空心硅纳米粒子光敏剂在光动力治疗胆管癌中的作用及机制
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摘要
目的：
1、制备负载光敏剂Photosan的空心氧化硅纳米粒子(以下简称纳米化Photosan),并初步探讨纳米化Photosan的药物安全性。
2、探索并比较Photosan及纳米化Photosan介导的光动力疗法(Photodynamic Therapy, PDT)在体外试验中对胆管癌的疗效。
3、探索并比较Photosan及纳米化Photosan介导的PDT在体内试验中对胆管癌的疗效。
4、探索并比较Photosan及纳米化Photosan介导的PDT抑制胆管癌生长的可能途径。
方法：
1、采用一步湿化学方法制备负载Photosan的空心氧化硅纳米粒子。
2、体外实验中,用MTT法检测Photosan及纳米化Photosan介导的PDT对人胆管癌QBC939细胞的杀伤作用。观察不同的光敏剂孵育时间、光敏剂浓度,及光照剂量对PDT效果的影响,并了解这两种光敏剂的暗毒性,比较其药效差异。用流式细胞计数实验进一步观察两种光敏剂应用于PDT的效果,并比较它们之间的差异。光学显微镜下观察PDT前后细胞的形态学改变。
3、建立裸鼠人胆管癌移植瘤模型,观察Photosan及纳米化Photosan介导的PDT对肿瘤的抑制作用及它们之间的差异。观察光敏剂对动物有无毒副作用。病理切片下观察PDT后肿瘤组织细胞的形态学改变。
4、检测裸鼠肿瘤PDT治疗后对其血管生成、侵袭能力及细胞凋亡的影响,并对比两种光敏剂间的区别。所用方法为用PCR、 western-blot、免疫组化等检测相关基因在mRNA和蛋白表达的水平的区别。
结果：
1、成功制备了负载Photosan的空心氧化硅纳米粒子,实现了光敏剂Photosan的纳米化。
2、MTT法结果显示Photosan及纳米化Photosan介导的PDT均对胆管癌细胞有明显的杀伤作用(P<0.05),而后者的杀伤作用更强(P<0.05)。光动力作用在一定范围内与光敏剂的孵育时间、光敏剂的浓度及光照剂量均呈正相关的关系。两种光敏剂均无明显的暗毒性,但两者具有不同的最佳作用参数。纳米化Photosan的最佳作用参数中,其所需的光敏剂浓度更低,孵育时间更短。Photosan及纳米化Photosan的流式细胞计数实验显示,使用各自的最佳作用参数时,后者的细胞杀伤效果明显强于前者(P<0.05)。两者对细胞杀伤的途径均以诱导凋亡为主,而后者中细胞坏死的比例高于前者。光学显微镜下亦可观察到纳米化Photosan对肿瘤细胞的杀伤作用更强,且坏死细胞的比例更高。
3、动物实验结果显示,在使用各自的最佳作用参数时,Photosan与纳米化Photosan介导的PDT均对胆管癌有明显的抑制作用(P<0.05),而后者作用更强(P<0.05)。病理切片结果亦显示,后者对肿瘤的杀伤效果更为明显。
4、Photosan及纳米化Photosan介导的PDT作用于胆管癌后,与肿瘤血管生成、侵袭能力相关的基因在mRNA及蛋白水平的表达均下调(P<0.05),与细胞凋亡相关的基因表达上调(P<0.05)。且后者相较于前者,对肿瘤侵袭能力及细胞凋亡的影响更为强烈(P<0.05)。
结论：
1、采用一步湿化学方法可制备出负载Photosan的空心氧化硅纳米粒子,实现光敏剂Photosan的纳米化,且纳米化Photosan无明显毒性。
2、Photosan及纳米化Photosan介导的PDT均对胆管癌细胞具有明显的杀伤作用,而后者的杀伤作用更强。光动力作用在一定范围内与光敏剂的孵育时间、光敏剂的浓度及光照剂量均呈正相关的关系。纳米化Photosan较之于普通Photosan,带来的光动力效应更强,达到有效治疗浓度的速率更快。
3、Photosan及纳米化photosan介导的PDT均在裸鼠人胆管癌移植瘤模型上证实具有明显的治疗作用,且Photosan纳米化后可缩短给药——光照时间间隔,增强光动力效应。
4、Photosan及纳米化Photosan介导的PDT治疗肿瘤的机制至少包括了三个方面,即抑制肿瘤的血管生成、降低肿瘤的侵袭能力以及诱发肿瘤细胞的凋亡。且纳米化Photosan减轻肿瘤的侵袭性以及诱发肿瘤细胞凋亡的能力均明显强于Photosan。
Aims:
1. Preparation Photosan-loaded hollow silica nanoparticles (Photosan-loaded HSNPs), and study the drug safety of Photosan-loaded HSNPs.
