Preparation Of Regulable AuNRs@MOFs-FA Nanocomposite For Single Laser Activated Synergistic PDT/PTT In Cervical Cancer

Cervical cancer is the second common female malignancies in China with the squamous cell carcinoma as the main histopathological type.In recent years,patients tend to be younger female.The treatments of cervical cancer mainly consist of surgery,chemotherapy and radiotherapy.Radical surgery makes females completely lose fertility,and their pregnancies are prone to be aborted after cervical cone resection,which is not suitable for young cervical cancer patients with the needs for procreation.Chemotherapy and radiotherapy have severe side effects,and can cause ovarian damage,which will reduce the quality of life for young patients.Therefore,safer and simultaneously effective treatment is needed.Photodynamic therapy(PDT),characterized by photosensitizers accumulated in the tumor tissue absorbing light and then reacting with the surrounding oxygen to generate excessive ROS,has caused widespread concern on account of the excellent selectivity,only acting on the region of laser irradiation,retaining organ anatomical and functional integrity,non-invasive operations,minor side effects,the advantages of the repeated treatment.PDT has been applied in treatment of young patients with high-risk HPV persistent infection and CIN diseases.PDT may be able to provide more security management strategies for CIN even early-stage cervican cancer of young patients with generational demands.Excessive ROS generated in PDT,mainly singlet oxygen(102),can cause irreversible damage to important cellular components and functional structures,and then result in cancer cell death.However,the hypoxic microenvironment of cervical cancer is not conducive to the generation of 1O2,and the most commonly used photosensitizer,porphyrin,has power cervical cancer target,which mainly limit the application of PDT for treating cervical cancer in clinic.It was found that H2O2 level was increased in tumor cells.Besides,the photothermal therapy(PTT),is an oxygen independent treatment method which ablates tumor cells through local hyperthermia generated by laser activated photothermal agents.Meanwhile,folate receptor alpha(FR?)is over-expressed in cervical cancer and the FR? overexpression is associated with the occurrence and progression of cervical cancer.Therefore,folic acid target,catalyzing the decomposition of intracellular increased H2O2 into O2,combined with PTT can effectively make up for the weakness of PDT in the treatment of cervical cancer.Usually,twice lasers was needed to play the role of synergistic PDT and PTT due to the spectral mismatch of photothermal material and photosensitizer,which is inexpedient to clinical operation.Realizing synergistic PDT and PTT effects under single laser irradiation is more conducive to clinical conversion.[Objective]This thesis aimed to overcome the weakness of hypoxic on PDT in cervical cancer,enhance cervical cancer targeting,regulate the photothermal and photodynamic materials to realize PDT and PTT by single laser source irradiating.Synthesized AuNRs@MOFs-FA core-shell nano-composite,which coated photosensitive materials,Fe3O-porphyrin-MOFs,with the ability of catalyzing the decomposition of H2O2 into O2,on the regulatory synthesis of AuNRs with the same absorption peak of porphyrin in 660 nm.The thickness of outer shell could be controlled successfully.Furthermore,folic acid was connected to the surface of MOFs to enhance cervical cancer targeting.Explore the photodynamic,catalytic and best photothermal efficacy of AuNRs@MOFs-FA with the specific thickness of shell under 660 nm laser irradiation,and then explore the anti-tumor effect of the AuNRs@MOFs-FA consisting of synergistic PDT/PTT effects on cervical cancer and the mechanism of cervical cancer cell death.[Method](1)According to seed-mediated method with some modifications,regulated the concentration of Ag+in growth solution to synthesize the specific AuNRs whose absorption peak matches with porphyrin.AuNRs was enriched with carboxyl functional groups by the formation of Au-S bond.Then coated AuNRs with Fe3O-porphyrin-MOFs by the formation of coordination bonds by metal node-Fe3O clusters and organic ligand-tetracarboxylic porphyrin,in the light of Layer-by-Layer synthesis.Finally,connect folic acid on the outer MOFs shell by the carboxyl group of folic acid ligand connecting with Fe3O clusters.AuNRs@MOFs-FA,a multifunctional nanocomposite with PTT effect,PDT effect in hypoxia and folic acid target under the single light source of 660 nm,was constructed,the thickness of MOFs shell could be regulated by controlling the number of layers.