Mesoporous Silica Nanoparticles Capped with Graphene Quantum

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Mesoporous Silica Nanoparticles Capped with Graphene Quantum Dots for Potential Chemo-Photothermal Synergistic Cancer Therapy Xianxian Yao, Zhengfang Tian, Jiaxing Liu, Yufang Zhu, and Nobutaka Hanagata Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b04189 • Publication Date (Web): 21 Dec 2016 Downloaded from http://pubs.acs.org on December 25, 2016

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Mesoporous Silica Nanoparticles Capped with Graphene Quantum Dots for Potential Chemo-Photothermal Synergistic Cancer Therapy

Xianxian Yaoa1, Zhengfang Tianb1, Jiaxing Liua, Yufang Zhua,b*, Nobutaka Hanagatac*

a

School of Materials Science and Engineering, University of Shanghai for Science and

Technology, 516 Jungong Road, Shanghai 200093, China. Email: [email protected] b

Hubei Key Laboratory of Processing and Application of Catalytic Materials, College

of Chemical Engineering, Huanggang Normal University, Huanggang 438000, China c

Nanotechnology Innovation Station, National Institute for Materials Science, 1-2-1

Segen, Tsukuba, Ibaraki 305-0047, Japan. Email: [email protected] 1

the first two authors contributed equally to this work.

1

ACS Paragon Plus Environment

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Abstract: In this study, mesoporous silica nanoparticles (MSNs) have been successfully capped with graphene quantum dots (GQDs) to form multifunctional GQD-MSNs with the potential for synergistic chemo-photothermal therapy. The structure, drug release behavior, photothermal effect, and synergistic therapeutic efficiency of GQD-MSNs to 4T1 breast cancer cells were investigated. The results showed that GQD-MSNs were monodisperse and had a particle size of 50–80 nm. Using doxorubicin hydrochloride (DOX) as a model drug, the DOX-loaded GQD-MSNs (DOX-GQD-MSNs) not only exhibited pH and temperature-responsive drug release behavior, but using near-infrared (NIR) irradiation, they efficiently generated heat to kill cancer cells. Furthermore, GQD-MSNs were biocompatible and were internalized by 4T1 cells. Compared with chemotherapy and photothermal therapy alone, DOX-GQD-MSNs were much more effective in killing 4T1 cells due to a synergistic chemo-photothermal effect. Therefore, GQD-MSNs may have promising applications in cancer therapy.

Keywords: Mesoporous silica, Graphene quantum dots, Controlled drug release, Photothermal effect, Synergistic therapy

2


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Introduction Mesoporous silica nanoparticles (MSNs) are widely considered to be efficient drug delivery nanocarriers due to their biocompatibility, high drug loading capacity, and easily controlled drug release [1-4]. To date, a variety of studies have demonstrated MSN-based controlled drug delivery systems using nanoparticles, supramolecular assembles, and biomolecules as “gate-keepers”, because these “gate-keepers” can respond to different stimuli including pH, temperature, light, redox activation, competitive binding, and enzymes [5-16]. A MSN-based controlled drug delivery system may enhance therapeutic efficiency and decrease side-effects of toxic anticancer drugs when compared to systemic administration [17], but chemotherapy with controlled drug delivery still cannot achieve optimal therapeutic efficacy due to unavoidable multidrug-resistance of cancer cells [18]. Recently, there have been multiple attempts to construct multifunctional MSNs for the combination of chemotherapy with other therapeutic modalities including photothermal therapy, magnetic hyperthermia, gene therapy, and radiotherapy [19-28]. Among them, photothermal therapy is a recently developed technique based on photothermal agents that strongly absorb near-infrared (NIR) light and convert it to heat to destroy tumor cells. Due to local treatment, this is a non-invasive and highly efficient therapeutic modality [29]. Therefore, it is likely that MSNs functionalized with photothermal agents have the potential for controlled drug delivery and photothermal therapy, and can thereby achieve synergistic effects of both therapeutic modalities to enhance therapeutic efficacy and decrease 3


