Accepted Manuscript Optimization of tetracycline hydrochloride adsorption on amino modified SBA-15 using response surface methodology Samaneh Hashemikia, Nahid Hemmatinejad, Ebrahim Ahmadi, Majid Montazer PII: DOI: Reference:
S0021-9797(14)00868-6 http://dx.doi.org/10.1016/j.jcis.2014.11.020 YJCIS 19987
To appear in:
Journal of Colloid and Interface Science
Received Date: Accepted Date:
19 July 2014 6 November 2014
Please cite this article as: S. Hashemikia, N. Hemmatinejad, E. Ahmadi, M. Montazer, Optimization of tetracycline hydrochloride adsorption on amino modified SBA-15 using response surface methodology, Journal of Colloid and Interface Science (2014), doi: http://dx.doi.org/10.1016/j.jcis.2014.11.020
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Optimization of tetracycline hydrochloride adsorption on amino modified SBA-15 using response surface methodology
Samaneh Hashemikia1, Nahid Hemmatinejad1*, Ebrahim Ahmadi2, Majid Montazer1 1
Department of Textile Engineering, Functional Fibrous Structures & Environmental Enhancement
(FFSEE), Amirkabir University of Technology (Tehran Polytechnic), P.O. Box 15875-4413, Tehran, Iran 2
Chemistry Department, University of Zanjan, P.O. Box 45195-313, Zanjan, Iran
*
Corresponding author e-mail and phone:
[email protected], +98-21-64542606
1
Abstract
Several researchers are focused on preparation of mesoporous silica as drug carriers with high loading efficiency to control or sustain the drug release. Carriers with highly loaded drug are utilized to minimize the time of drug intake. In this study, amino modified SBA-15 was synthesized through grafting with amino propyl triethoxy silane and then loaded with tetracycline hydrochloride. The drug loading was optimized by using the response surface method considering various factors including drug to silica ratio, operation time, and temperature. The drug to silica ratio indicated as the most influential factor on the drug loading yield. Further, a quadratic polynomial equation was developed to predict the loading percentage. The experimental results indicated reasonable agreement with the predicted values. The modified and drug loaded mesoporous particles were characterized by FT-IR, SEM, TEM, X-ray diffraction (XRD), elemental analysis and N2 adsorption-desorption. The release profiles of tetracycline-loaded particles were studied in different pH. Also, Higuchi equation was used to analyze the release profile of the drug and to evaluate the kinetic of drug release. The drug release rate followed the conventional Higuchi model that could be controlled by amino-functionalized SBA-15. Further, the drug delivery system based on amino modified SBA-15 exhibits novel features with an appropriate usage as an anti-bacterial drug delivery system with effective management of drug adsorption and release.
Keywords: Modified SBA-15, drug delivery, response surface methodology, tetracycline hydrochloride, loading
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1. Introduction Controlled drug delivery has been developed in polymer and inorganic materials based systems over the past years [1]. The diverse materials including inorganic silica, carbon based compounds and layered double hydroxides [2–5] as well as polymeric matrix [6–8] have been employed in the drug delivery systems. Recently, there has been an increased interest in amorphous colloidal and mesoporous silica materials as drug carriers in the field of controlled drug release, to meet the need for a prolonged control of drug administration [9– 16]. Mesoporous silica nano particles with a high surface area, large pore volume, uniform porosity, stable aqueous dispersion, excellent biocompatibility, in vivo biodegradability, and capability to be functionalized with different organic compounds, are the attractive candidates for a wide range of biomedical purposes, such as controlled drug delivery, bone tissue regeneration, cell tracking and immobilization of proteins or enzymes. They have been proposed as carriers for drug delivery in 2001 [17]. SBA-15 is an important mesoporous material with large, controlled pore size and highly ordered hexagonal topology [18]. It is more attractive for easy modification and release of large molecules [19]. Various drugs such as antibiotics have been immobilized in mesoporous silica [20]. Pure SBA-15 with silanol groups present on the channel walls simply form the weak intermolecular hydrogen bonds with drugs that are not strong enough to hold drugs and allow them to be released fast. Therefore, introduction of the functional groups on the SBA-15 surface to create the specific host– guest interactions with drugs is important for controlling drug delivery [2]. Accordingly, SBA-15 has been functionalized with alkyl triethoxy silanes for controlling the drug delivery [20-24]. A new technique to diminish the bacteriological risk is the sustain release of antibiotic drug into the infected organs [25]. Tetracycline is the most important broad spectrum antibiotic and tetracycline hydrochloride, a most common medicine, is a crystalline salt with
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bright yellow color. Tetracycline possesses a wide range of antimicrobial activity against Gram-positive and Gram-negative bacteria [26]. Moreover, tetracycline exhibits anticollagenase action, anti-inflammatory activity, and promotes the attachment of fibroblasts and connective tissue to root surfaces [27-28]. It has been used not only in human medicine for the treatment of infectious diseases but also as additives in animal feed to promote their growth [26]. Encapsulation of tetracycline hydrochloride prolongs its action and reduces the number of dosing. It also solves the problem of bitter taste [1]. There have been a few reports in sustain release of tetracycline [26-31]. Also, mesoporous silica has been used to immobilize antibiotics such as amoxicillin [20]. However, to the best of our knowledge, there is no report concerning usage of functionalized SBA-15 as carriers of tetracycline hydrochloride. The purpose of this study was to synthesize and modify SBA-15 with amino propyl triethoxy silane, and to optimize the effective variables on tetracycline
hydrochloride
loading
on
amino
functionalized
SBA-15
statistically.
