CN114376965A - 促进肿瘤血管正常化和放疗增敏的一氧化氮水凝胶及其制备方法 - Google Patents
促进肿瘤血管正常化和放疗增敏的一氧化氮水凝胶及其制备方法 Download PDFInfo
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Abstract
本发明提供了一种促进肿瘤血管正常化和放疗增敏的一氧化氮水凝胶及其制备方法，所述的水凝胶包括一段能形成水凝胶的多肽、β‑半乳糖保护的NO供体分子，所述的成胶多肽和β‑半乳糖保护的NO供体分子共价连接而成。首先，本发明合成成本低，所用到的原料为人体每天所必需的氨基酸，生物相容性好；其次，该水凝胶作为NO储存库，用于按需连续输送NO，显著解决了NO分子半衰期短的问题；最重要的是，该水凝胶仅在β‑半乳糖苷酶(β‑Gal)催化下释放NO，释放量可通过酶浓度精确控制。
Description
技术领域
本发明属于肿瘤领域，尤其是涉及一种促进肿瘤血管正常化和放疗增敏的一氧化氮水凝胶及其制备方法。
背景技术
在癌症放射治疗中，肿瘤部位低氧环境常导致实体肿瘤产生辐射耐药。一氧化氮(NO)是一种与血管相关的具有多种生物学功能的重要气体分子，对缺氧肿瘤有多种作用。首先，NO是一种强效的低氧细胞辐射增敏剂，因为它与氧具有类似的电子亲和能力，可与损伤DNA自由基结合固定损伤。除这一直接作用外，NO还可以通过一种间接的方式降低肿瘤缺氧，也就是使肿瘤微环境中的血管系统正常化。肿瘤血管系统的正常化可以增强肿瘤的血液灌注，最终增加肿瘤细胞的供氧量，使肿瘤对放疗敏感，因此，向肿瘤传递NO成为逆转肿瘤对放疗抗性的一种很有希望的方法。
由于直接使用NO气体在体内存在问题，学术界开发了一些外源性NO供体材料作为放疗增敏剂，有直接在溶液中自发释放NO分子的材料，也有一些在pH、温度、光照等刺激下释放NO的材料。尽管在NO递送上取得了这些成功，但这些研究均没有实现临床应用，NO复杂的功能和机理仍然需要更精确的递送材料设计。研究证明，不同的NO用量会导致完全不同甚至相反的生物学功能。除此之外，NO释放治疗的时间也很重要。在放射致敏的情况下，使肿瘤血管恢复正常减少缺氧，需要浓度NO持续刺激。然而，在放射性照射后需要大量的NO，才能对癌细胞产生最大的DNA损伤和细胞毒性，同时如此大的剂量也可能对正常细胞造成损伤。目前研发的NO释放材料能够集中在较短的时间内大量释放NO，但不能持续低剂量释放NO。目前还缺乏一种能够在数量和持续时间上精确控制并持续释放NO的递送材料用于放疗增敏。
发明内容
有鉴于此，本发明旨在提出一种促进肿瘤血管正常化和放疗增敏的一氧化氮水凝胶及其制备方法，一方面解决自发释放NO的NO供体的治疗效果受到NO极短半衰期的限制，另一方面解决目前NO供体在数量和时间上都缺乏持续的控释的问题。
为达到上述目的，本发明的技术方案是这样实现的：
促进肿瘤血管正常化和放疗增敏的一氧化氮水凝胶，所述的水凝胶包括一段能形成水凝胶的成胶多肽、β-半乳糖保护的NO供体分子，所述的成胶多肽和β-半乳糖保护的NO供体分子共价连接而成。
本发明设计了一种超分子水凝胶作为NO储存库，用于按需连续输送NO。超分子水凝胶由半乳糖保护的NO供体自组装而成，仅在β-半乳糖苷酶(β-Gal)催化下释放NO，其机理为：β-Gal去除SupraNO的半乳糖基团，解放NO供体，随后释放两分子NO。释放量可通过酶浓度精确控制。
该设计基于以下几个方面:首先，据报道，β-Gal被人类卵巢癌、黑色素瘤等多种癌细胞过度表达。因此，在肿瘤内注射该水凝胶后，肿瘤环境中的β-Gal会持续触发NO释放，提供局部低剂量的持续NO释放。其次，放疗后立即静脉注射大量β-Gal可释放大量NO。总之，该水凝胶的设计能够控制NO在体内暴露的位置、浓度和持续时间，同时具有促进低氧肿瘤中血管正常化和放疗增敏的双重功能。
