Research Article www.acsami.org
Controllable Drug Release System in Living Cells Triggered by Enzyme−Substrate Recognition Pengchang Liu,†,‡ Xiaoliang Wang,†,‡ Kalervo Hiltunen,‡,§ and Zhijun Chen*,‡,§ ‡
State Key Laboratory of Supramolecular Structure and Materials, Institute of Theoretical Chemistry, and International Joint Research Laboratory of Nano-Micro Architecture Chemistry, Jilin University, 2699 Qianjin Street, Chaoyang, Changchun, Jilin 130012, People’s Republic of China § Faculty of Biochemistry and Molecular Medicine and Biocenter Oulu, University of Oulu, FI-90014 Oulu, Finland S Supporting Information *
ABSTRACT: Vehicles can deliver the drug molecules into cells, yet immunoreaction of the commonly used capping agents and release triggers limit their biomedical use. This shortcoming might be circumvented through replacing these chemicals with certain biomolecules. Here, we show a new and facile way to encapsulate the drug delivery vehicles and release the cargos in a highly controllable manner via modulating supramolecular interactions between enzyme, substrate, and vehicle. The cargo release from the vehicles within cells can be achieved upon substrate treatment. Yeast cells were used, allowing for a fast and cost-effective way for imaging and morphological analysis. We believe this new platform can be readily extended to various carrier systems for different purposes based on shifting the recognition pattern of enzyme−substrate pairs. KEYWORDS: enzyme−substrate recognition, release control, delivery, living cells, yeast
1. INTRODUCTION Smart drug delivery and release systems have attracted much interest in chemical and biological research communities and have been gradually recognized as critical issues in medical science. To target and release drugs at given space−time in a highly controllable way, various materials, such as gold nanoparticles, polymers, and other inorganic nanomaterials, including mesoporous silica nanoparticles (MSNs), have been extensively studied and acted as vehicles.1−6 Multifunctional vehicles that can transport the drug molecules to the targeted location and/or response to environmental stimuli within the cells and tissues are highly favored.7−10 The intermolecular recognitions between drug molecules, vehicles, and capping agents are the key to generate stimuli−responsive and nearzero premature cargo release systems.11−15 Among the wellcharacterized delivery vehicles, MSNs have turn out to be an excellent candidate as a result of their high surface areas (900−1500 m2 g−1), large pore volume (0.5−1.5 cm3 g−1), tunable pore sizes, good biological compatibility, and low toxicity.16,17 Further, the MSN particles can readily function with a variety of agents (e.g., macrocyclic compounds, polymers, macromolecules, and nanoparticles) to build diverse stimuli− responsive controllable release systems.18−22 The functionalized MSNs can thus response to various external stimuli (e.g., pH, temperature, light, enzymes, and redox states).23−27 However, whether or not these exogenous molecules will bring about immunoreaction is seriously concerned. Very recently, the natural low-toxic-component-functionalized MSNs emerged as a new player in the MSN-based vehicle system for their © 2015 American Chemical Society
biocompatibility, low cytotoxicity, and other advantages, which may turn out to be more suitable for biomedical applications.28−30 In particular, proteins, nucleic acids, or other biomolecules, such as glutathione (GSH), are intriguing alternatives to the currently available capping and stimuli agents. However, designing and constructing biocompatible delivery systems that contain an in vivo release control function remain a challenge. To this end, we should work out a series of biocompatible capping−decapping pairs that can efficiently control cargo release and can be applied to different circumstances by shifting these pairs. In this report, we developed a new drug delivery and controllable release system based on the modulation of the supramolecular interactions between glutathione S-transferase (GST), GSH, and amino-functionalized MSN (MSN−NH2) nanocontainers. The GST enzyme is among the key enzymes involved in detoxification of prokaryotic and eukaryotic organisms via catalyzing the conjugation of the reduced form of GSH to its xenobiotic substrates. As a physiological substrate of the GST enzyme, GSH is a small sulfhydryl group (−SH)-containing peptide composed by glutamic acid, cysteine, and glycine (Figure S1). Interestingly, GSH is beneficial to health and widely distributed in various organs and tissues within the human body. In nature, the enzyme and substrate interaction frequently leads to the shift of the protein structure, which Received: September 21, 2015 Accepted: November 12, 2015 Published: November 12, 2015 26811
DOI: 10.1021/acsami.5b08914 ACS Appl. Mater. Interfaces 2015, 7, 26811−26818
Research Article
ACS Applied Materials & Interfaces appears to be also the case for GST and GSH (Figures 1 and 2 and Figure S2). Furthermore, the beneficial health effect of GSH hints that this small molecule can be a promising signal to trigger cargo release in drug delivery systems.
2. EXPERIMENTAL SECTION 2.1. Reagent and Equipment. Cetyltrimethylammonium bromide (CTAB), tetraethoxysilane (TEOS), and (3-aminopropyl)triethoxysilane (APTES) were purchased from Adamas Reagent Co., Ltd. (Shanghai, China). Fourier transform infrared (FTIR) spectra were measured using a Bruker Vertex 80 V spectrometer. Scanning electron microscopy (SEM) images were taken from a JEOL JSM 6700F instrument. Transmission electron microscopy (TEM) images were measured on a JEM-2100F instrument (TECNAI G2, Netherlands), with an accelerated voltage of 200 kV. N2 adsorption and desorption isotherms [Brunauer−Emmett−Teller (BET) and Barrett−Joyner− Halenda (BJH)] were obtained using a Micromeritics Gemini instrument. Ultraviolet−visible (UV−vis) spectra were analyzed using a UV3600 spectrophotometer (Shimadzu, Japan). ζ potential was measured by Zetasizer Nano ZS (Malvern Instruments). Small-angle powder X-ray diffraction (XRD) measurements were carried out using a Rigaku SmartLab III powder diffractometer. 2.2. MSN−OH Synthesis. MSN−OH was synthesized using a previously reported method.31 CTAB (1.0 g) and NaOH (3.5 mL, 2M) were dissolved in 240 mL of water and then heated to reach 80 °C. Tetraethoxysilane (TEOS, 5.0 mL) was added to the solution and vigorously stirred for 2 h at 80 °C. The solid nanoparticles were quickly separated, washed extensively using dH2O and methanol, and dried under vacuum overnight. Empty pores were obtained by solvent extraction of the CTAB template: nanoparticles (1 g) were suspended in MeOH (100 mL); then a concentrated aqueous solution of HCl (12 M, 6 mL) was added; and the mixture was heated under reflux overnight. The solvent-extracted nanoparticles were washed thoroughly with MeOH and collected by vacuum filtration to result in the final product. The nanoparticles were characterized by means of XRD, SEM, and TEM. 2.3. MSN−NH2 Synthesis. A total of 1.0 g of calcined MSN was suspensed in 100 mL of anhydrous toluene, followed by adding an excess amount of APTES (1 mL). After the mixture was stirred continuously for 12 h at 115 °C, it was then filtered and washed with toluene and ethanol before drying under vacuum overnight. The nanoparticles were characterized by means of XRD, BET/BJH, SEM, and TEM. 2.4. Protein Overexpression and Purification. For overexpression, the pGEX-6p-1-GST plasmid (GE Healthcare) was transformed into Escherichia coli BL21 gold (DE3) cells. The bacterial cells containing pGEX-6p-1-GST was inoculated into lysogeny broth (LB) media and incubated overnight (O/N) by shaking at 37 °C. The O/N culture was diluted and transferred into M9ZB media (10 g/L acid casein hydrolyzate, 5 g/L NaCl, 1 g/L NH4Cl, 3 g/L KH2PO4, 6 g/L Na2HPO4·12H2O, 0.246 g/L MgSO4, and 4 g/L glucose) and further incubated by shaking at 18 °C. When the OD600 of these bacterial cells reach approximately 0.8, isopropyl-β-D-thiogalactopyranoside (IPTG, 0.5 mM for a final concentration) was introduced into the media to induce the GST protein expression for 4 h. The cells were collected by centrifugation (5000g for 10 min), washed 3 times using phosphate-buffered saline (PBS) buffer, and stored in −70 °C before use. For protein purification, the bacterial cells were lysed using sonication (with 40% of intensity, 9.9 s on plus 9.9 s off, for 10 min in total) in binding buffer (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4 at pH 7.3). The cell lysate was centrifuged at 14000g for 60 min. The supernatant was filtered through 0.45 μm filter (purchased from Jinteng Laboratory Equipment Co., Ltd., Tianjin, China) and loaded onto a binding buffer pre-equilibrated Glutathione Sepharose 4B column. The column was washed with 10× volumes of binding buffer to remove the unspecific binding of contaminating proteins. The GST-containing fractions were eluted from the column using elution buffer (50 mM Tris−HCl and 10 mM reduced glutathione
