| Objective Breast cancer is one of the most common malignant tumors in women with annually increased incidence.At present,traditional breast cancer treatments include surgery,chemotherapy and radiation therapy.Nevertheless,there are problems of incomplete treatment,easy recurrence,severe side effects,damage to normal tissues and easy multi-drug resistance,so it is urgent to develop breast cancer treatment with good safety and high treatment efficiency.Photothermal therapy(PTT)is a therapeutic modality with high selectivity and extremely low invasiveness,demonstrating excellent therapeutic efficacy in the treatment of superficial tumors such as breast cancer.PTT is the method of using photosensitizer to convert light energy into heat energy under near-infrared(NIR)laser irradiation,therefore raising local temperature to kill tumor cells.In addition,PTT can also induce immunogenic cell death(ICD)though releasing tumor associated antigens(TAAs)and damage associated molecular patterns(DAMPs),and then activate the immune response.However,the immune stimulation is transient,incapable of inducing systemic immune response.Therefore,lignin supramolecular self-assembly containing photosensitizer indocyanine green(ICG)and aluminum adjuvant were prepared for the photothermal-imniunotherapy of breast cancer.Methods Lignin supramolecular self-assembly comprising photosensitizer ICG and aluminum adjuvant were prepared by one-step method(LS-Al-ICG).LS-Al-ICG was characterized by transmission electron microscopy,Malvern particle size analyzer,ultraviolet-visible near-infrared spectrometer,X-ray energy dispersive spectrometer,X-ray photoelectron spectrometer and infrared spectrometer.The drug loading(DL)and encapsulation efficiency(EE)of ICG and Al were measured via ultraviolet-visible near-infrared spectrometer and inductively coupled plasma,respectively.Molecular docking simulation was conducted to clarify the self-assembly mechanism of LS-Al-ICG.The photothermal conversion ability of LS-Al-ICG was evaluated by thermal infrared camera.The in vitro release profile was investigated through dialysis method.The hemolytic toxicity of LS-Al-ICG was evaluated by hemolytic assay.In cytological evaluation,the confocal laser scanning microscope was used to evaluate the cellular uptake of LS-Al-ICG in 4T1 cells.The in vitro antitumor effect of LS-Al-ICG was evaluated by CCK-8 kit and live/dead cell kit.The release of DAMPs in 4T1 cells treated by different methods was analyzed by confocal laser scanning microscope and ATP kit.The maturity of bone marrow-derived dendritic cells was analyzed by flow cytometry,and ELISA kit was used to detect the levels of related cytokines in culture medium.To study the in vivo antitumor effect,bilateral 4T1 breast cancer mouse model was established.The in vivo distribution of LS-AL-ICG after intravenous injection was investigated by small animal imager.The in vivo photothermal ability was monitored via a thermal infrared camera.Combined with PD-1 inhibitor,the in vivo antitumor efficacy and biosafety of LS-Al-ICG were evaluated by the volume of primary and distant tumor,body weight,survival rate,H&E and TUNEL staining.Antitumor mechanism in vivo,the degree of immune activation in vivo was evaluated by flow cytometry to analyze the maturity of dendritic cells in tumor draining lymph nodes and primary tumor,the proportion of T lymphocytes in spleen,and ELISA kit to detect the level of relevant cytokines in serum.Results The as-prepared lignin supramolecular self-assembly showed regular circular shape and uniform distribution under TEM,with particle size of 8.3 ± 2.0 nm,Zeta potential of-20.7 ± 0.7 mV and PDI of 0.215 ± 0.014.The absorption spectrum showed that LS-Al-ICG had the characteristic absorption peak of ICG,which redshifted to 808 nm.The results of X-ray energy dispersive spectrometer showed that the atoms of carbon,oxygen,sulfur and aluminum accounted for 69.0%,28.1%,1.6%and 1.3%,respectively,and the results of X-ray photoelectron spectrometer further confirmed the element composition of LS-Al-ICG and the payload of Al3+.The result of FTIR spectra showed that LS-Al-ICG was successfully self-assembled.LS-Al-ICG was stable in serum-containing medium.The DL and EE of ICG were 7.10 ± 0.12%and 35.70 ± 0.62%,respectively,and those of Al were 14.29± 0.54%and 11.85 ±0.45%,respectively,which were determined by ultraviolet-visible near-infrared spectrometer and inductively coupled plasma.The results of molecular docking simulation demonstrated that Al3+initiates the self-assembly process through coordination bonds formed with sulfonate groups of LS and ICG,which induced the formation of a hydrogen bond between the sulfonate group of LS and the hydroxyl group of ICG.The results of photothermal evaluation implied that the photothermal effect of LS-Al-ICG was concentration-dependent and time-dependent.In vitro release results showed that LS-Al-ICG had good pH-sensitive property.The results of hemolysis showed that LS-Al-ICG had good biocompatibility.In cytological evaluation,the results of confocal laser scanning microscope indicated the increased cellular uptake of LS-Al-ICG and morphological change of 4T1 cells caused by NIR.The results of CCK-8 and staining of live/dead cell experiments displayed that LS-Al-ICG showed good biocompatibility in HUVEC and effective antitumor effect in vitro in combination with NIR laser irradiation.The expression levels of CRT and HMGB1 in 4T1 cells and the content of extracellular ATP verified that LS-Al-ICG combined with NIR laser irradiation could induce immunogenic death of tumor cells.The co-incubation experiment of tumor cell culture medium after PTT treatment with immature primary DCs verified that DCs maturity and the secretion of related cytokines was increased.In bilateral 4T1 breast cancer mouse model,the results of in vivo distribution and photothermal conversion experiments showed that,compared with free ICG,LS-Al-ICG showed enhanced accumulation at the tumor site and exhibited excellent in vivo photothermal ability.In vivo anti-tumor results showed that LS-Al-ICG inhibited the primary tumor and delayed the growth of distant tumor under the irradiation of 808 nm NIR laser,which improved the survival rate of mice.LS-Al-ICG mediated PTT and immunotherapy sginificantly enhanced the antitumor effect in combination with PD-1 inhibitor.No obvious decline in body weight was observed during the treating period,and no damage to major organs was observed via histopathology analysis,demonstrating the excellent biosafety of LS-Al-ICG.In vivo antitumor mechanism studies showed that photothermal-immunotherapy could effectively promote the maturation of dendritic cells,then the proportion of cytotoxic T cells and helper T cells in spleen and the level of TNF-α,IFN-y and IL-2 was increased.Conclusion In this study,lignin supramolecular self-assembly has been successfully prepared,with small and uniform particle size;it has pH-sensitive properties,good in vitro photothermal conversion ability and biocompatibility.The cytological evaluation showed that LS-Al-ICG combined with NIR laser irradiation had good antitumor effect.In addition,ICD promoted the maturation of DCs by releasing DAMPs and increased the expression levels of immune-related cytokines.The results of in vivo antitumor and mechanism studies showed that LS-Al-ICG were enriched in tumor site after intravenous injection with excellent photothermal effect.When LS-Al-ICG-mediated photothermal-immunotherapy combined with PD-1 inhibitor,the growth of primary and distant tumor was effectively inhibited by activating the in vivo immune response,showing good therapeutic effect for breast cancer. |
PDF Full Text Request