pH/hypoxia programmable triggered cancer photo-chemotherapy based on a semiconducting polymer dot hybridized mesoporous silica framework

We have integrated the pH/hypoxia-triggered Fe(iii)-banoxantrone (AQ4N) prodrug and semiconducting polymer dots (SPs) for programmable triggered cancer photothermal-chemotherapy.


Materials.
Banoxantrone (AQ4N), dopamine-HCl, tetraethylorthosilicate (TEOS), cetyltrimethyl ammonium chloride (CTAC) and poly [2,6-(4,4-bis (2-ethylhexyl Preparation of PMSF nanoparticle. Firstly, 1 mL of tetrahydrofuran (TEA) mixture containing PCPDTBT (0.25 mg) was carefully injected into 10 mL DI water rapidly, then followed by sonication for 1.5 min using a microtip probe sonicator (40 W output) to prepare SPs [19] . Afterwards, the mixture was stirred at room temperature in dark overnight (600 rpm/min) to evaporate TEA. Subsequently, CTAC (2g) and triethanolamine (20 mg) were carefully dissolved in 20 mL DI-water (at room temperature), and the prepared SPs were then added into above mixture with vigor stirring for 1hr. Afterwards, the reaction mixture was heated to 80 o C and then further stirred for 1hr; then, tetraethylorthosilicate (TEOS, 1.5 mL) was slowly and carefully added into mixture with stirring. After 2 hours of incubation under stirring, the color of reaction mixture changed from ivory to wathet / blue with the formation of precipitates. Subsequently, the precipitates were collected, and then washed with 50 mL methanol (with 1 wt% NaCl) for three times to remove CTAC 24 . The final products of PMSF were then stored in ethyl alcohol for further usage.
Preparation of Mn-APPMSF. PPMSF was prepared by improving our previous method 27 .
Briefly, 1 mg PMSF was added into 50 mL Tris-HCl solutions (10 mM, pH 8.5), then incubated for 2hrs. After centrifugation and washing with DI water and ethyl alcohol for twice, the obtained PPMSF was then mixed with PEG-(NH 2 ) 2 and FeCl 3 solution at pH 7.4 for 2hrs, and then 0.5 mg/mL of AQ4N was added. After incubation for 24hrs, the obtained APPMSF was further washed twice with DI water and obtained by centrifugation for 15 min.
After that, APPMSF was mixed with MnCl 2 solution overnight. Subsequently, the obtained Mn-APPMSF was centrifuged for 15 min, then washed 2 times with DI water. Then, the final products were dispersed in PBS buffer (1mL, pH 7.4) for further usage. The amounts of Mn(II) and Fe(III) bound to Mn-APPMSF were further determined by ICP-MS) (Thermo, USA). Quantification of AQ4N was then measured by absorbance of supernatant at 600 nm.
The photothermal conversion efficiency, η, was calculated using Equation 1 described by our previous reports 32 , where h is the heat transfer coefficient, A is the surface area of the container, T max is the equilibrium temperature, T Surr is ambient temperature of the surroundings, ΔT max =T max -T Surr , I is incident laser power (1 W cm -2 ), and A λ is the absorbance of PMSF or PPMSF at 670 nm. Q s is the heat associated with the light absorbance of the solvent, which is measured independently to be 25.2 mW by using deionized water without nanoparticles as the solvent.
(1) max (1 10 ) The value of hA is derived according to Equation 2: Where τ s is the sample system time constant, m D and C D are the mass (1 g) and heat capacity (4.2 J g -1 ) of deionized water used as the solvent, respectively.
In order to obtain the hA, herein introduce θ, which is defined as the ratio of ΔT to ΔT max : pH sensitive drug release of Mn-APPMSF. The pH responsive release study was investigated as follows: Mn-APPMSF (0.5 mg, PPMSF; 48 μg/mL, AQ4N) was dissolved into PBS buffers (1 mL) with various pH values (pH 5.0 and 7.4); at certain time intervals, 0.5 mL supernatant was carefully taken and analyzed to calculate the amounts of released drug by measuring the supernatant absorbance at 600 nm after the centrifugation at 13000 rpm/min for 10 min. To keep the constant volume, 0.5 mL PBS with corresponding pH values was further added after each sampling.

