CN113398281B - 一种金纳米花多肽复合物、其制备方法以及其在肿瘤诊疗中的应用 - Google Patents
一种金纳米花多肽复合物、其制备方法以及其在肿瘤诊疗中的应用 Download PDFInfo
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- CN113398281B CN113398281B CN202110789491.2A CN202110789491A CN113398281B CN 113398281 B CN113398281 B CN 113398281B CN 202110789491 A CN202110789491 A CN 202110789491A CN 113398281 B CN113398281 B CN 113398281B
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Abstract
本发明涉及生物医药技术领域,尤其涉及一种金纳米花多肽复合物、其制备方法以及其在肿瘤诊疗中的应用。本发明以普鲁士蓝类似物纳米颗粒为内核,以温度敏感的脂质体为保护剂,在室温下一步原位合成金纳米花,并在金纳米花上修饰靶向肿瘤的多肽,获得的金纳米花多肽复合物具有较高的光热转换能力和稳定性,可装载治疗基因,能够同时用于温度控制的基因治疗和较低功率密度下的光热治疗。
Description
技术领域
本发明涉及生物医药技术领域,尤其涉及一种金纳米花多肽复合物、其制备方法以及其在制备抗肿瘤药物中的应用。
背景技术
胰腺癌是最致命的实体恶性肿瘤,5年生存率仅为5%(Nature ReviewsGastroenterology&Hepatology 2020,17,153)。目前,胰腺癌的首选治疗方式是外科手术,但80%的胰腺癌确诊时已存在转移,处于无法进行手术治疗的阶段。虽然一些癌症治疗方式,如化疗、放疗和靶向治疗在临床治疗中普遍使用,但它们在控制肿瘤转移和治疗后复发方面的效果并不理想。因此,开发具有肿瘤靶向性、降低对正常组织的毒性,克服肿瘤细胞多耐药性的新型高效的癌症治疗手段得到了广泛的关注和深入的研究。
自美国国立卫生研究院1990年5月批准第一个临床基因治疗试验以来,基因治疗技术(Gene Therapy)的发展突飞猛进。至2013年10月,64.2%基因治疗案例的主要目标都是癌症的基因治疗(http://www.wiley.co.uk/genmed/clinical)。大多数胰腺肿瘤都存在某种致癌基因的改变,其中,K-Ras基因的突变率为80-95%,突变的K-Ras持续激发下游的信号通路,导致胰腺癌发生侵袭与转移(Cancermetastasis reviews 2021,40,355)。K-Ras的突变是胰腺癌变的关键驱动因素,但仍然是一个具有挑战性的基因治疗靶点,其中一个比较重要的原因是外源治疗基因易被细胞内的生物酶消化降解而影响基因治疗的效果。所以,设计和研发可以保护治疗基因,并将治疗基因靶向递送到胰腺癌组织的基因载体是实现胰腺癌基因治疗的关键。阳离子脂质体作为安全有效的非病毒基因载体被广泛用于基因治疗。针对部分阳离子脂质体基因递送效率低和基因释放可控性差的问题,我们小组分别设计合成了单一脂质体保护的金纳米颗粒(DODAB-AuNPs)和双脂质体保护的纳米金颗粒(DODAB/DOPE-AuNPs),在提高基因递送效率之余,实现了治疗基因的刺激(pH和负电磷脂)调控释放(Biomaterials 2008,29,3617;small 2015,19,2333)。
