- Email: [email protected]
Biothiol-triggered, self-disassembled silica nanobeads for intracellular drug delivery
Acta Biomaterialia xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Acta Biomaterialia journal homepage: www.elsevier.com/locate/actabiomat
Brief communication
Biothiol-triggered, self-disassembled silica nanobeads for intracellular drug delivery Xin-Chun Huang a, Li-Bang Wu a, Jen-Fang Hsu b, Shinsuke Shigeto a,b, Hsin-Yun Hsu a,b,⇑ a b
Department of Applied Chemistry, National Chiao-Tung University, No. 1001 Ta-Hsueh Road, Hsinchu 30010, Taiwan Institute of Molecular Science, National Chiao-Tung University, No. 1001 Ta-Hsueh Road, Hsinchu 30010, Taiwan
a r t i c l e
i n f o
Article history: Received 14 November 2014 Received in revised form 4 May 2015 Accepted 10 May 2015 Available online xxxx Keywords: Drug delivery Silica nanoparticles Sol–gel processes Self-disassembly Biothiols
a b s t r a c t Silica-based nanomaterials have demonstrated great potential in biomedical applications due to their chemical inertness. However, the degradability and endosomal trapping issues remain as rate-limiting barriers during their innovation. In this study, we provide a simple yet novel sol–gel approach to construct the redox-responsive silica nanobeads (ReSiNs), which could be rapidly disassembled upon redox gradient for intracellular drug delivery. The disulfide-linked scaffold of the nanobead was synthesized by employing the dithiobis-(succinimidyl propionate) to bridge (3-aminopropyl)-trimethoxysilane. Such silica matrix could be efficiently disrupted in response to intracellular glutathione, resulting in drug release and collapse of entire nanocarrier. Moreover, the ReSiNs exhibited insignificant cytotoxicity before and after the degradation. These results indicated the potential of using ReSiNs as a novel silica-based, biothiol-degradable nanoplatform for future drug delivery. Ó 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
1. Introduction Nanomaterial-based drug delivery systems (DDSs) have shown great potential in theranostic applications [1–3]. The use of metals and non-metals, polymers, liposomes, micelles, and nanoclusters had been found in many studies [4–8] for smart drug delivery. Despite the immense advancement in the field, there are still huge gaps from bench to the bedside and several critical concerns shall be considered when designing the nanocarriers. It has been reported that the surface-modified nanocarriers with a diameter in the range of 20–200 nm can escape from renal filtration [9– 11] and show enhanced permeability and retention (EPR) effects in targeted cancer therapy [10,12,13]. On the other hand, the degradation and clearance of the constructed nanomaterials in the body should occur in a reasonable period after drug release at the target tissue site [14]. In this regard, recent study has shown promising targeted delivery and uncaging of nanoparticles into fragments for subsequent renal clearance by employing the photouncaging ligands to enable light triggering [15]. Alternatively, some micelle systems with cleavable linkers were incorporated to allow responsive degradation upon internal stimuli such as pH or redox status [16]. But nevertheless, external light-based systems ⇑ Corresponding author at: Department of Applied Chemistry, National Chiao-Tung University, No. 1001 Ta-Hsueh Road, Hsinchu 30010, Taiwan. Tel.: +886 (0)3 5712121x56556; fax: +886 (0)3 5723764. E-mail address: [email protected] (H.-Y. Hsu).
may suffer from the rapid intensity attenuation of the short-wavelength excitation in tissues; conversely, the chemical instability caused by oxidation and the difficulties in precise size control due to vesicle fusion frequently occurred in the polymeric micelle-based nanoplatforms. In the past decades, amorphous [17,18] and mesoporous silica nanoparticles [19,20] have become one of the most promising materials in nanomedicine. The silica matrix has shown several intrinsic advantages over conventional organic polymers, including high mechanical resistance, chemical inertness with minimal cargo interaction, and biosafety, which has been recognized by the U.S. Food and Drug Administration [21,22]. Silica nanoparticles with tunable particle diameter and morphology have been studied in sol–gel process and water in oil microemulsion, in addition, the chemical modification of its surface can be easily achieved using silane coupling reagents to enable a variety of surface functionalities (hydroxyl/amino/thiol/carboxyl groups) and targeting ligand modification [23]. However, synthesis of mesoporous silica nanoparticles often requires the employment of surfactants such as cetyltrimethylammonium bromide (CTAB) to form mesostructured silica shell. Removal of surfactants is necessary to improve biocompatibility. In addition, the hampered intracellular bioavailability by the endosomal uptake pathway and the degradability of silica nanocomposites remain as challenges to fast dissolving DDSs [24]. Although several sophisticated designs have been shown to facilitate the endosomal escape by modulating the
http://dx.doi.org/10.1016/j.actbio.2015.05.006 1742-7061/Ó 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
Please cite this article in press as: X.-C. Huang et al., Biothiol-triggered, self-disassembled silica nanobeads for intracellular drug delivery, Acta Biomater. (2015), http://dx.doi.org/10.1016/j.actbio.2015.05.006
2
X.-C. Huang et al. / Acta Biomaterialia xxx (2015) xxx–xxx
surface chemistry [25], incorporating a photosensitizer [26], or conjugating endosomolytic peptides [27] to the nanocomposites, limited researches alter the chemically inert silica scaffold by manipulating the bonding linkage, creating the functionalities in structure itself to simplify the synthetic routes. Besides, the feasibility of such materials for subsequent intracellular degradation also had been rarely considered. It is well known that disulfide bonds can be cleaved by reducing agents (e.g., dithiothreitol, DTT), and they are sensitive to the reduced milieu. One of such redox-active biothiols in cells is glutathione (GSH), which is often present in the millimolar range in the cytoplasm in contrast to their concentration in the extracellular milieu (2–20 lM) [28]. The significant redox potential difference between the interior and exterior of the cell could be exploited as a physiological stimulus for subcellular delivery of therapeutic biomolecules. In this regard, we developed a disulfide-rich silica-based scaffold for the nanocarrier to achieve redox-facilitated drug release and enhance its self-degradability. Once these drug nanocarriers were taken up by the targeted cells, disulfide bonds in the silica matrix could be reduced by intracellular thiols, enabling the drug release and collapse of the nanobeads into fragmented residuals for rapid removal.
electrostatic interactions. The mixture was washed three times with PBS by centrifugation and stored in PBS at 4 °C. 2.4. ReSiN characterization
2. Materials and methods
A field-emission scanning electron microscope (FE-SEM, JSM-7401F, JOEL) and an ultra-high resolution transmission electron microscope (JEOL JEM-ARM200F Cs-corrected Field Emission) were employed to characterize the morphology of ReSiNs. Raman scattering spectrum, generated by a He–Ne laser (632.8 nm, 11 mW), was collected under RT (60 s/spectrum) using an oil-immersion objective (100, NA 1.3). Ten spectra were recorded for each sample and those that did not show spiky feature (i.e., cosmic rays) were averaged to obtain the mean spectra (No background subtraction was performed). 29Si NMR spectra of ReSiN were analyzed using DSX400WB NMR Spectrometer (Bruker, Germany). The silica concentration was determined by ICP-AES (Model S-35, Kontron, Germany). The degradation percentage was calculated as follows: ReSiN degradation (%) = average Si content of treated group/average Si content of control group (ReSiN dissolved in 1 M KOH) 100%. Zeta potentials were recorded using Zetasizer Nano ZS (Malvern Instruments, UK). Fluorescence intensity was obtained on a microplate fluorescence reader (BioTek FL 800).
2.1. Reagents
2.5. Cytotoxicity assay and cell imaging
Dithiobis-succinimidyl propionate (DTSP), ethanol, 3-aminopropyl-trimethoxysilane (APTMS), 3-mercaptopropyl-tri methoxysilane (MPTMS), hydrochloric acid solution, ammonium hydroxide solution, 5-carboxy-tetramethylrhodamine (5-TAMRA), potassium hydroxide, and Tris (base) were purchased from Sigma–Aldrich. Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), Dulbecco’s phosphate-buffered saline (PBS), and penicillin/streptomycin were purchased from Biowest (Nuaillé, France). CellLightÒ Lysosomes-GFP was supplied by Life Technologies (ThermoFisher, Massachusetts, USA).
Hep G2 cells (1 104 cells/well) were cultured in 96-well plates containing DMEM supplemented with 10% FBS and 1% penicillin/streptomycin at 37 °C under 5% CO2. After 12 h, the culture medium was removed, and the cells were washed three times with PBS. Next, the cells were incubated in 100 lL of fresh medium with 1, 10, 20, 100, 200, or 400 lg mL 1 ReSiNs or ReSiN@TAMRA@Tf nanocarriers for 24 h. The cytotoxicity of degraded ReSiN and ReSiN@TAMRA@Tf fragments was evaluated by incubating the cells for an additional 24 h with 100 lL of culture medium containing 10 mM GEE. The culture medium was then discarded, and cells were washed twice with PBS. The culture medium (50 lL) containing 5 mg mL 1 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo lium bromide (MTT) was applied to the cells, followed by incubation for 3 h. The supernatant was removed, and the formazan precipitate was dissolved in DMSO for 10 min. Absorption (Abs) at 570 nm was measured using a UV–Vis microplate reader (Cary 100 Varian, Palo Alto, CA, USA). Cell viability was determined as follows: cell viability (%) = mean Abs of treated group/mean Abs of control 100%. Confocal images were obtained by using Leica TCS SP5 confocal laser scanning microscope.
2.2. Preparation of ReSiNs The synthesis of ReSiNs was carried out using the sol–gel process. Firstly, DTSP (2.00 mg, 5 lmole) was dissolved in 50 lL of dimethyl sulfoxide (DMSO). Next, HCl(aq) (1.00 M, 5 lL) and APTMS (6.3 lL, 35 lmole) were added to the DTSP solution, followed by sonication for 40 min. Tris–HCl buffer (1 M, pH 8.0, 50 lL) was added to the reaction mixture, followed by sonication for an additional 10 min. The mixture was then added dropwise to an ethanol solution, followed by the addition of 50 lL of 0.25% ammonium hydroxide (NH4OH) and 100 lL of ddH2O. After incubation for 6 h at room temperature (RT), the product was purified by centrifugation (12,000g, 10 min) and washed three times with ethanol. ReSiNs were finally suspended in water and dried by lyophilization.
2.6. Statistical analysis The statistical analyses were performed using OriginPro 8 Software (OriginLab Corp., Northampton, MA). The Mann– Whitney U test with a p-value less than 0.05 was reported as significant.
