【摘要】：目的： 脂質(zhì)體已經(jīng)成為生物醫(yī)藥技術(shù)領(lǐng)域里，最主要的藥物、基因、影像劑的重要載體，是一種非病毒載體類(lèi)載體，在基因傳遞中具有顯著優(yōu)勢(shì)。但仍然存在生物大分子負(fù)載率低、攝取效率低、胞內(nèi)釋放性能欠佳、內(nèi)涵體逃逸障礙等問(wèn)題。因此，本文重在設(shè)計(jì)一種多能高效的生物大分子輸送載體。 本文合成了能高效提高轉(zhuǎn)染效率的DC-Chol，以及賦予脂質(zhì)體pH敏感性能的CHEMS，對(duì)其化學(xué)結(jié)構(gòu)進(jìn)行表征。將DSPE-PEG(2000)與CPP共軛連接，得到導(dǎo)向性脂材DSPE-PEG-CPP，以提高脂質(zhì)體對(duì)細(xì)胞的攝取能力。構(gòu)建了一種能使siRNA沖破內(nèi)涵體逃逸障礙，避免被溶酶體滅活的CPP修飾載壓縮物脂質(zhì)體CL2，和一種CPP修飾的非載壓縮脂質(zhì)體CL1，并對(duì)其進(jìn)行表征和初步的評(píng)價(jià)。 方法：1膽固醇氯甲酸酯和過(guò)量N，N-二甲基乙二胺分別溶于無(wú)醇氯仿中，0-4℃冰水浴條件下反應(yīng)。TLC監(jiān)測(cè)反應(yīng)進(jìn)程，待膽固醇氯甲酸酯反應(yīng)完全后終止反應(yīng)（約2h），旋蒸揮干溶劑，干燥后用熱絕對(duì)乙醇重結(jié)晶兩次純化并真空干燥。2膽固醇與過(guò)量的丁二酸酐溶于20mL甲苯， DMAP作為催化劑，加熱回流反應(yīng)3h。旋轉(zhuǎn)蒸發(fā)揮干溶劑，干燥后重結(jié)晶兩次，得到灰色產(chǎn)品。3Mal-PEG-DSPE用氯仿-甲醇（3:1）溶解，旋轉(zhuǎn)成膜，HBS(pH6.5)溶液3.0mL水化，和CPP肽溶液（5mg溶于HBS(pH6.5)2.0mL）混合，攪拌反應(yīng)48h，加入半胱氨酸適量繼續(xù)攪拌4h，透析2d，冷凍干燥。4空白脂質(zhì)體單因素考察，通過(guò)固定膜材總量；DOPE:CHEMS比例的篩選；添加Chol對(duì)制劑的影響；水化液選擇；超聲時(shí)間的選擇，最終確定空白脂質(zhì)體制備方法。5構(gòu)建非壓縮載藥脂質(zhì)體（NL1/CL1）和載壓縮物脂質(zhì)體（NL2和CL2），并進(jìn)粒徑、電位和電鏡表征。 制備方法如下： 分別以DSPE-PEG、DSPE-PEG（CPP）成膜，DEPC水溶液水化，超聲得膠束M1、M2。 非壓縮載藥脂質(zhì)體（NL1/CL1）：以SPC/Chol/DC-Chol為脂材成膜，含siRNA的DEPC溶液水化脫膜，水浴超聲10-15min，得到普通載藥脂質(zhì)體。通過(guò)后插入法分別與膠束M1和M2孵育4h，，得NL1和CL1。 載壓縮物脂質(zhì)體（NL2和CL2）：以DOPE/CHEMS/Chol為脂材成膜，DEPC水化液脫膜，超聲得空白脂質(zhì)體，與siRNA壓縮物混合孵育后,冰浴條件調(diào)至pH7.4-7.8，再通過(guò)后插入法制備N(xiāo)L2和CL2。6以流式細(xì)胞術(shù)分析包載siRNA的NL1＆CL1和包載壓縮siRNA的NL2＆CL2處理的MCF-7細(xì)胞，以陽(yáng)性細(xì)胞百分?jǐn)?shù)和胞內(nèi)平均熒光強(qiáng)度（MFI）為考察指標(biāo)，評(píng)價(jià)細(xì)胞攝取效率。 結(jié)果：1合成DC-Chol：白色固體粉末1.621g,產(chǎn)率達(dá)到60.3%;熔點(diǎn):107-108℃;TLC(氯仿-甲醇=65：35（v/v）,10%H2SO4顯色)， Rf=0.56，顯示為純品;質(zhì)譜圖中m/z501.6，與DC-Chol的理論計(jì)算值501.80（M+H+）相符；1HNMR：δ,2.301（s，6H）；δ，2.495（t，2H）； δ，3.305，3.296（q，2H）；δ，5.365（s，1H)。證明產(chǎn)物結(jié)構(gòu)正確。2合成CHEMS：將無(wú)水乙醇重結(jié)晶干燥后，得到淺灰色固體粉末0.49g，產(chǎn)率78%；熔點(diǎn)：173-176℃；TLC測(cè)定（氯仿：甲醇（10:1，v/v），新生成斑點(diǎn)Rf=0.10，顯示為純品；質(zhì)譜圖中有離子峰m/z485.5，與CHEMS的理論計(jì)算值為485.5（M-H+）相符。證明產(chǎn)物結(jié)構(gòu)正確。3合成DSPE-PEG-CPP：得到白色絮狀物；通過(guò)Ellman試劑測(cè)得CPP反應(yīng)率為84.4%；MS+檢測(cè)，分子離子峰m/z4469.17，與設(shè)計(jì)肽平理論計(jì)算值相對(duì)應(yīng)。證明產(chǎn)物為目標(biāo)化合物為DSPE-PEG-CPP。4通過(guò)單因素考察，確定脂質(zhì)體制備工藝： 精密量取DOPE、CHEMS、Chol按5:2:2（mol/mol/mol）成膜，加入37℃DEPC水溶液2.0mL水化，超聲10-15min，得到帶有淡藍(lán)色乳光的半透明體系，測(cè)粒徑。5構(gòu)建了非壓縮載藥脂質(zhì)體（NL1/CL1）和載壓縮物脂質(zhì)體（NL2和CL2），并進(jìn)粒徑、電位和電鏡表征。 NL1、CL1的粒徑分別為226.8nm、262.4nm；后者PdI較小，粒徑分布窄;Zeta分別為37.0mV、55.7mV，體系穩(wěn)定；電鏡下觀察，粒徑約200nm左右。 