CN106995442B - 靶向荧光染料及其应用 - Google Patents
靶向荧光染料及其应用 Download PDFInfo
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- CN106995442B CN106995442B CN201710298171.0A CN201710298171A CN106995442B CN 106995442 B CN106995442 B CN 106995442B CN 201710298171 A CN201710298171 A CN 201710298171A CN 106995442 B CN106995442 B CN 106995442B
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Abstract
本发明公开一种靶向荧光染料及其应用。所述靶向荧光染料具有通式I的结构。基于该靶向荧光染料,本发明进一步公开以该染料进行表面修饰的载药羟基磷灰石及其制备方法。通过该方法制得的靶向复合材料可灵敏识别并结合在大多数肿瘤细胞膜中高度表达的谷氨酰胺转氨酶,通过内吞方式穿过细胞膜,实现抗癌药物的缓慢靶向释放。不仅能够提高抗癌药物的包覆率,并且有效地区分了正常与肿瘤细胞,进而有效提高抗癌药物的释放效率和局部浓度并降低抗癌药物的使用剂量和毒副作用,该复合材料能够快速渗透到肿瘤组织内,结合细胞毒性,在癌症治疗领域有着重大的研究价值和应用前景。
Description
技术领域
本发明涉及生物纳米材料领域和精细化工领域一类荧光染料、其制法和用途,具体涉及一种靶向肿瘤细胞表面谷氨酰胺转氨酶的萘酰亚胺衍生物类荧光染料及其应用。
背景技术
癌症已经成为威胁人类生命的主要疾病之一。据统计,全球每年新发癌症患者人数高达数千万人次,死亡人数在六百万左右,危害严重,治疗困难;目前,越来越多 的国家政府和科学界投入了大量的精力和财力用于癌症的治疗和预防。因此,发展能 够更加有效地治疗这类疾病的手段,对促进人类健康具有重大意义。
抗肿瘤药物化疗是除外科手术之外最重要的癌症治疗手段,包括传统化疗和靶向治疗两种方法。传统化疗是指利用抗肿瘤药物(细胞毒性物质)干扰体内细胞有丝分 裂,杀死快速分裂的细胞。即在杀伤肿瘤细胞的同时,又杀伤正常组织的细胞,尤其 是杀伤人体中生长发育旺盛的血液、淋巴组织细胞等。而这些细胞与组织是人体重要 的免疫防御系统,因此传统化疗往往伴随着很大程度上的毒副作用。
靶向治疗主要是基于传统化疗的基础上,通过瘤内注射,载体修饰等手段,实现化疗药物对于肿瘤的识别,降低正常组织细胞的摄入,从而大大提高传统化疗的治疗 效果。瘤内注射虽然简便,大多数情况下由于实际肿瘤部位无法精确探知,所以并不 适用。目前,更多是利用能够识别肿瘤细胞膜上特定分子信号的特殊配体来修饰药物 载体外层,主要包括小分子靶向染料、缩氨酸(如环状五肽cRGD)、铁传递蛋白和某 些抗体。γ-谷氨酰转肽酶(GGT)是一种糖蛋白性的膜腱酶。肝脏中的GGT主要分布在 肝细胞毛细胆管内侧和整个胆管系统,GGT合成增多或胆管系统病变,胆汁排泄受阻 时均可引起血清GGT升高。而血清GGT有癌胚特性,在原发性肝癌(PHC)实体肿 瘤组织生长时,释放到外周血液中的甚至高达600U/L以上。即肝癌患者GGT活性显 著升高,尤其是恶性肿瘤肝转移及肝癌手术复发时更明显,往往大于正常的几倍甚至 几十倍,阳性率可达90%。当肿瘤切除后,GGT可降至正常,复发时又升高,故动态 观察可监测肝癌疗效、判断预后。
另一方面,目前多数小分子抗癌药水溶性较差,临床上通常使用表面活性剂对其进行乳化,但在血液循环过程中其稳定性较差,基本上不具有缓释或控释性能,而且 存在血液半衰期短、毒副作用依然很大等众多缺点。因此,研究新型的高效药物运输 载体已成为生物医药、肿瘤治疗领域的关键。以无机纳米材料作为药物载体用于靶向 药物输送系统,近年来得到迅速发展。无机药物载体如二氧化硅、四氧化三铁等,虽 然药物负载能力高,化学稳定性和生物相容性好,但若在体内过量积聚,会对正常组 织造成损伤,目前已有文献报道体内过量积聚的二氧化硅对肺和肝的损伤尤为明显。 羟磷灰石(HAP)是脊椎动物骨骼和牙齿的主要无机组成成分,具有优良的生物相容性 和生物降解性、安全低毒、稳定的化学结构和载药性广等优点。此外,在酸性条件下 (pH3.5-6),羟磷灰石易水解,且对正常细胞无毒副作用,是一种有潜力的药物载体。