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multidrug-resistance of cancer cells. To date, much effort has been made to functionalize MSNs with photothermal agents, such as Au, CuS nanoparticles, and carbon dots (C-Dots), for biomedical applications in controlled drug delivery, photothermal therapy, and imaging [30-36]. For example, Wang et al. prepared uniform Janus Au-mesoporous silica nanoparticles with a superior surface plasmon resonance wavelength and high surface area, and doxorubicin hydrochloride (DOX)-loaded Janus nanoparticles were more toxic to liver cancer cells by NIR irradiation when compared to chemotherapy or photothermal therapy alone [30]. Zhou et al. reported the use of biocompatible C-Dots as caps to attach the outlets of MSNs for the design of intelligent on-demand drug delivery and as an optical imaging system, which exhibited pH-sensitive drug delivery and the potential for noninvasive tracking of the therapeutic agent delivery in vivo [31]. Graphene and its derivatives are flexible two-dimensional carbon nanosheets, and are new biomaterials used in biomedical applications due to their novel physical properties, chemical modulatability, good biocompatibility, and highly functional surface [37-38]. Graphene quantum dots (GQDs) maintain the intrinsic layered structure of graphene, but with a smaller lateral size; they contain abundant carboxylic and hydroxyl groups, which are useful for surface functionalization and biomedical applications [39]. In addition, GQDs can effectively convert NIR light energy into heat, potentially acting as a photothermal agent [40]. Recently, several studies have demonstrated the functionalization of MSNs with graphene, graphene 4


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oxide (GO), and GQDs for biomedical applications [41-47]. For example, Tang et al. reported an aptamer-targeting photoresponsive drug delivery system by non-covalent assembly of a Cy5.5-AS1411 aptamer conjugation on the surface of GO-wrapped MSNs. Drug release could be triggered by laser irradiation due to the expansion of GO sheets and the vibration of GO and MSNs caused by the local temperature increase [41]. Wang et al. constructed a targeting peptide-modified mesoporous silica-coated graphene sheet with the potential for synergistic chemo-photothermal therapy, which showed heat, pH-responsive, sustained release behavior, and efficient heat transformation from NIR light [42]. Chen et al. capped luminescent GQDs onto the nanopores of MSNs through an acid-cleavable acetal bond, and the GQDs-capped MSNs (GQD-MSNs) could be used for intracellular drug delivery and simultaneous cell imaging [43]. Therefore, it can be speculated that GQD-MSNs would be promising for synergistic chemo-photothermal therapy due to MSNs function as a nanocarrier for controlled drug release, and GQDs function as photothermal agents for photothermal therapy. However, previous studies have revealed that the construction strategies of GQD-MSNs are complicated and not easy to control. In addition, there are only a few reports describing the potential application in synergistic chemo-photothermal therapy. In this study, the MSNs have been successfully capped with GQDs to form multifunctional GQD-MSNs by the simple interaction between the carboxylic and hydroxyl groups of GQDs and amino groups on MSNs (Scheme 1). The hydrogen bonds and electrostatic force between GQDs and MSNs are relatively weak and are 5


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easily disrupted when the pH value decreases [19]. Thus, the pH-sensitivity of GQDs capping may contribute to controlled drug release from GQD-MSNs. In addition, capping with GQDs provides GQD-MSNs with a photothermal effect. Furthermore, in vitro cytotoxicity, cell uptake, and synergistic therapeutic efficiency of the GQD-MSNs were investigated using 4T1 cells, a breast cancer line as a model cellular system.

Experimental methods Materials Hexadecyltrimethylammonium 3-aminopropyltriethoxysilane

p-toluenesulfonate

(APTES)

were

purchased

(CTAT) from

and

Sigma-Aldrich.

