有機金屬框架納米載藥系統(tǒng)的構(gòu)建
發(fā)布時間：2018-07-29 14:04
【摘要】：金屬-有機骨架(Metal-Organic Frameworks, MOFs)是近年來新型的多孔式有機-無機雜化的功能性的材料。與其他功能性材料相比,M O F s具有結(jié)構(gòu)可變性和易功能化的特征,可同時實現(xiàn)較高的載藥量和適宜的藥物控制釋放的能力。本課題構(gòu)建了納米有機金屬框架Cu-BTC載藥系統(tǒng)并成功將抗腫瘤代表性藥物5-氟尿嘧啶(5-FU)包封于載體中,初步探索了有機金屬框架納米載藥系統(tǒng)構(gòu)建的可行性及規(guī)律。本文首先考察載體Cu-BTC的制備,以粒徑、載體形態(tài)、分散性為指標,單因素考察法分別考察溶劑熱法和配位調(diào)控法的影響因素。結(jié)果表明,本文所用的配位調(diào)控法制備出的載體粒徑均為納米級,且分散度較好。下一步對配位調(diào)控法中反應物濃度、攪拌速度、滴定時間用正交試驗進一步優(yōu)化,優(yōu)化后確定最佳因素條件為:反應物濃度為二倍濃度(即:醋酸銅0.36g,苯甲酸4g,均苯三甲酸0.8g)、滴定時間為1.5h、攪拌速度為1000轉(zhuǎn)/分鐘。綜合試驗結(jié)果得出最優(yōu)的制備工藝為:將0.36g醋酸銅和4g苯甲酸(調(diào)控劑)溶于30mL 正丁醇中,將0.8g均苯三甲酸溶于30mLDMF溶液當中,攪拌兩種溶液至完全溶解。室溫攪拌,同時將配體溶液滴入到金屬鹽溶液中(邊滴加邊攪拌),滴定時間約為1.5h,然后攪拌30min。攪拌速度為1000r/min,將產(chǎn)物離心,用乙醇洗滌2次,超聲分散。所得產(chǎn)物常溫干燥,稱量。對所合成的納米載體進行了 XRD定性鑒別,結(jié)果表明樣品X射線衍射圖譜和由單晶結(jié)構(gòu)模擬的Cu-BTC標準物質(zhì)十分吻合。SEM和粒徑分布儀查看載體形態(tài),測定粒徑分布。結(jié)果SEM測得載體粒徑大小在50～1 OOnm之間, 而粒徑分布儀測定粒徑在270nm左右。這是由于載體分散于水中時,水分子包裹在載體周圍,使粒徑增大。此外,從SEM圖譜可見載體形態(tài)呈規(guī)整的圓球形,不粘連,分散性好。TEM和氮氣吸附試驗描述了載體中孔徑的大小及分布,經(jīng)證實,載體中孔徑大小約為2到5nm。適宜載入粒徑小于此范圍的藥物。熱重分析曲線證明160℃以內(nèi)為最佳活化溫度,當溫度升至310℃左右時,框架結(jié)構(gòu)發(fā)生改變,故本載體有較高的熱穩(wěn)定性,與文獻報道的熱穩(wěn)定性基本吻合[84]。最后,我們對載體在血液中的溶血性進行考察,相關(guān)圖片證明,載體具有較好的血液相容性。本課題用響應面法考察最佳載藥工藝,首先以載藥量為指標,通過單因素法篩選載藥的影響因素及各因素水平值的最優(yōu)區(qū)間。相關(guān)數(shù)據(jù)及圖譜表明,藥載比、載藥時間、乙醇濃度對載藥量影響較大。以這三個因素為自變量,通過響應面法考察最優(yōu)載藥工藝。三維曲面圖和等高線圖得到優(yōu)化后條件為乙醇濃度70%;藥載比7:1;載藥時間96h時,載藥量最高。工藝驗證結(jié)果,載藥量均達到30%以上,證明優(yōu)化后的工藝穩(wěn)定,操作簡便,結(jié)果可信。對載藥后有機金屬框架進行表征,SEM表征結(jié)果顯示載藥前后載體大小及形態(tài)均無明顯改變,說明載藥過程對載體形態(tài)無影響;XRD廣角衍射表征結(jié)果載藥前后圖譜無明顯差異;小角衍射可見載藥后載體孔道發(fā)生變化,證明藥物載入載體的孔道中;差示掃描量熱法中DTA曲線證明載藥過程對納米有機金屬骨架的熱穩(wěn)定性不產(chǎn)生影響。體外釋放試驗是通過對比正向動態(tài)透析法和反向動態(tài)透析法得到的,并分別以零級動力學方程、一級動力學方程和Higuchi方程對上述兩種方法測得的體外釋藥數(shù)據(jù)進行擬合。結(jié)果表明,兩種方法測得的數(shù)據(jù)均較符合一級動力學方程,兩種方法釋放曲線均顯示出在剛開始的1小時表現(xiàn)為突釋,這是由于吸附在載體表面的藥物經(jīng)擴散進入釋放介質(zhì)所致,并隨后出現(xiàn)平穩(wěn)的釋放效果。MTT法測定FITC-5-FU-Cu-BTC體外細胞毒性,結(jié)果表明,空載體的毒性和5-FU藥物的毒性相當,FITC-Cu-BTC毒性較高;熒光倒置顯微鏡測定細胞對FITC-5-FU-Cu-BTC的攝取可知,載藥載體可被細胞吸收,并隨著樣品濃度的增加吸收的量明顯加大;由流式細胞儀測定的結(jié)果可見,FITC-5-FU-Cu-BTC細胞攝取過程呈飽和性和溫度依賴性,初步推測是通過內(nèi)吞途徑被細胞攝取的。
[Abstract]:Metal-Organic Frameworks (MOFs) is a new porous organic-inorganic hybrid functional material in recent years. Compared with other functional materials, M O F s has the characteristics of structural variability and functionalization, which can simultaneously achieve higher drug loading and appropriate drug control release ability. This topic has been constructed. The nano organic metal frame Cu-BTC drug loading system was successfully encapsulated in the carrier of the antitumor drug 5- fluorouracil (5-FU). The feasibility and regularity of the construction of the organic metal frame nano drug loading system were preliminarily explored. First, the preparation of the carrier Cu-BTC was investigated with the particle size, the carrier morphology and dispersion as the index, and the single factor investigation method was used. The influence factors of the solvent heat method and coordination regulation method are not investigated. The results show that the size of the carrier prepared by the coordination method in this paper is all nanoscale, and the degree of dispersion is good. The next step is to optimize the concentration of reactants, stirring speed and titration time in the coordination control method with orthogonal test. The concentration is two times concentration (i.e., copper acetate 0.36g, 4G benzoic acid, 0.8g of benzoic acid three formic acid), the titration time is 1.5h, and the stirring speed is 1000 rpm. The optimum preparation process is that 0.36g copper acetate and 4G benzoic acid (regulators) are dissolved in 30mL Ding Chunzhong, and 0.8g mean three formic acid is dissolved in 30mLDMF