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Article Cite This: ACS Omega 2019, 4, 6097−6105
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Enzyme-/Redox-Responsive Mesoporous Silica Nanoparticles Based on Functionalized Dopamine as Nanocarriers for Cancer Therapy Dandan Zhu, Chunlin Hu, Yuan Liu, Feng Chen, Zhen Zheng, and Xinling Wang* School of Chemistry and Chemical Technology, State Key Laboratory of Metal Matrix Composites, Shanghai Jiao Tong University, Shanghai 200240, China
ACS Omega 2019.4:6097-6105. Downloaded from pubs.acs.org by 95.85.71.242 on 04/01/19. For personal use only.
S Supporting Information *
ABSTRACT: Mesoporous silica nanoparticles (MSNs) have been proved potential nanocarriers for efficient stimuli-triggered drug delivery systems (DDSs) in cancer therapy. We have developed a novel pepsin enzyme-responsive MSN based on mussel mimetic coating of dopamine for controlled release of drug delivery via a facile onepot strategy. Herein, to improve drug release efficiency for cancer therapy, a novel dual-sensitive (enzyme- and redox-) MSN based on functionalized dopamine containing both peptide and disulfide bonds was designed and prepared. In detail, cystine-dopamine (Cy-DA) was newly synthesized and used as a monomer to selfpolymerize into a coating on MSNs via simple a one-pot reaction to give rise to a chemotherapy drug [e.g., doxorubicin (DOX)] delivery system poly-Cy-DA-MSN. The in vitro cellular cytotoxicity tests of normal Marc-145 cells and HeLa cells showed that poly-Cy-DA-MSN was nontoxic, highly biocompatible, and suitable as drug carriers in controlled release DDS (CDDS). Because of the peptide and disulfide bonds in Cy-DA, the as-prepared DOX-loaded poly-Cy-DA-MSNs exhibited improving enzyme- and redox-responsive release property. Confocal laser scanning microscopy results showed that DOX@ poly-Cy-DA-MSNs transported by the cells through endocytosis mechanism and the cancer drug DOX were released mainly in the cytoplasm and also in cell nucleus. These results showed that poly-Cy-DA-MSN is a promising double-responsive CDDS for sustained cancer therapy.
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INTRODUCTION Mussels can secrete a particular protein which would be strongly adhesive to various materials with dramatic binding strength, even in harsh environments.1 It was revealed by a series of investigations that 3,4-dihydroxyphenylalanine is significant for the extraordinarily robust adhesion nature of mussels.2−4 On the basis of these studies, Messersmith et al. developed a facile method to modify substrates via selfpolymerization behavior of dopamine in aqueous solution of alkaline.5 Polydopamine has been proven to be able to easily deposit on various substrates, including superhydrophobic materials,6 with durable stability and controllable film thickness. Studies have revealed that polydopamine presents good performance in optics,7 electricity,8 magnetics,9,10 and adhesive property.11−15 Furthermore, polydopamine could be applied as a platform for further chemical modification because of its special chemical structure which would be combined with many functional groups such as imine, amine, and catechol groups.16 The metal ion chelating17,18 and redox activity19 of polydopamine have also drawn considerable attention in manufacturing organic−inorganic hybrid materials. Till now, lots of studies have reported that polydopamine coating on substrates could promote cell adhesion and proliferation, showing good biocompatibility and nontoxicity to cell.20,21 As melanin,22 polydopamine-coated nanoparticles degraded after incubation with hydrogen peroxide.23 Langer’s group has © 2019 American Chemical Society
reported that polydopamine implants incubated in vivo were almost fully degraded after 8 weeks.24 Because of these excellent functions and properties, polydopamine was widely employed in surface modification,25−28 hybrid energy storage materials,29,30 and biological materials.31−38 Polydopamine shows good biocompatibility and nontoxicity to cells, making it a promising candidate for drug delivery system (DDS). Zhou et al. fabricated polydopamine nano/ microcapsules33 and subsequently found that the uptake of rhodamine 6G by polydopamine microcapsules showed pHselective property.39 Afterward, acetyl methacrylate and (ethylene glycol) methyl acrylate were grafted onto the polydopamine capsule surface, giving rise to a pH and temperature-sensitive controlled release systems.40 Despite its highly selective drug loading ability, these systems still presented unsatisfactory drug release performance in aqueous solution. On the basis of this limitation, Caruso reported a pHresponsive DDS prepared by covalently functionalizing the polydopamine capsule surfaces with a cleavable poly(methacrylic acid)−doxorubicin (DOX) conjugate, leading to dramatic pH-induced DOX release in aqueous solution.41 Received: September 27, 2018 Accepted: December 6, 2018 Published: April 1, 2019 6097