(2)The nanocomposite was characterized by absorption spectroscopy and TEM,and the generation of heat and 1O2 under the 660 nm laser irradiation was investigated.The nanocomposite with the best PTT effect(Au/MOFD)in this shell was selected to investigate the PDT efficiency and the property of catalyzing decomposition of H2O2 into O2.(3)hSCC cell-CaSki were used as the research model to investigate the uptake of AuNRs@MOFs-FA,the biosecurity of AuNRs@MOFs-FA without irradiation,the killing effect and the mechanism of tumor cell death under irradiation.(4)Establish subcutaneous hSCC bearing model in nude mice,then divide the mice operated with directly injecting AuNRs@MOFs-FA into the tumor following irradiating into experimental group,injecting PBS into the tumor without irradiation into control group.Verified the PTT effect in vivo through infrared thermal imaging.Observed and recorded the growing status and tumor growth of nude mice in the experimental and control group,then calculated the tumor-inhibiting rate of experimental group,and preliminarily evaluated the safety of the treatment.[Result](1)AuNRs with longitudinal LSPRs mainly at 655-665 nm were successfully prepared,and the average longitudinal length of AuNRs was 52 ± 5 nm.By regulating the number of layers(Fe3O-porphyrin as a whole layer),MOFs-FA shell coated the AuNRs with different thickness were formed,2.59 ± 0.38 nm(two layers),5.43 ± 0.81 nm(three layers),and 8.12 ±0.80 nm(four layers),respectively.The absorption spectrum notarized the specific Q band of porhypins-MOFs.(2)The temperature detection experiment showed that AuNRs@MOFs-FA(Au@MOFD)with 5.43±0.81 nm MOFs-shell has the best PTT effect under 660 nm irradiation.The 1O2 detection experiment confirmed that Au@MOFD could generate enough 102,and the production of heat and 1O2 increased along with the rise of laser intensity.The catalytic experiment of H2O2 proved that Au@MOFD could catalyze the decomposition of H2O2 into O2 and improve the solubility of O2 in water.(3)In vitro cell experiments showed that intracellular Au@MOFD was in the cytoplasm and distributed most evenly after incubating for 48 hours.WST-1 cell proliferation assay showed that when Au@MOFD was less than 1.0 nM,the cells could proliferate normally as same as the control group without irradiation.When irradiated by 660 nm laser,the survival rate of CaSki cells decreased with the increase of Au@MOFD.In the range of 0.4 nM to 0.6 nM,the cell survival rate decreased from 23%to 7%,and the killing effect was obvious.Fluorescence imaging of dead/living cells showed that most of CaSki cells were death upon the laser direct irradiation,and cells survived when incubated by Au@MOFD alone or irradiated by laser alone.The apoptosis staining imaging showed that the CaSki cells underwent early apoptosis under low laser intensity irradiation,while the cells showed late apoptosis or necrosis under high laser intensity irradiation.(4)Animal experiments showed that Au@MOFD had good PTT effect in vivo.After irradiated by 660 nm laser for 5 min,the temperature of tumor site would exceed 50?.The tumor inhibition rate of the experimental group with Au@MOFD and laser irradiation was 93.66%.There was no significant difference of important organ sections between the experimental,the control and normal nude mice.[Conclusion]The controllability of core and shell was successfully achieved by the combination application of seed-mediated method and Layer-by-Layer synthesis.Fe3O-porphyrin-MOFs was successfully coated in AuNRs and connected with folic acid to form a multi-functional nanocomposite,AuNRs@MOFs-FA.Under single laser-660 nm laser irradiation,AuNRs@MOFs-FA could play the role of PTT and PDT effect,and could catalyze the decomposition of high level of H2O2 in tumor cells into O2 for the synthesis of 102.This multifunctional nanocomposite provided a safer and more effective strategy for phototherapy of cervical cancer under single laser irradiation.