2.3. Preparation of ReSiN@TAMRA@Tf 3. Results and discussion Briefly, 0.11 mg of ReSiNs was dispersed in 600 lL of EtOH, and 2.4 nmole 5-TAMRA was added to the suspension at RT, followed by sonication for 4 h. To remove free 5-TAMRA, the product was centrifuged at 12,000g for 10 min and washed three times with ethanol. After centrifugation, the supernatant was discarded, and purified ReSiN@TAMRA was obtained. Next, ReSiN@TAMRA was suspended in 1 mg mL 1 holo-transferrin solution (dissolved in PBS solution, pH 7.4), which was incubated at 4 °C overnight. Transferrin was adsorbed at the surface of ReSiN@TAMRA via
The synthetic process of the redox-responsive silica nanobeads (ReSiNs) was developed based on the fabrication principle of sol– gel system [29] using the crosslinker dithiobis-succinimidyl propionate (DTSP) to bridge (3-aminopropyl)-trimethoxysilane (Fig. 1). The morphology and inner structure of ReSiN were analyzed by transmission electron microscopy (TEM) with a point-EDX scan as shown in figure Fig. 2a and b. No crystalline structure was observed in TEM images and the sulfur/silicon molar ratio
Please cite this article in press as: X.-C. Huang et al., Biothiol-triggered, self-disassembled silica nanobeads for intracellular drug delivery, Acta Biomater. (2015), http://dx.doi.org/10.1016/j.actbio.2015.05.006
X.-C. Huang et al. / Acta Biomaterialia xxx (2015) xxx–xxx
3
Fig. 1. Schematic illustrations of the proposed route after the cellular uptake of ReSiNs (upper panel) and preparation of ReSiNs (lower panel) (5-TAMRA: 5-carboxytetramethylrhodamine N-succinimidyl ester; Tf: transferrin; DMSO: dimethyl sulfoxide; APTMS: 3-aminopropyl-trimethoxysilane).
increased from the surface (S: Si = 1: 8.7 toward the core (S: Si = 1: 2) of ReSiN. The statistical diameter of ReSiN was approximately 143.3 ± 38.2 nm, as estimated by scanning electron microscopy (SEM, Fig. 2c), and this value was within the optimal diameter range of nanoparticle-based DDSs for enhanced permeability and retention effects on targeted cancer cells. Raman spectroscopy was employed to characterize the fabricated nanobeads (Fig. 2d, S1). Two strong peaks at 509 cm 1 and 615 cm 1 have been observed in aggregates of ReSiNs (Fig. 2d, black), which correspond to the stretching vibrational mode of the aryl disulfide (S–S and C–S, respectively). The same peaks were also observed in the Raman spectrum of pure DTSP (Fig. 2d, red). In addition, 3-mercap topropyl-trimethoxysilane (MPTMS) silica nanoparticles, used as thiolated silane control material to monitor the S–H stretching
vibration at 2570 cm 1 (see Fig. 2d, blue), were synthesized by the sol–gel process. No apparent S–H stretch band was observed in the spectrum of the ReSiN powder, indicating that the disulfide bonds of ReSiN remained intact after the synthetic process. We evaluated the silanol functionality in ReSiN by solid NMR spectroscopy (Fig. 2e). A strong T3 signal corresponding to silicon atoms with a single alkyl group and a small shoulder T2 peak were both observed in 29Si-NMR spectrum, revealing that the silane networks were stably formed. The degradability of ReSiNs was evaluated by measuring the released silica from ReSiNs (14.6 lg for each condition) after the DTT treatment (10 lM–10 mM in PBS, pH 7.4) at 37 °C for 1 h using inductively coupled plasma-atomic emission spectroscopy (ICP-AES). As shown in Fig. 3a, approximately 22.5 ± 5.8% of the silica fragments were released from ReSiNs. In
Please cite this article in press as: X.-C. Huang et al., Biothiol-triggered, self-disassembled silica nanobeads for intracellular drug delivery, Acta Biomater. (2015), http://dx.doi.org/10.1016/j.actbio.2015.05.006
4
X.-C. Huang et al. / Acta Biomaterialia xxx (2015) xxx–xxx
Fig. 2. (a) TEM image, (b) point-EDX scan, and (c) SEM image of ReSiN. (d) Raman spectra of APTMS droplet (green trace), DTSP powder (red trace), MPTMS powder (blue trace), and ReSiN aggregate (black trace). Samples were deposited on a glass substrate for spectral measurement. Raman spectra were obtained with an exposure time of 60 s using a confocal Raman microspectrometer equipped with a He–Ne laser (632.8 nm, 11 mW at the sample point) as the excitation source [48,49]. Ten spectra were recorded for each sample and those that did not show spiky features were averaged to obtain the mean spectra. No background subtraction was performed. The optical image displays ReSiN aggregates, and an asterisk indicates the position at which the Raman spectra were measured. (e) Solid-NMR spectrum of ReSiN ((SiO)2Si(OAlk)2 (T2); (SiO)3Si(OAlk) (T3); Alk = alkyl group). (f) Zeta potentials of the bare ReSiNs, ReSiN@TAMRA, and ReSiN@TAMRA@Tf nanocomposites. The surface of ReSiNs was functionalized by APTMS, rendering positive charges on the surface. Insignificant change in the surface charge was found after 5-TAMRA labeling. Holo-transferrin was conjugated to the surface of ReSiNs via electrostatic interaction for enabling specific targeting of ReSiNs to Hep G2 cells. All samples were analyzed in 1 mM PBS (pH 7.4) at RT.
contrast, no silica fragment was detected in the supernatant of MPTMS nanoparticles, which were subject to the same DTT treatments, after incubation for 1 h. SEM was employed to monitor
the morphological changes of ReSiNs incubated with 10 mM DTT at room temperature (RT) for 0.5, 1.5, 2, and 2.5 h (Fig. 3b). An increase in the deformation level of ReSiNs was observed with an
Please cite this article in press as: X.-C. Huang et al., Biothiol-triggered, self-disassembled silica nanobeads for intracellular drug delivery, Acta Biomater. (2015), http://dx.doi.org/10.1016/j.actbio.2015.05.006
X.-C. Huang et al. / Acta Biomaterialia xxx (2015) xxx–xxx
5
Fig. 3. (a) Degradability of ReSiNs and MPTMS nanoparticles (control) determined by ICP-AES. Briefly, 14.6 lg mL 1 ReSiNs and 21.9 lg mL 1 MPTMS nanoparticles were separately incubated with DTT at indicated concentrations (in PBS) at 37 °C for 1 h. The suspensions were centrifuged at 12000g for 10 min, and supernatants were collected and diluted with 10 mL of 0.5% HNO3 for measuring the silica content by ICP-AES. The release percentage of silica from the two types of materials was calculated and normalized according to the initial input. (ND: not detectable, the concentration of silica was lower than the detection limit of 0.026 ppm). (b) SEM images showing the distribution and morphology of ReSiNs. Briefly, 0.12 lg of ReSiNs was dried on the glass substrate, and 3 lL of 10 mM DTT was dropped onto the ReSiN-loaded glass substrate, followed by incubation for indicated time periods at RT. After each hour of incubation, the DTT droplet was discarded, followed by washing with DI water for three times and replenishing the glass substrate with DTT before SEM measurement. ReSiNs incubated in H2O for 2.5 h were employed as control to confirm the stability of ReSiNs in the absence of DTT. All the washing steps were consistent with the conditions of using DTT to avoid bias. The black bar represents 1 lm. (c) Release profiles of 5-TAMRA from the ReSiN@TAMRA@Tf nanocarriers in the presence or in the absence of 10 mM DTT in PBS (pH 7.4) at 37 °C. The fluorescence intensity of the supernatant was determined at the indicated time points using a microplate fluorescence reader.
increase in the incubation time, and the degraded ReSiN fragments could be easily washed away from the glass substrate afterward. The degradability of ReSiN under acidic environment was also investigated. ReSiNs were incubated in 10 mM DTT buffered by the pH gradient at 37 °C for 1 h (Fig. S2). The degradation was decreased with decreasing pH, from 20.92 ± 1.56% (pH 7) to 5.34 ± 0.73% (pH 3). Current data indicated that the disulfide cleavage predominantly comes from thiol-disulfide exchange that requires the presence of thiolate anion (pKa = 8.3), which is unlikely to deprotonate under acidic environment. We also studied the redox-responsive degradation by MALDI-TOF-MS to analyze the ReSiN fragments after 10 mM DTT or 1 M KOH treatments. The results have identified significantly different characteristic peaks in mass spectra (Fig. S3), indicating that the mechanism of degradation by DTT was different from KOH-triggered alkaline hydrolysis. To further clarify the feasibility of using ReSiNs as redox-degradable drug nanocarriers, we labeled the nanobead surface with 5-carboxy-tetramethylrhodamine (5-TAMRA), a model drug, via N-succinimidyl ester to form thiol non-responsive bond. This design enabled us to further confirm the rapid silica matrix degradation upon redox potential by tracking intracellular fluorescence distribution. The diffused 5-TAMRA fluorescence can be observed only in the case that ReSiNs are degraded in the cells. The negatively charged holo-transferrin (Tf) was chosen as the tumor-targeting molecule due to its specificity for the over-expressed transferrin receptors in carcinoma cells (e.g., Hep
G2 cells). In addition, the relatively positively charged ReSiN surface can be shielded by the transferrin to prevent nonspecific binding to the cell surface, minimizing internalization rate and short half-life in blood circulation. A series of surface modification steps were evaluated by zeta potential measurements; and SEM analysis revealed that the modification process did not alter the morphology of the beads (Fig. S4). Because of the excess amount of APTMS employed in the sol–gel reaction, it was expected that the bead surface was composed of polymerized APTMS, which provided positively charged amino groups (–NH2) and enabled the modification of ReSiNs with 5-TAMRA via N-succinimidyl ester. The maximum loading capacity of 5-TAMRA on ReSiN (ReSiN@TAMRA) was approximately 4.65 milligrams per gram of ReSiNs. As the transferrin has a measured pI = 5.5, it is negatively charged at PBS-buffered (pH 7.4) solution. The shift in zeta potential from +38.6 mV to 13.3 mV indicated that the modification with holo-transferrin influenced the surface charge of ReSiNs (Fig. 2f). Approximate 322 ± 86 transferrin molecules were adsorbed on single ReSiN@TAMRA by estimating the consumed proteins from the initial reaction stock (Fig. S5). In vitro study of drug release from ReSiN@TAMRA@Tf nanocarriers was conducted in PBS buffer (pH 7.4) at 37 °C with or without (w/o) DTT (Fig. 3c). Approximately 14.08% of 5-TAMRA was released within 4 h when it was exposed to 10 mM DTT, and sustained drug release was observed thereafter. It was proposed that the rapid release of fluorescent molecules from ReSiNs was due to the disassembly of the disulfide-linked scaffold by DTT
Please cite this article in press as: X.-C. Huang et al., Biothiol-triggered, self-disassembled silica nanobeads for intracellular drug delivery, Acta Biomater. (2015), http://dx.doi.org/10.1016/j.actbio.2015.05.006
6
X.-C. Huang et al. / Acta Biomaterialia xxx (2015) xxx–xxx
reduction. In contrast, only a small amount of 5-TAMRA (3.96%) was released in the absence of the reducing agent. As 5-TAMRA was covalently labeled to the nanocarrier, this minute amount of fluorescence leakage might be due to the partial hydrolysis of silane. The cytotoxicity of degraded ReSiN and ReSiN@TAMRA@Tf fragments was evaluated by incubating the cells for an additional 24 h with culture medium containing 10 mM glutathione ethyl ester (GEE). The cytotoxicity assay revealed that negligible effect of the treatment with either bare ReSi or functionalized ReSiN@TAMRA@Tf suspension on Hep G2 cell viability was observed at the concentration as high as 0.4 mg mL 1 before and after ReSi degradation (Fig. 4a). We also examined the cell viability
by external addition of the degraded ReSiN suspension (pre-treated with DTT). Insignificant toxicity was found in cells incubated with degradation products having free, reduced SH functional groups (Fig. S6). Finally, the confocal microscope was employed to monitor the ReSiN@TAMRA@Tf trafficking in the Hep G2 cells after incubation for 24 h w/o GEE (1 mM). Glutathione disulfide (GSSH) is maintained in a reduced state by enzymes such as glutathione reductase, and the intracellular GSH level is strictly regulated by NADH/NAD+, NADPH/NADP+, and thioredoxinred/thioredoxinox levels. GEE was introduced to stimulate the cellular glutathione production, and ReSiN@TAMRA@Tf was degraded in the presence
Fig. 4. (a) Evaluation of ReSiN cytotoxicity in Hep G2 cells by MTT assay. Bare ReSiNs and ReSiN@TAMRA@Tf nanocarriers at indicated concentrations were incubated separately with Hep G2 cells for 24 h, followed by washing with PBS for three times before the assay. To estimate the toxicity of degraded ReSiNs and the nanocomposites, Hep G2 cells were treated with 10 mM GEE for an additional 24 h. (b) Internalization and degradation of ReSiN@TAMRA@Tf nanocarriers in Hep G2 cells with and without (w/o) 1 mM GEE. Hep G2 cells (4 104 cells) were grown on a glass substrate in 24-well plates and treated with 1.2 lg mL 1 ReSiNs for 24 h, followed by washing with PBS for three times. Enhanced cellular glutathione level caused by GEE triggered significant release of TAMRA inside Hep G2 cells. Confocal fluorescent images were captured at 24 h after GEE treatment. (Control: cell only, without any treatments). (c) Statistical evaluation of intra- and extracellular TAMRA fluorescence of 20 cells shown in the confocal image (Fig. S) for determining the drug release triggered by GEE (***p < 0.001).
