CN114748414A - 一种共负载化疗药和纳米粒的海藻酸钠水凝胶复合体系及其制备方法和应用 - Google Patents
一种共负载化疗药和纳米粒的海藻酸钠水凝胶复合体系及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种共负载化疗药和纳米粒的海藻酸钠水凝胶复合体系及其制备方法和应用,包括将PCL‑PEG‑PCL、疏水性IMQ溶于有机溶剂,去除有机溶剂形成薄膜,再经水化超声,形成均一的胶束纳米粒溶液;DSPE‑PEG5000‑Mannose、DSPE‑PEG2000‑Mal、MPLA溶于有机溶剂,去除有机溶剂后形成磷脂薄膜;将胶束纳米粒溶液加入磷脂薄膜后经水化超声、浓缩,得到脂质聚合物杂化纳米粒溶液;将化疗药盐酸阿霉素、Indoximod(IND)及脂质聚合物杂化纳米粒溶液混合后加到海藻酸钠粉末中,震荡混合;本发明复合体系能够缓慢释放盐酸阿霉素和IND,脂质聚合物杂化纳米粒通过促进树突状细胞的成熟及抗原提呈过程进一步促进T细胞的活化,从而抑制原位瘤和远端瘤的生长。
Description
技术领域
本发明涉及生物技术领域,具体涉及一种共负载化疗药和纳米粒的海藻酸钠水凝胶复合体系及其制备方法和应用。
背景技术
近年来,癌症的发病率与死亡率逐年升高,已经成为一种严重威胁人类健康的疾病。目前常用的传统肿瘤治疗方法包括手术、化疗、放疗等,但均具有一定的局限性:手术能切除大部分的肿瘤组织,但风险高、易复发;化疗有严重的毒副作用且容易使肿瘤细胞产生耐药性;放疗在杀伤肿瘤组织的同时会损害部分正常的组织细胞,对于较大的肿瘤无法完全消除。
与此同时,免疫疗法作为一种新兴的肿瘤治疗策略迅速发展,为许多癌症患者带来新的希望。与传统的化疗、放疗直接杀伤肿瘤细胞的方式不同,免疫疗法通过激活体内免疫系统间接攻击和杀伤肿瘤细胞,从而引发高效的抗肿瘤免疫反应。因此,免疫疗法不仅能消除局部和远端转移肿瘤,减少脱靶效应,还能通过机体的免疫记忆效应预防肿瘤的复发。目前在临床免疫治疗策略主要有:免疫检查点阻断治疗、肿瘤疫苗和嵌合抗原受体CAR-T细胞治疗。然而单独的免疫治疗仍然存在很多缺点,如成本高、治疗周期长、治疗反应率低和免疫相关不良反应的个体差异大等,从而限制了其临床应用。因此为了提高疗效,减轻副作用,化疗免疫的联合治疗逐渐被应用于癌症的治疗。此时则需要一种理想的递送策略实现化疗药物与免疫佐剂的共递送。
发明内容
本发明的目的在于提供一种共负载化疗药和纳米粒的海藻酸钠水凝胶复合体系及其制备方法和应用,该复合体系以天然的海藻酸钠为基质,负载化疗药、Indoximod及脂质聚合物杂化纳米粒,其中脂质聚合物杂化纳米粒的疏水内核包载IMQ,磷脂层镶嵌MPLA,载体外表面修饰马来酰亚胺功能化磷脂以捕获化疗产生的的肿瘤抗原,甘露糖配体用于靶向抗原提呈细胞,从而形成一种通过化疗联合免疫治疗的方法有效治疗恶性黑色素瘤的复合体系。
为了实现本发明的上述目的,特采用以下技术方案:
本发明第一方面提供一种共负载化疗药和纳米粒的海藻酸钠水凝胶复合体系的制备方法,所述制备方法包括如下步骤:
(a)将PCL-PEG-PCL、疏水性IMQ溶于有机溶剂,然后,去除有机溶剂形成薄膜,薄膜再经水化、超声、超滤后形成均一的胶束纳米粒溶液;
(b)DSPE-PEG5000-Mannose、DSPE-PEG2000-Mal、MPLA溶于有机溶剂,去除有机溶剂后形成磷脂薄膜;
(c)将胶束纳米粒溶液加入磷脂薄膜后经水化、超声、超滤浓缩,得到脂质聚合物杂化纳米粒溶液;
(d)将化疗药、Indoximod及脂质聚合物杂化纳米粒溶液混合后加到海藻酸钠粉末中,震荡混合,即得共负载化疗药和纳米粒的海藻酸钠水凝胶复合体系。
与单独使用化疗药物或免疫佐剂相比,共负载化疗药和纳米粒的海藻酸钠水凝胶复合体系将产生1+1>2的抗肿瘤效果。首先从水凝胶中释放的化疗药物通过非特异性杀伤肿瘤可以消除大部分原位肿瘤,并引发免疫原性细胞死亡、肿瘤相关抗原释放及损伤相关分子模式高表达;继而负载免疫佐剂的纳米粒捕获肿瘤相关抗原并靶向递送至抗原提呈细胞,在抗原与佐剂的联合作用下活化的树突状细胞将抗原提呈给效应T细胞,引起抗原特异性T细胞的快速增殖与活化,从而对剩余的原位肿瘤细胞及转移的肿瘤产生杀伤。同时该体系的双持续释放特点使其如免疫系统的发电站,源源不断地提供肿瘤抗原和免疫佐剂来刺激免疫细胞的增殖和活化,从而对肿瘤细胞产生长期有效的抑制作用。
优选地,所述有机溶剂选自二氯甲烷和甲醇中的至少一种;更优选地,疏水性IMQ溶于二氯甲烷和甲醇的混合溶剂中,且二氯甲烷和甲醇的体积比为1∶(0.6~1.5)。
优选地,所述PCL-PEG-PCL的分子量为10000~20000;
优选地,所述PCL-PEG-PCL为PCL4000-PEG8000-PCL4000。
优选地,所述步骤(a)中,PCL-PEG-PCL与疏水性IMQ的质量比为(20~25)∶1;
水化温度为65~75℃,时间为5~8h;
超声时间为10~20min;
超滤为采用10KDa超滤管以3000~5000rpm离心1~3h。
优选地,所述步骤(b)中,DSPE-PEG5000-Mannose与PCL-PEG-PCL的质量比为1∶(50~70);
DSPE-PEG2000-Mal与PCL-PEG-PCL的质量比为1∶(10~40);
MPLA与PCL-PEG-PCL的质量比为(1~5)∶1000。
优选地,所述步骤(c)中,水化温度为25~40℃,时间为1~2h;
超声时间为4~10min。
优选地,超滤浓缩至PCL-PEG-PCL的浓度为30~40mg/mL。
