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. 2020 Jun 25;49(3):364–374. [Article in Chinese] doi: 10.3785/j.issn.1008-9292.2020.06.03

叶酸修饰壳聚糖纳米载药胶束的制备及其体外抗肿瘤效果研究

Synthesis of folate modified chitosan-based nanomicelles and its in vitro anti-tumor activity

Lu LIU 1,2, Guojun HUANG 1, Hongzhen BAI 1, Guping TANG 1,*
PMCID: PMC8800751  PMID: 32762172

Abstract

Objective

To design and synthesize folate-modified pH-responsive chitosan-based nanomicelles and investigate the in vitro anti-tumor activity of the drug-loaded micelles.

Methods

CHI-DMA was obtained by reductive amination reaction of aldehyde-based chitosan and hydrophilic amine compounds, and CHI-DMA-LA was obtained by condensation reaction with lauric acid; FA-CHI-DMA-LA was obtained after modification with folic acid (FA). The drug-loaded nanomicelles FA-CHI-DMA-LA/DOX were assembled by solvent change method. The physicochemical properties of polymers were characterized by hydrogen nuclear magnetic resonance and transmission electron microscope. The particle size and surface potential were determined by dynamic light scattering method. Folic acid access rate, doxorubicin (DOX) loading rate and entrapped efficiency were measured by UV-vis spectrophotometer. The drug release properties of DOX-loaded micelles in vitro were monitored by fluorescence spectrophotometer at different pHs (7.4, 6.5, 5.0). The cytotoxicity against human oral cancer KB cells was detected by MTT assay. Fluorescence microscope and flow cytometry were applied to investigate the phagocytosis of DOX-loaded micelles on KB cells.

Results

FA-CHI-DMA-LA was synthesized. The particle sizes of FA-CHI-DMA-LA-1 and FA-CHI-DMA-LA-2 micelles which used for the subsequent experiments were (73±14) nm and (106±15) nm, zeta potential were (15.59±1.98) mV and (21.20±2.35) mV, respectively. The drug loading rates of drug-loaded micelles FA-CHI-DMA-LA-1/DOX and FA-CHI-DMA-LA-2/DOX are (4.08±1.12)%and (4.12±0.44)%, respectively. In vitro drug release is pH-responsive, with cumulative release of DOX up to 37%and 36%at pH 5.0, which is about 1.5 times higher than that of pH 7.4. For FA-CHI-DMA-LA micelles with 1.25 to 125 μg/mL concentration, the survival rate of KB cells is more than 70%after incubation for 24 hours. The cell uptake of FA-CHI-DMA-LA/DOX micelles was enhanced compared to CHI-DMA-LA/DOX, and the cell uptake was higher in incubation without FA medium than that with FA. Compared with free DOX or CHI-DMA-LA/DOX, FA-CHI-DMA-LA/DOX nanomicelles showed higher cyctoxicity to KB cells, especially the FA-CHI-DMA-LA-2/DOX nanomicelles, the cell survival rate was about 17% after incubation for 24 hours.

Conclusion

FA-modified chitosan-based nanomicelle with good biocompatibility was successfully prepared, which exhibits tumor microenvironmental pH responsive drug release and tumor targeting.

Keywords: Drug delivery systems, Nanoparticles, Antineoplastic agents, Folic acid, pH responsive

