Application Of Targeting Peptide-modified Polypeptide Nanogels With Drug For Bladder Cancer Intravesical Chemotherapy

Background:Bladder Cancer is one of the most common malignant tumors of the urinary system in clinic.The frequent recurrence and disease progression of bladder cancer after surgical resection is the main difficulty in current treatment.Postoperative intravesical instillation of chemotherapy drugs directly is an effective auxiliary method for preventing recurrence.However,due to some factors such as insufficient selectivity,short retention time,and poor permeability,the conventional therapy cannot achieve the expected effect of preventing the recurrence and progression of BC.In recent years,the application of nanotechnology in the field of biopharmaceuticals has provided new ideas and options for the development of intravesical instillation drugs.Some studies have designed and prepared a variety of nano-drugs to improve the effect of perfusion therapy,but there are a series of problems that the drugs act on non-target sites and cannot penetrate deep into the tumor,and poor biocompatibility limits the clinical translation.In order to break through this bottleneck,a new functional polypeptide material is urgently needed.This material must have many advantages,such as good biocompatibility,biodegradability and easy functional modification.Objective:In this work,we prepared a reduction-responsive PLZ4-poly(ethylene glycol)-poly(L-phenylalanine-co-L-cystine)nanocarriers(PLZ4-poly(ethylene glycol)-poly(L-phenylalanine-co-L-cystine),PLZ4-PEG-P(LP-co-LC)),and loaded with Epirubicin(EPI)nanoparticle drug-loading system to improve tumor targeting,mucoadhesion and permeability of EPI,while achieving precisely controlled intracellular drug release to optimize bladder cancer perfusion therapy.Method:In order to overcome the challenges in the current bladder cancer treatment,we synthesised targeting EPI-loaded nanoparticles,and then we assessed the characterization,the physicochemical properties,and the anti-tumor effects of nano-drugs in in vitro and in in vivo.The specific research plan includes the following three aspects:(1)The amino-terminated maleimide-polyethylene glycol(Mal-PEG-NH₂)and the amino-terminated polyethylene glycol monomethyl ether(m PEG-NH₂)were mixed in a certain proportion,and L-phenylalanine NCA(LP NCA)and L-cystine NCA(LC NCA)subjected to ring-opening polymerization The amphiphilic polyethylene glycol-poly(L-phenylalanine-co-L-cystine)(PEG-P(LP-co-LC))was synthesized by ring-opening polymerization(ROP)and deprotection reaction.In the production,PEG provided a hydrophilic segment,and P(LP-co-LC)is a hydrophobic and reduction-responsive polypeptide.Subsequently,cysteine-terminated PLZ4(bladder cancer targeting peptides,Targeting peptides,TPs)were modified to the maleimide end of the prepared nanoparticle by Michael addition reaction.The chemotherapeutic drug EPI was loaded into the above-mentioned nanoparticle core by nanoprecipitation technology to prepare a highly selective polypeptide drug-loaded nanogel PLZ4-NP/EPI.And then,the physicochemical properties of the prepared drug-loaded nanoparticles were systematically characterized,such as drug-loading performance,particle size,morphology and so on.(2)Confocal laser scanning microscope(CLSM)was used to assess the cell endocytosis and intracellular drug release behavior of PLZ4-NP/EPI in bladder cancer T24 cells qualitatively,and validated the endocytosis of the drug using flow cytometry.The standard methylthiazolyl tetrazolium(MTT)method was used to detect the potential cytotoxicity of PLZ4-NP/EPI;the cell clone formation assay was used to detect the effect of PLZ4-NP/EPI on the proliferation ability of bladder cancer cells;The plate scratch healing test was used to detect the effect of PLZ4-NP/EPI on the migration ability of bladder cancer cells.(3)CLSM was used to evaluate mucoadhesiveness and permeability permeability of PLZ4-NP/EPI.The tissue distribution of PLZ4-NP/EPI was analyzed by a multifunctional microplate reader.The SD rat orthotopic bladder cancer model was induced and the antitumor effect of PLZ4-NP/EPI was systematically evaluated.Cystography assessed the size and the development of bladder cancer.The body weight of experimental animals was monitored and recorded periodically to evaluate the systemic toxicity of PLZ4-NP/EPI.In order to further accurately evaluate the antitumor activity of PLZ4-NP/EPI,the histopathological examination and immunohistochemical staining of the bladder were performed after the end of intravesical instillation chemotherapy.Finally,the safety of nanomedicines was evaluated by biochemical indications and histopathological detection of major organ.Results:Part I:The experimental results showed that PLZ4 successfully modified the surface of NP/EPI with suitable hydrodynamic diameter,stable existence and reduction responsiveness.In addition,the drug loading rate can meet the basic requirements of subsequent experiments.Part II:The experimental results showed that PLZ4-NP/EPI can significantly enhance the uptake of EPI and increase the accumulation of EPI in cells in vitro.In addition,PLZ4-NP/EPI reduced the half-inhibitory concentration of the drug,while the half-inhibitory concentration of NP/EPI and Free EPI on cells was higher than that of PLZ4-NP/EPI.Meanwhile,PLZ4-NP/EPI could significantly inhibit the proliferation and migration of bladder cancer cells,while NP/EPI and Free EPI had weaker inhibitory effects on bladder cancer cells than PLZ4-NP/EPI.Part III:The experimental results showed that PLZ4-NP/EPI enhanced the adhesion and permeability of the drug,prolonged the retention time of the drug in the bladder,and had a stronger anticancer effect on bladder cancer than NP/EPI and Free EPI.By observing the H&E pathological results of each organ and analyzing the results of serum components,it was shown that PLZ4-NP/EPI was less toxic to animals.Conclusions:1.The targeting peptide-modified reduction-responsive carrier PLZ4-NP and its drug-loaded nanoparticles PLZ4-NP/EPI were successfully synthesized;2.Bladder cancer targeting peptide PLZ4 enhanced the ability of PLZ4-NP/EPI to recognized bladder cancer,and enhanced the adhesion and penetration of EPI to bladder cancer tissue;3.PLZ4-NP/EPI can better treat bladder cancer and had a good application prospect.