CN114146166B - 一种多功能诊疗一体化纳米复合探针及其制备方法和应用 - Google Patents
一种多功能诊疗一体化纳米复合探针及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种多功能诊疗一体化纳米复合探针及其制备方法和应用，该纳米复合探针将阿霉素(DOX)和19F信号源全氟‑15‑冠‑5‑醚(PFCE)通过疏水‑疏水相互作用与赖氨酸铜和油胺共修饰的聚琥珀酰亚胺两亲高分子(PSILys‑Cu‑Lys/OAm)进行组装，再通过聚赖氨酸(PLL)调节静电作用吸附葡萄糖氧化酶(GOx)，进而成功构建了多功能纳米复合体系DFPPG。本发明提供的纳米复合探针DFPPG在100μg/mL时，对4T1细胞具有78％的抑制率，相比于其他对照组具有最优的抗癌活性；且小鼠活体肿瘤部位19F磁共振成像信号在数小时内呈现高对比度影像并在24h后有明显减弱的现象，表明探针具有优良的肿瘤成像示踪性能和适宜的体内代谢行为，具有良好生物相容性。
Description
技术领域
本发明属于纳米材料技术领域，特别是涉及一种多功能诊疗一体化纳米复合探针及其制备方法和应用。
背景技术
肿瘤生长难抑制、易转移等特性严重影响了传统肿瘤治疗方式的疗效。单一疗法通常难以取得良好的抗肿瘤效果，目前的临床研究趋势已经逐渐转向多模式联合治疗的策略，以实现协同增强疗效。成像诊断对于肿瘤治疗来说也是非常重要的辅助手段，将MRI与多模式联合治疗两种功能复合于同一体系中，使构建的多功能纳米探针同时具备肿瘤成像及治疗的双重功能。在治疗过程中，无需分别使用这两种功能的材料，避免大剂量带来不必要的毒性，以及防止不同性质药物混合造成的干扰，这对于提高肿瘤治疗效率同时降低毒副作用具有非常重要的意义。
化学动力学治疗(CDT)作为一种新兴的癌症治疗手段，无需外界能量激发即可通过催化H2O2产生毒性·OH杀死癌细胞。但是肿瘤微环境中低表达的H2O2限制了CDT抗癌效果。DOX作为化学治疗的药物，在血液循环过程中会造成很大的副作用。
发明内容
本发明提供了一种多功能诊疗一体化纳米复合探针及其制备方法和应用。
本发明所采用的技术方案是：
一种多功能诊疗一体化纳米复合探针，利用阿霉素和19F信号源全氟-15-冠-5-醚通过疏水-疏水相互作用与赖氨酸铜和油胺共修饰的聚琥珀酰亚胺两亲高分子进行组装，再通过聚赖氨酸调节静电作用吸附葡萄糖氧化酶，构建纳米复合体系。
其中，纳米复合探针是粒径为157.20±50nm的颗粒。
其中，纳米复合探针的溶液的浓度为400μg/mL至4000μg/mL。
一种多功能诊疗一体化纳米复合探针的制备方法，包括以下步骤：
步骤一：赖氨酸铜配合物的制备：在40mL超纯水中通过超声溶解0.04mol的L-赖氨酸盐酸盐，待溶液至澄清透明后将准确称取的10mmol碱式碳酸铜，在60℃水浴条件下缓慢加入，溶液逐渐变为蓝色，在650rpm条件下搅拌2h；反应结束后趁热过滤，取得深蓝色澄清滤液；将过滤后的溶液通过减压蒸馏除去部分水分，直至瓶壁出现蓝色固体析出迹象，停止操作静置一段时间；待瓶中固体大量析出后，将其转移至布氏漏斗中抽滤，多次用乙醇洗涤滤饼，得到蓝色固体产物Lys-Cu-Lys并将其烘干后干燥保存；