靶向给药系统是指药物通过载体经局部给药或全身血液循环而选择性地浓集定位于靶器官、靶组织、靶细胞或细胞内结构的给药系统 [ 1] 。其中叶酸受体介导的靶向给药系统是目前最受关注的肿瘤靶向治疗研究技术之一 [ 2- 3] 。叶酸受体是一类糖基磷脂酰肌醇连接的跨膜糖蛋白，相对分子质量为38 000~40 000，在小鼠白血病L1210细胞、小鼠肺癌M109细胞、人宫颈癌Hela细胞、人口腔癌KB细胞等肿瘤细胞表面过表达 [ 4- 9] 。叶酸(folic acid, FA, 合成物中均用简称代替，下同)是一种人体必需的维生素，对叶酸受体具有很强的亲和力。通过叶酸受体介导的胞吞作用，叶酸的喋酸部分埋在叶酸受体内部，谷氨酸部分则暴露在外部，且叶酸受体的功能未受影响 [ 10- 11] 。正常体液的酸碱度值为7.4，肿瘤组织间隙一般为6.3~6.8，而肿瘤细胞核内环境一般为5.0~5.5 [ 12] 。叶酸受体的构型在胞内弱酸性环境中发生改变，释放叶酸修饰的给药系统，同时受体回到细胞膜表面，再转运其他叶酸分子或叶酸修饰的给药系统 [ 2] 。近来，以叶酸受体作为药物释放靶点的研究已成为新型药物释放领域研究的热点之一，并有多个药物进入临床研究阶段 [ 13] 。叶酸-去乙酰基长春碱单酰肼缀合物vintafolide(EC145)对实体瘤的Ⅱ期临床研究于2017年完成 [ 14] 。EC20和EC145对实体瘤、非小细胞肺癌的Ⅰ期临床研究已于2019年完成 [ 15- 16] 。叶酸抗体偶联药物mirvetuximab soravtansine(IMGN853)是首个叶酸受体α靶向的抗体药物偶联物，在治疗叶酸受体α阳性铂耐药卵巢癌Ⅲ期临床中失败，但其对于复发性乳腺癌、复发性输卵管癌、复发性卵巢癌、复发性原发腹膜癌、复发性子宫内膜癌等适应证目前处于Ⅰ期临床研究招募阶段 [ 17] 。

壳聚糖(chitosan, CHI)由自然界广泛存在的几丁质脱乙酰化而得，这种天然高分子具有生物官能性、相容性、安全性、微生物降解性等优良性能，被广泛用于药物载体 [ 18- 24] 。本研究制备了叶酸、月桂酸(lauric acid, LA)、氨基化合物 N′-(3-氨丙基)- N, N-二甲基-1, 3-丙二胺(DMAPAPA)共修饰的FA-CHI-DMA-LA聚合物及其胶束，用于输送抗肿瘤药物阿霉素(doxorubicin, DOX)，考察聚合物胶束的理化性能、载药性能及体外释药行为，并进一步研究聚合物胶束的细胞毒性及细胞摄取情况，以期获得具有肿瘤微环境酸碱度响应的药物输送载体。

1 材料与方法

1.1 细胞、试剂和仪器

KB细胞购自中国科学院上海生科院细胞资源中心，于37 ℃、5%二氧化碳浓度的细胞培养箱中培养，取对数生长期细胞进行实验。壳聚糖(相对分子质量为10 000~30 000)、高碘酸钠、DMAPAPA、乙酸、硼氰化钠、月桂酸、1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDCI)、4-二甲氨基吡啶(DMAP)、 N-羟基丁二酰亚胺(NHS)、叶酸、阿霉素、噻唑蓝(MTT)、无EDTA胰蛋白酶等试剂为美国Sigma-Aldrich公司产品；氘代二甲基亚砜(DMSO- d 6)为上海百灵威化学技术有限公司产品；其他试剂均为分析纯；不含叶酸的RPMI-1640培养基为美国Gibco公司产品；FBS为杭州四季青生物工程材料有限公司产品；透析袋为上海源叶生物科技有限公司产品。核磁共振仪(AVANCE Ⅲ 500)为德国Bruker公司产品；倒置荧光显微镜(Eclipse TE200)为日本Nikon公司产品；激光共聚焦扫描显微镜(Radiance 2100)、酶标仪(Model 550)为美国Bio-Rad公司产品；紫外可见分光光度计(U-2800)为日本Hitachi公司产品；恒温加热磁力搅拌器(85-2型)为上海志威电器有限公司产品；电子天平(FA-1004)为上海衡平仪器仪表厂产品；细胞培养箱(Thermo Scientific 3110系列)为美国Thermo Fisher Scientific公司产品；荧光分光光度计(RF-5301PC)为日本Shimadzu公司产品；超声波细胞粉碎机(JY92-DN)为宁波新芝生物科技有限公司产品；流式细胞仪为美国Beckman Coulter公司产品。

1.2 FA-CHI-DMA-LA的合成

如 图 1所示，壳聚糖经高碘酸钠开环得醛基化壳聚糖，再与亲水性的胺类化合物DMAPAPA经还原胺化反应得CHI-DMA，进一步与疏水性的月桂酸经缩合反应得两亲嵌段的酯CHI-DMA-LA。壳聚糖中多余CH 2OH基团与叶酸的 γ-COOH发生缩合反应，得FA-CHI-DMA-LA。

图1.

