Study On The Synergistic Anti-glioma Effect Of Paclitaxel-shikonin Microemulsion System With AS1411 Aptamer And Hyaluronic Aci

Part Ⅰ: Optimization of preparation and characterization of shikonin-docetaxel-coloaded microemulsionObjective In this part,we analyzed the emulsification,optimal mass ratio between components,entrapment efficiency and stability of shikonin and docetaxel-coloaded microemulsion from the perspective of preparation process,and optimization of preparation technology.Methods Shikonin and docetaxel were employed as entrapment drugs,and the optimal excipients were determined through screening the oil phase,surfactant and co-surfactant;the emulsification of shikonin and docetaxel-coloaded microemulsion was investigated by pseudo-ternary phase diagram,and the optimal preparation technology was established by testing various parameters,such as particle size,zeta potential,stability,appearance and entrapment efficiency.Results Shikonin and docetaxel-coloaded microemulsion prepared with 1944 CS as oil phase,HS15 as surfactant and PEG400 as cosurfactant displayed the smallest particle size,and the highest stability within 7 days.The area of microemulsion on the pseudo-ternary phase diagram is the largest when the Km value was set as 3/1,and the area of microemulsion with 1944 CS as oil phase was the largest under such Km value.After the comparison of each prescription,we found that the particle size of the microemulsion was the smallest,and the entrapment efficiency of both DTX and SKN were higher than 80% once the mass ratio of these two drugs was set as 1/1.Conclusion The optimal excipients and technology of shikonin and docetaxel-coloaded microemulsion were thoroughly studied,and the physicochemical properties of the microemulsion under the optimal preparation process met the requirements of pharmaceutics.Part Ⅱ Preparation and characterization of AS1411&hyaluronic acid-modified shikonin and docetaxel-coloaded microemulsionObjective To investigate the preparation technology and physicochemical properties of AS1411&hyaluronic acid-modified shikonin and docetaxel-coloaded microemulsion from the perspective of target ligand modification and microemulsion preparation.Methods Shikonin and docetaxel were employed as two entrapment drugs,AS1411 aptamer and hyaluronic acid were used as two tumor-targeted ligands,and 1944 CS,HS15 and PEG400 were screened as oil phase,surfactant and cosurfactant,respectively,hyaluronic acid-modified shikonin and docetaxel-coloaded microemulsion was prepared by one-step emulsion method,and then the AS1411 aptamer was covalently conjugated outside of microemulsion by using EDC/NHS classical method.The morphology,particle size,zeta potential,drug release characteristics and entrapment efficiency of the microemulsion were investigated by means of transmission electron microscope,dynamic light scattering particle sizemeter and high performance liquid chromatography,respectively.Results The screening results of target ligand density showed that 10%(wt%)of HA and 180 n M of AS1411 aptamer contributed to the high density of target ligands.The modification of target ligands and the entrapment of various drugs had no significant effect on the morphology and pharmaceutics behavior of the microemulsion.The entrapment efficiency of the two drugs was the highest when the SKN/DTX mass ratio was 1/1 and the total drug/carriers mass ratio was 1/36,respectively.The morphology and drug release behavior of AS1411&hyaluronic acid-modified shikonin and docetaxel-coloaded microemulsion both changed significantly in the presence of HAase,and the microemulsion had high tolerance to temperature,p H and long-term storage.Conclusion AS1411&hyaluronic acid-modified shikonin and docetaxel-coloaded microemulsion was successfully prepared,and the physicochemical properties were characterized thoroughly.The properties of the microemulsion met the requirements of pharmaceutics.Part Ⅲ: Anti-tumor effect of AS1411&hyaluronic acid-modified shikonin and docetaxel-coloaded microemulsion in vitro and BBB permeabilityObjective The purpose of this part is to verify the synergistic anti-tumor effect of AS1411&hyaluronic acid-modified shikonin and docetaxel-coloaded microemulsion in vitro,the advantages of double-targeted modification