CN105477630B - 一种同时实现促进树突细胞迁移至淋巴结和多模式成像的方法 - Google Patents
一种同时实现促进树突细胞迁移至淋巴结和多模式成像的方法 Download PDFInfo
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Abstract
本发明公开了一种同时实现促进树突细胞迁移至淋巴结和多模式成像的方法，由成熟树突细胞(mDCs)携带磁性荧光纳米颗粒在外加磁场下实现。所述磁性荧光纳米颗粒是由油酸酯‑磁性氧化铁颗粒、两种磷脂、近红外探针和一种具有脂质结合能力的抗原融合肽通过自组装有机结合形成。本发明能有效提高DC活体迁移至淋巴组织的效率，促进DC摄取抗原肽后的肿瘤防治功效，以及可以进行活体多模式成像检测。
Description
技术领域
本发明属于生物科学领域，特别涉及一种促进树突细胞迁移至淋巴组织，并能对该迁移过程进行多模式成像监测的方法。
背景技术
DC疫苗是通过采用病人自体的单核细胞在体外培养诱导生成DC，然后负载相应的肿瘤抗原并刺激成熟而获得。若要高效地发挥其治疗作用，成熟的DC(mature DC，mDC)必须迁移至病人的淋巴结组织并与幼稚T细胞相互作用。已有大量研究证据表明，DC所产生的体内免疫反应与携带相关肿瘤抗原的成熟DC迁移至次级淋巴组织的效率成正相关。虽然近期的临床预实验显示DC疫苗具有良好的治疗效果，但是仅有不到2％的注射进入体内的DC到达次级淋巴组织。因此，若要提升DC疫苗治疗功效，其中关键的一步在于如何提高DC在体的迁移效率。目前的研究主要集中在通过探索DC在体迁移的分子机制来解决DC迁移效率低的问题。Fontecha等人发现采用肿瘤坏死因子(TNF)或白细胞介素(IL-1α)等炎症因子预免疫小鼠，可以增强其淋巴管内皮细胞对于趋化因子CCL21的表达，从而引导DC细胞更高效地迁移。Weber等人于2013年1月在Science上发表文章，指出通过构建模型证实了DC在体迁移主要取决于淋巴管内皮细胞表达的CCL21的浓度。阐明DC的迁移机制在一定程度上可以指导今后的DC疫苗设计，但是采用炎症因子预刺激存在以下问题：1)DC迁移率仍较低(＜3％)；2)在DC接种量较高时(＞2×106)，预刺激基本不起作用；3)高剂量的炎症因子在体给药存在一定的不良反应；4)预刺激可以提高体内CCL21的浓度梯度水平，但无法实现对DC迁移的直接控制。因此，迫切需要开发更加高效、安全和可控的技术手段来提高DC的活体迁移效率。
磁力牵引是借用磁体异极相吸的原理而产生的一种方法，目前在生物医学领域已展现出良好的应用前景。Susan P.Foy合成了一种装载氧化铁的纳米颗粒(magneticnanoparticle，MNP)经小鼠尾静脉给药后，利用外加磁场提高了MNP在肿瘤组织中的蓄积。Alain Luciani将摄取MNP的Huh7肝癌细胞经脾脏注射到小鼠体内，发现利用磁力牵引可以显著性地提高Huh7在肝脏外周血管周围的数量。目前还未有报道利用磁力牵引来提高DC的活体迁移效率。目前的研究已表明磁力牵引具有特异性好、安全和可控等优点，因此有望解决DC活体迁移效率低的难题。
另外，DC疫苗的优化要求建立非创伤性的成像手段来动态地监测DC迁移至淋巴组织的过程，从而实现在治疗过程中快速评价DC疫苗的效果。将这些造影剂标记DC主要是通过纳米运输载体实现，包括量子点、磁性纳米颗粒和聚合物纳米材料等。其中，采用纳米载体携带多种造影剂(尤其是近红外和MRI探针)进行多模式成像可以兼顾成像检测灵敏度和空间分辨率。为了满足以上需求，纳米颗粒的设计必须满足以下要求：1)生物相容性好；2)能被DC高效地摄取；3)能同时包裹肿瘤抗原、近红外和磁性MRI探针；4)在应用于肿瘤治疗时，携带的肿瘤抗原能被优先递送到DC的胞浆中而不是内体/溶酶体中。
发明内容
