【摘要】：透明質(zhì)酸納米微乳經(jīng)皮藥物釋放體系，在皮膚滲透、定量給藥、藥物緩釋、延長(zhǎng)藥效和淺表層病灶區(qū)定位方面體現(xiàn)了諸多優(yōu)勢(shì)。10,11-亞甲二氧基喜樹(shù)堿（MD-CPT）是喜樹(shù)堿衍生物的代表，具有很高的生物活性，能夠有效地阻礙DNA復(fù)制，阻止有絲分裂，抑制細(xì)胞增殖，是良好的抗腫瘤制劑。但是，MD-CPT的疏水性、內(nèi)酯環(huán)不穩(wěn)定性等理化性質(zhì)限制了其體內(nèi)應(yīng)用。透明質(zhì)酸納米微乳作為MD-CPT的載體，可以克服MD-CPT的水難溶性、促進(jìn)機(jī)體吸收、提高生物利用度、增加藥物靶向性、減少毒副作用、維持血藥濃度、延長(zhǎng)半衰期和提高藥效。瘢痕疙瘩（KD）被認(rèn)為是一種良性腫瘤，其特征是成纖維細(xì)胞過(guò)度增殖，且受刺激易復(fù)發(fā)和加重病情，因此本文將載MD-CPT透明質(zhì)酸納米微乳應(yīng)用于瘢痕疙瘩的修復(fù)研究，建立了一種溫和無(wú)創(chuàng)的經(jīng)皮給藥治療途徑。 對(duì)透明質(zhì)酸進(jìn)行修飾，，成功制備了雙親性透明質(zhì)酸單甘脂衍生物（HA-GMS）。通過(guò)紅外、核磁檢測(cè)證實(shí)了單甘酯成功地交聯(lián)到HA多糖主鏈上，以FITC為熒光標(biāo)記，以乙二胺為鏈接壁，利用硫脲鍵將FITC共價(jià)修飾在EDA-HA-GMS多糖主鏈上，得到FITC-HA-GMS，可以對(duì)HA-GMS載體材料進(jìn)行定位示蹤與定量分析。溶血實(shí)驗(yàn)表明，HA-GMS的溶血率均小于5%，符合生物醫(yī)用材料對(duì)溶血率的要求，具有較好的血液相容性。通過(guò)MTT法檢測(cè)了HA-GMS的細(xì)胞毒性。 采用超聲乳化法，成功制備了不同組分配比的載MD-CPT的HANs納米微乳，平均粒徑是50~200nm，zeta電位絕對(duì)值是10~20mV，帶負(fù)電，均一性較好。通過(guò)透射電鏡觀察，納米粒均為完整的球形，大小均一，分布均勻，幾乎沒(méi)有聚集的現(xiàn)象，穩(wěn)定性良好。通過(guò)體外模擬實(shí)驗(yàn)，考察了HANs納米微乳在皮膚表面的穩(wěn)定性，HANs納米微乳在pH4.0~7.4形態(tài)幾乎沒(méi)有變化，在pH7.4是納米微乳表現(xiàn)出不規(guī)則的形狀，而且部分顆粒出現(xiàn)崩解的現(xiàn)象，說(shuō)明HANs納米微乳在皮膚弱酸性環(huán)境下可以有效地遞送藥物。 MD-CPT/HANs納米微乳用于透皮擴(kuò)散研究，角質(zhì)層透過(guò)試驗(yàn)：0~4h呈時(shí)間依賴性遞增，達(dá)到平衡時(shí)累積滲透量Qt為660.7±20.5μg/cm2，明顯高于對(duì)照組。全皮滲透性測(cè)定：Qt為51.7±9.5μg/cm2明顯低于角質(zhì)層透過(guò)量，隨時(shí)間呈S型變化趨勢(shì)。不同HA-GMS：MD-CPT質(zhì)量配比制得的載藥納米微乳的透皮擴(kuò)散效率不同。Rhodamine B標(biāo)記HA-GMS，利用熒光顯微鏡觀察到載藥納米乳透過(guò)角質(zhì)層到達(dá)真皮層的擬動(dòng)態(tài)過(guò)程，4h內(nèi)，部分載藥納米微乳已經(jīng)到達(dá)真皮層，實(shí)現(xiàn)透皮。 血藥濃度與尿樣檢測(cè)，證明了HANs納米載體對(duì)MD-CPT具有緩釋的作用，多余的MD-CPT進(jìn)入了血液循環(huán)，參與體內(nèi)的新陳代謝，最終被排出體外，不會(huì)成為機(jī)體負(fù)擔(dān)。活體成像研究體內(nèi)分布，進(jìn)一步驗(yàn)證了MD-CPT在給藥區(qū)皮下組織的滯留時(shí)間較長(zhǎng)，藥物濃度較高，形成了局部臨時(shí)藥物貯庫(kù)，可以不斷滲透到淺表層病灶區(qū)，實(shí)現(xiàn)持續(xù)給藥、延長(zhǎng)藥效的目的。另外，藥物含量：腎肝脾，進(jìn)一步說(shuō)明MD-CPT參與了新陳代謝，并避開(kāi)了對(duì)喜樹(shù)堿類藥物十分敏感的消化道等組織器官。 