2013 Silica nanorattle with enhanced protein loading A potential vaccine adjuvant

Silica nanorattle with enhanced protein loading: A potential vaccine adjuvant

Tianlong Liu, Huiyu Liu, Changhui Fu, Linlin Li, Dong Chen, Yanqi Zhang, Fangqiong Tang ?

Laboratory of Controllable Preparation and Application of Nanomaterials, Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing 100190, PR China

a r t i c l e i n f o Article history:

Received 30 December 2012 Accepted 6March 2013

Available online 22 March 2013 Keywords:

Silica nanorattle Protein Vaccine Adjuvant

a b s t r a c t

Nanopar t icles are excellent carriers for drug and protein, and have the potential to be used in vaccine delivery system. Here, we prepared different structures silica nanoparticles such as silica nanorattles (SNs),mesoporous silica nanop a rticles (MSNs)and solid silica nanoparticl e s (SSNs),and chosen ovalbu- min (OVA)as model protein to study the potential app l ication of silica nanoparticl e s in protein vaccine delivery system. The results showed that silica nanoparticles were ef?cient in protein loading and depen- dent on structure, size and incubation medium. According to the three structure particles ,SNs were favorabl e to be used as protein carriers. Furthermore, we proved low cytotoxic i ty of silica nanorattle on RAW 264.7 cell line and biocompatibility in vivo .In addition, SNs was capable to up-regulate the humoral immunity reaction when mice were vaccinated with SNs–OVA formulation. Taken together, SNs was excellent carriers for protein vaccine and has the potential to be used as adjuvant.

ó2013 Elsevier Inc. All rights reserved.

1. Introduction

Vaccines, known as an effective weapon for human disease pre- vention, are playing an increasingly important role in the ?ght against infectious disease. According to the division of preparation, vaccines can be divided into classic vaccine and modern vaccine [1,2].The former include inactivated ,attenuated and subunit vac- cine which be made from some ingredients of natural organism. Modern vaccine refer to those vaccine apply modern biological technology, such as subunit vaccines, gene delete vaccines, recom- binant and synthetic peptide vaccine. Classic vaccines have some ingredients like inactivated or low activity microorganism will in- duce adverse effects [3,4].Although modern vaccine showing more safety than classic types, such like recombinant and synthetic pep- tide vaccine, the less ef?cient immunity protect effect limits their broader applications [5,6].Furthermore, recombinant and syn- thetic peptide vaccine face many challenges because of their bad stability, short half-life and vulnerability to interfere n ce by endog- enous substances [7,8].It is necessary to provide suitable carrier for prevention and modify of antigen protein in modern vaccine formulation .

Adjuvant has advantages in enhancing vaccine immunity pro- tect effect. Since 1925, ?rst discovered of some other substances (metal salt, lecithin and saponin)can improve the antigen speci?c immune response by Ramon, all sorts of different material were

used as adjuvant [9–11].However ,there were little numbers of clinical cases. With the booming of nanotechno l ogy, the applica- tion of nanomaterials as adjuvant has aroused more concern [12–16].Local in?ammation and systemic immune response caused by nanoparticles as foreign matter actually can be considered as an immunopot e ntiation effect. Therefore, it is important to investi- gate the loading of proteins in nanomaterials and their bio-effect in vivo [17].Mesopor o us materials are of great interest to nanoscience and nanotechno l ogy due to their unique porous structures as well as their tailorable adsorption, catalytic, conductive, and magnetic propertie s [18,19].Among the ordered mesoporous materials, mes- oporous silica nanoparticl e s (MSNs)have become a type of very popular materials with potential applications ranging from adsorp- tion and catalysis to nanotechno l ogy and biotechnolo g y [20,21].However ,the storage capacity for the conventional mesoporous materials is relatively low, and also the irregular bulk morphology is not perfect for delivery [22].To overcome these problems, one strategy is to synthesize mesoporous hollow silica nanoparticl e s with penetrating pore channels from outside to the inner hollow capacity. Recently ,we reported a ?exible,scalable and robust method to prepare rattle-typ e mesoporous silica hollow spheres named as silica nanorattles (SNs).SNs are emerging as new and promising class of nanoparti c les that developed for drug delivery system due to their special structure and functions [23–25].In pre- vious study, we found that low lethal toxicity of SNs when intrave- nous injection at single dose. We also found that kupffer cells (KCs)are the major target cells when SNs entered the blood stream [26].These previous research provide advantages of SNs for potential applicati o n in adjuvant. However, loading and releasing ef?ciency of SNs on antigen and potential adverse effect of SNs on immune

