CN117045619B - 一种共载蜂毒溶瘤肽和腺苷a2ar受体抑制剂的脂质体-聚合物纳米粒及制备和应用 - Google Patents
一种共载蜂毒溶瘤肽和腺苷a2ar受体抑制剂的脂质体-聚合物纳米粒及制备和应用 Download PDFInfo
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Abstract
本发明提供了一种共载蜂毒溶瘤肽和腺苷A2AR受体抑制剂的脂质体‑聚合物纳米粒,为核壳结构,内层是以两亲性阳离子聚合物作为载体,载体中包裹蜂毒溶瘤肽PalAno和腺苷A2AR受体抑制剂CPI‑444,外层为连接有CD44靶向肽A6的脂质双分子层。本发明还提供了上述脂质体‑聚合物纳米粒的制备方法和在制备治疗肿瘤的药物中的用途。本发明能够特异性靶向三阴性乳腺癌细胞和黑色素瘤细胞,蜂毒溶瘤肽PalAno可导致肿瘤细胞裂解,激活机体抗肿瘤免疫响应,CPI‑444用于克服溶瘤过程中ATP代谢产物腺苷导致的免疫抑制。本发明将在靶向溶瘤的同时重塑免疫抑制微环境,实现两种药物的高效协同。
Description
技术领域
本发明属于生物医药领域,具体地说,是一种共载蜂毒溶瘤肽和腺苷A2AR受体抑制剂的脂质体-聚合物纳米粒及制备和应用。
背景技术
肿瘤免疫治疗是调动机体自身免疫系统、激活免疫细胞杀伤肿瘤的一种治疗方法,该疗法革命性地改变了肿瘤治疗的理念,已经成为继手术治疗、放疗和化疗之后的第四大肿瘤治疗手段,以PD-1/L1为代表的免疫检查点抑制剂是目前最令人瞩目的肿瘤免疫疗法,并在临床应用中取得了巨大的进步。然而,因肿瘤突变负荷低、免疫检查点蛋白表达差异、肿瘤免疫抑制微环境等因素,癌症患者对免疫检查点抑制剂的整体响应率仅约20%。因此积极探索高效、低毒、特异性强且不易产生耐药的新型肿瘤免疫疗法,对于提高癌症患者的生存率和生存质量具有重要的前景和意义。
溶瘤免疫治疗是通过溶瘤病毒或溶瘤肽识别和裂解肿瘤细胞,诱导肿瘤细胞发生免疫原性细胞死亡(ICD),有效增强肿瘤的免疫原性,从而激活机体抗肿瘤免疫反应的新型免疫治疗手段。相较于溶瘤病毒,溶瘤肽的自身免疫原性和制备成本更低,安全性更高,目前已有多个溶瘤肽类药物进入美国临床试验阶段。
相比于其他诱导ICD的药物,溶瘤肽对肿瘤细胞膜的裂解作用可在短时间释放大量的三磷酸腺苷(ATP),用于招募和激活抗原呈递细胞,继而激活抗肿瘤免疫应答。然而肿瘤微环境中(TME)中的负反馈机制-腺苷能轴(CD39/CD73通路)会将ATP代谢为腺苷,腺苷通过与树突状细胞、巨噬细胞和T细胞等免疫细胞表面的A2AR受体相互作用,从而形成免疫抑制TME,严重限制溶瘤肽的免疫治疗效果。另外,溶瘤肽在目前的临床试验中主要采取瘤内注射的方式给药,亦限制了其在深部和转移部位肿瘤中的临床应用。
CPI-444是一种具有较高选择性的A2AR抑制剂(Ki=3.54nM),其结构式如下所示,
。目前正在开展多个临床试验。CPI-444可通过阻断A2AR发挥抗肿瘤作用,有效提高癌症患者的估计总生存期率,但A2AR广泛分布于各个组织中,具有重要的生理功能,抑制正常组织中的A2AR可能导致毒副作用。肿瘤靶向纳米药物技术的发展为溶瘤肽和CPI-444等组织选择性低的药物的递送提供了有效解决方法,因此,可构建共载溶瘤肽和腺苷A2AR受体抑制剂的肿瘤靶向纳米药物系统。
CD44靶向肽A6,其氨基酸序列为Ac-KPSSPPEEC-NH2,A6肽能够特异性地与三阴性乳腺癌、黑色素瘤和多发性骨髓瘤等肿瘤细胞表面过表达的CD44受体结合,也已被应用于作为靶向配体特异性地向肿瘤细胞递送药物。
