Article pubs.acs.org/Langmuir
A Biodegradation Study of SBA-15 Microparticles in Simulated Body Fluid and in Vivo Youngjin Choi,† Jung Eun Lee,‡ Jung Heon Lee,§ Ji Hoon Jeong,*,‡ and Jaeyun Kim*,† †
School of Chemical Engineering, Sungkyunkwan University, Suwon 440-746, Republic of Korea School of Pharmacy, Sungkyunkwan University, Suwon 440-746, Republic of Korea § School of Advanced Materials Science and Engineering, Sungkyunkwan University, Suwon 440-746, Republic of Korea ‡
S Supporting Information *
ABSTRACT: Mesoporous silica has received considerable attention as a drug delivery vehicle because of its large surface area and large pore volume for loading drugs and large biomolecules. Recently, mesoporous silica microparticles have shown potential as a three-dimensional vaccine platform for modulating dendritic cells via spontaneous assembly of microparticles in a specific region after subcutaneous injection. For further in vivo applications, the biodegradation behavior of mesoporous silica microparticles must be studied and known. Until now, most biodegradation studies have focused on mesoporous silica nanoparticles (MSNs); here, we report the biodegradation of hexagonally ordered mesoporous silica, SBA-15, with micrometer-sized lengths (∼32 μm with a high aspect ratio). The degradation of SBA-15 microparticles was investigated in simulated body fluid (SBF) and in mice by analyzing the structural change over time. SBA-15 microparticles were found to degrade in SBF and in vivo. The erosion of SBA-15 under biological conditions led to a loss of the hysteresis loop in the nitrogen adsorption/desorption isotherm and fingerprint peaks in small-angle X-ray scattering, specifically indicating a degradation of ordered mesoporous structure. Via comparison to previous results of degradation of MSNs in SBF, SBA-15 microparticles degraded faster than MCM-41 nanoparticles presumably because SBA-15 microparticles have a pore size (∼8 nm) and a pore volume larger than those of MCM-41 mesoporous silica. The surface functional groups, the residual amounts of organic templates, and the hydrothermal treatment during the synthesis could affect the rate of degradation of SBA-15. In in vivo testing, previous studies focused on the evaluation of toxicity of mesoporous silica particles in various organs. In contrast, we studied the change in the physical properties of SBA-15 microparticles depending on the duration after subcutaneous injection. The pristine SBA-15 microparticles injected into mice subcutaneously slowly degraded over time and lost ordered structure after 3 days. These findings represent the possible in vivo use of microsized mesoporous silica for drug delivery or vaccine platform after local injection.
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INTRODUCTION Mesoporous silica materials with a large surface area, a large pore volume, and controllable pore structures have been intensively studied as tools for drug delivery systems.1−13 Mesoscale pores are highly preferred in drug delivery carriers as the sizes of most biomolecules are in this range.10,13−17 To be used in in vivo application, biodegradation of mesoporous silica must be considered to prevent toxicity and unwanted accumulation in tissues. There have been several studies of degradation of mesoporous silica, but most degradation studies have focused on mesoporous silica nanoparticles (MSNs) because of their potential use in targeted intravenous delivery.15,16,18−20 Most MCM-41 type MSNs (∼100 nm in size) were tested in most biodegradation studies using diverse physiological conditions. These studies showed mesoporous silica nanoparticles degrading in distilled water, simulated body fluid (SBF), and cell culture medium over time.11,13,14,21,22 The degradation of MSNs depends on chemical and physical © 2015 American Chemical Society
properties, including pore size, surface area, composition, and surface functional groups.13,17,21−23 In contrast to systemic delivery systems using MSNs to target specific cells and tissues, local delivery systems based on subcutaneous or intramuscular injection of microsized mesoporous silica particles form a local depot that can hold high concentrations of the bioactive molecules that are slowly released to achieve the desired level of the bioactive molecules in local tissue or bloodstream.24−27 For example, SBA-15 mesoporous silica was incorporated with bioactive glass as a form of scaffold and improved the efficiency of the drug carrier.25 Lei et al. demonstrated the in vivo local delivery of therapeutic immunoglobulin to tumors by subcutaneously injecting functionalized SBA-15 microparticles loaded with Received: April 10, 2015 Revised: May 20, 2015 Published: May 26, 2015 6457
DOI: 10.1021/acs.langmuir.5b01316 Langmuir 2015, 31, 6457−6462
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75 °C for 6 h, and then the white powder was collected by filtration. The functionalized SBA-15 was washed with ethanol and distilled water and dried at 100 °C. To prepare carboxyl-functionalized SBA-15, the amine-modified SBA-15 was dispersed in 500 mL of 2 wt % succinic anhydride in DMF and the mixture solution was stirred for 24 h at room temperature. SBA-15 functionalized with carboxylic groups was filtered, washed, and dried. Preparation of Simulated Body Fluid (SBF). SBF was prepared according to the previous report.13 To prepare 1 L of SBF, 7.996 g of NaCl, 0.35 g of NaHCO3, 0.224 g of KCl, 0.228 g of K2HPO4·3H2O, and 0.305 g of MgCl2·6H2O were dissolved in 750 mL of deionized water. Then, 3.34 mL of hydrochloric acid (concentrated solution) was added to the solution; 0.278 g of CaCl2, 0.071 g of Na2SO4, and 6.057 g of tris(hydroxymethyl)aminomethane were sequentially dissolved in the solution. Finally, the solution was diluted to 1 L and adjusted to pH 7.4 with hydrochloric acid. The prepared SBF was filtered with 0.25 μm steritop before use. In Vitro Degradation Study. SBA-15 powder was dispersed in SBF at concentrations of 0.1, 0.5, and 1 mg/mL. The solutions were shaken at 150 rpm and 37 °C. At designated time points (2 h, 4 h, 8 h, 24 h, 72 h, 10 days, and 14 days), an aliquot was collected and centrifuged at 11000 rpm for 10 min. Then the supernatant was collected and analyzed via inductively coupled plasma atomic emission spectroscopy (ICP-AES) to measure the amount of silicon that leached from the particles. The precipitate was washed with distilled water and analyzed via small-angle X-ray scattering (SAXS), transmission electron microscopy (TEM), and the Brunauer− Emmett−Teller (BET) method. In Vivo Degradation Study. To test in vivo degradation of SBA15, 10 mg of SBA-15 dispersed in 200 μL of PBS was injected subcutaneously into the right flank of Balb/c mice. PBS was sterilized with a 0.2 μm filter. The injected SBA-15 was retrieved at desired time points (3, 7, 10, 14, and 28 days), washed with PBS and water, and dried at room temperature. The retrieved SBA-15 was analyzed via SAXS and TEM. To observe the change of the nodules, 10 mg of SBA15 dispersed in 200 μL of PBS was subcutaneously injected into the right flank of Blab/c mice (n = 3). The size of the nodule was measured 1, 7, 9, and 14 days after injection in live mice. The volume (V) of the nodule was calculated with the equation V = 0.5AB2 (A is the longer length of the nodule and B the shorter length of the nodule). Characterization. TEM images were obtained with a JEOL JEM3010 electron microscope. Prior to measurements, the material was supersonically dispersed in ethanol, and the suspended particulates were deposited onto a perforated carbon film supported on a copper grid. Nitrogen adsorption−desorption isotherms of mesoporous silica samples were measured on a Micromeritics ASAP 2000 apparatus at liquid nitrogen temperature (77 K). The pore size distribution was calculated from the adsorption branch of the isotherm using the Barret−Joyner−Hallenda (BJH) model. SAXS was performed with Anton Paar’s SAXSess. An inductively coupled plasma atomic emission spectrometer (ICP-AES, Shimadzu ICPS-1000IV, JAPAN) was used for a quantitative analysis.
