用于癌症标志物早期诊断的免疫磁性微球的制备及评价

【作者】 王海青；

【导师】 宋存先； 俞玫；

【作者基本信息】 中国协和医科大学 ， 生物医学工程， 2008， 博士

【摘要】 恶性肿瘤在我国已成为导致人们死亡的第一位疾病,要攻克肿瘤,诊断是关键的一步,现有的肿瘤诊断和筛查技术要么价格昂贵、要么灵敏度低,往往是在肿瘤发生到一定阶段才能发现,存在严重不足。一种解决思路是利用抗原抗体反应的特异性,研发一种新型免疫磁性微球,将其作为肿瘤标志物的捕获探针,对人体血液或体液中的肿瘤标志物进行捕获,再利用放大探针将捕获的标记物信号进行放大并检测,以期能进一步提高肿瘤标志物检测的灵敏度,简化检测步骤,降低检测成本。本课题组对上述思路中的免疫磁性微球进行制备,以人源IgG模拟肿瘤标志物,采用2～3μm功能化磁性微球与抗体偶联,制备了具有结合活性的免疫磁性微球。采用¹²⁵I标记抗原及抗体,通过对放射性同位素的检测对偶联工艺进行定量评价,考察了各影响因素对抗体偶联效果的影响。对所制备的免疫磁性微球进行了初步性能评价。市售磁性微球表面功能基数量少,品种单一,限制了磁性微球的进一步多功能化。多糖微球表面功能基团种类丰富,易于改性,且表面功能基团分布密集,可以引入更多抗体。我们以普鲁兰为例,优化多糖微球的合成条件,对免疫微球的进一步改进打下基础。磁性微球可以通过磁场富集,用于体外检测试剂载体,在疾病诊断上有重要用途。也可用于药物载体,通过磁场定位达到靶向给药的目的。为了拓展体内检测试剂载体的研究,我们设计合成一类具有pH/温度双重敏感性的、可生物降解的两亲性聚丙烯酰胺衍生物-co-聚氨基酸聚合物,通过高分子自组装的方法形成纳米微球,作为下一步合成多重靶向药物载体的前期基础。本文具体研究内容如下:一、磁性微球与抗体的偶联方法的考察选用2～3μm SiO₂磁性微球,分别连接有不同间隔臂的胺基、羧基等功能基,与带有荧光标记的抗体通过不同的方法进行偶联,再与带有荧光标记抗原进行吸附,通过激光共聚焦检测偶联情况,对磁球与抗体、抗体偶联情况进行定性研究。选定出工艺简便、偶联效果好的偶联方法。二、磁性微球-抗体与抗原吸附定量考察及其稳定性考察采用放射性同位素标记的方法进行定量研究。对选定的偶联方法进行进一步工艺优化,得出最佳的偶联条件,并对免疫磁性微球进行稳定性考察。在此基础上逐步降低抗原的浓度,确定抗原最小检测浓度。三、蛋白G定向偶联磁性微球与抗体的工艺研究EDC化学偶联方法是将抗体无规则地连接在磁性微球上,而采用蛋白G可通过对IgG的Fc区的结合,实现抗体与磁性微球的定向偶联。考察抗体定向偶联条件,并研究了pH值对抗体检测回收率的影响。四、普鲁兰微球的制备工艺的考察以正庚烷-四氯化碳为油相、Span-80为乳化剂,采用反相乳化法制备粒径可控的普鲁兰微球。考察普鲁兰浓度、水油比、搅拌速度、乳化剂用量等因素对微球的大小、形态的影响。为下一步制备各种功能化磁性微球并与SiO₂微球进行比较研究,奠定实验基础。五、温度PH敏感性材料P（NiPAAm-co-DMAA）-co-P（L-Ala）的制备通过原子转移自由基聚合（ATRP）合成一种带有活性-NH₂基团的温度敏感性亲水型共聚物P（NiPAAm-co-DMAA）,并将其作为引发剂,引发丙氨酸的阴离子聚合。所合成的P（NiPAAm-co-DMAA）-co-P（L-Ala）的PDI在1.3左右。聚合物可以通过自组装的方式形成纳米胶束。利用透射电镜（TEM）研究了胶束的粒径分布和形态。结果表明,胶束大小在200～300nm左右,具有明显的核壳结构。共聚物的最低临界溶解温度（LCST）为45.5℃。温度低于LCST时,聚合物溶解形成胶束;高于LCST时,胶束解离,聚合物不溶。聚合物对温度的响应是快速而可逆的。综上所述,本论文制备了免疫磁性微球,并讨论了磁性微球上的功能基团、偶联剂、间隔臂、以及其他实验因素对偶联效果的影响,其抗体偶联量最高为31.33±2.16μg/0.1mg磁性微球,抗原吸附量最高为34.78±0.36μg/0.1mg磁性微球。所制免疫磁性微球对人源IgG的检测极限水平可达50pg/ml,对人源IgG吸附率为65.96±6.99%。且稳定性较好,在37℃60转/分钟摇动条件下存放5天,抗体脱落量仅有13%。考察了蛋白G法定向连接抗体工艺,其抗体连接量最高为1.26±0.03μg/0.1mg磁性微球,抗原吸附量为2.45±0.10μg/0.1mg磁性微球。相比非定向化学连接工艺来说,定向连接工艺的抗体利用率较高,可有效地结合抗原,但抗体连接量较低。本实验为免疫磁性微球在肿瘤标记物早期诊断方面的应用提供了参考。另外,本论文还研究粒径可控的普鲁兰微球以及温度pH敏感纳米胶束P（NiPAAm-co-DMAA）-co-P（L-Ala）的制备工艺,为下一步制备多功能化检测载体提供了参考。更多还原

