~(125)I-UdR-壳聚糖纳米微粒的研制及其治疗兔肝癌的研究
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摘要
目的
1.通过制备高脱乙酰度低分子量壳聚糖（Chitosan，CS），研制得到壳聚糖纳米微粒（Chitosan nanoparticles，CS-NP）作为具有肝癌靶向作用的药物载体，装载肿瘤内放射治疗药物~(125)I-脱氧尿嘧啶核苷酸（~(125)I-UdR）后成为具有肿瘤靶向性和缓释性能的~(125)I-UdR-壳聚糖纳米微粒（5-[~(125)I]Iodo-2’-Deoxyuridine-Chitosan-Nanoparticles，~(125)I-UdR-CS-NP）。确定最佳工艺条件并鉴定其理化性质、生物相容性和药物释放特性，分析~(125)I-UdR-CS-NP在新西兰兔体内的药代动力学、组织分布和内照射细胞生物学效应。
2.分析验证~(125)I-UdR-CS-NP对兔肝癌原位模型的被动靶向性，特别是通过肝动脉介入给药方式所能达到的肝癌靶向能力。
3.观察分析CS-NP进入肿瘤细胞途径及其在亚细胞水平的分布、转运、降解和药物释放方式。
方法
1.采用间歇水解法和辐射降解法制备高脱乙酰度低分子量壳聚糖，并分析其红外光谱。
2.离子交联法制备CS-NP，并通过正交试验优化其制备工艺。采用激光粒度分析仪、透射电子显微镜和zeta电位仪分析鉴定CS-NP的粒径、分散度、形态和表面性状以及zeta电势等物理性状和表征。
3.通过MTT法和流式细胞仪检测肝癌细胞和正常肝组织细胞与CS-NP共同孵育后的存活分数和凋亡率，以评价其生物相容性。
4.离子交联法制备~(125)I-UdR-CS-NP，并通过单因素分析法确定其最佳工艺条件。测量其粒径、分散度，并用透射电镜观察其形貌。
5.采用动态透析法测量~(125)I-UdR-CS-NP的体外药物累积释放率，并绘制累积释放曲线，观察比较载药量与释药特性的关系，确认其是否具备长效缓释特性。
6.分析外周静脉注射~(125)I-UdR-CS-NP后实验兔的药代动力学特性和药物组织分布特征。
7.观察分析实验兔注射~(125)I-UdR-CS-NP后的血液生化指标和骨髓图片的变化，评价~(125)I-UdR-CS-NP在生物体内的毒副作用。
8.离子交联法制备异硫氰酸荧光素标记壳聚糖纳米微粒（FITC-CS-NP），并采用激光粒度分析仪、透射电子显微镜和zeta电位仪分析鉴定FITC-CS-NP的粒径、分散度、形态和表面性状以及zeta电势等物理性状和表征。
9.体外细胞摄取实验分析肝癌细胞和正常肝组织细胞对~(125)I-UdR-CS-NP的摄取量及其时间效应和剂量效应，以验证~(125)I-UdR-CS-NP的肝癌靶向性。
10.采用激光共聚焦显微镜观察FITC-CS-NP在肝癌细胞和正常肝细胞中不同时段的分布量和位置，验证CS-NP作为药物载体的肝癌靶向性。
11.采用ROI区域时间序列扫描技术连续定量分析FITC-CS-NP进入肝癌细胞和正常肝组织细胞的过程。
12.采用MTT法、流式细胞仪观察肝癌细胞和正常肝细胞经~(125)I-UdR-CS-NP作用后的存活分数和增殖指数。
13.用中性单细胞凝胶电泳技术检测肝癌细胞和正常肝细胞经~(125)I-UdR-CS-NP作用后2h的DNA链断裂程度和作用后24h的DNA损伤修复能力，以评估~(125)I-UdR-CS-NP对肿瘤细胞的杀伤效应。
14.超声引导下采用经皮穿刺术在实验兔肝左叶种植VX2瘤块，制备兔肝癌原位模型，并做病理学鉴定。
15.兔肝癌原位模型在CT引导下采用Seldinger微导管肝动脉插管术灌注~(125)I-UdR-CS-NP，SPECT分时段观察~(125)I-UdR-CS-NP在兔体内的分布及其对原位肝癌的靶向性。测量并分析~(125)I-UdR-CS-NP在肿瘤和其他组织中的分布，以验证其肿瘤靶向性。
16.将用药后48h的肿瘤组织做病理切片后进行Tunel染色，观察细胞凋亡率改变，以评价~(125)I-UdR-CS-NP的抑瘤效果。
17.激光共聚焦显微镜观察37℃和4℃条件下FITC-CS-NP及其原料FITC-CS长链分子进入肿瘤细胞的行为特征，以观察FITC-CS-NP进入细胞的耗能情况。
18.采用CPZ、Fil、Amil分别抑制以clathrin-经典途径、caveolae-途径和巨胞饮三条纳米微粒进入细胞的主要途径后，定量分析~(125)I-UdR-CS-NP进入肝癌细胞HepG2的量，以分析其内化途径类型。并用激光共聚焦显微镜观察验证FITC-CS-NP通过不同途径进入肝癌细胞的跨膜过程及其在细胞内的分布特征。
19.采用多色荧光染色技术观察FITC-CS-NP在亚细胞水平的动态分布、转运和降解的行为特征。
结果
1.间歇水解法和辐射降解法制备的壳聚糖脱乙酰度为93.062±2.384%，分子量3kDa，外观为深黄色粉末，在PH=7.0的环境中可完全溶解，呈深黄色澄清透亮的液体。红外图谱表明经碱处理后壳聚糖的酰胺键被水解，更多游离氨基暴露，脱乙酰度得到明显提高。
2.成功制备CS-NP，正交试验结果表明，对CS纳米微粒粒径的影响力从大到小依次为CS分子量>TPP浓度>搅拌速度>CS浓度。优化后的制备工艺条件为CS浓度1g/L，搅拌速度600rpm，TPP浓度2g/L，CS分子量3kDa，所制得的CS-NP平均粒径为70.39±5.12nm，PDI0.16±0.012，zeta电位为+32.5±3.3。透射电镜观察其外观为规整的球形，表面平整，大小均匀，分散度较好。
3. MTT法和流式细胞仪结果都表明CS-NP对人正常肝细胞株HL-7702、QSG-7701无细胞毒性；当CS-NP浓度≥200μg/mL时对于人肝癌细胞系HepG2、SMMC-7721有一定的细胞毒性。
4.成功制备~(125)I-UdR-CS-NP。优化后的制备条件为投药量1.2～2.4mg/mL，PH5.5，所制得的~(125)I-UdR-CS-NP平均粒径175.8nm。与空白CS-NP相比，其性状会略有改变，表现为粒径增大且均一性下降，结构较疏松，表面形貌略欠规整。
5.~(125)I-UdR-CS-NP在PH5.3到7.4环境下的释放曲线符合Higuchi方程，为长效制剂特征，具有明显的缓释作用。
6.~(125)I-UdR-DLN经外周静脉用药后药代动力学符合二室模型特征，第一时相和第二时相半衰期分别为1.1±0.69h和1.5±0.16h，曲线下面积(AUC0-∞)为11.2±1.4mg/L·h。与~(125)I-UdR相比，~(125)I-UdR-CS-NP的生物分布倾向于肝脾，通过肾脏的排泄呈现明显的延迟现象，骨髓及胃肠道黏膜的药物分布量较少。