发明内容
针对现有技术中的上述技术问题制备和应用,所述的这种共载蜂毒溶瘤肽和腺苷A2AR受体抑制剂的脂质体-聚合物纳米粒及制备和应用要解决现有技术中的药物对于治疗肿瘤的效果不佳的技术问题。
本发明的第一个目的是针对现有技术中的不足,提供一种共载蜂毒溶瘤肽和腺苷A2AR受体抑制剂的脂质体-聚合物纳米粒。
本发明的第二个目的是针对现有技术中的不足,提供如上所述的脂质体-聚合物纳米粒的用途。
为实现上述第一个目的,本发明采取的技术方案是:
一种共载蜂毒溶瘤肽和腺苷A2AR受体抑制剂的脂质体-聚合物纳米粒,为核壳结构,内层是以两亲性阳离子聚合物作为载体,载体中包裹蜂毒溶瘤肽PalAno和腺苷A2AR受体抑制剂CPI-444,所述的蜂毒溶瘤肽PalAno的结构如下:
CPI-444的结构式如下所示,
外层为连接有CD44靶向肽A6的脂质双分子层。
本发明还提供了上述的一种共载蜂毒溶瘤肽和腺苷A2AR受体抑制剂的脂质体-聚合物纳米粒的制备方法:
(1)称取甲氧基聚乙二醇-聚(β-氨基酯)(mPEG-PAE)、蜂毒溶瘤肽PalAno和CPI-444,加入到甲醇和二氯甲烷的混合有机溶剂中,再加入纯水,超声形成乳剂,旋蒸去除有机溶剂,得到同时载有蜂毒溶瘤肽PalAno和CPI-444的阳离子聚合物;
(2)称取二棕榈酰磷脂酰胆碱DPPC、胆固醇Chol和磷脂聚乙二醇马来酰亚胺DSPE-PEG-Mal,加入到三氯甲烷中,而后旋蒸去除有机溶剂,在容器底部形成脂质双分子层薄膜,向该容器中加入步骤(1)中得到的阳离子聚合物和透明质酸溶液,水化,得到的纳米粒通过200nm的聚碳酸酯膜挤出8~20次,加入CD44靶向肽A6,得到共载蜂毒溶瘤肽PalAno和CPI-444的脂质体-聚合物纳米粒CA@TLM。
进一步,所述的mPEG-PAE、PalAno和CPI-444质量比为1-20:1-3:1-2。
进一步,所述的mPEG-PAE、PalAno和CPI-444质量比为18:2:2。
进一步,所述的DPPC、Chol和DSPE-PEG-Mal摩尔比为77.5:20:1-5。
进一步,所述的DPPC、Chol和DSPE-PEG-Mal摩尔比为77.5:20:2.5。
进一步,所述的脂质双分子层薄膜和阳离子聚合物的质量比为0.5-2:1。
进一步,所述的脂质双分子层薄膜和阳离子聚合物的质量比为1:1。
进一步,所述的PalAno载样量在脂质体-聚合物纳米粒中的质量百分比大于或等于4.11%,所述的CPI-444载样量在脂质体-聚合物纳米粒中的质量百分比大于或等于1.95%。
本发明还提供了一种蜂毒溶瘤肽PalAno,其结构如下所示:
Anoplin是一种具有净正电荷和疏水氨基酸的两亲性α-螺旋多肽,来源于蜂毒肽,其能够通过静电吸附作用于肿瘤细胞,导致肿瘤细胞膜的快速裂解。中国专利202010035278.8公开了一种构象锁定蜂毒肽Anoplin衍生物的制备方法与抗肿瘤应用,在Anoplin肽段侧链形成碳氢支架以稳定多肽的α-螺旋构象,达到构象锁定的目的。其中Ano-3具有显著提升的α-螺旋构象、酶稳定性和体内外抗肿瘤活性。为了使Ano-3能够包载于脂质体-聚合物纳米粒中,我们在Ano-3的基础上进一步进行结构修饰,并将两个棕榈酸连接至Ano-3的N末端,即得本发明的蜂毒溶瘤肽PalAno。
为实现上述第二个目的,本发明采取的技术方案是:
如上任一所述的脂质体-聚合物纳米粒在制备治疗肿瘤疾病的药物中的应用。