荧光染料的识别或传感功能是近年发展起来的。相比于传统的检测手段,荧光染料具有选择性,灵敏度高,实时原位检测,动态可视化检测等优点,被广泛应用在各 个领域。在生物医学领域,荧光染料可被应用在细胞、组织或体内,用于疾病的早期 诊断和病理分析,如基因诊断(DNA测序、基因重组)、生物分析(核酸识别、蛋白质标 记、离子及活性氧等小分子检测),荧光成像(细胞,组织及活体成像)及医疗诊断(免疫检 测、血液分析、肿瘤标志物检测)等。因此,设计以GGT为靶标的荧光染料用于检测 GGT含量异常具有重要意义;将这类荧光染料与羟磷灰石纳米药物载体结合,在肿瘤 靶向治疗和示踪具有实际应用价值。
发明内容
本发明的目的在于提供一种对癌细胞靶向识别示踪且酸敏响应性的羟磷灰石纳米 药物复合体,用于靶向表面谷氨酰转肽酶过表达的癌细胞,从而实现癌细胞选择性识别示踪及抗癌药物的缓慢靶向释放。
为此,本发明首先公开一种靶向荧光染料,具有通式I的结构:
通式I中,
R1和R2各自独立地选自:H、NO2、COOH、NH2、OH、C1-6烷氧基、C1-6烷基氨 基、C1-6酰胺基、卤素或C1-6卤代烷基;
R3是N或O。
进一步,本发明提供上述靶向荧光染料的制备方法,该制备方法中:
(1)当R3=N时所述方法包括如下步骤:
①式II的化合物与式III的化合物在125℃条件下按照摩尔比0.1-1000:1反应4-8h,制备式IV的化合物;
反应溶剂优选为冰醋酸,反应摩尔比优选为0.1-500:1,更优选为0.1-100:1,最优选为0.1-20:1;
②式IV的化合物与Zn粉在90℃条件下按照摩尔比0.1-100:1下反应3-6h,反 应体系加入适量无水CaCl2结晶固体,制备式V的化合物;
反应溶剂优选为乙醇,摩尔比优选为0.1-50:1,更优选为0.1-20:1。
③式V的化合物与式X的化合物在室温下按照摩尔比0.1-1000:1下反应24-72h,加入0.05当量催化剂EDCl和HOBT,制备式XI的化合物(即式I的化合物,R3=N);
反应溶剂优选为DMF与二氯甲烷体积比0.1-100:1的混合溶剂,更优选为0.1-20:1;
(2)当R3=O时所述方法包括如下步骤:
①式VI的化合物与式III的化合物在125℃条件下按照摩尔比0.1-1000:1反应4-8h,制备式VII化合物。
反应溶剂优选为冰醋酸,摩尔比优选为0.1-100:1,更优选为0.1-20:1。
②式VII的化合物与乙醇钠在85℃条件下按照摩尔比1:0.1-1000反应6-24h,制备式VIII化合物;
反应溶剂优选为甲醇,加入0.1-0.5当量无水Cu2SO4,反应优选进行氮气保护, 摩尔比优选1:0.1-200,更优选1:0.1-50,最优选1:0.1-20;
式VIII与过量的HI水溶液中反应2-5h,制备式IX化合物。
反应溶剂优选为乙醇水溶液,条件优选为回流反应。
③式IX的化合物与式X的化合物在室温下按照摩尔比0.1-1000:1下反应24-72 h,加入0.05当量催化剂EDCl和HOBT,制备式XII化合物(即式I的化合物,R3=O)。
反应溶剂优选为DMF与二氯甲烷体积比0.1-100:1的混合溶剂,更优选为0.1- 20:1。
进一步,本发明公开一种靶向荧光染料修饰的载药羟基磷灰石,所述的靶向荧光染料即上述本发明的靶向荧光染料(具有通式I的结构)。
再一方面,本发明还公开上述靶向荧光染料修饰的载药羟基磷灰石的制备方法,包括下述步骤:(1)制备载药羟基磷灰石(D@HAP);(2)以靶向荧光染料对载药羟 基磷灰石进行表面修饰。
