發(fā)布時間：2018-04-25 20:24

  本文選題：mPEG-PCL-PGA + 聚合物囊泡��； 參考：《山東大學(xué)》2014年碩士論文

【摘要】：高分子合成化學(xué)的快速發(fā)展使得合成的生物可降解聚合物在生物醫(yī)藥領(lǐng)域得到了廣泛的應(yīng)用。聚酯類聚合物如聚乳酸(PLA)、聚己內(nèi)酯(PCL)、聚丙交酯-乙交酯共聚物(PLGA)等都是比較常見的合成的聚合物嵌段,這些材料合成簡單,對人體毒性較低,具有生物可降解性,因此作為藥物傳遞載體有著較為廣闊的應(yīng)用前景。隨著藥物傳遞系統(tǒng)對載體要求的日益提高,近年來聚肽嵌段引起人們的廣泛關(guān)注,此類聚合物降解后的單體可被生物體利用,且隨著外界環(huán)境如pH、溫度的變化可以改變自身的二級構(gòu)象,引起嵌段水溶性的改變,引起聚集體結(jié)構(gòu)的聚集-解聚行為或者聚合物聚集體形態(tài)的改變。 兩親性聚合物由親水嵌段及疏水嵌段構(gòu)成,其組成中親疏水嵌段的比例不同,組裝體的形態(tài)(膠束、納米粒、囊泡)也各不相同。聚合物囊泡與細(xì)胞結(jié)構(gòu)相似,是由兩親性聚合物自組裝形成的完整閉合雙層結(jié)構(gòu)。相比于膠束和納米粒,囊泡的內(nèi)部為親水腔,能夠?qū)⒌鞍追肿拥扔H水性藥物包裹起來,避免外界環(huán)境對藥物活性的影響。與具有類似結(jié)構(gòu)的脂質(zhì)體相比,聚合物囊泡的膜較厚,體系穩(wěn)定,而脂質(zhì)體的組成成分磷脂易氧化發(fā)生內(nèi)容物的泄漏。此外,聚合物囊泡的性質(zhì)可通過改變聚合物嵌段的種類和長度進行調(diào)節(jié)。 鹽酸多柔比星是臨床上治療多種腫瘤的一線藥物,廣泛用于治療急性白血病、淋巴癌、軟組織和骨肉癌、兒童惡性腫瘤及多種成人實體瘤,尤其對乳腺癌和肺癌的治療效果較為明顯,但是應(yīng)用多柔比星的患者會出現(xiàn)各種各樣的副作用如惡心、嘔吐、骨髓抑制等,最為嚴(yán)重的是對病人的心臟毒性。為了降低鹽酸多柔比星的毒副作用,常將藥物包載于載體中進而降低其在正常組織中的分布,使較多的藥物運送到腫瘤部位。為了實現(xiàn)藥物的腫瘤靶向,可以利用腫瘤部位pH值低的特點,將鹽酸多柔比星包載于pH敏感載體中,使其在腫瘤部位能夠釋放較多的藥物。而且已有報道稱,將鹽酸多柔比星包裹于聚合物囊泡中,能降低其心臟毒性。 本課題以mPEG、ε-CL、L-谷氨酸-γ-芐酯-N-羧酸酐為原料合成三嵌段聚合物mPEG-PCL-PGA,根據(jù)文獻報道選擇合適的親、疏水鏈段比例,使合成的聚合物在水溶液中可以自發(fā)形成囊泡。聚乙二醇(mPEG)作為親水嵌段能夠抵抗血漿蛋白吸附,其伸展在囊泡表面能夠賦予囊泡“隱形”的特點,避免網(wǎng)狀內(nèi)皮系統(tǒng)的吞噬,達到長循環(huán)的目的；聚己內(nèi)酯(PCL)作為疏水嵌段,各單體間其通過易水解的酯鍵相連接,其結(jié)構(gòu)相對柔軟,容易被體內(nèi)酶分解代謝；聚谷氨酸(PGA)嵌段在中性條件下,呈現(xiàn)水溶性的無規(guī)卷曲構(gòu)象(coil),而在酸性條件下則會呈現(xiàn)水溶性差的α螺旋(a-helical)構(gòu)象,PGA嵌段具有pH敏感性從而賦予其所形成的聚合物囊泡具有pH敏感性,實現(xiàn)在腫瘤部位(pH較低)釋藥的目的。 本論文的主要研究內(nèi)容如下： 1.三嵌段pH敏感聚合物mPEG-PCL-PGA的合成、表征及聚集行為研究 利用開環(huán)聚合反應(yīng)分三大步合成了三嵌段pH敏感聚合物mPEG-PCL-PGA。首先以單甲氧基聚乙二醇為大分子起始物,ε-己內(nèi)酯為單體,辛酸亞錫為催化劑,開環(huán)聚合生成mPEG-PCL;由于一級胺是引發(fā)谷氨酸單體聚合最合適的物質(zhì),因此將mPEG-PCL末端的羥基與氨基端BOC(叔丁氧羰基)保護的苯丙氨酸通過酯鍵相連,然后水解掉BOC保護基,露出氨基；以此氨基為引發(fā)劑,與谷氨酸活性單體γ-芐基-L-谷氨酸-N-羧酸酐(NCA-BLG)發(fā)生聚合,水解掉γ-芐基,得到目標(biāo)聚合物。聚合物的結(jié)構(gòu)通過核磁共振氫譜與傅里葉紅外光譜進行驗證；表面張力法測定表面張力等溫線,由曲線拐點測出聚合物的臨界聚集濃度為3.6mg/mL,此聚合物可以使水的表面張力降到46mN/m。2%磷鎢酸溶液負(fù)染色透射電子顯微鏡表明溶液中聚集體是形態(tài)比較規(guī)整的球形單層囊泡。 2.載藥聚合物囊泡的制備、性質(zhì)及體外釋放行為研究 本實驗采用納米沉淀法(nanoprecipitation)制備載藥聚合物囊泡,負(fù)染色法觀察載藥囊泡的形態(tài)；并考察不同藥物與聚合物質(zhì)量比對囊泡的載藥量和包封率的影響,根據(jù)囊泡穩(wěn)定性及藥物終濃度選擇藥物/聚合物最適的比例為1.5：10(w/w),此時囊泡的載藥量為(10.4±0.5)%,包封率為(78.7±1.4)%；測得囊泡粒徑大小為(180.9±7.6)nm,zeta電勢為-(38.5±0.3)mV。不同pH條件下的體外釋放實驗表明,與原料藥溶液相比,囊泡中鹽酸多柔比星的釋放更加緩慢,且在酸性條件下(pH5.0和6.5)較中性條件下藥物的釋放要快,釋放量要高,說明此載藥聚合物囊泡具有pH敏感性。 3.載藥聚合物囊泡的體外抗腫瘤實驗 本實驗采用WST-1法研究聚合物材料的毒性,原料藥溶液和載藥囊泡溶液對人乳腺癌細(xì)胞(MCF-7)和人肺癌細(xì)胞(A549)的細(xì)胞毒性作用。最高濃度聚合物材料(500μg/mL)在各時間點對MCF-7細(xì)胞增殖的抑制率如下：(25.7±1.5)%(72h)、(17.1±3.4)%(48h)、(12.8±2.7)%(24h),對A549的抑制率如下：(36.3±1.7)%(72h)、(19.6±3.4)%(48h)、(11.3±2.7)%(24h)；各組對細(xì)胞的殺傷力均與濃度和時間成正相關(guān)性,各濃度載藥囊泡溶液組對兩種細(xì)胞的殺傷力均比原料藥組強,鹽酸多柔比星原料藥溶液對MCF-7的抑制率為(86.7±4.1)%(72h)、(79.9±7.3)%(48h)、(71.2±4.9)%(24h),而載藥囊泡對MCF-7的抑制率為(93.4±3.4)%(72h)、(89.7±2.3)%(48h)、(79.0±3.4)%(24h)；鹽酸多柔比星原料藥溶液對A549的抑制率為(84.2±2.2)%(72h)、(76.5±6.7)%(48h)、(70.2±3.0)%(24h),載藥囊泡的對A549的抑制率為(86.3±0.9)%(72h)、(84.4+7.2)%(48h)、(75.0±2.5)%(24h)。