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So far, among various drug carriers, porous silica nanoparticles [mesoporous silica nanoparticles (MSNs)] present special features, such as good chemical and thermal stability, large surface area, controllable particle size, high pore volume, great loading capacity, excellent biocompatibility, and ease of surface modification.42 Owing to the abundant active silanol groups (Si−OH) on the MSN surface, stimuli-responsive DDSs could be easily constructed via modification with various nanovalves or attachments of different functional groups. In our previous work, we developed a facile one-pot strategy, which avoided generally used complicated preparation methods, and prepared a pepsin enzyme-responsive controlled release DDS (CDDS) which successfully combined the advantages of dopamine and MSNs together.43 Hence, to enhance the drug release performance and specificity of drug delivery of this novel one-pot prepared dopamine/MSN-based DDS, a novel dual-responsive DDS (enzyme- and redox-) was developed in this work. Because of high concentration of glutathione (GSH) in tumor tissues, the redox-sensitive property was exhibited and realized through the redox reaction between GSH and disulfide bond. Thereby, a pseudo tripeptide Cy-DA molecule, carrying disulfide and peptide bonds, was designed and synthesized successfully. Poly-Cy-DA coating was deposited on MSN surfaces through self-polymerization with model drug molecules (rhodamine B, DOX) trapped in MSN pores via the one-pot reaction, giving rise to a developed dual-responsive MSN drug release system (poly-Cy-DA-MSNs) with high loading efficiency (85% for RhB and 88% for DOX). The resulting RhB/DOX@poly-CyDA-MSNs showed a good redox- and enzyme-responsive drug release behavior when exposed to GSH and pepsin, respectively. The biocompatibility and anticancer activity in vitro were evaluated by using corresponding normal Marc-145 cells and HeLa cells, revealing that the dual-responsive polyCy-DA-MSNs are nontoxic, highly biocompatible, and suitable to be utilized as drug carriers in a controlled DDS. Furthermore, confocal laser scanning microscopy (CLSM) demonstrated that DOX@poly-Cy-DA-MSNs might be taken up by the cells through endocytosis and DOX was released in endocytic compartments.
Scheme 1. Synthetic Route for Cystine-Dopamine (Cy-DA): (1) N-Hydroxysuccinimide, DCM/THF, EDC-HCl, 0 °C, 6.5 h; (2) Dopamine, MeOH, 0 °C, 6.5 h; and (3) HCl/EA, rt, 4 h
tively. The blank sample without the drug model was dominated as poly-Cy-DA-MSNs. Energy dispersive X-ray (EDX)-loaded transmission electron microscopy (TEM) was employed to verify the successful insitu polymerization of Cy-DA on the MSN surface. From the TEM images in Figure 1, it is evident that poly-Cy-DA-MSNs
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RESULTS AND DISCUSSION Fabrication and Characterization of Poly-Cy-DAMSNs and RhB@poly-Cy-DA-MSNs. In our previous work, a facile one-pot strategy was developed to fabricate the drug delivery nanocarrier system based on MSNs and enzymeresponsive lysine-dopamine. Here, a new functionalized dopamine molecule Cy-DA (with an additional disulfide bond) was designed and synthesized for a novel dual-triggered drug release system. The detailed synthetic route for Cy-DA is shown in Scheme 1. The chemical structures of (Boc)2-Cy-DA and Cy-DA were confirmed by 1H NMR, 13C NMR (Figures S1 and S2), and element analysis (Table S1). The highresolution time-of-flight mass spectrometry (TOF-MS) result of Cy-DA is shown in Figure S3, indicating the successful synthesis of the designed Cy-DA molecule. The target dual-responsive drug release system was prepared via in situ polymerization of Cy-DA on the MSN surface with RhB or DOX as the drug model molecule by the one-pot strategy under a weak alkaline condition. The corresponding as-prepared drug release nanomaterials were named as RhB@ poly-Cy-DA-MSNs and DOX@poly-Cy-DA-MSNs, respec-
Figure 1. TEM images of MSNs (a), poly-Cy-DA-MSNs (b) with a scale bar of 100 nm, and size distributions of MSNs and poly-Cy-DAMSNs obtained from the DLS measurement (c).