7.静脉注射~(125)I-UdR-CS-DLN剂量高至7.4MBq（6mg），未发现血液生化指标明显异常，也无明显骨髓抑制现象，而~(125)I-UdR对照组2d时骨髓象表明~(125)I-UdR对增殖阶段的造血细胞抑制作用较强。表明~(125)I-UdR-CS-DLN明显减轻了对骨髓造血细胞的抑制作用。
8.体外细胞摄取实验表明~(125)I-UdR-CS-NP对上述2株肝癌细胞具有明显的靶向性，进入肝癌细胞的量是正常肝细胞的10倍，并具有时间效应和剂量效应。加药后30min或剂量大于185kBq/mL时细胞内药物的放射性强度都会达到饱和。
9.成功制备异硫氰酸荧光素标记的CS-NP（FITC-CS-NP），平均粒径158.02±12.30nm，zeta电势为+28.8±3.1。透射电镜观察外形呈较为规整的球形，表面结构较疏松。
10.在细胞摄取实验中，FITC-CS-NP可被上述2株肝癌细胞可大量摄取，且在细胞浆内呈不均匀分布；而FITC-CS-NP进入2株正常肝细胞明显减少，更多的是吸附在细胞膜上，30min后开始解离。
11. ROI时序扫描曲线表明FITC-CS-NP与肝癌细胞HepG2接触后完成了细胞膜吸附-跨膜内化-进入细胞浆的过程，细胞膜吸附FITC-CS-NP量的峰值时间为15min，胞浆内FITC-CS-NP荧光峰值时间为30min。FITC-CS-NP同样可以在15min内大量吸附在正常肝细胞HL-7702表面，但随后进入细胞的量很少，且过程明显减慢。
12.当浓度≥37kBq/mL时，~(125)I-UdR-CS-NP作用后肝癌细胞的存活率明显低于正常肝细胞。流式细胞仪分析细胞周期结果表明，~(125)I-UdR-CS-DLN和~(125)I-UdR对增殖期的细胞都有杀伤作用，并造成细胞增殖周期阻滞在G1期。而~(125)I-UdR-CS-DLN更对DNA合成后进行有丝分裂的G2/M期HepG2细胞也有明显的杀伤作用。
13.不论是肝癌细胞HepG2还是正常肝细胞HL-7702，经~(125)I-UdR-CS-DLN作用后2h的DNA损伤程度都明显高于同等剂量~(125)I-UdR组。药物作用24h后各组的彗星实验结果表明，~(125)I-UdR-CS-DLN组的DNA损伤修复能力远低于~(125)I-UdR组。另外，~(125)I-UdR-CS-DLN组中，肝癌细胞株HepG2的DNA损伤后修复能力明显低于HL-7702。
14.成功制备VX2肿瘤兔肝原位模型，均为肝左叶单发肿瘤病灶，平均直径9.2±1.3mm。
15. Seldinger微导管经股动脉成功超选择至肝左动脉，灌注~(125)I-UdR-CS-NP后，SPECT分时段显像表明~(125)I-UdR-CS-DLN凭借Seldinger微导管的精确定位和肝脏首过效应，可大量聚积在肝脏和瘤体内，2h时肝脏组织中的~(125)I-UdR-CS-DLN已排泄至全身平均水平，而瘤体内依然有明显的~(125)I-UdR-CS-DLN浓聚，到24h时ROI感兴趣区T/NT比值达4.49±1.22。表明~(125)I-UdR-CS-DLN在兔体内实验中对肝原位肿瘤有明显的被动靶向性，且具备缓释特征。用药后48h时的药物组织分布表明瘤内放射性强度超过正常肝组织的12倍，是肌肉组织的50倍左右，且表现出更长的药物缓释时间，进一步证明了~(125)I-UdR-CS-DLN在兔活体中对肝原位肿瘤的被动靶向性和缓释特征。
16.介入给药后48h时肿瘤组织Tunel染色结果表明，~(125)I-UdR-CS-DLN可致肿瘤细胞发生明显的凋亡，其肿瘤杀伤能力明显高于同等剂量的~(125)I-UdR。
17.激光共聚焦显微镜观察结果表明，FITC-CS-NP进入肿瘤细胞主要通过一类温度依赖的，需要消耗细胞能量的主动摄取机制。
18.肝癌细胞摄取~(125)I-UdR-CS-DLN是多种途径共同参与的，以巨胞饮途径为主（43%），clathrin-经典途径其次（34%），caveolae-途径仅占12%。这三种胞饮机制相互独立，不存在协同作用，但存在一定的代偿作用。激光共聚焦显微镜进一步通过形态和定量手段验证了FITC-CS-NP通过三条胞吞途径进入肝癌细胞的跨膜过程，并观察不同胞吞内体在亚细胞水平的分布规律。
19.采用多色荧光染色技术分时段观察结果表明经clathrin-经典途径内吞的FITC-CS-NP跨膜较快，入胞后向细胞核移动并与溶酶体融合，最终次级溶酶体在核周释放出FITC-CS-NP；经巨胞饮途径内吞的FITC-CS-NP入胞较慢，跨膜后散在分布于细胞质中，可驻留较长时间，有利于药物的延迟释放，达到细胞内缓释的目的。
结论
1.首次将~(125)I-UdR装载入纳米药物载体，成为靶向缓释制剂。采用优化工艺条件制备的壳聚糖纳米微粒粒径均匀，形貌规整，生物相容性好；释放曲线符合Higuchi方程，为长效制剂特征，具有明显的缓释作用。
2.首次将肝动脉介入给药技术用于提高壳聚糖纳米微粒的被动靶向性。体内外实验证明~(125)I-UdR-CS-DLN具有明显的肝癌被动靶向性；经肝动脉介入给药后~(125)I-UdR-CS-DLN可大量聚积于肝肿瘤内，48h时瘤内放射性强度是正常肝组织的12倍。同时，~(125)I-UdR-CS-DLN在体内外实验中都显示出比~(125)I-UdR更强的肿瘤杀伤能力。
3.证明~(125)I-UdR-CS-DLN主要通过巨胞饮和clathrin-经典途径进入肿瘤细胞，巨胞饮体可在细胞质内实现较长时间驻留；观察并较早证实了通过clathrin-途径进入细胞质的FITC-CS-NP在与溶酶体融合后会导致溶酶体破裂而逃逸。并首次观察到这种逃逸现象集中发生在细胞核周围，这一发现有利于提高药物进入细胞核内的量。
总之，本次研制的~(125)I-UdR-CS-DLN具有明显的肝癌细胞靶向性和更强的肿瘤杀伤能力，通过肝动脉介入给药可大幅提高其瘤内药物积聚量，在肝癌细胞内可驻留较长时间，并有部分微粒可在核周集中释放。故~(125)I-UdR-CS-DLN有望成为肿瘤亚细胞水平内放射治疗的新剂型，达到更好的治疗效果。
Objective
1. To prepare high deacelation degree and low molecular weight Chitosan, andfurther development chitosan nanoparticles and~(125)I-UdR-Chitosan nanoparticles. Theoptimum process conditions were established, and the physical and chemical properties,biocompatibility and drug release properties were identified. Further more, thepharmacokinetics and tissue distribution of~(125)I-UdR-CS-NP in New Zealand rabbitwere analyzed.
