CN119303109A - 一种基于叶酸靶向的脂质体纳米粒子及其制备方法和应用 - Google Patents
一种基于叶酸靶向的脂质体纳米粒子及其制备方法和应用 Download PDFInfo
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Abstract
本发明属于纳米药剂技术领域，公开了一种基于叶酸靶向的脂质体纳米粒子及其制备方法和应用。该制备方法，包括以下步骤：将磷脂、胆固醇、聚乙二醇、叶酸修饰的聚乙二醇、荧光染料溶于溶剂中，旋转蒸发形成均匀薄膜；将纳米四氧化三铁制成水化液；将水化液加入薄膜中进行水化处理，直至无固态物；再加入microRNA进行RNA装载，制得脂质体纳米粒子。本发明提供的脂质体纳米粒子能够整合多个功能物质到同一个纳米载体中，实现多模态诊断和治疗的协同作用，进而实现集成化的癌症诊疗方案，提升治疗效果并减少副作用。
Description
技术领域
本发明属于纳米药剂技术领域，具体涉及一种基于叶酸靶向的脂质体纳米粒子及其制备方法和应用。
背景技术
随着肿瘤治疗技术的不断进步，纳米技术在癌症诊断和治疗中的应用逐渐引起了广泛关注。纳米粒子因其独特的物理化学性质，如高表面积、粒径可调、表面功能化容易等，成为理想的药物载体和诊疗平台。近年来，多功能纳米粒子在肿瘤诊疗一体化领域的应用研究取得了重要进展，这些纳米粒子不仅可以实现肿瘤的成像诊断，还能够直接用于治疗，从而大大提高了治疗的精准性和效果。
由于肿瘤细胞表面通常过表达叶酸受体，因此叶酸被广泛应用于肿瘤的靶向治疗。通过将叶酸与纳米粒子结合，可以增强纳米粒子在肿瘤组织中的富集效应，提高治疗的特异性和有效性。其中，纳米四氧化三铁(Fe3O4)由于其良好的磁性和可作为磁共振成像(MRI)对比剂的特性，是目前研究的热点。同时，荧光染料在光热治疗和荧光成像中的应用前景也非常广阔。
此外，microRNA(miRNA)作为一种内源性非编码RNA，能够调控基因表达并参与多种生物学过程。microRNA-101-3p被证明在多种肿瘤中具有抑制细胞增殖和迁移的功能，因此在肿瘤治疗中具有潜在的应用价值。然而，将这些功能物质整合到同一个纳米载体中，以实现多模态诊断和治疗的协同作用，目前仍面临巨大挑战。具体的挑战有：(1)复合材料的稳定性：将不同功能的纳米材料有效复合，并保持其在体内的稳定性，是一个技术难题。复合材料需要在体内环境中保持物理和化学稳定，才能保证其成像和治疗效果。(2)功能化的精准控制：在纳米载体上准确地结合叶酸、荧光染料和miRNA-101-3p，需要精确控制每个成分的负载量和释放行为，以确保有效性和安全性。(3)体内分布和排除：纳米载体在体内的分布和排除过程可能影响治疗效果和副作用。例如，纳米粒子可能在肝脏、脾脏等器官积累，从而影响其在肿瘤中的靶向效果。(4)成像与治疗的协调性：纳米载体在多模态成像(如MRI和荧光成像)中的表现需要与其治疗功能(如光热治疗)相协调。不同模式的成像和治疗效果往往会互相影响。(5)优化治疗和成像窗口：确保在治疗时能够有效成像，并在成像时能够维持治疗效果，这要求纳米载体具有良好的功能稳定性。
因此，亟需提供一种纳米粒子，能够整合功能物质到同一个纳米载体中，以期实现集成化的癌症诊疗方案，提升治疗效果并减少副作用。
发明内容
本发明旨在至少解决上述现有技术中存在的技术问题之一。为此，本发明提出了一种基于叶酸靶向的脂质体纳米粒子及其制备方法和应用。本发明提供的脂质体纳米粒子能够整合多个功能物质到同一个纳米载体中，实现多模态诊断和治疗的协同作用，进而实现集成化的癌症诊疗方案，提升治疗效果并减少副作用。
