| Ligustrazine is a main active ingredient of traditional Chinese medicine Ligusticum chuanxiong Hort.and an active alkaloid--tetramethylpyrazine,which can improve microcirculation,inhibit platelet aggregation,promote blood circulation and remove blood stasis.In recent years,its anti-tumor effect has attracted much attention,with effects in inhibiting the growth and metastasis of tumors and inducing apoptosis and autophagy of tumor cells.However,ligustrazine hydrochloride has a short half-life period and low bioavailability in vivo.Its common preparations need to be administrated frequently,and drug concentration in blood fluctuates greatly,which brings inconvenience to patients.Its sustained-release preparations can enter the body in micro-particles,and thus alleviating adverse reactions,reducing toxic and side effects,and improving efficacyPoly(lactic-co-glycolic acid)(PLGA)is a degradable functional organic compound with high molecular weight,which can be finally decomposed into carbon dioxide and water.It does not harm the human body and has good film-forming property.Therefore,it is often used as the preferred carrier in the preparation of microspheres.Chitooligosaccharides(COS)are the only cationic alkaline amino oligosaccharides with positive charge in nature.They have the advantages of good water solubility,absorption ability and biocompatibility.Moreover,they play an important role in human immune regulation,anti-tumor,blood lipid reduction,antioxidation.Modification of PLGA with cationic groups can improve the cell adhesion and absorption of drugs,and solve drug sudden release,short half-life period,unstable site for drug delivery in vivo and frequent drug deliveryThe main research and Results of this subj ect include:1.Prepare ligustrazine hydrochloride microspheres and process studyLigustrazine hydrochloride microspheres modified with COS were prepared by emulsion-solvent evaporation method.Formated w/o/w compound emulsion,then magnetic stirred,volatile organic solvent and high speed centrifugal collection.By the method of EDC and NHS activation,covalent linkage PLGA’s terminal carboxyl group and COS’s free amino acids.Prepared ligustrazine hydrochloride microspheres modified with COS in the end.The influence of formulation and manufacture was studied by the single factor experiments;the optimal formulation was verified by Orthogonal experimental.The optimum prescription was 50g/L PLGA,30g/L Ligustrazine,internal water phase:oil phase=1:10,external water phase:oil phase=4:1.The surface of Ligustrazine microspheres prepared with optimal prescription was with a good appearance,almost no adhesions,encapsulation efficiency was over 60%.At the same time,A content determination method was established for the ligustrazine hydrochloride in the preparations.The method has high accuracy,good stability and good precision.It can be used for the determination of ligustrazine hydrochloride.2.Surface characteristics of ligustrazine hydrochloride microspheres modified with cosThe particle size and morphology of ligustrazine hydrochloride-loaded microspheres were observed by scanning electron microscopy.The solidified microspheres were collected by high-speed centrifugation,washed three times with deionized water,freeze-dried,placed on silicon wafers and sprayed with gold under vacuum.The size and morphology of microspheres were observed using TM3000 desktop scanning electron microscope.Ligustrazine hydrochloride-loaded microspheres were uniformly distributed with uniform size.No obvious adhesion between individuals was observed.The surface was smooth,in a complete spherical shape,with high encapsulation efficiency and drug loading.3.The release rule in vitro of ligustrazine hydrochloride microspheresUnmodified ligustrazine hydrochloride-based microsphere suspension in precision quantity(10 ml),chitooligosaccharide-modified microsphere suspension(10 ml)and equal quality(10 ml)of ligustrazine hydrochloride injection solution were collected respectively,and placed in the prepared dialysis bags.After sealing,suspensions were placed in phosphate buffer solution(pH7.4),stirred in water bath at constant temperature.With the drug was gradually released,sustained-release solution was collected and fluid was infused at intervals.The release of ligustrazine hydrochloride injection in the dissolution medium was faster.Unmodified ligustrazine hydrochloride-loaded microspheres showed sudden release at the early release stage The sudden release of modified microspheres reduced and stabilized at 24 h,with cumulative release of 78.23%,showing a certain sustained release.4.Cell experiment in vitroThe cytotoxicity of ligustrazine hydrochloride-loaded microspheres was investigated by CCK-8 assay using human breast cancer MCF-7 cells and human lung adenocarcinoma A549 cells.Previous experiments showed that the optimum concentration of ligustrazine hydrochloride was 0.054 mg/ml and the optimum reaction time was 24 h.The concentration of ligustrazine hydrochloride in MCF-7 and A549 cells was determined by HPLC,revealing a higher concentration in MCF-7 cells than A549 cells,46.77 μg/ml and 30.38 μg/ml,respectively.Under the confocal scanning laser microscope,it was observed that cells treated with chitooligosaccharide-modified microspheres presented more obvious fluorescence than those treated without chitooligosaccharide-modified microspheres and the ligustrazine control group.The fluorescence intensity of MCF-7 cells was higher than that of A549 cells.Conclusion:1.Chitooligosaccharide-modified ligustrazine hydrochloride-loaded micro spheres were prepared by emulsion-solvent evaporation method.The process was simple and easy,the conditions were mild,the encapsulation efficiency and drug loading were high,and the structure was stable.The microspheres could be preserved for a long time at room temperature2.The morphology of microsphere surface was observed by scanning electron microscopy,showing that the microspheres were smooth,round and controllable in size.The average particle size was 180.5 μm,the drug loading was 19.05%,and the encapsulation efficiency was 68.16%.3.The drug release in vitro was investigated before and after microsphere modification.Because the modified microspheres were encapsulated with chitooligosaccharides,the drug released after slow degradation of chitooligosaccharides.Therefore,sudden release reduced4.Because of their low molecular weight,chitooligosaccharides are easier to enter cells.In vitro cell experiments show that chitooligosaccharide-modified microspheres have higher targeting ability to tumor cells,providing reference for the development and application of ligustrazine targeting preparations. |
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