DOI:
10.1039/C6RA03543D
(Paper)
RSC Adv., 2016,
6, 25480-25484
A drug release switch based on protein-inhibitor supramolecular interaction†
Received
7th February 2016
, Accepted 1st March 2016
First published on 2nd March 2016
Abstract
In this report, we describe a new system in which mesoporous silica nanoparticles (MSNs) are gated with α-chymotrypsin A protein (CTRA) and the cargoes within the vehicles are released in the presence of phenylmethanesulfonyl fluoride (PMSF), a canonical inhibitor of CTRA. This cargo release switch is based on the specific interaction between CTRA and PMSF as well as structural changes upon their supramolecular complex formation. This host–guest gating system works smoothly both in vitro and within cells. This type of bio-switch may be extended to other drug carrier systems by using diverse protein–inhibitor pairs that exist in nature.
1. Introduction
Drug delivery systems have attracted much attention during the last few decades. Designing and building drug carriers that can deliver cargoes into cells and release them in a controllable manner has become an important issues in the biomedical research. To this end, various delivery platforms such as nanogels, polymer, micelles, inorganic materials, and bio-molecule based carriers have been constructed and showed inspirational outcomes.1–8
Mesoporous silica nanoparticles (MSNs) are among the best studied and well characterized nano-carriers that can function as vehicles for drug delivery. MSNs contain large pore volume, high surface areas, and tunable pore sizes, as well as fine biocompatibility.9–13 In particular, through interacting with suitable chemical agents such as polymers and nanoparticles, one can build smart systems containing distinct stimuli–responsive nano-gates.14–19 These advanced drug release systems are highly attractive in the field of biomaterials and drug delivery. Notably, the properties of supramolecular interactions between the carriers and capping agents largely determine the nature stimuli–responsive cargo release systems.20–30
An open question concerning MSN based carriers is the cytotoxicity of the systems, which is sometimes caused by certain chemical capping agents. To enhance the biocompatibility of the smart MSN materials, low toxic compounds have been proposed to sever as gating agents. Recently, we used enzymes and their substrates to act as capping and stimuli agents for MSNs.31 Nevertheless, this system requires high concentration of substrates to trigger cargo release, which is a disadvantage. To overcome this problem, herein we developed a new platform that allows the cargo release in the presence of low concentration of stimuli agents. In the new system, α-chymotrypsin A protein (CTRA) and its inhibitor phenylmethanesulfonyl fluoride (PMSF) are served as capping and decapping agents, respectively (Scheme 1). The specific supramolecular interaction between the enzyme and its inhibitor triggers the cargo release under sophisticated environment such as cellular context.
 |
| Scheme 1 Schematic illustration of PMSF–CTRA–MSN drug delivery system. (a) The cargo release can be triggered through either specific interaction between PMSF and CTRA or pH modulation, which disassemble CTRA–MSN. (b) The CTRA–MSNs can be internalized by cells and release the cargoes when PMSF is present. | |
2. Experimental section
2.1 Reagent
Chemicals: (3-aminopropyl)-triethoxysilane (APTES), cetyltrimethylammonium bromide (CTAB), and tetraethoxysilane (TEOS) were purchased from Adamas Reagent (Shanghai, China). CTRA and PMSF were obtained from Kayo (Shanghai, China) and Genview (Beijing, China), respectively. Doxorubicin (DOX) and propidium iodide (PI) were ordered from Dingguo Changsheng Biotechnology (Beijing, China). Double distilled water (dH2O) was used for all the experiments.
2.2 MSN–OH and MSN–NH2 synthesis
MSN–OH nanoparticle synthesis and their NH2 functionalization were carried out according to previously reported method.32–35 The synthesized nanoparticles were characterized by using Ultraviolet-visible (UV-vis), Fourier transform infrared (FTIR) spectroscopy, ζ potential analyzer, scanning electron microscopy (SEM), transmission electron microscopy (TEM), Brunauer–Emmett–Teller and Barrett–Joyner–Halenda (BET/BJH), and small-angle powder X-ray diffraction (XRD). UV-vis spectra were measured using a UV-3600 spectrophotometer (Shimadzu, Japan). FTIR spectra were analyzed using a Bruker Vertex 80 V spectrometer. ζ Potential was measured by Zetasizer Nano ZS (Malvern Instruments). SEM images were taken from a JEOL JSM 6700F instrument. TEM images were collected on a JEM-2100F instrument (TECNAI G2, Netherlands) with an accelerated voltage of 200 kV. N2 adsorption and desorption isotherms (BET/BJH) were obtained using a Micromeritics Gemini instrument. XRD measurements were carried out using a Rigaku SmartLab III powder diffractometer.