优选地,所述步骤(d)中,化疗药、Indoximod(IND)和PCL-PEG-PCL的质量比为(1~2)∶(0.5~1)∶20;
海藻酸钠的终浓度为20~25mg/mL。
优选地,所述化疗药为盐酸阿霉素。
本发明第二方面提供一种上述制备方法制得的共负载化疗药和纳米粒的海藻酸钠水凝胶复合体系。
本发明第三方面提供一种上述共负载化疗药和纳米粒的海藻酸钠水凝胶复合体系在制备化疗联合免疫治疗的抗肿瘤药物中的应用。
本发明第四方面提供一种化疗联合免疫治疗的抗肿瘤药物,所述抗肿瘤药物包括上述共负载化疗药和纳米粒的海藻酸钠水凝胶复合体系。
在本发明中共负载化疗药和纳米粒的海藻酸钠水凝胶复合体系包括三部分:化疗药DOX、IDO抑制剂IND和脂质聚合物杂化纳米粒;其中阿霉素(DOX)是一种广谱的抗肿瘤抗生素,通过抑制核酸合成干扰肿瘤细胞的增殖,是目前临床上应用较为广泛的化疗药物;IND是一种吲哚胺2,3-双加氧酶(IDO)途径抑制剂,通过充当色氨酸模拟物干扰IDO介导的色氨酸信号通路,逆转IDO信号通路,抑制调节性T细胞的活性,促进效应T细胞的增殖,从而增强对肿瘤的杀伤作用;脂质聚合物杂化纳米粒内部核心为聚合物纳米胶束用于包载疏水性的IMQ,胶束表面用马来酰亚胺功能化磷脂DSPE-PEG2000-Mal、甘露糖靶向磷脂DSPE-PEG5000-Mannose进行修饰并嵌合磷脂类TLR4免疫激动剂MPLA;IMQ是一种TLR7/8激动,是一种已被FDA批准的很有前途的树突状细胞(DC)激活剂;甘露糖受体在树突状细胞表面丰富表达,通过纳米粒表面修饰的甘露糖配体可以实现对树突状细胞的靶向递送;MPLA是第一种被批准作为疫苗佐剂使用的TLR4激动剂,研究表明基于TLR4位于细胞膜的表面,TLR7/8位于细胞膜表面和内体,并且二者具有不同的信号传导机制,使得MPLA与IMQ的联合使用可明显促进DC成熟和抗原提呈,从而进一步引发强大的抗肿瘤免疫效应;天然的海藻酸钠溶液经瘤内原位注射后响应体内一定浓度的钙离子形成原位凝胶,缓慢释放的盐酸阿霉素通过抑制细胞周期进展杀伤肿瘤,经马来酰亚胺修饰的NPs捕获肿瘤裂解产生的抗原形成原位疫苗,然后纳米疫苗通过甘露糖靶向递送到树突状细胞,进一步促进DC的成熟与抗原提呈,从而引发机体产生强大的抗肿瘤免疫效应,最终实现对肿瘤的化疗联合免疫治疗。
与现有技术相比,本发明的有益效果至少包括:
(1)本发明采用天然的海藻酸钠为聚合物基质制备的水凝胶具有良好的生物相容性,体内易降解,响应肿瘤内的钙离子形成的水凝胶可以实现化疗药盐酸阿霉素、IDO抑制剂的缓慢释放,从而对原位肿瘤产生持续的杀伤作用。
(2)本发明采用稳定性和生物相容性良好的PCL-PEG-PCL为载体材料制备包载疏水性IMQ的胶束纳米粒,胶束表面用马来酰亚胺功能化磷脂DSPE-PEG2000-Mal、甘露糖靶向磷脂DSPE-PEG5000-Mannose进行修饰并嵌合磷脂类TLR4免疫激动剂MPLA,从而制备出粒径较小、分布均匀,具有抗原捕获性能并能程序化共递送多组分的脂质聚合物杂化纳米粒。
(3)本发明制备的脂质聚合物杂化纳米粒不仅对骨髓树突状细胞(BMDCs)有良好的生物安全性,还能促进BMDCs的成熟,上调其表面共刺激分子的表达及细胞因子的分泌。
(4)本发明制备的共负载化疗药和纳米粒的海藻酸钠水凝胶复合体系通过药物和纳米粒的双持续释放实现药物的程序化交付;一方面,水凝胶中负载的化疗药盐酸阿霉素和IDO抑制剂IND通过缓慢释放来持续地杀伤原位肿瘤细胞;另一方面释放的脂质聚合物杂化纳米粒通过捕获原位肿瘤裂解产生的抗原形成共载抗原与双佐剂的疫苗并靶向递送到树突状细胞,通过促进树突状细胞的成熟及抗原提呈过程来促进T细胞的活化,进一步促进肿瘤坏死因子TNF-α,细胞杀伤因子IFN-γ的产生,从而抑制原位瘤和远端瘤的生长。
(5)本发明的共负载化疗药和纳米粒的海藻酸钠水凝胶复合体系不仅制备方法简单,操作周期短,可重复性强;而且载体材料价廉易得,生物相容性好,作为一种化疗联合免疫治疗肿瘤的复合体系有很好的的临床转化潜力。
附图说明
为了更清楚地说明本发明具体实施方式或现有技术中的技术方案,下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍。在所有附图中,类似的元件或部分一般由类似的附图标记标识。附图中,各元件或部分并不一定按照实际的比例绘制。
图1为本发明实例1中制备的脂质聚合物杂化纳米粒的粒径分布图、粒径稳定性图、IMQ的紫外吸收光谱图及抗原捕获前后的粒径电位变化图;
图2为本发明实施例1中制备的脂质聚合物杂化纳米粒对树突状细胞的毒性结果图、促成熟作用的柱状统计图及经诱导后BMDCs的细胞因子分泌柱状统计图;
图3为本发明实例1中制备的负载化疗药和纳米粒的海藻酸钠水凝胶的体外成胶实验图、扫描电镜图及流变特性图;
图4为实施例3制备的GEL-IND-DOX水凝胶中负载的化疗药DOX和IDO抑制剂IND在pH 7.4和pH 6.5的PBS溶液中的体外释放曲线图;
图5为实施例3和实施例4制备的载药水凝胶和游离药物与B16F10细胞共孵育后的细胞存活率图、细胞周期分布统计图;
图6为实施例4制备的GEL-DOX和Free DOX与B16F10共孵育后的细胞摄取共聚焦图;
图7为本发明的治疗方案及经治疗后C57BL/6荷瘤小鼠的近端、远端抑瘤曲线、生存曲线及体重曲线图;
图8为经本发明不同制剂诱导C57BL/6荷瘤小鼠脾细胞内CD4+ T和CD8+ T细胞增殖的流式图及柱状统计图;
图9为本发明不同制剂促进C57BL/6荷瘤小鼠脾细胞内CD4+ T和CD8+ T细胞活化的流式图及柱状统计图;
