| Objective:The high incidence of prostate cancer seriously threatened the health of men worldwide,especially older men,and given rise to huge economic losses.Reactive oxygen species（ROS）as a DNA damage factor can increase the instability of the tumor genome and help kill tumors.Excessive ROS production in normal cells can also lead to disease.Therefore,the development of precise ROS generators and targeting strategies can improve therapeutic effectiveness and reduction of toxic side effects are crucial.Prostate-specific membrane antigen（PSMA）is overexpressed in prostate cancer and is an ideal target for targeted therapy of prostate cancer.This paper designed and prepared a new type of nanocomposite that amplifies the generation of ROS in tumor cells through chemical cascade reactions,and used anti-PSMA nanobodies as a targeting molecule to modify the nanocomposite to prepare a new drug with targeted therapy for prostate cancer,and evaluate its anti-prostate effect in vitro Cancer effect,providing new therapeutic strategies for targeted therapy of prostate cancer.Methods:1.Synthesis and Characterization of DOX/Mn O2@PDA-GOX/PEG nanocomposite:（1）Preparation of DOX/Mn O2@PDA nanocomposite by template method,electrostatic interaction grafting of glucose oxidase（GOX）and polyethylene glycol（PEG）polymer to prepare DOX/Mn O2@PDA-GOX/PEG nanocomposite.（2）The basic physical properties of nanocomposites were analyzed by TEM,DLS,UV-vis and FT-IR.（3）Evaluate the ROS amplification cascade catalytic effect and biocompatibility of nanocomposites by drug release,optical glutathione（GSH）consumption,ROS generation,hydrogen peroxide（H2O2）generation,p H change,hemolysis rate,cytotoxicity experiments.2.Preparation of anti-PSMA nanobodies:（1）Anti-PSMA nanobodies were panned using the natural phage nanobodies library,and three rounds of affinity panning were performed with PSMA as the target antigen.Individual positive clones were identified by ELISA and analyzed by gene sequencing.（2）The screened positive cloned gene sequence was inserted into the p ET28a prokaryotic expression vector,and transformed into Escherichia coli BL21,and the expression of the recombinant protein was induced by isopropyl-β-D-thiogalactopyranoside（IPTG）,and carried out using a Ni column.Affinity purification,verification of purified products by SDS-PAGE.（3）Specificity of anti-PSMA nanobodies and prostate cancer cells detected by flow cytometry.3.Anti-prostate cancer effect of DOX/Mn O2@PDA-GOX/VHH nanocomposite in vitro:（1）The prepared anti-PSMA nanobodies was used as the targeting agent of the nanocomposite to construct DOX/Mn O2@PDA-GOX/VHH nanocomposite with prostate cancer targeting.The targeting effect of nanocomposite on prostate cancer cells was evaluated by cell uptake experiments.（2）CCK-8 and life/death staining experiments were used to evaluate the anti-tumor effects of nanocomposite in vitro.（3）Changes in intracellular ROS,changes in mitochondrial membrane potential,ATP levels and Changes in GSH content to explore the anti-tumor mechanism of nanocomposite.Results:1.Synthesis and Characterization of DOX/Mn O2@PDA-GOX/PEG nanocomposite:（1）TEM and mapping showed that MSN-Mn O2 nanocomposites were successfully prepared.UV-vis spectra showed that DOX was successfully loaded into MSN-Mn O2 nanocomposites.FT-IR showed that DOX/Mn O2@PDA-GOX/PEG nanocomposites were successfully synthesized.DLS detection showed that the average particle size of the complex is 390 nm,and the Zeta potential is-5 m V.（2）The simulated tumor cell environment,the DOX/Mn O2@PDA-GOX/PEG nanocomposite can release DOX in response to GSH and acidic environment,and the solution in the presence of glucose,the nanocomposite can generate ROS through a chemical cascade catalytic reaction.（3）The hemolysis rate of the Mn O2@PDA-GOX/PEG nanocomposite is less than 5%and the MSN@PDA-PEG nanocomposite has negligible toxicity to normal cells and prostate cancer.2.Preparation of anti-PSMA nanobodies:（1）After three rounds of affinity panning,four PSMA nanobodies sequences VHH1,VHH2,VHH3,and VHH4 were screened.（2）The VHH1 failed to expression protein,while the VHH2,VHH3 and VHH4 proteins were expressed.The purity of anti-PSMA nanobodies with high and relative molecular mass（Mr）of about 15000.（3）The results of flow cytometry showed that VHH3 had the highest affinity to prostate cancer.3.Anti-prostate cancer effect of DOX/Mn O2@PDA-GOX/VHH nanocomposite in vitro:（1）The DOX/Mn O2@PDA-GOX/VHH nanocomposite modified by anti-PSMA nanobodies is more easily phagocytized by prostate cancer cells.（2）Nanocomposite have good antitumor effects and low normal cytotoxicity.（3）Nanocomposite induces intracellular ROS production,damages mitochondrial membrane potential,reduces intracellular ATP level and restores GSH-induced cytotoxicity,thereby achieving anti-prostate cancer effect.Conclusion:In this paper,anti-PSMA nanobodies were used as nanocomposite targeting molecules to amplify the generation of ROS in tumor cells through chemical cascade reactions and cooperate with chemotherapy to target prostate cancer.It has good anti-tumor effect in vitro. |
PDF Full Text Request