pH敏感聚（原酸酯—氨酯）藥物載體的合成、表征及體內(nèi)外抗腫瘤評估
發(fā)布時(shí)間：2018-07-29 05:50
【摘要】：生物降解性聚合物納米藥物載體因具有較高的載藥率和包封率,良好的體內(nèi)藥物分布及降低藥物毒副作用的能力,延長藥物的體內(nèi)循環(huán)時(shí)間等優(yōu)點(diǎn)而被廣泛應(yīng)用于抗腫瘤藥物傳遞。如今,研究者們已經(jīng)開發(fā)了一系列高分子材料用于抗腫瘤藥物傳遞。其中生物降解性聚氨酯作為一種人工合成的生物相容性高分子材料,因其制備方法簡單、高效和優(yōu)越的生物醫(yī)學(xué)性能而被廣泛應(yīng)用于各種生物醫(yī)學(xué)領(lǐng)域,主要包括藥物緩控系統(tǒng)和組織工程支架。傳統(tǒng)的聚氨酯通過引入降解性聚酯或聚碳酸酯而被賦予降解性,然而這些聚氨酯的降解速率較慢,往往不能滿足抗腫瘤藥物傳遞的要求。為了提高傳統(tǒng)聚氨酯的降解速率,研究者們將一些環(huán)境響應(yīng)性的化學(xué)鍵(例如:pH敏感縮酮鍵、縮醛鍵、腙鍵和還原敏感的二硫鍵等)引入到了聚氨酯的主鏈中。原酸酯鍵作為一種微酸敏感的化學(xué)鍵,已被廣泛應(yīng)用到抗腫瘤納米藥物載體中。然而主鏈含原酸酯的pH敏感聚氨酯的制備及其在抗腫瘤納米藥物載體領(lǐng)域的應(yīng)用還未見報(bào)道。本論文通過一種酸敏感的原酸酯二氨基單體和兩種分子量不同的聚己內(nèi)酯二醇(Fw=530和2000)活性酯在溫和的條件下縮聚得到2種pH敏感聚(原酸酯-氨酯)(POEU1和POEU2),并通過水包油(O/W)單層乳液技術(shù)分別制備了具有不同酸降解速率的納米粒子(NP1和NP2)。通過核磁以及凝膠滲透色譜(GPC)檢測聚(原酸酯-氨酯)的結(jié)構(gòu)以及分子量,結(jié)果表明POEU1和POEU2結(jié)構(gòu)正確,數(shù)均分子量(Mn)分別為2.2×104和4.09104。透射電鏡(TEM)以及動(dòng)態(tài)光衍射儀(DLS)結(jié)果表明,兩種納米粒子粒徑分布在200 nm左右且都為球形結(jié)構(gòu)。體外降解結(jié)果表明兩種納米粒子在生理?xiàng)l件(pH 7.4)下非常穩(wěn)定,而在弱酸性條件下(pH 5.0)持續(xù)降解并伴隨著粒徑的不斷減小。另外,在pH 5.0條件下,兩種納米粒子(NP1和NP2)的降解速率可被聚合物的疏水性調(diào)控。在192 h后,由疏水性較弱的POEU1制備的納米粒子(NP1)完全降解,而由疏水性較強(qiáng)的POEU2制備的納米粒子(NP2)僅降解了 83.41%。由POEU1和POEU2制備的納米粒子(NP1和NP2)可高效的包載抗腫瘤藥物阿霉素(DOX),得到了兩種載藥納米粒子,分別命名為NP1/DOX和NP2/DOX。體外藥物釋放實(shí)驗(yàn)結(jié)果表明:NP1/DOX和NP2/DOX具有顯著地酸加速藥物釋放行為,并且釋放速率可以被聚合物的疏水性調(diào)控。在pH 5.0,由疏水性強(qiáng)的POEU2制備的NP2/DOX釋放速率慢于由疏水性相對弱的POEU1制備的NP1/DOX。通過激光共聚焦掃描顯微鏡(CLSM)以及流式細(xì)胞儀分別定性和定量檢測了人神經(jīng)母細(xì)胞瘤細(xì)胞(SH-SY5Y)和鼠源肝癌細(xì)胞(H22)對NP1/DOX和NP2/DOX的攝取情況,結(jié)果表明NP1/DOX和NP2/DOX可被SH-SY5Y和H22細(xì)胞有效地?cái)z取并釋放出阿霉素進(jìn)入細(xì)胞核。通過MTT法檢測空白納米粒子NP1和NP2以及載藥粒子NP1/DOX和NP2/DOX與SH-SY5Y和H22細(xì)胞共培養(yǎng)24 h后的細(xì)胞存活率。結(jié)果顯示,對于空白納米粒子,即使在最大濃度1mg/mL,細(xì)胞的存活率都在90%以上,表明空白納米粒子NP1和NP2基本無毒。然而,NP1/DOX和NP2/DOX可以有效地殺死癌細(xì)胞SH-SY5Y和H22,并呈現(xiàn)出濃度依賴性殺傷作用,即隨著載藥粒子濃度升高,SH-SY5Y和H22細(xì)胞存活率降低。由人神經(jīng)母細(xì)胞瘤細(xì)胞形成的3維多細(xì)胞球(3D SH-SY5Y MCTS)作為一種體外腫瘤模型被用于模擬NP1/DOX和NP2/DOX對腫瘤的滲透和生長抑制作用。結(jié)果表明,隨著NP1/DOX和NP2/DOX與SH-SY5Y MCTS共培養(yǎng)時(shí)間的延長,相比于裸藥(DOX),NP1/DOX和NP2/DOX對SH-SY5Y MCTS表現(xiàn)出了更好的滲透以及更高的生長抑制作用。由鼠源肝癌細(xì)胞H22建立的肝癌小鼠模型被用來檢測兩種載藥納米粒子NP1/DOX和NP2/DOX在體內(nèi)各器官及腫瘤組織的藥物分布,毒副作用以及抗腫瘤效果。體內(nèi)藥物分布結(jié)果表明,與裸藥(DOX)相比,包載在納米粒子中的阿霉素具有更長的血液循環(huán)時(shí)間,更有效的腫瘤富集以及更低的心臟毒性。體內(nèi)抗腫瘤結(jié)果表明,在小鼠處理的7天內(nèi),生理鹽水組以及空白納米粒子組(NP1和NP2)的小鼠腫瘤不斷增大,7天后腫瘤體積增加了 8-9倍,然而裸藥(DOX)組以及兩種載藥粒子組(NP1/DOX和NP2/DOX)的小鼠腫瘤被明顯抑制,且NP1/DOX 和 NP2/DOX 抑制率(72.68%和 70.37%)高于裸藥(DOX)組(62.61%)。最后利用組織切片技術(shù)進(jìn)一步評估NP1/DOX和NP2/DOX的毒副作用以及抗腫瘤效果,結(jié)果顯示,相比于生理鹽水組,裸藥(DOX)組和兩種載藥粒子(NP1/DOX和NP2/DOX)組小鼠的腫瘤組織細(xì)胞數(shù)目顯著下降并出現(xiàn)了不同程度的壞死,并且這一現(xiàn)象在NP1/DOX和NP2/DOX給藥組更為嚴(yán)重。另外裸藥(DOX)組的小鼠心臟壞死嚴(yán)重,然而NP1/DOX和NP2/DOX組小鼠的心臟基本正常且也沒有其他器官毒性。綜上所述,這種生物相容性良好的pH敏感聚(原酸酯-氨酯)在抗腫瘤藥物傳遞領(lǐng)域具有良好的應(yīng)用前景。