优选地，所述的成胶多肽为包括氨基酸序列GFFY、FFG的多肽、或其多肽的活性片段、类似物或衍生物；
优选地，所述的成胶多肽通式为R-GFFY、R-GFFYG、R-GFFYGG、R-GFFYGGG、R-FF、R-FFG、R-FFGG、R-FFGGG中的一种，其中，R代表H、乙酸(Ac-)、萘乙酸(Nap-)或9-芴甲氧羰基(Fmoc-)；
更优选地，所述的成胶多肽还包括炔基，所述炔基位于多肽的C端或其中一个氨基酸侧链上。
炔基能与NO供体分子的叠氮基团反应，包括端位炔烃，也可以是非端炔烃，炔烃只能位于多肽的C端或者某个氨基酸侧链上，不能在多肽主链的其他位置。
优选地，所述的GFFY中的FFY全部为L构型或全部为D构型，FFG中的FF全部为L构型或全部为D构型。
优选地，所述的β-半乳糖保护的NO供体分子的结构式为：
优选地，所述的水凝胶在β-半乳糖苷酶(β-Gal)的催化下释放NO。
优选地，在肿瘤内注射该水凝胶后，肿瘤环境中的β-半乳糖苷酶(β-Gal)会持续触发NO释放，提供局部低剂量的持续NO释放；
放疗后立即静脉注射β-半乳糖苷酶(β-Gal)可释放大量NO。
本发明的另一目的在于提供所述的水凝胶的制备方法，包括如下步骤：
S1：多肽固相合成方法合成成胶多肽；
S2：使用Click chemistry方法将β-半乳糖保护的NO供体分子连接到成胶多肽上。
本发明的再一目的在于提供所述的水凝胶在制备癌症放射治疗药物中的应用。
相对于现有技术，本发明所述的一氧化氮水凝胶具有以下有益效果：
(1)本发明合成成本低，所用到的原料为人体每天所必需的氨基酸，生物相容性好；
(2)该水凝胶作为NO储存库，用于按需连续输送NO，显著解决了NO分子半衰期短的问题；
(3)该超分子水凝胶仅在β-半乳糖苷酶(β-Gal)催化下释放NO，释放量可通过酶浓度精确控制。
所述制备方法与上述水凝胶相对于现有技术所具有的优势相同，在此不再赘述。
附图说明
构成本发明的一部分的附图用来提供对本发明的进一步理解，本发明的示意性实施例及其说明用于解释本发明，并不构成对本发明的不当限定。在附图中：
图1为水凝胶光学照片；
图2为NO凝胶内部纳米纤维电子显微镜图；
图3为成胶分子3的化学结构式和酶催化释放NO的原理图；
图4为NO凝胶(1mg/ml)在小鼠血浆中酶控释放NO测定图；
图5为NO凝胶(5mg/ml)在PBS缓冲液中酶控释放NO测定图；
图6为乏氧情况下，B16细胞在不同组别处理下放疗后克隆形成(存活率)统计图；
图7为NO凝胶与放疗联合治疗小鼠黑色素瘤后肿瘤体积统计图；
图8为NO水凝胶与放疗联合治疗小鼠黑色素瘤后肿瘤照片；
图9为NO凝胶促进肿瘤部位血管正常化(外周细胞覆盖比例)统计结果。
具体实施方式
除有定义外，以下实施例中所用的技术术语具有与本发明所属领域技术人员普遍理解的相同含义。以下实施例中所用的试验试剂，如无特殊说明，均为常规生化试剂；所述实验方法，如无特殊说明，均为常规方法。
下面结合实施例及附图来详细说明本发明。
实施例1
(1)化合物3的合成
具体步骤如下：
1)称取0.5mmol 2-cl-Trt树脂于固相合成器中，加入10mL的无水二氯甲烷(以下用DCM表示)，放置在摇床上摇晃5min，使2-Cl-Trt树脂充分溶胀；
2)用洗耳球把DCM从装有2-Cl-Trt树脂的固相合成器中压除干净；
3)将0.75mmolFmoc保护的氨基酸溶解在10mL的无水DCM里，加入0.75mmol的DIEPA，然后转移到上述固相合成器中，再补加0.75mmol的DIEPA，在室温下反应1h；
4)封闭：用洗耳球除去固相合成器中的反应液，然后用10mL无水DCM洗涤，每次1min，共洗5次，加入配好的体积比为无水DCM∶DIEPA∶甲醇＝17∶1∶2的溶液20mL，在室温下反应10min；
