Macrophage-mediated tumor homing of hyaluronic acid nanogels loaded with polypyrrole and anticancer drug for targeted combinational photothermo-chemotherapy

Rationale: Development of nanosystems that can be integrated with macrophages (MAs), an emerging carrier system, for effective tumor therapy remains to be challenging. We report here the development of MAs specifically loaded with hyaluronic acid (HA) nanogels (NGs) encapsulated with a photothermal agent of polypyrrole (PPy) and anticancer drug doxorubicin (DOX) (HA/DOX@PPy NGs) for tumor homing and combination photothermo-chemotherapy. Methods: Cystamine dihydrochloride-crosslinked HA NGs were first prepared through a double emulsification method, then loaded with PPy via an in-situ oxidization polymerization and physically encapsulated with DOX. The created HA/DOX@PPy NGs were well characterized and subjected to be endocytosed by MAs (MAs-NGs). The MAs-mediated tumor-homing property, phenotype changes and photothermal performance of MAs-NGs were investigated in vitro, and a subcutaneous tumor model was also established to confirm their targeting capability and enhanced antitumor therapy effect in vivo. Results: The generated hybrid NGs possess a size around 77 nm and good colloidal stability, and can be specifically endocytosed by MAs without appreciably affecting their normal biofunctionalities. In particular, NG-loaded MAs display excellent in-vitro cancer cell and in-vivo tumor homing property. Systemic administration of the MAs-NGs leads to the significant inhibition of a subcutaneous tumor model through combination photothermo-chemotherapy under laser irradiation. Conclusions: The developed hybrid HA-based NG nanosystem incorporated with PPy and DOX fully integrates the coordination and heating property of PPy to regulate the optimized DOX release in the tumor region with the assistance of MA-mediated tumor homing, providing a promising cell therapy strategy for enhanced antitumor therapy.


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imaging, a typical sample of HA@PPy NGs in aqueous solution (1 mg/mL, 10 μL) was deposited onto a carbon-coated copper grid and air-dried before measurements. The particle size distribution was calculated using Image J 1.40 G software (http://rsb.info.nih.gov/ij/download.html). For each sample, at least 300 particles from different SEM or TEM images were randomly selected and analyzed.
Fourier transform infrared (FTIR) spectra were collected using a Nexus 670 spectrophotometer (Thermo Nicolet Corporation, Madison, WI) with an attenuated total reflectance (ATR) technique.
Solid samples were mixed with KBr crystals, grinded fully and tableted before measurements. Thermal gravimetric analysis (TGA) was carried out using a TG 209 F1 thermo gravimetric analyzer (NETZSCH Instruments Co., Ltd., Selb/Bavaria, Germany) at a heating rate of 10 °C/min under N2 atmosphere with a temperature ranging from 50 to 800 °C.

