CN116602938A - 一种层状双金属氢氧化物纳米颗粒在制备射频消融增强剂中的应用 - Google Patents
一种层状双金属氢氧化物纳米颗粒在制备射频消融增强剂中的应用 Download PDFInfo
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Abstract
本发明公开了一种层状双金属氢氧化物纳米颗粒在制备射频消融增强剂中的应用，本发明首次将层状双金属氢氧化物纳米颗粒与射频消融联合使用，在RFA杀伤肿瘤后，LDHs进入肿瘤组织吸附抗原，原位形成疫苗，激发机体产生针对性免疫杀伤，达到抑制肿瘤的效果，有效提高了射频消融的疗效；同时，将免疫药物或质粒药物联合使用，利用LDHs多联药物的递送、药物缓慢释放，进一步刺激免疫细胞、质粒细胞产生IFN‑beta/TNF‑α/IL‑12等因子，促进免疫细胞/免疫系统的进一步活化，提高免疫杀伤效果，进一步提高RFA治疗效力、产生高效、持久的免疫治疗效果。
Description
(一)技术领域
本发明属于射频消融技术领域，特别是涉及一种层状双金属氢氧化物纳米颗粒在制备射频消融增强剂中的应用。
(二)背景技术
作为介入治疗中最主要的治疗手段，射频消融(RFA)治疗可以通过加热组织导致细胞死亡，从而达到缩小体内肿瘤、结节或其他类别生长物的个体大小。因此，RFA被广泛用于疾病的治疗中，涵盖良性和恶性肿瘤、以及其他例如腿部慢性静脉功能不全、慢性疼痛等。基于RFA在治疗肝脏癌症中良好的结果，RFA也于2000年逐步被应用到肺癌的临床治疗当中，并展现出良好的治疗效果[1]。有研究发现，RFA治疗不宜外科切除手术的Ⅰ期NSCLC病人，可以取得较好的远期治疗效果，将5年生存率及中位生存期提高至42％和59个月[2]。虽然，微创介入肺肿瘤RFA治疗具备创伤小、治疗效果良好及恢复快等优势，但是该治疗方案也存在潜在的风险。例如，癌变组织成分的异质性(例如肿瘤组织密度、纤维化或钙化等差异)及血液流动，将引起RFA在肿瘤消融过程中电导率和热导率的变化，进而影响对癌组织的加热效果，最终造成不完全消融。因此，在RFA治疗后约有31–42％的患者出现局部进展，并伴有后期的肿瘤复发[3]。虽然RFA的重复使用可以一定程度抑制首次治疗后的局部进展，但该方式仅针对部分类别的肿瘤，并且有限物理消融无法很好控制后期复发[4]。由此可见，单一RFA治疗无法满足根除肺癌的治疗效果。以射频消融(RFA)为主的新兴介入治疗，被大量应用到肺癌及肝癌的治疗当中，虽然RFA可以帮助癌症病人有效延长生存期，然而单一物理治疗无法有效避免术后的肿瘤复发。因此，临床迫切需求一种具备高效肿瘤消除效果的介入治疗手段。
为了进一步提高RFA的治疗效果，临床常用治疗手段包括化疗、靶向及免疫治疗都被尝试应用到RFA潜在联合治疗方案的研究中[5-7]。有研究发现，RFA与顺铂(CDDP)及紫杉醇(PTX)联合使用，可以有效抑制肿瘤、延长接种了VX2肺癌肿瘤兔子的中位生存时间至120天(RFA治疗组为30天)[5]。同时，临床实验也表明RFA具备可作为辅助治疗方案，提高全身化疗治疗肺转移癌症的效果(P<0.05)[6]。此外，靶向药物--安洛替尼(Anlotinib)与RFA的联用，可以显著地抑制小鼠模型中肺癌肿瘤的生长[7]。虽然，RFA与化疗、靶向治疗的联合使用可以提高针对肺癌的治疗效果，但化疗和靶向药物所带来的副作用将对病人的身心健康产生严重的影响[8]。
相对比，其余肿瘤特异性治疗例如免疫治疗，尤其是治疗性肿瘤疫苗可以提供更为高效、高性价比及安全的肿瘤治疗方式。其中，免疫激动剂的递送可以有效激树突状细胞(DCs)及其他抗原呈递细胞(APCs)(例如巨噬细胞、单核细胞、B细胞和NK细胞)、增强免疫反应，故被广泛用作肿瘤疫苗佐剂进行使用。然而，由于治疗药物短暂的体内持续时间，RFA的联用治疗仅能一定程度延缓肿瘤生长，无法介导高效、持久的肿瘤免疫杀伤效果。因而，具备缓释特性，且拥有附加佐剂特性的载体将有望解决这些问题。