2. Study and compare the effect of photodynamic therapy (PDT) with Photosan and Photosan-loaded HSNPs on cholangiocarcinoma in vitro.
3. Study and compare the effect of PDT with Photosan and Photosan-loaded HSNPs on cholangiocarcinoma in vivo.
4. Study and compare the possible ways of PDT with Photosan and Photosan-laoded HSNPs inhibiting the growth of cholangiocarcinoma.
Methods:
1. Prepare the Photosan-loaded hollow silica nanoparticles.
2. In vitro experiments, test the killing effect of PDT with Photosan or Photosan-loaded HSNPs for human cholangiocarcinoma QBC939cells using MTT assay. Observe the influence of PDT effects with different photosensitizer incubation times, different concentrations of photosensitizers and different light dose, and study the dark toxicity of the2photosensitizers as well as compare the differences of their effects. Flow cytometry analysis further investigate the PDT effect of2photosensitizers, and compare the difference between them. Using an optical microscope to observe the morphological changes of the cells before and after PDT.
3. Establish the human cholangiocarcinoma in nude mice xenograft model, and observe the differences of inhibiting effect of PDT with Photosan of Photosan-loaded HSNPs. Observe whether the photosensitizers have toxic or side effects on animals. Observe the morphological changes of tumor cells before and after PDT by biopsy.
4. Detect the impacts of PDT for mice's tumors on vascular generation, invasion capacity and cell apoptosis, and compare the differences between the two photosensitizers. The methods were detecting the differences in the level of mRNA and protein expression of related genes using PCR, Western-blot, and immunohistochemistry.
Results:
1. Prepare the Photosan-loaded hollow silica nanoparticles successfully.
2. MTT assay shows that the PDT both with Photosan and Photosan-loaded HSNPs have obvious killing effect on cholangiocarcinoma cells (P<0.05), and the latter one has even better killing effect (P<0.05). Within a certain range, the effect of PDT shows a positive relationship with the photosensitizer incubation time, the concentration of photosensitizer and light dose. There is no obvious dark toxicity on both two kinds of photosensitizers. But these two kinds of photosensitizers have different optimal interaction parameters. The optimal interaction parameter of Photosan-loaded HSNPs has lower photosensitizer concentration and shorter incubation time. Flow cytometry assay with Photosan or Photosan-loaded HSNPs shows that the latter has obviously stronger cell killing effect to the former in the condition of they both using their own optimal interaction parameters (P<0.05). Both of them kill the cells through the way of apoptosis. But the latter has higher proportion of cell necrosis to the former. With an optical microscope we can also find that the killing effect on tumor cells of Photosan-loaded HSNPs is stronger, and the percentage of necrotic cells is higher.
3. Animal experiments show that PDT with Photosan or Photosan-loaded HSNPs both have significantly inhibition effect to cholangiocarcinoma by using their own optimal interaction parameters (P<0.05), and the latter one is even stronger than the former (P<0.05). Biopsy findings also show that the latter has more obvious anti-tumor effect.
4. The genes expression at mRNA and protein levels relating to tumor angiogenesis, invasion in cholangiocarcinoma were reduced (P<0.05), and the apoptosis-related genes were up-regulated after PDT with Photosan or Photosan-loaded HSNPs (P<0.05). Compared to the former, the latter has more strongly influence on the tumor invasion ability and tumor cell apoptosis (P<0.05)
Conclusions:
1. Photosan-loaded HSNPs can be prepared by one-step wet chemical-based synthetic route. HSNPs have no significant toxicity.
2. Photosan and Photosan-loaded HSNPs both have obvious killing effect on cholangiocarcinoma cells, while the latter's killing effect is stronger. In a certain range, the effect of PDT shows a positive relationship with the photosensitizer incubation time, the concentration of photosensitizer and light dose. Comparing to Photosan, Photosa-loaded HSNPs bring stronger PDT effect, faster rate to achieve effective therapeutic concentration.
3. PDT with Photosan and Photosan-loaded HSNPs both proved to have a significant therapeutic effect on nude mice xenograft model of human cholangiocarcinoma.
4. The possible approachs of PDT with Photosan and Photosan-loaded HSNPs in inhibiting the growth of cholangiocarcinoma are:inhibition of tumor angiogenesis, reduction of tumor invasion ability, as well as induction of tumor cells apoptosis, while the latter reduce tumor invasion ability and induce apoptosis of tumor cells more significantly.
