CN113827546A - 一种含阿霉素与免疫佐剂组合药物脂质体的注射用水凝胶及其制备方法 - Google Patents
一种含阿霉素与免疫佐剂组合药物脂质体的注射用水凝胶及其制备方法 Download PDFInfo
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- CN113827546A CN113827546A CN202010570256.1A CN202010570256A CN113827546A CN 113827546 A CN113827546 A CN 113827546A CN 202010570256 A CN202010570256 A CN 202010570256A CN 113827546 A CN113827546 A CN 113827546A
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Abstract
本发明提供了一种含化药阿霉素及免疫佐剂组合的药物脂质体的注射用水凝胶制剂,药物水凝胶用于实体肿瘤的原位给药治疗。本发明中阿霉素与免疫佐剂共载的脂质体可以缓慢从水凝胶中释放,开展针对肿瘤的化疗‑免疫联合治疗作用,达到抑制肿瘤生长及转移的目的。
Description
技术领域
本发明涉及一种用于肿瘤化疗协同免疫治疗的水凝胶药物制剂,具体为是一种含阿霉素与免疫佐剂组合药物脂质体的注射用水凝胶及其制备方法。
背景技术
肿瘤临床治疗包括手术、化药治疗(化疗)、放射治疗(放疗)、靶向治疗和免疫治疗。在一般情况下,化疗是肿瘤治疗不可或缺的手段。
化药治疗通常采用全身给药,不仅仅是杀伤肿瘤细胞;对于人体正常细胞也具有明确的毒副作用,如恶心、脱发、造血毒性、造血祖细胞从骨髓动员到外周血的减少、贫血、骨髓抑制、全血细胞减少、血小板减少、中性粒细胞减少、淋巴细胞减少、白细胞减少。这些副作用常常限制了化疗药物的施用剂量和用药频率。
阿霉素(英文名称 adriamycin,又称多柔比星)是一种常见的抗肿瘤化药,其抗瘤谱较广,适用于急性白血病(淋巴细胞性和粒细胞性)、恶性淋巴瘤、乳腺癌、支气管肺癌(未分化小细胞性和非小细胞性)、卵巢癌、软组织肉瘤、成骨肉瘤、横纹肌肉瘤、尤文肉瘤、母细胞瘤、神经母细胞瘤、膀胱癌、甲状腺癌、***癌、头颈部鳞癌、睾丸癌、胃癌、肝癌等。但其临床毒副作用亦十分明显,除骨髓抑制,胃肠道毒性以及脱发外,还可引起严重的 心脏毒性,表现为各种心率失常,累积量大时可发生不可逆的心肌损伤乃至充血性心力衰竭。
然而,在肿瘤化疗过程中依然会产生耐药、抗肿瘤效力不足等问题,导致仅通过单一药物或制剂难以抑制肿瘤细胞生长。目前,化疗可以与其他治疗方式(如手术、放疗、靶向治疗和免疫治疗)一起联用,形成针对肿瘤的综合治疗。联合化疗的综合治疗往往可以产生协同效应,均较单一治疗药物或治疗形式产生更好的抗肿瘤效果、更低的毒副作用。
发明内容
本发明旨在提供一种含化药阿霉素及免疫佐剂组合的药物脂质体的注射用水凝胶,其优点在于,通过肿瘤原位注射水凝胶,缓慢释放化药阿霉素与免疫佐剂共载的脂质体,开展针对肿瘤的化疗-免疫联合治疗,可在阿霉素有效杀伤肿瘤细胞同时,免疫佐剂提升免疫***对肿瘤细胞死亡产生相关抗原的提呈效率,引发更强的人体对肿瘤特异性免疫反应,达到抑制肿瘤生长及转移的目的。
本发明的目的可以通过以下方法以实现:
一种含共载阿霉素与免疫佐剂组合药物脂质体的注射用水凝胶及其制备方法,其每100g制剂的原料配方为:
