Biodegradable Metal Ion-Doped Mesoporous Silica Nanospheres

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Biodegradable metal ions doped mesoporous silica nanospheres stimulate anti-cancer Th1 immune response in vivo Xiupeng Wang, Xia Li, Atsuo Ito, Yu Sogo, Yohei Watanabe, Noriko Tsuji, and Tadao Ohno ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b16118 • Publication Date (Web): 01 Dec 2017 Downloaded from http://pubs.acs.org on December 4, 2017

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ACS Applied Materials & Interfaces

Biodegradable Metal Ions Doped Mesoporous Silica Nanospheres Stimulate Anti-cancer Th1 Immune Response in vivo Xiupeng Wang1*, Xia Li1, Atsuo Ito1, Yu Sogo1, Yohei Watanabe2, Noriko M. Tsuji2, Tadao Ohno3 1

Health Research Institute, Department of Life Science and Biotechnology, National Institute of

Advanced Industrial Science and Technology (AIST), Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan 2

Biomedical Research Institute, Department of Life Science and Biotechnology, National

Institute of Advanced Industrial Science and Technology (AIST), Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan 3

School of Life Dentistry at Tokyo, The Nippon Dental University, Fujimi, Chiyoda-ku Tokyo

102-0071, Japan

ABSTRACT: Modern vaccines usually require accompanying adjuvants to increase the immune response to antigens. Aluminum compounds (alum) are the most commonly used adjuvants in human vaccinations for infection diseases. However, alum adjuvants are non-degradable, cause side effects due to the persistence of alum at injection sites, and are rather ineffective for cancer

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immunotherapy, which requires the Th1 immune response. Recently, we have shown that a plain mesoporous silica (MS) adjuvant can stimulate Th1 anti-cancer immunity for cancer vaccines. Herein, MS nanospheres doped with Ca, Mg and Zn (MS-Ca, MS-Mg and MS-Zn) showed significantly higher degradation rates than pure MS. Moreover, MS-Ca, MS-Mg and MS-Zn nanospheres enhanced anti-cancer immune response and increased the CD4+ and CD8+ T cell populations in spleen compared with alum. The MS-Ca, MS-Mg and MS-Zn nanospheres with improved biodegradability and excellent ability to induce Th1 anti-cancer immunity show potential for clinical application as cancer immunoadjuvants.

KEYWORDS: mesoporous silica, adjuvant, cancer immunotherapy, doping, Th1 immune, degradation 1. Introduction Synthesis of a suitable adjuvant that overcomes the poor immunogenicity and/or high degradability associated with cancer antigens is an important challenge in cancer vaccine development 1. Alum is the most usually used adjuvant in human vaccines 2-3. Alum is generally considered non-degradable and can cause serious side effects owing to its persistence at injection sites. These side effects include granulomatous inflammation, sclerosis, erythema, subcutaneous nodules and urticaria 4-7. Moreover, alum provokes the Th2 immune response and is rather ineffective for cancer immunotherapy, which requires the Th1 immune response 3, 8-9. Therefore, an adjuvant with excellent biodegradability and Th1 anti-cancer immunity is desirable for cancer immunotherapy. Mesoporous silica (MS)-based particles are promising among the various cancer immunoadjuvants because of their adjustable morphology, particle sizes and surface properties,