本发明所提供的靶向荧光染料以及在其基础上所构建的靶向荧光染料修饰的载药 羟基磷灰石靶向谷氨酰胺转氨酶。基于谷氨酰胺转氨酶在大多数肿瘤中高度表达,而靶向荧光染料的谷氨酸酰胺区域同肿瘤细胞表面的谷氨酰胺转氨酶亲和力很强;荧光 染料修饰的载药羟基磷灰石经由靶向荧光染料区域与谷氨酰胺转氨酶识别结合,并以 内吞方式穿过细胞膜,从而靶向表面谷氨酰胺转氨酶过表达的肿瘤细胞,实现抗癌药 物的缓慢靶向释放。因此,本发明再一方面提供所述的靶向荧光染料修饰的载药羟基 磷灰石在制备肿瘤示踪及治疗制剂中的应用。
与现有技术相比,本发明的靶向荧光染料修饰的载药羟基磷灰石在如下几个方面都有突出的优势:(1)设计合成的荧光染料I对谷氨酰胺转氨酶具有专一、快速识别 的效果,能够对谷氨酰胺转氨酶过表达的肿瘤细胞与谷氨酰胺转氨酶低表达的正常细 胞进行区分,从而通过荧光信号有效区分肿瘤与正常细胞,在肿瘤细胞中发出极强的 黄色的荧光,实现纳米材料对肿瘤部位的识别与示踪。(2)羟磷灰石(HAP)是脊椎 动物骨骼和牙齿的主要无机组成成分,具有优良的生物相容性和生物降解性、安全低 毒、稳定的化学结构和载药性广等优点。随着生物体内pH的变化,有机药物载体如 脂质体、聚合微球等不稳定,易发生结构的变化,不能有效进行药物释放。无机药物 载体如二氧化硅、四氧化三铁等虽然药物负载能力高,化学稳定性和生物相容性好, 但若在体内过量积聚,会对正常组织造成损伤。与这些无机纳米材料相比,羟基磷灰 石有着极为优异的性能;此外,在酸性条件下(pH3.5-6),羟磷灰石易发生酸解,且对 正常细胞无毒副作用,是一种有潜力的药物载体。(3)本发明所述的药物载体是将抗 癌药物和羟磷灰石纳米颗粒进行共掺杂,而非仅仅将抗癌药物修饰在纳米颗粒表面, 从而提高了抗癌药物的包覆率。(4)掺杂抗癌药物的羟磷灰石纳米颗粒表面修饰上靶 向荧光染料后,就如同在该纳米载体表面增加了一个开关,从而有效地降低了该纳米 载体在血液循环过程中的被体内正常组织,血液内含物等的吸附,提高了纳米载体在 肿瘤部位的聚集,进而提高了抗癌药物的释放效率和局部浓度并降低抗癌药物的使用 剂量和毒副作用等,在癌症治疗领域有着重大的研究价值和应用前景。(5)谷氨酰胺 转氨酶在大多数肿瘤中高度表达,在正常组织内低表达,而靶向肿瘤细胞萘酰亚胺衍 生物类荧光染料I与谷氨酰胺转氨酶有着极高的结合能力,荧光染料与酶结合,并可通过内吞方式穿过细胞膜。因此,本发明制备的I@D@HAP纳米药物载体对肿瘤细胞 具有很好的主动响应性能,也就是说该纳米药物载体具有良好的谷氨酰胺转氨酶特异 靶向性,从而实现抗癌药物的靶向缓慢释放。
附图说明
本发明附图11幅:
图1是实施例2.1HAP、DOX@HAP、DFA1@DOX@HAP纳米颗粒的透射电镜 (TEM)图。其中,(a)、(b)、(c)分别为HAP、DOX@HAP、DFA1@DOX@HAP在200nm 下的电镜图。其形态为棒状,平均长度是60-130nm,平均直径15-50nm,不易聚集, 分散性较好。
图2是实施例2.2的红外光谱(FTIR)图。其中,(a)为HAP,(b)为DOX@HAP, (c)为DFA1@DOX@HAP。
图3是实施例2.3HAP,DOX@HAP,DFA1@DOX@HAP纳米颗粒的Zeta电位 图。其中HAP,DOX@HAP,DFA1@DOX@HAP的浓度均为0.5mg/mL。
图4(a)是实施例2.4DFA1的时间响应曲线;图4(b)是实施例2.4DFA1的浓 度响应曲线;图4(c)是实施例2.4DFA1对氨基酸选择性实验;图4(d)是实施例2.4 DFA1对阴阳离子选择性实验;母液浓度为2.5mM,激发波长为440nm。
图5-7是实施例3DFA1@DOX@HAP纳米颗粒的荧光光谱图。DFA1@DOX@HAP 浓度分别为5mg/mL,荧光染料的最大吸收波长为440nm,最大发射波长为550nm; 抗癌药物的最大吸收波长为480nm,最大发射波长为590nm。
图7中,1-14物质分别为:1、空白;2、人血清蛋白;3、RNA;4、DNA;5、三 酰基甘油基水解酶;6、溶菌酶;7、蛋白激酶K;8、精氨酸;9、胶原;10、血红素; 11、牛血清蛋白;12、β-淀粉酶;13、酪氨酸;14、谷氨酸转氨酶GGT。
