CN107412787A - 一种用于光学治疗的光敏剂靶向纳米粒及其制备方法和应用 - Google Patents
一种用于光学治疗的光敏剂靶向纳米粒及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种用于光学治疗的光敏剂靶向纳米粒及其制备方法和应用,该靶向纳米粒的通式为HA‑PAMAM‑COOH‑ICG,其中HA为透明质酸,PAMAM为树枝状聚合物;COOH为羧基,ICG为吲哚菁绿。本发明的纳米粒的稳定性好,用药后通过靶向作用达到了药物局部浓度高的优点,基本无毒副作用,不仅可以进行荧光成像研究药物在肿瘤内蓄积情况,还可以作为光敏剂发挥PTT/PDT疗效。
Description
技术领域
本发明属于纳米生物医学技术领域,具体而言,涉及一种用于光学治疗的光敏剂靶向纳米粒及其制备方法和应用,尤其涉及一种以树枝状聚合物作为载体的吲哚菁绿靶向纳米粒子及其制备方法和应用。
背景技术
黑素瘤是由异常黑素细胞过度增生引发的皮肤肿瘤,恶性程度极高,预后较差,其病死率占皮肤肿瘤死亡病例的80%左右。近年来发病率不断增加,年增长率为3~5%,且具有年轻化的趋势(Siegel RL,Miller KD,Jemal A.Cancer statistics,2016.CA Cancer JClin.2016,66(1):7-30.)。临床上治疗黑素瘤常用的化疗药物有达卡巴嗪、替莫唑胺(TMZ)、铂类、紫杉醇、福莫司汀等,然而疗效并不令人满意。其中,占主导地位的达卡巴嗪有效率仅为7.5%~12.2%,患者中位生存期5~6月(Gogas H J,Kirkwood J M,Sondak VK.Chemotherapy for metastatic melanoma.Cancer.2007,109(3):455-464.)。替莫唑胺是一种新型的第2代口服烷化剂,因其可口服,生物利用度高,能透过血脑屏障,对脑转移有治疗和预防作用,被很多国家推荐用于黑素瘤一线治疗(Agarwala S S,Kirkwood JM.Temozolomide,a novel alkylating agent with activity in the central nervoussystem,may improve the treatment of advanced metastatic melanoma.Theoncologist.2000,5(2):144-151.)。TMZ通过DNA链甲基化,引起DNA单链或双链断裂,阻断DNA复制,最终导致肿瘤细胞死亡。然而,同其它化疗药物一样,TMZ的疗效有限,且由于缺少靶向性,TMZ在杀伤肿瘤细胞的同时对其他非肿瘤细胞也有杀伤作用,引起毒副作用。药代动力学显示口服TMZ后血浆药物浓度于1h内达峰,之后消除迅速,平均半衰期为1.8h。TMZ在生理环境下很容易降解,在肿瘤部位的酸性环境中反而较稳定(郑蓉,蒋德锡,廖心丹,等.温度及pH对替莫唑胺溶液稳定性的影响.华西药学杂志.2012,(05):550-551.)。此外,TMZ作用机制会启动细胞内修复机制,上调O6-甲基鸟嘌呤-DNA-甲基转移酶(MGMT)基因表达,后者通过自身活性中心的半胱氨酸残基与O6-甲基鸟嘌呤上的烷基基团共价结合并将其移除,从而使损伤的DNA修复,导致肿瘤细胞对TMZ耐药(Bradbury PA,Middleton MR.DNArepair pathways in drug resistance in melanoma.Anti-Cancer Drugs.2004,15(5):421-426.)。因此,如何提高TMZ的稳定性和敏感性,增强其靶向性从而降低其副作用是目前研究的热点与难点,也是提高黑素瘤整体疗效的关键。