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good biocompatibility, uniform pore structure and high surface areas 10-17. Most importantly, MS is not only an efficient carrier for immunostimulatory molecules but also an effective immunopotentiator by itself. An MS-immunostimulatory molecule-apatite composite adjuvant in combination with an autologous cancer antigen inhibited in vivo cancer growth compared with an alum adjuvant 18-20. MS loaded with OVA (a model cancer antigen) and immunostimulatory molecules (CpG oligodeoxynucleotides, granulocyte macrophage colony-stimulating factor (GM-CSF)) inhibited OVA-specific cancer growth from E.G7-OVA lymphoma cells in vivo 21. We currently demonstrated that hollow MS nanospheres free of immunostimulatory molecules exhibited strong anti-cancer immunity in vivo, significantly enhanced Th1 and Th2 immune responses and a high population of effector memory T cells in bone marrow 22. On the other hand, the degradation of silica is relatively slow owing to its stable Si-O-Si network structure. Ca-, Mg- and Zn-doped MS (MS-Ca, MS-Mg and MS-Zn) nanospheres prepared by doping metal elements into MS framework are believed to accelerate their degradation because the Si-O-M (M=Ca, Mg and Zn) network structure is much weaker than the Si-O-Si network structure 23-27. In addition, Ca, Mg and Zn are important elements in stimulating anti-cancer immunity as they stimulate the secretion of cytokines including GM-CSF, interleukin (IL)-2, 4, 6, 10 and 17, interferon (IFN)-γ, tumor necrosis factor (TNF)-α and antibodies, such as immunoglobulin G (IgG), IgG1 and IgG2a 28-40. Herein, MS-Ca, MS-Mg and MS-Zn nanospheres were synthesized to act as adjuvants with improved biodegradability and excellent ability to induce Th1 anti-cancer immunity for cancer immunotherapy. 2. Materials and methods


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Synthesis of MS, MS-Ca, MS-Mg and MS-Zn nanospheres MS nanospheres were synthesized by adding tetraethoxysilane (Wako, Japan) dropwise into 480 mL of cetyltrimethylammonium bromide aqueous solution supplemented with sodium hydroxide under vigorous stirring at 70oC. The ratio of tetraethoxysilane: cetyltrimethylammonium bromide: sodium hydroxide: water is 1mL: 0.2 g: 0.056 g: 96 mL, respectively. After stirring for 5 h, the precipitate was gathered after centrifugation, washing with ultrapure water and ethanol. The precipitate was dried at 80oC and calcined at 550oC for 5 h. MS-Ca, MS-Mg and MS-Zn nanospheres were synthesized in the same way as in the synthesis of MS nanospheres, except for immediately after the addition of TEOS, 200 mg of CaCl2·2H2O (Sigma-Aldrich), 210 mg of Mg(NO3)2·6H2O (Wako) and 224 mg of Zn(NO3)2·6H2O (Wako) were dissolved in 1 mL ultrapure water and added dropwise with vigorous stirring, respectively. Characterization of MS, MS-Ca, MS-Mg and MS-Zn nanospheres The MS, MS-Ca, MS-Mg and MS-Zn nanospheres was observed by a field-emission scanning electron microscope (S-4800, Hitachi) and a transmission electron microscope (TEM, EM-002B, TOPCON). The nanospheres were tested by a powder X-ray diffractometer with CuKα X-rays (RINT 2500, Rigaku) and an FTIR spectrometer (JASCO Corporation). The zeta potential and particle size were tested by a particle analyzer (Beckman Coulter) after dispersing the nanospheres in PBS(-) and ultrapure water, respectively. In vitro chicken egg ovalbumin (OVA) adsorption At first, MS, MS-Ca, MS-Mg or MS-Zn nanospheres (2 mg) was mixed with 1.5 mg/mL OVA (Sigma-Aldrich) in saline (0.1 mL) at 4oC for 12h. The supernatant was used to determine the