0021-9797/$ -see front matter ó2013 Elsevier Inc. All rights reserved. https://www.360docs.net/doc/5111663396.html,/10.1016/j.jcis.2013.03.005

?Corresponding author. Address: Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, No. 29, East Road Zhongguancun, Beijing 100190, PR China. Fax: +86 10 82543521.

E-mail address: tangfq@https://www.360docs.net/doc/5111663396.html, (F.Tang).

system or immune response should be determined before the kind of application.

Here, we examine d the loading and releasing of silica nanorattle with 110 nm on ovalbumin (OVA).In?uencing factors on protein loading and releasing such as nanoparti c les structure, size, temper- ature, medium and time were studied. In addition, we also evalu- ated the effect of SNs on immune cells and immune organs in vivo and in vitro .Finally, the effect of SNs on OVA antibody levels after vaccination by SNs–OVA formulation were investigated in this study.

2. Materials and methods

2.1. Materials

Tetraethylor t hosilane (T

E OS),N-[3-(trimethoxysilyl)pro-pyl]ethylen e diamine (TSD),3-aminoprop y ltrimethox y silane (APT-MS),3-Aminopro p yltriethoxysilane (APS),hydro?uoric acid (HF), NaOH, CuSO 4á5H2O)and KNaC 4H4O6á4H2O were obtained from Beijing Chemical Reagents Company (China).Potassium E DTA, BSA, OVA (grade V),complete freund adjuvant (CFA),hematoxy l in and eosin were purchased from Sigma. RAW264. 7cell line was gained from American Type Culture Collectio n(ATCC).E LISA kit of OVA antibody was obtained from Roche Ltd.

2.2. Fabrication and characteri z ation of silica nanoparticles

SNs, MSNs and solid silica nanoparticl e s (SSNs)were synthe- sized as previous described [27,28].MSNs and SSNs have been amino functionalized as positive charge control by 3-Aminopro p yl- triethoxysilane (APS)and remarked as MSNs-NH 2and SSNs-NH 2. Morphology and structure of the resulting nanoparti c les were observed with a JE OL-200CX transmis s ion electron microscope (TE M).Zeta potential and size distribution was characteri z ed by dynamic light scattering using a using the Malvern Zetasizer 3000HS at 25 °C.

2.3. Preparation of protein-capt u ring nanopariticles

To determine protein entrapment, 40 mg silica nanoparticles were diluted in 1.0 mL PBS (pH7.4).Then 1.0 mL of 2mg/mL OVA was added and the solution was stirred vigorously for 24 h. The silica nanoparticl e s were separated by centrifugation at 15,000 rpm for 8min. The concentr a tion of protein remained in the media was determined by biuret protein assay using UV–visible spectroscopy at 280 nm. The absorbance of the remained protein was converte d into the concentration with ?tted curve of OVA in PBS (Supporting informations, Fig. S1 A).The loading con- tent (LC)means the percent of loaded protein in total amount of protein plus carrier and entrapm e nt ef?ciency(EE)means the per- cent of loaded protein in total protein were calculated. All mea- surements were conducted in triplicate s and the mean values and standard deviation s were calculated. Morphology and struc- ture of the loaded OVA nanoparticles were observed with TE M. Zeta potential and size distribution was characteri z ed by dynamic light scattering using the Malvern Zetasizer 3000HS at 25 °C.