solution. Two solutions are completely dissolved. At room temperature, at the same time, the ligand solution is dripped into the metal salt solution (side drop and agitation), the titration time is about 1.5h, then the stirring speed of 30min. is 1000r/min, the product is centrifuged, 2 times washed with ethanol and ultrasonic dispersion. The product is dry at normal temperature and weighed. The synthesized nano carrier is XRD The results show that the X ray diffraction pattern of the sample and the Cu-BTC standard material simulated by the single crystal structure are in good agreement with the.SEM and the particle size distribution instrument to examine the carrier morphology and determine the particle size distribution. Results the size of the carrier particle size of the carrier is between 50~1 OOnm and the particle size distribution meter is about 270nm. This is due to the dispersion of the carrier in the water. In addition, the water molecules are wrapped around the carrier to make the particle size increase. In addition, it can be seen from the SEM map that the shape of the carrier is a regular round ball and does not adhere. The size and distribution of the pore size in the carrier is described by the good dispersibility.TEM and nitrogen adsorption test. It is proved that the size of the pore size of the carrier is about 2 to 5nm. and is suitable for carrying the drug with smaller size smaller than this range. The analysis curve proves that the optimum activation temperature is within 160 C. When the temperature rises to about 310 C, the frame structure changes. Therefore, the carrier has high thermal stability, which basically coincides with the thermal stability reported in the literature [84].. Finally, we examine the hemolysis of the carrier in the blood. The related pictures show that the carrier has better blood. The best drug loading process was investigated by response surface method. First, the influence factors of drug loading and the optimal range of the level of each factor were selected by the single factor method. The related data and Atlas showed that the drug load ratio, the time of drug loading and the concentration of ethanol had great influence on the drug loading. These three factors were the independent variables and passed the noise. The optimum drug loading process was investigated by surface method. The optimized conditions were ethanol concentration 70%, drug loading ratio 7:1, and drug loading time 96h, the drug loading was the highest. The results of the process validation were more than 30%, which proved that the optimized process was stable, the operation was simple and the results were credible. The organic metal frame after the drug loading was characterized, The results of SEM showed that the size and morphology of the carrier before and after the drug loading had no obvious changes, indicating that the drug loading process had no influence on the carrier morphology, and there was no obvious difference in the spectrum of the carrier before and after the XRD wide angle diffraction; the small angle diffraction showed that the carrier channel changes after the drug loading, which proved that the drug loaded into the channel of the carrier, and the DTA curve in the differential scanning calorimetry. It is proved that the drug loading process has no effect on the thermal stability of the nanoscale organometallic skeleton. In vitro release test is obtained by comparing the forward dynamic dialysis method and the reverse dynamic dialysis method, and the above two methods are fitted with zero order kinetic equation, first order dynamic equation and Higuchi equation. The results showed that the data obtained by the two methods were all in accordance