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OVA content with a BioRad protein assay kit (Bio-Rad Laboratories, Inc.). The amounts of OVA adsorbed onto the adjuvants were calculated from the difference in the OVA concentration before and after adsorption. In vitro and in vivo degradation The in vitro degradation behavior of the MS, MS-Ca, MS-Mg and MS-Zn nanospheres was studied by immersing nanospheres (10 mg) contained in a bag of dialysis membrane (Funakoshi Co., Ltd.) in citric acid-sodium citrate buffer at pH=5.1 (10mL) at 37oC. At certain time intervals, the entire buffer was collected, and at the same time, the nanospheres were reimmersed in 10 mL of the new buffer. The collected buffer was analyzed for different ions concentration including Ca, Mg, Zn and Si, by an inductively coupled plasm - atomic emission spectrometry (ICP-AES, Hitachi High-technologies) to calculate the degradation ratio of the nanospheres. The in vivo degradation behavior of the MS, MS-Ca, MS-Mg and MS-Zn nanospheres was studied by the subcutaneous injection of 0.1 mL of 50 mg/mL nanospheres into the back of mice. One day after injection, the tissue around the nanosphere injection site was collected, dissolved in NaOH and HCl, and analyzed using ICP-AES. The in vivo degradation ratio of MS, MS-Ca, MS-Mg and MS-Zn nanospheres was calculated from the difference in the Si, Ca, Mg and Zn concentration before and after injection, respectively. In vivo anti-cancer test


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The animal experimental protocol was approved by the ethical committee on experiments involving animals of AIST, Japan. The animal feeding and experiments were carried according to the AIST guidelines for animal experiments. OVA was used as the cancer antigen specific to E.G7-OVA lymphoma cells (CRL-2113™, ATCC®). Female C57BL/6J mice (CLEA Inc., 6 weeks old) were first divided into six groups named Alum-OVA, MS-OVA, MS-Ca-OVA, MS-Mg-OVA, MS-Zn-OVA and control (saline, Otsuka Normal Saline). Then the mice in each group (left flank) were subcutaneously injected with 0.1 mL of the corresponding adjuvants (Alum, MS, MS-Ca, MS-Mg or MS-Zn nanospheres (20 mg/mL)) mixed with OVA (1500 µg/mL) in saline at 4oC for 12h. At days 3 and 10, the mice (left flank) were further subcutaneously injected with the corresponding adjuvants. Mice in the control group were injected with saline in the same manner as described above. At day 14, mice were (right flank) subcutaneously injected with E.G7-OVA cells (0.1 mL of 5×106 cells/mL). Cancer growth was monitored for 4 weeks. Flow cytometry analysis Flow cytometry analysis was used to clarify the mechanisms of anti-cancer immunity induced by the adjuvants. Lymphocytes were collected from the inguinal lymph nodes following an in vivo anti-cancer test to form a single-cell suspension. Anti-CD16/CD32 antibody (2.4G2, BD Pharmingen) was used to prevent the non-specific staining. Anti-mouse CD4 and anti-mouse CD8α antibodies (BioLegend) were used to stain the cells for 30 min. Flow cytometry was performed for the cell suspensions using a FACSAria cell cytometer (BD Bioscience). Ex vivo immunogenic activity test


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The mice (base of tail and hind footpads) were subcutaneously immunized with 0.1 mL of MS, MS-Ca, MS-Mg or MS-Zn nanospheres (20 mg/mL) mixed with OVA (0.5 mg/mL) in saline at day 0. Mice subcutaneously immunized with 0.1 mL of OVA (0.5 mg/mL) in saline were used as controls. The mice were sacrificed at day 7. Draining lymph nodes were collected from the mice and gently grinded to form a single-cell suspension. The collected cells were cultured in RPMI1640 medium containing 10% fetal bovine serum and certain amounts of antibiotics (streptomycin, 100 µg/mL; penicillin, 100 U/mL). The cells were co-cultured with OVA for 3 days. IFN-γ and IL-4 cytokines in the cell culture media were analyzed using mouse ELISA kits (BD Pharmingen).

