彭盛峰,劉 偉,鄒立強,周 磊

(南昌大學食品科學與技術(shù)國家重點實驗室,江西南昌 330047)

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層層自組裝技術(shù)在脂質(zhì)體修飾中的應用

彭盛峰,劉 偉*,鄒立強,周 磊

(南昌大學食品科學與技術(shù)國家重點實驗室,江西南昌 330047)

脂質(zhì)體作為載體具有良好的生物相容性和生物降解性等優(yōu)點,在藥品和化妝品等領(lǐng)域中應用廣泛,但由于脂質(zhì)體的貯藏和消化穩(wěn)定性較差,限制了其在食品中的應用。利用層層自組裝技術(shù)將聚電解質(zhì)沉積在脂質(zhì)體表面形成一層保護膜是提高其貯藏和消化穩(wěn)定性的有效手段。本文從層層自組裝過程的影響因素、薄膜表征手段以及薄膜對脂質(zhì)體的保護作用等方面綜述了層層自組裝在脂質(zhì)體中的應用。

脂質(zhì)體,層層自組裝技術(shù),聚電解質(zhì)

層層自組裝技術(shù)(Layer-by-layer assembly technology,LBL)是指以分子間的靜電引力、氫鍵等為驅(qū)動力,將聚電解質(zhì)逐層沉積到基質(zhì)表面,構(gòu)建多層納米級薄膜的過程[1]。Decher等[2]在Langmuir-Blodgett技術(shù)[3]的基礎(chǔ)上將聚電解質(zhì)連續(xù)的分層堆積到硅片、玻璃等基質(zhì)表面,形成一層超薄的納米級薄膜,開創(chuàng)了LBL的先河。LBL具有許多優(yōu)點,它對基質(zhì)沒有特定限制,修飾材料來源廣泛,修飾條件溫和,不需要特殊的設(shè)備,操作簡單且能制備出不同性質(zhì)的結(jié)構(gòu)穩(wěn)定薄膜。Sukhorukov[4]等將LBL應用到微米級的膠體中,發(fā)現(xiàn)能提高微粒的穩(wěn)定性,緩釋效果,使微粒功能多樣化等,因此許多學者開始探索LBL在膠體微粒(空心微球[5]、脂質(zhì)體[6]、微膠囊[7]和微乳[8]等)中的應用。脂質(zhì)體是一種類似于細胞結(jié)構(gòu)的雙分子層薄膜,由于脂質(zhì)體能保護包裹的活性物質(zhì)而被應用于基因轉(zhuǎn)染、癌癥治療和化妝品等領(lǐng)域[9]。而脂質(zhì)體存在著保存期內(nèi)粒徑變大、絮凝、藥物滲漏以及口服后在胃腸道中易被分解的,限制了其在食品中的應用[9]。以LBL在脂質(zhì)體表面沉積聚電解質(zhì)形成一層保護膜能提高其貯藏穩(wěn)定性和消化穩(wěn)定性[6,10]。而LBL在脂質(zhì)體中的應用較少,本文歸納了LBL在脂質(zhì)體修飾中的應用,并對影響LBL制備過程中的因素、表征手段和LBL修飾脂質(zhì)體的作用進行綜述,為脂質(zhì)體的制備提供參考。

1 影響LBL的因素

在自組裝過程中,一般分為兩個步驟,即聚電解質(zhì)固定在基質(zhì)表面和聚電解質(zhì)緩慢松弛在基質(zhì)表面形成濃密的聚電解質(zhì)層[11]。影響自組裝的因素主要有聚電解質(zhì)濃度、溶液的pH和離子強度以及修飾完成后游離聚電解質(zhì)的分離。

1.1 聚電解質(zhì)

聚電解質(zhì)是指分子中有可離子化基團的聚合物,在極性溶劑如水中會電離,在水中形成帶電的高分子離子和小分子離子[12]。聚電解質(zhì)根據(jù)其來源將其分為天然聚電解質(zhì)和合成聚電解質(zhì)兩類[13],前者包括核酸蛋白質(zhì)和多糖(海藻酸和硫酸軟骨素鹽)、DNA、肝素、殼聚糖、纖維素硫酸紙、葡聚糖硫酸酯以及羧甲基纖維素等;后者包括聚丙乙烯磺酸鹽(PSS)、聚二甲基二烯丙基氯化銨(PDDA)、聚乙烯亞胺(PEI)、聚N-異丙烯酰(PNIPAM)、聚丙烯酸(PAA)、聚甲基丙烯酸(PMA)、聚乙烯基硫酸酯(PVS)和聚丙烯酰胺鹽酸鹽(PAH)等。根據(jù)聚電解質(zhì)所帶離子的電荷種類,可將其分為聚陽離子(如殼聚糖)和聚陰離子(如海藻酸鈉)。

