Chlorin e6 (Ce6), RGD peptide, 3-aminopropyltriethoxysilane (APTS), triethanolamine (TEA), cetyltrimethylammonium chloride (CTAC, 25 wt%), 1,2 distearoyl-sn-glycero-3–phosphoethanolamine-N-[amino(polyethylene-glycol)] with a PEG length of 2000 (DSPE-PEG2K-NH2 ), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), cholesterol were purchased from Xi’an ruixi Biological Technology Co., Ltd. Hydrogen hexachloroplatinate (IV) hexahydrate (H2PtCl6‚6H2O, 99%), sodium borohydride(NaBH4) were purchased from Beijing Hongke Chemical Products Co. Tetraethoxysilane (TEOS) and sodium hydroxide (NaOH) were purchased from Aladdin (Shanghai China). TH588 was purchased from Selleckchem (Houston, TX, USA). All other chemical reagents were analytical grade and do not require further purification.
Preparation of MSN-NH2
15g CTAC and 0.6g TEA were dissolved in 150ml water and incubated in a trimethyl silicone bath at 80°C, followed by intensive magnetic stirring at 200 rpm for 1 h until completely dissolved. Subsequently, 10ml of TEOS was added to the reaction system drop wise at the rate of two seconds per drop, the reaction was carried out at 80°C for 2h. After cooling, the precipitation was collected by centrifugation at 12000rpm for 10min, followed by alternately washed with water and ethanol for 3 times. The removal process of the template was further performed by dissolving the reaction product in an ethanol/hydrochloric acid solution and carried out at 70°C overnight. The MSN were centrifuged at 12000rpm for 10min, then washed alternately with ddH2O and ethanol for 3 times, and dissolved in water for later use. Finally, 50μL APTS and 100μl glacial acetic acid were added to the MSN aqueous solution and stirred at room temperature for 24h. After centrifugation at 12000rpm for 15min, precipitation was collected and vacuum freeze-dried to obtain MSN-NH2.
In situ growth of Pt NPs in nanochannels
150mg MSN-NH2 was dispersed in 20ml ddH2O, and 20mL H2PtCl6·6H2O aqueous solution was added into the NPs suspension and stirred at room temperature for 1h. NaBH4 was then added to the reaction system and stirred for another 1h. NPs were centrifuged at 12000rpm for 10min, washed with ddH2O for 3 times and dried in vacuum to obtain MSN-Pt.
Synthesis of [email protected]/TH588
For successful loading of Ce6, MSN-Pt (75mg) was dispersed in phosphate buffered saline (PBS), 5mg Ce6 (dissolved in 750μl DMSO solution) was added and stirred at room temperature for 24h in the dark. The mixture was centrifuged at 12000rpm for 20min, the supernatant was collected, and the precipitation was washed with PBS for 3 times. The loading process of TH588 is similar to that of Ce6, MSN-Pt/Ce6 75mg was dispersed in PBS, 7.5mg TH588 (20mg/mL,375μl DMSO solution) was added and stirred overnight at room temperature. The precipitation and supernatant were collected after centrifugation at 12000rpm for 20min. The absorbance of Ce6 and TH588 in the supernatant was detected by fluorescence spectrophotometer to determine the drug loading content and encapsulation efficiencies, respectively.
Fabrication of DSPE-PEG-RGD
In order to synthesize the RGD-modified phospholipid (DSPE-PEG-RGD), DSPE-PEG-NHS and RGD (molar ratio 3:1) were co-incubated in DMF solution for 24 h under nitrogen flow at 25°C. The excess RGD and DSPE-PEG-NHS were removed by dialysis (MWCO:3500) for 48 hours to achieve the purpose of separation and purification of the products. Finally, the products were freeze-dried and stored at -80°C for later use.
