Study Of A Supramolecular Self-assembling Peptide-based Hydrogel Drug Delivery System In The Treatment Of Rheumatoid Arthritis

Background:Rheumatoid arthritis(RA)is a systemic autoimmune disease that mainly involves the joints around the body,and may eventually lead to the loss of joint function and greatly affect the life of patients.Currently,the primary treatment of RA is still drug therapy,which has certain effects on delaying the progression of the disease and anti-inflammatory.However,its long-term effectiveness and side effects still exist,which has a negative impact on the treatment of RA.Recently,more and more biomaterials have been invented such as liposomes,hydrogels and carbon nanoparticles.Because of their excellent performance in biocompatibility,bio-responsiveness and adjustability,they have become a hotspot of drug delivery system.Among them,supramolecular self-assembling peptide-based hydrogels can be released slowly in the form of a mixture of nanostructures and small aggregates,so that the drug can be sustained released.Natural amino acids are L-type,and D-type amino acids are non-natural amino acids.The polypeptides composed of them have different biocompatibility,which can improve the selectivity of drugs,so as to reduce the side effects of drugs and improve the therapeutic effect of drugs.Therefore,supramolecular self-assembling peptide-based hydrogels have a promising application prospect.Purpose:1.To form a supramolecular self-assembling peptide-based hydrogel material(Nap-GFFY)and its D-configuration polypeptide drug delivery system in order to carry methotrexate(MTX);2.To verify the in vitro toxicity of this drug delivery system and its inhibitory effect on the proliferation and migration of RA fibroblast synoviocytes;3.To verify and explore the anti-inflammatory effect of this drug delivery system and its mechanism;4.To verify the toxicity and therapeutic effect of this drug delivery system in vivo.Methods:This experiment will be divided into three aspects:material characterization,cell experiment and animal experiment.Supramolecular self-assembling peptide-based hydrogel(Nap-GFFY)was prepared by Fmoc solid phase polypeptide synthesis(SPPS),and its D-configuration peptide was synthesized from the same D-configuration amino acids.The method of preparing MTX-loaded supramolecular self-assembling peptide hydrogel(MTX-GFFY)is also SPPS.The physical and chemical properties,such as material characterization,drug release and biological function were studied by transmission electron microscopy and mass spectrometry.MTT assay,scratch assay and Transwell assay were used to study the in vitro toxicity of this drug delivery system and its effect on the proliferation and migration of RA fibroblast synoviocytes.Real-time quantitative PCR(qPCR)and Western Blot were used to investigate its effect on phenotypic differentiation of macrophages.Finally,the antigen-induced arthritis(AIA)model mice were treated with this drug delivery system,the efficacy and safety of the treatment in vivo were evaluated by H&E staining,Safarin-O staining,symptom score,liver and kidney tissue sections,etc.Data were analyzed in SPSS 20.0,Image J,GraphPad Prism 8.0,PS 2018 and other software.Results:In the experiment,the supramolecular self-assembling peptide-based hydrogel and its D-configuration drug delivery showed significant sustained-release effect compared to L-type.This MTX-loaded drug delivery system especially its D-configuration can effectively inhibit the proliferation and migration of RA fibroblast synoviocytes without obvious in vitro toxicity.It had great anti-inflammatory effectiveness by inhibiting the differentiation of macrophages to M1 type.By the meantime,it didn't inhibit the differentiation of macrophages to M2 type.Thus,it wouldn't interfere the process of inflammation healing.Finally,in vivo experiments,we confirmed that this MTX-Loaded drug delivery system could effectively improve the clinical and pathological scores of arthritis model mice,without significant toxicity as well.It is worth noting that the drug delivery system of D peptide sequence is superior to that of natural L peptide.Conclusion:My experiment confirmed that the Nap-GFFY supramolecular self-assembling peptide-based hydrogel and especially its D-configuration drug delivery system loaded with MTX can effectively reduce the side effects of the target drug,as well as achieve the purpose of sustained release,thus to increase the drug effect in the treatment of RA.