针对癌细胞比正常细胞对高温更敏感的特点,人们开发了利用具有较高光热转换效率的材料,在外部光源(一般是近红外光)的照射下,将光能转化为热能,并在一定的温度下来杀死癌细胞的一种治疗方法:光热疗法(Photothermal Therapy,PTT)。PTT因为具有微创性、时空选择性和副作用小等优点而受到广泛关注。设计合成具有高光热转换能力的材料是实现高效光热治疗的需要解决的核心问题。在众多的光热材料中,金纳米粒子由于其具有较高的光稳定性,高效热转化能力和明确的表面化学性质,所以,近年来利用金纳米材料对各种肿瘤进行基因或者光热治疗受到了广泛关注(Angewandte Chemie-International Edition,2018,57,1491-1496)。Jochen Feldmann等提出了一种在掺杂阳离子脂质(DOPC)和阴离子脂质(DOPP)的中性磷脂(DOPC)脂质体膜上控制生长金纳米颗粒方法,考察了金纳米颗粒的分布以及聚集状态与脂质体电荷的关系。金纳米粒子的大小和数量以及它们在脂质体表面的聚集程度可以通过改变脂质体与前体浓度的比例或在脂质体中掺杂离子脂质体来控制。这些脂质体-纳米粒子可以通过光学产生局域加热以控制脂膜扩散。遗憾的是,这种脂质体-金纳米颗粒的合成过程耗时较长,大约需要1-2小时(Colloids and Surfaces A:Physicochemical and Engineering Aspects 2009,342,92)。Marek Romanowski等研究人员开发了金包脂质体的新方法,将三种磷脂:DPPC、MPPC和DPPE-PEG 2000以荧光素按照一定的比例分散磷酸盐缓冲盐水中,得到一定浓度的含有荧光素的脂质体溶液,加入抗坏血酸和氯金酸后,在脂质体表面还原金纳米颗粒,所形成的金纳米颗粒的分布密度决定了脂质体的等离子体共振的光谱位置。当用波长与等离子体共振波长相匹配的激光对溶液进行照射时,溶液吸收的光能被转化为热能,此时,受激的热敏脂质体将释放其预装载的荧光素(Advanced Materials 2009,21,2334)。可见,当携带的荧光分子脂质体-金纳米颗粒受到一定波长的激光照射后,金纳米颗粒将光能转换成热能,脂质体的亚稳态特性使其在一定温度(脂质体的相变温度)下产生渗漏,可释放其装载的荧光分子,以达到控制释放的目的(Nano Letters 2019,19,1821-1826)。然而,设计和合成胰腺癌靶向的、较高光热转换能力、刺激响应能力,且同时能光声、CT、光热等多模式的纳米基因载体仍具有一定的挑战性。
目前纳米材料制备方法还存在以下问题:制备过程复杂,耗时较长,部分材料纳米尺寸较大或者毒性较强,不适于生物体内治疗;部分材料只具备单一的治疗功能,治疗普适性和效果较差;有些材料只有单一治疗或者成像能力,很难达成成像指导的精准治疗,实现诊断与治疗一体化;多数材料不具备刺激响应的能力,不能实现装载药物或者基因的控释释放和治疗的时空控制,或装载效率较低,进而影响基因治疗效果。
发明内容
本发明提供了一种金纳米花多肽复合物、其制备方法以及其在肿瘤诊疗中的应用。该金纳米花多肽复合物粒径较小,具有较高的光热转换能力和稳定性,制备时间短、工艺简单,毒性低,生物相容性好。
为了实现上述发明目的,本发明提供以下技术方案:
本发明提供一种金纳米花多肽复合物,包括:
内核,所述内核为普鲁士蓝类似物纳米颗粒;和
在所述内核上原位生长的脂质体保护的金纳米颗粒和靶向肿瘤的多肽。
本发明以普鲁士蓝类似物纳米颗粒为内核,以温度敏感的脂质体为保护剂,在室温下一步原位合成金纳米花,并在金纳米花上修饰靶向肿瘤的多肽,获得的金纳米花多肽复合物具有较高的光热转换能力和稳定性。既有光热治疗能力,也有基因递送能力和多模式成像的能力,可以实现热刺激响应的光热/光声/CT三模态成像引导的靶向胰腺癌的光热及基因协同治疗。
在一个具体实施例中,本发明通过光热性能稳定性实验及光热刺激释放效率实验发现,所述金纳米花多肽复合物中,脂质体一方面提高了金纳米花多肽复合物的生物相容性;另一方面,其本身具有很多活性位点,可以作为靶向分子链接的桥梁,提高材料在靶向位置的聚集效率;最重要的是,材料到达肿瘤部位后,通过激光照射调节温度可实现药物的控制释放。