Tetraethyl orthosilicate (TEOS), triethanolamine (TEA), N-Hydroxysuccinimide (NHS), 1-ethyl-3-(3-dimethylaminopropyl)

carbodiimide

hydrochloride

(EDC),

and

methylbenzene were obtained from Sinopharm Chemical Reagent Co. Ltd. HEPES buffer, DOX, and PBS (pH 7.4) were bought from Sangon Biotech (Shanghai) Co., Ltd. Graphene quantum dots (GQDs, 1 mg/ml) were purchased from Nanjing XFNANO Co. Ltd. Ultrapure water was obtained from Millipore pure water system. All chemicals were of analytical-reagent grade and used without further purification. Synthesis of GQD-MSNs and DOX-loaded GQD-MSNs MSNs were synthesized using a sol–gel process according to a previously reported method with some modifications [48]. Generally, 1.71 g of CTAT and 1.0 g of TEA were added into 150 ml of H2O at 80 °C for 30 min and stirred using a magnetic stir bar. Subsequently, 10 ml of TEOS was added into the mixed solution 6


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and incubated at 80 °C for an additional 2 h. Then, the white precipitates were collected by centrifugation, washed several times with H2O, and dried in vacuum at 60 °C for 12 h. Finally, MSNs were obtained after calcination of the dried nanoparticles at 650 °C for 6 h to remove the surfactant. To modify MSNs with amino groups, 0.5 g of MSNs were homogeneously dispersed in 80 ml of methylbenzene at 100 °C for 2 h and stirred using a magnetic stir bar. Subsequently, 0.75 ml of APTES was added to the MSN suspension and slowly stirred for 20 h at 120 °C. The treated nanoparticles were collected by centrifugation, and were extensively washed with methylbenzene to remove the unreacted APTES. Finally, the aminated MSNs (MSN–NH2) were obtained after drying in vacuum at 60 °C for 12 h. To cap GQDs on MSN-NH2, GQDs (20 ml, 1mg/ml) were activated by EDC (654 μl, 2.8 mM) and NHS (132 μl, 2.8 mM) aqueous solution. Subsequently, 20 mg of MSN-NH2 was slowly added into the activated GQDs solution. After the reaction mixture was incubated at 4 °C by agitation overnight in dark conditions, GQDs-capped MSN-NH2 (GQD-MSNs) were collected by centrifugation and washed with H2O to remove residual EDC, NHS, and GQDs. Finally, GQD-MSNs were lyophilized and stored at 4 °C. To synthesize DOX-loaded GQD-MSNs (DOX-GQD-MSNs), DOX was loaded into MSN-NH2 before GQDs capping on MSNs. Generally, MSN-NH2 (20 mg) was dispersed in a DOX solution in PBS (10 ml, 0.5 mg/ml). After 24 h of agitation in dark conditions, DOX-MSN-NH2 was collected by centrifugation, and was washed twice 7


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with PBS to remove DOX adsorbed on the surface of MSN-NH2. To estimate the DOX loading capacity, all supernatants were collected and the residual DOX amount was measured by UV-Vis analysis at 481 nm. First, a calibration curve was recorded using a NanoDrop 2000C spectrophotometer. Then, GQDs were capped on the DOX-MSN-NH2 complex to form DOX-GQD-MSNs using the same process for GQDs capping on MSN-NH2 . Characterization The wide-angle X-ray diffraction (WAXRD) patterns were obtained on a D8 ADVANCE powder diffractometer using Cu Kα1 radiation (1.5405 Å). Scanning electron microscopy (SEM) was carried out using an FEI Quanta 450 field emission scanning electron microscope. Transmission electron microscopy (TEM) images were obtained using a Tecnai G2 F30 transmission electron microscope at an acceleration voltage of 300 kV. Fourier transform infrared (FTIR) spectra were recorded on a LAM750(s) spectrometer in transmission mode. N2 adsorption–desorption isotherms were obtained on a Micromeritics Tristar 3020 automated surface area and pore size analyzer. Brunauer–Emmett–Teller (BET) and Barrett–Joyner–Halenda (BJH) methods were used to determine the surface area, the pore size distribution and the pore volume. UV–Vis absorption spectra were measured on a NanoDrop 2000C spectrophotometer. Thermogravimetric analysis (TGA) was performed on STA 449 F3 thermal analyzer with N2 atmosphere with a flow rate of 20 ml/min and a heating rate of 10 °C/min. The photothermal effect of the samples was measured on an infrared thermal imaging system with a diode laser (808 nm). 8


