光热/pH响应B-CuS-DOX纳米药物用于化疗-光热协同治疗肿瘤

基于纳米材料的化疗-光热协同治疗是一种高效的肿瘤治疗方式, 但如何构建具有高载药量与良好光热转换性能的纳米药物依然面临挑战。本研究通过超声剥离法制备二维硼(boron, B)纳米片, 进一步在其表面原位负载超小粒径硫化铜(CuS)纳米颗粒和化疗药阿霉素(DOX), 形成B-CuS-DOX纳米药物。B-CuS具有高的DOX药物装载能力(864 mg/g)和优异的光热转化性能(在808 nm处的光热转换效率为55.8%), 同时可实现pH及近红外激光双重刺激响应而释放药物。细胞实验表明在808 nm近红外光的照射下, B-CuS-DOX展示了良好的化疗-光热协同治疗效果。本研究构建的纳米药物有望为体内肿瘤治疗提供一种有效的化疗-光热协同治疗策略。

Abstract

Nanoparticles-based drug delivery system for synergetic chemo-photothermal therapy is an efficient strategy for tumor therapy. However, construction of nanodrugs with high drug loading capacity and good photothermal conversion property is still a great challenge. In this work, two-dimensional boron nanosheets were prepared by ultrasonic liquid exfoliation method, which were further loaded with ultra-small copper sulfide (CuS) nanoparticles and doxorubicin (DOX) to obtain B-CuS-DOX nanocomposites. The prepared B-CuS-DOX exhibited high drug loading capacity (864 mg/g) and excellent photothermal conversion efficiency (55.8%). In addition, B-CuS-DOX exhibited pH and laser--responsive drug release behavior. In vitro results demonstrated that the prepared B-CuS-DOX achieved a good synergistic effect of chemotherapy and photothermal therapy. This work is expected to provide an effective chemo-photothermal synergistic therapy strategy for tumor therapy.

化疗是目前临床上应用最广泛的癌症治疗方法 [1] 。然而, 非特异性的化疗存在较大的副反应, 严重影响了患者的生存质量 [2] 。此外, 化疗往往受限于多药耐药性, 导致治疗效果不佳 [3, 4] 。因此, 开发更为高效而安全的肿瘤治疗手段成为研究者们关注的热点。纳米医学的发展为研发能提高临床疗效并减轻副作用的药物提供了新的策略。纳米药物递送系统 是指利用纳米颗粒作为药物载体, 以实现化疗药物的靶向定点递送及释放, 从而减少化疗引起的全身副作用 [5, 6, 7] 。然而, 单一的化疗对肿瘤治疗效果有限, 迫切需要将化疗与其他治疗方式联合使用以提高肿瘤治疗疗效、减轻毒副作用和耐药性 [8] 。

光热治疗(photothermal therapy, PTT)以其侵袭性小和有效性高的优势而逐渐成为一种有潜力的治疗方式 [9, 10, 11] 。PTT通过使用光热转换试剂在激光照射下将光能转化成热能杀死癌细胞 [12, 13] 。研究表明, 化疗与PTT联合治疗肿瘤效果高于单一化疗或PTT, 且能降低副作用 [6, 14 - 15] 。近年来, 科学家们开发了许多优良的光热转换剂并在细胞、动物水平验证了其良好的光热治疗肿瘤效果。各种无机纳米材料, 例如不同类型的金纳米结构(金纳米棒 [16] 、金纳米笼 [17] 、金纳米壳 [18] 等)、碳纳米材料(石墨烯 [19] 、碳纳米管 [20])具有良好的肿瘤光热治疗效果, 但是其在体内的不可降解性和潜在的长期毒性限制了它们的临床转化 [21] 。有机纳米材料(如吲哚菁绿、聚苯胺和聚吡咯等)作为光热转换药物也得到了研究者们的广泛关注, 但其稳定性较差, 且较低的光热转换效率和光漂白现象也制约了它们在肿瘤诊疗中的应用 [22] 。最近, 超薄二维硼(boron, B)纳米片由于具有良好的生物相容性、体内低毒可降解性、比表面积大, 可增加对药物的负载能力等优势, 作为一种新型药物递送载体引起了人们极大的研究兴趣 [23] 。同时, 超小硫化铜(CuS)纳米颗粒具有强的近红外光吸收、优良的摩尔消光系数和高的光热转化效率 [24] 。如何通过将超小CuS原位生长在B纳米片来提高B纳米片的光热转换效率, 同时作为药物载体实现化疗与PTT对肿瘤进行协同治疗, 值得深入探索。

