Surface functionalized mesoporous silica nanoparticles with natural proteins for reduced immunotoxicity Zhong Luo,1,2* Yan Hu,1* Renlong Xin,2 Beilu Zhang,1 Jinghua Li,1 Xingwei Ding,1 Yanhua Hou,1 Li Yang,1 Kaiyong Cai1 1
Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400044, People’s Republic of China 2 College of Material Science and Engineering, Chongqing University, Chongqing 400044, People’s Republic of China Received 14 October 2013; revised 19 November 2013; accepted 26 November 2013 Published online 00 Month 2013 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.a.35049 Abstract: Mesoporous silica nanoparticles (MSNs) present themselves as one of the most promising nano-carriers for drug delivery. To reduce their immunotoxicities, in this study, natural proteins of gelatin (Gel), bovine serum albumin (BSA), and lysozyme (Lys) were employed as end-caps of MSNs by using succinic anhydride as an intermediate linker, thus leading to fabrication of MSNs/protein nanocomposites, respectively. Furthermore, combined techniques of SEM, TEM, FTIR, and zeta potential instruments were utilized to monitor the construction processes of MSNs/protein nanocomposites, respectively. Finally, the immunotoxicities of those nanocomposites to macrophage cells (RAW264.7 cells) were investigated in detail, i.e., cell morphology, cell viability, nitric oxide (NO) production, reactive oxygen species (ROS),
and acid phosphatase activity (ACP) as well as inflammation cytokine expressions (tumor necrosis factor-a and interleukin1b). All results suggest that macrophages were activated after uptaking nanoparticles of SiO2 and MSNs, which subsequently induced severe inflammation responses in vitro. In contrast, the inflammation responses of MSNs nanocomposites were reduced dramatically after end-capping with those natural proteins. Overall, this study accumulates knowledge for the development of MSNs-based drug delivery systems C 2013 Wiley Periodicals, Inc. J with reduced immunotoxicity. V Biomed Mater Res Part A: 00A:000–000, 2013.
Key Words: mesoporous silica nanoparticles, immunotoxicity, inflammation responses, macrophages, natural proteins
How to cite this article: Luo Z, Hu Y, Xin R, Zhang B, Li J, Ding X, Hou Y, Yang L, Cai K. 2013. Surface functionalized mesoporous silica nanoparticles with natural proteins for reduced immunotoxicity. J Biomed Mater Res Part A 2013:00A:000–000.
INTRODUCTION
Recently, the convergence of nanotechnology, materials, biology, and medicine accelerates the development of nanomedicine in the clinical applications, which displays great potential for human healthcare, particularly in cancer therapy.1–4 Although an increasing number of powerful and targeted therapeutic treatments (e.g., nano-scale drug delivery systems) and diagnostic techniques (e.g., biomedical imaging agents) have been exploited, it still might be first interacted with immune systems of a host in vivo. Therefore, it is necessary to reveal the toxicity of nano-scale formulations to immune systems,5 which was currently defined as a concept of “immunotoxicity.”6 Actually, the immune systems are the natural barriers to the allogeneic materials in vivo.5 In clinical application, once a nano-scale drug delivery system is administered, it would delicately remain in the host’s body and thus inevitably interact with the immune systems.
Mesoporous silica nanoparticles (MSNs) were developed to be one of the most promising nano-carriers for anticancer drug delivery, mainly owing to their good biocompatibility, large surfaces areas, and uniform mesoporous structures.7–9 Previously, various types of MSNs-based controlled drug delivery systems were exploited, including inorganic nanoparticles/MSNs systems,10–12 molecular switches/MSNs systems,13–15 and biomacromolecules/MSNs systems, etc.16–19 However, both inorganic nanoparticles/ MSNs systems and molecular machines/systems might suffer from cytotoxicity and adverse biological interactions since they were xenobiotics.20,21 The cell viability and functions (e.g., cell adhesion and migration) were greatly affected after uptaking of nanoparticles.22 In contrast, biomacromolecules/MSNs controlled-drug delivery systems presented themselves as one of the most promising candidates for future clinic application since biomacromolecules had good biocompatibility and limited immune reactivity.23
Additional Supporting Information may be found in the online version of this article. *These authors contributed equally to this work. Correspondence to: K. Cai; e-mail: [email protected] Contract grant sponsor: Natural Science Foundation of China; contract grant number: 21274169, 31200712 Contract grant sponsor: Fundamental Research Funds for the Central Universities; contract grant numbers: CQDXWL-2013-Z002, CDJZR 10238801, “111” project B06023
C 2013 WILEY PERIODICALS, INC. V
1
FIGURE 1. Schematic illustration of macrophage inflammation responses to silica nanoparticles, mesoporous silica nanoparticles, and MSNs/ protein nanocomposites. Endosome refers to a membrane-bound compartment within cells, acting as a reservoir for transferring substances from plasma membrane to lysosome. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Previous studies revealed that cytotoxicities of nanomateirals and their effects on cellular functions highly depended on their physiochemical properties and chemical compositions.24,25 As end-capping agents, biomacromolecules were generally immobilized onto the exterior surfaces of mesoporous silica nanoparticles (MSNs), which played an important role in mediating the interactions between MSNs/biomacromolecules nanocomposites and cells/tissues.26 During the past decades, a diversity of biomacromolecules was exploited as efficient end-capping agents of MSNs (such as biotin-avidin,27 antibody,28 insulin,19 and DNA29). Besides, our group also constructed a series of pH- and redox-responsive MSNs/biomacromolecules smart drug delivery systems by using gelatin,16 bovine serum albumin (BSA),17 and collagen18 as end-capping agents. Those systems were proved to be efficient carriers for intracellular drug delivery via targeted cellular uptake or specific endocytosis mechanisms.16–19 Nevertheless, the immunotoxicity of those systems were not investigated. To guarantee the successful clinic applications of MSNs-based drug delivery systems, potential effects of those nano-scale systems on immune systems of a host definitely should be evaluated. In immune systems, macrophages play a key role in the regulation of “foreign body reactions,” including inflammation responses.30 Thus, it is urgently necessary to evaluate the immunotoxicity of MSNs-based controlled drug delivery systems to macrophages except for the routine cytocompatibility assays. Previously, Lunov et al. demonstrated that carboxydextran coating of magnetic nanoparticles could reduce the apoptosis of macrophages.31 In another study, Seo et al.
2
LUO ET AL.