近年来,纳米技术在肿瘤的诊断和治疗上的研究发展快速,越来越多的研究人员将研究焦点放于纳米技术的生物医学应用上,利用纳米粒独特的结构和理化性质为恶性肿瘤的诊断和治疗提供新的策略和手段。目前研究较多的用作药物载体的纳米材料有脂质体、聚合物胶束、树枝状聚合物、二氧化硅、金纳米粒等,其中聚酰胺-胺(polyamidoamine,PAMAM)是一种人工合成的纳米级大分子化合物。Tomalia等(Tomalia D,Baker H,DewaldJ,et al.A New Class of Polymers:Starburst-Dendritic.Polymer Journal.1985,17(1):117-132.)首次研究的树枝状高分子PAMAM,是以乙二胺为核,通过Michael加成反应和对酯基的酰胺化反应这两个重复的步骤合成的。但是PAMAM表面的正电荷对细胞具有一定的细胞毒性和溶血毒性(Imae T,Hamaguchi S.Network of sodium hyaluronate withnano-knots junction of poly(amido amine)dendrimer[J].Carbohydratepolymers.2012,88(1):352-360.),因此需要进一步修饰才能更好的发挥作用。Kim等(KimT,Seo H J,Choi J S,et al.PAMAM-PEG-PAMAM:novel triblock copolymer as abiocompatible and efficient gene delivery carrier.Biomacromolecules.2004,5(6):2487-2492.)用PEG修饰PAMAM来降低其正电荷毒性,细胞活力实验证明修饰PEG后的PAMAM毒性明显降低。然而,PEG的存在会导致纳米粒的粒径急剧增大(Jiang G,Li R,TangJ,et al.Formulation of temozolomide-loaded nanoparticles and their targetingpotential to melanoma cells.Oncology reports,2016.),影响纳米载体的被动靶向性和肿瘤渗透性。另外,经PEG修饰后的载体会在一定程度上降低药物的释放,且对PAMAM本身的酸敏感性及质子海绵效应造成一定的影响,这将降低药物的抗肿瘤效果。再者,纳米载体负载化疗药物用于抗肿瘤治疗在近几十年里取得了较大的成绩,但是单一治疗模式仍然有很多不足,例如药物释放不良、需要多次给药、疗效差等。将两种或多种不同的肿瘤治疗方法结合起来,不仅能彼此促进、提高疗效,还能减少副作用,其中,联合光学治疗与化疗已得到广泛的关注与研究。同传统的治疗手段相比,光学治疗具有创伤小、可定点治疗、毒副作用少、无耐药性和可多次重复治疗的优点。而利用具有较强组织穿透能力的近红外光的光学治疗联合化疗是纳米医学领域中重要的研究方向。
吲哚菁绿是经美国FDA批准可以用于临床近红外成像的有机小分子,属于一种良好的光敏剂,在NIR照射下可以产生单线态氧发挥肿瘤杀伤作用。然而,吲哚菁绿在生理介质中不稳定,容易与培养基或者血液中的蛋白质发生非特异性吸附而被清除(ChristianDA,Cai S,Bowen DM,et al.Polymersome carriers:from self-assembly to siRNA andprotein therapeutics.Eur J Pharm Biopharm.2009,71(3):463-474.),且其同样不具有靶向性,因此不能特异性的进入病灶局部,这些缺点限制了其应用。
发明内容
鉴于现有技术的不足,本发明的目的在于提供一种不仅可以进行荧光成像研究药物在肿瘤内蓄积情况,还可以作为光敏剂发挥PTT/PDT疗效的光敏剂靶向纳米粒及其制备方法和应用。该光敏剂靶向纳米粒的稳定性好,用药后通过靶向作用达到了药物局部浓度高的优点,同时基本无毒副作用。
为了提高光敏剂靶向纳米粒的生理稳定性和用药后的肿瘤局部浓度,本发明人通过大量试验研究并不懈努力,创造性地采用聚酰胺-胺树状大分子作为载体,使用丁二酸酐来对PAMAM进行修饰,并将HA包裹在载体表面,所获得的纳米载体将吲哚菁进行装载,最终获得了如下技术方案:一种用于光学治疗的光敏剂靶向纳米粒,该靶向纳米粒的通式为HA-PAMAM-COOH-ICG,其中HA为透明质酸,PAMAM为树枝状聚合物;COOH为羧基,ICG为吲哚菁绿。
