本文選題：CD147單抗 + α-常春藤皂苷　； 參考：《蘇州大學(xué)》2014年碩士論文

【摘要】：目的：以殼聚糖（Chitosan，CS）為載體，以水難溶性藥物-常春藤皂苷（-Hederin，-Hed）為模型藥物，制備-常春藤皂苷殼聚糖納米粒（-Hed-CS-NP， HCS），再以肝腫瘤細(xì)胞表面高度表達(dá)的CD147單抗為靶頭，將其偶聯(lián)修飾于納米粒表面制備CD147介導(dǎo)的-常春藤皂苷殼聚糖納米粒（-Hed-CS-CD147-NP， αHCD），從而通過納米粒的緩釋效果和單抗介導(dǎo)的主動(dòng)靶向性達(dá)到減毒增效的作用。 方法：（1）以平均粒徑、多分散指數(shù)（Polydisperse Index，P.I.）和包封率（EntrapmentEfficiency，EE）為綜合指標(biāo)，采用乳化-溶劑擴(kuò)散法制備HCS，并采用單因素試驗(yàn)和正交試驗(yàn)設(shè)計(jì)，考察納米粒處方和制備工藝。紅外（FT-IR）、X射線衍射（XRD）、差示掃描量熱分析（DSC）等對(duì)αHCS納米粒進(jìn)行表征，同時(shí)系統(tǒng)考察納米粒的體外釋放行為及其相關(guān)藥劑學(xué)性質(zhì)。（2）采用EDC/NHS酰胺化反應(yīng)制備αHCD，并對(duì)納米粒進(jìn)行粒徑測(cè)定、FT-IR和XRD表征；采用BCA試劑盒和ELISA法分別建立CD147單抗的含量和活性測(cè)定方法；依據(jù)HepG2細(xì)胞對(duì)不同單抗修飾量納米粒的攝取結(jié)果，選擇合適的單抗修飾量。（3）以未載藥的CD147單抗介導(dǎo)的殼聚糖納米粒（VoidCS-CD147-NP，VCD）為對(duì)照，MTT法考察CD147單抗對(duì)肝腫瘤細(xì)胞活性的抑制作用、-Hed及其納米粒對(duì)HepG2、SMMC-7721、HSC的細(xì)胞毒作用；采用流式細(xì)胞術(shù)檢測(cè)原料藥及其納米粒對(duì)HepG2細(xì)胞的凋亡效果和對(duì)其細(xì)胞周期的影響。(4)流式細(xì)胞術(shù)方式考察HepG2在2h和24h內(nèi)αHCS和αHCD的攝取率，HSC細(xì)胞作為對(duì)照細(xì)胞考察兩種納米粒的攝取差異性，并通過加入過飽和CD147進(jìn)行納米粒的競(jìng)爭性抑制試驗(yàn)；多種細(xì)胞內(nèi)吞抑制劑對(duì)兩種納米粒的攝取機(jī)制進(jìn)行探討，并考察兩種納米粒在體內(nèi)的攝取分布過程。（5）分別以生理鹽水組為陰性對(duì)照，環(huán)磷酰胺為陽性對(duì)照，HepG2細(xì)胞裸鼠腫瘤模型腹腔注射給予-Hed及其納米粒后，考察其體內(nèi)藥效學(xué)；采用小動(dòng)物活體成像技術(shù)，動(dòng)態(tài)考察納米粒在荷HepG2細(xì)胞裸鼠體內(nèi)的組織分布及其腫瘤靶向性。 結(jié)果：（1）乳化溶劑擴(kuò)散法制備的αHCS納米粒粒徑分布均勻，平均粒徑（92.7±2.77）nm，P.I.（0.134±0.036），包封率（75.63±4.06）%；透射電鏡觀察納米粒外觀形態(tài)圓整；FT-IR、XRD、DSC表征結(jié)果表明藥物已分散于殼聚糖納米粒中；體外釋放顯示殼聚糖納米粒具有一定的緩釋特性。（2）CD147能通過酰胺反應(yīng)偶聯(lián)修飾到αHCS表面；與αHCS的粒徑相比，αHCD的粒徑變化較��；偶聯(lián)所得αHCS中CD147單抗飽和修飾量為（6.55±0.234）μg/mgNP，結(jié)合不同修飾量對(duì)納米粒攝取結(jié)果影響、單抗的活性和過多修飾量可能產(chǎn)生的空間位阻效應(yīng)等因素，本實(shí)驗(yàn)最終確定修飾量為1.5μg/mgNP，且單抗活性保留率為（57.07±0.60）%。