與HSF、HUVECS、MCF-7、N2a等不同細(xì)胞株作用比較，證明了MD-CPT對(duì)細(xì)胞生長(zhǎng)抑制、細(xì)胞吞噬和細(xì)胞周期調(diào)節(jié)都是有選擇的，效果明顯傾向于分裂活躍的增生性細(xì)胞。 MD-CPT對(duì)瘢痕成纖維細(xì)胞（KF）的抑制呈濃度依賴性。KF對(duì)FITC-HANs-MD-CPT細(xì)胞吞噬是一個(gè)時(shí)間依賴性的動(dòng)態(tài)的過(guò)程，由胞外擴(kuò)散到胞內(nèi)再到細(xì)胞核內(nèi)。將MD-CPT/HANs作用于瘢痕皮膚，角質(zhì)層變得膨脹、疏松，局部呈現(xiàn)篩孔的結(jié)構(gòu)，促進(jìn)滲透。熒光顯微鏡證明了Rhodamine-HANs-MD-CPT透過(guò)瘢痕疙瘩表皮直達(dá)真皮層的病變部位。 MD-CPT/HA-GMS作用KF細(xì)胞12h，處于S期的細(xì)胞增加，24h，48h，處于S與G2/M的細(xì)胞均急劇減少，超過(guò)90%的細(xì)胞處于G0/G1期，停止有絲分裂，出現(xiàn)了老化和凋亡的趨勢(shì)。實(shí)時(shí)熒光定量RT-PCR的結(jié)果，PAI-1表達(dá)水平下調(diào)，但是TGF-β、Smad3、Smad7上調(diào)，推測(cè)TGF-β/SMAD—PAI-1被Smad7阻斷。Western Blot顯示Smad2/3磷酸化水平顯著下降，印證了Smad7通過(guò)阻止Smad2/3磷酸化進(jìn)而阻斷TGF-β/SMAD信號(hào)通路，使下游基因PAI-1表達(dá)水平下降，從而抑制瘢痕疙瘩再生。 綜上所述，通過(guò)體外、體內(nèi)實(shí)驗(yàn)評(píng)價(jià)了雙親性的透明質(zhì)納米微乳經(jīng)皮給藥體系的生物安全性，皮膚滲透性，細(xì)胞特異性及藥代動(dòng)力學(xué)特性，并應(yīng)用于治療瘢痕。研究表明，喜樹(shù)堿衍生物通過(guò)透明質(zhì)酸納米微乳經(jīng)皮給藥有望成為瘢痕修復(fù)的治療途徑。
[Abstract]:Hyaluronic acid nano-microemulsion transdermal drug delivery system has many advantages in skin penetration, quantitative drug delivery, drug sustained release, prolonged efficacy and superficial lesion localization. 10,11-methylenedioxycamptothecin (MD-CPT) is a representative of camptothecin derivatives with high biological activity, which can effectively hinder DNA replication and prevent filaments. Hyaluronic acid nanoemulsion as the carrier of MD-CPT can overcome the water insolubility of MD-CPT, promote the body absorption, improve bioavailability, increase drug targeting, reduce toxicity and side effects. Keloids (KD) are considered to be benign neoplasms characterized by excessive proliferation of fibroblasts and prone to recurrence and aggravation by stimulation. In this paper, MD-CPT-loaded hyaluronic acid nanoemulsions were applied to keloid repair and a mild non-invasive skin graft was established. Route of administration.