showed clear coated polymers on surfaces (Figure 1b) compared with MSNs, which presented a typical well-ordered hexagonal structure with uniform pore sizes (Figure 1a). In addition, the average diameter of poly-Cy-DA-MSNs was 180 ± 30 nm, larger than MSNs (160 ± 20 nm), further demonstrating the polymerization and coating of Cy-DA onto MSNs. DLS measurement was also carried out, giving consistent results with TEM studies. The DLS curves of pure MSNs and poly-Cy-DA-MSNs (Figure 1c) both exhibited narrow unimodal size distribution, with an average diameter of approximately 162.7 and 211.3 nm, respectively. In the DLS measurement, the zeta potential was −33.2 mV for pure MSNs, owing to the negatively charged silanol groups, and increased to −22.3 mV for poly-Cy-DA-MSNs, revealing a 6098
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density decrease of free silanol groups,45 which the surfacecoated poly-Cy-DA could explain. The surface element changes before and after in situ polymerization of Cy-DA on the MSN surface were investigated by EDX. Discriminating elements N and S were chosen as the comparing object, of which the EDX spectra are shown in Figure S4. The Si and S mapping results indicated that MSNs were uniformly covered by poly-Cy-DA coating.46 The corresponding element percentages are summarized in Table 1. There were no traces of N and S for MSNs at all, whereas poly-Cy-DA-MSNs carried elements N and S distinctly.
exhibited a IV isotherm type with H3-type hysteresis loops. The relevant pore size and Brunauer−Emmett−Teller (BET) surface area values are summarized in Table 2. The BET surface, pore size, and pore volume of MSNs are 689.20 m2 g−1, 3.99 nm, and 0.59 cm3 g−1, respectively. After the surface was covered with poly-Cy-DA coating, these values decreased to 453.41 m2 g−1, 3.05 nm, and 0.48 cm3 g−1, respectively. The adsorption/desorption data indicated that MSNs were successfully covered by the poly-Cy-DA coating and the drug model was effectively blocked in the MSN pores. In Vitro Release Behavior of RhB@poly-Cy-DA-MSNs. The target drug release system was designed with dualresponse property, resulting from disulfide bond and dopamine moiety of Cy-DA. The responsive releasing behavior of disulfide bond in RhB@poly-Cy-DA-MSNs was investigated upon different concentrations of GSH, which could lead to breakage of disulfide bond through redox. The relationship between the amount of the released rhodamine dye and the GSH concentration is shown in Figure 4a. It was obviously observed that very small amount of RhB was released when the solution was rotated for 96 h in phosphate buffer saline (PBS) (pH = 7.49) without GSH, indicating the efficient confinement of RhB to the MSNs pores led by poly-Cy-DA coating. While GSH was added, especially at a concentration of 20 mM, the disulfide bonds of the poly-Cy-DA coating were cleaved more rapidly and the release of drug model accelerated, giving rise to an increasing concentration of RhB in the solutions. The poly-Cy-DA coating was essentially a peptide mimetic polymer produced by self-polymerization from a pseudo tripeptide Cy-DA; therefore, this peptide mimetic polymer poly-Cy-DA can be degraded when exposed to pepsin. To determine the pepsin-induced degradation behavior of the poly-Cy-DA coating, release experiments were conducted for different concentrations of pepsin at pH = 1.7. The relationship between the amount of the released RhB and the concentration of pepsin is shown in Figure 4b. In the absence of pepsin, only very small amounts of the dye were released into the solution when it was shaken for 96 h in PBS (pH = 1.7), which manifested the efficient confinement of the dye to the MSN pores resulted from covered poly-Cy-DA coating. As the pepsin concentration increased, the amido bond of poly-Cy-DA coating was cleaved more rapidly, leading to accelerated model drug release. It was interesting that the released amount of RhB in PBS at pH 1.7 was higher than that at pH 7.4. This phenomenon was due to the hydroxyl groups of MSNs reacted with catechol units of dopamine to form a complex compound, which could also be cleaved by H+. When the nanocarrier was exposed to the GSH environment, it showed excellent drug release property with a release efficiency
