阿糖胞苷囊泡型磷脂凝胶脑部植入剂的研究
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摘要
脑胶质瘤发病率高,手术难以完全切除,需要术后化疗,但传统的给药方式很难使高浓度药物到达肿瘤部位,具有高死亡率及高原位复发率的特点。本研究首次将阿糖胞苷制成囊泡型磷脂凝胶,作为术后局部缓释化疗制剂用于脑胶质瘤综合治疗,其优势在于：该制剂具有缓慢释放的特性,长时间作用于肿瘤细胞达到抑制肿瘤复发的作用；成功避开血脑屏障,使较高浓度的药物直接作用于肿瘤切除部位,有效杀灭残存的肿瘤细胞；磷脂作为其主要载体材料具有较好的生物相容性,且无毒性。
在处方前研究中,建立了阿糖胞苷(Ara-C)、磷脂及溶血磷脂的体外含量测定方法。Ara-C在不同水性介质中的溶解度均大于452.7mg/ml,油水分配系数小,亲脂性弱,随着pH的增加而降低；其脂质体／水分配系数为2.51±0.53,推测该药具有较好的细胞膜亲和力。
从制剂的外观、粒径、包封率、体外释放、流变学等方面研究了阿糖胞苷囊泡型磷脂凝胶(Ara-C VPGs)的性质及灭菌稳定性。透射电镜观察,其分散后的结构大多数为小单层脂质体；冷冻蚀刻电镜观察凝胶内部真实结构为相邻囊泡紧密堆积形成了独特的三维网状立体结构,在结构上区别于脂质体和脂质体凝胶。作为载体材料的磷脂浓度分别为300、400、500mg/g时,Ara-C VPGs的包封率分别为31.65%、53.99%、72.12%,432h体外累计释药分别为48.93%、37.37%、23.22%。此结果表明在水相体积不变时,随着磷脂浓度的增加,包封的体积增大而使得包封率升高；体外释放明显减慢,其原因为磷脂浓度增加,使粘度明显增大,减慢囊泡之间和囊泡内的药物向释放介质的扩散过程,结果释放明显减慢。通过高压均质法制备的Ara-C VPGs,经pH7.4的PBS再分散后粒径和包封率分别由灭菌前的119.6±66.2nm、32.6±2.1%灭菌后增加到165.6±71.9nm、62.6±2.3%,推测可能的原因是囊泡融合使粒径增大,包封体积增加导致包封率增加。体外药物释放由灭菌前80h累积释放79.13%,灭菌后降低至432h累积释放74.76%;在流变学研究中,灭菌后粘弹性加强,流动性降低。粘度的变化与粒径的分布密切相关,多分散系数越大粘度越低,由于小粒径囊泡类似于球珠可以润滑大粒径囊泡,从而降低体系粘度。灭菌后多分散系数比灭菌前小,因此,粘度增强,药物释放减慢。总之,经过高压灭菌后,Ara-C VPGs的囊泡粒径增大,包封率增加,体外释放缓慢,粘弹性加强,性质更加稳定。然而,0.05g Ara-C VPGs中药物含量灭菌前为1.01±0.04mg,灭菌后降低至0.73±0.06mg,溶血磷脂灭菌前为1.39±0.03%,灭菌后增加至2.32±0.01%。为了提高制剂中药物和磷脂的稳定性,尝试在处方中加入不同的稳定剂。研究发现加入亚硫酸钠能同时增加阿糖胞苷和磷脂的灭菌稳定性。推测其原因为灭菌的过程中亚硫酸钠电子转移产生SO3-·,通过自由基的反应能大大的缩减HO·的产生,抑制了Ara-C和磷脂的降解。
改变囊泡的表面电荷结构,通过与聚合物静电吸附,形成具有特殊性质的囊泡,可以改变其体内外性质。对于聚谷氨酸吸附-十八胺修饰复合型VPGs (OCA-PGlu VPGs)和聚赖氨酸吸附-胆固醇硫酸钠修饰复合型VPGs(CHss-PLys VPGs),从透射电镜观察可以看出在囊泡的表面包上一层衣膜,粒径在100～200nm,衣膜厚度约为10nm; zeta电位与采用E80磷脂制备的VPGs是一致的,推测PGlu和PLys通过静电作用分别吸附含OCA和CHss的VPGs的囊泡表面。体外释放研究Ara-C VPGs、OCA-PGlu VPGs和CHss-PLys VPGs432h累积释放分别为37.37%、45.65%、47.03%；在流变学研究中,CHss-PLys VPGs和OCA-PGlu VPGs粘弹性较Ara-C VPGs明显加强,推测聚氨基酸的链段以网状交织在囊泡的周围,在应变力作用下,形变量明显变大,其中弹性模量和粘性模量随着角频率变化的曲线形状没有改变,这个曲线非常接近福格特模型的蠕变现象。尽管粘弹性明显加强,但是药物释放反而加快,因此,从增加粘度的角度而减慢药物的释放是解释不通的。然而,推测其原因为在37℃囊泡膜上的磷脂分子具有流动性,聚电解质增加膜的渗透性,导致药物释放加快。