3. Results and discussion Physicochemical characterization of MS, MS-Ca, MS-Mg and MS-Zn nanospheres Monodispersed MS nanospheres with diameter about 100 nm and mesopores with diameter about 4 nm were synthesized by a one-pot route. All the samples had a similar size and morphology, indicating that the Ca-, Mg- and Zn-doping had no obvious influence on the size and morphology of the nanospheres (Figure 1). The MS, MS-Ca, MS-Mg and MS-Zn nanospheres had narrow hydrodynamic size distributions with average diameters of 104±24 nm, 103±26 nm, 107±53 nm and 99±21 nm, respectively (Figures 2 a-d), which were in accordance with the TEM and SEM images. The broad peak (15-30o) in the XRD patterns and the Si-O bands (470, 800 and 1093-1120 cm-1) in the FTIR spectra indicated the MS, MS-Ca, MS-Mg and MS-Zn were mainly composed of amorphous silica. No obvious difference was observed in the XRD patterns and FTIR spectra among the MS, MS-Ca, MS-Mg and MS-Zn nanospheres owing


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to both the low Ca, Mg and Zn contents in the nanospheres and the moderate heat treatment temperature of 550oC used to remove the templates (Figures 2 e, f). The successful synthesis of MS-Ca, MS-Mg and MS-Zn nanospheres was proved by chemical analyses using ICP-AES. The Ca/Si, Mg/Si and Zn/Si molar ratios of the MS-Ca, MS-Mg and MS-Zn nanospheres were 10.3%±0.9%, 10.4%±0.9% and 10.7%±0.1%, respectively (Table 1). The MS, MS-Ca, MS-Mg and MS-Zn nanospheres were negatively charged in PBS(-) with zeta potential values of approximately -17, -28, -25 and -34 mV, respectively (Figure 2 g). The higher zeta potentials of MS-Ca, MS-Mg and MS-Zn nanospheres than that of the MS nanospheres indicates improved dispersibility and stability against aggregation in solution. The MS, MS-Ca, MS-Mg and MS-Zn nanospheres efficiently adsorbed 48±25 to 73±5 µg/mg of OVA (Figure 2 h). Degradation rate of MS, MS-Ca, MS-Mg and MS-Zn nanospheres in vitro and in vivo The MS-Ca, MS-Mg and MS-Zn nanospheres showed significantly higher degradation rates than the pure MS nanospheres in vitro and in vivo (Figures 3, S1 and S2). The MS, MS-Ca, MS-Mg and MS-Zn nanospheres exhibited in vitro degradation rates of 15.1±0.1, 26.3±2.1, 27.7±0.6 and 33.4±1.4% at 37oC (Figure 3 a) and in vivo degradation rates of 24.0±6.7, 50.8±10.2, 52.8±1.0 and 56.3±8.2% one day after subcutaneous injection (Figure 3 b), respectively. The MS-Zn nanospheres showed the highest degradation rate among all the groups. Pure silica materials are composed of [SiO4] tetrahedra with bridging oxygen in a stable Si-O-Si network structure. For the MS-Ca, MS-Mg and MS-Zn nanospheres, the presence of M (M=Ca, Mg and Zn) in the silica network structure introduces non-bridging oxygen via the [SiO4] tetrahedra, which results in relatively weak Si-O-M (M=Ca, Mg and Zn) connections. Therefore, the hydrolysis of the silica network is accelerated by the presence of non-bridging oxygens in the


