替莫唑胺/紫杉醇复合缓释系统的构建及其抗胶质瘤性能的实验研究
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摘要
研究背景
胶质瘤是颅内最常见恶性肿瘤,是欧美成年人群癌症致死的第四位死因,目前主要治疗方案为手术切除加术后放化疗。但患者术后生存中期仍小于2年。胶质瘤转移少见,大约超过90%的复发为瘤腔局部2cm内的复发,这就为控制胶质瘤生长提供了思路,即局部用药。术后一定时间内维持肿瘤局部一定浓度的治疗药物可能是使肿瘤静止甚至消失、预防复发的有效方法。理想的局部化疗体系能够提供有效浓度药物的持续释放,且组织相容性好,局部瘢痕和神经毒性小。目前研究较多的是以生物可降解材料为载体的缓释系统。Gliadel(?) wafer是第一个用于临床的可降解的局部缓释化疗系统,是以PCPP:SA (20:80)为载体的含有3.85%卡莫司汀(BCNU)的小碟片。临床应用证实它可以明显提高患者生存中期,给胶质瘤患者带来新的希望。但目前它只被美国FDA批准用于复发性胶质瘤的治疗,因为大部分肿瘤都含有一种DNA修复蛋白AGT,可以使肿瘤对BCNU产生抗药性。因此需要探寻其它有效药物和更安全有效的缓释载体。
替莫唑胺是目前胶质瘤化疗药中单药疗效最高的化疗药物。它无需酶催化,即可转化为活性化合物MTIC[5-(3-甲基三氮烯-1-)咪唑-4-酰胺],直接发挥烷基化DNA,诱导细胞凋亡的作用。因此替莫唑胺是局部缓释用药的良好选择。最近较多研究表明替莫唑胺局部应用优于全身用药。
紫杉醇是一种微管稳定剂,临床上对多种肿瘤有良好的治疗效果。但由于血脑屏障的存在,紫杉醇对脑胶质瘤的治疗效果不理想。紫杉醇全身用药副作用较大,有研究表明利用可降解材料局部缓释紫杉醇能有效抑制大鼠胶质瘤的生长。因此紫杉醇也是局部缓释治疗脑胶质瘤的理想药物。
当前用于制作缓释载体的材料多为人工合成聚合物,如聚乳酸(Polylactic acid, PLA)、聚乙醇酸(Polyglycolic acid, PGA)及其共聚物(PLGA)等。Wafer即采用聚酸酐(PCPP-SA)作为缓释载体。在应用过程中发现这类聚合物存在一定缺点,如亲水性差,细胞贴附力较弱,酸性降解产物引起无菌性炎症,在实验中观察到局部脑组织内炎细胞浸润及脑水肿明显。此外,在一些临床试验中发现,此类降解材料的组织相容性较差,周围瘢痕形成明显,影响缓释药物的弥散,降解的碎片游动,有导致脑室系统堵塞的危险。而PPC是一种不释放酸性物质的新型聚合物,最终降解产物是CO2和水,大大减少了材料对脑组织的局部刺激作用,而且与脑组织有很好的组织相容性,因此PPC是用于神经系统药物缓释系统载体的理想材料。
目前用于制作缓释系统的方法很多,如盐释析法、膜复合法、相色分离法、乳液冷冻干燥法、水相冷冻干燥法等。由于局部组织间压力及脑脊液的流动,微球容易游动,很难聚集在瘤腔内,而且采用微球表结构,药物突释现象严重,容易产生局部神经毒性,造成组织水肿及坏死等,因此微球结构很难应用于脑部治疗。电纺丝方法是一种制备高分子纳米/微米纤维的有效方法。电纺丝制作的膜状材料具有很好的韧性,断裂伸长率高,满足了对缓释系统的力学性能要求。电纺丝的制备过程条件温和,可以较为精确的控制纤维膜的微观结构、孔隙率和组成,在空间结构上具有仿生特点,很好的模拟了人体组织中细胞外基质,可以用于脑局部缓释系统的制备。
前期研究表明药物的释放速度和突释现象随载药量的增大而增加,短期内药物的集聚可能导致神经毒性、脑水肿等不良反应,因此通过本研究,我们拟设计一种较低载药量的复合缓释系统。通过携带两种有协同作用的药物,在缓释系统载药量较低的情况下,仍然可以达到有效抑制肿瘤的效果。我们采用电纺丝技术,将含有微球结构的乳液进行电纺丝操作,制备出微球包被于纺丝纤维内部的具有壳核结构的复合系统,分别携带替莫唑胺和紫杉醇两种药物,分析制备过程中的各种参数,各样品的形态特征及药物释放曲线,并通过体外体内实验分析它的安全性及有效性,希望能用于脑胶质瘤的临床治疗。
本实验共分三部分进行：
第一部分药物缓释系统的构建及其参数特征的测定；
第二部分药物缓释系统体外抗C6胶质瘤细胞的实验研究；
第三部分复合缓释系统体内抗大鼠胶质瘤性能的实验研究。
第一部分药物缓释系统的构建及其参数特征的测定
研究目的
在本部分中拟制备替莫唑胺电纺丝缓释系统、紫杉醇微球缓释系统及替莫唑胺/紫杉醇复合缓释系统,分析制作参数、形态特征以及释放曲线。研究方法
1.制备缓释系统：用电纺丝方法制作替莫唑胺纺丝缓释系统,用乳化-离子交联法制作紫杉醇微球,再将微球悬液通过电纺丝装置制作成复合缓释系统。紫杉醇微球载药量不变,通过改变替莫唑胺的载药量使复合缓释系统内替莫唑胺和紫杉醇的比例约为2:1、1:1、1:2。
2.扫描电镜观察：用扫描电镜观察替莫唑胺纺丝缓释系统、紫杉醇微球缓释系统及复合缓释系统的表面形态特征。
3.热分析：用差示扫描量热法分析替莫唑胺纺丝缓释系统和紫杉醇微球缓释系统中药物的物理形态。
4.药物包封率、载药量的测定：将制备的替莫唑胺纺丝缓释系统、紫杉醇微球缓释系统及复合缓释系统,完全溶解后用紫外分光光度法测定药物含量。载药量=系统中药物总量/系统总质量×100%包封率=系统中药物总量/实验加入药物总量×100%5.药物释放曲线的测定：准确称取替莫唑胺纺丝缓释系统、紫杉醇微球缓释系统及复合缓释系统样品,放入离心管后固定于水浴恒温摇床中,条件37℃,100 rpm/min。在1h、3h、6h、12h、24h、72h、120h、168h、240h和280h后,检测残余样品中药物含量,从而计算累计释放药物百分比,得到释放时间-累计释药百分比的药物释放曲线。研究结果
1.替莫唑胺/PPC纺丝缓释系统的制作条件比较严格,只有在PPC浓度为8wt%、纺丝距离为15-20cm、纺丝电压为10-15kV时才能流畅顺利的进行电纺丝操作。采用乳化离子交联法可以制作出直径较为均匀的紫杉醇微球。制作复合纺丝缓释系统时的电压为12kV,接收距离为15cm。
2.通过扫描电镜观察,纺丝纤维均匀光滑,平均直径为3μm,微球平均直径为7.2μm,复合缓释系统呈串珠样结构,微球包被在纺丝纤维内。
3.从DSC分析可以看出,单纯替莫唑胺和紫杉醇的放热峰值分别为207.5℃和223.0℃,而缓释系统的DSC曲线中没有放热峰值,说明药物是以高温稳定的晶型或无定形态的状态存在于材料内,而不是以颗粒状态。
4.微球缓释系统中紫杉醇的载药量为3.9%,包封率为65.3%,在复合缓释系统中的载药量为2.1%,包封率为94.5%,替莫唑胺在复合系统中的载药量分别为1.2%、2.3%、4.1%,包封率分别为97.6%、96.1%、95.7%。微球缓释系统中紫杉醇的包封率低,可能因为有部分紫杉醇从微球中迁移出来,溶于有机溶剂中,而在复合缓释系统中包封率增加是因为溶于有机溶剂中的紫杉醇通过电纺丝操作留在了纺丝纤维中,同时说明电纺丝方法优于微球方法。
5.微球缓释系统中的紫杉醇可以持续释放8天左右,前期突释现象明显,而在复合系统中紫杉醇的释药时间约为12天,突释现象有所减轻。说明经纺丝纤维包被以后可以减轻微球缓释系统的突释现象,同时能延长药物释放时间。替莫唑胺在复合系统中的释药时间约为12天,载药量越大,前期释放速度越快,因此载药量低有利于药物平稳释放。结论
1.PPC材料可以作为药物缓释系统载体进行电纺丝操作。
2.替莫唑胺和紫杉醇是以高温稳定的晶型或无定形态的状态存在于缓释系统内。
3.复合缓释系统的核壳结构可以减轻微球缓释系统的突释现象,延长药物释放时间。
4.所制备的各种缓释系统均可以长时间稳定释放替莫唑胺和紫杉醇。
第二部分药物缓释系统体外抗C6胶质瘤细胞的实验研究
研究目的
分析替莫唑胺/PPC缓释系统、紫杉醇/海藻酸钙微球缓释系统及替莫唑胺/紫杉醇复合缓释系统体外对C6胶质瘤细胞的细胞毒性作用,讨论复合缓释系统中两种药物的理想比例。
研究方法
1.培养C6胶质瘤细胞：用含10%胎牛血清的DMEM培养液,在5%CO2,湿度90%,温度37℃条件下培养。
2.细胞毒性实验：将C6胶质瘤细胞移至96孔板,每孔10000个细胞。准确称取样品经钴60消毒后与细胞共培养,分别于1、2、3天后检测。分组：1组：2:1复合样品；2组：1:1复合样品；3组：1:2复合样品；4组：替莫唑胺纺丝膜；5组：紫杉醇微球；6组：PPC/海藻酸组；7组：空白对照,每组分三份。