聚電解質(zhì)需要達到一定的濃度,才能改變脂質(zhì)體表面的帶電正負值,這個濃度稱為最小臨界濃度,臨界濃度是由聚電解質(zhì)的溶解度和表面電荷密度決定的[11]。在聚電解質(zhì)的濃度高于臨界濃度后,聚電解質(zhì)濃度的大小不影響吸附作用,而平均粒徑則隨聚電解質(zhì)濃度的增加呈指數(shù)遞增[6]。

1.2 離子強度和pH

由于聚合物單體間的同種電荷相互排斥,聚電解質(zhì)在溶液中的構(gòu)象更為舒展,尺寸更大,而小分子尤其是強電解質(zhì)的加入會屏蔽聚電解質(zhì)之間的帶電基團間的電荷作用力,使得由電荷排斥引起的舒展減弱,分子尺寸縮小,構(gòu)象由舒展線性收縮成球狀線團[14-15]。因此隨著離子濃度的增加,吸附的薄膜厚度也隨之增加,粒徑會顯著上升。而達到一定濃度后,聚電解質(zhì)溶液變成渾濁的固體,不再具有吸附作用[16]。

當聚電解質(zhì)表面的電荷濃度低于一定值時,自組裝過程不能發(fā)生,即“臨界電荷密度效應”[17]。pH的變化會改變?nèi)蹙垭娊赓|(zhì)的電離度,從而改變其表面的電荷密度,影響自組裝進行。Krasemann L[18]指出,pH是兩個聚電解質(zhì)的pKa值的平均值時,吸附效果最好,即pH最佳=(pKa聚陽離子+pKa聚陰離子)/2,常磊[19]等測量在不同的pH條件下PAA和PAcrNPP在不同基質(zhì)上的吸附量,發(fā)現(xiàn)在pH5.5時,聚電解質(zhì)吸附量大,正好是PAA和PAcrNPP兩者的pKa的均值,與Krasemann,L的理論相吻合。

1.3 分離方法

為了避免由于聚合物不足導致其與脂質(zhì)體之間交聯(lián)或絮凝,通常將待修飾的脂質(zhì)體溶液逐滴滴入到聚電解質(zhì)溶液中,并持續(xù)攪拌[20]。同時,為了避免帶相反電荷的聚電解質(zhì)之間發(fā)生交聯(lián)生成聚電解質(zhì)復合物,因此在進行下一層的修飾之前,需要除去未結(jié)合的游離聚電解質(zhì)。

目前常用的技術(shù)為超速離心法[21],該法具有操作簡單、高效等優(yōu)點,但卻存在耗能、復水難、復水后脂質(zhì)體凝聚使平均粒徑變大等缺點。另一種常用的技術(shù)是超濾,Keiji[10]等將聚賴氨酸和聚天冬氨酸修飾在脂質(zhì)體表面,通過多次超濾的方式將脂質(zhì)體從修飾劑溶液中分離,Fukui[22]等用多次超濾將修飾完成后的脂質(zhì)體從殼聚糖和葡聚糖溶液中分離,超濾具有處理溫和、高選擇性、低能耗等優(yōu)點,然而隨著過濾的進行,大分子逐漸聚積在膜表面,并堵塞膜孔,降低過濾通量,即使增加壓力,也只能將聚積層壓緊,而不能改善過濾狀態(tài)[23]。與之相比,切向流過濾技術(shù)就克服了上述的缺點。該技術(shù)料液流動方向與過濾方向呈垂直方向的過濾形式,沿濾膜表面流動的料液會將膜表面由于濃差極化而形成的凝膠層“掃掠”清除,從而維持膜的過濾狀態(tài),保證小分子通過。切向流過技術(shù)具有較好的濃縮和分餾功能,能在除去未結(jié)合的聚電解質(zhì)的同時改變其濃度或置換緩沖液,該方法具有快速、高效、節(jié)能等優(yōu)點[24],但一些天然聚電解質(zhì)如殼聚糖等粘度很高,容易粘附在膜表面造成膜堵塞,因此其應用有限。Monika[25]等利用凝膠過濾層析技術(shù)分離游離殼聚糖,凝膠過濾具有條件溫和、操作簡單和對高分子聚合物分離效果好的優(yōu)點,但存在收率較低、耗時和分離后脂質(zhì)體濃度降低等缺點。由于聚電解質(zhì)與脂質(zhì)體或聚電解質(zhì)之間的作用力較弱,超濾過程中會伴隨著聚電解質(zhì)解吸附的作用,因此超濾過程應盡量減少超濾的次數(shù)和時間。