Synthesis of [email protected]/[email protected], [email protected]/[email protected]
Liposome shell was prepared by thin film hydration method. Briefly, cholesterol, DPPC, DSPE-PEG-RGD, DSPE-PEG, and DOTAP (molar ratio: 40:50:3:3:6) were placed in a round flask containing 30 mL chloroform. The organic solvent was removed in a rotary evaporator under a vacuum environment of 40°С and a thin lipid film was obtained at the bottom of the flask. The flask was then dried in a vacuum desiccator for 24 h until the organic solvent completely evaporated. Subsequently, 15ml of deionized water was added to the flask and the liposome sample was obtained by ultrasonic oscillation at 30°C for 15min. The prepared MSN-Pt/Ce6/TH588 and liposome sample were dispersed in 25ml PBS and continuously stirred in the dark for 12h. The mixture was then centrifuged at 12000 rpm for 10 min to collect the precipitate (MSN-Pt/Ce6/[email protected]). Non-RGD-targeted liposome encapsulated MPCT was synthesized in a similar method as described previously without the addition of DSPE-PEG-RGD. All nanoproducts were re-suspended in saline or PBS for later use.
The particle size and morphology of NPs at each stage of synthesis process were characterized by transmission electron microscopy (TEM, JEOL, JEM F200) and scanning electron microscopy (SEM, ZEISS, Gemini 300). Size distribution, zeta potential and polydispersity index (PDI) of NPs were monitored by Malvern zeta sizer Nano-ZS90. The UV-vis spectra of the samples were obtained by using a UV-vis spectrophotometer (Perkin-Elmer, Lambda Bio40).
Drug release in vitro
To explore the drug release kinetics and pH responsive release properties of [email protected], two kinds of NPs (MPCT, [email protected]) with equal amounts were immersed in different aqueous solutions (pH=7.4, 5.0) at room temperature, respectively. The supernatant of the samples was extracted at different time points, and the release amount of TH588 and Ce6 was determined by UV-vis spectrophotometry. All the release tests were repeated three times in parallel and the average of the results were taken.
The cytotoxicity of [email protected] NPs was evaluated by Cell Counting Kit-8 (CCK-8) assay. Specifically, HOS cells were seeded into 96-well plates at a density of 5000 cells per well and co-incubated with fresh medium at 37°C in 5% CO2 for 24h. Next, the cells were treated with a series of increasing concentrations of [email protected] (0, 6.25, 12.5, 25, 50, 100, 200 μg/ml) for 24h and 48h. Then, 10μl of CCK8 solution was added to each well, and incubate for 2h. The absorbance values at the 450nm test wave were determined using an enzyme immunoassay analyzer (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The experiment was performed in quintuplicate.
The PDT-CHT combination treatment efficacy of [email protected] was monitored by CCK8 assay. Generally, HOS cells were seeded into a 96-well plate (5000 cells per well) and cultured in humidified 5%CO2 at 37°C for 24 h until completely attached. Subsequently, the cells were washed once with PBS, fresh medium containing a range of concentrations of free Ce6, [email protected] or [email protected] was then added. After co-incubation with HOS cells for 6h, the cells were irradiated with 660nm laser (400 mW cm-2) for 10 minutes and incubated for another 24h. Finally, the HOS cell viabilities were determined by CCK8 method in vitro.
Calcein AM/PI staining
Calcein AM/PI staining was used to further investigate the antitumor effect of [email protected] in vitro. HOS cells were inoculated in 24-well plates (5×104 cells per well) and cultured at 37°C with 5% CO2 for 24 hours until completely adherent. TH588(250μL, 20μg/ml), Ce6(250μL, 20μg/ml), [email protected] (250μL, equivalent MSN concentration: 20μg/ml), [email protected] (250μL, equivalent MSN concentration: 20μg/ml) were added to displace the medium. PBS was added as negative control group. After incubation for 4 h, the treatment group was irradiated with a 660nm laser with an energy density of 2.5W cm-2 for 5 min. Finally, Calcein AM/PI detection solution was added and incubated in dark for 30 minutes at 37°C. After incubation, the staining effect was observed under an inverted fluorescence microscope.
