European Journal of Pharmaceutical Sciences 72 (2015) 12–20
Contents lists available at ScienceDirect
European Journal of Pharmaceutical Sciences journal homepage: www.elsevier.com/locate/ejps
Redox-responsive mesoporous silica as carriers for controlled drug delivery: A comparative study based on silica and PEG gatekeepers Ying Wang, Ning Han, Qinfu Zhao, Ling Bai, Jia Li, Tongying Jiang, Siling Wang ⇑ Department of Pharmaceutics, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenhe District, Shenyang, Liaoning Province 110016, PR China
a r t i c l e
i n f o
Article history: Received 6 November 2014 Received in revised form 15 January 2015 Accepted 11 February 2015 Available online 18 February 2015 Keywords: Redox-responsive Mesoporous silica nanoparticles Gatekeepers Controlled drug delivery Biocompatibility
a b s t r a c t Hybrid mesoporous silica nanoparticles (MSNs) modified with polymer polyethylene glycol (PEG) through the biodegradable disulfide bonds were prepared to achieve ‘on demand’ drug release. In this system, PEG chains were chosen as the representative gatekeepers that can block drugs within the mesopores of MSNs. After the addition of glutathione (GSH), the gatekeepers were removed from the pore outlets of MSNs, followed by the release of encapsulated drugs. In this research, the effects of grafting density of gatekeepers on the drug release and biocompatibility of silica carriers were also investigated. First, PEG modified MSNs were prepared by the condensation reaction between the carboxyl groups of MSN and the hydroxyl of PEG. The structure of the resultant MSN-SS-PEG was characterized by transmission electron microscopy (TEM), nitrogen adsorption/desorption isotherms analysis and Fourier transform infrared spectroscopy (FTIR). Rhodamine B (RhB) as the model drug was loaded into MSNs. The in vitro assay results indicated that RhB was released rapidly after the addition of 10 mM GSH; M1-SS-PEG had the best capping efficiency compared with M0.5 and M1.5 groups. Moreover, hemolysis assay, serum protein adsorption and cell viability test indicated that with the increase of PEG grafting density, the biocompatibility of silica carriers increased. Ó 2015 Elsevier B.V. All rights reserved.
1. Introduction In the past decades, many studies have focused on controlled drug delivery systems (CDDSs) since they can achieve site-selective, autonomously-modulated release (Zhu et al., 2005, 2007; Chu et al., 2013; Simões et al., 2014). It is highly desirable that ideal CDDSs should deliver therapeutic drugs without any premature release during the blood circulation or in normal tissues prior to reaching the special site and release drugs rapidly after being taken up by affected tissues (Zhang et al., 2012, 2013). Therefore, designing CDDSs which can release cargos on demand to protect healthy organs and reduce unexpected side effect is becoming very crucial. In recent years, mesoporous silica nanomaterials have attracted much attention as drug delivery carriers due to their unique advantages, such as high specific surface area and large pore volume, homogeneous controllable particle size, easily modified surface and good biocompatibility (Wang et al., 2013; Zhang et al., 2010, 2014; Mellaerts et al., 2008). Moreover, mesoporous silicaassisted drug delivery systems have increasingly focused on stimuli-responsive CDDSs that can release encapsulated drugs from
⇑ Corresponding author. Tel./fax: +86 24 23986348. E-mail address: [email protected] (S. Wang). http://dx.doi.org/10.1016/j.ejps.2015.02.008 0928-0987/Ó 2015 Elsevier B.V. All rights reserved.
silica carriers at the expected site in response to various stimuli including pH, redox potential, temperature, photoirradiation, and biomacromolecule (Yang et al., 2012; Meng et al., 2010; Zhou et al., 2007; He et al., 2012; Bernardos et al., 2010). Many available functionalized mesoporous silica nanoparticles (MSNs) have been developed to regulate drug release by capping the outlets of mesopore or encapsulating drugs within the porous channels of MSNs. In general, mesopore-entrapped drugs cannot leach out from the MSNs unless the drug delivery system is exposed to certain stimuli that can trigger the opening of the pore entrances. Among various stimuli used for CDDSs, redox potential has emerged as an appealing trigger. It is demonstrated that glutathione (GSH) is the most abundant reducing agent in vivo and the concentration of GSH has a significant difference between the extracellular (2 lM) and intracellular (10 mM) environment. Furthermore, intracellular GSH levels in the tumor tissues are at least 4-fold higher than those in normal tissues (Saito et al., 2003). Since disulfide bonds can be reduced by GSH, redox potentials based on the disulfide bonds are expected as a stimulus to achieve ‘on demand’ drug release. Recently, a few redox-responsive CDDSs using MSNs as carriers have been reported. For example, Liu et al. (2008) reported a disulfide cross-linked polymeric network as a gatekeeper that can be removed by GSH, thus allowing the release of encapsulated drugs. Cui and co-workers have
Y. Wang et al. / European Journal of Pharmaceutical Sciences 72 (2015) 12–20