伴随着新兴纳米技术的发展，纳米科技为癌症提供了一系列更具潜力的诊疗方式。对比传统药物，纳米材料拥有诸多的优势，可以通过药物递送(例如免疫激动剂等)同时兼具多种治疗功能，有效地解决传统肿瘤单一的治疗方式。其中，部分纳米材料，例如铝盐佐剂类型纳米材料，可以作为免疫佐剂形成高效的肿瘤治疗性疫苗，对肿瘤产生特异性的免疫治疗。因此，兼具递药及免疫刺激特性的纳米材料将是理想的RFA联用药物体系。
Reference:
[1]T.Hiraki,H.Gobara,T.Iguchi,H.Fujiwara,Y.Matsui,S.Kanazawa,Radiofrequency ablation for early-stage nonsmall cell lung cancer,Biomed ResInt,2014(2014)152087.
[2]M.Lanuti,A.Sharma,H.Willers,S.R.Digumarthy,D.J.Mathisen,J.A.Shepard,Radiofrequency ablation for stage I non-small cell lung cancer:management of locoregional recurrence,Ann Thorac Surg,93(2012)921-927；discussion 927-988.
[3]S.W.Kwan,K.E.Mortell,A.D.Talenfeld,M.C.Brunner,Thermal ablationmatches sublobar resection outcomes in older patients with early-stage non-small cell lung cancer,J Vasc Interv Radiol,25(2014)1-9e1.
[4]T.Hiraki,H.Mimura,H.Gobara,Y.Sano,H.Fujiwara,H.Date,S.Kanazawa,Repeat radiofrequency ablation for local progression of lung tumors:does ithave a role in local tumor control,J Journal of Vascular InterventionalRadiology,19(2008)706-711.
[5]A.Ueki,T.Okuma,S.Hamamoto,K.Kageyama,K.Murai,Y.Miki,Combinationtherapy involving radiofrequency ablation and targeted chemotherapy withbevacizumab plus paclitaxel and cisplatin in a rabbit VX2 lung tumor model,BMC Res Notes,11(2018)251.
[6]T.C.Chua,K.Thornbury,A.Saxena,W.Liauw,D.Glenn,J.Zhao,D.L.Morris,Radiofrequency ablation as an adjunct to systemic chemotherapy for colorectalpulmonary metastases,Cancer,116(2010)2106-2114.
[7]W.Zhou,Y.Gao,Y.Tong,Q.Wu,Y.Zhou,Y.Li,Anlotinib enhances theantitumor activity of radiofrequency ablation on lung squamous cellcarcinoma,Pharmacol Res,164(2021)105392.
[8]R.M.Navari,M.Aapro,Antiemetic prophylaxis for chemotherapy-inducednausea and vomiting,New England Journal of Medicine,374(2016)1356-1367.
