CN111249480B - 一种红细胞膜包覆的d-a型超小分子spn纳米材料及其制备方法与应用 - Google Patents
一种红细胞膜包覆的d-a型超小分子spn纳米材料及其制备方法与应用 Download PDFInfo
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Abstract
本发明提供了一种红细胞膜包覆的D‑A型超小分子SPN纳米材料及其制备方法与应用。设计合成的D‑A型有机半导体聚合物材料SP有着良好的光热转换效率，以及较低的生物毒性。通过共沉淀法合成的超小纳米颗粒SPN，在肿瘤部位有着较深的渗透深度以及可在体内快速代谢，避免了代谢过慢对机体造成的潜在毒副作用。通过在其表面包覆一层红细胞膜，既改善了纳米材料的生物相容性又可以延长纳米材料在体内的循环时间，进而提高纳米材料在肿瘤部位的富集。通过上述功能的有机整合，高效的实现肿瘤的光热治疗和光声成像一体化。
Description
技术领域
本发明涉及一种红细胞膜包覆的D-A型超小分子SPN纳米材料及其制备方法，以及其在光声成像和光热消融中的应用。
背景技术
有机光热转换材料作为一种重要的光热转换材料，具有很多独特的应用，其中最重要的就是用于光热治疗。700—1100nm的近红外区域是重要的“生物窗口”。生物体本身对这个区域的光吸收很少，因此这个区域的光具有非常好的生物组织穿透性。而有机光热转换材料对因其优异的光学性质以及好的生物相容性被广泛应用于生物成像、生物传感和治疗等，相比于无机纳米粒子(如金纳米棒)能够更有效地将吸收的光能高效的转换为热能，实现其在光声成像和光热消融领域的应用。
小分子有机化合物具有良好的体内生物相容性以及低毒性的特点，例如美国食品和药物管理局(FDA)批准的商业染料吲哚菁绿(ICG) 已经得到很好的临床应用，但容易在外界激光照射下导致特征的近红外吸收和荧光信号减弱或消失，且如图4c容易光漂白，难以改性修饰及靶向性差等缺点，吲哚菁绿等小分子光热试剂的进一步应用受到了的限制。研发具有高光热转换效率、生物相容性好、光稳定及靶向性的有机小分子光热诊断试剂显得越来越重要。共轭小分子具有化学结构明确、合成简便和易纯化的优点，因此共轭小分子纳米粒子有望具有较好的生物相容性和稳定的光热性质，CN108864106B公布合成了近红外二区有机小分子荧光探针，进一步通过纳米共沉淀方法制备得到具有良好水溶性、生物相容性及靶向性的纳米颗粒DPP-BT-R2， DSPE-mPEG2000修饰后的纳米颗粒DPP-BT-R2外层为亲水的PEG 链，该纳米颗粒具有优良的近红外二区荧光成像及光声成像能力、光动力及光热转换性能，可以用于近红外二区荧光成像、光声成像指导下的肿瘤靶向光热与光动力联合治疗，从而显著提高治疗效果，且基于光动力治疗(PDT)及光热治疗(PTT)，可以预防降低对正常组织的毒副作用及肿瘤耐药性；其成像效果优异、毒副作用小、疗效显著，作为新型成像探针及肿瘤靶向联合治疗试剂具有较好的临床应用前景。尽管如此，该纳米探针以小分子发光基团组成，如范曲立在论文“All-in-One Phototheranostics:Single LaserTriggers NIR-II Fluorescence/Photoacoustic Imaging Guided Photothermal/Photodynamic /Chemo Combination Therapy.”的第3页及图2d中记载的“激光功率密度增加时，DPP-BT纳米颗粒在414nm处的吸收强度急剧下降”即易受光漂白影响，浓度为1mg/ml时在600～700nm的吸收强度低于 0.20a.u，CN10600825B公布的DPP-R NPs在600～700nm的吸收强度低于0.70a.u，平均尺寸120～130nm。