developed a novel polyethylene glycol (PEG) modified MSN-based CDDS for the first time, in which PEG chains were linked to the MSNs through disulfide bonds. In this system, PEG shell can block drugs tightly and open the pore outlets upon GSH conditions (Cui et al., 2012). More recently, Liang Chen and co-workers (Chen et al., 2014) also used PEG as a gatekeeper for GSH-mediated drug delivery system. In the research, they used SBA-15 (3D cubic MSN) as the drug carrier and compared it with MCM-41 (2D hexagonal MSN). The results obtained suggested that both types of MSNs-assisted CDDSs can provide controlled drug release. In the previous studies, researchers have focused on designing various gatekeepers based on MSNs instead of considering the grafting density of gatekeepers. Zhong Luo and Kaushik Patel et al. have used collagen and cyclodextrine as gatekeepers to realize controlled drug release, but the grafting density of gatekeepers has not been investigated in their studies (Luo et al., 2011; Patel et al., 2008; Zhang et al., 2014). In our research, for the first time, we investigated the effects of grafting density of gatekeepers on the drug release and the biocompatibility of silica carriers. Here, PEG chains were still chosen as the representative gatekeepers due to their good biocompatibility and suited molecular weight to cap the entrance of MSNs. Moreover, PEG modified MSNs have the advantages such as prolonging the blood circulation time, greatly improving the enhanced permeability and retention (EPR) effect and preventing the endocytosis by the reticuloendothelial system (RES) Meng et al., 2011. We have developed an innovative and convenient approach to prepare PEG-linked MSNs with disulfide bonds for smart drug delivery (Fig. 1). In this system, 2,20 -dipyridyl disulfide was first covalently bonded with thiol-functionalized MSN to yield MSN-SS-Py, which can be further reacted with 3-mercaptopropionic acid to achieve MSN-SS-COOH nanoparticles. Then, the PEG chains were covalently combined to above MSN-SS-COOH nanoparticles to obtain the redox-responsive MSNs (MSNSS-PEG). The redox-responsive drug delivery system has a good capping efficiency and the influence of surface modification of MSNs on their biocompatibility has also been investigated. 2. Experimental 2.1. Materials 3-Mercaptopropyltrimethoxysilane (MPTMS), 3-mercaptopropionic acid, rhodamine B (RhB), methoxypolyethylene glycol (PEG,
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5000), 2,20 -dipyridyl disulfide, N-(3-Dimethylaminopropyl)N0 -ethylcarbodiimide hydrochloride (EDCHCl), 3-(4,5)dimethylthiahiazo(-z-y1)-3,5-diphenytetrazoliumromide (MTT), 4-dimethylaminopyridine (DMAP), glutathione (GSH) were purchased from Aladdin Chemistry, Co. (Shanghai). Cetyltrimethylammonium bromide (CTAB), tetraethyl orthosilicate (TEOS), anhydrous ethanol, methanol, N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), hydrochloric acid (HCl, 36.5%), sodium hydroxide (NaOH), potassium phosphate monobasic (KH2PO4) were purchased from Shan Dong Yu Wang Reagent Company (China). All the reagents were used as received. 2.2. Characterization techniques Transmission electron microscopy (TEM) images of nanoparticles were performed on Tecnai G2 20 (FEI, USA) microscope at an acceleration voltage of 200 kV. Fourier transform infrared spectroscopy (FTIR) spectra were recorded on a Bruker IFS 55 (Switzerland). N2 adsorption–desorption isotherms were conducted on a SA3100 surface area analyzer (Beckman Coulter, USA). X-ray Photoelectron Spectroscopy (XPS) was performed on ESCALAB250 (Thermo VG, USA) spectrometer with an Al Ka monochromatic source (1486.6 eV, 150 W). Thermogravimetric analysis (TGA) was carried out by a TGA-50 instrument (Shimadzu, Japan) at a heating rate of 10 °C/min under a nitrogen purge of 40 mL/min. 2.3. Synthesis of MSN-SH MSN-SH was prepared according to the reported method (Mortera et al., 2009). Firstly, CTAB (1 g) was dissolved in 480 mL of deionized water. Then, 3.5 mL of 2 M NaOH solution was introduced to CTAB solution under vigorous stirring at 80 °C. After that, TEOS (5 mL) was added dropwise to the above solution and followed by addition of different amount of MPTMS (0.5 mL, 1 mL and 1.5 mL, respectively). Then, the mixture was stirred for 2 h at 80 °C. To remove the template CTAB, the resultant particles were dispersed in a solution of HCl (18 mL, 37.4%) and methanol (320 mL), and refluxed for 24 h. After that, an ATS AH110D homogenizer (ATS Engineer Inc., Shanghai, China) was performed to homogenize the resulting precipitate. The MSN-SH particles were collected by centrifugation (6000 rpm, 15 min), washed with methanol and dried under vacuum for 12 h. 2.4. Synthesis of MSN-SS-COOH MSN-SH (1 g) and excessive 2,20 -dipyridyl disulfide were first dispersed in 25 mL of anhydrous ethanol containing acetic acid (1 mL). The mixture was stirred at room temperature for 24 h. The resultant MSN-SS-Py was isolated by centrifugation and washed unreacted 2,20 -dipyridyl disulfide with anhydrous ethanol. Under the nitrogen atmosphere, 0.8 mL of acetic acid was added to a DMF (20 mL) solution of MSN-SS-Py, followed by addition of excessive 3-mercaptopropionic acid. The reaction was kept under stirring at 40 °C for 24 h. The resulting MSN-SS-COOH was separated by centrifugation, washed with water and anhydrous ethanol and dried under vacuum (He et al., 2013). 2.5. Synthesis of MSN-SS-PEG
Fig. 1. Schematic illustration of drug loading and release.
MSN-SS-COOH (0.2 g), abundant EDCI and DMAP (-SS-COOH/ EDCI/DMAP molar ratio was 6:12:1) were dispersed in 20 mL of deionized water under stirring for 2 h, followed by addition of PEG (0.1 g). After the mixture was stirred for 24 h, the resulting MSN-SS-PEG was collected by centrifugation, washed extensively with water and anhydrous ethanol and finally dried under vacuum.
