Study On Environmental Responsive GNPep-modified Doxorubicin Liposomes

As an anthracycline compound,doxorubicin(DOX)can inhibit the synthesis of RNA and DNA,and has significant anti-tumor effect.However,its clinical application has been severely limited due to the lack of tumor targeting and serious toxic side effects,such as bone marrow hematopoietic inhibition,renal function impairment,and dose-dependent cardiotoxicity.In order to enhance the therapeutic efficacy of DOX and reduce its toxic side effects,a variety of DOX-containing nanosystems,such as liposomes,nanoparticles,and polymer micelles,have been investigated,with liposomes being the most widely reported.In addition,the polyethylene glycol molecules can form a water layer around the lipid bilayer,and the modified cloaking liposomes can significantly extend the blood circulation time,thus improving the efficiency of drug delivery.GNPeptide(GNPep)is an antimicrobial peptide containing amino acid sequences recognized and cut by matrix metalloproteinases,which can be used to modify liposomes to achieve rapid release of drugs in the tumor microenvironment and improve the drug concentration in tumor sites.In this paper,an enzyme-sensitive functional polymer with MMPase recognition and cleavage was synthesized from PEG and peptide.Using it to modify lipid bilayer and the enzyme sensitive doxorubicin liposome was constructed with liposome as a carrier and DOX as model drug.The lipid carrier was constructed by thin film dispersion method,and the liposomes were characterized by particle size,polydispersion coefficient.The osmotic effect of DOX liposomes in tumor spheroid was studied by three dimensional laser confocal test.In vivo biological distribution in animal tumor models was studied by in vivo imaging technique.Antitumor effect was studied orthotopic tumor model.The results showed that DOX liposomes could achieve drug release in the MMP-sensitive environment,and has anti-tumor effect at the same time.Part one Preparation and characterization of liposomesObjective: To construct peptide modified enzyme-sensitive doxorubicin liposomes from synthase-sensitive functional polymers.Methods: MMP-sensitive functional liposomes were prepared by modifying doxorubicin liposomes by conjugating peptide and PEG through Michael addition reaction.The liposomes were characterized by particle size,polydispersion coefficient.Results: The particle size of doxorubicin liposomes was in the range of116-197 nm,and the distribution was more uniform(PDI < 0.2).Conclusions: The particle size and distribution of liposomes prepared in this part meet the requirements of subsequent tests.Part two Evaluation of permeability of liposomesObjective: To establish a spheroid of MCF-7 and investigate the spheroid osmosis of liposomes.Methods: Spheroid of MCF-7 was constructed,and different enzyme concentrations were set.The drug penetration was observed by laser confocal microscope.Results: With the prolongation of time,the fluorescence intensity of doxorubicin was gradually enhanced.After adding exogenous collagenase,the fluorescence intensity of doxorubicin penetrated to the center of tumor sphere.Conclusion: Enzyme-sensitive agents can stimulate the release of responsive drugs by MMPs and penetrate into the tumor spheroid.Part three Study on the distribution of liposomes in vivoObjective: To study the distribution of doxorubicin liposomes in subcutaneous tumor animal model.Methods: The liposomes were fluorescently labeled by DIR and SCA,and injected into the animals through the tail vein.In vivo imaging technology was used to observe the distribution of the preparation in the animals at different time points.Results: The retention of liposomes was observed in subcutaneous tumor animal model.Conclusion: Enzyme-sensitive liposomes(PEG-G-LP-DOX)can accumulate in tumor sites and respond to drug release under conditions of high expression of matrix metalloproteinases.Part four In vivo pharmacodynamics and preliminary safety evaluation of liposomesObjective: An orthotopic tumor model was established to investigate the inhibitory effect of enzyme sensitive doxorubicin liposomes on tumor growth and tumor metastasis,and to evaluate the safety in vivo.Methods: A 4T1 tumor model was established.The animals were randomly divided into four groups and injected with normal saline,free DOX,common preparation(PEG-LP-DOX)and enzyme sensitive preparation(PEG-G-LP-DOX)through tail vein.The changes in tumor volume and body weight of mice after administration were monitored.Blood biochemical analysis and histopathological analysis were performed after administration.The number of pulmonary nodules was counted to evaluate the anti-tumor metastasis ability of different administration forms.Results: Compared with free drug and normal liposome group,enzyme-sensitive liposome group(PEG-G-LP-DOX)showed significantly enhanced tumor growth inhibition and anti-tumor metastasis effect.The blood biochemical analysis of the enzyme sensitive preparation group showed no abnormally elevated blood indexes,no obvious pathological changes in tissue sections,and the number of pulmonary nodules significantly decreased.Conclusion: The enzyme-sensitive liposome(PEG-G-LP-DOX)constructed in this study showed strong inhibition of tumor growth and metastasis,and had high safety in vivo.