Please cite this article in press as: X.-C. Huang et al., Biothiol-triggered, self-disassembled silica nanobeads for intracellular drug delivery, Acta Biomater. (2015), http://dx.doi.org/10.1016/j.actbio.2015.05.006
X.-C. Huang et al. / Acta Biomaterialia xxx (2015) xxx–xxx
of GEE (Fig. 4b, and c), resulting in the diffused 5-TAMRA fluorescence in the culture milieu (The fluorescence intensity of 5-TAMRA had been examined in buffer under a pH 3–7 gradient to rule out its potential intensity enhancement upon pH, Fig. S7). We also compared the transferrin-mediated targeting efficacy in Hep G2 cell line (with high transferrin receptor expression) and a non-targeted breast adenocarcinoma MCF-7 cell line (with relatively low transferrin receptor expression) by flow cytometry (Fig. S8). The TAMRA-positive Hep G2 cells were significantly more than MCF-7. The result indicated that the cell selectivity of ReSiN@TAMRA@Tf toward Hep G2 was potentially through the specific recognition of functionalized transferrin with its receptor at Hep G2 cells, leading to the differential delivery in two types of cells. Subsequent labeling of lysosomes also demonstrated that the localization of lysosome fluorescence (green) and ReSiN@TAMRA@Tf fluorescence (red) had little signal overlap (Fig. S9), revealing the occurrence of endosomal escape. It has been reported that transferrin and transferrin receptor can be recycled in endosomes [30,31]. Presumably, these molecules could be retrieved from the ReSiN@TAMRA@Tf nanocarriers in endosomes. Although additional investigations are necessary for an understanding the underlying intracellular trafficking mechanisms, the proposed endosomal escape of silica-based nanocarriers in the present study can be explained by the proton sponge hypothesis of the deprotonated silanol groups [25], which may interrupt the charge balance of the system, as well as the modulation of the endocytic pathways by various alternative routes, for instance, macropinosome leakage [32]. Furthermore, the reduced microenvironment has been also found in the endocytotic pathway. The enzyme gamma interferon-inducible lysosomal thiol-reductase (GILT) and the excess level of cysteine in a lysosome favor the reduction of disulfide bonds [33]. Accordingly, the destabilization of redox-responsive ReSiNs led to efficient intracellular drug release and simultaneous degradation of carriers in the cytoplasm upon the cellular stimulation with biothiols. The design of novel redox-responsive drug nanocarriers for cancer theranostics by taking advantage of the redox gradient existing in the intracellular milieu of carcinomas has received considerable attention. Most of the contemporary silica-based, biothiol-mediated release systems are established with similar setups. Cap or gatekeeper molecules such as collagen [34], DNA [35,36], polymer molecules [37,38], gold nanoparticles [39], and cyclodextrin [40,41] have been employed to functionalize the surface of mesoporous silica nanoparticles using disulfide linkers for enabling the redox-responsive off/on drug release. However, few studies [42,43] have focused on exploiting the inherent properties of silica materials to achieve self-modulations in the intracellular milieu and to improve cytosolic bioavailability. The necessity to develop effective strategies for improving intracellular delivery is self-evident. In this study, we altered the chemically inert silica scaffold by introducing the cleavable disulfide linkage, creating the functionalities in structure itself to enable rapid, redox-triggered active degradation of silica nanocomposite rather than passive hydrolysis in most of other reported studies. In addition, surfactants were not employed during the synthesis which excluded an essential concern in biocompatibility of most mesoporous silica nanocomposites. As the main focus of this study is to provide new aspect in silica matrix design, varying the disulfide bond number and the spacing in the silica matrix may enable fine-tuning of the release profile. The substitution of TAMRA dye molecule by anticancer agent doxorubicin (DOX) in ReSiN has confirmed its compatible anticancer activity to the free form drug. Interestingly, we also observed a superior cell-killing performance of immobilized doxorubicin to free type at high concentration (Fig. S10). Although detailed mechanism requires further elucidation, this nanocomposite can be of
7
benefit in treating some drug-resistant cells which are insensitive to chemotherapy under repeated high dosage remedy. Drug-resistant carcinoma has been the major reason leading to the failure of chemotherapy. The high expression level of glutathione-S-transferases and its ligand GSH were observed in these cells and inhibition of the enzyme or depletion of GSH improved drug efficacy [44]. Our disulfide-rich silica matrix was likely to also play the role in depleting the intracellular GSH during its degradation. To enable better understanding of degradability, the clearance and the cytotoxicity of ReSiN-based nanocarrier, further investigation in either zebrafish [45] or mouse [46] models shall be employed in our future work. According to current drug loading capacity and the required dose of DOX to achieve anti-tumor effect, approximate 1.2–5 mg (in 1 mL culture medium) and 8.6–43 mg ReSiN nanobead (suspended in 0.5–1 mL saline for intraperitoneal injection) will be sufficient respectively for zebrafish and in tumor-bearing mice models. Comparing with traditional mice models, transparent zebrafish embryos and adults additionally allow for in vivo visualization of cancer growth and progression at single-cell resolution which may facilitate our ReSiN-based DDS monitoring. As doxorubicin is a well-known anti-cancer drug but with severe cardiotoxicity, as already demonstrated in a cell model in vitro, not only the anti-cancer activity of DOX shall be evaluated, but the comparison between the cardiotoxicity of the constructed DOX-loaded nanocomposite and that of free DOX also will be performed and investigated using a recently established zebrafish pseudodynamic 3D imaging technique [47] for a detailed understanding of the pharmacological response of cardiovascular function in vivo.
4. Conclusion In summary, we constructed a novel ReSiN-based DDS capable of redox-facilitated drug release and self-degradation after drug release. The disulfide-linked scaffold in the silica matrix could be disrupted by intracellular thiols, resulting in drug release and collapse of the ReSiNs. The results obtained indicated the potential in vivo application of the ReSiN-based drug carriers in fast dissolving DDSs. But nevertheless, poor porous structure has been a bottleneck in the current study which limits the drug loading capacity. The doping of DOX or photosensitizers in the silica matrix can be an alternative strategy for cancer therapy. Future studies should be conducted to improve these aspects, enhancing the therapeutic efficacy of the devised DDS for in vivo application.
5. Competing interest disclosure The authors declare no competing financial interest. Acknowledgements This work has been supported by the Ministry of Science and Technology of Taiwan (Grants No. MOST103-2113-M-009-006) and the Ministry of Education, Taiwan (‘Aim for the Top University Plan’ of National Chiao Tung University).
Appendix A. Figures with essential colour discrimination Certain figures in this article, particularly Figs. 1–4, are difficult to interpret in black and white. The full colour images can be found in the on-line version, at doi: http://dx.doi.org/10.1016/j.actbio. 2015.05.006.
