CN110538151A - 近红外光响应的纳米脂质体及其制备方法与在肿瘤协同治疗中的应用 - Google Patents
近红外光响应的纳米脂质体及其制备方法与在肿瘤协同治疗中的应用 Download PDFInfo
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Abstract
发明公开了一种近红外光响应的纳米脂质体及其制备方法与在肿瘤协同治疗中的应用。近红外光响应的纳米脂质体通过薄膜水化法获得负载有上转换@黑磷的脂质体,并为其表面共价结合巯基修饰的核酸适配体。该方法制备的纳米脂质体优点表现在:具有近红外光收集的纳米脂质体可以高效地将近红外光能转化为化学能和热能,实现了肿瘤靶向光动及光热协同治疗,解决了光诊断治疗在临床转化应用中单一模式治疗效果差、组织穿透性低、选择识别能力差等的局限性。近红外光响应的纳米脂质体在体内实验中具有很高的生物兼容性、更强的肿瘤特异性聚集能力、更高的肿瘤杀伤作用。
Description
技术领域
本发明属于医用纳米材料领域,具体涉及一种近红外光响应的纳米脂质体及其制备方法与在肿瘤协同治疗中的应用。
背景技术
癌症因其高发病率及高致死率,已成为人类健康的主要威胁之一。然而由于存在个体差异和癌细胞易转移等特点,肿瘤治疗无法得到根治。传统肿瘤治疗方法,如化疗、放疗和手术切除,在治疗期间通常存在各种副作用,如给患者带来巨大的创伤。目前,温和的非侵入性治疗能有效的破坏肿瘤且对正常组织损伤最小而受到广泛关注。其中,光热力学治疗(PTT)及光动力学治疗(PDT)是恶性肿瘤非侵入性治疗中的两个主要方法。在PTT中,近红外(NIR)光通过纳米材料转化为局部热能,诱导细胞热疗导致癌细胞凋亡或坏死。在PDT中,光敏剂在适当的波长照射下产生细胞毒性单线态氧(1O2)或活性氧引起局部细胞死亡和组织破坏。由于PTT及PDT具有相似的光诱导条件,而且为了克服单一治疗在临床转化应用中固有的局限性(如光照射时间长、功率大,光敏剂易猝灭,氧气含量低等),迫切需要开发一种光响应的纳米复合物能将两者结合以达到协同治疗的目的。
脂质体是具有双分子层结构的封闭囊泡。作为最成功的纳米药物递送平台之一,脂质体已被用于传递各种各样的小分子、基因、蛋白质、甚至纳米粒子等。目前,脂质体作为成像剂载体已广泛应用于许多现有的医学成像技术,包括荧光、磁共振、超声和核磁共振成像应用。作为一种药物递送平台,脂质体在临床中也得到了广泛的应用。迄今为止,已经有几种脂质体产品被批准用于治疗多种疾病,包括真菌感染、疼痛管理、甲型肝炎、流感和各种癌症。与其他递送系统相比,脂质体具有优异的生物相容性、生物降解性、较低的毒性、尺寸可控和表面功能化等特点。对脂质体组成、对载体分子种类及对多种识别试剂表面功能化的控制,使体内疾病标志物的早期检测、医学成像模式的增加及癌症治疗的改善等取得了很大进展。近年来,许多策略利用肿瘤微环境的多种特点,侧重于将响应分子整合到脂质体中实现pH、酶促、温度和超声波等触发释放系统,以提高载体分子在肿瘤间质的生物利用度。由于光激发的无创性治疗和远程时空控制的优势,开发光响应纳米脂质体已成为一个重要的课题。