本研究拟制备一种具有良好光热转化效率的CuS与负载的B纳米片纳米药物, 并将其作为载体负载化疗药物阿霉素(DOX)得到一个兼具高载药量、良好光热转换性能和生物相容性的纳米药物(B-CuS-DOX), 用于药物递送和协同化疗/光热治疗。该纳米药物利用了B纳米片和CuS纳米粒协同增强光热转换效率, 同时实现DOX的高效装载及光热和pH双重刺激响应药物释放, 从而达到良好的化疗-光热协同治疗肿瘤的效果。

1 实验方法

1.1 B-CuS的制备

将硼粉(0.5 g)分散在100 mL的 N -甲基-2-吡咯烷酮(NMP)和乙醇的混合溶剂(1 : 1, V / V)中, 冰浴超声(500 W)5 h后离心(3000 r/min)10 min。弃去沉淀物, 将上清液以12000 r/min的速度继续离心20 min, 乙醇洗涤3次。接着, 将产物进行热氧化处理, 取100 mg上述制备的B纳米片放入陶瓷容器中, 以5 ℃/min的升温速率升温至650 ℃, 在氧气氛围下加热2 h使B纳米片表面被氧化生成氧化硼(B 2 O 3)。之后用上述的液相剥离法结合超声剥离得到B纳米片。下一步, 将CuCl 2 溶液(50 mmol/L, 5 mL)分散在B纳米片分散液中, 室温搅拌2 h后, 向反应体系中滴加Na 2 S溶液(50 mmol/L, 3 mL), 继续搅拌15 min后转移到80 ℃的水浴中, 反应10 min, 将产物用分子量为10 kDa的纤维素膜透析3 d, 冷冻干燥得到B-CuS纳米片。

1.2 测试药物的负载及释放量

药物负载实验: 将DOX溶液(0.1~1.0 mg/mL)与B-CuS分散液(1.0~5.0 mg/mL)混合, 室温搅拌过夜。将混合溶液离心(10000 r/min)除去未负载的DOX, 并用PBS反复洗涤离心后得到B-CuS-DOX载药复合物。同时, 收集所有上清液和洗涤液, 采用UV-Vis-NIR测定DOX的吸光度并根据公式计算药物的负载量

药物负载量=(DOX的总质量-上清液DOX的质量)/复合物的质量\times100%

药物释放试验: 将B-CuS-DOX分散液装入透析袋(MWCO: 3500 Da)中, 分别浸入装有不同pH(pH 5.0或7.4)磷酸缓冲液(PBS)的离心管中, 并置于摇床中振荡(150 r/min)。激光照射试验组用808 nm近红外激光(0.5 W/cm 2)照射5 min。在不同时间点从离心管中取出1 mL液体并补充同等体积的新鲜缓冲液继续振荡。采用UV-Vis-NIR法测定溶液在485 nm处的吸光度。通过建立的药物标准曲线计算不同条件下DOX的累积释放量, 从而得到药物DOX的释放曲线。

药物释放率=(释放的DOX质量/复合物中DOX的总质量)\times100%

1.3 材料表征

采用JEM-2100F透射电子显微镜(TEM)对所得的纳米材料进行形貌观察。采用Zeta粒度分析仪(Nano ZS90, Malven Instrument Ltd)测定材料合成过程中各个阶段产物的粒径和Zeta电位。采用X射线光电子能谱(XPS)对样品中各元素的化学价态进行表征。使用UV-Vis-NIR记录不同纳米颗粒的吸收光谱。

1.4 B-CuS光热性能测试

将200 μL不同浓度(0、100、200和500 μg/mL)的B-CuS分散液置于EP管中, 采用808 nm激光(0.5 W/cm 2)照射5 min, 通过Fotric-225热红外成像仪记录材料的温度变化曲线。随后, 测定不同功率密度(0.25、0.5、0.75和1.0 W /cm 2)激光照射B-CuS分散液(200 μg/mL)的升温曲线。此外, 将样品进行多次激光“开-关”的循环照射以测定材料光热稳定性, 并根据文献报道的方法计算B-CuS的光热转换效率(η) [23] 。