Figure 1. Schematic illustration of drug loading and GST capping of MSN−NH2 and their response to the environmental stimuli. The release of drug molecules can be triggered by either adding GSH molecules or shifting pH values. at pH 8.0). The eluted fractions were dialyzed again overnight at 4 °C. The pooled purified GST proteins were divided into 50 μL aliquots in the EP tubes, incubated in liquid nitrogen for 5 min, and then transferred into a −80 °C freezer before use. 2.5. Drug Loading and GST Encapsulation toward MSN− NH2. MSN−NH2 (0.01 g) was dispersed in 1 mL of Tris−HCl solution (0.02 mM, pH 8) of rhodamine 6G (Rh6G, 1 mM) under stirring for 24 h at room temperature for cargo loading. The mixture was separated with centrifugation and washed with dH2O at least 5 times. Then, 3 mg/mL GST was added, and the mixture was further stirred for an additional 1 h, which makes the interactions of GST with the MSN−NH2 pore gate. The Rh6G-loaded MSN−NH2@Rh6G− GST was washed thoroughly with Tris−HCl solution (pH 8) until no dye leakage was visible under UV light. 2.6. Drug Release. Rh6G-loaded and GST-gated nanoparticles were added in the dialysis bag, and Tris−HCl solution was added in the cuvette. The release of Rh6G to the solution was monitored by the UV absorbance at 530 nm in the presence of different amounts of GSH or under different pH values. The volumes of the release media were kept unchanged in the detection process. 2.7. Fluorescence Images. The yeasts were cultured in the yeast extract peptone dextrose (YPD) medium overnight at 30 °C under continuous shaking until reaching a density of 108 cells/mL. Then, aliquots of 1 mL of the cell suspension were centrifuged and resuspended in 100 μL of YPD medium containing 5 mg/mL MSN− GST−doxorubicin (DOX) and incubated for 6 h at room temperature. For the cargo release experiment, these cells are treated with GSH. Intracellular release of DOX was monitored using confocal fluorescence microscopy. 2.8. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide (MTT) Assay. The yeast cells were cultured in YPD media at 30 °C for 17 h under continuous shaking. A total of 1 mL of the diluted cells (OD600 = 1.0) was centrifuged, and the pellets were resuspended in 100 μL of YPD media. MSN−NH2−DOX@GST particles were added to the cell suspensions to reach a final 26812
DOI: 10.1021/acsami.5b08914 ACS Appl. Mater. Interfaces 2015, 7, 26811−26818
Research Article
ACS Applied Materials & Interfaces
Figure 2. (A) SEM and (inset) TEM images of the representatives of MSN−NH2. (B) XRD patterns of MSN−OH (blue), MSN−NH2 (red), and MSN−NH2−Rh6G (black). (C) ζ potentials of MSN−NH2 (red), MSN−OH (green), and Rh6G-loaded MSNs (MSN−NH2−Rh6G) (blue). (D) ζ potentials of pure GSH (75 mM) (red) and GST enzyme in the presence of different contents of GSH [from left to right: 0 mM (green), 10 mM (blue), 25 mM (bluish), 50 mM (violet), and 75 mM (yellow)]. GST crystal structures were shown (E and F): (E) apo form, PDB 13GS and (F) holo form, PDB 1PL1. concentration of 5 mg/mL, followed by 6 h of incubation at room temperature. These samples were then washed 3 times using 1× phosphate-buffered saline (PBS) buffer and resuspended in 100 μL of 1× PBS buffer. GSH was added to the solutions to reach a final concentration of 75 mM and incubated for different periods of time. Cells were then centrifuged to remove the supernatants and resuspended in 100 μL of YPD media. A total of 10 μL of MTT solution (5 mg/mL) was mixed with the cell samples at room temperature for 2 h, followed by centrifugation to collect the pellets. The pellets were then dissolved in 100 μL of dimethyl sulfoxide (DMSO) and measured at OD = 490 nm using an Elx808 microplate reader (BioTek).
turns the electric charge of GST from negative to positive in the presence of GSH. The release of GST−GSH complexes from MSN−NH2 results in the discharge of cargo (e.g., drug molecules) from the pores of the vehicles. Moreover, this setup also responses to other environmental changes, such as pH and salt conditions, that affect the supramolecular interaction between these assembly modules, making it a multiple-response system. Importantly, this new drug delivery/controllable release system is proven to work both in vitro and in living cells (see below). MSNs functionalized with hydroxyl groups (MSN−OH) were synthesized using the sol−gel approach.31 The hydroxyl groups on the surface of MSN−OH can be converted to amino groups via APTES incubation.32,33 The overall structures of the synthesized MSN−OH and MSN−NH2 nanoparticles were characterized using SEM and TEM. The SEM and TEM images of MSN−NH2 and MSN−OH particles suggest that the diameter of these particles is approximately 100 nm (Figure 2A and Figure S3). The MSNs were further characterized using XRD. Both MSN−OH and MSN−NH2 particles (Rh6G-loaded) show the microcrystalline structure with the Bragg peaks of (100), (110), and (200) (Figure 2B), suggesting that the amino functionalization does not impair the crystalline structure of the silica nanoparticles. No significant difference can be recognized concerning the appearance of MSNs before and after surface amino modification. However, the surface
3. RESULTS AND DISCUSSION The encapsulation strategy for the MSN nanocontainer is based on the supramolecular assembly of GST on the outer layer of the MSN−NH2 particles under the physiological condition (Figure 1). The negatively charged GST [isoelectric point (pI) = 5.9] readily interacts with positively charged MSN−NH2 through non-covalent weak force, resulting in the pore closing of the nanoparticles. When GSH is present, it accesses the substrate binding pocket of the GST enzyme through specific intermolecular recognition, leading to a shift of the structure ensemble. This transition results in the rise of the electric charge of GST−GSH complexes, triggering the departure of the complex from the MSN−NH2 nanoparticles (Figure 1). The high efficiency of this logic gate forms a close−open switch that 26813
DOI: 10.1021/acsami.5b08914 ACS Appl. Mater. Interfaces 2015, 7, 26811−26818
Research Article
ACS Applied Materials & Interfaces
Figure 4. (A) Release of Rh6G from MSN−NH2@GST in the presence of different concentrations of GSH and/or at different pH conditions. (B−D) Release profile of Rh6G from MSN−NH2− Rh6G@GST at pH 4.9 and/or in the presence of (B) 10 mM, (C) 25 mM, or (D) 50 mM GSH (red line). (E−G) Release profile of Rh6G from MSN−NH2−Rh6G@GST at pH 5.9 (black line) and/or in the presence of (E) 10 mM, (F) 25 mM, or (G) 50 mM GSH (red line).
Figure 3. Release profiles of Rh6G from MSN−NH2−Rh6G@GST in the presence of (A) different contents of GSH or at (B) different pH values. Controlled release of Rh6G from the vehicles by (C) adding different contents of GSH (OFF denotes that the GSH concentration is 0, and ON denotes that GSH is present) or (D) shifting pH values (OFF denotes that the pH value is 7.4, and ON denotes that the pH is other values, as indicated). The dissociation of GST with MSN−NH2 in the presence of (E) different concentrations of GSH and at (F) different pH values, as shown by SDS−PAGE. (G) SDS−PAGE of the pellet (inset = supernatant) of the MSN−OH−GST mixture after centrifugation, following the incubation with different amounts of GSH. (H) Release profiles of Rh6G from MSN−NH2−Rh6G@GST in the presence of different contents of salt (NaCl).