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Fig. 2. TEM images of (A) MSNs and (B) MSN-SS-PEG nanoparticles.
Fig. 3. Nitrogen adsorption/desorption isotherms and pore size distribution of MSNs.
2.6. Drug loading and release studies
2.7. Hemolysis assay
Typically, MSN-SS-COOH (0.2 g) were first dispersed in 5 mL of deionized water, followed by adding a certain amount of EDCI and DMAP (-SS-COOH/EDCI/DMAP molar ratio was 6:12:1) under stirring for 1.5 h to achieve carboxyl active ester for further connecting with PEG. Then, 5 mL of RhB (4 mg/mL) solution was loaded into above MSN-SS-COOH under stirring for 6 h. After that, PEG (0.2 g) was added to the above solution, stirred for 12 h, centrifuged and washed extensively to achieve RhB-loaded MSN-SS-PEG (called as MSN-SS-PEG@RhB). Finally, MSN-SS-PEG@RhB was dried under vacuum. To investigate the redox-responsive drug release of different grafted amounts of MSN-SS-PEG systems, RhB-loaded MSN-SSPEG (25 mg) was dissolved into PBS (pH 7.4) with/without 10 M GSH, shaking at 150 rpm (37 °C). At predetermined time intervals, 3 mL of PBS was taken out for UV–Vis measurement (UV-2000, Unico, USA) and then replaced with an equal volume of fresh buffer. The cumulative released amount was determined from the absorbance at 554 nm according to a standard curve of RhB in the same buffer.
The rabbit red blood cells (RBCs) were obtained by centrifugation and suction to remove the serum from the blood and further washed five times with sterile normal saline solution. Following the last wash, the cells were diluted to 2/100 of their volume with normal saline solution. Then 2 mL of the diluted RBCs suspension was mixed with: (a) 2 mL nanoparticles which were dispersed into normal saline solution at the different concentrations ranging from 40 to 800 lg/mL; (b) 2 mL of normal saline solution as a negative control; (c) 2 mL of distilled water as a positive control. Then the mixtures were kept still for 4 h at room temperature. Finally, the mixtures were centrifuged for 10 min at 1000 r/min and the absorbances of supernatant at 541 nm were recorded by the UV–vis absorption spectrophotometer. 2.8. Serum protein adsorption 60 mg of BSA was completely dissolved in 100 mL of distilled and deionized water under stirring mildly. 0.8 g of NaCl, 0.02 g of KCl, 0.115 g of Na2HPO4 and 0.02 g of KH2PO4 were dissolved in
Y. Wang et al. / European Journal of Pharmaceutical Sciences 72 (2015) 12–20
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3. Result and discussion 3.1. Synthesis and characterization of MSN-SS-PEG
Fig. 4. Size distribution of MSN, MSN-SS-COOH and MSN-SS-PEG.
500 mL of distilled and deionized water to prepare phosphate buffered saline (PBS). Pure MSN and M-SS-PEG were dispersed into 5 mL of PBS solutions at a concentration of 1 mg/mL, and then 5 mL of BSA solutions were added. Meanwhile, a control group was prepared by mixing 5 mL of PBS and 5 mL BSA solutions. After shaking at 150 rpm (37 °C) for 4 h, the mixed solutions were centrifugalized, and then the supernatant was collected. The concentrations of residual BSA were determined by the dying method with coomassie brilliant blue. 0.5 mL of collected supernatant, 0.5 mL of coomassie brilliant blue and 2 mL of distilled water were mixed to measure the absorbances of residual BSA at 555 nm. A calibration curve was drawn by using BSA solutions with a series of concentrations. BSA adsorbed on MSN and MSN-SS-PEG were calculated according to
BSA adsorbance ð%Þ ¼ ðC b Ce Þ 6 V=m
ðA:1Þ
where Cb and Ce were initial BSA concentration in a control group and the residual BSA concentration after BSA adsorption; V was the total solution volume (10 mL); m was the weight of pure MSN or MSN-SS-PEG added into solutions.