1.5 B-CuS的细胞毒性测试

将4T1细胞(1\times10 5 /孔)接种于96孔板中培养12 h。待细胞贴壁后, 加入100 µL含有不同浓度B-CuS的新鲜培养基继续孵育24 h或48 h。PBS洗涤3次后, 采用标准的CCK-8法计算细胞存活率。

1.6 细胞对B-CuS-DOX摄取

将4T1细胞接种于共聚焦培养皿中, 孵育12 h使其贴壁。实验分组: a)Control; b)Free DOX; c)B- CuS-DOX; d)B-CuS-DOX+NIR。对于NIR照射组, 细胞与纳米材料孵育2 h后以激光0.5 W/cm 2 照射5 min后继续培养4 h, PBS清洗细胞3次, 用4%的多聚甲醛固定细胞30 min, 采用DAPI染色后用激光扫描共聚焦显微镜(CLSM)观察细胞状态。

1.7 B-CuS-DOX的化疗-PTT协同治疗效果

细胞增殖和毒性检测: 实验分组如下: a)Control; b)B-CuS; c)Free DOX; d)B-CuS+NIR; e)B-CuS-DOX; f)B-CuS-DOX+NIR。对于NIR激光照射组, 细胞孵育4 h后在808 nm的激光(0.5 W/cm 2)下照射5 min。细胞经不同治疗方式处理后采用CCK-8法进行细胞增殖和毒性检测。此外, 细胞经过不同治疗处理后, 加入钙黄绿素-AM/PI染色后进行CLSM观察。

流式细胞仪检测细胞凋亡: 4T1细胞(5\times10 5 /孔)接种在6孔板中培养24 h。实验分组处理如上所述。随后, 消化离心细胞, PBS溶液洗2次, 离心收集细胞后用流式凋亡染色试剂盒(Annexin V-FITC/PI染液)检测细胞凋亡。

2 结果与讨论

2.1 B-CuS-DOX的制备与表征

首先采用文献报道的超声剥离法制备B纳米片 [23], 进一步采用原位生长的策略, 在B纳米片的表面可控生长超小CuS纳米粒, 形成二维B-CuS纳米药物。如 图1 (A,B)所示, 制备的B纳米材料呈片状结构。B-CuS 的TEM照片显示B纳米片上存在大量CuS纳米粒, 表明成功制备了B-CuS。采用动态光散射粒径测试不同纳米复合物的水合粒径, B纳米片约为110 nm, CuS 纳米粒约为15 nm, B-CuS纳米粒约为139 nm, B-CuS-DOX水合粒径约为152 nm(图1 (C))。此外, 图1 (D,E)中XPS分析显示, B纳米片两个归属于B-B键的低结合能峰(187.5和188.4 eV)。进一步, 考察了所制备的B-CuS纳米药物在生理环境中的分散稳定性, 如 图1 (F)所示, B-CuS的粒径在15 d内没有发生明显变化, 表明其具有良好的分散稳定性。 图1 (G)的UV-Vis-NIR光谱证明B纳米片在全光谱具有较好的吸收, 与CuS结合后(1098 nm)会进一步提高B-CuS在近红外区域的吸收。此外, B-CuS-DOX的光谱中出现了DOX的特征吸收峰(485 nm), 证明DOX的成功负载。 图1 (H)显示B-CuS的电位为-11.2 mV。由于氨基的存在, DOX负载后, B-CuS-DOX的电动电势变为13.2 mV, 推测DOX(带正电)是通过静电吸附的方式负载在B-CuS(带负电)纳米片上。 图1 (I)表明B-CuS对DOX的负载能力在一定药物浓度内随着DOX浓度的增大而增强, 并具有较高的药物装载量(864 mg/g), 显示了B-CuS对DOX具有良好的装载能力。