进一步优选地,如上所述用于光学治疗的光敏剂靶向纳米粒,其中的树枝状聚合物为以乙二胺为核的第1-10代聚酰胺-胺树状大分子。
再进一步优选地,如上所述用于光学治疗的光敏剂靶向纳米粒,其中的树枝状聚合物为以乙二胺为核的第4-6代聚酰胺-胺树状大分子。再进一步优选地,所述用于光学治疗的光敏剂靶向纳米粒,其中的树枝状聚合物为以乙二胺为核的第5代聚酰胺-胺树状大分子。
再一方面,本发明还提供了一种上用于光学治疗的光敏剂靶向纳米粒的制备方法,该方法包括如下2个步骤:
(1)PAMAM-COOH的合成;将PAMAM溶解于甲醇中,旋干,并加入无水DMSO重新溶解,形成反应体系;取丁二酸酐溶于无水DMSO中,滴加入反应体系内,室温搅拌12-24h,使用Mw=3000-4000Da的透析袋透析2-4天后,冷冻干燥,得到产物PAMAM-COOH。
(2)HA-PAMAM-COOH-TMZ-ICG的合成:将PAMAM-COOH与ICG共溶于去离子水中,得到A液;将HA共溶于去离子水中,得到B液;B液置于功率为150-300W的探头超声下,一边超声一边将A液缓慢加到B液中,超声5-20min后,将超声后得到的液体超滤,超滤后用去离子水洗1-4次,冻干,得到用于光学治疗的光敏剂靶向纳米粒HA-PAMAM-COOH-ICG。
与现有技术相比,本发明采用聚酰胺-胺树状大分子作为载体,使用丁二酸酐来对PAMAM进行修饰,将PAMAM表面的大部分氨基变为更加安全的羧基,从而降低其毒性。红外光谱和核磁氢谱结果显示,PAMAM表面的氨基成功地被羧基取代,取代率为67%,降低了PAMAM的正电荷毒性,DLS结果显示纳米载体的Zeta电位由40.1mV变为12.3mV,而其粒径大小并没有发生明显的变化。为了进一步减少纳米载体表面的正电荷,同时提高其稳定性和药物包封的效率,我们将HA包裹在载体表面。DLS和TEM结果表明,HA包封的载体大小均匀,呈单分散状态,Zeta电位进一步减小,变为-6.4mV。ICG的包封率和载药率分别为99.87%和28.55%,升温曲线和单线态氧测定HPCI纳米粒在808nm波长近红外激光照射下能使温度升高和单线态氧产量增加;成像实验显示HPCI纳米粒对肿瘤具有良好的靶向作用。
为了研究合成的纳米粒对肿瘤的靶向性,我们构建了人黑素瘤A375移植肿瘤模型,尾静脉注射HPCI后进行荧光成像,实验结果显示其可以在肿瘤内部高浓度、长时间蓄积,并在其他脏器无明显蓄积。有文献报道,ICG在乳腺癌荷瘤裸鼠体内的循环时间约为24h,大部分都聚集在肝脏中。Mundra等[36]分别研究了游离ICG和ICG胶束在A375移植瘤裸鼠体内蓄积情况,结果显示在24h时,游离ICG组在肿瘤内荧光强度很弱,而在肝脏和肾脏内荧光信号较强。本发明的实验中,在剥离下来的肿瘤中,72h时HPCI在肿瘤仍有明显蓄积,而在其他脏器中量很少,进一步说明该体系的靶向性良好。
为了评价本实验中的纳米粒的联合化疗和光学治疗的疗效以及有无其他毒副作用,我们进行了荷瘤裸鼠的体内治疗实验。通过肿瘤生长曲线和肿瘤照片结果显示,对照组肿瘤以较快速度持续生长,NIR组在治疗第1周肿瘤生长有所抑制,我们推测是由于激光照射使肿瘤局部温度轻度升高,这在热成像实验结果得到证明,升高的温度会影响肿瘤细胞代谢而抑制生长,之后肿瘤生长加速。TMZ组同样是在第一周内肿瘤生长缓慢,随后肿瘤生长明显加速,说明单次给药后,TMZ能发挥部分治疗作用,但随着其在体内很快被代谢清除,血药浓度下降,肿瘤继续生长。HPCT组总体趋势与TMZ相似,但最终肿瘤体积明显小于TMZ组,说明HPCT在体内可长时间持续释药,在一定程度上抑制肿瘤生长。HPCI+NIR组肿瘤生长明显受到抑制,表明PTT/PDT作用显著,但由于治疗深度有限,因此单次治疗并不能杀死所有的癌细胞,治疗结束后一段时间有复发趋势。HPCT+HPCI+NIR组在治疗结束时肿瘤大部分消退,疗效优于HPCI+NIR组和HPCT单独治疗组,我们推测当激光照射后HPCI发挥PTT/PDT作用杀伤肿瘤细胞,温度升高可进一步促进载体内TMZ的释放,继续杀死未被PTT/PDT杀死的肿瘤细胞,实现化疗和PTT/PDT联合治疗作用。纳米药物的安全性问题制约着其在生物医学方面的应用。
附图说明