3.细胞活性检测：采用cck-8法检测细胞活性。按照试剂说明书操作。将cck-8加入各孔后,37℃下培养1小时候,用自动酶标仪检测各孔OD值。细胞活力(%)=实验组OD值/对照组OD值×100%。
研究结果
1.空白纺丝膜和微球材料组的细胞活性在99%以上,说明所用载体材料对C6细胞没有细胞毒性作用。
2.第1、2、3天时的细胞活性1组分别为52.4±6.3%、27.9±5.3%、5.5±2.4%,2组分别为56.3±4.2%、29.3±6.2%、9.1±5.1%,3组分别为75.7±2.9%、52.1±4.9%、22.9±2.5%,4组分别为86.7±5.1%、66.4±4.3%、35.4±2.7%,5组分别为80.0±4.2%、63.1±4.5、29.5±3.4%。采用SPSS13.0统计分析软件进行方差分析,1组与2组间P>0.05,差异无统计学意义,1组与3组、2组与3组、1组与4组、1组与5组间P<0.05,差异有统计学意义。虽然2组缓释系统中替莫唑胺的载药量比1组的低,但抑制C6细胞的效果没有显著性差异,说明复合缓释系统中替莫唑胺和紫杉醇的比例约为1:1时,两种药物可以达到较为理想的协同效应。同时可以看出各个缓释系统均能持续抑制C6胶质瘤细胞的活性,而且复合缓释系统优于单药物缓释系统。
结论
1.PPC和海藻酸材料对C6胶质瘤细胞无细胞毒性作用。
2.本研究所制备的各种药物缓释系统均能持续抑制C6胶质瘤细胞的活性。
3.本研究所制备的复合药物缓释系统比单药物缓释系统更能有效抑制C6胶质瘤细胞的活性。
4.替莫唑胺和紫杉醇质量比约为1:1的复合缓释系统在保持较低载药量的同时又可以达到有效抑制C6胶质瘤细胞的效果,是较为理想的局部缓释系统。
第三部分复合缓释系统体内抗大鼠胶质瘤性能的实验研究
研究目的
观察讨论替莫唑胺/紫杉醇复合缓释系统在大鼠脑内局部应用的安全性及对大鼠原位C6胶质瘤的治疗效果。所采用复合缓释系统中替莫唑胺和紫杉醇的载药量分别为2.3%和2.1%。
研究方法
1.构建大鼠胶质瘤模型：采用立体定向技术将C6胶质瘤细胞注入SD大鼠大脑皮层,共31只。1周后行MRI检查,生长肿瘤者分两组：治疗组(10只)、对照组(8只),1只处死取标本。另设正常大鼠组(4只)。
2.植入缓释膜片：扩大骨窗,将lmg药物缓释膜片植入治疗组肿瘤内。对照组进行相同操作,但不植入膜片,3周后再行脑MRI检查。同时将膜片植入正常大鼠脑内,于第3、第7天行脑MRI检查。
3.HE染色：将大鼠脑组织及肿瘤组织做石蜡切片,行HE染色。光镜下观察组织变化。
4.胶质瘤组织中增殖细胞核抗原(PCNA)蛋白的表达：采用链霉亲和素-生物素-过氧化氢酶复合物法(SABC)检测肿瘤组织中PCNA表达。每张切片随机选取10个视野(400倍),计数PCNA阳性细胞数和总细胞数。PCNA阳性率=阳性细胞数/总细胞数×100%
5.肿瘤组织内细胞凋亡的检测：采用末端脱氧核苷酸转移酶介导的末端标记技术(TUNEL)试剂盒检测肿瘤组织细胞的凋亡。按照试剂盒说明书操作。观察组织内阳性细胞的表达。
研究结果
1.正常大鼠植入膜片后第3、7天行脑MRI检查,未见脑组织水肿。从HE染色看,第3天时可见脑组织表面约0.8mm处细胞间质水肿,少量淋巴细胞浸润,第7天水肿略有减轻,约0.5mm,仍有少量炎细胞浸润。大鼠在饲养过程中未出现饮食异常、抽搐等表现。说明该复合缓释系统在脑中的局部反应轻微,未导致严重水肿等并发症,安全性高。
2.制作肿瘤模型31只,术后死亡7只,磁共振显示肿瘤未生长者5只,形成肿瘤者19只,成瘤率为61.3%。光镜下HE染色观察肿瘤细胞呈假栅栏状或珊瑚状排列,密集成群,早期肿瘤组织与脑组织边界清楚,晚期可见肿瘤细胞侵入脑实质,边界模糊。经治疗后肿瘤组织内可见坏死灶。
3.接种1周后,大鼠脑胶质瘤体积平均为122.1±4.5mm3,经治疗3周后体积为184.0±5.4mm3,肿瘤增长率为50.8%,对照组肿瘤体积平均为341.3±11.4mm3,肿瘤增长率为180.0%,两组间差异有统计学意义(P<0.0001),说明该复合缓释系统可以有效抑制肿瘤生长。
4.治疗组大鼠生存中期为42.5天,对照组生存中期为28.5天,两组间差异有统计学意义(P<0.05)。说明经过该复合缓释系统治疗,大鼠生存期明显延长。
5.治疗前、对照组和治疗组大鼠脑肿瘤组织中PCNA阳性表达率分别为65.4±5.7%、68.6±6.3%、15.3±5.2%,治疗前与对照组间无明显差异(P>0.05),对照组与治疗组间差异有统计学意义(P<0.05),治疗组的PCNA阳性细胞明显减少。
6.对照组肿瘤组织大部分无凋亡细胞表达,偶可见少量凋亡细胞,而治疗组肿瘤内可见大量凋亡细胞,尤其膜片周围瘤组织。
结论
1.本研究所制备的替莫唑胺/紫杉醇复合缓释系统有良好的组织相容性,安全性高,在脑局部应用无不良反应。
2.本研究所制备的替莫唑胺/紫杉醇复合缓释系统能有效抑制大鼠胶质瘤的生长,降低肿瘤细胞增殖,诱导肿瘤细胞凋亡,显著提高了大鼠的生存中期。
创新性及局限性
1.创新点
(1)采用一种微米级核壳结构复合纤维作为缓释系统支架,该复合纤维结合了微球与电纺丝纤维的优点,可以同时携带两种药物。
(2)复合缓释系统携带替莫唑胺和紫杉醇两种有协同作用的药物,在药物载药量较低时,仍可以达到理想的抑制肿瘤效果,并分析讨论了复合缓释系统内两种药物的合理配比。
2.局限性
(1)本研究制备的缓释系统的药物持续释放时间相对较短,未能达到理想时间,这与载体材料降解速率、药物与材料结合力、纺丝纤维直径等有关。
(2)由于大鼠脑体积小,颅内切除肿瘤困难,未能采用切除后大鼠胶质瘤模型进行动物实验,因此本研究动物实验部分只能证明该复合缓释系统可以有效抑制肿瘤生长,对控制肿瘤复发的效果仍不能确定,需要进一步研究。
Background
Malignant glioma is the most common type of primary brain tumor in adults. Approximately 5 new cases per 100,000 in population are diagnosed each year. The current standard treatment for malignant glioma consists of surgical resection followed by radiation therapy and aggressive systemic chemotherapy. However, the prognosis for patients with maglignant glioma is relatively poor with a median survival of<2 years. Autopsy studies indicate that 90% of recurrent gliomas occur within 2 centimeters and metastasize rarely, so local chemotherapy provides an alternative drug delivery method for the treatment of brain tumors. Desirable local chemotherapy system could provide sustained and effective drug concentration with well histocompatibility and less neurotoxicity. The present research about local chemotherapy system is focus on biodegradable materials. Gliadel(?) wafer is the first biodegradable chemotherapy system used to treat glioma. The wafer consists of polycarboxyphenoxypropane and sebacic acid in a 20:80 molar ratio and contains 3.85% BCNU. It has showed some success in improving survival of patients with malignant glioma. However, it is only used to treat recurrent glioma by FDA because AGT, a DNA-repair protein found in the majority of brain tumors. So we need to find other effective drugs and safe biodegradable vehicle.