2.4. In vitro release of OVA

For protein release assay, the SN–OVA samples were placed in 20 mL of release medium (PBS;5% glucose solution or pure water), stirring at given temperature (4,25 and 37 °C).2mL of the release medium was taken at given time intervals. The concentr a tion of OVA was determined by biuret protein assay using UV–visible spectroscopy at 280 nm. 2.5. In vitro cell viability and cell apoptosis

Mouse macrophage- l ike RAW264. 7cells were maintained in high-gluc o se Dulbecco’s modi?ed E agle’s medium (DME M),sup- plemente d with 10% FBS at 37 °C in a humidi?ed atmosphere of 5% CO2. The cell culture media were supplemented with 100 units/mL penicillin, 100 l g/mL streptomycin. The cytotoxicity of SNs was evaluated by MTT viability assay. SNs were dissolved using DMSO. For 24 h detection, the cells were seeded at a density of 8?104 cell/mL on 96-well plates. After incubating the cells with silica nanorattles for 24 h, MTT (3-(4,5-dimethylthiazol- 2- yl)-2,5-diphenyltetrazol i um bromide)(?nal concentration of 0.5 mg/mL)was added to each well. After 4h of incubation at 37°C, colorimetric measureme n ts were performed at 570 nm on a scanning multiwell spectrometer. Data were expressed as mean ±standard error of mean of at least six independen t experime n ts.

Cell apoptosis was determined with ?uorescence activated cell sorter (FACS)and assessed with an annexin V detection kit. After incubation with SNs for 24 h, RAW 264.7 cells were stained with annexin V and PI, and assessed by FACS as described previously. Brie?y,cells were washed with binding buffer and incubate d with annexin V and PI at room temperat u re for 10 min, then analysed immedia t ely by FACS.

2.6. In vivo effect of SNs on immune system

All animal experiments were performed in compliance with the local ethics committee. Female BALB/c mice (provided by Vital River Laboratory Animal Technology Co. Ltd., Beijing),aged 6–

8weeks, were used in the experime n ts. E very ?ve mice were housed in stainless steel cages containing sterile paddy husk as bedding in ventilated animal rooms. They were acclimated in the controlle d environment (temperature:22 ±1°C; humidity: 60 ±10% and light: 12 h light/dark cycle)with free access to water and a commercial laboratory complete food. SNs was sterilized by UV irradiation for 2h, then suspended in sterile 5% glucose solu- tion and sonicated for 15 min before being loaded into 1mL syrin- ges under sterile conditions. An endotoxin assay showed that there was no detectable gram negative endotoxin on any of the particle at a concentratio n of 1mg/mL (the detection limit was less than 0.1 EU/mL).BALB/c mice were intraperitonea l ly injected with SNs suspensi o n in 5% glucose at 10, 25 and 50 mg/kg levels once a week for 4weeks. Intraperiton e al injections of sterile 5% glucose injec- tion were also given to mice as controls. Mortality and clinical manifest a tion was recorded. On day 14 after the last injection, the mice were sacri?ced,and the serum and organs were recov- ered. The spleen and thymus were excised and weighed accurately. The coef?cients of spleen and thymus to body weight were calcu- lated as the ratio of tissues (wet weight, mg)to body weight (g). Spleen and thymus tissues recovered from the necropsy were ?xed

in 10% formalin, embedded in paraf?n,sectioned, and stained with hematoxy l in and eosin (HE)for histological examination using standard techniques. After hematoxylineo s in staining, the slides were observed and photos were taken using optical microscope (Olympus X71, Japan).All the identity and analysis of the pathol- ogy slides were blind to the pathologi s t.