以SD大鼠为动物模型,通过脑内局部定位给药,用UPLC-MS/MS法测定Ara-C VPGs大鼠脑内植入给药后在不同时间、不同区域脑组织中的药物浓度,研究该制剂中的药物在脑内的分布情况。结果显示,在脑内注射VPGs后28天,Ara-C浓度在距离植入部位5mm范围内仍然能高于0.1μg/ml的治疗浓度,表明Ara-C VPGs在脑内有明显的缓释效应。脑组织是不规则的球体,具有流动性的Ara-C VPGs在脑内的分布不同于其他的固体植入剂在脑组织中的扩散过程,这个过程可能为Ara-C VPGs逐渐被脑脊液稀释成无数的囊泡并随着脑脊液循环进入侧脑室,沿着侧脑室的边缘及侧脑室的脉络丛向脑组织深部扩散。由于脑脊液中有丰富的磷脂酶,VPGs中的磷脂最终在脑内被磷脂酶降解,而药物在脑脊液中被代谢。荧光显微镜观察验证药物在脑内转运过程及分布情况。安全性研究中,VPGs在植入正常大鼠脑内第3、7天后,对脑组织切片HE染色观察,发现出现轻度的脑水肿和少量的炎性细胞的浸润,未见脑组织坏死,局部反应轻微,安全性相对较高。
体外细胞实验结果表明该制剂对C6和U87-MG胶质瘤细胞均有较明显的体外抑瘤效应,呈剂量依赖性,其中OCA-PGlu VPGs组抑制细胞生长作用最强,与荧光显微镜观察体外细胞摄取情况是一致的；对鼠C6胶质瘤细胞抑制效应优于人U87-MG胶质瘤细胞。体内抑瘤实验结果表明：与空白VPGs组相比,Ara-C VPGs对皮下C6鼠胶质瘤有较明显的抑制效应,抑瘤率达到95.87%(p<0.01),明显强于对照组溶液剂的抑瘤率45.52%(p>0.05)；而对于U87-MG裸鼠皮下移植瘤动物模型,与空白VPGs组相比,在相同剂量下,各VPGs组均有明显的肿瘤抑制作用(p<0.01),顺序为OCA-PGlu VPGs组(抑瘤率为36.82%)>Ara-C VPGs组(抑瘤率为34.93%)>CHss-PLys VPGs组(抑瘤率为28.58%),Ara-C溶液抑瘤率为16.02%,无明显抑制作用(p>0.01),与体外细胞抑制效应及细胞摄取情况的结果是一致的。其原因一方面是具有促渗透作用的PGlu和PLys能促进药物进入肿瘤细胞,另一方面是当OCA-PGlu VPGs与肿瘤细胞接触时,由于聚氨基酸被蛋白酶降解,暴露了囊泡表面OCA的正电荷,与带负电的肿瘤细胞膜之间通过静电的作用,增加细胞的摄取；然而对于CHss-PLys VPGs,囊泡表面的聚氨基酸被蛋白酶降解,暴露囊泡表面CHss的负电荷与带负电性的肿瘤细胞相互排斥,不被细胞摄取；每组动物体重变化均较小,毒性均较低。
研究显示Ara-C VPGs能提高肿瘤部位的药物浓度,延长抗肿瘤药物与肿瘤细胞的接触时间,改善阿糖胞苷的脑内分布行为和抗肿瘤疗效。其性质稳定,安全有效,适于脑内植入给药,为阿糖胞苷脑部缓释给药系统的研究提供科学的依据。
Glioblastoma multiforme (GBM), a malignancy of the glial cells of the brain, is the most frequent primary brain tumor and is of high incidence rate. Removing all tumor cells is impossible through surgical operation. Additionally, conventional chemotherapeutic drugs are difficult to go across the blood brain barrier (BBB) and reach the effective concentration of treatment in the brain. So it is of high fatality rate and in situ recurrence rate. In this paper, this is first attempt to develop vesicular phospholipids gels loaded with cytarabine for GBM chemotherapeutic at the original tumor site after surgical operation. Some advantages are manifested: VPGs are semisolid phospholipids dispersions with high concentration of phospholipids intended for long time sustained release properties; VPGs were implanted in the site of removed tumor and bypass the BBB; phospholipids, as the main component, are of good biocompatibility and no toxicity.