阿霉素0.5g~5g
免疫佐剂50mg~2.5g
中性磷脂240mg~30g
正电荷磷脂2mg~10g
胆固醇10mg~15g
含聚乙二醇亲水性A嵌段、含聚酯疏水性B嵌段所组成的双亲性嵌段共聚物5g~30g;
维生素E 0.25mg~480mg
磷酸二氢钠100mg~2000mg
蔗糖 2~10g
注射用水定容至100g
其中,阿霉素和免疫佐剂的重量比例为0.1∶1-2∶1,阿霉素和中性磷脂的重量比例为0.1∶1-1∶1,正电荷磷脂和中性磷脂的摩尔比例为0.05∶5-1∶5,胆固醇和中性磷脂的摩尔比例为0.1∶1-1∶1。
所述的含共载阿霉素与免疫佐剂组合药物脂质体的注射用水凝胶,其中的脂质体包含中性磷脂,选自氢化大豆卵磷脂、蛋黄卵磷脂、氢化蛋黄卵磷脂、双硬脂酸卵磷脂、大豆卵磷脂、双软脂酸卵磷脂或双肉豆寇酸卵磷脂;
优选地,所述中性磷脂为氢化大豆卵磷脂。
所述的含共载阿霉素与免疫佐剂组合药物脂质体的注射用水凝胶,其中的脂质体中包含免疫佐剂,选自负电荷免疫佐剂CpG、PolyIC、PolyICLC、MPL、MPL-TDM、脂多糖中的一种或多种;
优选地,所述免疫佐剂为CpG和脂多糖质量1:1配比。
所述的含共载阿霉素与免疫佐剂组合药物脂质体的注射用水凝胶,其中的脂质体包含带正电荷磷脂,选自DOTAP,DOTMA,DOSPA,DODMA,Dlin-MC3-DMA,Dlin-KC2-DMA,c12-200;
优选地,所述正电荷磷脂为DOTAP。
所述的含共载阿霉素与免疫佐剂组合药物脂质体的注射用水凝胶,其中的双亲性嵌段共聚物:
(a)含有10-90wt%的聚乙二醇聚合物为亲水性A嵌段;
(b)含有90-10wt%的聚酯聚合物为疏水性B嵌段,其单体结构为DL-丙交酯、L-丙交酯、乙交酯、ε-己内酯、δ-戊内酯中的任意一种;
(c)AB二者嵌段共聚物的构成包括ABA、BAB型的三嵌段共聚物、AB型的二嵌段共聚物、A-g-B和B-g-A型的接枝共聚物中的至少一种。
所述的含共载阿霉素与免疫佐剂组合药物脂质体的注射用水凝胶,其制剂形态为可注射性胶态溶液或混悬液。
所述的含共载阿霉素与免疫佐剂组合药物脂质体的注射用水凝胶的制备方法,包含下列步骤:
步骤一:制备空白脂质体:将中性磷脂、正电荷磷脂、胆固醇和维生素E按照质量份数和比例加入于体积质量浓度4-10%的乙醇水溶液中,升温使之溶解均匀形成脂质溶液,备用;另配制浓度250mM的硫酸铵水溶液并升温至55-65℃,并加热条件下将上述脂质溶液高速注入其中,所形成乳液用高射流挤出设备或物理挤压过相应孔径的微孔膜来制得粒径范围在100nm空白脂质体,再3%-20%的蔗糖溶液置换空白脂质体的溶剂体系;
步骤二:制备共载阿霉素与免疫佐剂组合药物脂质体:将阿霉素与免疫佐剂按比例溶于注射用水或浓度为3%-20%的蔗糖溶液,加热到40-70℃后与步骤一中得到的空白脂质混合均匀,并在40-70℃下保温一段时间,制得共载阿霉素与免疫佐剂的脂质体;
步骤三:制备含共载阿霉素与免疫佐剂组合药物脂质体的注射用水凝胶:取PH为7的注射用水适量,加入聚乙二醇为亲水嵌段、聚酯为疏水嵌段组成的双亲性嵌段共聚物,室温匀速搅拌获得一定浓度的嵌段共聚物溶液;再加入步骤二中得到的共载阿霉素与免疫佐剂的脂质体,并充分混合均匀并用注射用水调节最终质量,制得含共载阿霉素与免疫佐剂组合药物脂质体的注射用水凝胶。
步骤四:除菌、分装、保存:将步骤三中制得的含共载阿霉素与免疫佐剂组合药物脂质体的注射用水凝胶加压经过0.45μm微孔膜过滤除菌,分装即得成品,成品置于2-8℃下保存。