Please cite this article in press as: X.-C. Huang et al., Biothiol-triggered, self-disassembled silica nanobeads for intracellular drug delivery, Acta Biomater. (2015), http://dx.doi.org/10.1016/j.actbio.2015.05.006
8
X.-C. Huang et al. / Acta Biomaterialia xxx (2015) xxx–xxx
Appendix B. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.actbio.2015.05. 006. References [1] M.E. Davis, Z. Chen, D.M. Shin, Nanoparticle therapeutics: an emerging treatment modality for cancer, Nat. Rev. Drug Discov. 7 (2008) 771–782. [2] R.K. Jain, T. Stylianopoulos, Delivering nanomedicine to solid tumors, Nat. Rev. Clin. Oncol. 7 (2010) 653–664. [3] O. Veiseh, J.W. Gunn, M.Q. Zhang, Design and fabrication of magnetic nanoparticles for targeted drug delivery and imaging, Adv. Drug Deliv. Rev. 62 (2010) 284–304. [4] G.F. Paciotti, D.G.I. Kingston, L. Tamarkin, Colloidal gold nanoparticles: a novel nanoparticle platform for developing multifunctional tumor-targeted drug delivery vectors, Drug Dev. Res. 67 (2006) 47–54. [5] I.I. Slowing, B.G. Trewyn, S. Giri, V.S.Y. Lin, Mesoporous silica nanoparticles for drug delivery and biosensing applications, Adv. Funct. Mater. 17 (2007) 1225– 1236. [6] N. Nasonkla, E. Bey, J. Ren, H. Ai, C. Khemtong, J.S. Guthi, et al., Multifunctional polymeric micelles as cancer-targeted, MRI-ultrasensitive drug delivery systems, Nano Lett. 6 (2006) 2427–2430. [7] K.S. Soppimath, D.C. Tan, Y.Y. Yang, PH-triggered thermally responsive polymer core-shell nanoparticles for drug delivery, Adv. Mater. 17 (2005) 318–323. [8] S. Dromi, V. Frenkel, A. Luk, B. Traughber, M. Angstadt, M. Bur, et al., Pulsedhigh intensity focused ultrasound and low temperature-sensitive liposomes for enhanced targeted drug delivery and antitumor effect, Clin. Cancer Res. 13 (2007) 2722–2727. [9] A. Albanese, P.S. Tang, W.C. Chan, The effect of nanoparticle size, shape, and surface chemistry on biological systems, Annu. Rev. Biomed. Eng. 14 (2012) 1– 16. [10] F. Danhier, O. Feron, V. Préat, To exploit the tumor microenvironment: passive and active tumor targeting of nanocarriers for anti-cancer drug delivery, J. Controlled Release 148 (2010) 135–146. [11] M.J. Ernsting, M. Murakami, A. Roy, S.-D. Li, Factors controlling the pharmacokinetics, biodistribution and intratumoral penetration of nanoparticles, J. Controlled Release 172 (2013) 782–794. [12] A.K. Iyer, G. Khaled, J. Fang, H. Maeda, Exploiting the enhanced permeability and retention effect for tumor targeting, Drug Discov. Today 11 (2006) 812– 818. [13] Y. Matsumura, H. Maeda, A new concept for macromolecular therapeutics in cancer chemotherapy: mechanism of tumoritropic accumulation of proteins and the antitumor agent smancs, Cancer Res. 46 (1986) 6387–6392. [14] E. Markovsky, H. Baabur-Cohen, A. Eldar-Boock, L. Omer, G. Tiram, S. Ferber, et al., Administration, distribution, metabolism and elimination of polymer therapeutics, J. Controlled Release 161 (2012) 446–460. [15] E.S. Shibu, K. Ono, S. Sugino, A. Nishioka, A. Yasuda, Y. Shigeri, et al., Photouncaging nanoparticles for MRI and fluorescence imaging in vitro and in vivo, ACS Nano 7 (2013) 9851–9859. [16] H. Wei, R.-X. Zhuo, X.-Z. Zhang, Design and development of polymeric micelles with cleavable links for intracellular drug delivery, Prog. Polym. Sci. 38 (2013) 503–535. [17] Y.H. Jin, A.Z. Li, S.G. Hazelton, S. Liang, C.L. John, P.D. Selid, et al., Amorphous silica nanohybrids: synthesis, properties and applications, Coord. Chem. Rev. 253 (2009) 2998–3014. [18] H. Nabeshi, T. Yoshikawa, K. Matsuyama, Y. Nakazato, K. Matsuo, A. Arimori, et al., Systemic distribution, nuclear entry and cytotoxicity of amorphous nanosilica following topical application, Biomaterials 32 (2011) 2713–2724. [19] J.M. Rosenholm, C. Sahlgren, M. Linden, Multifunctional mesoporous silica nanoparticles for combined therapeutic, diagnostic and targeted action in cancer treatment, Curr. Drug Targets 12 (2011) 1166–1186. [20] D. Tarn, C.E. Ashley, M. Xue, E.C. Carnes, J.I. Zink, C.J. Brinker, Mesoporous silica nanoparticle nanocarriers: biofunctionality and biocompatibility, Acc. Chem. Res. 46 (2013) 792–801. [21] P.P. Adiseshaiah, J.B. Hall, S.E. McNeil, Nanomaterial standards for efficacy and toxicity assessment, Wires Nanomed. Nanobi. 2 (2010) 99–112. [22] A.A. Burns, J. Vider, H. Ow, E. Herz, O. Penate-Medina, M. Baumgart, et al., Fluorescent silica nanoparticles with efficient urinary excretion for nanomedicine, Nano Lett. 9 (2008) 442–448. [23] V. Biju, Chemical modifications and bioconjugate reactions of nanomaterials for sensing, imaging, drug delivery and therapy, Chem. Soc. Rev. 43 (2014) 744–764. [24] X. Huang, C.S. Brazel, On the importance and mechanisms of burst release in matrix-controlled drug delivery systems, J Controlled Release 73 (2001) 121–136.
[25] N. Gouda, K. Miyata, R.J. Christie, T. Suma, A. Kishimura, S. Fukushima, et al., Silica nanogelling of environment-responsive PEGylated polyplexes for enhanced stability and intracellular delivery of siRNA, Biomaterials 34 (2013) 562–570. [26] S.A. Mackowiak, A. Schmidt, V. Weiss, C. Argyo, C. von Schirnding, T. Bein, et al., Targeted drug delivery in cancer cells with red-light photoactivated mesoporous silica nanoparticles, Nano Lett. 13 (2013) 2576–2583. [27] C.E. Ashley, E.C. Carnes, K.E. Epler, D.P. Padilla, G.K. Phillips, R.E. Castillo, et al., Delivery of small interfering RNA by peptide-targeted mesoporous silica nanoparticle-supported lipid bilayers, ACS Nano 6 (2012) 2174–2188. [28] R. Cheng, F. Feng, F. Meng, C. Deng, J. Feijen, Z. Zhong, Glutathione-responsive nano-vehicles as a promising platform for targeted intracellular drug and gene delivery, J. Controlled Release 152 (2011) 2–12. [29] W. Stöber, A. Fink, E. Bohn, Controlled growth of monodisperse silica spheres in the micron size range, J. Colloid Interface Sci. 26 (1968) 62–69. [30] A. Dautry-Varsat, A. Ciechanover, H.F. Lodish, PH and the recycling of transferrin during receptor-mediated endocytosis, Proc. Natl. Acad. Sci. 80 (1983) 2258–2262. [31] V. Biju, T. Itoh, M. Ishikawa, Delivering quantum dots to cells: bioconjugated quantum dots for targeted and nonspecific extracellular and intracellular imaging, Chem. Soc. Rev. 39 (2010) 3031–3056. [32] Y. Zhang, L. Hu, D. Yu, C. Gao, Influence of silica particle internalization on adhesion and migration of human dermal fibroblasts, Biomaterials 31 (2010) 8465–8474. [33] B. Arunachalam, U.T. Phan, H.J. Geuze, P. Cresswell, Enzymatic reduction of disulfide bonds in lysosomes: characterization of a gamma-interferoninducible lysosomal thiol reductase (GILT), Proc. Natl. Acad. Sci. 97 (2000) 745–750. [34] Z. Luo, K. Cai, Y. Hu, L. Zhao, P. Liu, L. Duan, et al., Mesoporous silica nanoparticles end-capped with collagen: redox-responsive nanoreservoirs for targeted drug delivery, Angew. Chem. Int. Ed. 50 (2011) 640–643. [35] X. Ma, K.T. Nguyen, P. Borah, C.Y. Ang, Y. Zhao, Functional silica nanoparticles for redox-triggered drug/ssDNA Co-delivery, Adv. Health Mater. 1 (2012) 690– 697. [36] C.-L. Zhu, X.-Y. Song, W.-H. Zhou, H.-H. Yang, Y.-H. Wen, X.-R. Wang, An efficient cell-targeting and intracellular controlled-release drug delivery system based on MSN-PEM-aptamer conjugates, J. Mater. Chem. 19 (2009) 7765–7770. [37] Y. Cui, H. Dong, X. Cai, D. Wang, Y. Li, Mesoporous silica nanoparticles capped with disulfide-linked PEG gatekeepers for glutathione-mediated controlled release, ACS Appl. Mater. Interf. 4 (2012) 3177–3183. [38] Y. Wang, Y. Yan, J. Cui, L. Hosta-Rigau, J.K. Heath, E.C. Nice, et al., Encapsulation of water-insoluble drugs in polymer capsules prepared using mesoporous silica templates for intracellular drug delivery, Adv. Mater. 22 (2010) 4293– 4297. [39] S. Giri, B.G. Trewyn, M.P. Stellmaker, V.S.Y. Lin, Stimuli-responsive controlledrelease delivery system based on mesoporous silica nanorods capped with magnetic nanoparticles, Angew. Chem. Int. Ed. 44 (2005) 5038–5044. [40] H. Kim, S. Kim, C. Park, H. Lee, H.J. Park, C. Kim, Glutathione-induced intracellular release of guests from mesoporous silica nanocontainers with cyclodextrin gatekeepers, Adv. Mater. 22 (2010) 4280–4283. [41] Q. Zhang, F. Liu, K.T. Nguyen, X. Ma, X. Wang, B. Xing, et al., Multifunctional mesoporous silica nanoparticles for cancer-targeted and controlled drug delivery, Adv. Funct. Mater. 22 (2012) 5144–5156. [42] Z. Tao, B.B. Toms, J. Goodisman, T. Asefa, Mesoporosity and functional group dependent endocytosis and cytotoxicity of silica nanomaterials, Chem. Res. Toxicol. 22 (2009) 1869–1880. [43] I. Slowing, B.G. Trewyn, V.S.Y. Lin, Effect of surface functionalization of MCM41-type mesoporous silica nanoparticles on the endocytosis by human cancer cells, J. Am. Chem. Soc. 128 (2006) 14792–14793. [44] D.M. Townsend, K.D. Tew, The role of glutathione-S-transferase in anti-cancer drug resistance, Oncogene 22 (2003) 7369–7375. [45] S. Deng, Q. Wu, Y. Zhao, X. Zheng, N. Wu, J. Pang, et al., Biodegradable polymeric micelle-encapsulated doxorubicin suppresses tumor metastasis by killing circulating tumor cells, Nanoscale 7 (2015) 5270–5280. [46] P.D. Ottewell, H. Mönkkönen, M. Jones, D.V. Lefley, R.E. Coleman, I. Holen, Antitumor effects of doxorubicin followed by zoledronic acid in a mouse model of breast cancer, J. Natl. Cancer Inst. 100 (2008) 1167–1178. [47] K.-Y. Lin, W.-T. Chang, Y.-C. Lai, I. Liau, Toward functional screening of cardioactive and cardiotoxic drugs with zebrafish in vivo using pseudodynamic three-dimensional imaging, Anal. Chem. 86 (2014) 2213– 2220. [48] C.-K. Huang, H.-o. Hamaguchi, S. Shigeto, In vivo multimode Raman imaging reveals concerted molecular composition and distribution changes during yeast cell cycle, Chem. Commun. 47 (2011) 9423–9425. [49] N. Noothalapati Venkata Hemanth, S. Shigeto, Stable isotope-labeled raman imaging reveals dynamic proteome localization to lipid droplets in single fission yeast cells, Chem Biol 19 (2012) 1373–1380.