磁分離技術(shù)是利用組分磁敏感性的差異,借助外磁場將物質(zhì)進行磁場處理,從而達到強化分離效果的一種快速、高效的分離技術(shù)[26]。磁分離技術(shù)常被用于分離細胞和生物大分子如核酸等[27]。da Silva Gomes[28]等以磁分離技術(shù)將磁性脂質(zhì)體從溶液中分離,再用新的緩沖液分散,進行下一步修飾。由于作用時間短,對脂質(zhì)體薄膜結(jié)構(gòu)基本沒有影響,且磁性脂質(zhì)體本身就具有靶向性等功能,因此以磁性脂質(zhì)體作為分離手段具有良好的發(fā)展前景。

2 LBL薄膜的結(jié)構(gòu)表征

脂質(zhì)體的平均粒徑隨修飾層數(shù)的增加而增加,且在前兩次修飾時粒徑變化較為明顯,平均粒徑的增加是其表面膜變厚和聚電解質(zhì)與脂質(zhì)體之間橋連的共同結(jié)果[29]。然而Haidar等在脂質(zhì)體表面交替修飾殼聚糖和海藻酸鈉時發(fā)現(xiàn),第一層修飾后,其平均粒徑由180nm變?yōu)?45nm,而第二層修飾后發(fā)現(xiàn)其粒徑不升反降,由345nm降為210nm左右,隨著層數(shù)的增加,粒徑則趨于平穩(wěn)。這種現(xiàn)象可能是相對較短的海藻酸鈉分子容易在長鏈殼聚糖分子之間擴散形成一層致密的網(wǎng)狀結(jié)構(gòu)造成的[30]。粒徑是脂質(zhì)體的重要性質(zhì),文獻表明較大的微粒(<5μm)會被淋巴細胞攝取而較小的微粒(<500nm)則會被上皮細胞以內(nèi)吞的方式攝取[31]。ζ-電位是脂質(zhì)體體系穩(wěn)定性的另一個重要參數(shù)。電位絕對值越大,膜表面的電荷量越高,微粒間的排斥力就越大,體系就越穩(wěn)定[32]。LBL需要脂質(zhì)體膜表面先帶有電荷,再利用靜電作用,將帶相反電荷的聚電解質(zhì)沉積在膜表面,隨著聚電解質(zhì)在脂質(zhì)體表面的沉積,表面電位會隨之改變正負值。脂質(zhì)體的粒徑和電位一般是以動態(tài)光散射法測定的,如Zou[33]等使用美國Nicomp 380 ZLS激光粒度儀測定脂質(zhì)體的平均粒徑和表面電位,Tamaru[34]等使用英國馬爾文納米粒度儀及Zeta電位分析儀測定了脂質(zhì)體的平均粒徑和表面電位。

表1 LBL層層自組裝技術(shù)在脂質(zhì)體修飾中的應用Table1 Layer-by-Layer self-assembly technology for the modification of liposome

除粒徑和電位外,可以通過光譜學和電鏡等對脂質(zhì)體修飾進行表征。如紅外圖譜顯示修飾前后脂質(zhì)體的特征峰偏移情況,并用來分析脂質(zhì)體與聚電解質(zhì)的作用力和作用方式[6]。原子力顯微鏡(AFM)一般用于驗證動態(tài)光散射法(DLS)測量的粒徑結(jié)果,但由于樣品制備和測量過程中的影響,其結(jié)果與DLS有一定的差異,而原子力顯微鏡無法識別包覆在脂質(zhì)體表面的聚合物[28],因此AFM能給出的信息有限。相比于AFM,透射電鏡(TEM)可以更為直觀的觀察出脂質(zhì)體的微觀結(jié)構(gòu)包括粒徑,Fujimoto[10]等用AFM觀察脂質(zhì)體表面的輪廓判斷多聚賴氨酸和聚天冬氨酸成功的沉積在脂質(zhì)體表面形成保護膜。然而AFM仍然不能給出關(guān)于脂質(zhì)體表面聚電解質(zhì)的層數(shù)和分布等信息。激光共聚焦顯微鏡(CLSM)可以清楚的觀察到熒光標記的聚電解質(zhì)在脂質(zhì)體表面的分布,但由于其分辨率有限,只能測量粒徑較大的微粒,限制了它在脂質(zhì)體中的應用[35]。