图10为本发明不同制剂抑制脾内调节性T细胞(Treg)增殖的流式图及柱状统计图;
图11为本发明不同制剂治疗后C57BL/6荷瘤小鼠脾细胞内分泌IFN-γ的T细胞比例的流式图和柱状统计图;
图12为本发明不同制剂治疗后C57BL/6荷瘤小鼠脾细胞内分泌TNF-α的T细胞比例的流式图和柱状统计图;
图13为本发明不同制剂治疗后C57BL/6荷瘤小鼠脾细胞内巨噬细胞分型的流式图和柱状统计图。
具体实施方式
下面将结合实施例对本发明技术方案的实施例进行详细的描述。以下实施例仅用于更加清楚地说明本发明的技术方案,因此只作为示例,而不能以此来限制本发明的保护范围。
需要注意的是,除非另有说明,本申请使用的技术术语或者科学术语应当为本发明所属领域技术人员所理解的通常意义。
实施例1
本实施例为一种共负载化疗药和纳米粒的海藻酸钠水凝胶复合体系的制备方法,该制备方法包括如下步骤:
(a)将1mg疏水性TLR7/8激动剂IMQ溶于1mL混合溶剂(甲醇与二氯甲烷体积比1:1),然后加入3mL二氯甲烷和20mg PCL4000-PEG8000-PCL4000,经37℃旋转蒸发有机溶剂后在烧瓶底形成均匀的薄膜,经真空干燥6h后水化并于冰浴条件下以30%的功率超声10min,得到均一的胶束纳米粒溶液,最后经超滤后去除游离的药物;水化选用去离子水作为溶剂于65℃水化5h,超滤选择10KDa的超滤管于4000rpm离心3次(每次30~40min);
(b)将0.4mg DSPE-PEG5000-Mannose、2mg DSPE-PEG2000-Mal、20μgTLR4激动剂MPLA溶于4mL二氯甲烷,经37℃旋转蒸发有机溶剂后形成一层均匀的磷脂薄膜,然后真空干燥6h;
(c)将胶束纳米粒溶液加入干燥后的磷脂薄膜后于37℃水化2h,于冰浴中超声形成甘露糖靶向并具有抗原捕获功能的脂质聚合物杂化纳米粒(记为NP-Mal)溶液,之后进行超滤浓缩到0.62mL(PCL4000-PEG8000-PCL4000等效浓度为32mg/mL),其中超声的功率为25%,超声时间为4min;
(d)将0.75mg IND加入到0.5mL去离子水经超声溶解成为均一透明的IND溶液,将1.5mg盐酸阿霉素、IND溶液和浓缩后得到的0.62mL脂质聚合物杂化纳米粒溶液混合后加到28mg海藻酸钠粉末中,经震荡溶解后形成共负载化疗药和纳米粒的海藻酸钠水凝胶复合体系。
实施例2
本实施例为一种共负载化疗药和纳米粒的海藻酸钠水凝胶复合体系的制备方法,该制备方法与实施例1中的制备方法基本相同,区别仅在于步骤(b)中未添加DSPE-PEG2000-Mal。
实施例3
本实施例为一种负载化疗药阿霉素和IDO抑制剂IND的海藻酸钠水凝胶的制备方法,制备方法包括如下步骤:
将0.75mg IND加入到0.5mL离子水经超声溶解成为均一透明的IND溶液,加0.62mL去离子水溶解1.5mg盐酸盐酸阿霉素,二者混合后加到28mg海藻酸钠粉末中,经震荡溶解后形成共负载化疗药和纳米粒的海藻酸钠水凝胶复合体系。
实施例4
本实施例为一种负载化疗药的海藻酸钠水凝胶的制备方法,该制备方法与实施例3中的制备方法基本相同,区别仅在于未添加IND。
实验例5
分别按照实施例1~4制备得到水凝胶复合体系,其中实施例1制备得到的水凝胶复合体系简称为GEL-ID-NP-Mal,实施例2制备得到的水凝胶复合体系简称为GEL-ID-NP,实施例3制备得到的水凝胶复合体系简称为GEL-ID,实施例4制备得到的水凝胶复合体系简称为GEL-D;
1、采用马尔文粒度仪对实施例1中步骤(c)制备的脂质聚合物杂化纳米粒的粒径、电位、稳定性进行表征;结果如图1中A、B所示;
由图1的A、B可知,本发明制备的脂质聚合物杂化纳米粒的平均粒径为165.8±9.01nm,粒径分布为0.300±0.05,电位值为-11.6±0.78mV,纳米粒在PBS溶液中可稳定保存8天,粒径仍未发生明显改变,可见该纳米粒具有良好的稳定性,有利于长期储存和运输。
2、本发明实施例1中脂质聚合物杂化纳米粒的载药量和包封率的表征:
将实施例1中(c)的脂质聚合物杂化纳米粒冷冻干燥,称取1mg冻干粉末溶于1mL二甲基亚砜(DMSO)中作为样品,称取IMQ标准品溶于DMSO中超声溶解配成200μg/mL的溶液,然后采用倍比稀释法配制不同浓度的标准溶液;紫外扫谱确定IMQ的最大吸收峰,使用紫外分光光度计测量样品溶液和标准品的吸光度,并采用标准曲线法测定样品中IMQ的量,并计算脂质聚合物杂化纳米粒中IMQ的载药量和包封率;
载药量公式如下:
载药量(%)=脂质聚合物杂化纳米粒中的药物质量/脂质聚合物杂化纳米粒的质量×100%
包封率(%)=脂质聚合物杂化纳米粒中的药物质量/药物投药量×100%
如图1中C所示,IMQ与脂质聚合物杂化纳米粒均在331nm处有最大吸收峰,证明脂质聚合物杂化纳米粒成功包载了IMQ,计算得出实施例1中制备的脂质聚合物杂化纳米粒的IMQ载药量为3.74±0.08wt%;包封率为83.00±1.83%,因此脂质聚合物杂化纳米粒表现出对IMQ有较高的装载能力,可能是由于两亲性的三嵌段共聚物PCL-PEG-PCL进一步增加了疏水药物IMQ的溶解度。
3、本发明实施例1中制备的脂质聚合物杂化纳米粒的抗原捕获作用的评估:
将B16F10细胞在大培养皿中培养长满(约107个细胞),加入含20μg/mL的DOX完全培养基,培养4h后,吸弃培养基,并用PBS洗涤2次,加入PBS(7-8mL)孵育48h后,以200g离心5min以除去不溶的细胞碎片得到含肿瘤细胞裂解抗原的上清液。分别加入23mg纳米粒NP或NP-Mal(纳米粒NP与NP-Mal区别在于实施例1的步骤(b)中未添加DSPE-PEG2000-Mal)与如上所述制备的含抗原的上清液一起温育。具体而言,将每种制剂(23mg)与来自107个细胞的肿瘤抗原混合,震荡孵育后(室温,20h),使用100KDa超滤管于5000g离心15min,并用去离子水洗涤纳米粒2次,得到与肿瘤抗原共孵育后的纳米粒(NP@pro、NP-Mal@pro)。同时测定纳米粒与肿瘤裂解抗原孵育前后的粒径和电位,以评估纳米粒的抗原捕获作用。
如图1的D-F所示,NP-Mal组纳米粒与抗原孵育后粒径和电位均发生显著变化,粒径由孵育前的142nm变为306nm,平均电位由-11.6mV变为1.1mV,而NP组纳米粒的粒径和电位只发生较小的变化,因此纳米粒表面修饰Mal基团赋予了纳米粒抗原捕获功能。
4、本发明实施例1制备的脂质聚合物杂化纳米粒对DCs的毒性评估:
将处于对数生长期的DC 2.4细胞稀释成105个/mL,然后以每孔10000个的数量接种于96孔板。孵育24h细胞贴壁后,吸去培养基,分别以5、10、15、20、40、80μg/mL的IMQ等效浓度加入脂质聚合物杂化纳米粒(NP-Mal),将96孔板放入培养箱孵育24h,取出96孔板,吸弃孔中液体,每孔加100μLPBS洗2次,然后每孔中加入100μL混有MTT的完全培养液,培养箱中孵育30min后待对照组变成紫色,于酶标仪上490nm处测吸光度值,根据细胞存活率公式计算DC的存活率。
细胞存活率=(实验组OD值-空白组OD值)/(阴性对照组OD值-空白组OD值)
如图2中A所示,不同IMQ浓度的脂质聚合物杂化纳米粒孵育24h后的DC存活率,在IMQ浓度5~80μg/mL的范围内,DC的存活率大于90%,证明该发明制备的脂质聚合物杂化纳米粒对DC细胞是安全无毒的。
5、本发明制备的脂质聚合物杂化纳米粒对于促进BMDCs成熟和活化的作用:
将B16F10细胞在大培养皿中培养长满(约107个细胞),加入含20μg/mL的DOX完全培养基,培养4h后,吸弃培养基,并用PBS洗涤2次,加入PBS(7-8mL)孵育48小时。孵育后,收集上清液并以200g离心5分钟除去不溶的细胞碎片得到肿瘤细胞裂解抗原。
纳米粒(约23mg)与如上所述制备的含抗原的上清液一起温育。具体而言,将每种制剂与来自107个细胞的肿瘤抗原混合。震荡孵育后(室温,20h),使用100KDa超滤管于5000g离心15min,并用去离子水洗涤纳米粒2次,得到与肿瘤抗原共孵育后的纳米粒(NP@pro、NP-Mal@pro)。
将6~8周的小鼠处死,提取腿骨中的BMDCs,裂解红细胞后用BMDCs专用培养基重悬后加入到6孔板中,于37℃,5%CO2条件下培养至第6天,然后分别加入Free IMQ+MPLA、NP@pro、NP-Mal、NP-Mal@pro(IMQ等效浓度15μg/mL)以及不加任何刺激的对照组(PBS组),继续在细胞培养箱中培养24h后收集上层半贴壁的细胞进行分析。将各组BMDCs收集至1.5mL离心管中,加入PBS离心(450g,5min)洗涤两次,用100μL的1640培养基重悬,分别向离心管中加入PerCP-anti-CD11c,PE-anti-CD40以及FITC-anti-CD86。在4℃下孵育30min后,用PBS洗涤2次,用0.4mL组织固定液重悬于流式管中过200目细胞筛,上流式细胞分析仪测定BMDCs表面共刺激因子CD86和CD40的表达水平。
脂质聚合物杂化纳米粒对BMDCs成熟的诱导,通过测定与纳米粒共孵育24h的BMDCs分泌的细胞因子水平来判断。向培养至第6天的BMDCs中加入Free IMQ+MPLA、NP@pro(与肿瘤细胞裂解物共孵育后的NP)、NP-Mal、NP-Mal@pro(与肿瘤细胞裂解物共孵育后的NP-Mal)(IMQ等效浓度15μg/mL)以及不加任何刺激的对照组(PBS组),继续在细胞培养箱中(37℃,5%CO2)培养24h后收集细胞离心(450g,5min)并收集上清。随后用ELISA试剂盒通过绘制标准曲线计算BMDCs分泌的肿瘤坏死因子TNF-α、及细胞杀伤因子IFN-γ的浓度。
抗原提呈细胞(APC)尤其树突状细胞(DC)作为连接先天性免疫和适应性免疫的桥梁,在调节机体免疫系统正确识别特异性抗原和提高效应T细胞的功能方面起着重要作用。成熟的DC细胞表面高表达CD40、CD86等共刺激分子,继而活化CD4+ T细胞和CD8+ T细胞为效应T细胞,诱导机体产生强大的免疫效应。因此,通过分析脂质聚合物杂化纳米粒上调BMDCs表面CD40、CD86的情况,来研究其对BMDCs的促成熟作用。如图2中B显示,与PBS组相比,NP-Mal组上调CD40、CD86的水平明显提高,与抗原共孵育后NP-Mal@pro组CD40+CD86+水平进一步提高,表现了最好的诱导BMDCs成熟的作用,说明NP-Mal通过捕获肿瘤相关抗原能进一步促进BMDCs的成熟。
成熟的DC通过释放细胞因子调节机体的免疫反应,抑制肿瘤的生长。通过不同的制剂与BMDCs体外培养24h来检测活化的DC能否促进细胞因子的分泌。如图2中C所示,NP-Mal@pro组与BMDCs孵育后比NP-Mal、NP@pro、Free组产生更高水平的TNF-α,且NP-Mal@pro组产生的TNF-α是NP-Mal的1.36倍;如图2中D所示NP-Mal@pro组诱导产生最高水平的细胞杀伤因子IFN-γ,是NP-Mal组的2.29倍,Free组的3.34倍。这些Th1型细胞因子可以促进DCs的成熟并且有利于CD8+ T的细胞激活。因此NP-Mal@pro通过Mal基团的抗原捕获作用及免疫佐剂IMQ和MPLA的免疫调节作用,能显著提高BMDC对细胞因子TNF-α及细胞杀伤因子IFN-γ的分泌。