FA-CHI-DMA-LA的合成

DMAPAPA：氨基化合物 N’-(3-氨丙基)- N, N-二甲基-1, 3-丙二胺；EDCI：1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐；DMAP：4-二甲氨基吡啶；NHS：N-羟基丁二酰亚胺.

图1

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1.2.1 醛基化壳聚糖的合成

避光条件下，500 mL单口瓶中加入壳聚糖6.2 g，再加入酸碱度4.0的醋酸-醋酸钠缓冲溶液320 mL，室温搅拌溶解，缓慢加入高碘酸钠6.6 g，室温避光搅拌反应4 h后加入乙二醇1.67 mL，继续搅拌1 h除去未反应的高碘酸钠，透析膜(截留相对分子质量8000~14 000)透析3 d，透析完毕，过滤，滤液于低温冰箱预先冷冻，再在冷冻干燥机上脱水干燥，得白色固体醛基化壳聚糖。

1.2.2 CHI-DMA的合成

250 mL单口瓶中加入醛基化壳聚糖8.89 mmol，溶解于含1%乙酸的去离子水溶液120 mL，搅拌溶解，然后缓慢滴加DMAPAPA液体44.47 mmol，室温搅拌反应24 h，滴加0.1 mol/L盐酸溶液调节酸碱度至5.0，缓慢加入硼氰化钠44.47 mmol，继续反应36 h。透析膜(截留相对分子质量7000)透析3 d，透析完毕后过滤，滤液于低温冰箱预先冷冻，再在冷冻干燥机上脱水干燥，得白色固体CHI-DMA。

1.2.3 CHI-DMA-LA的合成

含25 mL DMSO的100 mL单口瓶中分别加入CHI-DMA 22.4 μmol、月桂酸112 μmol、EDCI 110 μmol及DMAP催化量，室温搅拌反应24 h后用透析膜(截留相对分子质量7000)透析3 d后过滤，滤液于低温冰箱预先冷冻，再在冷冻干燥机上脱水干燥，得白色固体CHI-DMA-LA。

1.2.4 FA-CHI-DMA-LA的合成

含50 mL DMSO的100 mL单口瓶中加入CHI-DMA-LA 1 mmol、叶酸0.033 mmol(叶酸与CHI-DMA-LA投料摩尔比为1:30)、EDCI 0.11 mmol、NHS 0.11 mmol及催化量DMAP，室温搅拌反应24 h后用透析膜(截留相对分子质量7000)，去离子水透析3 d，透析完毕，过滤，滤液于低温冰箱预先冷冻，再在冷冻干燥机上脱水干燥，得黄色固体产物FA-CHI-DMA-LA，记为FA-CHI-DMA-LA-1。调整叶酸与CHI-DMA-LA投料摩尔比分别为1:20和1:10，得FA-CHI-DMA-LA-2和FA-CHI-DMA-LA-3。

1.3 FA-CHI-DMA-LA及其胶束理化性质的测定

1.3.1 氢核磁共振测定FA-CHI-DMA-LA结构

称取5 mg待测物，溶解于0.5 mL DMSO- d 6中，室温下用氢核磁共振仪测定样品结构，用Bruker Topspin软件处理数据。

1.3.2 紫外分光光度法测定FA-CHI-DMA-LA叶酸接入率

叶酸溶于0.1 mol/L氢氧化钠溶液配制成不同叶酸浓度的标准溶液，在波长283 nm处测定吸光度值并绘制标准曲线。测定已知浓度FA-CHI-DMA-LA的吸光度值，根据标准曲线得叶酸浓度，进一步计算FA-CHI-DMA-LA中叶酸的接入率。

