Construction Of A Dual-responsive Degradation Nanoplatform For Tumor Photothermal-antiangiogenic Synergistic Therapy

Objectives:A hollow spherical Mn Ca P nanoparticle was prepared,and Mn Ca P-MNPs-Thd（MMT）nanoplatforms with photothermal and antiangiogenic effects were obtained after loading endogenous photothermal therapeutic agents melanin nanoparticles（MNPs）and antiangiogenic drug thalidomide（Thd）.Under the stimulation of tumor weak acid microenvironment and near-infrared laser,the hollow spherical nanoplatform degraded and loaded Thd,Ca²⁺and Mn²⁺ions were released and realized the photothermal therapy（PTT）combined antiangiogenesis of tumor guided by photoacoustic imaging（PAI）and magnetic resonance imaging（MRI）.Methods:1.Preparation and Characterization of nanoplatformsHollow spherical Mn Ca P nanoparticles（NPs）were prepared by the biomineralization method.After loading endogenous MNPs and Thd throughπ-πadsorption,Mn Ca P-MNPs-Thd nanoplatforms were obtained.The morphology of nanoplatforms was viewed by transmission electron microscopy（TEM）and the size was determined by dynamic light scattering（DLS）.Surface electrical properties were analyzed by zeta potential and UV-vis-NIR was used to evaluate the absorption spectrum.Besides,Fourier-transform infrared（FT-IR）,thermogravimetric analysis（TGA）and nitrogen adsorption were performed to characterize the physical and chemical properties of nanoplatforms.The released ions and drug were detected under the stimulation of weak acid（p H 5.5,6.5 and 7.4）and laser irradiation to analyze the degradation performance.Different concentrations of MMT NPs irradiated by 808 nm laser to measure the temperature for evaluating photothermal performance.The PAI and MRI signal at different concentrations were measured to evaluate the imaging performance in vitro.2.Evaluation of In vitro performanceCCK-8 assay was used to evaluate the cytotoxicity of the nanoplatforms to 4T1 cells,and FITC fluorescence staining was used to evaluate the cellular uptake.The therapeutic effect of the nanoplatforms in vitro under different stimulus conditions was evaluated using live and dead cell staining assays.Changes in mitochondrial membrane potential and the intracellular Ca²⁺concentration were detected separately by a mitochondrial membrane potential assay kit with JC-1 dye and Fluo-4 AM working solution.3.Evaluation of In vivo performanceThe subcutaneous tumor model of nude mice was established,MRI and PAI in vivo were performed to analyze the imaging performance of MMT NPs before and after administration.Various indicators of mice were detected regularly within 14 days after treatment to evaluate the tumor treatment effect of nanoplatforms.Platelet endothelial cell adhesion molecule-1（CD31）and vascular endothelial growth factor（VEGF）were evaluated by immunohistochemical staining of tumor sites to assess the antiangiogenesis effect of MMT NPs.The blood biochemical analysis of liver and kidney function and H&E staining of main organs after treatment to evaluate the biological safety.Results:1.TEM and DLS results show that the synthesized nanoplatforms have a hollow spherical structure with homogeneous diameter distribution.The size of MMT NPs increased from 164 nm to 220 nm after the hollow spherical Mn Ca P NPs connected with MNPs and Thd.The potential also increased from negative gradually.The UV-vis-NIR characteristic absorption peak and the characteristic peak of FT-IR proved the successful preparation of MMT NPs.Under the dual-stimulation of the weak acid and laser irradiation,the nanoplatform showed a trend of gradual degradation,and increasing drug and ions released with enhancing of the acidity and the laser stimulation will promote the release performance.The solution temperature of the MMT NPs gradually increased in a concentration-dependent and power-dependent manner under the irradiation of 808 nm laser,indicating that the nanoplatform has excellent photothermal properties.The PAI and MRI of MMT NPs with different concentrations showed that the signal intensity is dependent on concentration,suggesting the good performance of PAI and MRI.2.After connecting the endogenous photothermal conversion agent MNPs with hollow Mn Ca P NPs,the cytotoxicity test showed that the cell viability remained above 80%even at the concentration of 1000μg m L^-1,indicating the low toxicity and good biocompatibility.The cytotoxicity is significantly enhanced after loading Thd,demonstrating the killing effect of Thd.Cell uptake experiments showed that the uptake of MMT NPs by 4T1 increased with time going by.Live and dead cell experiments showed the outstanding therapeutic effect of MMT NPs against tumor cells under the cooperative stimulation of weak acid and laser irradiation.The dual stimulation of weak acid and laser irradiation had the greatest impact on mitochondrial membrane potential and intracellular calcium concentration.3.MRI and PAI showed a significant signal of the tumor site after intratumorally injected MMT NPs due to good imaging performance in vivo.Studies on the therapeutic effect of the tumor showed that the tumor almost disappeared in mice after 14 days of treatment under the synergistic effect of photothermal and anti-angiogenesis.Meanwhile,mitochondrial damage caused by Ca²⁺ions release can also enhance the therapeutic effect of the tumor.Immunohistochemical staining analysis showed that MMT NPs reduced the expression of CD31 and VEGF in the tumor site,and then inhibited angiogenesis in the tumor site.The results of blood biochemical analysis and H&E analysis showed that the nanoplatform had few side effects on the normal tissues with good biocompatibility.Conclusion:To sum up,dual-responsive hollow spherical Mn Ca P NPs were fabricated by biomineralization,and then progressively loaded MNPs and Thd to form MM NPs and MMT NPs to achieve the synergetic treatment of tumor guided by dual-mode imaging.Under the synergetic stimulation of tumor weak acid environment and NIR laser irradiation,the hollow spherical structure nanoparticles could disintegrate and release different functional components which perform their respective roles.The released Mn²⁺and MNPs could mediate tumor MRI and PAI,which could lay the foundation for tumor treatment.Simultaneously,under the irradiation of 808 nm laser,the released MNPs could produce favorable photothermal performance.In combination with PTT behavior from MNPs,antiangiogenic effects of Thd,as well as mitochondrial damage caused by intracellular Ca²⁺overload,nanoplatforms achieve efficient tumor synergistic therapy.Besides,with the characteristics of non-toxicity and biodegradability,the MMT nanoplatform provide a promising strategy for clinic translation.