Study On The Mastoparan Optimized Peptide-paclitaxel Conjugate

Drugs cross the biological barrier and enter cells across the membrane,which is the premise for most active substances to exert their efficacy.According to the Biopharmaceutical Classification System,in addition to class 1 high solubility-high permeability drugs,Class 2 low solubility-high permeability drugs,class 3 high solubility-low permeability drugs,class 4 low solubility-low permeability drugs reduce their biological activity in vivo because of their properties.Especially the low permeability of class 3 and 4 drugs,makes it difficult to cross the cell membrane and limis their development.Therefore,improving the transmembrane ability of drugs is one of the hotspots in pharmacy research.Mastoparan,is derived from wasp venom and is a cationic polypeptide with 14 amino acids,which has strong biofilm interference ability and is a potential cell-penetrating peptide.However,its further development is limited due to its high hemolysis and cytotoxicity.The key to solving such problems is to study the structural properties of mastoparan and to further grasp the structure-activity relationship of such peptides.According to the relationship between the characteristics of charge,hydrophobicity and amphiphilicity of mastoparan and its biological function,we propose that charge is the attractive force and initial driving force for the initial binding of the peptide to the cell membrane;hydrophobicity and amphipathicity are the keys to influencing the formation of the α-helical conformation of the polypeptide in the membrane and giving the driving force for penetration.Therefore,this subject designed a series of modified peptides of mastoparan to regulate the polypeptide sequence and change its charge,hydrophobicity and hydrophobic torque,so as to reduce the hemolysis of mastoparan and retain its membrane penetration ability.Through the investigation of related experiments in vitro,the optimized peptides with less hemolysis but strong transmembrane function were screened out,and further coupled with paclitaxel through chemical bonds to enhance its transmembrane ability,so as to improve the oral bioavailability of paclitaxel.Details as follows:First,we designed 15 modified mastoparan with different properties,prepared them by solid-phase synthesis,and identified their structures and purity by mass spectrometry and liquid phase.Using in vitro cytotoxicity and hemolytic toxicity as evaluation indicators,the obtained two modified peptides LK4 and LM1 had lower hemolytic toxicity and stronger cell membrane interference ability.Further microstructure observation found that they can directly form the form of α-helix penetration or assemble and penetrate outside the cell membrane to interfere with the biofilm,and play a role in improving the transmembrane.Next,we used succinic acid as a linking molecule to cross-link paclitaxel and peptides to prepare paclitaxel-polypeptide drug conjugates(PPCs),and systematically characterized their physicochemical properties such as particle size potential,microscopic morphology,and chemical stability.The results showed that LK4-PTX mainly existed in the form of nanoparticles with a particle size of 300 nm,while LM1-PTX could assemble to form nanofibers with a particle size of about 1500 nm.Further research found that the two had good stability in artificial gastric juice,pH 6.5,pH 7.4 and artificial intestinal juice,and had a certain ability to prevent the degradation and destruction of the digestive tract.Characterized by circular dichroism spectroscopy,polypeptides can form a distinct α-helix conformation in a hydrophobic environment,which is the structural basis for penetrating cell membranes.In the in vitro uptake experiment of Caco-2 cells,through qualitative observation and quantitative research,LK4 and LM1 both showed a certain ability to promote cell uptake,and LK4 had a stronger penetration ability.In the Caco-2 monolayer cell model penetration experiment,the apparent permeability coefficient(Papp)of LK4-PTX and LM1-PTX reached 4.78 times and 3.29 times,respectively,with the commercially available Taxol preparation as a control.Finally,we studied the pharmacokinetics of PTX-LK4 and PTX-LM1 in rats with intravenous Taxol preparation and oral Taxol as reference preparations.The results showed that the absolute oral bioavailability of PTX-LK4 and PTX-LM1 were 25.85%and 15.33%,respectively.Compared with the Taxol preparation,the bioavailability of the PTX-LK4 and PTX-LM1 groups was increased by 4.25 times and 2.25 times,respectively.In summary,based on the structure-activity relationship of Mastoparan,this project innovatively designed 15 modified peptides,and screened 2 optimized peptides with low toxicity and high transmembrane ability,which were coupled with paclitaxel,and the pharmacokinetics of rats was improved.The results of scientific studies showed that it successfully improved the bioavailability of paclitaxel.