1、制备负载光敏剂Photosan的空心氧化硅纳米粒子(以下简称纳米化Photosan),并初步探讨纳米化Photosan的药物安全性。
2、探索并比较Photosan及纳米化Photosan介导的光动力疗法(Photodynamic Therapy, PDT)在体外试验中对胆管癌的疗效。
3、探索并比较Photosan及纳米化Photosan介导的PDT在体内试验中对胆管癌的疗效。
4、探索并比较Photosan及纳米化Photosan介导的PDT抑制胆管癌生长的可能途径。
方法：
1、采用一步湿化学方法制备负载Photosan的空心氧化硅纳米粒子。
2、体外实验中,用MTT法检测Photosan及纳米化Photosan介导的PDT对人胆管癌QBC939细胞的杀伤作用。观察不同的光敏剂孵育时间、光敏剂浓度,及光照剂量对PDT效果的影响,并了解这两种光敏剂的暗毒性,比较其药效差异。用流式细胞计数实验进一步观察两种光敏剂应用于PDT的效果,并比较它们之间的差异。光学显微镜下观察PDT前后细胞的形态学改变。
3、建立裸鼠人胆管癌移植瘤模型,观察Photosan及纳米化Photosan介导的PDT对肿瘤的抑制作用及它们之间的差异。观察光敏剂对动物有无毒副作用。病理切片下观察PDT后肿瘤组织细胞的形态学改变。
4、检测裸鼠肿瘤PDT治疗后对其血管生成、侵袭能力及细胞凋亡的影响,并对比两种光敏剂间的区别。所用方法为用PCR、 western-blot、免疫组化等检测相关基因在mRNA和蛋白表达的水平的区别。
结果：
1、成功制备了负载Photosan的空心氧化硅纳米粒子,实现了光敏剂Photosan的纳米化。
2、MTT法结果显示Photosan及纳米化Photosan介导的PDT均对胆管癌细胞有明显的杀伤作用(P<0.05),而后者的杀伤作用更强(P<0.05)。光动力作用在一定范围内与光敏剂的孵育时间、光敏剂的浓度及光照剂量均呈正相关的关系。两种光敏剂均无明显的暗毒性,但两者具有不同的最佳作用参数。纳米化Photosan的最佳作用参数中,其所需的光敏剂浓度更低,孵育时间更短。Photosan及纳米化Photosan的流式细胞计数实验显示,使用各自的最佳作用参数时,后者的细胞杀伤效果明显强于前者(P<0.05)。两者对细胞杀伤的途径均以诱导凋亡为主,而后者中细胞坏死的比例高于前者。光学显微镜下亦可观察到纳米化Photosan对肿瘤细胞的杀伤作用更强,且坏死细胞的比例更高。
3、动物实验结果显示,在使用各自的最佳作用参数时,Photosan与纳米化Photosan介导的PDT均对胆管癌有明显的抑制作用(P<0.05),而后者作用更强(P<0.05)。病理切片结果亦显示,后者对肿瘤的杀伤效果更为明显。
4、Photosan及纳米化Photosan介导的PDT作用于胆管癌后,与肿瘤血管生成、侵袭能力相关的基因在mRNA及蛋白水平的表达均下调(P<0.05),与细胞凋亡相关的基因表达上调(P<0.05)。且后者相较于前者,对肿瘤侵袭能力及细胞凋亡的影响更为强烈(P<0.05)。
结论：
1、采用一步湿化学方法可制备出负载Photosan的空心氧化硅纳米粒子,实现光敏剂Photosan的纳米化,且纳米化Photosan无明显毒性。
2、Photosan及纳米化Photosan介导的PDT均对胆管癌细胞具有明显的杀伤作用,而后者的杀伤作用更强。光动力作用在一定范围内与光敏剂的孵育时间、光敏剂的浓度及光照剂量均呈正相关的关系。纳米化Photosan较之于普通Photosan,带来的光动力效应更强,达到有效治疗浓度的速率更快。
3、Photosan及纳米化photosan介导的PDT均在裸鼠人胆管癌移植瘤模型上证实具有明显的治疗作用,且Photosan纳米化后可缩短给药——光照时间间隔,增强光动力效应。
4、Photosan及纳米化Photosan介导的PDT治疗肿瘤的机制至少包括了三个方面,即抑制肿瘤的血管生成、降低肿瘤的侵袭能力以及诱发肿瘤细胞的凋亡。且纳米化Photosan减轻肿瘤的侵袭性以及诱发肿瘤细胞凋亡的能力均明显强于Photosan。
Aims:
1. Preparation Photosan-loaded hollow silica nanoparticles (Photosan-loaded HSNPs), and study the drug safety of Photosan-loaded HSNPs.