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Drug release from DOX-GQD-MSNs To investigate the DOX release behavior from DOX-GQD-MSNs, 10 mg of DOX-GQD-MSNs was dispersed in 5 ml PBS with pH 5.0 or pH 7.4 by shaking in dark conditions. The temperature of the PBS solution was maintained at 37 °C or 50 °C. After a predetermined time interval, the release suspension was centrifuged and an aliquot (3 μl) of supernatant was removed for UV-Vis analysis to estimate the amount of released DOX, and replaced with the same volume of fresh PBS for continuous drug release. Photothermal effect of DOX-GQD-MSNs Generally, DOX-GQD-MSNs were dispersed in water at a concentration of 0, 1, 2.5, 5, 10, and 20 mg/ml, respectively. Subsequently, 1 ml of the DOX-GQD-MSN suspension in each well of a 96-well plate was irradiated for 10 min by a diode laser (808 nm) with a power of 2.5 W/cm2 at a distance of 1.0 cm. During irradiation, an infrared thermometer was used to record the temperature change of the DOX-GQD-MSNs suspension every 30 s. The temperature changes were transferred to a computer through an optical fiber and recorded as heating curves. The infrared thermal images of the DOX-GQD-MSNs suspension were recorded every 2 min. Cell culture The 4T1 breast cancer cell line was cultured in Dulbecco’s modified Eagle’s medium (DMEM) and supplemented with 10 % fetal bovine serum (FBS), 100 units/ml penicillin and 100 mg/ml streptomycin. Cells were incubated in 5 % CO2 atmosphere at 37 °C. 9


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In vitro cytotoxicity of GQD-MSNs In vitro cytotoxicity of GQD-MSNs was evaluated using a Cell Counting Kit-8 (CCK-8) assay. 4T1 cells were seeded into a 96-well plate at a density of 5 × 104 cells per well. After the cells were cultured for 12 h the medium was removed and the GQD-MSNs suspension (in DMEM) was added to the 96-well plate. The final concentrations of GQD-MSNs were 0, 25，50, 100, and 200 μg/ml respectively, and the final medium volume in each well was 100 μl. After the cells were incubated for 24 h, 10 μl of CCK-8 solution was added into each well, and the cells were incubated for an additional 2 h. The absorbance at 450 nm was then measured using a microplate reader (ELX800 BioTek). Cytotoxicity was expressed as the percentage of viable cells compared with that of untreated control cells. Cell uptake of DOX-GQD-MSNs For the investigation of cell uptake of DOX-GQD-MSNs, 1 × 105 4T1 cells were seeded in a 35 mm Petri dish with a glass bottom for 12 h to allow cell attachment to the bottom. Subsequently, the cells were washed with PBS twice, and 1.5 ml of DOX-GQD-MSNs suspension (100 μg/ml in DMEM) was added to the Petri dish. After incubation

of

cells

for

4

h,

1

ml

of

methanolic

solution

of

4',6-diamidino-2-phenylindole (DAPI, 1.5 μg/ml) was added into the Petri dish and incubated at 37 °C for 15 min to stain the nuclei. Then the cells were washed thrice with PBS to remove residual nanoparticles and dead cells. Finally, the cells were observed using a confocal laser scanning microscope (CLSM, Olympus, FV 1000). Chemo-photothermal therapy of 4T1 cells by DOX-GQD-MSNs 10


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4T1 cells were seeded in a 96-well plate at a density of 5 × 104 cells per well. After cell culture for 12 h, the cells were washed with PBS twice, and the DOX–GQD-MSNs suspension (100 μg/ml in DMEM) was added to each well. As controls, a solution of free DOX and the GQD-MSNs suspension with the same concentrations as the DOX-GQD-MSNs suspension were used. After incubation of cells for 8 h, the cells in each well were irradiated for 3 min using an 808-nm laser at a distance of 1.0 cm and diode laser power of 2.5 W/cm2. After irradiation, the cells were cultured for 4 h and then observed via fluorescence microscopy. Then, 10 μl of CCK-8 solution was added into each well, and the cells were incubated for an additional 2 h to measure cell viability.

