载ADM-PLGA微球的纳米羟基磷灰石胶原多孔支架的实验研究

发布时间：2018-01-04 08:42

本文关键词：载ADM-PLGA微球的纳米羟基磷灰石胶原多孔支架的实验研究　出处：《南方医科大学》2015年硕士论文　论文类型：学位论文

【摘要】：研究背景：骨肉瘤是人类在15-20岁这一时期内多发的常见恶性肿瘤。骨肉瘤患者在20世纪70年代之前主要通过手术进行治疗,但是在行手术切除肿瘤之后患者的生存率不足20%。目前我国作为世界第一人口大国,因骨肿瘤致死或造成残疾、功能障碍人数也逐年增多,位居世界各国之首。关于骨肿瘤的手术治疗,其主要问题不仅在于手术切除后常常残存部分未完全切除的肿瘤组织,这是一个复发的极大隐患,而且还有一个很大的弊端就是在手术切除肿瘤病灶的同时切除了病灶处的负重骨骼；某些发生在特定部位的肿瘤,特别是脊柱肿瘤,因为其发生部位的特殊造成了其常常难以切除,此外,肿瘤组织的跳跃转移以及在手术中这些组织与正常组织结构的接触带来的医源性播散,这些都有可能引起手术后肿瘤的局部复发。因此,在骨肿瘤切除术后往往需要进行化疗,在临床上目前使用最广泛的传统的化疗药物比如阿霉素、顺铂、长春碱类这些药物不仅具有抗肿瘤活性,也因为它们起效的非选择性对人体内的各个组织和器官带来了相应的毒副作用,常规治疗剂量即可对人体产生明显的骨髓移植、心脏毒性、胃肠道反应等毒副作用,这些毒副作用降低了患者的治疗的依从性和生活质量,容易引起患者对治疗的抗拒,通常化疗药物具有时间和剂量的依赖,高药物浓度,较长的半衰期常常可以带来更好的抗肿瘤效果,但是剂量的提高和用药时间的延长通常会带来更加严重甚至致命的毒副作用。在漫长的临床实践中人们渴望获得一种可以提高局部药物浓度,延长药物作用时间的新的化疗药物来减少化疗相关毒副作用,提高化疗效率。随着制药技术的进步,局部缓释药物的概念被提出,其思路是缓释药物,延长药物作用时间并且局部靶向给药,提高药物的选择性；而随着骨组织工程技术的发展,将局部缓释药物与仿生的人工骨材料相结合制备成为在局部植入后可以靶向选择性给药并且能够修复手术造成的负重骨骼缺损,这种思路为骨肿瘤患者的康复带来了新的希望。药物局部缓释系统在肿瘤的治疗方面具有良好的应用前景,这种系统是将一系列具有良好生物相容性、可降解性的无机或有机材料与一种或者是几种传统的化疗药物相结合,通过不同的给药途径植入肿瘤切除术后的原病灶处或者是病灶周围的组织内,由此达到类似于靶向给药的目的。药物局部缓释给药系统对于局部的残存的肿瘤细胞、病灶可以作用较长的时间,药物不需要通过生物代谢,肿瘤细胞在一个较长的时间内暴露在较高浓度的化疗药物下,显著提高了化疗药物的治疗效率,同时相对于传统给药途径来说其避免了长时间的全身给药诱发的各种毒副反应。晚期癌症患者或是不能耐受传统化疗的患者,药物缓释系统植入可以增大给药剂量,局部释放的药物可对肿瘤长时间的直接起效,这对提高晚期患者的生活质量以及延长寿命也有一定的帮助。但是目前的传统的药缓释系统存在着许多的不足之处：1.缓释系统生物相容性差,植入后产生明显的异物排斥反应；2.缓释系统的载药率、封包率低,复合困难；3.复合的药物剂量偏低,持续释放时间不理想；4.复合后容易分解,稳定性差。随着21世纪纳米技术的兴起,极大地推动各个交叉学科的进步,纳米技术的引入为局部药物缓释系统安全性和有效性的提高起到了明显的推动作用。纳米颗粒(nano-particle)有着独特的表征和理化性质,小的尺寸、体积使这类微粒更容易穿透细胞屏障进入组织间隙,显著的提高了被细胞吞噬的能力,此外纳米颗粒还可以增加药物的稳定性和可溶性,同时在改善肾脏的清除率方面可以起到重要作用。在肿瘤组织中的迟滞效应(EPR效应)的基础上,纳米药物可以选择性、特异性的通过配体诱导的细胞表面的胞吞作用增加药物的摄入,在进一步提高疗效的同时减少肿瘤的耐药,通过减少对非目标部位细胞组织器官的损伤,来降低毒性。此外因为纳米材料有其独特的小尺寸、表面或界面效应,纳米级别的羟基磷灰石这类材料可以有效地诱导骨组织再生和细胞生长,因此在骨传导性能上其与传统的毫米、微米材料相比较具有明显优势。局部药物缓释理念,随着骨支架修复材料、抗肿瘤药物的发展,在纳米医学的新生土壤之上,有望继续发挥优势,开发出性能更佳的抗肿瘤纳米缓释包囊和支架材料。我们课题组构建了载ADM-PLGA微球的纳米羟基磷灰石胶原多孔支架(ADM-PLGA-NHAC),并对其表征和骨修复能力及抗肿瘤活性进行了相关检测。研究目的：通过制备搭载了阿霉素(ADM)、聚乳酸-羟弪基乙酸共聚物(PLGA)纳米缓释微球的纳米羟基磷灰石/胶原多孔支架(ADM-PLGA-NHAC),并进行相关实验检测这种材料的理化性质、释药特点以及验证其体内、体外的抗肿瘤能力和骨修复能力,为本材料的临床实验打下坚实基础,为临床骨肉瘤的治疗提供新的思路。研究方法：1.复乳-溶剂挥发法加工阿霉素(ADM)-聚乳酸-羟基乙酸共聚物(PLGA)缓释微球；纳米羟基磷灰石/胶原多孔支架(NHAC)通过冷冻干燥法以纳米羟基磷灰石及胶原为原料进行制备,在NHAC的制备过程中加入ADM-PLGA微球制得载ADM-PLGA纳米微球的纳米羟基磷灰石／胶原多孔支架(ADM-PLGA-NHAC).制备完成后材料在体外通过透析袋扩散法及制备浸提液进行材料的药物释放动力学实验和抗肿瘤实验,再通过扫描电镜(SEM)、激光粒径仪等对微球和支架的表征进行相关测试。2.新西兰大白兔的临界骨缺损模型选择来作为本实验的动物模型,造模完成后的动物随机分为3组：A组骨缺损不予处理作为空白对照组；B组骨缺损处以NHAC填塞；C组骨缺损处植入载ADM-PLGA-NHAC。在手术完成后的第8周及12周处死动物,并对股骨标本行X线检查,所得X线结果根据Lane-Sandhu标准对骨缺损的愈合情况进行评价；X线检查完成后对各组的骨标本进行Micro-CT扫描,扫描图像导入电脑进行3维重建,之后再将扫描数据导入Micro-CT自带的分析软件进行BMD值分析,对骨缺损修复情况进行定量的分析；骨标本行组织学检测(HE、MASSON染色)及扫描电镜(SEM)检测根据结果判断标本的骨修复情况及材料降解情况。3.将MG63细胞接种于裸鼠皮下,成瘤后裸鼠皮下肿瘤以尖刀片随机植入材料,实验终止时处死全部裸鼠,肿瘤剥离、称重并测量肿瘤长、宽计算肿瘤体积。并将肿瘤重量、肿瘤体积进行比较；将裸鼠皮下肿瘤按照标准程序制作石蜡切片(厚度4umm),行HE染色观察肿瘤转移、坏死以及心脏毒性,进行肿瘤组织TUNEL检测并计算坏死率。4.所有的统计分析均由SPSS 13.0软件完成。具体数据均用均值±标准差(X±SD)表示,多组间资料采用单因素方差分析(One-way ANOVA),组间比较采用LSD法(least significant difference),若方差不齐则采用Dunnett's T3法进行检验；P值0.05认为差异有统计学意义。研究结果：1.本课题组由乳化挥发法制得的ADM-PLGA纳米微球呈圆球形,平均粒径约为319.5nm±11.17nm,其载药率为(6.42+0.28)%。制备的ADM-PLGA-NHAC复合支架,其孔径经测试约为100-200um,孔隙率可达到约(82.3±4.6)%,材料多孔而且空洞的内壁十分粗糙,有利于细胞黏附及营养交换。释药曲线显示药物并未出现明显的突释现象,24h内药物释放率约为17.6%,本材料在28d内能持续缓慢释放ADM,且释放浓度比较稳定。通过活-死染色及CCK8法检测材料的抗肿瘤效果,结果显示ADM-PLGA-NHAC材料的浸提液在体外随着浓度和时间的增长可以明显抑制MG63细胞的生长。2.建立新西兰兔股骨髁骨缺损模型后植入材料,在手术后的第8周及12周取材进行X线摄片分析、Micro-CT扫描3维重建并进行BMD值分析,结果显示：空白对照组骨缺损未愈合而载药的ADM-PLGA-NHAC支架组与未载药物的NHAC支架组骨愈合程度无明显差异。在石蜡切片组织学检测中显示A组(空白对照组)未见明显新骨形成；B组(NHAC组)可见尚未降解的填充材料与散在其间的大量不规则的新骨组织,新生骨组织开始融合；C组(ADM-PLGA-NHAC组)材料降解程度与B组接近,但不规则的新骨组织数量及新生骨组织融合程度差于B组。