The enhanced immune response of hepatitis B virus DNA vaccine using SiO2@LDH nanoparticles as an adjuvant

Biomaterials xxx (2013) 1e13

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The enhanced immune response of hepatitis B virus DNA vaccine using SiO2@LDH nanoparticles as an adjuvant Jin Wang a, Rongrong Zhu a, Bo Gao a, Bin Wu a, Kun Li a, Xiaoyu Sun a, Hui Liu b, **, Shilong Wang a, * a b

Tenth People’s Hospital, School of Life Science and Technology, Tongji University, 1239 Siping Road, Shanghai 200092, PR China Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai 200438, PR China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 24 July 2013 Accepted 17 September 2013 Available online xxx

Various approaches have been used to improve systemic immune response to infectious disease or virus, and DNA vaccination has been demonstrated to be one of these effective ways to elicit protective immunity against pathogens. Our previous studies showed that layered double hydroxides (LDH) nanoparticles could be efficiently taken up by the MDDCs and had an adjuvant activity for DC maturation. To further enhance the immune adjuvant activity of LDH, coreeshell structure SiO2@LDH nanoparticles were synthesized with an average diameter of about 210 nm. And its high transfection efficiency in vitro was demonstrated by using GFP expression plasmid as model DNA. Exposing SiO2@LDH nanoparticles to macrophages caused a higher dose-dependent expression of IFN-g, IL-6, CD86 and MHC II, compared with SiO2 and LDH respectively. Furthermore, in vivo immunization of BALB/c mice indicated that, DNA vaccine loaded-SiO2@LDH nanoparticles not only induced much higher serum antibody response than naked DNA vaccine and plain nanoparticles, but also obviously promoted T-cell proliferation and skewed T helper to Th1 polarization. Additionally, it was proved that the caveolae-mediated uptake of SiO2@LDH nanoparticles by macrophage lead to macrophages activation via NF-kB signaling pathway. Our results indicate that SiO2@LDH nanoparticles could serve as a potential non-viral gene delivery system. Ó 2013 Elsevier Ltd. All rights reserved.

Keywords: Layered double hydroxides DNA vaccine Hepatitis B surface antigen Adjuvant Immunology Macrophages

1. Introduction Chronic hepatitis B virus (HBV) infection remains as a fatal worldwide public health problem [1,2]. And more than 370 million people worldwide were chronically infected with HBV [3]. Compared with currently available therapies which failed to completely control viral replication in most patients, DNA vaccination was supposed to be a promising strategy for activating immune responses against HBV infection [4]. Although DNA vaccines were of great interest in anti-infection of HBV, the effects were not satisfactory in clinical trials [5,6], mostly due to the difficulties associated with DNA stability and delivery, resulting in inefficient antigen presentation. Therefore, effective vaccine vectors are desirable which can protect DNA vaccine from damage in unfavorable conditions after systemic or mucosal administration, and increase its uptake by antigen-presenting cells to facilitate the

* Corresponding author. Tel.: þ86 21 659 82595; fax: þ86 21 659 82286. ** Corresponding author. Tel.: þ86 21 818 75463; fax: þ86 21 655 62400. E-mail addresses: [email protected] (H. Liu), [email protected], [email protected] (S. Wang).

induction of potent immune responses. There were three primary mechanisms of adjuvant function: stabilization of antigen, delivery of antigen, and activation of innate immunity. For instance, efficient and potent immune responses were induced by DNA vaccine adsorbed onto anionic poly (lactic-co-glycolic acid) microspheres, chitosan or anionic wax nanoparticles [7e15]. However, in most of these systems the presence of adsorbed surfactants or polymeric stabilizers may give rise to irreproducibility of the vaccine formulation and premature release of the charged molecules and the antigen during preparation of the formulation and vaccine administration, which leads to variable efficacy as well as undesirable toxic effects of the free charged molecules [16]. Moreover, the size of the microsphere may be not suitable for macrophage phagocytosis and produce limited immune response, thus leading to further criticism for future clinical development. Layered double hydroxide (LDH), commonly known as hydrotalcite-like materials and anionic clays, was a family of layered nanomaterial with wide applications in catalysts, absorption, pharmaceutics, and photochemistry [17,18]. LDH was already used as antacids [19] and pH-sensitive drug carrier [20], mainly due to the hydrolysis behavior in acidic media and anionic exchange capacity which make them attractive as potential drug vectors for

0142-9612/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biomaterials.2013.09.060