reported that gelatin molecules could improve the activities of immune cells with enhanced immunomodulatory property.32 Those studies implied that surface engineering of nanoparticles with appropriate components could efficiently reduce their immunotoxicity, in turn optimizing them as efficient carriers for drug delivery. Herein, gelatin (Gel), bovine serum albumin (BSA), and lysozyme (Lys) were immobilized onto the exterior surfaces of MSNs via succinic anhydride as an intermediate linker to fabricate MSNs/protein nano-composites systems, respectively. We hypothesized that the natural proteins end-capping of MSNs could efficiently reduce their inflammation responses to macrophages (shown in Fig. 1). It is well known that gelatin, a derivative of the extracellular matrix component of collagen, was widely used in biomedical fields.16 BSA is a serum albumin protein that interacts with the biological functions of a host.18 Meanwhile, lysozyme is an abundant component of natural products,30 such as egg, milk, etc. It has been widely used in the anti-inflammation fields as well. Thus, we hypothesized that macrophages would be activated acutely when exposed to either bare silica nanoparticles or MSNs, leading to severe inflammation responses and dysfunction of the immune system (Fig. 1). MATERIALS AND METHODS
Materials Tetraethylorthosilicate (TEOS), N-hydroxysuccinimide (NHS), dimethyl sulfoxide (DMSO), 3-(4,5)-dimethylthiahiazo(-z-y1)3,5-di-phenytetrazoliumromide (MTT), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide-HCl (EDC), lysozyme (Lys), gelatin (Gel), and bovine serum albumin (BSA) were
MSN-BASED DRUG DELIVERY SYSTEMS WITH REDUCED IMMUNOTOXICITY
ORIGINAL ARTICLE
purchased from Sigma–Aldrich Co. (Beijing, China). Phalloidin and 40 ,6-diamidino-2-phenylindole (DAPI) were obtained from Invitrogen Co. (Beijing, China). N-cetyltrimethylammonium bromide (CTAB) and succinic anhydride (SA) were provided by Alfa Aesar (Tianjin, China). Other chemicals were supplied by Oriental Chemicals Co. Ltd. (Chongqing, China). Synthesis of silica nanoparticles and mesoporous silica nanoparticles (MSNs) Silica nanoparticles were synthesized according to a previous study.31 Briefly, tetraethoxysilane (TEOS, 10 mL) and ammonium hydroxide (10 mL) were dissolved into ethanol/ water (500 mL, v/v 5 7:1) mixture solution with gentle stirring for 2 h. After filtration and configuration, the white product was rinsed with ethanol and distilled water for six times each. Finally, the sample was dried at 60 C for 6 h to get silica nanoparticles. MSNs were synthesized according to previous studies.15–18 Briefly, reagents of N-cetyltrimethyl-ammonium bromide (CTAB, 1.0 g) and sodium hydroxide (0.28 g) were dissolved in distilled water (480 mL) and heated to 80 C. Then, TEOS (5 g) was slowly added to the mixture solution within 2 h under stirring until a white precipitant was formed. After filtration and centrifugation, the resulting product was rinsed with excessive distilled water and methanol for six times each. Finally, the sample was dried at 60 C for 6 h to get MSNs. Amination of MSNs with 3-aminopropyl trimethoxysilane (APTS-MSNs) To prepare amino-functionalized MSNs, the as-synthesized MSNs (1.0 g) were dispersed in anhydrous toluene (80 mL) containing 3-aminopropyl trimethoxysilane (APTS, 0.75 mL) under vigorous stirring. Then, the mixture was re-fluxed gently for 24 h. The resulting products were filtered and washed with double-distilled water and methanol each for five times. Then, samples were dried at 50 C under high vacuum ( MSNs-BSA > MSNs-Gel > MSNsLys (Fig. 10). The results further suggest that macrophages were severely activated by bare SiO2 nanoparticles or MSNs.
FIGURE 8. Cell viability assay. RAW264.7 cells were co-cultured with SiO2, MSNs, MSNs-Gel, MSNs-BSA, and MSNs-Lys nanoparticles for 24 h and 48 h, respectively (n 5 6).
FIGURE 9. Production of ROS in RAW264.7 macrophages co-cultured with SiO2, MSNs, MSNs-Gel, MSNs-BSA, and MSNs-Lys nanoparticles for 24 h and 48 h, respectively (n 5 6).
JOURNAL OF BIOMEDICAL MATERIALS RESEARCH A | MONTH 2013 VOL 00A, ISSUE 00
11
FIGURE 10. NO production of RAW264.7 macrophages co-cultured with SiO2, MSNs, MSNs-Gel, MSNs-BSA, and MSNs-Lys nanoparticles for 24 h and 48 h, respectively (n 5 6).
However, surface conjugations of gelatin, BSA, and lysozyme to MSNs were beneficial for reducing the NO production of macrophages.
Inflammation cytokines secretions. Finally, we measured the expression levels of inflammation factors of interleukin1b (IL-1b) and tumor necrosis factor a (TNF-a) secreted by macrophages after incubation with different nanoparticles for 24 h and 48 h, respectively. It is well known that both IL-1b and TNF-a are the most important markers for indicating the inflammation responses of cells and/or tissues in vivo.49–52,55 In this study, we used an enzyme linked immunosorbent assay (ELISA) kit to monitor the amount of IL-1b and TNF-a produced by macrophages. There was no clear difference in the expression levels of both IL-1b and TNF-a between all nanoparticles groups after incubation for 24 h. However, the groups treated with silica nanoparticles and MSNs displayed much higher expression of both IL-1b and TNF-a in RAW264.7 cells when the incubation time extended to 48 h (Fig. 12). RAW264.7 cells incubated with SiO2 nanoparticles and MSNs displayed 5.9-fold and 4.2-fold IL-1b production compared to that of the TCPS group [Fig. 12(A)]. On the other hand, RAW264.7 cells treated with silica nanoparticles and MSNs showed 4.9-fold and 3.8-fold of TNF-a expression than that of TCPS [Fig. 12(B)]. However, the expression levels of both IL-1b and TNF-a in RAW264.7 cells treated with MSNs/protein nanoparticles
Acid phosphatase activity. Fourth, we evaluated the acid phosphatase (ACP) activity of macrophages after incubation with various nanoparticles for 24 h and 48 h, respectively. ACP is one of the important enzymes that reflect inflammation responses in vivo.33 The ACP activity was directly indicated by the production of p-nitrophenol in this study. Silica nanoparticles and MSNs induced significantly higher (p < 0.01) ACP activities than other groups after incubation for 24 h. The average ACP activities of macrophages decreased in the order of MSNs > SiO2 > MSNs-BSA > MSNsLys > MSNs-Gel (Fig. 11). The same trend was also found after incubation for 48 h. The results imply that the immunotoxicity of macrophages were reduced after immobilization of gelatin, BSA, and lysozyme onto the surface of MSNs, respectively.
FIGURE 11. Acid phosphatase activity of RAW264.7 macrophages cocultured with SiO2, MSNs, MSNs-Gel, MSNs-BSA, and MSNs-Lys nanoparticles for 24 h and 48 h, respectively (n 5 6).
12
LUO ET AL.
FIGURE 12. Expressions of (A) IL-1b and (B) TNF-a of RAW264.7 macrophages co-cultured with SiO2, MSNs, MSNs-Gel, MSNs-BSA, and MSNs-Lys nanoparticles for 24 h and 48 h, respectively (n 5 6).
