吳興軍,魏英英,何文濤，施 萍，袁建超

(西北師范大學(xué) 化學(xué)化工學(xué)院，甘肅省高分子材料重點(diǎn)實(shí)驗(yàn)室，甘肅 蘭州 730070)

pH響應(yīng)型苯硼酸酯綴合物的制備、表征和智能釋放研究

吳興軍,魏英英,何文濤，施 萍，袁建超*

(西北師范大學(xué) 化學(xué)化工學(xué)院，甘肅省高分子材料重點(diǎn)實(shí)驗(yàn)室，甘肅 蘭州 730070)

以葡聚糖(Dextran)作為主鏈,將羅丹明B(RhB,熒光智能釋放藥物模型/示蹤顯像)、3-羥基苯硼酸(PBA)酯化后,綴合到葡聚糖上,形成一種新型pH響應(yīng)的高分子綴合物.由于該高分子綴合物中存在硼酸酯鍵,使得其負(fù)載模型藥物到達(dá)腫瘤細(xì)胞的溶酶體之后釋放出羅丹明B,不僅降低了藥物毒性,還能夠示蹤藥物的遞送過程.體外模擬釋放結(jié)果表明,綴合物在癌細(xì)胞的溶酶體/內(nèi)涵體 (pH 5)內(nèi)能很好地釋放出羅丹明B,而在藥物輸送過程(pH 7.4)中有很少的羅丹明B釋放出來.另外,高分子載體葡聚糖的存在,使聚合物體現(xiàn)出良好的生物相溶性,從而減少了對正常組織的傷害,在抗癌藥物的智能釋放方面具有很好的應(yīng)用前景.

高分子綴合物;pH響應(yīng);硼酸酯鍵;智能釋放

刺激響應(yīng)材料對于各種應(yīng)用顯示出巨大的潛力,因?yàn)樗鼈兊奈锢砘瘜W(xué)性質(zhì)可以根據(jù)生物系統(tǒng)中的內(nèi)部變化或外部變化而經(jīng)歷動態(tài)變化.外部或生理刺激,如光[1],溫度[2],pH[3-5],氧化還原性[6-7],超聲波[8],磁力[9],酶[10],已被測試用于觸發(fā)藥物釋放.刺激響應(yīng)性聚合物和納米材料已應(yīng)用于基因或藥物遞送[11]. 由于腫瘤細(xì)胞以異常高的速率增殖,導(dǎo)致糖酵解的高速率并積聚乳酸，降低環(huán)境pH值.此外,還研究了細(xì)胞器(內(nèi)涵體和溶酶體,pH 4.0～6.0)的酸性環(huán)境,用于設(shè)計(jì)細(xì)胞內(nèi)釋放的pH響應(yīng)納米載體[12].針對腫瘤細(xì)胞(內(nèi)涵體/溶酶體)內(nèi)的弱酸性環(huán)境,pH觸發(fā)的抗癌藥物智能釋放越來越多的應(yīng)用于藥物的靶向傳遞,以提高抗癌藥物的治療效率并減少其毒副作用.

早期含硼酸的大分子已被用于許多生物醫(yī)學(xué)領(lǐng)域，包括用于糖反應(yīng)性水凝膠，傳感器和納米材料中，通常以糖尿病的檢測和治療為目標(biāo)，其需要不斷監(jiān)測血糖水平和主動胰島素管理. 如今科學(xué)研究發(fā)現(xiàn)，硼酸與二醇形成硼酸酯鍵后，顯示出pH敏感性[13]. 1,2-二酚成功地用于中性溶液中形成硼酸酯,并且可以在溫和的酸性條件下水解[14]. 硼酸酯可以合成智能釋放的藥物載體，起到藥物緩釋的作用，被廣泛應(yīng)用于抗腫瘤治療.

葡聚糖是一種多糖用于藥物輸送的天然替代物，因?yàn)樗哂写罅康姆磻?yīng)性基團(tuán)，可控的分子量，可生物降解的特征[15]. 葡聚糖還具有生物活性,其中β-葡聚糖的活性最強(qiáng)[16-17],葡聚糖作為藥物載體，具有很好的生物相容性，分解后可作為人體供能的單糖存在，無任何毒性.同時葡聚糖還可以激活機(jī)體的免疫系統(tǒng)從而提高免疫力[18]. 通常，通過疏水改性手段制備基于葡聚糖的納米膠束.

以羅丹明B作為一種藥物模型/示蹤劑連接到3-羥基苯硼酸上,再與葡聚糖相連，制備了一種生物相容性好的pH響應(yīng)性藥物載體.在腫瘤細(xì)胞內(nèi)的溶酶體中,載藥綴合物的硼酸酯鍵斷裂[19],羅丹明B得以釋放(圖1);并進(jìn)行了體外pH引發(fā)的藥物模型釋放.

