| Etoposide（ETP）is a carbohydrate metabolism derivative of podophyllotoxin,a plant component,which is effective for lung cancer,malignant lymphoma,testicular tumors,malignant germ cell tumor,rhabdomyosarcoma and other tumors.Tumors have therapeutic effects.However,existing etoposide preparations have problems such as poor water solubility(58.7μg·m L-1),easy metabolic inactivation,low bioavailability,and dose-limiting blood toxicity.In addition,the excipients added to etoposide injection,such as absolute ethanol,phenethyl alcohol,polysorbate 80 etc,may also cause hypersensitivity reactions in the body.Based on this,it is of great significance to construct a new etoposide delivery system for improving the solubility,bioavailability and targeted sustained-release properties of etoposide.The research group has established the preparation method of folic acid-targeted curcumin nano-mixed micelles and the UPLC method to determine the analysis method of the drug content in the micelles.The research has confirmed that the micelle preparation has obvious solubilization and slowing down of hydrophobic drugs.The release effect,and the safety are greatly improved,and the anti-tumor effect is significant.On the basis of the previous research results and this study used Pluronic（P123）and methoxypolyethylene glycol-polylactic acid（m PEG-PLA）as carrier materials for the first time,and successfully prepared etoposide-loaded nano-mixed micelle preparations（ETP m PEG-PLA/P123）through self-assembly technology.Further sur FAce FA targeted modification to obtain FA-ETPm PEG-PLA/P123 micelles.The main research methods and results of this topic are as follows:1.Establishment of ETP in vitro UPLC analysis methodThe Waters BEH C18 column（100 mm×2.1 mm,1.7μm）.Mobile phase acetonitrile:water=50:50（v/v）;Detection wavelength284nm;Volume flow 0.3m L/min;The sample volume 1μL;Column temperature 35℃.Follow the above chromatographic injection.Analytical method verification includes specificity,linearity,precision,density,repeatability and accuracy.The results showed that the concentration of etoposide was linear in the range of 5.0-80.0μg·m L-1.The specificity,precision,repeatability,stability and accuracy were good,and RSDs were all less than 2%.2.Preparation and optimization of ETP m PEG-PLA/P123 micellesETP m PEG-PLA/P123 micelles were prepared,and the formulation was optimized by single FActor investigation and central design-response sur FAce method.The functional relationships of m PEG-PLA mass ratio,drug dosage and hydration volume on encapsulation efficiency（EE）,drug loading（DL）and particle size were established.The optimal formulation process was m PEG-PLA:P123=38:62,the dosage is 5mg,and the hydration volume is 6m L.The actual average encapsulation efficiency of the micelle is 87.4%,the average drug loading is 4.19%,and the particle size is116.6 nm.The absolute values of both experimental and predicted values are less than 5%。3.The physicochemical properties of ETP m PEG-PLA/P123 micellar preparations and its in vitro releaseThe ETP m PEG-PLA/P123 micellar formulation was prepared with the optimal prescription,and the physicochemical properties and release behavior were evaluated.The particle size is 115.6nm,the PDI is 0.216,and the Zeta potential is-16.3m V.Under the TEM microscope,The preparation is round and evenly dispersed.The critical micelle concentration of m PEG-PLA/P123 was 2.5×10-3g·L-1,which indicated that the m PEG-PLA/P123 micelles had good stability.DSC results showed that etoposide in the micelle may be in the form of amorphous or disordered crystalline phase.In addition,in vitro release experiments showed that etoposide could be released slowly and continuously from micelles,and the release amount reached 80%within 48 hours,which has a certain sustained release effect.4.Synthesis and characterization of FA-P123Using the hydroxyl group of P123 and the carboxyl group of FA to undergo an esterification reaction,FA-P123 is synthesized,and the reaction product is obtained by dialysis and purification.The synthetic product FA-P123 was characterized by IR,UV and HNMR.In the UV spectrum,FA-P123 has peak absorption at the wavelengths of 256nm,283nm and365nm,which was similar to the UV-vis spectrum of FA.The infrared spectra of FA-P123 showedνC=O at 1727nm,the existence of ester bond could be inferred,and the esterification reaction was proved to have occurred successfully.Compared with the 1HNMR spectra of P123 and FA-P123,a new peak appeared,and the new peak was attributed to folic acid,which can be inferred that the targeted compound synthesis was successful.The average yield of FA-P123 was 35.53%.5.Preparation and evaluation of physicochemical properties of FA-ETPm PEG-PLA/P123 micellesThe FA-ETP m PEG-PLA/P123 micelles were prepared,and encapsulation efficiency was 56.53%,and drug loading was 2.74%.The particle size of FA-ETP m PEG-PLA/P123 is 105.8nm,which is larger than that of the blank micelle,and the absolute value of Zeta potential was13.7m V,which illustrated that the micelle has better stability.Under the TEM microscope,the preparation was round and uniformly.The CMC value was 4.31×10-3g·L-1.In the DSC chart of FA-ETP m PEG-PLA/P123micelles,no exothermic peak of etoposide was found,which can be inferred that etoposide inside the micelles may be in the form of dispersed amorphous or disordered crystalline phase.FA-ETP m PEG-PLA/P123micellar solution released slowly,and within 48 hours,the release rate of etoposide was only 67.84%.It showed that the micellar preparation has a significant slow-release effect.6.In vitro cell experiment6.1 Cytotoxicity testUsing human non-small cell lung cancer cell line（H1299 cells）as a model,the MTT method is used to investigate the inhibitory effect of the different preparation on tumor cells.The IC50 points of Etoposide solution,ETP m PEG-PLA/P123 micellar solution,etoposide injection and FA-ETP m PEG-PLA/P123 micellar solution are 59.61μg·m L-1,14.83μg·m L-1,1.54μg·m L-1and 6.64μg·m L-1.Compared with etoposide solution group,ETP m PEG-PLA/P123 micelles group,etoposide injection group and FA-ETP m PEG-PLA/P123 micelles group had stronger cytotoxicity（P<0.001,P<0.001,P<0.001）.Compared with etoposide injection group,FA-ETP m PEG-PLA/P123 micelles group had stronger inhibitory effect on H1299 cells（P<0.001）.6.2 Cell uptake experimentConfocal laser microscopy and protein precipitation were used to investigate the effect of H1299 cell on different micelles.The results showed that the uptake were related to the concentration of C6 and administration time of FA-C6 m PEG-PLA/P123 micelles.With the increase of the time of administration and the concentration of C6,the intracellular fluorescence intensity increases,and the cell uptake increases.Under the same Coumarin-6 concentration,the ability of cells to take up C6ranged from large to small were FA-C6 m PEG-PLA/P123 micelles、FA-C6m PEG-PLA/P123+FA and C6 m PEG-PLA/P123 micelles.As mentioned above,etoposide was used as a model drug to prepare etoposide by the thin film hydration method.ETP m PEG-PLA/P123micelles,using single FActor investigation and star point design-response sur FAce method,were successfully constructed.On this basis,perform sur FAce folic acid Targeting to obtain FA-ETP m PEG-PLA/P123 micelles with small particle size,high drug loading and good stability,In vitro cell experiments showed that Drug-loaded micelles prepared by FA-ETP m PEG-PLA/P123 and ETP m PEG-PLA/P123 have good anti-tumor effect and cell uptake ability. |
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