MSN-BASED DRUG DELIVERY SYSTEMS WITH REDUCED IMMUNOTOXICITY
ORIGINAL ARTICLE
were much lower than those of bare silica nanoparticles and MSNs. Compared to TCPS, MSNs-Gel, MSNs-BSA, and MSNs-Lys samples only shows 1.7-fold, 1.2-fold, and 1.7-fold of IL-1b expression in RAW264.7 cells [Fig. 12(A)] whereas 2.8-fold, 2.4-fold, and 2.9-fold of TNF expression [Fig. 12(B)]. The results further suggest that macrophages were severely activated by bare silica nanoparticles or MSNs. The expression levels of inflammation factors of MSNs were reduced apparently after the immobilization of gelatin, BSA, and lysozyme. Generally, there are approximately 4–10 3 103 56 inflammation-related cells in 1 mL of human blood. The common values of IL-1b and TNF-a in the serum of a normal human are 10 6 4.3 pg/mL and 3.8 6 0.2 pg/mL, respectively.57,58 By conversion, we could get an estimated result that the normal expressions of IL-1b and TNF-a are lower than 1000 6 430 pg/mL/106 cells and 380 6 20 pg/mL/106cells, respectively. After end-capping with natural proteins, it was interesting to note that the expression of TNF-a was reduced from 482.06 6 59.52 pg/mL/106 cells for bare MSNs (higher than normal level) to 352.35 6 168.22 pg/mL/106 cells for MSNs-Gel, 310.85 6 64.83 pg/mL/106 cells for MSNs-BSA, and 370.58 6 73.6 pg/mL/106 cells for MSNs-Lys nanocomposites, respectively. All those concentrations were lower than the reference value of normal human (380 6 20 pg/mL/106cells). Although the expression of IL-1b for bare MSNs (96.32 6 3.07 pg/mL/106 cells) was under the reference value of normal human, different proteins end-capping still reduced their IL-1b expressions for MSNs-Gel, MSNs-BSA, and MSNs-Lys nanocomposites. The results imply that systematic investigations should be performed in the future to clarify its influence on other preinflammatory factors, such as IL-2, IL-6, IL-8, IL-9, etc. Although inflammation reaction assay with macrophages is essential in the immunotoxicity evaluation of nanotechnology-based delivery systems before their clinical application, it would coordinate with other assessments (compatibility tests of nanoparticles with blood and inflammation reaction assay in vivo) to get a comprehensive understanding of their feasibility. Taken together, we confirmed our hypothesis that natural proteins end-capping of MSNs could reduce their inflammation responses to macrophages. However, the feasibility of those MSNs’ drug delivery systems should be further evaluated in vivo. On the other hand, to overcome the disadvantage of the un-biodegradable property of MSNs, it is essentially urgent and necessary to exploit biodegradable MSNs for their clinical applications in the future.59,60
CONCLUSIONS
In summary, the immunotoxicity of silica nanoparticles, MSNs, and MSNs/proteins nanocomposites to macrophages (RAW.246.7 cells) were investigated in detail. We found that macrophages were severely activated by bare silica nanoparticles or MSNs. The production/expression levels of NO, ROS, acid phosphatase, and inflammation factors of IL-1b and TNF-a by macrophages in turn led to negative effects on cell behaviors. However, the inflammation responses of MSNs were significantly reduced by employing natural pro-
teins (gelatin, bovine serum protein, and lysozyme) as endcapping agents. This finding provides new insight into the design of MSNs/protein drug delivery systems with reduced inflammation responses (immunity) for potential clinical applications, such as cancer therapy. REFERENCES 1. Peer D, Karp JM, Hong S, Farokhzad OC, Margalit R, Langer R. Nanocarriers as an emerging platform for cancer therapy. Nat Nanotechnol 2007;2:751–760. 2. Wolfgang JP. Complex colloidal assembly. Science 2011;334: 1359–1360. 3. Chen ZL, Li Y, Liu WW, Zhang DZ, Zhao YY, Yuan B, Jiang XY. Patterning mammalian cells for modeling three types of naturally occurring cell-cell interactions. Angew Chem Int Ed 2009;48:8303– 8305. 4. Wang XJ, Wang C, Cheng L, Lee ST, Liu Z. Noble metal coated single-walled carbon nanotubes for applications in surface enhanced Raman scattering imaging and photothermal therapy. J Am Chem Soc 2012;134:7414–7422. 5. Dobrovolskaia MA, Germolec DR, Weaver JL. Evaluation of nanoparticle immunotoxicity. Nat Nanotechnol 2009;4:411–414. 6. Dobrovolskaia MA, McNeil SE. Immunological properties of engineered nanomaterials. Nat Nanotechnol 2007;2:469–478. 7. Li LL, Tang FQ, Liu HY, Liu TL, Hao NJ, Chen D, Teng X, He JQ. In vivo delivery of silica nanorattle encapsulated docetaxel for liver cancer therapy with low toxicity and high efficacy. ACS Nano 2010;4:6874–6882. 8. Chen Y, Chu C, Zhou Y, Ru YF, Chen HR, Chen F, He QJ, Zhang YL, Zhang LL, Shi JL. Reversible pore-structure evolution in hollow silica nanocapsules: Large pores for siRNA delivery and nanoparticle collecting. Small 2011;7:2935–2944. 9. Ambrogio MW, Thomas CR, Zhao YL, Zink JI, Stoddart JF. Mechanized silica nanoparticles: A new frontier in theranostic nanomedicine. Acc Chem Res 2011;44:903–913. 10. Lai CY, Trewyn BG, Jeftinija DM, Jeftinija K, Xu S, Jeftinija S, Lin VYS. A mesoporous silica nanosphere-based carrier system with chemically removable CdS nanoparticle caps for stimuliresponsive controlled release of neurotransmitters and drug molecules. J Am Chem Soc 2003;125:4451–4459. 11. Sun X, Zhao Y, Lin VS, Slowing II, Trewyn BG. Luciferase and luciferin co-immobilized mesoporous silica nanoparticle materials for intracellular biocatalysis. J Am Chem Soc 2011;133:18554–18557. 12. Ma M, Chen HR, Chen Y, Wang X, Chen F, Cui XZ, Shi JL. Au capped magnetic core/mesoporous silica shell nanoparticles for combined photothermo-/chemo-therapy and multimodal imaging. Biomaterials 2012;33:989–998. 13. Yan H, Teh C, Sreejith S, Zhu LL, Kwok A, Fang WQ, Ma X, Nguyen KT, Korzh V, Zhao YL. Functional mesoporous silica nanoparticles for photothermal—Controlled drug delivery in vivo. Angew Chem Int Ed 2012;51:8373–8377. 14. Zhao YL, Li Z, Kabehie S, Botros YY, Stoddart JF, Zink JI. pHoperated nanopistons on the surfaces of mesoporous silica nanoparticles. J Am Chem Soc 2010;132:13016–13025. 15. Meng H, Xue M, Xia T, Zhao YL, Tamanoi F, Stoddart JF, Zink JI, Nel AE. Autonomous in vitro anticancer drug release from mesoporous silica nanoparticles derivatized with pH-sensitive supramolecular nanovalves. J Am Chem Soc 2010;132:12690–12697. 16. Luo Z, Cai KY, Hu Y, Zhao L, Liu P, Duan L, Yang WH. Mesoporous silica nanoparticles end-capped with collagen: Redoxresponsive nanoreservoirs for targeted drug delivery. Angew Chem Int Ed 2011;50:640–643. 17. Hu Y, Cai KY, Luo Z, Jandt KD. Layer-by-layer assembly of bestradiol loaded mesoporous silica nanoparticles on titanium substrates and its implication for bone homeostasis. Adv Mater 2010; 22:4146–4150. 18. Luo Z, Cai KY, Hu Y, Zhang BL, Xu DW. Cell-specific intracellular anticancer drug delivery from mesoporous silica nanoparticles with pH-sensitivity. Adv Healthcare Mater 2012;1:321–325. 19. Zhao Y, Trewyn BG, Slowing II, Lin VS. Mesoporous silica nanoparticle-based double drug delivery system for glucose-
JOURNAL OF BIOMEDICAL MATERIALS RESEARCH A | MONTH 2013 VOL 00A, ISSUE 00
13
20.
21.
22.
23.
24.
25. 26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36. 37.
38.
39.