2.7. Vaccinati o n and ELISA for anti-OVA Abs

A total of 2mg/mL OVA or SN–OVA in PBS was mixed for injec- tion at 1:1 (v:v),with the complete Freund’s adjuvant (CFA)adju- vants following the manufactur e r’s recomme n dations for optimal immunog e nicity. Female BALB/c mice were divided into four groups injected with different materials as CFA–OVA,SN–OVA, CFA–SN–OVA and PBS–OVA containing 10 l g ovalbumi n on days

T. Liu et al. /Journal of Colloid and Interface Science 400 (2013)168–174169

0, 1, and 2. On day 21, plasma was collected to assess antigen- speci?c antibody responses. Antigen- s peci?c IgG and subclass IgG1 antibody levels were determined by enzyme- l inked immuno- sorbent assay (ELISA).The ELISA plates were coated with 10 l g/mL OVA and incubate d overnight at 4°C. Non-speci ?c Antibody bind- ing was minimize d by incubating the plates with 4% blocking solu- tion at 37 °C for 2h. Plasma dilutions were added to the antigen coated plates and incubated at 37 °C for a further 2h. The coated plates were then washed with PBS containing 0.05% Tween 20 and incubate d with a horseradish peroxidase conjugat e d goat anti-mouse IgG solution at 37 °C for 2h. The color reaction was developed with tetramethylbenzi d ine, stopped with 2N H2SO4, and quantitated by measuring OD 450minus OD 655using a micro- plate reader.

2.8. Statistical analysis

Results were expressed as mean ±standard deviation (S.D). Multigroup comparisons of the means were carried out by one- way analysis of variance (ANOVA)test using SPSS 14.0 (SPSS Inc., Chicago, IL).The statistical signi?cance for all tests was set at p<0.05.

3. Results and discussion

3.1. Preparation and characterization of silica nanoparticle s tential of SNs. Potential of amino functionalized MSN and SSNs were 27.4 ±

4.5 and 24.8 ±3.2 mv, respectively .

3.2. Effect of nanoparti c le structure and size on OVA loading ef?ciency

To ?nd the optimum loading conditions of OVA, we investiga t ed varied factors including particles structure ,size, concentr a tions of OVA, media, temperat u re and entrap time. Different structure as silica nanorattles, mesporous silica nanoparticl e s and solid silica nanoparti c les were used for the study. Three sizes 70, 110 and 260 nm of SNs was used to detect the size effect on protein loading.

40 mg of SNs was added in different proportions of OVA (20:1; 10:1; 5:1; 2.5:1; 1:1; 1:2.5; 1:5)diluted in PBS pH 7.4 in ?nal vol- ume of 2mL (v/v).Furthermore, different media like PBS, 5% glu- cose solution, reaction temperature 4°C and 37 °C and 0.5, 1, 2, 4 and 24 h entrap time were considered .

Under the same condition s, amount of OVA which loaded on SNs was greater than that of MSNs and SSNs. LC of MSNs and SSNs were 5.14% and 3.5%, respectively. According to the positive con- trol, LC of MSNs-NH 2and SSNs-NH 2were 10.25% and 6.8%. LC of SNs was 2.87, 2.17 and 4.22 times than MSN, SSNs-NH 2and SSNs as 14.76%, respectivel y. (Fig. 2A).There was no signi?cant differ- ence between SNs and MSN-NH 2.No obvious difference of LC was founded between 70, 110 and 260 nm SNs. (Fig. 2B).

3.3. Effect of concentratio n of OVA, media and temperature on loading ef?ciency

TEM images of SNs, MSNs and SSNs in 5% glucose solution. Three nanoparticles, SNs (A),MSNs (C),SSNs (D),all had well monodispersion in the dispersant. OVA did not affect the monodispersion of SNs and OVA loaded on SNs could be observed (B,black arrow).

170T. Liu et al. /Journal of Colloid and Interface Science 400 (2013)168–174

loading was a dynamical change process and 4h was the appropri- ate entrap time. The absorbance of the remained OVA was con- verted into the concentratio n with ?tted curve of OVA in glucose and water (Supporting information s, Fig. S1 B and C).Incubation media and temperature did not signi?cantly affect the loading of protein on nanocarrier s. In the conclusion, 110 nm diameter, 1.5 ratio of protein and particles, 5% glucose solution as media, 4h and 4°C were the optimization condition s for proteins loading. 3.4. Effect of media and temperature on releasing