附图说明
图1是本发明含阿霉素和佐剂CpG共载脂质体注射用PLGA-PEG-PLGA 水凝胶(ADR/CpG-Lip)PLGA1870-Gel(胶体材料为PLGA(75/25)1870-PEG1500-PLAG(75/25)1870嵌段共聚物)的电镜照片。
图2是本发明含阿霉素和佐剂CpG共载脂质体注射用PLGA-PEG-PLGA 水凝胶(ADR/CpG-Lip)PLGA1704-Gel(胶体材料为PLGA(75/25)1704-PEG1500 -PLAG(75/25)1704嵌段共聚物)的电镜照片。
图3是本发明含阿霉素与佐剂PolyICLC共载脂质体注射用PCL-PEG-PCL水凝胶(ADR/PolyICLC-Lip)PLC2000-Gel(胶体材料为PCL2000-PEG1500- PCL2000嵌段共聚物)的透射电镜照片。
图4是本发明含阿霉素与佐剂PolyICLC共载脂质体注射用PCL-PEG-PCL水凝胶(ADR/PolyICLC-Lip)PLC3400-Gel(胶体材料为PCL3400-PEG1500- PCL3400嵌段共聚物)的透射电镜照片。
图5是本发明含阿霉素与佐剂脂多糖共载脂质体注射用PDLLA-PEG-PDLLA水凝胶(ADR/LPS-Lip)PDLLA-Gel(胶体材料为PDLLA(1500-2000)-PEG(1000-1500)-PDLLA(1500 -2000)嵌段共聚物)的透射电镜照片。
图6是本发明各种含阿霉素和免疫佐剂共载脂质体注射用水凝胶实例的化药阿霉素体外释放曲线。
图7是本发明各种含阿霉素和免疫佐剂共载脂质体注射用水凝胶实例治疗HepG2肝癌移植瘤动物模型的肿瘤生长曲线。
图8是本发明各种含阿霉素和免疫佐剂共载脂质体注射用水凝胶实例治疗7721乳腺癌移植瘤动物模型的肿瘤生长曲线。
图9是本发明各种含阿霉素和免疫佐剂共载脂质体注射用水凝胶实例治疗7721乳腺癌移植瘤动物模型的取出肿瘤重量。
图10是是本发明各种含阿霉素和免疫佐剂共载脂质体注射用水凝胶实例治疗HepG2肝癌移植瘤动物模型的动物体重变化。
具体实施方式
下面结合实施例对本发明作进一步的描述。
实施例1:制备含阿霉素与CpG共载脂质体的PLGA-PEG-PLGA(聚乙交酯丙交酯-聚乙二醇-聚乙交酯丙交酯)水凝胶,简称(ADR/CpG-Lip)PLGA-Gel
制剂处方(50g定量):
阿霉素100mg
CpG 10mg
双硬脂酸卵磷脂(DSPC)640mg
DOTAP 140mg
PLGA-PEG-PLGA嵌段共聚物 6g(PLGA(75/25)1870-PEG1500-PLAG(75/25)1870或PLGA(75/25)1704-PEG1500 -PLAG(75/25)1704)
胆固醇190mg
维生素E 2.4mg
磷酸二氢钠160mg
蔗糖 1g
注射用水定量至50g
制剂工艺如下:
根据配方选用DSPC、DOTAP、胆固醇和维生素E在4%乙醇溶液中混合,并升温至65℃,形成脂质溶液;另配制浓度250mM的硫酸铵溶解阿霉素、CpG并升温至60℃;并加热条件下将脂质溶液快速注入其中,形成乳液,用微射流高压均质机制得粒径范围在100nm左右空白脂质体。再5%的蔗糖溶液透析置换空白脂质体的溶剂体系。另外将阿霉素以及免疫佐剂溶于5%的蔗糖溶液,加热到50℃后与空白脂质体悬浮液混合均匀,并在50℃下保温3个小时,制得共载阿霉素与免疫佐剂的脂质体。