进一步,所述的肿瘤为三阴性乳腺癌和黑色素瘤。
本发明是以脂质体-聚合物为载体,CD44靶向肽A6为靶向配体,蜂毒溶瘤肽PalAno和腺苷A2AR受体抑制剂CPI-444为模型药物,构建新型纳米药物-A6多肽修饰的共载蜂毒溶瘤肽和腺苷A2AR受体抑制剂的脂质体-聚合物纳米粒(CA@TLM)。
本发明优点在于:
1、本发明基于溶瘤肽裂解肿瘤后,ATP代谢产物腺苷导致的免疫抑制,将腺苷A2AR受体抑制剂共同递送,通过阻断腺苷与免疫细胞表面的A2AR受体相互作用,克服腺苷导致的免疫抑制微环境。
2、本发明的CA@TLM对肿瘤细胞具有靶向性。
3、纳米粒装载的蜂毒溶瘤肽PalAno未表现出对红细胞的毒性作用,而可以高效地杀伤三阴性乳腺癌4T1和MDA-MB-231细胞和黑色素瘤B16F10细胞。
4、本发明的CA@TLM显著抑制BALB/c小鼠原位4T1三阴性乳腺癌和C57小鼠皮下B16F10黑色素瘤的生长,溶瘤免疫效果显著增强。
5、本发明的脂质体-聚合物纳米粒能够特异性靶向三阴性乳腺癌细胞和黑色素瘤细胞,蜂毒溶瘤肽PalAno可导致肿瘤细胞裂解,激活机体抗肿瘤免疫响应,CPI-444用于克服溶瘤过程中ATP代谢产物腺苷导致的免疫抑制。
6、本发明将在靶向溶瘤的同时重塑免疫抑制微环境,实现两种药物的高效协同,具有较好的应用前景。
附图说明
图1为蜂毒溶瘤肽PalAno的质谱图。
图2为CA@TLM透射电镜(A),动态光散射粒径(B)和zeta电位(C)。
图3为CA@TLM对红细胞的作用。
图4为A6修饰介导4T1和MDA-MB-231细胞对纳米粒的靶向摄取。
图5为CA@TLM对4T1、MDA-MB-231和B16F10细胞的杀伤作用。
图6为CA@TLM显著抑制BALB/c小鼠原位4T1三阴性乳腺癌的生长。
图7为CA@TLM显著抑制C57小鼠皮下B16F10黑色素瘤的生长。
具体实施方式
以下结合具体实施方式,进一步阐明本发明。应理解,以下实施例仅用于说明本发明而非用于限定本发明的范围。此外应理解,在阅读了本发明记载的内容之后,本领域技术人员可以对本发明作各种改动或修改,这些等价形式同样落于本申请所附权利要求书所限定的范围。
蜂毒溶瘤肽PalAno(实施例1)、CPI-444(上海喀露蓝科技有限公司)、mPEG-PAE(西安瑞禧生物科技有限公司)、A6多肽(希施生物科技(上海)有限公司)
实施例1蜂毒溶瘤肽PalAno的合成方法
称取667mg氨基树脂(取代度0.3mmol/g)放入多肽合成管中,于室温加入5mL二氯甲烷溶胀20min,然后用水泵抽干溶剂。向上述合成管中加入5mL N,N-二甲基甲酰胺洗涤树脂后,向合成管中加入7mL 20%哌啶/N,N-二甲基甲酰胺溶液脱去氨基树脂上的Fmoc保护基,于35℃晃动反应10min,抽干反应液。再向合成管中加入7mL 20%哌啶的N,N-二甲基甲酰胺溶液,于35℃晃动反应10min,抽干反应液。重复上述洗涤树脂,即3次N,N-二甲基甲酰胺、3次二氯甲烷、3次N,N-二甲基甲酰胺,用量及方法如上所述。称取Fmoc-Leu-OH(353mg,1mmol)放入10mL离心管中,再向上述离心管中加入2-肟氰乙酸乙酯(142mg,1mmol)、N,N'-二异丙基碳二亚胺(155μL,1mmol),加入7mL N,N-二甲基甲酰胺混匀活化一分钟。将活化好的氨基酸加入到固相合成管中,于60℃反应20min后洗涤。然后脱去第一个氨基酸Leu的Fmoc保护基,方法同上。