步骤二：赖氨酸铜和油胺共修饰聚琥珀酰亚胺高分子的制备方法：配制A液：称取1.5g PSI放入100mL圆底烧瓶中，用量筒量取28mL DMF加入烧瓶；配制B液：称取1.2g先前合成的Lys-Cu-Lys，并通过超声溶解于12mL水中；搅拌升温至A液澄清，随后快速加入B液，在90℃条件下反应5h；反应完毕后静置冷却，待产物析出，用丙酮离心洗涤数次，弃去上清除去水分，真空干燥除去残留丙酮和水，得到固体产物PSILys-Cu-Lys；
与上述反应条件相似，将干燥后的PSILys-Cu-Lys超声分散在32mL DMF中，在90℃条件下搅拌均匀后再用移液枪加入2.3mL OAm，然后将混合物在100℃，650rpm/min搅拌5h；反应完毕后冷却至室温，并向反应液中加入大量甲醇沉淀产物；9000rpm，8min离心处理之后，蒸干多余的甲醇液体；向沉淀中加入二氯甲烷溶解，定量PSILys-Cu-Lys/OAm(300mg/mL)储备液；
步骤三：DOX·HCl疏水改性：准确称取20mgDOX·HCl并与20μL三乙胺一起加入三氯甲烷中室温搅拌12h，通过酸碱反应去除盐酸盐；为了去除未反应的DOX·HCl，反应完毕后加入适量H2O进行萃取纯化；静置取下层氯仿相，于4℃冰箱中储存备用(2mg/mL)；
步骤四：DOX/F@PSILys-Cu-Lys/OAm(DFP)纳米探针的制备：将60mgPSILys-Cu-Lys/OAm，0.5mg DOX(疏水)，10μL PFCE加入二氯甲烷中并定容至1mL，然后将二氯甲烷溶液充分振荡后迅速加入到10mL NaOH水溶液(0.5mM)中，在冰浴条件下间断超声乳化6min，得到粉色均一乳液；将乳液在每分钟200rpm条件下于室温中搅拌4h，除去易挥发的二氯甲烷；待溶液变为澄清的橙红色溶液后，离心15min得到纳米探针DOX/F@PSILys-Cu-Lys/OAm(DFP)并分散在1mL水中；
步骤五：DOX/F@PSILys-Cu-Lys/OAm@PLL-GOx(DFPPG)的制备：将制得的DFP和4mg正电高分子聚合物聚赖氨酸先后加入水中室温搅拌4h，离心收集包裹PLL的纳米颗粒DOX/F@PSILys-Cu-Lys/OAm@PLL(DFPP)；负电位GOx通过静电吸附包裹于纳米颗粒表面，通过超声将正电纳米颗粒均匀分散在水中，在搅拌条件下加入2mM GOx水溶液，并在室温下温和搅拌2h；反应完毕后，先用3,500rpm离心3min去除大颗粒，随后再用15,000rpm的转速离心收取沉淀，得到纳米复合探针DOX/F@PSILys-Cu-Lys/OAm@PLL-GOx(DFPPG)；随后将所得产物分散于1mL水中，避光储存在4℃冰箱中备用。
一种多功能诊疗一体化纳米复合探针的应用，Lys-Cu-Lys作为CDT催化单元在酸性条件下催化H2O2产生毒性羟基自由基(·OH)，杀死肿瘤细胞；而GOx可以原位催化消耗葡萄糖实现肿瘤的饥饿疗法，同时产生大量葡萄糖酸和H2O2，葡萄糖酸导致更酸性的肿瘤环境和原位生成更多的H2O2进而有效增强CDT疗效；DOX作为广谱抗癌药，在pH较低的肿瘤微环境中释放，产生化学治疗作用。
本发明的优点如下：
Lys-Cu-Lys作为类芬顿反应催化剂，催化H2O2生成·OH，实现CDT。同时释放DOX进行化学治疗。本发明提供的纳米复合探针DFPPG在100μg/mL时，对4T1细胞具有78％的抑制率，相比于其他对照组具有最优的抗癌活性。在4T1荷瘤小鼠的肿瘤治疗中，注射纳米复合探针组20天后肿瘤均得到良好的抑制。通过对小鼠的器官切片分析表明在治疗过程中DFPPG不会对小鼠的健康产生不良影响，具有良好的生物相容性。通过对体外/体内MR成像性能的研究结果分析，纳米复合探针DFPPG在体外及肿瘤部位均能够观察到明显的19F MRI信号。且根据24h后肿瘤部位信号有所减弱的现象，看出探针在体内部分代谢，具有良好生物相容性。本发明提供的纳米复合探针DFPPG有望作为一种兼具19F磁共振成像和治疗的多功能复合纳米平台，在纳米生物医学领域具有潜在的开发应用前景。