MSN-SH was synthesized according to the previous method by a one-pot method (Mortera et al., 2009). Subsequently, 2,20 -dipyridyl disulfide and 3-mercaptopropionic acid were used to obtain MSN-SS-COOH by the disulfide exchange reaction. Then, PEG was covalently grafted onto external surface of MSNs by the condensation reaction between the carboxyl groups of MSN and the hydroxyl of PEG to get MSN-SS-PEG. In this research, PEG was regarded as a significant gatekeeper to impede the drug release. In order to compare the drug release properties of MSNs with different amount of grafted gatekeepers, 0.5 mL, 1 mL and 1.5 mL of MPTMS were used respectively to obtain the thiol-modified MSNs (called as M0.5-SH, M1-SH, M1.5-SH), carboxyl-modified MSNs with cleavable disulfide bonds (donated as M0.5-SS-COOH, M1-SS-COOH, M1.5-SS-COOH) and PEG-grafted MSNs (called as M0.5-SS-PEG, M1-SS-PEG, M1.5-SS-PEG). The structures of MSNs and MSN-SS-PEG were monitored by transmission electron microscopy (TEM), Brunauer–Emmett– Teller (BET) analysis and Barret–Joyner–Halenda (BJH) analysis, and dynamic light scattering (DLS). As displayed in Fig. 2A, the mean diameter of synthesized MSNs was about 100 nm with 2– 3 nm of the mesopore size. A highly ordered mesoporous network could be clearly seen in the image. In Fig. 2B, the mesoporous network of MSN-SS-PEG was still observed indicating that the polymer PEG did not destroy the mesoporous property of MSNs. The surface area and average pore diameter of MSNs and MSN-SSPEG were measured by BET nitrogen adsorption–desorption isotherms and BJH pore size distribution analysis. As shown in Fig. 3, the surface area and cumulative pore volume of pure MSN were calculated to be 1268.7 m2 g1 and 1.1 cm3 g1, respectively, and the average pore size was about 2.7 nm, indicating that these MSNs could be used for loading drugs and functionalization. Then, pure MSNs were modified by adding different amount of MPTMS, 2,20 -dipyridyl disulfide and 3-mercaptopropionic acid. In Table 1, it is also found that the surface area and cumulative pore volume of different thio-modified MSNs and carboxyl-modified MSNs were still high enough to host drugs. Although the average pore sizes of M1.5-SH and M1.5-SS-COOH were microporous with the increase of modified agent, the surface area and pore volume were still suited
2.9. Cell viability studies Experiments were performed using a human MCF-7 breast cancer cells. The cells were cultured at 37 °C under 5% CO2 atmosphere using RPMI 1640 containing 10% fetal bovine serum (FBS). About 2 104 cells/well were seeded into 96-well plates in 100 lL of culture medium with 10% FBS. The MCF-7 cells were incubated for 24 h, and then exposed for additional 24 h to various silica carriers with concentration of ranging from 50 to 1000 lg/mL that were prepared in normal saline solution. Then, 50 lL of 2 mg/mL sterile MTT solution was added to each well and the plate was incubated for another 4 h, allowing live cells to reduce MTT into dark-blue formazan. After that, the medium was replaced with 150 lL of DMSO and the plate was incubated at room temperature for 10 min on a shaking platform. Then, the absorbance was measured at a wavelength of 492 nm using a microplate reader (KHB ST-360, JingGong Industrial Co., Ltd., Shanghai, People’s Republic of China). The relative cell viability (%) was calculated by
Cell viability ð%Þ ¼
AbsðtestÞ 100% AbsðblankÞ
ðA:2Þ
where Abs(test) is the absorbance of test samples, and Abs(blank) is the absorbance of blank sample.
Fig. 5. XPS spectra for MSN and MSN-SH.
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Fig. 6. FTIR spectra of MSN, MSN-SH, M-SS-COOH and M-SS-PEG. The inset shows the area corresponding to the SH stretching vibrational mode (2800–2500 nm).
area of survey spectra. The analyses showed that with the increase of MPTMS, the sulfur content of M0.5-SH, M1-SH and M1.5-SH was 1.9%, 3.66%, 6.56%, respectively. According to the sulfur content of each MSN-SH, molar ratios of a disulfide exchange and condensation reaction can be obtained. FTIR spectroscopy was also used to characterize the samples. As shown in Fig. 6, vibrational peaks at 3420 cm1 was observed in the pure MSN spectrum, which was assigned to the hydroxyl groups. Vibrational peaks at lower frequencies were attributed to various vibrational modes of SiAOH and SiAOASi. More importantly, a band at 2566 cm1 consistent with stretching vibrations of thiol group was observed from the spectrum of MSN-SH, which is missing in the spectrum of pure MSN and MSN-SS-COOH. Compared with MSN-SH, an adsorption peak appeared at
Fig. 7. Zeta potential of M-SH, M-SS-COOH and M-SS-PEG.
to encapsulate drugs. After further functionalization, the surface area and pore volume of M-SS-PEG were decreased obviously, indicating that PEG was modified on the surface of MSNs. Then, the hydrodynamic diameter (Dh) and size distribution of the samples were measured by DLS. As shown in Table 2 and Fig. 4, the diameter of MSN was 229.2 nm, larger than what was observed from TEM because of different principles between these two characterizations. Generally, TEM gives the size of particles in a dried state while DLS results in the hydrodynamic size of particles. MSN-SS-COOH and MSN-SS-PEG showed a larger Dh than that of MSNs due to the modification of carboxyl groups and PEG chains, induced by carboxyl group-solvent interactions and chain-solvent interactions (Cui et al., 2012). XPS was used to investigate the thiol-functionalized MSNs to verify the introduction and modifying amount of thiol groups. As shown in Fig. 5, compared with pure MSN, XPS survey spectra of MSN-SH confirmed the presence of S apart from Si, C, and O, validating successful functionalization. Moreover, the sulfur content of different MSN-SH was analyzed by calculating the peak
Fig. 8. TGA curves of M0.5-SS-PEG, M1-SS-PEG and M1.5-SS-PEG.
Y. Wang et al. / European Journal of Pharmaceutical Sciences 72 (2015) 12–20
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Fig. 9. Cumulative release of RhB from (A) M1-SS-PEG, (B) M0.5-SS-PEG, (C) M1.5-SS-PEG and (D) M1-SS-PEG with different concentrations of GSH.