Results and discussion SEM and TEM images of the MSNs, GQD-MSNs, and DOX-GQD-MSNs are shown in Fig. 1. Similar to the previously reported results [43], MSNs were spherical and highly monodisperse. The particle size of the MSNs was in the range of 50–60 nm, and a dendritic mesoporous structure can be clearly observed on the MSNs. Particle size of MSNs measured by dynamic laser scatting (DLS) was relatively narrow, and the average particle size was estimated to be 84 nm (see the supporting information). On the other hand, both GQD-MSNs and DOX-GQD-MSNs were highly monodisperse, but a very thin layer of nanosheets was grafted on the surfaces of the nanoparticles and blocked the mesopores, which might be attributed to capping of the GQDs on MSNs. The average particle sizes of the GQD-MSNs and DOX-GQD-MSNs were 94 and 11


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95 nm respectively (see the supporting information), which is a little larger than that of MSNs suggesting GQDs grafting on MSN-NH2 and DOX-MSNs. From the elemental mapping of the GQD-MSNs (Fig. 1G), elemental C was homogenously distributed on GQD-MSNs, similar to the distributions of Si and O elements, suggesting capping of GQDs on MSNs. To further confirm the formation of the DOX-GQD-MSNs, WAXRD, FTIR spectra, N2 physisorption, and UV-Vis spectra were applied in this study. Fig. 2A shows WAXRD patterns of the MSN-NH2, DOX-MSN-NH2, GQD-MSNs and DOX-GQD-MSNs. Before capping with GQDs, MSN-NH2 and DOX-MSN-NH2 only showed a broad diffraction peak at 2θ = 15–30° on the WAXRD patterns, suggesting an amorphous structure. In contrast, for both GQD-MSNs and DOX-GQD-MSNs, a well-resolved diffraction peak at 2θ = 15–20° assigned to GQDs can be observed on the WAXRD pattern except for that of amorphous silica, which indicate that GQDs were capped onto MSN-NH2 and DOX-MSN-NH2. As shown on FTIR spectra (Fig. 2B), the bending vibration of N-H groups at the peaks of 1492 and 1560 cm−1, and the stretching vibration of C-H groups at the peaks of 2800–2900 cm−1 were clearly observed on the spectrum for MSN-NH2, indicating successful functionalization of amino groups on MSNs. For GQDs, there are several peaks at 1725 cm−1, 1630 cm−1, and 1200 cm−1, which are attributed to the C=O stretching vibration from the carbonyl, C-OH bending vibration, and C-O-C stretching vibration respectively, suggesting the potential to react with the amino groups on MSN-NH2. Moreover, the FTIR spectra of GQD-MSNs and DOX-GQD-MSNs exhibited a new stretching vibration of –CONH– at 12


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1580 cm−1, but the C=O stretching vibration from the carbonyl at 1725 cm−1 disappeared, which indicated capping of the GQDs on MSN-NH2 and DOX-MSN-NH2 due to the interaction between the carboxyl groups of GQDs and amino groups on MSN-NH2 and DOX-MSN-NH2. N2 physisorption results of the MSNs, MSN-NH2, DOX-MSN-NH2, and DOX-GQD-MSNs are shown in Fig. 3. Compared to MSNs, MSN-NH2 still exhibit type IV isotherm characteristics although the N2 adsorption amount decreased sharply, suggesting that MSN-NH2 maintained a mesoporous structure, but the surface area and pore volume decreased from 728 m2/g and 1.39 cm3/g to 390 m2/g and 0.82 cm3/g, respectively. Additionally, the mesopore size also decreased from 3.1 nm to 2.1 nm. However, DOX molecules may diffuse into the mesoporous channels of MSN-NH2 due to the small molecular size of DOX. After DOX loading and capping with GQDs, the N2 adsorption amount further decreased, and the surface area and pore volume decreased to 196 m2/g and 0.69 cm3/g for DOX-MSN-NH2 and 156 m2/g and 0.16 cm3/g for the DOX-GQD-MSNs, respectively. Furthermore, there was almost no pore size distribution in the mesoporous range, which might be attributed to DOX loading in the mesoporous channels and the capping effect of GQDs. Fig. 4A shows UV-Vis absorbance spectra of the GQDs, DOX, MSN-NH2, DOX-MSN-NH2 and DOX-GQD-MSNs suspensions. The absorbance peak at 481 nm for DOX-MSN-NH2 and DOX-GQD-MSNs is attributed to the characteristic absorbance of DOX, suggesting DOX loading in MSN-NH2 and GQD-MSNs. When the DOX concentration was 0.5 mg/ml, the DOX loading amount in the DOX-GQD-MSNs was 13