At preformulation study, determination methods of Ara-C, phospholipids and lysophosphatidylcholine (LPC) were established. The specificity, precision and reproducibility of the method were good. The solubility of Ara-C in different aqueous media are larger than452.7mg/ml; oil/water partition coefficient is low with poor lipophilicify and declines slightly with the increasing of pH, the value is0.1686for50mg/L Ara-C in pH7.4PBS; and liposome/water partition coefficient is2.51±0.53and is of better ablility of cell membrane affinity.
Ara-C VPGs were prepared and characterized in term of the appearance, particle size, entrapment efficiency (EE), in vitro release, and rheologic propperties. The structure of small unilamellar vesicles was observed by a Transmission Electron Microscope (TEM). Micrograph of Freeze-fracture electron microscopy (FF-TEM) showed that each vesicle was tightly packed between neighbor-vesicles to be three-dimensional network structure, the structure is different from liposome and liposome gels. With the lipid concentration of300、400、500mg/g, EE of Ara-C VPGs were31.65%、53.99%、72.12%, respectively; in vitro cumulative release were 48.93%、37.37%、23.22%in432h, respectively. It showed that EE are increasing and in vitro release are slow with the increase of lipid concentration, it supposed that entrapment volume is increasing with the increase of lipid concentration and aqueous phase volume is constant, viscosity is increasing with increase of lipid concentration can lead to release slowly. The sterilization stability of cytarabine (Ara-C) loaded vesicular phospholipids gels (VPGs) were also examined. The particle size of VPGs after redispersion was119.6±66.2nm, and EE was32.6±2.1%. Comparatively, after autoclaved sterilization, increased particle size and EE were obtained as165.6±71.9nm and62.6±2.3%, respectively. In vitro cumulative release was from79.13%in80h for nonautoclaved VPGs to74.76%in432h for autoclaved VPGs. Viscoelasticity were strengthened. The change in viscosity and particle size distribution is closely related, it means that, being of larger polydispersity of particle size distribution, the viscosity is lower at the same lipid concentration. The reason might be that the smaller particles like ball bearings lubricate the larger ones. As a result, after sterilization polydispersity is smaller than that of nonautoclaved. Additionally, the increased viscosity can slow drug release. In total, after sterilization, the vesicle is integrity, EE is increased, in vitro release is slow, viscoelasticity was strengthened, the properties become more stable. And characteristics of in vitro drug release were slowed remarkablely. Also, the viscoelasticity was reinforced with clearly decreased fluidity. However, reduced Ara-C and increased LPC were observed. The content of Ara-C in VPGs was1.01±0.04mg, after sterilization the content declined to0.73±0.06mg; LPC in autoclaved VPGs was increased to2.32±0.01%from1.39±0.03%for non autoclaved VPGs. In order to enhance the stability of Ara-C and phospholipids, stabilizers were added and among them addition of sodium sulfite showed best effects with high stability of both Ara-C and phospholipids. It supposed that SO3-? free radicals producing from sodium sulfite during the process of sterilization can reduce the generation of HO?free radicals greatly, which inhibit the degradation of Ara-C and phospholipids.
VPGs are with specific surface characteristics through the modification of the surface structure and change its in vitro and in vivo properties. For complexation of charged polyelectrolyte modified VPGs, TEM observed that the surface of vesicle was coated with a layer of film, particle size is about100nm. Zeta potential has no obvious different from Ara VPGs, which confirm that polyelectrolyte was adsorbed on the surface of charged VPGs with electrostatic interaction. In vitro cumulative release percent of CHss-PLys VPGs,OCA-PGlu VPGs and VPGs in432h is47.03%、45.65%、37.37%, respectively. It indicated that polyelectrolyte adsorbed-charged modified VPGs can speed up in vitro drug release. In the research of rheology, these parameters indicated that viscoelasticity of polyelectrolyte adsorbed-charged modified VPGs was strengthened. It supposed that the segment of polyelectrolyte interwove with mesh style around the vesicles, variables of shape are significantly larger under strain force, G'and G" has no change on the shape of the curve as a function angular frequency, the curve is approach to the creep phenomenon of the Voigt model. Despite the viscoelasticity are significantly strengthened, yet drug release were faster. So, it is inexplicable that from the point of the view of the increase in viscosity can slow release. However, We speculated that, for phospholipid molecules in the vesicular film with fluidity at37℃, polyelectrolyte can increase the permeability of the membrane, lead to drug release accelerating.
