粉防己碱-PLGA纳米粒的制备及其对骨质疏松模型小鼠的治疗作用

Preparation of tetrandrine-PLGA nanoparticles and its therapeutic effect on osteoporosis model mice

目的 粉防己碱(tetrandrine,TET)最近被证明是治疗骨质疏松症的药物。但是,它很难溶于水,只可溶于某些有机溶剂,因此,其临床应用受到限制。然而,当TET被聚乳酸-羟基乙酸共聚物[poly (lactic-co-glycolic) acid,PLGA]纳米粒包覆之后,可以解决其水溶性不足问题。所以,拟制备载TET的PLGA纳米粒,并构建去势骨质疏松小鼠模型,研究TET-PLGA纳米粒(PLGA@TET NPs)对骨质疏松小鼠的治疗作用。方法 采用纳米沉淀法制备PLGA@TET NPs,考察其包封率、载药量及粒径。采用双侧卵巢切除法构建骨质疏松症小鼠模型,12周龄C57BL/6雌性小鼠随机分为4组(每组8只):假手术组、单纯卵巢切除组、去卵巢+TET组、去卵巢+PLGA@TET NPs组。各治疗组尾静脉分别注射含同等剂量TET的100μl磷酸盐缓冲液(phosphate buffer solution,PBS),单纯卵巢切除组和假手术组尾静脉注射100μl的PBS,每周一次,连续8周。Micro-CT检测骨小梁体积百分数(percent trabecular bone volume/per tissue volume,BV/TV),骨小梁数量(trabecular number,Tb.N)及骨小梁分离度(trabecular separation,Tb.Sp),以评价小鼠骨质疏松程度。用HE染色观察肝、肾组织损伤情况。抗酒石酸酸性磷酸酶(tartrate-resistant acid phosphatase,TRAP)染色检测破骨细胞活性。结果 纳米沉淀法制备的PLGA@TET NPs粒径约为236 nm,呈规则的球形。TET的包封率和载药量分别为(85.34±0.12)%及(7.86±0.01)%。此外,Micro-CT结果显示,与单纯卵巢切除组相比,去卵巢+TET组及去卵巢+PLGA@TET NPs组的BV/TV及Tb.N均增加(P <0.01),而Tb.Sp降低(P <0.01);并且去卵巢+TET组及去卵巢+PLGA@TET NPs组的Tb.N差异有统计学意义(P <0.05)。HE染色结果显示,各组织切片肝肾未见明显损害,说明纳米粒没有明显毒性。TRAP染色结果显示,单纯卵巢切除组破骨细胞活性增加,去卵巢+TET组,去卵巢+PLGA@TET NPs组破骨细胞活性降低,并且纳米粒组和TET组比较差异有统计学意义(P <0.05)。结论 制备的PLGA@TET NPs粒径较均一,包封率及载药量高。动物实验结果显示PLGA@TET NPs治疗组和游离TET组均可治疗骨质疏松症,但PLGA@TET NPs治疗组较游离的TET组可以更好的预防或治疗骨质疏松症。

Objective Tetrandrine(TET)has recently been shown to be a option for the treatment of osteoporosis.However,its clinical application is limited because of its solubleness in water.The problem of insufflcient water solubility can be solved when TET is coated with poly(lactic-co-glycolic acid,PLGA)nanospheres.Therefore,we intend to prepare PLGA nanospheres loaded with TET and establish an ovariectomized mouse model to study the therapeutic effect of tetrandrine-PLGA nanospheres(PLGA@TET NPs)on osteoporotic mice.Methods PLGA@TET NPs were prepared by the nano-precipitation method,and their encapsulation efflciency,drug loading capacity,and particle size were investigated.The osteoporotic mouse model was established by bilateral ovariectomy.12-week-old C57BL/6 female mice were randomly divided into four groups(n=8 in each group):sham operation group,simple ovariectomized group,ovariectomized+TET group,ovariectomized+PLGA@TET NPs group.100μl phosphate buffer solution(PBS)containing the same dose of TET was injected into the caudal vein of each treatment group,and 100μl of PBS was injected into the caudal vein of the simple ovariectomized group and sham operation group once a week for eight weeks.Micro-CT was performed to detect the percent trabecular bone volume/per tissue volume(BV/TV),trabecular number(Tb.N),and trabecular separation(Tb.Sp)to evaluate the degree of osteoporosis in mice.HE staining was used to observe the injury of the liver and kidney.The activity of osteoclasts was detected by tartrate-resistant acid phosphatase(TRAP)staining.Results The particle size of PLGA@TET NPs prepared by the nano-precipitation method was about 236 nm,which was regular spherical.Tetrandrine's encapsulation efflciency and drug loading were(85.34±0.12)%and(7.86±0.01)%,respectively.In addition,Micro-CT results showed that compared with the simple ovariectomized group,the BV/TV and Tb.N in the ovariectomized+TET group and the ovariectomized+PLGA@TET NPs group increased(P 0.01),while the Tb.Sp decreased(P 0.01),with a signiflcant difference in Tb.N between the ovariectomized+TET group and the ovariectomized+PLGA@TET NPs group(P 0.05).The HE staining results showed no apparent damage to the liver and kidney in each tissue section,indicating that the nanoparticles had no apparent toxicity.TRAP staining showed that osteoclast activity increased in the ovariectomized group,decreased in ovariectomized+TET group and ovariectomized+PLGA@TET NPs group,and there were signiflcant differences between the nanoparticle group and TET group(P 0.05).Conclusions The prepared PLGA@TET NPs have a uniform particle size,high encapsulation efflciency,and high drug loading.The results of animal experiments showed that both the PLGA@TET NPs treatment and free tetrandrine treatment have effects on osteoporosis.Still,the PLGA@TET NPs treatment could prevent or treat osteoporosis better than the free tetrandrine.