Temozolomide(TM) is one of the most effective antineoplastic agents for malignant glial tumors. TM is converted to its active metabolite 3-methyl-(triazen-1-yl) imidazole-4-carboxamide (MTIC) at physiological pH. MTIC induces apoptosis of cells due to alkylation of DNA at the 06 and N7 positions of guanine. It is evident that local delivery of temozolomide by biodegradable polymers is superior to oral administration in a rodent glioma model.
Paclitaxel targets microtubules and has been clinically effective against a variety of human cancers, that shows cytotoxicity against glioma in vitro. However, it is prevented from entering brain and penetrating intact blood-brain barrier poorly. Biodegradable polymer implant was developed to deliver paclitaxel against experimental malignant glioma and found to be efficient in a rat model.
Most work on electrospinning biodegradable polymers has focused on synthetic materials, notably PLA, PGA, PLGA, and PCL. However, these materials have some disadvantages, such as low hydrophilicity, and aseptic inflammation because of their acid degradation products. Poly-propylene carbonate (PPC) is a degradable material formed from copolymerization of propylene oxide and carbon dioxide. It has low local irritant effect in brain tissue and good histocompatibility with brain. So PPC is the ideal material as release vehicle in brain.
Various techniques have been designed to prepare controlled drug delivery systems, including solvent casting/salt leaching, membrane lamination, phase separation, emulsion freezing/drying, and hydrogel freezing/drying. Due to very high interstitial pressure and circulation of cerebrospinal fluid, microspheres have the risk of being expelled out of the target site. The other disadvantage of using microspheres is the high initial burst due to the presence of the drug on the surface which might lead to undesired neurotoxicity. Electrospinning is an attractive approach for the fabrication of fibrous biomaterials. Electrospun mats have larger specific surface areas and smaller pore size for polymer degradation and drug diffusion compared to other controlled drug delivery systems.
Therefore, the aim of this study was to designe a high effective local drug delivery system with low loading rate. We combine electrospinning with microspheres delivering temozolomide and paclitaxel, avoiding the possibility of neurotoxicity. The preparation parameters and the safety and effectivity of the system were analyzed.
This study includes three parts:
PartⅠThe fabrication and study of the biodegradable implants
PartⅡCytotoxic study of the implants against glioma C6 cells in vitro
PartⅢThe study of combined implant against glioma in rats
PartⅠThe fabrication and study of the biodegradable implants
Objective
To fabricate the drug delivery systems and investigate the preparation parameters, physical characteristics and release properties
Methods
1. Preparation of the drug delivery system:Temozolomide fibers were fabricated by electrospinning, and paclitaxel microspheres were prepared using emulsifying-solvent evaporation method. Then emulsion of microspheres was electrospinned into combined mats. The ratio of temozolomide and paclitaxel was 2:1, 1:1,1:2 respectively, through holding the loading rate of paclitaxel and changing the temozolomide.