Table 1 The property characterization of M-SH, M-SS-COOH and M-SS-PEG. Sample
MPTMS (mL)
SBET (m2/g)
Vt (cm3/g)
Dp (nm)
MSN M0.5-SH M1-SH M1.5-SH M0.5-SS-COOH M1-SS-COOH M1.5-SS-COOH M0.5-SS-PEG M1-SS-PEG M1.5-SS-PEG
0.00 0.50 1.00 1.50 0.50 1.00 1.50 0.50 1.00 1.50
1268.7 1268.5 940.50 907.90 1030.0 778.70 775.40 877.00 226.80 65.000
1.10 0.80 0.58 0.50 1.00 0.56 0.62 0.43 0.47 0.16
2.7 2.3 2.2
Contents lists available at ScienceDirect
European Journal of Pharmaceutical Sciences journal homepage: www.elsevier.com/locate/ejps
Redox-responsive mesoporous silica as carriers for controlled drug delivery: A comparative study based on silica and PEG gatekeepers Ying Wang, Ning Han, Qinfu Zhao, Ling Bai, Jia Li, Tongying Jiang, Siling Wang ⇑ Department of Pharmaceutics, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenhe District, Shenyang, Liaoning Province 110016, PR China
a r t i c l e
i n f o
Article history: Received 6 November 2014 Received in revised form 15 January 2015 Accepted 11 February 2015 Available online 18 February 2015 Keywords: Redox-responsive Mesoporous silica nanoparticles Gatekeepers Controlled drug delivery Biocompatibility
a b s t r a c t Hybrid mesoporous silica nanoparticles (MSNs) modified with polymer polyethylene glycol (PEG) through the biodegradable disulfide bonds were prepared to achieve ‘on demand’ drug release. In this system, PEG chains were chosen as the representative gatekeepers that can block drugs within the mesopores of MSNs. After the addition of glutathione (GSH), the gatekeepers were removed from the pore outlets of MSNs, followed by the release of encapsulated drugs. In this research, the effects of grafting density of gatekeepers on the drug release and biocompatibility of silica carriers were also investigated. First, PEG modified MSNs were prepared by the condensation reaction between the carboxyl groups of MSN and the hydroxyl of PEG. The structure of the resultant MSN-SS-PEG was characterized by transmission electron microscopy (TEM), nitrogen adsorption/desorption isotherms analysis and Fourier transform infrared spectroscopy (FTIR). Rhodamine B (RhB) as the model drug was loaded into MSNs. The in vitro assay results indicated that RhB was released rapidly after the addition of 10 mM GSH; M1-SS-PEG had the best capping efficiency compared with M0.5 and M1.5 groups. Moreover, hemolysis assay, serum protein adsorption and cell viability test indicated that with the increase of PEG grafting density, the biocompatibility of silica carriers increased. Ó 2015 Elsevier B.V. All rights reserved.
1. Introduction In the past decades, many studies have focused on controlled drug delivery systems (CDDSs) since they can achieve site-selective, autonomously-modulated release (Zhu et al., 2005, 2007; Chu et al., 2013; Simões et al., 2014). It is highly desirable that ideal CDDSs should deliver therapeutic drugs without any premature release during the blood circulation or in normal tissues prior to reaching the special site and release drugs rapidly after being taken up by affected tissues (Zhang et al., 2012, 2013). Therefore, designing CDDSs which can release cargos on demand to protect healthy organs and reduce unexpected side effect is becoming very crucial. In recent years, mesoporous silica nanomaterials have attracted much attention as drug delivery carriers due to their unique advantages, such as high specific surface area and large pore volume, homogeneous controllable particle size, easily modified surface and good biocompatibility (Wang et al., 2013; Zhang et al., 2010, 2014; Mellaerts et al., 2008). Moreover, mesoporous silicaassisted drug delivery systems have increasingly focused on stimuli-responsive CDDSs that can release encapsulated drugs from
⇑ Corresponding author. Tel./fax: +86 24 23986348. E-mail address: [email protected] (S. Wang). http://dx.doi.org/10.1016/j.ejps.2015.02.008 0928-0987/Ó 2015 Elsevier B.V. All rights reserved.
silica carriers at the expected site in response to various stimuli including pH, redox potential, temperature, photoirradiation, and biomacromolecule (Yang et al., 2012; Meng et al., 2010; Zhou et al., 2007; He et al., 2012; Bernardos et al., 2010). Many available functionalized mesoporous silica nanoparticles (MSNs) have been developed to regulate drug release by capping the outlets of mesopore or encapsulating drugs within the porous channels of MSNs. In general, mesopore-entrapped drugs cannot leach out from the MSNs unless the drug delivery system is exposed to certain stimuli that can trigger the opening of the pore entrances. Among various stimuli used for CDDSs, redox potential has emerged as an appealing trigger. It is demonstrated that glutathione (GSH) is the most abundant reducing agent in vivo and the concentration of GSH has a significant difference between the extracellular (2 lM) and intracellular (10 mM) environment. Furthermore, intracellular GSH levels in the tumor tissues are at least 4-fold higher than those in normal tissues (Saito et al., 2003). Since disulfide bonds can be reduced by GSH, redox potentials based on the disulfide bonds are expected as a stimulus to achieve ‘on demand’ drug release. Recently, a few redox-responsive CDDSs using MSNs as carriers have been reported. For example, Liu et al. (2008) reported a disulfide cross-linked polymeric network as a gatekeeper that can be removed by GSH, thus allowing the release of encapsulated drugs. Cui and co-workers have
Y. Wang et al. / European Journal of Pharmaceutical Sciences 72 (2015) 12–20
