,,2

(1.浙江工業(yè)大學 藥學院,浙江 杭州 310014;2.沈陽藥科大學 藥學院,遼寧 沈陽 110016)

微球(microspheres)是指藥物溶解或者分散在高分子材料基質(zhì)中形成的骨架型微小球狀實體.微球的材料按來源分可分為天然高分子材料,如明膠(gelatin)、海藻酸鈉(alginate)、殼聚糖(chitosan)等;半合成高分子材料,如甲基纖維素(MC)、乙基纖維素(EC)、羥丙甲纖維素(HPMC)等;合成高分子材料,如聚乳酸(PLA)、聚乳酸-聚乙醇酸共聚物(PLGA)等[1].合成材料中以PLGA最為常用,PLGA由乳酸(lactic acid, LA)和羥基乙酸(glycolic acid, GA)以不同比例聚合而來,具有良好的生物相容性與可降解性、成囊和成膜的性能,獲美國食品藥品管理局(FDA)和歐洲藥監(jiān)局批準用于人體各種給藥系統(tǒng)[2].包封率是指制劑包封的藥物占總投藥量的百分比,一般要求不小于80%.Tu等[3]以明膠與海藻酸鈉混合物為囊材,通過乳化/內(nèi)部凝膠化方法制備了載枯草芽孢桿菌SL-13的微球,調(diào)節(jié)囊材混合物中明膠的比例至1.5%時,達最大包封率為93.44%.Filipovic-Grcic等[4]以低、中、高分子量的殼聚糖和羥丙基甲基纖維素(HPMC)及其混合物為囊材,運用噴霧干燥技術(shù)制備微球.實驗結(jié)果表明:當使用低分子量的殼聚糖或者HPMC進行載藥時,微球的包封率最高.Oz等[5]以W1/O/W2乳化法制備艾塞那肽多糖微球時,發(fā)現(xiàn)包封率主要取決于多糖的乙?；潭?乙?；潭仍礁?兩者間的氫鍵作用與疏水相互作用增強,使親和力增強,包封率提高.以殼聚糖為載體材料制備載親水性藥物的微球時,殼聚糖分子量越大,親水性減弱,包封率越低[6].

雖然近年來微球的研究取得了較大的進展,但其制備仍然面臨載藥量與包封率低下的問題,影響微球制劑的應(yīng)用.筆者主要回顧以往合成高分子微球制劑研究中各種提高包封率的辦法,進行歸納、概括與分析,為將來制備高包封率的合成高分子微球制劑提供參考與可行性辦法,并對其前景作出展望.

1 微球的制備方法

乳化-溶劑揮發(fā)法制備合成高分子材料微球,是由乳化作用形成的乳狀液中除去揮發(fā)性有機溶劑、乳滴固化形成微球的過程,可分為簡單乳化法和復(fù)乳法.O/W簡單乳化法[7-8],主要適用于疏水性藥物.而對于親水性和兩親性藥物主要采用W1/O/W2復(fù)乳法[9],但許多研究[5,10-11]發(fā)現(xiàn),運用S/O/W法、W/O1/O2復(fù)乳化法或S/O1/O2復(fù)乳化法制備載藥微球,比W1/O/W2復(fù)乳法的包封率顯著提高.Nafea等[9]采用W/O1/O2復(fù)乳化法,連續(xù)相為非水溶性的液態(tài)石蠟,藥物溶解度減小,損失減少,包封率增加.Yang等[12]通過S/O1/O2復(fù)乳化法制備載BSA-PEG的PLGA微球,該方法制備而來的微球包封率大于90%.當所載藥物為蛋白質(zhì)、多肽等時,S/O/W法有利于維持它們的生物活性[13].其他的微球制備方法如:噴霧干燥法[14]是指將聚合物溶解于有機溶劑中,藥物充分分散或溶解其中,進行噴霧、溶劑揮發(fā)形成微球,該法對藥物、聚合物和溶解度要求不高,適于工業(yè)化生產(chǎn),但使用有機溶劑較多;在超臨界流體法[15-16]中,聚合物和藥物均需溶于超臨界狀態(tài)的二氧化碳,該法適用于低分子量的藥物,不需有機溶劑,較環(huán)保;相分離法[17]是指將高分子溶于、藥物溶解或混懸于某有機溶劑中,然后加入與有機溶劑互溶但不能溶解聚合物和藥物的非溶劑,使聚合物因溶解度降低迅速發(fā)生凝聚,實現(xiàn)固液分離,得到載藥微球,該法適用于親水性藥物.