細(xì)胞攝取實驗表明：MCF-7對鹽酸多柔比星原料藥溶液及囊泡溶液的攝取無差別,A549對載藥囊泡的攝取量高于對原料藥的攝取。 4.載藥聚合物囊泡的體內(nèi)藥動學(xué)研究 本實驗以Wistar大鼠為實驗動物,尾靜脈給藥,沉淀血漿蛋白后,內(nèi)標(biāo)法測定血藥濃度。所測各時間點藥物濃度數(shù)據(jù)采用DAS2.0軟件處理,以統(tǒng)計矩原理分析各組藥動數(shù)據(jù)得統(tǒng)計矩參數(shù)。注射載藥囊泡大鼠藥動曲線下面積AUC(2081.621),高于原料藥組(1591.873)；且在大鼠體內(nèi)的體內(nèi)平均滯留時間(MR7)增加了一倍,分別為1.897h(PolyDox)和0.948(Dox)；載藥囊泡與鹽酸多柔比星的表觀分布容積(V)分別為0.015L/kg與0.008L/kg,載藥囊泡的的表觀分布容積提高一倍多；同時載藥囊泡在藥物體內(nèi)的清除率低于鹽酸多柔比星原料藥。 綜上所述,本論文合成了pH敏感的三嵌段聚合物mPEG-PCL-PGA,其在水中可形成穩(wěn)定的聚合物囊泡,用此囊泡包載抗癌藥物鹽酸多柔比星,減少了藥物的毒副作用,提高了藥物在體內(nèi)的濃度并使其較多的釋放于腫瘤部位,增強了對腫瘤細(xì)胞的殺傷力,為開發(fā)安全有效的pH敏感藥物載體提供了一定的研究基礎(chǔ)。
[Abstract]:The rapid development of synthetic polymer chemistry makes synthetic biodegradable polymers widely used in the field of biological medicine. Polyester polymers such as polylactic acid (PLA), polyhexyl ester (PCL), polylactone - glycolide copolymer (PLGA) are common polymer blocks, which are simple in synthesis and to human toxicity. Because of its low sex and biodegradability, it has a broad application prospect as a carrier of drug delivery. With the increasing requirements of the drug delivery system, polypeptides have attracted wide attention in recent years. The monomer after the degradation of this kind of polymer can be used by the raw body, and with the external environment such as pH, the temperature changes. It can change the two level conformation of its own and cause the change of water solubility in the block, which causes the aggregation and depolymerization of the aggregate structure or the change of the morphology of the polymer aggregates.
The two amphiphilic polymers are composed of hydrophilic block and hydrophobic block, and the proportion of hydrophobic block in its composition is different. The morphology of the assembly (micelle, nanoparticle, vesicle) is different. The polymer vesicles are similar to the cell structure, which are formed by the self-assembly of two amphiphilic polymers. Compared with micelles and nanoparticles, vesicles It is a hydrophilic cavity in which the hydrophilic drugs such as protein molecules can be wrapped up to avoid the influence of the external environment on the drug activity. Compared with the liposomes with similar structure, the membrane of the polymer vesicles is thicker and the system is stable, and the composition of the liposomes is easily oxidized by the phospholipids. In addition, the properties of the polymer vesicles are accessible. Adjust the type and length of polymer block.