developed a novel polyethylene glycol (PEG) modified MSN-based CDDS for the first time, in which PEG chains were linked to the MSNs through disulfide bonds. In this system, PEG shell can block drugs tightly and open the pore outlets upon GSH conditions (Cui et al., 2012). More recently, Liang Chen and co-workers (Chen et al., 2014) also used PEG as a gatekeeper for GSH-mediated drug delivery system. In the research, they used SBA-15 (3D cubic MSN) as the drug carrier and compared it with MCM-41 (2D hexagonal MSN). The results obtained suggested that both types of MSNs-assisted CDDSs can provide controlled drug release. In the previous studies, researchers have focused on designing various gatekeepers based on MSNs instead of considering the grafting density of gatekeepers. Zhong Luo and Kaushik Patel et al. have used collagen and cyclodextrine as gatekeepers to realize controlled drug release, but the grafting density of gatekeepers has not been investigated in their studies (Luo et al., 2011; Patel et al., 2008; Zhang et al., 2014). In our research, for the first time, we investigated the effects of grafting density of gatekeepers on the drug release and the biocompatibility of silica carriers. Here, PEG chains were still chosen as the representative gatekeepers due to their good biocompatibility and suited molecular weight to cap the entrance of MSNs. Moreover, PEG modified MSNs have the advantages such as prolonging the blood circulation time, greatly improving the enhanced permeability and retention (EPR) effect and preventing the endocytosis by the reticuloendothelial system (RES) Meng et al., 2011. We have developed an innovative and convenient approach to prepare PEG-linked MSNs with disulfide bonds for smart drug delivery (Fig. 1). In this system, 2,20 -dipyridyl disulfide was first covalently bonded with thiol-functionalized MSN to yield MSN-SS-Py, which can be further reacted with 3-mercaptopropionic acid to achieve MSN-SS-COOH nanoparticles. Then, the PEG chains were covalently combined to above MSN-SS-COOH nanoparticles to obtain the redox-responsive MSNs (MSNSS-PEG). The redox-responsive drug delivery system has a good capping efficiency and the influence of surface modification of MSNs on their biocompatibility has also been investigated. 2. Experimental 2.1. Materials 3-Mercaptopropyltrimethoxysilane (MPTMS), 3-mercaptopropionic acid, rhodamine B (RhB), methoxypolyethylene glycol (PEG,
13
5000), 2,20 -dipyridyl disulfide, N-(3-Dimethylaminopropyl)N0 -ethylcarbodiimide hydrochloride (EDCHCl), 3-(4,5)dimethylthiahiazo(-z-y1)-3,5-diphenytetrazoliumromide (MTT), 4-dimethylaminopyridine (DMAP), glutathione (GSH) were purchased from Aladdin Chemistry, Co. (Shanghai). Cetyltrimethylammonium bromide (CTAB), tetraethyl orthosilicate (TEOS), anhydrous ethanol, methanol, N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), hydrochloric acid (HCl, 36.5%), sodium hydroxide (NaOH), potassium phosphate monobasic (KH2PO4) were purchased from Shan Dong Yu Wang Reagent Company (China). All the reagents were used as received. 2.2. Characterization techniques Transmission electron microscopy (TEM) images of nanoparticles were performed on Tecnai G2 20 (FEI, USA) microscope at an acceleration voltage of 200 kV. Fourier transform infrared spectroscopy (FTIR) spectra were recorded on a Bruker IFS 55 (Switzerland). N2 adsorption–desorption isotherms were conducted on a SA3100 surface area analyzer (Beckman Coulter, USA). X-ray Photoelectron Spectroscopy (XPS) was performed on ESCALAB250 (Thermo VG, USA) spectrometer with an Al Ka monochromatic source (1486.6 eV, 150 W). Thermogravimetric analysis (TGA) was carried out by a TGA-50 instrument (Shimadzu, Japan) at a heating rate of 10 °C/min under a nitrogen purge of 40 mL/min. 2.3. Synthesis of MSN-SH MSN-SH was prepared according to the reported method (Mortera et al., 2009). Firstly, CTAB (1 g) was dissolved in 480 mL of deionized water. Then, 3.5 mL of 2 M NaOH solution was introduced to CTAB solution under vigorous stirring at 80 °C. After that, TEOS (5 mL) was added dropwise to the above solution and followed by addition of different amount of MPTMS (0.5 mL, 1 mL and 1.5 mL, respectively). Then, the mixture was stirred for 2 h at 80 °C. To remove the template CTAB, the resultant particles were dispersed in a solution of HCl (18 mL, 37.4%) and methanol (320 mL), and refluxed for 24 h. After that, an ATS AH110D homogenizer (ATS Engineer Inc., Shanghai, China) was performed to homogenize the resulting precipitate. The MSN-SH particles were collected by centrifugation (6000 rpm, 15 min), washed with methanol and dried under vacuum for 12 h. 2.4. Synthesis of MSN-SS-COOH MSN-SH (1 g) and excessive 2,20 -dipyridyl disulfide were first dispersed in 25 mL of anhydrous ethanol containing acetic acid (1 mL). The mixture was stirred at room temperature for 24 h. The resultant MSN-SS-Py was isolated by centrifugation and washed unreacted 2,20 -dipyridyl disulfide with anhydrous ethanol. Under the nitrogen atmosphere, 0.8 mL of acetic acid was added to a DMF (20 mL) solution of MSN-SS-Py, followed by addition of excessive 3-mercaptopropionic acid. The reaction was kept under stirring at 40 °C for 24 h. The resulting MSN-SS-COOH was separated by centrifugation, washed with water and anhydrous ethanol and dried under vacuum (He et al., 2013). 2.5. Synthesis of MSN-SS-PEG
Fig. 1. Schematic illustration of drug loading and release.
MSN-SS-COOH (0.2 g), abundant EDCI and DMAP (-SS-COOH/ EDCI/DMAP molar ratio was 6:12:1) were dispersed in 20 mL of deionized water under stirring for 2 h, followed by addition of PEG (0.1 g). After the mixture was stirred for 24 h, the resulting MSN-SS-PEG was collected by centrifugation, washed extensively with water and anhydrous ethanol and finally dried under vacuum.