附图说明
图1是本发明的实施例中DFPPG的透射电镜及粒径分布实验数据图；
图2是本发明的实施例中不同条件下的类芬顿反应实验数据图；
图3是本发明的实施例中不同条件下催化glu实验数据图；
图4是本发明的实施例中体外药物释放能力评估实验数据图；
图5是本发明的实施例中探针的癌细胞抑制性能实验数据图；
图6是本发明的实施例中不同治疗方式处理后4T1荷瘤小鼠的肿瘤体积及解剖实验数据图；
图7是本发明的实施例中体外19F MRI实验数据图；
图8是本发明的实施例中体内19F MRI实验数据图。
具体实施方式
下面将采用具体实施例来进一步描述本发明。然而，本发明并不受这些具体实施例的限制。
实施例1
纳米复合探针DFPPG的制备
1)赖氨酸铜配合物(Lys-Cu-Lys)的制备：
在40mL超纯水中通过超声溶解0.04mol的L-赖氨酸盐酸盐(L-Lys)，待溶液至澄清透明后将准确称取的10mmol碱式碳酸铜，在60℃水浴条件下缓慢加入，溶液逐渐变为蓝色，在650rpm条件下搅拌2h。反应结束后趁热过滤，取得深蓝色澄清滤液。将过滤后的溶液通过减压蒸馏除去部分水分，直至瓶壁出现蓝色固体析出迹象，停止操作静置一段时间。待瓶中固体大量析出后，将其转移至布氏漏斗中抽滤，多次用乙醇洗涤滤饼，得到蓝色固体产物Lys-Cu-Lys并将其烘干后干燥保存。
2)赖氨酸铜和油胺共修饰聚琥珀酰亚胺高分子(PSILys-Cu-Lys/OAm)的制备：
配制A液：称取1.5g PSI放入100mL圆底烧瓶中，用量筒量取28mL DMF加入烧瓶。配制B液：称取1.2g先前合成的Lys-Cu-Lys，并通过超声溶解于12mL水中。搅拌升温至A液澄清，随后快速加入B液，在90℃条件下反应5h。反应完毕后静置冷却，待产物析出，用丙酮离心洗涤数次，弃去上清除去水分，真空干燥除去残留丙酮和水，得到固体产物PSILys-Cu-Lys。
与上述反应条件相似，将干燥后的PSILys-Cu-Lys超声分散在32mL DMF中，在90℃条件下搅拌均匀后再用移液枪加入2.3mL OAm，然后将混合物在100℃，650rpm/min搅拌5h。反应完毕后冷却至室温，并向反应液中加入大量甲醇沉淀产物。9000rpm，8min离心处理之后，蒸干多余的甲醇液体。向沉淀中加入二氯甲烷溶解，定量PSILys-Cu-Lys/OAm(300mg/mL)储备液。
3)DOX/F@PSILys-Cu-Lys/OAm@PLL-GOx(DFPPG)纳米复合探针的制备：
DOX·HCl疏水改性：准确称取20mg DOX·HCl并与20μL三乙胺(TEA)一起加入三氯甲烷中室温搅拌12h，通过酸碱反应去除盐酸盐。为了去除未反应的DOX·HCl，反应完毕后加入适量H2O进行萃取纯化。静置取下层氯仿相，于4℃冰箱中储存备用(2mg/mL)。