(三)发明内容
本发明目的是提供一种层状双金属氢氧化物纳米颗粒在制备射频消融增强剂中的应用，在射频消融治疗后，施用层状双金属氢氧化物纳米颗粒和抗肿瘤药物，解决了射频消融效果有限、效应时长短及肿瘤易复发等难点问题。
本发明采用的技术方案是：
本发明提供一种层状双金属氢氧化物纳米颗粒溶液在制备射频消融(RFA)增强剂中的应用，将层状双金属氢氧化物纳米颗粒溶液与射频消融联合使用，提高射频消融效率；所述层状双金属氢氧化物纳米颗粒溶液是由氯化镁、氯化铝和氢氧化钠与水加热制成的镁铝纳米颗粒溶液。
优选的，所述层状双金属氢氧化物纳米颗粒溶液按如下方法制备：含0.2～0.3M氯化镁(优选0.2M)和0.1～0.15M氯化铝(优选0.1)的水溶液，在搅拌条件下，加入0.15～0.3M氢氧化钠水溶液(优选0.15M)，并在室温下200rpm搅拌10mins，离心(优选5000g离心5-10min)，沉淀用去离子水洗后(优选重复3次)，用去离子水重悬，加入水热合成反应釜中，于100℃反应10-20小时，反应产物即为层状双金属氢氧化物纳米颗粒溶液；所述氯化镁和氯化铝摩尔比例为1：0.33-0.75，优选1:0.33；所述氯化镁和氯化铝的水溶液与氢氧化钠水溶液体积比为1:1-10，优选1:4；所述氯化镁和氯化铝的水溶液与重悬用去离子水用量体积比为1:2-4，优选1:3。
优选的，所述增强剂中层状双金属氢氧化物纳米颗粒溶液的用量为66.5微克/100mm3肿瘤。
优选的，所述增强剂为层状双金属氢氧化物纳米颗粒溶液与抗肿瘤药物混合的纳米药物复合物，所述抗肿瘤药物包括环鸟苷酸-腺苷酸(cGAMP)、CpG寡脱氧核苷酸(CpG)或pIL-12质粒中的一种或两种的混合。
优选的，所述增强剂是将层状双金属氢氧化物纳米颗粒溶液与抗肿瘤药物混合，振荡1-2分钟后，在40Hz下超声5分钟后，继续震荡1-2分钟，形成纳米药物复合物；所述抗肿瘤药物与层状双金属氢氧化物纳米颗粒溶液中层状双金属氢氧化物纳米颗粒质量比为1:5-50，当抗肿瘤药物为环鸟苷酸-腺苷酸(cGAMP)时，抗肿瘤药物与层状双金属氢氧化物纳米颗粒质量比优选为1:15；当抗肿瘤药物为CpG寡脱氧核苷酸(CpG)时，抗肿瘤药物与层状双金属氢氧化物纳米颗粒质量比优选为1:7.5；当抗肿瘤药物为pIL-12质粒时，抗肿瘤药物与层状双金属氢氧化物纳米颗粒质量比优选为1:30；当抗肿瘤药物为CpG寡脱氧核苷酸和pIL-12质粒时，CpG寡脱氧核苷酸和pIL-12质粒与层状双金属氢氧化物纳米颗粒质量比为1:1:30。
本发明所述层状双金属氢氧化物纳米颗粒溶液在制备射频消融(RFA)增强剂中的应用方法是：将RFA治疗与所述层状双金属氢氧化物纳米颗粒溶液或层状双金属氢氧化物纳米颗粒溶液的抗肿瘤药物复合物联合使用，即在RFA治疗后，加入所述层状双金属氢氧化物纳米颗粒溶液或层状双金属氢氧化物纳米颗粒溶液的抗肿瘤药物复合物，对肿瘤RFA治疗后，起到促进治疗效果的作用。所述免疫药物用量以免疫药物用量为准。
与现有技术相比，本发明有益效果主要体现在：本发明首次将层状双金属氢氧化物纳米颗粒(即镁铝纳米颗粒)与射频消融联合使用，在RFA杀伤肿瘤后，LDHs进入肿瘤组织吸附抗原，原位形成疫苗，激发机体产生针对性免疫杀伤，达到抑制肿瘤的效果，有效提高了射频消融的疗效；同时，将免疫药物或质粒药物联合使用，利用LDHs多联药物的递送、药物缓慢释放，进一步刺激免疫细胞、质粒细胞产生IFN-beta/TNF-α/IL-12等因子，促进免疫细胞/免疫系统的进一步活化，提高免疫杀伤效果，进一步提高RFA治疗效力、产生高效、持久的免疫治疗效果。通过LDHs与RFA的联合治疗，诱导形成原位肿瘤治疗性疫苗的研发。