目前尚且沒有技術(shù)能很好的表征脂質(zhì)體表面的膜的結(jié)構(gòu),只能綜合粒徑、電位和電鏡圖的信息判斷自組裝的成功與否。

3 LBL對脂質(zhì)體的影響

表面修飾技術(shù)是為了解決脂質(zhì)體在保存期內(nèi)粒徑變大、絮凝、藥物滲漏以及給藥后在體內(nèi)脂質(zhì)體破裂、包封藥物快速滲漏等問題。以表面活性劑(曲通X-100)處理修飾前后的脂質(zhì)體,對比其透明度,修飾后的脂質(zhì)體穩(wěn)定性較好,這主要是因為聚電解質(zhì)在脂質(zhì)體表面形成的薄膜能保護脂質(zhì)體,提高其物理穩(wěn)定性[10,22,28,36],Haidar[30]等比較了脂質(zhì)體殼聚糖-海藻酸鹽修飾三層和五層的脂質(zhì)體凍干前后平均粒徑和表面電位的變化,發(fā)現(xiàn)表面修飾后的脂質(zhì)體凍干前后平均粒徑和表面電位基本沒變化,而未修飾的脂質(zhì)體,凍干再復水后粒徑急劇增大,可能出現(xiàn)了絮凝沉淀。除了提高其穩(wěn)定性外,體外消化實驗表面LBL對脂質(zhì)體的緩釋效果也有提高[22],這是聚電解質(zhì)薄膜能阻止胰酶對磷脂的降解,從而提高其緩釋效果。

除了提高貯藏和消化穩(wěn)定性外,通過LBL也能賦予脂質(zhì)體一些特殊的性能。Ziyad S[30]發(fā)現(xiàn)隨著脂質(zhì)體表面的聚電解的增加,包裹的水溶性蛋白也隨之增加,在聚電解質(zhì)達到10層時,包封率高達80%,LBL或?qū)⒊蔀榭刂扑苄运幬锇饴实募夹g(shù)手段。Fujimoto[10]發(fā)現(xiàn)沉積在脂質(zhì)體第一層的聚賴氨酸量直接決定藥物的釋放特性;Fukui[22]用低分子殼聚糖作為陽離子修飾劑,葡聚糖硫酸酯或DNA作為陰離子修飾劑,殼聚糖單層修飾的脂質(zhì)體具有非常好的緩釋效果,再修飾一層葡聚糖硫酸酯或DNA沒有顯著提高,在60℃條件下,DNA解螺旋,使脂質(zhì)體表面的聚電解質(zhì)殼結(jié)構(gòu)破壞,脂質(zhì)體藥物釋放迅速,使脂質(zhì)體具有溫敏性。在脂質(zhì)體表面修飾具有特定性能的聚電解質(zhì)使脂質(zhì)體功能多樣化是LBL發(fā)展趨勢。

4 展望

LBL利用靜電引力、氫鍵、配位鍵等作用力將聚陽離子和聚陰離子交替沉積到脂質(zhì)體表面,形成一層保護膜,能提高其貯藏及消化穩(wěn)定性。該技術(shù)具有操作簡單、條件溫和、材料來源廣泛且能通過控制修飾參數(shù)(聚電解質(zhì)種類、修飾層數(shù)、溶液pH及離子強度等)來調(diào)節(jié)脂質(zhì)體的表面性質(zhì)(平均粒徑、表面電位、表面粗糙度)、理化穩(wěn)定性以及功能多樣性等優(yōu)點。隨著更多的聚電解質(zhì)被提取和合成,LBL構(gòu)建的脂質(zhì)體體系將得到更大的發(fā)展。

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Layer-by-Layer self-assembly technology for the modification of liposome

PENG Sheng-feng,LIU Wei*,ZOU Li-qiang,ZHOU Lei

(State Key Laboratory of Food Science and Technology,Nanchang University,Nanchang 330047,China)

Liposomes were commonly investigated as vehicles and utilized in medicine and cosmetic industries because of their good biocompatibility and biodegradability. Nonetheless,their poor stability during storage and gastrointestinal environment greatly limited their application. One of the strategies was to modify the liposomes with polyelectrolytes using Layer-by-layer assembly technology. In this review,the factors that influenced the self-assembly process,the characterization of Layer-by-layer constructs and the contribution of LbL self-assembly to liposome were discussed.

liposomes;layer-by-layer self-assembly technology;polyelectrolyte

2014-08-07

彭盛峰(1992-)，男，碩士研究生，研究方向：食物(含生物質(zhì))資源開發(fā)與利用。

*通訊作者:劉偉(1972-)，男，博士，教授，研究方向：食品高新技術(shù)與資源綜合利用。

國家自然科學基金(21266021)；研究生創(chuàng)新計劃(YC2014-S056)。

TS201.4

A

:1002-0306(2015)09-0391-05

10.13386/j.issn1002-0306.2015.09.076