6、水凝胶的理化性质表征(体外成胶性、流变特性和扫描电镜表征):
分别将游离的盐酸阿霉素溶液、GEL-DOX、GEL-DOX-NP-Mal用1mL注射器注射到1.8mM的钙离子溶液中,使其转变为水凝胶,并用相机记录不同时间点的照片(如图3中A所示);然后将形成的海藻酸钠水凝胶取出,放置到流变仪上测定储能模量与损耗模量随剪切速率的变化以研究形成的水凝胶的流变特性(如图3中C、D所示);将形成的海藻酸钠水凝胶用液氮淬冷并冻干,再次用液氮淬冷,切成扫描电镜所需的厚度,然后使用透射电镜对其形貌进行表征,结果如图3中B所示。
由图3中A可知,在含有钙离子的溶液中注入游离的盐酸阿霉素溶液后红色液体迅速扩散,而GEL-DOX及GEL-DOX-NP前体溶液在钙离子溶液中迅速转化为凝胶;图3中C和3中D的流变学实验结果进一步证明在应变为1%时,一定的剪切速率范围内(0.01~10rad/s),水凝胶的储能模量G’和损耗模量G”趋于稳定,对频率的依赖性较弱,且储能模量G’一直大于损耗模量G”,且在应力0.1~100Pa的范围内,水凝胶的储能模量G’和损耗模量G”也趋于稳定,对应力的依赖性也较弱,且储能模量G’一直大于损耗模量G”,说明在一定的外力作用下水凝胶的结构仍能保持着较稳定的凝胶状态;由图3中B的扫描电镜图中可以看出形成的水凝胶具有三维多孔网状结构,孔的形状为类圆形,内部连通性良好,有利于游离药物的释放。
7、本发明实施例3制备得到的GEL-ID中IND和DOX的体外释放:
将实施例3制备得到的GEL-ID溶胶样品加入到分子量8000-14000的透析袋中,然后置于1.8mM的钙离子溶液中室温过夜以形成稳定的水凝胶,第二天取出后放到有不同pH的PBS的缓冲液中在摇床上震荡进行释放实验。其中每组3个平行样,释放液分别为pH 7.4和6.5的PBS缓冲液,体积为20mL,考察不同pH对水凝胶中药物释放的影响;摇床的温度为37℃,转速为120rpm,一段时间后(0.5h、1h、2h、3h、4h、6h、8h、10h、12h、14h、1d、2d、3d、4d、5d、6d)取出全部释放液,并补充相应的20mL释放液。利用紫外分光光度计在780nm处测定DOX的吸光度,结合游离DOX在释放液中的标准曲线计算水凝胶DOX-ID中DOX在不同时间点的累积释放量并绘制DOX的释放曲线,如图4中A所示。利用高效液相法,使用紫外检测器,以水/乙腈:75/25,v/v;0.1%三氟乙酸为流动相,以225nm为检测波长测定不同浓度的IND标准溶液以绘制标准曲线,并测定不同时间点的释放液中IND的含量并绘制IND的释放曲线,如图4中B所示。
由图4可知,DOX和IND均以稳定的速度持续释放,一方面微酸环境加速了DOX和IND的释放,在pH 6.5的PBS溶液中DOX和IND的释放速度更快;另一方面DOX比IND释放得更加完全,可能是由于DOX的水溶性比IND好,更容易通过扩散从水凝胶的网状结构中释放。
8、本发明实施例3和实施例4制备的载药水凝胶对B16F10细胞的毒性考察:
将处于对数生长期的B16F10细胞稀释成105个/mL,然后以每孔10000个的数量接种于96孔板。孵育24h细胞贴壁后,吸去培养基,分别以DOX等效浓度0.0125、0.125、0.625、1.25、2.5、5、10μg/mL,IND等效浓度0.00625、0.0625、0.3125、0.625、1.25、2.5、5μg/mL加入不同浓度的样品,其中对于游离药物组(Free DOX、Free IND、Free IND+DOX)直接加入100μL含等效药物浓度的培养基,水凝胶组(Blank GEL、GEL-DOX、GEL-IND、GEL-IND-DOX)先加入100μL含钙离子的培养基,再加入10μL样品,将96孔板放入培养箱孵育24h,取出96孔板,吸去孔中液体,每孔加100μL PBS洗2次,然后每孔中加入100μL混有CCK8的1640完全培养液,培养箱中孵育30min后待对照组变成橙色,于酶标仪上450nm处测吸光度值,根据细胞存活率公式计算与不同药物制剂孵育的B16F10细胞的存活率,具体如图5中A所示。
细胞存活率=(实验组OD值-空白组OD值)/(阴性对照组OD值-空白组OD值)
如图5中A所示,Blank组的细胞存活率接近100%,25mg/mL的天然的海藻酸钠对细胞的生存完全没有影响,证明该材料具有良好的生物相容性;GEL-IND和Free IND组细胞的存活率均大于60%,随IND的浓度的增大,对B16F10细胞的存活产生轻度的抑制作用;而GEL-DOX、GEL-IND-DOX、Free DOX、Free IND+DOX等含有化疗药DOX的实验组明显抑制B16F10的细胞生存,相同药物浓度下,Free IND+DOX的细胞毒性大于GEL-IND-DOX,FreeDOX的细胞毒性大于GEL-DOX,这是由于通过水凝胶负载药物保证了DOX的缓慢释放,短时间内水凝胶组的DOX并未完全释放,故而对B16F10细胞产生的杀伤作用不如游离组强;更重要的的是GEL-IND-DOX组比GEL-DOX组对B16F10细胞产生了更强的细胞毒性,证明DOX和IND的联合使用增强了水凝胶复合体系对肿瘤细胞的抑制作用。
本实施例3、4制备的GEL-ID、GEL-D体外抑制B16F10细胞增殖机制研究:
将B16F10细胞以每孔2×105个细胞接种在6孔板中,然后用不同的药物制剂(FreeDOX、Blank GEL、GEL-DOX、GEL-IND、GEL-IND-DOX)使用与上述用来分析细胞摄取相同的方法处理细胞12h;然后用冷PBS洗涤细胞两次,胰蛋白酶处理后收集细胞;收集到的细胞用PBS洗涤,用PBS重悬;然后用90%乙醇固定细胞悬液,最终乙醇浓度为70%;细胞在4℃固定过夜后,用冷PBS洗涤细胞以去除残留的乙醇,并按照试剂盒方案用PI染色。
细胞周期是一系列介导细胞分裂的可控过程。由于DOX与DNA相互作用并抑制大分子的生物合成,因此研究了载入水凝胶中的DOX是否保留了干扰B16F10细胞的细胞周期进程的能力。如图5中B流式细胞术的定量分析柱状图显示,经Free DOX、GEL-DOX、GEL-IND-DOX处理的细胞具有非常相似的细胞周期谱,有更高比例的细胞被阻滞在G2/M期,并显著减少了处于S期细胞数量。这表明GEL-DOX和GEL-IND-DOX具有抑制细胞周期进展的能力。
9、本实施例4制备的GEL-DOX的B16F10摄取情况考察:
将处于对数生长期的B16F10细胞稀释成4×105个/mL,以每孔2×105个细胞接种于激光共聚焦皿,每孔体积500μL。过夜后,将激光共聚焦培养皿中的培养基吸弃,Free DOX组加入2mL用培养基稀释的游离DOX(1.25μg/mL),GEL-DOX组先加入2mL含钙离子培养再加入100μL的GEL-DOX(25μg/mL),使培养基中等效DOX浓度为1.25μg/mL;培养一定时间(1h、4h、12h),吸弃培养基,用1mL PBS洗涤细胞2次;用碧云天的免疫染色固定液室温固定细胞10分钟;用碧云天的免疫染色洗涤3次,每次约5分钟;加入Hochest染色液1mL室温培养20min,用PBS洗涤3次,最后加入300μLPBS去激光共聚焦观察,观察结果如图6所示。
如图6所示,与药物孵育1h后游离DOX组的B16F10细胞中有明显的DOX荧光,而GEL-DOX组的细胞中未发现DOX的荧光;与药物孵育4h后,游离DOX组细胞中的DOX荧光明显增强,而GEL-DOX组出现微弱的荧光;最终与药物孵育12h后,游离组与GEL-DOX的荧光均增强,但游离组荧光强度仍大于GEL-DOX。说明GEL-DOX通过水凝胶的三维网状结构保证了DOX的缓慢释放,所以短时间内(1h、4h)游离组细胞内的DOX荧光明显强于GEL-DOX组,到12h GEL-DOX组释放到培养基中的DOX显著增加,使得细胞内DOX荧光显著提高,因此游离组与GEL-DOX组的荧光强度差异减小。
10、本发明实施例1制备的负载化疗药和纳米粒的海藻酸钠水凝胶复合体系的体内抑廇效果研究:
在6~8周的C57BL/6的右侧近后肢部位接种1×106的B16F10细胞,构建原位肿瘤模型,6天后于左侧近后肢部位接种5×105的B16F10细胞,构建远端肿瘤模型;待肿瘤长到75mm3左右(第7天)将小鼠随机分成6组,每组6只:PBS、Free(DOX+IND+IMQ+MPLA)、GEL-D、GEL-ID、GEL-ID-NP、GEL-ID-NP-Mal。在第7天、11天、15天各组分别在原位瘤内注射药物75μL(DOX 5mg/kg,IND 2.5mg/kg,IMQ 2.5mg/kg,MPLA 67μg/mL),自给药后开始每两天记录一次肿瘤体积,任一侧肿瘤体积大于4000mm3时小鼠被视为死亡,并绘制小鼠的生存曲线和体重变化曲线。
肿瘤体积计算公式如下:
肿瘤体积(mm3)=(长径×短径2)/2
如图7中B所示,PBS组小鼠原位肿瘤生长迅速,在近端瘤接种17天后平均肿瘤体积接近3000mm3,Free组轻微抑制了肿瘤生长,GEL-D和GEL-ID组更好地抑制肿瘤生长,这是由于海藻酸钠水凝胶对药物的长效缓释作用增强了药物的抗肿瘤效果;共负载化疗药和纳米粒的海藻酸钠水凝胶复合体系GEL-ID-NP和GEL-ID-NP-Mal组较大程度地抑制肿瘤生长,在接种肿瘤31天后GEL-ID-NP组平均肿瘤体积小于400mm3,GEL-ID-NP-Mal组平均肿瘤体积小于200mm3,显示出极好的抑制肿瘤生长的效果。此外,由图7中D所示,与其他各组相比GEL-ID-Mal显著提高了B16F10荷瘤小鼠的生存率(50%)。
同时通过对比各组远端肿瘤的生长情况来评估各组制剂的免疫效果。如图7中C所示,Free组和GEL-D组对远端瘤的生长抑制效果不明显,说明单独的化疗诱导产生的免疫效果较弱,不足以压制远端瘤的生长;GEL-ID组增加了IDO抑制剂调节肿瘤免疫抑制微环境,因此与GEL-D组相比能更好地抑制远端瘤生长;共负载化疗药和纳米粒的海藻酸钠水凝胶复合体系GEL-ID-NP和GEL-ID-NP-Mal组由于靶向纳米粒对DC等树突状细胞的免疫激活作用,能最大程度抑制远端瘤生长,其中GEL-ID-NP-Mal组在整个实验期50%的小鼠未发生远端肿瘤。
11、本发明制备的负载化疗药和纳米粒的海藻酸钠水凝胶复合体系的体内抗肿瘤免疫机制的研究:
在6~8周的C57BL/6的右侧近后肢部位接种1×106的B16F10细胞,构建原位肿瘤模型,6天后(给药前1天)于左侧近后肢部位接种5×105的B16F10细胞,构建远端肿瘤模型;待肿瘤长到75mm3左右将小鼠随机分成6组,每组6只(第7天):PBS、Free(DOX+IND+IMQ+MPLA)、GEL-D、GEL-ID、GEL-ID-NP、GEL-ID-NP-Mal。在第7天、11天、15天各组分别在原位瘤内注射药物75μL(DOX 5mg/kg,IND 2.5mg/kg,IMQ 2.5mg/kg,67ug/mL),在初次给药后10天处死小鼠,切除脾脏,进行研磨过滤,加入红细胞裂解液裂解2min,加入10倍体积的PBS终止裂解,450g离心5min,加入PBS重悬分到1.5mL小管中进行染色,部分脾细胞加入完全培养基重悬到12孔板中培养用于测定上清中的细胞因子TNF-α、细胞杀伤因子IFN-γ的分泌及T细胞胞内TNF-α和IFN-γ的水平。