2.3 Cargo loading, CTRA encapsulation, and cargo release test
For cargo loading, 0.01 g of MSNs–NH2 were dispersed in 1 mL of Tris–HCl (0.02 mM, pH = 8) containing rhodamine 6G (Rh 6G, 1 mM). The solution was mixed under stirring for 24 hours at room temperature. The resulting cargo containing MSN–NH2 was washed for minimal 5 times by using centrifugation. For CTRA encapsulation, 1 mg mL−1 CTRA was added into the mixture followed by stirring for 1 hour. The CTRA encapsulated CRTA–MSN–NH2@Rh 6G was washed thoroughly with Tris–HCl (pH = 8) until no fluorescent signal (Rh 6G) can be detected from the supernatant. For cargo release experiment, CTRA–MSN–NH2@Rh 6G was transferred into a dialysis bag (3500 dalton cut off), which was incubated in a Tris–HCl buffer containing cuvette. The cargo (Rh 6G in this case) release to the Tris–HCl solution under different conditions was monitored at 530 nm by using UV-vis spectroscopy.
2.4 Yeast culture and fluorescence images
The yeast cells were inoculated in the yeast extract peptone dextrose (YPD) media for shaking incubation at 30 °C until reaching a density of 108 cells per mL. Cells from 1 mL of the suspension were resuspended in 100 μL of YPD containing 5 mg mL−1 of CTRA–MSN–NH2@cargo followed by 6 hour incubation at room temperature before confocal fluorescence microscopic analysis. The cargo release within cells was triggered by adding of PMSF into the cultural media for 30 minutes.
3. Results and discussion
3.1 Synthesis and particle characterization
MSNs containing hydroxyl or amino groups (MSN–OH and MSN–NH2) were synthesized according to previously published protocols.32–35 The amino functionalization was demonstrated by FTIR spectra (Fig. S1†). The synthesized MSN nanoparticles show a diameter of approximately 100 nm (Fig. 1a, S2 and S3†) and contain microcrystalline structure (Fig. 1b). ζ-Potential analysis showed that the surfaces of MSN–OH and MSN–NH2 nanoparticles are negatively and positively charged, respectively (Fig. 1c). These porous MSN nanoparticles contain a surface area of 1093 m2 g−1 with an average pore diameter of 2 nm (Fig. 1d).
 |
| Fig. 1 (a) SEM and TEM (inset) characterization of MSN–NH2. (b) XRD spectra of MSN–OH (black), MSN–NH2 (red), and MSN–NH2@Rh 6G (blue). The diffraction peaks at 100, 110, and 200 indicate these MSN materials contain microcrystalline structure before and after amino-modifications. (c) Zeta potential analysis shows that the surface of MSN–OH (red) is negatively charged, while those of MSN–NH2 and MSN–NH2@Rh 6G are positively charged. Therefore, negatively charged CTRA could interact with MSN–NH2@Rh 6G through electrostatic interactions, thus encapsulating the Rh 6G-containing MSN–NH2. (d) Nitrogen desorption curve of MSN–NH2. The surface area and mean pore size of MSN–NH2 are 1093 m2g−1 and 2 nm, respectively. | |
3.2 The drug loading and release
The gate system of MSN–NH2 nanoparticles is based on the structural shift of CTRA when either recognizing PMSF molecules or environmental pH decreasing (Fig. 3a and b). Notably, CTRA is a classic enzyme with well documented effective inhibitors and is widely used in biomedical and clinical research. The well-established inhibitor of this protein is PMSF (Scheme 1), which specifically recognizes CTRA and triggers its morphological changes (Fig. S4†). Moreover, the surface change of CTRA shifts upon pH modulation (the isoelectronic point of CTRA is approximately 6.1) (Fig. S5†).