图1为PC纳米粒合成和鉴定结果图。A:载药纳米粒的合成过程;B:PAMAM和PC的1HNMR图谱;C:PAMAM、PC和HPC的红外光谱图。
图2为PAMAM、PC、HPCT纳米粒的表征结果图。DLS测定PAMAM(A)、PC(B)、HPCT(C)的水合粒径和Zeta电位;HPCT的透射电镜照片(D)。
图3为TMZ的含量测定结果图。A:TMZ和MTIC的紫外吸收光谱;B:TMZ的标准曲线;C:MTIC的标准曲线。
图4为HPCT在不同条件下的释药曲线结果图。
图5为HPCI纳米粒的升温曲线和单线态氧产生量的测定结果图。A:不同浓度的HPCI在NIR照射下的升温曲线;B:水、ICG(15μM)和HPCI(ICG含量为15μM)在NIR照射下的升温曲线;C:ICG(5μM)和HPCI(ICG含量为5μM)在NIR照射下的单线态氧产量。
图6为CCK-8法测定纳米粒对A375细胞的杀伤作用。A:不同浓度TMZ对A375细胞的杀伤作用;B:合成的纳米粒对A375细胞的杀伤作用。
图7为HPCI经静脉注射后在荷瘤裸鼠体内分布和组织蓄积结果图。A:HPCI在裸鼠体内不同时间的分布B:72h时HPCI在裸鼠各脏器中的蓄积。
图8为HPCT和HPCI单独或联合治疗对黑素瘤的治疗效果图。A:对照组和联合治疗组的热成像照片;B:肿瘤生长曲线;C:各组肿瘤剥离后照片;D:治疗结束后裸鼠照片。
具体实施方式
下面通过具体实施方式对本发明作进一步详细说明。但本领域技术人员将会理解,下列实施例仅用于说明本发明,而不应视为限定本发明的范围。另外,实施例中未注明具体技术操作步骤或条件者,均按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者,均为可以通过市购获得的常规产品。
实施例1:PAMAM-COOH的合成及表征
PAMAM表面的氨基所带的正电荷使其具有一定的细胞毒性,为了使其具有更好的生物相容性,可对其进行表面修饰。本研究拟使用丁二酸酐来对PAMAM进行改性,将PAMAM表面的大部分氨基变为更加安全的羧基,从而得到PC纳米粒(图1A)。
合成方法为:将1.5mL PAMAM-G5.0(290mg,0.01mmol)的甲醇溶液置于单口烧瓶中,旋干,并加入1mL无水DMSO重新溶解。使丁二酸酐(640mg,6.4mmol)溶于1mL无水DMSO中,滴加入反应体系内,室温搅拌24h。使用Mw=3500Da的透析袋透析三天后,冷冻干燥,得到产物PAMAM-COOH(PC)。利用傅里叶红外光谱仪分析产物的红外光谱测定聚合物官能团,以固体溴化钾粉末作为稀释剂,压片测试。使用核磁共振仪分析测定聚合物的核磁氢谱(1HNMR),以D2O为溶剂,TMS为内标。
核磁氢谱结果显示,在2.3ppm处出现了丁二酸酐的-CH2-的质子振动峰。根据峰面积比计算得出丁二酸酐的取代度为67%(图1B)。红外光谱结果显示,与PAMAM相比,PC在1257cm-1处出现了羧基的伸缩振动峰,表明了羧基已被成功修饰到纳米粒表面;为了增加纳米粒的稳定性,提高其对药物的包封能力,我们在其表面修饰HA,合成了HA-PAMAM-COOH(HPC)。红外光谱显示HPC的光谱与PC相比,在1084和1045cm-1处出现了HA上羟基的伸缩振动峰,说明了成功将HA包裹在PC的表面(图1C)。以上结果表明,我们成功合成了PC纳米粒,并且可以实现HA对纳米粒的修饰。
实施例2:HA-PAMAM-COOH-TMZ、HA-PAMAM-COOH-ICG和HA-PAMAM-COOH-TMZ-ICG的合成及表征
将5mg PC(实施例1制备)与2mg TMZ共溶于0.5mL去离子水中,得到A液,将3mg TMZ与5mg HA共溶于2mL去离子水中,得到B液。B液置于功率为180W的探头超声下,一边超声一边将A液滴加到B液中,超声10min后,使用30K超滤管将超声后得到的液体用6000rpm/min的转速超滤,用去离子水洗浓缩液三次后,冻干得到载TMZ的纳米粒(HA-PAMAM-COOH-TMZ,HPCT)。