Effect of media and temperature on releasing rate of SNs–OVA was investigated in this study. 5% glucose solution was the most suitable one among the three candidat e media (glucose,PBS and pure water).The cumulative OVA release curve for SNs in PBS sol- vent basically started with a burst effect of release and followed with a control release (Fig. 3A).There was low release of OVA in pure water till 12 days (Fig. 3C).Unlike the two solvents above, burst release did not occur in glucose and releasing started at 5days followed with ef?cient releasing till 12 days reached 87% (Fig.3B).Temperature had a certain in?uence on OVA releasing in different solvents, especially in glucose. Releasing data was signif- icant declined at 4°C compare d with 37 °C in glucose.

Nanomateri a ls which probably used as adjuvant should meet the following requiremen t s: 1, Particle size should be less than 100 nm for ef?cient traversing biological barriers, 2, good biocom- patibility and high clearance from body, 3, sustained release of antigen and maintain a constant and effective blood concentratio n, 4, mass production for commercial purpose, 5, be adapt to surface modify for target location. Progresses in SBA-15 over the last dec- ade offered exciting opportun i ties in the developmen t of adjuvant. SBA-15 was the ?rst mesoporous silica nanoparti c les which used for adjuvant application [29,30].In 2006, Lucildes et al. demon- strated that SBA-15 behaves as an adjuvant inducing a better re- sponse in BALB/c immunized with E. coli bacterial protein or a complex proteins spectrum of Micrurus venom in comparison to a commonly used adjuvant for animal application, such as the complex oil incomplete freund adjuvant [31].

In this study, we found the high loading ef?ciency of OVA on SNs. Furthermore, the condition of the binding reaction and the releasing were optimized. Three aspects of loading reaction were studied, including nanoparticl e s characters, protein concentration and reaction condition s such as temperature ,media and so on. Our results indicated that structure had great effect on protein binding on nanoparti c les. Silica nanorattles had greatest loading ef?ciency among three silica nanoparticles, may be attributed to positive surface charge. Although LC of amino functionalized nano- particles such as MSN-NH 2and SSN-NH2 ,has been raised com- pared with unfunctionaliz e d particles, but still lower than SNs with the similar positive potential. And the SSNs-NH 2was the low- est one, so this result indicated that the hollow structure and the positive charge surface both play important roles in protein load- ing. We also found that particle size had some effect on protein loading, but the changes were not signi?cant.Ratios of protein and SNs had great in?uence on the protein binding and the optimal ratio was 1.5 at both optimization LC and EE. Entrap time was the most important among varied reaction conditions, media and tem- perature did not have a signi?cant effect on LC.

3.5. In vitro study of SNs on RAW26

4.7 cell lines

Cytotoxic i ty of SNs was determined by cell viability and apop- tosis on murine macrophage cell lines in vitro .MTT assay result indicated that there was no observed change of the viability when cells exposed SNs at 6.25–400l g/mL concentratio n s (Fig. 4A). FACS result showed that apoptosis rate of cells increased when the concentratio n over 100 l

g/mL, but the increase was not Effect of structure (A),particle size (B),ratio of OVA and SNs (C)and media or temperature (D)on LC or EE of OVA loading on nanoparticles.

statistically signi?cant(Fig. 4B).These results indicated that there was no adverse effect of SNs on murine macrophage cells.

3.6. In vivo study of SNs on immune organs

On 14 day after the last injection ,the mice were sacri?ced and the weights of body, spleen and thymus tissues were collected .No obvious differenc e s were found in the body weights of groups. The coef?cient of spleen at 25 mg/kg was slightly declined, but was not statistical l y signi?cant.Coef?cients of thymus at all dose showed no remarkabl e change after administrat i on of SNs (Fig. 4C). found no abnormality in any of spleen and thymus organs in the SNs injected mice at three doses (Fig. 5).Compare d with control animals, spleen red pulp and white pulp remained normal regions when the mice received SNs. Morphology of thymus also showed there was no apparent damage. These resulted suggested that the benign immune system biocompatib i lity of SNs in vivo .