immunoglobulin.26 Recently, mesoporous silica microparticles (SBA-15) have shown potential as three-dimensional vaccine platforms to modulate dendritic cells through spontaneous assembly of microparticles in a specific region after subcutaneous injection.28 In these applications, the concentration of silica microparticles would be very high at the local injection site, and thus, clearance and biodegradation behavior should be investigated prior to clinical applications and further in vivo research. There are a few reports on the biosafety of microsized mesoporous silica particles after local injection.29 For example, Hudson et al. showed the toxicity and biocompatibility of mesoporous silica particles depending on injection locations and silica dose in animals.29 On the basis of increasing potentials of microsized mesoporous silica particles for the local delivery system and vaccine platform, demands for investigation of biodegradability of mesoporous silica microparticles after local injection are increasing. In this paper, we investigate the degradation behaviors of microsized mesoporous silica, SBA-15, in simulated body fluid (SBF) and in mice after subcutaneous injection to mimic a local delivery system. The average length of SBA-15 particles used was 31.7 μm (Figure S1 of the Supporting Information). SBA15 lost their ordered mesoporous structures under a physiological condition as a function of time. The effects of several factors affecting the degradation rates, such as the surface functional group, the residual amounts of the organic template, and hydrothermal treatment, were investigated. Furthermore, the pore structure of the subcutaneously injected SBA-15 microparticles was analyzed at designated time points and was found to slowly degrade over time. The SBA-15 lost ordered structure, as in in vitro work, allowing possible applications of microsized mesoporous silica as a biodegradable local delivery system.
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MATERIALS AND METHODS
Materials. Poly(ethylene glycol)-poly(propylene glycol)-poly(ethylene glycol) (P123, Aldrich), tetraethyl orthosilicate (TEOS, Aldrich), hydrochloric acid (35.0−37.0%, Samchun), sodium chloride (NaCl, 99.0%, Samchun), sodium hydrogen carbonate (NaHCO3, Kanto), potassium chloride (KCl, Kanto), potassium phosphate dibasic trihydrate (K2HPO4·3H2O, Sigma-Aldrich), magnesium chloride hexahydrate (MgCl2·6H2O, Junsei), calcium chloride (CaCl2, anhydrous, 96.0%, Samchun), sodium sulfate (Na2SO4, anhydrous, powder, Junsei), tris(hydroxymethyl)aminomethane (99.0%, Samchun), 3-aminopropyltrimethoxysilane (APTMS, Aldrich), 2-[methoxy(polyethyleneoxy)propyl]9−12 trimethoxysilane (Gelest), N,N-dimethylformamide (DMF, Sigma-Aldrich), and succinic anhydride (Aldrich) were purchased and used without further purification. Synthesis of SBA-15. SBA-15 was synthesized using triblock copolymer as a structure-directing agent. In a typical synthesis, 4 g of P123 was dissolved in 130 mL of distilled water and 20 mL of 12 M hydrochloric acid by being stirred at 40 °C. A volume of 9.2 mL of TEOS was added to the P123 solution, and the mixture solution was vigorously stirred at 1000 rpm for 20 h at 40 °C. The solution was transferred to a polypropylene bottle and held at 100 °C for 24 h for the hydrothermal treatment (H.T). The precipitate was filtered, washed with water and ethanol, and dried at 100 °C. The removal of organic template, P123, was achieved by either extraction in acidic ethanol or calcination at 550 °C for 5 h. Each extraction was performed in ethanol and hydrochloric acid at 70 °C for 5 h. Descriptions of the samples are specified in Table S1 of the Supporting Information. Surface Modification of SBA-15. To prepare amine-modified SBA-15, 1 g of SBA-15 was dispersed in 500 mL of ethanol and 6 mmol of APTMS was added. The mixture was stirred and refluxed at
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RESULTS AND DISCUSSION We first investigated SBA-15 microparticle degradation in SBF solutions, to mimic physiological body fluid. Calcined SBA-15 was prepared at three different concentrations (0.1, 0.5, and 1 mg/mL in SBF) and incubated at 37 °C while being shaken. SBA-15 microparticles sunk to the bottom of the bottle despite being continuously shaken. This condition may mimic the local assembly of SBA-15 microparticles subcutaneously injected in vivo. ICP-AES analysis of the supernatant of SBA-15 dispersion clearly showed all concentration conditions degraded over time (Figure 1). The degradation rates were dependent on the concentration of SBA-15. The lower concentration of SBA-15 in SBF resulted in a higher silica degradation rate. For example, SBA-15 at a concentration of 0.1 mg/mL rapidly degraded (up 6458
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degradation of MCM-41 type mesoporous silica nanoparticles in SBF.13 The silica walls were degraded quickly after immersion in SBF because of the corrosion reaction with cations (Ca2+ and Mg2+). The subsequent deposition of magnesium and calcium silicate on the silica surface prevented the degradation reaction from the cations (Ca2+ and Mg2+) in SBF. This protection layer and corrosion reaction of silica species led to the equilibrium phase, which was seen in the slow degradation rate after the initial fast degradation. The difference in final degradation percentages of SBA-15 microparticles (Figure 1) may be due to the saturation limit of silicon species in SBF.13 To mimic dynamic in vivo conditions, the media should be in an unsaturated state in the period of degradation. On the basis of this result, a concentration of 0.1 mg/mL was chosen for further degradation tests using SBA-15 microparticles with various parameters such as different residues of surfactants, hydrothermal treatment, and surface functionality. To further examine the physical changes of the mesopores of SBA-15 microparticles, the retrieved SBA-15 particles (0.5 mg/ mL) were further analyzed with nitrogen sorption analysis and SAXS after 3 and 10 days (3d and 10d, respectively). Nitrogen sorption analysis revealed that the hysteresis loops of SBA-15 microparticles immersed in SBF (Figure 3a) were observed at a relative pressure higher than that in the pristine SBA-15 sample (control). The pore size analysis from the adsorption branch shows a broader pore size distribution in incubated samples than in the pristine sample (Figure 3b). The pore size of SBA15 increased from 7.44 to 8.4 nm after 3 days, probably because of the loss of silica walls in the pores as observed via TEM (Figure 2). SBA-15 microparticles incubated for 10 days showed a pore size distribution with two peaks at 7.32 and 36.5 nm (Table 1), which likely resulted from the repetitive deposition and degradation of silica at equilibrium. Sequential deposition of silica species on silica walls resulted in a smaller pore size, and continuous degradation of silica induced a larger pore size.11 Small-angle X-ray scattering shows that the