将5mg PC(实施例1制备)与0.15mg ICG共溶于0.5mL去离子水中,得到A液,将5mgHA溶于2mL去离子水中,得到B液。B液置于功率为180W的探头超声下,一边超声一边将A液滴加到B液中,超声10min后,使用30K超滤管将超声后得到的液体用6000rpm/min的转速超滤,用去离子水洗浓缩液三次后,冻干得到载ICG的靶向纳米粒(HA-PAMAM-COOH-ICG,HPCI)。
纳米粒形态使用场发射透射电子显微镜测定。用动态散射粒度仪测定纳米粒水合直径、多分散指数(PDI)和Zeta电位等参数。
我们在PC的基础上分别成功包封TMZ和ICG合成HPCT、HPCI、HPCTI纳米粒(图2)。DLS结果显示PAMAM的平均水合粒径为5.46nm,其Zeta电位为40.1mV,多分散指数(PDI)为0.308(图2A);PC的平均水合粒径为5.80nm,Zeta电位为12.3mV,PDI为0.27(图2B);HPCT的平均水合粒径为7.43nm,其Zeta电位为-6.43mV,PDI为0.213(图2C)。该结果表明载药后的HPCT纳米粒较PAMAM和PC在粒径上有轻微的增加,其表面电荷由正值变为负值,进一步说明了HA修饰成功。此外HPCT的PDI值更小,说明了HA能够使纳米粒更均匀的分散在溶液中。TEM结果显示HPCT呈球形,大小较为均匀,呈单分散状态,无明显团聚,与DLS测定结果一致(图2D)。HPCI的表征结果同HPCT相似(未列出)。
实施例3:TMZ含量测定
为了精确测定药物TMZ的含量,我们测定了TMZ的降解曲线。将一定量的TMZ溶于pH=7.4的PBS中,配成50μg/mL的溶液,放于37℃的摇床上,速度为150rpm/min。间隔一定的时间点,取出来200μL液体,测定200-400nm的紫外吸收光谱。
由于TMZ在水溶液中不稳定,会逐渐降解为化合物MTIC(5-(3-甲基三氮烯-1-基)咪唑-4-酰胺),因此需要建立TMZ和MTIC的测定方法。紫外吸收法测定结果显示TMZ的最强吸收峰位于328nm,其降解产物MTIC的最强吸收波长为266nm(图3A),而HPCT纳米粒在此波长处无吸收,不会干扰TMZ的测定。因此我们可以使用328nm和266nm波长处的吸光强度作为药物定量的标准。为了进一步研究TMZ的稳定性,我们测定TMZ在不同时间的吸收谱。结果表明,TMZ在6h的时候已经完全转化为MTIC,而6~24h在266nm处的吸光度并未发生明显变化,表明MTIC是比较稳定的。配置一系列浓度的药物,绘制标准曲线,以吸光度A对浓度C进行线性回归处理,得回归方程:A=0.0591C+0.1243,R2=0.9988(TMZ)和A=0.0591C+0.135,R2=0.9997(MTIC)(图3B、3C)。
实施例4:包封率和载药率的测定
通过紫外可见吸收光谱仪测定上清液中328nm和266nm处的光吸收,然后使用差减法来确定TMZ含量。通过多功能连续光谱酶标仪测定上清液中780nm处的光吸收,并使用差减法来确定ICG含量。
纳米粒包封率(EE)和载药率(DL)计算公式如下:
根据公式计算HPCT中TMZ的包封率和载药率分别为44.91%和16.64%,HPCI中ICG的包封率和载药率分别为99.87%和28.55%。
实施例5:HPCT在不同条件下对TMZ的释放动力学研究
将含有1.5mg TMZ的溶液使用pH=2.0的PBS分散为3mL,放入透析袋中(其中pH=5.0+Haase组的透析袋中加入1mg的HA酶)。将透析袋分别放入50mL离心管中,并在离心管中加入pH=5.0和pH=7.4的PBS溶液40mL。将离心管放入37℃,150rpm的摇床上,模拟释药。在不同的时间点取出1mL释放液后,加入1mL新鲜的对应pH的PBS。测定不同时间点所取液体在328nm和266nm处的吸收,计算得到对应时间点的释放量。
释药曲线显示,在72h时,TMZ在pH=7.4的条件下释放量(32%)略高于在pH=5.0环境中的释放量(27%),但是在酸性环境中添加了透明质酸酶之后,TMZ的释放量明显上升,72h时约为38%(图4)。
实施例6:激光照射下的升温曲线和单线态氧产生的测定
为了检测所合成的HPCI纳米粒的光热疗和光动力治疗能力,对HPCI纳米粒进行了升温曲线和单线态氧水平测定。