GST recombinant protein (Figure S6 and Sequences S1 and S2) was overexpressed and purified as described in the Experimental Section. GST proteins are negatively charged at the physiological condition (150 mM NaCl at pH 7.4; Figure 2D), which readily encapsulate positively charged Rh6G-loaded MSN−NH2 nanoparticles (Figure 2C) via intermolecular weak interaction. These supramolecular interactions can be modulated by different environmental stimuli, such as GSH and pH conditions (see below). The quantitive cargo release profile of MSN−NH2−Rh6G@ GST toward different environmental stimuli was investigated using a classic dialysis approach (Figure S7).12 Briefly, 10 mg of GST-capped MSN−NH2−Rh6G was placed in a dialysis bag and incubated in a cuvette containing 3 mL of 1× PBS buffer (pH 7.4) supplemented with different concentrations of GSH. When GSH binds to GST, the conformation and surface charge of the enzyme are shifted. This allosteric effect decaps MSN− NH2−Rh6G, resulting in the release of cargo molecules. The release profile of the cargo molecules (Rh6G) can be monitored using UV−vis absorbance spectroscopy. Rh6G molecules are
electric charge of MSNs shifted from negative to positive after APTES modification, as shown by ζ-potential analysis (Figure 2C). The nitrogen adsorption−desorption isotherm indicates the surface area of 1093 m2 g−1 and porous structure of MSN−NH2, with an average pore diameter of 2 nm (Figure S4). FTIR spectra were applied to monitor the degree of hydroxylation and amination functionalization of the MSN particles. In addition to the emerged peak at approximately 2930 cm−1, which corresponds to the −CH2− group, two characteristic peaks from the vibration of the amino groups, localized at 1550 and 1490 cm−1, are present in the spectra of MSN−NH2, in contrast to that of MSN−OH particles, confirming the reaction success (Figure S5). 26814
DOI: 10.1021/acsami.5b08914 ACS Appl. Mater. Interfaces 2015, 7, 26811−26818
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Figure 5. Confocal laser scanning microscopy (CLSM) images of (A−C) yeast cells, (D−F) yeast cells treated with DOX, (G−I) yeast cells treated with DOX-loaded MSN−NH2−DOX@GST, or (J−L) yeast cells treated with DOX-loaded MSN−NH2−DOX@GST followed by GSH incubation. The bar refers to 20 μm. The inset denotes the enlarged images (the bar refers to 5 μm). Left, middle, and right lanes denote for bright field, red fluorescence, and overlapped images, respectively.
Similarly, this system is highly controllable via changing the pH conditions. No cargo release from the vehicles can be detected at pH 7.4 (0−10 min in Figure 3D). When pH switches to 5.9, a substantial amount of Rh6G is released (30%) (10−20 min in Figure 3D). The cargo release approaches nearly maximum (98%), while pH is 3.9, indicating that the pore gate is entirely opened at this point. A new approach was developed here to visualize the association and dissociation of GST with Rh6G-loaded MSN−NH2 using a combination of centrifugation and sodium dodecyl sulfate−polyacrylamide gel electrophoresis (SDS−PAGE). Briefly, the encapsulated vehicles (MSN−NH2−Rh6G@GST) were incubated at different concentrations of GSH and/or at different pH conditions for 30 min. The separated supernatants and pellets after centrifugation were collected and subjected to SDS−PAGE to visualize the remaining proteins that bind to MSN−NH2−Rh6G. It was shown that the amount of GST associating with MSN−NH2−Rh6G was gradually decreased following the incubation of the MSN−NH2−Rh6G@GST complex with a higher concentration of GSH (Figure 3E), indicating that GST detaches from the MSNs. Similarly, the release of GST from the particles can be detected when the
released from the GST-gated MSN vehicles following the increase of the GSH concentration (Figure 3A). The cargo release of this system can also be triggered by changing the environmental pH values (Figure 3B). It is known that the pI of GST is 5.9, meaning that the surface charge of this protein shifts from negative to positive when the environmental pH changes from a neutral to an acidic condition. These changes decrease the strength of the supramolecular interaction between the GST protein and MSN−NH2, resulting in the departure of GST from the surface of the nanoparticles, leading to the cargo release. This supramolecular recognition nature between the enzyme (GST) and substrate (GSH) makes this system highly controllable depending upon the content of GSH in the solution. In the absence of GSH, hardly any cargo release can be detected (0−10 min in Figure 3C). The cargo molecules are released (28%) when the GSH concentration approaches 10 mM (10−20 min in Figure 3C). The release is ceased by putting the system back to neutralized 1× PBS buffer (pH 7.4). When the GSH concentration is 25 mM, Rh6G is released up to 42% (30−40 min in Figure 3C). Finally, the cargo molecules can be discharged to the maximum (almost 100%) when the concentration of GSH reaches 75 mM. 26815
DOI: 10.1021/acsami.5b08914 ACS Appl. Mater. Interfaces 2015, 7, 26811−26818
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ACS Applied Materials & Interfaces
Figure 6. CLSM images of yeast cells treated by (A−C) GSH, (D−F) GSH−PI, (G−I) PI, or (J−O) PI-loaded MSN−NH2−PI@GST [in the (J−L) absence or (M−O) presence of GSH]. The bar refers to 10 μm. The inset denotes the enlarged images (the bar refers to 5 μm).
in living cells, modulating the salt concentration is not highly practicable. Therefore, we propose that GSH and pH are the two keys to open the gate of this GST-capped vehicle. The synergistic effect of these two triggering factors for cargo release is summarized (panels A−G of Figure 4). Notably, approximately 30% of cargo molecules are released at pH 5.9, while the GSH concentration reaches 10 mM, resulting in 30% release (panels A and E of Figure 4). When the GSH concentration is 10 mM at pH 5.9, 70% release of cargo can be achieved (Figure 4A). To evaluate whether this new drug delivery and controllable release system works in vivo/ex vivo or not, these GSTencapsulated vesicles containing DOX were incubated with yeast (Saccharomyces cerevisiae) cells and subjected to imaging analysis. As shown in confocal fluorescence microimages (Figure 5), this system successfully delivers DOX into the
system was incubated with low pH solutions (Figure 3F). Moreover, GST was solely present in the supernatant instead of associating with the vehicles when MSN−NH2 was replaced by MSN−OH (Figure 3G), suggesting that GST does not interact with MSN−OH nanoparticles. The supramolecular force that encapsulates the MSN particles is associated with the amino groups of the functionalized vehicles. The intermolecular interaction between GST and MSN−NH2 can also be modulated by other factors, such as the salt concentration. While no cargo release can be detected at the physiological condition (140 mM NaCl), a high salt concentration of the environmental condition results in the Rh6G release (Figure 3H). When the salt concentration is 250 mM, the cargo is inclined to leave the vehicles. When the salt concentration reaches 550 mM, the nanogate of the system is almost completely opened (Figure 3H). However, considering the situation 26816
DOI: 10.1021/acsami.5b08914 ACS Appl. Mater. Interfaces 2015, 7, 26811−26818
Research Article
ACS Applied Materials & Interfaces
Notably, GSH treatment by itself did not make any detectable harm to the yeast cells (panels A−C of Figure 6), indicating that the deleterious effect was caused by released DOX.
eukaryotic cells and releases the cargo upon GSH treatment. The delivery efficiency of the MSN−NH2−DOX@GST system is assumably much higher than that of the self-penetration of DOX through the cell membrane (panels A−I of Figure 5), because the majority of cells show significant morphological changes when the DOX molecules are released from the vehicles upon GSH treatment (panels J−L of Figure 5). The morphological changes may be associated with the apoptosis of the yeast cells. Therefore, we propose that yeast cells might be an interesting alternative platform to evaluate the drug delivery and release efficiency of vehicles and capping systems in addition to widely used mammalian cell cultural systems. There are several reasons and/or advantages of using yeast as a new platform to evaluate drug delivery and release. First of all, both yeast and mammalian cells are eukaryotic cells containing similar subcellular structures and cell signaling pathways, albeit the size of the yeast cells are smaller. Second, in comparison to mammalian-cell-related research, which requires a standard tissue culture room with high cost, yeast cells can be cultured in a regular laboratory with much cheap expenses. Third, the simplicity of the subcellular structure of yeast cells allows us to have a relatively quick and easy investigation toward their changes upon drug release. Because DOX can cross the cell membrane by itself possibly through diffusion (panels D−F of Figure 5),34 other cargo molecules become necessary to further evaluate the new capping−delivery−release system. Propidium iodide (PI) is a membrane-impermeable fluorescent molecule and cannot enter living cells by itself.35 Indeed, PI is excluded from the viable yeast cells (panels A−I of Figure 6). MSN−NH2−PI@GST successfully delivers PI molecules into the yeast cells (panels J−L of Figure 6) and dispersed these cargo molecules into the cytoplasm in the presence of GSH (panels M−O of Figure 6). The viability of yeast cells after different treatments was evaluated using the MTT assay (Figure 7). Yeast cells survived
4. CONCLUSION In this report, we developed for a new MSN capping and uncaping system based on the modulation of the supramolecular interaction between MSN, GST enzyme, and its substrate. The electrostatic interaction between MSN−NH2 and GST enzyme at physiological conditions closes the pores of the MSN vehicles successfully, while the enzyme and substrate interaction between GST and GSH leads to opening of the nanogate. The supramolecular nature of this system makes it subtile to environmental changes and highly controllable. Indeed, mild changes in either the substrate concentration or pH condition effectively trigger the cargo release. The advantages of this system also lie in its feasibility of manipulation in living cells. Given the diverse enzyme−substrate pairs existing in nature, we believe that our approach can be readily extended to various drug delivery and controllable release systems for different and specific purposes via converting the enzyme− substrate combination. In addition, a SDS−PAGE-based system was described for a visual analysis of detached corona from vehicles. We also propose that yeast may be a new and handy model system for the evaluation of drug delivery and controllable cargo release under physiological conditions.