一些实施方案中,所述普鲁士蓝类似物纳米颗粒为掺杂Mn、Co离子的普鲁士蓝类似物纳米颗粒;所述脂质体为温度敏感的脂质体;所述多肽为cRGD。本发明中,cRGD的序列为cyclo(Arg-Gly-Asp-d-Phe-Cys)。
本发明金纳米花多肽复合物中修饰的多肽为cRGD,可以特异性识别表面高表达αvβ3整合素受体的癌细胞,并且由于材料中含有Fe、Mn、Co、Au,使其成为光声及CT成像造影剂,具有成像和疾病诊断能力,可用于精准的指导治疗。
本发明中,所述金纳米花多肽复合物的平均水合粒径为148nm。
本发明还提供了所述的金纳米花多肽复合物的制备方法,其特征在于,包括:
步骤1:在普鲁士蓝类似物纳米颗粒表面原位生长脂质体和金纳米颗粒,获得金纳米花;
步骤2:在所述金纳米花上修饰靶向肿瘤的多肽,获得金纳米花多肽复合物。
一些实施方案中,步骤1具体为:向脂质体溶液中依次加入普鲁士蓝类似物纳米粒和氯金酸溶液,脂质体保护的金纳米颗粒在普鲁士蓝表面进行原位生长;将反应液离心,弃上清,沉淀物清洗后加水,得金纳米花溶液。
具体地,所述原位反应在搅拌条件下进行,所述搅拌为100~500rpm搅拌5~30min;所述脂质体溶液的浓度为2.5-6mg/mL;所述氯金酸的浓度为50-120mM。一些具体实施例中,所述脂质体溶液的浓度为5mg/mL;所述氯金酸的浓度为100mM。
本发明中,所述脂质体溶液的制备方法如下:
将双十八烷基二甲基溴化铵(DODAB)和二棕榈酰磷脂酰胆碱(DPPC)溶于氯仿中,加入相当于DODAB和DPPC总重量的10-30%的DSPE-PEG2000,再加入相当于DSPE-PEG2000质量的5-30%的DSPE-PEG2000-Maleimide,震荡混匀,获得混合液;向所述混合液中通入氮气吹干并真空干燥,加入PBS,于40-60℃下超声至体系澄清透明。
本发明步骤2中:
所述修饰具体为:将金纳米花与靶向肿瘤的多肽混合,2-10℃下旋转震荡6-20h;
所述靶向肿瘤的多肽的用量为DSPE-PEG2000质量的5-30%。
本发明还提供了所述的制备方法制得的金纳米花多肽复合物。
其中,所述金纳米花多肽复合物的水合平均粒径为148nm。
本发明还提供了金纳米花多肽复合物作为基因载体在制备抗肿瘤药物中的应用。
其中,所述肿瘤为表面高表达αvβ3整合素受体的癌症,具体包括胰腺癌、肝癌、肺癌、胃癌、乳腺癌和宫颈癌。一些具体实施例中,所述肿瘤为胰腺癌。
本发明还提供一种抗肿瘤药物,包括本发明所述的金纳米花多肽复合物和负载于所述金纳米花多肽复合物上的基因药物。
一些实施方案中,本发明负载基因药物的金纳米花多肽复合物(LPBGD+siRNA)水合平均粒径为149nm。
一些实施方案中,所述基因药物为siRNA和/或DNA。
本发明一种金纳米花多肽复合物包括:内核,所述内核为普鲁士蓝类似物纳米颗粒;和包覆在所述内核上的脂质体、金纳米颗粒和靶向肿瘤的多肽。本发明至少具有如下有益效果:
(1)本发明金纳米花多肽复合物经过6个升温降温周期循环后,溶液仍能达到46.6℃,光热转换效率为62.52%,具有较高的光热转换能力和稳定性。
(2)仅用2分钟即可制得所述金纳米花多肽复合物,制备时间短、制备工艺简单,产品粒径较小,毒性低,生物相容性好;
(3)本发明金纳米花多肽复合物修饰的靶向肿瘤的多肽为cRGD多肽时,可以特异性识别表面高表达αvβ3整合素受体的癌细胞,并且由于材料中含有Fe、Mn、Co、Au,使其成为光声及CT成像造影剂,具有成像和疾病诊断能力,用于精准的指导治疗;
(4)在所述金纳米花多肽复合物上负载基因药物(如siRNA)后具有基因及光热协同治疗能力,并且通过热刺激响应的能力,可以进一步促使更多的siRNA释放,进一步提高协同治疗效果。
附图说明
图1示金纳米花的合成过程;(a)为金纳米花制备过程中原位反应0s,30s,60s,90s和120s时的电子照片;(b)为与(a)图对应的透射电镜图像,标尺为100nm;(c)LPBGD的透射电镜图像;(d)Lipo-Au的透射电镜图像;