Please cite this article in press as: X.-C. Huang et al., Biothiol-triggered, self-disassembled silica nanobeads for intracellular drug delivery, Acta Biomater. (2015), http://dx.doi.org/10.1016/j.actbio.2015.05.006
Contents lists available at ScienceDirect
Acta Biomaterialia journal homepage: www.elsevier.com/locate/actabiomat
Brief communication
Biothiol-triggered, self-disassembled silica nanobeads for intracellular drug delivery Xin-Chun Huang a, Li-Bang Wu a, Jen-Fang Hsu b, Shinsuke Shigeto a,b, Hsin-Yun Hsu a,b,⇑ a b
Department of Applied Chemistry, National Chiao-Tung University, No. 1001 Ta-Hsueh Road, Hsinchu 30010, Taiwan Institute of Molecular Science, National Chiao-Tung University, No. 1001 Ta-Hsueh Road, Hsinchu 30010, Taiwan
a r t i c l e
i n f o
Article history: Received 14 November 2014 Received in revised form 4 May 2015 Accepted 10 May 2015 Available online xxxx Keywords: Drug delivery Silica nanoparticles Sol–gel processes Self-disassembly Biothiols
a b s t r a c t Silica-based nanomaterials have demonstrated great potential in biomedical applications due to their chemical inertness. However, the degradability and endosomal trapping issues remain as rate-limiting barriers during their innovation. In this study, we provide a simple yet novel sol–gel approach to construct the redox-responsive silica nanobeads (ReSiNs), which could be rapidly disassembled upon redox gradient for intracellular drug delivery. The disulfide-linked scaffold of the nanobead was synthesized by employing the dithiobis-(succinimidyl propionate) to bridge (3-aminopropyl)-trimethoxysilane. Such silica matrix could be efficiently disrupted in response to intracellular glutathione, resulting in drug release and collapse of entire nanocarrier. Moreover, the ReSiNs exhibited insignificant cytotoxicity before and after the degradation. These results indicated the potential of using ReSiNs as a novel silica-based, biothiol-degradable nanoplatform for future drug delivery. Ó 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
1. Introduction Nanomaterial-based drug delivery systems (DDSs) have shown great potential in theranostic applications [1–3]. The use of metals and non-metals, polymers, liposomes, micelles, and nanoclusters had been found in many studies [4–8] for smart drug delivery. Despite the immense advancement in the field, there are still huge gaps from bench to the bedside and several critical concerns shall be considered when designing the nanocarriers. It has been reported that the surface-modified nanocarriers with a diameter in the range of 20–200 nm can escape from renal filtration [9– 11] and show enhanced permeability and retention (EPR) effects in targeted cancer therapy [10,12,13]. On the other hand, the degradation and clearance of the constructed nanomaterials in the body should occur in a reasonable period after drug release at the target tissue site [14]. In this regard, recent study has shown promising targeted delivery and uncaging of nanoparticles into fragments for subsequent renal clearance by employing the photouncaging ligands to enable light triggering [15]. Alternatively, some micelle systems with cleavable linkers were incorporated to allow responsive degradation upon internal stimuli such as pH or redox status [16]. But nevertheless, external light-based systems ⇑ Corresponding author at: Department of Applied Chemistry, National Chiao-Tung University, No. 1001 Ta-Hsueh Road, Hsinchu 30010, Taiwan. Tel.: +886 (0)3 5712121x56556; fax: +886 (0)3 5723764. E-mail address: [email protected] (H.-Y. Hsu).
may suffer from the rapid intensity attenuation of the short-wavelength excitation in tissues; conversely, the chemical instability caused by oxidation and the difficulties in precise size control due to vesicle fusion frequently occurred in the polymeric micelle-based nanoplatforms. In the past decades, amorphous [17,18] and mesoporous silica nanoparticles [19,20] have become one of the most promising materials in nanomedicine. The silica matrix has shown several intrinsic advantages over conventional organic polymers, including high mechanical resistance, chemical inertness with minimal cargo interaction, and biosafety, which has been recognized by the U.S. Food and Drug Administration [21,22]. Silica nanoparticles with tunable particle diameter and morphology have been studied in sol–gel process and water in oil microemulsion, in addition, the chemical modification of its surface can be easily achieved using silane coupling reagents to enable a variety of surface functionalities (hydroxyl/amino/thiol/carboxyl groups) and targeting ligand modification [23]. However, synthesis of mesoporous silica nanoparticles often requires the employment of surfactants such as cetyltrimethylammonium bromide (CTAB) to form mesostructured silica shell. Removal of surfactants is necessary to improve biocompatibility. In addition, the hampered intracellular bioavailability by the endosomal uptake pathway and the degradability of silica nanocomposites remain as challenges to fast dissolving DDSs [24]. Although several sophisticated designs have been shown to facilitate the endosomal escape by modulating the
http://dx.doi.org/10.1016/j.actbio.2015.05.006 1742-7061/Ó 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
Please cite this article in press as: X.-C. Huang et al., Biothiol-triggered, self-disassembled silica nanobeads for intracellular drug delivery, Acta Biomater. (2015), http://dx.doi.org/10.1016/j.actbio.2015.05.006
2
X.-C. Huang et al. / Acta Biomaterialia xxx (2015) xxx–xxx
surface chemistry [25], incorporating a photosensitizer [26], or conjugating endosomolytic peptides [27] to the nanocomposites, limited researches alter the chemically inert silica scaffold by manipulating the bonding linkage, creating the functionalities in structure itself to simplify the synthetic routes. Besides, the feasibility of such materials for subsequent intracellular degradation also had been rarely considered. It is well known that disulfide bonds can be cleaved by reducing agents (e.g., dithiothreitol, DTT), and they are sensitive to the reduced milieu. One of such redox-active biothiols in cells is glutathione (GSH), which is often present in the millimolar range in the cytoplasm in contrast to their concentration in the extracellular milieu (2–20 lM) [28]. The significant redox potential difference between the interior and exterior of the cell could be exploited as a physiological stimulus for subcellular delivery of therapeutic biomolecules. In this regard, we developed a disulfide-rich silica-based scaffold for the nanocarrier to achieve redox-facilitated drug release and enhance its self-degradability. Once these drug nanocarriers were taken up by the targeted cells, disulfide bonds in the silica matrix could be reduced by intracellular thiols, enabling the drug release and collapse of the nanobeads into fragmented residuals for rapid removal.
electrostatic interactions. The mixture was washed three times with PBS by centrifugation and stored in PBS at 4 °C. 2.4. ReSiN characterization
2. Materials and methods
A field-emission scanning electron microscope (FE-SEM, JSM-7401F, JOEL) and an ultra-high resolution transmission electron microscope (JEOL JEM-ARM200F Cs-corrected Field Emission) were employed to characterize the morphology of ReSiNs. Raman scattering spectrum, generated by a He–Ne laser (632.8 nm, 11 mW), was collected under RT (60 s/spectrum) using an oil-immersion objective (100, NA 1.3). Ten spectra were recorded for each sample and those that did not show spiky feature (i.e., cosmic rays) were averaged to obtain the mean spectra (No background subtraction was performed). 29Si NMR spectra of ReSiN were analyzed using DSX400WB NMR Spectrometer (Bruker, Germany). The silica concentration was determined by ICP-AES (Model S-35, Kontron, Germany). The degradation percentage was calculated as follows: ReSiN degradation (%) = average Si content of treated group/average Si content of control group (ReSiN dissolved in 1 M KOH) 100%. Zeta potentials were recorded using Zetasizer Nano ZS (Malvern Instruments, UK). Fluorescence intensity was obtained on a microplate fluorescence reader (BioTek FL 800).
2.1. Reagents
2.5. Cytotoxicity assay and cell imaging
Dithiobis-succinimidyl propionate (DTSP), ethanol, 3-aminopropyl-trimethoxysilane (APTMS), 3-mercaptopropyl-tri methoxysilane (MPTMS), hydrochloric acid solution, ammonium hydroxide solution, 5-carboxy-tetramethylrhodamine (5-TAMRA), potassium hydroxide, and Tris (base) were purchased from Sigma–Aldrich. Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), Dulbecco’s phosphate-buffered saline (PBS), and penicillin/streptomycin were purchased from Biowest (Nuaillé, France). CellLightÒ Lysosomes-GFP was supplied by Life Technologies (ThermoFisher, Massachusetts, USA).
Hep G2 cells (1 104 cells/well) were cultured in 96-well plates containing DMEM supplemented with 10% FBS and 1% penicillin/streptomycin at 37 °C under 5% CO2. After 12 h, the culture medium was removed, and the cells were washed three times with PBS. Next, the cells were incubated in 100 lL of fresh medium with 1, 10, 20, 100, 200, or 400 lg mL 1 ReSiNs or ReSiN@TAMRA@Tf nanocarriers for 24 h. The cytotoxicity of degraded ReSiN and ReSiN@TAMRA@Tf fragments was evaluated by incubating the cells for an additional 24 h with 100 lL of culture medium containing 10 mM GEE. The culture medium was then discarded, and cells were washed twice with PBS. The culture medium (50 lL) containing 5 mg mL 1 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo lium bromide (MTT) was applied to the cells, followed by incubation for 3 h. The supernatant was removed, and the formazan precipitate was dissolved in DMSO for 10 min. Absorption (Abs) at 570 nm was measured using a UV–Vis microplate reader (Cary 100 Varian, Palo Alto, CA, USA). Cell viability was determined as follows: cell viability (%) = mean Abs of treated group/mean Abs of control 100%. Confocal images were obtained by using Leica TCS SP5 confocal laser scanning microscope.
2.2. Preparation of ReSiNs The synthesis of ReSiNs was carried out using the sol–gel process. Firstly, DTSP (2.00 mg, 5 lmole) was dissolved in 50 lL of dimethyl sulfoxide (DMSO). Next, HCl(aq) (1.00 M, 5 lL) and APTMS (6.3 lL, 35 lmole) were added to the DTSP solution, followed by sonication for 40 min. Tris–HCl buffer (1 M, pH 8.0, 50 lL) was added to the reaction mixture, followed by sonication for an additional 10 min. The mixture was then added dropwise to an ethanol solution, followed by the addition of 50 lL of 0.25% ammonium hydroxide (NH4OH) and 100 lL of ddH2O. After incubation for 6 h at room temperature (RT), the product was purified by centrifugation (12,000g, 10 min) and washed three times with ethanol. ReSiNs were finally suspended in water and dried by lyophilization.
2.6. Statistical analysis The statistical analyses were performed using OriginPro 8 Software (OriginLab Corp., Northampton, MA). The Mann– Whitney U test with a p-value less than 0.05 was reported as significant.