Doxorubicin is a first-line drug in the clinical treatment of various tumors. It is widely used in the treatment of acute leukemia, lymphadenocarcinoma, soft tissue and bone meat cancer, children's malignant tumors and many adult solid tumors, especially for breast and lung cancer, but patients with doxorubicin will have a variety of side effects such as Nausea, vomiting, bone marrow suppression, and so on, the most serious is the heart toxicity of the patient. In order to reduce the toxic and side effects of doxorubicin hydrochloride, the drug is often loaded in the carrier to reduce its distribution in the normal tissue, so that more drugs are transported to the tumor site. In order to achieve the target of the tumor, the pH value of the tumor site can be used low. It is characterized by carrying doxorubicin in the pH sensitive carrier to release more drugs at the tumor site. And it has been reported that doxorubicin can be wrapped in a polymer vesicle and can reduce its cardiac toxicity.
The three block polymer mPEG-PCL-PGA was synthesized from mPEG, epsilon -CL, L- glutamic acid - gamma benzyl -N- carboxylic anhydride. According to the literature, it was reported that the suitable parent, the proportion of hydrophobic segments, was selected to form the vesicles spontaneously in aqueous solution. Polyethylene glycol (mPEG) was used as a hydrophilic block to resist plasma protein adsorption. The surface of the vesicles can give the vesicles "stealth", avoiding the phagocytosis of the reticuloendothelial system and reaching the goal of a long cycle. Polyhexyl ester (PCL) is used as a hydrophobic block, and each monomer is connected through a hydrolytic ester bond, its structure is relatively soft and easy to be metabolize in vivo, and polyglutamic acid (PGA) block is under neutral condition. The water-soluble random curl conformation (coil) shows a conformation of poor water-soluble alpha helix (A-helical) under acidic conditions, and PGA block has pH sensitivity, thus giving its formed polymer vesicles with pH sensitivity, which can be achieved at the tumor site (lower pH).
The main contents of this paper are as follows:
1. synthesis, characterization and aggregation behavior of three block pH sensitive polymer mPEG-PCL-PGA
The three block pH sensitive polymer mPEG-PCL-PGA. was synthesized by open ring polymerization in three steps. First, monomethoxy polyethylene glycol was used as large molecular initiator, epsilon hexyl ester as monomer, and stannous octanoate was used as the catalyst to produce mPEG-PCL. As the first order amine was the most suitable substance for the polymerization of glutamic acid monomer, the end of mPEG-PCL