Study On Environmental Responsive Doxorubicin Liposome Mediated By PFPep

In recent years,nanoparticles have been shown to significantly improve the solubility of drugs,reduce the systemic toxicity of anti-tumor drugs,and ultimately improve the therapeutic effect.Importantly,nanoparticles can be passively targeted to solid tumor tissue through enhanced permeability and retention(EPR)effects.However,the frequent occurrence of low EPR effects,especially in clinical treatment,affects the delivery of nanoparticles based on EPR effects,resulting in insufficient accumulation of drugs in solid tumors.Therefore,how to improve the bioavailability of drugs through the rational design of nanoparticles has aroused great interest.Due to the diversity of specific enzymes in the tumor microenvironment,enzyme-responsive nanoparticles have become a good candidate for designing intelligent drug delivery systems.Matrix metalloproteinases(MMPs)are known to be essential for extracellular matrix degradation.In normal cells,MMPs play a silencing role,while in tumor cells,MMPs are active and overexpressed and play a critical role in tumor invasion and metastasis.Therefore,MMPs can be used as a specific target for tumor therapy.Pore-forming peptide is an effective functional transmembrane agent,which can be inserted into the lipid bilayer through self-assembly to form a pathway for drug release.The surface of nanoparticles can be covered with hydrophilic polymer polyethylene glycol(PEG)to extend the cycle time of the nanocercarrier in vivo.However,several studies have reported that this protective layer blocks drug release and cell uptake.To solve these problems,a separable PEG conjugate and an enzyme-activated PEG-PFPep conjugate under MMPs have been developed.Based on these facts,a new multi-functional lipid carrier was prepared by using doxorubicin(DOX)as the model drug and liposome(LP)as the carrier,and MMPs cleasable PEG-PFPep conjugate was modified on the surface of liposome.The particle size,multi-dispersion coefficient and encapsulation rate were characterized.The uptake of liposomes in 2D cells at different enzyme concentrations was investigated by laser confocal microscopy and flow cytometry.An in vitro 3D tumor model was constructed to investigate the growth inhibition of liposomes on tumor spheres.The distribution of liposomes in vivo and anti-tumor ability were investigated in tumor-bearing nude mice.Part one Preparation and characterization of liposomesObjective: Blank liposomes(LP and LP-EP),DOX liposomes(LP-DOX and LP-DOX/Di D)and enzyme-sensitive PEG-PFPep conjugated DOX liposomes(LP-EP-DOX and LP-EP-DOX/Di D)were prepared and characterized.Methods: Di D was coated in the hydrophobic region of liposomes by membrane dispersion method to prepare blank lipid carrier.DOX was encapsulated by ammonium sulfate gradient method.The drug delivery target liposome modified by enzyme-sensitive PEG-PFPep combination were prepared by one-step membrane formation.Results: The particle sizes of the six liposomes were uniform,ranging from 80 nm to 150 nm,PDI was less than or equal to 0.4,and the encapsulation rates were all greater than 70%.Conclusions: In this part,six liposomes were successfully constructed,whose particle size,potential and encapsulation rate all meet the standards,which can be used in subsequent experiments.Part two Horizontal study of two-dimensional cell model in vitroObjective: The enzyme sensitivity of liposomes was preliminarily evaluated by measuring the DOX fluorescence intensity in two-dimensional cells in vitro.Methods: The fluorescence intensity of DOX in cells was observed by laser confocal microscopy and flow cytometry.The DOX fluorescence intensity at different time and at different enzyme concentrations was compared to investigate the enzyme sensitivity and cell uptake of the preparation.Results: Liposomes are effective in releasing drugs,which enter the cytoplasm and are eventually concentrated in the nucleus.The fluorescence intensity of LP-EP-DOX in HT-1080 cells was significantly stronger than that in other groups,and in the presence of MMPs,LP-EP-DOX could increase the uptake of DOX in tumor cells.Conclusions: In this part,the cellular uptake capacity of the preparation was investigated by two-dimensional cell model in vitro,and the results showed that LP-EP-DOX had strong enzymatic sensitivity.Part three Horizontal study of 3D cell model in vitroObjective: The inhibition of free DOX,LP-DOX and LP-EP-DOX on tumor globules was investigated with 3D MCF-7 tumor globules as the model.Methods: An in vitro 3D MCF-7 tumor spheroids model was constructed by hanging drop method,and the tumor spheroids was incubated with the preparation for 0 to 7 days.The volume of the tumor spheroids was calculated by Motic software,and the volume changes of the tumor spheroids were compared to investigate the inhibitory effect of the preparation on the tumor spheroids.Results: Tumor globules in the untreated group kept growing at a steady rate,and DOX liposomes could effectively inhibit the growth of tumor globules.In the presence of MMPs,the tumor globules inhibition was the strongest in the LP-EP-DOX group,and the tumor globules volume was reduced to about 70%.Conclusions: In this part,the inhibition effect of the preparation was investigated by 3D tumor spheroids model in vitro.The results showed that LP-EP-DOX had the strongest tumor spheroids inhibition ability and obvious enzyme sensitivity.Part four Antitumor study of liposomes in vivoObjective: Taking tumor-bearing nude mice as the model,the distribution of the preparation in tumor-bearing nude mice was investigated,and the targeting,anti-tumor effect and biological safety of the preparation in tumor-bearing nude mice were evaluated.Methods: The fluorescence intensity of the preparation in tissues and organs of tumor-bearing nude mice was observed by small animal imager.By observing the changes of tumor volume and histopathological sections of tumor-bearing nude mice,the antitumor proliferation inhibition and systemic toxicity of the preparation were investigated.Results: LP-EP-SCA&Di R has a higher fluorescence intensity at the HT-1080 tumor site,which allows for better accumulation at the HT-1080 tumor site.LP-EP-DOX significantly inhibited the growth of tumor volume in tumor-bearing nude mice,and caused no obvious damage to the normal tissues and organs of tumor-bearing nude mice.Conclusion: In this part,the model of tumor-bearing nude mice was used to evaluate the anti-tumor effect of liposomes.The results showed that LP-EP-DOX showed the strongest tumor proliferation inhibition ability and significantly reduced systemic toxicity.