我们用上述方法依次接入Fmoc-Leu-OH,Fmoc-Thr(tBu)-OH,Fmoc-S5-OH(即Fmoc-(S)-2-(4-pentenyl)Ala-OH),Fmoc-Ile-OH,Fmoc-Arg(Pbf)-OH,Fmoc-Lys(Boc)-OH,Fmoc-S5-OH,Fmoc-Trp(Boc)-OH,Fmoc-Trp(Boc)-OH,Fmoc-Lys(Fmoc)-OH,棕榈酸。得到接好线性多肽的树脂之后,用10mL 1,2-二氯乙烷在固相合成管中洗涤树脂,重复三次。加入5mL Grubbs一代催化剂的1,2-二氯乙烷溶液(10mmol/L),35℃反应2小时,抽干重复上述反应一次,洗涤树脂,干燥。向多肽合成管中加入15mL切割试剂(95%三氟乙酸,2.5%,水,2.5%三异丙基硅烷,体积比),室温振荡2小时。将切割试剂过滤至50mL离心管中,再用5mL三氟乙酸润洗树脂,用氩气将离心管中的三氟乙酸吹干。然后向该离心管中,加入35mL冰乙醚,振荡沉淀,用3500转/分的离心机离心3min,倾倒出上清溶液,重复上述沉淀过程三次,风干即得粗肽。最后经凝胶柱纯化后冻干得到蜂毒溶瘤肽PalAno,其结构如下:
质谱数据见图1。
实施例2CA@TLM的构建
将甲氧基聚乙二醇-聚(β-氨基酯)mPEG-PAE(18mg)、实施例1的蜂毒溶瘤肽PalAno(2mg)和CPI-444(2mg)溶解于1mL混合有机溶剂中(甲醇:二氯甲烷=1:20,体积比),缓慢加入1mL纯水,利用超声细胞破碎仪在100W功率下超声1min(4s开/2s关),用旋转蒸发仪除去有机溶剂,得到共载蜂毒溶瘤肽PalAno和CPI-444的阳离子聚合物。
称取二棕榈酰磷脂酰胆碱DPPC(15.832mg)、胆固醇Chol(2.152mg)和磷脂聚乙二醇马来酰亚胺DSPE-PEG-Mal(2.016mg),加入到5mL三氯甲烷中,而后旋蒸去除有机溶剂,在容器底部形成脂质双分子层薄膜.向该容器中加入前述步骤得到的共载蜂毒溶瘤肽PalAno和CPI-444的阳离子聚合物和2.2mL透明质酸水溶液(5mg),在55℃下水化20min,得到的纳米粒通过200nm的聚碳酸酯膜挤出10次,加入A6多肽(氨基酸序列为Ac-KPSSPPEEC-NH2)(0.70mg),室温振荡4h得到共载PalAno和CPI-444的脂质体-聚合物纳米粒CA@TLM。
观察纳米粒表面形态,测定纳米粒的粒径、zeta电位、包封率、载药量等特性。粒径和zeta电位采用Nano-2S90 Malvern马尔文粒径仪测定。纳米粒的药物包封率是指单位重量纳米粒制剂中被载入纳米粒中的药物重量与体系中药物重量比,其计算方法为:包封率=(纳米粒中试剂药物量/纳米粒中理论投药量)×100%。纳米粒的载药量是指单位重量的纳米粒中所含药物的量,其计算方法为:载药量=(纳米粒中所含药物重量/纳米粒总重量)×100%。
图2A为CA@TLM透射电镜图像,图2B为动态光散射粒径135.33nm,图2C为zeta电位-15.33mV。纳米系统蜂毒溶瘤肽PalAno包封率为96.37%,载样量在脂质体-聚合物纳米粒中的质量百分比为4.11%,CPI-444包封率为44.83%,载样量在脂质体-聚合物纳米粒中的质量百分比为1.95%。
实施例3CA@TLM对红细胞的安全性
