【作者】 王东新；

【导师】 杨斌盛； 李英奇；

【作者基本信息】 山西大学 ， 无机化学， 2014， 博士

【摘要】 由于环境污染日益严重和人们不良的生活习惯,导致癌症的高发病率,以及现代医学对于癌症治疗的低效,导致其高的死亡率。在传统的癌症治疗方法中,化学疗法被认为是最简便有效的方法之一。但是由于癌症确诊较晚,以及抗癌药物多为小分子化合物,血液给药后快速扩散,分布于全身,在杀死肿瘤细胞的同时也对分裂较快的正常细胞有较大的毒副作用,且多次给药后肿瘤细胞易产生多药耐药性,特别是固态肿瘤,从而导致化疗的失败。随着纳米技术的发展,纳米医药为肿瘤的诊断和治疗提供了新的思路。纳米技术在影像医学诊断中的应用可提高其检测灵敏度,如磁共振成像技术、超声成像技术及x-射线计算断层摄影技术等,近期随着活体成像技术和多光子激发扫描技术等的发展,推动了荧光成像在肿瘤诊断中的应用,这些新技术的发展使肿瘤的早期确诊成为可能。此外利用肿瘤新生血管的不完整结构导致纳米颗粒在肿瘤附近的高透过高滞留效应,可将抗癌药物制备成纳米药物或装载在纳米载体上可达到被动靶向治疗肿瘤的效果,进一步在纳米颗粒表面修饰一些与肿瘤细胞或新生血管具有特异性结合的配体,得到主动靶向到肿瘤的目的,即靶向输送药物体系。该体系可以降低抗癌药物的用量和毒副作用,同时也可抑制肿瘤细胞的多药耐药性,提高治疗效率。现研究的纳米载体材料主要有脂质体、高分子聚合物、碳纳米管、胶束、纳米微球及一些天然高分子化合物等。最近,具有生物相容性好、光学与化学性质稳定、合成技术成熟和表面易于修饰等诸多优点的纳米钻石在生物领域的应用成为当前研究的热点,以纳米钻石作为药物载体和荧光成像试剂还处于研究初期。本论文以爆炸合成的纳米钻石(ND)作为载体材料,聚乙二醇(PEG)作为交联剂,转铁蛋白(Tf)为靶向分子,对ND进行修饰制备得到靶向肿瘤细胞的纳米载体。以阿霉素(DOX)为药物模型,正常人肝细胞(L-02细胞)和肝癌细胞(HepG2细胞)为细胞模型,对纳米载药体系与细胞的相互作用和载药体系在细胞内的药物缓释及靶向性进行了研究。具体研究内容如下：1.使用电导返滴定法对强酸氧化处理后的ND表面羧酸含量进行测定,计算得到每个粒径为～140 nm的ND粒子表面含有3.36x 105个羧基。2.对ND物理吸附行为进行了研究,结果表明,Cit3-可以加强ND对DOX的吸附,同时验证了Cl-可以调控DOX从ND上的解离；对Tf的吸附行为符合Langmuir等温吸附,在PBS (pH7.4)中最大吸附量为(176.46±2.13)μg/mg,在pH(5.5)等于Tf的等电点附近有最大吸附,可能是因为Tf在等电点时溶解度最小,与ND的疏水作用力增大,吸附能力增强。利用激光共聚焦和流式细胞仪对ND和ND-Tf内吞到HepG2细胞的差异进行了定性和定量分析,结果得到ND-Tf比ND容易内吞到细胞中,利于可发荧光的ND在胞内成像。3.对ND进行PEG修饰后可提高其分散性,利于细胞内吞。对细胞内吞ND-PEG-DOX纳米载药体系进行了研究,证明HepG2细胞内吞ND-PEG-DOX的速率比游离DOX慢2倍,但是进入细胞的DOX量明显比游离的DOX内吞量增加,并降低了DOX的细胞毒性。使用流式细胞仪测定细胞内吞ND-PEG-DOX后胞内ND的荧光强度与侧向角的变化呈一致的趋势,故可以使用侧向角的变化来判断细胞内吞纳米颗粒的量。使用激光共聚焦显微镜观察细胞内吞ND-PEG-DOX的过程,表明FND-PEG-DOX对DOX具有缓释效应。细胞内吞ND-PEG-DOX的机理为网格蛋白介导的内吞过程,与DOX的自由扩散进入细胞的机理不同。4.以Tf为靶向分子,成功构建了ND-PEG-Tf纳米靶向药物载体,使用流式细胞仪测定细胞内ND的荧光强度表明细胞内吞ND-PEG-Tf是时间和浓度依赖的。细胞内吞ND-PEG-Tf的量与细胞表而表达TfR的量密切相关,利用Fe3+存在下游离Tf的抑制试验证明细胞内吞ND-PEG-Tf是由TfR介导的。依据高表达TfR的肿瘤细胞和低表达TfR的正常细胞差异,ND-PEG-Tf可选择性的进入肿瘤细胞,靶向输送药物。使用MTT法对ND-PEG-Tf-DOX纳米载药体系的细胞毒性进行了研究,结果表明ND-PEG-Tf-DOX对TfR表达较高的肿瘤细胞具有较大的细胞毒性,而与游离DOX的细胞毒性相比,ND-PEG-Tf-DOX可减小对TfR表达较低的正常细胞的毒性,即减小DOX的毒副作用。因此ND-PEG-Tf作为靶向药物输送载体对肿瘤的治疗具有潜在的应用。5.对HepG2细胞内吞ND-PEG-Tf纳米颗粒后的生长曲线证明ND-PEG-Tf不影响细胞的生长,同时使用流式细胞仪和激光共聚焦显微镜测定得到细胞内的ND-PEG-Tf会随细胞分裂将其传到子细胞中,随着分裂次数的增多,导致胞内ND-PEG-Tf纳米颗粒量的减少。结果表明ND-PEG-Tf是生物相容的,可作为纳米药物载体用于靶向输送药物。更多还原

【Abstract】 More serious environmental pollution and some bad habits of people lead to a high incidence of cancer, as well as cancer treatment is low efficient nowadays, which induce its high mortality rate. In the conventional methods of cancer treatment, chemotherapy is considered one of the most simple and effective methods. However, it often causes the failure of chemotherapy in particular for solid tumors, due to the more hysteretic diagnosis of cancer, the side effect of small molecule anti-cancer drug and multidrug resistance of cancer cells. The small molecule anti-cancer drug is rapidly diffused the whole body after blood administration. Simultaneously, not only cancer cells are killed, but also normal cells division faster are given great