Chinese Journal of Tissue Engineering Research ›› 2026, Vol. 30 ›› Issue (8): 1940-1951.doi: 10.12307/2026.607
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Construction of pH/near-infrared laser stimuli-responsive drug delivery system and its application in treatment of oral squamous cell carcinoma
Min Changqin1, Huang Ying2
- 1Chengdu Wenjiang District People’s Hospital, Chengdu 611100, Sichuan Province, China; 2Hospital of Chengdu Office of the People’s Government of Tibet Autonomous Region, Chengdu 610041, Sichuan Province, China
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Received:2024-12-09Accepted:2025-03-19Online:2026-03-18Published:2025-07-16 -
Contact:Huang Ying, Attending physician, Hospital of Chengdu Office of the People’s Government of Tibet Autonomous Region, Chengdu 610041, Sichuan Province, China -
About author:Min Changqin, MS, Attending physician, Chengdu Wenjiang District People’s Hospital, Chengdu 611100, Sichuan Province, China
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Min Changqin, Huang Ying. Construction of pH/near-infrared laser stimuli-responsive drug delivery system and its application in treatment of oral squamous cell carcinoma[J]. Chinese Journal of Tissue Engineering Research, 2026, 30(8): 1940-1951.
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2.1 纳米羟基磷灰石-聚多巴胺-阿霉素复合体的性能表征结果 使用液氮冻干纳米羟基磷灰石、纳米羟基磷灰石-聚多巴胺和纳米羟基磷灰石-聚多巴胺-阿霉素复合体后喷金，利用扫描电镜测试3组材料形貌，结果如图1A所示，3组材料呈均匀的棒状结构。 透射电镜观察结果也显示纳米羟基磷灰石、纳米羟基磷灰石-聚多巴胺和纳米羟基磷灰石-聚多巴胺-阿霉素复合体呈均匀的棒状结构，见图1B，粒径分别为(123.67±4.36)，(129.13±6.44)，(137.29± 9.51) nm，证实聚多巴胺和阿霉素成功包覆在纳米羟基磷灰石载体上。 采用动态激光粒度分析仪检测样品的Zeta电位和水合粒径，结果如图1C所示。纳米羟基磷灰石和纳米羟基磷灰石-聚多巴胺均呈负电荷，Zeta电位分别为(-18.63±1.09) mV和(-31.07±0.76 ) mV，阿霉素溶液的Zeta电位为(27.57±0.85) mV，纳米羟基磷灰石-聚多巴胺-阿霉素复合体的电位为(20.60±1.33) mV。