本发明的目的是为解决上述问题而提供了一种促进树突细胞迁移至淋巴组织，并能对该迁移过程进行多模式成像监测的方法，利用该方法能有效提高DC的肿瘤防治功效。
本发明所采用的技术方案是：
一种同时实现促进树突细胞迁移至淋巴结和多模式成像的方法，所述方法由成熟树突细胞(mDCs)携带磁性荧光纳米颗粒在外加磁场下实现。
进一步地，所述磁性荧光纳米颗粒是由油酸酯-磁性氧化铁颗粒、两种磷脂、近红外探针和一种具有脂质结合能力的抗原融合肽通过自组装有机结合形成。
进一步地，所述的磷脂为DMPC和MHPC。
进一步地，所述的近红外探针为ICG分子。
进一步地，所述的具有脂质结合能力的抗原融合肽是由α螺旋多肽、连接序列和抗原肽以共价键的形式串连而成。
进一步地，所述α螺旋多肽的氨基酸序列为FAEKFKEAVKDYFAKFWD，所述连接序列的氨基酸序列为GSG，所述抗原肽的氨基酸序列为KVPRNQDWL、KTWGQYWQV、SIINFEKL或ISQAVHAAHAEINEAGR。
进一步地，所述外加磁场为磁铁产生或者恒定外加磁场。
本发明提供的一种同时实现促进树突细胞迁移至淋巴结和多模式成像的方法，具有以下优点：
(1)生物相容性好：本发明方法中所选用的材料，磷脂、ICG、氧化铁颗粒和抗原融合肽，这些原材料都已用于临床或临床试验，具有良好的生物相容性。
(2)操作工艺简单，有望应用于临床。
(3)能大幅提高DC的活体迁移效率。
(4)能同时进行DC的活体多模示踪和肿瘤免疫治疗：mDCs摄取磁性荧光纳米颗粒后，其活体分布可以采用近红外光学成像和磁共振成像进行示踪；由于磁性荧光纳米颗粒含有抗原融合肽，因此可以用于肿瘤的预防和治疗。
(5)采用磁力牵引能显著增强mDCs的抗癌功效。
(6)功能可扩展：该系统除了直接应用于DC疫苗外，还可以在其表面装载免疫佐剂直接作为纳米疫苗，同时实现在体免疫细胞的激活、抗原高效递送、磁力牵引和多模成像。
附图说明
图1为所述磁性荧光纳米颗粒的透射电子显微镜成像图像和动态激光光散射(DLS)图。
图2为磁性荧光纳米颗粒的磁共振信号和荧光信号成像图。
图3为mDCs摄取磁性荧光纳米颗粒的流式细胞图。
图4为摄取磁性荧光纳米颗粒对DC生存率的影响。
图5为摄取磁性荧光纳米颗粒的mDCs的荧光成像图。
图6为摄取磁性荧光纳米颗粒的mDCs的普鲁士蓝染色图。
图7为磁力牵引对DC迁移能力的影响研究。
图8为小鼠足垫回输mDCs在24后的多模式成像(近红外和磁共振)图。
图9为mDCs后的近红外荧光成像图以及磁力牵引实施24小时后的荧光成像图。
图10为实施磁力牵引前后小鼠腿弯淋巴结荧光成像对比图。
图11为流式检测含磁性荧光纳米颗粒的DC在促进特异性淋巴细胞增值方面的能力。
图12为在有无磁场作用下DC抗肿瘤疫苗功效的对比研究。
具体实施方式
本发明实施例提供一种同时实现促进树突细胞迁移至淋巴结和多模式成像的方法。具体步骤如下：
1)mDCs的制备：DC的制备来源性骨髓提取分离，mDCs的制备采用常规培养方法获得，可以携带肿瘤抗原，也可以不含肿瘤抗原。DC可以是来源于骨髓提取，也可以是DC2.4细胞系。
2)mDCs携带多肽抗原和磁性物质：mDCs与一种磁性荧光纳米颗粒在37℃下孵育6小时即可完成。
所述磁性荧光纳米颗粒是由油酸酯-氧化铁颗粒、两种磷脂、近红外探针和一种具有脂质结合能力的抗原融合肽通过自组装有机结合形成。
所述油酸酯-氧化铁颗粒是油酸修饰氧化铁后的纳米颗粒，该物质已经投入商业化生产(见http://www.nanoeast.net/cxnmklfzxs.html，油酸修饰的四氧化三铁磁性纳米颗粒(OA@Fe3O4，共沉淀法或高温热解法)
【产品名称】
油酸修饰的四氧化三铁磁性纳米颗粒
【英文名称】
OA coated Fe3O4 nanoparticles
【成分】
油酸修饰的Fe3O4磁性纳米颗粒
【特点】
油溶性，可分散在环己烷、氯仿、四氢呋喃等溶剂中.