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Fig. 2. TEM images of (A) MSNs and (B) MSN-SS-PEG nanoparticles.
Fig. 3. Nitrogen adsorption/desorption isotherms and pore size distribution of MSNs.
2.6. Drug loading and release studies
2.7. Hemolysis assay
Typically, MSN-SS-COOH (0.2 g) were first dispersed in 5 mL of deionized water, followed by adding a certain amount of EDCI and DMAP (-SS-COOH/EDCI/DMAP molar ratio was 6:12:1) under stirring for 1.5 h to achieve carboxyl active ester for further connecting with PEG. Then, 5 mL of RhB (4 mg/mL) solution was loaded into above MSN-SS-COOH under stirring for 6 h. After that, PEG (0.2 g) was added to the above solution, stirred for 12 h, centrifuged and washed extensively to achieve RhB-loaded MSN-SS-PEG (called as MSN-SS-PEG@RhB). Finally, MSN-SS-PEG@RhB was dried under vacuum. To investigate the redox-responsive drug release of different grafted amounts of MSN-SS-PEG systems, RhB-loaded MSN-SSPEG (25 mg) was dissolved into PBS (pH 7.4) with/without 10 M GSH, shaking at 150 rpm (37 °C). At predetermined time intervals, 3 mL of PBS was taken out for UV–Vis measurement (UV-2000, Unico, USA) and then replaced with an equal volume of fresh buffer. The cumulative released amount was determined from the absorbance at 554 nm according to a standard curve of RhB in the same buffer.
The rabbit red blood cells (RBCs) were obtained by centrifugation and suction to remove the serum from the blood and further washed five times with sterile normal saline solution. Following the last wash, the cells were diluted to 2/100 of their volume with normal saline solution. Then 2 mL of the diluted RBCs suspension was mixed with: (a) 2 mL nanoparticles which were dispersed into normal saline solution at the different concentrations ranging from 40 to 800 lg/mL; (b) 2 mL of normal saline solution as a negative control; (c) 2 mL of distilled water as a positive control. Then the mixtures were kept still for 4 h at room temperature. Finally, the mixtures were centrifuged for 10 min at 1000 r/min and the absorbances of supernatant at 541 nm were recorded by the UV–vis absorption spectrophotometer. 2.8. Serum protein adsorption 60 mg of BSA was completely dissolved in 100 mL of distilled and deionized water under stirring mildly. 0.8 g of NaCl, 0.02 g of KCl, 0.115 g of Na2HPO4 and 0.02 g of KH2PO4 were dissolved in
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3. Result and discussion 3.1. Synthesis and characterization of MSN-SS-PEG
Fig. 4. Size distribution of MSN, MSN-SS-COOH and MSN-SS-PEG.
500 mL of distilled and deionized water to prepare phosphate buffered saline (PBS). Pure MSN and M-SS-PEG were dispersed into 5 mL of PBS solutions at a concentration of 1 mg/mL, and then 5 mL of BSA solutions were added. Meanwhile, a control group was prepared by mixing 5 mL of PBS and 5 mL BSA solutions. After shaking at 150 rpm (37 °C) for 4 h, the mixed solutions were centrifugalized, and then the supernatant was collected. The concentrations of residual BSA were determined by the dying method with coomassie brilliant blue. 0.5 mL of collected supernatant, 0.5 mL of coomassie brilliant blue and 2 mL of distilled water were mixed to measure the absorbances of residual BSA at 555 nm. A calibration curve was drawn by using BSA solutions with a series of concentrations. BSA adsorbed on MSN and MSN-SS-PEG were calculated according to
BSA adsorbance ð%Þ ¼ ðC b Ce Þ 6 V=m
ðA:1Þ
where Cb and Ce were initial BSA concentration in a control group and the residual BSA concentration after BSA adsorption; V was the total solution volume (10 mL); m was the weight of pure MSN or MSN-SS-PEG added into solutions.