5)用洗耳球除去固相合成器中的反应液，先用无水DCM洗涤，每次DCM用量10mL、洗涤时间1min，共洗5次，再用N,N-二甲基甲酰胺(以下用DMF表示)洗涤，每次DMF用量10mL、洗涤时间1min，共洗5次，加入10mL含体积百分比为20％的哌啶的DMF，反应25min，再用10mL含体积百分比为20％的哌啶的DMF反应5min，然后用DMF洗涤，每次DMF用量10mL、洗涤时间1min，共洗5次，进行下一步反应；
6)加入的第二个Fmoc保护的氨基酸1mmol、HBTU 1.5mmol、DIEPA2mmol和10mlDMF，把配好的溶液加入到上述固相合成器中，反应2h；
7)重复步骤5)和6)的方法依次加入需要的氨基酸或封端基团(2-萘乙酸)；然后用DMF洗涤5遍，二氯甲烷洗5遍，进行下步反应；
8)按95％TFA，2.5％TIS，2.5％H2O体积百分比组成的溶液10mL加入到上述固相合成器中，反应半小时(或者TFA与DCM的体积比为1∶99，配制成体积百分比浓度为1％的TFA溶液，取该TFA溶液每次3mL加入到上述固相合成器中，共加十次，每次反应时间为1min)，把产物从2-cl-Trt树脂上切下，真空浓缩，除去溶剂，得到粗品，之后用HPLC分离提纯得到Nap-GFFYG。
9)将1.0mmol(651.7mg)的Nap-GFFYG、1.1mmol(416.9mg)的HBTU和2.2mmol(284.4mg)的DIPEA溶液溶解在2mlDMF中，然后将1.1mmol(60.5mg)的丙炔胺加入到上述溶液中。室温(25℃)搅拌过夜后，反应液直接送入高效液相色谱纯化，得到化合物1。
(2)化合物3的合成
将过量的化合物1(0.2mmol,137.76mg)溶于10ml的dd-H2O中，与化合物2(0.1mmol,37.7mg)加入5ml的dd-H2O溶液中。把混合物搅拌一下，得到一种清澈的溶液。随后加入1mL含CuSO4(12.5mg,0.05mmol)和抗坏血酸钠(19.8mg,0.1mmol)的水溶液，引发Click反应。在氮气气氛下，室温(25℃)搅拌24小时，产物3通过HPLC分离提纯得到。
(3)NO水凝胶的形成
称取5.0mg纯化后的化合物3，置于2毫升的玻璃瓶中，加入1mL PBS溶液(pH＝7.4)，用碳酸钠溶液将其pH值调节至7.4，加热至沸腾使化合物完全溶解，冷却到室温之后即得透明可倒置水凝胶，水凝胶的光学照片见图1。图1为使用化合物3在小瓶底部制备水凝胶后，倒置小瓶，该水凝胶在小瓶倒置之后不会从小瓶底部流下，说明其具有一定的粘弹性，与流体不同。将水凝胶涂附于铜网用透射电子显微镜观察(见图2)，可以看到直径为10nm左右的长纤维，表明化合物自组装形成有序的纳米结构。
(4)NO水凝胶在酶催化下释放NO
将50uL含有不同浓度β-gal(0，0.2和2U/mL)的小鼠血浆，或者50uL含有不同浓度β-gal(0，0.3，3和30U/L)的PBS缓冲液，加入50uL水凝胶(化合物3浓度为1mg/mL，溶液为PBS)的顶部。通过Griess试剂盒测量如图4和5中所示时间点从水凝胶释放的NO量。如图4所示，水凝胶在没有酶的血浆中不释放NO，在含有0.2U/mLβ-gal的血浆中经过30个小时累计释放了22nmol的NO，在含有2U/mL的血浆中经过30小时累计释放32nmol NO。如图5所示，水凝胶在没有酶的PBS缓冲液中不释放NO，在含有0.3，3和30U/Lβ-gal的PBS中经10个小时分别累计释放了5，7和11nmol NO。
(5)NO水凝胶加酶提高乏氧情况下肿瘤细胞对放疗的敏感性
将黑色素瘤细胞系B16细胞在乏氧条件下培养24小时，之后分别加入正常培养基，含有2U/mLβ-gal的培养基，含有1mg/mL水凝胶和2U/mLβ-gal的的培养基处理24小时，之后进行γ射线照射。由图6所示，不加酶与加酶组在2，4，6Gy剂量的射线照射下存活率均相同，说明β-gal酶对B16肿瘤细胞本身没有毒性。肿瘤细胞被水凝胶加酶处理后，在2，4，6Gy剂量的射线照射下存活率均低于不处理组，说明水凝胶加酶释放NO提高了放疗对肿瘤细胞的杀伤。