12周末时A组在骨缺损边缘处可见少量不规则新生骨组织,而B组、C组均可见较多的成熟骨组织和骨类似组织融合成片且大量的新生骨质与胶原广泛融合且呈片状,局部仍可见少量尚未降解的填充材料,两组均未见明显炎性细胞浸润及组织坏死。3.裸鼠处死后以肉眼观察肺组织有无转移瘤,对剥离干净的肿瘤进行称重、测量体积,分别进行HE染色及TUNEL检测,结果显示ADM-PLGA-NHAC组的抑瘤效果最好,各组均肺部无转移,而在腹腔注射阿霉素组裸鼠出现心脏毒性,而其他各组心脏未见明显异常。研究结论：1.我们课题组将纳米级别的化疗药物缓释囊与纳米级别的羟基磷灰石相结合,通过乳化挥发法、冷冻干燥法研制出了载ADM-PLGA纳米囊的纳米羟基磷灰石/胶原多孔支架并在体外通过一系列理化检测证明了其具有良好粒径、孔隙率、载药率、缓释性能及抗肿瘤能力；2.通过建立新西兰兔股骨髁骨缺损模型并植入我们制备的ADM-PLGA-NHAC材料,在8周后及12周后分别进行X线、Micro-CT以及组织学等检测,结果充分证实了载ADM-PLGA纳米囊的纳米羟基磷灰石／胶原多孔支架具有良好的骨缺损修复能力；3.我们建立了裸鼠的异种骨肉瘤模型,皮下肿瘤长至一定程度后我们通过将材料植入肿瘤内部,通过进行瘤重、肿瘤体积的比较以及肿瘤、心脏的组织学相关检测分析证明了我们研制的这种材料在裸鼠体内具有良好的抗肿瘤效果,并且与传统化疗药物相比较能够显著降低其毒副作用。
[Abstract]:Background: osteosarcoma is the most common malignant tumors in multiple human 15-20 years old in this period. In patients with osteosarcoma before 1970s through surgery, but after undergoing surgical resection of the tumor survival rate is less than 20%. with China as the world's most populous country, due to bone tumor death or disability, the number of dysfunction also increased year by year, ranked first in the world. Surgical treatment of bone tumor, the main problem lies not only in the operation is often part of the remaining complete resection of tumor resection, which is a great risk of a relapse, but there are a lot of malpractice is the surgical removal of the tumor lesions and resection of the lesions the weight of bone; something occurring in specific parts of the tumor, especially spinal tumor, because of its special location caused this is often difficult to resection. Outside, skip metastasis of the tumor tissue and the tissue and normal tissue structure contact in patients with iatrogenic dissemination, these are likely to cause local recurrence after surgery of tumors. Therefore, in the bone after tumor resection often need chemotherapy, in current clinical use of traditional chemotherapy drugs such as the most widely doxorubicin, cisplatin, Changchun alkaloids of these drugs not only have antitumor activity, but also because of various tissues in the human body and organs are non selective has brought the corresponding side effects, the dose can be produced by bone marrow transplantation, obvious to the human body cardiac toxicity, side effects such as gastrointestinal reactions, these poison the side effects were reduced on treatment compliance and quality of life, easy to cause the patient to resist treatment, usually rely on chemotherapy with time and dose, high drug concentration, longer Half life can often lead to better anti-tumor effect, but the prolonged dose increased and medication time often leads to more serious or even fatal side effects. In clinical practice in the long people eager to obtain a high local drug concentration, prolong the drug for new chemotherapy drugs to reduce the time of chemotherapy related toxicity effect of chemotherapy efficiency. With pharmaceutical technology, the concept of local drug delivery is put forward, the idea is to release drug, prolong drug action and local drug target, improve the selection of drugs; and with the development of tissue engineering technology, the local drug delivery and biomimetic artificial bone material combination in the preparation of local implantation can become the selective targeting of drug and surgery to repair the defect caused by the bone weight, this idea for the rehabilitation of patients with bone tumor A new