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MS-Ca, MS-Mg and MS-Zn nanospheres 23-27. In addition, the amorphous structure of silica after calcining below 550oC to remove the templates is believed to facilitate their degradation 15. The amorphous phase (Figure 2 e), the regular mesoporous arrangement (Figure 1 b) and the relatively weak Si-O-M (Ca, Mg and Zn) network in the MS-Ca, MS-Mg and MS-Zn nanospheres account for their improved degradability. MS-OVA, MS-Ca-OVA, MS-Mg-OVA and MS-Zn-OVA showed superior anti-cancer immunity in vivo The anti-cancer effects associated with the MS-OVA, MS-Ca-OVA, MS-Mg-OVA and MS-ZnOVA were evaluated as shown in Figures 4 and S3. The mice without immunization and those immunized with Alum-OVA exhibited rapid cancer growth with a cancer volume of about 10981288 mm3 at day 19 (Figure 4 b). In contrast, the immunization with MS-OVA, MS-Ca-OVA, MS-Mg-OVA and MS-Zn-OVA greatly inhibited cancer growth in mice, resulting in a cancer volume of about 108-292 mm3 at day 19 (Figure 4 b). The percentages of the mice without immunization and those immunized with Alum-OVA, MS-OVA, MS-Ca-OVA, MS-Mg-OVA and MS-Zn-OVA with cancer size smaller than 15 mm were 0%, 20%, 60%, 60%, 60% and 80%, respectively (Figure 4 c). MS, MS-Ca, MS-Mg and MS-Zn nanospheres accelerated CD4+ and CD8+ T cells proliferation, Th1 and Th2 cytokines secretion To clarify the mechanisms of MS, MS-Ca, MS-Mg and MS-Zn nanosphere-mediated anti-cancer immunity, lymphocytes were collected and analyzed by a flow cytometry following the in vivo anti-cancer test (Figure 5). MS-OVA, MS-Ca-OVA, MS-Mg-OVA and MS-Zn-OVA nanospheres significantly increased the population of CD4+ and CD8+ T cells in the lymphocytes


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of mice compared with those without immunization. MS-Zn-OVA nanospheres significantly increased CD4+ and CD8+ T cell populations in the lymphocytes of mice compared with mice immunized with Alum-OVA. The mice immunized with MS-Zn-OVA had the highest percentages of CD4+ and CD8+ T cells in their lymphocytes among all the groups. The ex vivo immunoadjuvant activity of the MS, MS-Ca, MS-Mg and MS-Zn nanospheres is shown in Figure 6. The MS-OVA-, MS-Ca-OVA-, MS-Mg-OVA- and MS-Zn-OVA-treated mice exhibited significantly higher cytokines secretion including Th1-type IFN-γ and Th2-type IL-4 in spleen ex vivo, when co-cultured with 0.1 mg/mL OVA, than the mice treated with OVA. The MS-Zn-OVA-administrated mice exhibited the highest OVA-specific IFN-γ secretion by lymphocytes ex vivo among all the groups. The anti-cancer Th1 immunity of MS has been reported in recent studies. MS nanoparticles adsorb biomolecules to promote their cellular uptake by immune cells, increase the number of CD86+ cells, promote cytokine secretion from DCs in vitro, increase immunoglobulin M (IgM), IgG, IgG1, IgG2a and IgA levels in serum, promote lymphocyte proliferation, enhance population of effector memory CD4+ and CD8+ T cells and stimulate anti-cancer immunity in vivo 18-22, 41-47. Additionally, Ca-, Mg- and Zn-containing substances have been observed to stimulate anti-cancer immunity. For instance, calcium phosphate stimulates the production of IgG, IgG1, IgG2a, TNF-α and IL-6 and promotes Th1 immune responses 28-31. Zn promotes antigen-presenting cells activity and Th1 cell response 32-33. ZnO nanoparticles promote the secretion of IL-2, 4, 6, 10, 12 and 17, TNF-α and IFN-γ in vitro 34-36, 38. Zn, Mg-containing tricalcium phosphate-based adjuvants promote IL-2, TNF-α, IL-12, GM-CSF and IFN-γ secretion and the anti-cancer efficacy of mice compared with tricalcium phosphate-based adjuvants 33. In this study, MS-Zn nanospheres showed the highest anti-cancer Th1 immune


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response in vivo among all the groups, which may be explained by the synergistic effect of Zn and MS. Conclusions Monodispersed MS, MS-Ca, MS-Mg and MS-Zn nanospheres with a uniform diameter of about 100 nm were prepared and used as cancer immunoadjuvants. MS-Ca, MS-Mg and MS-Zn nanospheres showed significantly higher degradation rates than pure MS nanospheres in vitro and in vivo. Moreover, MS-Zn-OVA-administrated mice showed the highest anti-cancer immunity in vivo, CD4+ and CD8+ T cell populations in lymphocytes in vivo and Th1 cytokine secretion ex vivo among all the groups. The MS-Ca, MS-Mg and MS-Zn nanospheres, particularly MS-Zn nanospheres are promising for cancer immunotherapy owing to their high ability to induce Th1 anti-cancer immunity and improved biodegradability.