[Abstract]:Biodegradable polymer nanomaterials are widely used in antitumor drug delivery because of their high drug loading rate and encapsulation efficiency, good drug distribution in the body and the ability to reduce drug side effects and prolong the circulation time of drugs. Biodegradable polyurethane, in which biodegradable polyurethane is a synthetic biocompatible polymer material, is widely used in various biomedical fields because of its simple preparation method, high efficiency and superior biomedical properties, including drug slow control system and tissue engineering scaffold. Degrading polyesters or polycarbonate are endowed with degradability, but the degradation rate of these polyurethanes is slow and often fails to meet the requirements for the transfer of antitumor drugs. In order to improve the degradation rate of traditional polyurethane, researchers have some environmental responsive chemical bonds (such as pH sensitive ketal bond, acetal bond, hydrazone bond and reduction sensitive two sulfur). Bonds, etc., are introduced into the main chain of polyurethane. As a kind of acid sensitive chemical bond, the original ester bond has been widely used in anti-tumor nano drug carriers. However, the preparation of pH sensitive polyurethane and its application in the field of anti-tumor nanoscale drug carriers have not yet been reported. The two amino monomers and two kinds of polyhexyl glycol (Fw=530 and 2000) active esters with different molecular weights were polycondensation under mild conditions to obtain 2 pH sensitive poly (POEU1 and POEU2), and nanoparticles (NP1 and NP2) with the rate of inhomogeneous degradation were prepared by the monolayer emulsion of water oil (O/W). The structure and molecular weight of poly (tryptophone) were detected by gel permeation chromatography (GPC). The results showed that the structure of POEU1 and POEU2 was correct. The number of molecular weights (Mn) were 2.2 x 104 and 4.09104. transmission electron microscopy (TEM) and dynamic light diffractometer (DLS). The results showed that the particle size distribution of two nanoparticles was around 200 nm and all were spherical. The results show that the two nanoparticles are very stable under the physiological conditions (pH 7.4), while under the weak acid condition (pH 5), the degradation rate and the particle size decrease continuously. In addition, the degradation rate of the two nanoparticles (NP1 and NP2) can be controlled by the hydrophobicity of the polymer under the condition of pH 5. After 192 h, the weak hydrophobic POEU1 is prepared. The nanoparticles (NP1) were completely degraded, and the nanoparticles (NP2), prepared by the hydrophobic POEU2, only degraded the nanoparticles (NP1 and NP2) prepared by POEU1 and POEU2 (NP1 and NP2), which could efficiently encapsulate the antitumor drug adriamycin (DOX). Two kinds of drug loaded nanoparticles were obtained, named as NP1/DOX and NP2/DOX. drug release experimental nodes, respectively. The results showed that NP1/DOX and NP2/DOX significantly accelerated the drug release behavior, and the release rate could be regulated by the hydrophobicity of the polymer. In pH 5, the release rate of NP2/DOX prepared by the hydrophobic POEU2 was slower than the NP1/DOX. which was prepared by the relatively weak hydrophobic POEU1 through the stimulated confocal scanning microscope (CLSM) and the flow cells. The uptake of NP1/DOX and NP2/DOX by human neuroblastoma cells (SH-SY5Y) and mouse derived hepatoma cells (H22) was detected and quantified. The results showed that NP1/DOX and NP2/DOX could be effectively absorbed and released by SH-SY5Y and H22 cells and released adriamycin into the