图8是实施例4DFA1@DOX@HAP在不同pH值下的药物释放过程。不同pH值 下的药物累积释放率图。激发波长为480nm。
图9是实施例5DFA1@DOX@HAP纳米颗粒的细胞毒性图。其中,a-f分别为COS- 7、HL-7702、HCT-116、MCF-7,HeLa以及A2780细胞,g图为6种细胞的细胞毒 性柱状比较图。DFA1@DOX@HAP浓度为5mg/mL,激发波长为480nm,细胞孵育时 间为24h。
图10是实施例6DFA1@DOX@HAP在不同细胞、不同时间的荧光共聚焦显微成 像图。其中,选择COS-7、HL-7702、HCT-116、MCF-7,HeLa以及A2780这六种细 胞,以时间间隔为15min进行单光子显微共聚焦成像。DFA1@DOX@HAP浓度为 5mg/mL,激发波长为488nm。
图11是实施例7DFA1@DOX@HAP在裸鼠瘤体冰冻切片中的组织成像实验。其 中,裸鼠瘤体为皮下种植HepG2细胞,切片与材料母液孵育时间为4h。 DFA1@DOX@HAP浓度为5mg/mL,激发波长为405nm和488nm。
具体实施方式
本发明旨在提供对肿瘤细胞靶向识别示踪且酸敏响应性的功能性靶向荧光染料及 其与载药羟磷灰石所构成的功能复合体,并且提供这些产品的制备方法。本发明中所述及的靶向均表示针对肿瘤细胞表面高表达的谷氨酰胺转氨酶的靶向。
如无特殊说明,本说明书中所使用的符号GGT均代表谷氨酰胺转氨酶,I代表靶 向GGT的萘酰亚胺衍生物类荧光染料,DFA1代表R1,R2为氢原子,X为氮原子,R3为氮原子的小分子染料,D代表所载的药物,DOX代表阿霉素,HAP代表羟基磷灰 石,D@HAP代表载药羟基磷灰石,DFA1@D@HAP代表修饰靶向GGT的萘酰亚胺 衍生物类荧光染料DFA1的载药羟基磷灰石。
首先,本发明中所述的靶向荧光染料,具有通式I的结构:
通式I中,R1和R2各自独立地选自:H、NO2、COOH、NH2、OH、C1-6烷氧基、 C1-6烷基氨基、C1-6酰胺基、卤素或C1-6卤代烷基;作为优选地,选自H、NO2、NH2、 COOH、OH、OCH3、OC2H5、Br、Cl;进一步优选,选自H、NO2、OH、OCH3、OC2H5、 Br,更优选H、NO2、OCH3;最优选为H。
通式I中,R3是N或O。优选为N。
当上述优选条件的组合,所得到的通式I的化合物,是本发明中关于靶向荧光染料的优选化合物。该组合可举例为,当R1和R2均为H,而R3是N时,所获得的化合 物视为本发明代表性的优选化合物,具备如下DFA1的结构描述:
使上述本发明的靶向荧光染料与载药羟基磷灰(D@HAP)石结合,获得靶向荧光 染料修饰的载药羟基磷灰石。具体一些,是通过靶向荧光染料与载药羟基磷灰石在乙 醇-水体系中充分接触制得。该说明书中所描述的靶向荧光染料的结构、基团选择与优 选均适用于该复合体中的靶向荧光染料结构区域。根据所述,则最为优选的靶向荧光 染料修饰的载药羟基磷灰石可描述为DFA1修饰的载药羟基磷灰石,记为DFA1@ D@HAP。
根据现有技术中修饰的载药羟基磷灰石的制备方法,可经适应性调整获得本发明的靶向荧光染料修饰的载药羟基磷灰石的制备方法,该方法可概括性描述为:靶向荧 光染料与载药羟基磷灰石在乙醇-水体系中充分接触。
更为具体地,所述的制备方法包括包括下述步骤:
(1)制备载药羟基磷灰石(D@HAP);
(2)以靶向荧光染料对载药羟基磷灰石进行表面修饰。
进一步具体的实施方式中,上述步骤(1)是由水可溶性钙盐、磷酸盐及药物在乙醇-水体系中,于室温条件下反应制备载药羟基磷灰石(D@HAP)。其中,所述的可溶 性钙盐优选但不限于Ca(NO3)2·4H2O;所述的可溶性磷酸盐优选但不限于(NH4)2HPO4。
另一方面,该所述载药羟基磷灰石中述及的药物选自:阿霉素、紫杉醇、顺铂、 吉西他滨、氟尿嘧啶、盖诺、表阿霉素、环磷酰胺、长春新碱、博莱霉素、多帕菲或 其衍生物。尤其优选优选阿霉素(DOX)、盐酸阿霉素(DOX·HCl)或其衍生物。其可 举例但不限于:阿霉素、吡喃阿霉素、表柔比星、米托蒽醌。