The ultra performance liquid chromatography tandem mass spectrometry method (UPLC-MS/MS) has been developed for determination of Ara-C in biological sample. The brain tissue biodistribution study showed that drugs of Ara-C VPGs and solutions can penetrate into brain tissue5mm depth from implant site. The concentration of Ara-C with Ara-C VPGs intracerebral injection at28days is still higher than0.1ug/ml therapeutic concentrations, it showed that Ara-C VPGs can maintain therapeutic effects against glioma for28days. Ara-C VPGs were of significant sustained release properties and irregular biodistribution in brain tissue. Drug concentrations in brain tissue with Ara-C VPGs implanting were higher than that of Ara-C solutions injecting in normal SD rats. At the same time, flourescence sections were used to observe the relative distribution of drug in brain tissues, and the results of observation is in agreement with that of UPLC-MS/MS. In the safety study, the Ara-C VPGs were implanted into brain with infiltration of few inflammatory cells in 3to7days. No necrosis appears in brain tissue. Ara-C VPGs are of higher safety.
The in vitro cytotoxicity of Ara-C VPGs was examined by microculture tetrazolium (MTT) colorimetric assay using two tumor cell lines, C6glioma cell line and human U87-MG glioma cell line. Cellular uptake of FITC formulations was assessed in C6and U87-MG glioma cells using Fluorescence Microscopy. As a result, Ara-C VPGs had a significant in vitro cytotoxicity against these two cell lines, which was concentration-dependent. OCA-PGlu VPGs exhibited more obvious cytotoxicity than any other formations. The result is consistent with that of cellular uptake observed with Fluorescence Microscopy.
The in vivo antitumor effect of Ara-C VPGs was assessed using C6glioma-bearing wistar rats and human astrocytoma U87-MG tumor-bearing nude mice as models respectively. Experiment showed that tumor inhibition rate (95.87%) of Ara-C VPGs group on C6glioma-bearing wistar rats (p<0.01) was higher than that of Ara-C solutions group (45.52%)(p>0.05) with the same dose. However, for U87-MG glioma-bearing nude mice, all VPGs group showing tumor inhibition rate was higher(p<0.01)than that of Ara-C solutions group(p>0.01)with the same dose. Tumor inhibition rate of OCA-PGlu VPGs group (36.82%) is highest, Ara-C VPGs group (34.93%) is second, CHss-PLys VPGs group (28.58%) is minimum. This result was in agreement with the result of in vitro cytotoxicity and cellular uptake. The reason was supposed that, usually polyelectrolyte can promote drug permeation into cell, at the same time, OCA with positive charge on the surface of vesicle, have strong electrostatic interaction with a negatively charged cancer cellular membrane, this can contribute to cellular uptake. Yet, for CHss-PLys VPGs, CHss with Negative charge on the surface of vesicle, due to strong electrostatic repulsion with a negatively charged cancer cellular membrane, Vesicles can't be uptaked. Meanwhile no obvious decrease were observed for the body weight of tumor-bearing wistar rats or nude mice. No obvious toxicity occurred in all group.
Ara-C VPGs was of good physical and chemical stability and safe as implant in brain. Enhancing the concentration of drug and extending the action time of drug at the tumor region were successfully achieved. Moreover, the Ara-C VPGs can improve the brain distribution behavior and in vivo antitumor activity. This research provides scientific basis for development of Ara-C brain sustained released delivery system.