而目前ICG在700～800nm的吸收强度1.3a.u，因此光声信号较弱，限制了有机小分子荧光探针在光声成像和光热治疗方面的应用。同时，尽管表面修饰有PEG组分，但作为外来物质在体内应用过程中仍存在较强免疫原性，易被免疫系统清除的问题。CN108250222A公布的小分子发光材料3BDTBDD和5BDTBDD在500～600nm的吸收强度较好，但仍低于0.9a.u，且吸收区域不在近红外一区范围内，CN109943319A公布的DPP-BDT如该专利的图5所示，一定浓度DPP-BDT纳米粒子在不同功率光照下 DPBF的414nm紫外吸收强度随时间变化图，吸收强度急剧下降，即易受光漂白影响，且DPP-BDT在远高于本申请中的纳米粒子的浓度下(1mg/ml)才具有较好的吸收强度。
Second Near-Infrared Conjugated Polymer Nanoparticles forPhotoacoustic Imaging and PhotothermalTherapy.Zhigang Xie利用贺泳霖等人设计合成的TBDOPV-DT分子开展了光热治疗和光声成像等相关研究，该探针表面也是修饰的外源PEG组分，存在易被免疫系统识别清除、血液循环时间短的问题。另外，该探针尺寸较大(250nm) 左右，不利于在肿瘤部位渗透和机体内快速清除。
Q.Miao,Y.Lyu,D.Ding,K.Pu,Adv.Mater.2016,28,3662；K.Pu, A.J.Shuhendler,J.Rao,Angew.Chem.,Int.Ed.2013,52,10325；A.J. Shuhendler,K.Pu,L.Cui,J.P.Uetrecht,J.Rao,Nat.Biotechnol.2014, 32,373.等研究二酮吡咯基SPN的结构-性能关系，证明了SPN的光声(PA)亮度与其光热转换效率有着相应的关系，据我们所知包括聚吡咯np(光热转换效率45％)、多巴胺-黑色素np(光热转换效率 40％)、杂环导电聚合物PPDSnp(光热转换效率31.4％)、聚(环戊二噻吩alt-二酮吡咯)SPNs(光热转换效率20％)。基于结构/组分- 性能关系来设计和制备用于提高光声亮度或提高PTT效率的spn的工作相对较少。
CN106943378A公开了通过红细胞膜的包裹作用，可提高小分子水溶性药物在高聚物纳米粒子中的包封率，红细胞膜包裹的纳米粒，具有更好的缓释效果，被动靶向，降低药物毒性，没有溶血性从而可以用于静脉注射的优势。本申请中DSPE-mPEG2000修饰和红细胞膜包裹的技术方案结合，与小分子相比，我们合成的聚合物光稳定性强，不易泄露，同时又包裹有红细胞膜，可以延长其在机体内的血液循环时间，便于在肿瘤部位高效富集。
发明内容
本发明旨在解决目前用于近红外一区的SPN纳米材料光热转化效率低，光声信号弱，易受光漂泊和对RONS分解敏感，尺寸较大不易渗透和排出，易被免疫系统识别清除、血液循环时间短的问题，因此提供一种在肿瘤部位高富集和渗透性强、且在机体内可代谢的诊疗一体化纳米探针，用于肿瘤的光声成像(PAI)以及光热消融治疗 (PTT)的研究。
本发明采用的技术方案是：
一种红细胞膜包覆的D-A型超小分子SPN纳米材料，由D-A型有机半导体聚合物材料SP与双亲分子DSPE-mPEG2000通过纳米共沉淀法自组装形成超小分子纳米颗粒SPN，随后在纳米颗粒SPN表面包覆一层红细胞的细胞膜得到平均粒径为2～5nm的纳米粒，所述的聚合物材料SP具有分子式III所示的分子结构，