2. Physical characteristics:The size, distribution and surface morphology of the fibers and microspheres were examined using a scanning electron microscope after gold coating.
3. Thermal analysis of drugs in matrices:Thermal analysis was performed using differential scanning calorimetry (DSC) to characterize the physical status of drugs in matrices.
4. The drug encapsulation efficiency (EE) and loading rate were calculated according to the following equations: Loading rate=WM/WT×100 Encapsulation efficiency=WM/Dt×100 , where WM is the amount of drug in systems, Dt is the amount of drug used for the preparation, and WT is the weight of systems.
5. The drugs releasing study in vitro:the systems were immersed into 20ml PBS buffer in tube incubated at 37℃and 100 rpm in a shaking water bath. At each specified time interval,1h、3h、6h、12h、24h、72h、120h、168h、240h和280h, drugs in solution were detected by UV analysis. Then the time-cumulative drug released plots would be produced.
Results
1. When PPC was 8%, distance was 15-20cm and voltage was 10-15kV, electrospinning could be performed normally. The TM fibers were smooth and uniform only at 8% by weight. The paclitaxel microspheres have proper size when the stirring rate is 500 rpm and concentration of alginate is 1%. The emulsion of microspheres was electrospinned into the beads-in-string structure at 15cm and a voltage of 12kV.
2. SEM images showed that the diameters of the fibers and microspheres were 3μm and 7.2μm. The surface of fibers and microspheres was smooth and uniform.
3. Pure TM and paclitaxel showed an endothermic melting peak at 207.5℃and 223.0℃, but no peak was seen at temperatures of 100-250℃for the samples. It is evident that the drugs were in an amorphous or disordered-crystalline phase of a molecular or a solid solution state in the matrix.
4. The loading rate and encapsulation efficiency of paclitaxel in microspheres were 3.9% and 65.3%,2.1% and 94.5% in combined fibers. The loading rate and encapsulation efficiency of TM in combined fibers were 1.2%,2.3%,4.1% and 97.6%, 96.1%,95.7%. The EE of paclitaxel in microspheres is lower, because that it could dissolve in dichloromethane in preparation of microspheres. However, this part of paclitaxel stays in fibers during process of electrospinning.
5. The microspheres released paclitaxel for about 8 days with obvious initial burst, and paclitaxel could be released for about 12 days with low initial burst. Because it could prolong the release time and reduce the initial burst when the microspheres were covered in fibers. TM was released for about 12 days from combined fibers. The initial release rate was faster with higher loading rate.
Conclusion
1. PPC could fabricate the drug delivery system by electrospinning.
2. TM and paclitaxel were in an amorphous or disordered-crystalline phase of a molecular or a solid solution state in the matrix.
3. Combined delivery systems could reduce initial burst of paclitaxel microspheres and prolong its release time.
4. Temozolomide and paclitaxel could be released stably for long time from delivery systems.
PartⅡCytotoxic study of the implants against glioma C6 cells in vitro
Objective
To investigate the effect of implants against glioma C6 cells in vitro and the best ratio of two drugs
Methods
1. C6 cell culture:Cells were maintained in tissue culture in Dulbecoo's minimum essential medium with 10% fetal bovine serum, in atmosphere of 5% CO2, and 90% relative humidity at 37℃。
2. Cytotoxicity test:C6 cells were transferred to a 96-well plate to ensure 1×104 cells per well. The following experimental groups were studied. (1) 2:1 combined fibers; (2) 1:1 combined fibers; (3) 1:2 combined fibers; (4) TM/PPC fibers; (5) paclitaxel microspheres; (6) PPC and alginate; (7) control group, each group in triplicate.
3. To detect cell viability:The cytotoxicity assay was conducted by Cell Counting Kit-8(cck-8). The plates were detected through microplate reader. Cell viability was determined according to the following equation:
Cell viability (%)=Abs test cells/Abs control cells×100
Results
1. The cells viability of group (6) and (7) is over 99%. It is evident that the material PPC and alginate has no cytotoxicity to C6 glioma cells.
2. The cells viability of group (1) at 1,2,3 days is 52.4±6.3%、27.9±5.3%、 5.5±2.4%, group (2) is 56.3±4.2%、29.3±6.2%、9.1±5.1%,group (3) is 75.7±2.9%、52.1±4.9%、22.9±2.5%, group (4) is 86.7±5.1%、66.4±4.3%、35.4±2.7%, and group (5) is 80.0±4.2%、63.1±4.5、29.5±3.4%. Statistical analysis shows that there are no statistical difference between group (1) and group (2)(P>0.05), but there are obvious difference between group (1) and (3), group (2) and group (3), group (1) and group (4), group (1) and group (5)(P<0.05). All kinds of implants could inhibit the C6 glioma cells continuously.
Conclusion
1. The material PPC and alginate has no cytotoxicity to C6 glioma cells.
2. Combined implants could produce stronger cytotoxicity than single drug implants with low loading rate.
3. When the ratio of TM and paclitaxel was about 1:1, the synergistic effect was obvious.
PartⅢThe study of combined implants against glioma in rats
Objective
To investigate the effect of combined implants (1:1) against glioma in rat model and the security used in brain
Methods
1. Combined fiber mats were implanted in the surface of hemisphere of four Sprague Dawley rats. The rats were scanned by MRI at 3,7 days.
2. Preparation of rat glioma model:31 male (200-250g) Sprague Dawley rats were implanted in C6 cells. All rats should be scanned by MRI after a week. The rats with tumors were divided to two groups:treatment group (n=10) and control group (n=8).
3. The sections were subjected to hematoxylin and eosin attaining for histological examination.
4. Proliferation cell nuclear antigen (PCNA) was used to evaluate the proliferation of tumor. PCNA-positive cells were determined by randomly counting 10 fields of the section and were expressed as a percentage of normal nuclei.
5. To evaluate apoptotic activity, the TUNEL technique was used.
Results
1. There was no brain edema on MRI after the mats were implanted in hemisphere. In microscope, the range of edema is about 0.8mm at 3 day, and 0.5mm at 7day, accompanying with few lymphocyte infiltrating.
2. The volume of tumor was 122.1±4.5mm3 at first week. Three weeks after treatment, the volume of treatment group tumor was 184.0±5.4mm3, and the growth rate was 50.8%. The control group was 341.3±11.4mm3 and 180.0%. Significant differences were observed between two groups (P<0.0001).
3. The median survival time of treatment group was 42.5 days, and the control group is 28.5 days. Two groups had significantly difference (P<0.05). It is evident that the combined fibers could prolong the survival of rats.
4. The percentage of PCNA-positive were 65.4±5.7%、68.6±6.3%、15.3±5.2% with initial tumor, control and treatment group. The PCNA-positive cells decreased obviously after cure.
5. The treatment group shows many apoptotic cells, and the control group has no expression of apoptotic cells.
Conclusion
1. The combined fibers have well histocompatibility and security.
2. The combined fibers could inhibit the growth of tumor effectively, decrease proliferation of tumor cells, induce apoptosis of tumor cells, and prolong the rat median survival time.