在处方前研究中,建立了阿糖胞苷(Ara-C)、磷脂及溶血磷脂的体外含量测定方法。Ara-C在不同水性介质中的溶解度均大于452.7mg/ml,油水分配系数小,亲脂性弱,随着pH的增加而降低；其脂质体／水分配系数为2.51±0.53,推测该药具有较好的细胞膜亲和力。
从制剂的外观、粒径、包封率、体外释放、流变学等方面研究了阿糖胞苷囊泡型磷脂凝胶(Ara-C VPGs)的性质及灭菌稳定性。透射电镜观察,其分散后的结构大多数为小单层脂质体；冷冻蚀刻电镜观察凝胶内部真实结构为相邻囊泡紧密堆积形成了独特的三维网状立体结构,在结构上区别于脂质体和脂质体凝胶。作为载体材料的磷脂浓度分别为300、400、500mg/g时,Ara-C VPGs的包封率分别为31.65%、53.99%、72.12%,432h体外累计释药分别为48.93%、37.37%、23.22%。此结果表明在水相体积不变时,随着磷脂浓度的增加,包封的体积增大而使得包封率升高；体外释放明显减慢,其原因为磷脂浓度增加,使粘度明显增大,减慢囊泡之间和囊泡内的药物向释放介质的扩散过程,结果释放明显减慢。通过高压均质法制备的Ara-C VPGs,经pH7.4的PBS再分散后粒径和包封率分别由灭菌前的119.6±66.2nm、32.6±2.1%灭菌后增加到165.6±71.9nm、62.6±2.3%,推测可能的原因是囊泡融合使粒径增大,包封体积增加导致包封率增加。体外药物释放由灭菌前80h累积释放79.13%,灭菌后降低至432h累积释放74.76%;在流变学研究中,灭菌后粘弹性加强,流动性降低。粘度的变化与粒径的分布密切相关,多分散系数越大粘度越低,由于小粒径囊泡类似于球珠可以润滑大粒径囊泡,从而降低体系粘度。灭菌后多分散系数比灭菌前小,因此,粘度增强,药物释放减慢。总之,经过高压灭菌后,Ara-C VPGs的囊泡粒径增大,包封率增加,体外释放缓慢,粘弹性加强,性质更加稳定。然而,0.05g Ara-C VPGs中药物含量灭菌前为1.01±0.04mg,灭菌后降低至0.73±0.06mg,溶血磷脂灭菌前为1.39±0.03%,灭菌后增加至2.32±0.01%。为了提高制剂中药物和磷脂的稳定性,尝试在处方中加入不同的稳定剂。研究发现加入亚硫酸钠能同时增加阿糖胞苷和磷脂的灭菌稳定性。推测其原因为灭菌的过程中亚硫酸钠电子转移产生SO3-·,通过自由基的反应能大大的缩减HO·的产生,抑制了Ara-C和磷脂的降解。
改变囊泡的表面电荷结构,通过与聚合物静电吸附,形成具有特殊性质的囊泡,可以改变其体内外性质。对于聚谷氨酸吸附-十八胺修饰复合型VPGs (OCA-PGlu VPGs)和聚赖氨酸吸附-胆固醇硫酸钠修饰复合型VPGs(CHss-PLys VPGs),从透射电镜观察可以看出在囊泡的表面包上一层衣膜,粒径在100～200nm,衣膜厚度约为10nm; zeta电位与采用E80磷脂制备的VPGs是一致的,推测PGlu和PLys通过静电作用分别吸附含OCA和CHss的VPGs的囊泡表面。体外释放研究Ara-C VPGs、OCA-PGlu VPGs和CHss-PLys VPGs432h累积释放分别为37.37%、45.65%、47.03%；在流变学研究中,CHss-PLys VPGs和OCA-PGlu VPGs粘弹性较Ara-C VPGs明显加强,推测聚氨基酸的链段以网状交织在囊泡的周围,在应变力作用下,形变量明显变大,其中弹性模量和粘性模量随着角频率变化的曲线形状没有改变,这个曲线非常接近福格特模型的蠕变现象。尽管粘弹性明显加强,但是药物释放反而加快,因此,从增加粘度的角度而减慢药物的释放是解释不通的。然而,推测其原因为在37℃囊泡膜上的磷脂分子具有流动性,聚电解质增加膜的渗透性,导致药物释放加快。
以SD大鼠为动物模型,通过脑内局部定位给药,用UPLC-MS/MS法测定Ara-C VPGs大鼠脑内植入给药后在不同时间、不同区域脑组织中的药物浓度,研究该制剂中的药物在脑内的分布情况。结果显示,在脑内注射VPGs后28天,Ara-C浓度在距离植入部位5mm范围内仍然能高于0.1μg/ml的治疗浓度,表明Ara-C VPGs在脑内有明显的缓释效应。脑组织是不规则的球体,具有流动性的Ara-C VPGs在脑内的分布不同于其他的固体植入剂在脑组织中的扩散过程,这个过程可能为Ara-C VPGs逐渐被脑脊液稀释成无数的囊泡并随着脑脊液循环进入侧脑室,沿着侧脑室的边缘及侧脑室的脉络丛向脑组织深部扩散。由于脑脊液中有丰富的磷脂酶,VPGs中的磷脂最终在脑内被磷脂酶降解,而药物在脑脊液中被代谢。荧光显微镜观察验证药物在脑内转运过程及分布情况。安全性研究中,VPGs在植入正常大鼠脑内第3、7天后,对脑组织切片HE染色观察,发现出现轻度的脑水肿和少量的炎性细胞的浸润,未见脑组织坏死,局部反应轻微,安全性相对较高。