10.15541/jim20200394.F001

图1B纳米片、B-CuS和B-CuS-DOX的表征Characterization of B nanosheets, B-CuS and B-CuS-DOX TEM images of (A) B-CuS and (B) B-CuS; (C) Diameter of B-CuS-DOX; (D) XPS survey spectra of B nanosheets; (E) The selective XPS survey spectra corresponding to B1s spectra; (F) Hydrodynamic size change of B-CuS-DOX dispersed in saline, medium containing fetal bovine serum (FBS), human simulated body fluid (SBF) for 15 d; (G) UV-Vis-NIR spectra and (H) Zeta potential of B nanosheets, B-CuS and B-CuS-DOX; (I) Histogram of the relationship between DOX drug loading and DOX concentration. DOX: doxorubicin

Fig. 1

2.2 B-CuS光热性能

进一步探讨B-CuS的光热转换性能, 随着 808 nm激光(0.5 W/cm 2) 照射时间的延长, 浓度为200 μg/mL的B-CuS分散液的温度逐渐升高, 照射5 min后, 溶液温度从22.3 ℃上升到50.4 ℃。B-CuS具有明显的剂量、激光辐照时间和激光功率密度依赖性(图2 (A-C))。接着, 通过808 nm激光重复辐照B-CuS分散水溶液5次, 可以看到溶液的升温趋势没有明显的变化, 表明所制备的B-CuS具有良好的光热稳定性。如 图2 (D-E)所示, 通过文献报道的方法 [23] 计算B-CuS在808 nm激光照射下的光热转换效率(η)为55.8%, 以上结果表明所制备的B-CuS具有高的光热转换效率和光热稳定性。

10.15541/jim20200394.F002

图2B-CuS的光热转化性能Photothermal performance of B-CuS

(A) The temperature change of B-CuS dispersion with different concentrations within 5 min of laser irradiation with a power density of 0.5 W/cm²; (B) The temperature change of B-CuS (200 μg/mL) dispersion under different laser power densities for 5 min; (C) Thermal images of B-CuS dispersions with different concentrations after irradiation within 5 min with a laser power density of 0.5 W/cm²; (D) B-CuS photothermal curve under five laser “on-off” cycles; (E) The linear regression equation of the negative natural logarithm of B-CuS heating and cooling time and temperature, θ=ΔT/ΔT_max

Fig. 2

2.3 B-CuS-DOX体外pH及光热响应药物释放性能

设计具有刺激响应的药物递送系统对于化疗疗效的提高及减轻副作用具有显著意义 [24, 25] 。纳米药物可通过对肿瘤内部的微环境(如弱酸性、乏氧、高组织液压力等)响应或者外部装置刺激触发(超声、激光等)来完成药物在肿瘤内部的定点释放 [25] 。B-CuS-DOX到达肿瘤部位后可通过微酸环境响应性释放DOX。如 图3 所示, B-CuS-DOX在pH=7.4的PBS溶液中孵育24 h 后, DOX释放率为12.4%, 表明其在生理环境中具有良好的稳定性。而在pH=5.0下, DOX释放率为32.8%。NIR激光照射会进一步加速DOX释放, 在pH=5.0条件下DOX的释放率增高到68.6%, 说明光热会促进药物的释放, 其主要原因可能是在NIR激光的照射下, B-CuS产生的热量使得DOX与B-CuS之间的作用力减弱而释放药物 [26] 。以上结果证明B-CuS-DOX可在pH和光热双响应下可控释放药物, 减轻化疗的副作用。

10.15541/jim20200394.F003

图3B-CuS-DOX在不同pH的PBS溶液及在有无NIR激光照射下的药物释放曲线Drug release curves of B-CuS-DOX in PBS solution with different pH and with or without NIR laser irradiation

Fig. 3

2.4 细胞对B-CuS-DOX的摄取

采用CLSM观察4T1 细胞对B-CuS-DOX的特异性摄取和细胞内药物释放行为。如 图4 所示, B-CuS-DOX组红色荧光信号明显强于游离DOX组, 证明DOX的有效负载及细胞内的响应性释放。在NIR激光照射下, B-CuS-DOX组表现出更强的荧光, 说明NIR引起的高热不仅可以增强细胞对纳米颗粒的摄取, 而且可以促进DOX在胞内释放。这主要是因为局部温和的光热可以提高细胞膜的通透性, 从而促进了肿瘤细胞对纳米颗粒的摄取, 从而提高化疗和PTT效果 [27, 28, 29] 。