Furthermore, the drug delivery properties of the drug loaded modified and unmodified SBA15 was investigated and the results thoroughly discussed. Various experiments with different variables including drug to silica ratio (drug/silica), operation time, and temperature were designed through Box-Behnken Design. The drug loading percentage was studied as the experimental response. The correlation between variables and responses were found in polynominal equations, using response surface method (RSM). Finally, the optimum variables and responses were obtained based on the statistical method. The drug loading was then carried out on modified and unmodified SBA-15 at the optimized conditions followed with examination of the drug delivery properties in different pH.
4
2. Experimental 2.1. Materials Poly (ethylene glycol)-block-poly (propylene glycol)-block-poly (ethylene glycol) [Pluronic P123, [(EO)20(PO)70(EO)20]] was purchased from Aldrich. Tetraethyl orthosilicate (TEOS) and 3-(triethoxysilyl) propan-1-amine (APTES), toluene, n-hexane, ethanol and dichloromethane were purchased from Merck (Germany). Tetracycline hydrochloride was prepared from Sina-darou Co., Iran. Toluene, n-hexane and dichloromethane were dried before use and distilled water was used during the experiments.
2.2.Synthesis and functionalization of SBA-15 SBA-15 was prepared as described previously [32]. Briefly, 4.0 g pluronic 123 as a structure-directing agent was dissolved in 30.0 mL distilled water and 116.0 mL HCl 2 M with stirring at 40 °C. 8.5 mL TEOS was then added into the solution stirred for 20 h and remained overnight at 80 °C. The solid product was filtered, washed, and dried at 110 ºC for 18 h. The template of the resultant sample was removed from the as-made mesoporous material by calcination at 550°C for 6 h. The synthesized SBA-15 was then modified. 3.0 g dried SBA-15 (pre-dried at 110 ºC for 24 h) was introduced into 50 mL dry toluene containing 11 mmol (3-Aminopropyl) triethox ysilane (APTES). The mixture was refluxed at 110 ºC for 48 h under N2. The product was washed with toluene, n-hexane and dichloromethane followed by drying at 25 ºC for 24 h and the sample named as SBA-15-NH2. The synthesis and modification procedure is shown in Fig.1.
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2.3.Experimental design RSM was applied to optimize the drug loading of SBA-15. The optimization was designed based on a three-factor Box-Behnken Design with a total of 17 experimental runs involved 3 factorial points and 5 replicates at the center points. Box-Behnken Design (BBD) is a class of rotatable or nearly rotatable second-order design based on three-level incomplete factorial designs. The number of experiments (N) required for the development of BBD is defined as N=2k (k - 1) + C0, where k is the number of factors and C0 is the number of central points. Basically this optimization method consists of the following steps: performing the statistically designed experiment, estimating the regression coefficients in a mathematical model, predicting the response, and checking the adequacy of the model. BBD has been applied for optimization of several chemical, physical, and biological processes [33]. Based on the preliminary experiments and our previous studies, three parameters including time (x1), temperature (x2) and drug to silica ratio (x3) were identified as responsible factors for loading. In view of the feasible loading of SBA-15, the ranges of the factors were determined as follows: loading time (1, 2.5, 4 h), temperature (30, 40, 50 °C), and ratio of drug to silica (0.5, 1, 1.5). The experimental runs for Box-Behnken are reported in Table 1. The response could be related to the selected variables by a second-order polynomial model. In this study, a second-order polynomial was used to generate the response surfaces (Eq. 1).