体外细胞实验结果表明该制剂对C6和U87-MG胶质瘤细胞均有较明显的体外抑瘤效应,呈剂量依赖性,其中OCA-PGlu VPGs组抑制细胞生长作用最强,与荧光显微镜观察体外细胞摄取情况是一致的；对鼠C6胶质瘤细胞抑制效应优于人U87-MG胶质瘤细胞。体内抑瘤实验结果表明：与空白VPGs组相比,Ara-C VPGs对皮下C6鼠胶质瘤有较明显的抑制效应,抑瘤率达到95.87%(p<0.01),明显强于对照组溶液剂的抑瘤率45.52%(p>0.05)；而对于U87-MG裸鼠皮下移植瘤动物模型,与空白VPGs组相比,在相同剂量下,各VPGs组均有明显的肿瘤抑制作用(p<0.01),顺序为OCA-PGlu VPGs组(抑瘤率为36.82%)>Ara-C VPGs组(抑瘤率为34.93%)>CHss-PLys VPGs组(抑瘤率为28.58%),Ara-C溶液抑瘤率为16.02%,无明显抑制作用(p>0.01),与体外细胞抑制效应及细胞摄取情况的结果是一致的。其原因一方面是具有促渗透作用的PGlu和PLys能促进药物进入肿瘤细胞,另一方面是当OCA-PGlu VPGs与肿瘤细胞接触时,由于聚氨基酸被蛋白酶降解,暴露了囊泡表面OCA的正电荷,与带负电的肿瘤细胞膜之间通过静电的作用,增加细胞的摄取；然而对于CHss-PLys VPGs,囊泡表面的聚氨基酸被蛋白酶降解,暴露囊泡表面CHss的负电荷与带负电性的肿瘤细胞相互排斥,不被细胞摄取；每组动物体重变化均较小,毒性均较低。
研究显示Ara-C VPGs能提高肿瘤部位的药物浓度,延长抗肿瘤药物与肿瘤细胞的接触时间,改善阿糖胞苷的脑内分布行为和抗肿瘤疗效。其性质稳定,安全有效,适于脑内植入给药,为阿糖胞苷脑部缓释给药系统的研究提供科学的依据。
Glioblastoma multiforme (GBM), a malignancy of the glial cells of the brain, is the most frequent primary brain tumor and is of high incidence rate. Removing all tumor cells is impossible through surgical operation. Additionally, conventional chemotherapeutic drugs are difficult to go across the blood brain barrier (BBB) and reach the effective concentration of treatment in the brain. So it is of high fatality rate and in situ recurrence rate. In this paper, this is first attempt to develop vesicular phospholipids gels loaded with cytarabine for GBM chemotherapeutic at the original tumor site after surgical operation. Some advantages are manifested: VPGs are semisolid phospholipids dispersions with high concentration of phospholipids intended for long time sustained release properties; VPGs were implanted in the site of removed tumor and bypass the BBB; phospholipids, as the main component, are of good biocompatibility and no toxicity.
At preformulation study, determination methods of Ara-C, phospholipids and lysophosphatidylcholine (LPC) were established. The specificity, precision and reproducibility of the method were good. The solubility of Ara-C in different aqueous media are larger than452.7mg/ml; oil/water partition coefficient is low with poor lipophilicify and declines slightly with the increasing of pH, the value is0.1686for50mg/L Ara-C in pH7.4PBS; and liposome/water partition coefficient is2.51±0.53and is of better ablility of cell membrane affinity.
