compared to the controls in LPS-stimulated Raw 264.7 cells. Cells activated by LPS (10 ng/mL) without NPs treatment served as the controls. Indicated values were mean ± SD (n ¼ 3). *P < 0.05, ***P < 0.001.
mRNA expression in colonic tissues by 79.4%, which were superior to MTC-13 and MTC-21 NPs. UC was characterized by sustained weight loss and shortened colon length. As illustrated in Fig. 7C and D, the body weight and colon length of mice without any treatment dramatically decreased by 19% and 37% after 8-day exposure to DSS, respectively. By contrast, oral administration of MTC NPs effectively alleviated the weight loss within 5% (Fig. 7C) and the colon shortening within 16% (Fig. 7D). MTC-4 NPs exerted the superiority in prevention of colon shortening among all NPs as evidenced by the minimal reduction of 7%. H&E stained colonic sections of UC control mice exhibited various histological damages as compared with those of healthy mice, including depletion of crypt and goblet cells, abnormality or loss of epithelial cells, marked infiltration of mononuclear cells, and irregular mucosal structure (Fig. 8) [20]. Sections from MTC-4 NPstreated mice revealed histological characteristics resembling those of healthy mice, while MTC-13 and MTC-21 NPs-treated mice showed relatively poor therapeutic efficacy. 4. Discussion Due to the non-selectivity and hydrophilic nature of siRNA [3], numerous works had been focused on the rational design of mannose-modified vectors for macrophage-targeting treatment of inflammatory diseases [7,8]. However, little emphasis had been laid on the optimization of ligand density to improve gene silencing efficiency. Therefore, the study herein was aimed at exploring the effects of mannose density of polymeric NPs on cellular uptake and RNAi efficiency both in vitro and in vivo. MTC conjugate-based delivery systems have shown significantly superior cellular uptake and gene silencing to trimethyl chitosancysteine NPs via mannose ligand-mediated macrophage-targeting in our previous work [2,7], thus representing an ideal platform for us to elucidate the correlation between the ligand density and cellular uptake as well as RNAi efficiency of polymeric NPs. On the basis of prior reports that mannose density of about 5% was necessary for sufficient MR-mediated internalization [21,22], we successfully synthesized a series of MTC conjugates with mannose densities of 4%, 13%, and 21% through adjusting the feed mass ratio of MPITC to TMC, alternatively to examine whether a mannose density above 5% would further increase cellular uptake and gene
transfer efficiency. MTC conjugates and siRNA self-assembled into NPs through ionic gelation with anionic TPP. All NPs possessed similar particle sizes and preferable protection of siRNA integrity in physiological fluids. This might probably be attributed to the complete complexation of MTC conjugates towards anionic siRNA, thus preventing the approach of nucleases during delivery to the macrophages. To clarify the effects of mannose density on the structural stability of MTC NPs, polyanion heparin was employed to simulate the attack of anionic molecules to positively-charged NPs in physiological fluids. MTC NPs with higher mannose density displayed stronger resistance to heparin, thereby possessing more stable structure (Fig. 2B). This phenomenon might be explained by the following two reasons. On one hand, substitution of primary amino groups with mannose residues would diminish positively-charged groups, leading to weak electrostatic interaction with polyanion heparin [16]. On the other hand, uncharged and hydrophilic mannose residues with plentiful hydroxyl groups would likely orientate towards the surface of MTC NPs and link with each other, forming a corona structure which was able to resist the approach of competitive molecules [23]. Moreover, the aforementioned dense corona composed of mannose residues might prevent the dissociation of siRNA from MTC NPs via steric hindrance and retain the cargoes inside the core of MTC NPs. Consequently, slower siRNA release rate was noted in MTC NPs with higher mannose density (Fig. 3B). Additionally, the above-mentioned charge shielding and steric hindrance effects might contribute to the phenomenon that zeta potentials decreased with the increase of mannose density, which would further exert an implication on the cellular uptake by macrophages (Table 2). Since mannose conjugation was aimed at promoting the uptake of MTC NPs by macrophages, we went on to investigate the effects of mannose density on the in vitro internalization by Raw 264.7 cells of MTC NPs (Fig. 4AeC). In contrast to MTC-4 and MTC-13 NPs, as expected, MTC-21 NPs bearing higher mannose residues were more readily internalized into Raw 264.7 cells, which was in line with previous results [10,22]. The highest cellular uptake level of MTC-21 NPs was assumed to majorly result from the enhanced MR-mediated internalization, as evidenced by the maximum uptake inhibition in the presence of free mannose (Fig. 4C). Meanwhile, the strongest structural stability and slowest siRNA
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Fig. 6. In vivo gene silencing of MTC NPs containing TNF-a siRNA in acute hepatic injury mouse model at the siRNA dose of 30 mg/kg. (A) TNF-a production in the serum evaluated by ELISA assay. Indicated values were mean ± SD (n ¼ 6). *P < 0.05. (B) TNF-a mRNA levels in mouse liver, spleen, and lung determined by Real-Time PCR. Indicated values were mean ± SD (n ¼ 3). *P < 0.05. Acute hepatic injury mice induced by LPS (12.5 mg/kg) and D-GalN (1.25 g/kg) with the treatment of PBS served as the controls. (C) H&E stained liver sections for histopathological analyses. Bar represented 500 mm.
