本文選題：膽固醇基普魯蘭 + 異硫氰酸熒光素��； 參考：《北京協(xié)和醫(yī)學(xué)院》2013年博士論文

【摘要】：作為天然高分子材料之一的多糖具有良好的生物相容性和安全性,疏水改性多糖通過自組裝方式形成具有獨(dú)特的“核殼結(jié)構(gòu)”的納米粒,在體內(nèi)具有長(zhǎng)循環(huán)、主體穩(wěn)定性和被動(dòng)靶向性等特點(diǎn),在藥物傳輸領(lǐng)域有著潛在的應(yīng)用前景。了解納米粒與細(xì)胞的相互作用對(duì)于在細(xì)胞層次上理解生命體的生理過程、藥物的作用機(jī)制、基因治療的機(jī)理等具有重要的科學(xué)意義和實(shí)用價(jià)值,同時(shí)也可為構(gòu)建更加安全有效的納米藥物載體提供依據(jù)。 本研究采用異硫氰酸熒光素(FITC, fluorescein isothiocyanate)標(biāo)記膽固醇基普魯蘭(CHSP, cholesterol-modified pullulan)并制備納米粒,討論了納米粒濃度、孵育時(shí)間以及溫度對(duì)細(xì)胞攝取的影響,研究了CHSP納米粒的體外HepG2細(xì)胞的攝取機(jī)制以及亞細(xì)胞分布。同時(shí),評(píng)價(jià)了CHSP納米粒作為阿霉素(DOX, doxorubin)藥物載體的細(xì)胞毒性,研究了HepG2細(xì)胞對(duì)載藥納米粒的攝取以及對(duì)藥物的亞細(xì)胞分布進(jìn)行定位和定量分析,為肝癌治療提供了研究基礎(chǔ)和理論依據(jù)。主要研究?jī)?nèi)容及結(jié)果如下： 1. FITC標(biāo)記的CHSP (FITC-CHSP)合成及自組裝納米粒的制備和表征 合成了FITC-CHSP并通過紅外進(jìn)行表征,結(jié)果表明,FITC實(shí)現(xiàn)了接枝成功。采用透析法制備了FITC-CHSP自組裝納米粒,并利用透射電鏡(TEM)和動(dòng)態(tài)光散射粒度分析儀(DLS)對(duì)納米粒的形態(tài)、粒徑及粒徑分布進(jìn)行了表征,研究結(jié)果表明,制備的FITC-CHSP納米粒呈規(guī)則的球形,平均粒徑為63.0±1.9nm。 2. FITC-CHSP自組裝納米粒的體外HepG2細(xì)胞攝取機(jī)制以及亞細(xì)胞分布 CHSP納米粒的體外細(xì)胞毒實(shí)驗(yàn)表明：CHSP納米粒對(duì)HepG2細(xì)胞無明顯的細(xì)胞毒性。分別采用激光掃描共聚焦顯微鏡(CLSM)和熒光光度法對(duì)HepG2細(xì)胞攝取CHSP納米粒進(jìn)行觀察和定量,系統(tǒng)研究了納米粒濃度、孵育時(shí)間、孵育溫度對(duì)納米粒攝取的影響。結(jié)果表明,HepG2細(xì)胞攝取納米粒的過程是能量依賴的過程,呈現(xiàn)出濃度依賴性、時(shí)間依賴性和溫度依賴性。內(nèi)吞抑制實(shí)驗(yàn)表明：網(wǎng)格蛋白介導(dǎo)的內(nèi)吞途徑以及巨胞飲途徑共同參與了CHSP納米粒的入胞過程。納米粒的亞細(xì)胞分布實(shí)驗(yàn)表明：在研究的孵育時(shí)間(4h)內(nèi),并沒有發(fā)現(xiàn)CHSP納米粒進(jìn)入高爾基體和內(nèi)質(zhì)網(wǎng)。當(dāng)納米粒與細(xì)胞孵育30min時(shí),沒有納米粒定位于溶酶體中。但是,隨著孵育時(shí)間的延長(zhǎng),大量納米粒分布于溶酶體中,且定位于溶酶體中的納米粒逐漸向細(xì)胞核周區(qū)域移動(dòng)。 3.載阿霉素CHSP納米粒的制備及體外藥物釋放 以DOX為模型藥物,采用透析法制備了載DOX的CHSP納米粒,并用TEM和DLS對(duì)載藥納米粒進(jìn)行表征,結(jié)果證實(shí),載藥納米粒呈球形,隨著藥物與載體投料比的增加,納米粒的粒徑也增大(185.6-226.4nm)。采用紫外-可見分光光度法測(cè)得載藥納米粒的載藥量在10.31-30.79%范圍內(nèi),可通過調(diào)整起始藥物投料比對(duì)載藥量調(diào)控,包封率為71.2-88.3%。載藥納米粒的體外釋放與釋放介質(zhì)的pH值有關(guān),在低pH環(huán)境(pH5.0)中藥物釋放較快,72h內(nèi)的累積釋放量為62.0%,但在pH6.8或pH7.4的釋放介質(zhì)中,藥物釋放依次減慢,72h的累積釋放量分別為46.4%和30.1%。 4.載阿霉素CHSP納米粒的體外HepG2細(xì)胞攝取研究 載藥納米粒的體外細(xì)胞毒實(shí)驗(yàn)結(jié)果表明,不同濃度的游離DOX和載藥納米粒與HepG2細(xì)胞分別孵育72h,二者對(duì)HepG2細(xì)胞的生長(zhǎng)均顯示出一定的抑制作用,并且隨著DOX濃度的增加,二者對(duì)HepG2細(xì)胞生長(zhǎng)的抑制作用均增強(qiáng)。根據(jù)量效曲線計(jì)算得到游離DOX的IC50為2.36μg/mL,而載DOX的納米粒的IC50為0.99μg/mL表明,載DOX的納米粒對(duì)HepG2細(xì)胞的毒性更大。采用流式細(xì)胞儀對(duì)HepG2細(xì)胞攝取的DOX進(jìn)行定量,結(jié)果表明,隨著孵育時(shí)間的增加,HepG2細(xì)胞攝取DOX的量也增大。當(dāng)孵育時(shí)間大于2h,HepG2細(xì)胞攝取載藥納米粒的量要多于游離DOX。采用CLSM以及定量分析軟件分別對(duì)藥物的亞細(xì)胞分布進(jìn)行定位和定量分析結(jié)果表明,由于游離藥物與載藥納米粒的攝取機(jī)制不同,孵育30min,游離DOX主要分布在細(xì)胞核中,并且隨著孵育時(shí)間的增加,細(xì)胞以及細(xì)胞核中的DOX也增多。