2. To validate the passive targeting property of~(125)I-UdR-CS-NP to in situ livercancer models, especially the improvement of liver targeting ability through hepaticartery interventional delivering drug.
3. To observe the internalization pathways and distribution, transshipment anddegradation of CS-NP in subcellular level.
Mehtod
1. High deacelation degree and low molecular weight Chitosan was preparedusing intermittent hydrolysis and radiation degradation method, and its infraredspectrum was analyzed.
2. Using ion cross-linking method for preparation of CS-NP, and optimizing thepreparation techniques through the orthogonal test.
3. The physical properties and characterization of CS-NP were analyzed usingtransmission electron microscope, laser particle size analyzer and zeta potentiometer.
4. The biocompatibility of CS-NP was evaluated through the MTT method andflow cytometry instrument.
5. Using the ion cross-linking method for the preparation of~(125)I-UdR-CS-NP, andthrough the single factor analysis method to determine the optimum process conditions.
6. The in vitro drug release properties of~(125)I-UdR-CS-NP were analyzed by dynamic dialysis method.
7. The pharmacokinetic characteristics and drug organization distribution of~(125)I-UdR-CS-NP injected via peripheral vein of the rabbits were analyzed.
8. The side effects of~(125)I-UdR-CS-NP were analyzed using in vivo experiments.
9. Rabbit in situ liver cancer models were prepared using ultrasound-guidedpercutaneous puncture technology.
10. The in vitro cell uptake experiments were performed to verify the liver cancertargeting ability, including the comparisons on the uptake volume of~(125)I-UdR-CS-NPand time effection, dose effection between the liver cancer cells and normal liver cells.
11. The fluorescein isothiocyanate marked chitosan nanoparticles were preparedusing ion cross-linking method.
12. As drug carrier, the liver cancer targeting property of CS-NP were validated,through continuous observing the dynamic distribution of FITC-CS–NP in liver cancercells and normal liver cells with laser confocal microscope.
13. The process of liver cancer cells and normal liver cells internalizedFITC-CS–NP were dynamically record via the time sequence scanning technology.
14.~(125)I-UdR-CS-NP were perfused in hepatic artery of rabbits with liver cancer insitu model using Seldinger micro catheter under the CT guided, and the distribution andliver targeting property were observed in deferent period by SPECT.
15. The organization distribution of~(125)I-UdR-CS-NP been perfused in rabbithepatic artery were analyzed to verify its tumor targeting property.
16. When the FITC-CS–NP were uptake under different temperature, the cells'behavior characteristics were observed by Laser confocal microscope.
17. The transmembrane process of FITC-CS-NP entering into hepatoma cellsthrough different ways and its distribution characteristics within the cell was observedwith the help of laser scanning confocal microscopy.
18. The dynamic distribution, transportation and degradation process ofFITC-CS-NP in subcellular level was observed by adopting multicolor stainingtechnology.
Result
1. The deacetylation degree of chitosan prepared by intermittent hydrolysis andradiation degradation was93.062±2.384%, with the molecular weight of3kDa. It was dark yellow powder, which can be completely dissolved in water of PH7.0, to presentdark yellow clarified translucent liquid. IR spectra showed that the processed chitosanamide bond was hydrolyzed with more exposure of free amino groups, while the degreeof deacetylation was markedly improved.
2. The CS-NP was successfully prepared. The orthogonal experiment showed thatthe influences on CS nano-particle size were arranged in a decreasing manner, the CSmolecular weight, TPP concentration, stirring speed and CS concentration. Optimizedpreparation technological condition had CS concentration of1g/L, stirring speed of600rpm, TPP concentration of2g/L, CS molecular weight of3kDa, average preparedparticle diameter of CS-NP of70.39±5.12nm, PDI of0.16±0.012, and zeta potentialof+32.5±3.3. Its appearance through TEM observation presented a regular sphere withsmooth surface, uniformed size, and good dispersity.
3. MTT method and flow cytometry results showed that CS-NP had no celltoxicity on human normal liver cell line HL-7702and QSG-7701; when the CS-NPconcentration was equal or greater than200μg/mL, there was certain toxicity for thehuman hepatoma cell line HepG2and SMMC-7721.
4.~(125)I-UdR-CS-NP was successfully prepared in the study. The optimizedpreparation condition required reagent quantity of1.2-2.4mg/mL, PH5.5, and theaverage particle diameter of the prepared~(125)I-UdR-CS-NP was175.8nm. Compared tothe blank CS-NP, its traits was slightly changed with the increase of the particle size anddecrease of uniformity, loose of structure, and irregular of the surface morphology.
5. Dissolution curve of the~(125)I-UdR-CS-NP in PH5.3to7.4fit Higuchi equation,with the characteristics of long-acting preparation and distinct sustained releasefunction.