（3）MTT實(shí)驗(yàn)結(jié)果表明未載藥單抗修飾的殼聚糖納米粒VCD及本實(shí)驗(yàn)所用CD147單抗修飾量均對(duì)肝腫瘤細(xì)胞無細(xì)胞毒作用，原料藥及其納米粒對(duì)HepG2、 SMMC-7721的IC50值為αHCDHCS-Hed，以HSC細(xì)胞為對(duì)照，提示與單抗介導(dǎo)的主動(dòng)靶向性有關(guān)；通過細(xì)胞凋亡實(shí)驗(yàn)結(jié)果與HepG2細(xì)胞MTT實(shí)驗(yàn)相一致；在細(xì)胞周期實(shí)驗(yàn)中發(fā)現(xiàn)細(xì)胞在S期的百分比有所增加。（4）HepG2細(xì)胞對(duì)納米粒攝取具有時(shí)間依賴性，且αHCD的攝取明顯高于HCS，另外HSC對(duì)兩種納米粒攝取無差異，結(jié)合過飽和競(jìng)爭抑制實(shí)驗(yàn)中αHCD攝取率明顯下降，說明αHCD納米粒對(duì)肝腫瘤細(xì)胞具有更好的親和性；從攝取抑制劑實(shí)驗(yàn)結(jié)果可發(fā)現(xiàn)載藥納米粒的內(nèi)吞攝取過程屬于能量依賴型，需通過形成有被小窩內(nèi)陷和網(wǎng)格蛋白介導(dǎo)的內(nèi)吞進(jìn)入細(xì)胞，且與細(xì)胞膜結(jié)構(gòu)穩(wěn)定性和流動(dòng)性息息相關(guān)。倒置熒光顯微鏡結(jié)果顯示納米粒1h時(shí)大多聚集在細(xì)胞膜表面，3h時(shí)細(xì)胞攝入納米粒明顯增多。（5）體內(nèi)藥效學(xué)實(shí)驗(yàn)中， HCS（79.02%）和αHCD（88.43%）納米粒的抑瘤率遠(yuǎn)高于原料藥中劑量組（53.83%）；納米粒組抑瘤率均在70%以上，且存在劑量依賴性。近紅外熒光成像實(shí)驗(yàn)結(jié)果發(fā)現(xiàn)，尾靜脈給藥1h后，兩種納米粒在腫瘤部位和肝臟可見較為集中的熒光分布，在6h時(shí)，腫瘤部位熒光強(qiáng)度有所減弱，12h后納米粒熒光開始減弱。 結(jié)論：乳化溶劑擴(kuò)散法制備的αHCS大小分布均勻、外觀形態(tài)圓整、包封率高，有緩釋特性，體外體內(nèi)藥效學(xué)實(shí)驗(yàn)表明空白納米粒與細(xì)胞具有生物相容性，， αHCD對(duì)肝腫瘤細(xì)胞的毒副作用的增強(qiáng)和肝腫瘤細(xì)胞對(duì)該納米粒攝取的增多與CD147單抗介導(dǎo)的主動(dòng)靶向性有關(guān)。綜上所述， αHCD具有應(yīng)用潛能并有望成為一種新型的難溶性藥物的載體。
[Abstract]:Objective: using Chitosan (CS) as the carrier and using the water-insoluble drug -Hederin (-Hed) as the model drug, the -Hed-CS-NP (HCS) of ivy saponins (-Hed-CS-NP, HCS) was prepared, and then the CD147 mAb expressed in the surface of the liver tumor cells was used as the target, and the coupling modified to the nanoparticles surface to prepare the CD147 mediated ivy. Saponins chitosan nanoparticles (-Hed-CS-CD147-NP, alpha HCD) can achieve the effect of attenuation and synergism through the sustained release effect of nanoparticles and the active targeting mediated by monoclonal antibody.