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ASSOCIATED CONTENT
* Supporting Information S
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.5b08914. Molecular formula of GSH, CD spectra and crystal structures of GST, SEM, FTIR spectra, amino acid and cDNA sequence of GST, plasmid map, SDS−PAGE, and release profiles (PDF)
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AUTHOR INFORMATION
Corresponding Author
*Telephone: +86-186-8663-6807. Fax: +86-431-8519-3421. E-mail: [email protected]. Author Contributions †
Pengchang Liu and Xiaoliang Wang contributed equally to this work. Funding
This work was supported by the National Natural Science Foundation of China (NSF, 21372097) and a CIMO grant from Finland. Notes
Figure 7. Yeast cell viability as shown using the MTT assay. Yeast cells were incubated with DOX (red), MSN−NH2−DOX−GST (green), or MSN−NH2−DOX−GST followed by GSH treatment (blue). The MTT assay was carried out at different time points.
The authors declare no competing financial interest.
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quite well when treated with DOX (98 and 88% viability at 30 and 60 min, respectively). Similarly, cells are still rather active after incubation with MSN−NH2−DOX−GST (99 and 94% viability at 30 and 60 min, respectively). Strikingly, when the MSN−NH2−DOX−GST-treated sample were followed by incubation with GSH, the viability of the cells were significantly decreased (65 and 57% viability at 30 and 60 min, respectively), suggesting that DOX was efficiently delivered into the yeast cells by MSN vehicles and released in the presence of GSH. 26817
NOMENCLATURE MSN = mesoporous silica nanoparticle GSH = glutathione GST = glutathione S-transferase TEM = transmission electron microscopy SEM = scanning electron microscopy XRD = small-angle powder X-ray diffraction SDS−PAGE = sodium dodecyl sulfate−polyacrylamide gel electrophoresis Rh6G = rhodamine 6G DOX = doxorubicin DOI: 10.1021/acsami.5b08914 ACS Appl. Mater. Interfaces 2015, 7, 26811−26818
Research Article
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[5] arenes for On-command Cargo Release. Small 2013, 9, 3224− 3229. (19) Feng, L.; Li, H.; Qu, Y.; Lü, C. Detection of TNT Based on Conjugated Polymer Encapsulated in Mesoporous Silica Nanoparticles Through FRET. Chem. Commun. 2012, 48, 4633−4635. (20) Zhang, Y.; Yuan, Q.; Chen, T.; Zhang, X.; Chen, Y.; Tan, W. DNA-capped Mesoporous Silica Nanoparticles as an Ion-responsive Release System to Determine the Presence of Mercury in Aqueous Solutions. Anal. Chem. 2012, 84, 1956−1962. (21) Wang, X.; Chen, H.; Zheng, Y.; Ma, M.; Chen, Y.; Zhang, K.; Zeng, D.; Shi, J. Au-nanoparticle Coated Mesoporous Silica Nanocapsule-based Multifunctional Platform for Ultrasound Mediated Imaging, Cytoclasis and Tumor Ablation. Biomaterials 2013, 34, 2057−2068. (22) Lee, J.; Kim, H.; Han, S.; Hong, E.; Lee, K. H.; Kim, C. Stimuliresponsive Conformational Conversion of Peptide Gatekeepers for Controlled Release of Guests from Mesoporous Silica Nanocontainers. J. Am. Chem. Soc. 2014, 136, 12880−12883. (23) Popat, A.; Liu, J.; Lu, G. Q.; Qiao, S. Z. A pH-responsive Drug Delivery System Based on Chitosan Coated Mesoporous Silica Nanoparticles. J. Mater. Chem. 2012, 22, 11173−11178. (24) Sun, J. T.; Yu, Z. Q.; Hong, C. Y.; Pan, C. Y. Biocompatible Zwitterionic Sulfobetaine Copolymer-coated Mesoporous Silica Nanoparticles for Temperature-responsive Drug Release. Macromol. Rapid Commun. 2012, 33, 811−818. (25) Zhang, Q.; Liu, F.; Nguyen, K. T.; Ma, X.; Wang, X.; Xing, B.; Zhao, Y. Multifunctional Mesoporous Silica Nanoparticles for Cancertargeted and Controlled Drug Delivery. Adv. Funct. Mater. 2012, 22, 5144−5156. (26) Yuan, Q.; Zhang, Y.; Chen, T.; Lu, D.; Zhao, Z.; Zhang, X.; Li, Z.; Yan, C. H.; Tan, W. Photon-manipulated Drug Release from a Mesoporous Nanocontainer Controlled by Azobenzene-modified Nucleic Acid. ACS Nano 2012, 6, 6337−6344. (27) Popat, A.; Ross, B. P.; Liu, J.; Jambhrunkar, S.; Kleitz, F.; Qiao, S. Z. Enzyme-responsive Controlled Release of Covalently Bound Prodrug from Functional Mesoporous Silica Nanospheres. Angew. Chem., Int. Ed. 2012, 51, 12486−12489. (28) Wu, S.; Huang, X.; Du, X. Glucose- and pH-responsive Controlled Release of Cargo from Protein-gated Carbohydratefunctionalized Mesoporous Silica Nanocontainers. Angew. Chem. 2013, 125, 5690−5694. (29) Schlossbauer, A.; Kecht, J.; Bein, T. Biotin-avidin as a Proteaseresponsive Cap System for Controlled Guest Release from Colloidal Mesoporous Silica. Angew. Chem. 2009, 121, 3138−3141. (30) Chen, M.; Huang, C.; He, C.; Zhu, W.; Xu, Y.; Lu, Y. A Glucose-responsive Controlled Release System Using Glucose Oxidase-gated Mesoporous Silica Nanocontainers. Chem. Commun. 2012, 48, 9522−9524. (31) Zhao, Y.; Trewyn, B. G.; Slowing, I. I.; Lin, V. S.-Y. Mesoporous Silica Nanoparticle-based Double Drug Delivery System for Glucoseresponsive Controlled Release of Insulin and Cyclic AMP. J. Am. Chem. Soc. 2009, 131, 8398−8400. (32) Park, H. S.; Kim, C. W.; Lee, H. J.; Choi, J. H.; Lee, S. G.; Yun, Y. P.; Kwon, I. C.; Lee, S. J.; Jeong, S. Y.; Lee, S. C. A Mesoporous Silica Nanoparticle with Charge-convertible Pore Walls for Efficient Intracellular Protein Delivery. Nanotechnology 2010, 21, 225101. (33) Chu, C. W.; Kirby, D. P.; Murphy, P. D. Interactions of Aminosilane with Alumina and Silica Substrates Deposited from Nonaqueous and Aqueous Media. J. Adhes. Sci. Technol. 1993, 7, 417− 434. (34) Bian, X.; McAllister-Lucas, L. M.; Shao, F.; Schumacher, K. R.; Feng, Z. W.; Porter, A. G.; Castle, V. P.; Opipari, A. W., Jr. NF-κB Activation Mediates Doxorubicin-induced Cell Death in N-type Neuroblastoma Cells. J. Biol. Chem. 2001, 276 (52), 48921−48929. (35) Nagy, S.; Jansen, J.; Topper, E. K.; GadellaA, B. M. Triple-stain Flow Cytometric Method to Assess Plasma- and Acrosome-membrane Integrity of Cryopreserved Bovine Sperm Immediately after Thawing in Presence of Egg-yolk Particles. Biol. Reprod. 2002, 68, 1828−1835.