孵育30min,載藥納米粒主要分布在胞漿中；1h后部分載藥納米粒分布于溶酶體中,細(xì)胞核中DOX顯著增加,并且隨著孵育時(shí)間的增加,細(xì)胞、溶酶體以及細(xì)胞核中的DOX也增加。孵育4h時(shí),載藥納米粒實(shí)驗(yàn)組細(xì)胞以及細(xì)胞核中的DOX均多于游離DOX實(shí)驗(yàn)組。在研究的孵育時(shí)間(4h)內(nèi),載藥納米粒實(shí)驗(yàn)組以及游離DOX實(shí)驗(yàn)組細(xì)胞分布在高爾基體、內(nèi)質(zhì)網(wǎng)以及線粒體中的DOX量很少,隨著孵育時(shí)間的增加,在上述細(xì)胞器中的含量無明顯變化。 綜上所述,CHSP納米粒具有良好的生物相容性,能通過網(wǎng)格蛋白介導(dǎo)的內(nèi)吞以及巨胞飲途徑快速進(jìn)入HepG2細(xì)胞,同時(shí)HepG2細(xì)胞對(duì)載DOX的CHSP納米粒的攝取能力要比游離DOX強(qiáng),載藥納米粒對(duì)HepG2細(xì)胞的毒性明顯大于游離DOX,提示與游離DOX相比,載藥納米�？稍谳^低劑量下達(dá)到較高療效。CHSP納米粒有望成為一種新型的肝癌治療藥物載體。
[Abstract]:As one of the natural polymer materials, polysaccharides have good biocompatibility and safety. Hydrophobic modified polysaccharides form a unique "nuclear shell structure" nanoparticles by self-assembly. They have a long cycle in the body, the stability of the body and the passive targeting, and have potential applications in the field of drug delivery. The interaction between nanoparticles and cells is of great scientific and practical value for understanding the physiological processes of the life body at the cellular level, the mechanism of drug action, the mechanism of gene therapy and so on. It also provides a basis for the construction of a more safe and effective nano drug carrier.
In this study, FITC (fluorescein isothiocyanate) was used to mark the cholesterol Kip Ruland (CHSP, cholesterol-modified pullulan) and prepare nanoparticles. The effect of nanoparticle concentration, incubation time and temperature on cell uptake was discussed. The uptake mechanism of HepG2 cells in vitro and subcells of CHSP nanoparticles were studied. At the same time, the cytotoxicity of CHSP nanoparticles as a drug carrier for DOX (doxorubin) was evaluated. The uptake of drug loaded nanoparticles by HepG2 cells and the location and quantitative analysis of the subcellular distribution of drugs were studied. The basis and theoretical basis for the treatment of liver cancer were provided. The main contents and results were as follows:
Preparation and characterization of 1. FITC labeled CHSP (FITC-CHSP) synthesized and self assembled nanoparticles
The FITC-CHSP was synthesized and characterized by IR. The results showed that the grafting was successful by FITC. The FITC-CHSP self assembled nanoparticles were prepared by dialysis method. The morphology, particle size and particle size distribution of the nanoparticles were characterized by transmission electron microscopy (TEM) and dynamic light scattering particle size analyzer (DLS). The results showed that the prepared FITC-CHSP was prepared. The nanoparticles were regular spheres with an average particle size of 63 + 1.9nm..