100μL鼠红细胞以4%(V/V)的浓度接种于96孔板中,加入100μL含有CA@TLM的培养基(PalAno浓度范围为0.1μM-30μM),空白对照加100μL PBS,阳性对照加100μL 0.2%Triton X-100,每种孔设置3个复孔。加药后在37℃培养箱中孵育1h。离心(4℃、1000g)15min,使用酶标仪测定上清液570nm处的吸光值A。溶血率计算方法为:溶血率%=(A样本-A对照)/(A阳性-A对照)×100%。由图3的结果可以看出,蜂毒溶瘤肽PalAno在10μM和30μM浓度下明显导致溶血,而CA@TLM在测试浓度下无明显溶血作用。***p<0.001。
实施例4A6修饰介导4T1和MDA-MB-231细胞对纳米粒的靶向摄取。
采用流式细胞仪考察4T1和MDA-MB-231细胞对纳米粒的摄取情况,采用前述纳米药物相同制备方法,制备得到载有香豆素6的(C6,荧光标记)靶向纳米药物(C6@TLM)和非靶向纳米药物(C6@LM),并对C6含量进行定量。4T1和MDA-MB-231细胞以5×104/孔浓度接种于12孔板,24h后吸去原培养基并加入1mL/孔含有纳米药物培养基,其中C6浓度设置为0.2μg/mL。孵育不同时间后用PBS冲洗细胞2次,消化重悬,于流式细胞仪中检测细胞对纳米粒的摄取情况。
为进一步考察纳米粒的细胞摄取是由A6多肽和肿瘤细胞表面表达的CD44受体介导的内吞。另外设置一组对照,在加入纳米粒之前,用50eq的A6多肽事先与细胞孵育时间2h,再加入C6@TLM。实验结果见图4,由图可见纳米粒经A6修饰后可显著提升其对靶细胞的靶向性和摄取能力。*p<0.05,**p<0.01,***p<0.001。
实施例5CA@TLM的抗增殖活性
将4T1、MDA-MB-231和B16F10细胞以5×103/孔浓度接种于96孔板,24h后加入含有纳米药物培养基(实施例1的蜂毒溶瘤肽PalAno浓度范围为0.1μM-30μM),继续培养24h。采用LIVE/DEAD细胞活性检测试剂盒和CCK-8法测定细胞活力。其中LIVE/DEAD细胞活性检测试剂盒能通过钙黄绿素染活细胞,PI染死细胞,荧光显微镜拍摄下可直观地观察活细胞和死细胞的数量。
实验设置蜂毒溶瘤肽PalAno作为对照。实验结果见图5,蜂毒溶瘤肽PalAno经纳米系统包载后仍可剂量依赖性杀伤肿瘤细胞。*p<0.05,**p<0.01,***p<0.001。
实施例6CA@TLM对三阴性乳腺癌的治疗作用评价
6.1模型建立
雌性BALB/c小鼠,右腹股沟第四对乳房垫接种4T1细胞(1×106),约3天形成肿瘤。
6.2评价CA@TLM的体内抗肿瘤作用
实验分组:①空白组;②C@TLM组;③A@TLM组;④CA@TLM组。按优化方案给药。评价指标为:①以瘤体积500mm3为终点,解剖出小鼠肿瘤,比较各组肿瘤大小;②跟踪记录实验过程中小鼠一般健康状况,饮食、饮水和体重变化,比较副作用。
图6为CA@TLM显著抑制BALB/c小鼠原位4T1三阴性乳腺癌生长。静脉给予纳米药物(剂量为PalAno 20mg/kg,CPI-444 10mg/kg),连续观察肿瘤体积和小鼠体重。(A)4T1肿瘤体积生长曲线。与其他各组相比,CA@TLM抑制肿瘤生长的作用最强(n=5)。(B)小鼠体重监测(n=5)。**p<0.01,***p<0.001。
实施例7CA@TLM对黑色素瘤的治疗作用评价
7.1模型建立
雌性C57小鼠,右腹股沟皮下接种B16F10细胞(5×105),约5天形成肿瘤。
7.2评价CA@TLM的体内抗肿瘤作用
实验分组:①空白组;②C@TLM组;③A@TLM组;④CA@TLM组。按优化方案给药。评价指标为:①以瘤体积1500mm3为终点,解剖出小鼠肿瘤,比较各组肿瘤大小;②跟踪记录实验过程中小鼠一般健康状况,饮食、饮水和体重变化,比较副作用。
