本文關(guān)鍵詞：基于熱敏脂質(zhì)體及低密度脂蛋白模型的納米載體靶向藥物遞送系統(tǒng)研究　出處：《中國人民解放軍軍事醫(yī)學(xué)科學(xué)院》2017年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 長春新堿 阿霉素 熱敏脂質(zhì)體 人工低密度脂蛋白 擴(kuò)散能力 靶向效率

【摘要】：背景在腫瘤治療領(lǐng)域,主動靶向藥物遞送系統(tǒng)早已引起各國的廣泛關(guān)注。雖然該研究方向的論文發(fā)表量呈指數(shù)增加,卻基本沒有上市產(chǎn)品�；谖锢砘瘜W(xué)和生物學(xué)原理分析,通過思考分子與納米粒子在水介質(zhì)中擴(kuò)散能力的差異,以及納米載體在體內(nèi)的尋靶過程,已有很多研究工作剖析了主動靶向藥物遞送系統(tǒng)理論中存在的某些誤區(qū),揭示出主動靶向修飾的納米載體有時并不能夠按照載體設(shè)計初衷提高對腫瘤組織靶向效率的缺陷。EPR效應(yīng)只適用于藥物分子和具有足夠擴(kuò)散能力的納米載體。目的為真正提高納米載體對于腫瘤組織的靶向效率,提高成藥性,我們將目光聚焦于依靠環(huán)境特異性響應(yīng)的靶向釋藥納米載體(以熱敏感脂質(zhì)體為模型)和具有強(qiáng)擴(kuò)散能力的主動靶向納米載體(以低密度脂蛋白為模型),這兩種最具有可行性的靶向策略。本研究中旨在研究制備長春新堿及阿霉素共載藥熱敏脂質(zhì)體(VD-TSL)和長春新堿人工低密度脂蛋白(VCR-a LDL),并進(jìn)行一系列的物理化學(xué)性質(zhì)評價,體外細(xì)胞評價,以及體內(nèi)活體成像、藥效學(xué)研究,說明這兩種靶向策略可以顯著提高納米載體的腫瘤組織靶向效率。內(nèi)容與方法第一部分:首先建立VCR與DOX同步分析的含量測定液相色譜檢測方法,并進(jìn)行了一系列方法學(xué)驗證,接著采用薄膜分散法制備空白脂質(zhì)體,p H梯度主動載藥法實現(xiàn)VCR和DOX的同步載藥,制備得到VD-TSL。以形態(tài)學(xué)研究、粒徑和zeta電位、外觀、熱敏特性、包封率、磷脂分析、穩(wěn)定性等為主要評價指標(biāo),進(jìn)行評價。通過MTT法測定各組制劑結(jié)合加熱對于兩個細(xì)胞系的毒性,并利用激光共聚焦顯微鏡觀察細(xì)胞攝取情況。建立裸鼠異種移植腫瘤模型,結(jié)合體外腫瘤部位局部加熱,對VD-TSL進(jìn)行了活體成像體內(nèi)靶向分布研究,并對藥效學(xué)及毒性進(jìn)行了初步評價。第二部分:首先研究處方工藝,利用薄膜分散一步載藥法,制備100nm和35nm的VCR-a LDL,隨之進(jìn)行粒徑、zeta電位、包封率、形態(tài)學(xué)研究及穩(wěn)定性等一系列物理化學(xué)性質(zhì)評價。為比較兩種粒徑VCR-a LDL的擴(kuò)散能力,我們進(jìn)行了MTT單層細(xì)胞毒性研究、MTT擴(kuò)散倒置細(xì)胞毒性研究、激光共聚焦觀察腫瘤餅和腫瘤球模型載體滲透作用實驗。接著建立了裸鼠異種移植腫瘤模型,進(jìn)行VCR-a LDL的3D活體成像實驗、組織分布研究及藥效學(xué)評價。結(jié)果第一部分:制備得到平均粒徑在85nm左右的VD-TSL,zeta電位約為零;透射電鏡下觀察,外觀圓整;包封率達(dá)95%以上;制備多批樣品,處方工藝重現(xiàn)性好;-20℃條件下放置6個月,VD-TSL穩(wěn)定性良好。體外細(xì)胞毒性評價和攝取實驗均表明VCR與DOX具有協(xié)同作用,同時加熱還可以促進(jìn)熱敏脂質(zhì)體進(jìn)入腫瘤細(xì)胞內(nèi)部,即包載藥物的熱敏脂質(zhì)體與加熱也有協(xié)同增效的作用。裸鼠異種移植模型活體成像實驗也證明了以上結(jié)論。藥效學(xué)實驗結(jié)果表明,VDTSL結(jié)合腫瘤組織局部加熱,具有很好的腫瘤抑制能力,抑瘤率達(dá)到了70%以上。同時,VD-TSL組裸鼠活動能力優(yōu)于注射液組,且體重下降幅度最小,即顯著降低了裸鼠的全身毒性。第二部分:制備了兩個不同粒徑的具有脂溶性核心和親水性外殼結(jié)構(gòu)的長春新堿人工低密度脂蛋白(VCR-a LDL),其中間粒徑分別為100nm左右和35nm左右,呈正態(tài)分布,zeta電位基本為零。透射電鏡下觀察,可見VCR-a LDL的粒徑大小分布均勻,外觀圓整,并具有明顯的殼核結(jié)構(gòu)。不論100nm還是35nm VCR-a LDL,藥物包封率均能夠達(dá)到95%以上,且穩(wěn)定性良好。細(xì)胞毒性MTT實驗中兩種粒徑VCR-a LDL的細(xì)胞抑制率并無顯著性差異,但MTT擴(kuò)散倒置細(xì)胞毒性研究中,35nm VCR-a LDL擴(kuò)散能力明顯強(qiáng)于100nm VCR-a LDL,細(xì)胞抑制率更強(qiáng)。激光共聚焦顯微鏡下觀察熒光標(biāo)記的不同粒徑a LDL對于體外腫瘤餅和腫瘤球模型的滲透作用,可見35nm a LDL的滲透能力雖沒有熒光分子強(qiáng),但顯著強(qiáng)于100nm a LDL。3D活體成像實驗中,與注射液組和100nm VCR-a LDL組相比,35nm VCR-a LDL組的腫瘤組織靶向滯留作用最強(qiáng)。藥效學(xué)實驗中,35nm VCR-a LDL組腫瘤抑制率最強(qiáng),同時裸小鼠體重下降幅度最小,全身毒性最小。結(jié)論本研究成功研發(fā)制備了VD-TSL和VCR-a LDL,二者具有不同的靶向腫瘤釋藥原理,同時均具有良好的體外物理化學(xué)性質(zhì),及體內(nèi)腫瘤組織靶向作用。工藝穩(wěn)定重現(xiàn)性好。VD-TSL中結(jié)合了VCR與DOX的協(xié)同作用,以及載藥熱敏脂質(zhì)體與加熱效應(yīng)的協(xié)同作用,是具有雙重協(xié)同作用的環(huán)境控制靶向載體。VCR-a LDL中,a LDL本身自帶主動靶向腫瘤的功能,通過考察100nm和35nm a LDL的靶向作用差異,說明減小粒徑可以提高其擴(kuò)散能力,能夠充分利用EPR效應(yīng)實現(xiàn)腫瘤組織被動靶向作用,才有基礎(chǔ)進(jìn)一步發(fā)揮主動靶向作用。