(四)附图说明
图1、LDHs投射电镜图(比例尺200nm)。
图2、LDHs的水合粒径检测图。
图3、cGAMP-LDHs吸附的朗格缪尔吸附曲线。
图4、CpG-LDHs琼脂糖凝胶吸附图。
图5、pIL-12-LDHs琼脂糖凝胶吸附图。
图6、肝癌模型肿瘤生长曲线。
图7、肝癌模型各组的肿瘤生长曲线。
图8、肺癌模型肿瘤生长曲线。
图9、肺癌模型各组的肿瘤生长曲线。
图10、肺癌模型各组小鼠生存率曲线图。
图11、肺癌模型各组小鼠体重变化曲线图。
(五)具体实施方式
下面结合具体实施例对本发明进行进一步描述，但本发明的保护范围并不仅限于此：
本发明实施例所述室温是指25-30℃。
实施例1、层状双金属氢氧化物纳米颗粒药物复合物
1、层状双金属氢氧化物纳米颗粒(LDHs)
配制10mL包含0.2M氯化镁和0.1M氯化铝的水溶液，在搅拌条件下，加入40mL的0.15M氢氧化钠水溶液，并在室温下200rpm搅拌10mins。5000g离心5-10min，沉淀用去离子水洗后，重复3次，用40ml去离子水重悬，加入水热合成反应釜中，于100℃反应10小时，反应产物即为层状双金属氢氧化物纳米颗粒(LDHs)溶液，简称LDHs溶液，干燥至恒重，获得层状双金属氢氧化物纳米颗粒，并进行透射电镜(TEM)和水合粒径(DLS)检测，结果见图1及图2所示，平均粒径110～120nm。同时，根据恒重质量获得层状双金属氢氧化物纳米颗粒溶液质量含量为10.0mg/mL。
2、LDHs药物复合物：
cGAMP-LDHs复合物：将步骤1制备的LDHs溶液滴加到装有1mg/mL的环鸟苷酸-腺苷酸(cGAMP)水溶液的ep管中，使得cGAMP与LDHs质量比分别为1:5、1:10、1：15、1:20，振荡1-2分钟后，在40Hz下超声5分钟后，继续震荡1-2分钟，形成cGAMP-LDHs复合物溶液。
CpG-LDHs复合物：将步骤1制备的LDHs溶液分别滴加到装有1mg/mL的CpG寡脱氧核苷酸(CpG)水溶液的ep管中，使得CpG与LDHs质量比分别为1：5、1:7.5、1:10、1:12.5、1:15，振荡1-2分钟后，在40Hz下超声5分钟后，继续震荡1-2分钟，形成不同比例的CpG-LDHs复合物溶液。
pIL12-LDHs复合物：将步骤1制备的LDHs溶液分别滴加到装有1mg/mL的pIL-12质粒(pIL-12)水溶液的ep管中，使得pIL12与LDHs质量比分别为1：10、1:20、1:30、1:40、1:50，振荡1-2分钟后，在40Hz下超声5分钟，后继续震荡1-2分钟，形成不同比例的pIL12-LDHs复合物溶液。
CpG-pIL12-LDHs复合物：将步骤1制备的LDHs溶液滴加到装有含1mg/mL的CpG和1mg/mL的pIL-12质粒水溶液的ep管中，使得CpG、pIL12与LDHs质量比为1:1:30，振荡1-2分钟后，在40Hz下超声5分钟，后继续震荡1-2分钟，形成CpG-pIL12-LDHs复合物溶液。
3、载药验证