然而,将光响应的脂质体应用于临床上进行光协同治疗仍存在以下几个问题。首先,作为PTT的光热剂及PDT的光敏剂,通常需要不同的激发波长分别用于产生热量和活性氧,两种激光连续的照射不仅延长了治疗时间给患者带来更多副作用,而且两种激光聚焦在同一位置使得治疗难度提高、治疗过程复杂。其次,许多光敏系统的光收集已经研究了几十年,而大多数光敏剂具有比较大的带隙(>1.7eV),因此需要紫外线或可见光激活。由于46%的太阳光是属于NIR光范围的,所以一半以上的太阳光无法被利用被激活。然而,目前有关NIR光收集的体系报道很少。再次,由于缺乏分子识别靶向性,光热剂及光敏剂在递送过程中仅基于增强渗透滞留效应被传递到肿瘤位点,容易分布于各个器官中导致正常细胞及组织损伤,而且光热剂及光敏剂生物兼容性差、难以穿透细胞膜,导致其药代动力学及药效动力学差,如何选择具有生物兼容性的靶向载体将光热剂及光敏剂高效地传递到肿瘤细胞又是一大挑战。
发明内容
本发明的目的在于克服现有技术存在的缺点与不足,提供一种近红外光响应的纳米脂质体及其制备方法与在肿瘤协同治疗中的应用。
为实现上述目的,本发明主要提供了下述技术方案:
第一方面,本发明提供一种近红外光响应的纳米脂质体,其特征在于:所述纳米脂质体具有均匀的球形结构,直径范围为100-200nm;所述纳米脂质体在近红外光照射下具有上转换发光性质并发出绿光及红光的可见光;所述纳米脂质体在近红外光照射下具有光敏剂与光热剂的特点并产生单线态氧及热量。
第二方面,本发明提供一种上述近红外光响应的纳米脂质体的方法,其特征在于:通过薄膜水化法获得负载有上转换@黑磷即UCNPs@BPQDs的脂质体,并为其表面连接巯基修饰的AS1411核酸适配体;所述UCNPs@BPQDs是将具有羧基的UCNPs-PAA与氨基官能化的BPQDs-NH2通过经典的EDC/NHS偶联形成酰胺键结合,包括如下步骤:
(1)二棕榈酰磷脂酰胆碱、胆固醇和1,2-二硬脂酰-sn-甘油基-3-磷酸乙醇胺-N-(马来酰亚胺-(聚乙二醇)-2000)按照摩尔比为100:50:5分散在氯仿溶液中,利用旋转蒸发仪得到一层薄薄的磷脂膜;所述磷脂膜形成的参数为120rpm、45℃旋转蒸发30min;
(2)向步骤(1)获得磷脂膜的烧瓶中加入UCNPs@BPQDs溶液,并放置于37℃水浴中水化10min,离心去除未负载上的UCNPs@BPQDs,最后利用脂质体挤出器以获得尺寸均匀的UCNPs@BPQDs@Mal-Lip,即UBML溶液;
(3)向步骤(2)获得的UBML溶液中按照摩尔比10:1加入SH-Apt溶液,4℃避光孵育过夜反应,得到近红外光响应的纳米脂质体UCNPs@BPQDs@Apt-Lip,即UBAL溶液;所述反应通过SH-Apt与Mal-Lip之间的硫醇-马来酰亚胺交联化学结合。
进一步地,所述UCNPs@BPQDs的合成包括如下步骤:
A.将分散在甲苯溶液中的UCNPs纳米颗粒缓慢加入烧瓶中并在氩气保护下反应1h;然后将溶液加热至240℃的继续反应1.5h;冷却至室温后加入乙醇得到UCNPs-PAA沉淀;
B.将机械剥离获得的BPQDs与PEG-NH2分散在去离子水中,超声处理30min并在冰/水浴下搅拌4h;最后离心除去过量的PEG-NH2得到BPQDs-NH2;所述步骤(2)中所述的离心转速及时间分别为12000rpm、20min;
C.将步骤A中合成的UCNPs-PAA分散到2-(N-吗啉代)乙磺酸缓冲液中,然后加入EDC和NHS活化羧基;离心并用水洗涤后,将UCNPs-PAA重新置于PBS缓冲液中,在超声处理下加入BPQDs-NH2溶液,在摇床中30℃反应12h;通过离心获得UCNPs@BPQDs纳米颗粒;所述MES及PBS缓冲液的pH分别为5.5、7.2;所述羧基活化时间为15-30min。
第三方面,本发明还提供一种上述的近红外光响应的纳米脂质体在肿瘤光动及光热协同治疗中的应用。该近红外光响应的纳米脂质体可用于肿瘤光动及光热协同治疗的方法具体包括如下步骤:
(1)购买4周龄雌性Balb/c裸鼠进行肿瘤接种,将乳腺癌细胞皮下注射到每只雌性Balb/c小鼠的右上肢。
(2)肿瘤细胞注射后第5天肿瘤生长明显。为了评估不同处理对肿瘤生长的抑制作用,将携带皮下4T1肿瘤的雌性Balb/c小鼠随机分组,并在不同的条件下分别进行肿瘤内注射:I,DPBS;II,NIR;III,UBAL;IV,BPQDs@Apt-Lip(BAL)+NIR;V,UBAL+NIR。
(3)注射后,将小鼠麻醉并用NIR激光照射肿瘤。在激光照射后,通过红外热成像相机同时监测肿瘤的温度。
(4)每2天测量肿瘤大小及小鼠体重,将这些数据绘制为时间的函数。
(5)在第14天,处死小鼠,取小鼠的肿瘤及主要器官通过苏木精-伊红(H&E)染色检查损伤情况。
上述方法中步骤(4)所述的肿瘤大小计算公式如下:体积(V)=(肿瘤长度)×(肿瘤宽度)2/2。
所述UBAL喂食一个月期间没有发现体重变化的显着差异,主要器官(心脏,肝脏,脾脏,肺和肾)的组织没有明显的病理异常,该近红外光响应的纳米脂质体具有较高的体内生物相容性。
所述UBAL通过纳米药物增强的渗透与滞留效应以及核酸适配体与核仁素的特异性识别和结合效应,在肿瘤部位具有明显的荧光信号,并且信号强度在2h达到最大值,具有更好的肿瘤内部富集能力。
所述UBAL在NIR光照射下显着产生活性氧且具有高效的光热转换能力,可以有效地损伤肿瘤组织,抑制肿瘤生长。
本发明具有以下优点和有益效果:
(1)本发明使用的单个激光进行肿瘤的协同治疗使得治疗时间短、治疗过程简单及治疗难度降低。
(2)本发明提供的近红外光响应能增加组织穿透性,在深层肿瘤组织治疗中具有巨大的实用前景。
(3)本发明为近红外光响应的纳米脂质体增加了靶向肿瘤细胞的核酸适配体,避免正常细胞及组织损伤,减少治疗过程中的副作用
(4)本发明的近红外光响应的纳米脂质体具有良好的生物兼容性,可将光热剂及光敏剂高效递送到肿瘤组织,且表现出显著的治疗效果,因此它非常适于肿瘤组织的成像及治疗应用。
附图说明
图1为UBAL的制备过程及其在肿瘤光动及光热协同治疗的示意图。
图2为UBAL的透射电子显微镜图。
图3为UBAL的原子力显微镜图及图中相应部位的高度分布。
图4为UBAL的近红外光响应性能。
其中,a.BPQDs的紫外-可见光谱、UCNPs及UBAL的上转换光谱;b.Er3
+
从4F9/2跃迁发
射波长为662nm的荧光衰减曲线。
图5为UBAL的光动及光热性质。
其中,a.通过检测DPBF在410nm处漂白程度评估UBAL在808nm激光(1.5Wcm-2)照射
下产生1O2的能力;b.不同浓度UBAL在808nm激光(1.5Wcm-2)照射下的光热曲线。
图6为UBAL的选择性识别功能。
其中,流式细胞术分析FAM标记的UBAL/UBCL选择性识别MCF-7细胞结果;流式细胞
术分析FAM标记的UBAL/UBCL选择性识别HEK293细胞结果。
图7为BAL或UBAL孵育的MCF-7细胞在808nm激光照射不同时间下的相对生存活力。
其中,数据表示为平均值±SD(*P<0.05,**P<0.01)。
图8为BAL或UBAL孵育的MCF-7细胞在808nm激光照射不同时间下的共聚焦荧光图像。
其中,Calcein-AM(活细胞,绿色荧光)和PI(死细胞,红色荧光)染色。比例尺,100μ
m。
图9为UBAL的体内长期生物兼容性研究。
其中,a.不同剂量的UBAL处理的小鼠血液中肝功能标志物分析。丙氨酸氨基转移
酶(ALT),碱性磷酸酶(ALP)和天冬氨酸氨基转移酶(AST)的血液水平作为肝功能标志物。b.