Photothermal conversion property.
To evaluate the photothermal conversion property of the HA@PPy NGs, the temperature changes of the NG solution at different concentrations (0, 0.125, 0.5, 1 and 2 mg/mL, respectively) were recorded in real-time when exposed to an 808-nm laser device (Shanghai Xilong Optoelectronics Technology Co. Ltd., Shanghai, China). Typically, 0.3 mL of an aqueous HA@PPy NG solution at different concentrations was laser irradiated for 5 min under an output power density of 1.0 W/cm 2 . The real-time temperature changes of corresponding samples were recorded every 5 s by an online DT-8891E thermocouple thermometer (Shenzhen Everbest Machinery Industry Co., Ltd., Shenzhen, China). To quantify the photothermal conversion property of the HA@PPy NGs, a heating and cooling cycle of the sample was carried out. The photothermal conversion efficiency (η) of the HA@PPy NGs was calculated according to the literature protocols [1,2]. Furthermore, to evaluate the photothermal stability of the NGs, 5 heating and cooling (laser on-off) cycles were performed to record the temperature changes.
DOX release from NGs. To investigate the DOX release from the HA/DOX@PPy NGs under different pHs with or without NIR laser irradiation, the HA/DOX@PPy NGs dispersed in phosphate buffered saline (1 mg/mL, pH 7.4) or sodium citrate buffer (pH 5.5 with or without laser) were placed in a regenerated cellulose dialysis bag with an MWCO of 10,000 to have a volume of 1 mL, submerged S-5 into 9 mL of the corresponding buffer medium at 37 ℃ and kept shaking constantly. For the pH 5.5 + Laser group, the NG solution in the dialysis bag was irradiated for 10 min with an 808-nm laser at a power density of 1 W/cm 2 before the dialysis experiment. At each scheduled time interval, 1 mL of the outer phase buffer medium was taken out and subsequently supplemented with 1 mL of the corresponding buffer to maintain the constant volume of outer phase buffer medium. The DOX absorption at 490 nm was recorded by UV-vis spectrometry to determine the concentration of released DOX according to the corresponding absorption-concentration calibration curve at different pHs.
In vitro cytotoxicity to MAs. The conventional Cell Counting Kit-8 (CCK-8) assay was utilized to evaluate the in vitro cytotoxicity of the HA/DOX@PPy NGs to MAs. RAW264.7 cells were seeded into 96-well plates at a density of 1 × 10 4 cells per well and incubated overnight at 37 ℃ and 5% CO2.
Afterwards, the medium in each well was replaced with fresh medium (100 μL) containing free DOX or HA/DOX@PPy NGs at different DOX concentrations (5, 10, 15, 20, 30, 40 and 80 μg/mL, respectively), and the cells were continuously cultured for another 24 h. Then, the cells were rinsed with PBS for three times, incubated with 100 μL of FBS-free DMEM supplemented with 10% CCK- For the concentration-dependent macrophage loading, fresh medium containing the NGs with different DOX concentrations (10, 20 or 40 μg/mL) was utilized to treat the RAW264.7 cells for 4 h.
After that, the cells in each well were washed with PBS for 3 times, collected, counted and lysed by 3 cycles of freeze-thawing process in liquid nitrogen. The released DOX was measured by UV-vis spectrometry as described above and the loaded DOX content per cell was calculated by subtracting the background absorption of cells treated with PBS.
Furthermore, the uptake of HA/DOX@PPy NGs by MAs was validated by Confocal laser scanning microscope (CLSM, ZEISS LSM 700, Jena, Germany) through observation of the DOX fluorescence. Briefly, 1×10 5 RAW264.7 cells were seeded into each confocal dish and cultured overnight at 37 o C and 5% CO2. After that, the HA/DOX@PPy NGs with a DOX concentration of 20 μg/mL were chosen to treat the cells for 4 h and 12 h, respectively. Free DOX and PBS were used as the positive and negative controls, respectively. DAPI was used to stain the MA nuclei before observation according to the standard protocols reported in our previous work [3].
Cell migration assay. Cell migration assay through a transwell system was used to investigate whether the NG-loaded MAs (MAs-HA/DOX@PPy NGs, for short, MAs-NGs) remained their tumorhoming property in vitro. MAs without NGs were used as control. The transwell system was equipped was performed after exposed to an 808-nm laser at an output power density of 1 W/cm 2 or 1.5 W/cm 2 .

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The thermal images were recorded by an FLIR infrared thermal camera (IRS Systems Inc., Shanghai, In each treatment, 5 × 10 6 cells dispersed in 100 μL of PBS were intravenously injected to each mouse for MAs-NGs and MAs-NGs + Laser groups, and the injected DOX dosage in each group was kept consistent at 42.9 μg per mouse (1.95 mg/kg DOX for each mouse), which is significantly higher than the lowest typical injection dose (1 mg/kg) reported in the literature [4]. For PTT, the tumor of each mouse was irradiated by an 808-nm laser (1.5 W/cm 2 , for 5 min) at 2 and 24 h post-injection, respectively for the HA/DOX@PPy NGs + Laser and MAs-NGs + Laser groups. The tumor volume S-11 and body weight of each mouse were measured every other day for 15 days. At the 15 th day, one mouse in each group was sacrificed and the tumor was removed from the sacrificed mouse for hematoxylin & eosin (H&E) and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining. The remaining mice were used to record the survival rate. The tumor volume (V) was calculated according to the formula of V = tumor length × (width) 2 /2.
In vivo biosafety examination. First, to examine the histological changes of the mice, major organs including heart, liver, spleen, lung, and kidney from each mouse of different groups were harvested at 15 th day post-treatment, fixed in 4% paraformaldehyde, embedded into paraffin and sectioned into thin slices before H&E staining. After that, the specimens were observed by inverted optical microscopy (Nikon Corporation, Tokyo, Japan).
Statistical analysis. Experimental data were given as the mean ± standard deviation (n ≥ 3). Oneway analysis of variance method was adopted to evaluate the significance of the experimental data between groups. A p value of 0.05 was selected as the significance level, and all data were marked as (*) for p < 0.05, (**) for p < 0.01, and (***) for p < 0.001, respectively.