Y= +∑ +∑ +∑ +
6
(1)
Where Y represents the predicted responses, xi and xj are the coded values of independent variables, ß0 is the intercept coefficient, ßi is the linear coefficients, ßii is the squared coefficients, and ßij is the interaction coefficients [33-34].
2.4.Verification of the results The maximum predicted loading percentage and the optimal conditions were obtained using the second-order polynomial model of RSM. The practical acquired loading percentage was achieved under the optimal conditions. The experimental and predicted acquired results were compared in order to confirm the validity of the model. 2.5.Tetracycline hydrochloride loading The adsorption of pure tetracycline hydrochloride (TC) was studied by using UV-visible spectrophotometer. The TC was loaded via post-impregnation as reported in Table 1 by using three drug to silica ratios of 0.5, 1, and 1.5 and soaking 30 mg SBA-15-NH2 in 3 mL drug solution in distilled water. Finally, the drug loaded solid samples were centrifuged at 4000 rpm for 15 min and washed with distilled water followed by drying at room temperature. The loading percentage was achieved by measuring UV-Vis absorbance at 276 nm [35]. The drug loading percentage of the washed sample were then (DL %) calculated by using Eq. 2;
DL%=
(2)
7
The weight of loaded drug on silica particles was obtained by calculating drug concentration using UV-Vis absorbance of the diluted drug solution and calibration curve.
2.6.In vitro release of TC
In vitro release profile of TC loaded silica was determined dynamically. The SBA-15 in specific sample weight was placed into dialysis bag in buffer solutions of simulated body fluid (PBS, pH 7.2-7.4), acetate buffer (pH 4.8) and hydrochloric acid/potassium chloride buffer (pH 1.2) incubated at 37 ºC under stirring with 100 rpm. Sampling was performed at appropriate time intervals when 4 mL aliquots were taken and replaced with the same volume of the fresh medium. Drug concentrations were determined using the UV-Vis at 276 nm.
2.7.Physico-chemical characterization A Shimadzu UV-1650PC UV-visible spectrophotometer and a FT-IR (Perkin-Elmer, 580 B) in the range between 400 and 4000 cm-1at room temperature from KBr pellets was used. The microscopic pictures were taken by scanning electron microscopy (SEM, Philips XL30, Netherlands). Transmission electron microscopy (TEM) images were obtained with a Philips EM 208. Nitrogen adsorption–desorption isotherms were obtained at 77 K and vapor pressure of 88.51 kPa using an OMNISORP (TM) 100CX VER 1 G adsorption apparatus. Samples were out gassed at 393K for at least 8 h in vacuum prior to measurements. Carbon, hydrogen and nitrogen contents of the samples were determined using a Vario EL III elemental analyzer. The small angle XRD patterns from 0.8 to 5 were recorded with X’Pert Pro MPD (PANalytical) using Cu (Kα = 1.54 Å) radiation.
8
3. Results and discussion 3.1. Scanning electron microscopy (SEM) The mesoporous silica with high specific surface area is an excellent substrate for drug delivery, catalysts and mesoreactors. SBA-15 were synthesized in different morphologies including rope-like, doughnut-like and fiber-like among which rope-like with relatively uniform length of ~1μm is favorable for the mass transfer in adsorption and catalysis [18]. Plenty researches have been worked on the rope like SBA-15 in the field of drug delivery systems [18]. The synthesized SBA-15, in this research, indicates a rope-like morphology with relatively length of ~1 μm as shown in Fig. 2a-b. Functionalized SBA-15 (Fig. 2c-d) and drug loading (Fig. 2e-f) indicated similar morphology as SBA-15. This proved no influence effect of functionalization and drug loading on the particles morphology.
3.2. N2 adsorption–desorption isotherm The nitrogen adsorption/desorption isotherms of SBA-15, SBA-15-NH2 and SBA-15NH2-TC in Fig. 3 indicated typical type IV isotherms according to IUPAC classification showing the characteristic of mesoporous materials isotherms. A steep curve in the capillary condensation regime in isotherm of pure SBA-15 indicates a narrow pore-size distribution and uniform structure of the synthesized SBA-15(Fig. 3a). However, modification and drug loading of SBA-15 leads to gradual increase in the amount adsorbed over the capillary and the shape of the isotherm slightly changes. This implies the uniformity of the SBA-15 decreased to some extent. The capillary condensation region of the adsorption/desorption isotherm for SBA-15-NH2 and SBA-15-NH2-TC is shifted to a lower P/Po value than for normal SBA-15. This indicates smaller pores due to the surface 9
modification and drug loading into the pores. The obtained results are confirmed by the smaller pore diameter and pore volume of SBA-15-NH2 and SBA-15-NH2-TC as given in Table 2.
