Construction And Preliminary Evaluation Of Doxorubicin-loaded Liposomes With Gatekeeper

A targeted drug delivery system can deliver an effective amount of drug molecule to target tissue and target cells,but the “zero release” of the drug is the key to reducing its toxic side effects before reaching the target site.Accordingly,many smart drug delivery systems with gatekeeper have been designed to stabilize drug molecules under normal physiological conditions by gatekeepers.In the tumor microenvironment(such as low pH,high concentration of glutathione,reactive oxygen species and enzymes)or with exogenous factors(such as magnetic field,ultrasound and light),the gatekeeper can be shed or changed to achieve a stimulating release of the drug.Gatekeepers include polyethylene glycols(PEG),gold nanoparticles,quantum dots,β-cyclodextrin,ε-polylysine,etc.When PEG is used as a gatekeeper,it has the two following characteristics: 1.Space repulsion effect: it can block the pore of the pore-forming peptide.2.Spatial repulsion effect combined with hydrophilic properties: the drug-loading system can avoid the clearance by the reticuloendothelial system(RES),increasing the blood circulation time of the carrier in the body,and facilitating the enrichment of the vector to the tumor site.The pore-forming peptide is a linear peptide composed of a plurality of amino acids and has hydrophobic side chains which can be inserted into the lipid bilayer via hydrophobic interaction,which forms a pathway for drug leakage.The engineered pore-forming peptides have matrix metalloproteinases(MMPs)substrate and are therefore enzyme sensitive.In view of the characteristics of PEG and pore-forming peptide,PEG was attached to the pore-forming peptide by addition reaction,and modified into liposome surface,and doxorubicin(DOX)was used as a model drug to construct a nanocarrier with a gatekeeper.The pore-forming peptide and PEG co-modified DOX-loaded liposomes were prepared by membrane hydration.The particle size,polydispersity coefficient and encapsulation efficiency were characterized.Enzyme sensitivity performance was evaluated using type IV collagenase,and preliminary evaluation of in vitro activity was performed using a human breast cancer cell model(MCF-7).The results showed that the doxorubicin-loaded liposome with gatekeeper(D-PEG-1-LP-DOX)constructed in this paper has good enzyme-sensitive drug release properties and exhibited high in vitro activity against MCF-7 cells,which laid the foundation of subsequent envaluation based on cells and in vivo model.Part one Preparation and characterization of lipid carriersObjective: To synthesize a series of functional polymers and monitor the reaction by thin layer chromatography(TLC).The DOX liposome(D-PEG-LP-DOX)co-modified with the pore-forming peptide and PEG was prepared,and the lipid carrier was characterized.Methods: Series of functional polymers were synthesized by conjugation of peptides with five kinds of PEG,respectively.During the reaction,the reaction solution was quantitatively sampled to the same GF 254 silica gel plate to monitor the progress.D-PEG-LP-DOX was prepared by thin film hydration method and characterized by particle size,PDI and encapsulation efficiency.Results: The results of TLC showed that the peptide was completely connected with PEG and the target functional polymer was synthesized.The prepared lipid carrier has uniform appearance color,the particle size is in the range of 80 ~ 135 nm,the PDI is between 0.23 and 0.37,and the encapsulation efficiency of most preparation groups is 75 ~ 90%.Conclusion: The doxorubicin-loaded lipid carrier with co-modified pore-forming peptide and PEG was successfully prepared in this experiment.The preparation method is simple and reproducible.The prepared liposome has suitable particle size,uniform distribution and high encapsulation efficiency,and can be used for subsequent experiments.Part two Enzyme-sensitivity evaluation of lipid carrierObjective: To establish an evaluation method for enzyme-sensitive release of drugs and to screen out the optimum carriers with obvious enzyme-sensitive characteristics.Methods: On the basis of dialysis,the DOX leakage was investigated,in the absence or presense of collagenase.The fluorescence intensity of DOX was measured by a microplate reader,the DOX concentration was calculated by the standard curve method,and the cumulative release rate of DOX was calculated to investigate the release of DOX in the carrier.Results: The series of lipid carriers here,showed certain collagenasesensitive drug release properties totally,and the highest enzyme sensitivity was found in D-PEG-1-LP-DOX.The cumulative release rate of D-PEG-1-LP-DOX at 6 h without collagenase was 15.3%.After the addition of collagenase,the release level of DOX is significantly improved,and the cumulative release rate was more than 96% at 24 h.Conclusion: The D-PEG-1-LP-DOX was confirmed to be the DOX-loaded lipsome with ideal enzyme-sensitive property.Part three Preliminary evaluation of in vitro activity of lipid carrierObjective: To evaluate in vitro activity of the constructed lipid carrier.Methods: The constructed D-PEG-LP-DOX was evaluated based on MCF-7 cells.Exposed to DOX-loaded liposomes with various surface modification and collagenase of different concentration,the survival rate was determined by MTT assay.Results: Following the addition of collagenase,the viability of MCF-7 cells exposed to D-PEG-1 ~ 5-LP-DOX was reduced significantly.Among them,the D-PEG-1-LP-DOX group exhibited the highest in vitro activity.Conclusion: The enzyme sensitivity of D-PEG-1-LP-DOX was confirmed to be highest based on the preliminary evaluation of the in vitro activity.