10.15541/jim20200394.F004

图4B-CuS-DOX的细胞摄取行为<i>In vitro</i> cellular uptake of B-CuS-DOX

(A) CLSM images of 4T1 cells treated with free DOX and B-CuS-DOX with or without laser irradiation; (B) Quantitative analysis of fluorescence intensity of DOX uptake by 4T1 cells in each treatment. Scale bar: 15 μm; *: p < 0.05. DOX: doxorubicin

Fig. 4

2.5 B-CuS-DOX的化疗-PTT协同治疗效果

B-CuS具有良好的光热转换性能和药物负载能力, 在化疗和PTT协同治疗肿瘤中有重要的作用。B-CuS-DOX介导的化疗-PTT协同杀死肿瘤细胞的效果存在剂量依赖性。经过不同浓度B-CuS处理4T1细胞后, 即使浓度达到500 μg/mL, 细胞增殖与毒性检测结果也没有发生明显的变化。 图5 (A)显示, 各组细胞存活率在90%以上, 说明在实验浓度范围内, B-CuS具有良好的细胞相容性。 图5 (B)结果表明, 化疗(B-CuS-DOX)和光热治疗(B-CuS+NIR)联合治疗效率根据公式 [30] 计算为56%, 高于单一化疗组(癌细胞死亡率: 37.7%)和单一光热治疗组(癌细胞死亡率: 29.3%), 但低于B-CuS-DOX+NIR组对4T1细胞的杀伤效果(癌细胞死亡率: 77.8%), 说明化疗和PTT协同可产生最佳的癌细胞杀伤效果(图5 (C))。此外, 通过钙黄绿素-AM和PI双染色评价不同治疗方式效果(图6), 与单纯治疗组(化疗组或PTT组)相比, 协同治疗组(B-CuS-DOX+NIR)显示出了大量的红色荧光信号(红色荧光代表肿瘤细胞死亡), 进一步直观证明其显著的化疗与PTT协同治疗效果。

10.15541/jim20200394.F005

图5B-CuS-DOX对细胞的化疗-PTT协同治疗效果评价Evaluation of B-CuS-DOX on cell chemotherapy-PTT synergistic therapy (DOX: doxorubicin)

(A) Cell survival rate of 4T1 cells incubated with different concentrations of B-CuS for 24 and 48 h; (B) Cell survival rate of 4T1 cells treated with different experimental groups; (C) Therapeutic efficacy of PTT, chemotherapy, chemo-photothermal synergistic therapy and additive therapy. *: p<0.05

Fig. 5

10.15541/jim20200394.F006

图64T1细胞存活(calcein-AM染色, 绿色荧光)和死亡(PI双染色, 红色荧光)的荧光显微照片。Observation of 4T1 cells stained with calcein AM and PI under confocal microscope (scale: 50 μm). DOX: doxorubicin

Fig. 6

2.6 流式细胞仪检测细胞凋亡

进一步采用流式细胞仪检测不同处理方式诱导肿瘤细胞凋亡的效果。如 图7 所示, 对照组和B-CuS组细胞凋亡率分别为7.02%、10.29%; 与单独DOX组(35.27%)、B-CuS-DOX组(23.95%)及B-CuS+ NIR(24.82%)的细胞凋亡率相比, 协同治疗组(B-CuS- DOX+NIR)表现出更强的肿瘤细胞杀伤效果(51.69%), 进一步证实其显著的杀伤效果。综上所述, B-CuS-DOX可以实现良好的化疗-PTT协同治疗肿瘤的效果。

10.15541/jim20200394.F007

图7流式细胞仪检测4T1细胞经不同治疗方式处理后的细胞凋亡结果Detection of apoptosis of 4T1 cells after different treatments by flow cytometry (DOX: doxorubicin)

Fig. 7

3 结论

本研究基于二维B纳米片制备了一种智能型的B-CuS-DOX纳米药物。该纳米药物通过引入CuS提高了B纳米片的光热转化效率, 同时进一步增强了化疗效果。B-CuS-DOX具有pH和光热双重刺激响应DOX释放行为, 且实现了光热协同化疗高效杀死肿瘤细胞。本工作构建的B-CuS-DOX纳米药物可以作为一种智能型的化疗-PTT平台用于肿瘤的高效治疗。此研究为构建新型基于二维纳米片的刺激响应性药物递送系统提供了思路。

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