Preparation Of Targeted Peptide Modified SN38 Nanoprodrugs And Their Antitumor And Antiangiogenic Effects In Vitro And In Vivo

Nanomedicine-based tumor-targeted therapy has become a promising approach to overcome the lack of specificity of conventional chemotherapy drugs and to provide clinicians with the ability to overcome the shortcomings of current cancer treatment methods.Drug delivery systems can be used to specifically target tumors and improve the therapeutic index and pharmacokinetic properties of anticancer drugs.Nanomedicines are submicron sized carrier materials designed to improve the biodistribution of chemotherapeutic drugs for systemic administration.Nanoformulations are designed to improve the balance between efficacy and toxicity of systemic chemotherapy interventions.Polymer nanomicelles are usually selfassembled by amphiphilic block copolymers or graft copolymers to form nanoshells with core-shell structures.Micelle,polyethylene glycol as the hydrophilic segment of the polymer,can effectively increase the biocompatibility of the micelle,and can reduce the removal of the micelles in the reticuloendothelial system,prolong the blood circulation time of the drug,etc.Hydrophobic anti-tumor drugs act as hydrophobic segments and help to significantly increase the amount of anti-tumor drugs.Camptothecin(CPT)is a highly cytotoxic quinoline alkaloid with high antitumor activity.However,the water solubility of SN38 is extremely poor,which makes it impossible to directly apply to clinical applications.The construction of tumor-targeted nanomedicine has become a hotspot and focus in the field of tumor therapy in recent years.In this study,a targeting peptide-modified SN38 prodrug micellar drug delivery system was constructed,and the PEG segment was selected as the hydrophilic moiety.The hydrophobic SN38 molecule C10 is linked to PEG and can form amphiphilic nanomicelles by self-assembly in aqueous solution.A7R(ATWLPPR)is a highly expressed blood vessel on tumor neovascularization,mimetic blood vessels and tumor cells.Endothelial Growth Factor Receptor 2(VEGFR2),and neuropilin(NRP-1)have high affinity linear peptides for the purpose of targeting tumor cells.A7RC-PEG-SN38 nanomicelles are novel,highly efficient,and Safe anticancer drugs have potential clinical application prospects and are worthy of further research.This research mainly includes the following research contents:1.Synthesis and characterization of A7RC-PEG-SN38.OPSS-PEG2000-OH was used as linker,and SN38 and A7 RC polypeptides were ligated at both ends.The synthesized product had an amphiphilic structure and could self-assemble to form micelles in aqueous solution.It was confirmed by experimental study in this chapter that SN38 was determined by NMR.The characteristic peaks of OPSS-PEG2000-OH and A7RC-PEG-SN38 confirm the successful synthesis of the final product.The particle size of the sample is determined by dynamic light scattering.A7RC-PEG-SN38 can form micelle particles of about 100 nm in aqueous solution.The surface of these nanomedicines is modified by PEG.Because of the charge shielding effect of PEG,the potential is relatively low.The morphology of the nanomedicine is observed by SEM.It is a relatively uniform sphere,and the structure of the outer shell and the inner core can be clearly seen.The fetal blood serum was used to simulate the human blood environment.After the incubation for 32 h,the particle size distribution showed some changes.In summary,A7RC-PEG-SN38 micelles can be further studied in vivo by intravenous administration.2.Anti-tumor and anti-angiogenic effects of A7RC-PEG-SN38 in vitro.The cytotoxicity of A7RC-PEG-SN38 on MDA-MB-231 and MCF-7 cell lines was investigated,and the cellular uptake effect was qualitatively and quantitatively analyzed.For MDA-MB-231 cell line,A7 RC polypeptide The introduction of the cell has greatly enhanced its cell killing ability.For the MCF-7 cell line,the cell viability of the targeted peptide group is only slightly lower than that of the non-targeted group.Confocal experiments demonstrate the green fluorescence intensity of the A7RC-PEG-SN38 group.The strongest,flow-through proved that the uptake rate of the MDA-MB-231 cell line targeting group was as high as that after the incubation for 4 h,and the uptake rate of the MCF-7 cell line targeting group was only ··· The ROS level of the drug and the mitochondrial membrane potential were examined.The target group treated MDAMB-231 cell line had the highest ROS content,showing the strongest green fluorescence,and consistent with the mitochondrial membrane potential detection results.The red fluorescence intensity was the weakest and the green fluorescence was the strongest,indicating that the apoptotic cells were the highest.The inhibitory effect on angiogenesis was further investigated.The HUVEC cell line was selected.The final result showed that the inhibitory effect of the targeted drug-administered group was CPT-11 group.1.45 times that of PEG-SN38 1.79 times the targeted group demonstrated a significant effect of inhibiting angiogenesis,the above experiments show that,A7RC-PEG-SN38 can be efficiently taken up by cells,it has superior antitumor activity worthy of further study.3.Antitumor and antiangiogenic effects of A7RC-PEG-SN38 in vivo.This chapter firstly investigated the hemolytic properties of drugs,and then established an ectopic tumor-bearing model of breast cancer in nude mice to evaluate the anti-tumor effect of the drug in vivo.The hemolytic results indicated that CPT-11,PEG-SN38,A7RC-PEG-SN38 were In the range of 5-100 μg/mL,the hemolysis rate was less than 5%.In vivo anti-tumor test results,the A7RC-PEG-SN38 group had superior anti-tumor activity and inhibition compared with the CPT-11 group and the PEG-SN38 group.The tumor volume increase effect was 1.92 times and 1.64 times,respectively.The biodistribution experiment showed that the group with the highest cumulative drug content in the tumor site was A7RC-PEG-SN38 group,which further proved that the people should be able to effectively deliver the drug to the tumor.Regional enhanced uptake,in vivo anti-tumor experiments After the final measurement of tumor completion,the heart,liver,spleen,lung,kidney,colon,small intestine of nude mice were sampled and pathologically analyzed,which proved that in addition to CPT-11,the colon and small intestine were produced.There were no significant differences between the toxic and side effects,PEG-SN38 and A7RC-PEG-SN38 sections and the blank group,indicating that PEG-SN38 and A7RC-PEG-SN38 had no obvious side effects.The expression level of antigen CD31 was analyzed by immunohistochemistry to reflect the amount of new blood vessel production.Finally,compared with other groups,the A7RC-PEG-SN38 group had the lowest antigen expression,indicating that the targeting group can effectively inhibit the formation of new blood vessels.The above experiments prove that the A7RC-PEG-SN38 group has good safety and significant anti-tumor effect in vivo,and it is worthy of further research.