Study On Enzyme Sensitive Doxorubicin Liposome Modified By PFPep

Conventional cancer chemotherapy interventions could easily lead to the distribution of drug molecules throughout the body,which in turn has strong toxic side effects on normal human tissues.Targeted drug delivery system could deliver drug molecules to tumor specifically,reduce the leakage of drug molecules in normal tissues and organs,reduce toxic side effects,and improve therapeutic effects.The tumor microenvironment(TEM)provides a suitable and complex environment for tumor,which is conducive to tumor growth,invasion and metastasis.Matrix metalloproteinases(MMPs)are expressed highly proteases in tumor microenvironment,but expressed weakly in normal tissues.Therefore,matrix metalloproteinases are widely recognized as the target of intelligent drug delivery systems.Pore-forming peptides(PFPep),with an α-helical structure,can form pores with lipid through hydrophobic interactions self-assembly,which facilitates the release of drugs through the pores and kills tumor cells.The polypeptide has an amino acid sequence that can be specifically recognized and hydrolyzed by MMPs,so it is sensitive to MMPs.Polyethylene glycols(PEG)are modified on the surface of liposomes to block the PFPep.At the same time,PEG avoid the recognition and clearance of the drug-carrying system by the reticuloendothelial system(RES),prolong the circulation time of the drug-carrying system in the body,which is conducive to the enrichment in tumor sites.In this article,based on the characteristics of PFPep and PEG,PEG was attached to the surface of porogenic peptides through Michael addition reaction to form functional polypeptides,which could be identified and cleaved by MMPs(Enzyme-activated PEG-PFPep conjugate,PEG-P).Using PEG-P,phospholipids,etc.as raw materials.The blank liposomes were prepared by the thin-film dispersion-hydration method.The liposomes containing drug doxorubicin(DOX)was encapsulated by the ammonium sulfate gradient method.The particle size,polydispersity Index(PDI),zeta potential(Zeta)and encapsulation efficiency(EE)are evaluation indicators to characterize liposomes.In vitro three-dimensional(3D)tumor model was constructed to investigate the permeability of liposomes.The subcutaneous tumor-bearing nude mouse model was subjected to fluorescence imaging to investigate the distribution of liposomes in vivo.Orthotopic tumor-bearing mouse models were used to investigate the liposome anti-tumor ability and preliminary toxicity.Part one Preparation and characterization of liposomesObjective: DOX liposomes(LP-DOX),enzyme-sensitive DOX liposomes(PEG-P-LP-DOX,PEG-P-LP-DOX&Di D)co-modified with PFPep and PEG,and SCA&DIR liposomes(PEG-P-LP-SCA&Di R,LP-SCA&Di R),were prepared and characterized.Methods: The functional polypeptide(PEG-P)was synthesized by Michael addition reaction between the thiol of porogenetic peptide and the maleimide group of PEG.LP-DOX,PEG-P-LP-DOX,PEG-P-LP-DOX&Di D were prepared by thin-film dispersion-hydration method,and PEG-P-LP-SCA&Di R,LP-SCA&Di R were prepared by ethanol injection method.Results: The particle size of liposomes was uniform,and in the range of100～200 nm,PDI ≤ 0.25,EE > 90%.Conclusion: The particle size,potential and encapsulation efficiency of the liposomes constructed in this part meet the standards,which can be used in subsequent experiments.Part two Study of three-dimensional(3D)tumor model in vitroObjective: The enzyme sensitivity and permeability of liposomes were evaluated by observing the dynamic process of liposomes entering the 3D cell spheroid.Methods: The hanging drop method was used to construct the MCF-7tumor spheroid model in vitro and HT-1080 tumor spheroid model,and the laser confocal microscope was used to observe the penetration of DOX in the3 D cell spheroid.Results: In the presence of MMPs,DOX could separate from PEG-P-LP-DOX&Di D and infiltrate into tumor spheroid.Conclusions: In this part,the enzyme-sensitive properties of the preparation were investigated by 3D tumor spheroid model in vitro.The results showed that PEG-P-LP-DOX&Di D had obvious enzyme-sensitive drug release characteristics,and could penetrate into the tumor spheroid.Part three In vivo distribution of enzyme-sensitive liposomesObjective: To investigate the distribution of preparation in tumor-bearing mice and evaluate the targeted accumulation ability of preparation in tumor-bearing mice by using subcutaneous nude mice.Methods: MCF-7&HT-1080 subcutaneous tumor-bearing nude mouse model was established to observe the fluorescence distribution of PEG-P-LP-SCA&Di R in nude mice and to investigate the targeted accumulation and enzyme sensitivity of the preparation in vivo.Results: PEG-P-LP-SCA&Di R was retained in the tumor site of nude mice,and enzyme-sensitive drug release was achieved better in HT-1080 tumor site.Conclusions: MCF-7&HT-1080 subcutaneous tumor-bearing nude mouse model experiments confirmed that the enzyme-sensitive liposomes constructed in this study can be enriched in tumor sites and released more in HT-1080 tumor with high expression of MMPs.Part four Preliminary evaluation of pharmacodynamics and safety of enzyme-sensitive liposomes in vivoObjective: To evaluate the antitumor activity and anti-metastasis ability of 4T1-Luc in situ tumor-bearing mice,preliminary toxicity analysis of the preparation was conducted.Methods: After the tumor-bearing mice were treated with lipsomes by the tail vein administration,the tumor volume was monitored with a vernier caliper to investigate the anti-tumor proliferation ability of the preparation.By observing lung tumor metastasis nodules,the anti-tumor metastasis ability of the preparation was investigated.H&E staining analysis was performed on organs and tumors.biochemical analysis and use of balance to monitor the weight change of mice was carried out to investigate the toxicity of the preparation.Results: PEG-P-LP-DOX significantly inhibited the growth of tumor volume and lung metastasis with the strongest tumor inhibition ability.PEG-P-LP-DOX did not cause obvious damage to normal tissues and organs of tumor bearing mice.Conclusions: The enzyme-sensitive DOX liposome PEG-P-LP-DOX constructed in this study,can effectively inhibit the orthotopic tumors growth,inhibit tumor proliferation,resist tumor metastasis,and reduce systemic toxicity significantly.