6. After peripheral intravenous administration, the pharmacokinetics of~(125)I-UdR-DLN was in line with the characteristics of the two-compartment model. Thehalf-life of the first phase and the second phase were1.1±0.69h and1.5±0.16hrespectively, and the area under the curve (AUC0-∞) was11.2±1.4mg/L h. Comparedto~(125)I-UdR, the biological distribution of~(125)I-UdR-CS-NP tended to be on the liver andspleen, showing obvious delay by renal excretion, and its distribution of marrow andgastrointestinal mucosa was less.
7. Intravenous injection of~(125)I-UdR-CS-DLN was as high as7.4MBq (6mg) indosage, with no significant abnormality of blood biochemical indicators or distinct bone marrow suppression found. While the bone marrow of the control group of the~(125)I-UdRshowed that the~(125)I-UdR had an inbibitional effect on the hematopoietic cells ofproliferation phase in2d, which indicated that~(125)I-UdR-CS-DLN could significantlyreduce the inhibitory effect on bone marrow hematopoietic cells.
8. The in-vitro cells uptake experiment revealed that~(125)I-UdR-CS-NP had a cleartargeting on the above two hepatoma cells, the amount of which entering to thehepatoma cells was10times of normal liver cells, presenting time effect and doseeffects. The intracellular drug radioactivity intensity would reach saturation30min afterdosing or when the dose was greater than185kBq/mL.
9. The CS-NP (FITC-CS-NP) labeled by isothiocyanate fluorescein wassuccessfully prepared, with an average particle size of158.02±12.30nm, and zetapotential of+28.8±3.1. The observation of its appearance by electron microscopy wasregular sphere with its surface structure being loose.
10. In the cellular uptake experiment, FITC-CS-NP can be largely absorbed by theabove two hepatoma cells and unevenly distributed within the cytoplasm; whileFITC-CS-NP entering to the two normal liver cells was significantly reduced, more ofthem were adsorbed on the cell membrane, and dissociated after30min.
11. ROI scanning curve demonstrated that the FITC-CS-NP, after contacting withhepatoma cell HepG2, completed processes of cell membrane adsorption,transmembrane internalization and entering cytoplasm. The peak time of cell membraneadsorbing FITC-CS-NP was15min, and that of intracytoplasmic FITC-CS-NPfluorescence was30min. FITC-CS-NP can also be largely absorbed on the surface ofnormal liver cells HL-7702within15min, however, the amount entering the cells wassmaller with clear slowing down of the process.
12. Liver VX2tumor model was established successfully in rabbit, all of whichwere left lobe of liver tumor lesions with an average diameter of9.2±1.3mm.
13. Micro-catheter using Seldinger technique made super-selection to the lefthepatic artery through the arteria cruralis. After infusion of~(125)I-UdR-CS-DLN, SPECTday parting image showed that the~(125)I-UdR-CS-DLN could be massively accumulatedin the liver and tumor by virtue of the precise positioning of Seldinger micro-catheterand liver first-pass effect.~(125)I-UdR-CS-DLN in the liver tissue could spread over thewhole body to maintain at an average level after2h, while the tumor body stillpresented clear~(125)I-UdR-CS-DLNconcentration, and the T/NT ratio in the ROI interest area reached4.49±1.22, which indicated that the~(125)I-UdR in-CS-DLN had clearpassive targeting on the in-situ liver tumor of the rabbit living body with sustainedrelease characteristics.
14.48h after hepatic artery infusion of~(125)I-UdR-CS-DLN, the drug tissuedistribution revealed that the radioactivity intensity in the tumor was over12times morethan that of the normal liver tissue, and about50times more than the muscle tissue,exhibiting longer drug release time, which further proved the passive targeting andsustained release characteristics of~(125)I-UdR-CS-DLN in rabbit living body on in-situliver tumor. However, the gastrointestinal mucosa drug was slightly higher or lowerthan~(125)I-UdR.
15. As indicated by the laser scanning confocal microscopy results, the entering ofFITC-CS-NP to tumor cells was mainly through the temperature-dependent activeuptake mechanisms requiring cellular energy consumption.
16. Uptaking of~(125)I-UdR-CS-DLN by hepatoma cell involved a variety of ways,mainly by the giant endocytosis pathway for43%, and then the clathrin-classicalpathway for34%. The caveolae-pathway accounted for only12%. The three pinocytosismechanisms were independent with no synergy, but with a certain amount ofcompensation.
17. Laser scanning confocal microscopy verified the function of three endocytosismeans for FITC-CS-NP entering to the hepatoma cells by morphological andquantitative means. Moreover, the locations of endocytic vesicles distributed in theinner cell membrane and cytoplasm can also be observed.
18. The observation by the multicolor fluorescence staining techniques in dayparting showed that the endocytic FITC-CS-NP by clathrin-classical pathway had fastertransmembrane speed to move towards the nucleus after endocytosis and integrate withthe lysosomes. The secondary lysosomes ultimately released the FITC-CS-NP or debrisaround the nuclear; endocytic FITC-CS-NP by giant endocytosis pathway had slowendocytosis speed, and can be scattered in the cytoplasm after transmembrane for a longtime, which was conducive to the delayed release of drugs for the purpose of sustainedintracellular release.
Conclusion
1. This paper firstly loads~(125)I-UdR into nano-drug carrier to be the targeting sustained release preparation. The chitosan nano-uniform particle prepared by optimizedtechnological condition is with uniformed size, regular morphology, as well as goodbiocompatibility; the release curve fits the Higuchi equation, with the characteristics oflong-acting preparation and distinct sustained release function.
2. The paper creatively applies the administration technology of hepatic arteryintervention into the improvement of the passive targeting of nanoparticles. In vitro andin vivo experiments demonstrated that~(125)I-UdR-CS-DLN had obvious hepatomapassive targeting. After the administration by hepatic artery intervention,~(125)I-UdR-CS-DLN can be largely accumulated in liver tumor, and the T/NT can be upto12within48h.
3.~(125)I-UdR-CS-DLN enters to the tumor cells mainly through large pinocytosisand clathrin-classical pathway, and the giant pinocytosis body can reside in thecytoplasm for a long time. It was first observed in the study that the FITC-CS-NPentering into the cytoplasm via clathrin-pathway can be centralized and released aroundthe nucleus after its integration and digestion by the lysosomes.
All in all, the development of~(125)I-UdR-CS-DLN here has an obvious targeting ofHEPATOMA cells, whose intratumoral drug accumulation can be improvedsignificantly through the administration by hepatic artery intervention. It can stay in thehepatoma cells for a long time with some of the particles released around the nucleus.Therefore,~(125)I-UdR-CS-DLN is expected to become the new formulation of the internalradiotherapy of tumor subcellular level for the achievement of better therapeutic effect.