图2示金纳米花复合物的表征;(a)复合材料的傅里叶变换红外光谱;材料的(b)水合粒径分布、(c)紫外吸收光谱图和(d)Zeta电势;
图3示琼脂糖凝胶电泳迁移实验评估LPBGD对DNA的负载能力;第1泳道为阳性对照(200ng DNA),第2-6泳道中为不同体积的LPBGD(体积分别为3μL、6μL、9μL、12μL、15μL)与200ng DNA的复合物;M:DNA Marker;
图4示Lipo-Au/ROXDNA和LPBGD/ROXDNA加入细胞,4h后的共聚焦荧光成像图片;定量分析Lipo-Au/ROXDNA和LPBGD/ROXDNA递送效率的流式细胞仪采集的图像;
图5示(a)6次升温和降温的光热稳定性测定;(b)LPBGD在降温过程时间与-ln(θ)的线性曲线;(c)35℃起始,以1℃/5分钟的速率升温至60℃,用紫外分光光度计测溶液的紫外吸收;(d)材料对200ng siRNA的负载效率(Binding efficiency)及释放效率(Releaseefficiency);
图6示不同组别处理Panc-1细胞24h和48h后,细胞的相对存活率;
图7示体外PAI和CT成像能力测试;体外不同浓度材料的(a)光声成像强度值及材料溶液的光声成像图片和(b)CT成像强度值及材料的CT成像图片;
图8示活体的治疗效果研究(n=5);荷载Panc-1肿瘤的小鼠在治疗14天期间,各个组别肿瘤体积的变化情况(a)以及对照组及实验组小鼠治疗后肿瘤的照片(b);L表示用808nm的激光器(功率密度为1.2W/cm2)辐照10分钟。
具体实施方式
本发明提供了一种金纳米花多肽复合物、其制备方法以及其在肿瘤诊疗中的应用。本领域技术人员可以借鉴本文内容,适当改进工艺参数实现。特别需要指出的是,所有类似的替换和改动对本领域技术人员来说是显而易见的,它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述,相关人员明显能在不脱离本发明内容、精神和范围内对本文的方法和应用进行改动或适当变更与组合,来实现和应用本发明技术。
基因治疗(gene therapy):基因治疗是指将外源基因导入目标细胞,用以修正因为基因缺陷和异常导致的疾病,达到治疗疾病的目的。
光热治疗(photothermal therapy):光热治疗法是将具有较高光热转换效率的材料注射入人体内部,利用靶向性识别技术聚集在肿瘤组织附近,并在外部光源(一般是近红外光)的照射下将光能转化为热能来杀死癌细胞的一种治疗方法。
温度响应释放(temperature response release):由于材料具有温度敏感性,在环境达到一定温度时,材料的状态改变,进而释放其负载的分子。
本发明提供一种金纳米花多肽复合物的制备方法,包括:步骤1:在普鲁士蓝类似物纳米颗粒表面原位生长脂质体保护的金纳米颗粒,获得金纳米花;
步骤2:在所述金纳米花上修饰靶向肿瘤的多肽,获得金纳米花多肽复合物。
一些具体实施例中,所述金纳米花多肽复合物的制备方法具体包括:
(1)普鲁士蓝类似物PBA的制备
首先,将0.64-0.96mmol铁氰化钾加入到32-48mL 60-90mg/mL的PVP溶液中,搅拌均匀作为A溶液,接下来将0.16-0.24mmol氯化锰和氯化钴(摩尔比1:1)加入到PVP溶液中,搅拌均匀作为溶液B,将溶液B缓慢加入溶液A中,搅拌均匀后,加入64-96mL盐酸溶液(0.008-0.012M),搅拌至澄清后,转移至高压反应釜,80℃恒温高压反应釜反应或者水浴锅反应12-24h,得到的普鲁士蓝类似物用乙醇及二次水分别洗涤3次后备用。
(2)脂质体的制备
取不同比例的DODAB/DPPC(3:7-7:3)溶解在2mL的氯仿中,再加入相当于DODAB/DPPC总质量的10-30%的DSPE-PEG2000,加入相当于DSPE-PEG2000质量的5-30%的DSPE-PEG2000-Maleimide,震荡混合均匀后,通入氮气吹干,并进一步用真空干燥箱对其进行干燥处理。然后,取4-6mL PBS加入干燥后的磷脂中,并于40-60℃的环境中进行超声至溶液澄清透明。
