CN116120232B - 一种dna-螺烯纳米药物及其制备方法和用途 - Google Patents
一种dna-螺烯纳米药物及其制备方法和用途 Download PDFInfo
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Abstract
本发明提供了一种DNA‑螺烯纳米药物，其结构式如下所示：本发明还提供了上述的一种DNA‑螺烯纳米药物的制备方法，先制备苯并[4]螺烯溴化盐；然后将含有非甲基化的胞嘧啶磷酸鸟嘌呤二核苷酸的寡聚脱氧核苷酸和苯并[4]螺烯溴化盐自组装得到DNA‑螺烯纳米药物。本发明还提供了上述的DNA‑螺烯纳米药物在制备用于治疗肾细胞癌的药物中的用途。本发明的纳米药物制备方法简单，水溶性良好以及选择性肿瘤抑制疗效明显，具有高效、低毒的性能。
Description
技术领域
本发明属于生物医药领域，涉及一种治疗肿瘤的药物，具体来说是一种DNA-螺烯纳米药物及其制备方法和用途。
背景技术
肾细胞癌(Renal cell carcinoma,RCC)是泌尿系统最常见的恶性肿瘤之一【HuaX,Chen J,Ge S,Xiao H,Zhang L,and Liang C.Integrated analysis of the functionsof RNA binding proteins in clear cell renal cell carcinoma.Genomics,2021,113(1Pt 2):850-860.】，外科手术治疗通常是RCC早期患者的首选治疗方法，术后5年生存率可达60％【Jabaji RB,Fischer H,Kern T,and Chien GW.Trend of Surgical Treatment ofLocalized Renal Cell Carcinoma.Perm J,2019,23:18-108.】。然而20％的RCC患者即使是经过手术、放化疗等治疗仍会出现复发转移，发展成转移性RCC【Ball MW.Surgicalmanagement of metastatic renal cell carcinoma.Discov Med,2017,23(129):379-387.】。对于转移性RCC患者，其预后极差，5年生存率小于10％【Mickisch GH.Principlesof nephrectomy for malignant disease.BJU Int,2002,89(5):488-495.】。转移性RCC的一线治疗主要依赖细胞因子(IFN-α和IL-2)和靶向药物(VEGF/VEGFR抑制剂和mTOR抑制剂)，但由于其严重的不良反应和靶向治疗介导的耐药性限制了临床应用【Xu W,AtkinsMB,and McDermott DF.Checkpoint inhibitor immunotherapy in kidney cancer.NatRev Urol,2020,17(3):137-150.】。因此开发兼具安全性、高疗效和防复发的RCC治疗手段仍然是RCC治疗的前沿研究方向。
随着肿瘤免疫治疗手段的快速发展，已逐渐成为继手术治疗、放射治疗、化疗和靶向治疗之后的“第五大”肿瘤治疗方法【McNutt M.Cancer immunotherapy.Science,2013,342(6165):1417.】，其作用机制是通过重新启动并维持肿瘤-免疫循环，恢复机体正常的抗肿瘤免疫反应，从而达到控制与清除肿瘤的效果。
鉴于各种肿瘤免疫微环境中免疫细胞状态的差异，当前免疫治疗的适用范围和受益群体依然很少。因此，通过外源性递送免疫刺激物有助于提高免疫治疗疗效。传统化疗在杀伤肿瘤细胞时，也可以因为肿瘤细胞死亡从而触发炎症反应，继而改变肿瘤微环境中的免疫状态。这意味着联合应用免疫刺激物和化疗可以相互促进治疗疗效，用于肿瘤的治疗。
发明内容
针对现有技术中的上述技术问题，本发明提供了一种DNA-螺烯纳米药物及其制备方法和用途，所述的这种DNA-螺烯纳米药物及其制备方法和用途要解决现有技术中的药物对于治疗肾细胞癌的效果不佳的技术问题。
本发明提供了一种DNA-螺烯纳米药物(CpG-benzo[4]helicenium)，其结构式如下所示：
本发明还提供了上述的一种DNA-螺烯纳米药物的制备方法，包括如下步骤：