以PH为7的注射用水33.3g室温条件匀速搅拌溶解PLGA(75/25)1870-PEG1500-PLAG(75/25)1870或PLGA(75/25)1704-PEG1500 -PLAG(75/25)1704嵌段共聚物,获得18wt%的嵌段共聚物溶液后再向其中加入共载阿霉素与免疫佐剂的脂质体,充分混合均匀后用磷酸氢二钠调节溶液PH至7,并注射用水调节最终质量至50g,形成含共载阿霉素与免疫佐剂组合药物脂质体的水凝胶。最后,将所得含共载脂质体的水凝胶加压经过0.45μm微孔膜过滤除菌,分装即得成品,成品可在2-8℃下保存保存至使用。并通过电镜观测(ADR/CpG-Lip)PLGA1870-Gel和(ADR/CpG-Lip)PLGA 1704-Gel的表观形态,详见图1、2。
实施例2:制备含阿霉素与PolyICLC共载脂质体的PCL-PEG-PCL(聚己内酯-聚乙二醇-聚己内酯)水凝胶,简称(ADR/PolyICLC-Lip)PCL-Gel
制剂处方(50g定量):
阿霉素100mg
PolyICLC 20mg
氢化大豆卵磷脂(HSPC)760mg
Dlin-MC3-DMA 220mg
胆固醇220mg
PCL-PEG-PCL 6.4g(PCL2000-PEG1500- PCL2000或PCL3400-PEG1500- PCL3400)
维生素E 2.0mg
蔗糖 2g
磷酸二氢钠240mg
注射用水定量至50g
制剂工艺如下:
根据配方选用HSPC、Dlin-MC3-DMA、胆固醇和维生素E在4%乙醇溶液中混合均匀升温至55℃,形成脂质溶液;另配制浓度250mM的硫酸铵溶解阿霉素和PolyICLC并升温至60℃;并加热条件下将脂质溶液快速注入其中,形成乳液,用微射流高压均质机制得粒径范围在150nm左右空白脂质体。再5%的蔗糖溶液透析置换空白脂质体的溶剂体系。另外将阿霉素以及免疫佐剂溶于5%的蔗糖溶液,加热到50℃后与空白脂质体悬浮液混合均匀,并在50℃下保温3个小时,制得共载阿霉素与免疫佐剂的脂质体。以PH为7的注射用水32g在室温条件匀速搅拌溶解PCL2000-PEG1500- PCL2000或PCL3400-PEG1500- PCL3400嵌段共聚物,获得20wt%的嵌段共聚物溶液后再向其中加入共载阿霉素与免疫佐剂的脂质体,充分混合均匀后用磷酸氢二钠调节溶液PH至7,并注射用水调节最终质量至50g,形成含共载阿霉素与免疫佐剂组合药物脂质体的水凝胶。最后,将所得含共载脂质体的水凝胶加压经过0.45μm微孔膜过滤除菌,分装即得成品,成品可在2-8℃下保存保存至使用。并通过电镜观测(ADR/PolyICLC-Lip)PLC2000-Gel和(ADR/PolyICLC-Lip)PCL3400-Gel的表观形态,详见图3、4。
实施例3:制备含阿霉素与脂多糖共载脂质体的PDLLA-PEG-PDLLA(聚D,L-丙交酯-聚乙二醇-聚D,L-丙交酯)水凝胶,简称(ADR/LPS-Lip)PDLLA-Gel
制剂处方(50g定量):
阿霉素100mg
脂多糖35mg
双硬脂酸磷脂酰甘油(DSPG)160mg
蛋黄磷脂酰甘油(EPG)240mg
c12-200 220mg
胆固醇220mg
维生素E 1.6mg
PDLLA(1500-2000)-PEG(1000-1500)-PDLLA(1500-2000) 8.5g
磷酸二氢钠200mg
注射用水定容至所需容量
制剂工艺如下:
根据配方选用DSPG、EPG、c12-200、胆固醇和维生素E在4%乙醇溶液中混合均匀升温至58℃,形成脂质溶液;另配制浓度250mM的硫酸铵溶解阿霉素和脂多糖并升温至60℃,并加热条件下将脂质溶液快速注入其中,形成乳液,用微孔膜基础设备来制得粒径范围在120nm左右空白脂质体。再5%的蔗糖溶液透析置换空白脂质体的溶剂体系。另外将阿霉素以及免疫佐剂溶于5%的蔗糖溶液,加热到50℃后与空白脂质体悬浮液混合均匀,并在50℃下保温2个小时,制得共载阿霉素与免疫佐剂的脂质体。以PH为7的注射用水28.3g在室温条件匀速搅拌溶解PDLLA(1500-2000)-PEG(1000-1500)-PDLLA(1500-2000)嵌段共聚物,获得30wt%的嵌段共聚物溶液后再向其中加入共载阿霉素与免疫佐剂的脂质体,充分混合均匀后用磷酸氢二钠调节溶液PH至7,并注射用水调节最终质量至50g,形成含共载阿霉素与免疫佐剂组合药物脂质体的水凝胶。最后,将所得含共载脂质体的水凝胶加压经过0.45μm微孔膜过滤除菌,分装即得成品,成品可在2-8℃下保存保存至使用。并通过电镜观测(ADR/LPS -Lip)PDLLA-Gel的表观形态,详见图5。
实施例4:检测含阿霉素与免疫佐剂组合药物脂质体的注射用水凝胶的性质参数
采用激光粒径仪测实施例1~3所制备ADR/CpG-LPS-Lip、ADR/PolyICLC-Lip、ADR/MPL-TDM-Lip溶液的粒径以及zeta电位值;通过高效液相色谱方法(HPLC,美国Agilent公司;色谱条件:十八烷基硅烷键合硅胶为填充剂;以十二烷基硫酸钠溶液-乙腈-甲醇为流动相;检测波长254nm;参比试剂:盐酸多柔比星对照品和盐酸表柔比星对照品,流速1ml/min;柱温25℃),测定上述脂质体中阿霉素含量,计算获得脂质体中阿霉素的包封率,公式:阿霉素的包封量 = MADR包封/MADR投入×100%,结果详见表1。
表1. 脂质体的性质参数(n = 3)。
水凝胶组别 | 脂质体粒径(nm) | 水凝胶中脂质体粒径(nm) | 阿霉素包封率(%) |
(ADR/CpG-Lip)PLGA1870-Gel | 110.5 ± 10.6 | +128.0 ± 12.9 | 90.2 ± 1.5 |
(ADR/CpG-Lip)PLGA1704-Gel | 105.3 ± 11.4 | +111.6 ± 13.2 | 91.1 ± 2.3 |
(ADR/PolyICLC-Lip)PLC2000-Gel | 110.7 ± 12.2 | +115.4 ± 12.8 | 89.0 ± 2.8 |
(ADR/PolyICLC-Lip)PLC3400-Gel | 115.5 ± 15.2 | +121.4 ± 10.9 | 88.1 ± 4.0 |
(ADR/LPS-Lip)PDLLA-Gel | 112.6 ± 13.4 | +124.1 ± 14.5 | 89.4 ± 3.7 |
实施例5:检测含阿霉素与免疫佐剂组合药物脂质体的注射用水凝胶的体外药物释放曲线
将实施例1~3所制备(ADR/CpG-Lip)PLGA1870-Gel、(ADR/CpG-Lip)PLGA1704-Gel、(ADR/PolyICLC-Lip)PLC2000-Gel、(ADR/PolyICLC-Lip)PLC3400-Gel、(ADR/LPS-Lip)PDLLA-Gel溶液个2ml分别放置于300KD透析袋中,并置于1000毫升PBS溶液(PH=7.4)的烧杯中进行恒温水浴震摇(温度37℃,震摇速率110 rpm),并开始从零计时。在设定时间点(第0、2、5、30、120、240、360小时),吸取透析袋中的溶液,利用高效液相色谱方法检测阿霉素的含量(检测参数如实施例4)。计算药物阿霉素的体外释放速率。结果如实施图6所示,各种阿霉素脂质体的药物累计释放15天期间达到缓释效果。