1.通过制备高脱乙酰度低分子量壳聚糖（Chitosan，CS），研制得到壳聚糖纳米微粒（Chitosan nanoparticles，CS-NP）作为具有肝癌靶向作用的药物载体，装载肿瘤内放射治疗药物~(125)I-脱氧尿嘧啶核苷酸（~(125)I-UdR）后成为具有肿瘤靶向性和缓释性能的~(125)I-UdR-壳聚糖纳米微粒（5-[~(125)I]Iodo-2’-Deoxyuridine-Chitosan-Nanoparticles，~(125)I-UdR-CS-NP）。确定最佳工艺条件并鉴定其理化性质、生物相容性和药物释放特性，分析~(125)I-UdR-CS-NP在新西兰兔体内的药代动力学、组织分布和内照射细胞生物学效应。
2.分析验证~(125)I-UdR-CS-NP对兔肝癌原位模型的被动靶向性，特别是通过肝动脉介入给药方式所能达到的肝癌靶向能力。
3.观察分析CS-NP进入肿瘤细胞途径及其在亚细胞水平的分布、转运、降解和药物释放方式。
方法
1.采用间歇水解法和辐射降解法制备高脱乙酰度低分子量壳聚糖，并分析其红外光谱。
2.离子交联法制备CS-NP，并通过正交试验优化其制备工艺。采用激光粒度分析仪、透射电子显微镜和zeta电位仪分析鉴定CS-NP的粒径、分散度、形态和表面性状以及zeta电势等物理性状和表征。
3.通过MTT法和流式细胞仪检测肝癌细胞和正常肝组织细胞与CS-NP共同孵育后的存活分数和凋亡率，以评价其生物相容性。
4.离子交联法制备~(125)I-UdR-CS-NP，并通过单因素分析法确定其最佳工艺条件。测量其粒径、分散度，并用透射电镜观察其形貌。
5.采用动态透析法测量~(125)I-UdR-CS-NP的体外药物累积释放率，并绘制累积释放曲线，观察比较载药量与释药特性的关系，确认其是否具备长效缓释特性。
6.分析外周静脉注射~(125)I-UdR-CS-NP后实验兔的药代动力学特性和药物组织分布特征。
7.观察分析实验兔注射~(125)I-UdR-CS-NP后的血液生化指标和骨髓图片的变化，评价~(125)I-UdR-CS-NP在生物体内的毒副作用。
8.离子交联法制备异硫氰酸荧光素标记壳聚糖纳米微粒（FITC-CS-NP），并采用激光粒度分析仪、透射电子显微镜和zeta电位仪分析鉴定FITC-CS-NP的粒径、分散度、形态和表面性状以及zeta电势等物理性状和表征。
9.体外细胞摄取实验分析肝癌细胞和正常肝组织细胞对~(125)I-UdR-CS-NP的摄取量及其时间效应和剂量效应，以验证~(125)I-UdR-CS-NP的肝癌靶向性。
10.采用激光共聚焦显微镜观察FITC-CS-NP在肝癌细胞和正常肝细胞中不同时段的分布量和位置，验证CS-NP作为药物载体的肝癌靶向性。
11.采用ROI区域时间序列扫描技术连续定量分析FITC-CS-NP进入肝癌细胞和正常肝组织细胞的过程。
12.采用MTT法、流式细胞仪观察肝癌细胞和正常肝细胞经~(125)I-UdR-CS-NP作用后的存活分数和增殖指数。
13.用中性单细胞凝胶电泳技术检测肝癌细胞和正常肝细胞经~(125)I-UdR-CS-NP作用后2h的DNA链断裂程度和作用后24h的DNA损伤修复能力，以评估~(125)I-UdR-CS-NP对肿瘤细胞的杀伤效应。
14.超声引导下采用经皮穿刺术在实验兔肝左叶种植VX2瘤块，制备兔肝癌原位模型，并做病理学鉴定。
15.兔肝癌原位模型在CT引导下采用Seldinger微导管肝动脉插管术灌注~(125)I-UdR-CS-NP，SPECT分时段观察~(125)I-UdR-CS-NP在兔体内的分布及其对原位肝癌的靶向性。测量并分析~(125)I-UdR-CS-NP在肿瘤和其他组织中的分布，以验证其肿瘤靶向性。
16.将用药后48h的肿瘤组织做病理切片后进行Tunel染色，观察细胞凋亡率改变，以评价~(125)I-UdR-CS-NP的抑瘤效果。
17.激光共聚焦显微镜观察37℃和4℃条件下FITC-CS-NP及其原料FITC-CS长链分子进入肿瘤细胞的行为特征，以观察FITC-CS-NP进入细胞的耗能情况。
18.采用CPZ、Fil、Amil分别抑制以clathrin-经典途径、caveolae-途径和巨胞饮三条纳米微粒进入细胞的主要途径后，定量分析~(125)I-UdR-CS-NP进入肝癌细胞HepG2的量，以分析其内化途径类型。并用激光共聚焦显微镜观察验证FITC-CS-NP通过不同途径进入肝癌细胞的跨膜过程及其在细胞内的分布特征。
19.采用多色荧光染色技术观察FITC-CS-NP在亚细胞水平的动态分布、转运和降解的行为特征。
结果
1.间歇水解法和辐射降解法制备的壳聚糖脱乙酰度为93.062±2.384%，分子量3kDa，外观为深黄色粉末，在PH=7.0的环境中可完全溶解，呈深黄色澄清透亮的液体。红外图谱表明经碱处理后壳聚糖的酰胺键被水解，更多游离氨基暴露，脱乙酰度得到明显提高。
2.成功制备CS-NP，正交试验结果表明，对CS纳米微粒粒径的影响力从大到小依次为CS分子量>TPP浓度>搅拌速度>CS浓度。优化后的制备工艺条件为CS浓度1g/L，搅拌速度600rpm，TPP浓度2g/L，CS分子量3kDa，所制得的CS-NP平均粒径为70.39±5.12nm，PDI0.16±0.012，zeta电位为+32.5±3.3。透射电镜观察其外观为规整的球形，表面平整，大小均匀，分散度较好。
3. MTT法和流式细胞仪结果都表明CS-NP对人正常肝细胞株HL-7702、QSG-7701无细胞毒性；当CS-NP浓度≥200μg/mL时对于人肝癌细胞系HepG2、SMMC-7721有一定的细胞毒性。
4.成功制备~(125)I-UdR-CS-NP。优化后的制备条件为投药量1.2～2.4mg/mL，PH5.5，所制得的~(125)I-UdR-CS-NP平均粒径175.8nm。与空白CS-NP相比，其性状会略有改变，表现为粒径增大且均一性下降，结构较疏松，表面形貌略欠规整。
5.~(125)I-UdR-CS-NP在PH5.3到7.4环境下的释放曲线符合Higuchi方程，为长效制剂特征，具有明显的缓释作用。
6.~(125)I-UdR-DLN经外周静脉用药后药代动力学符合二室模型特征，第一时相和第二时相半衰期分别为1.1±0.69h和1.5±0.16h，曲线下面积(AUC0-∞)为11.2±1.4mg/L·h。与~(125)I-UdR相比，~(125)I-UdR-CS-NP的生物分布倾向于肝脾，通过肾脏的排泄呈现明显的延迟现象，骨髓及胃肠道黏膜的药物分布量较少。
7.静脉注射~(125)I-UdR-CS-DLN剂量高至7.4MBq（6mg），未发现血液生化指标明显异常，也无明显骨髓抑制现象，而~(125)I-UdR对照组2d时骨髓象表明~(125)I-UdR对增殖阶段的造血细胞抑制作用较强。表明~(125)I-UdR-CS-DLN明显减轻了对骨髓造血细胞的抑制作用。
