【作者】 李民；

【导师】 郑启昌；

【作者基本信息】 华中科技大学 ， 普外科， 2013， 博士

【摘要】 目的：制备具有聚集诱导发光（AIE）特性的壳聚糖（CS）纳米颗粒，评价纳米颗粒的理化特性、AIE特性和光稳定性以及生物亲和性，并探讨其在活细胞长程示踪标记中的应用价值。方法：通过酰胺化反应将四苯基乙烯衍生物（TPEITC）化学连接到CS主链上生成TPE修饰的壳聚糖（TPE-CS），采用核磁（NMR）、红外光谱（FTIR）分析检测TPE的接枝率。经离子交联法制备负载TPE分子的CS的纳米颗粒（TPE-CS NPs），采用动态光散射（DLS）测定纳米颗粒的水合粒径、分散性，激光多普勒电泳法检测纳米颗粒的表面电位，透射电镜（TEM）观察纳米颗粒的直径和形态。采用荧光分光光度仪分析TPE-CS NPs的发光特性和pH值对其的影响。MTT法评价TPE-CS NPs的细胞毒性。通过不同浓度的TPE-CS NPs进行HeLa细胞染色以观察该纳米颗粒对细胞成像的浓度影响，并采用共聚焦激光扫描显微镜（CLSM）连续激发染色细胞以计算该纳米颗粒的光稳定性。TPE-CS NPs入胞机制研究通过叠氮钠（NaN3）阻断三磷酸腺苷（ATP）的合成，抑制纳米颗粒的进入胞内，采用CLSM或者流式细胞仪（FCM）检测细胞内荧光强度。TPE-CS NPs的出胞作用研究通过标记的HeLa细胞和未标记的3T3细胞共培养，在CLSM下观察纳米颗粒的荧光分布情况。经TPE-CS NPs标记的HeLa细胞持续培养，每代观察一次荧光强度变化，直至细胞内荧光消失，以判断TPE-CS NPs对细胞的示踪时间。结果：通过TPEITC的异硫氰酸酯基和CS的胺基发生加成反应，成功合成了TPE-CS。1H NMR分析显示CS中不存在的芳香环的化学位移峰出现在TPE-CS中，经积分面积计算出TPE的接枝率为4.13%。FTIR检测结果显示TPEITC中的NCS在2045cm-1处的特征峰在产物TPE-CS中已消失。通过离子交联法成功制备出TPE-CS NPs，DLS检测显示纳米颗粒的水合粒径为170nm，分散性好，在TEM下观察可见纳米颗粒成球形、粒径均一且单分散。PE-CS NPs的表面电位随着溶液的pH值升高而逐渐降低，但在pH=7.0时仍为正电荷。TPE-CS在酸性溶液（pH2.5）中不发光，而TPE-CS NPs因TPE分子被固定在CS分子中而发出强荧光。当pH升高至7.0时，两种的荧光强度均增强，但是TPE-CS NPs的仍强于TPE-CS。细胞的荧光强度随着培养基中TPE-CS NPs的浓度增加而增强，且在连续激发30min后，细胞的荧光强度仅下降不到25%。细胞经NaN3预处理后，细胞内ATP的合成被阻断，限制了细胞的吞噬作用。与未经处理的细胞相比，细胞的荧光强度较弱。标记的HeLa细胞和未标记的3T3细胞共培养24h后，CLSM下观察纳米颗粒大部分仍在HeLa细胞内，仅有极少量荧光出现在3T3细胞的区域。在细胞长程示踪研究中，可见随着培养时间的延长，细胞内的荧光强度呈下降的趋势，但是染色后第7天仍可见荧光。结论：TPE-CS NPs呈球形、直径均一、分散性好且表面带正电荷，具有典型的AIE特性。TPE-CS NPs的细胞毒性低，并可通过细胞吞噬作用进入细胞内，且仅少量的纳米颗粒经出胞作用被排出到胞外，可长时间示踪活细胞。因此，TPE-CS NPs有望成为一种长程示踪活细胞的纳米荧光探针，并可进一步应用于监测药物或者基因的负载和释放。目的设计和制备对生物素受体高表达的肿瘤细胞特异性结合的负载聚集诱导发光（AIE）特性的荧光硅胶纳米颗粒（FSNPs）。评估该纳米颗粒的理化特性和生物亲和性，并检测其对生物素受体表达上调的肿瘤细胞的靶向成像。方法：通过溶胶-凝胶反应制备出负载噻咯衍生物1的荧光硅胶纳米颗粒（FSNP-1），然后在其表面进行胺基化修饰（FSNP-1-NH2）。FSNP-1-NH2表面的胺基与生物素的羧基发生酰胺化反应，使生物素化学结合在纳米颗粒表面（FSNP-1-biotin）。采用红外光谱仪分析纳米颗粒表面的生物素基团，采用透射电镜（TEM）、扫描电镜（SEM）观察纳米颗粒的直径及形态，采用X射线光电子能谱（XPS）和能量色谱X射线光谱（EDX）分析纳米颗粒的化学成分，热重分析（TGA）检测纳米颗粒的热稳定性并计算表面生物素分子的含量。通过细胞形态变化、细胞活性、细胞凋亡和细胞内活性氧自由基（ROS）等一系列检测来评价FSNP-1-biotin的生物亲和性。以生物素受体低表达的人正常肝细胞LO2对照，对生物素受体高表达的人宫颈癌细胞HeLa和人肝癌细胞BEL-7402进行细胞成像，观察进入细胞内的纳米颗粒的荧光强度，并行透射电镜观察纳米颗粒进入细胞的过程。用FSNP-1-biotin标记HeLa细胞后，观察其标记细胞的时间并用其示踪肿瘤细胞迁移过程。结果：通过溶胶-凝胶反应成功制备出FSNP-1，并通过化学反应修饰了生物素分子。红外光谱显示生物素分子的羰基（C=O）出现在FSNP-1-biotin上。TEM和SEM可见FSNP-1-biotin呈球形、粒径均一且分散性好。XPS和EDX分析显示生物素分子中的硫元素出现在FSNP-1-biotin中。前体分子1在乙醇溶液中无荧光，而纳米颗粒形成后其分子的旋转受到硅胶基质的限制，从而发出强烈的荧光。经FSNP-1-biotin处理后的细胞，细胞形态无显著性改变，仅出现少量多核细胞，细胞质区域出现少量吞噬空泡。CCK-8和台盼蓝检测显示在浓度低于100μg/mL时，与对照组比较，毒性效应无统计学差异（P>0.05），而当浓度达到100μg/mL时，对细胞具有明显的毒性作用（P<0.05）。FSNP-1-biotin对细胞凋亡和细胞内ROS水平在低浓度（40μg/mL）时无明显影响（P>0.05），而在高浓度（80μg/mL）时在一定程度上促进细胞发生凋亡和提高细胞内ROS水平（P<0.05）。FSNP-1-biotin靶向进入生物素受体高表达的肿瘤细胞中，并选择性标记细胞质区域。这些纳米颗粒通过受体介导的细胞吞噬作用进入细胞后，可长时间存留在细胞内，在细胞染色后7天仍可见荧光。FSNP-1-biotin标记肿瘤细胞后，可追踪肿瘤细胞迁移过程并且能定量分析细胞的迁移能力。结论：FSNP-1-biotin有望成为新型的肿瘤细胞示踪探针，靶向标记生物素受体高表达的肿瘤细胞，研究肿瘤的生物学特性或者进一步优化用于活体肿瘤组织靶向成像。更多还原

【Abstract】 Objective：To fabricate chitosan (CS) nanoparticles with aggregation-induced emission(AIE) characteristics, assess the physical and chemical properties, AIE features,photostability and biocompatibility of these nanoparticles and explore the application valuein long-term cell tracking of live cells.Methods: The TPE modified CS (TPE-CS) was synthesized through addition reactionbetween the isothiocyanate groups on TPEITC and amines on CS. The products weredetected through NMR and FTIR, and the substitution degree of TPE was calculated. Then,the CS nanoparticles hybridized with TPE molecules (TPE-CS NPs) were fabricated byionic gelation method. The hydrodynamic diameter and dispersity of TPE-CS NPs were measured by dynamical light scattering (DLS) and the zeta potential of these nanoparticleswas detected by laser doppler electrophoresis. The diameter