pH響應(yīng)的PEG化基因傳遞體系

劉亞杰, 張　鵬, 杜建委, 王幽香

(浙江大學(xué)高分子合成與功能構(gòu)造教育部重點(diǎn)實(shí)驗(yàn)室, 高分子科學(xué)與工程學(xué)系, 杭州 310027)

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pH響應(yīng)的PEG化基因傳遞體系

劉亞杰, 張鵬, 杜建委, 王幽香

(浙江大學(xué)高分子合成與功能構(gòu)造教育部重點(diǎn)實(shí)驗(yàn)室, 高分子科學(xué)與工程學(xué)系, 杭州 310027)

摘要制備了苯甲酰亞胺鍵偶聯(lián)的聚乙二醇化(PEG化)聚乙烯亞胺(mPEG-CHN-PEI), 并以還原無pH響應(yīng)特性mPEG-PEI作為對(duì)照. 研究結(jié)果表明, PEG鏈段的引入并未影響聚乙烯亞胺與脫氧核糖核酸(DNA)分子的締合, 形成了尺寸為80 nm, 表面電位約為10 mV的基因超分子組裝體, 具有很好的生理鹽穩(wěn)定性. 在模擬溶酶體的酸性環(huán)境下, 苯甲酰亞胺鍵有效斷裂, 顯示出很好的pH響應(yīng)特性. HepG2細(xì)胞培養(yǎng)結(jié)果表明, 由于PEG鏈段有效屏蔽了組裝體表面的正電荷, PEG化組裝體的細(xì)胞毒性和內(nèi)吞效率顯著降低, 但溶酶體酸性條件使苯甲酰亞胺鍵斷裂, 有利于組裝體逃離溶酶體, 因此mPEG-CHN-PEI依然具有很高的基因轉(zhuǎn)染效率, 實(shí)現(xiàn)了基因載體細(xì)胞外穩(wěn)定傳遞、 細(xì)胞內(nèi)響應(yīng)解離并高效轉(zhuǎn)染的功能.

關(guān)鍵詞非病毒基因載體; 聚乙烯亞胺; pH響應(yīng); 聚乙二醇化

基因治療是將人的正常基因或有治療作用的基因通過一定方式導(dǎo)入人體靶細(xì)胞, 以糾正基因缺陷或發(fā)揮治療作用, 從而達(dá)到治療疾病目的的生物醫(yī)學(xué)新技術(shù)[1]. 基因治療技術(shù)的發(fā)展有賴于安全有效的基因載體[2,3].Boussif等[4,5]以聚乙烯亞胺(PEI)作為非病毒載體, 其獨(dú)特的“質(zhì)子海綿”效應(yīng)有利于逃離溶酶體并進(jìn)行高效轉(zhuǎn)染, 成為非病毒基因載體的黃金標(biāo)準(zhǔn). 由于聚陽離子/基因組裝體在體內(nèi)易發(fā)生聚集, 從而被網(wǎng)狀內(nèi)皮系統(tǒng)清除, 導(dǎo)致轉(zhuǎn)染效率降低[6]. 開發(fā)細(xì)胞外穩(wěn)定傳遞、 細(xì)胞內(nèi)響應(yīng)解離并高效轉(zhuǎn)染的基因傳遞體系是當(dāng)前的研究熱點(diǎn)[7].

1實(shí)驗(yàn)部分

1.1試劑與儀器

支化聚乙烯亞胺(bPEI, 重均分子量為2.5×104)、 聚乙二醇甲醚(mPEG, 數(shù)均分子量為5×103)和對(duì)甲酰基苯甲酸和四甲基偶氮唑鹽(MTT),Aldrich-Sigma公司; 二環(huán)己基碳二亞胺(DCC)、 4-二甲氨基吡啶(DMAP)和硼氫化鈉(NaBH4), 阿拉丁試劑(上海)有限公司; 魚精蛋白脫氧核糖核酸(鈉鹽)、 N-2-羥乙基哌嗪-N-2-乙磺酸(Hepes)和Cy3-DNA, 生工生物工程(上海)股份有限公司;Dulbecco’smodifiedeaglemedium(DMEM)高糖培養(yǎng)基,Gibco公司; 胎牛血清(FBS), 杭州四季青生物工程材料有限公司. 美國Mercury-300MHz核磁共振波譜儀(NMR); 英國Malvern公司ZetasizerNanoZS型光散射粒徑分布儀(DLS); 日本NEC公司JEM-1200EX型透射電子顯微鏡(TEM); 美國BectonDickinson公司FACSCalibur型流式細(xì)胞儀; 美國Bio-Rad公司550型酶標(biāo)儀及凝膠成像系統(tǒng); 德國LeicaTSSP5型激光共聚焦顯微鏡(CLSM).