不同剂量的UBAL处理的小鼠血液中肾功能标志物分析。肌酸酐(CREA),血尿素氮(BUN)和尿
酸(UA)的血液水平作为肾功能标志物。
图10为在不同剂量的UBAL处理的小鼠主要器官(心脏,肝脏,脾脏,肺和肾)的组织学检查。比例尺,200μm。
图11为UBAL或UBCL对荷瘤小鼠的肿瘤靶向聚集成像。
图12为荷瘤小鼠注射不同样品后在808nm激光照射(1.5W cm-2)下的肿瘤部位红外热成像图。
图13为UBAL的肿瘤的抑制效果。
其中,a.肿瘤体积变化曲线。b.处死后的小鼠肿瘤组织照片。
图14为UBAL对肿瘤抑制后各器官的苏木精-伊红(H&E)染色。
具体实施方式
下面结合实施例及附图对本发明做进一步详细的描述,但本发明的实施方式不限于此。
实施例1:基于上转换@黑磷近红外光响应的纳米脂质体(UCNPs@BPQDs@Apt-Lip,UBAL)的制备及表征:
(1)在100mL三口烧瓶中加入30mL二甘醇(DEG)和300mg PAA,在真空条件下加热到110℃,将分散在甲苯溶液中的100mg UCNPs纳米颗粒缓慢加入烧瓶中并在氩气保护下反应1h。然后将溶液加热至240℃并继续反应1.5h。冷却至室温后加入乙醇得到UCNPs-PAA沉淀。
(2)将200mg块状BP加入到300mL N-甲基-2-吡咯烷酮(NMP)中。在氩气保护下,用超声波细胞粉碎仪(超声频率:19-25kHz)在冰/水浴中超声处理8h(2s,间隔0.1s)再持续超声10h(2s,间隔为4s)得到棕色溶液。7000rpm下离心20min以除去未剥离的BP,将含BPQDs的上清液以12000rpm离心20min,收集沉淀并重悬于水溶液中。将1mg BPQDs与5mg PEG-NH2分散在5mL去离子水中,超声处理30min并在冰/水浴下搅拌4h。最后离心除去过量的PEG-NH2得到BPQDs-NH2。
(3)将10mg上述步骤(1)中合成的UCNPs-PAA分散到10mL 2-(N-吗啉代)乙磺酸(MES)缓冲液(10mM,pH=5.5)中,然后加入10mM EDC和25mM NHS,30℃下振荡15min。离心并用水洗涤后,将UCNPs-PAA重新置于10mL PBS缓冲液(10mM,pH=7.2)中,在超声处理下将2.5mg BPQDs-NH2加入溶液中,在摇床中30℃反应12h。通过离心获得UCNPs@BPQDs纳米颗粒。
(4)二棕榈酰磷脂酰胆碱(DPPC)、胆固醇和1,2-二硬脂酰-sn-甘油基-3-磷酸乙醇胺-N-(马来酰亚胺-(聚乙二醇)-2000)(DSPE-PEG(2000)-Mal)按照摩尔比为100:50:5分散在氯仿溶液中,利用旋转蒸发仪并设置相关参数,120rpm、45℃旋转蒸发30min,得到一层薄薄的磷脂膜。
(5)向步骤(4)获得磷脂膜的烧瓶中加入2mL 10mg UCNPs@BPQDs的PBS溶液,并放置于37℃水浴中水化10min,4000r/min离心10min去除未负载在Mal-Apt中的UCNPs@BPQDs,最后选择400nm的聚碳酸酯膜、利用脂质体挤出器以获得尺寸均匀的UBML溶液。
(6)向步骤(5)获得的UBML溶液中按照摩尔比10:1加入SH-Apt溶液,4℃避光孵育过夜,得到UBAL近红外光响应的纳米脂质体。
UBAL近红外光响应的纳米脂质体的构建及其在肿瘤治疗中的应用示意图如图1所示。将得到的UBAL水溶液进行透射电子显微镜(TEM)及原子力显微镜(AFM)表征。TEM表征结果(图2)显示UBAL具有均匀球形结构,直径约为100到200nm之间,且均显示出UCNPs@BPQDs成功的负载在脂质体里面。AFM图像(图3)也显示了UBAL的球形形貌,测量高度约为50nm。