【用途】
用于掺杂水包油纳米乳、修饰纳米脂质体、构建磁性纳米药物等。
【技术参数】
TEM尺寸：约10nm
饱和磁化强度：约75emu/g Fe。
优选地，所述磷脂为DMPC(1,2-dimyristoyl-sn-glycero-3-phosphocholine)和MHPC(lipids 1-myristoyl-2-hydroxy-sn-glycero-3-phosphocholine)。
优选地，所述近红外探针为ICG分子(吲哚氰绿/心脏绿)。
所述具有脂质结合能力的抗原融合肽，是由α螺旋多肽、连接序列和抗原肽以共价键的形式串连而成，所述α螺旋多肽的氨基酸序列为FAEKFKEAVKDYFAKFWD，所述连接序列的氨基酸序列为GSG，所述抗原肽的氨基酸序列为KVPRNQDWL、KTWGQYWQV、ISQAVHAAHAEINEAGR或其它抗原多肽序列。
3)磁场作用下促进DCs活体迁移至淋巴组织：本发明使用圆形的磁铁N35(40×10mm；Gauss,11.7-12.1k)用来引导含磁性荧光纳米颗粒的mDCs的迁移，但不局限于磁铁发出的磁场，恒定的磁场也可以用于上述应用。
下面结合附图和实施方式对本发明做进一步详细的说明。
实施例1
组成磁性荧光纳米颗粒的抗原融合多肽，该肽是由α螺旋多肽(R4F)、连接序列和抗原肽(AP)以共价键的形式串连而成。其中α螺旋多肽的氨基酸序列为：FAEKFKEAVKDYFAKFWD，连接序列的氨基酸序列为GSG，抗原肽的氨基酸序列为：KVPRNQDWL。该多肽的氨基酸序列为序列表中SEQ ID NO.1所述。
制备磁性荧光纳米颗粒，其步骤为：
磁性荧光纳米颗粒的制备：称取DMPC和羟丙基甲基纤维素醚(MHPC)各5mg分别置于EP管中，然后在EP管中加入200μl的三氯甲烷进行溶解，将溶解好的DPMC和MHPC转移至体积为约为400μl含氧化铁的氯仿溶液中(10mg/mL)，再按氯仿：甲醇＝20:1的比例加入甲醇进行混合。取约6ml的超纯水放入一个小的玻璃瓶中，加热至80℃左右，然后将混合好的溶液逐滴慢慢加入玻璃瓶中，边搅拌边加入，此过程持续约30分钟，加完溶液之后，继续搅拌30min，使甲醇和三氯甲烷完全蒸发掉。然后，停止加热，室温下冷却，将其转移至10ml离心管中，在4℃和3000rpm条件下离心10分钟，去掉底部沉淀，收集上清液，此过程重复三次。分别称取荧光染料ICG 5mg和多肽R4F-AP 8mg，其中ICG用500μl的ddH2O进行溶解，R4F-AP用2ml的ddH2O溶解。然后把溶解好的ICG、R4F-AP和FAM溶液加入到上面溶液中，避光放置4℃冰箱中过夜。将此混合物在4℃、3000rpm条件下浓缩至约2ml时，加满PBS再进行浓缩3次，最后纳米颗粒的体积约为3ml左右。在无菌操作台上用无菌过滤器过滤，避光保存在4℃冰箱中。
磁性荧光纳米颗粒的纳米粒径图参见图1。图1(左)为磁性荧光纳米颗粒的电镜图，显示该材料为近球形且分散性较好。图1(右)为磁性荧光纳米颗粒的水合粒径图，显示为30nm左右。图2为磁性荧光纳米颗粒的近红外和磁共振成像图，表明该材料具备多模成像的特性。这些实验结果表明，磁性荧光纳米颗粒是一种单分散性、粒径均一且具有多模成像特性的纳米颗粒。
实施例2