in tumoral uptake,the inhibitory effect of SKN on decrease tumoral stem-like effect and the high permeability of microemulsion system to blood brain barrier(BBB).Methods The synergism of antiproliferation of AS1411&hyaluronic acid-modified shikonin and docetaxel-coloaded microemulsion against U87 glioma cells was evaluated by different mass ratios of DTX and SKN,as well as different microemulsions.The U87 cellular uptake was studied after C6 was used to label the tested microemulsions.The apoptosis induction,as well as intracellular fluorescence,were investigated by using a flow cytometer.U87 stem-like cell spheres were prepared by the suspension culture method.The spherical-formation and stem cell markers were monitored after treatment with different microemulsion through bright-filed imaging and mono-antibody staining method,respectively.The in vitro BBB model was established based on human brain microvascular endothelial cells and used to investigate the potential BBB penetration of each microemulsion.Results Both DTX/SKN with a mass ratio of 1/1 and AS1411&hyaluronic acid-modified shikonin and docetaxel-coloaded microemulsion had a significantly synergistic antiproliferative effect against U87 cells.Combinational AS1411 and hyaluronic acid modification significantly enhanced the cellular uptake and apoptosis induction of the microemulsion.Besides,the microemulsion system assisted SKN to inhibit the aggregation of U87 stem-like cells and significantly decreased the expression of CD133 in the cell spheres.The AS1411&hyaluronic acid-modified shikonin and docetaxel-coloaded microemulsion has a potent BBB penetration ability.Importantly,double-targeted modification,microemulsion system and small size were involved in the boosted BBB penetration potentially.Conclusion AS1411&hyaluronic acid-modified shikonin and docetaxel-coloaded microemulsion strongly inhibit the growth of glioma cells and significantly reduce the marker of stem-like cells,exhibiting a promising potential in penetrating BBBPart Ⅳ Pharmacokinetics and anti-glioma efficacy of AS1411&hyaluronic acid-modified shikonin and docetaxel-coloaded microemulsionObjective The aim of this part is to investigate the pharmacokinetics of AS1411&hyaluronic acid-modified shikonin and docetaxel-coloaded microemulsion,evaluate the effect of double-targeted modification on selective biodistribution,and verify the advantage of design strategy on anti-glioma efficacy in vivo.Methods The curve of concentration-time,as well as pharmacokinetic parameters,in the plasma,tumor,brain and liver samples were investigated after different treatments,respectively.The subcutaneous U87 tumor-bearing and orthotopic U87tumor-bearing models were established.Di R was used as the fluorescence probe to qualitatively and semi-quantitatively investigate the biodistribution of various microemulsions in the brain and tumors,respectively.Besides,the drug accumulation in the tumor,brain,liver and spleen was investigated by using HPLC after single administration with different formulations.The growth of tumors,tumor inhibition rate,survival curve and body weight were investigated to evaluate in vivo anti-tumor efficacy,as well as the potential mechanism of enhancement on therapies.Results Compared with the control group,the retention,circulation and absorption in blood,brain tissue,liver and tumor samples were significantly improved after treatment with AS1411&hyaluronic acid-modified shikonin and docetaxel-coloaded microemulsion.The brain and tumor accumulation of probe were both significantly higher in the microemulsion group than that the control group on orthotopic and subcutaneous tumor model.The designed microemulsion can significantly inhibit the tumor growth,improve the survival rate and inhibit the proliferation of tumor cells in the subcutaneous U87-bearing tumor model.Meanwhile,it was also able to significantly inhibit the tumor fluorescence signal of orthotopic U87 glioma nude mouse model,prolong the survival time and reduce the expression of CD133 in tumor tissue.Conclusion The pharmacokinetic parameters and biodistribution of AS1411&hyaluronic acid-modified shikonin and docetaxel-coloaded microemulsion are significantly improved,and the anticancer efficacy also receives an obvious promotion as designed.