(3)脂质体包覆的类普鲁士蓝@金纳米花(Lipo-PBA-Au)的制备
取不同体积PBA溶液(5mg/mL)和28-43μL HAuCl4(100mM)依次加入至5-10mL脂质体的PBS溶液(pH=7.2-7.4),加入43-65μL抗坏血酸溶液或者不同体积硼氢化钠或者柠檬酸钠后,以速度为100-500rpm搅拌5-30分钟后,所形成的墨蓝色溶液即为Lipo-PBA-Au溶液。12000rpm离心8分钟,弃上清,加入一定量的二次水清洗,摇匀,重复清洗2-3次,加入5mL二次水,置于4℃冰箱中备用。
(4)靶向蛋白cRGD的连接
称取相当于DSPE-PEG2000质量的5-30%质量的cRGD加入Lipo-PBA-Au中,2-10℃下搅拌6-20h后,去离子水洗两次后收集产物Lipo-PBA-Au-cRGD(命名为LPBGD)。
本发明采用的试剂材料皆为普通市售品,皆可于市场购得。
下面结合实施例,进一步阐述本发明:
实施例1本发明金纳米花复合物的制备
(1)普鲁士蓝类似物PBA的制备
首先,将0.8mmol铁氰化钾加入到40mL75mg/mL的PVP溶液中,搅拌均匀作为A溶液,接下来将0.2mmol氯化锰和氯化钴(摩尔比1:1)加入到PVP溶液中,搅拌均匀作为溶液B,将溶液B缓慢加入溶液A中,搅拌均匀后,加入80mL盐酸溶液(0.01M),搅拌至澄清后,转移至高压反应釜,80℃恒温高压反应釜反应20h,得到的普鲁士蓝类似物用乙醇及二次水分别洗涤3次后备用。
(2)脂质体的制备
取不同比例的DODAB/DPPC(5:5)溶解在2mL的氯仿中,再加入相当于DODAB/DPPC总质量的12%的DSPE-PEG2000,加入相当于DSPE-PEG2000质量的5%的DSPE-PEG2000-Maleimide,震荡混合均匀后,通入氮气吹干,并进一步用真空干燥箱对其进行干燥处理。然后,取5mLPBS加入干燥后的磷脂中,并于40-60℃的环境中进行超声至溶液澄清透明。
(3)脂质体包覆的类普鲁士蓝@金纳米花(Lipo-PBA-Au)的制备
取5μLPBA溶液(5mg/mL)和36μL HAuCl4(100mM)依次加入至5mL脂质体的PBS溶液(pH=7.2-7.4),加入54μL抗坏血酸溶液或者不同体积硼氢化钠或者柠檬酸钠后,以速度为100-200rpm搅拌5分钟后,所形成的墨蓝色溶液即为Lipo-PBA-Au溶液。12000rpm离心8分钟,弃上清,加入一定量的二次水清洗,摇匀,重复清洗2-3次,加入5mL二次水,置于4℃冰箱中备用。
(4)靶向蛋白cRGD的连接
称取相当于DSPE-PEG2000质量的cRGD加入Lipo-PBA-Au中,4℃下搅拌12h后,去离子水洗两次后收集产物Lipo-PBA-Au-cRGD(命名为LPBGD)。
如图1(a,b)为Lipo-PBA-Au的合成过程,由图1可以看出金纳米花溶液颜色的变化以及形貌的变化。图1(c)为LPBGD的透射电镜图像,(d)为没有PBA模板,只有脂质体所生成的磷脂金纳米颗粒(Lipo-Au)的透射电镜图像。可以看到,对于不加PBA为内核的材料图1d,根本不能形成纳米花的结构,所以,PBA为LPBGD提供的必不可少的生长起点和模板支撑。并且后续对于纳米花LPBGD和Lipo-Au的光热性能的研究发现,LPBGD的光热转换效率为62.52%,Lipo-Au的光热转换效率为51.33%。所以,本发明以PBA为内核,脂质体为模板的金纳米花LPBGD具有更高光热转换效率的新型结构的纳米复合材料。
由图2a可以看到,本发明制得的金纳米花多肽复合物LPBGD的平均水合粒径148nm。相对于Lipo-PBA-Au,连接靶向肽RGD的Lipo-PBA-Au-RGD(LPBGD)的傅里叶变换红外光谱图(FT-IR)在1690cm-1处多出一个酰胺基团的特征吸收峰(–NH–C=O),由于脂质体中马来酰亚胺基团与RGD中的巯基迈克尔加成缩合反应后生成稳定的酰胺键。如图2b所示,PBA、LPBGD和LPBGD+siRNA的水合动力学直径分别为140nm、148nm和149nm。