2.3. Preparation of ReSiN@TAMRA@Tf 3. Results and discussion Briefly, 0.11 mg of ReSiNs was dispersed in 600 lL of EtOH, and 2.4 nmole 5-TAMRA was added to the suspension at RT, followed by sonication for 4 h. To remove free 5-TAMRA, the product was centrifuged at 12,000g for 10 min and washed three times with ethanol. After centrifugation, the supernatant was discarded, and purified ReSiN@TAMRA was obtained. Next, ReSiN@TAMRA was suspended in 1 mg mL 1 holo-transferrin solution (dissolved in PBS solution, pH 7.4), which was incubated at 4 °C overnight. Transferrin was adsorbed at the surface of ReSiN@TAMRA via
The synthetic process of the redox-responsive silica nanobeads (ReSiNs) was developed based on the fabrication principle of sol– gel system [29] using the crosslinker dithiobis-succinimidyl propionate (DTSP) to bridge (3-aminopropyl)-trimethoxysilane (Fig. 1). The morphology and inner structure of ReSiN were analyzed by transmission electron microscopy (TEM) with a point-EDX scan as shown in figure Fig. 2a and b. No crystalline structure was observed in TEM images and the sulfur/silicon molar ratio
Please cite this article in press as: X.-C. Huang et al., Biothiol-triggered, self-disassembled silica nanobeads for intracellular drug delivery, Acta Biomater. (2015), http://dx.doi.org/10.1016/j.actbio.2015.05.006
X.-C. Huang et al. / Acta Biomaterialia xxx (2015) xxx–xxx
3
Fig. 1. Schematic illustrations of the proposed route after the cellular uptake of ReSiNs (upper panel) and preparation of ReSiNs (lower panel) (5-TAMRA: 5-carboxytetramethylrhodamine N-succinimidyl ester; Tf: transferrin; DMSO: dimethyl sulfoxide; APTMS: 3-aminopropyl-trimethoxysilane).
increased from the surface (S: Si = 1: 8.7 toward the core (S: Si = 1: 2) of ReSiN. The statistical diameter of ReSiN was approximately 143.3 ± 38.2 nm, as estimated by scanning electron microscopy (SEM, Fig. 2c), and this value was within the optimal diameter range of nanoparticle-based DDSs for enhanced permeability and retention effects on targeted cancer cells. Raman spectroscopy was employed to characterize the fabricated nanobeads (Fig. 2d, S1). Two strong peaks at 509 cm 1 and 615 cm 1 have been observed in aggregates of ReSiNs (Fig. 2d, black), which correspond to the stretching vibrational mode of the aryl disulfide (S–S and C–S, respectively). The same peaks were also observed in the Raman spectrum of pure DTSP (Fig. 2d, red). In addition, 3-mercap topropyl-trimethoxysilane (MPTMS) silica nanoparticles, used as thiolated silane control material to monitor the S–H stretching
vibration at 2570 cm 1 (see Fig. 2d, blue), were synthesized by the sol–gel process. No apparent S–H stretch band was observed in the spectrum of the ReSiN powder, indicating that the disulfide bonds of ReSiN remained intact after the synthetic process. We evaluated the silanol functionality in ReSiN by solid NMR spectroscopy (Fig. 2e). A strong T3 signal corresponding to silicon atoms with a single alkyl group and a small shoulder T2 peak were both observed in 29Si-NMR spectrum, revealing that the silane networks were stably formed. The degradability of ReSiNs was evaluated by measuring the released silica from ReSiNs (14.6 lg for each condition) after the DTT treatment (10 lM–10 mM in PBS, pH 7.4) at 37 °C for 1 h using inductively coupled plasma-atomic emission spectroscopy (ICP-AES). As shown in Fig. 3a, approximately 22.5 ± 5.8% of the silica fragments were released from ReSiNs. In
Please cite this article in press as: X.-C. Huang et al., Biothiol-triggered, self-disassembled silica nanobeads for intracellular drug delivery, Acta Biomater. (2015), http://dx.doi.org/10.1016/j.actbio.2015.05.006
4
X.-C. Huang et al. / Acta Biomaterialia xxx (2015) xxx–xxx
Fig. 2. (a) TEM image, (b) point-EDX scan, and (c) SEM image of ReSiN. (d) Raman spectra of APTMS droplet (green trace), DTSP powder (red trace), MPTMS powder (blue trace), and ReSiN aggregate (black trace). Samples were deposited on a glass substrate for spectral measurement. Raman spectra were obtained with an exposure time of 60 s using a confocal Raman microspectrometer equipped with a He–Ne laser (632.8 nm, 11 mW at the sample point) as the excitation source [48,49]. Ten spectra were recorded for each sample and those that did not show spiky features were averaged to obtain the mean spectra. No background subtraction was performed. The optical image displays ReSiN aggregates, and an asterisk indicates the position at which the Raman spectra were measured. (e) Solid-NMR spectrum of ReSiN ((SiO)2Si(OAlk)2 (T2); (SiO)3Si(OAlk) (T3); Alk = alkyl group). (f) Zeta potentials of the bare ReSiNs, ReSiN@TAMRA, and ReSiN@TAMRA@Tf nanocomposites. The surface of ReSiNs was functionalized by APTMS, rendering positive charges on the surface. Insignificant change in the surface charge was found after 5-TAMRA labeling. Holo-transferrin was conjugated to the surface of ReSiNs via electrostatic interaction for enabling specific targeting of ReSiNs to Hep G2 cells. All samples were analyzed in 1 mM PBS (pH 7.4) at RT.
contrast, no silica fragment was detected in the supernatant of MPTMS nanoparticles, which were subject to the same DTT treatments, after incubation for 1 h. SEM was employed to monitor
the morphological changes of ReSiNs incubated with 10 mM DTT at room temperature (RT) for 0.5, 1.5, 2, and 2.5 h (Fig. 3b). An increase in the deformation level of ReSiNs was observed with an
Please cite this article in press as: X.-C. Huang et al., Biothiol-triggered, self-disassembled silica nanobeads for intracellular drug delivery, Acta Biomater. (2015), http://dx.doi.org/10.1016/j.actbio.2015.05.006
X.-C. Huang et al. / Acta Biomaterialia xxx (2015) xxx–xxx
5
Fig. 3. (a) Degradability of ReSiNs and MPTMS nanoparticles (control) determined by ICP-AES. Briefly, 14.6 lg mL 1 ReSiNs and 21.9 lg mL 1 MPTMS nanoparticles were separately incubated with DTT at indicated concentrations (in PBS) at 37 °C for 1 h. The suspensions were centrifuged at 12000g for 10 min, and supernatants were collected and diluted with 10 mL of 0.5% HNO3 for measuring the silica content by ICP-AES. The release percentage of silica from the two types of materials was calculated and normalized according to the initial input. (ND: not detectable, the concentration of silica was lower than the detection limit of 0.026 ppm). (b) SEM images showing the distribution and morphology of ReSiNs. Briefly, 0.12 lg of ReSiNs was dried on the glass substrate, and 3 lL of 10 mM DTT was dropped onto the ReSiN-loaded glass substrate, followed by incubation for indicated time periods at RT. After each hour of incubation, the DTT droplet was discarded, followed by washing with DI water for three times and replenishing the glass substrate with DTT before SEM measurement. ReSiNs incubated in H2O for 2.5 h were employed as control to confirm the stability of ReSiNs in the absence of DTT. All the washing steps were consistent with the conditions of using DTT to avoid bias. The black bar represents 1 lm. (c) Release profiles of 5-TAMRA from the ReSiN@TAMRA@Tf nanocarriers in the presence or in the absence of 10 mM DTT in PBS (pH 7.4) at 37 °C. The fluorescence intensity of the supernatant was determined at the indicated time points using a microplate fluorescence reader.
increase in the incubation time, and the degraded ReSiN fragments could be easily washed away from the glass substrate afterward. The degradability of ReSiN under acidic environment was also investigated. ReSiNs were incubated in 10 mM DTT buffered by the pH gradient at 37 °C for 1 h (Fig. S2). The degradation was decreased with decreasing pH, from 20.92 ± 1.56% (pH 7) to 5.34 ± 0.73% (pH 3). Current data indicated that the disulfide cleavage predominantly comes from thiol-disulfide exchange that requires the presence of thiolate anion (pKa = 8.3), which is unlikely to deprotonate under acidic environment. We also studied the redox-responsive degradation by MALDI-TOF-MS to analyze the ReSiN fragments after 10 mM DTT or 1 M KOH treatments. The results have identified significantly different characteristic peaks in mass spectra (Fig. S3), indicating that the mechanism of degradation by DTT was different from KOH-triggered alkaline hydrolysis. To further clarify the feasibility of using ReSiNs as redox-degradable drug nanocarriers, we labeled the nanobead surface with 5-carboxy-tetramethylrhodamine (5-TAMRA), a model drug, via N-succinimidyl ester to form thiol non-responsive bond. This design enabled us to further confirm the rapid silica matrix degradation upon redox potential by tracking intracellular fluorescence distribution. The diffused 5-TAMRA fluorescence can be observed only in the case that ReSiNs are degraded in the cells. The negatively charged holo-transferrin (Tf) was chosen as the tumor-targeting molecule due to its specificity for the over-expressed transferrin receptors in carcinoma cells (e.g., Hep
G2 cells). In addition, the relatively positively charged ReSiN surface can be shielded by the transferrin to prevent nonspecific binding to the cell surface, minimizing internalization rate and short half-life in blood circulation. A series of surface modification steps were evaluated by zeta potential measurements; and SEM analysis revealed that the modification process did not alter the morphology of the beads (Fig. S4). Because of the excess amount of APTMS employed in the sol–gel reaction, it was expected that the bead surface was composed of polymerized APTMS, which provided positively charged amino groups (–NH2) and enabled the modification of ReSiNs with 5-TAMRA via N-succinimidyl ester. The maximum loading capacity of 5-TAMRA on ReSiN (ReSiN@TAMRA) was approximately 4.65 milligrams per gram of ReSiNs. As the transferrin has a measured pI = 5.5, it is negatively charged at PBS-buffered (pH 7.4) solution. The shift in zeta potential from +38.6 mV to 13.3 mV indicated that the modification with holo-transferrin influenced the surface charge of ReSiNs (Fig. 2f). Approximate 322 ± 86 transferrin molecules were adsorbed on single ReSiN@TAMRA by estimating the consumed proteins from the initial reaction stock (Fig. S5). In vitro study of drug release from ReSiN@TAMRA@Tf nanocarriers was conducted in PBS buffer (pH 7.4) at 37 °C with or without (w/o) DTT (Fig. 3c). Approximately 14.08% of 5-TAMRA was released within 4 h when it was exposed to 10 mM DTT, and sustained drug release was observed thereafter. It was proposed that the rapid release of fluorescent molecules from ReSiNs was due to the disassembly of the disulfide-linked scaffold by DTT
Please cite this article in press as: X.-C. Huang et al., Biothiol-triggered, self-disassembled silica nanobeads for intracellular drug delivery, Acta Biomater. (2015), http://dx.doi.org/10.1016/j.actbio.2015.05.006
6
X.-C. Huang et al. / Acta Biomaterialia xxx (2015) xxx–xxx
reduction. In contrast, only a small amount of 5-TAMRA (3.96%) was released in the absence of the reducing agent. As 5-TAMRA was covalently labeled to the nanocarrier, this minute amount of fluorescence leakage might be due to the partial hydrolysis of silane. The cytotoxicity of degraded ReSiN and ReSiN@TAMRA@Tf fragments was evaluated by incubating the cells for an additional 24 h with culture medium containing 10 mM glutathione ethyl ester (GEE). The cytotoxicity assay revealed that negligible effect of the treatment with either bare ReSi or functionalized ReSiN@TAMRA@Tf suspension on Hep G2 cell viability was observed at the concentration as high as 0.4 mg mL 1 before and after ReSi degradation (Fig. 4a). We also examined the cell viability
by external addition of the degraded ReSiN suspension (pre-treated with DTT). Insignificant toxicity was found in cells incubated with degradation products having free, reduced SH functional groups (Fig. S6). Finally, the confocal microscope was employed to monitor the ReSiN@TAMRA@Tf trafficking in the Hep G2 cells after incubation for 24 h w/o GEE (1 mM). Glutathione disulfide (GSSH) is maintained in a reduced state by enzymes such as glutathione reductase, and the intracellular GSH level is strictly regulated by NADH/NAD+, NADPH/NADP+, and thioredoxinred/thioredoxinox levels. GEE was introduced to stimulate the cellular glutathione production, and ReSiN@TAMRA@Tf was degraded in the presence
Fig. 4. (a) Evaluation of ReSiN cytotoxicity in Hep G2 cells by MTT assay. Bare ReSiNs and ReSiN@TAMRA@Tf nanocarriers at indicated concentrations were incubated separately with Hep G2 cells for 24 h, followed by washing with PBS for three times before the assay. To estimate the toxicity of degraded ReSiNs and the nanocomposites, Hep G2 cells were treated with 10 mM GEE for an additional 24 h. (b) Internalization and degradation of ReSiN@TAMRA@Tf nanocarriers in Hep G2 cells with and without (w/o) 1 mM GEE. Hep G2 cells (4 104 cells) were grown on a glass substrate in 24-well plates and treated with 1.2 lg mL 1 ReSiNs for 24 h, followed by washing with PBS for three times. Enhanced cellular glutathione level caused by GEE triggered significant release of TAMRA inside Hep G2 cells. Confocal fluorescent images were captured at 24 h after GEE treatment. (Control: cell only, without any treatments). (c) Statistical evaluation of intra- and extracellular TAMRA fluorescence of 20 cells shown in the confocal image (Fig. S) for determining the drug release triggered by GEE (***p < 0.001).