实施例6:测定含阿霉素与免疫佐剂组合药物脂质体的注射用水凝胶提高肿瘤微环境中的肿瘤特异性CD8+T细胞数量增加
利用HepG2肝癌以及7721乳腺癌细胞在C57BL6小鼠中建立肝癌或乳腺癌移植瘤动物模型,待肿瘤体积100立方毫米时,随机(如下)分组,分别在1、8、11、15、18、22、25天给药进行治疗,其中单次给药剂量阿霉素2mg/kg,同种免疫佐剂剂量相同。第26天时,处死小鼠并取出肿瘤进行流式细胞术分析,通过SIINFEKL-Kb四聚体染色来检测肿瘤微环境中的肿瘤特异性CD8+T细胞数量(肿瘤抗原特异性T细胞:CD3+/ CD8+/SIINFEKL-H2Kb tetramer+)。肿瘤微环境中的CD8+ T激活状态通过用颗粒酶B染色进行分析:
分组1:生理盐水组
分组2:阿霉素脂质体
分组3:阿霉素脂质体+皮下注射CpG组
分组4:阿霉素脂质体+皮下注射PolyICLC组
分组5:阿霉素脂质体+皮下注射LPS组
分组6:肿瘤原位注射(ADR/CpG-Lip)PLGA1870-Gel组
分组7:肿瘤原位注射(ADR/CpG-Lip)PLGA1704-Gel组
分组8:肿瘤原位注射(ADR/PolyICLC-Lip)PLC2000-Gel组
分组9:肿瘤原位注射(ADR/PolyICLC-Lip)PLC3400-Gel组
分组10:肿瘤原位注射(ADR/LPS-Lip) PDLLA-Gel组
通过SIINFEKLH2Kb四聚体同源抗原流式检测,肿瘤特异性T细胞在生理盐水组别处于低水平状态,与用生理盐水处理的对照组中分离得到的CD8+ T细胞(设定为1)相比,各治疗组别中小鼠肿瘤微环境中提取的CD8+ T细胞如下表2,可见阿霉素与免疫佐剂组合药物脂质体显著增加肿瘤微环境CD8+ T细胞数量(p<0.05,n = 5):
表2. 各治疗组别中肿瘤微环境中CD8+ T细胞相对数量(设参比生理盐水组为1,*与同种类皮下注射佐剂相比*p<0.05,n = 5)。
注射组别 | HepG2肝癌移植瘤模型 | 7721乳腺癌移植瘤模型 |
生理盐水组 | 1 | 1 |
阿霉素脂质体 | 1.05 ± 0.03 | 1.10 ± 0.02 |
阿霉素脂质体+皮下注射 CpG | 1.22 ± 0.03 | 1.39 ± 0.08 |
阿霉素脂质体+ 皮下注射PolyICLC | 1.29 ± 0.04 | 1.28 ± 0.12 |
阿霉素脂质体+皮下注射 LPS | 1.25 ± 0.06 | 1.26 ± 0.08 |
肿瘤原位注射(ADR/CpG-Lip)PLGA1870-Gel | 1.75 ± 0.18* | 1.75 ± 0.12* |
肿瘤原位注射(ADR/CpG-Lip)PLGA1704-Gel | 1.69 ± 0.17* | 1.69 ± 0.11* |
ADR/MPL-TDM-Lip | 1.79 ± 0.13* | 1.65 ± 0.15* |
肿瘤原位注射(ADR/PolyICLC-Lip)PLC2000-Gel | 1.68 ± 0.16* | 1.68 ± 0.11* |
肿瘤原位注射(ADR/LPS-Lip) PDLLA-Gel组 | 1.79 ± 0.15* | 1.72 ± 0.21* |