8.体外细胞摄取实验表明~(125)I-UdR-CS-NP对上述2株肝癌细胞具有明显的靶向性，进入肝癌细胞的量是正常肝细胞的10倍，并具有时间效应和剂量效应。加药后30min或剂量大于185kBq/mL时细胞内药物的放射性强度都会达到饱和。
9.成功制备异硫氰酸荧光素标记的CS-NP（FITC-CS-NP），平均粒径158.02±12.30nm，zeta电势为+28.8±3.1。透射电镜观察外形呈较为规整的球形，表面结构较疏松。
10.在细胞摄取实验中，FITC-CS-NP可被上述2株肝癌细胞可大量摄取，且在细胞浆内呈不均匀分布；而FITC-CS-NP进入2株正常肝细胞明显减少，更多的是吸附在细胞膜上，30min后开始解离。
11. ROI时序扫描曲线表明FITC-CS-NP与肝癌细胞HepG2接触后完成了细胞膜吸附-跨膜内化-进入细胞浆的过程，细胞膜吸附FITC-CS-NP量的峰值时间为15min，胞浆内FITC-CS-NP荧光峰值时间为30min。FITC-CS-NP同样可以在15min内大量吸附在正常肝细胞HL-7702表面，但随后进入细胞的量很少，且过程明显减慢。
12.当浓度≥37kBq/mL时，~(125)I-UdR-CS-NP作用后肝癌细胞的存活率明显低于正常肝细胞。流式细胞仪分析细胞周期结果表明，~(125)I-UdR-CS-DLN和~(125)I-UdR对增殖期的细胞都有杀伤作用，并造成细胞增殖周期阻滞在G1期。而~(125)I-UdR-CS-DLN更对DNA合成后进行有丝分裂的G2/M期HepG2细胞也有明显的杀伤作用。
13.不论是肝癌细胞HepG2还是正常肝细胞HL-7702，经~(125)I-UdR-CS-DLN作用后2h的DNA损伤程度都明显高于同等剂量~(125)I-UdR组。药物作用24h后各组的彗星实验结果表明，~(125)I-UdR-CS-DLN组的DNA损伤修复能力远低于~(125)I-UdR组。另外，~(125)I-UdR-CS-DLN组中，肝癌细胞株HepG2的DNA损伤后修复能力明显低于HL-7702。
14.成功制备VX2肿瘤兔肝原位模型，均为肝左叶单发肿瘤病灶，平均直径9.2±1.3mm。
15. Seldinger微导管经股动脉成功超选择至肝左动脉，灌注~(125)I-UdR-CS-NP后，SPECT分时段显像表明~(125)I-UdR-CS-DLN凭借Seldinger微导管的精确定位和肝脏首过效应，可大量聚积在肝脏和瘤体内，2h时肝脏组织中的~(125)I-UdR-CS-DLN已排泄至全身平均水平，而瘤体内依然有明显的~(125)I-UdR-CS-DLN浓聚，到24h时ROI感兴趣区T/NT比值达4.49±1.22。表明~(125)I-UdR-CS-DLN在兔体内实验中对肝原位肿瘤有明显的被动靶向性，且具备缓释特征。用药后48h时的药物组织分布表明瘤内放射性强度超过正常肝组织的12倍，是肌肉组织的50倍左右，且表现出更长的药物缓释时间，进一步证明了~(125)I-UdR-CS-DLN在兔活体中对肝原位肿瘤的被动靶向性和缓释特征。
16.介入给药后48h时肿瘤组织Tunel染色结果表明，~(125)I-UdR-CS-DLN可致肿瘤细胞发生明显的凋亡，其肿瘤杀伤能力明显高于同等剂量的~(125)I-UdR。
17.激光共聚焦显微镜观察结果表明，FITC-CS-NP进入肿瘤细胞主要通过一类温度依赖的，需要消耗细胞能量的主动摄取机制。
18.肝癌细胞摄取~(125)I-UdR-CS-DLN是多种途径共同参与的，以巨胞饮途径为主（43%），clathrin-经典途径其次（34%），caveolae-途径仅占12%。这三种胞饮机制相互独立，不存在协同作用，但存在一定的代偿作用。激光共聚焦显微镜进一步通过形态和定量手段验证了FITC-CS-NP通过三条胞吞途径进入肝癌细胞的跨膜过程，并观察不同胞吞内体在亚细胞水平的分布规律。
19.采用多色荧光染色技术分时段观察结果表明经clathrin-经典途径内吞的FITC-CS-NP跨膜较快，入胞后向细胞核移动并与溶酶体融合，最终次级溶酶体在核周释放出FITC-CS-NP；经巨胞饮途径内吞的FITC-CS-NP入胞较慢，跨膜后散在分布于细胞质中，可驻留较长时间，有利于药物的延迟释放，达到细胞内缓释的目的。
结论
1.首次将~(125)I-UdR装载入纳米药物载体，成为靶向缓释制剂。采用优化工艺条件制备的壳聚糖纳米微粒粒径均匀，形貌规整，生物相容性好；释放曲线符合Higuchi方程，为长效制剂特征，具有明显的缓释作用。
2.首次将肝动脉介入给药技术用于提高壳聚糖纳米微粒的被动靶向性。体内外实验证明~(125)I-UdR-CS-DLN具有明显的肝癌被动靶向性；经肝动脉介入给药后~(125)I-UdR-CS-DLN可大量聚积于肝肿瘤内，48h时瘤内放射性强度是正常肝组织的12倍。同时，~(125)I-UdR-CS-DLN在体内外实验中都显示出比~(125)I-UdR更强的肿瘤杀伤能力。
3.证明~(125)I-UdR-CS-DLN主要通过巨胞饮和clathrin-经典途径进入肿瘤细胞，巨胞饮体可在细胞质内实现较长时间驻留；观察并较早证实了通过clathrin-途径进入细胞质的FITC-CS-NP在与溶酶体融合后会导致溶酶体破裂而逃逸。并首次观察到这种逃逸现象集中发生在细胞核周围，这一发现有利于提高药物进入细胞核内的量。
总之，本次研制的~(125)I-UdR-CS-DLN具有明显的肝癌细胞靶向性和更强的肿瘤杀伤能力，通过肝动脉介入给药可大幅提高其瘤内药物积聚量，在肝癌细胞内可驻留较长时间，并有部分微粒可在核周集中释放。故~(125)I-UdR-CS-DLN有望成为肿瘤亚细胞水平内放射治疗的新剂型，达到更好的治疗效果。
Objective
1. To prepare high deacelation degree and low molecular weight Chitosan, andfurther development chitosan nanoparticles and~(125)I-UdR-Chitosan nanoparticles. Theoptimum process conditions were established, and the physical and chemical properties,biocompatibility and drug release properties were identified. Further more, thepharmacokinetics and tissue distribution of~(125)I-UdR-CS-NP in New Zealand rabbitwere analyzed.