CD4+ T细胞和CD8+ T增殖和活化水平测定:处死小鼠后切除脾脏,使用无菌筛网在1640中进行研磨过滤,离心后加入红细胞裂解液2min,加入10倍体积的PBS终止裂解,加入PBS洗1次,离心后加入抗体FITC-anti-CD3e,APC-anti-CD4,PE-anti-CD8,Percp-cy5.5-anti-CD69抗体室温孵育30min,PBS洗1次,加入组织固定液重悬过筛上流式,检测T细胞的增殖和活化水平;检测结果如图8、图9所示;
脾内调节性T细胞的比例测定:按上述步骤处理脾细胞后,加入APC-anti-CD4抗体于4℃孵育30min,PBS洗1次后,加入固定破膜液室温孵育30-60min,之后加入透化液洗1次,再加入抗体PE-anti-Foxp3室温孵育30min,加入PBS洗1次,然后加入PBS重悬过筛上流式分析;分析结果如图10所示。
脾内T细胞分泌TNF-α和IFN-γ水平的测定:按上述步骤处理脾细胞后加入完全培养基重悬到12孔板中,培养2-6h后并加入肿瘤细胞裂解物(TDPA),继续培养50h后加入1×的PMA和离子霉素,继续培养10h后加入BFA培养6h,之后450g离心收集脾细胞;PBS洗1次,加入抗体Percp-cy5.5-anti-CD8a、PE-anti-CD4,4℃孵育30min;PBS洗1遍,加IC fixation bμffer(细胞内固定液)涡旋,室温避光孵育60min;每管加permeabilization bμffer(透化液)洗1次;透化液稀释FITC-anti-IFN-γ,APC-anti-TNF-α,室温避光孵育70min;PBS洗一次,并加PBS重悬过筛上流式,流式分析结果如图11、12所示。
脾内巨噬细胞分型测定:按上述步骤处理脾细胞后,加入Percp-cy5.5-anti F4/80,PE-anti-CD11B抗体于4℃孵育30min;加入PBS洗一次,每管加IC fixation bμffer(细胞内固定液)涡旋,室温避光孵育60min;加1×permeabilization buffer(透化液)洗1次,再加入透化液稀释的APC-anti-CD206抗体打孔边偶联抗体,室温避光孵育60min;透化液洗1次,PBS重悬过筛上流式,流式分析结果如图13所示。
由图8可知,GEL-ID-NP-Mal组的辅助性T细胞比例(CD3+CD4+T细胞)比例(23.5%)明显高于PBS(9.5%)、Free(14.9%)、GEL-D(13.7%)、GEL-ID(14.1%)组,略高于GEL-ID-NP组(19.9%);同时GEL-ID-NP-Mal组细胞毒性T细胞(CD3+CD8+ T细胞)的比例(21.7%)明显高于PBS组(8.0%)、Free(12.2%)、GEL-D(10.7%),略高于GEL-ID(12.8%)、GEL-ID-NP(15.4%)组。结果表明,相比于游离的药物(Free IMQ+MPLA+DOX+IND)及单纯的化疗GEL-D,具有化疗免疫联合治疗作用的水凝胶复合体系GEL-ID-NP和GEL-ID-NP-Mal的治疗能更好地促进体内细胞毒性T细胞和辅助性T细胞的增殖,同时GEL-ID-NP-Mal组由于Mal基团的抗原捕获作用实现肿瘤相关抗原(TAA)与佐剂(IMQ和MPLA)的共递送,因而显示了比无捕获作用纳米粒GEL-ID-NP组更好的促进T细胞增殖的作用。
CD69是一种免疫细胞泛表达的标志,在淋巴细胞活化早期诱导细胞表面受体的结合,参与免疫细胞的活化和分化。由图9可知,与PBS组(14.7%)相比GEL-ID-NP-Mal组(20.3%)辅助性T细胞的CD69表达水平显著提高,GEL-D(16.8%)、GEL-ID(17.5%)组略有提高,GEL-ID-NP(18.3%)组进一步提高但仍低于GEL-ID-NP-Mal组;同时GEL-ID-NP-Mal组的细胞毒性T细胞也具有最高水平的CD69表达(10.88%)并显著高于PBS组和Free组。结果表明本发明制备的负载化疗药和纳米粒的海藻酸钠水凝胶复合体系不仅能很好地促进细胞毒性T细胞和辅助性T细胞的增殖,还能促进其表面活化分子CD69的表达,从而促进T细胞的活化和分化过程,加速体内的抗肿瘤免疫进程。
调节性T细胞(Treg)是一类发挥免疫抑制功能的CD4+ T(CD4+Foxp3+)细胞,当其富集于肿瘤组织时能通过免疫无能和免疫抑制两大功能促进肿瘤的免疫逃逸,介导肿瘤免疫耐受,从而加速肿瘤的发展进程。为了研究各组制剂对小鼠体内Treg细胞增殖的影响,收集初次治疗10天后各组小鼠的脾细胞进行免疫荧光染色,分析各组脾内Treg细胞(CD4+Foxp3+T细胞)水平。
如图10所示,与PBS组(p<0.002)、Free(p<0.03)、GEL-D(p<0.002)相比,GEL-ID-NP-Mal组显著抑制Treg(CD4+Foxp3+)细胞的增殖,在所有组中有最低比例的Treg细胞(11.2%)。
干扰素IFN可通过抑制肿瘤细胞增生和抑癌基因表达,促进肿瘤细胞凋亡和调节免疫等多种方式抑制肿瘤的发生发展,有研究表明II型干扰素IFN-γ可抑制肿瘤血管生成从而抑制肿瘤的转移。IFN-γ可由活化的T细胞产生,我们通过细胞内因子染色评估了CD4+T细胞和CD8+ T细胞分泌IFN-γ的水平。如图11所示,GEL-ID-NP-Mal组的CD4+IFN-γ+ T细胞的比例是其他组的2.7~3倍,CD8+IFN-γ+ T细胞是其他组的3~7.4倍,说明GEL-ID-NP-Mal能更有效地诱导CD4+ T细胞和CD8+ T细胞分泌IFN-γ,从而增强效应T细胞的杀伤作用,诱导有效的抗肿瘤免疫应答。