hope. The local drug delivery system has a good application prospect in the treatment of tumors, this system is a series of good biocompatibility, biodegradability of inorganic or organic materials with chemotherapy drugs one or several traditional combination, through different routes of administration into the tumor resection after the original lesions or lesions around the organization, so as to achieve similar to the targeted drug delivery. Local drug sustained-release drug delivery system for residual tumor cells in the local lesions, can be a long time, drugs are not needed by the biological metabolism of tumor cells exposed to higher concentrations of drug in chemotherapy for a long time, significantly improve the efficiency of the treatment of chemotherapy, and compared with the traditional way of administration, avoid all kinds of adverse reactions induced by systemic administration of long time late. Cancer patients or intolerant of conventional chemotherapy, drug delivery system can increase the implantation dose, drug release can be directly effect on the local tumor in long time, it will also be helpful to improve the living quality of patients with advanced and prolong the life. But the traditional drug delivery system deficiencies many of the 1. release system has poor biocompatibility and implantation have obvious reject reaction; 2. delivery system drug loading rate, packet rate is low, the composite difficulty; 3. composite dose is low, sustained release time is not ideal; 4. composite easy decomposition, poor stability. With the development of nanotechnology in twenty-first Century. Greatly promote interdisciplinary progress, introduction of nanotechnology for drug delivery system and the effectiveness and reliability has played a significant role in promoting. Nanoparticles (nano-par Ticle) has a unique characterization and physicochemical properties, small size, the volume of the particles more easily penetrate the cell barrier into the tissue space, improve the ability by phagocytosis, in addition of nano particles can also increase the stability of the drug and at the same time improve the solubility, scavenging rate of kidney can play an important role. The hysteresis effect in tumor tissues (EPR effect) on the basis of nano drugs can selectively, endocytosis specific surface by ligand induced cells increased drug intake, reduce tumor drug resistance at the same time to further improve the efficacy, by reducing cellular non target organs damage. To reduce the toxicity. In addition because the nano materials have the unique small size, surface and interface effect, nano hydroxyapatite level of this kind of material can effectively induce bone tissue regeneration and cell So in the growth, osteoconductive with the traditional millimeter, micron material has obvious advantages compared. Local drug delivery concepts with bone scaffold materials, the development of anticancer drugs, on new soil nano medicine, is expected to continue to take advantage of a better anti-tumor sustained-release capsule and nano stent materials. Our group constructed nano hydroxyapatite collagen porous scaffolds loaded ADM-PLGA microspheres (ADM-PLGA-NHAC), and its characterization and anti tumor activity and bone repair ability were tested. Objective: through preparation with adriamycin (ADM), polylactic acid hydroxy group hydroxyl acetic acid copolymer (PLGA) / nano hydroxyapatite