Table 1. EDX Results of MSNs and Poly-Cy-DA-MSNs MSNs element CK Si K OK NK SK
poly-Cy-DA-MSNs
weight percentage
atomic percentage
weight percentage
atomic percentage
23.53 40.27 36.19
34.64 25.35 40.00
14.96 48.55 26.07 0.53 9.90
38.24 34.92 32.92 0.76 6.24
The results presented above demonstrated the successful in situ polymerization of Cy-DA on MSN surfaces. We further investigated the loading capability of the as-prepared RhBloaded RhB@poly-Cy-DA-MSNs via the one-pot strategy. Figure 2a shows powder X-ray patterns of the solid MSNs and RhB@poly-Cy-DA-MSNs. Compared with MSNs, which showed typical MCM-41 characteristic peaks, RhB@poly-CyDA-MSNs presented decreased intensity at those peaks corresponding to d100, d110, and d200, respectively, caused by MSNs being filled with rhodamine molecules and covered by poly-Cy-DA coating.47 In addition, Figure 2b presents the thermogravimetric analysis (TGA) curves, which could reflect the amount of polymer and loaded RhB. RhB@poly-Cy-DAMSNs yielded a weight loss of approximately 5.5% from 25 to 300 °C, which was attributed to desorption of physically adsorbed water. The decline in the temperature range from 300 to 800 °C could be ascribed to the decomposition of the poly-Cy-DA coating on the MSN surface and loaded rhodamine, whereas the MSNs exhibited an obvious lower weight loss. The TGA results also indicated that this novel drug carrier presented high thermal stability that decomposition started at a temperature higher than 300 °C. The N2 adsorption−desorption isotherms of MSNs and RhB@poly-Cy-DA-MSNs are shown in Figure 3. It was observed that the MSNs and RhB@poly-Cy-DA-MSNs
Figure 2. (a) X-ray diffraction (XRD) patterns and (b) TGA curves of MSNs and RhB@poly-Cy-DA-MSNs. 6099
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Figure 3. Nitrogen adsorption−desorption isotherms for MSNs and RhB@poly-Cy-DA-MSNs: (a) BET curves and (b) pore size distribution.
Table 2. Summary of Porous Properties of MSNs and RhB@ poly-Cy-DA-MSNs sample MSNs RhB@poly-Cy-DA-MSNs
SBET/(m2 g−1) pore size (nm) 689.20 453.41
3.99 3.05
Scheme 2. Construction of Dual-Responsive MSN-Based Drug Delivery System
Vt (cm3 g−1) 0.59 0.48
the solution of PBS pH = 7.4, PBS pH = =7.4 with 20 mM GSH (6.6 mmol/mL), PBS pH = 1.7, and PBS pH = 1.7 with 40 U pepsin (13.3 μmol/mL) for 72 h, respectively. X-ray photoelectron spectroscopy (XPS) was first used to investigate the chemical compositions of glass surfaces before and after poly-Cy-DA coating deposition. Table 3 summarizes Table 3. Surface Atom Percentages of the Glass and Cy-DA Coating on the Glass atom percentage (100%)
Figure 4. In vitro releasing curves of RhB from RhB@poly-Cy-DAMSNs at different concentrations of (a) GSH (1.6 mmol/mL, 6.6 mmol/mL) and (b) pepsin (3.3 μmol/mL, 13.3 μmol/mL).
sample
C 1s
O 1s
N 1s
S 2p
glass poly-Cy-DA
52.05 71.03
45.39 20.86
2.36 5.74
2.37
the surface atom percentages. As Figure 5a shows, after polyCy-DA deposition, the C 1s content of poly-Cy-DA- modified glass decreased to 52.5% from 71.2%, whereas the N 1s and S 1s percentages were enhanced from 2.36 to 5.74% and from 0 to 2.37%, respectively. Figure 5b exhibits the component peaks for glass surfaces at 284.9, 286.1, and 288.2 eV, corresponding to carbon species of C−C, C−O, and CO, respectively. After coated with poly-Cy-DA (Figure 5c), the XPS spectrum showed component peaks at 284.6, 285.2, 286.2, 286.8, 288.0, and 288.8 eV, corresponding to carbon species of C−C, C−S, C−N, C−O, CO, and O−CO, respectively.46,50 In the S 2p spectrum of poly-Cy-DA (Figure 5d), the S 2p3/2 (163.6 and 164.8 eV) and 2p1/2 (164.3 and 165.5 eV) spin−orbit levels with an energy separation of 1.2 eV were attributed to S−S bond and S−O species, respectively.51 The other small peaks at 167.9 and 169.2 eV were ascribed to the sulfate species which were formed by the oxidation of sulfur in air.