2. Study and compare the effect of photodynamic therapy (PDT) with Photosan and Photosan-loaded HSNPs on cholangiocarcinoma in vitro.
3. Study and compare the effect of PDT with Photosan and Photosan-loaded HSNPs on cholangiocarcinoma in vivo.
4. Study and compare the possible ways of PDT with Photosan and Photosan-laoded HSNPs inhibiting the growth of cholangiocarcinoma.
Methods:
1. Prepare the Photosan-loaded hollow silica nanoparticles.
2. In vitro experiments, test the killing effect of PDT with Photosan or Photosan-loaded HSNPs for human cholangiocarcinoma QBC939cells using MTT assay. Observe the influence of PDT effects with different photosensitizer incubation times, different concentrations of photosensitizers and different light dose, and study the dark toxicity of the2photosensitizers as well as compare the differences of their effects. Flow cytometry analysis further investigate the PDT effect of2photosensitizers, and compare the difference between them. Using an optical microscope to observe the morphological changes of the cells before and after PDT.
3. Establish the human cholangiocarcinoma in nude mice xenograft model, and observe the differences of inhibiting effect of PDT with Photosan of Photosan-loaded HSNPs. Observe whether the photosensitizers have toxic or side effects on animals. Observe the morphological changes of tumor cells before and after PDT by biopsy.
4. Detect the impacts of PDT for mice's tumors on vascular generation, invasion capacity and cell apoptosis, and compare the differences between the two photosensitizers. The methods were detecting the differences in the level of mRNA and protein expression of related genes using PCR, Western-blot, and immunohistochemistry.
Results:
1. Prepare the Photosan-loaded hollow silica nanoparticles successfully.
2. MTT assay shows that the PDT both with Photosan and Photosan-loaded HSNPs have obvious killing effect on cholangiocarcinoma cells (P<0.05), and the latter one has even better killing effect (P<0.05). Within a certain range, the effect of PDT shows a positive relationship with the photosensitizer incubation time, the concentration of photosensitizer and light dose. There is no obvious dark toxicity on both two kinds of photosensitizers. But these two kinds of photosensitizers have different optimal interaction parameters. The optimal interaction parameter of Photosan-loaded HSNPs has lower photosensitizer concentration and shorter incubation time. Flow cytometry assay with Photosan or Photosan-loaded HSNPs shows that the latter has obviously stronger cell killing effect to the former in the condition of they both using their own optimal interaction parameters (P<0.05). Both of them kill the cells through the way of apoptosis. But the latter has higher proportion of cell necrosis to the former. With an optical microscope we can also find that the killing effect on tumor cells of Photosan-loaded HSNPs is stronger, and the percentage of necrotic cells is higher.
3. Animal experiments show that PDT with Photosan or Photosan-loaded HSNPs both have significantly inhibition effect to cholangiocarcinoma by using their own optimal interaction parameters (P<0.05), and the latter one is even stronger than the former (P<0.05). Biopsy findings also show that the latter has more obvious anti-tumor effect.
4. The genes expression at mRNA and protein levels relating to tumor angiogenesis, invasion in cholangiocarcinoma were reduced (P<0.05), and the apoptosis-related genes were up-regulated after PDT with Photosan or Photosan-loaded HSNPs (P<0.05). Compared to the former, the latter has more strongly influence on the tumor invasion ability and tumor cell apoptosis (P<0.05)
Conclusions:
1. Photosan-loaded HSNPs can be prepared by one-step wet chemical-based synthetic route. HSNPs have no significant toxicity.
2. Photosan and Photosan-loaded HSNPs both have obvious killing effect on cholangiocarcinoma cells, while the latter's killing effect is stronger. In a certain range, the effect of PDT shows a positive relationship with the photosensitizer incubation time, the concentration of photosensitizer and light dose. Comparing to Photosan, Photosa-loaded HSNPs bring stronger PDT effect, faster rate to achieve effective therapeutic concentration.
3. PDT with Photosan and Photosan-loaded HSNPs both proved to have a significant therapeutic effect on nude mice xenograft model of human cholangiocarcinoma.
4. The possible approachs of PDT with Photosan and Photosan-loaded HSNPs in inhibiting the growth of cholangiocarcinoma are:inhibition of tumor angiogenesis, reduction of tumor invasion ability, as well as induction of tumor cells apoptosis, while the latter reduce tumor invasion ability and induce apoptosis of tumor cells more significantly.
引文
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