In Situ Self-assembly Polypeptide Combined With Gemcitabine Prodrug For Pancreatic Cancer Treatment

Background:Pancreatic ductal adenocarcinoma(PDAC)is the most malignant tumor with increasing incidence rate in recent years.Moreover,the five-year survival rate of pancreatic cancer is below 5%.Most of the patients with local diseases have no identifiable symptoms or signs.Only less than 20%of patients with pancreatic cancer can undergo radical resection,and most patients can only receive cytotoxic chemotherapy.Gemcitabine has been used as a first-line chemotherapy drug for decades.Due to its serious side effects and chemotherapy resistance,gemcitabine has not achieved statisfying results in clinical treatment.Therefore,it is imperative to explore novel therapies for advanced pancreatic cancer.As drug carriers,nanomaterials show great advantages in tumor treatment,which attract clinical doctors'interest.Polypeptide micelles can self assemble into nanoparticles in water.Its hydrophobic core can load a variety of drugs.However,the hydrophilicity of gemcitabine makes it difficult to be loaded into the core.In order to solve this problem,we designed and prepared a reduction-responsive gemcitabine prodrug(LA-SS-GEM).Its hydrophobic stearic acid side chain containing disulfide bond can increase its hydrophobicity and make it successfully loaded into the core of nanoparticles.The reductive microenvironment in tumor cells can break the disulfide bond,sulfhydryl attack the carbonyl carbon as a nucleophilic reagent,produce intramolecular cyclization,remove the five membered ring,and finally release gemcitabine completely.In this work,methoxy poly(ethylene glycol)-poly(L-alanin-co-phosphorylation tyrosine)(mPEG_2k-b-P(PO(OH)₂-Tyr-co-Ala)was designed and perpared.The phosphate group can be removed under the catalysis of alkaline phosphatase(ALP)which is highly expressed in tumor sites.After that,the micelles can be self-assembled?-sheet nanofibers.This process can specifically kill tumor cells,limit the movement of tumor cells,and release the loaded drugs.The drug-loaded nanoparticles(NPs/LA-SS-GEM)was prepared by loading LA-SS-GEM into mPEG_2k-b-P(PO(O H)₂-Tyr-co-Ala.It can kill PDAC cells,limit the metastasis of PDAC,and reduce the side effects of treatment,thus achieving the treatment of PDAC.Objective:To explore the physical and chemical properties of NPs/LA-SS-GEM and further explore the effect of the preparation on the proliferation and movement of PDAC cells.Finally,in vivo experiments were conducted to verify the anti-tumor effect of NPs/LA-SS-GEM,thus providing a theoretical basis for developing new therapeutic agents for PDAC.The specific research scheme is divided into the following three parts:Part I:We designed and parpered amphiphilic polyethylene glycol poly(L-alanine-co-phosphorylated L-tyrosine)(mPEG_2k-b-P(PO(OH)₂-Tyr-co-Ala)micelles by ring opening polymerization and reduction-responsive gemcatibine prodrug(LA-SS-GEM).The drug-loaded nanoparticles(NPs/LA-SS-GEM)and the negative control(NPs/LA-GEM)were obtained by loading the prodrug into the core of the micelle by nano precipitation method.The physicochemical properties of the preparation were systematically characterized,including particle size,drug loading rate and drug loading efficiency,self-assembly under enzyme catalysis and the characteristic of drug release.Part II:The ability of different cells to micellar endocytosis was measured by laser confocal microscope and flow cytometry.Furthermore,the effects of different preparations on cell viability,F-actin,cell cycle,apoptosis,cell migration and cell invasion were measured.At the same time,the nanofibers formed in the cells were observed by electron microscope.Part III:The antitumor effect of NPs/LA-SS-GEM was systematically evaluated by constructing the subcutaneous transplanted tumor model of C57BL/6N mice.The toxic side effects of the preparation were evaluated by recording the changes of animal weight and normal organ sections.The antitumor effect of NPs/LA-SS-GEM was determined by histopathology and immunohistochemistry,and the morphology of nanofibers was observed by electron microscope.Finally,the possible anti-tumor mechanism was explored by metabonomics.Results and conclusion:In this work,we found that NPs/LA-SS-GEM with a diameter of about 80 nm has high drug loading rate and drug loading efficiency.It can self assemble to form nanofibers under the catalysis of ALP.At the same time,the prodrug can quickly release gemcitabine under the action of glutathione.In in vitro experiments,NPs/LA-SS-GEM can specifically kill pancreatic cancer cells without obvious toxic side effects on normal pancreatic ductal cells.At the same time,it can form nanofibers inside and outside the cells,which can affect the formation of F-actin and cause apoptosis.Interestingly,NPs/LA-SS-GEM can limit the increase of cell migration caused by free gemcatibine,and plays a significant role in limiting drug-induced tumor metastasis.