1)按照物料比称取苯并[4]螺烯和1-溴丙烷，苯并[4]螺烯和1-溴丙烷的摩尔比为1:10，加入有机溶剂中溶解，在65℃反应48小时即得到苯并[4]螺烯溴化盐；
2)按照物料比称取含有非甲基化的胞嘧啶磷酸鸟嘌呤二核苷酸的寡聚脱氧核苷酸和苯并[4]螺烯溴化盐，含有非甲基化的胞嘧啶磷酸鸟嘌呤二核苷酸的寡聚脱氧核苷酸和苯并[4]螺烯溴化盐的摩尔比为1：20，在25℃反应24小时即得到DNA-螺烯纳米药物。
具体的，所述的有机溶剂为CHCl3。
本发明还提供了上述的DNA-螺烯纳米药物在制备用于治疗肾细胞癌的药物中的用途。
本发明基于苯并[4]螺烯的季铵化修饰的方法，增加了水溶性，其衍生物能够有效结合DNA，选择性抑制多种RCC细胞的生长。合成方法是在65℃和有机溶剂中，苯并[4]螺烯和1-溴丙烷摩尔比为1:10反应48小时。反应产物可以通过真空除溶剂、极性有机溶剂溶解和非有机溶剂再沉淀，离心等方法进行纯化。如将固体溶解在CH2Cl2中，并通过加入Et2O沉淀，离心收集固体。将溶解-沉淀-离心过程重复三次，然后将固体保持在真空烘箱中以除去残留的溶剂，得到产物benzo[4]helicenium。在25℃，CpG DNA和benzo[4]helicenium摩尔比为1：20反应24小时即得到CpG-benzo[4]helicenium。
将CpG-benzo[4]helicenium与骨髓巨噬细胞进行免疫刺激效果评估，具有明显的免疫刺激活性。
将CpG-benzo[4]helicenium与RCC细胞株ACHN进行抗肿瘤药效评估，具有明显的肿瘤细胞抑制效果。
苯并[4]螺烯是一类具有非平面结构的稠环芳烃的衍生物，通过对苯并[4]螺烯进行季铵化可以明显增加分子的水溶性以及和DNA的结合能力，benzo[4]helicenium除了具有优异的选择性杀伤肾癌细胞而不影响正常细胞功能外，还能刺激免疫细胞产生免疫炎症因子。CpG DNA具有较强的免疫激活功能，因此将CpG DNA与benzo[4]helicenium通过静电相互作用自组装形成纳米药物，不仅可以提升其在肿瘤部位的富集，还可以发挥免疫/化疗协同作用治疗肾癌。
本发明发现benzo[4]helicenium可以有效治疗RCC，并能激活炎症反应，诱导免疫反应相关细胞因子的表达，而CpG DNA具有免疫刺激活性【Wagner M,Poeck H,Jahrsdoerfer B,Rothenfusser S,Prell D,Bohle B,Tuma E,Giese T,Ellwart JW,Endres S,and Hartmann G.IL-12p70-dependent Th1 induction by human B cellsrequires combined activation with CD40 ligand and CpG DNA.J Immunol,2004,172(2):954-963.】，因此两者可以发挥免疫与化疗的协同作用。另一方面，benzo[4]helicenium通过与CpG DNA构建成纳米颗粒能提升其在肿瘤部位的富集。因此利用CpG DNA与benzo[4]helicenium来构建兼具免疫和化疗双重作用的DNA-螺烯纳米药物CpG-benzo[4]helicenium来选择性杀伤肿瘤细胞，具有高效、低毒的性能。
本发明的抗肿瘤纳米药物CpG-benzo[4]helicenium是一种高效、低毒的肿瘤免疫/化学治疗的药物。Benzo[4]helicenium能够与DNA结合，继而阻止肿瘤细胞DNA复制、诱导肿瘤细胞DNA损伤并抑制肿瘤细胞DNA修复，能够高效地杀伤多种肾癌细胞。最关键的是benzo[4]helicenium在杀伤肾癌细胞的同时，对正常细胞无明显毒性，具有优异的选择性。除此以外，benzo[4]helicenium还刺激免疫细胞产生免疫炎症因子，因此将其与具有免疫激活功能的CpG DNA通过静电相互作用自组装形成纳米药物，不仅可以提升其在肿瘤部位的富集，还可以发挥免疫/化疗协同作用治疗肾癌。
本发明与现有技术相比，其技术进步是显著的。本发明的药物构建方法简便，CpGDNA与benzo[4]helicenium是通过静电相互作用自组装形成纳米颗粒，不需要任何试剂的参与，形成纳米颗粒后也无需反复清洗来清除试剂，这有助于提高药物的荷载效率。而且CpG DNA赋予了CpG-benzo[4]helicenium纳米药物免疫刺激性，而benzo[4]helicenium不仅能刺激免疫细胞产生免疫炎症因子，而且能直接杀伤RCC细胞，因此CpG-benzo[4]helicenium纳米药物能发挥免疫/化疗双重疗效，从而高效杀伤RCC。以CpG-benzo[4]helicenium为代表的DNA-螺烯纳米药物可以作为一种新型免疫/化疗药物研发的方向，有助于解决RCC易于复发的问题。本发明的CpG-benzo[4]helicenium可以用于制备静脉注射剂型药剂，治疗肾癌。