2. To validate the passive targeting property of~(125)I-UdR-CS-NP to in situ livercancer models, especially the improvement of liver targeting ability through hepaticartery interventional delivering drug.
3. To observe the internalization pathways and distribution, transshipment anddegradation of CS-NP in subcellular level.
Mehtod
1. High deacelation degree and low molecular weight Chitosan was preparedusing intermittent hydrolysis and radiation degradation method, and its infraredspectrum was analyzed.
2. Using ion cross-linking method for preparation of CS-NP, and optimizing thepreparation techniques through the orthogonal test.
3. The physical properties and characterization of CS-NP were analyzed usingtransmission electron microscope, laser particle size analyzer and zeta potentiometer.
4. The biocompatibility of CS-NP was evaluated through the MTT method andflow cytometry instrument.
5. Using the ion cross-linking method for the preparation of~(125)I-UdR-CS-NP, andthrough the single factor analysis method to determine the optimum process conditions.
6. The in vitro drug release properties of~(125)I-UdR-CS-NP were analyzed by dynamic dialysis method.
7. The pharmacokinetic characteristics and drug organization distribution of~(125)I-UdR-CS-NP injected via peripheral vein of the rabbits were analyzed.
8. The side effects of~(125)I-UdR-CS-NP were analyzed using in vivo experiments.
9. Rabbit in situ liver cancer models were prepared using ultrasound-guidedpercutaneous puncture technology.
10. The in vitro cell uptake experiments were performed to verify the liver cancertargeting ability, including the comparisons on the uptake volume of~(125)I-UdR-CS-NPand time effection, dose effection between the liver cancer cells and normal liver cells.
11. The fluorescein isothiocyanate marked chitosan nanoparticles were preparedusing ion cross-linking method.
12. As drug carrier, the liver cancer targeting property of CS-NP were validated,through continuous observing the dynamic distribution of FITC-CS–NP in liver cancercells and normal liver cells with laser confocal microscope.
13. The process of liver cancer cells and normal liver cells internalizedFITC-CS–NP were dynamically record via the time sequence scanning technology.
14.~(125)I-UdR-CS-NP were perfused in hepatic artery of rabbits with liver cancer insitu model using Seldinger micro catheter under the CT guided, and the distribution andliver targeting property were observed in deferent period by SPECT.
15. The organization distribution of~(125)I-UdR-CS-NP been perfused in rabbithepatic artery were analyzed to verify its tumor targeting property.
16. When the FITC-CS–NP were uptake under different temperature, the cells'behavior characteristics were observed by Laser confocal microscope.
17. The transmembrane process of FITC-CS-NP entering into hepatoma cellsthrough different ways and its distribution characteristics within the cell was observedwith the help of laser scanning confocal microscopy.
18. The dynamic distribution, transportation and degradation process ofFITC-CS-NP in subcellular level was observed by adopting multicolor stainingtechnology.
Result
1. The deacetylation degree of chitosan prepared by intermittent hydrolysis andradiation degradation was93.062±2.384%, with the molecular weight of3kDa. It was dark yellow powder, which can be completely dissolved in water of PH7.0, to presentdark yellow clarified translucent liquid. IR spectra showed that the processed chitosanamide bond was hydrolyzed with more exposure of free amino groups, while the degreeof deacetylation was markedly improved.
2. The CS-NP was successfully prepared. The orthogonal experiment showed thatthe influences on CS nano-particle size were arranged in a decreasing manner, the CSmolecular weight, TPP concentration, stirring speed and CS concentration. Optimizedpreparation technological condition had CS concentration of1g/L, stirring speed of600rpm, TPP concentration of2g/L, CS molecular weight of3kDa, average preparedparticle diameter of CS-NP of70.39±5.12nm, PDI of0.16±0.012, and zeta potentialof+32.5±3.3. Its appearance through TEM observation presented a regular sphere withsmooth surface, uniformed size, and good dispersity.
3. MTT method and flow cytometry results showed that CS-NP had no celltoxicity on human normal liver cell line HL-7702and QSG-7701; when the CS-NPconcentration was equal or greater than200μg/mL, there was certain toxicity for thehuman hepatoma cell line HepG2and SMMC-7721.
4.~(125)I-UdR-CS-NP was successfully prepared in the study. The optimizedpreparation condition required reagent quantity of1.2-2.4mg/mL, PH5.5, and theaverage particle diameter of the prepared~(125)I-UdR-CS-NP was175.8nm. Compared tothe blank CS-NP, its traits was slightly changed with the increase of the particle size anddecrease of uniformity, loose of structure, and irregular of the surface morphology.
5. Dissolution curve of the~(125)I-UdR-CS-NP in PH5.3to7.4fit Higuchi equation,with the characteristics of long-acting preparation and distinct sustained releasefunction.