14
responsive controlled release of insulin and cyclic AMP. J Am Chem Soc 2009;131:8398–8400. Huang XL, Li LL, Liu TL, Hao NJ, Liu HY, Chen D, Tang FQ. The shape effect of mesoporous silica nanoparticles on biodistribution, clearance, and biocompatibility in vivo. ACS Nano 2011;5: 5390–5399. Zhao F, Zhao Y, Liu Y, Chang X, Chen C, Zhao Y. Cellular uptake, intracellular trafficking, and cytotoxicity of nanomaterials. Small 2011;7:1322–1337. Zhang Y, Hu L, Yu D, Gao C. Influence of silica particle internalization on adhesion and migration of human dermal fibroblasts. Biomaterials 2010;31:8465–8474. Franz S, Rammelt S, Scharnweber D, Simon JC. Immune responses to implants—A review of the implications for the design of immunomodulatory biomaterials. Biomaterials 2011;32: 6692–6709. Mao Z, Zhou X, Gao C. Influence of structure and properties of colloidal biomaterials on cellular uptake and cell functions. Biomater Sci 2013;1:896–911. Hu L, Mao Z, Gao C. Colloidal particles for cellular uptake and delivery. J Mater Chem 2009;19:3108–3115. Hartono SB, Gu WY, Kleitz F, Liu J, He LZ, Middelberg APJ, Yu CZ, LU GQ, Qiao SZ. Poly-l-lysine functionalized large pore cubic mesostructured silica nanoparticles as biocompatible carriers for gene delivery. ACS Nano 2012;6:2104–2117. Schlossbauer A, Kecht J, Bein T. Biotin-avidin as a proteaseresponsive cap system for controlled guest release from colloidal mesoporous silica. Angew Chem Int Ed 2009;48:3092–3095. n ~ ez R, Maquieira A, Marcos Climent E, Bernardos A, Martınez-Ma MD, Pastor-Navarro N, Puchades R, Sancenon F, Soto J, Amoros P. Controlled delivery systems using antibody-capped mesoporous nanocontainers. J Am Chem Soc 2009;131:4075–4080. Schlossbauer A, Warncke S, Gramlich PM, Kecht J, Manetto A, Carell T, Bein T. A programmable DNA-based molecular valve for colloidal mesoporous silica. Angew Chem Int Ed 2010;49:4734– 4737. Muszanska AK, Busscher HJ, Herrmann A, van der Mei HC, Norde W. Pluronic-lysozyme conjugates as anti-adhesive and antibacterial biofunctional polymers for surface coating. Biomaterials 2011; 32:6333–6341. € cker C, Tron K, Lunov O, Syrovets T, B€ uchele B, Jiang X, Ro Nienhaus GU, Walther P, Mailander V, Landfester K, Simmet T. The effect of carboxydextran-coated superparamagnetic iron oxide nanoparticles on C-jun N-terminal kinase-mediated apoptosis in human macrophages. Biomaterials 2010;31:5063–5071. Seo YC, Choi WY, Lee CG, Cha SW, Kim YO, Kim JC, Drummen GPC, Lee HY. Enhanced immunomodulatory activity of gelatinencapsulated rubus coreanus miquel nanoparticles. Int J Mol Sci 2011;12:9031–9056. Hayman AR, Cox TM. Purple acid phosphatase of the human macrophage and osteoclast-characterization, molecular properties, and crystallization of the recombinant di-iron-oxo protein secreted by baculovirus-infected insect cells. J Biol Chem 1994; 269:1294–1300. € ber W, Fink A, Bohn E. Controlled growth of monodisperse Sto silica spheres in the micron size range. J Colloid Interface Sci 1968;26:62–69. € tting C. Surface€ ldenhaupt J, Gerwert K, Ko Pinkerneil P, Gu attached polyhistidine-tag proteins characterized by FTIR difference spectroscopy. Chem Phys Chem 2012;13:2649–2653. Barth A. The infrared absorption of amino acid side chains. Prog Biophys Mol Biol 2000;74:141–173. Kong J, Yu S. Fourier transform infrared spectroscopic analysis of protein secondary structures. Acta Biochim Biophys Sin 2007;39: 549–559. Zhang Q, Liu F, Nguyen KT, Ma X, Wang XJ, Xing BG, Zhao YL. Multifunctional mesoporous silica nanoparticles for cancertargeted and controlled drug delivery. Adv Fun Mater 2012;22: 5144–5156. Creager MS, Jenkins JE, Thagard-Yeaman LA, Brooks AE, Jones JA, Lewis RV, Holland GP, Yarger JL. Solid-state NMR comparison of various spiders’ dragline silk fiber. Biomacromolecules 2010;11:2039–2043.
LUO ET AL.
40. Folkman J, Moscona A. Role of cell shape in growth control. Nature 1978; 273:345–349. 41. Lee S, Choi J, Shin S, Im YM, Song J, Kang SS, Nam TH, Webster TJ, Kim SH, Khang D. Analysis on migration and activation of live macrophages on transparent flat and nanostructured titanium. Acta Biomater 2011;7:2337–2344. 42. Yue H, Wei W, Yue ZG, Lv PP, Wang LY, Ma GH, Su ZG. Particle size affects the cellular response in macrophages. Eur J Pharm Sci 2010;41:650–657. 43. Chiou WF, Shum AYC, Peng CH, Chen CF, Chou CJ. Piperlactam S suppresses macrophage migration by impeding F-actin polymerization and filopodia extension. Eur J Pharmacol 2003;458:217–225. 44. Cai KY, Hou YH, Hu Y, Zhao L, Luo Z, Shi Y, Lai M, Yang WH, Liu P. Correlation of the cytotoxicity of TiO2 nanoparticles with different particle sizes on a sub-200-nm scale. Small 2011;7:3026–3031. 45. Kumari S, Mg S, Mayor S. Endocytosis unplugged: multiple ways to enter the cell. Cell Res 2010;20:256–275. 46. Luo Z, Cai KY, Hu Y, Li JH, Ding XW, Zhang BL, Xu DW, Yang WH, Liu P. Redox-responsive molecular nanoreservoirs for controlled intracellular anticancer drug delivery based on magnetic nanoparticles. Adv Mater 2012;24:431–435. 47. Orr G, Panther DJ, Phillips JL, Tarasevich BJ, Dohnalkova A, Hu DH, Teeguarden JG, Poundsl JG. Submicrometer and nanoscale inorganic particles exploit the actin machinery to be propelled along microvilli-like structures into alveolar cells. ACS Nano 2007;1:463–475. 48. Chung TH, Wu SH, Yao M, Lu CW, Lin YS, Hung Y, Mou CY, Chen YC, Huang DM. The effect of surface charge on the uptake and biological function of mesoporous silica nanoparticles in 3T3L1 cells and human mesenchymal stem cells. Biomaterials 2007; 28:2959–2966. 49. Hirst SM, Karakoti AS, Tyler RD, Sriranganathan N, Seal S, Reilly CM. Anti-inflammatory properties of cerium oxide nanoparticles. Small 2009;5:2848–2856. 50. Park MV, Neigh AM, Vermeulen JP, de la Fonteyne LJ, Verharen HW, JJ, van Loveren H, de Jong WH. The effect of particle size on Briede the cytotoxicity, inflammation, developmental toxicity and genotoxicity of silver nanoparticles. Biomaterials 2011;32:9810–9817. 51. Jeong YS, Oh WK, Kim S, Jang J. Cellular uptake, cytotoxicity, and ROS generation with silica/conducting polymer core/shell nanospheres. Biomaterials 2011;32:7217–7225. 52. Morishige T, Yoshioka Y, Inakura H, Tanabe A, Yao X, Narimatsu S, Monobe Y, Imazawa T, Tsunoda S, Tsutsumi Y, Mukai Y, Okada N, Nakagawa S. The effect of surface modification of amorphous silica particles on NLRP3 inflammasome mediated IL1beta production, ROS production and endosomal rupture. Biomaterials 2010;31:6833–6842. 53. Fujisawa A, Kambe N, Saito M, Nishikomori R, Tanizaki H, Kanazawa N. Disease-associated mutations in CIAS1 induce cathepsin B-dependent rapid cell death of human THP-1 monocytic cells. Blood 2007;109:2903–2911. 54. Willingham SB, Bergstralh DT, O’Connor W, Morrison AC, Taxman DJ. Microbial pathogen-induced necrotic cell death mediated by the inflammasome components CIAS1/cryopyrin/NLRP3 and ASC. Cell Host Microbe 2007;2:147–159. 55. Park EJ, Park K. Oxidative stress and pro-inflammatory responses induced by silica nanoparticles in vivo and in vitro. Toxicol Lett 2009;184:18–25. 56. http://www.nursingtimes.net/nursing-practice/clinical-zones/haemat ology/routine-blood-tests/201124.article; http://www.webhealthcent re.com/HealthyLiving/lab_test_blood.aspx. 57. Darabos N, Hundric-Haspl Z, Haspl M, Markotic A, Darabos A, Moser C. Correlation between synovial fluid and serum IL-1b levels after ACL surgery—Preliminary report. Int Orthop 2009;33:413–418. 58. Sun A, Wang YP, Chia JS, Liu BY, Chiang CP. Treatment with levamisole and colchicine can result in a significant reduction of IL-6, IL-8 or TNF-a level in patients with mucocutaneous type of Behcet’s disease. J Oral Pathol Med 2009;38:401–405. 59. Park JH, Gu L, von Maltzahn G, Ruoslahti E, Bhatia SN, Sailor MJ. Biodegradable luminescent porous silicon nanoparticles for in vivo applications. Nat Mater 2009;8:331–336. 60. Mitchell KKP, Liberman A, Kummel AC, Trogler WC. Iron(III)doped, silica nanoshells: A biodegradable form of silica. J Am Chem Soc 2012;134: 13997–14003.