另一方面,所述的步骤(2)是在室温条件下,靶向荧光染料与载药羟基磷灰石在乙醇-水体系中充分接触。该接触可举例描述为:室温条件(25℃)下,靶向荧光染料与 载药羟基磷灰石在乙醇-水体系中搅拌反应10~30h。
上述方法所制备的载药羟基磷灰石是棒状材料,平均长度是60-130nm,平均直径15-50nm。
进一步的具体实施方式中,上述靶向荧光染料修饰的载药羟基磷灰石的制备方法包括如下步骤:
(1)向100ml浓度为15~30mg/ml、pH为10.5±0.5的Ca(NO3)2·4H2O的乙醇溶液 中缓慢滴加20~50ml的浓度为6~10mg/ml的药物-水溶液,搅拌5~15min后,滴加50 ml浓度为60~100mg/ml的(NH4)2HPO4水溶液,继续搅拌1~2h;混合物在室温条件下 (25℃下)反应72-96h后,高速离心,沉淀物洗涤干燥,得到载药羟基磷灰石;
(2)将质量比1:100~5:50的200mg靶向荧光染料、载药羟基磷灰石的混合物加 入到50~100ml含25%(v/v)乙醇的去离子水溶液中以1000r/min搅拌0.5~2h,继 续以600r/min搅拌12~24h后,高速离心,沉淀物洗涤干燥,得到靶向荧光染料修饰 的载药羟基磷灰石。
上述本发明的方法制得的表面修饰靶向荧光染料的载药羟基磷灰石基于谷氨酰胺 转氨酶靶向且酸敏响应性羟磷灰石纳米药物载体被用于靶向传输抗癌药物,所得纳米药物载体的结构验证、大小和形态等基本性能分别采用傅里叶红外光谱分析仪(FTIR)、Zeta电位分析仪、透射电子显微镜(TEM)来测定。其对抗癌药物负载能力的大小则 采用紫外可见分光光度计、荧光显微镜来进行验证,并通过体外细胞实验及组织成像 来检测该纳米药物载体的靶向治疗效果。
此外,该纳米药物载体还通过细胞实验来评价这一可定向传输抗癌药物到肿瘤细胞的靶向体系,即采用人肝细胞(HL-7702)、非洲绿猴肾细胞(COS-7)、人结肠肿瘤 细胞(HCT-116)、乳腺肿瘤细胞(MCF-7)、宫颈肿瘤细胞(HeLa)、人卵巢肿瘤细胞 (A2780)来进行体外的细胞毒性实验,以测定表面修饰靶向荧光染料的载药羟基磷 灰石对不同细胞的细胞毒性。其中COS-7,HL-7702为正常细胞,后者有一定GGT表 达,HCT-116、MCF-7,HeLa以及A2780为肿瘤细胞,将两组进行对比实验。
下述非限制性实施例用于对本发明做进一步的说明,但不应当理解为对本发明内容任何形式的限定。
实施例1.DFA1@DOX@HAP的制备
1.1载药羟基磷灰石(DOX@HAP)的合成
采用沉淀水热法结合,称取30mg盐酸阿霉素,1.490g的Ca(NO3)2·4H2O和0.495g的(NH4)2HPO4分别溶于2mL的超纯水,30mL的无水乙醇和2mL的超纯水中,并将 Ca(NO3)2·4H2O的乙醇溶液pH调节至10.5左右。在这种碱性环境下,边搅拌边缓慢 滴加盐酸阿霉素的水溶液,稳定10min后,继续滴加(NH4)2HPO4水溶液,滴加完毕 后,继续搅拌1h,然后将浑浊液倒入反应釜中,25℃条件下室温反应72h,然后高速 离心,用超纯水和无水乙醇多次洗涤产物,60℃下真空干燥12h,研磨,即得样品。
1.2修饰靶向荧光染料的载药羟基磷灰石(DFA1@DOX@HAP)
称取10mg靶向荧光染料DFA1和200mg载药羟基磷灰石DOX@HAP溶于50ml 的含25%(w/w)的乙醇去离子水中,先搅拌0.5~2h,继续以600r/min搅拌12~24h 后,高速离心,沉淀物洗涤干燥,得到表面修饰靶向荧光染料的载药羟基磷灰石 (DFA1@DOX@HAP)。
实施例2.表面修饰靶向荧光染料的载药羟基磷灰石基本性能的测试
2.1表面修饰靶向荧光染料的载药羟基磷灰石大小和形态的测试
实施例1所得表面修饰靶向荧光染料的载药羟基磷灰石的形态大小采用透射电子显微镜来观察确定,测试结果见图1。从透射电子显微镜图片可以明显看出,通过共 沉淀的方法合成的DFA1@DOX@HAP纳米颗粒,其形态为棒状,平均长度是60-130 nm,平均直径15-50nm,不易聚集,分散性较好。
