Application Of Functionalized Polylysine Carrier In Intracellular Delivery Of Peptides

Peptides have great potential in the treatment of diseases such as cancer and vascular disease,and there are more than 100 FDA-approved peptides.Compared with small molecule drugs,peptides have the advantages of easy synthesis,low immunogenicity,high specificity and excellent biocompatibility.Despite these advantages,peptides have a short half-life due to readily hydrolysis by proteases,and lack the ability to cross cell membranes due to entrapment by lysosomes,resulting in low bioavailability.At present,there is a lack of effective technology for peptide delivery across the biological barriers that prevent peptide delivery into cells.To break down these barriers,many attempts have been made,including modifying the molecular structure of peptides to extend the half-life and improve protease resistance,and using cell-transmembrane peptides,liposomes,polymers,and nanoparticles to enhance the cell uptake,endosomal escape and tissue penetration of peptides.In the first chapter of this paper,the progress of peptide-based therapeutics and the strategies to improve the stability and intracellular delivery of peptides were discussed.Despite great progresses in this area,the development of an efficient and robust delivery method for intracellular peptide delivery remains a daunting task.ε-Polylysine(PLL)has good water solubility,biocompatibility and degradability.Here,PLL was selected as the polymeric scaffold to fabricate carriers for intracellular peptide delivery.In the second chapter,a fluorophilic interaction driven collaborative assembly(FIDCA)strategy is proposed to deliver peptides that cannot cross the cell membrane into living cells.The model peptide was first labeled with a fluorous tag bearing 13 fluorine atoms(Tag 13F)by a disulfide bond,and then co-assembled with fluorinated PLL(PLL-13F)to form nanoparticles through a fluorophilic interaction.By this technique,30 peptides with different charge characteristics and polarity were effectively delivered into cells,and the delivery efficiency was significantly higher than that of control materials.In addition,the bioactive peptides delivered by this method remain bioactive after cell internalization.Polypeptide nanoparticles with anticancer activity prepared by this strategy also effectively inhibit tumor growth in a bladder cancer model.In chapter 3,an intracellular peptide delivery strategy based on dynamic chemical bond is developed.PLL was used as the polymer scaffold,and 3,4-dihydroxy benzaldehyde(CAT)was modified on PLL to generate a kind of cationic polycatechols.Based on the material toxicity screening and peptide delivery efficiency,the PLL conjugated with 11 CAT moieties(SPC2)was used for later studies.SPC2 can efficiently bind and deliver phenylboronic acid modified peptides by boronate-catechol bond and can maintain the biological activity of cargo peptide after intracellular delivery.Furthermore,SPC2 can efficiently deliver hydrazine modified peptides by using 4-formylphenylboronic acid(4-FPBA)as a bifunctional anchor,via the formation of boronate-catechol and hydrazine linkages.Besides,SPC2 efficiently delivered a 4-nitrophenyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl carbonate(NBC)functionalized peptide with reactive oxygen species(ROS)responsiveness inside cells to achieve traceless peptide release.In summary,the current study provides several efficient and robust methods for the delivery of peptides into cytosol of living cells.