release in MTC-21 NPs would lead to less premature release, which might also contribute to their preferable uptake amount. Interestingly, the lower mannose density of MTC-4 NPs did not result in decreased internalization compared with MTC-13 NPs. A possible explanation for this phenomenon would be the strong electrostatic interactions between negatively charged cell membrane and MTC-4 NPs with the highest positive charges among all NPs [24]. TNF-a was elicited by LPS-activated macrophages, and served as an important regulator of inflammation [2]. Therefore it was selected as the therapeutic target in this investigation. MTC-21 NPs performed better in attenuating TNF-a production and TNF-a mRNA expression in LPS-stimulated Raw 264.7 cells as compared to MTC-4 and MTC-13 NPs. And MTC-4 NPs mediated superior TNF-a knockdown compared to MTC-13 NPs (Fig. 5). The tendency of in vitro silencing efficiency with respect to mannose density was consistent with in vitro cellular uptake results. Unlike plasmid DNA,
the dissociation of siRNA from cationic polymeric vector was much easier due to its short length and rigid nature, e.g. the majority of siRNA was released within 8 h for all NPs in our study (Fig. 3B). Moreover, disulfide reduction of MTC NPs would occur under the high concentration of intracellular glutathione. Taken together, siRNA would be rapidly dissociated from MTC NPs and interact with target mRNA once entry into the macrophages. Therefore, the in vitro gene silencing efficiencies of MTC NPs might be closely related to their uptake amounts. Due to the greater complexity of physiological surroundings and transport processes following oral administration than in vitro conditions, the correlation between ligand density and in vivo cellular uptake as well as RNAi efficacy could not be accurately predicted by in vitro results. Hence, the in vivo uptake of MTC NPs in PECs was examined after oral gavage to mice. The highest in vivo uptake amounts by PECs were noted in MTC-4 NPs rather than
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Fig. 7. In vivo gene silencing and the therapeutic efficacy of MTC NPs containing TNF-a siRNA in the UC mouse model. (A) Inhibition of TNF-a secretion in the colonic tissues measured by ELISA. Indicated values were mean ± SD (n ¼ 6). *P < 0.05, **P < 0.01. (B) Attenuation of TNF-a mRNA in the colonic tissues detected by Real-Time PCR. Indicated values were mean ± SD (n ¼ 3). *P < 0.05. (C) Changes of body weight over time in mice treated with or without 3% DSS in drinking water. Indicated values were mean ± SD (n ¼ 6). (D) Changes of colon length over time in mice treated with or without 3% DSS in drinking water. Indicated values were mean ± SD (n ¼ 6). *P < 0.05.
MTC-13 and MTC-21 NPs (Fig. 4D), which was consistent with the results of in vitro cellular uptake by PECs (Fig. S3A). In contrast to the highly-efficient in vitro internalization by Raw 264.7 cells, the poor in vitro uptake by PECs of MTC-21 NPs was observed, which might be due to their differences in mannose density on the cell membranes. Receptor density has been proven to exert an important influence on ligand-receptor interaction [25]. Compared with Raw 264.7 cells (leukemic macrophages) containing abundant MR, there probably exists limited MR on the cell membranes of PECs (primary macrophages). Such speculation was confirmed by competitive inhibition experiment in the presence of free mannose wherein MR-mediated endocytosis in PECs was saturated in the case of MTC-4 NPs with the lowest ligand density (Fig. S3B). Owing to the saturation of ligand-receptor recognition, increasing mannose density in MTC NPs from 4% to 21% would cause decreased surface charges and thus weaken electrostatic interaction with cell membranes rather than enhanced MR-mediated endocytosis, thereby reducing in vitro and in vivo internalization by PECs of MTC-21 NPs. Moreover, steric hindrance effect stemming from excessive mannose residues on the surface of NPs would presumably be amplified when the binding sites for mannose were limited. For instance, overcrowding could prevent ligand molecules
from obtaining the correct orientation essential for the receptor recognition, while lowering the mannose density enhanced their congruency to MR, thus providing more effective binding and uptake by macrophages. Accordingly, the highest in vivo cellular uptake was found in MTC-4 NPs while further increasing mannose density would exert adverse effects on in vivo internalization of MTC NPs (Fig. 4D), which was consistent with the previous results that the mannose density of about 5% would be sufficient for MR-mediated gene delivery to macrophages [21,22]. Inflammation models of acute liver injury and chronic UC, characterized by excessive secretion of TNF-a from macrophages, are related to discrepant target macrophages (systemic vs. local) and focal tissues (liver vs. colon), which might have distinct demands on the mannose density for highly enhanced therapeutic efficacy. These models were consequently established to evaluate disease-specific effects of mannose density on in vivo RNAi efficiency and anti-inflammatory efficacy mediated by orally delivered MTC NPs (Fig. 6 and 7). Considering the detailed pathogenesis of inflammation models adopted and the relatively slow absorption of siRNA via oral administration, to assure the high possibility for uncovering the effects of mannose density on in vivo RNAi and antiinflammation efficacy, the prophylactic treatment regimens
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Fig. 8. H&E stained colon sections for histopathological analyses. Bar represented 100 mm.