胶质瘤是颅内最常见恶性肿瘤,是欧美成年人群癌症致死的第四位死因,目前主要治疗方案为手术切除加术后放化疗。但患者术后生存中期仍小于2年。胶质瘤转移少见,大约超过90%的复发为瘤腔局部2cm内的复发,这就为控制胶质瘤生长提供了思路,即局部用药。术后一定时间内维持肿瘤局部一定浓度的治疗药物可能是使肿瘤静止甚至消失、预防复发的有效方法。理想的局部化疗体系能够提供有效浓度药物的持续释放,且组织相容性好,局部瘢痕和神经毒性小。目前研究较多的是以生物可降解材料为载体的缓释系统。Gliadel(?) wafer是第一个用于临床的可降解的局部缓释化疗系统,是以PCPP:SA (20:80)为载体的含有3.85%卡莫司汀(BCNU)的小碟片。临床应用证实它可以明显提高患者生存中期,给胶质瘤患者带来新的希望。但目前它只被美国FDA批准用于复发性胶质瘤的治疗,因为大部分肿瘤都含有一种DNA修复蛋白AGT,可以使肿瘤对BCNU产生抗药性。因此需要探寻其它有效药物和更安全有效的缓释载体。
替莫唑胺是目前胶质瘤化疗药中单药疗效最高的化疗药物。它无需酶催化,即可转化为活性化合物MTIC[5-(3-甲基三氮烯-1-)咪唑-4-酰胺],直接发挥烷基化DNA,诱导细胞凋亡的作用。因此替莫唑胺是局部缓释用药的良好选择。最近较多研究表明替莫唑胺局部应用优于全身用药。
紫杉醇是一种微管稳定剂,临床上对多种肿瘤有良好的治疗效果。但由于血脑屏障的存在,紫杉醇对脑胶质瘤的治疗效果不理想。紫杉醇全身用药副作用较大,有研究表明利用可降解材料局部缓释紫杉醇能有效抑制大鼠胶质瘤的生长。因此紫杉醇也是局部缓释治疗脑胶质瘤的理想药物。
当前用于制作缓释载体的材料多为人工合成聚合物,如聚乳酸(Polylactic acid, PLA)、聚乙醇酸(Polyglycolic acid, PGA)及其共聚物(PLGA)等。Wafer即采用聚酸酐(PCPP-SA)作为缓释载体。在应用过程中发现这类聚合物存在一定缺点,如亲水性差,细胞贴附力较弱,酸性降解产物引起无菌性炎症,在实验中观察到局部脑组织内炎细胞浸润及脑水肿明显。此外,在一些临床试验中发现,此类降解材料的组织相容性较差,周围瘢痕形成明显,影响缓释药物的弥散,降解的碎片游动,有导致脑室系统堵塞的危险。而PPC是一种不释放酸性物质的新型聚合物,最终降解产物是CO2和水,大大减少了材料对脑组织的局部刺激作用,而且与脑组织有很好的组织相容性,因此PPC是用于神经系统药物缓释系统载体的理想材料。
目前用于制作缓释系统的方法很多,如盐释析法、膜复合法、相色分离法、乳液冷冻干燥法、水相冷冻干燥法等。由于局部组织间压力及脑脊液的流动,微球容易游动,很难聚集在瘤腔内,而且采用微球表结构,药物突释现象严重,容易产生局部神经毒性,造成组织水肿及坏死等,因此微球结构很难应用于脑部治疗。电纺丝方法是一种制备高分子纳米/微米纤维的有效方法。电纺丝制作的膜状材料具有很好的韧性,断裂伸长率高,满足了对缓释系统的力学性能要求。电纺丝的制备过程条件温和,可以较为精确的控制纤维膜的微观结构、孔隙率和组成,在空间结构上具有仿生特点,很好的模拟了人体组织中细胞外基质,可以用于脑局部缓释系统的制备。
前期研究表明药物的释放速度和突释现象随载药量的增大而增加,短期内药物的集聚可能导致神经毒性、脑水肿等不良反应,因此通过本研究,我们拟设计一种较低载药量的复合缓释系统。通过携带两种有协同作用的药物,在缓释系统载药量较低的情况下,仍然可以达到有效抑制肿瘤的效果。我们采用电纺丝技术,将含有微球结构的乳液进行电纺丝操作,制备出微球包被于纺丝纤维内部的具有壳核结构的复合系统,分别携带替莫唑胺和紫杉醇两种药物,分析制备过程中的各种参数,各样品的形态特征及药物释放曲线,并通过体外体内实验分析它的安全性及有效性,希望能用于脑胶质瘤的临床治疗。
本实验共分三部分进行：
第一部分药物缓释系统的构建及其参数特征的测定；
第二部分药物缓释系统体外抗C6胶质瘤细胞的实验研究；
第三部分复合缓释系统体内抗大鼠胶质瘤性能的实验研究。
第一部分药物缓释系统的构建及其参数特征的测定
研究目的
在本部分中拟制备替莫唑胺电纺丝缓释系统、紫杉醇微球缓释系统及替莫唑胺/紫杉醇复合缓释系统,分析制作参数、形态特征以及释放曲线。研究方法
1.制备缓释系统：用电纺丝方法制作替莫唑胺纺丝缓释系统,用乳化-离子交联法制作紫杉醇微球,再将微球悬液通过电纺丝装置制作成复合缓释系统。紫杉醇微球载药量不变,通过改变替莫唑胺的载药量使复合缓释系统内替莫唑胺和紫杉醇的比例约为2:1、1:1、1:2。
2.扫描电镜观察：用扫描电镜观察替莫唑胺纺丝缓释系统、紫杉醇微球缓释系统及复合缓释系统的表面形态特征。
3.热分析：用差示扫描量热法分析替莫唑胺纺丝缓释系统和紫杉醇微球缓释系统中药物的物理形态。
4.药物包封率、载药量的测定：将制备的替莫唑胺纺丝缓释系统、紫杉醇微球缓释系统及复合缓释系统,完全溶解后用紫外分光光度法测定药物含量。载药量=系统中药物总量/系统总质量×100%包封率=系统中药物总量/实验加入药物总量×100%5.药物释放曲线的测定：准确称取替莫唑胺纺丝缓释系统、紫杉醇微球缓释系统及复合缓释系统样品,放入离心管后固定于水浴恒温摇床中,条件37℃,100 rpm/min。在1h、3h、6h、12h、24h、72h、120h、168h、240h和280h后,检测残余样品中药物含量,从而计算累计释放药物百分比,得到释放时间-累计释药百分比的药物释放曲线。研究结果
1.替莫唑胺/PPC纺丝缓释系统的制作条件比较严格,只有在PPC浓度为8wt%、纺丝距离为15-20cm、纺丝电压为10-15kV时才能流畅顺利的进行电纺丝操作。采用乳化离子交联法可以制作出直径较为均匀的紫杉醇微球。制作复合纺丝缓释系统时的电压为12kV,接收距离为15cm。
2.通过扫描电镜观察,纺丝纤维均匀光滑,平均直径为3μm,微球平均直径为7.2μm,复合缓释系统呈串珠样结构,微球包被在纺丝纤维内。
3.从DSC分析可以看出,单纯替莫唑胺和紫杉醇的放热峰值分别为207.5℃和223.0℃,而缓释系统的DSC曲线中没有放热峰值,说明药物是以高温稳定的晶型或无定形态的状态存在于材料内,而不是以颗粒状态。
4.微球缓释系统中紫杉醇的载药量为3.9%,包封率为65.3%,在复合缓释系统中的载药量为2.1%,包封率为94.5%,替莫唑胺在复合系统中的载药量分别为1.2%、2.3%、4.1%,包封率分别为97.6%、96.1%、95.7%。微球缓释系统中紫杉醇的包封率低,可能因为有部分紫杉醇从微球中迁移出来,溶于有机溶剂中,而在复合缓释系统中包封率增加是因为溶于有机溶剂中的紫杉醇通过电纺丝操作留在了纺丝纤维中,同时说明电纺丝方法优于微球方法。
5.微球缓释系统中的紫杉醇可以持续释放8天左右,前期突释现象明显,而在复合系统中紫杉醇的释药时间约为12天,突释现象有所减轻。说明经纺丝纤维包被以后可以减轻微球缓释系统的突释现象,同时能延长药物释放时间。替莫唑胺在复合系统中的释药时间约为12天,载药量越大,前期释放速度越快,因此载药量低有利于药物平稳释放。结论
1.PPC材料可以作为药物缓释系统载体进行电纺丝操作。
2.替莫唑胺和紫杉醇是以高温稳定的晶型或无定形态的状态存在于缓释系统内。
3.复合缓释系统的核壳结构可以减轻微球缓释系统的突释现象,延长药物释放时间。
4.所制备的各种缓释系统均可以长时间稳定释放替莫唑胺和紫杉醇。
第二部分药物缓释系统体外抗C6胶质瘤细胞的实验研究
研究目的
分析替莫唑胺/PPC缓释系统、紫杉醇/海藻酸钙微球缓释系统及替莫唑胺/紫杉醇复合缓释系统体外对C6胶质瘤细胞的细胞毒性作用,讨论复合缓释系统中两种药物的理想比例。
研究方法
1.培养C6胶质瘤细胞：用含10%胎牛血清的DMEM培养液,在5%CO2,湿度90%,温度37℃条件下培养。
2.细胞毒性实验：将C6胶质瘤细胞移至96孔板,每孔10000个细胞。准确称取样品经钴60消毒后与细胞共培养,分别于1、2、3天后检测。分组：1组：2:1复合样品；2组：1:1复合样品；3组：1:2复合样品；4组：替莫唑胺纺丝膜；5组：紫杉醇微球；6组：PPC/海藻酸组；7组：空白对照,每组分三份。
3.细胞活性检测：采用cck-8法检测细胞活性。按照试剂说明书操作。将cck-8加入各孔后,37℃下培养1小时候,用自动酶标仪检测各孔OD值。细胞活力(%)=实验组OD值/对照组OD值×100%。
研究结果
1.空白纺丝膜和微球材料组的细胞活性在99%以上,说明所用载体材料对C6细胞没有细胞毒性作用。
