葡萄多组分重组囊泡的制备及其载多肽性能研究

Preparation and characterization of restructured vesicles of grape-derived multicomponents for active peptides loading

目的分离葡萄不同组分并重组构建功能囊泡,考察其装载多肽类药物的性能。方法采用乙醇为溶剂提取葡萄多酚(GP),通过单因素和正交试验优化GP的提取工艺。大孔树脂吸附法纯化GP。正交试验考察蜂毒肽(Mel)与GP的复合条件,制备纳米复合物(GPMC)。蔗糖密度梯度离心法提取葡萄囊泡(GDVs),用于装载优化的GPMC,得到GPMC-GDVs。考察GPMC-GDVs在PBS、DMEM、10%FBS中的稳定性。MTT法评价其对SMMC-7721及Hep G2细胞的毒性。结果优化的GP提取工艺:60%乙醇为提取溶剂,料液比1∶10,提取温度50℃,提取50 min,提取2次。GP纯化条件:4 mg/m L GP粗提物上样7 BV(树脂床体积),水洗5 BV,60%乙醇洗脱5 BV。GPMC-GDVs制备工艺:室温下,将0.4 mg/m L GDVs缓慢滴入等体积含Mel质量浓度为2 mg/m L的GPMC溶液中,孵育30 min。制备的GPMC-GDVs在PBS、DMEM、10%FBS中稳定性良好。MTT结果显示,GPMC-GDVs具有明显的肿瘤抑制作用。结论通过对GP与GDVs组分的分离重组,可制备含药囊泡,并能用于活性多肽的装载,在多肽类药物递送和抗肿瘤领域具有较好的应用前景。

Objective To reconstruct the functional vesicles by using the components isolated from grapes, and investigate its properties of the loading of peptides. Methods Grape polyphenols（GP） were extracted by ethanol, and the extraction technology of GP was optimized by single factor and orthogonal experiments. GP was purified by macroporous resin adsorption and the purification technology. The preparation of GP and melittin（Mel） complexes（GPMC） were determined by orthogonal tests, and Sucrose density gradient centrifugation was used to extract grape-derived vesicles（GDVs） for loading complex GPMC to get GPMC-GDVs. The stability of GPMC-GDVs in PBS, DMEM, and 10% FBS were investigated. The cytotoxicity of GPMC and GPMC-GDVs on SMMC-7721 or Hep G 2 cells was investigated by MTT method. Results The extraction process of GP was as follows： the extraction solvent was 60% ethanol solution, the heating temperature was 50 ℃, the solid-liquid ratio was 1∶10, the extraction time was 50 min and extract 2 times. The purification conditions of GP were as follows： 4 mg/m L GP crude sample volume was 7 times of bed volume（BV）, the washing dosage was 5 BV, and the elution volume of 60% ethanol was 5 BV. The preparation of GPMC-GDVs was as follows： 0.4 mg/m L GDVs was slowly dripped into GPMC solution with equal volume of Mel containing 2 mg/m L and incubated at room temperature for 30 min. As-prepared GPMC-GDVs had good stability in PBS, DMEM, and 10% FBS. The results of MTT method showed that GPMC-GDVs had a better tumor inhibitive effect. Conclusion By extracting the components of grape GP and GDVs and reorganizing the structures, the vesicles can be prepared for the loading of active melittin, which has a good application prospect in the field of delivery and anti-tumor effect of polypeptide drugs.