over 70% at less than 100 h under 20 mM GSH condition (Scheme 2). The drug release behavior was effectively accelerated upon increased concentration of both GSH and pepsin. These release experiment results proved that the as-prepared RhB@ poly-Cy-DA-MSNs via the one-pot strategy were endowed with obvious dual-sensitive property to GSH and pepsin. In addition, according to reports, the GSH concentration in tumor cells is 4 times that in normal cells (0.5−10 mM).48,49 Therefore, the release performance upon GSH demonstrated that poly-Cy-DA-MSN is very suitable for cancer therapy. The model drug release behavior was further investigated via a visual simulation experiment. Four glass slides were soaked into 150 mL of Tris 8.5 with 5 mg/mL of Cy-DA. As Figure 6a shows, a yellow coating was deposited on the glass slides after 24 h, followed by being washed three times with Tris 8.5 solution. Then, the four glass slides were put into beakers with 6100
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Figure 5. (a) XPS spectra of clean glass and poly-Cy-DA coating on glass; (b,c) C 1s peak-fitting curves of clean glass and poly-Cy-DA-glass; and (d) S 2p peak-fitting curves of poly-Cy-DA-glass.
These XPS results indicated that poly-Cy-DA was successfully deposited on glass surfaces. Furthermore, from Figure 6b, compared with the blank sample (Figure 6b1), the yellow coatings faded away upon solutions containing GSH and pepsin, demonstrating the responsive feature of poly-Cy-DA upon GSH and pepsin. The
above results further suggested that the poly-Cy-DA coating could retain RhB molecules in MSN pores and exhibit redoxresponsive, pepsin-inspired, and pH-responsive behavior. Thereby, it was supposed that this DDS is promising not only for cancer therapy but also for stomach therapy. Cytotoxicity Assay. The in vitro cell cytotoxicity of MSNs and poly-Cy-DA-MSNs to normal Marc-145 cells was investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Figure 7a shows that after incubation for 24 h, both MSNs and poly-Cy-DA-MSNs showed no obvious cytotoxic effect against Marc-145 cells at 0.5 μg/mL. The cell viability was about 82% after 24 h of incubation, even when the concentration of poly-Cy-DA-MSNs was high up to 50 μg/mL. These results indicated that both MSNs and polyCy-DA-MSNs were nontoxic at low concentrations and almost nontoxic even at high concentrations. Thereby, the as-prepared poly-Cy-DA-MSNs were highly biocompatible and promising candidates as drug delivery nanocarriers in CDDS. For DOX@poly-Cy-DA-MSNs, DOX was stored in the MSN pores through capping of poly-Cy-DA coating by this one-pot strategy. Figure 7b shows the in vitro cellular cytotoxicity of DOX and DOX@poly-Cy-DA-MSNs to HeLa cancer cells at different concentrations. It was observed that with increasing concentrations of DOX and DOX@poly-CyDA-MSNs, the cell viability of HeLa cells dramatically decreased. Similar to DOX, DOX@poly-Cy-DA-MSN was also effective at killing the cancer cells but with a lower efficiency, which might result from the slow sustained DOX releasing from DOX@poly-Cy-DA-MSNs triggered by the GSH condition of cancer cells. The cellular uptake of MSNs and DOX@poly-Cy-DA-MSNs by HeLa cells was further investigated by CLSM (Figure 8) and a fluorescence microscope (Figure S6). After the HeLa cells were cultured at 37 °C for 24 h, DOX@poly-Cy-DAMSNs were then added into the culture medium with 20 μg/
Figure 6. (a) Glass slides deposited with yellow poly-Cy-DA coating; (b) glass slides with yellow poly-Cy-DA coating after being soaked into different solutions for 72 h: (1) PBS pH = 7.4, (2) PBS pH = 7.4 with 20 mM GSH (6.6 mmol/mL), (3) PBS pH = 1.7, and (4) PBS pH = 1.7 with 40 U pepsin (13.3 μmol/mL). The blue lines in the picture are the liquid level indicators. 6101
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Figure 7. (a) Cell viabilities of Marc-145 cells after incubated with different concentrations of MSNs and poly-Cy-DA-MSNs for 24 h; (b) cell viabilities of HeLa cells after cultivated with different concentrations of DOX and DOX@poly-Cy-DA-MSNs for 24 h [data are expressed as the average ±standard deviation (n = 3)].