从结果看出,LPBGD在制备过程中,粒径稍有增大,这应该是由于金纳米线在PBA表面缠绕的结果,而与siRNA复合后对粒径的影响几乎不大,比较适合在生物体系中应用。图2c中,PBA的紫外吸收峰为739nm左右,Lipo-Au和Lipo-PBA-Au-RGD(LPBGD)都有广谱的紫外吸收,也进一步证明了材料的光热能力,尤其LPBGD在近红外808nm处有更高的吸收,这也意味着LPBGD有更好的光热性能,利于近红外光激发的光热治疗。图2d所示为各种纳米粒子的zeta电势变化,单纯的PBA的zeta电位为-20.6mV,在阳离子脂质体包覆并缠绕金纳米线后,Lipo-PBA-Au的电位为+47.9mV。而连接上靶向蛋白后,LPBGD的电位为+44.6mV。当负载上带负电荷的siRNA后,LPBGD+siRNA纳米配合物的zeta电位显著下降为+37.3mV。
实施例2本发明金纳米花多肽复合物负载DNA能力的测试
LPBGD的理化性质及细胞的内吞作用与LPBGD和DNA的负载比例有着很大的关系。为了研究本发明金纳米花多肽复合物的DNA装载能力,本实施例进行了琼脂糖凝胶(1%)电泳实验(图3)。
将200ng DNA分别与不同体积的实施例1的LPBGD混合(3μL,6μL,9μL,12μL,15μL),室温放置15分钟,然后进行琼脂糖凝胶电泳,确定LPBGD与DNA的最佳负载比例。图3中泳道1为单纯DNA,随着LPBGD的体积的增加(第2-6泳道),可以看到DNA的条带越来越浅,直至LPBGD的体积增加至9μL或以上时,DNA条带几乎完全消失,说明此时DNA与LPBGD已经可以完全结合,形成稳定的LPBGD/DNA的纳米复合物。
实施例3靶向能力测试
首先使用共聚焦荧光成像(CLSM)和流式细胞分析仪(FCM)对实施例1的LPBGD的靶向能力进行测试,将Panc-1细胞以10000个/孔的密度接种于96孔板,并在含有10%胎牛血清的DMEM培养基中培养12h。根据琼脂糖凝胶迁移实验结果,提前4h将200ng带红色荧光的ROXDNA加入12μL的实施例1的LPBGD或Lipo-PBA-Au(60μg/mL),在室温下共同孵育15min后,加到细胞中,在561nm激发的共聚焦激光扫描显微镜下对Panc-1细胞进行成像。同时,收集递送了4h后的Panc-1细胞,用PBS洗涤3次后,用流式细胞仪分析细胞内的荧光强度来进行靶向递送的定量分析。对于递送4h之后的Panc-1细胞用Hoechst 33342进行细胞核染色15分钟,随后用4%的多聚甲醛在细胞培养箱中固定15分钟,PBS洗两次后即可进行细胞成像。如图4所示,带有靶向的LPBGD/ROXDNA在细胞中孵育4h后,其荧光强度明显更高,这充分证明了靶向分子对Panc-1细胞的亲和力。此外通过流式细胞分析仪(FCM)结果来看,LPBGD/ROXDNA与Lipo-Au/ROXDNA(无靶向多肽修饰)的递送效率分别为81.9%和65.1%,充分说明本发明LPBGD纳米粒子具有癌细胞的靶向性。
实施例4光热能力与光稳定性测试
为了研究本发明金纳米花多肽复合物(LPBGD)的光热能力与光稳定性,将1mL不同浓度的实施例1的LPBGD溶液置于无色透明比色皿中,用0.3W/cm2的功率密度激光辐照1mLLPBGD溶液10分钟,并降至室温,每10s读取一次温度,循环6个周期。红外热成像图用FLIRC2热像相机记录。并计算光热转换效率(η)。如图5a所示,LPBGD经过6个升温降温周期循环后,溶液仍能达到46.6℃,说明LPBGD材料在多次照射后依然可以保持稳定的光热能力。另外,如图5b,通过计算得LPBGD的光热转换效率为62.52%。
上述实验结果表明,本发明LPBGD具有较高的光热转换效率和良好光热稳定性,适合用作光热治疗材料。