NL2、CL2粒徑分別為289.8nm、293.2nm；兩組制劑PdI都很小，粒徑分布窄；Zeta分別為-29.6mV、-29.2mV，脂質(zhì)體表面帶負(fù)電，體系趨于穩(wěn)定;電鏡下觀察，粒徑約200nm左右。6采用流式細(xì)胞分析術(shù)分析經(jīng)藥物制劑處理的細(xì)胞懸液 非壓縮制劑組N-L和C-L進(jìn)入細(xì)胞的熒光強(qiáng)度X-mean分別為9.1,10.4，是陰性對(duì)照組和游離siRNA組的20倍左右，由此說(shuō)明CPP修飾非壓縮載藥脂質(zhì)體（C-L制劑）、普通修飾非壓縮載藥脂質(zhì)體（N-L）均提高了細(xì)胞攝取率，CPP修飾非壓縮載藥脂質(zhì)體具有更高的細(xì)胞攝取率。 載壓縮物制劑組N-L’、C-L’陽(yáng)性細(xì)胞百分比為6.4%、7.9%，其陽(yáng)性細(xì)胞百分?jǐn)?shù)均有顯著變化，提高細(xì)胞攝取效率約10倍，表明載壓縮物制劑均提高了細(xì)胞攝取效率，CPP修飾載壓縮物脂質(zhì)體具有更高的細(xì)胞攝取率。 結(jié)論： 實(shí)驗(yàn)結(jié)果表明，DC-Chol以及CHEMS合成路線(xiàn)可靠，產(chǎn)率分別為60.3%、78%，產(chǎn)品純度較高，重現(xiàn)性高。 由Ellman試劑判斷反應(yīng)進(jìn)程，質(zhì)譜測(cè)定得出的分子離子峰推測(cè)產(chǎn)物結(jié)構(gòu)，成功地將CPP共軛連接到DSPE-PEG（2000）中的PEG遠(yuǎn)端，得到增強(qiáng)細(xì)胞穿透能力的導(dǎo)向性脂材DSPE-PEG（2000）-CPP。 成功地制備了四組遞送siRNA的遞送納米脂質(zhì)載體CPP修飾非壓縮載藥脂質(zhì)體（NL1）、普通修飾非壓縮載藥脂質(zhì)體（NL2）、普通修飾在壓縮物脂質(zhì)體（CL1）、CPP修飾載壓縮物脂質(zhì)體（CL2）。 四組制劑均提高了細(xì)胞攝取效率。普通脂質(zhì)體與CPP修飾的脂質(zhì)體的細(xì)胞攝取相對(duì)較高，但差異并不顯著。導(dǎo)致這個(gè)結(jié)果的可能的原因是，如DSPE-PEG-CPP合成產(chǎn)物中有較多剩余脂材原料，反應(yīng)條件有待進(jìn)一步優(yōu)化，產(chǎn)物純度有待提高。
[Abstract]:Purpose: The liposome has become one of the most important carriers of medicine, gene and image agent in the technical field of biological medicine, However, there are still some questions about the low loading rate of the biological macromolecule, the low uptake efficiency, the poor in-cell release performance, the obstacle of the escape of the connotation, etc. Therefore, this paper focuses on the design of a multi-functional and efficient biological macromolecule transport carrier In this paper, we synthesize the DC-Chol which can improve the efficiency of the transfection, and the CHEMS which gives the pH-sensitive properties of the liposomes to the chemical structure. Line characterization. The DSPE-PEG (2000) was co-linked to the CPP to obtain a guided lipid, DSPE-PEG-CPP, to increase the uptake of the liposome to the cells. The invention provides a CPP modified carrier liposome CL2 which can cause the siRNA to break through the connotation body to escape, avoid the CPP modified carrier liposome CL2 which is inactivated by the lysosome, and a CPP modified non-loaded compressed liposome CL1, The method comprises the following steps:1-cholesterol chloroformate and an excess of N, N-dimethyl ethylenediamine are respectively dissolved in the alcohol-free chloroform, and the ice water bath at the temperature of 0-4 DEG C under conditions, the reaction is monitored by TLC, the reaction process is monitored by TLC, the reaction is terminated after the reaction