Research On Nanoparticle System Of “Swithching Mode” Photodynamic Therapy For Tumors Based On Molecular Recognition

The tumor is a serious threat to human health.Currently,the main treatment methods for tumors include surgical treatment,chemotherapy and radiotherapy.PDT is a form of therapy in which the photosensitizer（PS）produces reactive oxygen species（ROS）via transforming the energy to oxygen under the irradiation of light and damages tumor cells.It has become a research hotspot because of its mild nature,non-invasion and small side effects.However,there are still some problems in the field of PDT,such as the poor water solubility of PS,the low permeability of PS the photosensitizer through the biological barriers the distribution in the tumor efficiently,the phototoxicity to normal organ and tumor microenvironment induced-efficiency reduction.Therefore,to improve the treatment efficiency of PDT and decrease the phototoxicity are the key problems to be solved.At present,the researches focus on the tumor-targeting delivery of PS,the mechanism of tumor treatment of PDT,and the synergistic effect of PDT with other therapy methods.Except for the efficiency of PDT,phototoxicity is also a major obstacle to the wide application of PDT.Therefore,this paper intends to construct a PDT system based on molecular recognition,to improve the PDT efficiency and reduce the phototoxicity of PDT using the concept of molecule activated-switch.In recent years,MnO₂ has been increasingly applied in nanocarriers due to its controlled particle size,the ability to react with reductive GSH,and the ability to catalyze the production of oxygen from hydrogen peroxide.Due to the advantages of steady quality,strong photosensitivity,low toxicity,and rapid metabolism in the body,the second-generation PS,hematoporphyrin monomethyl ether（HMME）,has been widely used clinically for PDT of tumors.Aptamer（APT）of specific sequence has been established to recognize specific tumor cell.In order to endow the PDT system with switching effect,this study utilized mesoporous manganese dioxide nanoparticles（MMnNPs）to quench the efficient ROS generation of PS and construct a switch to PDT.To ensure the PDT switch to respond specifically to tumor cells,hairpin structured APT with both molecular recognition and conformational change capability was used as intelligent sensors for the switch of the PDT system to achieve tumor-specific activation.When the PDT system is in normal tissue,hairpin-structured APT does not recognize the cell,without conformational change or HMME release;instead,HMME was still loaded in the MMnNPs in a quenched state to avoid phototoxicity;When the PDT system is in the tumor tissues,hairpin-structured APT recognizes the MUC-1 on the surface of MCF-7 cells,resulting in conformation change and HMME release.At the same time,high concentrations of hydrogen peroxide in the tumor produces oxygen under the catalysis of manganese dioxide,to offer oxygen source for the generation of ROS.Besides,MnO₂ depletes the GSH in the cells via oxidizing reaction,reducing its effect on the generated ROS,thus,via clearing the upstream and downstream barrier of PDT in the tumor,effective PDT treatment of tumors was established.This research applied ultrasonic recrystallization method to achieve efficiently HMME loading in MMnNPs,with a drug loading rate as high as 88.7%.The hairpin-structured APT,which recognizes the protein MUC-1 on the surface of breast cancer cells（MCF-7）,was used as a gated molecule to block the pores of MMnNPs,increasing biocompatibility and target-function of MCF-7 cells at the same time.The nanostructure was characterized,including its morphology,zeta potential,pore size,and feasibility as a switching material.The results of TEM showed that MMnNPs had uniform particle size,of around 200nm.The XPS results showed that the valence state of Mn was+4.According to the nitrogen adsorption results,the specific surface area of MnO₂ was 123.9982 m²/g,with an average pore diameter of3.23039 nm,and the pore diameter was suitable,to load the drug.The ROS test results showed that MnO₂ had a quenching effect on HMME production of ROS.The experimental results of drug release showed that only when the MUC-1 protein existed,the channel could be opened and HMME could be released.The above results indicated that MMnNPs/HMME-DNA with switching characteristics had been successfully constructed.In the anti-tumor activity experiments in vitro,we selected MCF-7,a kind of breast cancer cell,as a test subject and normal breast cell Hs578Bst acted as the control group to explore the anti-tumor effect of MMnNPs/HMME-DNA under 532nm laser irradiation.The results showed that the carrier material MnO₂ had little toxicity on the two kinds of cells,while the formulation had a significant inhibition rate on MCF-7 cells and inconspicuous inhibition on Hs578Bst cells.Compared with free HMME,the growth inhibition of MCF-7 was enhanced with MMnNPs/HMME-DNA;In the uptake experiment,strong red fluorescence appeared in the cytoplasm of MCF-7 cells in the group treated with MMnNPs/HMME-DNA,indicating that it could release HMME in MCF-7 cells.However,red fluorescence in the Hs578Bst cells was very weak,indicating that HMME could not be released in Hs578Bst cells.In the results of apoptosis test experiments,the apoptosis rate of MCF-7 group is much higher than that of the Hs578Bst group,while there was no significant difference in apoptotic rates between the two cell treated with HMME,indicating that the MMnNPs/HMME-DNA had a certain selectivity for MCF-7 and Hs578Bst.It can be calculated from the change of cell GSH content that the system could reduce the intracellular GSH content and improve treatment for tumors.In the anti-tumor activity experiments in vivo,MCF-7 was selected to establish animal model in nude mice,and the anti-tumor activity of MMnNPs/HMME-DNA in animals was examined.The results of tissue distribution showed that MMnNPs/HMME-DNA could prolong the circulation time of IR783 in the nude compared with the free IR783 group,and have a good targeting effect,so that IR783can effectively accumulate in the tumor site.Immunofluorescence results showed that MnO₂ could improvemend the condition of tumor hypoxia to a certain extent;the results of anti-tumor activity experiments in vivo showed that the tumor cell activated MMnNPs/HMME-DNA could effectively inhibit tumor growth,and the effect was significantly stronger than that of HMME treatment spiked alone.The results of TUNEL apoptosis results further proved that the system could effectively inhibit tumor growth and promoted apoptosis in tumor tissue cells.GSH content in tumor tissues was detected and compared with saline group and the results showed that the content of GSH decreased by nearly 50%.In summary,the studies of the MMnNPs/HMME-DNA bsed tumor cell activated PDT system in vitro and in vivo showed that APT enabled the delivery system to achieve"switch controllability,"and HMME could specifically released in tumor tissue and effectively produce ROS under laser irradiation,killing tumor cells specifically.It is expected to be applied in cancer treatment.