toxicity. With the development of nanotechnology, nanomedicine provides a new way and idea of cancer diagnosis and treatment. Nanotechnology application in medical diagnostic imaging can improve the detection sensitivity, such as magnetic resonance imaging, ultrasound imaging and X-ray computed tomography so on. Recently the development of in vivo imaging technology and multi-photon excitation scanning technology promote the application of fluorescence imaging in cancer diagnosis. The developments of these new technologies become possible for the earlier diagnosis of the tumor. In addition, the incomplete structure of tumor angiogenesis makes nanoparticles aggregate near the tumor through enhanced permeability and retention effect. The anticancer drugs prepared into nanodrugs or loaded on nanocarriers can achieve the passive targeting tumor therapy. Furthermore, some nanoparticles modified on the surface by the ligand can bind to specific angiogenesis or cancer cells to obtain the active targeting tumor therapy. The targeting delivery drug system can reduce the dosage and side effects of anticancer drugs, and also inhibit the multidrug resistance of tumor cells to improve treatment efficiency. The materials as nanocarriers investigated at present mainly include, but are not limited to liposomes, polymers, carbon nanotubes, micelles and nanoparticles so on. Recently, owing to its biocompatibility, chemical stability, optical properties, surface functionalization capabilities and easy synthesis, nanodiamond has been emerged as a new nanomedicine candidate material. The researches of the biological application of nanodiamonds are focused on drug carriers and fluorescence imaging agents and are in their infancy.In this paper, the targeting nanocarrier was constucted from detonation nanodiamond used as a carrier material, polyethylene glycol as a crosslinking agent, and transferrin as a targeting molecule, respectively. The interaction between the loading drug nanocarrier and the cells, the slow-sustained releasing drug from nanocarriers in the cellul and the targeting of nanocarriers are investigated using doxorubicin (DOX) as a model drug, normal liver cells (L-02 cells) and hepatoma cells (HepG2 cells) as cell models, respectively. The main contents are as follows:1. The amount of surface carboxylic acid on oxidized is measured by conductometric backward titration and calculated 3.36×105 carboxylic groups per ND particle (the size～140 nm).2. The results of physical adsorption behavior of ND indicate that Cit3- can enhance the adsorption of DOX onto ND, and the Cl- can regulate the releasing DOX from ND. The adsorption behavior of Tf on ND is coincident with Langmuir model which is a kind of isothermy absorbed behavior. The maximum adsorption amount of DOX on ND is (176.46±2.13) μg/mg in PBS (pH 7.4) and the