图7为CA@TLM显著抑制C57小鼠皮下B16F10黑色素瘤生长。静脉给予纳米药物(剂量为PalAno 20mg/kg,CPI-444 10mg/kg),连续观察肿瘤体积和小鼠体重。(A)B16F10肿瘤体积生长曲线。与其他各组相比,CA@TLM抑制肿瘤生长的作用最强(n=6)。(B)小鼠体重监测(n=6)。***p<0.001。
以上所述的仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员,在不脱离本发明方法的前提下,还可以做出若干改进和补充,这些改进和补充也应视为本发明的保护范围。
Claims (8)
1.一种共载蜂毒溶瘤肽和腺苷A2AR受体抑制剂的脂质体-聚合物纳米粒,其特征在于,为核壳结构,内层是以两亲性阳离子聚合物作为载体,所述的两亲性阳离子聚合物为甲氧基聚乙二醇-聚(β-氨基酯),载体中包裹蜂毒溶瘤肽PalAno和腺苷A2AR受体抑制剂CPI-444,所述的甲氧基聚乙二醇-聚(β-氨基酯)、蜂毒溶瘤肽PalAno和CPI-444质量比为18:2:2;所述的蜂毒溶瘤肽PalAno的结构如下:
,CPI-444的结构式如下所示,
,外层为连接有CD44靶向肽A6的脂质双分子层,CD44靶向肽A6的氨基酸序列为Ac-KPSSPPEEC-NH2。
2.权利要求1所述的一种共载蜂毒溶瘤肽和腺苷A2AR受体抑制剂的脂质体-聚合物纳米粒的制备方法,其特征在于包括如下步骤:
(1)称取甲氧基聚乙二醇-聚(β-氨基酯)、蜂毒溶瘤肽PalAno和CPI-444,所述的甲氧基聚乙二醇-聚(β-氨基酯)、蜂毒溶瘤肽PalAno和CPI-444质量比为18:2:2,加入到甲醇和二氯甲烷的混合有机溶剂中,再加入纯水,超声形成乳剂,旋蒸去除有机溶剂,得到同时载有蜂毒溶瘤肽PalAno和CPI-444的阳离子聚合物;
(2)称取二棕榈酰磷脂酰胆碱、胆固醇和磷脂聚乙二醇马来酰亚胺,加入到三氯甲烷中,而后旋蒸去除有机溶剂,在容器底部形成脂质双分子层薄膜;
(3)向该容器中加入步骤(1)中得到的阳离子聚合物和透明质酸溶液,水化,得到的纳米粒通过200 nm的聚碳酸酯膜挤出8~20次,加入CD44靶向肽A6,CD44靶向肽A6的氨基酸序列为Ac-KPSSPPEEC-NH2,得到共载蜂毒溶瘤肽PalAno和CPI-444的脂质体-聚合物纳米粒CA@TLM。
3.根据权利要求2所述的一种共载蜂毒溶瘤肽和腺苷A2AR受体抑制剂的脂质体-聚合物纳米粒的制备方法,其特征在于,所述的二棕榈酰磷脂酰胆碱、胆固醇和磷脂聚乙二醇马来酰亚胺的摩尔比为77.5:20:1-5。
4.根据权利要求3所述的一种共载蜂毒溶瘤肽和腺苷A2AR受体抑制剂的脂质体-聚合物纳米粒的制备方法,其特征在于,所述的二棕榈酰磷脂酰胆碱、胆固醇和磷脂聚乙二醇马来酰亚胺的摩尔比为77.5:20:2.5。
5.根据权利要求2所述的一种共载蜂毒溶瘤肽和腺苷A2AR受体抑制剂的脂质体-聚合物纳米粒的制备方法,其特征在于,所述的脂质双分子层薄膜和甲氧基聚乙二醇-聚(β-氨基酯)的质量比为0.5-2:1。
6.根据权利要求5所述的一种共载蜂毒溶瘤肽和腺苷A2AR受体抑制剂的脂质体-聚合物纳米粒的制备方法,其特征在于,所述的脂质双分子层薄膜和甲氧基聚乙二醇-聚(β-氨基酯)的质量比为1:1。
7.根据权利要求1所述的一种共载蜂毒溶瘤肽和腺苷A2AR受体抑制剂的脂质体-聚合物纳米粒的制备方法,其特征在于,蜂毒溶瘤肽PalAno载样量在脂质体-聚合物纳米粒中的质量百分比等于4.1%,所述的CPI-444载样量在脂质体-聚合物纳米粒中的质量百分比等于1.9%。