将步骤2制备的各个复合物溶液在20000g离心20分钟，回收上清。
以步骤1制备的LDHs溶液为阳性对照，通过紫外吸收光谱检测cGAMP-LDHs复合物溶液的上清中的cGAMP(258nm)的吸光度，除以阳性对照组吸光度即游离的药物比例，并进一步计算吸附效率，拟合朗格缪尔吸附曲线，结果见图3所示，结果表明cGAMP和LDHs在比例达到1：15的时候能被完全吸附。
CpG-LDHs复合物溶液的上清通过琼脂糖电泳的条带强弱来判断吸附情况，分别以CpG、LDHs为对照，结果见图4所示，结果表明CpG和LDHs在比例达到1：7.5的时候能完全被吸附上。
pIL12-LDHs复合物溶液的上清通过琼脂糖电泳的条带强弱来判断吸附情况，以pIL12为对照，结果见图5所示，结果表明pIL-12和LDHs在比例达到1：30的时候能被完全吸附上。
实施例2、层状双金属氢氧化物纳米颗粒药物复合物与RFA联用
1、小鼠肿瘤模型的构建
肝癌模型小鼠：购买6-8周大小的C57/B6雌鼠，在小鼠的背部皮下注射200万的Hepa1-6细胞，经过10-14天，形成皮下肿瘤150～200mm2，构建得到肝癌(HepG1-6)模型小鼠。
肺癌模型小鼠：购买6-8周大小的C57/B6雌鼠，在小鼠的背部皮下注射200万的LLC细胞，经过10-14天，形成皮下肿瘤250～400mm3，构建得到肺癌(LLC)模型小鼠。
2、实验分组及给药
将上述步骤1构建的肿瘤模型小鼠分别分为无处理组(Control)、RFA组、RFA+LDHs药物复合物组(RFA+LDHs-cGAMP、RFA+CpG-LDHs、RFA+IL12-LDHs、RFA+CpG-IL12-LDHs)、RFA+LDHs组、RFA+药物组(RFA+CpG、RFA+IL12、RFA+CpG+IL12)。
对肝癌模型小鼠和肺癌模型小鼠，除无处理组外，其他各组分别运用迈德S-1500射频消融仪进行RFA(5w，20s)，RFA之后立即进行肿瘤内注射各组药物。
无处理组和RFA组分别注射20μL的PBS；
RFA+LDHs药物复合物组注射20μL的实施例1步骤2制备的LDHs药物复合物溶液，其中CpG用量为2.5μg/100mm3肿瘤(10μg每只小鼠)，cGAMP用量为2.5μg/100mm3肿瘤(10μg每只小鼠)，pIL-12质粒用量为2.5μg/100mm3肿瘤(10μg每只小鼠)，CpG-IL12中CpG用量为2.5μg/100mm3肿瘤(10μg每只小鼠)+pIL-12质粒用量为2.5μg/100mm3肿瘤(10μg每只小鼠)；
RFA+LDHs组注射20μL实施例1步骤1制备的LDHs溶液，用量为每只小鼠250μg。
RFA+药物组，CpG用量为2.5μg/100mm3肿瘤(10μg每只小鼠)g，cGAMP用量为2.5μg/100mm3肿瘤(10μg每只小鼠)，pIL-12质粒用量为2.5μg/100mm3肿瘤(10μg每只小鼠)，CpG-IL12中CpG用量为2.5μg/100mm3肿瘤(10μg每只小鼠)+pIL-12质粒用量为2.5μg/100mm3肿瘤(10μg每只小鼠)。
注射后，饲养14-21天，两天测量一次肿瘤的体积以及小鼠的重量。
肝癌模型的小鼠肿瘤生长曲线见图6和图7，结果表明RFA+LDHs相对于单独的RFA具有明显抑制肿瘤生长的效果，而RFA+cGAMP-LDHs复合物组相对于RFA+LDHs有明显的抑制肿瘤生长的效果，加强了RFA+LDHs对肿瘤的抑制效果。