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(1) Zhao, E.; Zhao, Z.; Wang, Z.; Yang, C.; Chen, C.; Gao, L.; Feng, Q.; Hou, W.; Gao, M.; Zhang, Q. Surface Engineering of Gold Nanoparticles for in Vitro siRNA Delivery. Nanoscale 2012, 4, 5102− 5109. (2) Wang, H.; Han, A.; Cai, Y.; Xie, Y.; Zhou, H.; Long, J.; Yang, Z. Multifunctional Biohybrid Hydrogels for Cell Culture and Controlled Drug Release. Chem. Commun. 2013, 49, 7448−7450. (3) Xiao, D.; Jia, H.; Zhang, J.; Liu, C. W.; Zhuo, R. X.; Zhang, X. Z. A Dual-responsive Mesoporous Silica Nanoparticle for Tumortriggered Targeting Drug Delivery. Small 2014, 10, 591−598. (4) Tong, W.; She, S.; Xie, L.; Gao, C. High Efficient Loading and Controlled Release of Low-molecular-weight Drugs by Combination of Spontaneous Deposition and Heat-Induced Shrinkage of Multilayer Capsules. Soft Matter 2011, 7, 8258−8265. (5) Song, W.; Tang, Z.; Li, M.; Lv, S.; Yu, H.; Ma, L.; Zhuang, X.; Huang, Y.; Chen, X. Tunable pH-Sensitive Poly(β-amino ester)s Synthesized from Primary Amines and Diacrylates for Intracellular Drug Delivery. Macromol. Biosci. 2012, 12, 1375−1383. (6) Yang, X.; Liu, X.; Liu, Z.; Pu, F.; Ren, J.; Qu, X. Near-infrared Light-triggered, Targeted Drug Delivery to Cancer Cells by Aptamer Gated Nanovehicles. Adv. Mater. 2012, 24, 2890−2895. (7) Geng, J.; Li, M.; Wu, L.; Chen, C.; Qu, X. Mesoporous Silica Nanoparticle-based H2O2 Responsive Controlled-release System Used for Alzheimer’s Disease Treatment. Adv. Healthcare Mater. 2012, 1, 332−336. (8) Chen, Z.; Li, Z.; Lin, Y.; Yin, M.; Ren, J.; Qu, X. Bioresponsive Hyaluronic Acid-Capped Mesoporous Silica Nanoparticles for Targeted Drug Delivery. Chem. - Eur. J. 2013, 19, 1778−1783. (9) Tian, Y.; Qi, J.; Zhang, W.; Cai, Q.; Jiang, X. Facile, One-pot Synthesis, and Antibacterial Activity of Mesoporous Silica Nanoparticles Decorated with Well-Dispersed Silver Nanoparticles. ACS Appl. Mater. Interfaces 2014, 6, 12038−12045. (10) Zhang, Z.; Ding, J.; Chen, X.; Xiao, C.; He, C.; Zhuang, X.; Chen, L.; Chen, X. Intracellular pH-sensitive Supramolecular Amphiphiles Based on Host−guest Recognition Between Benzimidazole and β-Cyclodextrin as Potential Drug Delivery Vehicles. Polym. Chem. 2013, 4, 3265−3271. (11) Chen, C.; Geng, J.; Pu, F.; Yang, X.; Ren, J.; Qu, X. Polyvalent Nucleic Acid/Mesoporous Silica Nanoparticle Conjugates: Dual Stimuli-responsive Vehicles for Intracellular Drug Delivery. Angew. Chem., Int. Ed. 2011, 50, 882−886. (12) Sun, Y. L.; Zhou, Y.; Li, Q. L.; Yang, Y. W. Enzyme-responsive Supramolecular Nanovalves Crafted by Mesoporous Silica Nanoparticles and Choline-sulfonatocalix [4]arene[2]pseudorotaxanes for Controlled Cargo Release. Chem. Commun. 2013, 49, 9033−9035. (13) Lv, R.; Yang, P.; He, F.; Gai, S.; Yang, G.; Dai, Y.; Hou, Z.; Lin, J. An Imaging-Guided Platform for Synergistic Photodynamic/ Photothermal/Chemo-Therapy with pH/Temperature-Responsive Drug Release. Biomaterials 2015, 63, 115−127. (14) Lv, R.; Zhong, C.; Li, R.; Yang, P.; He, F.; Gai, S.; Hou, Z.; Yang, G.; Lin, J. Multifunctional Anticancer Platform for Multimodal Imaging and Visible Light Driven Photodynamic/Photothermal Therapy. Chem. Mater. 2015, 27 (5), 1751−1763. (15) Lv, R.; Yang, G.; He, F.; Dai, Y.; Gai, S.; Yang, P. LaF3:Ln Mesoporous Spheres: Controllable Synthesis, Tunable Luminescence and Application for Dual-Modal Chemo-/Photo-Thermal Therapy. Nanoscale 2014, 6, 14799−14809. (16) Li, Q. L.; Sun, Y.; Sun, Y. L.; Wen, V.; Zhou, Y.; Bing, Q. M.; Isaacs, L. D.; Jin, Y.; Gao, H.; Yang, Y. W. Mesoporous Silica Nanoparticles Coated by Layer-by-Layer Self-assembly Using Cucurbit [7] uril for in Vitro and in Vivo Anticancer Drug Release. Chem. Mater. 2014, 26, 6418−6431. (17) Pan, L.; He, Q.; Liu, J.; Chen, Y.; Ma, M.; Zhang, L.; Shi, J. Nuclear-targeted Drug Delivery of TAT Peptide-conjugated Monodisperse Mesoporous Silica Nanoparticles. J. Am. Chem. Soc. 2012, 134, 5722−5725. (18) Sun, Y. L.; Yang, Y. W.; Chen, D. X.; Wang, G.; Zhou, Y.; Wang, C. Y.; Stoddart, J. F. Mechanized Silica Nanoparticles Based on Pillar 26818
DOI: 10.1021/acsami.5b08914 ACS Appl. Mater. Interfaces 2015, 7, 26811−26818
Controllable Drug Release System in Living Cells Triggered by Enzyme−Substrate Recognition Pengchang Liu,†,‡ Xiaoliang Wang,†,‡ Kalervo Hiltunen,‡,§ and Zhijun Chen*,‡,§ ‡
State Key Laboratory of Supramolecular Structure and Materials, Institute of Theoretical Chemistry, and International Joint Research Laboratory of Nano-Micro Architecture Chemistry, Jilin University, 2699 Qianjin Street, Chaoyang, Changchun, Jilin 130012, People’s Republic of China § Faculty of Biochemistry and Molecular Medicine and Biocenter Oulu, University of Oulu, FI-90014 Oulu, Finland S Supporting Information *
ABSTRACT: Vehicles can deliver the drug molecules into cells, yet immunoreaction of the commonly used capping agents and release triggers limit their biomedical use. This shortcoming might be circumvented through replacing these chemicals with certain biomolecules. Here, we show a new and facile way to encapsulate the drug delivery vehicles and release the cargos in a highly controllable manner via modulating supramolecular interactions between enzyme, substrate, and vehicle. The cargo release from the vehicles within cells can be achieved upon substrate treatment. Yeast cells were used, allowing for a fast and cost-effective way for imaging and morphological analysis. We believe this new platform can be readily extended to various carrier systems for different purposes based on shifting the recognition pattern of enzyme−substrate pairs. KEYWORDS: enzyme−substrate recognition, release control, delivery, living cells, yeast
1. INTRODUCTION Smart drug delivery and release systems have attracted much interest in chemical and biological research communities and have been gradually recognized as critical issues in medical science. To target and release drugs at given space−time in a highly controllable way, various materials, such as gold nanoparticles, polymers, and other inorganic nanomaterials, including mesoporous silica nanoparticles (MSNs), have been extensively studied and acted as vehicles.1−6 Multifunctional vehicles that can transport the drug molecules to the targeted location and/or response to environmental stimuli within the cells and tissues are highly favored.7−10 The intermolecular recognitions between drug molecules, vehicles, and capping agents are the key to generate stimuli−responsive and nearzero premature cargo release systems.11−15 Among the wellcharacterized delivery vehicles, MSNs have turn out to be an excellent candidate as a result of their high surface areas (900−1500 m2 g−1), large pore volume (0.5−1.5 cm3 g−1), tunable pore sizes, good biological compatibility, and low toxicity.16,17 Further, the MSN particles can readily function with a variety of agents (e.g., macrocyclic compounds, polymers, macromolecules, and nanoparticles) to build diverse stimuli− responsive controllable release systems.18−22 The functionalized MSNs can thus response to various external stimuli (e.g., pH, temperature, light, enzymes, and redox states).23−27 However, whether or not these exogenous molecules will bring about immunoreaction is seriously concerned. Very recently, the natural low-toxic-component-functionalized MSNs emerged as a new player in the MSN-based vehicle system for their © 2015 American Chemical Society
biocompatibility, low cytotoxicity, and other advantages, which may turn out to be more suitable for biomedical applications.28−30 In particular, proteins, nucleic acids, or other biomolecules, such as glutathione (GSH), are intriguing alternatives to the currently available capping and stimuli agents. However, designing and constructing biocompatible delivery systems that contain an in vivo release control function remain a challenge. To this end, we should work out a series of biocompatible capping−decapping pairs that can efficiently control cargo release and can be applied to different circumstances by shifting these pairs. In this report, we developed a new drug delivery and controllable release system based on the modulation of the supramolecular interactions between glutathione S-transferase (GST), GSH, and amino-functionalized MSN (MSN−NH2) nanocontainers. The GST enzyme is among the key enzymes involved in detoxification of prokaryotic and eukaryotic organisms via catalyzing the conjugation of the reduced form of GSH to its xenobiotic substrates. As a physiological substrate of the GST enzyme, GSH is a small sulfhydryl group (−SH)-containing peptide composed by glutamic acid, cysteine, and glycine (Figure S1). Interestingly, GSH is beneficial to health and widely distributed in various organs and tissues within the human body. In nature, the enzyme and substrate interaction frequently leads to the shift of the protein structure, which Received: September 21, 2015 Accepted: November 12, 2015 Published: November 12, 2015 26811
DOI: 10.1021/acsami.5b08914 ACS Appl. Mater. Interfaces 2015, 7, 26811−26818
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ACS Applied Materials & Interfaces appears to be also the case for GST and GSH (Figures 1 and 2 and Figure S2). Furthermore, the beneficial health effect of GSH hints that this small molecule can be a promising signal to trigger cargo release in drug delivery systems.