intensity of the (100) peak decreased significantly and (110) and (200) peaks almost vanished after treatment with SBF for 10 days, representing the loss of hexagonal ordering (Figure 3c). Altogether, these results verified the mesostructures of SBA-15 being destroyed in a physiological environment by degradation of the silica walls. The other parameter that could affect degradation of SBA-15 could be the residual amount of P123 in SBA-15. P123 can act as an inhibitor of the degradation reaction as it can interrupt the interaction between the silica wall and the cations in SBF.13 To consider the effect of P123, SBA-15 was prepared after different numbers of extractions in an acidic ethanolic solution. Thermogravimetric analysis (TGA) shows the residual amounts of P123 in SBA-15 gradually decreased as the extraction number increased (Figure S3 of the Supporting Information). As-synthesized SBA-15 without extraction (Extracted #0) contained P123 around 30 wt % of the composite, while the P123 amounts decreased to 15 and 10% after the first extraction (Extracted #1) and the second extraction (Extracted #2), respectively. As a control with no residual P123, calcined SBA-15 (Calcined) biodegradation was also tested. Overall degradation patterns were similar in all samples (Figure 4a). However, the final degradation percentages depended on the residual amounts of P123. As-synthesized SBA-15 (Extracted #0) showed the lowest level of degradation (63.3%). The first extraction (Extracted #1) and the second extraction (Extracted #2) showed 70.0 and 91.6% silica degradation, respectively.
Figure 1. Degradation behavior of SBA-15 in SBF depending on different concentrations (0.1, 0.5, and 1 mg/mL).
to 91.4% in 24 h), while the 0.5 mg/mL sample degraded to 51.9% in 24 h. SBA-15, at a concentration of 1.0 mg/mL, showed only 16.2% degradation in 24 h. The degradation profiles of SBA-15 microparticles are steeply maximized and reached a plateau by 24 h in all three samples. To evaluate the changes in mesostructured SBA-15 in SBF, the SBA-15 microparticles (1.0 mg/mL) were retrieved from SBF at designated time points and analyzed by TEM (Figure 2). The
Figure 2. TEM images of SBA-15 that have been immersed in SBF at a concentration of 1.0 mg/mL. The samples were retrieved at (a) 0, (b) 1, (c) 3, and (d) 7 days.
TEM images show that one-dimensionally elongated mesopores of SBA-15 collapsed over time. The initiation of the destruction of the pore structure was clearly observed on day 3 (Figure 2c). Nitrogen sorption analysis of retrieved SBA-15 microparticles from 0.5 and 1.0 mg/mL conditions after 3 days in SBF shows that SBA-15 at a lower concentration was more degraded (Figure S2 and Table S2 of the Supporting Information). This degradation behavior of SBA-15 microparticles is similar to that seen in a previous study of 6459
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Figure 3. (a) Nitrogen sorption isotherms, (b) pore size distributions obtained from the adsorption isotherm, and (c) SAXS of SBA-15 incubated in a SBF solution (0.5 mg/mL) for 0, 3, and 10 days.
to 9.57 nm after hydrothermal treatment as the hydrophobicity of P123 increases at high temperatures and the resulting expansion of P123 micelle leads to a larger pore size.30−32 In contrast, the silica wall thickness was reduced from 4.83 to 2.50 nm in SBA-15 prepared via hydrothermal treatment, because of the shrinkage of the silica wall at high temperatures during hydrothermal treatment.30−32 Figure 4b shows the degradation rate of SBA-15 microparticles with (H.T) and without (W/O H.T) hydrothermal treatment, showing that there was no significant difference in degradation rate between the two. Both samples reached maximal degradation in 24 h. In spite of the difference in silica wall thickness, degradation for 7 days led to a similar silica degradation level of 98% in both SBA-15 samples. It is well-known that the microporosity of silica walls decreased after hydrothermal treatment,30−32 which may compromise the effect of a thinner silica wall and a broader surface area of hydrothermally treated SBA-15 on the possible enhanced degradation compared to the control sample. To evaluate the effect of a functionally modified silica surface on the degradation behavior of SBA-15, SBA-15 samples with a surface of hydroxyl (OH), amine (NH2), and carboxylic (COOH) groups were tested (Figure 4c). COOH-SBA-15 showed the lowest degradation percentage, 70.7%, while OHSBA-15 and NH2-SBA-15 showed 91.5 and 86.6%, respectively, after 14 days in SBF. As COOH-SBA-15 was prepared using NH2-SBA-15 by coupling succinic acid, the silica surface was covered with longer alkyl chains than NH2-SBA-15. These longer alkyl chains on the silica surface reduced the pore size and surface area compared to those of NH2-SBA-15 [from 7.17 to 6.27 nm and from 343 to 278.2 m2/g, respectively (Table S4
Table 1. Physical Properties of SBA-15 before and after Their Incubation in SBF for 3 and 10 Days (0.5 mg/mL) time
surface area (m2/g)
pore volume (cm3/g)
pore size (nm)
wall thickness (nm)
control 3 days 10 days
1165 565.3 479.7
1.38 1.38 1.41
7.44 8.40 7.32−26.5
3.88 2.92
This result indicates that P123 protects the silica wall from degradation. In the addition, calcined SBA-15 showed 80.0% silica degradation, which is middle of the range of Extracted #1 and Extracted #2, which can be explained in terms of further condensation of silica at high temperatures.13 Previous results showed calcined MSNs at a concentration of 0.1 mg/mL degraded to 30% after SBF treatment for 15 days.13 In contrast, calcined SBA-15 microparticles degraded to 80% presumably because the SBA-15 type microparticles have a larger pore size and pore volume that can induce a high flux of SBF in pores and trigger the faster erosion reaction. The hydrothermal treatment during the synthesis of mesoporous silica materials is known to enhance hydrothermal stability because of the loss of microporosity in the silica wall and denser siloxane bonding (Si−O−Si).30−32 To test the effect of hydrothermal treatment on the degradation behavior, SBA15 synthesized with and without hydrothermal treatment at 100 °C for 24 h was prepared (H.T and W/O H.T, respectively). The pore size and wall thickness of two SBA-15 samples were analyzed via nitrogen sorption analysis and SAXS (Table S3 of the Supporting Information). The pore size increased from 4.89
Figure 4. Effects of (a) the amounts of surfactant residue in SBA-15 pores controlled by the number of ethanol extractions, (b) the hydrothermal treatment during the synthesis of SBA-15, and (c) the surface functional groups on the degradation behavior of SBA-15. All starting concentrations of SBA-15 in SBF were 0.1 mg/mL. 6460
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Figure 5. (a) Optical images of nodules resulted from the injection of SBA-15 over time (3, 10, and 28 days). (b) Change in volume of the nodules after subcutaneous injection of SBA-15 (10 mg) in the right flank of mice.