式III：
(n＝1，2……)
其中D为：
A为：
R1、R2为-CH2CH(R3)(R4)，R3和R4是C1～C20烷基。
进一步地，所述R1为
所述R2为
超小尺寸有利于纳米颗粒在体内代谢和肿瘤部位渗透；红细胞膜可以延长纳米颗粒在体内的循环时间，进而提高其在肿瘤部位的富集效果。
上述的D-A型有机半导体聚合物材料SP的合成方法，包括以下步骤：
S01.将Pd2(dba)3和P(o-tolyl)3分别添加到含等摩尔量的式I化合物和式II化合物的甲苯溶液中，将式I化合物和式II化合物的甲苯溶液等体积混合；
S02.在氮气保护下将混合物加热至110℃，搅拌反应48h后，冷却至室温，倒入甲醇中搅拌，过滤收集黑色沉淀；
S03.将黑色沉淀物在索氏提取器中分别用甲醇和二氯甲烷各洗涤得到纯化产物；
S04.用热氯仿在萃取器中萃取纯化产物，除去溶剂后得到的墨绿色固体即为D-A型有机半导体聚合物材料SP；
所述式I化合物为：
所述式II化合物为
所述的D-A型有机半导体聚合物材料SP的合成路线如下：
上述的纳米粒的合成方法，包括以下步骤：
J01.将D-A型有机半导体聚合物材料SP和DSPE-mPEG200按照质量比为1～3:5～9分别溶解在的四氢呋喃中，然后将两种溶液混匀后在超声的条件下加入到的超纯水中，然后搅拌过夜，除去溶液中的四氢呋喃，超滤离心除去多余DSPE-mPEG2000，即得到超小分子纳米颗粒SPN；
J02.将RBCM与超小分子纳米颗粒SPN按照质量比为0.2～1：1～3 混匀后通过挤膜器机械挤压，依次通过400nm，200nm的滤膜最后得到纳米粒。
优选地，所述RBCM采用采用以下方法制备：取健康SD大鼠外周血全血，在4℃，1000g的加速度下离心10min，用pH为7.4 的冰磷酸缓冲盐溶液洗涤3次，取红细胞，在稀释4倍的磷酸缓冲盐溶液中再次悬浮于冰浴中3小时，在4℃，16000g的加速度下离心 30min，取粉红色层RBCM用冰磷酸缓冲盐溶液洗涤，直到RBCM 变成粉红色，最后用BCA试剂盒对RBCM进行蛋白定量，并在-80℃保存备用。
本发明中红细胞膜(RBCM)包覆半导体纳米颗粒SPN形成的纳米粒的尺寸在2 nm，是一种超小的纳米颗粒，表面的红细胞膜可以改善纳米颗粒的生物相容性，延长纳米颗粒在体内的循环时间进而提高纳米颗粒在肿瘤部位的富集效率。可以在肿瘤部位有着较深的渗透深度以及在体内有着较快的代谢速率。
本发明涉及所述的红细胞膜包覆的超小分子SPN纳米材料的应用，同时用于光声成像和近红外一区荧光成像。D-A型半导体聚合物在近红外区域有着良好的吸收能力，具有优良的光热转换效率，可以实现光热治疗和光声成像一体化。
本发明的纳米粒(SPN@RBCM)解决了目前的部分SPN材料光声成像信号弱，另一部分SPN材料吸收峰不在近红外一区，造成癌症诊断与治疗不同步，容易错过治疗的最佳时期，从而影响肿瘤的治疗效果和治愈情况等问题，以及D-A型有机半导体聚合物材料易受光漂泊和对RONS分解敏感，尺寸较大不易渗透和排出，易被免疫系统识别清除、血液循环时间短的问题，构建了一种长循环、机体可经肾脏从体内排出、肿瘤高富集与渗透的新型一体化的纳米探针，实现肿瘤部位高效光热成像和光热治疗一体化的目的。
本发明所构建的SPN@RBCM纳米探针可通过任何已知的递送方法方式给药：全身递送(静脉注射)，动脉内，肿瘤内，胃肠外，肺腔内，局部、或局部给药的区域递送形式。
本发明的有益效果主要体现在：
本发明所构建的SPN@RBCM纳米探针，以有机半导体纳米材料 SPN为基础，本发明构建的D-A π共轭结构，可降低分子的HOMO 和LUMO能级，同时π离域结构有利于电子在分子中传输，两者共同作用可增强探针的近红外吸收。