本发明提供了一种基于叶酸靶向的脂质体纳米粒子的制备方法。
具体地，一种基于叶酸靶向的脂质体纳米粒子的制备方法，包括以下步骤：
将磷脂、胆固醇、聚乙二醇、叶酸修饰的聚乙二醇、荧光染料溶于溶剂中，旋转蒸发形成均匀薄膜；将纳米四氧化三铁制成水化液；将所述水化液加入薄膜中进行水化处理，直至无固态物；再加入microRNA进行RNA装载，制得基于叶酸靶向的脂质体纳米粒子。
优选地，所述磷脂、所述胆固醇、所述聚乙二醇、所述叶酸修饰的聚乙二醇的质量比为(50-60)：(30-40)：(8-10)：(0.5-2)。进一步优选地，所述磷脂、所述胆固醇、所述聚乙二醇、所述叶酸修饰的聚乙二醇的质量比为(52-56)：(32-40)：(8-10)：(0.5-1.5)。通过精确控制每个成分的负载量和释放行为，以确保脂质体纳米粒子的有效性和安全性。
优选地，所述磷脂包括卵磷脂、磷脂酰肌醇中的至少一种。
优选地，所述荧光染料包括IR-780、IR-1061、IR-820中的至少一种。进一步优选地，所述荧光染料为IR-780。IR-780与四氧化三铁的吸收波长差异较大，且IR-780无磁共振信号，能够很好地规避治疗时其他组分的干扰，提高成像与治疗的协调性。
优选地，所述荧光染料的质量占所述磷脂、所述胆固醇、所述叶酸修饰的聚乙二醇、所述聚乙二醇、所述microRNA、所述纳米四氧化三铁的总质量的1％-10％；进一步优选地，所述荧光染料的质量占所述磷脂、所述胆固醇、所述叶酸修饰的聚乙二醇、所述聚乙二醇、所述microRNA、所述纳米四氧化三铁的总质量的3％-7％。
优选地，所述溶剂包括三氯甲烷、二氯甲烷、无水乙醇中的至少一种。
优选地，所述磷脂、所述胆固醇、所述聚乙二醇、所述叶酸修饰的聚乙二醇和所述荧光染料的总质量与所述溶剂的质量比为1:250-300。
优选地，所述旋转蒸发的过程为：在35-45℃、真空90-110mbar下旋转蒸发30-60分钟直至形成均匀薄膜。
优选地，所述水化液的制备过程为：将纳米四氧化三铁溶液按照体积比1:(90-110)加入水中，超声混匀8-12分钟，制成水化液。所述纳米四氧化三铁溶液的质量分数为15％-25％，优选为20％。
优选地，所述水化处理的过程为在55-65℃下，超声处理5-10分钟，直至无固态物。
优选地，所述microRNA包括microRNA-101-3p。
优选地，所述RNA装载的过程为吸取microRNA储存液加入经水化处理的体系中，于2-4℃摇床上放置10-14小时。
在以上制备过程中，卵磷脂自组装成双层膜结构，形成脂质体的主要框架溶剂用于溶解磷脂，方便其在合成过程中均匀分布；并通过蒸发有机溶剂，磷脂将沉积为薄膜，准备进行下一步水化。水在薄膜水化过程中，水或缓冲溶液用于重新膨胀磷脂薄膜，形成脂质体囊泡。水化后，磷脂双层自组装成封闭的囊泡结构，封闭内部的水相药物，形成脂质体。纳米四氧化三铁作为药物或活性成分被装载到脂质体中，以便通过脂质体递送。叶酸用于功能化脂质体表面，以增强其靶向性、稳定性或生物相容性。聚乙二醇能够增加脂质体的稳定性，防止其在存储或体内环境中聚集或降解。
本发明还提供了一种基于叶酸靶向的脂质体纳米粒子。
具体地，一种基于叶酸靶向的脂质体纳米粒子，由上述制备方法制得；包括内核纳米四氧化三铁，包覆所述内核纳米四氧化三铁的脂质双分子层，位于所述脂质双分子层内部的荧光染料，嵌入所述脂质双分子层的PEG、叶酸修饰的PEG，以及吸附于所述脂质双分子层外部的microRNA。