 |
| Fig. 2 The CTRA crystal structures ((a) apo form: PDB 1EQ9; (b) holo form: PDB 1EQ9). (c) The Rh 6G release profile of CTRA–MSN–NH2@Rh 6G in the presence of different concentration of PMSF. The competitive binding of PMSF with CTRA triggers the release of Rh 6G from the pore of MSNs. (d) The Rh 6G release profile of CTRA–MSN–NH2@Rh 6G at different pHs. (e) The release of cargo molecules from this system is strictly PMSF concentration dependent and highly controllable and reversible. (f) The release of cargo molecules from this system is strictly pH dependent and highly controllable and reversible. (g) The synergistic effect of both PMSF and pH. At low pH (e.g., pH = 3.9), the dominant factor of the cargo release of this system is pH; while in the presence of high concentration of PMSF (e.g., 7.5 mM), the dominant factor turns out to be PMSF. | |
 |
| Fig. 3 CFM images show CTRA–MSN–NH2 delivers DOX into yeast cells. PMSF triggers the release of DOX, which leads to cell death. (a–d) Depict red channel; (e–h) depict the bright filed. (i–l) Depict overlay images. Yeast cells serve as control (a, e and i). Yeast cells are incubated with DOX (b, f and j). Yeast cells are incubated with CTRA–MSN–NH2@DOX (c, g and k). Yeast cells are incubated with CTRA–MSN–NH2@DOX followed by PMSF treatment (7.5 mM) (d, h and i). Bar refers to 20 μm (inset: 5 μm). | |
These two factors work synergistically toward CTRA, enabling this protein a specific and sensitive switch (Fig. S6†). When negatively charged CTRA was incubated with positively charged cargo-loaded MSN–NH2 under natural pH condition, the pores of MSNs were closed. The allosteric transition of CTRA upon substrate binding leads to the cargo release. Alternatively, when pH of the environment is lower than 6.1, CTRA becomes positively charged. The electrostatic repulsion between CTRA and MSN–NH2 triggers the departure of protein followed by the release of cargo molecules.
The quantitive cargo release profile of CTRA–MSN–NH2@Rh 6G was analyzed by using a classic dialysis approach complemented with UV-vis absorbance spectroscopy.32,36 Rh 6Gs are released from the vehicles following the increase of the PMSF concentration (Fig. 2c). This cargo release can be also achieved following the decrease of environmental pH as shown by a serial analysis (Fig. 2d). This supramolecular nature makes this system highly modulatable by changing the PMSF content in the solution. The cargo release shifts from 0 to 14% when PMSF concentration changing from 0 to 1 mM (Fig. 2c and S7†). When PMSF concentration reach 2.5 mM (5.0 mM, 7.5 mM), Rh 6Gs can be released up to 29% (60%, 100%) (Fig. 2c, S8 and S9†). Similarly, this system is highly controllable via changing the pHs. When pH reaches 5.9, ca. 42% of Rh 6G is released (Fig. 2d). This cargo release can be further improved to higher degrees (70%, 100%) by incubating the system to lower pHs (pH 4.9, pH 3.9) (Fig. 2d). Taking together, the cargo release of this CTRA-capped MSN vehicle can be modulated by the content of PMSF and pH of the solutions. The synergistic effect of these two factors toward CTRA–MSN–NH2@Rh 6G is summarized (Fig. 2g). For instance, 42% cargo molecules are released at pH 5.9 in the absence of PMSF, while addition of 5 mM PMSF to the solution result in 70% releases (Fig. 2g and S9†). When the PMSF concentration reaches 7.5 mM (pH 5.9), nearly 100% cargo release can be achieved. Similarly, 70% cargo molecules are released at pH 4.9 in the absence of PMSF, while addition of 1.0 mM (2.5 mM, 5 mM) PMSF to the solution result in 80% (90%, 100%) releases (Fig. S10–12†). It's worthy to note that the cargo release system is highly controllable and reversible toward PMSF content and/or pHs of the solutions (Fig. 2e and f).