of the Supporting Information)]. These differences may explain the different degradation rates between the two samples. All surface-modified SBA-15 microparticles showed a surface area, a pore size, and a level of degradation lower than those of pristine SBA-15 (Figure 4c and Table S4 of the Supporting Information), demonstrating that the functional groups can slow the rate of degradation of SBA-15 by interfering with the corrosion of the silica wall by cations in SBF, including Ca2+ and Mg2+.13,22 For in vivo application of SBA-15 microparticles for local sustained delivery of bioactive molecules, in vivo degradation behavior must be considered. The in vivo experiment was conducted with the subcutaneous local injection of 10 mg of SBA-15 microparticles dispersed in PBS into Balb/c mice. Injected SBA-15 induced a small nodule in the injection site (Figure 5a). To calculate the volume of nodules, we used the general formula for tumor volume (mentioned in Materials and Methods) because of the similarity of the nodule shape to subcutaneously xenografted tumors. The nodule size induced by injected SBA-15 was measured at designed times (Figure 5b). After injection of SBA-15, the volume of the nodule was gradually reduced from 336.9 to 150.5 mm3 over time. The decrease in nodule size may indicate the degradation of SBA-15 microparticles in vivo (Figure 5). Although the study was continued for 28 days, we could not retrieve SBA-15 microparticles from the nodules probably because the silica was mostly degraded. To study the change in the physical properties of SBA-15 microparticles depending on the duration after subcutaneous injection, the injected SBA-15 was retrieved at different time points (3, 7, 10, and 14 days) and characterized via TEM and SAXS. As the inner contents in the retrieved nodule contained the SBA-15 microparticles and infiltrated host cells (Figure S4 of the Supporting Information), SBA-15 microparticles were separated from the cells prior to analysis. TEM images show that the pore structure of SBA-15 was destroyed over time in the mice (Figure 6a−c). SAXS analysis of the sample retrieved on day 3 post-injection shows the intensity of the characteristic (100) peak to be significantly decreased and (110) and (200) peaks almost disappeared after 3 days in the animal (Figure 6d). Taken together, these results indicate SBA-15 microparticles degraded in vivo and lose their structural integrity over time as in the in vitro experiment.
Figure 6. (a−c) TEM images and (d) SAXS showing in vivo degradation of SBA-15 over time. The subcutaneously injected SBA-15 was retrieved from the mice on days (a) 0, (b) 3, and (c) 7 and analyzed by TEM. SAXS was measured for the pristine SBA-15 (---) and SBA-15 retrieved from mice 3 days after injection ().
microparticles with various surface functional groups and synthesis methodologies can affect the degradation and rate of degradation of SBA-15 in SBF. Furthermore, the in vivo experiment showed that SBA-15 microparticles degrade in the animal and pore structure deformation occurs as a function of time. These results provide basic information about the application of mesoporous silica microparticles for drug delivery; controllable biodegradation can be further investigated in association with the desired drug release profile.
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ASSOCIATED CONTENT
S Supporting Information *
Additional data (nitrogen sorption analysis, TGA curve, and optical microscopic image) and tables containing surface areas, pore volumes, pore sizes, and silica wall thicknesses of SBA-15. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.5b01316.
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CONCLUSION In summary, we show the degradation behavior of SBA-15 microparticles in vitro and in vivo. The mesoporous silica 6461
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AUTHOR INFORMATION
Corresponding Authors
*E-mail: [email protected]. *E-mail: [email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by a grant funded by the National Research Foundation under the Ministry of Science, ICT & Future Planning, Republic of Korea (Grant 2010-0027955), and a grant from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (Grant HI14C0211).