Please cite this article in press as: X.-C. Huang et al., Biothiol-triggered, self-disassembled silica nanobeads for intracellular drug delivery, Acta Biomater. (2015), http://dx.doi.org/10.1016/j.actbio.2015.05.006
X.-C. Huang et al. / Acta Biomaterialia xxx (2015) xxx–xxx
of GEE (Fig. 4b, and c), resulting in the diffused 5-TAMRA fluorescence in the culture milieu (The fluorescence intensity of 5-TAMRA had been examined in buffer under a pH 3–7 gradient to rule out its potential intensity enhancement upon pH, Fig. S7). We also compared the transferrin-mediated targeting efficacy in Hep G2 cell line (with high transferrin receptor expression) and a non-targeted breast adenocarcinoma MCF-7 cell line (with relatively low transferrin receptor expression) by flow cytometry (Fig. S8). The TAMRA-positive Hep G2 cells were significantly more than MCF-7. The result indicated that the cell selectivity of ReSiN@TAMRA@Tf toward Hep G2 was potentially through the specific recognition of functionalized transferrin with its receptor at Hep G2 cells, leading to the differential delivery in two types of cells. Subsequent labeling of lysosomes also demonstrated that the localization of lysosome fluorescence (green) and ReSiN@TAMRA@Tf fluorescence (red) had little signal overlap (Fig. S9), revealing the occurrence of endosomal escape. It has been reported that transferrin and transferrin receptor can be recycled in endosomes [30,31]. Presumably, these molecules could be retrieved from the ReSiN@TAMRA@Tf nanocarriers in endosomes. Although additional investigations are necessary for an understanding the underlying intracellular trafficking mechanisms, the proposed endosomal escape of silica-based nanocarriers in the present study can be explained by the proton sponge hypothesis of the deprotonated silanol groups [25], which may interrupt the charge balance of the system, as well as the modulation of the endocytic pathways by various alternative routes, for instance, macropinosome leakage [32]. Furthermore, the reduced microenvironment has been also found in the endocytotic pathway. The enzyme gamma interferon-inducible lysosomal thiol-reductase (GILT) and the excess level of cysteine in a lysosome favor the reduction of disulfide bonds [33]. Accordingly, the destabilization of redox-responsive ReSiNs led to efficient intracellular drug release and simultaneous degradation of carriers in the cytoplasm upon the cellular stimulation with biothiols. The design of novel redox-responsive drug nanocarriers for cancer theranostics by taking advantage of the redox gradient existing in the intracellular milieu of carcinomas has received considerable attention. Most of the contemporary silica-based, biothiol-mediated release systems are established with similar setups. Cap or gatekeeper molecules such as collagen [34], DNA [35,36], polymer molecules [37,38], gold nanoparticles [39], and cyclodextrin [40,41] have been employed to functionalize the surface of mesoporous silica nanoparticles using disulfide linkers for enabling the redox-responsive off/on drug release. However, few studies [42,43] have focused on exploiting the inherent properties of silica materials to achieve self-modulations in the intracellular milieu and to improve cytosolic bioavailability. The necessity to develop effective strategies for improving intracellular delivery is self-evident. In this study, we altered the chemically inert silica scaffold by introducing the cleavable disulfide linkage, creating the functionalities in structure itself to enable rapid, redox-triggered active degradation of silica nanocomposite rather than passive hydrolysis in most of other reported studies. In addition, surfactants were not employed during the synthesis which excluded an essential concern in biocompatibility of most mesoporous silica nanocomposites. As the main focus of this study is to provide new aspect in silica matrix design, varying the disulfide bond number and the spacing in the silica matrix may enable fine-tuning of the release profile. The substitution of TAMRA dye molecule by anticancer agent doxorubicin (DOX) in ReSiN has confirmed its compatible anticancer activity to the free form drug. Interestingly, we also observed a superior cell-killing performance of immobilized doxorubicin to free type at high concentration (Fig. S10). Although detailed mechanism requires further elucidation, this nanocomposite can be of
7
benefit in treating some drug-resistant cells which are insensitive to chemotherapy under repeated high dosage remedy. Drug-resistant carcinoma has been the major reason leading to the failure of chemotherapy. The high expression level of glutathione-S-transferases and its ligand GSH were observed in these cells and inhibition of the enzyme or depletion of GSH improved drug efficacy [44]. Our disulfide-rich silica matrix was likely to also play the role in depleting the intracellular GSH during its degradation. To enable better understanding of degradability, the clearance and the cytotoxicity of ReSiN-based nanocarrier, further investigation in either zebrafish [45] or mouse [46] models shall be employed in our future work. According to current drug loading capacity and the required dose of DOX to achieve anti-tumor effect, approximate 1.2–5 mg (in 1 mL culture medium) and 8.6–43 mg ReSiN nanobead (suspended in 0.5–1 mL saline for intraperitoneal injection) will be sufficient respectively for zebrafish and in tumor-bearing mice models. Comparing with traditional mice models, transparent zebrafish embryos and adults additionally allow for in vivo visualization of cancer growth and progression at single-cell resolution which may facilitate our ReSiN-based DDS monitoring. As doxorubicin is a well-known anti-cancer drug but with severe cardiotoxicity, as already demonstrated in a cell model in vitro, not only the anti-cancer activity of DOX shall be evaluated, but the comparison between the cardiotoxicity of the constructed DOX-loaded nanocomposite and that of free DOX also will be performed and investigated using a recently established zebrafish pseudodynamic 3D imaging technique [47] for a detailed understanding of the pharmacological response of cardiovascular function in vivo.
4. Conclusion In summary, we constructed a novel ReSiN-based DDS capable of redox-facilitated drug release and self-degradation after drug release. The disulfide-linked scaffold in the silica matrix could be disrupted by intracellular thiols, resulting in drug release and collapse of the ReSiNs. The results obtained indicated the potential in vivo application of the ReSiN-based drug carriers in fast dissolving DDSs. But nevertheless, poor porous structure has been a bottleneck in the current study which limits the drug loading capacity. The doping of DOX or photosensitizers in the silica matrix can be an alternative strategy for cancer therapy. Future studies should be conducted to improve these aspects, enhancing the therapeutic efficacy of the devised DDS for in vivo application.
5. Competing interest disclosure The authors declare no competing financial interest. Acknowledgements This work has been supported by the Ministry of Science and Technology of Taiwan (Grants No. MOST103-2113-M-009-006) and the Ministry of Education, Taiwan (‘Aim for the Top University Plan’ of National Chiao Tung University).
Appendix A. Figures with essential colour discrimination Certain figures in this article, particularly Figs. 1–4, are difficult to interpret in black and white. The full colour images can be found in the on-line version, at doi: http://dx.doi.org/10.1016/j.actbio. 2015.05.006.