was made. The phenylalanine protected by the terminal hydroxyl group and the amino terminal BOC (TERT oxo carbonyl) is connected through the ester bond and then hydrolyzed out of the BOC protection group to expose the amino group. The amino group is used as an initiator to polymerize the glutamic acid active monomer gamma benzyl -L- glutamic acid -N- carboxylic anhydride (NCA-BLG) and hydrolyze the gamma benzyl group to get the target polymer. The structure of the polymer passes through the core. The magnetic resonance hydrogen spectrum and Fourier infrared spectrum are verified. Surface tension method is used to determine the surface tension isotherm, and the critical concentration of polymer is measured from the curve point of 3.6mg/mL. The polymer can reduce the surface tension of water to 46mN/m.2% phosphotungstic acid solution negative dyeing transmission electron microscope, indicating that the aggregates in the solution are compared. Regular spherical monolayer vesicles.
Preparation, properties and in vitro release behavior of 2. drug loaded polymer vesicles
The drug loaded polymer vesicles were prepared by nano precipitation method (nanoprecipitation). The morphology of the carrier vesicles was observed by negative staining, and the effects of the mass ratio of different drugs and polymers on the drug loading and encapsulation efficiency of the vesicles were investigated. The optimum proportion of the vesicle stability and the final drug concentration of the drug / polymer was 1.5:10 (w/w). The loading capacity of the vesicles was (10.4 + 0.5)%, the encapsulation rate was (78.7 + 1.4)%, the size of the vesicles was (180.9 + 7.6) nm, and the release experiment of zeta electric potential - (38.5 + 0.3) mV. in different pH conditions showed that the release of doxorubicin in the vesicles was more slow compared with the solution of the API, and it was more neutral in acid conditions (pH5.0 and 6.5). The drug release was fast and the release rate was high, indicating that the drug loaded polymer vesicles had pH sensitivity.
In vitro antitumor experiment of 3. drug loaded polymer vesicles
In this experiment, the toxicity of the polymer material, the solution of the drug and the solution of the vesicle on the cytotoxicity of human breast cancer cells (MCF-7) and human lung cancer cells (A549) were studied by WST-1 method. The inhibition rate of the maximum concentration of polymer material (500 mu g/mL) on the proliferation of MCF-7 cells at all time points was as follows: (25.7 + 1.5)% (72h), (17.1 + 3.4)% (48h), (12.8). The inhibition rate of A549 was as follows: (24h) as follows: (36.3 + 1.7)% (72h), (19.6 + 3.4)% (48h) and (11.3 + 2.7)% (24h). The cell