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estimated to be 48 μg/mg by UV-Vis analysis according to the different DOX concentrations in the initial solution and residual supernatant. As shown in Fig. 4B, TGA showed that the weight loss before 100 oC belongs to the evaporation of physically adsorbed H2O, and the weight loss at the temperature range between 200 and 250 °C for GQD-MSNs and DOX-GQD-MSNs is attributed to the decomposition of GQDs. Additionally, DOX-GQD-MSNs had a much higher weight loss (3.73 %) than GQD-MSNs except of the weight losses of physically adsorbed H2O and GQDs, suggesting DOX loading of DOX-GQD-MSNs. The DOX loading amount was calculated to be 43.5 μg/mg, which is close to the result by UV-Vis analysis. On the other hand, the DOX loading capacity increased with the increase of the DOX concentration, and the maximal DOX loading capacity of 55.8 μg/mg was achieved at a DOX concentration of 0.75 mg/ml (see the supporting information). Therefore, MSN-NH2 can load DOX and be capped by GQDs to form a multifunctional nanoplatform with the potential for synergistic chemo-photothermal therapy. To evaluate drug release behavior in response to pH and temperature changes, DOX-GQD-MSNs were dispersed in the release medium (pH 7.4 or 5.0) at 37 °C or 50 °C, respectively. Fig. 5 shows the cumulative DOX release from the DOX-GQD-MSNs under different conditions. DOX release from the DOX-GQD-MSNs was slow in the medium with a pH of 7.4, and only 7.4 % and 13.6 % of DOX were released within 24 h at 37 and 50 °C, respectively. In contrast, the DOX-GQD-MSNs exhibited a much faster DOX release rate when the pH of the release medium decreased to 5.0, and the released DOX achieved 48.6 % and 69.1 % at 37 and 50 °C, 14


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respectively. We conclude that the DOX-GQD-MSNs exhibited pH-dependent drug release behavior, which might be attributed to the weakening of hydrogen bonds and electrostatic interactions between DOX, GQDs, and MSNs when the pH value of the release medium decreases. However, the DOX release rate was a little higher at 50 °C than at 37 °C in media with either pH. This may be because the temperature increase weakened the interaction between DOX, GQDs, and MSNs, and thereby accelerated

DOX

release.

Therefore,

DOX-GQD-MSNs,

with

pH-

and

temperature-responsive drug release, may enhance the delivery efficiency. GQDs can convert NIR light energy into thermal energy [34]. Capping with GQDs endows the GQD-MSNs with photothermal effects upon NIR irradiation. As shown in Fig. 6A, the infrared thermal images of the GQD-MSNs suspension visually confirmed the rapid temperature increase by NIR irradiation within a short period. To investigate the photothermal effect of the GQD-MSNs, GQD-MSNs suspensions with different concentrations were irradiated by NIR (λ = 808 nm, 2.5 W/cm2). We observed that the NIR irradiation only slightly increased the temperature of pure H2O within 10 min, but the temperature increased with increasing GQD-MSNs concentration in suspension by NIR irradiation, confirming the photothermal effect for the GQD-MSNs. At a concentration of 10 mg/ml, the temperature of the GQD-MSNs suspension increased from 30 °C to 55 °C within 10 min of irradiation. Furthermore, the GQD-MSNs suspension was irradiated by 808 nm laser for five times, the photothermal effect did not decrease yet (see the supporting information), which indicates that the GQD-MSNs had good photothermal stability, and showed 15