实施例7:含阿霉素与免疫佐剂组合药物脂质体的注射用水凝胶对HepG2肝癌及7721乳腺癌细胞移植瘤小鼠模型的治疗效果
利用HepG2肝癌以及7721乳腺癌细胞在C57BL6小鼠中建立乳腺癌移植瘤动物模型,待肿瘤体积150立方毫米时,每5只随机(如下)分组,分别在1、8、11、15、18、22、25天给药进行治疗,其中单次给药剂量阿霉素为2mg/kg,同种免疫佐剂剂量相同。然后在不同时间点用游标卡尺测定肿瘤体积,肿瘤体积的计算公式:肿瘤体积 =(长×宽2)/2。绘制肿瘤生长曲线(如图7、8),并在7721乳腺癌实验终点处死小鼠取出肿瘤称重(图9),结果说明阿霉素与免疫佐剂组合药物脂质体显示出良好的抗肿瘤活性,有效提升肿瘤微环境的CD8+ T细胞数量与激活状态,在动物体内实现肿瘤抑制效果:
分组1:生理盐水+CpG、PolyICLC、LPS混合佐剂组组
分组2:阿霉素脂质体
分组3:肿瘤原位注射(ADR/CpG-Lip)PLGA1870-Gel水凝胶组
分组4:肿瘤原位注射(ADR/CpG-Lip)PLGA1704-Gel水凝胶组
分组5:肿瘤原位注射(ADR/PolyICLC-Lip)PLC2000-Gel水凝胶组
分组6:肿瘤原位注射(ADR/PolyICLC-Lip)PLC3400-Gel水凝胶组
分组7:肿瘤原位注射(ADR/LPS-Lip) PDLLA-Gel水凝胶组。
实施例8:含阿霉素与免疫佐剂组合药物脂质体的注射用水凝胶治疗期间动物模型体重变化(毒副作用)
皮下接种HepG2肝癌在C57BL6小鼠中建立乳腺癌移植瘤动物模型,待肿瘤体积150立方毫米时,每5只随机(如下)分组,分别在1、8、11、15、18、22、25天给药进行治疗,其中单次给药剂量阿霉素为2mg/kg,同种免疫佐剂剂量相同。然后每个5天时间点用电子天平测定小鼠体重,绘制动物模型治疗期间体重变化曲线,体重重结果如图10所示,含阿霉素与免疫佐剂组合药物脂质体的注射用水凝胶的毒副作用较阿霉素组小:
分组1:生理盐水+CpG、PolyICLC、LPS混合佐剂组组
分组2:阿霉素脂质体
分组3:肿瘤原位注射(AD R/CpG-Lip)PLGA1870-Gel水凝胶组
分组4:肿瘤原位注射(ADR/CpG-Lip)PLGA1704-Gel水凝胶组
分组5:肿瘤原位注射(ADR/PolyICLC-Lip)PLC2000-Gel水凝胶组
分组6:肿瘤原位注射(ADR/PolyICLC-Lip)PLC3400-Gel水凝胶组
分组7:肿瘤原位注射(ADR/LPS-Lip) PDLLA-Gel水凝胶组。
虽然本发明已以较佳实施例揭示如上,然其并非用以限定本发明,任何本领域技术人员,在不脱离本发明的精神和范围内,当可作些许的修改和完善,因此本发明的保护范围当以权利要求书所界定的为准。
Claims (10)
1.一种含共载阿霉素与免疫佐剂组合药物脂质体的注射用水凝胶的制备方法,其特征在于,该注射用水凝胶包括脂质体,所述脂质体包括中性磷脂、免疫佐剂和正电荷磷脂,每100g该注射用水凝胶的重量配比为:
阿霉素0.5g~5g;
免疫佐剂50mg~2.5g;
中性磷脂240mg~30g;
正电荷磷脂2mg~10g;
胆固醇10mg~15g;
含聚乙二醇亲水性A嵌段、含聚酯疏水性B嵌段所组成的双亲性嵌段共聚物5g~30g;
维生素E 0.25mg~480mg
磷酸二氢钠100mg~2000mg
蔗糖 1~10g
剩余为注射用水;
