An Intracellular Peptide Delivery Technology Based On Double Dynamic Chemical Bonds And Its Application

In recent years,technology research and disease treatment based on bioactive peptides have been developed rapidly,and the growth rate of peptide based drug approval,sales and clinical use exceeded the average level of the global pharmaceutical market.Compared with proteins,peptides have the advantages of small molecular weight,easy to synthesis and relatively low cost.The biological activity of peptides is comparable to that of proteins under certain conditions,and the biological tolerance and safety are excellent.However,bioactive peptides composed of amino acid chains are easily degraded by enzymes in organism,resulting in a very short half-life of 2-30 minutes.Moreover,most peptides do not have the ability to penetrate across cell membranes,and therefore the targets of peptide drugs are limited outside the cell,and a large number of potential targets located inside cell cannot be effectively utilized.The clinical application and transformation of peptide drugs are severely restricted by their membrane impermeability.At present,the intracellular delivery technology of peptide is mainly divided into two categories.One is covalently modifying lipid molecules or cell-penetrating peptides on cargo peptides to endow them with cell membrane penetrating ability.The other is using carriers,such as polymers,inorganic nanoparticles,and liposomes to load and deliver peptides into cytosol.However,peptides have great differences in hydrophobicity,net charges and molecular weight,and it is difficult to develop a carrier to meet the delivery requirements for all peptide sequences.Even if the peptides can be delivered into the cells,the peptides should be escaped from the endolysosomes to avoid being degraded by the lysosomal enzymes.Most current strategies for intracellular peptide delivery have poor universality,high production cost and complexity,and may affect the biological activity of peptides when the peptides were covalently conjugated to the carriers.These problems limit the potential application of bioactive peptides in therapy.The key to solve this problem is the development of an efficient,safe,universal and facile technology for intracellular peptide delivery.In this study,an intracellular peptide delivery technology based on double dynamic covalent bonds was proposed.Firstly,a low molecular weight dendrimer was decorated with boronate moieties via a p-hydroxybenzylcarbamate spacer.The phydroxybenzylcarbamate spacer could be cleaved from the dendrimer when the boronate moieties were oxidized by reactive oxygen species(ROS)such as hydrogen peroxide.Then,an anchor molecule such as 3,4-dihydroxybenzaldehyde was connected on the surface of boronated polymer via a pH-responsive boronate-catechol bond.When the pH value of the surrounding environment is decreased,the boronate-catechol bond is cleaved.The aldehyde group on 3,4-dihydroxybenzaldehyde was further reacted with hydrazine-terminated peptides via the formation of pH-responsive acylhydrazone bond.When the peptide/polymer complex was internalized into cells,the two dynamic covalent linkages between polymer and peptide could be cleaved by the intracellular ROS and acidity,thus releasing the bound cargo peptides into the cytosol to exert their bioactivity.A peptide(KTCENLADTY)with potential anti-cancer activity was firstly selected as the model peptide.3,4-dihydroxybenzaldehyde was used as the anchor to connect the hydrazine-terminated peptide and NBC-modified dendrimer.The formed dynamic covalent bonds in the peptide/polymer conjugate were characterized by several techniques to confirm the ROS-and pH-responsiveness.Intracellular peptide delivery experiments confirmed that the method efficiently delivered the cargo peptides into different cell lines,and the delivery efficiency is dependent on peptide concentration and incubation time.Both hydrazine,NBC group and the anchol in the system are essential for efficient peptide delivery.The internalization of the peptide/polymer conjugate is mediated by clathrin-and lipid raft-mediated endocytosis.After cell internalization,the conjugate could efficiently escape from endolysosomes and release the cargo peptides into cytosols.Next,we tested a list of polyhydroxybenzaldehydes as potential anchors for the delivery system,and the results showed that the structures containing both catechol and aldehyde groups are critical for efficient peptide delivery.The delivery strategy is applicable for the delivery of various peptides,including bioactive ones.The peptides can maintain their biofuncitons after intracellular release.Finally,we tested the possibility of the peptide delivery system for cancer treatment,and the results suggested that the formulation can achieve efficient tumor inhibition in vivo.