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the potential for photothermal therapy. Here, the GQD-MSNs generated heat to increase the surrounding temperature due to the strong photothermal conversion capacity of GQDs by NIR irradiation. However, the surface temperature of the GQD-MSNs was much higher than that of the suspension, which may result in more cancer cell death after internalization of the GQD-MSNs by the cancer cells. To evaluate biological safety of the GQD-MSNs, 4T1 cells were used as a cellular system to investigate cytotoxicity of GQD-MSNs in vitro, measured by a Cell Counting Kit-8 (CCK-8) assay. As shown in Fig. 7, after incubation of 4T1 cells with the GQD-MSNs for 24 h, the cell viabilities were not significantly different between the concentrations from 0 to 200 μg/ml, which suggest that GQD-MSNs have good biocompatibility and may be used as nanocarriers for drug delivery. Cell uptake of DOX-GQD-MSNs was carried out by incubation of 4T1 cells with DOX-GQD-MSNs for 4 and 8 h. As shown in Fig. 8, a red fluorescence signal from DOX molecules was observed in the nuclei, which we attribute to DOX release from the DOX-GQD-MSNs before and after endocytosis by 4T1 cells, suggesting that 4T1 cells internalize DOX-GQD-MSNs. The drug release results in vitro indicated that more DOX is released from the DOX-GQD-MSNs under the acidic condition. Thus, the acidic microenvironment in the endosome/lysosome and cytosol of cancer cells (pH 4.5–6.0) could trigger DOX release when DOX-GQD-MSNs are internalized by the cells. In addition, the internalization of DOX-GQD-MSNs in cancer cells provides the possibility for local photothermal therapy. Therefore, the DOX-GQD-MSNs show great potential for synergistic chemo-photothermal therapy. 16


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To investigate synergistic effect of the DOX-GQD-MSNs on therapeutic efficiency, 4T1 cells were incubated for 8 h with a free DOX solution, GQD-MSNs, and DOX-GQD-MSNs, and followed by NIR irradiation for 3 min at 2.5 W/cm2. As shown in Fig. 9A, cell spreading morphology indicates that NIR irradiation did not induce cell apoptosis when 4T1 cells were not treated with the free DOX, GQD-MSNs, or DOX-GQD-MSNs. Without NIR irradiation, 4T1 cells incubated with GQD-MSNs had normal cell spreading morphology, suggesting no cell apoptosis occurred due to the biocompatibility of the GQD-MSNs. On the contrary, some 4T1 cells had a spherical morphology when incubated with free DOX and DOX-GQD-MSNs, indicating that chemotherapy caused apoptosis of the 4T1 cells. With NIR irradiation, 4T1 cells incubated with free DOX showed similar cell morphology to those without NIR irradiation, suggesting no photothermal effect for DOX molecules. However, most of the 4T1 cells incubated with the GQD-MSNs and DOX-GQD-MSNs showed spherical morphology after NIR irradiation, which indicated that NIR irradiation induced cell apoptosis significantly due to the photothermal effect of GQD-MSNs and DOX-GQD-MSNs. To further quantitatively evaluate the synergistic effect of DOX-GQD-MSNs on therapeutic efficiency, the viability of 4T1 cells after 8 h treatment by free DOX, GQD-MSNs, and DOX-GQD-MSNs, without and with 3 min NIR irradiation, was tested. As shown in Fig. 9B, cell viabilities of 63 % and 62 % were obtained after the treatment by free DOX with or without NIR irradiation. Obviously, the GQD-MSNs had negligible cytotoxicity without NIR irradiation, but cell viability decreased 17


