Study On Drug Delivery System Of H102 Peptide Loaded PEG-PLGA Nanoparticles

Objective:To prepare H102 peptide loaded PEG-PLGA nanoparticles(H102-NP)and to investigate its properties in vitro and in vivo.To study pharmacokinetics and brain targeting properties of H102 loaded PEG-PLGA nanoparticles and to evaluate its elementary safety.Method:Part 1,analytical technologies in vitro and vivo for the determination of H102 peptide were established by RP-HPLC and stability of H102 peptide in vitro and vivo was investigated.LC/MS analytical method was established to determine the drug concentration.Part 2,H102-NP was prepared by emulsion/solvent evaporation method.RP-HPLC analysis method in vitro and vivo were established for the determination of H102-NP and the encapsulation efficiency and drug loading were calculated.Main factors affecting the quality of nanoparticles were screened by single factor investigation and orthogonal design was used to optimize the formulation.The morphology of the nanoparticles was observed by transmission electron microscope,its mean diameter and zeta potential was determined by Zeta Potential/Particle Sizer.Stability of H102-NP in plasma and brain homogenate and in vitro release were investigated.The storage stability of H102-NP with different dispersant was performed.Part 3,LC/MS analysis method was established for the determination of drug concentration in plasma and brain tissue.Pharmacokinetics parameters were calculated from the drug concentration-time data in plasma.The drug concentration in brain tissue and organs was detected in mice.Part 4,MTT assay was used to evaluate the cytotoxicity of H102 peptide,polysorbate 80,H102-NP and PS-H102-NP.The initial toxicity test in mice was performed following intravenous injection of H102 peptide and PS-H102-NP including high,medium and low dose.To evaluate the toxicity,organ coefficients were calculated and histological changes were observed by fluorescence microscope through HE staining.Results:Part 1,the results of in vitro stability showed H102 peptide was kept stable in the citrate buffer sterile solution with pH between 5.5 and 7.5.The results of in vivo stability showed the half-life of H102 peptide in plasma was 20 min and could be degraded easily by various enzymes in plasma.Pharmacokinetics study showed that the half-life of H102 peptide was 6.5 min with a quick elimination in plasma and no drug could be detected at 60 min.The concentration of H102 peptide in brain decreased at 5 min and no drug was detected at 15 min.Part 2,the average size,Zeta potential,encapsulation efficiency and drug loading of H102-NP were found to be around 147 nm,-38 mV,64%and 0.7%.TEM images showed H102-NP had good dispersity with spherical appearance.Incubated in plasma and brain homogenate at 37? for 12 h,the degradation of H102-NP was about 5%.The cumulative release of H102-NP in PBS and plasma at 12 d was 93%and 95%,respectively.As the dispersant of H102-NP,polysorbate 80 solution could maintain the size of H102-NP for 90 days.When stored in solution for a long time,the content of H02-NP decreased.Part 3,the pharmacokinetics study showed the half-life of the two formulations was 68.4 min and 61.8 min,respectively,which was 10 times higher than that of H102 peptide solution.The Cmax and the AUC of PS-H102-NP was 1.33 and 1.42 times higher in brain than that of H102-NP with longer residence time and lower clearance,which indicated that PS-H102-NP could deliver more H102 peptide into brain and showed good brain-targeting effects.The distribution of H102-NP and PS-H102-NP mainly concentrated on liver and spleen and the uptake was increasing with the time,while a little was accumulated in other tissues.Part 4,the results of cytotoxicity showed H102 peptide had low cytotoxicity and polysorbate 80 below 2%had good safety.H102-NP and PS-H102-NP in high concentration had large cytotoxicity while that in medium and low concentration had low cytotoxicity.The results of toxicity test showed the H102 peptide group,the medium and low dose group did not exhibit obvious toxicity,but the high dose group exhibited large toxicity.Conclusion:H102-NP shows good stability and release properties in vitro and in vivo.It is ideal sustained-release preparations with long circulation in vivo.H102-NP coated with 1%polysorbate 80 has good brain targeting effects and low toxicity.These results provide strong experimental basis for the better application of H102 peptide for the treatment of Alzheimer's disease.