Self-actuated biomimetic nanocomposites for photothermal therapy and PD-L1 immunosuppression

Biomimetic nanocomposites are widely used in the biomedical field because they can effectively solve the problems existing in the current cancer treatment by realizing multi-mode collaborative treatment. In this study, we designed and synthesized a multifunctional therapeutic platform (PB/PM/HRP/Apt) with unique working mechanism and good tumor treatment effect. Prussian blue nanoparticles (PBs) with good photothermal conversion efficiency were used as nuclei and coated with platelet membrane (PM). The ability of platelets (PLTs) to specifically target cancer cells and inflammatory sites can effectively enhance PB accumulation at tumor sites. The surface of the synthesized nanocomposites was modified with horseradish peroxidase (HRP) to enhance the deep penetration of the nanocomposites in cancer cells. In addition, PD-L1 aptamer and 4T1 cell aptamer AS1411 were modified on the nanocomposite to achieve immunotherapy and enhance targeting. The particle size, UV absorption spectrum and Zeta potential of the biomimetic nanocomposite were determined by transmission electron microscope (TEM), Ultraviolet-visible (UV-Vis) spectrophotometer and nano-particle size meter, and the successful preparation was proved. In addition, the biomimetic nanocomposites were proved to have good photothermal properties by infrared thermography. The cytotoxicity test showed that it had a good killing ability of cancer cells. Finally, thermal imaging, tumor volume detection, immune factor detection and Haematoxilin-Eosin (HE) staining of mice showed that the biomimetic nanocomposites had good anti-tumor effect and could trigger immune response in vivo. Therefore, this biomimetic nanoplatform as a promising therapeutic strategy provides new inspiration for the current diagnosis and treatment of cancer.


The instrument.
The instrument. The product was characterized by transmission electron microscopy (TEM) (JEM-2100, JEOL). The fluorescence of the samples was measured using an F-4600 fluorescence spectrophotometer (Hitachi). The UV absorption spectra of the synthesized materials were measured using an Agilent UV-Vis spectrophotometer CARY-60. Mean particle size and Zeta potential were measured by dynamic light scattering (DLS), and confocal fluorescence imaging studies were performed using a laser scanning confocal microscope (LSCM) with an objective lens (×20) (Nikon C2 Plus). The cell viability assay was performed using BioTek Epoch full-wavelength microplate reader.

The preparation of the PM
10 mL of mouse whole blood was centrifuged at 1500 rpm for 10 min. The supernatant was separated into platelet-rich plasma (PRP). After PRP was centrifuged at 3000 rpm for 20 min, the precipitate was washed with PBS buffer and centrifuged repeatedly before platelets were obtained. Platelets were frozen at -80 ° C and thawed at room temperature, and this process was repeated three times. Membranes were obtained by centrifugation at 8000 rpm for 10 min, washed with PBS containing protease inhibitors, and sonicated for 5 min. The resulting PM was dispersed in 2 mL of PBS and stored at -20 ° C for later use.

Cell culture.
All 4T1 and L02 cells participating in the experiment were cultured at 37°C in a constant temperature and humidity chamber with 95% humidity and 5% CO2 concentration. The cell medium was DMEM containing 10% fetal bovine serum and 1% double antibody (penicillin-streptomycin).

CCK-8 analysis.
100 μL of 4T1 cell suspension at a concentration of 1×10 5 cells /mL was added to each well of a 96-well plate and precultured for 12 h in an incubator at 37 ° C. Then, 10 μL PB, PB/PM, PB/PM/Apt, PB/PM/HRP/Apt (PB: 400, 200, 100, 50, 10 μg/mL) were added to each well, and the incubation was continued for 4 h at 37℃. The supernatant was taken and 100 μL of fresh medium containing 10% CCK-8 solution was added to each well, taking care not to form bubbles in the Wells. The 96-well plates were incubated for an additional 35 min in an incubator, and finally the absorbance at 450 nm was determined using a microplate reader.

In vivo antitumor test.
All tumor-bearing mice were BALB/c nude mice aged 4-5 weeks. Each mouse was injected subcutaneously with 4T1 cells (3×10 7 cells/mouse). When the solid tumor size reached about 100 mm 3 , the tumor-bearing mice were divided into four groups according to the experimental requirements. The four groups of mice were treated with PBS, PBS + laser, PB/PM/HRP/Apt and PB/PM/HRP/Apt + laser, respectively. Tumor volume and body weight were recorded every other day. During the treatment, the temperature changes and infrared thermal imaging images of tumor in each group were obtained by infrared thermal imager. At the end of the treatment cycle, the experimental mice were euthanized, and the tumor tissues and major organs of the treated mice were obtained. Finally, the tumor, heart, liver, spleen, lung, and kidney were analyzed by H&E staining.