2.第1、2、3天时的细胞活性1组分别为52.4±6.3%、27.9±5.3%、5.5±2.4%,2组分别为56.3±4.2%、29.3±6.2%、9.1±5.1%,3组分别为75.7±2.9%、52.1±4.9%、22.9±2.5%,4组分别为86.7±5.1%、66.4±4.3%、35.4±2.7%,5组分别为80.0±4.2%、63.1±4.5、29.5±3.4%。采用SPSS13.0统计分析软件进行方差分析,1组与2组间P>0.05,差异无统计学意义,1组与3组、2组与3组、1组与4组、1组与5组间P<0.05,差异有统计学意义。虽然2组缓释系统中替莫唑胺的载药量比1组的低,但抑制C6细胞的效果没有显著性差异,说明复合缓释系统中替莫唑胺和紫杉醇的比例约为1:1时,两种药物可以达到较为理想的协同效应。同时可以看出各个缓释系统均能持续抑制C6胶质瘤细胞的活性,而且复合缓释系统优于单药物缓释系统。
结论
1.PPC和海藻酸材料对C6胶质瘤细胞无细胞毒性作用。
2.本研究所制备的各种药物缓释系统均能持续抑制C6胶质瘤细胞的活性。
3.本研究所制备的复合药物缓释系统比单药物缓释系统更能有效抑制C6胶质瘤细胞的活性。
4.替莫唑胺和紫杉醇质量比约为1:1的复合缓释系统在保持较低载药量的同时又可以达到有效抑制C6胶质瘤细胞的效果,是较为理想的局部缓释系统。
第三部分复合缓释系统体内抗大鼠胶质瘤性能的实验研究
研究目的
观察讨论替莫唑胺/紫杉醇复合缓释系统在大鼠脑内局部应用的安全性及对大鼠原位C6胶质瘤的治疗效果。所采用复合缓释系统中替莫唑胺和紫杉醇的载药量分别为2.3%和2.1%。
研究方法
1.构建大鼠胶质瘤模型：采用立体定向技术将C6胶质瘤细胞注入SD大鼠大脑皮层,共31只。1周后行MRI检查,生长肿瘤者分两组：治疗组(10只)、对照组(8只),1只处死取标本。另设正常大鼠组(4只)。
2.植入缓释膜片：扩大骨窗,将lmg药物缓释膜片植入治疗组肿瘤内。对照组进行相同操作,但不植入膜片,3周后再行脑MRI检查。同时将膜片植入正常大鼠脑内,于第3、第7天行脑MRI检查。
3.HE染色：将大鼠脑组织及肿瘤组织做石蜡切片,行HE染色。光镜下观察组织变化。
4.胶质瘤组织中增殖细胞核抗原(PCNA)蛋白的表达：采用链霉亲和素-生物素-过氧化氢酶复合物法(SABC)检测肿瘤组织中PCNA表达。每张切片随机选取10个视野(400倍),计数PCNA阳性细胞数和总细胞数。PCNA阳性率=阳性细胞数/总细胞数×100%
5.肿瘤组织内细胞凋亡的检测：采用末端脱氧核苷酸转移酶介导的末端标记技术(TUNEL)试剂盒检测肿瘤组织细胞的凋亡。按照试剂盒说明书操作。观察组织内阳性细胞的表达。
研究结果
1.正常大鼠植入膜片后第3、7天行脑MRI检查,未见脑组织水肿。从HE染色看,第3天时可见脑组织表面约0.8mm处细胞间质水肿,少量淋巴细胞浸润,第7天水肿略有减轻,约0.5mm,仍有少量炎细胞浸润。大鼠在饲养过程中未出现饮食异常、抽搐等表现。说明该复合缓释系统在脑中的局部反应轻微,未导致严重水肿等并发症,安全性高。
2.制作肿瘤模型31只,术后死亡7只,磁共振显示肿瘤未生长者5只,形成肿瘤者19只,成瘤率为61.3%。光镜下HE染色观察肿瘤细胞呈假栅栏状或珊瑚状排列,密集成群,早期肿瘤组织与脑组织边界清楚,晚期可见肿瘤细胞侵入脑实质,边界模糊。经治疗后肿瘤组织内可见坏死灶。
3.接种1周后,大鼠脑胶质瘤体积平均为122.1±4.5mm3,经治疗3周后体积为184.0±5.4mm3,肿瘤增长率为50.8%,对照组肿瘤体积平均为341.3±11.4mm3,肿瘤增长率为180.0%,两组间差异有统计学意义(P<0.0001),说明该复合缓释系统可以有效抑制肿瘤生长。
4.治疗组大鼠生存中期为42.5天,对照组生存中期为28.5天,两组间差异有统计学意义(P<0.05)。说明经过该复合缓释系统治疗,大鼠生存期明显延长。
5.治疗前、对照组和治疗组大鼠脑肿瘤组织中PCNA阳性表达率分别为65.4±5.7%、68.6±6.3%、15.3±5.2%,治疗前与对照组间无明显差异(P>0.05),对照组与治疗组间差异有统计学意义(P<0.05),治疗组的PCNA阳性细胞明显减少。
6.对照组肿瘤组织大部分无凋亡细胞表达,偶可见少量凋亡细胞,而治疗组肿瘤内可见大量凋亡细胞,尤其膜片周围瘤组织。
结论
1.本研究所制备的替莫唑胺/紫杉醇复合缓释系统有良好的组织相容性,安全性高,在脑局部应用无不良反应。
2.本研究所制备的替莫唑胺/紫杉醇复合缓释系统能有效抑制大鼠胶质瘤的生长,降低肿瘤细胞增殖,诱导肿瘤细胞凋亡,显著提高了大鼠的生存中期。
创新性及局限性
1.创新点
(1)采用一种微米级核壳结构复合纤维作为缓释系统支架,该复合纤维结合了微球与电纺丝纤维的优点,可以同时携带两种药物。
(2)复合缓释系统携带替莫唑胺和紫杉醇两种有协同作用的药物,在药物载药量较低时,仍可以达到理想的抑制肿瘤效果,并分析讨论了复合缓释系统内两种药物的合理配比。
2.局限性
(1)本研究制备的缓释系统的药物持续释放时间相对较短,未能达到理想时间,这与载体材料降解速率、药物与材料结合力、纺丝纤维直径等有关。
(2)由于大鼠脑体积小,颅内切除肿瘤困难,未能采用切除后大鼠胶质瘤模型进行动物实验,因此本研究动物实验部分只能证明该复合缓释系统可以有效抑制肿瘤生长,对控制肿瘤复发的效果仍不能确定,需要进一步研究。
Background
Malignant glioma is the most common type of primary brain tumor in adults. Approximately 5 new cases per 100,000 in population are diagnosed each year. The current standard treatment for malignant glioma consists of surgical resection followed by radiation therapy and aggressive systemic chemotherapy. However, the prognosis for patients with maglignant glioma is relatively poor with a median survival of<2 years. Autopsy studies indicate that 90% of recurrent gliomas occur within 2 centimeters and metastasize rarely, so local chemotherapy provides an alternative drug delivery method for the treatment of brain tumors. Desirable local chemotherapy system could provide sustained and effective drug concentration with well histocompatibility and less neurotoxicity. The present research about local chemotherapy system is focus on biodegradable materials. Gliadel(?) wafer is the first biodegradable chemotherapy system used to treat glioma. The wafer consists of polycarboxyphenoxypropane and sebacic acid in a 20:80 molar ratio and contains 3.85% BCNU. It has showed some success in improving survival of patients with malignant glioma. However, it is only used to treat recurrent glioma by FDA because AGT, a DNA-repair protein found in the majority of brain tumors. So we need to find other effective drugs and safe biodegradable vehicle.