肾小球电荷屏障研究的新进展.医学综述2014年6月第20卷第 12期指出分子有效半径小于2.0nm的中性物质可自由滤过肾小球， 2.0～4.2nm之间的各种物质随有效半径的增加，滤过量逐渐减少，本发明的SPN@RBCM纳米探针尺寸为2nm，小于现有技术，有利于从肾小球过滤屏障中排出，也有利于穿过细胞膜进入癌细胞。
本发明所构建的SPN@RBCM纳米探针，可以实现肿瘤光热治疗和光声成像一体化，避免了临床中癌症的诊断和治疗之间的时间差，使癌症治疗结果可视化，大大提高了治疗的有效性，为癌症的治愈提供了新的手段和希望。
附图说明
图1为SP的合成路线图；
图2为SPN@RBCM溶液的部分表征结果；(A)SPN超小纳米颗粒的透射电镜图像；(B)DLS测得SPN和SPN@RBCM的水合粒径分布图；(C)DLS测得SPN、RBCM、SPN@RBCM的表面电位图；(D) RBCM和SPN@RBCM经过考马斯亮蓝染色后的SDS-PAGE结果；
图3为SPN@RBCM溶液的体外光声性能的表征数据；(A)不同浓度的SPN@RBCM的光声信号强度；(B)不同浓度的SPN@RBCM 的光声信号强度与浓度的线性拟合图。
图4为SPN@RBCM溶液的体外光热性能的表征数据；(A)SP、SPN、 SPN@RBCM溶液的紫外吸收光谱图；(B)近红外激光照射不同浓度的SPN@RBCM溶液的升温曲线图；(C)近红外激光照射 SPN@RBCM与ICG溶液的升温-降温循环曲线图；(D)SPN@RBCM 与ICG溶液随着光照时间的变化，在808nm处紫外吸收变化图；(E) 近红外激光照射SPN@RBCM溶液的升温-降温曲线图；(F)降温曲线的时间和-lnθ的线性拟合图；由E图和F图可的SPN@RBCM的光热转换效率为59％。
图5为SPN@RBCM部分细胞水平的性能评估；(A)癌细胞(7721) 不同时间点对SPN@RBCM摄取的激光共聚焦图像；(B) SPN@RBCM对癌细胞系的杀伤效果图；(C)SPN@RBCM在体外构建的3D肿瘤球模型中的渗透效果图。
具体实施方法
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：
1.TTBDT-TBDOPV的合成
将Pd2(dba)3(0.0037mmol)和P(o-tolyl)3(0.0146mmol)作为催化剂分别添加到含有式(I)化合物TTBDT(0.122mmol)(购自苏州纳凯科技有限公司)和式(II)化合物TBDOPV(0.122mmol)(Wood,J. H.；Cox,L.2,5-Dihydroxy-p-benzenediaceticacid.Org.Synth.1946,26, 24-27.中的合成方法)的甲苯(6ml)溶液中。氮气保护下将混合物加热至110℃，搅拌反应48h后，冷却至室温，倒入甲醇中搅拌2h，过滤收集黑色沉淀。将黑色沉淀物在索氏提取器中分别用甲醇和二氯甲烷各洗涤24小时，用来除去催化剂，配体和为反应的单体，得到纯化的产物。最后用热氯仿在萃取器中萃取24小时，除去溶剂后得到的墨绿色固体即为SP。合成路线图如图1所示。
2.TTBDT-TBDOPV水溶性纳米粒子的制备：
称取D-A型半导体聚合物SP(TTBDT-TBDOPV)1mg，溶解在1mL 四氢呋喃中(THF)，再称取DSPE-mPEG2000 5mg溶解在 1mL的四氢呋喃中，然后将两者混匀加入到装有20mL超纯水的烧杯中，将烧杯置于超声破碎仪中，超声3min，室温搅拌过夜，超滤浓缩，超纯水洗涤3次，即得到SPN。SPN超小纳米颗粒的透射电镜图像如图2A所示。
3.SPN@RBCM纳米粒的制备：