Amphiphilic hyaluronic acid monoglyceride derivatives (HA-GMS) were successfully prepared by modification of hyaluronic acid. It was confirmed by IR and NMR that monoglyceride was successfully crosslinked onto the main chain of HA polysaccharide. FITC was labeled with fluorescence and linked with ethylenediamine. FITC was covalently modified on the main chain of EDA-HA-GMS by thiourea bond. Haemolysis test showed that the hemolysis rate of HA-GMS was less than 5%, which accorded with the requirement of biomedical materials for hemolysis rate and had good blood compatibility. The cytotoxicity of HA-GMS was detected by MTT method.
HANS nanoemulsions loaded with MD-CPT with different composition ratios were successfully prepared by ultrasonic emulsification. The average particle size was 50-200 nm, the absolute value of zeta potential was 10-20 mV, and the nanoparticles were negatively charged with good homogeneity. The stability of HANs nanoemulsion on the skin surface was investigated in vitro. The morphology of HANs nanoemulsion was almost unchanged at pH 4.0-7.4. The shape of HANs nanoemulsion was irregular at pH 7.4, and some particles disintegrated. The results showed that HANs nanoemulsion could deliver drugs effectively in weak acidic environment.
MD-CPT/HANs nanoemulsion was used in the study of transdermal diffusion. The cuticle penetration test showed that the cumulative penetration of MD-CPT/HANs nanoemulsion increased in a time-dependent manner from 0 to 4 hours. The cumulative penetration of Qt at equilibrium was 660.7+20.5 ug/cm 2, which was significantly higher than that of the control group. Rhodamine B labeled HA-GMS was used to observe the pseudo-dynamic process of drug-loaded nanoemulsions through the cuticle to the dermis by fluorescence microscopy. Within 4 hours, part of the drug-loaded nanoemulsions had reached the dermis and achieved transdermal penetration.
Blood concentration and urine samples showed that HANs nanocarriers could slow the release of MD-CPT. Excess MD-CPT entered the blood circulation, participated in metabolism in vivo, and eventually was excluded from the body. In vivo imaging study of the distribution of MD-CPT in the subcutaneous tissue of the drug delivery area further confirmed the longer retention time of MD-CPT. In addition, the content of MD-CPT in kidney, liver and spleen further indicates that MD-CPT participates in metabolism and avoids organs such as digestive tract which are sensitive to camptothecin.
Compared with HSF, HUVECS, MCF-7, N2a and other cell lines, MD-CPT has selective effects on cell growth inhibition, cell phagocytosis and cell cycle regulation, and the effect is obviously inclined to divide active proliferative cells.
MD-CPT inhibits scar fibroblasts (KF) in a concentration-dependent manner. KF phagocytosis of FITC-HANs-MD-CPT cells is a time-dependent dynamic process, from extracellular diffusion to intracellular and then to the nucleus. Microscopic examination showed that Rhodamine-HANs-MD-CPT penetrated the keloid epidermis directly to the dermal lesion site. KF cells in S phase increased at 12h, and decreased sharply at 24h and 48h. More than 90% of the cells in S and G2/M were in G0/G1 phase, stopped mitosis, and appeared the trend of aging and apoptosis. Western Blot showed a significant decrease in Smad2/3 phosphorylation, confirming that Smad7 blocked the TGF-beta/SMAD signaling pathway by inhibiting Smad2/3 phosphorylation, thereby inhibiting the downstream gene PAI-1 expression and thereby inhibiting scar formation. Lump regeneration.
In conclusion, in vitro and in vivo experiments were carried out to evaluate the bio-safety, skin permeability, cell specificity and pharmacokinetics of the amphiphilic hyaluronic nano-microemulsion transdermal delivery system and its application in the treatment of scars. Therapeutic approach.
【學(xué)位授予單位】：中國(guó)海洋大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R96

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