| Objective: In this study,osimertinib co-loaded astragaloside IV liposomes(LPs-OSI/AS)were constructed and their morphology,in vitro release and encapsulation properties were investigated.In vitro,the effects of LPs-OSI/AS on the proliferation of lung cancer cells,the uptake of liposomes by cells,and the changes of intracellular EMT-related genes were investigated by RT-q PCR.Finally,an in vivo experiment of subcutaneous lung cancer xenografts in nude mice was constructed,and the in vivo efficacy of LPs-OSI/AS was investigated from the tumor growth curve and the mouse growth curve.Method: To explore the optimization of the preparation,to investigate the effect of LPsOSI/AS on the proliferation of NCI-H1975 and NCI-H1975/OSIR cells in vitro,and the uptake of LPs-OSI/AS by cells,and to analyze the preparation to overcome drug-resistant cells and inhibit EMT mechanism.At the same time,drug-resistant mice bearing tumor were established to evaluate its pharmacodynamics.Results: First,the HPLC method for the determination of Osimertinib and Astragaloside IV was established.The HPLC methods of two drugs are accurate and reliable because of accuracy,specificity,good precision and high stability.Next,it is about the formulation,preparation process,quality evaluation,stability and in vitro release of LPs-OSI/AS.Firstly,the preparation prescription and method of LPs-OSI/AS were determined according to single factor screening,and the optimal ratio of the two drugs to be added was Astragaloside IV:Osimertinib = 2:1,and then ethanol injection method and sulfuric acid were selected.Co-carrier liposomes were prepared by amine gradient method,and egg yolk lecithin,cholesterol,and PEG2000-DSPE were determined to prepare LPs-OSI/AS.The most prescription ratio was determined to be: egg yolk lecithin: cholesterol: PEG2000-DSPE=10:1 :1.LPs-OSI/AS is a nanoformulation with light blue opalescence.The particle size was 96.92±12.04 nm,the PDI was 0.28±0.01,the encapsulation rate of astragaloside IV was 89.74±6.53%,and the encapsulation rate of osimertinib was 84.35±8.82%.LPs-OSI/AS has suitable particle size distribution,both osimertinib and astragaloside IV have high encapsulation efficiency and good stability.In addition,LPs-OSI/AS improved the solubility of astragaloside IV,which provided a basis for subsequent experiments.Then,after centrifuging LPs-OSI/AS at 15000 rpm for 30 min,no stratification was observed and after one month,the particle size was 121.60±11.3;PDI was 0.28±0.11,indicating the good stability that the prepared LPs-OSI/AS.The morphology of LPs-OSI/AS was observed by transmission electron microscope,and it was found that it had a double-layer spherical structure.The co-loaded liposomes and single-loaded liposomes prepared by differential calorimetry scanner were well encapsulated,and no leakage occurred.In vitro release evaluation investigated the release behavior of LPs-OSI/AS in PBS solution.The experimental results show that LPs-OSI/AS has obvious sustained release effect,and there is no burst release phenomenon.Finally,the effects of LPs-OSI/AS on cell proliferation were partially investigated in vitro,the uptake of LPs-OSI/AS by cells was qualitatively investigated,and the effects of LPs-OSI/AS on cell-related EMT and TGF-β genes were investigated by RT-q PCR.Nude mice subcutaneously transplanted tumor model of NCI-H1975 cells was constructed in vivo,and the in vivo efficacy was evaluated and the effect of LPs-OSI/AS on tumor proliferation was investigated.Cytotoxicity assay and cell clone formation assay evaluated the effects of free drug,simple combination drug and LPs-OSI/AS on the proliferation of NCI-H1975 and NCIH1975/OSIR cells.NCI-H1975/OSIR cells had no significant effect.OSI and the combination had inhibitory effects on NCI-H1975 cells,but no significant effect on NCI-H1975/OSIR cells,while LPs-OSI/AS had no significant effect on NCI-H1975 and NCI-H1975/OSIR cells.The proliferation inhibitory effect of OSIR cells far exceeds that of the simple combination drug,and the MTT range determined by the blank excipient has no obvious cytotoxic effect,indicating that LPs-OSI/AS has no significant effect on NCI-H1975 and NCI-H1975/OSIR cells.Has aproliferation inhibitory effect.The uptake of LPs-OSI/AS by NCI-H1975 and NCI-H1975/OSIR cells was qualitatively observed,and it was found that the uptake of co-loaded liposomes by cells was stronger than that of free coumarin,indicating that NCI-H1975 and NCI-H1975/OSIR cells Enhanced uptake of LPs-OSI/AS.RT-q PCR experiments were carried out to detect Ecadherin,Vimentin,TGFβ1 and TGFβ2 genes,and it was found that LPs-OSI/AS could increase E-cadherin and reduce the expression of Vimentin and TGFβ1/2,indicating that LPs-OSI/AS can increase the expression of Vimentin and TGFβ1/2 OSI/AS can reverse EMT.LPs-OSI/AS inhibits tumor growth,and the effect of tumor inhibition rate is better than that of single drug administration.Although the body weight of the mice did not change very much,the weight curve of the mice in the LPs-OSI/AS administration group kept rising,indicating that the LPsOSI/AS was well tolerated at the experimental dose.Conclusion: In this study,a scientific high-performance liquid chromatography detection method for astragaloside IV and osimertinib was successfully established.The LPs-OSI/AS drug delivery system was constructed and characterized.It was found that the prepared coloaded liposome had a double-layer spherical structure,good stability and encapsulation,no leakage and sustained release effect.In this study,the in vitro investigation of LPs-OSI/AS found that it can significantly inhibit the proliferation of NCI-H1975 and NCI-H1975/OSIR cells,increase the uptake of liposomes by cells,and inhibit the expression of EMT-related genes.In vivo experiments further demonstrated the inhibition of tumor growth by LPs-OSI/AS,indicating that it has good antitumor activity... |
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