1.3.3 透射电镜观察FA-CHI-DMA-LA胶束的形态

配制0.2 mg/mL FA-CHI-DMA-LA水溶液，吸取10 μL，滴入电镜用碳膜铜网上，放置于无尘环境中自然晾干，在透射电镜下观察样品形态并拍照。

1.3.4 动态光散射法测定FA-CHI-DMA-LA胶束的粒径和电位

配制0.2 mg/mL FA-CHI-DMA-LA水溶液，25 ℃下使用动态光散射测定胶束的粒径和表面电位。

1.4 透析法制备FA-CHI-DMA-LA/DOX载药胶束

CHI-DMA-LA和不同叶酸接入率的FA-CHI-DMA-LA分别溶于20 mL纯化水中，采用细胞破碎超声仪在冰浴下超声5 min(功率65 W，工作5 s停歇5 s)，并在该条件下缓慢滴入2 mL阿霉素二氯甲烷溶液(阿霉素投入量为载体质量的30%)得到水包油乳化液，滴完继续超声5 min以形成载药胶束溶液，室温搅拌过夜直到大部分二氯甲烷挥发完毕。纯化水透析(透析膜截留相对分子质量7000)6 h以除去残留二氯甲烷，过滤除去未包载的阿霉素，滤液于低温冰箱预冻，再在冷冻干燥机上脱水干燥，分别得CHI-DMA-LA/DOX和FA-CHI-DMA-LA/DOX载药胶束。

1.5 紫外分光光度法测定FA-CHI-DMA-LA/DOX的载药量及包封率

配制阿霉素标准溶液，在496 nm处测定吸光度值并绘制标准曲线。测定已知浓度CHI-DMA-LA/DOX和FA-CHI-DMA-LA/DOX载药胶束的吸光度值，根据标准曲线得阿霉素浓度，进一步计算载药胶束的载药量和包封率，载药量(%)=载药胶束中阿霉素的质量/载药胶束总质量×100%，包封率(%)=载药胶束中阿霉素的质量/初始投入阿霉素的总质量×100%。

1.6 透析法和荧光分光光度法测定载药胶束体外药物释放能力

将FA-CHI-DMA-LA/DOX载药胶束分别配制成1 mg/mL的水溶液，各取2 mL至透析袋中(截留相对分子质量1000)，分别放入40 mL酸碱度值为7.4、6.5、5.0的缓冲液中，37 ℃、80 r/min摇床上孵育。于不同的时间点采样1 mL，并补充等体积相应缓冲液，保持总体积不变。采用荧光分光光度法(E x=480 nm，E m=556 nm)检测样品浓度，计算累积释放百分率，绘制体外释放药物曲线。

1.7 MTT法测定FA-CHI-DMA-LA及其载药胶束的细胞毒性

将对数生长期的KB细胞接种于96孔板内，每孔1×10 4个细胞，37 ℃、5%二氧化碳培养箱孵育16 h，吸出培养液后分别加入FA-CHI-DMA-LA-1和FA-CHI-DMA-LA-2的无血清无叶酸培养液，孵育24 h后吸出培养液，每孔加入100 μL含有0.5 mg/mL MTT的无血清无叶酸培养液，再孵育4 h，小心吸去孔内培养液，每孔加入100 μL DMSO，置摇床上低速振荡10 min，使结晶物充分溶解。酶标仪测定570 nm吸收波长处的吸光度值，并计算细胞存活率。

分别以0.05、0.5、1、2、3、5 μg/mL阿霉素为阳性对照，进一步测定CHI-DMA-LA/DOX和FA-CHI-DMA-LA/DOX的细胞存活率，方法同上。

1.8 荧光倒置显微镜和流式细胞仪检测FA-CHI-DMA-LA的细胞吞噬能力

将对数生长期的KB细胞接种于24孔板中，5%二氧化碳、37 ℃下孵育24 h。吸去上清液，分别加入无叶酸和含叶酸(1 μg/mL)的无血清培养基溶液，孵育2 h。吸去上清液，分别加入阿霉素、CHI-DMA-LA/DOX、FA-CHI-DMA-LA-1/DOX和FA-CHI-DMA-LA-2/DOX溶液使得每孔阿霉素终浓度为5 μg/mL，继续孵育2 h，吸去溶液后洗涤，于荧光倒置显微镜下观察并拍照。