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targeting tissues and intracellular organelles [21,22]. Recently, LDHs have received considerable attentions as vaccine delivery system for their properties of low cytotoxicity, good biocompatibility and total protection of loaded DNA vaccines. Our previous studies also showed that LDH nanoparticles could be taken up by MDDCs efficiently, and have an adjuvant activity for DC maturation [23,24]. Macrophages, typical phagocytic cells that play an important role in body defenses, exert protective and pathogenic activities and have a function in both innate and adaptive immunity [25]. Besides, macrophages produce a number of reactive oxygen and nitric oxide (NO). Moreover, via secretion of immune modulating factors, macrophages make a considerable contribution to both local inflammation and other induced innate responses underlining the relevance of the cells in orchestrating immune defense mechanisms. Furthermore, macrophages can also act as APCs with an exquisite capacity to interact with T cells and participate in activation of adaptive immunity. They were proved to have an exceptional ability to initiate both primary and secondary immune responses in vivo by expressing co-stimulatory molecules such as CD40, CD80, CD86, and peptides loaded onto major histocompatibility complexes (MHC) class I and II, and creating cytokines (e.g. IL6, IL-12 and TNF-a) [26]. The activated macrophage played a key role in the initiation of the adaptive immune response, since fully activated macrophages were very potent antigen presenting cells to prime naive T cells [27]. Here, in order to enhance the adjuvant immune activity of inorganic vaccine delivery vector, we synthesized SiO2@LDH coree shell nanoparticles with mesoporous silica as core and LDH as shell, to activate macrophages and thereby enhance systemic immune responses in animals. SiO2@LDH coreeshell nanoparticles in this study were used as the delivery vehicle of HBVsAg DNA vaccine which encodes hepatitis B surface antigen protein. And the characteristics and advantage of SiO2@LDH nanoparticles as DNA vectors to activate macrophages were studied and related mechanism was discussed. 2. Materials and methods 2.1. Nanoparticles preparation Synthesis of SiO2@LDH Nanoparticles included: preparation of mesoporous silica (SiO2) nanoparticles; deposition of AlOOH on the surface of SiO2 spheres by layer-by-layer (LBL) method (termed SiO2@AlOOH); growth of LDH nanoplatelets on the surface of SiO2@AlOOH by an in situ growth technique [28]. The synthesis procedures were described in detail as follows: (1) Synthesis of mesoporous silica (SiO2) particles: 14.3 g (96 mmol) of trolamine (TEA) and 2 ml (9 mmol) of ethylsilicate (TEOS) were mixed in a 125 ml polypropylene bottle with a lid. The two-phase mixture was heated at 90  C for 20 min. As soon as the mixture was taken out, 26.7 g hexadecyltrimethyl ammonium bromide (CTAB, 2.5% wt) which had been preheated to 60  C was added immediately, and the final mixture was incubated with 600 rpm stir overnight at 23  C [29]. (2) Preparation of SiO2@AlOOH nanoparticles: according to the method reported, the AlOOH primer sol was prepared [30]. 11.3 g Aluminum isopropoxide (Al(OPr)3) was dissolved in 100 ml deionized water by stirring at 85  C for 20 min. HNO3 (1.0 M) was then dropped slowly into the solution to initiate the hydrolysis of Al(OPr)3 with pH from 3 to 4. The mixture was stirred at 85  C for 2 h and slowly cooled down to room temperature, then solid boehmite (AlOOH) could be obtained after water evaporation. After milling, 5.8 g boehmite was dissolved in 107 ml deionized water with 1 h stirring at 85  C. Then HNO3 (9.5 ml, 1.0 M) was slowly dropped into the solution, and the mix was refluxed gently with stirring for 6 h. AlOOH primer sol was prepared after slowly cooling down to room temperature process. Subsequently, SiO2 nanoparticles were dispersed in AlOOH primer sol for 1 h with vigorous agitation. Microspheres were washed thoroughly with ethanol. The resulting SiO2@AlOOH nanoparticles were dried in air for 30 min. The complete process (dispersion, withdrawing and drying) was repeated 10 times. (3) Preparation of SiO2@LDH nanoparticles: MgAl-LDH Nano platelet shell in situ crystallization on the surface of SiO2@AlOOH nanoparticles was performed. According to a typical procedure, 0.005 M of Mg (NO3)2$6H2O and 0.04 M of NH4NO3 were dissolved together in deionized water (total volume, 120 ml). 0.04 g SiO2@AlOOH nanoparticles were mixed with the above solution and incubated at 80  C for 24 h. After cooling, the

expected SiO2@MgAl-LDH microspheres were rinsed with ethanol and dried at room temperature. The pristine layered double hydroxides (LDH) were prepared according to the method in Ref. [23]. Preparation of nanoparticle-DNA complex was performed as follows: supercoiled plasmid pEGFP-N2 (BD Biosciences, USA), encoding green fluorescent protein (GFP), was transformed into Escherichia coli DH5a competent cells by standard molecular cloning methods. The pEGFP-N2 or pcDNA3-HBVsAg plasmid was extracted by using Qiagen maxiprep kit, and diluted in deionized water at a final concentration of 1 mg/ml. The nanoparticle-plasmid compound, in which the ratio (w/w) between plasmid DNA and nanoparticle was fixed at 1:40, was incubated with shaking at 37  C overnight. 2.2. Cytotoxicity test Human embryonic kidney (HEK) 293T cells were cultured in 96-well plates with 1  105 cells per well in which nutrient solution was composed of Dulbecco’s modified Eagle’s (DMEM) medium (Hclone No.1432) supplemented with 10% fetal bovine serum, L-glutamine (2 mmol/l) and penicillin/streptomycin (50 units/ml). Triplicate wells were treated with SiO2, LDH and SiO2@LDH at the concentration of 1, 10, 100 and 1000 mg/ml. The plates were incubated at 37  C in 5% CO2 for 24 h and 48 h respectively. Cells without any agents served as a blank control. The number of living cells was determined by MTT assay with 3-(4,5-dimethyl-thiazole-2-yl)-2,5phenyltetrazolium bromide. After cells were incubated with 20 mL of MTT (5 mg/ml) for 4 h at 37  C under a light-blocking condition, the medium was removed and 150 mL of DMSO was added into each well. Absorbance was measured at 490 nm using the ELx800 reader (BioTek Instruments, Inc, Winooski, VT). 2.3. DNase protection assay In order to determine the loading efficiency of SiO2@LDH nanoparticles and the ability of SiO2@LDH nanoparticles to protect DNA from endonuclease degradation, SiO2@LDH/plasmid was subjected to DNase treatment and analyzed by agarose gel electrophoresis. The nanoparticle-DNA compound was incubated together with DNase I for 1 h at 37  C respectively, and centrifuged at 2000 rpm for 5 min. The DNA was revered from the inorganic host by acidification and incubated at pH 2. Then an appropriate amount of DNA loading buffer was added to each sample. Agarose gel electrophoresis was run with a 1% agarose gel (TAE buffer, 1% ethidium bromide) at 100 V for 45 min and gels were subsequently imaged using a kodak gel documentation system. 2.4. Cellular transfection, cytokine ELISA and macrophages phenotype analysis The nanoparticle-plasmid compound was transfected into RAW264.7 cells for functional studies. RAW264.7 cells were cultured and maintained in DMEM medium supplemented with 10% fetal bovine serum at 37  C and 5% CO2, and seeded into 6 well plates when the confluence was 50e65%. Cells were incubated in media without serum for 24 h. Fetal bovine serum was added after 5 h post-treatment with the nanoparticle-pEGFP-N2 compound, and cells were incubated for 48 h at 37  C and observed under UV light in a fluorescence microscope. Then, the cells were transferred into polystyrene round-bottom tubes (5 ml) with 0.3 ml of PBS solution and maintained at 4  C for subsequent flow cytometry analysis. Secretion of cytokine from RAW264.7 cells in the presence of different nanoparticles (SiO2, LDH and SiO2@LDH), was analyzed by ELISA. The supernatants of RAW264.7, stimulated by different nanoparticles (SiO2, LDH and SiO2@LDH) under indicated conditions, were collected to analyze cytokine excretion using commercially available cytokine ELISA kits which is used for TNF-a, IFN-g, IL-6 and IL-12p70 analysis (R&D Systems, CA, USA). All operations were performed as described for the cytokine ELISA kits. The phenotype of RAW264.7 cells in the presence of different nanoparticles was analyzed. Nanoparticles were incubated with RAW264.7 cells for 24 h at 37  C. Control cells without nanoparticles were also treated similarly. Afterward, phenotype analysis of the RAW264.7 cells treated with SiO2, LDH and SiO2@LDH nanoparticles (40 mg/ml) respectively and control group was performed on a FACS cans flow cytometer (Becton Dickinson) after staining according to the manufacturers’ protocols with following mAbs, FITC-labeled anti-CD80, FITC-labeled anti-CD86, FITC-labeled anti-CD40 and FITC-labeled anti-MHC-II (all from BD Biosciences PharMingen, shanghai, China). 2.5. Immunization of mice Female BALB/c mice (6 weeks old) were purchased from the Laboratory Animal Center of Chinese Academy of Science, and housed in the pathogen-free animal facility of Tongji University. All animal-related procedures were performed according to protocols approved by the Tongji University Institute of Laboratory Animal Resources. Mice were divided into 5 groups (5 mice per group), each group was inguinal immunized as follows: Group I, PBS; Group II, pcDNA3-HBVsAg alone (100 mg plasmid into inguinal); Group III, SiO2 that entraps pcDNA3-HBVsAg (100 mg plasmid into inguinal); Group IV, LDH that entraps pcDNA3-HBVsAg (100 mg plasmid into inguinal); Group V, SiO2@LDH that entraps pcDNA3-HBVsAg (100 mg plasmid into inguinal) (Fig. 5A). Two weeks later, mice were boosted with the same immune