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significantly (32 %) with NIR irradiation, indicating the excellent photothermal therapeutic efficiency of the GQD-MSNs on cancer cells. However, it is noteworthy that the cell viability decreased (77 %) after incubation with the DOX-GQD-MSNs although no NIR irradiation was applied, which we attribute to the release of DOX from DOX-GQD-MSNs in cells, triggered by lower pH environment. When NIR irradiation was applied to 4T1 cells after incubation with DOX-GQD-MSNs, cell viability significantly decreased to 11 %, and it is much lower than that of the GQD-MSNs with NIR irradiation. Therefore, the results indicate that DOX-GQD-MSNs, as a multifunctional platform, can achieve synergistic therapeutic efficiency through chemo-photothermal therapy.

Conclusion In this study, we developed a MSNs-based multifunctional platform for synergistic chemo-photothermal therapy, which is composed of MSNs as nanocarriers and GQDs as caps and local photothermal agents. Monodisperse GQD-MSNs had a particle size of 50–80 nm. The DOX-loaded GQD-MSNs not only exhibited pH and temperature-responsive drug release behavior, but also efficiently generated heat to meet the temperature requirement of photothermal therapy. Using breast cancer 4T1 cells as a cellular system, we found that GQD-MSNs were internalized by the cells and had negligible cytotoxicity. Importantly, the DOX-GQD-MSNs had a synergistic chemo-photothermal effect and a significantly higher efficacy to kill cancer cells, compared to chemotherapy and photothermal 18


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therapy alone. Therefore, DOX-GQD-MSNs may raise the potential to improve the efficacy of cancer therapy.

Acknowledgements The authors gratefully acknowledge the support by National Natural Science Foundation of China (No. 51572172, 51102166), and the Scientific Development Project of University of Shanghai for Science and Technology (16KJFZ011).

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Scheme 1. Schematic illustration of the preparation process of the DOX-GQD-MSNs and synergistic chemo-photothermal therapy.

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Fig. 1. SEM and TEM images of the MSNs (A, D), GQD-MSNs (B, E) and DOX-GQD-MSNs (C, F); Elemental mapping of the GQD-MSNs (G).

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Fig. 2. (A) WAXRD patterns of the MSN-NH2, DOX-MSN-NH2, GQD-MSNs and DOX-GQD-MSNs; (B) FTIR spectra of GQDs, MSNs, MSN-NH2, DOX-MSN-NH2, GQD-MSNs, and DOX-GQD-MSNs.

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Fig. 3. (A) N2 adsorption-desorption isotherms of MSNs, MSN-NH2, DOX-MSN-NH2, and DOX-GQD-MSNs and (B) their corresponding pore size distributions.

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Fig. 4. (A) UV-Vis spectra before and after DOX loading and GQD capping on MSN-NH2; (B) TG analysis of MSNs, MSN-NH2, GQD-MSNs, and DOX-GQD-MSNs.

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Fig. 5. Cumulative DOX release from DOX-GQD-MSNs under different pH and temperature conditions.

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Fig. 6. (A) infrared thermal images of the GQD-MSNs suspension at a concentration of 10 mg/ml with 808 nm laser irradiation (2.5 W/cm2); (B) photothermal heating curves of the H2O and GQD-MSNs suspension with different concentrations evaluated by 808 nm laser irradiation (2.5 W/cm2).

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Fig. 7. Cell viabilities of 4T1 cells after incubation with different concentrations of GQD-MSNs as measured by a Cell Counting Kit-8 assay.

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Fig. 8. Confocal microscopy images of 4T1 cells after (A) 4 h and (B) 8 h incubation with DOX-GQD-MSNs at a concentration of 100 μg/ml.

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Fig. 9. Cell viability of 4T1 cells after 8 h incubation with free DOX, GQD-MSNs, and DOX-GQD-MSNs suspensions (DOX: 4.5 μg/ml, MSNs/GQDs: 100 μg/ml) without and with 3 min NIR irradiation, and then cultured in fresh DMEM for an additional 2 h.

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Mesoporous Silica Nanoparticles Capped with Graphene Quantum

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