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REFERENCES
(1) Zhao, Y.; Lin, L. N.; Chen, S. F.; Dong, L.; Yu, S. H. Templating Synthesis of Preloaded Doxorubicin in Hollow Mesoporous Silica Nanospheres for Biomedical Applications. Adv. Mater. (Weinheim, Ger.) 2010, 22, 5255−5259. (2) Choi, S. R.; Jang, D. J.; Kim, S.; An, S.; Lee, J.; Oh, E.; Kim, J. Polymer-coated spherical mesoporous silica for pH-controlled delivery of insulin. J. Mater. Chem. B 2014, 2, 616−619. (3) Zhang, J.; Yuan, Z. F.; Wang, Y.; Chen, W. H.; Luo, G. F.; Cheng, S. X.; Zhuo, R. X.; Zhang, X. Z. Multifunctional Envelope-Type Mesoporous Silica Nanoparticles for Tumor-Triggered Targeting Drug Delivery. J. Am. Chem. Soc. 2013, 135, 5068−5073. (4) Wang, Y.; Caruso, F. Mesoporous Silica Spheres as Supports for Enzyme Immobilization and Encapsulation. Chem. Mater. 2005, 17, 953−961. (5) Lu, Y.; Cao, G.; Kale, R. P.; Prabakar, S.; López, G. P.; Brinker, C. J. Microporous Silica Prepared by Organic Templating: Relationship between the Molecular Template and Pore Structure. Chem. Mater. 1999, 11, 1223−1229. (6) Li, Z.; Barnes, J. C.; Bosoy, A.; Stoddart, J. F.; Zink, J. I. Mesoporous silica nanoparticles in biomedical applications. Chem. Soc. Rev. 2012, 41, 2590−2605. (7) Lu, J.; Liong, M.; Zink, J. I.; Tamanoi, F. Mesoporous Silica Nanoparticles as a Delivery System for Hydrophobic Anticancer Drugs. Small 2007, 3, 1341−1346. (8) Piao, Y.; Burns, A.; Kim, J.; Wiesner, U.; Hyeon, T. Designed Fabrication of Silica-Based Nanostructured Particle Systems for Nanomedicine Applications. Adv. Funct. Mater. 2008, 18, 3745−3758. (9) Hoffmann, F.; Cornelius, M.; Morell, J.; Fröba, M. Silica-Based Mesoporous Organic-Inorganic Hybrid Materials. Angew. Chem., Int. Ed. 2006, 45, 3216−3251. (10) Chang, B.; Guo, J.; Liu, C.; Qian, J.; Yang, W. Surface functionalization of magnetic mesoporous silica nanoparticles for controlled drug release. J. Mater. Chem. 2010, 20, 9941−9947. (11) Gouze, B.; Cambedouzou, J.; Parrès-Maynadié, S.; Rébiscoul, D. How hexagonal mesoporous silica evolves in water on short and long term: Role of pore size and silica wall porosity. Microporous Mesoporous Mater. 2014, 183, 168−176. (12) Yang, P.; Gai, S.; Lin, J. Functionalized mesoporous silica materials for controlled drug delivery. Chem. Soc. Rev. 2012, 41, 3679− 3698. (13) He, Q.; Shi, J.; Zhu, M.; Chen, Y.; Chen, F. The three-stage in vitro degradation behavior of mesoporous silica in simulated body fluid. Microporous Mesoporous Mater. 2010, 131, 314−320. (14) Ma, Z.; Bai, J.; Wang, Y.; Jiang, X. Impact of Shape and Pore Size of Mesoporous Silica Nanoparticles on Serum Protein Adsorption and RBCs Hemolysis. ACS Appl. Mater. Interfaces 2014, 6, 2431−2438. (15) Mamaeva, V.; Sahlgren, C.; Lindén, M. Mesoporous silica nanoparticles in medicine: Recent advances. Adv. Drug Delivery Rev. 2013, 65, 689−702. 6462
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A Biodegradation Study of SBA-15 Microparticles in Simulated Body Fluid and in Vivo Youngjin Choi,† Jung Eun Lee,‡ Jung Heon Lee,§ Ji Hoon Jeong,*,‡ and Jaeyun Kim*,† †
School of Chemical Engineering, Sungkyunkwan University, Suwon 440-746, Republic of Korea School of Pharmacy, Sungkyunkwan University, Suwon 440-746, Republic of Korea § School of Advanced Materials Science and Engineering, Sungkyunkwan University, Suwon 440-746, Republic of Korea ‡
S Supporting Information *
ABSTRACT: Mesoporous silica has received considerable attention as a drug delivery vehicle because of its large surface area and large pore volume for loading drugs and large biomolecules. Recently, mesoporous silica microparticles have shown potential as a three-dimensional vaccine platform for modulating dendritic cells via spontaneous assembly of microparticles in a specific region after subcutaneous injection. For further in vivo applications, the biodegradation behavior of mesoporous silica microparticles must be studied and known. Until now, most biodegradation studies have focused on mesoporous silica nanoparticles (MSNs); here, we report the biodegradation of hexagonally ordered mesoporous silica, SBA-15, with micrometer-sized lengths (∼32 μm with a high aspect ratio). The degradation of SBA-15 microparticles was investigated in simulated body fluid (SBF) and in mice by analyzing the structural change over time. SBA-15 microparticles were found to degrade in SBF and in vivo. The erosion of SBA-15 under biological conditions led to a loss of the hysteresis loop in the nitrogen adsorption/desorption isotherm and fingerprint peaks in small-angle X-ray scattering, specifically indicating a degradation of ordered mesoporous structure. Via comparison to previous results of degradation of MSNs in SBF, SBA-15 microparticles degraded faster than MCM-41 nanoparticles presumably because SBA-15 microparticles have a pore size (∼8 nm) and a pore volume larger than those of MCM-41 mesoporous silica. The surface functional groups, the residual amounts of organic templates, and the hydrothermal treatment during the synthesis could affect the rate of degradation of SBA-15. In in vivo testing, previous studies focused on the evaluation of toxicity of mesoporous silica particles in various organs. In contrast, we studied the change in the physical properties of SBA-15 microparticles depending on the duration after subcutaneous injection. The pristine SBA-15 microparticles injected into mice subcutaneously slowly degraded over time and lost ordered structure after 3 days. These findings represent the possible in vivo use of microsized mesoporous silica for drug delivery or vaccine platform after local injection.
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INTRODUCTION Mesoporous silica materials with a large surface area, a large pore volume, and controllable pore structures have been intensively studied as tools for drug delivery systems.1−13 Mesoscale pores are highly preferred in drug delivery carriers as the sizes of most biomolecules are in this range.10,13−17 To be used in in vivo application, biodegradation of mesoporous silica must be considered to prevent toxicity and unwanted accumulation in tissues. There have been several studies of degradation of mesoporous silica, but most degradation studies have focused on mesoporous silica nanoparticles (MSNs) because of their potential use in targeted intravenous delivery.15,16,18−20 Most MCM-41 type MSNs (∼100 nm in size) were tested in most biodegradation studies using diverse physiological conditions. These studies showed mesoporous silica nanoparticles degrading in distilled water, simulated body fluid (SBF), and cell culture medium over time.11,13,14,21,22 The degradation of MSNs depends on chemical and physical © 2015 American Chemical Society