To test the blood biocompatibility of [email protected], hemolysis assay was performed. Venous blood was extracted from BALB/c mouse and centrifuged at 8000rpm for 5 min. The serum was discarded and 2ml PBS was added to resuspend the red blood cells (RBC). Then, 200µl RBC suspension was added to 800µl PBS, in which the concentration of [email protected] ranged from 12.5-400 µg/ml. Moreover, 200µl RBC suspension were added to 800µl PBS and deionized water as negative and positive controls, respectively. The mixture was shaken at 37°C for 2 hours and centrifuged at 12000rpm for 3 minutes. Finally, 8 treatment groups were photographed, 100µl supernatant was taken and placed in 96-well plate. The absorbance was measured by a microplate reader (Bio-Rad, Model 550, USA)
Cellular uptake experiments
To measure the efficiency of the cellular nanoparticle uptake, Human osteosarcoma HOS cells were cultured in a 24-well plate at 37°C and 5% CO2 for 24 h. After the complete application, the cells were washed once with PBS. Then, fresh medium containing different formulations were added to incubate for another 12 hours. The cells were then washed with PBS and stained with Hoechst 33342 for 15 min, followed by cell imaging with an inverted fluorescence microscope.
In vitro catalysis and ROS generation experiment
MPCT and [email protected] NPs were dissolved in 4ml 3% H2O2 solution or deionized water, respectively, and co-incubated for 30 minutes followed by detection using HI-2400 dissolved oxygen meter to demonstrate O2 generation. Subsequently, we further explored the catalytic performance of [email protected] The [email protected] was resuspended in H2O2 solutions of different concentrations (0, 5, 10, 20mM). The oxygen electrode probe was inserted into the solution and the change of O2 concentration in the solution was measured in real time within 15 min. To test the catalytic stability, H2O2 (1mM) solution was repeatedly added to the reaction system every 1h and continued to incubate with [email protected], followed by measuring the concentration of H2O2.
To detect ROS generation in cancer cells, five groups were set up (control, TH588, Ce6 + laser, [email protected] + laser, [email protected] + laser). The HOS cells (1×105 per well) were inoculated into 6-well plates and cultured for 24 hours until they adhered completely. Then, different formulations (TH588, Ce6, [email protected], [email protected]) were added into the corresponding wells according to the treatment of the above 5 groups. After the cells were incubated at 37°C for another 6 hours, the culture medium was replaced with fresh MEM again. The cells were irradiated with a 660nm, 500mW cm-2 laser for 5 min, and then co-incubated with the ROS probe: 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) for 30 min, followed by ROS fluorescence imaging.
Animal and tumor models
Female BALB/c nude mice (weight 15-19g, age 4-6 weeks) were provided by the Hebei Ex & Invivo Biotechnology Co. Ltd (Shijiazhuang, China). All animal experiments were carried out according to protocols approved by the Experimental Animal Ethics Committee of Hebei Ex & Invivo Biotechnology Co., Ltd. Each mouse was injected with 200μL PBS containing 6×106 HOS cells under the axilla. Vernier caliper was utilized to measure the volume of the tumor according to the formula: 1/2 × (width2) × length. HOS tumor-bearing mice can be used for further experiments after the tumor volume has reached about 80-100mm3.
In vivo imaging
[email protected] NPs (equivalent Ce6 amount :2mg/kg) was injected into tumor-bearing mice through the tail vein. Fluorescence imaging of mice was performed with the IVIS Lumina III imaging system (Perkin Elmer, Caliper Life Sciences, MA) at different time points under gas anesthesia. At the end of 24h fluorescence imaging, the mice were sacrificed, and their tumors and major organs were harvested for in vitro imaging analysis.
In vivo cancer treatment
Tumor-bearing mice were randomly divided into 5 treatment groups (n=3 each group): group1, PBS; group2, TH588; group3, Ce6 + laser; group4, [email protected] + laser; group5, [email protected] + laser. The mice in the 4, 5 groups received laser irradiation (660nm, 1 W cm-2) for 5 minutes. Body weight and tumor volume were monitored every other day for 14 consecutive days during treatment. Then, the mice were sacrificed and the tumor tissues were removed and weighed.
To further evaluate the efficacy of different treatment modalities, terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick-end labeling (TUNEL) staining was used to evaluate the apoptotic response of tumor tissues. Hematoxylin and eosin (H&E) and Ki67 staining were used to evaluate the efficacy of chemo-photodynamic combination therapy on tumor tissues. The main organs (heart, liver, spleen, lungs, kidneys) of mice were extracted, embedded in paraffin and sectioned for H&E staining to study the toxicity of NPs in vivo.
The values are expressed as mean ± standard deviation (SD). GraphPad Prism (version 184.108.40.206) was used to conduct the two-tailed Student’s t tests for multiple comparisons. P < 0.05 was considered statistically significant.