肿瘤坏死因子TNF可使多种肿瘤发生出血性坏死的物质,主要是由活化的巨噬细胞、T淋巴细胞以及NK细胞所产生的。TNF不仅可以直接杀伤或抑制肿瘤细胞,还可以通过对机体免疫功能的调节,促进T细胞以及其他杀伤细胞对肿瘤细胞的杀伤。我们通过细胞内因子染色评估了CD4+ T细胞和CD8+ T细胞分泌TNF-α的水平。如图12所示,GEL-ID-NP-Mal组具有最高水平的CD4+ TNF-α+ T细胞和CD8+TNF-α+ T,其中CD4+TNF-α+ T细胞水平是其他组的2~4.3倍,CD8+TNF-α+ T细胞是其他组的3.3~21.6倍,这显示GEL-ID-NP-Mal能极大程度地提高T淋巴细胞分泌TNF-α,从而通过直接杀伤或间接方式杀伤体内的肿瘤细胞,引发更有效的抗肿瘤免疫效应。
巨噬细胞可分为M1型(经典活化的巨噬细胞)和M2型(替代性活化的巨噬细胞),其中M1型巨噬细胞具有免疫防御和组织破坏的功能,有较强的裂解肿瘤、提呈抗原、促进T细胞抗肿瘤作用等功能;M2型巨噬细胞不仅缺乏前者的抗肿瘤功能,反而通过高表达多种促血管生成因子和生长因子刺激肿瘤细胞的增殖和血管生成,从而促进肿瘤的侵袭和转移。因此,评估治疗后小鼠体内M1和M2巨噬细胞的比例对于抗肿瘤免疫机制的研究具有重要意义。如图13所示,未经治疗的B16F10荷瘤小鼠(PBS组)脾内M1/M2巨噬细胞的比例为0.9,此时促进肿瘤增殖和转移的M2型巨噬细胞占主导地位;然而经过Free、GEL-D、GEL-ID组治疗的小鼠脾内M1/M2巨噬细胞的比例明显提高,分别为3.9、4.9和5.6;而化疗免疫联合治疗组GEL-ID-NP和GEL-ID-NP-Mal组进一步提高了M1/M2巨噬细胞的比例,分别为7.4和7.6。该结果表明化疗通过杀伤肿瘤细胞,诱导肿瘤细胞免疫原性死亡可在一定程度上促进巨噬细胞向M2型巨噬细胞分化,而化疗联合免疫治疗在诱导巨噬细胞的M2型分化方面的作用比单独化疗更胜一筹,这可能是由于化疗免疫联合治疗组GEL-ID-NP和GEL-ID-NP-Mal能更大程度地促进淋巴细胞分泌TNF-α和IFN-γ等细胞因子,从而促进巨噬细胞向M1型巨噬细胞的分化。
最后应说明的是:以上各实施例仅用以说明本发明的技术方案,而非对其限制;尽管参照前述各实施例对本发明进行了详细的说明,本领域的普通技术人员应当理解:其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分或者全部技术特征进行等同替换;而这些修改或者替换,并不使相应技术方案的本质脱离本发明各实施例技术方案的范围,其均应涵盖在本发明的权利要求和说明书的范围当中。
Claims (10)
1.一种共负载化疗药和纳米粒的海藻酸钠水凝胶复合体系的制备方法,其特征在于,包括如下步骤:
(a)将PCL-PEG-PCL、疏水性IMQ溶于有机溶剂,然后,去除有机溶剂形成薄膜,薄膜再经水化、超声、超滤后形成均一的胶束纳米粒溶液;
(b)DSPE-PEG5000-Mannose、DSPE-PEG2000-Mal、MPLA溶于有机溶剂,去除有机溶剂后形成磷脂薄膜;
(c)将胶束纳米粒溶液加入磷脂薄膜后经水化、超声、超滤浓缩,得到脂质聚合物杂化纳米粒溶液;
(d)将化疗药、Indoximod及脂质聚合物杂化纳米粒溶液混合后加到海藻酸钠粉末中,震荡混合,即得共负载化疗药和纳米粒的海藻酸钠水凝胶复合体系。
2.根据权利要求1所述的制备方法,其特征在于,所述PCL-PEG-PCL的分子量为10000~20000;
优选地,所述PCL-PEG-PCL为PCL4000-PEG8000-PCL4000。
3.根据权利要求1所述的制备方法,其特征在于,所述步骤(a)中,PCL-PEG-PCL与疏水性IMQ的质量比为(20~25)∶1;
水化温度为65~75℃,时间为5~8h;
超声时间为10~20min;
超滤为采用10KDa超滤管以3000~5000rpm离心1~3h。
4.根据权利要求1所述的制备方法,其特征在于,所述步骤(b)中,DSPE-PEG5000-Mannose与PCL-PEG-PCL的质量比为1∶(50~70);
DSPE-PEG2000-Mal与PCL-PEG-PCL的质量比为1∶(10~40);
MPLA与PCL-PEG-PCL的质量比为(1~5)∶1000。
5.根据权利要求1所述的制备方法,其特征在于,所述步骤(c)中,水化温度为25~40℃,时间为1~2h;
超声时间为4~10min;
超滤浓缩至PCL-PEG-PCL的浓度为30~40mg/mL。
6.根据权利要求1所述的制备方法,其特征在于,所述步骤(d)中,化疗药、Indoximod和PCL-PEG-PCL的质量比为(1~2)∶(0.5~1)∶20;
海藻酸钠的终浓度为20~25mg/mL。
7.根据权利要求1所述的制备方法,其特征在于,所述化疗药为盐酸阿霉素。
8.权利要求1~7任一所述的制备方法制得的共负载化疗药和纳米粒的海藻酸钠水凝胶复合体系。
9.权利要求8所述的共负载化疗药和纳米粒的海藻酸钠水凝胶复合体系在制备化疗联合免疫治疗的抗肿瘤药物中的应用。
10.一种化疗联合免疫治疗的抗肿瘤药物,其特征在于,包括权利要求8所述的共负载化疗药和纳米粒的海藻酸钠水凝胶复合体系。
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