properties, including pore size, surface area, composition, and surface functional groups.13,17,21−23 In contrast to systemic delivery systems using MSNs to target specific cells and tissues, local delivery systems based on subcutaneous or intramuscular injection of microsized mesoporous silica particles form a local depot that can hold high concentrations of the bioactive molecules that are slowly released to achieve the desired level of the bioactive molecules in local tissue or bloodstream.24−27 For example, SBA-15 mesoporous silica was incorporated with bioactive glass as a form of scaffold and improved the efficiency of the drug carrier.25 Lei et al. demonstrated the in vivo local delivery of therapeutic immunoglobulin to tumors by subcutaneously injecting functionalized SBA-15 microparticles loaded with Received: April 10, 2015 Revised: May 20, 2015 Published: May 26, 2015 6457
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75 °C for 6 h, and then the white powder was collected by filtration. The functionalized SBA-15 was washed with ethanol and distilled water and dried at 100 °C. To prepare carboxyl-functionalized SBA-15, the amine-modified SBA-15 was dispersed in 500 mL of 2 wt % succinic anhydride in DMF and the mixture solution was stirred for 24 h at room temperature. SBA-15 functionalized with carboxylic groups was filtered, washed, and dried. Preparation of Simulated Body Fluid (SBF). SBF was prepared according to the previous report.13 To prepare 1 L of SBF, 7.996 g of NaCl, 0.35 g of NaHCO3, 0.224 g of KCl, 0.228 g of K2HPO4·3H2O, and 0.305 g of MgCl2·6H2O were dissolved in 750 mL of deionized water. Then, 3.34 mL of hydrochloric acid (concentrated solution) was added to the solution; 0.278 g of CaCl2, 0.071 g of Na2SO4, and 6.057 g of tris(hydroxymethyl)aminomethane were sequentially dissolved in the solution. Finally, the solution was diluted to 1 L and adjusted to pH 7.4 with hydrochloric acid. The prepared SBF was filtered with 0.25 μm steritop before use. In Vitro Degradation Study. SBA-15 powder was dispersed in SBF at concentrations of 0.1, 0.5, and 1 mg/mL. The solutions were shaken at 150 rpm and 37 °C. At designated time points (2 h, 4 h, 8 h, 24 h, 72 h, 10 days, and 14 days), an aliquot was collected and centrifuged at 11000 rpm for 10 min. Then the supernatant was collected and analyzed via inductively coupled plasma atomic emission spectroscopy (ICP-AES) to measure the amount of silicon that leached from the particles. The precipitate was washed with distilled water and analyzed via small-angle X-ray scattering (SAXS), transmission electron microscopy (TEM), and the Brunauer− Emmett−Teller (BET) method. In Vivo Degradation Study. To test in vivo degradation of SBA15, 10 mg of SBA-15 dispersed in 200 μL of PBS was injected subcutaneously into the right flank of Balb/c mice. PBS was sterilized with a 0.2 μm filter. The injected SBA-15 was retrieved at desired time points (3, 7, 10, 14, and 28 days), washed with PBS and water, and dried at room temperature. The retrieved SBA-15 was analyzed via SAXS and TEM. To observe the change of the nodules, 10 mg of SBA15 dispersed in 200 μL of PBS was subcutaneously injected into the right flank of Blab/c mice (n = 3). The size of the nodule was measured 1, 7, 9, and 14 days after injection in live mice. The volume (V) of the nodule was calculated with the equation V = 0.5AB2 (A is the longer length of the nodule and B the shorter length of the nodule). Characterization. TEM images were obtained with a JEOL JEM3010 electron microscope. Prior to measurements, the material was supersonically dispersed in ethanol, and the suspended particulates were deposited onto a perforated carbon film supported on a copper grid. Nitrogen adsorption−desorption isotherms of mesoporous silica samples were measured on a Micromeritics ASAP 2000 apparatus at liquid nitrogen temperature (77 K). The pore size distribution was calculated from the adsorption branch of the isotherm using the Barret−Joyner−Hallenda (BJH) model. SAXS was performed with Anton Paar’s SAXSess. An inductively coupled plasma atomic emission spectrometer (ICP-AES, Shimadzu ICPS-1000IV, JAPAN) was used for a quantitative analysis.
immunoglobulin.26 Recently, mesoporous silica microparticles (SBA-15) have shown potential as three-dimensional vaccine platforms to modulate dendritic cells through spontaneous assembly of microparticles in a specific region after subcutaneous injection.28 In these applications, the concentration of silica microparticles would be very high at the local injection site, and thus, clearance and biodegradation behavior should be investigated prior to clinical applications and further in vivo research. There are a few reports on the biosafety of microsized mesoporous silica particles after local injection.29 For example, Hudson et al. showed the toxicity and biocompatibility of mesoporous silica particles depending on injection locations and silica dose in animals.29 On the basis of increasing potentials of microsized mesoporous silica particles for the local delivery system and vaccine platform, demands for investigation of biodegradability of mesoporous silica microparticles after local injection are increasing. In this paper, we investigate the degradation behaviors of microsized mesoporous silica, SBA-15, in simulated body fluid (SBF) and in mice after subcutaneous injection to mimic a local delivery system. The average length of SBA-15 particles used was 31.7 μm (Figure S1 of the Supporting Information). SBA15 lost their ordered mesoporous structures under a physiological condition as a function of time. The effects of several factors affecting the degradation rates, such as the surface functional group, the residual amounts of the organic template, and hydrothermal treatment, were investigated. Furthermore, the pore structure of the subcutaneously injected SBA-15 microparticles was analyzed at designated time points and was found to slowly degrade over time. The SBA-15 lost ordered structure, as in in vitro work, allowing possible applications of microsized mesoporous silica as a biodegradable local delivery system.
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MATERIALS AND METHODS
Materials. Poly(ethylene glycol)-poly(propylene glycol)-poly(ethylene glycol) (P123, Aldrich), tetraethyl orthosilicate (TEOS, Aldrich), hydrochloric acid (35.0−37.0%, Samchun), sodium chloride (NaCl, 99.0%, Samchun), sodium hydrogen carbonate (NaHCO3, Kanto), potassium chloride (KCl, Kanto), potassium phosphate dibasic trihydrate (K2HPO4·3H2O, Sigma-Aldrich), magnesium chloride hexahydrate (MgCl2·6H2O, Junsei), calcium chloride (CaCl2, anhydrous, 96.0%, Samchun), sodium sulfate (Na2SO4, anhydrous, powder, Junsei), tris(hydroxymethyl)aminomethane (99.0%, Samchun), 3-aminopropyltrimethoxysilane (APTMS, Aldrich), 2-[methoxy(polyethyleneoxy)propyl]9−12 trimethoxysilane (Gelest), N,N-dimethylformamide (DMF, Sigma-Aldrich), and succinic anhydride (Aldrich) were purchased and used without further purification. Synthesis of SBA-15. SBA-15 was synthesized using triblock copolymer as a structure-directing agent. In a typical synthesis, 4 g of P123 was dissolved in 130 mL of distilled water and 20 mL of 12 M hydrochloric acid by being stirred at 40 °C. A volume of 9.2 mL of TEOS was added to the P123 solution, and the mixture solution was vigorously stirred at 1000 rpm for 20 h at 40 °C. The solution was transferred to a polypropylene bottle and held at 100 °C for 24 h for the hydrothermal treatment (H.T). The precipitate was filtered, washed with water and ethanol, and dried at 100 °C. The removal of organic template, P123, was achieved by either extraction in acidic ethanol or calcination at 550 °C for 5 h. Each extraction was performed in ethanol and hydrochloric acid at 70 °C for 5 h. Descriptions of the samples are specified in Table S1 of the Supporting Information. Surface Modification of SBA-15. To prepare amine-modified SBA-15, 1 g of SBA-15 was dispersed in 500 mL of ethanol and 6 mmol of APTMS was added. The mixture was stirred and refluxed at