1.3基因超分子組裝體的制備

按同樣的方法制備mPEG-PEI/DNA與PEI/DNA超分子組裝體作為對(duì)照. 用凝膠電泳研究超分子組裝體的締合特性; 用光散射粒徑分布儀(DLS)測定超分子組裝體的粒徑及表面電位; 用TEM測定樣品的微觀形貌.

1.4基因超分子組裝體的穩(wěn)定性及pH響應(yīng)性實(shí)驗(yàn)

1.5細(xì)胞毒性

1.6體外細(xì)胞轉(zhuǎn)染

1.7細(xì)胞內(nèi)吞率

1.8胞內(nèi)分布

2結(jié)果與討論

Fig.1　1H NMR spectra of mPEG-CHO in CDCl3(a) and mPEG-CHN-PEI in D2O(b)

2.2基因超分子組裝體的基本特性

高效轉(zhuǎn)染的聚陽離子載體必須具有良好的DNA締合能力以保護(hù)其生物活性[17]. 采用凝膠電泳研究PEG化基因載體的DNA締合能力, 結(jié)果見圖2. 當(dāng)N/P比大于2時(shí),PEG化基因載體的DNA的自由條帶均消失, 表明PEG鏈段的引入并未明顯影響聚陽離子締合DNA的能力. 同時(shí), 聚陽離子締合DNA形成尺寸合適的納米粒子是聚陽離子高效轉(zhuǎn)染的基本條件[18]. 用DLS測定基因超分子組裝體的尺寸及表面電位, 結(jié)果示于圖3. 在N/P比為10和20時(shí),PEI能與DNA通過靜電組裝形成尺寸

Fig.2　Agarose gel electrophoresis assay of mPEG-CHN-PEI/DNA(A), mPEG-PEI/DNA(B), PEI/DNA(C) in 20 mmol/L Hepes buffer solution(pH=7.4, 20 mmol/L NaCl) with various N/P ratios

Fig.3　Particle sizes(A) and ζ-potentials(B) of different polyplexes in 20 mmol/L Hepes buffer solution(pH=7.4, 20 mmol/L NaCl) Error bars represent mean±SD for n=3. * Denotes statistically significant difference at p<0.05.

Fig.4　Typical TEM images of gene polyplexes at different N/P ratios All the polyplexes were prepared in 20 mmol/L Hepes buffer solution(pH=7.4, 20 mmol/L NaCl). (A) mPEG-CHN-PEI/DNA, N/P=10; (B) mPEG-CHN-PEI/DNA, N/P=20; (C) mPEG-PEI/DNA, N/P=10; (D) mPEG-PEI/DNA, N/P=20.

2.3基因超分子組裝體的穩(wěn)定性及pH響應(yīng)特性

Fig.5　Time-dependent change of the particle sizes under physiological salt(150 mmol/L NaCl) conditions(A) or culture media with 10% FBS(B)(t=0 means at prepared condition) and ξ-potentials of various polyplexes at pH=5.0 and 7.4(C) (A) a. PEI/DNA, pH=7.4; b. mPEG-CHN-PEI/DNA, pH=7.4; c. mPEG-PEI/DNA, pH=7.4; d. mPEG-CHN-PEI/DNA, pH=5.0; e. mPEG-PEI/DNA, pH=5.0. (B) a. mPEG-CHN-PEI/DNA in DMEM with 10%(volume fraction) FBS; b. mPEG-PEI/DNA in DMEM with 10%(volume fraction) FBS; c. PEI/DNA in DMEM with 10%FBS. (C) a. mPEG-CHN-PEI/DNA; b. mPEG-PEI/DNA. Error bars represent mean±SD for n=3. * Denotes statistically significant difference at p<0.05.

2.4細(xì)胞毒性及體外細(xì)胞轉(zhuǎn)染

采用MTT法測定組裝體對(duì)HepG2細(xì)胞的毒性[圖6(B)]. 由圖6(A)可見, 隨著N/P比的增大, 細(xì)胞的存活率降低; 在N/P比為10時(shí),PEI/DNA的細(xì)胞存活率為72%, 當(dāng)N/P比增大到20時(shí),PEI/DNA的細(xì)胞存活率僅為43%, 這是由于PEI的強(qiáng)正電性會(huì)擾動(dòng)細(xì)胞膜[20,21], 使PEI的細(xì)胞毒性增高. 在相同N/P比下,PEG化組裝體的細(xì)胞毒性顯著低于PEI/DNA組裝體的細(xì)胞毒性, 并且在N/P比為20時(shí)細(xì)胞存活率仍高于70%. 這是由于PEG殼層屏蔽表面的正電荷, 削弱了組裝體對(duì)細(xì)胞膜的擾動(dòng)作用及PEG的生物相容性, 使PEG化組裝體的細(xì)胞毒性降低, 這與Zhang等[22]的研究結(jié)果相符.