The pore size distribution was obtained from BJH adsorption data. The pore size distribution of TC loaded functionalized sample was broader than the pure SBA-15 and SBA-15-NH2 (Fig. 3b) indicating the effect of drug loading on broadening of the pore size distribution due to non-uniform adsorption of drugs into silica particles pores. The textural properties of the samples are summarized in Table 2 derived from nitrogen adsorption-desorption isotherm plot and pore size distribution curve. By introduction of amine functional groups, the BET surface areas gradually decrease from 489.00 m2g-1 for SBA-15 to 302.32 m2g-1 for SBA-15-NH2, and further to 154.57 m2g-1 for SBA-15-NH2-TC (Table 2). The reduction of pore size and pore volume proved the adsorbed drug loaded into the mesoporous structures. Accordingly, functionalization and drug loading leads to lower pore size and volume and also surface area of the samples. Therefore, the changes within the pore size, pore volume and surface area of the samples are within the expected results [20, 36-39].
3.3. X-ray diffraction
Fig. 4a represents the small angle X-ray diffraction (SAXRD) pattern of the samples. The SAXRD patterns of SBA-15, SBA-15-NH2 and SBA-15-NH2-TC exhibit three well-resolved 10
peaks. The peaks can be indexed as (100), (110), (200) associated with hexagonal symmetry with a high order, two-dimensional hexagonal p6mm mesostructure as reported previously [40-41]. After introducing 3-aminopropyl groups and tetracycline, the SAXRD patterns of parent SBA-15 silica are still remaining (Fig. 4a), indicated no influence on the mesoporous structure of parent SBA-15 through introduction of organic material. However, a lower intensity of (1 0 0) diffraction peak observed due to the contrast matching between the organic functional groups and inorganic framework [20, 42]. The diffraction peaks of (1 0 0), (1 1 0) and (2 0 0) related to SBA-15-NH2 and SBA-15NH2-TC slightly shifted to higher angles comparing with SBA-15 accounted for the contraction of the host framework during surface modification and drug loading inside the channels [40, 42]. All results indicate no damage to the high degree ordering of hexagonal symmetry structure after modification and drug loading. The amino modified and drug loaded particles (SBA-15-NH2 and SBA-15-NH2-TC) comparing to parent SBA-15 indicate thicker wall (bp) confirming the amino groups attachment and drug loading into SBA-15 mesochannels (Table 2). The results revealed that the functionalization and drug loading process were successful and did not damage the pore structure of the particles. Furthermore, they are in accord with BET analysis results.
3.4. Transmission electron microscopy (TEM) TEM images of SBA-15, APTES-functionalized SBA-15 and drug loaded particles in Fig. 4(b-d) clearly reveals that SBA-15 (Fig. 4b) and SBA-15-NH2 (Fig. 4c) have well-ordered arrays along the mesochannels axis suggesting the hexagonal mesostructure maintained during functionalization [42]. Transmission electron microscopy (TEM) is a powerful tool for
11
imaging nanostructures, yet its capability is limited with respect to the imaging of organic materials because of the intrinsic low contrast efficiency. Accordingly, the images of SBA15-NH2-TC with high magnification was not clear enough [43]. Although, the ordered periodic hexagonal arrays of channels cannot be clearly distinguished inside the particle in Fig. 4d, retaining some hexagonal mesochannels in its domain. Thus, TEM image of SBA15-NH2-TC (Fig. 4d) indicates the presence of drug molecules on the surface and inside the mesochannels of silica particles.
3.5. Elemental analysis Elemental analysis was applied to determine the nitrogen content of the samples (Table 2). Presence of three ethoxy groups in APTES means that they can react with the silica surface via one, two or all alkoxy groups. Although the condensation of the first ethoxy group of a triethoxysilane readily occurs, the condensation of the second and third groups attached to the same silicon atom become increasingly more difficult due to steric hindrance. Accordingly, the possibility of the entire reaction decreases [44]. Further, the C/N molar ratio should be 3 if the complete reaction occurred. However, the C/N molar ratio in SBA-15-NH2 sample was 4.7 obtaining from the elemental analysis data. This suggests the approximate stoichiometry between the reacted silanol groups of SBA-15 and the silylating agent of 2:1 indicating linkages of great number of aminopropyl silane chains to the pore wall surface only through tow Si–O–Si bond and two of three ethoxy group reacted with Si-OH groups of SBA-15 surface [45] (Fig. 5).
