Rational Design Of Glutathion-responsive Polymers As Carriers For Intracellular Delivery Of Biomacromolecules And In Vivo Therapy

In recent years,with the increasing proportion of biologics in the global market,biological macromolecular drugs have also achieved explosive development,and global pharmaceutical companies have invested more and more in the research and development of biopharmaceuticals.In the process of research and development of biomacromolecule drugs,the development of delivery carriers is a key point.In this paper,the author has successfully designed and synthesized a series of GSH-responsive Poly(disulfide)s by combining the knowledge of organic synthetic chemistry,materials science.These organic non-viral carriers were then successfully applied to a series of biomacromolecules intracellular delivery and treatment of diseases by combining the knowledge of biology and pharmacology.This paper mainly carries out the following two aspects of research:Part 1.In the past few years,due to the popularity of clustered regularly spaced short palindromic repeats(CRISPR)-Cas9 nuclease technology,the gene editing research has shown a blowout development.This emerging platform has shown tremendous promise in many applications,ranging from basic biology research to bioengineering,and healthcare.Moreover,the efficient delivery of biological macromolecules based on CRISPR-Cas9 genome editing has attracted more and more attentions from biomedical researchers.Delivery of CRISPR-Cas9-based genomeediting biomacromolecules is of particular interest for a wide range of biomedical applications,yet the short of efficient non-viral vectors that can mediate efficient in vitro and in vivo delivery has severely impeded CIRSPR-Cas9 towards therapeutic applications.Herein,we designed a series of poly(disulfide)s and found that the copolymerization of monomer 1(M1)containing cationic diethylenetriamine moieties and monomer 2(M2)containing guanidyl ligands generated an efficient delivery platform for different forms of CRISPR-Cas9-based genome editors,including Cas9-plasmid,Cas9-m RNA and RNP.The excellent delivery performance of designed poly(disulfide)s stems from delicate molecular structures to interact with genomeediting biomacromolecules,efficient intracellular delivery through thiol-mediated translocation pathway,and strong endosomal escape capability by ‘proton-sponge’effects.The degradation of poly(disulfide)s by intracellular glutathione not only promotes the timely release of CRISPR/Cas9 machineries into cytosol,but also minimizes the cytotoxicity that non-degradable polymeric carriers often encounter.These merits collectively account for the excellent ability of poly(disulfide)s to mediate different forms of CRISPR/Cas9 for their efficient genome-editing activities in vitro and in vivo.Part 2.On the basis of the part 1,we further synthesized monomer 2(M2)with guanidine ligand and monomer 3(M3)with phenylboronic acids(PBA),and synthesized a series of bifunctional modified poly(disulfide)s(PBA-CPDs)through by ring-opening copolymerization.The resulted poly(disulfide)s can directly complex a broad range of native,unmodified proteins and peptides via multiple non-covalent forces,regardless of their chemical structure,molecular weight and isoelectric point.The complexation between poly(disulfide)s and proteins can be predominantly internalized by cells via strain-promoted,thiol-mediated translocation,bypassing the classical endocytic pathway.The degradation of the poly(disulfide)is induced by rich intracellular glutathione,thereby timely releasing protein or peptide cargoes in their active form and minimize the cytotoxicity of the carrier.Of note,the surface coating of poly(disulfide)complexes by hyaluronic acid enables the systemic delivery of functional proteins(saporin),demonstrating their therapeutic potentials in vivo.