Please cite this article in press as: Wang J, et al., The enhanced immune response of hepatitis B virus DNA vaccine using SiO2@LDH nanoparticles as an adjuvant, Biomaterials (2013), http://dx.doi.org/10.1016/j.biomaterials.2013.09.060

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Fig. 1. TEM (A) and Zeta potential distribution (B) for SiO2, LDH and SiO2@LDH nanoparticles, respectively.

gene at an equivalent dose. Seven days after the third immunization, sera and spleens were harvested for antibody assay respectively.

2.6. Lymphocyte preparation, T-cell proliferation differentiation Mice were anesthetized 2 weeks after the last immunization, and the spleens were homogenized over 200 gage nylon mesh. Splenocytes were collected, treated with lysis buffer to eliminate red cells, washed, and resuspended in RPMI-1640 containing 10% FBS. Lymphocytes were derived from splenocytes by using nylon wool columns. Single-cell suspensions of lymphocytes were grown in 96-well plates (5  105 cells/well), stimulated in vitro with 5, 10 and 20 mg/ml HBVsAg peptides [IPQSLDSWWTSL, were synthesized and purification from GL Biochem (Shanghai) Ltd.] [31] or 5 mg/ml ConA (Sigma, Shanghai, China), and incubated at 37  C in 5% CO2 for 72 h. Cultures were incubated with 10 mL CCK-8 solution (Beyotime Institute of biotechnology, shanghai, China) for 4 h at 37  C. The absorbance was recorded at

450 nm. Spleen cells from immunized mice were grown at a starting concentration of 1  106 cells/ml in complete RPMI 1640 medium and stimulated for 6 h in the presence of 50 ng/ml phorbol 12-myristate 13-acetate, 1 mg/ml ionomycin, and 4 mg/ ml Brefeldin A (Sigma, USA). After washed with PBS, the cells were stained with FITC-conjugated anti-CD3 mAb and APC-conjugated anti-CD4 mAb (BD Biosciences PharMingen, China) for 30 min at 4  C, washed with PBS again, fixed with 4% paraformaldehyde, and permeabilized with PBS containing 0.5% saponin (both from BD, Shanghai, China). Cells were incubated with PE-labeled anti-interferon-g (IFN-g) or PE-labeled anti-interleukin 4(IL-4) (both from BD Shanghai, China) for 30 min at 4  C, washed with PBS, and analyzed by flow cytometry.

2.7. Detection of HBVsAg-specific IgG antibody pcDNA3-HBVsAg plasmid was purchased from Key Laboratory of Medical Molecular Virology (MOE & MOH), Fudan University. HBsAg antibodies were detected

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Fig. 2. MTT analysis of 293T cells survival ratio treated with SiO2, LDH and SiO2@LDH after 24 h (A) and 48 h (B), respectively. Error bar represented means of three independent experiments. by specific enzyme linked immunosorbent assays (ELISAs) in 96-well microtiter plates. HBVsAg peptide was diluted and dispensed in 2.5 mg/well into 96-well microtiter plates, followed by overnight incubation at 4  C. The plates were washed 5 times with PBS containing 0.1% Tween 20 (washing solution). Each well was then treated with 200 mL PBS containing1% BSA (solution A), incubated at 37  C for 60 min to block nonspecific binding, and washed 5 times with washing solution. After that, 100 mL sera diluted with solution A was added to each well. The plates were incubated for 60 min at 37  C and washed 5 times with washing solution. And then 100 mL horseradish peroxidase-labeled anti-mouse IgG (1:5000 dilution in solution A; American Qualex) solution was added as the second antibody. After incubation for 60 min at 37  C, the plates were washed 5 times with washing solution, and 100 mL o-phenylenediamine dihydrochloride substrate solution (Sumitomo ELISA Color Reagent Kit; Sumitomo Bakelite) was added and incubated for 15 min at room temperature. The enzyme reaction was stopped by stopping solution (Sumitomo ELISA Color Reagent Kit), and absorbance at 450 nm was measured with a microplate reader (Multiskan FC, ThermoFisher). Antibody titers were defined as the highest serum dilution that resulted in an absorbance value 2.5 times greater than that of negative sera control.