HepG2 cell uptake mechanism and subcellular distribution of 2. FITC-CHSP self assembled nanoparticles in vitro
In vitro cytotoxicity test of CHSP nanoparticles showed that CHSP nanoparticles had no obvious cytotoxicity to HepG2 cells. The HepG2 cells were observed and quantified by laser scanning confocal microscopy (CLSM) and fluoropphotometry respectively. The concentration of nanoparticles, incubation time and incubation temperature on nanoparticles uptake were studied. The results showed that the process of HepG2 cells uptake of nanoparticles was an energy dependent process, showing a concentration dependence, time dependence and temperature dependence. Endocytosis inhibition experiments showed that the endocytic pathway mediated by the gridin and the mega drink pathway participated in the cellular process of CHSP nanoparticles. The subcellular distribution of nanoparticles In the incubation time (4h), no CHSP nanoparticles were found to enter the Golgi bodies and endoplasmic reticulum. When the nanoparticles and cells incubated 30min, no nanoparticles were located in the lysosomes. However, a large number of nanoparticles were distributed in the lysosomes with the prolongation of incubation time, and the nanoparticles located in the lysosome gradually turned to the nucleus of the nucleus. Regional movement.
Preparation of 3. adriamycin CHSP nanoparticles and drug release in vitro
Using DOX as the model drug, the DOX loaded CHSP nanoparticles were prepared by dialysis method, and the drug loaded nanoparticles were characterized by TEM and DLS. The results showed that the drug loaded nanoparticles were spherical. With the increase of the ratio of drug to carrier, the particle size of nanoparticles increased (185.6-226.4nm). The loading of drug loaded nanoparticles was measured by UV visible spectrophotometry. In the range of 10.31-30.79%, the drug delivery ratio can be adjusted by adjusting the initial drug delivery ratio. The release of the encapsulated 71.2-88.3%. drug loaded nanoparticles in vitro is related to the pH value of the release medium. The release of drugs in the low pH environment (pH5.0) is faster and the cumulative release amount in 72h is 62%, but the release of drugs in the release medium of pH6.8 or pH7.4 is dependent on the release of drugs. The cumulative release of 72h was 46.4% and 30.1%. respectively.
HepG2 uptake of 4. adriamycin CHSP nanoparticles in vitro
In vitro cytotoxicity test of drug loaded nanoparticles showed that different concentrations of free DOX and drug loaded nanoparticles incubated 72h with HepG2 cells respectively. The two had a certain inhibitory effect on the growth of HepG2 cells, and with the increase of DOX concentration, the inhibition of the growth of HepG2 cells increased by the increase of the concentration of DOX. According to the volume effect curve, the results were calculated. The IC50 of free DOX was 2.36 mu g/mL, while IC50 loaded with DOX nanoparticles showed that the DOX loaded nanoparticles were more toxic to HepG2 cells. The flow cytometry was used to quantify DOX for HepG2 cells. The results showed that the HepG2 cells increased the amount of DOX as the incubation time increased. The amount of drug loaded nanoparticles was more than that of free DOX. using CLSM and quantitative analysis software to locate and quantify the subcellular distribution of drugs. The results showed that the free DOX was mainly distributed in the nucleus because of the different uptake mechanism between the free drug and the drug loaded nanoparticles, and the free DOX was mainly distributed in the nucleus, and as the incubation time increased, it was thinner. The number of DOX in cell and nucleus was also increased. The drug loaded nanoparticles were mainly distributed in the cytoplasm. After 1h, some drug loaded nanoparticles were distributed in the lysosome, and the DOX in the nucleus increased significantly. And as the incubation time increased, the cells, lysosomes and the DOX in the nucleus were also increased. When incubating 4h, the drug loaded nanoparticles experiment group cells and the incubating 4H. The DOX in the nucleus was more than that in the free DOX experiment group. In the incubation time (4h), the cells of the drug loaded nanoparticles and the free DOX experimental group were distributed in Golgi body, and the amount of DOX in the endoplasmic reticulum and mitochondria was very little.
To sum up, CHSP nanoparticles have good biocompatibility, can quickly enter HepG2 cells through the endocytic endocytosis and megagocytosis pathway, and the uptake of DOX CHSP nanoparticles by HepG2 cells is stronger than that of free DOX. The toxicity of drug loaded nanoparticles to HepG2 cells is significantly greater than that of free DOX, suggesting that compared with free DOX, the drug loaded nanoparticles are more toxic than free DOX. Drug loaded nanoparticles can achieve high efficacy at lower doses..CHSP nanoparticles are expected to become a new drug carrier for the treatment of liver cancer.

【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2013
【分類號(hào)】：R318.08

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