8.权利要求1的脂质体-聚合物纳米粒在制备治疗三阴性乳腺癌或者黑色素瘤的药物中的应用。
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Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN109288794A (zh) * | 2018-11-19 | 2019-02-01 | 上海交通大学 | 一种蜂毒素脂质体纳米制剂及其制备方法与应用 |
CN109692326A (zh) * | 2017-10-23 | 2019-04-30 | 华中科技大学 | 一种蜂毒脂质纳米颗粒的应用 |
CN112791192A (zh) * | 2020-12-31 | 2021-05-14 | 上海交通大学 | 炎性细胞靶向蜂毒素脂质体纳米制剂及其制备方法和应用 |
CN113368053A (zh) * | 2021-06-04 | 2021-09-10 | 苏州大学 | 一种装载溶瘤肽的聚合物囊泡及其与囊泡免疫佐剂、pd-1单抗的联合用药 |
CN114478707A (zh) * | 2022-03-08 | 2022-05-13 | 上海中医药大学 | 一种构象锁定蜂毒肽衍生物、偶联物及其制备和用途 |
KR20220097285A (ko) * | 2020-12-31 | 2022-07-07 | 주식회사 인스바이오팜 | 멜리틴 기반의 나노입자 복합체 및 이의 제조방법 |
-
2023
- 2023-07-14 CN CN202310868571.6A patent/CN117045619B/zh active Active
Patent Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN109692326A (zh) * | 2017-10-23 | 2019-04-30 | 华中科技大学 | 一种蜂毒脂质纳米颗粒的应用 |
CN109288794A (zh) * | 2018-11-19 | 2019-02-01 | 上海交通大学 | 一种蜂毒素脂质体纳米制剂及其制备方法与应用 |
CN112791192A (zh) * | 2020-12-31 | 2021-05-14 | 上海交通大学 | 炎性细胞靶向蜂毒素脂质体纳米制剂及其制备方法和应用 |
KR20220097285A (ko) * | 2020-12-31 | 2022-07-07 | 주식회사 인스바이오팜 | 멜리틴 기반의 나노입자 복합체 및 이의 제조방법 |
CN113368053A (zh) * | 2021-06-04 | 2021-09-10 | 苏州大学 | 一种装载溶瘤肽的聚合物囊泡及其与囊泡免疫佐剂、pd-1单抗的联合用药 |
CN114478707A (zh) * | 2022-03-08 | 2022-05-13 | 上海中医药大学 | 一种构象锁定蜂毒肽衍生物、偶联物及其制备和用途 |
Non-Patent Citations (1)
Title |
---|
可系统性给药的蜂毒肽脂质纳米颗粒及其抗黑色素瘤作用研究;黄川;博士电子期刊(第2016年第07期期);全文 * |
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