with the first order kinetic equation, and the release curves of the two methods showed a sudden release at the beginning of the first 1 hours, which was due to the diffusion of drugs adsorbed on the surface of the carrier and into the release medium, and then the.MTT method was used for the determination of FITC-5-FU-Cu-BTC cells in vitro. Toxicity, the results showed that the toxicity of the empty carrier was equal to the toxicity of the 5-FU drug, and the toxicity of the FITC-Cu-BTC was high. The fluorescence inversion microscope determined the uptake of FITC-5-FU-Cu-BTC by cells, and the amount of the carrier could be absorbed by the cell and increased with the increase of the concentration of the sample; the results measured by the flow cytometer can be seen, FITC-5-FU-Cu-B The uptake process of TC cells is saturated and temperature dependent. It is presumably presumed to be absorbed by cells through endocytosis pathway.
【學位授予單位】：黑龍江中醫(yī)藥大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R943
本文編號：2152911
[Abstract]:Metal-Organic Frameworks (MOFs) is a new porous organic-inorganic hybrid functional material in recent years. Compared with other functional materials, M O F s has the characteristics of structural variability and functionalization, which can simultaneously achieve higher drug loading and appropriate drug control release ability. This topic has been constructed. The nano organic metal frame Cu-BTC drug loading system was successfully encapsulated in the carrier of the antitumor drug 5- fluorouracil (5-FU). The feasibility and regularity of the construction of the organic metal frame nano drug loading system were preliminarily explored. First, the preparation of the carrier Cu-BTC was investigated with the particle size, the carrier morphology and dispersion as the index, and the single factor investigation method was used. The influence factors of the solvent heat method and coordination regulation method are not investigated. The results show that the size of the carrier prepared by the coordination method in this paper is all nanoscale, and the degree of dispersion is good. The next step is to optimize the concentration of reactants, stirring speed and titration time in the coordination control method with orthogonal test. The concentration is two times concentration (i.e., copper acetate 0.36g, 4G benzoic acid, 0.8g of benzoic acid three formic acid), the titration time is 1.5h, and the stirring speed is 1000 rpm. The optimum preparation process is that 0.36g copper acetate and 4G benzoic acid (regulators) are dissolved in 30mL Ding Chunzhong, and 0.8g mean three formic acid is dissolved in 30mLDMF solution. Two solutions are completely dissolved. At room temperature, at the same time, the ligand solution is dripped into the metal salt solution (side drop and agitation), the titration time is about 1.5h, then the stirring speed of 30min. is 1000r/min, the product is centrifuged, 2 times washed with ethanol and ultrasonic dispersion. The product is dry at normal temperature and weighed. The synthesized nano carrier is XRD The results show that the X ray diffraction pattern of the sample and the Cu-BTC standard material simulated by the single crystal structure are in good agreement with the.SEM and the particle size distribution instrument to examine the carrier morphology and determine the particle size distribution. Results the size of the carrier particle size of the carrier is between 50~1 OOnm and the particle size distribution meter is about 270nm. This is due to the dispersion of the carrier in the water. In addition, the water molecules are wrapped around the carrier to make the particle size increase. In addition, it can be seen from the SEM map