实施例1中制备得到的磁性荧光纳米颗粒对成熟DC(mDC)的标记能力见图3。图3显示磁性荧光纳米颗粒能对mDC进行高效荧光标记，且在50μg/mL时对mDC的形态没有大的影响(20％以内)，而实验选择的浓度为20μg/mL。图4显示了不同浓度的磁性荧光纳米颗粒与mDC共孵育后的细胞存活实验，结果表明在50μg/mL的浓度范围内，磁性荧光纳米颗粒对mDCs的生长没有显著性影响。摄取磁性荧光纳米颗粒的mDCs具有荧光特性(图5)，荧光强度随着细胞个数的增加而升高。同时，普鲁士蓝染色证明，摄取磁性荧光纳米颗粒的mDCs胞内含有大量的磁性氧化铁物质(图6)。这些结果表面磁性荧光纳米颗粒能赋予DC荧光和磁感应特性。
实施例3
实施例1中制备得到的磁性荧光纳米颗粒在有无磁场作用下促进DCs迁移能力的比较参见图7。该步骤如下：
1)消化DC2.4，接种细胞至六孔板中，每孔1×106个细胞，体积为2ml；2)在接种细胞的第二天加60μl的FMP-AP至其中一个孔中，另一个加60μl PBS作为对照，孵育时间为6小时；3)收集DC2.4，计数之后，调整细胞浓度至2×106个/mL；4)将1×106个上述细胞放入25cm2的细胞培养瓶中培养(体积为10ml)，并在培养瓶的一侧放置一块磁铁作用24小时；5)用剪刀剪下培养瓶的两侧(培养基覆盖的区域)，采用正置显微镜进行成像。
结果发现，在培养瓶测试中，放置磁铁24小时后迁移至磁铁端的壁上的DC细胞数量是对照组的9倍(图7)。这些实验结果均证实，采用磁力牵引能极大地增强DC的迁移能力。
实施例4
实施例1中制备得到的磁性荧光纳米颗粒在有无磁场作用下的迁移至淋巴组织的效率比较。
1)C57BL/6小鼠的预处理：
取5只C57BL/6小鼠，分为两组，其中实验组3只，对照组2只。在实验组小鼠右侧足垫处分别注射25μl(1ng/μL)TNF-α进行预刺激；在对照组的小鼠左侧和右侧足垫各注射25μl的(1ng/μL)TNF-α进行预刺激。
2)EGFP小鼠来源的BMDC的提取：
提取EGFP小鼠来源的BMDC，培养至第六天的时候，加入含有1μg/mL的LPS新鲜培养基10ml培养24小时,使其变为成熟DC。然后，收集细胞，经两遍清洗后，从新悬浮细胞至dish中，体积为5ml。在每个dish中加入100μl的FMP-AP共孵育6小时。
3)将DC由小鼠足垫处注射：
6小时后，收集DC，用无菌PBS洗两遍，计数并调整其浓度至2.4×107cell/mL。用异氟烷吸入麻醉剂使小鼠处于麻醉状态时，在实验组小鼠的右侧足注射体积50μl的DC(1.2×106个)。在对照组的2只小鼠的右侧足垫注射50μl的DC(1.2×106个)，并在其左侧足垫注射50μl PBS，最后分别在实验组小鼠的右侧大腿处套上一个磁铁，放置24小时。对照组不做处理。
4)采用近红外整体成像和MRI成像对腿弯淋巴结进行活体成像。
结果显示，含有磁性荧光纳米颗粒的mDCs经足垫注射，在经过磁场作用后，在腿弯淋巴结显示出较强的荧光信号和磁共振信号(图8)。通过荧光强度计算表明，在磁场作用下mDCs的迁移效率是未加磁场作用的16倍(图9)。在此实验中，进一步使用EGFP-mDCs代替没有荧光的mDCs，磁力牵引后将腿弯淋巴组织进行分离，继而进行活体显微成像，结果显示，磁力牵引组具有较强的EGFP荧光信号，表明该组织中具有大量的EGFP-mDCs存在，该结果与图8的活体成像结果基本一致(图10)。
实施例5
实施例1中制备得到的磁性荧光纳米颗粒在研究比较了mDC摄取单独抗原多肽gp100和装载gp100磁性荧光纳米颗粒运载(FMP-gp100)后的抗原提呈能力，主要是通过比较两者激活抗原特异性CD8+T细胞的能力。该实验主要从gp100多肽免疫的小鼠腹股沟淋巴结和脾脏中分离获取CD8+T细胞。如图11所示，T细胞的增值随着CD8+T/DC比例的增加而增加，并且FMP-gp100-DC较gp100-DC具有明显增强的促T细胞增值能力。