1、升温曲线测定
配制0μM、2μM、5μM、10μM、15μM、20μM的HPCI(ICG含量)的pH=7.4的PBS溶液,分别取0.5mL于1.5mL EP管中,初始温度为25℃。照射功率密度为8W/cm2,使用热像仪对不同照射时间的温度进行测定,照射时间为0、15s、30s、45s、60s、90s、150s、210s、270s、300s、330s、360s、390s、420s。记录温度值。
将15μM的游离ICG、HPCI(ICG 15μM)的pH=7.4的PBS溶液用8W/cm2的NIR进行照射不同时间,使用近红外热成像仪记录温度,绘制升温曲线。
2、单线态氧测定
配制5μM游离ICG和HPCI(含ICG 5μM)的pH=7.4的PBS溶液,和等体积的单线态氧捕获剂SOSG混合均匀。选择照射功率为8W/cm2分别照射0、0.5、1、2、3min。为保证光敏剂分子均匀分布,在照射过程中每隔1min涡旋一次。照射结束后震荡混匀。每组取200μL溶液加入到黑色荧光96孔板,酶标仪测定(激发波长:507nm;发射波长:531nm)并记录(实验全程在避光下进行)。
图5A结果显示,在近红外激光照射下,随着HPCI浓度的增加,溶液温度升高。在较低浓度时,温度升高并不明显,当浓度为15μM(ICG含量)时溶液温度可达51.7℃,因此我们选取该浓度用于后续实验。在相同ICG(15μM)浓度下,HPCI的升温能力与游离的ICG相近,并没有明显降低,表明将ICG包封于纳米载体中并未明显改变其热疗性质(图5B)。虽然HPCI的升温能力与ICG相似,但是HPCI的单线态氧产生能力却高于游离ICG(图5C)。这可能是因为有了HPC的保护,ICG在激光照射下的稳定性得到提高,这也说明HPCI应用于光动力治疗的效果要优于ICG。
实施例7:人黑素瘤A375细胞的复苏、培养、传代、细胞计数和细胞冻存
1、细胞复苏
(1)取出冻存于液氮中的人黑素瘤A375细胞冻存管,迅速放入37℃的水浴锅中,使其迅速融化,置于超净工作台中。
(2)无菌条件下打开冻存管,将细胞用移液枪转移至15mL无菌离心管中,加入DMEM高糖培养液4mL,轻轻混匀,1000rpm离心3min。弃上清液,加入5mL含10%FBS的DMEM高糖培养基,悬浮细胞。
(3)将细胞悬液转移至培养瓶中,放入37℃、5%CO2饱和湿度培养箱内培养。
(4)次日更换培养基,继续培养。倒置显微镜下观察细胞生长情况。
2、细胞培养
A375细胞常规培养于含10%胎牛血清、100U/mL青霉素和100μg/mL链霉素的DMEM培养液中,在37℃,5%CO2饱和湿度培养箱中培养。
3、细胞传代
倒置显微镜下见A375细胞贴壁生长至培养瓶面积80%~90%融合时,去掉原培养基,加入PBS液2mL漂洗细胞两次。加入0.25%胰蛋白酶消化细胞,显微镜下观察待细胞大部分变圆后,吸除消化液,加入培养液终止消化。吸管轻轻吹打细胞,制成单细胞悬液,按1:3比率,接种到培养瓶中,加入适量的培养液,置37℃、5%CO2饱和湿度培养箱培养。
4、细胞计数
细胞培养至饱和密度,消化细胞成单细胞悬液,1000rpm离心3min,弃上清,加入2mL含血清培养基,吹打混匀,20μL移液枪吸取一滴细胞悬液,从计数板边缘滴入。计数4个大方格内细胞数量。根据公式:细胞数目/每毫升悬液=(4个大方格的细胞数目/4)×104换算。
5、细胞冻存
(1)选取处于对数生长期的A375细胞,冻存前一天换培养液一次。
(2)用0.25%胰蛋白酶消化细胞,置离心管中,1000rpm×3min离心。
(3)弃上清,加入细胞冻存液,轻轻吹打使细胞重悬浮。
(4)每只冻存管内加入细胞悬液1mL,旋紧盖子,并在冻存管上标记。
(5)将冻存管4℃冰箱放置15min,然后迅速移入-20℃冰箱放置20min,最后-80℃冰箱过夜,第二天转移至液氮瓶内。
实施例8:CCK-8法测定HPCT和HPCI纳米粒对A375细胞的细胞毒性