2.2高级傅里叶变换红外光谱测定
取1-2mg表面修饰靶向荧光染料的载药羟基磷灰石样品在玛瑙研钵中与干燥的溴化钾粉末(A.R.级)混合并研磨成细粉末,装入模具中,在压片机上压制成片后在6700FTIR(Thermo Fisher,USA)上测试。测试参数为:光谱范围7800-350cm-1,分 辨率0.09cm-1,温度25℃。测试结果如图2所示,和HAP的FTIR光谱图比较, DOX@HAP和DFA1@DOX@HAP在1641cm-1,1590cm-1和1490cm-1处的峰是羧基 的伸缩振动和平面弯曲振动。此外,612cm-1处的峰是HAP羰基的伸缩振动,1002cm-1处的峰是HAP的PO4 3-特征峰。说明靶向荧光染料很好的修饰到材料的表面。
2.3Zeta电位的测定
Zeta电位的测量在纳米粒度及Zeta电位分析仪(Nanozs90,UK)上进行。测试 参数为:温度25℃,测试数遍取平均值。测试结果如图3所示,由于羟磷灰石几乎是 电中性的,故测其Zeta电位接近于零,与阿霉素掺杂后,电位变化不大;在掺杂阿霉 素的羟磷灰石材料表面修饰染料分子后,电位发生变化,Zeta值变大,表明靶向染料 分子已经成功的修饰在DOX@HAP纳米颗粒表面。
2.4荧光染料DFA1的光谱测试
称取一定质量的荧光染料DFA1,将称好的物质分别移入1.5ml的棕色容量瓶里面,用DMSO定容,配成浓度2.5mM的母液。采用量程为10μL微量进样器吸取3μL 母液,移入3ml测试体系石英池中,搅拌均匀,测其吸收和发射波长。测试参数为: 荧光染料的最大吸收波长为440nm,最大发射波长为550nm。
使用上述配置的母液进行选择性实验,选择不同的氨基酸以及阴阳离子进行检测, 检测时间为30min。测试参数为:荧光染料的最大吸收波长为440nm,最大发射波长 为550nm。
荧光染料DFA1的时间响应时间曲线和浓度滴定实验,如图4a,4b所示。荧光染 料与DFA1与GGT的反应时间为40min;浓度实验中,DFA1在15mg/ml GGT下即实 现饱和。在响应的氨基酸与阴阳离子选择性和竞争性实验中,如图4(c)和4(d) 所示,表明DFA1具备优异的选择性,对相对应得氨基酸和阴阳离子没有响应;再继续 加入GGT后,荧光信号发生了明显响应。综上所述,DFA1荧光染料是一个高选择性 和高灵敏性的GGT识别荧光染料。
实施例3.表面修饰靶向荧光染料的载药羟基磷灰石光谱性质测试
称取一定质量的DFA1@DOX@HAP,将称好的物质分别移入1.5ml的棕色容量 瓶里面,用DMSO定容,配成浓度分别为5mg/mL的母液。采用量程为10μL微量进 样器吸取3μL母液,移入3ml测试体系石英池中,搅拌均匀,测其吸收和发射波长。 如图5和图6的测试结果表明,当DOX@HAP的表面修饰上靶向肿瘤细胞表面的荧 光染料之后,阿霉素的荧光基本上被完全淬灭,究其原因是DOX@HAP被荧光染料完 全包覆,泄漏率降低。将材料母液进行GGT酶检测,逐次加入1-50mg GGT,检测时 间间隔为30min;当荧光信号不变后,降低pH 7.4到5.5,在保持搅拌的情况下检测 阿霉素荧光信号,时间间隔为10min。光谱测试在紫外分光光度计(AgIIlent 8453, USA)和荧光分光光度计(AgIIlent Cary EclIIpse,USA)上进行。测试参数为:荧光 染料的最大吸收波长为440nm,最大发射波长为550nm;抗癌药物的最大吸收波长为 480nm,最大发射波长为590nm。
使用上述配置的材料母液进行生物选择性实验,选择不同的生物活性物质进行检测,检测时间为30min,结果如图7所示。表明DFA1@DOX@HAP测试参数为:荧 光染料的最大吸收波长为480nm,最大发射波长为600nm。
实施例4.表面修饰靶向荧光染料的载药羟基磷灰石中药物释放测试