Confocal microscopy and flow cytometry studies. The cell uptake of Mn-APPMSF by
HepG2 cells was analyzed by using a confocal laser scanning microscope. In the typical experiment setting, the HepG2 cells (5 × 10 4 ) were seeding in 35-mm glass-bottom Petri dishes and cultured for another 24hrs at 37˚C in the incubator; afterwards, the Mn-APPMSF were added into the cell culture dish and further incubated for 0.5, 1, 1.5, 2 and 4hrs, respectively. Then, the treated cells were further washed for 3 times with PBS buffer (pH 7.4) and then fixed with 4% paraformaldehyde buffer for 15 min. Afterwards, 4',6-diamidino-2phenylindole (DAPI) was carefully added into cell culture dishes, and incubated for 15 min in dark at room temperature. Finally, the stained cells were analyzed by the confocal microscope (Carl Zeiss LSM 780, Germany), with 405 nm laser excitation for DAPI and 633 nm laser excitation for AQ4N.
To conduct the flow cytometry analysis, the HepG2 cells were carefully seeding to 6-well plates with the cell density of 5 × 10 5 cells/per well, and then the cells were incubated in incubator with 5% CO 2 and the humidity atmosphere for 24hrs. Afterwards, the original cell culture medium was carefully replaced by the fresh cell culture medium that contains Mn-APPMSF (0.02 mg/mL, PPMSF; 1.92 μg/mL, AQ4N); after further incubation for another 4hrs, the cells then were washed 3 times with PBS buffer and then irradiated by NIR laser (670 nm, power intensity of 1W/cm 2 ) for 5 min. After that, the cells was collected and dispersed in 1 mL PBS buffer. After that, cells were carefully filtered by the nylon mesh (40 microns) to remove cell aggregates before FACS analysis. The fluorescence measurement of intracellular AQ4N was conducted in FL4 channel (excitation at the 670 nm).
Antitumor efficacy of Mn-APPMSF in vitro. CCK8 (Cell Counting Kit) assay was applied to investigate antitumor effects of Mn-APPMSF against HepG2 cells. Briefly, cells were carefully seeded in the 96-well plate with the density of 1 × 10 4 cells/per well, and then incubated with 5% CO 2 in an incubator with humidity atmosphere for 24hrs. Afterwards, the original medium was discarded; subsequently the cells were further washed 3 times with PBS buffer to carefully remove dead cells, then followed by the incubation with PMSF, PPMSF and Mn-APPMSF for 4hrs, and subsequently washed 2 times with PBS to carefully remove non-internalized nanoparticles. Then the 96-well plate was irradiated with NIR laser (670 nm, power intensity of 1 W/cm 2 ) under aerobic or hypoxia condition, respectively. The cells only incubated with the culture medium were taken as untreated control. Cell viability was For antitumor efficiency analysis, the irradiation was carefully conducted after 4hrs of injection. Then, the therapeutic efficiency was carefully evaluated by monitoring the tumor volume change and body weight change in each group every 2 days, and over 12 days in total.
The tumor size was carefully measured by the caliper every other day after indicated treatment. Finally, tumor volume (V) was calculated by following equation: Here, the A is longer diameter and B is the shorter diameter (mm) of tumor, respectively.
We further investigated the PTT-enhanced hypoxia induced AQ4N activation at tumor site by detecting the metabolite of AQ4N (refer to as AQ4) through ESI-MS and HPLC analysis.
The experiments were performed as follows: After intravenous injection of Mn-APPMSF for 4hrs, the mice (n=3) was irradiated by 670 nm laser at the tumor site with the power intensity of (1 W/cm 2 ) for 5 min. The mice injected with Mn-APPMSF but without laser irradiation was taken as control. After 24hrs of irradiation, the tumors were excised, weighted and then crushed by sonifier in MeCN solution (containing 0.2% H 2 O 2 ) 10, 42, 43 . Afterwards, the tumor samples were homogenized using a pellet pestle, and centrifuged at 10000g for 5 min. Then, the supernatant was dried under vacuum evaporation, and afterwards reconstituted with DMSO. Subsequently, 20 μL of reconstituted supernatant was injected into the HPLC (Agilent 1260 Infinity, Agilent Technologies, Germany), and analyzed using Agilentzorbax Eclipes Plus C18 colum (4.6 mm x 100 mm, 3.5 um, Agilent Technologies, USA) by isocratic elution with 78% of 50 mM ammonium formate (pH 3.6) and 22% of acetonitrile.
The column temperature was maintained at 45 o C, and the flow-rate was maintained at 0.6 mL/min. Standards of AQ4 were prepared and diluted in DMSO. The AQ4 concentration was monitored by the absorption at 276 nm. The correlation between the peak area at the retention time of 3.27 min and the concentration of AQ4 was analyzed by linear regression (Y = 160.05x -5.7083), which showed a well-correlated linear relationship (R 2 = 0.9981); meanwhile, the calibration curve was prepared from 0.05 to 10 μg/mL. Moreover, the AQ4N activation at tumor site by hypoxia was further determined by electrospray lionization mass spectrometry (ESI-MS) 43 .

Histological evaluation and long-term toxicity examination.
To further carefully evaluate histological changes of the tumors after indicated treatment, a tumor-bearing mouse in each group were then sacrificed after 48hrs of treatment; afterwards, the tumors were collected, sectioned and then stained with Hematoxylin and eosin (H&E) for histopathology analysis.
Immunohistochemical staining of the tumor slices was performed by antibodies specific for HIF-1 (1:800) (H1alpha67, Novus Biologicals, Littleton, CO or 54, BD Bioscience) after indicated treatment for 24hrs. The long-term toxicity examination of mice was performed as follows: one tumor-bearing mouse in each group were sacrificed at the day 12 after indicated treatment, and major organs (including heart, liver, spleen, lungs and kidney) of the mouse were collected, then fixed in the 4% neutral formaldehyde, embedded in the paraffin, stained with the H&E, and finally observed under the Zeiss microscope (Axio Lab.A1).
All the data were shown as means ± SD through at least three experiments.             The content of metabolite AQ4 was detected by HPLC in tumor samples of Mn-APPMSF injected mice with or without laser irradiation, respectively. The statistical analysis was performed with the two-tailed paired Student's T-test, *p<0.05 (670 nm, 1 W/cm 2 ).