Further, significant increase in the nitrogen and carbon content after drug loading on the amino modified SBA-15 samples confirmed the adsorption of drug molecules into SBA-1512
NH2 particles. The calculated DL% for SBA-15-NH2-TC sample is around 43.1% which is in agreement with the UV-visible results.
3.6. FTIR spectra FTIR spectra of SBA-15 and SBA-15-NH2 carriers before and after TC loading are shown in Fig. 6. The asymmetric stretching vibrations (Si–O–Si) appear at 1075 cm-1 which is associated with the formation of a condensed silica network [39]. A peak centered at 478 cm−1 is assigned to Si–O–Si bending modes [35]. The broad peak at 3400 cm−1 is attributed to O–H stretching vibration mode. C-H stretching bands of propyl groups are appeared at 2897 cm−1 and 2980 cm−1 for SBA-15-NH2, [40-41]. Furthermore, the modification of SBA-15 with APTES results in the appearance of the band at 1560-1640 cm-1 are attributed to N–H bending vibrations of aminopropyl anchored on the surface of mesoporous support [17]. These characteristic bands confirmed the introduction of aminopropyl groups of APTES into SBA-15. The assignment of the main bands related to FTIR spectra of tetracycline can be found at 1675 cm−1for C= O, at 1650–1600 cm−1 for C= C stretching of aromatic ring, at 1460–1310 cm−1 for OH bending and at 1250–1200 cm−1 for C-C stretching and bending, N-H bending and C-N stretching [46–49, 35]. FTIR spectra of SBA-15-NH2 are very similar before and after drug loading. The characteristic bands of TC are identified in FTIR spectra of SBA-15NH2-TC such as 1675 cm−1 for C=O, 1650–1600 cm−1 and 3030 cm−1 for C=C and C-H stretching of aromatic ring beside the characteristic bands of SBA-15-NH2 such as asymmetric stretching vibrations (Si–O–Si) at 1075 cm-1 and C-H stretching bands of propyl groups at 2897 cm−1 and 2980 cm−1 [40-41]. This confirms the adsorption of drug on the surface of silica particles. The carbonyl bands at 1685 cm−1 seems to shift to lower wave 13
numbers as a result of hydrogen bonding between amino groups of SBA-15-NH2 and TC molecules.
3.7. Drug loading To evaluate the influences of loading time, temperature and drug/silica on DL%, the design matrix of experimental conditions with the corresponding responses was fitted to a polynomial model (Table 1) and a quadratic model was suggested. Therefore, the mathematical equation proposed for this response (in terms of coded values) is: Loading %= 27.80+0.29A -2.79B + 11.08C + 5.53AB -1.94AC - 5.07BC -0.58A2-4.12B21.18C2
(3)
Table 3 shows the ANOVA results of the regression model. The effect and importance of the variables can be determined by the sign and value of the coefficients [5051]. Thus, the negative coefficient of B (temperature) indicates an opposite influence on DL%, while the positive coefficient of C (drug/silica) reveals the linear effect. The coefficient of factor B (2.79) is smaller than the coefficient of factor C (11.08), which explains the importance of drug/silica rather than the temperature in the loading. Thus, the
14
main factor affecting the loading process is drug/silica followed by temperature. The time of loading has a minimum effect on the loading process. The significance of each coefficient was checked using F-test. Values of ‘‘Prob> F’’ less than 0.05 indicates that the model terms are significant [50]. Thus, the model F-value of 74.38 reveals the significance of model and there is only a 0.01% chance that a ‘‘Model Fvalue’’ this large could occur due to noise. The "Lack of Fit F-value" of 0.62 implies the Lack of Fit is not significant relative to the pure error. In this case B, C, AB, AC, BC, B2 are the significant model terms. In order to improve the model, the insignificant coefficients were eliminated and the final model was refined as Eq (4): Loading %=27.80-2.79B+11.08C+5.53AB-1.94AC-5.07BC-4.12B2
(4)
Accordingly, in the modified model, the insignificant factors were omitted. Table 4 shows the analysis of variance (ANOVA) for the Quadratic Model for the adjusted model of drug loading percentage. The accuracy and general ability of the above polynomial model could be evaluated by the coefficient of determination R2 and the adjusted R2. The coefficient of determination (R2) of 0.9836 with an adjusted R2 (0.9738) is in reasonable agreement with the predicted R2 (0.9599). The coefficient of determination R2 for DL% (see Table 4), indicates that the statistical model can explain 98.36% of the variability in the response and only about 1.64% of the total variation cannot be attributed to the independent variables. The R2 value is always between 0 and 1. The proximity of the R2 to 1.0 showed that the model is strong and it can predict the response carefully. A regression (R2), higher than 0.90, is usually considered as a very good correlation. The results in Tables 5 and 6 showed that the adequate precision and model F ratio improved due to the model adjustment. Table 4 indicates significance of all coefficients.
