Construction Of Polymeric Carriers For Intracellular Protein Delivery And Their Applications In Cancer Therapy

The intracellular targets of proteins are of great significance for the study of biological functions and the treatment of diseases,but the realization of these applications is limited by the lack of efficient intracellular delivery carriers.There are several important steps for effective intracellular protein delivery: effective binding of carriers with proteins,efficient internalization and endosomal escape,and timely intracellular release of proteins.Ideal protein delivery carriers that meet the above requirements are needed to improve the efficiency of intracellular delivery.In order to achieve this goal,traditional design concepts of carriers are often complicated,and it is inevitable to modify the carrier with a variety of functional units to endow the carrier with multifunctional characteristics.In this thesis,we propose a designing concept that polyamindoamine dendrimer(PAMAM)is used as a framework which can be modified with a single structural unit to realize the multi-functionalization.In the first part of this thesis,a polymeric material library was obtained by modifying tertiary amines bearing alkyl chains of varying lengths on the surface of PAMAM.The effects of tertiary amines on protein binding,endosomal escape and intracellular delivery were systematically analyzed.On this basis,in the second part of the thesis,we modified PAMAM with resiquimod(R848)adjuvant,a molecule possesses both structural function and biological activity,as a carrier to deliver protein antigen and tumor cell lysate for cancer immunotherapy.The specific research contents are as follows:1.A pH-sensitive intracellular protein delivery system based on the fifth generation polyamindoamine dendrimer G5-PAMAM was constructed.A polymeric material library was obtained by modifying tertiary amines bearing alkyl chains of varying lengths on the surface of PAMAM.Then bovine serum albumin and β-galactosidase were used as model proteins to study the effects of different tertiary amines on the structure-activity relationships including the stability of complex nanoparticles,cellular uptake,endosomal escape,hemolysis activity,and protein release.A PAMAM derivative(G5-C4)modified with N,N-dibutylaminoethyl moiety was screened for higher intracellular protein delivery efficiency.The results showed that the delivery efficiency was closely related to the pH-responsive feature of G5-C4.At the neutral pH,G5-C4 could enhance the protein binding through the hydrophobic effect of tertiary amine to obtain stable complex nanoparticles.Under the stimulation of acidic endosomal environment,the structure of G5-C4 tertiary amine underwent hydrophobic-to-hydrophilic transition as well as neutral-to-positive charged transition,which promoted the release of protein,enhanced the interaction between polymer and endosomal membrane to facilitate the endosomal escape,and effectively overcame the three obstacles to the intracellular protein delivery.In vivo tumor therapy experiment showed that delivery of ribonuclease A cytotoxic protein by G5-C4 could effectively inhibit tumor growth.2.Based on the in-depth understanding of the principle of intracellular protein delivery,an adjuvant-assisted intracellular delivery system for protein antigens was developed for cancer immunotherapy.R848 derivative was modified on the surface of G5-PAMAM to obtain G5-R848,a carrier that intergrated both the structural function and biological activity together for efficient generation of tumor vaccine.In terms of the structural function,R848 could enhance the binding ability of carrier with protein antigen through hydrophobic-hydrophobic and π-πstacking interactions to form stable nanocomplexes.The membrane-fusing property of the benzene ring in R848 could facilitate the internalization and endosomal escape of G5-R848/antigen complex.Disulfide bond in R848 derivative could promote the release of protein antigen through cytoplasmic reduction response.In terms of biological activity,R848 is an agonist of TLR7/8 and can be used as immune adjuvant to enhance the immune activity of antigens.In vitro and in vivo experiments demonstrated that the G5-R848 nanovaccine achieved efficient antigen cross-presentation and further stimulation of cellular immunity,resulting in the activation and proliferation of antigen-specific T cells.The minimalist nanovaccine consisting of G5-R848 and tumor antigens showed remarkable effects on tumor prevention and growth inhibition.G5-R848 could be combined with the cell lysate derived from the resected tumor tissues to form personalized tumor nanovaccine.The nanovaccine effectively inhibited postoperative tumor recurrence and metastasis,and prolonged the survival time of mice by itself or in combination with immune checkpoint inhibition therapy.