and morphology of TPE-CSNPs were observed using transmission electron microscope (TEM). The spectrofluorometerwas performed to analyze the light property of TPE-CS NPs and effect of pH value onfluorescent intensity. The cytotoxicity of TPE-CS NPs was assessed using MTT assay.HeLa cells were stained with various concentrations of TPE-CS NPs to investigate thedose-dependent effect, and the cells were continuously scanned using confocal laserscanning microscope (CLSM) to assess the photostability of TPE-CS NPs. The cells werepretreated with NaN3, which reduced the cellular synthesis of adenosine triphosphate(ATP), to inhibit the uptake of nanoparticles, and then observed the fluorescent intensityusing CLSM and flow cytometry (FCM). TPE-CS NPs labeled HeLa cells and unlabeled3T3cells were co-cultured and observed the distribution of fluorescence from thenanoparticles. The HeLa cells were stained with TPE-CS NPs and observed per day untilthe fluorescence disappeared, to assess the retention time of TPE-CS NPs in cells.Results: The TPE-CS was successfully synthesized through amidation reaction betweenthe isothiocyanate groups and amines.1H NMR spectra showed that the new peaks presentat the aromatic region emerged in the TPE-CS conjugates and the substitution degree ofTPE is calculated to be4.13%. FTIR spectrum showed the isothiocyanate bondcharacteristic peak at2045cm-1in TPEITC disappeared in the TPE-CS conjugates. TheTPE-CS NPs were fabricated through ionic gelation method. DLS revealed the averagehydrodynamic diameter of TPE-CS NPs is170nm with good dispersity and TEM imageshowed TPE-CS NPs were spherical shapes, uniform in diameter with monodispersity. Thezeta potential of TPE-CS NPs decreased with increase of pH value, but still kept positive atpH7.0. Nearly no fluorescence was recorded when TPE-CS was dissolved in pH2.5solution, while TPE-CS NPs emitted strong fluorescence owing to the restriction ofintramolecular rotation of TPE by CS chain. When pH value was adjusted to7.0, thefluorescent intensity of TPE-CS and TPE-CS NPs was enhanced, but the solution of TPE-CS NPs was still stronger. The fluorescence from cells was dose-dependent. The cellstreated with TPE-CS NPs were continuously scanned for30min, but the fluorescent signalsonly lost less than25%. The cells were pretreated with NaN3, blocking the synthesis ofATP, to inhibit the endocytosis. Compared with un-treated cells, the fluorescence of NaN3pretreated cells became weaker. After co-culturing TPE-CS NPs labeled HeLa cells andunlabeled3T3cells for24h, CLSM images showed the nanoparticles mainly distributed inHeLa cells and only scattered fluorescence is from3T3cell region. After labeled withTPE-CS NPs, the fluorescence became weaker with increase of subculture, but the cellsstill emitted light for7days.Conclusions: TPE-CS NPs possess spherical shape, uniform diameter, good dispersity andpositive potential with typical AIE characteristics. TPE-CS NPs with low cytotoxicity entercells through endocytosis. Only a small amount of TPE-CS NPs were excluded from cellsand TPE-CS NPs can trace the live cells for long period of time. Therefore, TPE-CS NPsare promising for a long-term live cell nanoprobe and further applied in monitoring theloading and release of drug and gene. Objective: To design and fabricate fluorescent silica nanoparticles (FSNPs) withaggregation-induced emission (AIE) characteristics, targeting to the tumor cells with over-expressed biotin receptors. To investigate the physical and chemical properties andbiocompatibility, and assess the targeted imaging to the tumor