2. EXPERIMENTAL SECTION 2.1. Reagent and Equipment. Cetyltrimethylammonium bromide (CTAB), tetraethoxysilane (TEOS), and (3-aminopropyl)triethoxysilane (APTES) were purchased from Adamas Reagent Co., Ltd. (Shanghai, China). Fourier transform infrared (FTIR) spectra were measured using a Bruker Vertex 80 V spectrometer. Scanning electron microscopy (SEM) images were taken from a JEOL JSM 6700F instrument. Transmission electron microscopy (TEM) images were measured on a JEM-2100F instrument (TECNAI G2, Netherlands), with an accelerated voltage of 200 kV. N2 adsorption and desorption isotherms [Brunauer−Emmett−Teller (BET) and Barrett−Joyner− Halenda (BJH)] were obtained using a Micromeritics Gemini instrument. Ultraviolet−visible (UV−vis) spectra were analyzed using a UV3600 spectrophotometer (Shimadzu, Japan). ζ potential was measured by Zetasizer Nano ZS (Malvern Instruments). Small-angle powder X-ray diffraction (XRD) measurements were carried out using a Rigaku SmartLab III powder diffractometer. 2.2. MSN−OH Synthesis. MSN−OH was synthesized using a previously reported method.31 CTAB (1.0 g) and NaOH (3.5 mL, 2M) were dissolved in 240 mL of water and then heated to reach 80 °C. Tetraethoxysilane (TEOS, 5.0 mL) was added to the solution and vigorously stirred for 2 h at 80 °C. The solid nanoparticles were quickly separated, washed extensively using dH2O and methanol, and dried under vacuum overnight. Empty pores were obtained by solvent extraction of the CTAB template: nanoparticles (1 g) were suspended in MeOH (100 mL); then a concentrated aqueous solution of HCl (12 M, 6 mL) was added; and the mixture was heated under reflux overnight. The solvent-extracted nanoparticles were washed thoroughly with MeOH and collected by vacuum filtration to result in the final product. The nanoparticles were characterized by means of XRD, SEM, and TEM. 2.3. MSN−NH2 Synthesis. A total of 1.0 g of calcined MSN was suspensed in 100 mL of anhydrous toluene, followed by adding an excess amount of APTES (1 mL). After the mixture was stirred continuously for 12 h at 115 °C, it was then filtered and washed with toluene and ethanol before drying under vacuum overnight. The nanoparticles were characterized by means of XRD, BET/BJH, SEM, and TEM. 2.4. Protein Overexpression and Purification. For overexpression, the pGEX-6p-1-GST plasmid (GE Healthcare) was transformed into Escherichia coli BL21 gold (DE3) cells. The bacterial cells containing pGEX-6p-1-GST was inoculated into lysogeny broth (LB) media and incubated overnight (O/N) by shaking at 37 °C. The O/N culture was diluted and transferred into M9ZB media (10 g/L acid casein hydrolyzate, 5 g/L NaCl, 1 g/L NH4Cl, 3 g/L KH2PO4, 6 g/L Na2HPO4·12H2O, 0.246 g/L MgSO4, and 4 g/L glucose) and further incubated by shaking at 18 °C. When the OD600 of these bacterial cells reach approximately 0.8, isopropyl-β-D-thiogalactopyranoside (IPTG, 0.5 mM for a final concentration) was introduced into the media to induce the GST protein expression for 4 h. The cells were collected by centrifugation (5000g for 10 min), washed 3 times using phosphate-buffered saline (PBS) buffer, and stored in −70 °C before use. For protein purification, the bacterial cells were lysed using sonication (with 40% of intensity, 9.9 s on plus 9.9 s off, for 10 min in total) in binding buffer (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4 at pH 7.3). The cell lysate was centrifuged at 14000g for 60 min. The supernatant was filtered through 0.45 μm filter (purchased from Jinteng Laboratory Equipment Co., Ltd., Tianjin, China) and loaded onto a binding buffer pre-equilibrated Glutathione Sepharose 4B column. The column was washed with 10× volumes of binding buffer to remove the unspecific binding of contaminating proteins. The GST-containing fractions were eluted from the column using elution buffer (50 mM Tris−HCl and 10 mM reduced glutathione
Figure 1. Schematic illustration of drug loading and GST capping of MSN−NH2 and their response to the environmental stimuli. The release of drug molecules can be triggered by either adding GSH molecules or shifting pH values. at pH 8.0). The eluted fractions were dialyzed again overnight at 4 °C. The pooled purified GST proteins were divided into 50 μL aliquots in the EP tubes, incubated in liquid nitrogen for 5 min, and then transferred into a −80 °C freezer before use. 2.5. Drug Loading and GST Encapsulation toward MSN− NH2. MSN−NH2 (0.01 g) was dispersed in 1 mL of Tris−HCl solution (0.02 mM, pH 8) of rhodamine 6G (Rh6G, 1 mM) under stirring for 24 h at room temperature for cargo loading. The mixture was separated with centrifugation and washed with dH2O at least 5 times. Then, 3 mg/mL GST was added, and the mixture was further stirred for an additional 1 h, which makes the interactions of GST with the MSN−NH2 pore gate. The Rh6G-loaded MSN−NH2@Rh6G− GST was washed thoroughly with Tris−HCl solution (pH 8) until no dye leakage was visible under UV light. 2.6. Drug Release. Rh6G-loaded and GST-gated nanoparticles were added in the dialysis bag, and Tris−HCl solution was added in the cuvette. The release of Rh6G to the solution was monitored by the UV absorbance at 530 nm in the presence of different amounts of GSH or under different pH values. The volumes of the release media were kept unchanged in the detection process. 2.7. Fluorescence Images. The yeasts were cultured in the yeast extract peptone dextrose (YPD) medium overnight at 30 °C under continuous shaking until reaching a density of 108 cells/mL. Then, aliquots of 1 mL of the cell suspension were centrifuged and resuspended in 100 μL of YPD medium containing 5 mg/mL MSN− GST−doxorubicin (DOX) and incubated for 6 h at room temperature. For the cargo release experiment, these cells are treated with GSH. Intracellular release of DOX was monitored using confocal fluorescence microscopy. 2.8. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide (MTT) Assay. The yeast cells were cultured in YPD media at 30 °C for 17 h under continuous shaking. A total of 1 mL of the diluted cells (OD600 = 1.0) was centrifuged, and the pellets were resuspended in 100 μL of YPD media. MSN−NH2−DOX@GST particles were added to the cell suspensions to reach a final 26812
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Figure 2. (A) SEM and (inset) TEM images of the representatives of MSN−NH2. (B) XRD patterns of MSN−OH (blue), MSN−NH2 (red), and MSN−NH2−Rh6G (black). (C) ζ potentials of MSN−NH2 (red), MSN−OH (green), and Rh6G-loaded MSNs (MSN−NH2−Rh6G) (blue). (D) ζ potentials of pure GSH (75 mM) (red) and GST enzyme in the presence of different contents of GSH [from left to right: 0 mM (green), 10 mM (blue), 25 mM (bluish), 50 mM (violet), and 75 mM (yellow)]. GST crystal structures were shown (E and F): (E) apo form, PDB 13GS and (F) holo form, PDB 1PL1. concentration of 5 mg/mL, followed by 6 h of incubation at room temperature. These samples were then washed 3 times using 1× phosphate-buffered saline (PBS) buffer and resuspended in 100 μL of 1× PBS buffer. GSH was added to the solutions to reach a final concentration of 75 mM and incubated for different periods of time. Cells were then centrifuged to remove the supernatants and resuspended in 100 μL of YPD media. A total of 10 μL of MTT solution (5 mg/mL) was mixed with the cell samples at room temperature for 2 h, followed by centrifugation to collect the pellets. The pellets were then dissolved in 100 μL of dimethyl sulfoxide (DMSO) and measured at OD = 490 nm using an Elx808 microplate reader (BioTek).