2.8. Cellular uptake mechanisms of particles The cellular uptake mechanisms of SiO2@LDH nanoparticles were performed by blocking uptake pathway with different treatments [32]. To block the energydependent endocytosis, cells were cultured at 4  C instead of 37  C. The cells were pre-incubated with NaN3 (10 mM) in PBS for 30 min at 37  C in order to deplete ATP. For hindering the clathrin-mediated pathway by hypertonic treatment, the cells

were pre-incubated for 30 min with sucrose (0.45 M) in PBS at 37  C. Additionally, cells were pretreated with mycostatin (5 mg/ml) in PBS for 30 min to block the caveolae-mediated pathway under mycostatin treatment. Afterward, all of the above cells were washed twice with PBS buffer, and incubated with 20 mg/ml SiO2@LDH in serum-free media at 37  C for 2 h. Treated cells were observed by a fluorescent microscopy imaging system after being washed several times with PBS. Treated RAW264.7 cells were washed three times with PBS, and harvested by trypsinization for quantitative characterization. After centrifugation, cell pellets were washed once and resuspended with PBS buffer. The cellular uptake of SiO2@LDH nanoparticles was quantitatively determined by flow cytometry. The experiment was triplicate with similar results and the mean value was displayed.

2.9. Western blotting Anti-NF-kB p65 and anti-IkB-a antibodies were from Cell Signaling Technology. RAW264.7 cells were treated with different concentrations of SiO2@LDH nanoparticles for 24 h. To determine the levels of protein expression in whole cell extracts or in the cytoplasm or nucleus of treated cells (1.5  106 cells in 1 ml of medium), cytosolic fraction and nuclear fraction were extracted by nucleoprotein and cytoplasmic protein extraction kit (purchased from KenGen Biotech, Nanjing, China). The protein concentration in supernatant was calculated using the BCA protein assay (KenGen, Nanjing, China). An aliquot of 60 mg total extract was mixed with protein-loading buffer containing 2-mercaptoethanol and boiled for 5 min before loading on a 10% SDS polyacrylamide gel. After electrophoresis, proteins were transferred into nitrocellulose membranes and blotted against the primary antibodies (antibodies used were against NF-kB p65, IkB-a, Lamin A and b-actin in

Fig. 3. Detection of SiO2@LDH nanoparticles loaded DNA about protection, release and delivery property. (A) DNase protection assay. Lane 1 shows SiO2@LDH nanoparticle loaded plasmid in the absence of DNase I digestion. Lane 2 shows SiO2@LDH particle loaded plasmid digested with DNase I for 1 h. Lane 3 shows SiO2@LDH loaded plasmid digested with DNase I for 1 h, followed by incubation at pH 2 for 1 h to recover from SiO2@LDH host. Lane 4 shows plasmid digested with DNase I for 1 h. Lane 5 shows undigested plasmid. Lane 6 shows Marker. Cell transfection assay. GFP expression after transfection with pEGFP-N2 (A,E), SiO2/pEGFP-N2 (B,F), LDH/pEGFP-N2 (C,G), and SiO2@LDH/pEGFP-N2 (D,H) against RAW264.7 cells was examined by fluorescence microscope (B) and flow cytometry (C) after 5 h treatment and 48 h incubation at 37  C. Green: GFP.

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J. Wang et al. / Biomaterials xxx (2013) 1e13 1:1000 dilutions). The membranes were washed with TBST [0.05% (v/v) Tween-20 in PBS pH 7.4] and incubated with HRP-conjugated secondary antibodies (1: 2000 dilutions) for 45 min. The protein bands were visualized by an enhanced chemiluminescence reaction (Amersham Pharmacia Biotech, Piscataway, NJ) as described [24]. 2.10. Statistical analysis Results were displayed as mean  SD. The statistical significance of differences between 2 groups was determined by student’s t-test, and differences between 2 or more groups were analyzed by one-factor analysis of variance (ANOVA). Data were considered statistically to be significant at P < 0.05 or 0.01.

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3. Results 3.1. SiO2@LDH nanoparticles characterization Plain LDH nanoparticles prepared here were thin hexagonal plate-like in shape with 80e100 nm in size and a zeta potential of 41.5 mV (Fig. 1). Mesoporous SiO2 nanoparticles were also synthesized via a solvothermal method as described above. The obtained mesoporous SiO2 nanoparticles have an average diameter of 50 nm determined by TEM, and its zeta potential was 15 mV.