ness of poly-Cy-DA coating through adjusting the reaction time, concentration of poly-Cy-DA, and pH value. The in vitro cellular cytotoxicity tests showed that poly-Cy-DA-MSNs are nontoxic, highly biocompatible, and suitable to be employed as drug nanocarriers in CDDS. In addition, CLSM results demonstrated that DOX@poly-Cy-DA-MSNs could be taken up by the cells through endocytosis mechanism and the DOX was efficiently released in endocytic compartments with a high GSH concentration and a low pH value. Therefore, it is believed that the MSN/functionalized dopamine-based dualresponsive DDS is a potential practical nanocarrier for cancer therapy and stomach diseases.
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EXPERIMENTAL SECTION Materials. n-Cetyltrimethylammonium bromide, tetraethoxysilane, DOX, rhodamine, Boc-L-cystine-OH, N-hydroxysuccinimide (NHS), dopamine HCl (DA-HCl), and 1-ethyl3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCHCl) were purchased from Adamas and used without further purification. Anhydrous ether, tetrahydrofuran (THF), ethyl acetate (EA), methanol, and triethylamine (TEA) were distilled under reduced pressure and dried before use. HCl and NaHCO3 were purchased from Sinopharm Chemical Reagent Co. RPMI-1640, fetal bovine serum (FBS), penicillin, and streptomycin were purchased from Gibco. Measurements. The NMR spectra were recorded at room temperature (rt) by a Mercury Plus 400 (Varian, Inc., USA) NMR spectrometer in methanol-d with the internal reference of tetramethylsilane (δ = 0). The surface area was measured by the BET method, and the pore size was calculated from the maxima of the pore size distribution curve carried out by the Barrett−Joyner−Halenda method using the adsorption branch of the isotherm. A Shimadzu-Kratos (AXIS Ultra) was used to measure the surface chemical composition. The XPS measurements were carried out at a base pressure of about 5 × 10−10 mbar using an Mg X-ray (1253.6 eV) source. A PerkinElmer (LAMBDA 35) UV−vis spectrometer was employed to measure the UV−vis spectrum. All measurements were conducted in quartz cuvettes. PBS solutions of pH 7.4 and pH 1.7 were used as blank samples. Powder XRD patterns were recorded by an X-ray diffractometer (D/MAX-2200/PC, Rigaku) equipped with Cu Kα radiation (40 kV, 20 mA) at a rate of 1.0°/min over a range of 0−6° (2θ). The morphology studies of nanoparticles were performed on a JEOL 2010F analytical electron microscope at 200 kV (TEM). The fluorescent images were taken on a confocal laser scanning microscope (LSM510 META, ZEISS).
Figure 8. CLSM images of cellular uptake of DOX@poly-Cy-DAMSN (20 μg/mL) and their intracellular drug release behavior after incubation with HeLa cells for 6, 12, and 24 h.
mL of DOX concentration. After the nucleus was stained with DAPI, the pretreated cells were observed using CLSM. As presented in Figure 8, very weak fluorescence was observed after 6 h. With a prolonged incubation time, the blue and red fluorescence became stronger, indicating more and more DOX release. After incubation for 24 h, the red fluorescence of DOX was observed mainly in the cytoplasm but also in the nucleus. The fluorescence microscope results of DOX@poly-Cy-DAMSNs showed the same results as CLSM images. These results indicated that DOX@poly-Cy-DA-MSNs could be taken up by HeLa cells through endocytosis mechanism and release DOX drug exposed to high GSH concentration and low pH environment in cytoplasm. Compared with our previous only enzyme-responsive nanocarrier system,43 this developed dualresponsive DOX@poly-Cy-DA-MSNs did not need pretreatment of pepsin to HeLa cells in cellular uptake experiments. Figure 8 shows that without pepsin treatment, DOX can also be released efficiently in endocytic compartments with an environment of low pH value and high GSH concentration of HeLa cells.