[Abstract]:The background in the field of cancer treatment, attention of active targeting drug delivery system has caused the world. Although the research papers has increased exponentially, but basically no listed products. Analysis of physical chemistry and biology based on the principle of differential diffusion ability in aqueous medium by thinking of molecules and nanoparticles, and the nanoparticles in in the targeting process, there has been a lot of research work on active targeting drug delivery system of certain errors in the theory, reveals the nano carrier to the modified active target is sometimes not according to the original design to improve the carrier for tumor targeting efficiency defects.EPR effect is only applicable to drug molecules and nanoparticles with sufficient diffusion capacity objective. To truly improve the nano carrier for tumor targeting efficiency, improve the resistance, we will focus on relying on the environment Drug targeting nano carrier specific response (by thermosensitive liposomes as model) and has strong diffusion ability of active targeting nanoparticles (with low density lipoprotein as a model of the two), the most feasible targeting strategy. Study on Preparation of vincristine and adriamycin were loaded thermosensitive liposomes to in this study, vincristine (VD-TSL) and artificial low density lipoprotein (VCR-a LDL), and evaluate the physical and chemical properties of a series of in vitro and in vivo evaluation, in vivo imaging, pharmacodynamics, which indicates that these two kinds of targeting strategies can significantly improve the tumor tissue to target nanoparticles content and method of efficiency. The first part: first establish content synchronization analysis of VCR and DOX were determined by liquid chromatography detection method, and conducted a series of method validation, then using blank liposomes, P H gradient active loading method VCR Synchronous loading and DOX, VD-TSL. was prepared by morphology, particle size and zeta potential, appearance, thermal properties, encapsulation efficiency, stability of phospholipid analysis, evaluation as the main evaluation index. Through the MTT method for the determination of each preparation combined with toxic heating for two cells, and the cells were observed using the uptake of confocal laser scanning microscopy. The establishment of nude mouse xenograft tumor model, combined with in vitro tumor local heating, VD-TSL for in vivo imaging in vivo targeting Distribution Research of pharmacodynamics and toxicity were evaluated. The second part: firstly, the formulation process, dispersion step loading method using thin film preparation. 100nm 35nM and VCR-a LDL, along with the particle size, zeta potential, encapsulation efficiency, stability and evaluation of morphological study and a series of physical and chemical properties. For the comparison of two kinds of particle diffusion diameter VCR-a LDL, we are MTT Study on monolayer cell toxicity, cytotoxicity of inverted MTT diffusion, laser confocal observation of tumor and tumor cake ball model carrier penetration experiment. Then we established nude mice xenograft model, 3D imaging experiments for VCR-a LDL, the study on tissue distribution and pharmacodynamic evaluation. Results the first part: the preparation of the average particle size about 85nm VD-TSL, the zeta potential is approximately zero; TEM round appearance; the entrapment efficiency was above 95%; the