此外,由于材料的光热敏感性,在达到一定温度后,脂质由凝胶相转变为液晶相。为检测本发明金纳米花复合物的相变温度,本实验通过紫外吸光度的方法检测材料的相变温度。
将0.8mL实施例1制得的LPBGD密封在石英比色皿中,按照1℃/5分钟的程序逐渐升温,并从35℃至60℃期间,每升温一度进行紫外吸收扫描。随后以温度为横坐标,吸光度为纵坐标拟合曲线,并对数据进行一阶求导,即可求得相变温度。对于负载效率及光热刺激释放效率实验,以200ng siRNA为阴性对照(NC)。负载效率是将足够量的材料与200ng siRNA共孵育15分钟后,离心测上清中剩余siRNA的浓度C1。光热刺激释放效率研究,是将负载了siRNA的LPBGD进行激光照射10分钟,立刻离心,测上清中siRNA的浓度C2。负载效率=(200-C1)/200;释放效率=C2/(200-C1)。如图5c,通过一阶求导,材料的相变温度确定为47℃。也就是说,当材料周围环境达到47℃以上时,材料所携带的药物应该会被释放出来。图5d的结果证明了上述假设,材料的负载效率可达94.89%,通过激光照射,使其温度高于相变温度以上,其释放效率为67.87%。上述结果说明:脂质体的包覆可以使纳米材料变成可控释放的纳米载体。
实施例5细胞活性及毒性实验
首先,将Panc-1细胞以10000个/孔的密度接种于96孔板中,培养过夜后,细胞分为7组:PBS,PBS+L,LPBGD,LPBGD+L,LPBGD+siRNANC,LPBGD+siRNA和LPBGD+siRNA+L。PBS+L,LPBGD+L和LPBGD+siRNA+L组,在孵育6h后用808nm激光照射。24h和48h后进行毒性检测。通过酶标仪获取450nm处的吸收强度并记录。如图6所示,相对于空白组,激光(Blank+L),材料(实施例1的LPBGD),基因阴性对照(LPBGD-siRNANC)对细胞的存活率几乎没有影响,而基因治疗(LPBGD-siRNA)和光热治疗(LPBGD+L)显著降低细胞活性,尤其在基因协同光热治疗(LPBGD-siRNA+L)48h后,仅有9.64%的细胞存活,说明以本发明金纳米花多肽复合物为载体的基因协同光热治疗的疗效显著。
实施例6光声成像实验
为了考察肿瘤中材料的富集情况以明确指导诊断及治疗,本实施例进行了光声成像实验。
根据操作说明,制作模拟活体小鼠的假体,将不同浓度的LPBGD(3.125,6.25,12.5,25,50,100μg/mL)打入到假体中进行光声成像(PAI,MSOT inVision128,iThermedical,德国),光声信号强度和光声图像使用制造商提供的软件进行处理。对扫描结果进行线性回归分析。同时,为了考察LPBGD纳米粒子的造影效率,我们测量了不同浓度LPBGD纳米粒子(0.0625,0.125,0.25,0.5,1,2mg/mL)的X-射线计算机断层成像(CT成像)能力。结果见图7。
如图7a,为体外用假体模拟体内光声成像情况的实验。随着假体中LPBGD浓度的加大,光声强度也随之成比例增大,以LPBGD浓度为横坐标,最大平均光声强度为纵坐标作图,其R2=0.9926,插图也很好的说明LPBGD可以产生光声信号,并且信号强度与浓度有良好的线性关系。图7b为LPBGD在体外的CT成像图片,及其浓度依赖的线性回归曲线,R2=0.9969可以看到随着LPBGD的浓度增大,其CT值也越来越大,说明LPBGD有CT成像的能力,并且成像效果与材料的浓度呈正相关。
实施例7体内抗肿瘤试验
当荷载Panc-1肿瘤的雌性BALB/c裸鼠的肿瘤体积达到100mm3时,随机分为7组(n=5):对照组:PBS、PBS+L、LPBGD(0.5mg/mL)、LPBGD-siRNANC(材料加阴性基因组)、LPBGD-siRNA(基因治疗组)、LPBGD+L(光热治疗组)、LPBGD-siRNA+L(协同治疗组)。注射剂量为200μL。静脉注射24h后,需要激光辐照组的小鼠用波长808nm的激光辐照10min,激光辐照密度为1.2W/cm2。在治疗期间每隔一天测量一次肿瘤的长(L)与宽(W)和小鼠的体重,肿瘤体积通过V=0.5×L×W2得到。以V/V0(V0为初始肿瘤体积)计算肿瘤相对体积。治疗14天后,收集各组小鼠的肿瘤、血清及主要脏器进行进一步研究。结果见图8。