在不同pH值下,如图8所示,药物的释放量不同,并且随着pH的不断降低,其 药物释放量不断增大,阿霉素的荧光不断增强。并且在同一pH值,不同时间内,药物 的释放量也不断增大,当pH=5.5时,其药物的最大释放量达到了80%以上,由此说 明,合成的该纳米载体可以有效地进行药物缓慢释放,从而达到治疗肿瘤细胞的效果。
实施例5.表面修饰靶向荧光染料的载药羟基磷灰石细胞毒性测试
本实验选用噻唑蓝MTT(3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐)分 析法,以细胞的存活率来评估染料对细胞的毒性大小。分别将对数生长期的COS-7, HL-7702、HCT-116、MCF-7,HeLa以及A2780细胞以每毫升1×105个细胞浓度接种 于96孔板中,每孔加入10μL,于37℃、5%CO2条件下培养24h,分别加入2.5 mg/L,5mg/L,10mg/L,15mg/L,20mg/L的药物载体DFA1@DOX@HAP,相同条 件下再培养24h后弃去原培养液,每孔加入MTT溶液(10μL,5mg/mL)孵育4h 后,小心吸掉孔内上清液并加入200μL的DMSO,待甲瓒结晶完全溶解后,在酶标仪 上测定570nm的吸光值OD,选择490nm作为参考波长,平行测定三次。细胞存活 率=(OD实验组-OD空白对照)/(OD阳性对照-OD空白对照)×100%,空白对照为不加药物载体组。
细胞毒性是衡量纳米载体用于活细胞成像性能好坏的一项重要指标。测试结果如图9所示,从中可以看出,10mg/L纳米载体与COS-7,HL-7702、HCT-116、MCF- 7,HeLa以及A2780细胞共培养24h之后,细胞存活率分别在83%、117%、34%、 29%、24%和25%左右,表明该纳米载体对正常细胞具有较低的细胞毒性,对谷氨 酰胺转氨酶过表达的肿瘤细胞具有高细胞毒性。
实施例6.表面修饰靶向荧光染料的载药羟基磷灰石体外吸收的细胞成像实验
实验过程中用到的细胞主要有人肝细胞(HL-7702)、非洲绿猴肾细胞(COS-7)、 人结肠肿瘤细胞(HCT-116)、乳腺肿瘤细胞(MCF-7)、宫颈肿瘤细胞(HeLa)、人卵 巢肿瘤细胞(A2780)。
细胞孵育:待贴壁生长的细胞长满瓶底,倒掉培养瓶中的培养液,并用PBS缓冲 溶液洗两次。加入适量胰酶进行消化,然后加入新鲜的培养基吹掉贴壁细胞,弃去部 分细胞悬液,再加入适量新鲜的培养基,放置在37℃,5%CO2的细胞孵育箱中培养。 细胞培养基为加入10%的胎牛血清和0.1%的硫酸庆大霉素的DMEM培养基。实验 操作过程中,在细胞培养皿中以每毫升2×105个细胞密度种上细胞,置于培养箱中培 养,待用。
进行细胞成像时,将12μL母液(10mg/mL)加入到细胞培养皿中,在37℃及 5%CO2条件下培养30min后小心地用PBS溶液冲洗除去未进入细胞的染料,加入2 mL新DMEM培养基后在Olympus FV1000-IX81激光共聚焦荧光显微镜下进行观察明 场和荧光图像。测试结果如图10所示,从中可以看出,纳米载体0.5h后几乎没有进 入谷氨酰胺转氨酶低表达的细胞(COS-7)。HL-7702、HCT-116、MCF-7,HeLa以及 A2780细胞有纳米载体摄入并具有强黄色荧光。在75min后,对于谷氨酰胺转氨酶过 表达的5种细胞中,正常细胞HL-7702中红色通道没有明显信号,表明羟基磷灰石中 的药物没有释放,而其余四种肿瘤细胞中纳米载体摄入量就大大增多,红色通道荧光 强度大大增强。以上实验结果表明,纳米载体DFA1@DOX@HAP能够特异性识别谷 氨酰胺转氨酶过表达的细胞,并且对肿瘤细胞有着极强的药物释放效率。
实施例7.表面修饰靶向荧光染料的载药羟基磷灰石与癌症组织切片的细胞成像实验
实验过程中用到的组织切片选自皮下肿瘤的裸鼠内,选取适量大小的瘤体进行冰冻切片。