Fig. 4. Activated macrophages induced by SiO2@LDH nanoparticles. (A and B) The macrophages phenotype analysis. The RAW264.7 cells were incubated with indicated concentrations of SiO2, LDH and SiO2@LDH nanoparticles (40 mg/ml) or LPS (1 mg/ml) at 37  C. After 24 h, cells were collected, and cell surface molecule expression was analyzed by flow cytometry. Red shaded histograms represent control cells in the absence of nanoparticles. (C) Cytokines level of macrophages induced by SiO2@LDH nanoparticles. The RAW264.7 were incubated with indicated concentrations of SiO2, LDH and SiO2@LDH nanoparticles or LPS (5 mg/ml) at 37  C. After 24 h, supernatants were collected and analyzed for the cytokines IFN-g, IL-6, IL-12p70 and TNF-a using ELISA. The data were the mean percentage of positive cells in comparison with untreated control RAW264.7 cells. Experiments were performed in triplicate and the statistical significance of the results was analyzed and indicated: *P < 0.05; **P < 0.01. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Based on SiO2 nanoparticles, AlOOH was deposited with Mg2þ on the surface as a kind of coating by an in situ growth procedure, and a shell of MgAl-LDH was subsequently formed. The results of X-ray diffraction pattern shows that SiO2@LDH nanoparticles was complex of LDH crystallize and mesoporous SiO2 (Fig. S1). Different from LDH and SiO2 nanoparticles, SiO2@MgAl-LDH nanoparticles were homogeneous spherical and dendritic observed by TEM, and had an average diameter of 210 nm and a zeta potential of 0.521 mV (Fig. 1A and B). In this case, SiO2@MgAl-LDH nanoparticles displayed dimensional coreeshell architecture and large surface area which can make it easier to combine DNA molecules than SiO2. Additionally, electrically neutral property could also avoid nonspecific impurities combination such as serum in vivo and enhance the delivery efficiency. 3.2. Biological safety of SiO2@LDH The biological safeties of SiO2, LDH and SiO2@LDH nanoparticles were tested by MTT assays. All the data showed that SiO2@LDH nanoparticles have no obvious cytotoxicity against HEK 293T cells. In the presence of different nanoparticles at various concentration and incubation time, HEK 293T cells viabilities were measured. After incubated with free LDH and SiO2@LDH nanoparticles for 24 h at different concentration, the ratio of living cells could be kept around 90%. In comparison, SiO2 nanoparticles showed certain cytotoxicity to HEK 293T cells when the concentration was up to 100 mg/ml (Fig. 2A), and cell survival ratio in 48 h was the similar trend as 24 h. Cells treated with 1 mg/ml SiO2@LDH nanoparticles for 48 h had a slight decreased viability (Fig. 2B). However, cell viability was not significantly time-dependent. The survival ratio maintained over 80% which indicated the irreversible damage threshold of cell membrane [33]. 3.3. DNA protection and in vitro gene expression Experiments were performed to reveal whether SiO2@LDH nanoparticles could prevent DNA from endonuclease degradation

and effectively release DNA. DNA and DNA-SiO2@LDH compound were treated with DNase I respectively and analyzed by running agarose gel. DNA molecules combined with SiO2@LDH nanoparticles did not migrate obviously (Fig. 3A, lane 1). And DNA molecules in DNA-SiO2@LDH compound treated with DNase I for 1 h could stay intact, which indicated that SiO2@LDH particles could protect DNA molecules from endonuclease degradation in vitro (Fig. 3A, lane 2). And when incubated at pH 2 for 1 h, DNA could release out from SiO2@LDH host (Fig. 3A, lane 3). However, naked DNA molecules were completely degraded by DNase I (Fig. 3A, lane 4). The DNA protection property of SiO2@LDH nanoparticles were consistent with previous studies [23,34], which suggested that SiO2@LDH nanoparticles could be used as a potential non-viral gene delivery system. GFP expressions mediated by pEGFP-N2, SiO2/pEGFP-N2, LDH/ pEGFP-N2 and SiO2@LDH/pEGFP-N2 were evaluated in RAW264.7 cells. As shown in Fig. 3B, SiO2@LDH/pEGFP-N2 (D, H) induced higher level of GFP expression than that of pEGFP-N2 (A, E), SiO2/ pEGFP-N2 (B, F) and LDH/pEGFP-N2 (C, G). Furthermore, the fluorescence intensity was also quantitatively detected by a flow cytometry. It was clearly observed that the SiO2@LDH could effectively improve the uptake of pEGFP-N2 by RAW264.7 cell and induce higher level of GFP expression compared with other in RAW264.7 cell (Fig. 3C). 3.4. SiO2@LDH nanoparticles induced macrophages highly expressed co-stimulatory molecules and cytokine Adhesion molecules played an important role in macrophage activation process. Whether uptake of SiO2@LDH nanoparticles by macrophages had a significant impact on surface markers expression and macrophages activation was analyzed by FACS analyses. Macrophages were incubated with LPS (1 mg/ml) as a positive control. LPSpulsed macrophages displayed a remarkable increase in CD86, CD80, CD40 and MHC class II expression on their surface [35]. In the same way, macrophages exposed to SiO2@LDH nanoparticles showed increased expression levels of CD86 and MHC-II in a dose-dependent

Fig. 5. Immunization of Mice and SiO2@LDH activated cellular immune response in HBV DNA vaccination. (A) Immunization scheme: native BALB/c mice were immunized in the inguinal area using the immunization scheme as shown with PBS, pcDNA3-HBVsAg and pcDNA3-HBVsAg loaded by SiO2, LDH and SiO2@LDH. (B) T cell proliferation analysis: 2 weeks after the last immunization of BALB/c mice, T cells were stimulated in vitro with HBsAg peptide (5 mg/ml, 10 mg/ml and 20 mg/ml). Concanavalin A (5 mg/ml) was used as positive control. T cell proliferation was analyzed by CCK8 method. BSA was used as negative control. (C and D) T-cell differentiation analysis: 2 weeks after the last immunization of BALB/c mice, T-cells were isolated. IFN-g and IL-4 production were induced by splenocytes. Splenocytes were stained with anti-mouse CD3-FITC/CD4-APC/IFN-g-PE for Th1 cytokine analysis (upper two lines), anti-mouse CD3-FITC/CD4-APC/IL-4-PE for Th2 cytokine analysis (lower two lines), and analyzed by flow cytometry. The geometric mean and standard deviation are shown for each group. The statistical significance of the results was analyzed and indicated: *P < 0.05; **P < 0.01.