MSN-BASED DRUG DELIVERY SYSTEMS WITH REDUCED IMMUNOTOXICITY
Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400044, People’s Republic of China 2 College of Material Science and Engineering, Chongqing University, Chongqing 400044, People’s Republic of China Received 14 October 2013; revised 19 November 2013; accepted 26 November 2013 Published online 00 Month 2013 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.a.35049 Abstract: Mesoporous silica nanoparticles (MSNs) present themselves as one of the most promising nano-carriers for drug delivery. To reduce their immunotoxicities, in this study, natural proteins of gelatin (Gel), bovine serum albumin (BSA), and lysozyme (Lys) were employed as end-caps of MSNs by using succinic anhydride as an intermediate linker, thus leading to fabrication of MSNs/protein nanocomposites, respectively. Furthermore, combined techniques of SEM, TEM, FTIR, and zeta potential instruments were utilized to monitor the construction processes of MSNs/protein nanocomposites, respectively. Finally, the immunotoxicities of those nanocomposites to macrophage cells (RAW264.7 cells) were investigated in detail, i.e., cell morphology, cell viability, nitric oxide (NO) production, reactive oxygen species (ROS),
and acid phosphatase activity (ACP) as well as inflammation cytokine expressions (tumor necrosis factor-a and interleukin1b). All results suggest that macrophages were activated after uptaking nanoparticles of SiO2 and MSNs, which subsequently induced severe inflammation responses in vitro. In contrast, the inflammation responses of MSNs nanocomposites were reduced dramatically after end-capping with those natural proteins. Overall, this study accumulates knowledge for the development of MSNs-based drug delivery systems C 2013 Wiley Periodicals, Inc. J with reduced immunotoxicity. V Biomed Mater Res Part A: 00A:000–000, 2013.
Key Words: mesoporous silica nanoparticles, immunotoxicity, inflammation responses, macrophages, natural proteins
How to cite this article: Luo Z, Hu Y, Xin R, Zhang B, Li J, Ding X, Hou Y, Yang L, Cai K. 2013. Surface functionalized mesoporous silica nanoparticles with natural proteins for reduced immunotoxicity. J Biomed Mater Res Part A 2013:00A:000–000.
INTRODUCTION
Recently, the convergence of nanotechnology, materials, biology, and medicine accelerates the development of nanomedicine in the clinical applications, which displays great potential for human healthcare, particularly in cancer therapy.1–4 Although an increasing number of powerful and targeted therapeutic treatments (e.g., nano-scale drug delivery systems) and diagnostic techniques (e.g., biomedical imaging agents) have been exploited, it still might be first interacted with immune systems of a host in vivo. Therefore, it is necessary to reveal the toxicity of nano-scale formulations to immune systems,5 which was currently defined as a concept of “immunotoxicity.”6 Actually, the immune systems are the natural barriers to the allogeneic materials in vivo.5 In clinical application, once a nano-scale drug delivery system is administered, it would delicately remain in the host’s body and thus inevitably interact with the immune systems.
Mesoporous silica nanoparticles (MSNs) were developed to be one of the most promising nano-carriers for anticancer drug delivery, mainly owing to their good biocompatibility, large surfaces areas, and uniform mesoporous structures.7–9 Previously, various types of MSNs-based controlled drug delivery systems were exploited, including inorganic nanoparticles/MSNs systems,10–12 molecular switches/MSNs systems,13–15 and biomacromolecules/MSNs systems, etc.16–19 However, both inorganic nanoparticles/ MSNs systems and molecular machines/systems might suffer from cytotoxicity and adverse biological interactions since they were xenobiotics.20,21 The cell viability and functions (e.g., cell adhesion and migration) were greatly affected after uptaking of nanoparticles.22 In contrast, biomacromolecules/MSNs controlled-drug delivery systems presented themselves as one of the most promising candidates for future clinic application since biomacromolecules had good biocompatibility and limited immune reactivity.23
Additional Supporting Information may be found in the online version of this article. *These authors contributed equally to this work. Correspondence to: K. Cai; e-mail: [email protected] Contract grant sponsor: Natural Science Foundation of China; contract grant number: 21274169, 31200712 Contract grant sponsor: Fundamental Research Funds for the Central Universities; contract grant numbers: CQDXWL-2013-Z002, CDJZR 10238801, “111” project B06023
C 2013 WILEY PERIODICALS, INC. V
1
FIGURE 1. Schematic illustration of macrophage inflammation responses to silica nanoparticles, mesoporous silica nanoparticles, and MSNs/ protein nanocomposites. Endosome refers to a membrane-bound compartment within cells, acting as a reservoir for transferring substances from plasma membrane to lysosome. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Previous studies revealed that cytotoxicities of nanomateirals and their effects on cellular functions highly depended on their physiochemical properties and chemical compositions.24,25 As end-capping agents, biomacromolecules were generally immobilized onto the exterior surfaces of mesoporous silica nanoparticles (MSNs), which played an important role in mediating the interactions between MSNs/biomacromolecules nanocomposites and cells/tissues.26 During the past decades, a diversity of biomacromolecules was exploited as efficient end-capping agents of MSNs (such as biotin-avidin,27 antibody,28 insulin,19 and DNA29). Besides, our group also constructed a series of pH- and redox-responsive MSNs/biomacromolecules smart drug delivery systems by using gelatin,16 bovine serum albumin (BSA),17 and collagen18 as end-capping agents. Those systems were proved to be efficient carriers for intracellular drug delivery via targeted cellular uptake or specific endocytosis mechanisms.16–19 Nevertheless, the immunotoxicity of those systems were not investigated. To guarantee the successful clinic applications of MSNs-based drug delivery systems, potential effects of those nano-scale systems on immune systems of a host definitely should be evaluated. In immune systems, macrophages play a key role in the regulation of “foreign body reactions,” including inflammation responses.30 Thus, it is urgently necessary to evaluate the immunotoxicity of MSNs-based controlled drug delivery systems to macrophages except for the routine cytocompatibility assays. Previously, Lunov et al. demonstrated that carboxydextran coating of magnetic nanoparticles could reduce the apoptosis of macrophages.31 In another study, Seo et al.
2
LUO ET AL.