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CONCLUSIONS In summary, a novel dual-responsive (enzyme- and redox-) drug delivery nanosystem based on MSNs was prepared from Cy-DA via the facile one-pot strategy. The RhB and DOX as model drugs could be loaded efficiently into the MSN pores. The as-prepared RhB/DOX@poly-Cy-DA-MSN was proved responsive to GSH and pepsin due to the disulfide and peptide bonds in poly-Cy-DA chains. The releasing speed of RhB/ DOX@poly-Cy-DA-MSNs could be controlled by the thick6102
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Synthesis of Cy-DA. Cy-DA was synthesized from cystine and dopamine. The detailed synthesis procedures are shown in Scheme 1 and the synthesis details were presented as follows. Synthesis of (Boc)2-Cystine-NHS [(Boc)2-Cy-NHS]. (Boc)2-L-cystine (0.005 mol) was dissolved in 100 mL of THF and dichloromethane (DCM) mixture solvent and then 0.015 mol of NHS was added into the flask (NHS in excess). EDC-HCl (0.015 mol) was dissolved in DCM at 0 °C (2 mol equivalent relative to NHS). About 30 min later, the reaction was warmed to rt and allowed to proceed for another 6 h under nitrogen atmosphere. Finally, a white precipitate was collected after filtration, thoroughly washed three times with deionized water to remove excess NHS and EDC-HCL, and dried in vacuum at 60 °C overnight. The yield of (Boc)2-Cy-NHS was about 62.9%. The product cannot be characterized by NMR because it is difficult to dissolve in the solvent. Synthesis of (Boc)2-Cy-DA [(Boc)2-Cy-DA]. DA-HCl was dissolved in 100 mL of methanol (dopamine-HCl in excess). The synthesized (Boc)2-Cy-NHS was added into a flask. Then, TEA was added into the solution (at a mole ratio of dopamineHCl/TEA/BCN = 3:3:1). The reaction proceeded for 6 h at rt under nitrogen atmosphere. Then, the reaction mixture was precipitated in 500 mL of 1 mol/L HCl solution. After precipitating in methanol/HCL two more times, the solid product was dried in vacuum and 5.7 g of light pink solid was obtained with a yield of about 71%. The 1H NMR of (Boc)2-Lcystine is shown in Figure S1. 1H NMR (400 MHz, CD3ODd4): δ (ppm) 6.67−6.52 (m, 3H, −Ar), 4.4 (m, 1H, −CH (COOH)−CH2−), 3.21−2.95 (m, 2H, −CH (COOH)− CH2), 1.45 (S, 9H, Boc). The organic element analyzer was employed to measure the elements N, S, and C of (Boc)2-Cy-DA. As shown in Table S1, the element measurement results were consistent with the theoretical results. All the results above proved that (Boc)2-CyDA was synthesized successfully. Synthesis of Cy-DA. The (Boc)2-Cy-DA synthesized above was dissolved in 50 mL of EA. After 100 mL of HCl/ EA solution was added, the reaction was conducted for 4 h at rt under nitrogen atmosphere. After reaction, the organic solvent was removed and the collected product was dried in vacuum at 60 °C for 24 h. The final product is a bis-HCl of CyDA with a yield of about 92%. The product was characterized by 1H NMR and 13C NMR as shown in Figure S2. 1H NMR (400 MHz, CD3OD-d4): δ (ppm) 4.37 (m, 1H, −CH(CONH)−CH2−), 3.35 (m, 2H, −CONH−CH2−CH2), 3.01−2.82 (m, 2H, −CH(CONH)−CH2), 2.65 (m, 2H, −CONH−CH2−CH2−), 13C NMR (400 MHz, CD3OD-d4): δ (ppm) 167.31 (−CO−NH−), 115.58−144.8 (−AR−), 51.92 (NH2−CH (−CH2)−), 41.51 (NH2−CH (−CH2)−), 38.25 (−NH−CH2−CH2−), 34.45 (−NH−CH2−CH2−). The 1H NMR and 13C NMR results proved that Cy-DA was successfully synthesized. ES MS (m/z): [M + H]+ found, 511.63. One-Pot Strategy Fabricated Drug-Loaded Poly-CyDA Film Coated MSNs. MSNs of MCM-41 were prepared according to a typical method.44 Fluorescein dye rhodamine B or DOX as a model drug (4 mg) was dissolved in 25 mL of Tris 8.5. After MCM-41 (200 mg) was added in the solution, the mixture was treated by ultrasonic treatment for 30 min and stirred for about 24 h at 25 °C. After 0.25 g of Cy-DA was added, the reaction system was stirred for another 24 h at 25 °C. The precipitate was collected by centrifugation and washed with PBS solution (pH = 7.4) to remove the redundant dye or