本发明还提供了上述基于叶酸靶向的脂质体纳米粒子的应用。
具体地，上述基于叶酸靶向的脂质体纳米粒子在制备肿瘤治疗药物中的应用。
本发明还提供了一种治疗肿瘤的药物。
具体地，一种治疗肿瘤的药物，包括上述基于叶酸靶向的脂质体纳米粒子。
本发明提供的基于叶酸靶向的脂质体纳米粒子，利用脂质体纳米载药系统的多重载药方式，将叶酸修饰的聚乙二醇与荧光染料嵌入脂质双分子层内，将纳米四氧化三铁装载于亲水核心，并将microRNA吸附于脂质体外部。本发明利用脂质体多载药位点的特点将Fe3O4和荧光染料二者根据溶解性的不同确定了最佳复合载药方式，所得脂质体纳米粒子光热稳定性、功能稳定性强；且因四氧化三铁与IR-780的吸收波长差异较大，IR-780无磁共振信号，能够很好地规避治疗时其他组分的干扰，实现双模态的成像和治疗，成像与治疗的协调性强。本发明利用叶酸对纳米粒子表面进行修饰，提高了脂质体纳米粒子对肿瘤细胞的靶向性，同时减少了对正常组织的附着。
相比现有技术，本发明的有益效果在于：
(1)多功能诊疗一体化：本发明通过将叶酸、荧光染料、纳米四氧化三铁和microRNA集成在一个纳米粒子载体中，实现了肿瘤的多模态诊疗一体化。该脂质体纳米粒子不仅具备靶向性强、成像效果好、治疗效率高的特点，还能够通过光热效应和基因调控作用进行有效的肿瘤治疗。
(2)靶向性增强：叶酸修饰的脂质体纳米粒子能够特异性识别并结合肿瘤细胞表面的叶酸受体，提高了脂质体纳米粒子在肿瘤组织中的富集效果，从而增强了治疗的靶向性，减少了对正常组织的副作用。
(3)协同治疗效果：通过将光热疗法、磁共振成像和基因调控三者结合，本发明的脂质体纳米粒子能够在肿瘤治疗中产生协同效应，不仅提高了肿瘤治疗的整体效果，还减少了单一治疗手段可能带来的副作用。
(4)创新性设计：本发明提供的脂质体纳米粒子设计独特，集成了多种功能于一体，并通过叶酸的靶向作用提高了治疗的特异性。这种创新设计能够克服现有技术的不足，在肿瘤治疗领域具有广泛的应用前景。
(5)安全性和生物相容性：通过优化纳米粒子的合成方法，本发明确保了所制备脂质体纳米粒子的安全性和生物相容性，降低了纳米材料在生物体内的潜在毒性和免疫反应风险。本发明提供的基于叶酸靶向的脂质体纳米粒子，具有肿瘤诊疗一体化领域的创新性和实用性，为癌症治疗提供了一种高效且低毒的新方法。
附图说明
图1为FA-Lipo-Fe3O4-Mir101@IR780脂质体纳米粒子的结构示意图；
图2为FA-Lipo-Fe3O4-Mir101@IR780脂质体纳米粒子的透射电镜图；
图3为FA-Lipo-Fe3O4-Mir101@IR780脂质体纳米粒子的zeta电位分布图；
图4为FA-Lipo-Fe3O4-Mir101@IR780脂质体纳米粒子的纳米粒径分布图；
图5为FA-Lipo-Fe3O4-Mir101@IR780脂质体纳米粒子的肉眼观测图；
图6为FA-Lipo-Fe3O4-Mir101@IR780脂质体纳米粒子的光热稳定性图；
图7为FA-Lipo-Fe3O4-Mir101@IR780脂质体纳米粒子及其他各粒子的吸光度测试图；
图8为FA-Lipo-Fe3O4-Mir101@IR780脂质体纳米粒子的磁性分布实验效果；
图9为不同浓度FA-Lipo-Fe3O4-Mir101@IR780脂质体纳米粒子(药物)在0.6W/cm2激光功率下光热效果图；
图10为不同浓度FA-Lipo-Fe3O4-Mir101@IR780脂质体纳米粒子(药物)在1.1W/cm2激光功率下光热效果图；
图11为不同浓度FA-Lipo-Fe3O4-Mir101@IR780脂质体纳米粒子(药物)在1.7W/cm2激光功率下光热效果图；