其中,阿霉素和免疫佐剂的重量比例为0.1∶1-2∶1,阿霉素和中性磷脂的重量比例为0.1∶1-1∶1,正电荷磷脂和中性磷脂的摩尔比例为0.05∶5-1∶5,胆固醇和中性磷脂的摩尔比例为0.1∶1-1∶1。
2.根据权利要求1所述的含共载阿霉素与免疫佐剂组合药物脂质体的注射用水凝胶,其特征在于,所述中性磷脂包括氢化大豆卵磷脂、蛋黄卵磷脂、氢化蛋黄卵磷脂、双硬脂酸卵磷脂、大豆卵磷脂、双软脂酸卵磷脂和双肉豆寇酸卵磷脂中的一种。
3.根据权利要求2所述的含共载阿霉素与免疫佐剂组合药物脂质体的注射用水凝胶,其特征在于,所述中性磷脂为氢化大豆卵磷脂。
4.根据权利要求1所述的含共载阿霉素与免疫佐剂组合药物脂质体的注射用水凝胶,其特征在于,所述免疫佐剂包括负电荷免疫佐剂CpG、PolyIC、PolyICLC、MPL、MPL-TDM、脂多糖中的至少一种。
5.根据权利要求4所述的含共载阿霉素与免疫佐剂组合药物脂质体的注射用水凝胶,其特征在于,所述免疫佐剂为CpG和脂多糖,CpG和脂多糖的质量比为1:1。
6.根据权利要求1所述的含共载阿霉素与免疫佐剂组合药物脂质体的注射用水凝胶,其特征在于,所述正电荷磷脂包括DOTAP,DOTMA,DOSPA,DODMA,Dlin-MC3-DMA,Dlin-KC2-DMA,c12-200中的一种。
7.根据权利要求6所述的含共载阿霉素与免疫佐剂组合药物脂质体的注射用水凝胶,其特征在于,所述正电荷磷脂为DOTAP。
8.根据权利要求1所述的含共载阿霉素与免疫佐剂组合药物脂质体的注射用水凝胶,其特征在于,所述双亲性嵌段共聚物:
(a)含有10-90wt%的聚乙二醇聚合物为亲水性A嵌段;
(b)含有90-10wt%的聚酯聚合物为疏水性B嵌段;
(c)AB二者嵌段共聚物的构成包括ABA、BAB型的三嵌段共聚物、AB型的二嵌段共聚物、A-g-B和B-g-A型的接枝共聚物中的至少一种。
9.根据权利要求1所述的含共载阿霉素与免疫佐剂组合药物脂质体的注射用水凝胶,其特征在于,该注射用水凝胶为可注射性胶态溶液或混悬液。
10.一种权利要求1-9中任一所述的含共载阿霉素与免疫佐剂组合药物脂质体的注射用水凝胶的制备方法,其特征在于,该方法包括下列步骤:
步骤一:制备空白脂质体:将中性磷脂、正电荷磷脂、胆固醇和维生素E按照质量份数和比例加入于体积质量浓度4-10%的乙醇水溶液中,升温至溶解均匀形成脂质溶液,备用;另配制浓度250mM的硫酸铵水溶液并升温至55-65℃,并加热条件下将上述脂质溶液高速注入其中,所形成乳液用高射流挤出设备或物理挤压过相应孔径的微孔膜来制得粒径范围在100nm空白脂质体,再3%-20%的蔗糖溶液置换空白脂质体的溶剂体系;
步骤二:制备共载阿霉素与免疫佐剂组合药物脂质体:将阿霉素与免疫佐剂按比例溶于注射用水或浓度为3%-20%的蔗糖溶液,加热到40-70℃后与步骤一中得到的空白脂质体混合均匀,并在40-70℃下保温一段时间,制得共载阿霉素与免疫佐剂的脂质体;
步骤三:制备含共载阿霉素与免疫佐剂组合药物脂质体的注射用水凝胶:取PH为7的注射用水适量,加入聚乙二醇为亲水嵌段、聚酯为疏水嵌段组成的双亲性嵌段共聚物,室温匀速搅拌获得一定浓度的嵌段共聚物溶液;再加入步骤二中得到的共载阿霉素与免疫佐剂的脂质体,并充分混合均匀并用注射用水调节最终质量,制得含共载阿霉素与免疫佐剂组合药物脂质体的注射用水凝胶。
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