选用对数生长期A375细胞以5×104/孔的密度接种于24孔板中,每孔1mL细胞悬液,37℃培养箱过夜孵育。实验分组:Ⅰ:Control,Ⅱ:NIR(不加任何药物),Ⅲ:HPCI,Ⅳ:HPCT,Ⅴ:ICG+NIR,Ⅵ:HPCI+NIR,Ⅶ:HPCT+HPCI+NIR。设置平行孔。细胞贴壁后,分别按以上分组加ICG、HPCI(含ICG 15μM)、HPCT(含TMZ 1mg/mL)以及HPCT+HPCI(含TMZ 1mg/mL,ICG15μM)处理24h后,用PBS轻洗3次,更换无药物的新鲜DMEM培养基,Ⅱ组、Ⅴ组、Ⅵ组和Ⅶ组用808nm激光器以8W/cm2的功率照射5min,继续培养24h后进行CCK-8比色实验。避光条件下每孔加入100μL CCK-8原液继续孵育2h,取相应孔中的液体到96孔板中用酶标仪测定450nm波长处每孔的O.D值,记录实验结果。实验重复3次。
实验结果表明,游离TMZ处理A375细胞24h后随着浓度增加细胞活力明显下降,浓度为1mg/mL时细胞基本全部死亡,细胞活力为1.7%(图6A)。而将其包封于纳米载体中,浓度为1mg/mL(TMZ含量)时细胞死亡较少,细胞活力为94.9%(图6B)。图6B中显示NIR和HPCI组均无明显细胞毒性(P>0.05),HPCI+NIR组具有较强的细胞杀伤能力,细胞活力为36.3%,且低于ICG+NIR组(48.9%,P<0.05)。与单独HPCI+NIR组和HPCT组相比,HPCT+HPCI+NIR组具有更强的肿瘤细胞杀伤能力,细胞活力为17.1%(P<0.05)。以上结果表明HPCT和HPCI纳米粒在激光照射下能发挥联合杀伤肿瘤作用。
实施例9:荧光成像监测HPI体内分布和蓄积
在进行体内抗肿瘤治疗前,首先对纳米粒在体内的过程进行研究,考察其对肿瘤是否有靶向性。将HPCI经尾静脉注射到荷瘤小鼠体内,使用多光谱荧光小动物活体成像系统监测不同时间HPCI在荷瘤裸鼠体内循环情况。试验方法如下:
BALB/c-nu裸鼠,雌性,6~8周龄,体重约19-21g,购于北京维通利华实验动物技术有限公司。裸鼠饲养于恒温(22~25℃)、恒定湿度的无特异病原体(SPF)级的屏障系统中。经高压灭菌的标准饲料和水供动物自由饮食。
取对数生长期A375细胞消化离心后收集,用PBS洗3次,1000rpm×5min,快速置于冰上,与提前解冻的matrigel 1:1混合,配成4×107/mL的混合液,取100μL接种于裸鼠右后腿。待肿瘤长至1000mm3,尾静脉注射HPCI(ICG 1.15mg/kg),于0h、1h、3h、6h、18h、24h、36h、48h、72h用多光谱荧光小动物活体成像系统(CRI Maestro 2)监测HPCI在荷瘤裸鼠体内的分布情况。72h时采用颈椎脱臼法处死裸鼠,解剖出各脏器并拍摄其荧光成像。
结果显示,药物经尾静脉注射后首先聚集于肝脏,3h时已经开始进入肿瘤,此后肿瘤内荧光逐渐增强,而肝脏内荧光变弱,至24h肿瘤内荧光强度最强,此后逐渐下降,但72h肿瘤内仍有药物蓄积(图7A)。72h后解剖出裸鼠各脏器,剥离肿瘤,成像结果显示,HPCI在肿瘤内部仍然有很强的荧光,在肝脏和肾脏中荧光强度较弱,在肺、心、脾中未检测到荧光(图7B)。上述结果表明HPCI在体内对肿瘤具有良好的靶向性,且NIR照射应于注射药物后24h时进行效果较好。
实施例10:荷瘤模型研究载药纳米粒的化疗和PTT/PDT联合治疗作用
为了进一步验证纳米粒在体内的联合抗黑素瘤疗效,我们构建了人人黑素瘤A375细胞的移植瘤模型,尾静脉注射药物,按如下实验方法中的方案进行处理:
BALB/c-nu裸鼠,雌性,6~8周龄,体重约19-21g,购于北京维通利华实验动物技术有限公司。裸鼠饲养于恒温(22~25℃)、恒定湿度的SPF级的屏障系统中。经高压灭菌的标准饲料和水供动物自由饮食。
实验分组:Ⅰ:Normal(不接肿瘤),Ⅱ:Control,Ⅲ:NIR,Ⅳ:TMZ,Ⅴ:HPCT,Ⅵ:HPCI+NIR,Ⅶ:HPCI+HPCT+NIR。