Temozolomide(TM) is one of the most effective antineoplastic agents for malignant glial tumors. TM is converted to its active metabolite 3-methyl-(triazen-1-yl) imidazole-4-carboxamide (MTIC) at physiological pH. MTIC induces apoptosis of cells due to alkylation of DNA at the 06 and N7 positions of guanine. It is evident that local delivery of temozolomide by biodegradable polymers is superior to oral administration in a rodent glioma model.
Paclitaxel targets microtubules and has been clinically effective against a variety of human cancers, that shows cytotoxicity against glioma in vitro. However, it is prevented from entering brain and penetrating intact blood-brain barrier poorly. Biodegradable polymer implant was developed to deliver paclitaxel against experimental malignant glioma and found to be efficient in a rat model.
Most work on electrospinning biodegradable polymers has focused on synthetic materials, notably PLA, PGA, PLGA, and PCL. However, these materials have some disadvantages, such as low hydrophilicity, and aseptic inflammation because of their acid degradation products. Poly-propylene carbonate (PPC) is a degradable material formed from copolymerization of propylene oxide and carbon dioxide. It has low local irritant effect in brain tissue and good histocompatibility with brain. So PPC is the ideal material as release vehicle in brain.
Various techniques have been designed to prepare controlled drug delivery systems, including solvent casting/salt leaching, membrane lamination, phase separation, emulsion freezing/drying, and hydrogel freezing/drying. Due to very high interstitial pressure and circulation of cerebrospinal fluid, microspheres have the risk of being expelled out of the target site. The other disadvantage of using microspheres is the high initial burst due to the presence of the drug on the surface which might lead to undesired neurotoxicity. Electrospinning is an attractive approach for the fabrication of fibrous biomaterials. Electrospun mats have larger specific surface areas and smaller pore size for polymer degradation and drug diffusion compared to other controlled drug delivery systems.
Therefore, the aim of this study was to designe a high effective local drug delivery system with low loading rate. We combine electrospinning with microspheres delivering temozolomide and paclitaxel, avoiding the possibility of neurotoxicity. The preparation parameters and the safety and effectivity of the system were analyzed.
This study includes three parts:
PartⅠThe fabrication and study of the biodegradable implants
PartⅡCytotoxic study of the implants against glioma C6 cells in vitro
PartⅢThe study of combined implant against glioma in rats
PartⅠThe fabrication and study of the biodegradable implants
Objective
To fabricate the drug delivery systems and investigate the preparation parameters, physical characteristics and release properties
Methods
1. Preparation of the drug delivery system:Temozolomide fibers were fabricated by electrospinning, and paclitaxel microspheres were prepared using emulsifying-solvent evaporation method. Then emulsion of microspheres was electrospinned into combined mats. The ratio of temozolomide and paclitaxel was 2:1, 1:1,1:2 respectively, through holding the loading rate of paclitaxel and changing the temozolomide.
2. Physical characteristics:The size, distribution and surface morphology of the fibers and microspheres were examined using a scanning electron microscope after gold coating.