3.3 Drug release behavior in yeast cells
As we demonstrated earlier, yeast cell (Saccharomyces cerevisiae) can be a nice alternative platform to evaluate cargo delivery and release events in a biological context,31 in addition to the widely used mammalian cell cultures.37 Fluorescent imaging analysis shows that CTRA–MSN–NH2 encapsulated DOX (CTRA–MSN–NH2@DOX) readily delivers DOX into the S. cerevisiae cells and releases it in the presence of PMSF (Fig. 3). Compared to yeast cells which don't show fluorescent signals (Fig. 3a, e and i), both DOX and CTRA–MSN–NH2@DOX treated cells show clear red fluorescence (Fig. 3b, f, g and c, g, k), suggesting the effective uptaking of the vehicles by the yeast cells. Interestingly, the majority of the CTRA–MSN–NH2@DOX containing cells show significant morphological changes when the DOX molecules are released from the vehicles upon PMSF treatment (Fig. 3d, h and i), suggesting a higher delivery efficiency than the self-penetration of DOX (Fig. 3b, f and j). Importantly, the morphological changes of the yeast hint the cell death. Yeast cells are also belong to eukaryotic organisms which contain similar subcellular structures and signaling pathways comparable to those of mammalian cells. The advantages of yeast system lie in the simplicity of their morphology allowing a quick investigation toward their cellular responses upon cargo release. Moreover, yeast cells can be maintained in a regular laboratory with much cheap expenses compared to mammalian systems, which demand a high standard tissue culture facility.
To further demonstrate the carrier capability of the CTRA–MSN–NH2 system, propidium iodide (PI) was loaded onto the MSN nanoparticles to form CTRA–MSN–NH2@PI. Notably, PI is a fluorescent molecule and cell membrane-impermeable, which cannot enter living cells without carrier's help.38 This property of PI was confirmed by the yeast system no matter PMSF is present or not (Fig. 4a, f, k and b, g, l and c, h, m). Encouragingly, CTRA–MSN–NH2@PI readily passes through the cell walls and plasma membranes of the yeast cells (Fig. 4d, i and n). Furthermore, in the presence of PMSF, PI molecules are released from the vehicles into the cytoplasm of the S. cerevisiae cells (Fig. 4e, j and o). These experiments demonstrated further the preferable vehicular properties of CTRA–MSN–NH2 concerning both the delivery and cargo release control.
 |
| Fig. 4 CLSM images of yeast cells ((a–e), fluorescent; (f–j), bright; (k–o), overlay). Yeast cells were incubated different agents as follows: PI (a, f and k), PMSF (b, g and l), PI–PMSF (c, h and m), CTRA–MSN–NH2@PI (d, i and n), CTRA–MSN–NH2@PI followed by PMSF treatment (7.5 mM) (e, j and o). The bar refers to 20 μm. | |
4. Conclusions
Among various capping and uncapping systems for drug carriers, enzyme-inhibitor combination (this study) represents a highly sensitive and specific manner in modulating cargo release in living cells. Compared to previously reported enzyme-substrate modulators such as GST–GSH pairs, enzyme-inhibitor switch shows much higher sensitivity and selectivity. Nowadays, plenty of information concerning proteins and their small molecule modulators including both inhibitors and activators is available from bioinformatic and related databases. We thus envision that one could easily design and build suitable synthetic bio-switches based upon these nature's inventories and apply them to drug delivery and release studies.
Nomenclature
MSN | Mesoporous silica nanoparticle |
CTRA | α-Chymotrypsin A |
PMSF | Phenylmethanesulfonyl fluoride |
XRD | Small-angle powder X-ray diffraction |
SEM | Scanning electron microscopy |
TEM | Transmission electron microscopy |
Rh 6G | Rhodamine 6G |
DOX | Doxorubicin |
PI | Propidium iodide |
Acknowledgements
This work was supported by the National Natural Science Foundation of China (NSF 21372097). We are grateful to Professor Yingwei Yang at Jilin University for inspirational discussion.
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Footnote |
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra03543d |
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