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Fig. 5. (continued).

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Fig. 5. (continued).

manner (Fig. 4A and B). However, there was no difference in the expression level of CD80 and CD40 when cells were treated with LDH. In addition, co-stimulatory molecules expression levels of macrophages which were treated with high concentration (40 mg/ml) of SiO2 nanoparticles were not up-regulated observably (Fig. 4A and B). This suggested that SiO2@LDH nanoparticles had great potential to be an adjuvant for macrophages activation. These results were confirmed by the quantification of the pro-inflammatory cytokines. The dosedependent increase of IFN-g and IL-6 in the supernatant of macrophages stimulated by SiO2@LDH nanoparticles was also observed. The expression of pro-inflammatory cytokine IFN-g in SiO2@LDH nanoparticles-treated group was even higher than LPS-treated group. However, compared with LDH nanoparticles the production amount of TNF-a and IL-12p70 did not significantly change when macrophages were treated with SiO2@LDH nanoparticles (Fig. 4C). The above results suggested that SiO2@LDH nanoparticles could play a vital role in inducing macrophages activation.

SiO2@LDH nanoparticles were shown to facilitate cellular immune responses to enhance Th1 cytokines secretion for HBV DNA vaccination. 3.6. Detection of HBVsAg-specific IgG antibody To characterize whether SiO2@LDH nanoparticles can serve as an adjuvant of HBV DNA vaccine, the expression of HBVSpecific IgG antibody in immunized BALB/c mice was analyzed. After immunization, serums were collected for HBVsAg-specific IgG antibody testing. We found that SiO2@LDH augmented stronger specific antibody compared with other groups (Fig. 6). The IgG antibody level increased significantly in BALB/c mice immunized with SiO2@LDH/pcDNA3-HBVsAg, compared with pcDNA3-HBVsAg, pcDNA3-HBVsAg/SiO2 and LDH/pcDNA3HBVsAg. There were no HBVsAg-specific IgG antibody detected in serums of immunized mice with PBS (Fig. 6). These results suggested that SiO2@LDH nanoparticles-based HBV DNA vaccine could enhance HBV-specific immune responses.

3.5. SiO2@LDH augmented Ag-specific T-cell proliferation and Th1 differentiation after boost

3.7. Mechanism of the endocytosis of SiO2@LDH by macrophages

HBV DNA immunization was a promising strategy for inducing cellular immunity [36,37]. T cell proliferation was determined in mice immunized by pcDNA3-HBVsAg loaded-SiO2@LDH nanoparticles. Lymphocyte proliferation was measured after 72 h by CCK-8 assay. As indicated in Fig. 5B, the results showed that SiO2@LDH/pcDNA3-HBVsAg induced much greater level of lymphocyte proliferation than pcDNA3-HBVsAg, SiO2/pcDNA3HBVsAg and LDH/pcDNA3-HBVsAg, indicating that vaccination with SiO2@LDH/pcDNA3-HBVsAg could effectively induce the proliferation of specific lymphocytes. Cytokines played a central role in the modulation of immune responses. To test Th1 (IFN-g) and Th2 cytokines (IL-4) in immunized BALB/c mice, splenocytes were prepared and intracellular stained and followed with flow cytometry analysis. As shown in Fig. 5C and D, the IL-4 positive cells presented no differences among these groups. However, the IFN-g was significant higher in BALB/c mice immunized with SiO2@LDH/pcDNA3-HBVsAg than that of other groups. In sum,

To further elucidate the cellular uptake pathways of SiO2@LDH nanoparticles, chemical inhibitors of specific uptake pathway were used. To date, four major cellular uptake pathways were identified [38,39], and listed as follows: (1) Phagocytosis, which was a main uptake pathway for macrophage. (2) Clathrin-mediated endocytosis, via which guest matters were internalized via clathrin-coated vesicles and subsequently transformed to endosomes. (3) Caveolaemediated endocytosis, which was characterized by the evolution of caveolae-derivatives of the subdomains of sphingolipid and cholesterol-rich cell membrane fractions. (4) Clathrin- and caveolae-independent endocytosis, which includes those pathways not usually classified by the above criteria, such as macropinocytosis, flotillin- or arf6-dependent pathways. All these pathways were energy-dependent and involved in active formation of membrane invaginations, membrane ruffles and transport vesicles. Low temperature (4  C) or the presence of NaN3 leads to obviously decreased intracellular fluorescent intensity compared with the

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Fig. 6. HBs-specific IgG Antibody titers (reciprocal serum dilution) induced by SiO2/ pcDNA3-HBsAg (SiO2/p), LDH/pcDNA3-HBsAg (LDH/p), SiO2@LDH/pcDNA3-HBsAg (SiO2@LDH/p), pcDNA3-HBsAg (plasmid) and PBS in BALB/C mice. Antibody titers were measured by ELISA using sera collected 2 weeks after the last DNA immunization. Each symbol represents an individual mouse serum sample. The geometric mean and standard deviation are shown for each group. The statistical significance of the results was analyzed and indicated: *P < 0.05; **P < 0.01.