turns the electric charge of GST from negative to positive in the presence of GSH. The release of GST−GSH complexes from MSN−NH2 results in the discharge of cargo (e.g., drug molecules) from the pores of the vehicles. Moreover, this setup also responses to other environmental changes, such as pH and salt conditions, that affect the supramolecular interaction between these assembly modules, making it a multiple-response system. Importantly, this new drug delivery/controllable release system is proven to work both in vitro and in living cells (see below). MSNs functionalized with hydroxyl groups (MSN−OH) were synthesized using the sol−gel approach.31 The hydroxyl groups on the surface of MSN−OH can be converted to amino groups via APTES incubation.32,33 The overall structures of the synthesized MSN−OH and MSN−NH2 nanoparticles were characterized using SEM and TEM. The SEM and TEM images of MSN−NH2 and MSN−OH particles suggest that the diameter of these particles is approximately 100 nm (Figure 2A and Figure S3). The MSNs were further characterized using XRD. Both MSN−OH and MSN−NH2 particles (Rh6G-loaded) show the microcrystalline structure with the Bragg peaks of (100), (110), and (200) (Figure 2B), suggesting that the amino functionalization does not impair the crystalline structure of the silica nanoparticles. No significant difference can be recognized concerning the appearance of MSNs before and after surface amino modification. However, the surface
3. RESULTS AND DISCUSSION The encapsulation strategy for the MSN nanocontainer is based on the supramolecular assembly of GST on the outer layer of the MSN−NH2 particles under the physiological condition (Figure 1). The negatively charged GST [isoelectric point (pI) = 5.9] readily interacts with positively charged MSN−NH2 through non-covalent weak force, resulting in the pore closing of the nanoparticles. When GSH is present, it accesses the substrate binding pocket of the GST enzyme through specific intermolecular recognition, leading to a shift of the structure ensemble. This transition results in the rise of the electric charge of GST−GSH complexes, triggering the departure of the complex from the MSN−NH2 nanoparticles (Figure 1). The high efficiency of this logic gate forms a close−open switch that 26813
DOI: 10.1021/acsami.5b08914 ACS Appl. Mater. Interfaces 2015, 7, 26811−26818
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Figure 4. (A) Release of Rh6G from MSN−NH2@GST in the presence of different concentrations of GSH and/or at different pH conditions. (B−D) Release profile of Rh6G from MSN−NH2− Rh6G@GST at pH 4.9 and/or in the presence of (B) 10 mM, (C) 25 mM, or (D) 50 mM GSH (red line). (E−G) Release profile of Rh6G from MSN−NH2−Rh6G@GST at pH 5.9 (black line) and/or in the presence of (E) 10 mM, (F) 25 mM, or (G) 50 mM GSH (red line).
Figure 3. Release profiles of Rh6G from MSN−NH2−Rh6G@GST in the presence of (A) different contents of GSH or at (B) different pH values. Controlled release of Rh6G from the vehicles by (C) adding different contents of GSH (OFF denotes that the GSH concentration is 0, and ON denotes that GSH is present) or (D) shifting pH values (OFF denotes that the pH value is 7.4, and ON denotes that the pH is other values, as indicated). The dissociation of GST with MSN−NH2 in the presence of (E) different concentrations of GSH and at (F) different pH values, as shown by SDS−PAGE. (G) SDS−PAGE of the pellet (inset = supernatant) of the MSN−OH−GST mixture after centrifugation, following the incubation with different amounts of GSH. (H) Release profiles of Rh6G from MSN−NH2−Rh6G@GST in the presence of different contents of salt (NaCl).
GST recombinant protein (Figure S6 and Sequences S1 and S2) was overexpressed and purified as described in the Experimental Section. GST proteins are negatively charged at the physiological condition (150 mM NaCl at pH 7.4; Figure 2D), which readily encapsulate positively charged Rh6G-loaded MSN−NH2 nanoparticles (Figure 2C) via intermolecular weak interaction. These supramolecular interactions can be modulated by different environmental stimuli, such as GSH and pH conditions (see below). The quantitive cargo release profile of MSN−NH2−Rh6G@ GST toward different environmental stimuli was investigated using a classic dialysis approach (Figure S7).12 Briefly, 10 mg of GST-capped MSN−NH2−Rh6G was placed in a dialysis bag and incubated in a cuvette containing 3 mL of 1× PBS buffer (pH 7.4) supplemented with different concentrations of GSH. When GSH binds to GST, the conformation and surface charge of the enzyme are shifted. This allosteric effect decaps MSN− NH2−Rh6G, resulting in the release of cargo molecules. The release profile of the cargo molecules (Rh6G) can be monitored using UV−vis absorbance spectroscopy. Rh6G molecules are
electric charge of MSNs shifted from negative to positive after APTES modification, as shown by ζ-potential analysis (Figure 2C). The nitrogen adsorption−desorption isotherm indicates the surface area of 1093 m2 g−1 and porous structure of MSN−NH2, with an average pore diameter of 2 nm (Figure S4). FTIR spectra were applied to monitor the degree of hydroxylation and amination functionalization of the MSN particles. In addition to the emerged peak at approximately 2930 cm−1, which corresponds to the −CH2− group, two characteristic peaks from the vibration of the amino groups, localized at 1550 and 1490 cm−1, are present in the spectra of MSN−NH2, in contrast to that of MSN−OH particles, confirming the reaction success (Figure S5). 26814
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Figure 5. Confocal laser scanning microscopy (CLSM) images of (A−C) yeast cells, (D−F) yeast cells treated with DOX, (G−I) yeast cells treated with DOX-loaded MSN−NH2−DOX@GST, or (J−L) yeast cells treated with DOX-loaded MSN−NH2−DOX@GST followed by GSH incubation. The bar refers to 20 μm. The inset denotes the enlarged images (the bar refers to 5 μm). Left, middle, and right lanes denote for bright field, red fluorescence, and overlapped images, respectively.