adsorption capacity is the maximum at the pH (5.5) which is equal to the isoelectric point of Tf, probably because the minimum solubility of Tf at the isoelectric point leads to increasing hydrophobic interactions with the ND. The difference of cellular uptake of ND and ND-Tf are analyzed qualitatively and quantitatively by laser scanning confocal microscope and flow cytometry. The results indicate that ND-Tf is endocytosed easier than ND and more beneficial in intracellular fluorescent imaging.3. ND can be covalently conjugated with polyethlene glycol (PEG) to increase its dispesity and stability, which is conducive to endocytosis. The cellular uptake of ND-PEG-DOX is approximately two times of free DOX, but which can enhance the DOX uptake as compared to DOX alone and decrease the cytotoxity of DOX. The change of fluorescent intensity of intracellular ND determined by flow cytometry is consistent to the side scatter. So the amount of intracellular nanoparticles can be determined by the side scatter. The process of releasing DOX from intracellular ND-PEG-DOX is tracked by laser scanning confocal microscope. The result indicates that DOX released from ND-PEG-DOX composites had a slow and sustained drug release capability. The mechanism of cellular uptake of ND-PEG-DOX is clathrin-mediated endocytosis, which is different from free DOX that is diffused into cell.4. Using Tf as a targeting molecule, the targeting delivery drug nanocarrier, ND-PEG-Tf is constructed. The results of intracellular fluorescent intensity of ND-PEG-Tf determined by flow cytometry indicate that the endocytosis of ND-PEG-Tf is time-and concentration-dependent. The amount of cellular uptake of ND-PEG-Tf is related to the expressing TfR level on cell surface. The inhibition experiment using free Tf and Fe3+ shows that the endocytosis of ND-PEG-Tf is a TfR-mediated pathway. So ND-PEG-Tf can selectively enter the tumor cells and target drug delivery according to the difference of TfR between normal cells (low-expressed) and tumor cells (over-expressed). The cytotoxity is measured by MTT assay. The result show that ND-PEG-Tf-DOX is more toxitic to over-expressed TfR tumor cells. Simultaneously, ND-PEG-Tf-DOX can reduce toxic to normal cells compared with free DOX. So ND-PEG-Tf can potentially be acted as targeting cancer cells and effective drug delivery for cancer therapy.5. ND-PEG-Tf can not affect cell growth which is demonstrated by the cell growth curve after the endocytosis of ND-PEG-Tf. The intracellular ND-PEG-Tf can be transmitted to the daughter cells, which is determined by flow cytometry and laser scanning confocal microscope. The amount of intracelluar ND-PEG-Tf is reduced with the increasing number of cell division. The results indicate that ND-PEG-Tf is biocompatible and can be acted as a targeting drug delivery nanocarrier.更多还原