Environment-responsive Polycations Used As Vaccine And Gene Delivery Systems And Cellular Uptake Mechanisms

Cationic polymers play an important role and become a hotspot in drug and gene delivery systems.The polycation can electrostatically interact with the negatively charged drugs and genes,and then deliver drugs or genes to the target cells or organs for the therapy of diseases.However,the conventinal polycation does not have the disease-specific ability of delivering drugs or genes,so the environmental-responsive polycation came into being.Environmental-responsive polycations have the role of environmental-specific depolymerization,such as pH,redox,temperature,light and magnetic conditions.According to the special microenvironment of different diseases,the different environmental-responsive polycation was prepared,and to used as delivery systems of drugs or genes to achieve better therapeutic effect of diseases.In this study,a redox-responsive hyperbranched poly(amide amine)(PAA)had been designed and used for drug delivery system in accordance with the particular environment of the nasal mucosa surface and tumor microenvironment.1.Redox-responsive biodegradable polycation poly(amido amine)used as intranasal vaccine delivery systemsPolycations have been widely used as mucosal vaccine delivery systems due to their permeation enhancement effect.Preferably,disulfide bonds-based redox-sensitive polycations could respond to the higher intracellular glutathione concentration and degrade in the cytoplasm,which are particularly suitable for antigen delivery.In this work,we evaluated the potential of redox-sensitive,biodegradable polycation poly(amido amine)(PAA)as mucosal vaccine carriers.From the primary studies with ovalbumin antigen,it is found that PAA could encapsulate antigen,enhance cellular uptake of antigen,stimulate maturation of DC2.4,prolong antigen residence in nasal cavity,and promote antigen permeation into nasal submucosal layer.Further,Balb/c mice were intranasally immunized with PAA-delivered recombinant hemagglutinin(HA)antigen protein of H7N9 influenza virus.The PAA/HA formulations induced significantly more potent systemic IgG response and mucosal IgA response,higher splenocyte proliferation activity,higher secretion levels of cytokines IFN-γ and IL-4 by splenocytes,more memory CD4+ and CD8+ T cells,and more DCs expressing MHC Ⅱ molecule.From the results,the redox-responsive polycation PAA as vaccine carriers helped elicit more potent cellular and humoral immune responses.Particularly,PAA induced much higher cellular immune response.The intelligent PAA could be developed as efficient mucosal vaccine delivery systems for clinical applications.2.iRGD-functionalized redox-responsive hyperbranched poly(amido amine)delivering EGFR siRNA for lung cancer therapyBio-safety and targeting ability of gene delivery systems are two critical aspects for efficient gene therapy of cancers.In the present study,redox-responsive poly(amido amine)(PAA)with good biocompatibility and biodegradation was synthesized and used as gene carrier materials.Then,iRGD as a tumor-specific tissue penetration peptide was conjugated to PAA through amidation reaction.The cytotoxicity and cellular uptake of PAA-iRGD were determined in H1299 cells,which indicated that PAA-iRGD had lower cytotoxicity and higher cellular uptake efficiency than PAA.Here,a siRNA specific to epidermal growth factor receptor(EGFR)over-expressed on lung cancer cell surface and often targeted in lung cancer treatment,was designed to silence EGFR(termed as siEGFR)and delivered by the gene carrier PAA-iRGD.EGFR gene silencing,apoptosis,anti-proliferation and anti-tumor effects of PAA-iRGD/siEGFR were evaluated in vitro and in vivo.PAA-iRGD/siEGFR displayed much higher gene silencing ability compared with PAA and polyethyleneimine(25 kDa),significantly inhibited the proliferation and migration of H1299 cells,and significantly elicited the apoptosis of the cells.Moreover,intravenously injected PAA-iRGD/siEGFR inhibited lung tumor growth in vivo.These results suggest that PAA-iRGD with good biocompatibility,biodegradation and targeting ability could be a promising gene delivery system for gene therapy of cancers.3.Biocompatibility and cellular uptake mechanisms of poly(N-isopropylacrylamide)in different cellsThermosensitive poly(N-isopropylacrylamide)(PNIPAM)is widely used in various biomedical applications including drug delivery systems,gene delivery systems,switching devices,sensors,diagnostic assays etc.To promote these clinical applications,it is essential to have a comprehensive understanding of the biosafety of PNIPAM and the interaction of PNIPAM with different cell lines,however,which has little research until now.In this work,we evaluated the biocompatibility of PNIPAM including cell viability,nitric oxide production and apoptosis of macrophages RAW264.7,HBE,A549 and HUVEC cells in the presence of PNIPAM.We have also examined the cellular uptake mechanisms of PNIPAM using endocytic inhibitors,and insighted into the intracellular co-localization of PNIPAM using confocal laser scanning microscope.The results showed that PNIPAM had good biocompatibility,and could be internalized by theses cells.It is macropinocytosis that PNIPAM could be internalized in RAW264.7 cells,and caveolae-mediated endocytosis in HBE,A549 and HUVEC cells.In addition,we also evidenced that intracellular PNIPAM was co-localizated with lysosome.The study provided important information for the development and clinical applications of PNIPAM in the biomedical field.