Similarly, this system is highly controllable via changing the pH conditions. No cargo release from the vehicles can be detected at pH 7.4 (0−10 min in Figure 3D). When pH switches to 5.9, a substantial amount of Rh6G is released (30%) (10−20 min in Figure 3D). The cargo release approaches nearly maximum (98%), while pH is 3.9, indicating that the pore gate is entirely opened at this point. A new approach was developed here to visualize the association and dissociation of GST with Rh6G-loaded MSN−NH2 using a combination of centrifugation and sodium dodecyl sulfate−polyacrylamide gel electrophoresis (SDS−PAGE). Briefly, the encapsulated vehicles (MSN−NH2−Rh6G@GST) were incubated at different concentrations of GSH and/or at different pH conditions for 30 min. The separated supernatants and pellets after centrifugation were collected and subjected to SDS−PAGE to visualize the remaining proteins that bind to MSN−NH2−Rh6G. It was shown that the amount of GST associating with MSN−NH2−Rh6G was gradually decreased following the incubation of the MSN−NH2−Rh6G@GST complex with a higher concentration of GSH (Figure 3E), indicating that GST detaches from the MSNs. Similarly, the release of GST from the particles can be detected when the
released from the GST-gated MSN vehicles following the increase of the GSH concentration (Figure 3A). The cargo release of this system can also be triggered by changing the environmental pH values (Figure 3B). It is known that the pI of GST is 5.9, meaning that the surface charge of this protein shifts from negative to positive when the environmental pH changes from a neutral to an acidic condition. These changes decrease the strength of the supramolecular interaction between the GST protein and MSN−NH2, resulting in the departure of GST from the surface of the nanoparticles, leading to the cargo release. This supramolecular recognition nature between the enzyme (GST) and substrate (GSH) makes this system highly controllable depending upon the content of GSH in the solution. In the absence of GSH, hardly any cargo release can be detected (0−10 min in Figure 3C). The cargo molecules are released (28%) when the GSH concentration approaches 10 mM (10−20 min in Figure 3C). The release is ceased by putting the system back to neutralized 1× PBS buffer (pH 7.4). When the GSH concentration is 25 mM, Rh6G is released up to 42% (30−40 min in Figure 3C). Finally, the cargo molecules can be discharged to the maximum (almost 100%) when the concentration of GSH reaches 75 mM. 26815
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Figure 6. CLSM images of yeast cells treated by (A−C) GSH, (D−F) GSH−PI, (G−I) PI, or (J−O) PI-loaded MSN−NH2−PI@GST [in the (J−L) absence or (M−O) presence of GSH]. The bar refers to 10 μm. The inset denotes the enlarged images (the bar refers to 5 μm).
in living cells, modulating the salt concentration is not highly practicable. Therefore, we propose that GSH and pH are the two keys to open the gate of this GST-capped vehicle. The synergistic effect of these two triggering factors for cargo release is summarized (panels A−G of Figure 4). Notably, approximately 30% of cargo molecules are released at pH 5.9, while the GSH concentration reaches 10 mM, resulting in 30% release (panels A and E of Figure 4). When the GSH concentration is 10 mM at pH 5.9, 70% release of cargo can be achieved (Figure 4A). To evaluate whether this new drug delivery and controllable release system works in vivo/ex vivo or not, these GSTencapsulated vesicles containing DOX were incubated with yeast (Saccharomyces cerevisiae) cells and subjected to imaging analysis. As shown in confocal fluorescence microimages (Figure 5), this system successfully delivers DOX into the
system was incubated with low pH solutions (Figure 3F). Moreover, GST was solely present in the supernatant instead of associating with the vehicles when MSN−NH2 was replaced by MSN−OH (Figure 3G), suggesting that GST does not interact with MSN−OH nanoparticles. The supramolecular force that encapsulates the MSN particles is associated with the amino groups of the functionalized vehicles. The intermolecular interaction between GST and MSN−NH2 can also be modulated by other factors, such as the salt concentration. While no cargo release can be detected at the physiological condition (140 mM NaCl), a high salt concentration of the environmental condition results in the Rh6G release (Figure 3H). When the salt concentration is 250 mM, the cargo is inclined to leave the vehicles. When the salt concentration reaches 550 mM, the nanogate of the system is almost completely opened (Figure 3H). However, considering the situation 26816
DOI: 10.1021/acsami.5b08914 ACS Appl. Mater. Interfaces 2015, 7, 26811−26818
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ACS Applied Materials & Interfaces
Notably, GSH treatment by itself did not make any detectable harm to the yeast cells (panels A−C of Figure 6), indicating that the deleterious effect was caused by released DOX.
eukaryotic cells and releases the cargo upon GSH treatment. The delivery efficiency of the MSN−NH2−DOX@GST system is assumably much higher than that of the self-penetration of DOX through the cell membrane (panels A−I of Figure 5), because the majority of cells show significant morphological changes when the DOX molecules are released from the vehicles upon GSH treatment (panels J−L of Figure 5). The morphological changes may be associated with the apoptosis of the yeast cells. Therefore, we propose that yeast cells might be an interesting alternative platform to evaluate the drug delivery and release efficiency of vehicles and capping systems in addition to widely used mammalian cell cultural systems. There are several reasons and/or advantages of using yeast as a new platform to evaluate drug delivery and release. First of all, both yeast and mammalian cells are eukaryotic cells containing similar subcellular structures and cell signaling pathways, albeit the size of the yeast cells are smaller. Second, in comparison to mammalian-cell-related research, which requires a standard tissue culture room with high cost, yeast cells can be cultured in a regular laboratory with much cheap expenses. Third, the simplicity of the subcellular structure of yeast cells allows us to have a relatively quick and easy investigation toward their changes upon drug release. Because DOX can cross the cell membrane by itself possibly through diffusion (panels D−F of Figure 5),34 other cargo molecules become necessary to further evaluate the new capping−delivery−release system. Propidium iodide (PI) is a membrane-impermeable fluorescent molecule and cannot enter living cells by itself.35 Indeed, PI is excluded from the viable yeast cells (panels A−I of Figure 6). MSN−NH2−PI@GST successfully delivers PI molecules into the yeast cells (panels J−L of Figure 6) and dispersed these cargo molecules into the cytoplasm in the presence of GSH (panels M−O of Figure 6). The viability of yeast cells after different treatments was evaluated using the MTT assay (Figure 7). Yeast cells survived
4. CONCLUSION In this report, we developed for a new MSN capping and uncaping system based on the modulation of the supramolecular interaction between MSN, GST enzyme, and its substrate. The electrostatic interaction between MSN−NH2 and GST enzyme at physiological conditions closes the pores of the MSN vehicles successfully, while the enzyme and substrate interaction between GST and GSH leads to opening of the nanogate. The supramolecular nature of this system makes it subtile to environmental changes and highly controllable. Indeed, mild changes in either the substrate concentration or pH condition effectively trigger the cargo release. The advantages of this system also lie in its feasibility of manipulation in living cells. Given the diverse enzyme−substrate pairs existing in nature, we believe that our approach can be readily extended to various drug delivery and controllable release systems for different and specific purposes via converting the enzyme− substrate combination. In addition, a SDS−PAGE-based system was described for a visual analysis of detached corona from vehicles. We also propose that yeast may be a new and handy model system for the evaluation of drug delivery and controllable cargo release under physiological conditions.
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ASSOCIATED CONTENT
* Supporting Information S
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.5b08914. Molecular formula of GSH, CD spectra and crystal structures of GST, SEM, FTIR spectra, amino acid and cDNA sequence of GST, plasmid map, SDS−PAGE, and release profiles (PDF)
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AUTHOR INFORMATION
Corresponding Author
*Telephone: +86-186-8663-6807. Fax: +86-431-8519-3421. E-mail: [email protected]. Author Contributions †
Pengchang Liu and Xiaoliang Wang contributed equally to this work. Funding
This work was supported by the National Natural Science Foundation of China (NSF, 21372097) and a CIMO grant from Finland. Notes
Figure 7. Yeast cell viability as shown using the MTT assay. Yeast cells were incubated with DOX (red), MSN−NH2−DOX−GST (green), or MSN−NH2−DOX−GST followed by GSH treatment (blue). The MTT assay was carried out at different time points.
The authors declare no competing financial interest.
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quite well when treated with DOX (98 and 88% viability at 30 and 60 min, respectively). Similarly, cells are still rather active after incubation with MSN−NH2−DOX−GST (99 and 94% viability at 30 and 60 min, respectively). Strikingly, when the MSN−NH2−DOX−GST-treated sample were followed by incubation with GSH, the viability of the cells were significantly decreased (65 and 57% viability at 30 and 60 min, respectively), suggesting that DOX was efficiently delivered into the yeast cells by MSN vehicles and released in the presence of GSH. 26817
NOMENCLATURE MSN = mesoporous silica nanoparticle GSH = glutathione GST = glutathione S-transferase TEM = transmission electron microscopy SEM = scanning electron microscopy XRD = small-angle powder X-ray diffraction SDS−PAGE = sodium dodecyl sulfate−polyacrylamide gel electrophoresis Rh6G = rhodamine 6G DOX = doxorubicin DOI: 10.1021/acsami.5b08914 ACS Appl. Mater. Interfaces 2015, 7, 26811−26818
Research Article
ACS Applied Materials & Interfaces
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