The increasing demand of new adjuvant provides broad spaces for the development of nanomat e rials as antigen delivery [32–34]. Although a dozen nanomaterial s have been investigated for dec- ades as potential adjuvant, few materials have entered clinical tri- als stage [35].The lack of research on bio-effect of nanomaterial s

ffects of medium and temperature on OVA releasing from SNs–OVA nanoparticles. 5% glucose solution (A)was the most suitable one among the three candidate media, the others were PBS (B)and pure water (C).Temperature had a certain in?uence on OVA releasing in different solvents, especially in glucose. Releasing date was signi?cant declined at 4°C.Fig. 4. Cytotoxicity and organ index in vivo of SNs. Different levels of SNs (0–g/mL)induced no decline of cell survival rate of RAW264.7 cell (A).Apoptosis rate were detected by FACS (B)and SNs caused a slight increase of apoptosis but not signi?cant.Repeated exposure of SNs did not affect organ index of spleen and thymus in mice (C).

and unclear adjuvant mechanism s were responsible for this situation.

Cytotoxicity of SNs in vitro on RAW264.7 cell line showed SNs that it was a pro m is i n g bi o ma t eria l with exc e lle n t bio c omp a tib i li t y. SNs did not induce decrease of cell viability or cell apoptosis. We previously reported that LD50 of SN was greater than 1000 mg/kg, which was much higher than that in

silica nanoparticles [26].In this study, we investiga

repeated dose SN on immune system. The results indicated that there were no obvious lesion of immune organs such as thymus and spleen after SN exposure.

3.7. ELISA assay of antibody of OVA

To assess the effect of silica particles on immune responses measure d the levels of anti OVA IgG antibody and anti OVA IgG1 antibody .The levels of OVA speci?c IgG antibody in mice exposed to SNS–OVA(OD=0.42)were signi?cantly higher than in those animals exposed to OVA alone (OD=0.11).The levels of anti OVA IgG induced by SNs–OVA collaborated with CFA (OD

slightly elevated than those without CFA (Fig. 6A).

ci?c IgG1 Antibody also followed the same changes (

IgG1 production is indicative of a Th2 type response. These results suggest that silica nanoratt l es can induce the production of antigen speci?c antibody responses including antigen-speci?c

immune responses.

Antigen uptake by antigen presenting cells (APCs)

by the association of antigens with nanoparticl e s. The adjuvant ef- fect of these particles appears to largely be a conseque

uptake into APCs [36,37].More importantly, particulate antigens In conclusio n, we investigated the possibilit y of SNs in protein vaccine adjuvant applications .SNs had high protein loading ef?-ciency, stability and releasing performance. The toxicity results of SNs in vivo and in vitro also indicate that SNs was a kind of biomater i als with benign biocomp a tibility. We also proved that SN enhanced antibody level of OVA after single dose vaccination. Further studies for SNs clinical application are in progress to

Histological analyses of tissues in silica nanorattles treated mice. Histological section of spleen (A–D)and thymus (E–H)stained with H&E. Data were representative of mice. The scale bar is 100 l m.

The levels of anti OVA IgG Ab (A)and anti OVA IgG1 Ab (B)were measured

by ELISA kits. The levels of OVA speci?c IgG Ab and IgG1 Ab in mice exposed to OVA

loaded SNs were signi?cantly higher than in those exposed to OVA alone. The levels

of anti OVA IgG induced by OVA loaded SNs collaborated with CFA were slightly

elevated than those without CFA but not signi?cant(??denotes statistical signi?-

cance for the comparison of control, ??p<0.01).

determine whether the novel nanotechnolog y can induce cellular or humoral immunity.

Acknowled g ments

The authors acknowledge ?nancial support from the National Natural Science Foundation of China (NSFC)(Nos.81201814, 81171454, 81000667, 30900349)and Beijing Nova Program (Z111103054511113).

Appendix A. Supplementar y material

Supplement a ry data associated with this article can be found, in the online version, at http://dx.doi.o r g/10.1016/j.jc i s.2013.03.005 . Reference

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