实施例6
比较研究了在有无磁场作用下，摄取磁性荧光纳米颗粒的mDCs的抗癌能力。具体方法如下
1)待DC成熟之后，收集mDC，并再用PBS洗两遍。计数之后，按照每个dish中加入5ml培养基、3×106个DC进行培养。然后在其中的三个dish中加入100μl PBS作为空白对照组进行孵育，其余的dish分别加入100μl的FMP-AP(铁的浓度为780μg/mL)与DC共孵育6小时。最后用PBS调节细胞浓度至2.4×107个/mL。
2)从小鼠足垫处注射体积为50μl，细胞数量为1.2×106个DC：其中5只小鼠注射PBS对照的DC，另外10只小鼠均注射孵育过FMP-AP的DC，且在其中5只鼠的大腿处放置一磁铁作用24小时，接着取下磁铁。
3)一个星期后，再以相同的方式免疫小鼠一次。
4)接种E.G7-OVA肿瘤细胞：在小鼠第二次免疫的一个星期后，在免疫过的一侧接近腿弯淋巴结处皮下接种数量为5×106、体积为100μl的E.G-7-OVA肿瘤细胞。
5)肿瘤体积的测量：待肿瘤体积长到肉眼可见的时候开始测量肿瘤的长、宽、高。然后每隔一天测量一次，测量周期为2周，2周后处死小鼠。肿瘤体积按照下面的公式进行计算：体积＝长×宽2/2。
如图12所示，空载DC的肿瘤体积随着天数的增加而逐渐增大，FMP-gp100-DC免疫的小鼠的肿瘤体积一直保持在较低的状态，说明了负载OVA多肽的DC能在体激活机体的免疫响应。值得注意的是，磁场作用能显著增强FMP-gp100-DC的抗癌功效，并且有2只小鼠(每组共5只小鼠)在第14天时肿瘤已完全消失，从而首次在活体上证实了通过磁力作用可以增强DC的肿瘤预防功效。
实施例7
实施例1中的抗原肽序列不只局限于KVPRNQDWL，其它抗原多肽也适用于该体系，比如KTWGQYWQV、SIINFEKL和ISQAVHAAHAEINEAGR。该促进树突细胞摄取抗原肽的多肽的氨基酸序列为序列表中SEQ ID NO.2、SEQ ID NO.3和SEQ ID NO.4所述。
以上实施例仅用以说明本发明的技术方案而非限制，尽管参照较佳实施例对本发明进行了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的精神和范围，其均应涵盖在本发明的权利要求范围当中。
<110> 华中科技大学
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Claims (3)
1.一种成熟树突细胞携带磁性荧光纳米颗粒在制备抗癌药物中的应用，所述应用由所述成熟树突细胞携带磁性荧光纳米颗粒在外加磁场下实现，其特征在于，所述磁性荧光纳米颗粒是由油酸酯-磁性氧化铁颗粒、两种磷脂、近红外探针和一种具有脂质结合能力的抗原融合肽通过自组装有机结合形成，所述磷脂为DMPC和MHPC，
所述的近红外探针为ICG分子；
所述的具有脂质结合能力的抗原融合肽是由α螺旋多肽、连接序列和抗原肽以共价键的形式串连而成；
所述α螺旋多肽的氨基酸序列为FAEKFKEAVKDYFAKFWD，所述连接序列的氨基酸序列为GSG，所述抗原肽的氨基酸序列为KVPRNQDWL、KTWGQYWQV、SIINFEKL或ISQAVHAAHAEINEAGR。
2.根据权利要求1所述的应用，其特征在于，所述DMPC和MHPC分别为5mg。
3.根据权利要求1所述的应用，其特征在于，所述外加磁场为磁铁产生或者恒定外加磁场。
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