肺癌模型的小鼠肿瘤生长曲线见图8和图9，结果表明，图8中单纯的LDHs与RFA联用就有良好的抑制肿瘤生长的功能。同时，图9中，RFA+LDHs药物复合物组相对于单独的RFA或者RFA+药物(CpG、pIL-12及CpG-pIL-12)具有明显优异的抑制肿瘤功能。
上述RFA组、RFA+LDHs药物复合物组(RFA+CpG-LDHs、RFA+IL12-LDHs、RFA+CpG-IL12-LDHs)、RFA+LDHs组、RFA+药物组(RFA+CpG、RFA+IL12、RFA+CpG+IL12)肺癌小鼠的生存率曲线图及小鼠体重图见图10、11所示，相对比，RFA治疗联合药物(免疫激活剂或是pIL-12)都无法很好的抑制肿瘤的生长，而RFA+LDHs能够明显抑制肿瘤细胞，肿瘤被完全抑制，且21天内没有复发，同时RFA+LDHs+药物的联合用药组(CpG、pIL-12及CpG-pIL-12)的肿瘤抑制效果明显优于RFA+LDHs，且肿瘤被完全抑制，21天内没有复发。
Claims (8)
1.一种层状双金属氢氧化物纳米颗粒溶液在制备射频消融增强剂中的应用。
2.如权利要求1所述的应用，其特征在于，所述层状双金属氢氧化物纳米颗粒溶液是由氯化镁、氯化铝和氢氧化钠与水加热制成的镁铝纳米颗粒溶液。
3.如权利要求2所述的应用，其特征在于，所述层状双金属氢氧化物纳米颗粒溶液按如下方法制备：含0.2～0.3M氯化镁和0.1～0.15M氯化铝的水溶液，在搅拌条件下，加入0.15～0.3M氢氧化钠水溶液，并在室温下200rpm搅拌10mins，离心，沉淀用去离子水洗后，用去离子水重悬，加入水热合成反应釜中，于100℃反应10-20小时，反应产物即为层状双金属氢氧化物纳米颗粒溶液。
4.如权利要求3所述的应用，其特征在于，所述氯化镁和氯化铝摩尔比例为1：0.33-0.75。
5.如权利要求3所述的应用，其特征在于，所述氯化镁和氯化铝的水溶液与氢氧化钠水溶液体积比为1:1-10；所述氯化镁和氯化铝的水溶液与重悬用去离子水用量体积比为1:2-4。
6.如权利要求3所述的应用，其特征在于，所述增强剂为层状双金属氢氧化物纳米颗粒溶液与抗肿瘤药物混合的纳米药物复合物，所述抗肿瘤药物包括环鸟苷酸-腺苷酸、CpG寡脱氧核苷酸或pIL-12质粒中的一种或两种的混合。
7.如权利要求6所述的应用，其特征在于，所述增强剂是将层状双金属氢氧化物纳米颗粒溶液与抗肿瘤药物混合，振荡1-2分钟后，在40Hz下超声5分钟后，继续震荡1-2分钟，形成纳米药物复合物；所述抗肿瘤药物与层状双金属氢氧化物纳米颗粒溶液中层状双金属氢氧化物纳米颗粒质量比为1:5-50。
8.如权利要求7所述的应用，其特征在于，当抗肿瘤药物为环鸟苷酸-腺苷酸时，抗肿瘤药物与层状双金属氢氧化物纳米颗粒质量比为1:15；当抗肿瘤药物为CpG寡脱氧核苷酸时，抗肿瘤药物与层状双金属氢氧化物纳米颗粒质量比为1:7.5；当抗肿瘤药物为pIL-12质粒时，抗肿瘤药物与层状双金属氢氧化物纳米颗粒质量比为1:30；当抗肿瘤药物为CpG寡脱氧核苷酸和pIL-12质粒时，CpG寡脱氧核苷酸和pIL-12质粒与层状双金属氢氧化物纳米颗粒质量比为1:1:30。
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