control group (Fig. 7A) which indicate the uptake of SiO2@LDH nanoparticles was an energy-dependent process. The ability of cellular uptake of SiO2@LDH nanoparticles was different when treated with corresponding clathrin- and caveolae-mediated endocytosis inhibitors, individually. Particularly, mycostatin was able to significantly reduce the uptake of SiO2@LDH nanoparticles by RAW264.7 cells. These results primarily demonstrate that SiO2@LDH nanoparticles are inclined to be internalized into cells via caveolae-mediated pathway. Moreover, flow cytometry was also used to quantify the amount of SiO2@LDH nanoparticles uptake by RAW264.7 cells. As shown in Fig. 7B, cellular uptake of SiO2@LDH nanoparticles was markedly reduced (>60%) by the treatment with NaN3 at 37  C or at 4  C, further demonstrating that the internalization process was energy-dependent endocytosis. When RAW264.7 cells were pre-incubated with 5 mg/ml mycostatin, the uptake efficiency was reduced to 76% compared with the untreated cells. However, pretreatment with sucrose did not specifically hinder the internalization of SiO2@LDH nanoparticles. These results indicated that the uptake of SiO2@LDH nanoparticles was mainly via the caveolae-mediated pathway. 3.8. The mechanism for activation of macrophage by SiO2@LDH The NF-kB signaling pathway played a vital role in immune responses. The production of cytokines could be regulated by NF-kB [40,41]. Our previous studies showed that LDH nanoparticles could be taken up by MDDCs effectively and increased the constitutive NF-kB expression in nucleus in a dose-dependent manner. We therefore investigated whether SiO2@LDH exerted a stimulatory effect on macrophages activation through the NF-kB signaling pathway. Interestingly, SiO2@LDH nanoparticles also increased constitutive NF-kB expression in nucleus in a dose-dependent manner (Fig. 8). Because translocation of NF-kB to nucleus is normally regulated by IkB-a degradation, we further explored whether the activation of NF-kB by SiO2@LDH nanoparticles was due to the increase of IkB-a degradation. A significant dose-dependent decrease in total IkB-a expression induces by different concentrations of SiO2@LDH nanoparticles could be observed from the result of Western blotting for IkB-a (Fig. 8).

Fig. 7. The mechanisms of cellular uptake of SiO2@LDH. (A) Fluorescent microscopy images of RAW264.7 cells treated with SiO2@LDH (20 mg/ml) for 2 h at 37  C (as control), at 4  C, and at 37  C while pretreated with NaN3 (10 mM), sucrose (0.45 M) and mycostatin (5 mg/ml), respectively. Green fluorescence shows location of the SiO2@LDH doped with FITC. The cell nuclei were stained with DIPA (blue). (B) Flow cytometry results show cellular internalization amount of SiO2@LDH under different inhibitors treatment. Cellular uptake was normalized against the control cells without any inhibitors treatment. Results were the mean value of three individual experiments and the statistical significance of the results was analyzed and indicated: **P < 0.01.

4. Discussion HBV infection is still one of the worldwide infectious diseases with serious long-term morbidities and mortalities [1], and useful strategies designed for anti-HBV are limited. However, HBV DNA vaccines offered an attractive option against HBV infection, and draw people’s attention for long time. Basically, an ideal vaccine should be capable of eliciting strong humoral and cellular immune

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Fig. 8. Effects of SiO2@LDH nanoparticles on the NF-kB signaling pathway in mice macrophages. (A) Western blot analysis for NF-kB protein and IkB-a protein from RAW264.7 cells treated or untreated by different concentrations of SiO2@LDH nanoparticles. (B) Densitometric measurement for NF-kB p65 protein and IkB-a protein levels normalized to internal control, respectively, and expressed as a relative number.

responses. DNA vaccine, as a kind of new vaccine, has been used for anti-HBV, but its immunogenicity still needs to be improved. Various strategies have been developed to transfect plasmid into cell lines in vitro [42], but few have been successfully adapted in vivo [43]. As DNA vaccine is easy to degrade and the immune responses are not high enough, several adjuvants have been used to enhance immune responses against HBV DNA vaccines and protect DNA from endonuclease. A DNA vaccine encoding HBVsAg with aluminum phosphate as adjuvant increased the numbers and affinity of antigen-specific IFN-g and IL-2 secreting T cells [44]. Amiloridea, a drug that was prescribed for hypertension treatment, could accelerate the entry of HBV DNA vaccine into antigen presenting cells (APCs) and enhance the development of full CD8 cytolysis function including the induction of high levels of antigen specific CTL [45]. Ubiquitin conjugation of hepatitis B virus core antigen DNA vaccine led to enhanced cell-mediated immune response in BALB/c mice [46]. Fms-like tyrosine kinase 3 ligand (Flt3L) was as an adjuvant that could augment humoral and cellular immune response of hepatitis B virus DNA vaccine by micro-needle vaccination [47]. There were also some reports about the usage of inorganic nanomaterial as DNA vaccine adjuvant to enhance immune response [7e15]. Currently, nanoparticles were of great scientific interest for exploiting as a kind of adjuvant in delivery of immunogens for vaccine development [23,48e51]. Nano-chitosan as Esat-6/3e-FL DNA vaccine vector could strongly induce Th1 and CTL immune responses and inhibit mycobacterium tuberculosis growth [7]. Intranasal delivery of cationic PLGA nano/micro particles loaded with various foot and mouth disease virus (FMDV) DNA vaccine formulations encoding IL-6 was used as a molecular adjuvant which enhanced the protective immunity against FMDV [52]. Moreover, our previous studies showed that LDH nanoparticles could be efficiently taken up by the MDDCs and had an adjuvant activity for DC maturation [23]. To enhance immune adjuvant activity of LDH nanoparticles, inorganic SiO2@LDH nanoparticles were synthesized and used as DNA vaccine delivery vector. With inorganic SiO2@LDH nanoparticles, the immunogenicity of an HBV DNA vaccine was enhanced by activating macrophages and inducing Th1 cell responses in BALB/c mice. SiO2@LDH nanoparticles were synthesized by in situ growth of LDH nanoplatelets on the surface of mesoporous silica. An average diameter of 210 nm and zeta potential of 0.521 mV proved that SiO2@LDH nanoparticles had the ability to easily bind with DNA compare with SiO2. Also, electrically neutral property could avoid nonspecific combination of impurities such as serum in vivo to enhance the delivery efficiency. The most important advantage of