Ara-C VPGs were prepared and characterized in term of the appearance, particle size, entrapment efficiency (EE), in vitro release, and rheologic propperties. The structure of small unilamellar vesicles was observed by a Transmission Electron Microscope (TEM). Micrograph of Freeze-fracture electron microscopy (FF-TEM) showed that each vesicle was tightly packed between neighbor-vesicles to be three-dimensional network structure, the structure is different from liposome and liposome gels. With the lipid concentration of300、400、500mg/g, EE of Ara-C VPGs were31.65%、53.99%、72.12%, respectively; in vitro cumulative release were 48.93%、37.37%、23.22%in432h, respectively. It showed that EE are increasing and in vitro release are slow with the increase of lipid concentration, it supposed that entrapment volume is increasing with the increase of lipid concentration and aqueous phase volume is constant, viscosity is increasing with increase of lipid concentration can lead to release slowly. The sterilization stability of cytarabine (Ara-C) loaded vesicular phospholipids gels (VPGs) were also examined. The particle size of VPGs after redispersion was119.6±66.2nm, and EE was32.6±2.1%. Comparatively, after autoclaved sterilization, increased particle size and EE were obtained as165.6±71.9nm and62.6±2.3%, respectively. In vitro cumulative release was from79.13%in80h for nonautoclaved VPGs to74.76%in432h for autoclaved VPGs. Viscoelasticity were strengthened. The change in viscosity and particle size distribution is closely related, it means that, being of larger polydispersity of particle size distribution, the viscosity is lower at the same lipid concentration. The reason might be that the smaller particles like ball bearings lubricate the larger ones. As a result, after sterilization polydispersity is smaller than that of nonautoclaved. Additionally, the increased viscosity can slow drug release. In total, after sterilization, the vesicle is integrity, EE is increased, in vitro release is slow, viscoelasticity was strengthened, the properties become more stable. And characteristics of in vitro drug release were slowed remarkablely. Also, the viscoelasticity was reinforced with clearly decreased fluidity. However, reduced Ara-C and increased LPC were observed. The content of Ara-C in VPGs was1.01±0.04mg, after sterilization the content declined to0.73±0.06mg; LPC in autoclaved VPGs was increased to2.32±0.01%from1.39±0.03%for non autoclaved VPGs. In order to enhance the stability of Ara-C and phospholipids, stabilizers were added and among them addition of sodium sulfite showed best effects with high stability of both Ara-C and phospholipids. It supposed that SO3-? free radicals producing from sodium sulfite during the process of sterilization can reduce the generation of HO?free radicals greatly, which inhibit the degradation of Ara-C and phospholipids.
VPGs are with specific surface characteristics through the modification of the surface structure and change its in vitro and in vivo properties. For complexation of charged polyelectrolyte modified VPGs, TEM observed that the surface of vesicle was coated with a layer of film, particle size is about100nm. Zeta potential has no obvious different from Ara VPGs, which confirm that polyelectrolyte was adsorbed on the surface of charged VPGs with electrostatic interaction. In vitro cumulative release percent of CHss-PLys VPGs,OCA-PGlu VPGs and VPGs in432h is47.03%、45.65%、37.37%, respectively. It indicated that polyelectrolyte adsorbed-charged modified VPGs can speed up in vitro drug release. In the research of rheology, these parameters indicated that viscoelasticity of polyelectrolyte adsorbed-charged modified VPGs was strengthened. It supposed that the segment of polyelectrolyte interwove with mesh style around the vesicles, variables of shape are significantly larger under strain force, G'and G" has no change on the shape of the curve as a function angular frequency, the curve is approach to the creep phenomenon of the Voigt model. Despite the viscoelasticity are significantly strengthened, yet drug release were faster. So, it is inexplicable that from the point of the view of the increase in viscosity can slow release. However, We speculated that, for phospholipid molecules in the vesicular film with fluidity at37℃, polyelectrolyte can increase the permeability of the membrane, lead to drug release accelerating.
The ultra performance liquid chromatography tandem mass spectrometry method (UPLC-MS/MS) has been developed for determination of Ara-C in biological sample. The brain tissue biodistribution study showed that drugs of Ara-C VPGs and solutions can penetrate into brain tissue5mm depth from implant site. The concentration of Ara-C with Ara-C VPGs intracerebral injection at28days is still higher than0.1ug/ml therapeutic concentrations, it showed that Ara-C VPGs can maintain therapeutic effects against glioma for28days. Ara-C VPGs were of significant sustained release properties and irregular biodistribution in brain tissue. Drug concentrations in brain tissue with Ara-C VPGs implanting were higher than that of Ara-C solutions injecting in normal SD rats. At the same time, flourescence sections were used to observe the relative distribution of drug in brain tissues, and the results of observation is in agreement with that of UPLC-MS/MS. In the safety study, the Ara-C VPGs were implanted into brain with infiltration of few inflammatory cells in 3to7days. No necrosis appears in brain tissue. Ara-C VPGs are of higher safety.
The in vitro cytotoxicity of Ara-C VPGs was examined by microculture tetrazolium (MTT) colorimetric assay using two tumor cell lines, C6glioma cell line and human U87-MG glioma cell line. Cellular uptake of FITC formulations was assessed in C6and U87-MG glioma cells using Fluorescence Microscopy. As a result, Ara-C VPGs had a significant in vitro cytotoxicity against these two cell lines, which was concentration-dependent. OCA-PGlu VPGs exhibited more obvious cytotoxicity than any other formations. The result is consistent with that of cellular uptake observed with Fluorescence Microscopy.