cells with over-expressedbiotin receptors.Methods: The FSNPs hybridized with silole derivative1were fabricated through sol-gelreaction, and then modified with amine groups on the surface of nanoparticles to obtainFSNP-1-NH2. The biotin molecules were decorated on the surface of FSNP-1-NH2byamidation reaction between amine groups and carboxyl groups of biotin, namelyFSNP-1-biotin. The biotin groups on the surface of nanoaparticles were analyzed by IRspectrum. The diameter and morphology of nanoparticles were observed using transmissionelectron microscope (TEM) and scanning electron microscope (SEM). The chemicalcompose was analyzed X-ray photoelectron spectroscopy (XPS) and energy dispersiveX-ray spectroscopy (EDX). Thermogravimetric analysis was measured to assess thethermal stability and calculate the ratio of biotin. The biocompatibility of FSNP-1-biotinwas investigated using cell morphological change, cell viability, cell apoptosis andintracellular reactive oxygen species (ROS) assays. Human cervical carcinoma HeLa cellsand human hepatocellular carcinoma BEL-7402cells with over-expressed biotin receptorswere stained with FSNP-1-biotin to observe the fluorescent intensity of cells. Humannormal liver LO2cells with low-expressed biotin receptors were used as a negative control.The uptake of nanoparticles was investigated using TEM. HeLa cells labeled withFSNP-1-biotin were continuously cultured to observe the retention time of thenanoparticles inside the cells and tracked to study the process of tumor cell migration.Results: The FSNP-1were successfully fabricated through sol-gel reaction and modifiedwith biotin molecules by amidation reaction. The IR spectrum showed the emergence ofcarbonyl group in the FSNP-1-biotin. TEM and SEM showed the nanoparticles werespherical in shape, uniform in diameters with narrow size distributions and possessed gooddispersity. XPS and EDX analysis demonstrated a amount of sulfur atom in biotin molecules was detected in the FSNP-1-biotin. Nearly no fluorescence signals wererecorded when the ethanol solution of precursor1was photoexcited, while thenanoparticles emitted strong fluorescence because the intramolecular rotation of1wasrestricted by silica network. After treating with FSNP-1-biotin, the cellular morphology didnot remarkably change, only small amount of multinucleate cells emerged and a littleamount of vacuole was observed in cytoplasmic region. Compared with control group,CCK-8and trypan blue assay showed low cytotoxicity at concentrations below100μg/mL,which is different significantly (P>0.05). At100μg/mL, the cytotoxicity of FSNP-1-biotinis remarkable (P<0.05). FSNP-1-biotin did not affect on the cell apoptosis and intracellularROS at the concentration of40μg/mL (P>0.05), but accelerated apoptosis and increasedthe level of intracellular ROS at the concentration of80μg/mL (P<0.05) to a certain extent.FSNP-1-biotin could target to the tumor cells with over-expressed biotin receptor andselectively stain cytoplasmic region. These nanoparticles entered cells throughreceptor-mediated endocytosis pathway and stayed inside cells. The fluorescence wasrecorded from the labeled cells, even up to7days. After stained with FSNP-1-biotin, thecells were tracked to observe the migration process and analyzed quantitatively the abilityof cell migration.Conclusions: The FSNP-1-biotin are promising for a novel tumor cell tracker, which targetto the tumor cells with over-expressed biotin receptor, to investigate the biologicalproperties of tumor and further apply in targeted imaging of tumor tissue in vivo.更多还原