of the chloroformate is complete (about 2 hours), the solvent is evaporated and dried, the solvent is recrystallized twice with hot absolute ethanol and dried in vacuum, and the cholesterol and the excess of the butylenic acid are dissolved in 20 mL of toluene and the DMAP As a catalyst, heat back The reaction was carried out for 3 h. The dry solvent was evaporated and recrystallized twice to obtain the gray product. The 3Mal-PEG-DSPE was dissolved with chloroform-methanol (3:1), the film was spin-formed, the HBS (pH 6.5) solution was hydrated, and the CPP peptide solution (5 mg dissolved in HBS (pH 6.5) 2.0 mL) was mixed, stirred for 48 h, and a proper amount of cysteine was added for stirring for 4 h. , dialyzing for 2d, freeze-drying.4 blank liposome single-factor investigation, through fixed membrane material total amount; DOPE: CHEMS ratio selection; addition of Chol on preparation; hydration liquid selection; ultrasound The preparation method of the blank liposome is finally determined by the time selection, and the non-compressed drug-loaded liposome (NL1/ CL1) and the carrier-loaded liposome (NL2 and CL2) are constructed, and the particle size and the potential are kept. And electron microscopy. The preparation method comprises the following steps: respectively forming DSPE-PEG and DSPE-PEG (CPP), and hydrating the DEPC aqueous solution, Acoustic micelle M1, M2. Non-compressed drug-loaded liposomes (NL1/ CL1): film-forming with SPC/ Chol/ DC-Chol, water-bath ultrasonic 10-15 mi, water bath ultrasound 10-15 mi, n, the general drug-carrying liposome is obtained, and the drug-carrying liposome is respectively incubated with the micelles M1 and M2 through the post-insertion method, and 4 hours to obtain NL1 and CL1, carrying the compressed object liposome (NL2 and CL2), taking DOPE/ CHEMS/ Chol as a lipid material to form a film, decoating the DEPC hydration liquid, performing ultrasonic to obtain a blank liposome, and mixing the liposome with the siRNA compact, and then carrying out ice bath The conditions were adjusted to pH 7.4-7.8, and then NL2 and CCL2 were prepared by the post-insertion method. The NL1 & CL1 and NL2 & CL2-treated MCF-7 cells of the encapsulated siRNA were analyzed by flow cytometry, and the percentage of positive cells and the intracellular mean fluorescence intensity (MFI) were The results were as follows:1. Synthesis of DC-Chol: white solid powder 1.621 g, yield of 60.3%; melting point:107-108 鈩

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