纳米羟基磷灰石、纳米羟基磷灰石-聚多巴胺和纳米羟基磷灰石-聚多巴胺-阿霉素复合体的水合粒径分别(156.12±8.91)，(164.79±10.38)，(171.82±7.72) nm。表面电荷和水合粒径的变化证实纳米羟基磷灰石-聚多巴胺-阿霉素复合体制备成功。 使用紫外-可见光-近红外分光光度计检测样品光谱，结果如图1D所示。纳米羟基磷灰石在200-800 nm段未见明显吸收峰，纳米羟基磷灰石-聚多巴胺在279 nm处出现吸收峰，证实聚多巴胺被成功修饰。与阿霉素溶液相比较(在483 nm处出现明显吸收峰)，纳米羟基磷灰石-聚多巴胺-阿霉素复合体在497 nm处出现更宽、更低的吸收峰，表明阿霉素被成功负载到纳米羟基磷灰石-聚多巴胺上。 X射线衍射结果如图1E所示。制备的纳米羟基磷灰石与羟基磷灰石标准卡片(JCPDS 09-0432)的经典六方相晶型相匹配，纳米羟基磷灰石-聚多巴胺-阿霉素复合体的结晶度下降，但未改变其晶相。纳米羟基磷灰石和纳米羟基磷灰石-聚多巴胺-阿霉素复合体在25.89°，31.75°，39.47°，46.64°，49.53°和53.31°处的衍射峰分别对应(002)(211)(212)(222)(213)和(004)晶面。 "
使用808 nm近红外激光照射不同质量浓度纳米羟基磷灰石-聚多巴胺-阿霉素复合体溶液，记录温度变化，结果如图2A所示。随着复合体质量浓度的增加，溶液温度升高，光热升温能力呈浓度依赖性增加，当复合体(含0.5 μg/mL阿霉素)被照射10 min时温度为46 ℃，大于肿瘤消融阈值42 ℃，因此后续选择此质量浓度进行后续实验。 为进一步优化激光照射条件，使用不同效率的808 nm近红外激光照射纳米羟基磷灰石-聚多巴胺-阿霉素复合体溶液，结果如图2B所示。随着激光功率的增加，溶液温度增加，照射10 min后，0，1，2，3 W/cm2激光照射后的溶液温度分别为41.2，46，49.7 ℃。由于41.2 ℃低于肿瘤消融阈值，并且文献报道温度在42-48 ℃范围内对肿瘤细胞有较强的杀伤作用而对正常细胞相对安全[25]，49.7 ℃远高于正常细胞的安全温度，因此选择808 nm近红外激光(2 W/cm2)照射纳米羟基磷灰石-聚多巴胺-阿霉素复合体(含0.5 μg/mL阿霉素)。 采用808 nm近红外激光以2 W/cm2的功率照射不同样品10 min，溶液温度变化如图2C所示。空白对照(水)组、纳米羟基磷灰石组和阿霉素组未见明显温度变化，纳米羟基磷灰石-聚多巴胺组和纳米羟基磷灰石-聚多巴胺-阿霉素复合体组随着照射时间的增加溶液温度逐渐升高，照射10 min后两者温度分别达到46.2 ℃和46.0 ℃(图2D)。 采用激光照射纳米羟基磷灰石-聚多巴胺-阿霉素复合体10 min后关掉10 min，循环4次，记录温度变化，结果如图2E所示。4次循环中溶液温度变化趋势和最高温度没有明显变化，具有光热稳定性。根据公式计算纳米羟基磷灰石-聚多巴胺-阿霉素复合体的光热转换效率约为38.7%。表1列举了常见光热试剂的光热转换效率及优缺点。综合评估光热试剂的光热转换效率、生物降解性和生物安全性等，纳米羟基磷灰石-聚多巴胺-阿霉素具有优异的临床应用前景。 "
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配制不同质量浓度的阿霉素溶液，通过紫外分光光度计检测吸光度值，并拟合得到阿霉素标准曲线：y=0.008 x+0.025(R2=0.999)，见图3A。利用阿霉素标准曲线，计算出纳米羟基磷灰石-聚多巴胺-阿霉素复合体的包封率和载药量分别为(72.27±3.29)%和(10.23±0.96) μg/mg。 浸泡于不同pH值PBS中及经808 nm近红外激光(2 W/cm2)照射后，纳米羟基磷灰石-聚多巴胺-阿霉素复合体的阿霉素释放率结果如图3B所示。纳米羟基磷灰石-聚多巴胺-阿霉素复合体可以持续释放144 h，前24 h药物快速释放，24 h后药物释放速度变慢；经过144 h，在pH=7.4，6.5，5.0 PBS中的药物释放率分别为(10.60±0.31)%，(29.96±1.16)%和(65.68±3.58)%。复合体中阿霉素的释放率随着浸泡液pH值的降低而增加，呈pH值刺激响应性释药；经过808 nm近红外激光照射后，复合体的药物释放率明显增加到(90.33±2.11)%，呈近红外激光刺激响应性释药。"