进一步检测了UBAL在近红外照射下能量转移、产生1O2及光热转换的性质。从图4中a中可以看出,BPQDs具有宽的吸收光谱,涵盖了UCNPs的整个发射区域。BPQD偶联到UCNPs表面后,UBAL整个发射区域的发射光强度减少,表明UCNPs向BPQDs的有效能量转移。在近红外激发下,UCNPs和UCNPs@BPQDs上转换光波长为662nm(4F9/2-4I15/2跃迁)处的发光寿命从0.59ms降低到0.47ms(图4中b),也进一步证实了能量传递过程。从图5中a中可以看出,随着辐照时间的增加,1O2敏感探针1,3-二苯异苯并呋喃(DPBF)的吸光度在410nm左右持续下降,说明UBAL有效产生1O2。从图5中b可以看出,浓度为500μg mL-1的UBAL在近红外光照射2min内温度从28℃明显增加到49℃。这些结果说明了UBAL近红外光响应的纳米脂质体在近红外光照射下具有很好的能量转移及转换功能,可以有效地产生1O2和热量。
由于表面锚定AS1411核酸适配体,利用流式细胞仪进一步检测了UBAL对靶细胞表面核仁素具有较强的结合亲和力和较好的选择性。表征结果如图6所示,FAM标记的UBAL孵育的乳腺癌细胞(MCF-7)荧光峰位移较大,具有很强的结合亲和力。然而,FAM标记的UCNPs@BPQDs@Ctrl-Lip(UBCL)对MCF-7细胞的亲和力较弱,荧光峰位移小。相比之下,UBAL或UBCL与人胚胎肾(HEK293)细胞孵育后荧光峰未发生右移。说明该UBAL近红外光响应的纳米脂质体仅对肿瘤靶细胞具有较强的结合亲和力,避免了正常细胞的非特异性识别作用。
由于具有良好的能量转换能力和细胞靶向能力,评估了UBAL在光动和光热协同治疗中的性能。如图7所示,当MCF-7细胞与UBAL在808nm照射(1.5Wcm-2)下孵育不同时间时,观察到MCF-7细胞的时间依赖性治疗效果。此外,如图8所示,Calcein-AM&PI活死细胞染色结果也显示,相比于UAL孵育的MCF-7细胞,当在808nm照射下用UBAL处理细胞10min时,几乎所有细胞都被杀死。说明UBAL近红外光响应的纳米脂质体具有显着的肿瘤细胞治疗效果。
实施例2:UBAL近红外光响应的纳米脂质体在体内的生物兼容性:
健康的雌性昆明鼠(20g)尾静脉分别注射100μL 5mg/kg、10mg/kg、25mg/kg的UBAL,100μL生理盐水作为阴性对照。每隔2天称量一次小鼠体重(小鼠体重-时间曲线)。28后杀死小鼠,收集每只小鼠0.8mL血液进行血常规和血生化分析,且收集每只小鼠主要的器官,10%中性福尔马林固定,石蜡包埋,切片厚度8μm,用苏木素和伊红染色,数字显微镜观察。
体内生物相容性可以进一步表征UBAL在临床转化的潜力。在喂食一个月后进行血液的常规检测,并通过苏木精和伊红(H&E)染色评估活组织检查。从图9可以看出,血液生化检查显示注射UBAL的小鼠的肝功能指数和肾功能指标与对照组相比没有显着差异和异常。此外,与对照小鼠相比,主要器官(心脏,肝脏,脾脏,肺和肾)的组织没有明显的病理异常(图10),这体现了UBAL近红外光响应的纳米脂质体的高组织相容性。这些结果清楚地表明UBAL近红外光响应的纳米脂质体具有高生物相容性。
实施例3:UBAL近红外光响应的纳米脂质体可用于肿瘤组织荧光成像、肿瘤光动及光热治疗:
(1)4周龄雌性Balb/c裸鼠(18-20g)进行肿瘤接种,将悬浮在DPBS中密度为1×107的乳腺癌细胞皮下注射到每只雌性Balb/c小鼠的右上肢。肿瘤细胞注射后第5天肿瘤生长明显。
(2)Balb/c裸鼠尾部静脉注射UBAL、UBCL(每只裸鼠100μL 5mg/mL),使用808nmNIR光作为激发源进行体内荧光成像,并用荧光成像系统进行不同时间的小鼠成像。如图11所示,当UBAL通过尾静脉注射到携带4T1肿瘤的小鼠中时,在肿瘤部位可以看到明显的荧光信号,并且信号强度在2h达到最大值。在注射UBCL的小鼠中信号强度较暗并且消失得更快,表明UBAL具有比UBCL更好的肿瘤累积能力。
(3)为了评估不同处理对肿瘤生长的抑制作用,在不同的条件下分别进行肿瘤内注射:I,DPBS;II,NIR;III,UBAL;IV,BAL+NIR;V,UBAL+NIR。注射后,将小鼠麻醉并用808nmNIR激光(1.5W cm-2)照射肿瘤5min。在激光照射后,通过红外热成像相机监测肿瘤部位的温度变化。如图12所示,与前三组相比,热成像数据显示IV组和V组小鼠的肿瘤温度迅速上升至50℃并显示肿瘤区域最严重的烧伤,表明高BAL和UBAL的肿瘤定位可以在NIR光照射下产生显着的光热转换。