Methods: (1) using the average particle size, Polydisperse Index, P.I. and EntrapmentEfficiency (EE) as a comprehensive index, HCS was prepared by emulsion solvent diffusion method, and the formulation and preparation process of nanoparticles were investigated by single factor test and orthogonal design. Infrared (FT-IR), X ray diffraction (XRD), differential scanning calorimetry analysis (DSC) to characterize alpha HCS nanoparticles and investigate the release behavior of nanoparticles in vitro and their related pharmaceutical properties. (2) the preparation of alpha HCD by EDC/NHS amidation, particle size determination, FT-IR and XRD characterization, and BCA reagent box and ELISA method, respectively, to determine the content and activity of CD147 McAbs; According to the results of HepG2 cells' uptake of different mAb modifier nanoparticles, the appropriate mAb modifier was selected. (3) the CD147 mAb mediated chitosan nanoparticles (VoidCS-CD147-NP, VCD) was used as the control, and the MTT method was used to investigate the inhibitory effect of CD147 mAb on the activity of liver tumor cells, and the cytotoxicity of -Hed and its nanoparticles on HepG2, SMMC-7721, HSC. The effect of flow cytometry was used to detect the effect of API and its nanoparticles on the apoptosis of HepG2 cells and its effect on its cell cycle. (4) flow cytometry was used to investigate the uptake of HepG2 in 2H and 24h, the uptake of alpha and alpha in 2H and 24h. HSC cells were used as control cells to investigate the difference in the uptake of the nanoparticles, and by adding a supersaturated CD147. The competitive inhibition test of nanoparticles, the uptake mechanism of two kinds of nanoparticles by multiple cell endocytosis inhibitors and the uptake of the two nanoparticles in the body were investigated. (5) the negative control of the saline group, cyclophosphamide as the positive control, and the intraperitoneal injection of the HepG2 nude mouse tumor model to -Hed and its nanoscale The pharmacodynamics of the nanoparticles in vivo were investigated and the tissue distribution and tumor targeting of nanoparticles in nude mice bearing HepG2 cells were dynamically investigated by using small animal living imaging techniques.
Results: (1) the particle size distribution of alpha HCS nanoparticles was evenly distributed, the average particle size (92.7 + 2.77) nm, P.I. (0.134 + 0.036), and encapsulation rate (75.63 + 4.06)%; transmission electron microscopy was used to observe the appearance of nanoparticles. The characterization results of FT-IR, XRD, and DSC showed that the drug was dispersed in chitosan nanoparticles, and the release of chitosan in vitro (2) CD147 can be modified to the surface of alpha HCS through amide reaction. Compared with the particle size of alpha HCS, the particle size of alpha HCD is smaller, and the saturation modification of CD147 mAb in alpha HCS is (6.55 + 0.234) mu g/mgNP, and the effects of different modifiers on the results of nanofilm uptake, the activity of McAbs and the excess modification In this experiment, the amount of space hindrance was 1.5 g/mgNP, and the retention rate of the monoclonal antibody was (57.07 + 0.60). (3) the results of MTT experiment showed that the chitosan nanoparticles VCD modified by unloaded McAb and the CD147 McAb modification used in this experiment had no cytotoxic effect on liver tumor cells. The IC50 value of rice grain to HepG2, SMMC-7721 is alpha HCDHCS-Hed and HSC cells as the control, which is related to the active targeting mediated by McAb; the results of apoptosis experiments are consistent with the MTT experiment of HepG2 cells, and the percentage of cells in S phase is increased in cell cycle experiments. (4) HepG2 cells have time for nanoparticles uptake. The uptake of alpha HCD was significantly higher than that of HCS, and there was no difference in the uptake of two nanoparticles in HSC. The uptake of alpha HCD decreased significantly in the combination of supersaturated competition inhibition experiments, indicating that the alpha HCD nanoparticles had better affinity to the liver tumor cells. The quantity dependent type should be entered into cells by the formation of endocytic endocytosis mediated by trift and gridin, which is closely related to the stability and fluidity of the cell membrane structure. The inverted fluorescence microscope results showed that most of the nanoparticles 1H aggregated on the surface of the cell membrane, and the cell intake nanoparticles increased significantly at 3H. (5) in the pharmacodynamic experiment, HCS (79.02) The tumor inhibition rate of%) and alpha HCD (88.43%) nanoparticles was much higher than that in the medium dose group (53.83%), and the tumor suppressor rate of the nanoparticles was more than 70%, and there was a dose dependence. The results of the near infrared fluorescence imaging test found that after the tail vein was given 1H, the two nanoparticles were more concentrated in the tumor site and the liver, and the tumor site was at 6h. The fluorescence intensity decreased, and the fluorescence of nanoparticles decreased after 12h.
Conclusion: the size distribution of alpha HCS in the emulsion solvent diffusion method is uniform, the appearance is round, the encapsulation efficiency is high, and it has the sustained release characteristics. In vitro, the pharmacodynamic experiment in vitro shows that the blank nanoparticles have biocompatibility with the cells, the enhancement of the toxic side effects of alpha HCD on the liver tumor cells and the increase of the liver tumor cells to the nanoparticles and CD147 single In conclusion, alpha HCD has potential for application and is expected to become a new carrier of insoluble drugs.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R943;R735.7

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