Fig.6　Cell cytotoxicity of HepG2 cells exposed to different nanoparticles with 1 μg DNA after incubation for 48 h(A) and in vitro transfection of the luciferase gene into HepG2 cells mediated by different nanoparticles for 48 h(B) (A) Error bars represent mean± SD for n=5; (B) error bars represent mean ±SD for n=3. * Denotes statistically significant difference at p<0.05.

Fig.7　Cellular uptake efficiency of different nanoparticles by HepG2 cells for incubation of 4 h Error bars represent mean±SD for n=3. * Denotes statistically significant difference at p<0.05.

2.5細(xì)胞內(nèi)吞及胞內(nèi)分布

為了研究PEG化組裝體轉(zhuǎn)染效率差異的機(jī)理, 采用Cy3-DNA作為示蹤分子, 采用流式細(xì)胞儀研究HepG2細(xì)胞對(duì)不同基因超分子組裝體的內(nèi)吞效率. 為清除內(nèi)吞后細(xì)胞表面附著的組裝體, 需要在內(nèi)吞后使用PBS清洗多次. 由圖7可見,PEG化組裝體的內(nèi)吞效率均低于20%, 顯著低于PEI/Cy3-DNA組裝體的內(nèi)吞效率, 這是由于PEG屏蔽了組裝體表面的正電荷, 阻礙了組裝體與細(xì)胞膜的靜電作用, 內(nèi)吞效率的降低影響了基因轉(zhuǎn)染效率. 對(duì)比2種PEG化組裝體的內(nèi)吞數(shù)據(jù)可知, 相同N/P比條件下,PEG化組裝體的內(nèi)吞效率無顯著性差異, 因此對(duì)于2種PEG化組裝體, 細(xì)胞內(nèi)吞并不是影響基因轉(zhuǎn)染效率的主要原因.

Fig.8　Intracellular distribution of Cy3-DNA polyplexed with mPEG-CHN-PEI(A1—A3), mPEG-PEI(B1—B3) and PEI(C1—C3) at an N/P ratio of 10 (A1—C1) Cy3; (A2—C2) Lyso-Tracker; (A3—C3) Cy3+Lyso-Tracker.

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(Ed.:W,Z)

?SupportedbytheNationalNaturalScienceFoundationofChina(Nos.21474087, 51273177).

doi:10.7503/cjcu20150907

收稿日期:2015-11-26. 網(wǎng)絡(luò)出版日期: 2016-03-16.

基金項(xiàng)目：國家自然科學(xué)基金(批準(zhǔn)號(hào): 21474087, 51273177)資助.

中圖分類號(hào)O631.3; Q782

文獻(xiàn)標(biāo)志碼A

pH-ResponsivePEGylatedGeneDeliverySystem?

LIUYajie,ZHANGPeng,DUJianwei,WANGYouxiang*

(Key Laboratory of Macromolecular Synthesis and Functionalization, Minstry of Education,Department of Polymer Science and Engineering, Zhejiang University, Hangzhou 310027, China)

AbstractThe pH-sensitive polyethylene glycol-grafted polyethylenimine(mPEG-CHN-PEI) was synthesized via benzoic imine linker according to the acidic environment of endosomal. The corresponding stable PEGylated polyethylenimine(mPEG-PEI) without pH-sensitivity was synthesized as control. The results showed that both PEGylated polycations could condense DNA into tight spherical nanoparticles about 80 nm in size and 10 mV in ξ-potential, which showed excellent stability under physiological conditions. The benzoic imine linkers were easily cleaved under the pH of 5, which was similar with the endosomal pH. Human hepatoblastoma cell line(HepG2) cell culture results indicated that the cytotoxicity and cellular uptake efficiency of both PEGylated gene nanoparticles decreased remarkably because of the shielding effect of PEG shell. Due to the cleavage of benzoic imine linker at acidic environment of endosomal, the PEG shell of mPEG-CHN-PEI/DNA polyplexes was detachable and lead to quick endosomal escape. So mPEG-CHN-PEI/DNA polyplexes showed higher gene expression than the mPEG-PEI/DNA polyplexes. Based on these results, we concluded that PEGylated polyplexes via benzoic imine linkers showed high extracellular stability, intracellular DNA release and effective gene transfection.

KeywordsNon-viral vector; Polyethylenimine; pH-Sensitive; PEGylation

聯(lián)系人簡介: 王幽香, 女, 博士, 副教授, 主要從事生物醫(yī)用高分子材料研究.E-mail:yx_wang@zju.edu.cn