附图说明
图1为benzo[4]helicenium的构建流程图；CHCl3为有机溶剂氯仿。
图2为本发明中实施例2中肾上皮细胞株HK2和肾癌细胞株ACHN、OS-RC-2、786-0、Caki-1用0～100μg mL-1浓度的benzo[4]helicenium处理24h后的细胞活力结果图；横坐标benzo[4]helicenium(μg mL-1)代表苯并[4]螺烯溴化盐的浓度，纵坐标Cell Viability代表活细胞百分比，IC50(μg mL-1)代表半抑制浓度，HK2为肾上皮细胞株，ACHN、OS-RC-2、786-0和Caki-1为肾癌细胞株。
图3为本发明中实施例3中CpG-benzo[4]helicenium纳米药物的透射电子显微镜图。
图4为本发明中实施例4中肾癌细胞株ACHN用10μg mL-1浓度的CpG-benzo[4]helicenium处理24h后的细胞活力结果图；横坐标Control代表对照组，CpG-benzo[4]helicenium代表实验组，纵坐标Cell Viability代表活细胞百分比，ACHN为肾癌细胞株，****代表p<0.0001，与对照组相比有显著统计学差异。
图5和图6分别为本发明中实施例5中骨髓巨噬细胞用CpG DNA、benzo[4]helicenium和CpG-benzo[4]helicenium处理后的IL-6和TNF-α的mRNA表达水平；横坐标Control代表对照组，CpG DNA、benzo[4]helicenium和CpG-benzo[4]helicenium代表3个实验组，纵坐标mRNA expression level代表基因的mRNA表达水平，IL-6代表白介素-6，TNF-α代表肿瘤坏死因子-α，****代表p<0.0001，与对照组相比有显著统计学差异。
具体实施方式
下面结合具体实施例对本发明进行详细说明。以下实施例将有助于本领域的技术人员进一步理解本发明，但不以任何形式限制本发明。所述方法如无特别说明均为常规方法。所述原材料如无特别说明均能从公开商业途径获得。应当指出的是，对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。
实施例1
如图1所示，本实施例涉及制备benzo[4]helicenium的方法，具体包括如下步骤：
将140mg苯并[4]螺烯(0.5mmol)悬浮在与3mL CHCl3和1.5mL 1-溴丙烷的混合溶液中，将混合物在65℃下搅拌48小时。随着反应进行，产生黄色固体，然后通过真空除去溶剂。将固体溶解在CH2Cl2中并通过加入Et2O沉淀，离心收集固体。将溶解-沉淀-离心过程重复三次，然后将固体保持在真空烘箱中以除去残留的溶剂。得到产物黄色固体135mg，产率67％。
该化合物表征结果下：
1H NMR(500MHz,DMSO-d6,295K):δ10.04(d,J＝8.6Hz,1H),9.58(d,J＝5.8Hz,1H),9.35(d,J＝8.8Hz,1H),9.10(d,J＝8.2Hz,1H),8.86(d,J＝9.4Hz,1H),8.77(d,J＝9.2Hz,1H),8.73(d,J＝9.4Hz,1H),8.48(d,J＝8.8Hz,1H),8.35(dd,J＝8.6,5.8Hz,1H),8.26(d,J＝9.2Hz,1H),8.21(d,J＝7.7Hz,1H),7.90–7.81(m,2H),5.19(t,J＝7.6Hz,2H),2.10(h,J＝7.3Hz,2H),1.05(t,J＝7.3Hz,3H).
13C NMR(125MHz,DMSO-d6,295K):δ146.66,145.89,139.47,137.17,131.57,131.27,130.38,129.54,128.60,128.32,128.08,127.72,127.59,127.09,126.92,126.18,125.66,125.11,124.08,122.01,116.84.