制备RBCM：取健康SD大鼠外周血全血，在4℃，1000g离心 10min，用冰PBS(磷酸缓冲盐溶液,pH＝7.4)洗涤3次，取红细胞，在 0.25×PBS中再次悬浮于冰浴中3小时。4℃，16000g离心30min，粉红色层为RBCM。用冰PBS洗涤，直到RBCM变成粉红色。最后用BCA试剂盒对RBCM进行蛋白定量，并在-80℃保存，备用。
将已定量的RBCM与已知浓度的SPN(质量比1:5)混匀后通过挤膜器机械挤压，依次通过400nm,200nm的滤膜最后得到 SPN@RBCM。通过酶标仪定量后，4℃避光保存，以备将来使用。 SPN的形貌使用透射电镜(TEM)表征进行表征，结果如图2A所示，该材料为球形纳米颗粒，尺寸范围为2～5nm；包裹红细胞膜后， SPN@RBCM、SPN和RBC-vesicles的聚集行为和表面电位通过DLS 表征，结果如图2B和2C所示，结果显示SPN@RBCM的表面电位在-34.8mV左右，与RBC-vesicles的表面电位更接近；此外，红细胞膜在SPN表面的成功包覆还可通过考马斯蓝色染色法进行检验，结果如图2D所示，SPN@RBCM与RBCM-Vesicles有着相同的蛋白条带，证实红细胞膜成功的包覆在纳米颗粒表面。
4.SPN@RBCM囊泡用于近红外一窗荧光成像和光声成像：
光声成像
取已知浓度的SPN@RBCM溶液稀释成10～60μg/mL，各取1mL，利用光声成像仪在808nm下检测各个浓度梯度的光声信号强度，结果如图3A所示，随着溶液浓度的升高，光声信号强度逐渐增强；线性拟合图如图3B所示，呈现了良好的线性关系。
近红外一区光吸收
取60μg/mL的SP四氢呋喃溶液和60μg/mL的SPN、 SPN@RBCM水溶液在酶标仪中检测300-1000nm之间的紫外吸收图谱。结果如图4A所示，SP、SPN、SPN@RBCM均在840nm处有着较强的吸收。
5.SPN@RBCM囊泡光热性能测试
光热性能测试
用功率0.8W/cm2 1000s的808nm激光照射浓度为0～60μg/mL 的SPN@RBCM溶液由红外热成像仪记录溶液温度随时间的变化趋势，如图4B，在SPN@RBCM溶液的浓度为60μg/mL时，溶液可在 600s内由22℃激增到55℃，1000s内增长到58℃，远高于人体正常体温，因此可以用于癌细胞的热杀伤，而SPN@RBCM溶液的浓度远低于现有的DPP-BT-R2材料，现有技术CN109943319A中DPP-BDT 在同样的浓度下，光热性能较本申请的材料弱。
抗光漂白能力测试
通过与现有的光热材料ICG进行对比表明SPN@RBCM具有良好的光热稳定性以及抗光漂白的能力，结果如图4C和4D所示，图 4C表明经过四个升温-降温循环，相比于ICG，SPN@RBCM依然维持着良好的升温效果；图4D表明经过激光照射15min，相比于ICG， SPN@RBCM在808nm处依然维持着良好的吸收；最后通过图4E和 4F计算得出SPN@RBCM的光热转换效率为59％。
6.SPN@RBCM部分细胞水平的性能评估
称取D-A型半导体聚合物SP(TTBDT-TBDOPV)1mg，溶解在1mL 四氢呋喃中(THF)，并向其中加入5mg/mL的细胞膜染料 DIO 10μL，再称取DSPE-mPEG2000 5mg溶解在1mL的四氢呋喃中，然后将两者混匀加入到装有20mL超纯水的烧杯中，将烧杯置于超声破碎仪中，超声3min，室温搅拌过夜，超滤浓缩，超纯水洗涤3 次，即得到带有荧光染料DIO的SPN。随后按照实施例4合成带有荧光染料DIO的SPN@RBCM。