Please cite this article in press as: X.-C. Huang et al., Biothiol-triggered, self-disassembled silica nanobeads for intracellular drug delivery, Acta Biomater. (2015), http://dx.doi.org/10.1016/j.actbio.2015.05.006
8
X.-C. Huang et al. / Acta Biomaterialia xxx (2015) xxx–xxx
Appendix B. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.actbio.2015.05. 006. References [1] M.E. Davis, Z. Chen, D.M. Shin, Nanoparticle therapeutics: an emerging treatment modality for cancer, Nat. Rev. Drug Discov. 7 (2008) 771–782. [2] R.K. Jain, T. Stylianopoulos, Delivering nanomedicine to solid tumors, Nat. Rev. Clin. Oncol. 7 (2010) 653–664. [3] O. Veiseh, J.W. Gunn, M.Q. Zhang, Design and fabrication of magnetic nanoparticles for targeted drug delivery and imaging, Adv. Drug Deliv. Rev. 62 (2010) 284–304. [4] G.F. Paciotti, D.G.I. Kingston, L. Tamarkin, Colloidal gold nanoparticles: a novel nanoparticle platform for developing multifunctional tumor-targeted drug delivery vectors, Drug Dev. Res. 67 (2006) 47–54. [5] I.I. Slowing, B.G. Trewyn, S. Giri, V.S.Y. Lin, Mesoporous silica nanoparticles for drug delivery and biosensing applications, Adv. Funct. Mater. 17 (2007) 1225– 1236. [6] N. Nasonkla, E. Bey, J. Ren, H. Ai, C. Khemtong, J.S. Guthi, et al., Multifunctional polymeric micelles as cancer-targeted, MRI-ultrasensitive drug delivery systems, Nano Lett. 6 (2006) 2427–2430. [7] K.S. Soppimath, D.C. Tan, Y.Y. Yang, PH-triggered thermally responsive polymer core-shell nanoparticles for drug delivery, Adv. Mater. 17 (2005) 318–323. [8] S. Dromi, V. Frenkel, A. Luk, B. Traughber, M. Angstadt, M. Bur, et al., Pulsedhigh intensity focused ultrasound and low temperature-sensitive liposomes for enhanced targeted drug delivery and antitumor effect, Clin. Cancer Res. 13 (2007) 2722–2727. [9] A. Albanese, P.S. Tang, W.C. Chan, The effect of nanoparticle size, shape, and surface chemistry on biological systems, Annu. Rev. Biomed. Eng. 14 (2012) 1– 16. [10] F. Danhier, O. Feron, V. Préat, To exploit the tumor microenvironment: passive and active tumor targeting of nanocarriers for anti-cancer drug delivery, J. Controlled Release 148 (2010) 135–146. [11] M.J. Ernsting, M. Murakami, A. Roy, S.-D. Li, Factors controlling the pharmacokinetics, biodistribution and intratumoral penetration of nanoparticles, J. Controlled Release 172 (2013) 782–794. [12] A.K. Iyer, G. Khaled, J. Fang, H. Maeda, Exploiting the enhanced permeability and retention effect for tumor targeting, Drug Discov. Today 11 (2006) 812– 818. [13] Y. Matsumura, H. Maeda, A new concept for macromolecular therapeutics in cancer chemotherapy: mechanism of tumoritropic accumulation of proteins and the antitumor agent smancs, Cancer Res. 46 (1986) 6387–6392. [14] E. Markovsky, H. Baabur-Cohen, A. Eldar-Boock, L. Omer, G. Tiram, S. Ferber, et al., Administration, distribution, metabolism and elimination of polymer therapeutics, J. Controlled Release 161 (2012) 446–460. [15] E.S. Shibu, K. Ono, S. Sugino, A. Nishioka, A. Yasuda, Y. Shigeri, et al., Photouncaging nanoparticles for MRI and fluorescence imaging in vitro and in vivo, ACS Nano 7 (2013) 9851–9859. [16] H. Wei, R.-X. Zhuo, X.-Z. Zhang, Design and development of polymeric micelles with cleavable links for intracellular drug delivery, Prog. Polym. Sci. 38 (2013) 503–535. [17] Y.H. Jin, A.Z. Li, S.G. Hazelton, S. Liang, C.L. John, P.D. Selid, et al., Amorphous silica nanohybrids: synthesis, properties and applications, Coord. Chem. Rev. 253 (2009) 2998–3014. [18] H. Nabeshi, T. Yoshikawa, K. Matsuyama, Y. Nakazato, K. Matsuo, A. Arimori, et al., Systemic distribution, nuclear entry and cytotoxicity of amorphous nanosilica following topical application, Biomaterials 32 (2011) 2713–2724. [19] J.M. Rosenholm, C. Sahlgren, M. Linden, Multifunctional mesoporous silica nanoparticles for combined therapeutic, diagnostic and targeted action in cancer treatment, Curr. Drug Targets 12 (2011) 1166–1186. [20] D. Tarn, C.E. Ashley, M. Xue, E.C. Carnes, J.I. Zink, C.J. Brinker, Mesoporous silica nanoparticle nanocarriers: biofunctionality and biocompatibility, Acc. Chem. Res. 46 (2013) 792–801. [21] P.P. Adiseshaiah, J.B. Hall, S.E. McNeil, Nanomaterial standards for efficacy and toxicity assessment, Wires Nanomed. Nanobi. 2 (2010) 99–112. [22] A.A. Burns, J. Vider, H. Ow, E. Herz, O. Penate-Medina, M. Baumgart, et al., Fluorescent silica nanoparticles with efficient urinary excretion for nanomedicine, Nano Lett. 9 (2008) 442–448. [23] V. Biju, Chemical modifications and bioconjugate reactions of nanomaterials for sensing, imaging, drug delivery and therapy, Chem. Soc. Rev. 43 (2014) 744–764. [24] X. Huang, C.S. Brazel, On the importance and mechanisms of burst release in matrix-controlled drug delivery systems, J Controlled Release 73 (2001) 121–136.
[25] N. Gouda, K. Miyata, R.J. Christie, T. Suma, A. Kishimura, S. Fukushima, et al., Silica nanogelling of environment-responsive PEGylated polyplexes for enhanced stability and intracellular delivery of siRNA, Biomaterials 34 (2013) 562–570. [26] S.A. Mackowiak, A. Schmidt, V. Weiss, C. Argyo, C. von Schirnding, T. Bein, et al., Targeted drug delivery in cancer cells with red-light photoactivated mesoporous silica nanoparticles, Nano Lett. 13 (2013) 2576–2583. [27] C.E. Ashley, E.C. Carnes, K.E. Epler, D.P. Padilla, G.K. Phillips, R.E. Castillo, et al., Delivery of small interfering RNA by peptide-targeted mesoporous silica nanoparticle-supported lipid bilayers, ACS Nano 6 (2012) 2174–2188. [28] R. Cheng, F. Feng, F. Meng, C. Deng, J. Feijen, Z. Zhong, Glutathione-responsive nano-vehicles as a promising platform for targeted intracellular drug and gene delivery, J. Controlled Release 152 (2011) 2–12. [29] W. Stöber, A. Fink, E. Bohn, Controlled growth of monodisperse silica spheres in the micron size range, J. Colloid Interface Sci. 26 (1968) 62–69. [30] A. Dautry-Varsat, A. Ciechanover, H.F. Lodish, PH and the recycling of transferrin during receptor-mediated endocytosis, Proc. Natl. Acad. Sci. 80 (1983) 2258–2262. [31] V. Biju, T. Itoh, M. Ishikawa, Delivering quantum dots to cells: bioconjugated quantum dots for targeted and nonspecific extracellular and intracellular imaging, Chem. Soc. Rev. 39 (2010) 3031–3056. [32] Y. Zhang, L. Hu, D. Yu, C. Gao, Influence of silica particle internalization on adhesion and migration of human dermal fibroblasts, Biomaterials 31 (2010) 8465–8474. [33] B. Arunachalam, U.T. Phan, H.J. Geuze, P. Cresswell, Enzymatic reduction of disulfide bonds in lysosomes: characterization of a gamma-interferoninducible lysosomal thiol reductase (GILT), Proc. Natl. Acad. Sci. 97 (2000) 745–750. [34] Z. Luo, K. Cai, Y. Hu, L. Zhao, P. Liu, L. Duan, et al., Mesoporous silica nanoparticles end-capped with collagen: redox-responsive nanoreservoirs for targeted drug delivery, Angew. Chem. Int. Ed. 50 (2011) 640–643. [35] X. Ma, K.T. Nguyen, P. Borah, C.Y. Ang, Y. Zhao, Functional silica nanoparticles for redox-triggered drug/ssDNA Co-delivery, Adv. Health Mater. 1 (2012) 690– 697. [36] C.-L. Zhu, X.-Y. Song, W.-H. Zhou, H.-H. Yang, Y.-H. Wen, X.-R. Wang, An efficient cell-targeting and intracellular controlled-release drug delivery system based on MSN-PEM-aptamer conjugates, J. Mater. Chem. 19 (2009) 7765–7770. [37] Y. Cui, H. Dong, X. Cai, D. Wang, Y. Li, Mesoporous silica nanoparticles capped with disulfide-linked PEG gatekeepers for glutathione-mediated controlled release, ACS Appl. Mater. Interf. 4 (2012) 3177–3183. [38] Y. Wang, Y. Yan, J. Cui, L. Hosta-Rigau, J.K. Heath, E.C. Nice, et al., Encapsulation of water-insoluble drugs in polymer capsules prepared using mesoporous silica templates for intracellular drug delivery, Adv. Mater. 22 (2010) 4293– 4297. [39] S. Giri, B.G. Trewyn, M.P. Stellmaker, V.S.Y. Lin, Stimuli-responsive controlledrelease delivery system based on mesoporous silica nanorods capped with magnetic nanoparticles, Angew. Chem. Int. Ed. 44 (2005) 5038–5044. [40] H. Kim, S. Kim, C. Park, H. Lee, H.J. Park, C. Kim, Glutathione-induced intracellular release of guests from mesoporous silica nanocontainers with cyclodextrin gatekeepers, Adv. Mater. 22 (2010) 4280–4283. [41] Q. Zhang, F. Liu, K.T. Nguyen, X. Ma, X. Wang, B. Xing, et al., Multifunctional mesoporous silica nanoparticles for cancer-targeted and controlled drug delivery, Adv. Funct. Mater. 22 (2012) 5144–5156. [42] Z. Tao, B.B. Toms, J. Goodisman, T. Asefa, Mesoporosity and functional group dependent endocytosis and cytotoxicity of silica nanomaterials, Chem. Res. Toxicol. 22 (2009) 1869–1880. [43] I. Slowing, B.G. Trewyn, V.S.Y. Lin, Effect of surface functionalization of MCM41-type mesoporous silica nanoparticles on the endocytosis by human cancer cells, J. Am. Chem. Soc. 128 (2006) 14792–14793. [44] D.M. Townsend, K.D. Tew, The role of glutathione-S-transferase in anti-cancer drug resistance, Oncogene 22 (2003) 7369–7375. [45] S. Deng, Q. Wu, Y. Zhao, X. Zheng, N. Wu, J. Pang, et al., Biodegradable polymeric micelle-encapsulated doxorubicin suppresses tumor metastasis by killing circulating tumor cells, Nanoscale 7 (2015) 5270–5280. [46] P.D. Ottewell, H. Mönkkönen, M. Jones, D.V. Lefley, R.E. Coleman, I. Holen, Antitumor effects of doxorubicin followed by zoledronic acid in a mouse model of breast cancer, J. Natl. Cancer Inst. 100 (2008) 1167–1178. [47] K.-Y. Lin, W.-T. Chang, Y.-C. Lai, I. Liau, Toward functional screening of cardioactive and cardiotoxic drugs with zebrafish in vivo using pseudodynamic three-dimensional imaging, Anal. Chem. 86 (2014) 2213– 2220. [48] C.-K. Huang, H.-o. Hamaguchi, S. Shigeto, In vivo multimode Raman imaging reveals concerted molecular composition and distribution changes during yeast cell cycle, Chem. Commun. 47 (2011) 9423–9425. [49] N. Noothalapati Venkata Hemanth, S. Shigeto, Stable isotope-labeled raman imaging reveals dynamic proteome localization to lipid droplets in single fission yeast cells, Chem Biol 19 (2012) 1373–1380.
Please cite this article in press as: X.-C. Huang et al., Biothiol-triggered, self-disassembled silica nanobeads for intracellular drug delivery, Acta Biomater. (2015), http://dx.doi.org/10.1016/j.actbio.2015.05.006