实施例2
本实施例涉及检测benzo[4]helicenium对肾癌细胞株的选择性杀伤效果，具体包括如下步骤：
取对数生长期的肾癌细胞株ACHN、OS-RC-2、786-0、Caki-1和肾上皮细胞株HK2，接种于96孔培养板(1×104个/孔)。在37℃、CO2体积分数为5％的细胞培养箱中培养过夜，待细胞贴壁后，加入0～100μg mL-1浓度的benzo[4]helicenium培养24h，每个浓度设置6个复孔，继续培养细胞24h后每孔加入10μl CCK-8工作液，置于培养箱中继续孵育2h。在酶联免疫检测仪上检测450nm波长处各孔的O.D值，计算细胞的存活率。细胞存活率(％)＝(实验组O.D值-空白对照组O.D值)/(对照组O.D值-空白对照组O.D值)×100％。
实验结果如图2所示，benzo[4]helicenium对肾上皮细胞株的半抑制浓度(IC50)明显高于肾癌细胞株，表明benzo[4]helicenium能选择性杀伤肾癌细胞株。
实施例3
本实施例涉及CpG-benzo[4]helicenium的构建及表征，具体包括如下步骤：
称取CpG DNA和benzo[4]helicenium，将25μL 100μM的CpG与500μL 100μM的benzo[4]helicenium在25℃下反应24小时。
实验结果如图3所示，CpG和benzo[4]helicenium在摩尔比1:20下能形成明显的纳米颗粒。
实施例4
本实施例涉及检测CpG-benzo[4]helicenium对肾癌细胞株的杀伤效果，具体包括如下步骤：
取对数生长期的肾癌细胞株ACHN，接种于96孔培养板(1×104个/孔)。在37℃、CO2体积分数为5％的细胞培养箱中培养过夜，待细胞贴壁后，加入10μg mL-1浓度的CpG-benzo[4]helicenium培养24h，每个浓度设置6个复孔，继续培养细胞24h后每孔加入10μl CCK-8工作液，置于培养箱中继续孵育2h。在酶联免疫检测仪上检测450nm波长处各孔的O.D值，计算细胞的存活率。细胞存活率(％)＝(实验组O.D值-空白对照组O.D值)/(对照组O.D值-空白对照组O.D值)×100％。
实验结果如图4所示，CpG-benzo[4]helicenium能明显杀伤肾癌细胞株ACHN。
实施例5
本实施例涉及检测CpG-benzo[4]helicenium对骨髓巨噬细胞的免疫刺激效果，具体包括如下步骤：
从BALB/c小鼠获得骨髓细胞，并用巨噬细胞集落刺激因子(MCSF)诱导分化成巨噬细胞，将CpG DNA、benzo[4]helicenium、CpG-benzo[4]helicenium纳米药物分别加入到巨噬细胞中，共培养24小时后收集细胞，通过RNA提取试剂盒提取细胞的总RNA，然后用逆转录试剂盒将RNA逆转录为cDNA，再进行实时荧光定量PCR检测IL-6和TNF-α的表达水平。
实验结果如图5和图6所示，benzo[4]helicenium和CpG-benzo[4]helicenium处理组IL-6和TNF-α的表达水平明显强于对照组和CpG DNA处理组，表明benzo[4]helicenium和CpG-benzo[4]helicenium均能对骨髓巨噬细胞产生免疫刺激，且CpG-benzo[4]helicenium略强于benzo[4]helicenium。
Claims (3)
1.一种化合物，其特征在于，其结构式如下所示：
。
2.一种DNA-螺烯纳米药物的制备方法，其特征在于包括如下步骤：
1）按照物料比称取苯并[4]螺烯和1-溴丙烷，苯并[4]螺烯和1-溴丙烷的摩尔比为1:10，加入有机溶剂中溶解，在65℃反应48小时即得到苯并[4]螺烯溴化盐；所述的苯并[4]螺烯溴化盐的结构式为：；
2）按照物料比称取含有非甲基化的胞嘧啶磷酸鸟嘌呤二核苷酸的寡聚脱氧核苷酸和苯并[4]螺烯溴化盐，含有非甲基化的胞嘧啶磷酸鸟嘌呤二核苷酸的寡聚脱氧核苷酸和苯并[4]螺烯溴化盐的摩尔比为1：20，在25℃反应24小时即得到DNA-螺烯纳米药物。
3.采用权利要求2所述的方法制备的DNA-螺烯纳米药物在制备用于治疗肾细胞癌的药物中的用途。
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