将带有荧光染料(DIO)的SPN@RBCM与癌细胞(SMMC-7721) 共孵育，观察不同时间点癌细胞对纳米颗粒的摄取情况如图5A所示，图5A表明，癌细胞在8h时对SPN@RBCM有着良好的摄取能力。
将50μg/mL的SPN@RBCM溶液与癌细胞共孵育8h后，用激光照射10min，继续培养24h后用AM-PI染色试剂染色癌细胞，观察 SPN@RBCM和空白对照组(仅加入PBS)对癌细胞的杀伤效果如图 5B所示(死细胞为红色图5B右下角，活细胞为绿色图5B)，图5B 表明，SPN@RBCM对癌细胞有着良好的杀伤效果。
通过在体外构建3D肿瘤球模型，将带有荧光染料DIO的 SPN@RBCM与PLGA@RBCM分别与肿瘤球共孵育，通过共聚焦显微镜观察纳米材料渗透深度的结果如图5C所示，图5C表明， SPN@RBCM在肿瘤球的渗透深度明显比PLGA@RBCM深，证明超小的纳米颗粒SPN@RBCM在肿瘤部位有着较深的渗透深度。
Claims (5)
1.一种红细胞膜包覆的D-A型超小分子SPN纳米材料，由D-A型有机半导体聚合物材料SP与双亲分子DSPE-mPEG2000通过纳米共沉淀法自组装形成超小分子纳米颗粒SPN，随后在纳米颗粒SPN表面包覆一层红细胞的细胞膜得到平均粒径为2～5nm的纳米粒，其特征在于，所述的D-A型有机半导体聚合物材料SP具有分子式III所示的分子结构，
式III：
(n＝1，2)
其中，R为
2.根据权利要求1所述的红细胞膜包覆的D-A型超小分子SPN纳米材料的制备方法，其特征在于，所述的D-A型有机半导体聚合物材料SP的合成方法，包括以下步骤：
S01.将Pd2(dba)3和P(o-tolyl)3分别添加到含等摩尔量的式I化合物和式II化合物的甲苯溶液中，将式I化合物和式II化合物的甲苯溶液等体积混合；
S02.在氮气保护下将混合物加热至110℃，搅拌反应48h后，冷却至室温，倒入甲醇中搅拌，过滤收集黑色沉淀；
S03.将黑色沉淀物在索氏提取器中分别用甲醇和二氯甲烷各洗涤得到纯化产物；
S04.用热氯仿在萃取器中萃取纯化产物，除去溶剂后得到的墨绿色固体即为D-A型有机半导体聚合物材料SP；
所述式I化合物为：
所述式II化合物为
所述的D-A型有机半导体聚合物材料SP的合成路线如下：
3.根据权利要求2所述的红细胞膜包覆的D-A型超小分子SPN纳米材料的制备方法，其特征在于，包括以下步骤：
J01.将D-A型有机半导体聚合物材料SP和DSPE-mPEG200按照质量比为1～3:5～9分别溶解在的四氢呋喃中，然后将两种溶液混匀后在超声的条件下加入到的超纯水中，然后搅拌过夜，除去溶液中的四氢呋喃，超滤离心除去多余DSPE-mPEG2000，即得到超小分子纳米颗粒SPN；
J02.将红细胞膜与超小分子纳米颗粒SPN按照质量比为0.2～1：1～3混匀后通过挤膜器机械挤压，依次通过400nm，200nm的滤膜最后得到纳米颗粒。
4.根据权利要求1所述的红细胞膜包覆的D-A型超小分子SPN纳米材料的制备方法，其特征在于，所述红细胞膜采用采用以下方法制备：取健康SD大鼠外周血全血，在4℃，1000g的加速度下离心10min，用pH为7.4的冰磷酸缓冲盐溶液洗涤3次，取红细胞，在稀释4倍的磷酸缓冲盐溶液中再次悬浮于冰浴中3小时，在4℃，16000g的加速度下离心30min，取粉红色层红细胞膜用冰磷酸缓冲盐溶液洗涤，直到红细胞膜变成粉红色，最后用BCA试剂盒对红细胞膜进行蛋白定量，并在-80℃保存备用。
5.一种权利要求1所述的红细胞膜包覆的D-A型超小分子SPN纳米材料在制备通过光声成像和近红外一区光热消融治疗肿瘤的药物中的应用。
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