3. Thermal analysis of drugs in matrices:Thermal analysis was performed using differential scanning calorimetry (DSC) to characterize the physical status of drugs in matrices.
4. The drug encapsulation efficiency (EE) and loading rate were calculated according to the following equations: Loading rate=WM/WT×100 Encapsulation efficiency=WM/Dt×100 , where WM is the amount of drug in systems, Dt is the amount of drug used for the preparation, and WT is the weight of systems.
5. The drugs releasing study in vitro:the systems were immersed into 20ml PBS buffer in tube incubated at 37℃and 100 rpm in a shaking water bath. At each specified time interval,1h、3h、6h、12h、24h、72h、120h、168h、240h和280h, drugs in solution were detected by UV analysis. Then the time-cumulative drug released plots would be produced.
Results
1. When PPC was 8%, distance was 15-20cm and voltage was 10-15kV, electrospinning could be performed normally. The TM fibers were smooth and uniform only at 8% by weight. The paclitaxel microspheres have proper size when the stirring rate is 500 rpm and concentration of alginate is 1%. The emulsion of microspheres was electrospinned into the beads-in-string structure at 15cm and a voltage of 12kV.
2. SEM images showed that the diameters of the fibers and microspheres were 3μm and 7.2μm. The surface of fibers and microspheres was smooth and uniform.
3. Pure TM and paclitaxel showed an endothermic melting peak at 207.5℃and 223.0℃, but no peak was seen at temperatures of 100-250℃for the samples. It is evident that the drugs were in an amorphous or disordered-crystalline phase of a molecular or a solid solution state in the matrix.
4. The loading rate and encapsulation efficiency of paclitaxel in microspheres were 3.9% and 65.3%,2.1% and 94.5% in combined fibers. The loading rate and encapsulation efficiency of TM in combined fibers were 1.2%,2.3%,4.1% and 97.6%, 96.1%,95.7%. The EE of paclitaxel in microspheres is lower, because that it could dissolve in dichloromethane in preparation of microspheres. However, this part of paclitaxel stays in fibers during process of electrospinning.
5. The microspheres released paclitaxel for about 8 days with obvious initial burst, and paclitaxel could be released for about 12 days with low initial burst. Because it could prolong the release time and reduce the initial burst when the microspheres were covered in fibers. TM was released for about 12 days from combined fibers. The initial release rate was faster with higher loading rate.
Conclusion
1. PPC could fabricate the drug delivery system by electrospinning.
2. TM and paclitaxel were in an amorphous or disordered-crystalline phase of a molecular or a solid solution state in the matrix.
3. Combined delivery systems could reduce initial burst of paclitaxel microspheres and prolong its release time.
4. Temozolomide and paclitaxel could be released stably for long time from delivery systems.
PartⅡCytotoxic study of the implants against glioma C6 cells in vitro
Objective
To investigate the effect of implants against glioma C6 cells in vitro and the best ratio of two drugs
Methods
1. C6 cell culture:Cells were maintained in tissue culture in Dulbecoo's minimum essential medium with 10% fetal bovine serum, in atmosphere of 5% CO2, and 90% relative humidity at 37℃。
2. Cytotoxicity test:C6 cells were transferred to a 96-well plate to ensure 1×104 cells per well. The following experimental groups were studied. (1) 2:1 combined fibers; (2) 1:1 combined fibers; (3) 1:2 combined fibers; (4) TM/PPC fibers; (5) paclitaxel microspheres; (6) PPC and alginate; (7) control group, each group in triplicate.
3. To detect cell viability:The cytotoxicity assay was conducted by Cell Counting Kit-8(cck-8). The plates were detected through microplate reader. Cell viability was determined according to the following equation:
Cell viability (%)=Abs test cells/Abs control cells×100
Results
1. The cells viability of group (6) and (7) is over 99%. It is evident that the material PPC and alginate has no cytotoxicity to C6 glioma cells.
2. The cells viability of group (1) at 1,2,3 days is 52.4±6.3%、27.9±5.3%、 5.5±2.4%, group (2) is 56.3±4.2%、29.3±6.2%、9.1±5.1%,group (3) is 75.7±2.9%、52.1±4.9%、22.9±2.5%, group (4) is 86.7±5.1%、66.4±4.3%、35.4±2.7%, and group (5) is 80.0±4.2%、63.1±4.5、29.5±3.4%. Statistical analysis shows that there are no statistical difference between group (1) and group (2)(P>0.05), but there are obvious difference between group (1) and (3), group (2) and group (3), group (1) and group (4), group (1) and group (5)(P<0.05). All kinds of implants could inhibit the C6 glioma cells continuously.
Conclusion
1. The material PPC and alginate has no cytotoxicity to C6 glioma cells.
2. Combined implants could produce stronger cytotoxicity than single drug implants with low loading rate.
3. When the ratio of TM and paclitaxel was about 1:1, the synergistic effect was obvious.
PartⅢThe study of combined implants against glioma in rats
Objective
To investigate the effect of combined implants (1:1) against glioma in rat model and the security used in brain
Methods
1. Combined fiber mats were implanted in the surface of hemisphere of four Sprague Dawley rats. The rats were scanned by MRI at 3,7 days.
2. Preparation of rat glioma model:31 male (200-250g) Sprague Dawley rats were implanted in C6 cells. All rats should be scanned by MRI after a week. The rats with tumors were divided to two groups:treatment group (n=10) and control group (n=8).
3. The sections were subjected to hematoxylin and eosin attaining for histological examination.
4. Proliferation cell nuclear antigen (PCNA) was used to evaluate the proliferation of tumor. PCNA-positive cells were determined by randomly counting 10 fields of the section and were expressed as a percentage of normal nuclei.
5. To evaluate apoptotic activity, the TUNEL technique was used.
Results
1. There was no brain edema on MRI after the mats were implanted in hemisphere. In microscope, the range of edema is about 0.8mm at 3 day, and 0.5mm at 7day, accompanying with few lymphocyte infiltrating.
2. The volume of tumor was 122.1±4.5mm3 at first week. Three weeks after treatment, the volume of treatment group tumor was 184.0±5.4mm3, and the growth rate was 50.8%. The control group was 341.3±11.4mm3 and 180.0%. Significant differences were observed between two groups (P<0.0001).
3. The median survival time of treatment group was 42.5 days, and the control group is 28.5 days. Two groups had significantly difference (P<0.05). It is evident that the combined fibers could prolong the survival of rats.
4. The percentage of PCNA-positive were 65.4±5.7%、68.6±6.3%、15.3±5.2% with initial tumor, control and treatment group. The PCNA-positive cells decreased obviously after cure.
5. The treatment group shows many apoptotic cells, and the control group has no expression of apoptotic cells.
Conclusion
1. The combined fibers have well histocompatibility and security.
2. The combined fibers could inhibit the growth of tumor effectively, decrease proliferation of tumor cells, induce apoptosis of tumor cells, and prolong the rat median survival time.
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