图12为不同浓度FA-Lipo-Fe3O4-Mir101@IR780脂质体纳米粒子(药物)下肿瘤细胞存活率；
图13为不同浓度FA-Lipo-Fe3O4-Mir101@IR780脂质体纳米粒子(药物)下肿瘤细胞抑制率。
具体实施方式
为了让本领域技术人员更加清楚明白本发明所述技术方案，现列举以下实施例进行说明。需要指出的是，以下实施例对本发明要求的保护范围不构成限制作用。
以下实施例中所用的原料、试剂或装置如无特殊说明，均可从常规商业途径得到，或者可以通过现有已知方法得到。
实施例
一种基于叶酸靶向的脂质体纳米粒子的制备方法，包括以下步骤：
(1)配料与溶解
将卵磷脂、胆固醇、聚乙二醇、叶酸修饰的聚乙二醇(购自上海芃硕生物科技有限公司)按照质量比54：36：9：1加入容器后，按照总质量5％加入IR-780。按固：液质量比1：280加入三氯甲烷，用100W探头式超声破碎仪超声处理至完全溶解，得到待旋蒸液。
(2)旋转蒸发
将待旋蒸液转移至500mL球形蒸发瓶中。在40℃、真空100mbar下旋转蒸发直至形成均匀薄膜。
(3)水化
将纳米四氧化三铁溶液(10-50nm，质量分数为20％)按照体积比1：99加入超纯水中，超声混匀10分钟，制成水化液。将水化液加入薄膜中，在60℃下水化。超声处理至无固态物，转移至避光离心管中定容至10mL。
(4)RNA装载
吸取50μL mir101-3p(microRNA)储存液加入步骤(3)离心管中。4℃摇床上放置12小时，得到FA-Lipo-Fe3O4-Mir101@IR780脂质体纳米粒子。
产品效果测试
(1)FA-Lipo-Fe3O4-Mir101@IR780脂质体纳米粒子的形态学表征。
图1为FA-Lipo-Fe3O4-Mir101@IR780脂质体纳米粒子的结构示意图，如图1所示，在脂质体纳米粒子中，内部核心装载亲水性纳米四氧化三铁，IR-780位于脂质形成的脂质双分子层内，PEG及叶酸(FA)修饰的PEG嵌入脂质双分子层并暴露出修饰后的主动靶向位点，microRNA吸附于脂质双分子层外部。
采用负染法电镜拍摄脂质体图片：通过负染法将脂质体样品在碳膜铜网上进行染色处理，然后使用透射电子显微镜(TEM)拍摄脂质体的形态和分布图像，以观察其形貌和结构特征。图2为FA-Lipo-Fe3O4-Mir101@IR780脂质体纳米粒子的透射电镜图，由图2可知，在电镜下脂质体呈现均匀的圆形，可看到脂质双分子层及内部的亲水核心。
测量纳米粒子电位：将纳米粒子悬浮在水相介质中，使用电泳光散射法测量其表面电位(ζ电位)，以评估粒子的表面电荷性质和稳定性。图3为FA-Lipo-Fe3O4-Mir101@IR780脂质体纳米粒子的zeta电位分布图，由图3可知，脂质体电位位于-30mv左右，具有较好的稳定性。
粒径分布测试：将脂质体纳米粒子分散在合适的溶液中，使用动态光散射仪(DLS)测量粒子在溶液中的布朗运动，并计算出脂质体纳米粒子的平均粒径及其分布。图4为FA-Lipo-Fe3O4-Mir101@IR780脂质体纳米粒子的粒径分布图，由图4可知，可见脂质体纳米粒子的大小处于100nm附近，具有十分理想的大小。
(2)FA-Lipo-Fe3O4-Mir101@IR780脂质体纳米粒子的理化性质。
图5为FA-Lipo-Fe3O4-Mir101@IR780脂质体纳米粒子的肉眼观测图，由图5可以观察到，制备的脂质体纳米粒子溶液澄清，证明脂质体纳米粒子均一性良好。