The in vivo antitumor effect of Ara-C VPGs was assessed using C6glioma-bearing wistar rats and human astrocytoma U87-MG tumor-bearing nude mice as models respectively. Experiment showed that tumor inhibition rate (95.87%) of Ara-C VPGs group on C6glioma-bearing wistar rats (p<0.01) was higher than that of Ara-C solutions group (45.52%)(p>0.05) with the same dose. However, for U87-MG glioma-bearing nude mice, all VPGs group showing tumor inhibition rate was higher(p<0.01)than that of Ara-C solutions group(p>0.01)with the same dose. Tumor inhibition rate of OCA-PGlu VPGs group (36.82%) is highest, Ara-C VPGs group (34.93%) is second, CHss-PLys VPGs group (28.58%) is minimum. This result was in agreement with the result of in vitro cytotoxicity and cellular uptake. The reason was supposed that, usually polyelectrolyte can promote drug permeation into cell, at the same time, OCA with positive charge on the surface of vesicle, have strong electrostatic interaction with a negatively charged cancer cellular membrane, this can contribute to cellular uptake. Yet, for CHss-PLys VPGs, CHss with Negative charge on the surface of vesicle, due to strong electrostatic repulsion with a negatively charged cancer cellular membrane, Vesicles can't be uptaked. Meanwhile no obvious decrease were observed for the body weight of tumor-bearing wistar rats or nude mice. No obvious toxicity occurred in all group.
Ara-C VPGs was of good physical and chemical stability and safe as implant in brain. Enhancing the concentration of drug and extending the action time of drug at the tumor region were successfully achieved. Moreover, the Ara-C VPGs can improve the brain distribution behavior and in vivo antitumor activity. This research provides scientific basis for development of Ara-C brain sustained released delivery system.
引文
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[1]M.F. Lin, J. DaVolio, R. Garcia. Cationic liposome-mediated incorporation of prostatic acid phosphatase protein into human prostate carcinoma cells, Biochemical and biophysical research communications,1993 (192):413-419.
[2]D. Volodkin, V. Ball, P. Schaaf, J.C. Voegel, H. Mohwald. Complexation of phosphocholine liposomes with polylysine. Stabilization by surface coverage versus aggregation, Biochimica et Biophysica Acta (BBA)-Biomembranes,2007 (1768):280-290.
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[4]I. Tsogas, D. Tsiourvas, C.M. Paleos, S. Giatrellis, G. Nounesis. Interaction of 1-arginine with dihexadecylphosphate unilamellar liposomes:the effect of the lipid phase organization, Chemistry and physics of lipids,2005 (134):59-68.
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[7]崔福德.药剂学.中国医药工业出版社,2002.08.
[1]B.A. Olsen. Hydrophilic interaction chromatography using amino and silica columns for the determination of polar Pharmaceuticals and impurities, Journal of Chromatography A,2001 (913):113-122.
[2]P.B. Storm, J.L. Moriarity, B. Tyler, P.C. Burger, H. Brem, J. Weingart. Polymer delivery of camptothecin against 9L gliosarcoma:release, distribution, and efficacy, Journal of neuro-oncology,2002 (56):209-217.
[3]S.H. Ranganath, Y. Fu, D.Y. Arifin, I. Kee, L. Zheng, H.S. Lee, P.K.H. Chow, C.H. Wang. The use of submicron/nanoscale PLGA implants to deliver paclitaxel with enhanced pharmacokinetics and therapeutic efficacy in intracranial glioblastoma in mice, Biomaterials, 2010 (31):5199-5207.
[4]S. Phuphanich, B. Maria, R. Braeckman, M. Chamberlain. A pharmacokinetic study of intra-CSF administered encapsulated cytarabine (DepoCyt(?)) for the treatment of neoplastic meningitis in patients with leukemia, lymphoma, or solid tumors as part of a phase III study, Journal of neuro-oncology,2007 (81):201-208.
[5]J. Siepmann, F. Siepmann, A. Florence. Local controlled drug delivery to the brain: mathematical modeling of the underlying mass transport mechanisms, International journal of pharmaceutics,2006 (314):101-119.
[6]B. Ong, S.H. Ranganath, L.Y. Lee, F. Lu, H.S. Lee, N.V. Sahinidis, C.H. Wang. Paclitaxel delivery from PLGA foams for controlled release in post-surgical chemotherapy against glioblastoma multiforme, Biomaterials,2009 (30):3189-3196.
[1]S. Karmakar, N.L. Banik, S.K. Ray. Curcumin suppressed anti-apoptotic signals and activated cysteine proteases for apoptosis in human malignant glioblastoma U87MG cells, Neurochemical research,2007 (32):2103-2113.
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