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RESULTS AND DISCUSSION We first investigated SBA-15 microparticle degradation in SBF solutions, to mimic physiological body fluid. Calcined SBA-15 was prepared at three different concentrations (0.1, 0.5, and 1 mg/mL in SBF) and incubated at 37 °C while being shaken. SBA-15 microparticles sunk to the bottom of the bottle despite being continuously shaken. This condition may mimic the local assembly of SBA-15 microparticles subcutaneously injected in vivo. ICP-AES analysis of the supernatant of SBA-15 dispersion clearly showed all concentration conditions degraded over time (Figure 1). The degradation rates were dependent on the concentration of SBA-15. The lower concentration of SBA-15 in SBF resulted in a higher silica degradation rate. For example, SBA-15 at a concentration of 0.1 mg/mL rapidly degraded (up 6458
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degradation of MCM-41 type mesoporous silica nanoparticles in SBF.13 The silica walls were degraded quickly after immersion in SBF because of the corrosion reaction with cations (Ca2+ and Mg2+). The subsequent deposition of magnesium and calcium silicate on the silica surface prevented the degradation reaction from the cations (Ca2+ and Mg2+) in SBF. This protection layer and corrosion reaction of silica species led to the equilibrium phase, which was seen in the slow degradation rate after the initial fast degradation. The difference in final degradation percentages of SBA-15 microparticles (Figure 1) may be due to the saturation limit of silicon species in SBF.13 To mimic dynamic in vivo conditions, the media should be in an unsaturated state in the period of degradation. On the basis of this result, a concentration of 0.1 mg/mL was chosen for further degradation tests using SBA-15 microparticles with various parameters such as different residues of surfactants, hydrothermal treatment, and surface functionality. To further examine the physical changes of the mesopores of SBA-15 microparticles, the retrieved SBA-15 particles (0.5 mg/ mL) were further analyzed with nitrogen sorption analysis and SAXS after 3 and 10 days (3d and 10d, respectively). Nitrogen sorption analysis revealed that the hysteresis loops of SBA-15 microparticles immersed in SBF (Figure 3a) were observed at a relative pressure higher than that in the pristine SBA-15 sample (control). The pore size analysis from the adsorption branch shows a broader pore size distribution in incubated samples than in the pristine sample (Figure 3b). The pore size of SBA15 increased from 7.44 to 8.4 nm after 3 days, probably because of the loss of silica walls in the pores as observed via TEM (Figure 2). SBA-15 microparticles incubated for 10 days showed a pore size distribution with two peaks at 7.32 and 36.5 nm (Table 1), which likely resulted from the repetitive deposition and degradation of silica at equilibrium. Sequential deposition of silica species on silica walls resulted in a smaller pore size, and continuous degradation of silica induced a larger pore size.11 Small-angle X-ray scattering shows that the intensity of the (100) peak decreased significantly and (110) and (200) peaks almost vanished after treatment with SBF for 10 days, representing the loss of hexagonal ordering (Figure 3c). Altogether, these results verified the mesostructures of SBA-15 being destroyed in a physiological environment by degradation of the silica walls. The other parameter that could affect degradation of SBA-15 could be the residual amount of P123 in SBA-15. P123 can act as an inhibitor of the degradation reaction as it can interrupt the interaction between the silica wall and the cations in SBF.13 To consider the effect of P123, SBA-15 was prepared after different numbers of extractions in an acidic ethanolic solution. Thermogravimetric analysis (TGA) shows the residual amounts of P123 in SBA-15 gradually decreased as the extraction number increased (Figure S3 of the Supporting Information). As-synthesized SBA-15 without extraction (Extracted #0) contained P123 around 30 wt % of the composite, while the P123 amounts decreased to 15 and 10% after the first extraction (Extracted #1) and the second extraction (Extracted #2), respectively. As a control with no residual P123, calcined SBA-15 (Calcined) biodegradation was also tested. Overall degradation patterns were similar in all samples (Figure 4a). However, the final degradation percentages depended on the residual amounts of P123. As-synthesized SBA-15 (Extracted #0) showed the lowest level of degradation (63.3%). The first extraction (Extracted #1) and the second extraction (Extracted #2) showed 70.0 and 91.6% silica degradation, respectively.
Figure 1. Degradation behavior of SBA-15 in SBF depending on different concentrations (0.1, 0.5, and 1 mg/mL).
to 91.4% in 24 h), while the 0.5 mg/mL sample degraded to 51.9% in 24 h. SBA-15, at a concentration of 1.0 mg/mL, showed only 16.2% degradation in 24 h. The degradation profiles of SBA-15 microparticles are steeply maximized and reached a plateau by 24 h in all three samples. To evaluate the changes in mesostructured SBA-15 in SBF, the SBA-15 microparticles (1.0 mg/mL) were retrieved from SBF at designated time points and analyzed by TEM (Figure 2). The
Figure 2. TEM images of SBA-15 that have been immersed in SBF at a concentration of 1.0 mg/mL. The samples were retrieved at (a) 0, (b) 1, (c) 3, and (d) 7 days.
TEM images show that one-dimensionally elongated mesopores of SBA-15 collapsed over time. The initiation of the destruction of the pore structure was clearly observed on day 3 (Figure 2c). Nitrogen sorption analysis of retrieved SBA-15 microparticles from 0.5 and 1.0 mg/mL conditions after 3 days in SBF shows that SBA-15 at a lower concentration was more degraded (Figure S2 and Table S2 of the Supporting Information). This degradation behavior of SBA-15 microparticles is similar to that seen in a previous study of 6459
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Figure 3. (a) Nitrogen sorption isotherms, (b) pore size distributions obtained from the adsorption isotherm, and (c) SAXS of SBA-15 incubated in a SBF solution (0.5 mg/mL) for 0, 3, and 10 days.