如图8为治疗14天期间,各组小鼠肿瘤体积的变化情况:与PBS组相比,LPBGD+siRNA、LPBGD+L和LPBGD+siRNA+L组的肿瘤明显变小,尤其在LPBGD+siRNA+L组中,多数肿瘤几乎被完全杀灭,而对照组(PBS+L、LPBGD和LPBGD+siRNANC),小鼠的肿瘤保持较高的生长速度。上述结果表明,本发明金纳米花多肽复合物可负载基因,可实现基因与光热协同治疗癌症,具有更好的癌症治疗效果。
以上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来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (13)
1.一种金纳米花多肽复合物,其特征在于,包括:
内核,所述内核为普鲁士蓝类似物纳米颗粒;和
在所述内核上,原位生长脂质体保护的金纳米颗粒,获得金纳米花,再修饰靶向肿瘤的多肽。
2.根据权利要求1所述的金纳米花多肽复合物,其特征在于,所述普鲁士蓝类似物纳米颗粒为掺杂Mn、Co离子的普鲁士蓝类似物纳米颗粒;所述脂质体为温度敏感的脂质体;所述多肽为cRGD。
3.根据权利要求1所述的金纳米花多肽复合物,其特征在于,所述金纳米花多肽复合物的平均水合粒径为148nm。
4.权利要求1~3任一项所述的金纳米花多肽复合物的制备方法,其特征在于,包括:
步骤1:在普鲁士蓝类似物纳米颗粒表面原位生长脂质体保护的金纳米颗粒,获得金纳米花;
步骤2:在所述金纳米花上修饰靶向肿瘤的多肽,获得金纳米花多肽复合物。
5.根据权利要求4所述的制备方法,其特征在于,步骤1具体为:向脂质体溶液中依次加入普鲁士蓝类似物和氯金酸溶液进行原位生长反应;将反应液离心,弃上清,沉淀物清洗后加水溶解,得金纳米花溶液。
6.根据权利要求5所述的制备方法,其特征在于,所述原位生长反应在搅拌条件下进行,所述搅拌为100~500rpm搅拌5~30min;所述脂质体溶液的浓度为2.5-6mg/mL;所述氯金酸的浓度为50-120mM。
7.根据权利要求5所述的制备方法,其特征在于,所述脂质体为PEG化的脂质体,所述脂质体溶液的制备方法如下:
将DODAB和DPPC溶于氯仿中,加入相当于DODAB和DPPC总重量的10-30%的DSPE-PEG2000,再加入相当于DSPE-PEG2000质量的5-30%的DSPE-PEG2000-Maleimide,震荡混匀,获得混合液;向所述混合液中通入氮气吹干并真空干燥,加入PBS,于40-60℃下超声至体系澄清透明。
8.根据权利要求7所述的制备方法,其特征在于,步骤2中,所述修饰具体为:
将金纳米花与靶向肿瘤的多肽混合,2-10℃下旋转震荡6-20h;
所述靶向肿瘤的多肽的用量为DSPE-PEG2000质量的5-30%。
9.权利要求4~8任一项所述的制备方法制得的金纳米花多肽复合物。
10.权利要求1~3任一项所述的金纳米花多肽复合物或权利要求4~8所述的金纳米花多肽复合物作为基因载体在制备抗肿瘤药物中的应用。
11.根据权利要求10所述的应用,其特征在于,所述肿瘤为表面高表达αvβ3整合素受体的癌症,包括胰腺癌、肝癌、肺癌、胃癌、乳腺癌和宫颈癌。
12.一种抗肿瘤药物,其特征在于,包括:
权利要求1~3任一项所述的金纳米花多肽复合物或权利要求4~8任一项所述的制备方法制得的金纳米花多肽复合物,和负载于所述金纳米花多肽复合物上的基因药物。
13.根据权利要求12所述的抗肿瘤药物,其特征在于,所述基因药物为siRNA和/或DNA。
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