6. After peripheral intravenous administration, the pharmacokinetics of~(125)I-UdR-DLN was in line with the characteristics of the two-compartment model. Thehalf-life of the first phase and the second phase were1.1±0.69h and1.5±0.16hrespectively, and the area under the curve (AUC0-∞) was11.2±1.4mg/L h. Comparedto~(125)I-UdR, the biological distribution of~(125)I-UdR-CS-NP tended to be on the liver andspleen, showing obvious delay by renal excretion, and its distribution of marrow andgastrointestinal mucosa was less.
7. Intravenous injection of~(125)I-UdR-CS-DLN was as high as7.4MBq (6mg) indosage, with no significant abnormality of blood biochemical indicators or distinct bone marrow suppression found. While the bone marrow of the control group of the~(125)I-UdRshowed that the~(125)I-UdR had an inbibitional effect on the hematopoietic cells ofproliferation phase in2d, which indicated that~(125)I-UdR-CS-DLN could significantlyreduce the inhibitory effect on bone marrow hematopoietic cells.
8. The in-vitro cells uptake experiment revealed that~(125)I-UdR-CS-NP had a cleartargeting on the above two hepatoma cells, the amount of which entering to thehepatoma cells was10times of normal liver cells, presenting time effect and doseeffects. The intracellular drug radioactivity intensity would reach saturation30min afterdosing or when the dose was greater than185kBq/mL.
9. The CS-NP (FITC-CS-NP) labeled by isothiocyanate fluorescein wassuccessfully prepared, with an average particle size of158.02±12.30nm, and zetapotential of+28.8±3.1. The observation of its appearance by electron microscopy wasregular sphere with its surface structure being loose.
10. In the cellular uptake experiment, FITC-CS-NP can be largely absorbed by theabove two hepatoma cells and unevenly distributed within the cytoplasm; whileFITC-CS-NP entering to the two normal liver cells was significantly reduced, more ofthem were adsorbed on the cell membrane, and dissociated after30min.
11. ROI scanning curve demonstrated that the FITC-CS-NP, after contacting withhepatoma cell HepG2, completed processes of cell membrane adsorption,transmembrane internalization and entering cytoplasm. The peak time of cell membraneadsorbing FITC-CS-NP was15min, and that of intracytoplasmic FITC-CS-NPfluorescence was30min. FITC-CS-NP can also be largely absorbed on the surface ofnormal liver cells HL-7702within15min, however, the amount entering the cells wassmaller with clear slowing down of the process.
12. Liver VX2tumor model was established successfully in rabbit, all of whichwere left lobe of liver tumor lesions with an average diameter of9.2±1.3mm.
13. Micro-catheter using Seldinger technique made super-selection to the lefthepatic artery through the arteria cruralis. After infusion of~(125)I-UdR-CS-DLN, SPECTday parting image showed that the~(125)I-UdR-CS-DLN could be massively accumulatedin the liver and tumor by virtue of the precise positioning of Seldinger micro-catheterand liver first-pass effect.~(125)I-UdR-CS-DLN in the liver tissue could spread over thewhole body to maintain at an average level after2h, while the tumor body stillpresented clear~(125)I-UdR-CS-DLNconcentration, and the T/NT ratio in the ROI interest area reached4.49±1.22, which indicated that the~(125)I-UdR in-CS-DLN had clearpassive targeting on the in-situ liver tumor of the rabbit living body with sustainedrelease characteristics.
14.48h after hepatic artery infusion of~(125)I-UdR-CS-DLN, the drug tissuedistribution revealed that the radioactivity intensity in the tumor was over12times morethan that of the normal liver tissue, and about50times more than the muscle tissue,exhibiting longer drug release time, which further proved the passive targeting andsustained release characteristics of~(125)I-UdR-CS-DLN in rabbit living body on in-situliver tumor. However, the gastrointestinal mucosa drug was slightly higher or lowerthan~(125)I-UdR.
15. As indicated by the laser scanning confocal microscopy results, the entering ofFITC-CS-NP to tumor cells was mainly through the temperature-dependent activeuptake mechanisms requiring cellular energy consumption.
16. Uptaking of~(125)I-UdR-CS-DLN by hepatoma cell involved a variety of ways,mainly by the giant endocytosis pathway for43%, and then the clathrin-classicalpathway for34%. The caveolae-pathway accounted for only12%. The three pinocytosismechanisms were independent with no synergy, but with a certain amount ofcompensation.
17. Laser scanning confocal microscopy verified the function of three endocytosismeans for FITC-CS-NP entering to the hepatoma cells by morphological andquantitative means. Moreover, the locations of endocytic vesicles distributed in theinner cell membrane and cytoplasm can also be observed.
18. The observation by the multicolor fluorescence staining techniques in dayparting showed that the endocytic FITC-CS-NP by clathrin-classical pathway had fastertransmembrane speed to move towards the nucleus after endocytosis and integrate withthe lysosomes. The secondary lysosomes ultimately released the FITC-CS-NP or debrisaround the nuclear; endocytic FITC-CS-NP by giant endocytosis pathway had slowendocytosis speed, and can be scattered in the cytoplasm after transmembrane for a longtime, which was conducive to the delayed release of drugs for the purpose of sustainedintracellular release.
Conclusion
1. This paper firstly loads~(125)I-UdR into nano-drug carrier to be the targeting sustained release preparation. The chitosan nano-uniform particle prepared by optimizedtechnological condition is with uniformed size, regular morphology, as well as goodbiocompatibility; the release curve fits the Higuchi equation, with the characteristics oflong-acting preparation and distinct sustained release function.
2. The paper creatively applies the administration technology of hepatic arteryintervention into the improvement of the passive targeting of nanoparticles. In vitro andin vivo experiments demonstrated that~(125)I-UdR-CS-DLN had obvious hepatomapassive targeting. After the administration by hepatic artery intervention,~(125)I-UdR-CS-DLN can be largely accumulated in liver tumor, and the T/NT can be upto12within48h.
3.~(125)I-UdR-CS-DLN enters to the tumor cells mainly through large pinocytosisand clathrin-classical pathway, and the giant pinocytosis body can reside in thecytoplasm for a long time. It was first observed in the study that the FITC-CS-NPentering into the cytoplasm via clathrin-pathway can be centralized and released aroundthe nucleus after its integration and digestion by the lysosomes.
All in all, the development of~(125)I-UdR-CS-DLN here has an obvious targeting ofHEPATOMA cells, whose intratumoral drug accumulation can be improvedsignificantly through the administration by hepatic artery intervention. It can stay in thehepatoma cells for a long time with some of the particles released around the nucleus.Therefore,~(125)I-UdR-CS-DLN is expected to become the new formulation of the internalradiotherapy of tumor subcellular level for the achievement of better therapeutic effect.
引文
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