将对数生长期的KB细胞接种于6孔板中，5%二氧化碳、37 ℃下孵育24 h。按照上述方法孵育完成后，用无EDTA胰蛋白酶消化，离心，弃上清液，加入PBS再次离心弃上清液，细胞内阿霉素的摄取能力以流式细胞仪显示荧光强度表示。

1.9 统计学方法

实验所得的数据釆用Excel 2016(for Windows)进行统计分析，多组间比较采用单因素方差分析， P＜0.05为差异具有统计学意义。

2 结果

2.1 FA-CHI-DMA-LA的结构

如 图 2所示，FA-CHI-DMA-LA的核磁共振氢谱中 δ=8.69、7.68、6.85处出现叶酸的特征峰，说明叶酸成功修饰在CHI-DMA-LA上。

图2.

FA-CHI-DMA-LA的核磁共振氢谱

箭头处 δ=8.69、7.68、6.85为叶酸的特征峰.

图2

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2.2 FA-CHI-DMA-LA的叶酸接入率

如 表 1所示，FA-CHI-DMA-LA-1、FA-CHI-DMA-LA-2和FA-CHI-DMA-LA-3中叶酸接入率分别为(6.92±0.9)%、(9.64±1.4)%和(21.7±3.1)%。结果可见，随着叶酸投料比例的增加，叶酸接入率也增加。

表1 FA-CHI-DMA-LA胶束的叶酸接入率、粒径和电位

Table 1 Folic acid access rate, particle sizes and zeta potential of FA-CHI-DMA-LA micelles   ( x ± s, n=3)

样品编号

叶酸接入率(%)

粒径(nm)

电位(mV)

CHI-DMA-LA

—

71±8

21.70±0.67

FA-CHI-DMA-LA-1

6.92±0.90

73±14

15.59±1.98

FA-CHI-DMA-LA-2

9.64±1.43

106±15

21.20±2.35

FA-CHI-DMA-LA-3

21.7±3.11

323±29

20.63±1.73
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“—”:无相关数据.

2.3 FA-CHI-DMA-LA胶束的粒径、电位及形态

不同叶酸接入率的FA-CHI-DMA-LA及CHI-DMA-LA均能形成纳米胶束，呈球形，分布较为均匀，无团聚现象，见 图 3。动态光散射测得FA-CHI-DMA-LA胶束的粒径和电位见 表 1。其结果大于透射电镜测量值, 且胶束的粒径随叶酸接入率的增加而增大。

图3.

FA-CHI-DMA-LA胶束的透射电镜图

CHI-DMA-LA接入叶酸后，粒径变大，胶束成球形，且粒径随着叶酸接入率增加而增大.

图3

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2.4 FA-CHI-DMA-LA/DOX胶束的载药量和包封率

CHI-DMA-LA/DOX、FA-CHI-DMA-LA-1/DOX、FA-CHI-DMA-LA-2/DOX和FA-CHI-DMA-LA-3/ DOX载药胶束的载药率分别为(9.23±0.16)%、(4.08±1.12)%、(4.12±0.44)%和(2.88±0.32)%，包封率分别为(10.36±0.29)%、(14.3±1.53)%、(14.6±1.29)%和(8.52±0.50)%。FA-CHI-DMA-LA-3/DOX的载药率和包封率相对较低，FA-CHI-DMA-LA-1/DOX和FA-CHI-DMA-LA-2/DOX的载药率和包封率接近。结果可见，载体材料的粒径可影响药物的载药量和包封率，粒径增大可能会阻碍药物的包载从而降低载药量和包封率。

2.5 FA-CHI-DMA-LA/DOX胶束的体外释药能力

FA-CHI-DMA-LA-3/DOX载药胶束在22 h时体外释放最大达48%。载药胶束在酸碱度7.4时药物释放最快，在酸碱度5.0时药物释放比酸碱度6.5时快，未表现出酸碱度响应；同时FA-CHI-DMA-LA-3/DOX的载药量和包封率较低，因此后续不再研究。FA-CHI-DMA-LA-1/DOX和FA-CHI-DMA-LA-2/DOX载药胶束在酸碱度5.0下药物释放最快，其体外释放在96 h时分别达36%和37%，释药初期均不存在突释现象，见 图 4。结果可见，FA-CHI-DMA-LA-1/DOX和FA-CHI-DMA-LA-2/DOX载药胶束的释药速率与酸碱度环境有关，酸碱度低时药物释放较快( P < 0.01)。

图4.