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Fig.7a demonstrates the effect of time and temperature at the center point of drug/silica on loading percentage. Increasing temperature led to lower DL%. Tanis et al. revealed that the adsorption of TC on iron oxides-coated quartz increased with increasing temperature because of the important role of the semi-chemisorption process in TC-adsorbent [52]. These are significantly different from the reported results by Turku et al. showed lower TC adsorption on silica by increasing temperature (physical adsorption) [53]. Li et al. indicated similar results as reported by Turku et al. [54]. Further, increasing temperature leads to more drugs in water rather than in porous silica as the solubility of TC increases in water at high temperature [55]. The interaction of time and drug/silica on loading percentage at constant temperature is shown in Fig. 7b. This shows rapid increase of DL% with higher drug/silica at different loading times especially at short time. After an adequate loading time, some loaded drugs release from the mesoporous silica leads to lower final loading [56]. Fig. 7c indicates the effect of drug/silica and temperature at 2.5 h on drug loading percentage. More drug/silica at different temperatures leads to the higher DL%, especially at low temperature.
Fig. 7d also indicates the loading percentage (the response) versus those of the empirical model. The predicted data of the response from the empirical model agreed well with the observed ones in the range of the operating variables. The model predicted the optimal values of the three variables; time=2.19 h, temperature=33 ºC and drug/silica=1.5 corresponding to DL%=41.9. 16
In order to confirm the optimized conditions practically, three additional experiments were performed using the predicted loading conditions. The mean value of loading was 42.3% agreed well with the predicted value. This demonstrates the validity of the response model. The statistical optimization used in this research showed the highest loading percentage comparing with other results on the loading of tetracycline in chitosan [35] and rectorite [46]. The drug loading was carried out at the optimum conditions for SBA-15 to compare the drug loading into modified and un-modified SBA-15. The loading percentage for unmodified SBA-15 was 20.6%, implying enhanced drug loading in the modified silica particles. Thus, the organophilicity of SBA-15 improves the drug loading in the presence of organic groups of amino propyl [32, 37]. Additionally, tetracycline possibly adsorbs to the surface of SBA-15NH2 by hydrogen bonding between NH2 groups of amino propyl groups and C=O and OH groups of the drug molecules (Fig. 3).
3.8. Drug release profile and the kinetic studies In–vitro drug release was performed in three buffer solutions under stirring at 37 °C and different pH. The plots of cumulative drug release against time are shown in Fig. 8a. The rate of drug release is lower for functionalized SBA-15 possibly due to the formation of strong hydrogen bonds between TC and SBA-15-NH2. SBA-15-NH2 shows the advantage of the gradual release over a longer time even after 48 h as demonstrated in Fig. 8a [57-58]. The release behavior of different particles are similar in PBS and pH=4.8, however the burst release was observed at pH=1.2 due to the changes of the zeta potential of TC in different pH. The release behavior is similar in pH=7.4 and pH=4.8 due to the zwitterion behavior of particles in pH 3.3 and 7.7. However, the dimethyl ammonium group of TC is protonating at
17
pH below 3.3 resulting in a cationic charge [58]. On the other hand, amine groups of SBA15-NH2 are protonating in acidic conditions. Thus, the repulsive forces between amine groups of SBA-15 and TC lead to sudden and burst release of drug from amino modified SBA-15.
Most of the works on the drug release kinetics from mesoporous carriers are explained using Higuchi model due to chemical and physical entrapment of the drug [59-63]. Thus, the drug release from mesoporous materials is diffusion control similar to other drug delivery systems such as biopolymers or hydrogels [2]. Higuchi was the first to derive an equation to describe the release of a drug from an insoluble matrix as the square root of a time-dependent process based on Fickian diffusion (Eq. 5).