DOX/F@PSILys-Cu-Lys/OAm(DFP)纳米探针的制备：将60mg PSILys-Cu-Lys/OAm，0.5mg DOX(疏水)，10μL PFCE加入二氯甲烷中并定容至1mL，然后将二氯甲烷溶液充分振荡后迅速加入到10mL NaOH水溶液(0.5mM)中，在冰浴条件下间断超声乳化6min，得到粉色均一乳液。将乳液在每分钟200rpm条件下于室温中搅拌4h，除去易挥发的二氯甲烷。待溶液变为澄清的橙红色溶液后，离心15min得到纳米探针DOX/F@PSILys-Cu-Lys/OAm(DFP)并分散在1mL水中。
DOX/F@PSILys-Cu-Lys/OAm@PLL-GOx(DFPPG)的制备：将制得的DFP和4mg正电高分子聚合物聚赖氨酸(PLL)先后加入水中室温搅拌4h，离心收集包裹PLL的纳米颗粒DOX/F@PSILys-Cu-Lys/OAm@PLL(DFPP)。负电位GOx通过静电吸附包裹于纳米颗粒表面，通过超声将正电纳米颗粒均匀分散在水中，在搅拌条件下加入2mM GOx水溶液，并在室温下温和搅拌2h。反应完毕后，先用3,500rpm离心3min去除大颗粒，随后再用15,000rpm的转速离心收取沉淀，得到纳米复合探针DOX/F@PSILys-Cu-Lys/OAm@PLL-GOx(DFPPG)。随后将所得产物分散于1mL水中，避光储存在4℃冰箱中备用。
实施例2
体外·OH产生能力评估
催化H2O2产生·OH能力评估：用TMB作为检测·OH的探针。将TMB溶解在丙酮溶液中(4mg/mL)备用。将5mM H2O2，0.5mg/mL DFPPG和1mM TMB分别加入不同pH值(7.4和5.0)的PBS缓冲溶液中。孵育2h后用紫外-可见分光光度计测试，得到每组的吸收曲线。
催化葡萄糖产生·OH活性评估：将1mM TMB加入有5mg/mL葡萄糖和0.5mg/mLDFPPG的水溶液中，并将不加葡萄糖的DFPPG水溶液作为对照组，混合均匀后于37℃培养箱中孵育，观察溶液颜色随时间的变化，并用吸收光谱记录。
实施例3
体外酶活性评估
配制不同浓度的β-D-葡萄糖溶液(0-2mM)，分别加入DFPPG和探针PTO，摇匀静置，测其在375nm处的吸光度。随后对GOx的催化能力进行检测，配制两组5mM葡萄糖溶液(每组n＝3)，分别加入DFPPG和游离GOx。每间隔30min用pH计测量一次体系的pH值并且用UV-Vis吸收光谱记录其在375nm处的吸光度。将所得吸光值代入标准曲线，得到H2O2浓度。
实施例4
体外药物释放能力评估
准备6个容量为100mL的烧杯并分成两组，分别加入70mL新鲜配制的两种不同pH值(7.4和5.0)的磷酸盐缓冲溶液(0.1M)，探究纳米探针DFPPG中负载的DOX的体外释放能力。为此，取1mL待测样品转移至透析袋中，将透析袋放入烧杯中，在37℃培养箱中避光匀速搅拌(350rpm)。在特定的时间间隔(如30min，1h，2h，5h，8h，10h，12h，24h，48h等)，从透析液中取出1mL用荧光发射光谱记录，同时补加等量对应相同pH的新鲜PBS溶液。通过使用先前建立的DOX标准曲线，将某一时间的591nm处荧光强度代入，计算得出该时间所取透析液中DOX的含量，并与总透析液体积相乘算得纳米复合物在该时间释放DOX的含量。根据计算不同时间点药物的释放量，绘制出DOX释放量与时间的曲线。
实施例5
探针的癌细胞抑制性能评估