FA-CHI-DMA-LA/DOX载药胶束在不同酸碱度下的体外药物释放曲线

与酸碱度7.4比较， * P < 0.01.

图4

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2.6 FA-CHI-DMA-LA胶束的细胞毒性

1.25~125 μg/mL浓度下FA-CHI-DMA-LA-1和FA-CHI-DMA-LA-2胶束作用24 h后KB细胞存活率均在70%以上，见 图 5，表明这两种胶束的细胞毒性均较小，生物相容性良好。

图5.

图5

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FA-CHI-DMA-LA胶束的细胞毒性试验结果

2.7 FA-CHI-DMA-LA/DOX载药胶束的细胞摄取能力

荧光倒置显微镜结果显示，与CHI-DMA-LA/DOX载药胶束比较，FA-CHI-DMA-LA/DOX载药胶束的细胞摄取增强，且后者在无叶酸培养基孵育下的细胞摄取相比含叶酸培养基孵育下增强，见 图 6。同样流式细胞检测结果显示，FA-CHI-DMA-LA/DOX相比阿霉素的细胞摄取增强，叶酸接入率较高的FA-CHI-DMA-LA-2/DOX相比FA-CHI-DMA-LA-1/DOX细胞摄取增强，接入叶酸的载药胶束在无叶酸培养基条件下(红线)的细胞内阿霉素荧光强度明显高于含叶酸培养基条件(蓝线)，见 图 7。可见载药胶束的细胞摄取随叶酸接入率增加而增强，无叶酸培养基下的细胞摄取相比含叶酸培养基下的细胞摄取增强，纯阿霉素不受培养基中叶酸含量影响。

图6.

荧光倒置显微镜下FA-CHI-DMA-LA/DOX载药胶束的细胞摄取

红色为阿霉素，亮度越高代表阿霉素的细胞摄取越多.叶酸修饰的载药胶束比无叶酸修饰载药胶束细胞摄取更多；叶酸修饰的载药胶束在无叶酸培养基条件下比含叶酸培养基条件下细胞摄取更多；无叶酸修饰载药胶束和游离阿霉素无明显区别.标尺=50 μm.

图6

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图7.

流式细胞仪检测FA-CHI-DMA-LA/DOX载药胶束的细胞摄取能力

游离阿霉素的细胞摄取在有/无叶酸培养基条件下无明显差异; 对于叶酸修饰的载药胶束，在无叶酸培养基条件下孵育(红色)比含叶酸培养基条件下孵育(蓝色)的荧光强度更大，细胞摄取更高.

图7

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2.8 FA-CHI-DMA-LA/DOX载药胶束的抗肿瘤细胞效果

与各浓度游离阿霉素比较，相应浓度CHI-DMA-LA/DOX和FA-CHI-DMA-LA/DOX载药胶束的细胞存活率降低；与CHI-DMA-LA/DOX载药胶束比较，FA-CHI-DMA-LA/DOX载药胶束的细胞存活率降低；与FA-CHI-DMA-LA-1/DOX载药胶束比较，FA-CHI-DMA-LA-2/DOX载药胶束的细胞存活率降低，给药24 h后细胞存活率约为17%，见 图 8。结果可见，叶酸接入后载药胶束细胞存活率降低，即抗肿瘤细胞效果增强，且细胞药效随着叶酸接入率的增加而增强。

图8.

FA-CHI-DMA-LA/DOX载药胶束抗肿瘤细胞能力

DOX：阿霉素.与DOX比较， * P < 0.05， ** P < 0.01.