reported that gelatin molecules could improve the activities of immune cells with enhanced immunomodulatory property.32 Those studies implied that surface engineering of nanoparticles with appropriate components could efficiently reduce their immunotoxicity, in turn optimizing them as efficient carriers for drug delivery. Herein, gelatin (Gel), bovine serum albumin (BSA), and lysozyme (Lys) were immobilized onto the exterior surfaces of MSNs via succinic anhydride as an intermediate linker to fabricate MSNs/protein nano-composites systems, respectively. We hypothesized that the natural proteins end-capping of MSNs could efficiently reduce their inflammation responses to macrophages (shown in Fig. 1). It is well known that gelatin, a derivative of the extracellular matrix component of collagen, was widely used in biomedical fields.16 BSA is a serum albumin protein that interacts with the biological functions of a host.18 Meanwhile, lysozyme is an abundant component of natural products,30 such as egg, milk, etc. It has been widely used in the anti-inflammation fields as well. Thus, we hypothesized that macrophages would be activated acutely when exposed to either bare silica nanoparticles or MSNs, leading to severe inflammation responses and dysfunction of the immune system (Fig. 1). MATERIALS AND METHODS
Materials Tetraethylorthosilicate (TEOS), N-hydroxysuccinimide (NHS), dimethyl sulfoxide (DMSO), 3-(4,5)-dimethylthiahiazo(-z-y1)3,5-di-phenytetrazoliumromide (MTT), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide-HCl (EDC), lysozyme (Lys), gelatin (Gel), and bovine serum albumin (BSA) were
MSN-BASED DRUG DELIVERY SYSTEMS WITH REDUCED IMMUNOTOXICITY
ORIGINAL ARTICLE
purchased from Sigma–Aldrich Co. (Beijing, China). Phalloidin and 40 ,6-diamidino-2-phenylindole (DAPI) were obtained from Invitrogen Co. (Beijing, China). N-cetyltrimethylammonium bromide (CTAB) and succinic anhydride (SA) were provided by Alfa Aesar (Tianjin, China). Other chemicals were supplied by Oriental Chemicals Co. Ltd. (Chongqing, China). Synthesis of silica nanoparticles and mesoporous silica nanoparticles (MSNs) Silica nanoparticles were synthesized according to a previous study.31 Briefly, tetraethoxysilane (TEOS, 10 mL) and ammonium hydroxide (10 mL) were dissolved into ethanol/ water (500 mL, v/v 5 7:1) mixture solution with gentle stirring for 2 h. After filtration and configuration, the white product was rinsed with ethanol and distilled water for six times each. Finally, the sample was dried at 60 C for 6 h to get silica nanoparticles. MSNs were synthesized according to previous studies.15–18 Briefly, reagents of N-cetyltrimethyl-ammonium bromide (CTAB, 1.0 g) and sodium hydroxide (0.28 g) were dissolved in distilled water (480 mL) and heated to 80 C. Then, TEOS (5 g) was slowly added to the mixture solution within 2 h under stirring until a white precipitant was formed. After filtration and centrifugation, the resulting product was rinsed with excessive distilled water and methanol for six times each. Finally, the sample was dried at 60 C for 6 h to get MSNs. Amination of MSNs with 3-aminopropyl trimethoxysilane (APTS-MSNs) To prepare amino-functionalized MSNs, the as-synthesized MSNs (1.0 g) were dispersed in anhydrous toluene (80 mL) containing 3-aminopropyl trimethoxysilane (APTS, 0.75 mL) under vigorous stirring. Then, the mixture was re-fluxed gently for 24 h. The resulting products were filtered and washed with double-distilled water and methanol each for five times. Then, samples were dried at 50 C under high vacuum ( MSNs-BSA > MSNs-Gel > MSNsLys (Fig. 10). The results further suggest that macrophages were severely activated by bare SiO2 nanoparticles or MSNs.
FIGURE 8. Cell viability assay. RAW264.7 cells were co-cultured with SiO2, MSNs, MSNs-Gel, MSNs-BSA, and MSNs-Lys nanoparticles for 24 h and 48 h, respectively (n 5 6).
FIGURE 9. Production of ROS in RAW264.7 macrophages co-cultured with SiO2, MSNs, MSNs-Gel, MSNs-BSA, and MSNs-Lys nanoparticles for 24 h and 48 h, respectively (n 5 6).
JOURNAL OF BIOMEDICAL MATERIALS RESEARCH A | MONTH 2013 VOL 00A, ISSUE 00
11
FIGURE 10. NO production of RAW264.7 macrophages co-cultured with SiO2, MSNs, MSNs-Gel, MSNs-BSA, and MSNs-Lys nanoparticles for 24 h and 48 h, respectively (n 5 6).
However, surface conjugations of gelatin, BSA, and lysozyme to MSNs were beneficial for reducing the NO production of macrophages.
Inflammation cytokines secretions. Finally, we measured the expression levels of inflammation factors of interleukin1b (IL-1b) and tumor necrosis factor a (TNF-a) secreted by macrophages after incubation with different nanoparticles for 24 h and 48 h, respectively. It is well known that both IL-1b and TNF-a are the most important markers for indicating the inflammation responses of cells and/or tissues in vivo.49–52,55 In this study, we used an enzyme linked immunosorbent assay (ELISA) kit to monitor the amount of IL-1b and TNF-a produced by macrophages. There was no clear difference in the expression levels of both IL-1b and TNF-a between all nanoparticles groups after incubation for 24 h. However, the groups treated with silica nanoparticles and MSNs displayed much higher expression of both IL-1b and TNF-a in RAW264.7 cells when the incubation time extended to 48 h (Fig. 12). RAW264.7 cells incubated with SiO2 nanoparticles and MSNs displayed 5.9-fold and 4.2-fold IL-1b production compared to that of the TCPS group [Fig. 12(A)]. On the other hand, RAW264.7 cells treated with silica nanoparticles and MSNs showed 4.9-fold and 3.8-fold of TNF-a expression than that of TCPS [Fig. 12(B)]. However, the expression levels of both IL-1b and TNF-a in RAW264.7 cells treated with MSNs/protein nanoparticles
Acid phosphatase activity. Fourth, we evaluated the acid phosphatase (ACP) activity of macrophages after incubation with various nanoparticles for 24 h and 48 h, respectively. ACP is one of the important enzymes that reflect inflammation responses in vivo.33 The ACP activity was directly indicated by the production of p-nitrophenol in this study. Silica nanoparticles and MSNs induced significantly higher (p < 0.01) ACP activities than other groups after incubation for 24 h. The average ACP activities of macrophages decreased in the order of MSNs > SiO2 > MSNs-BSA > MSNsLys > MSNs-Gel (Fig. 11). The same trend was also found after incubation for 48 h. The results imply that the immunotoxicity of macrophages were reduced after immobilization of gelatin, BSA, and lysozyme onto the surface of MSNs, respectively.
FIGURE 11. Acid phosphatase activity of RAW264.7 macrophages cocultured with SiO2, MSNs, MSNs-Gel, MSNs-BSA, and MSNs-Lys nanoparticles for 24 h and 48 h, respectively (n 5 6).
12
LUO ET AL.
FIGURE 12. Expressions of (A) IL-1b and (B) TNF-a of RAW264.7 macrophages co-cultured with SiO2, MSNs, MSNs-Gel, MSNs-BSA, and MSNs-Lys nanoparticles for 24 h and 48 h, respectively (n 5 6).