SiO2@LDH as a delivery system over other nano materials was their larger surface area. The extremely large areas made SiO2@LDH an ideal carrier for the delivery of large amount of DNA vaccine. The pH-sensitive dissolution of LDHs can achieve targeted drug release [20]. Similarly, DNA could be released from the vector in low-pH environments (Fig. 3A). On the other hand, SiO2@LDH nanoparticles did not show any cytotoxicity against normal cells (HEK 293T cells), and can totally protect DNA from DNases digestion (Fig. 3A). The activated macrophages could simultaneously secrete oxygen radicals and nitric oxide, both of which have potential antimicrobial activity. Additional changes in activated macrophages could further promote the immune responses. In this case, the expressions of MHC class molecules, CD80, CD86 and CD40 on the macrophage surface increased, which caused the cell to be more effectively presenting antigens to naïve T cells [53]. SiO2@LDH nanoparticles here were furthermore used for delivering DNA vaccine into cell and activating macrophage. The expression levels of co-stimulatory molecules (CD86, MHC-II, CD80 and CD40) and cytokines (IFN-g, IL-6, TNF-a and IL-12p70) were up-regulated (Fig. 4), which suggested that SiO2@LDH nanoparticles have the ability of inducing macrophages activation. Interestingly, more Th1 cytokines were secreted in BALB/c mice immunized with SiO2@LDH/pcDNA3-HBVsAg than that in other groups even in LDH/pcDNA3-HBVsAg group. Meanwhile, HBVsAb and T-cell responses both were strongly enhanced in mice immunized with SiO2@LDH/pcDNA3-HBVsAg (Fig. 5). SiO2@LDH was shown to enhance cellular immune responses and enhance the production of Th1 cytokines for HBV DNA vaccination. In our research, SiO2@LDH show advantages over LDH not only in delivering DNA into macrophage (Fig. 3B and C), but also in inducing macrophage activation as adjuvant. As the result from immunization of mice shows, SiO2@LDH loaded pcDNA3-HBVsAg show more distinct Ag-specific T-cell proliferation, Th1 differentiation and HBVsAg-specific IgG antibody expression, compare with LDH loaded pcDNA3-HBVsAg. The uptake pathway for internalization of particles by macrophage is mainly determined by the particle size. Generally, macrophage uptakes particles with diameter larger than 500 nm via phagocytosis, while 50e500 nm, mainly via endocytosis (including clathrin-, caveolae-mediated, non-mediated endocytosis etc) [54]. In this research, the average size of synthesized SiO2@LDH is about: 210 nm. Therefore, SiO2@LDH should be untaken by macrophage via endocytosis, which actually was confirmed by our results that the uptake of SiO2@LDH nanoparticles by macrophage was a caveolae-mediated pathway. There were several evidences

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Fig. 9. Schematic diagram shows the mechanisms underlying the activation of macrophage by SiO2@LDH nanoparticles. SiO2@LDH nanoparticles enter macrophages via caveolaemediated endocytosis, and subsequently activate NF-kB to activate macrophage.

showed that Caveolin-1 and Caveolin-2 were expressed in mouse macrophages [55]. Internalization of SiO2@LDH nanoparticles into the acidic environment (endosome and lysosome) lead to the collapse of material surface (LDH) and sustained vaccine release. On the other hand, SiO2@LDH nanoparticles could further influence other intracellular signaling pathways to achieve the macrophages activation. As one of the important pathway, NF-kB signaling pathway played a vital role in immune response, and cytokines production level could be regulated by NF-kB [40,41]. Our previous studies have shown that LDH nanoparticles could be taken up by the MDDCs and increased constitutive NF-kB expression in nucleus in a dose-dependent manner. We therefore wondered whether SiO2@LDH exerted a stimulatory effect on macrophages through the activation of NF-kB signaling pathway. Translocation of NF-kB to nucleus was normally regulated by IkB-degradation. Therefore, we examined whether the change of NF-kB results from the increased degradation of IkB-a. When cells were treated with different concentrations of SiO2@LDH nanoparticles, significant dose-dependent decreases in total IkB-a expression levels were detected (Fig. 8). Then, NF-kB, which has been inhibited by IkB-a, is released to translocate into nucleus and initiate the transcription of genes encoding various proinflammatory mediators (IFN-g, IL-6, TNF-a and IL-12p70) [56]. The mechanism of cellular uptake of LDH nanoparticles via clathrin-mediated endocytosis was already clear. Our research shows SiO2@LDH nanoparticles get into macrophages via caveolae-mediated but not clathrin-mediated endocytosis and subsequently activate NF-kB to activate macrophage (Fig. 9).

5. Conclusion Biodegradable SiO2@LDH nanoparticles were prepared in this study in order to find effective vaccine delivery systems and immunostimulants. In vitro cellular experiments showed that SiO2@LDH nanoparticles, as a DNA vector, not only protect DNA from degradation and exhibit high transfection efficiency, but also efficiently promote macrophage activation via NF-kB signaling pathway, compared with SiO2 and LDH. Animal level experiments showed that pcDNA3-HBsAg/SiO2@LDH nanoparticles immunized BALB/c mice had much higher serum antibody response than naked DNA vaccine and plain nanoparticles. Besides, pcDNA3-HBsAgSiO2@LDH nanoparticles also induced dramatically efficient T-cell proliferation and skewed T helper to Th1 polarization. Our results indicate that SiO2@LDH nanoparticles can serve as a promising DNA vaccine adjuvant.

Acknowledgments This work was financially supported by the 973 project of the Ministry of Science and Technology (Grant No. 2010CB912604), the International S&T Cooperation Program of China, (Grant No. 0102011DFA32980), the National Natural Science Foundation of China (Grant No. 81271694), the Science and Technology Commission of Shanghai Municipality (Grant no. 11411951500 and 12nm0502200), and the Fundamental Research Funds for the Central Universities.

Please cite this article in press as: Wang J, et al., The enhanced immune response of hepatitis B virus DNA vaccine using SiO2@LDH nanoparticles as an adjuvant, Biomaterials (2013), http://dx.doi.org/10.1016/j.biomaterials.2013.09.060

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Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.biomaterials.2013.09.060.

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