圖1 pH 響應(yīng)性綴合物的合成及細(xì)胞內(nèi)RhB釋放Fig.1 Synthesis of pH-responsive conjugates and intracellular RhB release

1 實(shí)驗(yàn)部分

1.1 試劑與儀器

二環(huán)己基碳二亞胺 (DCC, 99%), 4-二甲基氨基吡啶(DMAP, 99%), 三乙胺 (TEA, 99%), 羅丹明B (RhB, 99%), 3-羥基苯硼酸 (PBA,99%)和β-葡聚糖(Dex,相對分子量質(zhì)量20 000)購自阿拉試劑公司.二甲基亞砜(DMSO),四氫呋喃(THF)和二氯甲烷(CH2Cl2)購自北京化工廠.

Mercury plus-400 MHz 核磁共振波譜儀(Varian,美國); DUV-3700 紫外可見分光光度計(jì)(UV/Vis,日本島津公司).

1.2 RhB-PBA的合成

稱取羅丹明B (240 mg, 0.50 mmol)和N,N-二環(huán)己基碳二亞胺(110 mg, 0.58 mmol)溶于二氯甲烷(12 mL)中; 3-羥基苯硼酸(138 mg, 1.00 mmol)與 4-二甲氨基吡啶(140 mg, 1.15 mmol)溶解在四氫呋喃(4 mL)中;混合加入圓底燒瓶.將圓底燒瓶置于室溫反應(yīng)24 h.反應(yīng)液過濾除去生成的鹽,減壓蒸餾除溶劑后進(jìn)行硅膠柱層析[洗脫液為V(乙酸乙酯)∶V(甲醇)=6∶1]分離純化,得紅褐色固體RhB-PBA.

1.3 RhB-PBA-Dex的合成

稱取RhB-PBA(181 mg, 0.29 mmol)和葡聚糖(50 mg, 0.003 mmol)置于Schlenk瓶中,用二甲基亞砜溶解(15 mL),再加入氫化鈣(13 mg, 0.31 mmol)，抽真空充氮?dú)庋h(huán)3～5次,密封Schlenk瓶,于30 ℃ 反應(yīng)24 h.用冰乙醇沉淀,過濾,重復(fù)操作3次后,得橙色固體.

1.4 體外釋放

取兩份10 mg 綴合物，分別置于100 mL 的pH 7.4與100 mL的 pH 5.0的緩沖溶液中,透析48 h,按照一定的時間間隔(1，3，5 h 等)內(nèi),每次取3 mL 透析液測樣,并立即放回以維持緩沖液的體積.用 UV/Vis可見光譜在552 nm 處檢測透析液中羅丹明B的吸光度,并繪制體外釋放曲線.

2 結(jié)果與討論

2.1 綴合物的核磁共振氫譜表征

羅丹明B和3-羥基苯硼酸通過酯化,硅膠柱層析分離純化等一系列過程得到RhB-PBA.并用它與葡聚糖硼酯化合成RhB-PBA-Dex. RhB-PBA和 RhB-PBA-Dex的1H NMR譜圖如圖2和圖3所示.

圖2 RhB-PBA的核磁共振氫譜Fig.2 1H NMR spectrum of RhB-PBA

圖3 綴合物RhB-PBA-Dex的核磁共振氫譜Fig.3 1H NMR spectrum of RhB-PBA-Dex

圖2 中δ7.37(e)和7.15(f, g, h)處都為間羥基苯硼酸中苯環(huán)(C-H)的質(zhì)子吸收峰,8.39、7.81、7.77、7.67、6.84、6.71(a, b, c, d, i, g)處分別為RhB的苯環(huán)上質(zhì)子吸收峰,3.58處為- CH2-的質(zhì)子吸收峰,1.28處為- CH3-的質(zhì)子吸收峰,表明成功合成了RhB-PBA.圖3為共聚物RhB-PBA-Dex的1H NMR譜圖,δ4.72、3.76、3.67、3.28(a、b、c、d、e、f)處分別為Dex的質(zhì)子吸收峰,與圖1相比,綴合物RhB-PBA-Dex中其他質(zhì)子的位置基本保持不變.

2.2 pH響應(yīng)與羅丹明的體外藥物釋放研究

研究表明,硼酸酯是一種pH敏感的化合物，當(dāng)其所屬的化學(xué)環(huán)境從中性向酸性改變過程中,硼酸酯開始斷鍵,如綴合物從細(xì)胞質(zhì)逐漸進(jìn)入溶酶體中,酸性環(huán)境有所改變,從而硼酸酯斷裂,如圖4所示.