nano microspheres collagen scaffold (ADM-PLGA-NHAC), and the physicochemical properties of this material detection experiments, drug release characteristics and verify the in vivo, in vitro anti tumor The ability of tumor and bone repair capacity, a solid foundation for clinical trials of this material lay, to provide new ideas for clinical treatment of osteosarcoma. Methods: 1. double emulsion solvent evaporation process of adriamycin (ADM) - poly lactic acid glycolic acid (PLGA) microspheres; nano hydroxyapatite / collagen porous support (NHAC) by freeze drying method using nano hydroxyapatite and collagen as raw material for preparation of ADM-PLGA microspheres prepared with nano hydroxyapatite / collagen scaffold loaded ADM-PLGA nanoparticles in the preparation of NHAC (ADM-PLGA-NHAC). After preparation, in vitro drug materials by dialysis bag diffusion method and preparation of leaching material extract release kinetics experiment and anti tumor experiment, through scanning electron microscopy (SEM), critical bone characterization of laser particle size analyzer of microspheres and stent related test.2. New Zealand white rabbits. Loss model selection as the experimental animal model, animal model after completion of the randomly divided into 3 groups: group A bone defect not treated as blank control group; group B bone defect by NHAC tamponade; group C bone defect in ADM-PLGA-NHAC. implantation after surgery for eighth weeks and 12 weeks of dead animal, and X-ray examination of femur specimens, the X-ray results were evaluated according to Lane-Sandhu standard of bone defect healing; X-ray examination after the completion of each bone specimens were scanned with Micro-CT and scanned images into a computer for 3 dimensional reconstruction, then scan the data analysis software to import Micro-CT own BMD value analysis, quantitative analysis of bone defect repair; the detection of bone specimens (HE, tissue MASSON staining) and scanning electron microscope (SEM) detection according to the results of judgment and bone degradation.3. samples will be fine MG63 Cells were inoculated in nude mice subcutaneous tumor formation after subcutaneous tumors were implanted into the material with a sharp blade at the end of the experiment, all the mice were sacrificed and tumor, weighing and measuring the tumor length, width and calculate the tumor volume. The tumor weight and tumor volume were compared; the subcutaneous tumor in accordance with standard procedures of paraffin sections (thickness 4umm), observation of tumor metastasis by HE staining, necrosis and cardiac toxicity, tumor tissue TUNEL detection and statistical analysis of computing the necrosis rate of all.4. was completed by SPSS 13 software. The specific data are mean standard deviation (X + SD) said that the data among the groups using ANOVA (One-way, ANOVA) comparison between groups using the method of LSD (least significant difference), if the homogeneity of variance using Dunnett's T3 test; P value of 0.05 is considered statistically significant. Results: 1. the research group by emulsion solvent evaporation method ADM-PLGA nano microspheres are spherical, the average particle size is about 319.5nm + 11.17nm, the drug loading rate (6.42+0.28) ADM-PLGA-NHAC composite scaffolds prepared by%. After testing, the pore diameter is about 100-200um, the porosity can reach about (82.3 + 4.6)%, and the inner wall