(4)每2天通过卡尺测量肿瘤大小:体积=(肿瘤长度)×(肿瘤宽度)2/2,且每2天测量小鼠的体重,然后将这些数据绘制为与时间相关的曲线,评价UBAL与不同样品的体内肿瘤杀伤效果。在第14天,处死小鼠,取小鼠的肿瘤及主要器官(心脏,肝脏,脾脏,肺,肾和胃)。如图13中a所示,与前四组相比,用UBAL和NIR光(V组)治疗的小鼠显示出肿瘤体积增长最慢,证实了NIR光响应的UBAL近红外光响应的纳米脂质体的光动力学和光热治疗作用具有良好的抗肿瘤效率。如图13中b所示,从V组小鼠中分离的肿瘤具有最小的体积,清楚地证明NIR光调节的UBAL可以有效地抑制肿瘤生长。
(5)通过H&E染色检查不同样品组处理后的肿瘤组织的组织学变化。如图14所示,对于肿瘤组织,在V组小鼠的肿瘤组织上观察到细胞间隙的不规则扩大,这清楚地表明NIR光调节的UBAL近红外光响应的纳米脂质体可以有效地损伤肿瘤组织。
上面结合附图对本发明的实施方式作了详细说明,但是本发明并不限于上述实施方式,在本领域的普通技术人员所具备的知识范围内,还可以在不脱离本发明宗旨的前提下做出各种变化。
Claims (4)
1.一种近红外光响应的纳米脂质体,其特征在于:所述纳米脂质体具有均匀的球形结构,其直径范围为100-200nm;所述纳米脂质体在近红外光照射下具有上转换发光性质并发出绿光及红光的可见光;所述纳米脂质体在近红外光照射下具有光敏剂与光热剂的特点并产生单线态氧及热量。
2.一种制备如权利要求1所述近红外光响应的纳米脂质体的方法,其特征在于:通过薄膜水化法获得负载有上转换@黑磷即UCNPs@BPQDs的脂质体,并为其表面连接巯基修饰的AS1411核酸适配体;所述UCNPs@BPQDs是将具有羧基的UCNPs-PAA与氨基官能化的BPQDs-NH2通过经典的EDC/NHS偶联形成酰胺键结合,包括如下步骤:
(1)二棕榈酰磷脂酰胆碱、胆固醇和1,2-二硬脂酰-sn-甘油基-3-磷酸乙醇胺-N-(马来酰亚胺-(聚乙二醇)-2000)按照摩尔比为100:50:5分散在氯仿溶液中,利用旋转蒸发仪得到一层薄薄的磷脂膜;所述磷脂膜形成的参数为120rpm、45℃旋转蒸发30min;
(2)向步骤(1)获得磷脂膜的烧瓶中加入UCNPs@BPQDs溶液,并放置于37℃水浴中水化10min,离心去除未负载上的UCNPs@BPQDs,最后利用脂质体挤出器以获得尺寸均匀的UCNPs@BPQDs@Mal-Lip,即UBML溶液;
(3)向步骤(2)获得的UBML溶液中按照摩尔比10:1加入SH-Apt溶液,4℃避光孵育过夜反应,得到近红外光响应的纳米脂质体UCNPs@BPQDs@Apt-Lip,即UBAL溶液;所述反应通过SH-Apt与Mal-Lip之间的硫醇-马来酰亚胺交联化学结合。
3.根据权利要求2所述的近红外光响应的纳米脂质体的制备方法,其特征在于:所述UCNPs@BPQDs的合成包括如下步骤:
A.将分散在甲苯溶液中的UCNPs纳米颗粒缓慢加入烧瓶中并在氩气保护下反应1h;然后将溶液加热至240℃的继续反应1.5h;冷却至室温后加入乙醇得到UCNPs-PAA沉淀;
B.将机械剥离获得的BPQDs与PEG-NH2分散在去离子水中,超声处理30min并在冰/水浴下搅拌4h;最后离心除去过量的PEG-NH2得到BPQDs-NH2;所述步骤(2)中所述的离心转速及时间分别为12000rpm、20min;
C.将步骤A中合成的UCNPs-PAA分散到2-(N-吗啉代)乙磺酸缓冲液中,然后加入EDC和NHS活化羧基;离心并用水洗涤后,将UCNPs-PAA重新置于PBS缓冲液中,在超声处理下加入BPQDs-NH2溶液,在摇床中30℃反应12h;通过离心获得UCNPs@BPQDs纳米颗粒;所述MES及PBS缓冲液的pH分别为5.5、7.2;所述羧基活化时间为15-30min。
4.一种如权利要求1所述的近红外光响应的纳米脂质体在肿瘤协同治疗中的应用,其特征在于:所述协同治疗为肿瘤光动及光热协同治疗。
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