Qt= KHt1/2
(5)
Where Qt is the drug released in time t and KH is the release rate constant for Higuchi model [2]. Here, the kinetic parameters of TC release from modified and un-modified SBA-15 are evaluated by Higuchi model. The results of Higuchi model for SBA-15 and SBA-15-NH2 at pH=4.8 and 7.4 agreed well with the experimental data for drug release as shown in Table 5 and Fig. 8b. However, this model doesn’t fit with the experimental data at pH=1.2 because of the drug initial burst release. The value of KH revealed very high release rate for unmodified SBA-15 compared to the modified one. In-vitro study showed that the amino modification of SBA-15 could delay the release of TC as reported by others for various drugs [32, 40, 5657].This is very important in the field of controlled drug delivery systems. This system can be used locally by implantation or injection. The optimum pH for human blood and body tissues 18
is about 7.2. Thus, the gradual drug release from silica particles was carried out under the same pH. The application of local drug delivery system using implantation or injection has advantages over conventional drug therapies due to lower harmful side effects [64]. Application of silica particles with the size of 700 nm to 4 μm by injection method has been reported in the literature previously [65-67]. Accordingly, the synthesized SBA-15 with the particle size of 1~μm can be used by subcutaneous injection. Furthermore, mesoporous silica nano particles with excellent biocompatibility are the attractive candidates for a wide range of biomedical purposes, such as controlled drug delivery, bone tissue regeneration, cell tracking and immobilization of proteins or enzymes and the histopathological studies by researchers proved that impregnated reservoirs of SBA-15 exhibit benign local biocompatibility and macrophages showed little or no toxicity from SBA-15 particle [67-68].
4. Conclusions SBA-15 was synthesized and modified with (3-Aminopropyl) triethoxysilane and successfully loaded with tetracycline hydrochloride. The drug adsorption decreases specific surface area and specific pore volume of amino modified SBA-15. The percentage of drug loading on modified SBA-15 was optimized by response surface methodology. The maximum TC loading (42.3%) was achieved under the optimized loading conditions (time=2.19 h, temperature=33 ºC and drug/silica=1.5). In-vitro release studies revealed that the modified SBA-15 has higher loading percentage and slower release rate, especially at pH=7.4 and 4.8. However, the burst release occurs at pH=1.2 as a result of the repulsive forces between SBA-15-NH2 and TC. Overall, loading of tetracycline hydrochloride on modified SBA-15 prolongs its performance and decreases the number of dosing.
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Acknowledgment We gratefully acknowledge financial support from the Iran National Science Foundation (INSF). The research leading to these results has received funding from INSF under grant agreement number 92026485.
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24
Table captions
Table 1. Box-Behnken experimental design of the independent variables along with the observed values of the response (Y). Table 2. Structural parameters and Elemental analysis of SBA-15, SBA-15-NH2 and SBA-15-NH2-TC Table 3. Analysis of variance for drug loading percentages. Table 4. Analysis of variance for the adjusted model of drug loading percentage. Table 5. TC release kinetic parameters for modified and un-modified SBA-15 in different media
25
Figure captions Fig.1. Schematic of synthesis and amine functionalization of SBA-15 Fig. 2. SEM images of SBA-15 (a, b), SBA-15-NH2 (c, d), SBA-15-NH2-TC (e, f) Fig. 3. BET isotherms (A) and pore size distribution (B) of SBA-15, SBA-15-NH2 and SBA15-NH2-TC Fig. 4. (a) Small angle XRD pattern and TEM images of (b) SBA-15, (c) SBA-15-NH2 and (d) SBA-15-NH2-TC Fig. 5. Schematic of tetracycline loading on SBA-15-NH2 Fig. 6. FTIR spectra of SBA-15, SBA-15-NH2, tetracycline and SBA-15-NH2-TC Fig.7. Response surface and contour plots of the combined effects of (a) temperature and time (at drug/silica=1), (b) drug/silica and time (at temperature = 40 °C), (c) drug/silica and temperature (at time= 2.5 h) on loading percentages and (d) observed DL% vs. the predicted DL% Fig.8. Cumulative release percentage of tetracycline from SBA-15 and SBA-15-NH2 in different buffers (a) and Higuchi model of TC release from SBA-15 and SBA-15-NH2 at different pH (b)
26
27
28
29
30
31
32
33
34
Table 1. Box-Behnken experimental design of the independent variables along with the observed values of the response (Y).