DOX and poly-Cy-DA on RhB@poly-Cy-DA-MSNs or DOX@poly-Cy-DA-MSNs surfaces. The drug loading efficiency (DLE) and drug loading content (DLC) could be calculated by the following formula. The relative DLE and DLC are about 85 and 1.67% for RhB and about 88 and 1.74% for DOX, respectively. DLE % =
m(orginal RhB or DOX) − m(residual RhB or DOX) m(orginal RhB or DOX) × 100%
DLC % =
m(orginal RhB or DOX) − m(residual RhB or DOX) m(orginal RhB or DOX) + m(MSNs) × 100%
In Vitro Drug Release. To investigate the in vitro drug release behavior, 50 mg of RhB@poly-Cy-DA-MSNs was put into a dialysis bag and sealed and then put into a brown bottle with 100 mL of PBS solution with different concentrations of GSH and pepsin. The brown bottle was placed in a 37 °C constant temperature shock chamber and shaken at a speed of 100 rpm. At certain time intervals, 3 mL of the release medium was fetched to check the released drug concentration and then put back into the brown bottle. To measure the released RhB amount, the absorbance at 559 nm was recorded by a LAMBDA 35 UV−vis spectrometer (PerkinElmer Instruments) via an absorption spectrophotometer. Cell Culture. Hela (human cervix carcinoma cell line) and Marc-145 cells were incubated in RPMI-1640 containing 10% FBS (heat-inactivated FBS at 56 °C for 30 min) and antibiotics (50 units/mL streptomycin and 50 units/mL penicillin) at 37 °C under a humidified atmosphere containing 5% CO2. Cytotoxicity Assay. In order to investigate the in vitro cell assay, cancer drug DOX was loaded. The biocompatibility of the developed formulation was evaluated in normal cells like Marc-145 cells. Cancer cells (HeLa cells) were employed to evaluate the drug release and cancer cell-killing efficiency of this developed DDS. Briefly, HeLa/Marc-145 cells were seeded into 96-well plates at a concentration of 5 × 105 cells/mL in 100 μL of RPMI-1640 culture medium supplemented with 10% FBS, 100 μg/mL streptomycin, and 100 units/mL penicillin. After adding 100 μL of culture medium, the total volume was 200 μL. Cells were incubated for 24 h at 37 °C under a humidified atmosphere containing 5% CO2. DOX@ poly-Cy-DA-MSNs and poly-Cy-DA-MSNs were added with different concentrations into triplicate wells for each sample. Then, the cells were further cultured for another 24 h. Afterward, the incubated cells were washed and replenished with a fresh culture medium and further incubated for 2 h, followed by adding MTT to cells at a final concentration of 0.5 mg/mL. Then, 4 h of incubation of cells with MTT at 37 °C under 5% CO2 atmosphere was carried out. Dimethylformamide (100 μL) solution was added to each well to dissolve the resulting formazan crystals. The absorbance at 570 nm was measured by a microplate reader (BIO-RED). In Vitro Activity Analysis. HeLa cells were seeded at a concentration of 2 × 105 cells/mL in 2 mL of RPMI-1640 plus 10% FBS culture medium in a 35 mm × 10 mm Petri dish (corning). HeLa cells were allowed to adhere to the Petri dish for 24 h. The culture media were replaced with pH 7.4 RPMI1640 plus 10% FBS with 1% penicillin and streptomycin before particles were added. DOX@poly-Cy-DA-MSN (100 μL) with 6103
DOI: 10.1021/acsomega.8b02537 ACS Omega 2019, 4, 6097−6105
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a concentration of 20 μg/mL was added to each Petri dish. After incubation for 0, 6, 12, and 24 h, the cells were washed with PBS three times and investigated under CLSM (Zeiss, LSM-510).
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.8b02537. 1 H NMR spectrum and element analysis results of (Boc)2-Cy-DA; NMR and TOF-MS spectra of Cy-DA; propose cross-link of poly-Cy-DA; EDX spectrum of CyDA-MSNs; water contact angle of glass and poly-CyDA-modified glass; and fluorescence microscope of DOX@poly-Cy-DA-MSNs (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Xinling Wang: 0000-0001-7158-5737 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors are grateful for the project support from the National Science Fund of China (21274085) and the Shanghai Leading Academic Discipline Project (no. B202).
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