ASSOCIATED CONTENT Supporting Information The following files are available free of charge. Representative in vitro degradation study images; representative in vivo degradation study images; representative mouse tumor images (PDF) AUTHOR INFORMATION Corresponding Author


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*E-mail: [email protected] Funding Sources This study was supported in part by Nippon Sheet Glass Foundation for Materials Science and Engineering, JSPS KAKENHI Grant Numbers 17K01399. Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS We thank Ms. Hisako Sugino and Ms. Kazuko Yoshiyuki for their technical assistance. We thank Prof. Koji Tsuboi at University of Tsukuba for fruitful discussion. REFERENCES (1) Reed, S. G.; Orr, M. T.; Fox, C. B. Key Roles of Adjuvants in Modern Vaccines. Nat Med 2013, 19, 1597-1608. (2) Ren, H.; Zhang, Q. X.; Qie, L.; Baker, G. L. The Adjuvant Effect of Emerging Nanomaterials: A Double-Edged Sword. Acs Sym Ser 2013, 1150, 3-21. (3) Marrack, P.; Mckee, A. S.; Munks, M. W. Towards an Understanding of the Adjuvant Action of Aluminium. Nat Rev Immunol 2009, 9, 287-293. (4) Chong, H.; Brady, K.; Metze, D.; Calonje, E. Persistent Nodules at Injection Sites (Aluminium Granuloma) - Clinicopathological Study of 14 Cases with a Diverse Range of Histological Reaction Patterns. Histopathology 2006, 48, 182-188. (5) Marcoval, J.; Moreno, A.; Mana, J. Subcutaneous Sarcoidosis Localised to Sites of Previous Desensitizing Injections. Clin Exp Dermatol 2008, 33, 132-134. (6) Shabrawi-Caelen, E.; Poelt, P.; Aberer, W.; Aberer, E. Progressive Circumscribed Sclerosis a Novel Side-effect of Immunotherapy with Aluminium-adsorbed Allergen Extracts. Allergy 2009, 64, 965-967. (7) Ozden, M. G.; Kefeli, M.; Aydin, F.; Senturk, N.; Canturk, T.; Turanli, A. Y. Persistent Subcutaneous Nodules after Immunotherapy Injections for Allergic Asthma. J Cutan Pathol 2009, 36, 812-814. (8) Oka, H.; Emori, Y.; Ohya, O.; Kobayashi, N.; Sasaki, H.; Tanaka, Y.; Hayashi, Y.; Nomoto, K. An Immunomodulatory Arabinomannan Extracted from Mycobacterium Tuberculosis, Z-100,