Std
Run
Factor 1
Factor 2
Factor 3
Response
A:
B:
C:
Loading
Time(h)
Temperature(°C)
Drug/Silica (%)
SD
1
2
1
30
1
31.9
0.01
3
7
1
50
1
15.1
0.05
5
15
1
40
0.5
12.2
0.04
7
4
1
40
1.5
37.8
0.08
9
14
2.5
30
0.5
8.8
0.02
10
10
2.5
50
0.5
13.6
0.11
11
6
2.5
30
1.5
41.7
0.15
12
9
2.5
50
1.5
26.0
0.09
13
12
2.5
40
1
25.0
0.01
14
16
2.5
40
1
28.3
0.01
15
5
2.5
40
1
28.8
0.10
16
3
2.5
40
1
28.5
0.02
17
11
2.5
40
1
28.4
0.02
2
1
4
30
1
20.0
0.02
4
13
4
50
1
25.3
0.01
6
17
4
40
0.5
18.1
0.04
8
8
4
40
1.5
35.9
0.11
35
Table 2. Structural parameters and elemental analysis of SBA-15, SBA-15-NH2 and SBA-15-NH2TC. SaBET
Vbp
dcp
dd100
bep
C
H
N
(m2/g)
(ml/g)
(A°)
(A°)
(A°)
(wt. %)
(wt. %)
(wt. %)
SBA-15
489
0.85
74
91
31
<0.01
0.9
-
SBA-15-NH2
302
0.76
62
90
42
10.3
2.7
2.5
SBA-15-NH2-TC
154
0.42
54
88
48
24.6
2.6
3.5
Support
a:SBET, BET specific surface area; b:Vp, specific pore volume; c: dp, average pore diametere, obtained from adsorption BJH data, d: d100, XRD inter planar spacing; e: bp, pore wall thickness, bp= (a0 −dp), a0 = (2/√3) d100.
36
Table 3. Analysis of variance for drug loading percentages. Source
Sum of
df
Squares
Mean
F
p-value
Square
Value
Prob> F
significance
1368.17
9
152.02
74.38
< 0.0001
significant
A-time
0.66
1
0.66
0.32
0.5873
not significant
B-tempreture
62.38
1
62.38
30.52
0.0009
significant
C-drug/silica
982.13
1
982.13
480.51
< 0.0001
significant
AB
122.10
1
122.10
59.74
0.0001
significant
AC
15.05
1
15.05
7.37
0.03
significant
BC
102.82
1
102.82
50.30
0.0002
significant
1.44
1
1.44
0.70
0.4292
not significant
71.45
1
71.45
34.96
0.0006
significant
C
5.91
1
5.91
2.89
0.1329
not significant
Residual
14.31
7
2.04
-
-
-
Lack of Fit
4.54
3
1.51
0.62
0.6382
not significant
Pure Error
9.77
4
2.44
-
-
-
-
-
-
-
Model R-Squared: 0.9897 Adj R-Squared: 0.9763 Pred R-Squared: 0.9364 Adeq Precision: 29.457
A
2
B2 2
Correlation Total
1382.48 16
37
Table 4. Analysis of variance for the adjusted model of drug loading percentage. Source
Sum of
df
Squares
Mean
F
p-value
significance
Square
Value
Prob> F < 0.0001
significant
0.0004
significant
1359.84
6
226.64 100.09
B-tempreture
62.38
1
62.38
C-drug/silica
982.13
1
982.13 433.75
< 0.0001
significant
AB
122.10
1
122.1
53.93
< 0.0001
significant
AC
15.05
1
15.05
6.65
0.0275
significant
BC
102.82
1
102.82
45.41
< 0.0001
significant
2
B
75.34
1
75.34
33.27
0.0002
significant
Residual
22.64
10
2.26
-
-
-
Lack of Fit
12.88
6
2.15
0.88
0.5782
not significant
Pure Error
9.77
4
2.44
-
-
-
-
-
-
-
Model R-Squared: 0.9836 Adj R-Squared: 0.9738 Pred R-Squared: 0.9599 Adeq Precision: 33.451
Correlation Total
1382.48 16
27.55
Table 5. TC release kinetic parameters for modified and un-modified SBA-15 in different media
Silica
SBA-15
SBA-15-NH2
pH
Higuchi k
%R2
7.4
1.58
99.52
7.4
0.758
96.79
4.8
0.82
99.59
1.2
0.31
76.85
38
Graphical abstract
Highlights
•
Tetracycline hydrochloride loaded into amino modified SBA-15 optimized with RSM.
•
Drug to silica ratio was the main factor on the drug loading.
•
Drug release rate followed the conventional Higuchi model.
39