killing ability of each group was positively correlated with concentration and time, and the killing force of each concentration of vesicle solution group was stronger than that of the drug group, and the inhibitory rate of doxorubicin hydrochloride on MCF-7 was 86 (86). .7 + 4.1)% (72h), (79.9 + 7.3)% (48h), (71.2 + 4.9)% (24h), and the inhibition rate of drug vesicles to MCF-7 was (93.4 + 3.4)% (72h), (89.7 + 2.3)% (48h), (79 + 3.4)% (24h), and the inhibition rate of doxorubicin hydrochloride was (84.2 + 2.2)% (72h), (48h)% (24h)% (24h)). .9)% (72h), (84.4+7.2)% (48h), (75 + 2.5)% (24h). Cell uptake experiments showed that MCF-7 had no difference in the uptake of doxorubicin hydrochloride and vesicle solution, and the uptake of vesicles by A549 was higher than that of the drug.
Pharmacokinetic study of 4. drug loaded polymer vesicles in vivo
In this experiment, Wistar rats were used as experimental animals. After the tail vein was given, plasma protein was precipitated, the blood concentration was measured by internal standard. The data of drug concentration at each time point were treated by DAS2.0 software. The statistical moment principle was used to analyze the statistical moment parameters of the pharmacokinetic data of each group. The area under the dynamic curve under the dynamic curve of the injection of drug vesicles was AUC (2081.621), higher than the raw material. In the drug group (1591.873), the average retention time (MR7) in the body was doubled, 1.897h (PolyDox) and 0.948 (Dox), and the apparent volume (V) of the drug vesicles and doxorubicin (V) was 0.015L/kg and 0.008L/kg, respectively. The apparent volume of the vesicles carrying the vesicles was more than doubled, and the drug vesicles were loaded in the drug body. The internal scavenging rate is lower than the drug of doxorubicin hydrochloride.
To sum up, this paper synthesizes the pH sensitive three block polymer mPEG-PCL-PGA, which can form a stable polymer vesicle in water and encapsulate the anticancer drug doxorubicin in the water. It reduces the side effects of the drug, increases the concentration of the drug in the body and releases more of the drug in the tumor site, and strengthens the tumor cells. Lethality provides a basis for developing safe and effective pH sensitive drug carriers.

【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R943

【相似文獻】

相關(guān)期刊論文 前10條

1 陳龍;硬盤錄象機的最新技術(shù)進展[J];中國安防產(chǎn)品信息;2002年04期

2 ;法國輕工產(chǎn)品(之三)[J];上海輕工業(yè);1997年01期

3 韓濤,李富榮,張淵明,唐渝,周漢新;PLA-mPEG嵌段共聚物膠團的制備及其表面張力[J];應(yīng)用化學(xué);2005年04期

4 王原麗;李艷紅;;基于ARM7平臺的多媒體監(jiān)控系統(tǒng)研究[J];安防科技;2007年03期

5 張世杰,葉華,龐純,趙建青;PA6/MPEG共混物的制備及性能研究[J];塑料工業(yè);2004年02期

6 楊瑞娥,，

本文編號：1802823