nucleus. The MTT method was used to detect NP1 and NP2 and drug carrier particles by MTT method. The survival rates of NP1/DOX and NP2/DOX with SH-SY5Y and H22 cells were co cultured for 24 h. The results showed that the survival rate of the cells was more than 90% for the blank nanoparticles, even at the maximum concentration of 1mg/mL, indicating that the blank nanoparticles NP1 and NP2 were basically non-toxic. However, NP1/DOX and NP2/ DOX could effectively kill cancer cell SH-SY5Y and cells. There is a concentration dependent killing effect, that is, the survival rate of SH-SY5Y and H22 cells decreases with the increase of the concentration of drug particles. The 3 dimensional multicellular ball (3D SH-SY5Y MCTS) formed by the human neuroblastoma cells is used as an in vitro tumor model to simulate the infiltration and growth inhibition of NP1/DOX and NP2/DOX to the tumor. The results show that with NP1, with NP1 The co culture time of /DOX and NP2/DOX and SH-SY5Y MCTS was prolonged, compared with the naked drug (DOX), NP1/DOX and NP2/DOX showed better permeability and higher growth inhibition to SH-SY5Y MCTS. The mouse model of liver cancer, established by mouse hepatoma cell H22, was used to detect two kinds of drug loaded nanoparticles NP1/DOX and NP2/DOX in the organs and swelling in the body. The distribution, side effects, and antitumor effects of the tumor tissue. The drug distribution in vivo shows that adriamycin, which is loaded in the nanoparticles, has longer blood circulation time, more effective tumor enrichment and lower cardiac toxicity compared with the naked drug (DOX). In vivo antitumor results show that within 7 days of the mice treated, the physiological saline The mice tumor in the group and the blank nanoparticles group (NP1 and NP2) increased continuously, and the tumor volume increased by 8-9 times in 7 days. However, the tumor in the naked drug (DOX) group and the two carrier group (NP1/DOX and NP2/DOX) were obviously suppressed, and the inhibition rate of NP1/DOX and NP2/DOX (72.68% and 70.37%) was higher than that of the naked drug (62.61%) group (62.61%). Finally, the tissue was used in the tissue. The slicing technique further assessed the side effects and antitumor effects of NP1/DOX and NP2/DOX. The results showed that the number of tumor tissue cells in the naked drug (DOX) group and the two drug loaded particles (NP1/DOX and NP2/DOX) mice decreased significantly and had varying degrees of necrosis compared to the saline group, and this phenomenon was given in NP1/DOX and NP2/DOX. In addition, the mice in the DOX group had serious heart necrosis. However, the hearts of the NP1/DOX and NP2/DOX mice were basically normal and no other organ toxicity. In conclusion, the good biocompatible pH sensitive poly (tryptophone) has a good application prospect in the field of antitumor drug delivery.
【學(xué)位授予單位】：安徽大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R730.5
本文編號(hào)：2151726