to 9.57 nm after hydrothermal treatment as the hydrophobicity of P123 increases at high temperatures and the resulting expansion of P123 micelle leads to a larger pore size.30−32 In contrast, the silica wall thickness was reduced from 4.83 to 2.50 nm in SBA-15 prepared via hydrothermal treatment, because of the shrinkage of the silica wall at high temperatures during hydrothermal treatment.30−32 Figure 4b shows the degradation rate of SBA-15 microparticles with (H.T) and without (W/O H.T) hydrothermal treatment, showing that there was no significant difference in degradation rate between the two. Both samples reached maximal degradation in 24 h. In spite of the difference in silica wall thickness, degradation for 7 days led to a similar silica degradation level of 98% in both SBA-15 samples. It is well-known that the microporosity of silica walls decreased after hydrothermal treatment,30−32 which may compromise the effect of a thinner silica wall and a broader surface area of hydrothermally treated SBA-15 on the possible enhanced degradation compared to the control sample. To evaluate the effect of a functionally modified silica surface on the degradation behavior of SBA-15, SBA-15 samples with a surface of hydroxyl (OH), amine (NH2), and carboxylic (COOH) groups were tested (Figure 4c). COOH-SBA-15 showed the lowest degradation percentage, 70.7%, while OHSBA-15 and NH2-SBA-15 showed 91.5 and 86.6%, respectively, after 14 days in SBF. As COOH-SBA-15 was prepared using NH2-SBA-15 by coupling succinic acid, the silica surface was covered with longer alkyl chains than NH2-SBA-15. These longer alkyl chains on the silica surface reduced the pore size and surface area compared to those of NH2-SBA-15 [from 7.17 to 6.27 nm and from 343 to 278.2 m2/g, respectively (Table S4
Table 1. Physical Properties of SBA-15 before and after Their Incubation in SBF for 3 and 10 Days (0.5 mg/mL) time
surface area (m2/g)
pore volume (cm3/g)
pore size (nm)
wall thickness (nm)
control 3 days 10 days
1165 565.3 479.7
1.38 1.38 1.41
7.44 8.40 7.32−26.5
3.88 2.92
This result indicates that P123 protects the silica wall from degradation. In the addition, calcined SBA-15 showed 80.0% silica degradation, which is middle of the range of Extracted #1 and Extracted #2, which can be explained in terms of further condensation of silica at high temperatures.13 Previous results showed calcined MSNs at a concentration of 0.1 mg/mL degraded to 30% after SBF treatment for 15 days.13 In contrast, calcined SBA-15 microparticles degraded to 80% presumably because the SBA-15 type microparticles have a larger pore size and pore volume that can induce a high flux of SBF in pores and trigger the faster erosion reaction. The hydrothermal treatment during the synthesis of mesoporous silica materials is known to enhance hydrothermal stability because of the loss of microporosity in the silica wall and denser siloxane bonding (Si−O−Si).30−32 To test the effect of hydrothermal treatment on the degradation behavior, SBA15 synthesized with and without hydrothermal treatment at 100 °C for 24 h was prepared (H.T and W/O H.T, respectively). The pore size and wall thickness of two SBA-15 samples were analyzed via nitrogen sorption analysis and SAXS (Table S3 of the Supporting Information). The pore size increased from 4.89
Figure 4. Effects of (a) the amounts of surfactant residue in SBA-15 pores controlled by the number of ethanol extractions, (b) the hydrothermal treatment during the synthesis of SBA-15, and (c) the surface functional groups on the degradation behavior of SBA-15. All starting concentrations of SBA-15 in SBF were 0.1 mg/mL. 6460
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Figure 5. (a) Optical images of nodules resulted from the injection of SBA-15 over time (3, 10, and 28 days). (b) Change in volume of the nodules after subcutaneous injection of SBA-15 (10 mg) in the right flank of mice.
of the Supporting Information)]. These differences may explain the different degradation rates between the two samples. All surface-modified SBA-15 microparticles showed a surface area, a pore size, and a level of degradation lower than those of pristine SBA-15 (Figure 4c and Table S4 of the Supporting Information), demonstrating that the functional groups can slow the rate of degradation of SBA-15 by interfering with the corrosion of the silica wall by cations in SBF, including Ca2+ and Mg2+.13,22 For in vivo application of SBA-15 microparticles for local sustained delivery of bioactive molecules, in vivo degradation behavior must be considered. The in vivo experiment was conducted with the subcutaneous local injection of 10 mg of SBA-15 microparticles dispersed in PBS into Balb/c mice. Injected SBA-15 induced a small nodule in the injection site (Figure 5a). To calculate the volume of nodules, we used the general formula for tumor volume (mentioned in Materials and Methods) because of the similarity of the nodule shape to subcutaneously xenografted tumors. The nodule size induced by injected SBA-15 was measured at designed times (Figure 5b). After injection of SBA-15, the volume of the nodule was gradually reduced from 336.9 to 150.5 mm3 over time. The decrease in nodule size may indicate the degradation of SBA-15 microparticles in vivo (Figure 5). Although the study was continued for 28 days, we could not retrieve SBA-15 microparticles from the nodules probably because the silica was mostly degraded. To study the change in the physical properties of SBA-15 microparticles depending on the duration after subcutaneous injection, the injected SBA-15 was retrieved at different time points (3, 7, 10, and 14 days) and characterized via TEM and SAXS. As the inner contents in the retrieved nodule contained the SBA-15 microparticles and infiltrated host cells (Figure S4 of the Supporting Information), SBA-15 microparticles were separated from the cells prior to analysis. TEM images show that the pore structure of SBA-15 was destroyed over time in the mice (Figure 6a−c). SAXS analysis of the sample retrieved on day 3 post-injection shows the intensity of the characteristic (100) peak to be significantly decreased and (110) and (200) peaks almost disappeared after 3 days in the animal (Figure 6d). Taken together, these results indicate SBA-15 microparticles degraded in vivo and lose their structural integrity over time as in the in vitro experiment.
Figure 6. (a−c) TEM images and (d) SAXS showing in vivo degradation of SBA-15 over time. The subcutaneously injected SBA-15 was retrieved from the mice on days (a) 0, (b) 3, and (c) 7 and analyzed by TEM. SAXS was measured for the pristine SBA-15 (---) and SBA-15 retrieved from mice 3 days after injection ().
microparticles with various surface functional groups and synthesis methodologies can affect the degradation and rate of degradation of SBA-15 in SBF. Furthermore, the in vivo experiment showed that SBA-15 microparticles degrade in the animal and pore structure deformation occurs as a function of time. These results provide basic information about the application of mesoporous silica microparticles for drug delivery; controllable biodegradation can be further investigated in association with the desired drug release profile.
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ASSOCIATED CONTENT
S Supporting Information *
Additional data (nitrogen sorption analysis, TGA curve, and optical microscopic image) and tables containing surface areas, pore volumes, pore sizes, and silica wall thicknesses of SBA-15. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.5b01316.
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CONCLUSION In summary, we show the degradation behavior of SBA-15 microparticles in vitro and in vivo. The mesoporous silica 6461
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AUTHOR INFORMATION
Corresponding Authors
*E-mail: [email protected]. *E-mail: [email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by a grant funded by the National Research Foundation under the Ministry of Science, ICT & Future Planning, Republic of Korea (Grant 2010-0027955), and a grant from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (Grant HI14C0211).
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DOI: 10.1021/acs.langmuir.5b01316 Langmuir 2015, 31, 6457−6462