将贴壁的4T1细胞利用胰蛋白酶消化液将其脱壁，并吹打为均匀分散的状态，用DMEM将其稀释至所需浓度。向96孔板中加入密度为每孔5×104个4T1细胞(小鼠乳腺癌细胞)，体积约为每孔100μL，对肿瘤生长抑制性进行评估。在培养24h后，分别在每个孔中加入20μL不同浓度的DFPPG(0-100μg/mL)，与细胞培养基混合均匀，在37℃，5％CO2，95％相对湿度条件下孵育24h和48h。随后在避光条件下向每个孔中加入20μL新配制的MTT(5mg/mL)溶液，并在相同条件下继续孵育4h。MTT法的检测原理是活细胞内的酶能使MTT还原为不溶于水的紫色沉淀物甲臜，且被二甲基亚砜(DMSO)溶解后在492nm处有吸收。因此吸出培养液后向每孔加入100μL DMSO溶解甲臜。最后，避光振荡10min使其充分溶解，并通过酶标仪检测每个孔的吸光数值。未加DFPPG用作空白对照组，以PBS组的吸光度为标准，计算出细胞活性并绘制出不同浓度对应细胞活性图。
实施例6
荷瘤小鼠的化学/化学动力学/饥饿疗法协同治疗
选择乳腺癌荷瘤小鼠模型来评估探针的肿瘤抑制效果，培养小鼠约7-10天，通过游标卡尺测量肿瘤尺寸约为150-200mm3后，随机将小鼠分成5组(n＝5)。每组每次分别原位皮下注射体积约100μL的不同材料，分别为a)PBS，b)FP，c)DFP，d)FPPG和e)DFPPG剂量约为2mg/kg。间隔两天注射一次，间隔一天用游标卡尺测量肿瘤尺寸，计算肿瘤体积，治疗周期为20天。
实施例7
体外/体内19F MRI
体外19F MRI：用PBS(1×，pH＝7.4)配制约300μL不同浓度的探针(2.75-27.5mg/mL)装入PCR管中，使用MRI系统进行扫描采样，获得不同浓度下的MRI图像，读取19F NMR的信噪比(SNR)数值并建立其与材料浓度之间的线性关系。
体内19F MRI：饲养荷瘤小鼠至肿瘤体积约300mm3，原位注射100μL DFPPG NPs(27.5mg/mL)至小鼠肿瘤中。在不同的时间间隔(30min，4h，12h)将小鼠用异氟烷气体麻醉后进行19F MRI实验，获取不同时间的MRI图像。
综上所述，本发明通过纳米材料的可修饰性进行组装，将类芬顿反应催化单元赖氨酸铜(Lys-Cu-Lys)，抗癌药物阿霉素(DOX)和葡萄糖氧化酶(GOx)以及成像信号源全氟-15-冠-5-醚(PFCE)组装起来得到多组分的纳米复合材料DOX/F@PSILys-Cu-Lys/OAm@PLL-GOx(DFPPG)。此纳米材料在肿瘤微环境中，Lys-Cu-Lys在酸性环境中催化H2O2产生毒性高的·OH，诱导细胞死亡。GOx氧化葡萄糖产生葡萄糖酸和H2O2，降低了周围的pH并提高细胞内H2O2水平，起到增强CDT效果。增强的酸性环境导致DOX释放，产生化学治疗效果。包覆的PFCE分子赋予探针19F MRI能力。该纳米探针通过将化学动力学治疗、化学治疗和饥饿疗法及19FMRI集成于一体，实现多功能诊疗一体化。
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。
Claims (3)
1.一种多功能诊疗一体化纳米复合探针，其特征在于：利用阿霉素（DOX）和19F信号源全氟-15-冠-5-醚（PFCE）通过疏水-疏水相互作用与赖氨酸铜（Lys-Cu-Lys）和油胺（OAm）共修饰的聚琥珀酰亚胺两亲高分子进行组装，再通过聚赖氨酸（PLL）调节静电作用吸附葡萄糖氧化酶（GOx）；其中，纳米复合探针的制备方法包括以下步骤：