取对数生长期A375细胞消化离心后收集,用PBS洗3次,1000rpm×5min,快速置于冰上,与提前解冻的matrigel 1:1混合,配成4×107/mL的混合液,取100μL接种于BALB/c裸鼠右后腿。待肿瘤长至100cm3后将裸鼠随机分成6组,每组5只。进行尾静脉注射药物:Control组注射100μL生理盐水;TMZ组每只注射70mg/kg TMZ;HPCI组每只注射含ICG1.15mg/kg的HPCI;HPCT组每只注射含TMZ 70mg/kg的HPCT;联合治疗组每只注射含ICG1.15mg/kg的HPCI和TMZ 70mg/kg的HPCT。注射药物后24h用2%戊巴比妥钠进行腹腔注射麻醉,用808nm激光器进行肿瘤部位照射处理,1.5W/cm2,10min。照射过程中用近红外热成像仪检测温度。记录肿瘤生长情况,每两天称量体重和测量肿瘤体积,肿瘤体积(mm3)=长径×短径×短径/2。自给药开始连续观测14天,绘制肿瘤生长曲线。14天后眼球采血进行血生化检测,处死所有裸鼠,剥离移植瘤进行拍照,解剖出主要脏器(心、肝、脾、肺、肾),进行病理切片检测。
图8A的热成像照片显示注射生理盐水后进行NIR照射的荷瘤裸鼠肿瘤部位温度有轻微的升高,最高温度为41.6℃;而注射HPCI纳米粒并照射NIR的裸鼠肿瘤局部最高温度可迅速上升至49.2℃,此温度可以杀死肿瘤细胞。该结果进一步证明HPCI在体内能发挥热疗作用。图8B中肿瘤生长曲线显示,Control组肿瘤生长迅速,NIR组和TMZ组肿瘤生长速度被部分抑制,HPCT组肿瘤生长速度较游离TMZ组降低,HPCI+NIR组肿瘤生长速度明显降低,然而在治疗结束时有复发趋势;HPCT+HPCI+NIR组中的肿瘤则完全消失。图8C和8D分别为肿瘤剥离照片和裸鼠治疗结束后照片,各组肿瘤大小有差异,同肿瘤生长曲线结果一致。
另外,考察了合成的纳米载体治疗后对机体正常组织(心、肝、脾、肺、肾)的毒性,结果显示,同Normal组(未接种肿瘤)相比,联合治疗组对各器官均未产生损害。另外,眼球取血后进行了血生化检查,结果显示谷丙转氨酶、谷草转氨酶、总胆红素、肌酐、尿素氮、尿酸、磷酸肌酸同工酶的值均在正常范围内,说明治疗后无明显的系统毒性。以上结果表明载药纳米粒介导的联合治疗能有效治疗黑素瘤,且对荷瘤小鼠无系统毒性。
综上所述,本实验近红外染料吲哚菁绿的光学治疗联合化疗药物替莫唑胺构建纳米载体用于抗黑素瘤治疗,体外、体内实验成功地证明了其具有协同治疗作用,可为皮肤黑素瘤治疗提供新的治疗方法和治疗模式。
Claims (7)
1.一种用于光学治疗的光敏剂靶向纳米粒,其特征在于,该靶向纳米粒的通式为HA-PAMAM-COOH-ICG,其中HA为透明质酸,PAMAM为树枝状聚合物;COOH为羧基,ICG为吲哚菁绿。
2.根据权利要求1所述用于光学治疗的光敏剂靶向纳米粒,其特征在于,所述的树枝状聚合物为以乙二胺为核的第1-10代聚酰胺-胺树状大分子。
3.根据权利要求2所述用于光学治疗的光敏剂靶向纳米粒,其特征在于,所述的树枝状聚合物为以乙二胺为核的第4-6代聚酰胺-胺树状大分子。
4.根据权利要求3所述用于光学治疗的光敏剂靶向纳米粒,其特征在于,所述的树枝状聚合物为以乙二胺为核的第5代聚酰胺-胺树状大分子。
5.一种根据权利要求1所述用于光学治疗的光敏剂靶向纳米粒的制备方法,其特征在于,该方法包括如下步骤:
(1)PAMAM-COOH的合成;
(2)HA-PAMAM-COOH-ICG的合成:将PAMAM-COOH与ICG共溶于去离子水中,得到A液;将HA共溶于去离子水中,得到B液;B液置于功率为150-300W的探头超声下,一边超声一边将A液缓慢加到B液中,超声5-20min后,将超声后得到的液体超滤,超滤后用去离子水洗1-4次,冻干,得到用于光学治疗的光敏剂靶向纳米粒HA-PAMAM-COOH-ICG。
6.根据权利要求5所述用于光学治疗的光敏剂靶向纳米粒的制备方法,其特征在于,步骤(1)PAMAM-COOH的合成步骤为:将PAMAM溶解于甲醇中,旋干,并加入无水DMSO重新溶解,形成反应体系;取丁二酸酐溶于无水DMSO中,滴加入反应体系内,室温搅拌12-24h,使用Mw=3000-4000Da的透析袋透析2-4天后,冷冻干燥,得到产物PAMAM-COOH。
7.权利要求1所述的HA-PAMAM-COOH-ICG在制备光热且光动力治疗的光敏剂中的应用。
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