that the shape of the carrier is a regular round ball and does not adhere. The size and distribution of the pore size in the carrier is described by the good dispersibility.TEM and nitrogen adsorption test. It is proved that the size of the pore size of the carrier is about 2 to 5nm. and is suitable for carrying the drug with smaller size smaller than this range. The analysis curve proves that the optimum activation temperature is within 160 C. When the temperature rises to about 310 C, the frame structure changes. Therefore, the carrier has high thermal stability, which basically coincides with the thermal stability reported in the literature [84].. Finally, we examine the hemolysis of the carrier in the blood. The related pictures show that the carrier has better blood. The best drug loading process was investigated by response surface method. First, the influence factors of drug loading and the optimal range of the level of each factor were selected by the single factor method. The related data and Atlas showed that the drug load ratio, the time of drug loading and the concentration of ethanol had great influence on the drug loading. These three factors were the independent variables and passed the noise. The optimum drug loading process was investigated by surface method. The optimized conditions were ethanol concentration 70%, drug loading ratio 7:1, and drug loading time 96h, the drug loading was the highest. The results of the process validation were more than 30%, which proved that the optimized process was stable, the operation was simple and the results were credible. The organic metal frame after the drug loading was characterized, The results of SEM showed that the size and morphology of the carrier before and after the drug loading had no obvious changes, indicating that the drug loading process had no influence on the carrier morphology, and there was no obvious difference in the spectrum of the carrier before and after the XRD wide angle diffraction; the small angle diffraction showed that the carrier channel changes after the drug loading, which proved that the drug loaded into the channel of the carrier, and the DTA curve in the differential scanning calorimetry. It is proved that the drug loading process has no effect on the thermal stability of the nanoscale organometallic skeleton. In vitro release test is obtained by comparing the forward dynamic dialysis method and the reverse dynamic dialysis method, and the above two methods are fitted with zero order kinetic equation, first order dynamic equation and Higuchi equation. The results showed that the data obtained by the two methods were all in accordance with the first order kinetic equation, and the release curves of the two methods showed a sudden release at the beginning of the first 1 hours, which was due to the diffusion of drugs adsorbed on the surface of the carrier and into the release medium, and then the.MTT method was used for the determination of FITC-5-FU-Cu-BTC cells in vitro. Toxicity, the results showed that the toxicity of the empty carrier was equal to the toxicity of the 5-FU drug, and the toxicity of the FITC-Cu-BTC was high. The fluorescence inversion microscope determined the uptake of FITC-5-FU-Cu-BTC by cells, and the amount of the carrier could be absorbed by the cell and increased with the increase of the concentration of the sample; the results measured by the flow cytometer can be seen, FITC-5-FU-Cu-B The uptake process of TC cells is saturated and temperature dependent. It is presumably presumed to be absorbed by cells through endocytosis pathway.
【學位授予單位】：黑龍江中醫(yī)藥大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R943
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