测量纳米粒子光热稳定性：使用800nm激光仪以1.1W功率对纳米粒子溶液循环照射三次，每次冷却至室温后重新开始，记录每隔30秒的温度变化，评估其光热稳定性。图6为FA-Lipo-Fe3O4-Mir101@IR780脂质体纳米粒子的光热稳定性图，由图6可知，多次加热后脂质体纳米粒子升温性能无明显变化，说明其具有良好的光热稳定性。
测量纳米粒子吸收峰：通过紫外-可见光分光光度计扫描Fe3O4溶液、IR-780溶液、FA-Lipo-Fe3O4-Mir101@IR780脂质体纳米粒子溶液的吸收光谱，确定在特定波长下的最大吸收峰，以分析其光学特性。图7为FA-Lipo-Fe3O4-Mir101@IR780脂质体纳米粒子及其他各粒子的吸光度测试图；脂质体纳米粒子同时在两组分吸光区域存在吸收峰，说明各成分成功装载。
(3)FA-Lipo-Fe3O4-Mir101@IR780脂质体纳米粒子的磁性分布效果。
将进行细胞接种，将HepG2细胞以合适的密度接种在10cm培养皿中，并在培养基中培养至80％-90％汇合度。在进行磁性处理，在培养皿底部放置一个环形磁铁，确保磁场均匀分布。最后加入脂质体纳米粒子(药物)处理：加入含有磁性脂质体纳米粒子的药物溶液至培养皿中，保证脂质体纳米粒子(药物)覆盖细胞并受到磁场影响。将细胞在37℃、5％CO2环境下孵育24小时，孵育结束后进行结晶紫染色，以观察脂质体纳米粒子(药物)的磁性分布效果。具体过程如下在脂质体纳米粒子(药物)孵育24小时后，弃去培养基，用PBS缓冲液轻轻冲洗培养皿表面，重复多次，直至培养皿中看不到脂质体纳米粒子(药物)的颜色残留。然后使用4％多聚甲醛固定细胞10分钟，然后用PBS再次冲洗。再将1％结晶紫染色液加入培养皿中，染色10-15分钟，使细胞染色。并使用自来水或PBS冲洗培养皿，去除多余的结晶紫染色液，直至染液不再流出。最后在显微镜下观察并记录细胞分布和存活情况，特别关注脂质体纳米粒子(药物)分布区域的细胞存活情况。图8为FA-Lipo-Fe3O4-Mir101@IR780脂质体纳米粒子的磁性分布实验效果，其中，图8中A显示将纳米粒子加入下方黏贴磁铁的培养皿，进行细胞培养；图8中B显示去除磁铁后发现纳米粒子沿磁铁磁力线聚集；图8中C显示弃去培养基并用pbs多次冲洗，纳米粒子依然存在，说明已被细胞大量摄取；图8中D显示结晶紫染色后可见磁铁磁力线分布区域肿瘤细胞死亡明显。
(4)FA-Lipo-Fe3O4-Mir101@IR780脂质体纳米粒子不同浓度及不同功率光热效果。
将脂质体纳米粒子配置为不同浓度(10、30、50μg/mL)溶液，分别在0.6、1.1、1.7W/cm2激光功率下照射，每30s记录温度变化，评估光热效应。图9-图11分别为不同浓度FA-Lipo-Fe3O4-Mir101@IR780脂质体纳米粒子(药物)在0.6W、1.1W和1.7W/cm2激光功率下光热效果图，由图9-图11可知，FA-Lipo-Fe3O4-Mir101@IR780脂质体纳米粒子(药物)随着浓度及激光功率的增加，升温效果也有较为显著的提高，其光热效应优异。
(5)FA-Lipo-Fe3O4-Mir101@IR780脂质体纳米粒子体内抑瘤疗效。