preparation of samples, prescription and process reproducibility; for 6 months at -20 DEG C, VD-TSL stability is good. In vitro cytotoxicity and uptake experiments showed that VCR and DOX has a synergistic effect, but also can promote the heat sensitive liposomes into tumor cells, namely thermosensitive liposomes and heating ofencapsulated drugs have synergistic effect. A nude mouse xenograft model in vivo imaging experiment Also prove the above conclusion. Pharmacodynamics experimental results show that VDTSL combined with local heating of tumor tissue, have good tumor inhibition ability, the inhibition rate reached more than 70%. At the same time, the mice activity of VD-TSL is better than that of injection group, and the weight of the smallest decline, which significantly reduce the systemic toxicity of nude mice. The second part: the preparation of two different sizes with fat soluble vincristine artificial LDL core and a hydrophilic shell structure (VCR-a LDL), the particle sizes were 100nm and 35nM, normal distribution, zeta potential is zero. Transmission electron microscope, visible VCR-a LDL particle size distribution, roundness appearance, and has obvious core-shell structure. Both 100nm and 35nM VCR-a LDL, the drug entrapment rate can reach more than 95%, and good stability. The cytotoxicity of MTT in experiment two grain size VCR-a LDL There is no significant difference between the inhibition rate of cells, but the cytotoxicity in MTT diffusion of inverted 35nM, VCR-a LDL diffusion capacity is better than 100nm VCR-a LDL, cell inhibition rate is stronger. Under a laser scanning confocal microscope and fluorescence labeled with different sizes of a LDL for in vitro tumor infiltration and tumor cake ball model, penetration of visible 35nM a LDL has no strong fluorescence, but was significantly stronger than 100nm a LDL.3D in vivo imaging experiments, and the injection group compared with 100nm group LDL VCR-a, 35nM VCR-a LDL tumor target group to stay the strongest effect. The pharmacodynamics experiment, 35nM VCR-a LDL the tumor inhibition rate and the strongest, decrease of body weight of nude mice the smallest, and minimal systemic toxicity. Conclusion the successful development of VD-TSL and VCR-a LDL were prepared by two different tumor targeting drug release in vitro and principle, have good physical and chemical properties, And in vivo tumor targeting effect. Combined with the stable process to reproduce the synergetic effect of VCR and DOX of.VD-TSL, and the drug loaded thermosensitive liposomes and the heating effect is synergistic with dual synergistic effects of environmental control targeting carrier.VCR-a LDL, a LDL has active tumor targeting function the difference to 100nm and 35nM a by investigating the role of LDL target, indicating decreasing particle size can improve the diffusion ability, can realize the passive targeting of tumor tissue to make full use of the EPR effect, only the foundation of further play an active role in targeting.

【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R943

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本文編號：1380420