2 包封率影響因素與無機鹽的應(yīng)用及機理

2.1 影響因素與提高辦法

因合成高分子材料微球以PLGA微球居多,故主要以乳化-溶劑揮發(fā)法制備PLGA載藥微球為例進行討論.對于乳化-溶劑揮發(fā)法,包封率主要取決于藥物在分散相和連續(xù)相間的分布狀況[18]

微球的固化過程是影響包封率的一個重要階段,其過程分兩步,有機溶劑進入水相與溶劑的揮發(fā).隨著微球的固化,藥物被載體材料包載形成微球[40].凡影響微球固化速度的因素,均會影響包封率的大小,微球固化越快,藥物損失越少,包封率越高.例如有機溶劑的種類、連續(xù)相體積和溫度等.有機溶劑的水溶性越低,微球固化越慢,藥物損失越多,包封率越低[32,41];連續(xù)相體積越大,有機溶劑溶于外水相的速度越快,微球固化越快,藥物損失減少;另外在一定范圍內(nèi)的溫度越高,溶劑揮發(fā)越快,微球固化越快,藥物損失減少.

2.2 無機鹽對包封率的影響

2.2.1 無機鹽的應(yīng)用

Luan等[39]研究發(fā)現(xiàn):當W2相中NaCl的濃度為0.05 mol/L時,相比于不加NaCl,微球包封率由88.7%增大至99.0%.但當濃度繼續(xù)增大至0.5 mol/L時,包封率不再增加.Manoharan等[43]研究3種鋅鹽(氧化鋅、碳酸鋅和醋酸鋅)對胰島素包封率的影響,結(jié)果發(fā)現(xiàn)在沒有加入鋅鹽時,微球包封率為69%,當鋅鹽加入后,包封率顯著增加.Al-Maaieh等[38,44]采用O/W溶劑揮發(fā)法制備PLA微球時,研究不同濃度的Na2SO4,NaBr,NaClO4和NaSCN對離子型藥物——硫酸奎尼丁的水溶性和微球包封率的影響,發(fā)現(xiàn)鹽主要通過改變藥物和有機溶劑二氯甲烷在外水相的溶解度來影響微球包封率.

表1 微球包封率的影響因素與機理Table 1 Influence factors and mechanisms of encapsulation efficiency of microspheres

2.2.2 作用機理

無機鹽用于提高合成高分子微球包封率的作用機理主要分兩大方面:1) 無機鹽引起的滲透壓的變化;2) 無機鹽對藥物溶解度的影響.后者根據(jù)文獻資料,又可細分為同離子效應(yīng)、鹽析作用和新鹽的生成3種.其中滲透壓原理主要針對親水性藥物,同離子作用和鹽析作用主要影響疏水性藥物的固化過程.

對于滲透壓原理,油相在微球沒有完全固化時,像一張半透膜分隔內(nèi)外水相[45].分散相中的藥物導致其滲透壓常高于連續(xù)相,因此在兩相間形成的滲透壓差會促進藥物由分散相向連續(xù)相擴散,同時滲透壓的存在又會使連續(xù)相中的水分向內(nèi)分散遷移,形成的“孔道”進一步加劇藥物的損失,從而降低微球包封率.在微球固化過程中,在外水相加入無機鹽可以減小這種滲透壓,增大包封率.如周香蓮等[46]研究發(fā)現(xiàn)在外水相中加入NaCl,微球電鏡照片顯示表面孔洞明顯減少、內(nèi)部骨架致密.為了確認由于微球內(nèi)部內(nèi)外相物質(zhì)交換產(chǎn)生的“通道”的存在,又在微球固化完成后,在外水相中加入熒光染料硫磺素-T,觀察熒光染料進入微球內(nèi)部的情況.結(jié)果發(fā)現(xiàn):不加NaCl時微球內(nèi)部有大量的熒光,隨著外水相NaCl濃度的提高,微球內(nèi)部的熒光呈逐漸減少的趨勢.另外,一些非電解質(zhì)的加入,如山梨醇等,也具有增大滲透壓的作用,從而增大包封率[32].

1) 同離子效應(yīng)

在外水相中加入與離子型藥物具有相同離子的鹽能夠抑制藥物的溶解.藥物鹽溶解度可以用沉淀平衡常數(shù)(溶度積)Ksp°表示,它的大小反映了物質(zhì)的溶解能力,只受溫度影響,與其他因素無關(guān).藥物(MzXy)的解離表示為

Ksp°的計算公式為

Ksp°=(a(M+y))z(a(X-z))y=(M+y)z(X-z)y(γ±)z+y

式中:a為活度;γ±為平均離子活度系數(shù);M+y為陽離子的摩爾濃度;X-z為陰離子的摩爾濃度.

在電解質(zhì)溶液中,離子相互作用使得離子通常不能完全發(fā)揮其作用.離子實際發(fā)揮作用的濃度稱為有效濃度,或稱為活度(activity),顯然活度的數(shù)值通常比其對應(yīng)的濃度數(shù)值要小些.活度系數(shù)指物質(zhì)的化學活度對其濃度之比.由公式可知,當加入同離子的鹽時,其中某一離子的摩爾濃度增大,γ±不變,Ksp°不變,另一離子的摩爾濃度就要減小,即藥物溶解度減小[38].藥物在連續(xù)相中的溶解度減小,損失減少,包封率增大.