【Abstract】 As we all know,the human’s health and life are very badly threatened by the cancer. Diagnostic techniques are the key to conquering cancer.At present,early diagnostic technologies of tumor have different disadvantages,such as lack of sensitivity and specificity,or being too expensive.It’s often too late to cure when the tumor is final diagnosed.In order to improve the sensitivity on the diagnosis of the tumor,this paper try to designs one way to meet this goal:Immunomagnetic Microspheres（IMMS） were prepared,which were used to adsorb and concentrate the trace amounts of serum tumor markers.The signal of tumor markers was analyzed after being magnified by amplifier probe.This method is expected to improve the sensitivity,simplify the detection procedures and reduce the cost.Using human-IgG as the tumor marker model,IMMS was prepared by coupling the mouse-anti-human IgG with functionalized Magnetic Microspheres（MMS,2～3μm）.The antibody was labeled with ¹²⁵I.The coupling process was optimized by counting the radioactivity of ¹²⁵I-Ab on the MMS.All kinds of influence factors in the coupling reaction were investigated.Adsorption ability for human-IgG and stability of IMMS were evaluated preliminary.The amounts and species of functional group on the surface of the MMS sold on the markets were little,and the further multi-functionalized research was limited. Polysaccharides microspheres had a lot of functional groups on the surface,and were easier to be modified.We prepared pullulan microspheres and optimized the preparation conditions,providing basic data for the further improvement.Besides antibodies capture probe,MMS could also be used as drug carriers. Furthermore,we designed a pH/ temperature-sensitive material（P（NiPAAm -co-DMAA））, which could form nanometer-sized micelle by self-assembly.The major contents of this paper are shown as follows:1.The research of coupling process of the magnetic microspheres with antibody.First,2～3μm SiO₂ magnetic microspheres were coupled with Rhodamine antibody, then adsorbed the FITC antigen and made a sandwich complex.The sandwich complex was detected by the laser confocal scanning microscopy（LCSM）.The coupling effects were estimated by LCSM,and the coupling processes were optimized.2.The research of the quantity and stability of the magnetic microspheres and antibody.The antigen was labeled by ¹²⁵I through Ch-T technology,and the radioactivity in each sample was quantified in a gamma radiation counter.The coupling process was optimized.IMMS was prepared at the optimal conditions, and the lowest concentration of the antigen that could be detected was confirmed.The stability of the IMMS was investigated.3.The study of directional connection process between Ab and MMS.Ab was nondirectional coupled with the MMS by cross-linking agents（EDC or SPDP）.Protein G could bind with high affinity for the Fc region of IgG,and Ab was directional connected on the MMS by Protein G.The binding between protein G and Ab was reversible,it’s easy to elute Ab from the MMS by adjusting the pH of the solution.4.The preparation of the pullulan microspheresThe Pullulan microspheres were synthesized by inverse emulsion with glutaraldehyde as the cross-linking agent,Span80 as the emulsifying agent and the n-heptane /CCl₄ oil as the organic phase.The influence of the pullulan solution,dosage of oil phase/water phase,dosage of surfactants,dosage of cross-linking agent,and stirring speed on the size and shape of the microspheres were researched.It provided basic data for the preparation of pullulan magnetic microspheres.5.The preparation of P（NiPAAm-co-DMAA）-co-P（L-Ala）A pH-sensitive and temperature-sensitive polymer（P（NiPAAm -co-DMAA）） was synthesized by ATRP technology,which would be used as the initiator for the polymerization of L-Ala.The PDI of the polymer（P（NiPAAm -co-DMAA）-co-P（L-Ala）） is about 1.3.This amphipathic polymer could form nanometer-sized micelle by selfassembly. The particle diameter distribution and appearance of the micelle were detected by TEM.The micelle was core-shell structure,whose diameter was about 200～300nm. The LCST of the copolymer is 45.5℃.The micelle was formed when T≤45.5℃. Inversely it’s dissociated when T≥45.5℃.The responsiveness of the copolymer to the temperature is quickly and reversible.All in brief,IMMS was prepared used for the early diagnosis of tumor markers models.The antibody and antigen on the MMS was quantitively analyzed by counting the radioactivity of ¹²⁵I-Ab and ¹²⁵I-Ag.The effect of cross:linking agents,functional group,arm spaces of the functional group on the IMMS and other factors were investigated.The highest coupling amount of Ab on the MMS was 31.33±2.16μg/0.1 mg MMS,and the amount of Ag adsorbed on the IMMS was 34.78±0.36μg/0.1mg IMMS. The lowest concentration of the antigen that could be detected was 50pg/ml,and the adsorption efficient for human IgG was 65.96±6.99%。The stability of the IMMS is good,and the exfoliation amount was about 13%when the IMMS were rotated at 60r/min at 37℃for 5 days.The directional connection of Ab and MMS-protein G was investigated.The highest coupling amount of Ab was 1.26±0.03μg/0.1mgMMS,and the amount of Ag adsorbed on the IMMS was 2.45±0.10μg/0.1mgIMMS.Comparison to the non-oriented connection the utilization rate of the antibody was higher,but the amount of Ab on the MMS was lower.This paper provided reference data for the preparation of the IMMS used in the early diagnosis of the tumor markers.In addition,Pullulan microspheres and micelle of P（NiPAAm -co-DMAA）-co-P（L-Ala） were synthesized,which provided reference data for the preparation of multifunctional detection carrier.更多还原