测试FA-Lipo-Fe3O4-Mir101@IR780脂质体纳米粒子体内抑瘤疗效，具体过程如下：a.将HepG2肿瘤细胞以合适的密度接种在96孔板中，每孔接种100μL培养基中的细胞悬液，使每孔含有大约8000个细胞。将96孔板置于37℃、5％CO的培养箱中培养，直到细胞在孔内贴壁生长且达到适宜的状态(通常约24小时)。b.将纳米粒子药物按照10、20、30、50、100μg/mL的浓度梯度进行稀释。每个浓度配制一组。c.移去96孔板中的培养基，每孔加入100μL不同浓度的药物溶液，设置空白对照组(不含药物)和阴性对照组(不含细胞)。然后将96孔板返回培养箱中，继续培养24小时。d.24小时后，每孔加入10μL CCK-8试剂(细胞计数试剂盒)，轻轻混匀。继续在培养箱中孵育1-4小时，直至孔内液体颜色发生显著变化(从黄色变为橙红色)。e.使用酶标仪在450nm波长处测定各孔的吸光度(OD值)，记录数据。f.根据测得的吸光度(OD值)，计算每个浓度下细胞增殖抑制率。抑制率的计算公式为：
存活率(％)＝1-抑制率(％)。
g.通过对不同浓度FA-Lipo-Fe3O4-Mir101@IR780脂质体纳米粒子(药物)处理组的抑制率进行统计分析，评估药物的抗肿瘤效果，并绘制剂量-效应曲线。
图12为不同浓度FA-Lipo-Fe3O4-Mir101@IR780脂质体纳米粒子(药物)下肿瘤细胞存活率；图13为不同浓度FA-Lipo-Fe3O4-Mir101@IR780脂质体纳米粒子(药物)下肿瘤细胞抑制率。由图12、图13可知，随着药物浓度的增加，肿瘤细胞的存活率有较为显著地下降，药物有良好的抗肿瘤作用。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对本发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。
Claims (10)
1.一种脂质体纳米粒子的制备方法，其特征在于，包括以下步骤：
将磷脂、胆固醇、聚乙二醇、叶酸修饰的聚乙二醇、荧光染料溶于溶剂中，旋转蒸发形成均匀薄膜；将纳米四氧化三铁制成水化液；将所述水化液加入薄膜中进行水化处理，直至无固态物；再加入microRNA进行RNA装载，制得基于叶酸靶向的脂质体纳米粒子。
2.根据权利要求1所述的制备方法，其特征在于，所述磷脂、所述胆固醇、所述聚乙二醇、所述叶酸修饰的聚乙二醇的质量比为(50-60)：(30-40)：(8-10)：(0.5-2)。
3.根据权利要求1或2所述的制备方法，其特征在于，所述荧光染料包括IR-780、IR-1061、IR-820中的至少一种。
4.根据权利要求1或2所述的制备方法，其特征在于，所述溶剂包括三氯甲烷、二氯甲烷、无水乙醇中的至少一种。
5.根据权利要求4所述的制备方法，其特征在于，所述磷脂、所述胆固醇、所述聚乙二醇、所述叶酸修饰的聚乙二醇和所述荧光染料的总质量与所述溶剂的质量比为1:250-300。
6.根据权利要求1或2所述的制备方法，其特征在于，所述旋转蒸发的过程为：在35-45℃、真空90-110mbar下旋转蒸发30-60分钟直至形成均匀薄膜。
7.根据权利要求1或2所述的制备方法，其特征在于，所述RNA装载的过程为吸取microRNA储存液加入经水化处理的体系中，于2-4℃摇床上放置10-14小时。
8.一种脂质体纳米粒子，其特征在于，由权利要求1-7中任一项所述的制备方法制得；包括内核纳米四氧化三铁，包覆所述内核纳米四氧化三铁的脂质双分子层，位于所述脂质双分子层内部的荧光染料，嵌入所述脂质双分子层的PEG、叶酸修饰的PEG，以及吸附于所述脂质双分子层外部的microRNA。
9.权利要求8所述的基于叶酸靶向的脂质体纳米粒子在制备肿瘤治疗药物中的应用。
10.一种治疗肿瘤的药物，其特征在于，权利要求8所述的基于叶酸靶向的脂质体纳米粒子。
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