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(41) Vallhov, H.; Kupferschmidt, N.; Gabrielsson, S.; Paulie, S.; Stromme, M.; Garcia-Bennett, A. E.; Scheynius, A. Adjuvant Properties of Mesoporous Silica Particles Tune the Development of Effector T Cells. Small 2012, 8, 2116-2124. (42) Vallhov, H.; Gabrielsson, S.; Stromme, M.; Scheynius, A.; Garcia-Bennett, A. E. Mesoporous Silica Particles Induce Size Dependent Effects on Human Dendritic Cells. Nano Lett 2007, 7, 3576-3582. (43) Mercuri, L. P.; Carvalho, L. V.; Lima, F. A.; Quayle, C.; Fantini, M. C. A.; Tanaka, G. S.; Cabrera, W. H.; Furtado, M. F. D.; Tambourgi, D. V.; Matos, J. D. R.; Jaroniec, M.; Sant'Anna, O. A. Ordered Mesoporous Silica SBA-15: A New Effective Adjuvant to Induce Antibody Response. Small 2006, 2, 254-256. (44) Carvalho, L. V.; Ruiz, R. D.; Scaramuzzi, K.; Marengo, E. B.; Matos, J. R.; Tambourgi, D. V.; Fantini, M. C. A.; Sant'Anna, O. A. Immunological Parameters Related to the Adjuvant Effect of the Ordered Mesoporous Silica SBA-15. Vaccine 2010, 28, 7829-7836. (45) Wang, X.; Li, X.; Ito, A.; Yoshiyuki, K.; Sogo, Y.; Watanabe, Y.; Yamazaki, A.; Ohno, T.; Tsuji, N. M. Hollow Structure Improved Anti-Cancer Immunity of Mesoporous Silica Nanospheres In Vivo. Small 2016, 12, 3510-3515. (46) Guo, H. C.; Feng, X. M.; Sun, S. Q.; Wei, Y. Q.; Sun, D. H.; Liu, X. T.; Liu, Z. X.; Luo, J. X.; Yin, H. Immunization of Mice by Hollow Mesoporous Silica Nanoparticles as Carriers of Porcine Circovirus Type 2 ORF2 Protein. Virol J 2012, 9, 108. (47) Kupferschmidt, N.; Qazi, K. R.; Kemi, C.; Vallhov, H.; Garcia-Bennett, A. E.; Gabrielsson, S.; Scheynius, A. Mesoporous Silica Particles Potentiate Antigen-specific T-cell Responses. Nanomedicine-Uk 2014, 9, 1835-1846.


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Page 16 of 23

Table and Figures

Table 1 Ca/Si, Mg/Si and Zn/Si molar ratio of the MS-Ca, MS-Mg and MS-Zn nanospheres

Ca/Si

Mg/Si

Zn/Si

(%)

(%)

(%)

MS-Ca

10.3±0.9

-

-

MS-Mg

-

10.4±0.9

-

MS-Zn

-

-

10.7±0.1


16

Page 17 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 1. SEM (a) and TEM (b) images of MS, MS-Ca, MS-Mg and MS-Zn nanospheres.


17

60 20

40

10

20

0

80

120

160

200 240

0

b

40

80 30 60 20

40

10

20

0

80

120

60 20

40

10

20

0

80

120

160

200 240

0

Differential Number (%)

80 30

40

20 15 12

30

40

50 60 2θ (o)

g

60 20

40

10

20

0

80

120

MS-Zn MS-Mg MS-Ca MS

9 6 3 0 -100

-50

0 50 zeta potential (mV)

100

200 240

0


4000

80

160

Diameter (nm)

f

Absorbance (%)

70

100 80

OVA adsorbed (µg/mg)

Intensity (a.u.)


0

30

Diameter (nm)

e

200 240

d

100 Cumulative Number (%)


c

160

Diameter (nm)

Diameter (nm)

40

100

Cumulative Number (%)

80 30

Page 18 of 23


100


a


40

Intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60



80

3000 2000 1000 Wavenumbers (cm-1)

h

60 40 20 0

MS

MS-Ca MS-Mg

MS-Zn

Figure 2. Particle size distribution of MS (a), MS-Ca (b), MS-Mg (c) and MS-Zn (d); XRD patterns (e), FT-IR spectra (f) and zeta potential (g) of different nanospheres; OVA adsorption by different nanospheres in vitro (h).


18

a

40


30 20 * * *

10 0

0

* * *

1

Day

b * * * *

2

* *

3

In vivo degradation (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


In vitro degradation (%)

Page 19 of 23

80

*

*

*

60 40 20 0

MS

MS-Ca MS-Mg MS-Zn

Figure 3. In vitro (a) and in vivo (b) degradation of MS, MS-Ca, MS-Mg and MS-Zn nanospheres (n=4, p

Biodegradable Metal Ion-Doped Mesoporous Silica Nanospheres

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