2.2 纳米羟基磷灰石-聚多巴胺-阿霉素复合体的抗癌性能 采用CCK-8法检测各组样品对HSC-4细胞和L929细胞的毒性，结果如图4所示。不同质量浓度纳米羟基磷灰石和纳米羟基磷灰石-聚多巴胺与细胞共培养24 h后，HSC-4细胞和L929细胞的存活率明显高于90%，说明纳米羟基磷灰石和纳米羟基磷灰石-聚多巴胺无明显细胞毒性。不同质量浓度的阿霉素和纳米羟基磷灰石-聚多巴胺-阿霉素复合体与HSC-4细胞和L929细胞共培养后，随着阿霉素和纳米羟基磷灰石-聚多巴胺-阿霉素复合体质量浓度的增加，HSC-4细胞和L929细胞存活率下降，当阿霉素质量浓度为2 μg/mL时，阿霉素组和纳米羟基磷灰石-聚多巴胺-阿霉素复合体组HSC-4细胞存活率分别为(33.66±1.74)%和(47.17±2.26)%，L929细胞存活率分别为(30.32±1.67)%和(43.17±3.74)%。 将纳米羟基磷灰石、纳米羟基磷灰石-聚多巴胺、阿霉素和纳米羟基磷灰石-聚多巴胺-阿霉素复合体与HSC-4和L929细胞共培养12 h后，用808 nm近红外激光以2 W/cm2功率照射5 min后继续培养12 h，结果显示纳米羟基磷灰石-聚多巴胺-阿霉素复合体+激光组HSC-4细胞存活率(9.02±6.43)%，明显低于光热治疗组[纳米羟基磷灰石-聚多巴胺+激光组，(69.54±7.51%)]和化学治疗组[阿霉素组，(64.57±3.89)%]，说明光热-化学联合治疗组杀伤肿瘤的效果明显高于单一的光热治疗和化学治疗；纳米羟基磷灰石-聚多巴胺-阿霉素复合体+激光组L929细胞存活率(85.69±3.12)%高于化学治疗组(66.90±3.63)%。 以上结果表明，在808 nm近红外激光照射下，纳米羟基磷灰石-聚多巴胺-阿霉素复合体对肿瘤细胞具有较强的细胞毒性，而对正常细胞具有一定的保护作用。 "
采用流式细胞术考察纳米羟基磷灰石-聚多巴胺-阿霉素复合体对HSC-4细胞凋亡的影响，结果如图5所示。近红外激光组、纳米羟基磷灰石组、纳米羟基磷灰石+激光组和纳米羟基磷灰石-聚多巴胺组细胞凋亡率分别为(2.15±0.50)%，(2.53±0.11)%，(2.70±0.43)%和(3.72±0.06)%，与对照组细胞凋亡率(2.14±0.56)%比较无明显差异。单一光热治疗组(纳米羟基磷灰石-聚多巴胺+激光)和化学治疗组(阿霉素)细胞凋亡率增加到(10.18±0.42)%和(26.08±2.48)%，与对照组比较差异有显著性意义；此外，光热-化学联合治疗组(纳米羟基磷灰石-聚多巴胺-阿霉素复合体+激光)细胞凋亡率进一步增加，高达(55.93±2.79)%，明显高于任何单一治疗方式。"
2.3 药物摄取 采用流式细胞术检测不同时间点和激光照射下HSC-4细胞摄取纳米羟基磷灰石-聚多巴胺-阿霉素复合体的含量，结果如图6所示。随着培养时间的增加，药物摄取量逐渐增加，1，2，4，8，24 h的药物摄取量分别为(3.66±0.91)%，(6.46±1.28)%，(15.5±1.61)%，(25.93±2.15)%和(63.37±3.00)%。使用808 nm近红外激光照射后，药物摄取量进一步增加，24 h药物摄取量明显增加，高达(78.93±1.26)%。"
采用激光共聚焦观察药物的摄取和分布情况，结果如图7所示。随着培养时间的增加，细胞内红色荧光(阿霉素)荧光强度增加，1，6，12，24 h的红色荧光强度分别为515.33±185.53，1 191.67±78.05，1 486.33±97.03和2 147.67±75.05。使用808 nm近红外激光照射后，红色荧光强度进一步增强，24 h的红色荧光强度高达4 926.00±141.17。另外，大部分红色荧光与蓝色荧光(细胞核)发生重叠，说明纳米羟基磷灰石-聚多巴胺-阿霉素复合体入胞后释放阿霉素进入细胞核，杀伤肿瘤细胞。 "
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