2) 鹽 析

鹽析:由于無機鹽的加入,增大了非電解質(zhì)的活度系數(shù),從而降低了非電解質(zhì)的溶解度[47].有機溶劑作為非電解質(zhì),無機鹽的加入會對其產(chǎn)生鹽析作用,影響有機溶劑在外水相中的溶解度,從而對包封率造成影響.加入無機鹽,離子溶于水以后,由于離子的溶劑化效應(yīng),離子會和水分子結(jié)合形成溶劑殼層(Hydration shell).這種殼層結(jié)合會減少自由活動的水分子,即減少可供有機溶劑分子表面結(jié)合的水分子數(shù)量.水相與有機溶劑的親和力降低.如NaCl的加入增大了二氯甲烷的活度系數(shù),使二氯甲烷的溶解度減小[48].有機溶劑從乳滴中擴散進入水相的速度減慢,微球固化隨之減慢,包封率減小.

將離子按照其使蛋白質(zhì)變性能力的大小排序,得到了霍夫梅斯特序列(Hofmeister series).鹽離子不僅影響蛋白質(zhì)膠體的穩(wěn)定性,稀的鹽溶液還可以增加或減小蛋白質(zhì)的溶解度.在同等濃度下,按照對蛋白質(zhì)的溶解度改變程度的大小排列,得到了相同的離子序列.另外,除了蛋白質(zhì)溶液,離子對小分子或鹽溶液的穩(wěn)定性和溶解度,都有相同的序列[49].該序列如圖1所示.

圖1 霍夫梅斯特序列Fig.1 Hofmeister series

在對有機試劑在鹽溶液的溶解度研究中,無機鹽對各有機試劑的水溶性抑制程度通過霍夫梅斯特序列描述如下:苯(1/2 Na2SO4>NaCl>NaBr>NaClO4),氨和苯酚(1/2 Na2SO4>NaCl>NaBr),二丙酮醇(1/2 Na2SO4>NaCl>NaBr>NaSCN)[47].在制備小分子藥物載體微球時,由于鹽析作用,有機溶劑在水相中的溶解性降低,微球固化減慢,包封率隨之減小.而在制備載蛋白質(zhì)類微球時,鹽析作用使包封率增大.王亞軍等[50]報道鹽離子可以作為酶蛋白的保護劑,加入非特異性結(jié)合的離子,與蛋白質(zhì)帶電基團和偶極子產(chǎn)生鹽析作用,使酶的結(jié)構(gòu)更加緊湊,達到穩(wěn)定作用.故可在制備載蛋白微球時,通過加入鹽類來提高包封率的同時,也能提高其穩(wěn)定性.

3) 新鹽的生成

表2 硫酸奎尼丁在各鹽溶液中的溶解度Table 2 Solubility of quinidine sulfate in each salt solution

2.3 其他提高微球包封率的辦法

改變制備方法:噴霧干燥法,制得的微球的包封率可高達100%,有研究報道確定不同物料生產(chǎn)所適合的噴霧干燥塔捕粉結(jié)構(gòu),采用兩級旋風加布袋捕粉,產(chǎn)品得率達100%,無損失[51];靜電噴霧[52],該技術(shù)可一步成形,不需要額外添加模板或后處理步驟,藥物的損失減少,包封率普遍高達90%;3D打印制備微球[53],以藥物和載體材料溶于有機試劑的溶液為“打印油墨”,也可一步成形,另外,加入水溶性藥物使外水相飽和,使用藥物的飽和溶液也可減少藥物在固化過程中的損失.

3 結(jié) 論

微球制劑具有廣闊的應(yīng)用前景,其載藥范圍包括小分子化藥、蛋白質(zhì)、多肽和核苷酸等,給藥途徑多,能減輕毒副作用、提高療效和生物利用度等,能起到靶向作用,例如當微球粒徑小于10 μm時,能被巨噬細胞和樹突狀細胞攝取[54],且定位在淋巴結(jié)[55].使用特定的載體材料或?qū)Σ牧线M行修飾所制備得來的微球也能起到靶向作用,如帶正電荷的殼聚糖與表面很多負電荷的腫瘤細胞,兩者定向結(jié)合,使制劑具有靶向性[56].解決制備過程中包封率低這一主要問題對微球制劑的研發(fā)具有重要意義.藥物在分散相和連續(xù)相這兩相之間的分布狀況決定了微球的包封率.筆者簡述了影響乳化-溶劑揮發(fā)法制備微球的包封率的影響因素與機理,從滲透壓、同離子效應(yīng)、鹽析作用和新鹽的生成四個角度詳細闡述了無機鹽在提高微球包封率時發(fā)揮的作用,旨在微球的研究中,為解決包封率低下這一問題提供參考與建議.由于包封率受諸多因素的影響,故在實際操作中應(yīng)全面考慮聚合物、藥物、溶劑和工藝等多方面因素.相信鑒于微球制劑的諸多優(yōu)點與發(fā)展?jié)摿?未來的科學研究會充滿無限可能與挑戰(zhàn).

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