图8

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3 讨论

本研究设计并合成了叶酸修饰酸碱度响应壳聚糖纳米胶束材料FA-CHI-DMA-LA。采用叶酸与壳聚糖材料不同投料比，得到了不同叶酸接入率的载体材料，并通过核磁共振氢谱进行结构确认。透射电镜结果证实，所合成材料均能形成纳米胶束，呈球形，分布较为均匀，无团聚现象。当叶酸投料比越大，叶酸接入率越高，胶束粒径也越大，叶酸接入率较低的胶束粒径为50~150 nm，叶酸接入率高的胶束粒径约为400 nm，表面电位为+15~+22 mV，适合的胶束粒径及电位是细胞摄取的前提和保障 [ 25] 。

载药胶束有多种制备方法，主要包括物理包埋、化学键合和静电结合等。物理包埋法是指在形成胶束的过程中，疏水性药物与聚合物材料的疏水段通过疏水作用和氢键作用，药物直接进入聚合物胶束的疏水内核中，最大限度地保留了药物活性。物理包埋法又包括直接溶解法、透析法和薄膜水化法等几种制备方法。本研究结合了溶解法和透析法，将CHI-DMA-LA和FA-CHI-DMA-LA分别溶于纯化水中，采用超声仪在冰浴下超声5 min，并缓慢滴入阿霉素二氯甲烷溶液得到水包油乳化液，再超声5 min以形成载药胶束，室温搅拌过夜至大部分二氯甲烷挥发完毕，透析除去剩余二氯甲烷。所得材料均能包载疏水药物阿霉素。载体材料的粒径可影响药物的载药量和包封率。CHI-DMA-LA接入叶酸后，粒径增大，且粒径随着叶酸接入率增加而增大。CHI-DMA-LA/DOX的载药量和包封率相对较高，而FA-CHI-DMA-LA-3载药胶束(粒径约400 nm)的载药量约为2.88%，载体材料的粒径增大可能阻碍了药物的包载从而降低载药量和包封率。为了进一步提高载药量和包封率，今后可从以下方面考虑：①提高阿霉素的投料比；②调整材料和阿霉素溶液的浓度、搅拌时间及速度等；③本实验所用溶剂为二氯甲烷，与水不相溶，可考虑换成其他与水互溶的有机溶剂，如DMSO、四氢呋喃、二甲基甲酰胺等 [ 26] 。

药物体外释放方面，FA-CHI-DMA-LA-1/DOX和FA-CHI-DMA-LA-2/DOX在酸碱度5.0下释药率约为酸碱度7.4下的1.5倍，体现了药物释放的酸碱度响应。其原因为FA-CHI-DMA-LA是由壳聚糖中的CH 2OH与叶酸的 γ-COOH经缩合以酯键相连，在酸性条件下酯键断裂可释放出更多药物。因此，该载药胶束可能可以靶向酸碱度值比正常组织低的肿瘤组织，并在肿瘤组织中释放更多阿霉素。

细胞学实验表明，叶酸修饰壳聚糖胶束材料对KB细胞均有较低的细胞毒性，生物相容性良好。与CHI-DMA-LA/DOX载药胶束相比，FA-CHI-DMA-LA/DOX载药胶束细胞摄取有所增强，且随叶酸接入率增大而增强；FA-CHI-DMA-LA/DOX在无叶酸培养基下的细胞摄取相比含叶酸培养基下增强。可见KB细胞有叶酸受体表达，游离的叶酸与KB细胞表面的叶酸受体结合，占据了叶酸受体的位置，使得含叶酸的载药胶束进入细胞较少，因此含叶酸载药胶束对KB细胞具有一定靶向性。体外细胞药效试验显示，加药24 h后载药胶束的细胞药效较阿霉素优，尤其是载体材料中含有叶酸的时候，细胞活性明显下降。其中叶酸接入率高的载药胶束FA-CHI-DMA-LA-2/DOX细胞药效最优，可能是因为叶酸接入率高的载药胶束与KB细胞表面叶酸受体结合，使载药胶束更多进入细胞中，与细胞吞噬结果一致。

综合上述，FA-CHI-DMA-LA-2是理想的叶酸修饰酸碱度响应壳聚糖纳米胶束，具有良好的生物相容性，可促进肿瘤细胞的摄取，细胞药效明显增强，并表现出一定的抗肿瘤靶向效果。其载药胶束FA-CHI-DMA-LA-2/DOX也体现了良好的抗肿瘤靶向性及抗肿瘤效果，有望成为具有良好应用前景的靶向抗肿瘤药物。

Funding Statement

国家自然科学基金（51573161）；浙江省博士后科研项目（zj20180092）

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