MSN-BASED DRUG DELIVERY SYSTEMS WITH REDUCED IMMUNOTOXICITY
ORIGINAL ARTICLE
were much lower than those of bare silica nanoparticles and MSNs. Compared to TCPS, MSNs-Gel, MSNs-BSA, and MSNs-Lys samples only shows 1.7-fold, 1.2-fold, and 1.7-fold of IL-1b expression in RAW264.7 cells [Fig. 12(A)] whereas 2.8-fold, 2.4-fold, and 2.9-fold of TNF expression [Fig. 12(B)]. The results further suggest that macrophages were severely activated by bare silica nanoparticles or MSNs. The expression levels of inflammation factors of MSNs were reduced apparently after the immobilization of gelatin, BSA, and lysozyme. Generally, there are approximately 4–10 3 103 56 inflammation-related cells in 1 mL of human blood. The common values of IL-1b and TNF-a in the serum of a normal human are 10 6 4.3 pg/mL and 3.8 6 0.2 pg/mL, respectively.57,58 By conversion, we could get an estimated result that the normal expressions of IL-1b and TNF-a are lower than 1000 6 430 pg/mL/106 cells and 380 6 20 pg/mL/106cells, respectively. After end-capping with natural proteins, it was interesting to note that the expression of TNF-a was reduced from 482.06 6 59.52 pg/mL/106 cells for bare MSNs (higher than normal level) to 352.35 6 168.22 pg/mL/106 cells for MSNs-Gel, 310.85 6 64.83 pg/mL/106 cells for MSNs-BSA, and 370.58 6 73.6 pg/mL/106 cells for MSNs-Lys nanocomposites, respectively. All those concentrations were lower than the reference value of normal human (380 6 20 pg/mL/106cells). Although the expression of IL-1b for bare MSNs (96.32 6 3.07 pg/mL/106 cells) was under the reference value of normal human, different proteins end-capping still reduced their IL-1b expressions for MSNs-Gel, MSNs-BSA, and MSNs-Lys nanocomposites. The results imply that systematic investigations should be performed in the future to clarify its influence on other preinflammatory factors, such as IL-2, IL-6, IL-8, IL-9, etc. Although inflammation reaction assay with macrophages is essential in the immunotoxicity evaluation of nanotechnology-based delivery systems before their clinical application, it would coordinate with other assessments (compatibility tests of nanoparticles with blood and inflammation reaction assay in vivo) to get a comprehensive understanding of their feasibility. Taken together, we confirmed our hypothesis that natural proteins end-capping of MSNs could reduce their inflammation responses to macrophages. However, the feasibility of those MSNs’ drug delivery systems should be further evaluated in vivo. On the other hand, to overcome the disadvantage of the un-biodegradable property of MSNs, it is essentially urgent and necessary to exploit biodegradable MSNs for their clinical applications in the future.59,60
CONCLUSIONS
In summary, the immunotoxicity of silica nanoparticles, MSNs, and MSNs/proteins nanocomposites to macrophages (RAW.246.7 cells) were investigated in detail. We found that macrophages were severely activated by bare silica nanoparticles or MSNs. The production/expression levels of NO, ROS, acid phosphatase, and inflammation factors of IL-1b and TNF-a by macrophages in turn led to negative effects on cell behaviors. However, the inflammation responses of MSNs were significantly reduced by employing natural pro-
teins (gelatin, bovine serum protein, and lysozyme) as endcapping agents. This finding provides new insight into the design of MSNs/protein drug delivery systems with reduced inflammation responses (immunity) for potential clinical applications, such as cancer therapy. REFERENCES 1. Peer D, Karp JM, Hong S, Farokhzad OC, Margalit R, Langer R. Nanocarriers as an emerging platform for cancer therapy. Nat Nanotechnol 2007;2:751–760. 2. Wolfgang JP. Complex colloidal assembly. Science 2011;334: 1359–1360. 3. Chen ZL, Li Y, Liu WW, Zhang DZ, Zhao YY, Yuan B, Jiang XY. Patterning mammalian cells for modeling three types of naturally occurring cell-cell interactions. Angew Chem Int Ed 2009;48:8303– 8305. 4. Wang XJ, Wang C, Cheng L, Lee ST, Liu Z. Noble metal coated single-walled carbon nanotubes for applications in surface enhanced Raman scattering imaging and photothermal therapy. J Am Chem Soc 2012;134:7414–7422. 5. Dobrovolskaia MA, Germolec DR, Weaver JL. Evaluation of nanoparticle immunotoxicity. Nat Nanotechnol 2009;4:411–414. 6. Dobrovolskaia MA, McNeil SE. Immunological properties of engineered nanomaterials. Nat Nanotechnol 2007;2:469–478. 7. Li LL, Tang FQ, Liu HY, Liu TL, Hao NJ, Chen D, Teng X, He JQ. In vivo delivery of silica nanorattle encapsulated docetaxel for liver cancer therapy with low toxicity and high efficacy. ACS Nano 2010;4:6874–6882. 8. Chen Y, Chu C, Zhou Y, Ru YF, Chen HR, Chen F, He QJ, Zhang YL, Zhang LL, Shi JL. Reversible pore-structure evolution in hollow silica nanocapsules: Large pores for siRNA delivery and nanoparticle collecting. Small 2011;7:2935–2944. 9. Ambrogio MW, Thomas CR, Zhao YL, Zink JI, Stoddart JF. Mechanized silica nanoparticles: A new frontier in theranostic nanomedicine. Acc Chem Res 2011;44:903–913. 10. Lai CY, Trewyn BG, Jeftinija DM, Jeftinija K, Xu S, Jeftinija S, Lin VYS. A mesoporous silica nanosphere-based carrier system with chemically removable CdS nanoparticle caps for stimuliresponsive controlled release of neurotransmitters and drug molecules. J Am Chem Soc 2003;125:4451–4459. 11. Sun X, Zhao Y, Lin VS, Slowing II, Trewyn BG. Luciferase and luciferin co-immobilized mesoporous silica nanoparticle materials for intracellular biocatalysis. J Am Chem Soc 2011;133:18554–18557. 12. Ma M, Chen HR, Chen Y, Wang X, Chen F, Cui XZ, Shi JL. Au capped magnetic core/mesoporous silica shell nanoparticles for combined photothermo-/chemo-therapy and multimodal imaging. Biomaterials 2012;33:989–998. 13. Yan H, Teh C, Sreejith S, Zhu LL, Kwok A, Fang WQ, Ma X, Nguyen KT, Korzh V, Zhao YL. Functional mesoporous silica nanoparticles for photothermal—Controlled drug delivery in vivo. Angew Chem Int Ed 2012;51:8373–8377. 14. Zhao YL, Li Z, Kabehie S, Botros YY, Stoddart JF, Zink JI. pHoperated nanopistons on the surfaces of mesoporous silica nanoparticles. J Am Chem Soc 2010;132:13016–13025. 15. Meng H, Xue M, Xia T, Zhao YL, Tamanoi F, Stoddart JF, Zink JI, Nel AE. Autonomous in vitro anticancer drug release from mesoporous silica nanoparticles derivatized with pH-sensitive supramolecular nanovalves. J Am Chem Soc 2010;132:12690–12697. 16. Luo Z, Cai KY, Hu Y, Zhao L, Liu P, Duan L, Yang WH. Mesoporous silica nanoparticles end-capped with collagen: Redoxresponsive nanoreservoirs for targeted drug delivery. Angew Chem Int Ed 2011;50:640–643. 17. Hu Y, Cai KY, Luo Z, Jandt KD. Layer-by-layer assembly of bestradiol loaded mesoporous silica nanoparticles on titanium substrates and its implication for bone homeostasis. Adv Mater 2010; 22:4146–4150. 18. Luo Z, Cai KY, Hu Y, Zhang BL, Xu DW. Cell-specific intracellular anticancer drug delivery from mesoporous silica nanoparticles with pH-sensitivity. Adv Healthcare Mater 2012;1:321–325. 19. Zhao Y, Trewyn BG, Slowing II, Lin VS. Mesoporous silica nanoparticle-based double drug delivery system for glucose-
JOURNAL OF BIOMEDICAL MATERIALS RESEARCH A | MONTH 2013 VOL 00A, ISSUE 00
13
20.
21.
22.
23.
24.
25. 26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36. 37.
38.
39.