of the hollow porous material is very rough, there are celladhesion and nutrient exchange. The release curve showed that the drug did not appear obvious burst release phenomenon, 24h in the drug release rate is about 17.6%, the material in 28d during a sustained release of ADM, and the release concentration is relatively stable. Through the live - dead staining and CCK8 assay material anti-tumor effect, results showed that the leaching of growth.2. extract in vitro with the concentration and time of growth can significantly inhibit MG63 cells to establish rabbit bone defect model after implantation of ADM-PLGA-NHAC material, material for X-ray radiography in postoperative eighth weeks and 12 weeks Analysis of Micro-CT scan 3 dimensional reconstruction and BMD analysis results show that: the blank control group ADM-PLGA-NHAC stent group bone defect healing and drug loaded with NHAC stent group without drug loaded bone healing without obvious difference. In the study group A detection of paraffin tissue (blank control group) was no significant new bone formation; B group (group NHAC) and the filling material not visible degradation during the scattered in a large number of irregular new bone tissue, bone fusion; C group (group ADM-PLGA-NHAC) material degradation degree was close to B, but the number of irregular new bone tissue and bone fusion was worse than B group.12 group A at the weekend in the bone at the edge of a small amount of irregular new bone tissue, while in B group, C group showed more mature bone tissue and bone tissue and bone fusion were similar with a large number of collagen fusion and is in a sheet shape, local still A small amount of no degradation of filling materials, the two groups were no obvious inflammatory cell infiltration and tissue necrosis after.3. mice were sacrificed by visual observation of lung tissue without metastasis of the tumors were stripped clean, weighing, measurement of volume, respectively. HE staining and TUNEL detection, the results showed the best antitumor effect of ADM-PLGA-NHAC group, each group all lung metastasis, and after intraperitoneal injection of adriamycin group nude mice heart toxicity, heart and other groups had no obvious abnormalities. Conclusion: 1. our research group will nanoscale drug delivery capsule and chemotherapy combined with other nano hydroxyapatite, by emulsion solvent evaporation method, freeze-drying method developed nano hydroxyapatite / nano ADM-PLGA sac collagen porous scaffolds in vitro and through a series of physical and chemical testing proves that it has good porosity, particle size, drug loading, release property and anti tumor 2. ability; through the establishment of New Zealand rabbit femoral condyle bone defect model and we implanted ADM-PLGA-NHAC materials prepared respectively, X-ray in 8 weeks and 12 weeks after Micro-CT, and histological examination. The results proved that the nano hydroxyapatite / collagen scaffold loaded ADM-PLGA nanoparticles has the ability to repair bone defects is good; 3. we established a xenograft model of osteosarcoma in nude mice subcutaneous tumor, to a certain degree by our internal materials were implanted into the tumor, the tumor weight, tumor volume and tumor, cardiac histological correlation analysis proved that we developed this

【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R738.1

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