FOLATE-MODIFIED CHOLESTEROL BEARING PULLULAN, A NEW CANCER-TERGETED NANOPARTICLE DRUG CARRIER SYNTHESIS AND THEIR APPLICATION

在本研究中，我们合成了叶酸修饰的降胆固醇普鲁兰（FCHP），以开发各种抗癌药物的癌症特异性药物载体。以叶酸γ-2-氨基乙基酰胺和4-硝基氯甲酸酯活化的降胆固醇普鲁兰为原料，合成了FCHP。替代率估计为每糖苷单位10%。FCHP在PBS（或水）中容易溶解，单独或与CHP形成大分子胶团。FCHP和FCHP- chp纳米颗粒与阿霉素的结合稳定，比低于0.02:5 （DOX: FCHP或FCHP- chp， W/W）。然后，检测了几种FCHP、未修饰的含胆固醇普鲁兰（CHP）和阿霉素（DOX）混合物的癌症选择性细胞毒性。通过癌细胞-正常细胞混合培养系统（将表达高水平叶酸受体的人表皮样癌KB细胞和正常人胎儿肺成纤维细胞TIG-1-20细胞在同一培养皿中共培养）预先筛选几种纳米颗粒混合物后，通过细胞计数，用台锥蓝法分别定量了KB和TIG-1-20纳米颗粒混合物在单细胞培养系统中的细胞毒性作用。FCHP （50 mg/L）本身对人体细胞无细胞毒性作用。其中，在浓度为0.2 mg/L DOX的培养基中加入重量比为1:4:0.02的FCHP-CHP-DOX，对肿瘤KB细胞具有明显的选择性损伤，而相同的纳米颗粒混合物仅对正常成纤维细胞TIG-1-20细胞的生长有微弱的损伤。此外，FCHP-CHP-DOX（1:4: 0.02）复合物似乎比相同量的DOX本身更有效地导致KB细胞凋亡。

In the present study, we have synthesized folate-modified cholesterol-bearing pullulan (FCHP) to develop cancer-specific drug carrier for various anticancer reagents.FCHP was synthesized by the reaction of folic acid γ-2-aminoethylamide and 4-nitorophenyl chloroformate-activated cholesterol-bearing pullulan. The substitution ratio was estimated as 10% per glucoside units. FCHP was easily dissolved in PBS (or watcr) and formed macromolecular micelle either solely or with CHP. FCHP and FCHP-CHP nanoparticle seemed to stably bind doxorubicin a representative anticancer reagent, below the ratio of 0.02 : 5 (DOX : FCHP or FCHP-CHP, W/W).Then, several combinations of FCHP, unmodified cholesterol-bearing pullulan (CHP) and doxorubicin (DOX) mixture were tested for cancer selective cytotoxicity. After pre-screening of several nanoparticle mixtures by the cancer cells-normal cells mixed cultivation system (a human epidermoid cancer KB cells known as expressing a high level of folate receptor, and a normal human fetal lung fibroblast TIG-1-20 cells were co-cultured in the same dishes), the cytotoxic effects of the nanoparticle mixtures for KB and TIG-1-20 in single cell cultivation system were separately quantified by cell counting using trypan blue method. FCHP (50 mg/L) itself was proved to have no cytotoxic effects toward human cells. Representatvely, a combination of FCHP-CHP-DOX of 1 : 4 : 0.02 (weight ratio, added to the cultivation medium at the concentration of 0.2 mg/L DOX) gave sharp and selective damage against cancer KB cells, while the same nanoparticle mixture only weakly damaged the growth of normal fibroblast TIG-1-20 cells. In addition, FCHP-CHP-DOX (1 : 4 : 0.02) complex seemed to be able to lead KB cells to the cell apoptosis more effectively than the same amount of DOX itself.