14
responsive controlled release of insulin and cyclic AMP. J Am Chem Soc 2009;131:8398–8400. Huang XL, Li LL, Liu TL, Hao NJ, Liu HY, Chen D, Tang FQ. The shape effect of mesoporous silica nanoparticles on biodistribution, clearance, and biocompatibility in vivo. ACS Nano 2011;5: 5390–5399. Zhao F, Zhao Y, Liu Y, Chang X, Chen C, Zhao Y. Cellular uptake, intracellular trafficking, and cytotoxicity of nanomaterials. Small 2011;7:1322–1337. Zhang Y, Hu L, Yu D, Gao C. Influence of silica particle internalization on adhesion and migration of human dermal fibroblasts. Biomaterials 2010;31:8465–8474. Franz S, Rammelt S, Scharnweber D, Simon JC. Immune responses to implants—A review of the implications for the design of immunomodulatory biomaterials. Biomaterials 2011;32: 6692–6709. Mao Z, Zhou X, Gao C. Influence of structure and properties of colloidal biomaterials on cellular uptake and cell functions. Biomater Sci 2013;1:896–911. Hu L, Mao Z, Gao C. Colloidal particles for cellular uptake and delivery. J Mater Chem 2009;19:3108–3115. Hartono SB, Gu WY, Kleitz F, Liu J, He LZ, Middelberg APJ, Yu CZ, LU GQ, Qiao SZ. Poly-l-lysine functionalized large pore cubic mesostructured silica nanoparticles as biocompatible carriers for gene delivery. ACS Nano 2012;6:2104–2117. Schlossbauer A, Kecht J, Bein T. Biotin-avidin as a proteaseresponsive cap system for controlled guest release from colloidal mesoporous silica. Angew Chem Int Ed 2009;48:3092–3095. n ~ ez R, Maquieira A, Marcos Climent E, Bernardos A, Martınez-Ma MD, Pastor-Navarro N, Puchades R, Sancenon F, Soto J, Amoros P. Controlled delivery systems using antibody-capped mesoporous nanocontainers. J Am Chem Soc 2009;131:4075–4080. Schlossbauer A, Warncke S, Gramlich PM, Kecht J, Manetto A, Carell T, Bein T. A programmable DNA-based molecular valve for colloidal mesoporous silica. Angew Chem Int Ed 2010;49:4734– 4737. Muszanska AK, Busscher HJ, Herrmann A, van der Mei HC, Norde W. Pluronic-lysozyme conjugates as anti-adhesive and antibacterial biofunctional polymers for surface coating. Biomaterials 2011; 32:6333–6341. € cker C, Tron K, Lunov O, Syrovets T, B€ uchele B, Jiang X, Ro Nienhaus GU, Walther P, Mailander V, Landfester K, Simmet T. The effect of carboxydextran-coated superparamagnetic iron oxide nanoparticles on C-jun N-terminal kinase-mediated apoptosis in human macrophages. Biomaterials 2010;31:5063–5071. Seo YC, Choi WY, Lee CG, Cha SW, Kim YO, Kim JC, Drummen GPC, Lee HY. Enhanced immunomodulatory activity of gelatinencapsulated rubus coreanus miquel nanoparticles. Int J Mol Sci 2011;12:9031–9056. Hayman AR, Cox TM. Purple acid phosphatase of the human macrophage and osteoclast-characterization, molecular properties, and crystallization of the recombinant di-iron-oxo protein secreted by baculovirus-infected insect cells. J Biol Chem 1994; 269:1294–1300. € ber W, Fink A, Bohn E. Controlled growth of monodisperse Sto silica spheres in the micron size range. J Colloid Interface Sci 1968;26:62–69. € tting C. Surface€ ldenhaupt J, Gerwert K, Ko Pinkerneil P, Gu attached polyhistidine-tag proteins characterized by FTIR difference spectroscopy. Chem Phys Chem 2012;13:2649–2653. Barth A. The infrared absorption of amino acid side chains. Prog Biophys Mol Biol 2000;74:141–173. Kong J, Yu S. Fourier transform infrared spectroscopic analysis of protein secondary structures. Acta Biochim Biophys Sin 2007;39: 549–559. Zhang Q, Liu F, Nguyen KT, Ma X, Wang XJ, Xing BG, Zhao YL. Multifunctional mesoporous silica nanoparticles for cancertargeted and controlled drug delivery. Adv Fun Mater 2012;22: 5144–5156. Creager MS, Jenkins JE, Thagard-Yeaman LA, Brooks AE, Jones JA, Lewis RV, Holland GP, Yarger JL. Solid-state NMR comparison of various spiders’ dragline silk fiber. Biomacromolecules 2010;11:2039–2043.
LUO ET AL.
40. Folkman J, Moscona A. Role of cell shape in growth control. Nature 1978; 273:345–349. 41. Lee S, Choi J, Shin S, Im YM, Song J, Kang SS, Nam TH, Webster TJ, Kim SH, Khang D. Analysis on migration and activation of live macrophages on transparent flat and nanostructured titanium. Acta Biomater 2011;7:2337–2344. 42. Yue H, Wei W, Yue ZG, Lv PP, Wang LY, Ma GH, Su ZG. Particle size affects the cellular response in macrophages. Eur J Pharm Sci 2010;41:650–657. 43. Chiou WF, Shum AYC, Peng CH, Chen CF, Chou CJ. Piperlactam S suppresses macrophage migration by impeding F-actin polymerization and filopodia extension. Eur J Pharmacol 2003;458:217–225. 44. Cai KY, Hou YH, Hu Y, Zhao L, Luo Z, Shi Y, Lai M, Yang WH, Liu P. Correlation of the cytotoxicity of TiO2 nanoparticles with different particle sizes on a sub-200-nm scale. Small 2011;7:3026–3031. 45. Kumari S, Mg S, Mayor S. Endocytosis unplugged: multiple ways to enter the cell. Cell Res 2010;20:256–275. 46. Luo Z, Cai KY, Hu Y, Li JH, Ding XW, Zhang BL, Xu DW, Yang WH, Liu P. Redox-responsive molecular nanoreservoirs for controlled intracellular anticancer drug delivery based on magnetic nanoparticles. Adv Mater 2012;24:431–435. 47. Orr G, Panther DJ, Phillips JL, Tarasevich BJ, Dohnalkova A, Hu DH, Teeguarden JG, Poundsl JG. Submicrometer and nanoscale inorganic particles exploit the actin machinery to be propelled along microvilli-like structures into alveolar cells. ACS Nano 2007;1:463–475. 48. Chung TH, Wu SH, Yao M, Lu CW, Lin YS, Hung Y, Mou CY, Chen YC, Huang DM. The effect of surface charge on the uptake and biological function of mesoporous silica nanoparticles in 3T3L1 cells and human mesenchymal stem cells. Biomaterials 2007; 28:2959–2966. 49. Hirst SM, Karakoti AS, Tyler RD, Sriranganathan N, Seal S, Reilly CM. Anti-inflammatory properties of cerium oxide nanoparticles. Small 2009;5:2848–2856. 50. Park MV, Neigh AM, Vermeulen JP, de la Fonteyne LJ, Verharen HW, JJ, van Loveren H, de Jong WH. The effect of particle size on Briede the cytotoxicity, inflammation, developmental toxicity and genotoxicity of silver nanoparticles. Biomaterials 2011;32:9810–9817. 51. Jeong YS, Oh WK, Kim S, Jang J. Cellular uptake, cytotoxicity, and ROS generation with silica/conducting polymer core/shell nanospheres. Biomaterials 2011;32:7217–7225. 52. Morishige T, Yoshioka Y, Inakura H, Tanabe A, Yao X, Narimatsu S, Monobe Y, Imazawa T, Tsunoda S, Tsutsumi Y, Mukai Y, Okada N, Nakagawa S. The effect of surface modification of amorphous silica particles on NLRP3 inflammasome mediated IL1beta production, ROS production and endosomal rupture. Biomaterials 2010;31:6833–6842. 53. Fujisawa A, Kambe N, Saito M, Nishikomori R, Tanizaki H, Kanazawa N. Disease-associated mutations in CIAS1 induce cathepsin B-dependent rapid cell death of human THP-1 monocytic cells. Blood 2007;109:2903–2911. 54. Willingham SB, Bergstralh DT, O’Connor W, Morrison AC, Taxman DJ. Microbial pathogen-induced necrotic cell death mediated by the inflammasome components CIAS1/cryopyrin/NLRP3 and ASC. Cell Host Microbe 2007;2:147–159. 55. Park EJ, Park K. Oxidative stress and pro-inflammatory responses induced by silica nanoparticles in vivo and in vitro. Toxicol Lett 2009;184:18–25. 56. http://www.nursingtimes.net/nursing-practice/clinical-zones/haemat ology/routine-blood-tests/201124.article; http://www.webhealthcent re.com/HealthyLiving/lab_test_blood.aspx. 57. Darabos N, Hundric-Haspl Z, Haspl M, Markotic A, Darabos A, Moser C. Correlation between synovial fluid and serum IL-1b levels after ACL surgery—Preliminary report. Int Orthop 2009;33:413–418. 58. Sun A, Wang YP, Chia JS, Liu BY, Chiang CP. Treatment with levamisole and colchicine can result in a significant reduction of IL-6, IL-8 or TNF-a level in patients with mucocutaneous type of Behcet’s disease. J Oral Pathol Med 2009;38:401–405. 59. Park JH, Gu L, von Maltzahn G, Ruoslahti E, Bhatia SN, Sailor MJ. Biodegradable luminescent porous silicon nanoparticles for in vivo applications. Nat Mater 2009;8:331–336. 60. Mitchell KKP, Liberman A, Kummel AC, Trogler WC. Iron(III)doped, silica nanoshells: A biodegradable form of silica. J Am Chem Soc 2012;134: 13997–14003.
MSN-BASED DRUG DELIVERY SYSTEMS WITH REDUCED IMMUNOTOXICITY