步骤一：赖氨酸铜配合物的制备：在40mL超纯水中通过超声溶解0.04mol的L-赖氨酸盐酸盐，待溶液至澄清透明后，将准确称取的10mmol碱式碳酸铜，在60℃水浴条件下缓慢加入，溶液逐渐变为蓝色，在650rpm条件下搅拌2h；反应结束后趁热过滤，取得深蓝色澄清滤液；将过滤后的溶液通过减压蒸馏除去部分水分，直至瓶壁出现蓝色固体析出迹象，停止操作静置一段时间；待瓶中固体大量析出后，将其转移至布氏漏斗中抽滤，多次用乙醇洗涤滤饼，得到蓝色固体产物Lys-Cu-Lys并将其烘干后干燥保存；
步骤二：赖氨酸铜和油胺共修饰的聚琥珀酰亚胺两亲高分子的制备：配制A液：称取1.5g PSI放入100mL圆底烧瓶中，用量筒量取28mL DMF加入烧瓶；配制B液：称取1.2g步骤一合成的Lys-Cu-Lys，并通过超声溶解于12mL水中；搅拌升温至A液澄清，随后快速加入B液，在90℃条件下反应5h；反应完毕后静置冷却，待产物析出，用丙酮离心洗涤数次，弃去上清除去水分，真空干燥除去残留丙酮和水，得到固体产物PSILys-Cu-Lys；
将干燥后的PSILys-Cu-Lys超声分散在32mL DMF中，在90℃条件下搅拌均匀后再用移液枪加入2.3mL OAm，然后将混合物在100℃，650rpm搅拌5h；反应完毕后冷却至室温，并向反应液中加入大量甲醇沉淀产物；9000rpm，8min离心处理之后，蒸干多余的甲醇液体；向沉淀中加入二氯甲烷溶解，定量PSILys-Cu-Lys/OAm储备液为300mg/mL；
步骤三：DOX·HCl疏水改性：准确称取20mgDOX·HCl并与20μL三乙胺一起加入三氯甲烷中室温搅拌12h，通过酸碱反应去除盐酸盐；为了去除未反应的DOX·HCl，反应完毕后加入适量H2O进行萃取纯化；静置取下层氯仿相浓度为2mg/mL，于4℃冰箱中储存备用；
步骤四：DOX/F@PSILys-Cu-Lys/OAm的制备：将60mgPSILys-Cu-Lys/OAm，0.5mg疏水 DOX，10μLPFCE加入二氯甲烷中并定容至1mL，然后将二氯甲烷溶液充分振荡后迅速加入到10mL0.5mM NaOH水溶液中，在冰浴条件下间断超声乳化6min，得到粉色均一乳液；将乳液在200rpm条件下于室温中搅拌4h，除去易挥发的二氯甲烷；待溶液变为澄清的橙红色溶液后，离心15min得到纳米探针DOX/F@PSILys-Cu-Lys/OAm并分散在1mL水中；
步骤五：DOX/F@PSILys-Cu-Lys/OAm@PLL-GOx的制备：将制得的DOX/F@PSILys-Cu-Lys/OAm和4mg正电高分子聚合物聚赖氨酸先后加入水中室温搅拌4h，离心收集包裹PLL的纳米颗粒DOX/F@PSILys-Cu-Lys/OAm@PLL；通过超声将正电纳米颗粒DOX/F@PSILys-Cu-Lys/OAm@PLL均匀分散在水中，在搅拌条件下加入2mM GOx水溶液，并在室温下温和搅拌2h；反应完毕后，先用3,500rpm离心3min去除大颗粒，随后再用15,000rpm的转速离心收取沉淀，得到纳米复合探针DOX/F@PSILys-Cu-Lys/OAm@PLL-GOx；随后将所得产物分散于1mL水中，避光储存在4℃冰箱中备用。
2.根据权利要求1所述的一种多功能诊疗一体化纳米复合探针，其特征在于：所述的纳米复合探针是粒径为157.20±50nm的颗粒。
3.根据权利要求1所述的一种多功能诊疗一体化纳米复合探针，其特征在于：所述的纳米复合探针的溶液的浓度为400μg/mL至4000μg/mL。
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