圖4 硼酸醇的pH響應(yīng)示意圖Fig.4 Scheme of pH-response of borate ester

為了進(jìn)一步研究綴合物對RhB的緩釋行為,我們分別在pH=7.4(如圖5)和pH=5.0(如圖6)的緩沖溶液中模擬體外藥物釋放，釋放曲線如圖7所示.pH=7.4時,RhB的累積釋放率十分緩慢,48 h后累計(jì)釋放率僅為2.2%.而pH=5.0時,起初1 h內(nèi)RhB的累計(jì)釋放率為9.5%,出現(xiàn)明顯的暴釋現(xiàn)象, 48 h后累計(jì)釋放率高達(dá)60.0%.這一釋放結(jié)果說明綴合物對pH具有響應(yīng)性,可以對細(xì)胞內(nèi)酸性環(huán)境作出響應(yīng),實(shí)現(xiàn)細(xì)胞內(nèi)藥物的可控釋放,同時有利于提高藥物在腫瘤細(xì)胞中的累積,減少藥物毒副作用.

圖5 pH=7.4載藥綴合物的體外藥物釋放熒光光譜圖Fig.5 Release of RhB from RhB-PBA-Dex in buffer pH 7.4 (fluorescence spectra)

圖6 pH=5.0載藥綴合物的體外藥物釋放熒光光譜圖Fig.6 Release of RhB from RhB-PBA-Dex in buffer pH 5 (fluorescence spectra)

圖7 pH響應(yīng)性載藥綴合物的體外藥物釋放圖Fig.7 Release of RhB from RhB-PBA-Dex in buffers pH 5 and pH 7.4

硼酸酯鍵在中性溶液中十分的穩(wěn)定,累積釋放率不到3%;遠(yuǎn)遠(yuǎn)低于以前運(yùn)用的腙鍵pH響應(yīng)性藥物的釋放[20].當(dāng)在細(xì)胞外藥物載體受到影響時,傳統(tǒng)藥物載體受損,藥物釋放進(jìn)入正常細(xì)胞組織,損壞正常機(jī)體;然而本實(shí)驗(yàn)藥物載體RhB-PBA-Dex,很好地克服了傳統(tǒng)緩釋藥物的缺點(diǎn),當(dāng)硼酸酯鍵斷裂后，PBA具有靶向作用，能夠識別腫瘤細(xì)胞上表達(dá)的唾液酸[21]，從而使得藥物模型/示蹤顯像被拉入腫瘤中,無損于正常細(xì)胞組織.本研究是通過化學(xué)反應(yīng),形成了一種綴合物,而不是膠束.比起膠束的包覆藥物,此綴合物表現(xiàn)比較穩(wěn)定.

3 結(jié)論

合成了一種簡單的基于硼酸酯pH響應(yīng)的綴合物RhB-PBA-Dex.該綴合物在水溶液中具有良好的溶解性與穩(wěn)定性.pH=5.0時,連接PBA和Dex的硼酸酯鍵斷裂,導(dǎo)致RhB解離.綴合物的體外模擬釋放研究結(jié)果表明,pH=5.0時的藥物累計(jì)釋放率明顯較高,48 h內(nèi)RhB的累積釋放率達(dá)60.0%,而pH=7.4時RhB的累積釋放率為2.2%.總之,RhB-PBA-Dex綴合物為智能藥物載體系統(tǒng)提供了一個高效的平臺,在癌癥治療中有著極為廣闊的應(yīng)用背景.

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pH-sensitiveborateesterconjugate:preparation,characterizationandintelligentrelease

WU Xingjun, WEI Yingying, HE Wentao, SHI Ping, YUAN Jianchao*

(KeyLaboratoryofGansuPolymerMaterials,CollegeofChemistryandChemicalEngineering,NorthwestNormalUniversity,Lanzhou730070,Gansu,China)

Using Dextran (Dex) as a main chain rhodamine B (RhB) and 3-hydroxyphenylboronic acid (PBA) were esterified and then conjugated to dextran to form a novel pH-responsive polymer conjugate. Because of the presence of borate ester bonds in the polymer, the drug loaded conjugate can release rhodamine B in cancer cells, which not only reduces the toxicity of the drug, but also can be used to trace the drug delivery process. The results show that rhodamine B released well from the conjugate in lysosomes/endosomes of cancer cell (pH 5), and only little rhodamine B released in the process of drug delivery (pH 7.4). In addition, the presence of polymer carrier dextran made the polymer reflect a good bio-compatibility, thereby reducing the damage for normal tissue. The release of anti-cancer drugs would have a good application prospect.

polymer conjugate; pH response; borate ester bond; intelligent release

O632

A

1008-1011(2017)05-0639-06

2017-01-17.

國家自然科學(xué)基金資助項(xiàng)目(21364011, 20964003).

吳興軍(1989-), 男, 碩士生, 研究方向?yàn)榘邢蚩鼓[瘤高分子藥物、生物高分子材料.*

, E-mail:jianchaoyuan@nwnu.edu.cn.

[責(zé)任編輯：張普玉]