frequently reported, dosing started either before or on day 0, were therefore adopted for their effectiveness in siRNA delivery for attenuating inflammatory responses [1,2,4,5,7]. MTC-4 NPs presented robust anti-inflammation efficacy against both chronic UC and acute hepatic injury, which was likely due to their highest in vivo cellular uptake among all NPs (Fig. 4D). Compared to published vectors involving TNF-a knockdown for intestinal inflammation therapy, MTC-4 NPs demonstrated outstanding in vivo RNAi potency, wherein a total therapeutic dose of 150 mg siRNA/kg was 2e3 orders of magnitude less [26]. These results supported the strategy that optimization of ligand density was essential to achieve highly-performed therapeutic efficacy with minimal siRNA dose. It was intriguing to note that in spite of the unsatisfactory efficacy in UC model, MTC-13 NPs mediated efficient RNAi against acute hepatic injury. This inconsistent therapeutic outcome might be attributed to the distinct requirements for RNAi stemming from different types of inflammation (chronic UC vs. acute hepatic injury). The relatively quick siRNA release from MTC-13 NPs could lead to the rapidly increased intracellular concentration of free siRNA, which was beneficial to initiate prompt RNAi and thus offer timely protection against acute hepatic injury. However, such release behavior of MTC-13 NPs might compromise the long-time silencing efficiency. Therefore, their anti-inflammation efficacy against UC which preferred sustained TNF-a silencing was relatively inefficient. The discrepancy might also be due to the differences in the phenotype and functions of macrophages involved in relevant inflammation (systemic vs. local) and related tissues (liver vs. colon) [27]. As for MTC-21 NPs, poor in vivo RNAi efficiency and therapeutic effect were observed in both inflammation models, which was in accordance with the results of in vivo cellular uptake. The unsatisfactory RNAi performance of MTC-21 NPs supported the presumption that a higher ligand density above a certain optimum value would enhance the “off-target” effect, thus leading to decreased “selective delivery” of encapsulated drugs to target cells and lowered therapeutic efficacy [25].
5. Conclusions Through controlling the feed mass ratio of MPITC and TMC, a series of MTC conjugates with different mannose densities ranging from 4% to 21% were synthesized to underscore the effects of mannose density on cellular uptake and RNAi efficiency of polymeric NPs both in vitro and in vivo. MTC-4 NPs provided a relatively high in vitro and in vivo cellular uptake, resulting in the highly-performed in vitro and in vivo RNAi efficiency as well as antiinflammatory efficacy at a low dose of siRNA. Comparatively, MTC-21 NPs were correlated with supreme in vitro cellular uptake and gene knockdown, but their performance in in vivo RNAi remained unsatisfactory because of poor in vivo cellular uptake. MTC-13 NPs showed excellent TNF-a silencing in acute hepatic injury, whereas exerted disadvantageous influence on therapeutic efficacy of UC. Our data provided the in vivo evidence that polymeric carriers with lower ligand density would be more favorable for the treatment of inflammatory diseases than their counterparts with higher ligand density and the effects of ligand density were dependent on the type of inflammatory diseases. Therefore, the ligand density should be identified as a crucial parameter for screening targeted polymeric vehicles of siRNA for anti-inflammation therapy, and the idea could be further extended to other applications including cancer therapy.
Acknowledgements The authors are thankful for the financial support from the National Natural Science Foundation of China (No. 81072595).
Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.biomaterials.2015.02.044.
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