[Abstract]:Biodegradable polymer nanomaterials are widely used in antitumor drug delivery because of their high drug loading rate and encapsulation efficiency, good drug distribution in the body and the ability to reduce drug side effects and prolong the circulation time of drugs. Biodegradable polyurethane, in which biodegradable polyurethane is a synthetic biocompatible polymer material, is widely used in various biomedical fields because of its simple preparation method, high efficiency and superior biomedical properties, including drug slow control system and tissue engineering scaffold. Degrading polyesters or polycarbonate are endowed with degradability, but the degradation rate of these polyurethanes is slow and often fails to meet the requirements for the transfer of antitumor drugs. In order to improve the degradation rate of traditional polyurethane, researchers have some environmental responsive chemical bonds (such as pH sensitive ketal bond, acetal bond, hydrazone bond and reduction sensitive two sulfur). Bonds, etc., are introduced into the main chain of polyurethane. As a kind of acid sensitive chemical bond, the original ester bond has been widely used in anti-tumor nano drug carriers. However, the preparation of pH sensitive polyurethane and its application in the field of anti-tumor nanoscale drug carriers have not yet been reported. The two amino monomers and two kinds of polyhexyl glycol (Fw=530 and 2000) active esters with different molecular weights were polycondensation under mild conditions to obtain 2 pH sensitive poly (POEU1 and POEU2), and nanoparticles (NP1 and NP2) with the rate of inhomogeneous degradation were prepared by the monolayer emulsion of water oil (O/W). The structure and molecular weight of poly (tryptophone) were detected by gel permeation chromatography (GPC). The results showed that the structure of POEU1 and POEU2 was correct. The number of molecular weights (Mn) were 2.2 x 104 and 4.09104. transmission electron microscopy (TEM) and dynamic light diffractometer (DLS). The results showed that the particle size distribution of two nanoparticles was around 200 nm and all were spherical. The results show that the two nanoparticles are very stable under the physiological conditions (pH 7.4), while under the weak acid condition (pH 5), the degradation rate and the particle size decrease continuously. In addition, the degradation rate of the two nanoparticles (NP1 and NP2) can be controlled by the hydrophobicity of the polymer under the condition of pH 5. After 192 h, the weak hydrophobic POEU1 is prepared. The nanoparticles (NP1) were completely degraded, and the nanoparticles (NP2), prepared by the hydrophobic POEU2, only degraded the nanoparticles (NP1 and NP2) prepared by POEU1 and POEU2 (NP1 and NP2), which could efficiently encapsulate the antitumor drug adriamycin (DOX). Two kinds of drug loaded nanoparticles were obtained, named as NP1/DOX and NP2/DOX. drug release experimental nodes, respectively. The results showed that NP1/DOX and NP2/DOX significantly accelerated the drug release behavior, and the release rate could be regulated by the hydrophobicity of the polymer. In pH 5, the release rate of NP2/DOX prepared by the hydrophobic POEU2 was slower than the NP1/DOX. which was prepared by the relatively weak hydrophobic POEU1 through the stimulated confocal scanning microscope (CLSM) and the flow cells. The uptake of NP1/DOX and NP2/DOX by human neuroblastoma cells (SH-SY5Y) and mouse derived hepatoma cells (H22) was detected and quantified. The results showed that NP1/DOX and NP2/DOX could be effectively absorbed and released by SH-SY5Y and H22 cells and released adriamycin into the nucleus. The MTT method was used to detect NP1 and NP2 and drug carrier particles by MTT method. The survival rates of NP1/DOX and NP2/DOX with SH-SY5Y and H22 cells were co cultured for 24 h. The results showed that the survival rate of the cells was more than 90% for the blank nanoparticles, even at the maximum concentration of 1mg/mL, indicating that the blank nanoparticles NP1 and NP2 were basically non-toxic. However, NP1/DOX and NP2/ DOX could effectively kill cancer cell SH-SY5Y and cells. There is a concentration dependent killing effect, that is, the survival rate of SH-SY5Y and H22 cells decreases with the increase of the concentration of drug particles. The 3 dimensional multicellular ball (3D SH-SY5Y MCTS) formed by the human neuroblastoma cells is used as an in vitro tumor model to simulate the infiltration and growth inhibition of NP1/DOX and NP2/DOX to the tumor. The results show that with NP1, with NP1 The co culture time of /DOX and NP2/DOX and SH-SY5Y MCTS was prolonged, compared with the naked drug (DOX), NP1/DOX and NP2/DOX showed better permeability and higher growth inhibition to SH-SY5Y MCTS. The mouse model of liver cancer, established by mouse hepatoma cell H22, was used to detect two kinds of drug loaded nanoparticles NP1/DOX and NP2/DOX in the organs and swelling in the body. The distribution, side effects, and antitumor effects of the tumor tissue. The drug distribution in vivo shows that adriamycin, which is loaded in the nanoparticles, has longer blood circulation time, more effective tumor enrichment and lower cardiac toxicity compared with the naked drug (DOX). In vivo antitumor results show that within 7 days of the mice treated, the physiological saline The mice tumor in the group and the blank nanoparticles group (NP1 and NP2) increased continuously, and the tumor volume increased by 8-9 times in 7 days. However, the tumor in the naked drug (DOX) group and the two carrier group (NP1/DOX and NP2/DOX) were obviously suppressed, and the inhibition rate of NP1/DOX and NP2/DOX (72.68% and 70.37%) was higher than that of the naked drug (62.61%) group (62.61%). Finally, the tissue was used in the tissue. The slicing technique further assessed the side effects and antitumor effects of NP1/DOX and NP2/DOX. The results showed that the number of tumor tissue cells in the naked drug (DOX) group and the two drug loaded particles (NP1/DOX and NP2/DOX) mice decreased significantly and had varying degrees of necrosis compared to the saline group, and this phenomenon was given in NP1/DOX and NP2/DOX. In addition, the mice in the DOX group had serious heart necrosis. However, the hearts of the NP1/DOX and NP2/DOX mice were basically normal and no other organ toxicity. In conclusion, the good biocompatible pH sensitive poly (tryptophone) has a good application prospect in the field of antitumor drug delivery.
【學(xué)位授予單位】：安徽大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R730.5
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