MSN-SH was synthesized according to the previous method by a one-pot method (Mortera et al., 2009). Subsequently, 2,20 -dipyridyl disulfide and 3-mercaptopropionic acid were used to obtain MSN-SS-COOH by the disulfide exchange reaction. Then, PEG was covalently grafted onto external surface of MSNs by the condensation reaction between the carboxyl groups of MSN and the hydroxyl of PEG to get MSN-SS-PEG. In this research, PEG was regarded as a significant gatekeeper to impede the drug release. In order to compare the drug release properties of MSNs with different amount of grafted gatekeepers, 0.5 mL, 1 mL and 1.5 mL of MPTMS were used respectively to obtain the thiol-modified MSNs (called as M0.5-SH, M1-SH, M1.5-SH), carboxyl-modified MSNs with cleavable disulfide bonds (donated as M0.5-SS-COOH, M1-SS-COOH, M1.5-SS-COOH) and PEG-grafted MSNs (called as M0.5-SS-PEG, M1-SS-PEG, M1.5-SS-PEG). The structures of MSNs and MSN-SS-PEG were monitored by transmission electron microscopy (TEM), Brunauer–Emmett– Teller (BET) analysis and Barret–Joyner–Halenda (BJH) analysis, and dynamic light scattering (DLS). As displayed in Fig. 2A, the mean diameter of synthesized MSNs was about 100 nm with 2– 3 nm of the mesopore size. A highly ordered mesoporous network could be clearly seen in the image. In Fig. 2B, the mesoporous network of MSN-SS-PEG was still observed indicating that the polymer PEG did not destroy the mesoporous property of MSNs. The surface area and average pore diameter of MSNs and MSN-SSPEG were measured by BET nitrogen adsorption–desorption isotherms and BJH pore size distribution analysis. As shown in Fig. 3, the surface area and cumulative pore volume of pure MSN were calculated to be 1268.7 m2 g1 and 1.1 cm3 g1, respectively, and the average pore size was about 2.7 nm, indicating that these MSNs could be used for loading drugs and functionalization. Then, pure MSNs were modified by adding different amount of MPTMS, 2,20 -dipyridyl disulfide and 3-mercaptopropionic acid. In Table 1, it is also found that the surface area and cumulative pore volume of different thio-modified MSNs and carboxyl-modified MSNs were still high enough to host drugs. Although the average pore sizes of M1.5-SH and M1.5-SS-COOH were microporous with the increase of modified agent, the surface area and pore volume were still suited
2.9. Cell viability studies Experiments were performed using a human MCF-7 breast cancer cells. The cells were cultured at 37 °C under 5% CO2 atmosphere using RPMI 1640 containing 10% fetal bovine serum (FBS). About 2 104 cells/well were seeded into 96-well plates in 100 lL of culture medium with 10% FBS. The MCF-7 cells were incubated for 24 h, and then exposed for additional 24 h to various silica carriers with concentration of ranging from 50 to 1000 lg/mL that were prepared in normal saline solution. Then, 50 lL of 2 mg/mL sterile MTT solution was added to each well and the plate was incubated for another 4 h, allowing live cells to reduce MTT into dark-blue formazan. After that, the medium was replaced with 150 lL of DMSO and the plate was incubated at room temperature for 10 min on a shaking platform. Then, the absorbance was measured at a wavelength of 492 nm using a microplate reader (KHB ST-360, JingGong Industrial Co., Ltd., Shanghai, People’s Republic of China). The relative cell viability (%) was calculated by
Cell viability ð%Þ ¼
AbsðtestÞ 100% AbsðblankÞ
ðA:2Þ
where Abs(test) is the absorbance of test samples, and Abs(blank) is the absorbance of blank sample.
Fig. 5. XPS spectra for MSN and MSN-SH.
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Fig. 6. FTIR spectra of MSN, MSN-SH, M-SS-COOH and M-SS-PEG. The inset shows the area corresponding to the SH stretching vibrational mode (2800–2500 nm).
area of survey spectra. The analyses showed that with the increase of MPTMS, the sulfur content of M0.5-SH, M1-SH and M1.5-SH was 1.9%, 3.66%, 6.56%, respectively. According to the sulfur content of each MSN-SH, molar ratios of a disulfide exchange and condensation reaction can be obtained. FTIR spectroscopy was also used to characterize the samples. As shown in Fig. 6, vibrational peaks at 3420 cm1 was observed in the pure MSN spectrum, which was assigned to the hydroxyl groups. Vibrational peaks at lower frequencies were attributed to various vibrational modes of SiAOH and SiAOASi. More importantly, a band at 2566 cm1 consistent with stretching vibrations of thiol group was observed from the spectrum of MSN-SH, which is missing in the spectrum of pure MSN and MSN-SS-COOH. Compared with MSN-SH, an adsorption peak appeared at
Fig. 7. Zeta potential of M-SH, M-SS-COOH and M-SS-PEG.
to encapsulate drugs. After further functionalization, the surface area and pore volume of M-SS-PEG were decreased obviously, indicating that PEG was modified on the surface of MSNs. Then, the hydrodynamic diameter (Dh) and size distribution of the samples were measured by DLS. As shown in Table 2 and Fig. 4, the diameter of MSN was 229.2 nm, larger than what was observed from TEM because of different principles between these two characterizations. Generally, TEM gives the size of particles in a dried state while DLS results in the hydrodynamic size of particles. MSN-SS-COOH and MSN-SS-PEG showed a larger Dh than that of MSNs due to the modification of carboxyl groups and PEG chains, induced by carboxyl group-solvent interactions and chain-solvent interactions (Cui et al., 2012). XPS was used to investigate the thiol-functionalized MSNs to verify the introduction and modifying amount of thiol groups. As shown in Fig. 5, compared with pure MSN, XPS survey spectra of MSN-SH confirmed the presence of S apart from Si, C, and O, validating successful functionalization. Moreover, the sulfur content of different MSN-SH was analyzed by calculating the peak
Fig. 8. TGA curves of M0.5-SS-PEG, M1-SS-PEG and M1.5-SS-PEG.
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Fig. 9. Cumulative release of RhB from (A) M1-SS-PEG, (B) M0.5-SS-PEG, (C) M1.5-SS-PEG and (D) M1-SS-PEG with different concentrations of GSH.
Table 1 The property characterization of M-SH, M-SS-COOH and M-SS-PEG. Sample
MPTMS (mL)
SBET (m2/g)
Vt (cm3/g)
Dp (nm)
MSN M0.5-SH M1-SH M1.5-SH M0.5-SS-COOH M1-SS-COOH M1.5-SS-COOH M0.5-SS-PEG M1-SS-PEG M1.5-SS-PEG
0.00 0.50 1.00 1.50 0.50 1.00 1.50 0.50 1.00 1.50
1268.7 1268.5 940.50 907.90 1030.0 778.70 775.40 877.00 226.80 65.000
1.10 0.80 0.58 0.50 1.00 0.56 0.62 0.43 0.47 0.16
2.7 2.3 2.2
