In Vitro And In Vivo Evaluation Of GNPep-modified Doxorubicin Liposomes

Chemotherapy drugs have a low therapeutic index due to strong toxic side effects,which brings great treatment risks to cancer patients.The tumor microenvironment is the "soil" for the growth of cancer cells,which can promote tumor cell proliferation,invasion and metastasis,and protect tumor cells from immunological recognition.The growth rate of tumor cells is significantly higher than normal cells,and it is easier to invade surrounding tissues,which leads to abnormal expression of enzymes in the tumor microenvironment,including matrix metalloproteinases,cathepsins,phospholipases,and oxidoreductases.Targeting formulations designed based on the high expression of enzymes in tumor sites can promote the aggregation of drugs at target sites,increase the drug concentration in tumor sites,and reduce systemic toxicity.GNPep is a linear peptide with antibacterial activity.Based on the antibacterial mechanism of GNPep,a GNPep analog was designed,which contains an amino acid sequence that can be specifically recognized and hydrolyzed by matrix metalloproteinases(MMPs).Modification of liposomes with GNPep analogs can induce rapid release of drugs in liposomes and increase drug concentration in tumor sites.Polyethylene glycols(PEG)is a polymer material with both hydrophilicity and flexibility.It can form a hydration film on the surface of liposomes,reduce the adsorption of liposomes on plasma,and extend the liposomes in vivo circulation time.In this paper,functional peptides sensitive to enzymes were synthesized using GNPep analogues and PEG as raw materials,and it was modified on the lipid bilayer to construct doxorubicin(DOX)liposomes.Functional peptide-modified doxorubicin liposomes were prepared by the thin-film dispersion-hydration method.Encapsulation efficiency and particle size distribution were used as evaluation indicators for liposomes.When liposomes are administered to tumor cells and tumor-bearing mice,doxorubicin is specifically released in the tumor microenvironment where matrix metalloproteinases are highly expressed,thereby killing tumor cells and exerting their efficacy.We performed two-dimensional cell uptake test,three-dimensional including cytotoxicity test and animal tests(including in vivo distribution and evaluation of antitumor activity)to evaluate lipid.Part one Preparation and characterization of enzyme-sensitive liposomesObjective: Using GNPep analogues and PEG as raw materials to synthesize functional peptides.Preparing matrix metalloproteinase-sensitive DOX liposomes(PEG-G-LP-DOX)and characterize it.Methods: The thiol group in GNPep analog and maleimide group in PEG ndergo a Michael addition reaction to synthesize functional peptides.Modification of analogs to prepare enzyme-sensitive adriamycin-loaded liposomes PEG-G-LP-DOX,and their physicochemical properties such as stability,encapsulation efficiency,and particle size were investigated.Results: TLC results showed that the spots of raw peptide disappeared and the spots of functional peptide appeared.Doxorubicin liposome are relatively stable and meet the requirements for encapsulation efficiency.The particle size is less than 200 nm and the particle size peaks are normally distributed.Conclusion: In this subject,functional peptides were successfully synthesized.Doxorubicin liposomes modified with functional peptides were constructed and characterized.The prepared liposomes have high stability and uniform particle size distribution.Part two Evaluation of cell uptake by enzyme-sensitive liposomesObjective: To establish two-dimensional cell models of MMP-positive HT-1080 cells and MMP-negative MCF-7 cells.To observe the dynamic process of liposomes entering cells and evaluate cell uptake rate.Methods: Flow cytometry was used to quantitatively analyze the fluorescence value of cells and investigate the cell uptake rate.The confocal microscope was used to observe the distribution of adriamycin liposomes in cells.Results: The prepared functional liposomes showed different cell uptake rates under different collagenase concentrations.The cell uptake rate of PEG-G-LP-DOX in HT-1080 was higher than that of PEG-LP-DOX.With the addition of exogenous collagenase,the cell uptake rate is enhanced and the speed of DOX entering the cell is accelerated.Conclusion: In this section,the cell uptake rate and cell localization of the prepared enzyme-sensitive liposomes were investigated using flow cytometry and laser confocal microscopy.Under the action of collagenase,both the cell incorporation efficiency and cell uptake rate of PEG-G-LP-DOX were enhanced.Part three evaluation of cytotoxicity and penetrating capacity of enzyme-sensitive liposomesObjective: This section constructs three-dimensional cell sphere models of HT-1080 cells and MCF-7 cells,and examines the preparation's ability to inhibit cell sphere growth.Methods: The MCF-7 and HT-1080 cell spheroid models were constructed by the hanging drop method.Different dosing preparations and enzyme concentrations were set,and the volume changes of the spheroids after administration were measured to evaluate the cytotoxicity of PEG-G-LP-DOX.Results: Compared with PEG-LP-DOX,PEG-G-LP-DOX liposomes are more cytotoxic and significantly inhibit the growth of HT-1080 spheroids.After the addition of collagenase,PEG-G-LP-DOX liposomes significantly enhanced the growth inhibition ability of MCF-7 cells.Conclusion: PEG-G-LP-DOX has strong ability to inhibit cell growth.Part four Evaluation of enzyme-sensitive liposome distribution and antitumor activity in vivoObjective: To construct a tumor-bearing nude mouse model,investigate the distribution of PEG-G-LP-DOX in nude mice,and evaluate its efficacy and safety.Methods: Fluorescent dye-labeled PEG-G-LP liposomes were injected into nude mice bearing the tail vein,and the distribution of the preparations was observed using a three-dimensional imaging system.Nude mice bearing the tumor model were constructed and administered by tail vein injection.Changes in tumor volume and body weight of nude mice were measured.After the administration,the tissues and tumors of nude mice were sectioned and stained to observe the pathological changes.Results: The retention and release of fluorescent dye-labeled enzyme-sensitive liposomes were observed in the tumor site of nude mice;Enzyme-sensitive liposomes have the strongest tumor-suppressing ability,and tumor volume decreases significantly after administration.Conclusion: The enzyme-sensitive liposomes constructed in this project can be enriched in tumor sites and released in HT-1080 tumor sites where MMPs are highly expressed,which significantly inhibits tumor growth.