(6)NO水凝胶促进肿瘤放疗增敏
在C57小鼠皮下注射B16肿瘤细胞，当B16肿瘤体积达到约100mm3时，将荷瘤小鼠分为6组：PBS，NO水凝胶，NO水凝胶加酶，PBS加放疗，NO水凝胶加放疗，NO水凝胶加酶加放疗。对于NO水凝胶，NO水凝胶加放疗组，将50μL浓度为5mg/mL的NO水凝胶直接注射到肿瘤中，没有添加额外的β-Gal。对于NO水凝胶加酶，NO水凝胶加酶加放疗组，在接受放疗之前半小时，在肿瘤内注射相同量的NO水凝胶，然后静脉注射β-Gal(共4U溶于50μL PBS溶液中)。同样的治疗每隔2天一次，持续三次，第11天到达实验终点，在这过程中检测小鼠肿瘤体积。图7和图8结果表明NO水凝胶与NO水凝胶加酶两组处理方案，均可以提高肿瘤对放疗的敏感度，加强放疗对肿瘤的抑制作用，其中NO水凝胶加放疗组在所有组中显示出最佳的疗效，平均肿瘤体积最小。
第11天实验终点处死小鼠。取出各组肿瘤拍照并测量，结果显示NO水凝胶加酶加放疗组中的一只小鼠有一个非常小的肿瘤，两只小鼠的肿瘤已经消失，表明NO水凝胶加酶加放疗组抗肿瘤效果最好。
(7)NO水凝胶促进肿瘤部位血管正常化
将肿瘤组织进行冰冻切片免疫荧光染色，用血管标志物CD31和外周细胞标志物NG2共染色，CD31于NG2双阳性染色部位作为正常化血管标志，统计5张荧光照片上的正常血管密度并做图(见图9)。图9结果表明NO水凝胶加酶加放疗组的血管正常化程度最高。同时，无论加酶与否，NO水凝胶处理组均显示比PBS组更高的血管正常化程度。说明NO水凝胶可以促进肿瘤部位血管正常化。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
Claims (8)
1.促进肿瘤血管正常化和放疗增敏的一氧化氮水凝胶，其特征在于：所述的水凝胶包括一段能形成水凝胶的成胶多肽、β-半乳糖保护的NO供体分子，所述的成胶多肽和β-半乳糖保护的NO供体分子共价连接而成。
2.根据权利要求1所述的水凝胶，其特征在于：所述的成胶多肽为包括氨基酸序列GFFY、FFG的多肽、或其多肽的活性片段、类似物或衍生物；
优选地，所述的成胶多肽通式为R-GFFY、R-GFFYG、R-GFFYGG、R-GFFYGGG、R-FF、R-FFG、R-FFGG、R-FFGGG中的一种，其中，R代表H、乙酸(Ac-)、萘乙酸(Nap-)或9-芴甲氧羰基(Fmoc-)；
更优选地，所述的成胶多肽还包括炔基，所述炔基位于多肽的C端或其中一个氨基酸侧链上。
3.根据权利要求2所述的水凝胶，其特征在于：所述的GFFY中的FFY全部为L构型或全部为D构型，FFG中的FF全部为L构型或全部为D构型。
4.根据权利要求1所述的水凝胶，其特征在于：所述的β-半乳糖保护的NO供体分子的结构式为：
5.根据权利要求1所述的水凝胶，其特征在于：所述的水凝胶在β-半乳糖苷酶(β-Gal)的催化下释放NO。
6.根据权利要求5所述的水凝胶，其特征在于：在肿瘤内注射该水凝胶后，肿瘤环境中的β-半乳糖苷酶(β-Gal)会持续触发NO释放，提供局部低剂量的持续NO释放；
放疗后立即静脉注射β-半乳糖苷酶(β-Gal)可释放大量NO。
7.权利要求1所述的水凝胶的制备方法，其特征在于：包括如下步骤：
S1：多肽固相合成方法合成成胶多肽；
S2：使用Click chemistry方法将β-半乳糖保护的NO供体分子连接到成胶多肽上。
8.权利要求1-7任意一项所述的水凝胶在制备癌症放射治疗药物中的应用。
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