进行组织切片成像时,将12μL母液(10mg/mL)加入到细胞培养皿中,在37℃ 及5%CO2条件下培养4h后小心地用PBS溶液冲洗除去未进入细胞的染料,加入2 mL新DMEM培养基后在Olympus FV1000-IX81激光共聚焦荧光显微镜下进行观察明 场和荧光图像。测试结果如图11所示,从中可以看出在肝癌组织中,材料大量的吸附 在该组织表面,与癌细胞表面的GGT发生作用,从其所扫取的荧光曲线可以得出该染 料已经发生酸解,大量阿霉素得到释放;此外从组织深度成像来看,可以证明该材料 有效渗透入组织内部,可以通过红色通道观测内部阿霉素(Dox)释放,渗透深度约为 500um。以上实验结果表明,纳米载体DFA1@DOX@HAP能够特异性识别肿瘤部位 的谷氨酰胺转氨酶过表达的细胞,并且对肿瘤细胞有着极强的药物释放效率,能够很 快的渗透入组织内,渗透深度大约为0.5mm,证实纳米载体DFA1@DOX@HAP具有 应用到组织层面的能力。
Claims (9)
1.靶向荧光染料,具有通式I的结构:
通式I中,
R1和R2各自独立地选自:H、NO2、COOH、NH2、OH、C1-6烷氧基、C1-6烷基氨基、卤素或C1-6卤代烷基;
R3是NH或O。
2.根据权利要求1所述的靶向荧光染料,其特征在于,所述的R3是NH。
3.根据权利要求1所述的靶向荧光染料,其特征在于,所述的R1和R2各自独立地选自H、NO2、NH2、COOH、OH、OCH3、OC2H5、Br、Cl。
4.根据权利要求1所述的靶向荧光染料,是化合物DFA1:
5.权利要求1所述的靶向荧光染料的制备方法,其特征在于:
(1)当R3=NH时所述方法包括如下步骤:
①式II的化合物与式III的化合物在125℃条件下按照摩尔比0.1-1000:1反应4-8h,制备式IV的化合物;
②式IV的化合物与Zn粉在90℃条件下按照摩尔比0.1-100:1下反应3-6h,反应体系加入适量无水CaCl2结晶固体,制备式V的化合物;
③式V的化合物与式X的化合物在室温下按照摩尔比0.1-1000:1下反应24-72h,加入0.05当量催化剂EDCl和HOBT,制备式XI的化合物;
(2)当R3=O时所述方法包括如下步骤:
①式VI的化合物与式III的化合物在125℃条件下按照摩尔比0.1-1000:1反应4-8h,制备式VII化合物;
②式VII的化合物与乙醇钠在85℃条件下按照摩尔比1:0.1-1000反应6-24h,制备式VIII化合物;式VIII的化合物与过量的HI水溶液反应2-5h,制备式IX化合物;
③式IX的化合物与式X的化合物在室温下按照摩尔比0.1-1000:1下反应24-72h,加入0.05当量催化剂EDCl和HOBT,制备式XII化合物;
6.靶向荧光染料修饰的载药羟基磷灰石,其特征在于,所述的靶向荧光染料如权利要求1所述;所述的载药羟基磷灰石是棒状材料,平均长度是60-130nm,平均直径15-50nm;所述的载药羟基磷灰石通过靶向荧光染料与载药羟基磷灰石在乙醇-水体系中充分接触制得。
7.根据权利要求6所述的靶向荧光染料修饰的载药羟基磷灰石的制备方法,包括下述步骤:
(1)制备载药羟基磷灰石;
(2)以靶向荧光染料对载药羟基磷灰石进行表面修饰。
8.根据权利要求7所述的制备方法,包括如下步骤:
(1)向100ml浓度为15~30mg/ml、pH为10.5±0.5的Ca(NO3)2·4H2O的乙醇溶液中缓慢滴加20~50ml的浓度为6~10mg/ml的药物-水溶液,搅拌5~15min后,滴加50ml浓度为60~100mg/ml的(NH4)2HPO4水溶液,继续搅拌1~2h;混合物在室温条件下反应72-96h后,高速离心,沉淀物洗涤干燥,得到载药羟基磷灰石;
(2)将质量比1:100~5:50的200mg靶向荧光染料、载药羟基磷灰石的混合物加入到50~100ml含体积含量为25%乙醇的去离子水溶液中以1000r/min搅拌0.5~2h,继续以600r/min搅拌12~24h后,高速离心,沉淀物洗涤干燥,得到靶向荧光染料修饰的载药羟基磷灰石。
9.权利要求6所述的靶向荧光染料修饰的载药羟基磷灰石在制备肿瘤示踪及治疗制剂中的应用。
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