介孔二氧化硅包覆金纳米棒的肿瘤热疗—化疗联合治疗研究
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摘要
临床研究表明,热疗-化疗的联合治疗可以明显增强抗肿瘤疗效,主要由于热疗在杀死癌细胞的同时,有助于化疗药物进入肿瘤细胞和增加化疗药物的细胞毒性。但传统的热疗-化疗联合治疗技术很难达到理想效果,受限于化疗药物和热疗制剂很难同时到达肿瘤部位,协同发挥作用。当前,纳米技术的迅猛发展,纳米材料由于其纳米尺度所固有的独特物理性质,在肿瘤治疗方面可以发挥重要的作用。新型纳米系统的热疗-化疗联合治疗研究正成为肿瘤治疗的研究热点。
本课题设计并构建了一种新型的基于介孔二氧化硅包覆金纳米棒(GNRs@mSiO2)的肿瘤热疗-化疗联合治疗靶向递释系统。该系统可利用mSiO2孔道高效负载药物,通过近红外激光照射,纳米粒发热的同时能加快药物释放；经过PEG和RGD修饰后的纳米粒具有很好的生物相容性和很低的生物毒性,可避免单核巨噬系统的吞噬,并能主动靶向肿瘤细胞和组织。该系统能够同时发挥热疗-化疗的联合治疗效果,相比单纯的化疗或热疗,可显著提高抗肿瘤效果。
本文第一章为DOX-pGNRs@mSiO2-RGD的构建和表征。采用种子诱导生长法合成均一的长径比为3.92：1的GNRs纳米粒;然后利用GNRs表面CTAB作为模板剂,加入正硅酸乙酯(TEOS)形成Si02包覆GNRs的核壳结构；再用硝酸铵乙醇溶液通过离子交换去除CTAB模板剂,制备GNRs@mSiO2纳米粒。该纳米粒呈棒状结构,粒径长宽为80×50nm,有两个表面等离子共振(Surface plasmon resonance, SPR)峰,横向SPR峰约为530nm,纵向SPR峰约为800nm。纳米粒比表面积为327.42m2/g,孔体积为0.64cm3/g,平均孔道直径2.9nm。为了提高肿瘤靶向效率,对GNRs@mSiO2纳米粒表面进行了PEG和RGD修饰。
GNRs@mSiO2纳米粒可吸收近红外激光并将其转换成大量的热能,发热效果与纳米粒的浓度、激光照射强度、激光照射时间相关。由于合适的介孔孔道和巨大的比表面积,GNRs@mSiO2, pGNRs@mSiO2和pGNRs@mSiO2-RGD纳米粒在pH7.4的PBS溶液中,载DOX药量分别为28.1%士1.4%、25.2±1.5%和25.5±1.1%。DOX释放为pH依赖型,在酸性环境中释放较快；而近红外激光照射也可触发纳米粒快速释放药物。
第二章为pGNRs@mSiO2-RGD的细胞摄取和肿瘤靶向特性考察。生物透射电镜、荧光显微镜直观结果显示,A549细胞对pGNRs@mSiO2-RGD的摄取量大于pGNRs@mSiO2;且孵育时间越长,细胞摄取纳米粒越多DOX-pGNRs@mSiO2-RGD和DOX-pGNRs@mSiO2与A549孵育2h的流式结果表明,修饰RGD的纳米粒其DOX-positive细胞数为32.20%,而未修饰纳米粒仅为14.19%,说明RGD有助于GNRs@mSiO2的细胞内吞。用Lysotracker green和DAPI分别对细胞溶酶体和细胞核进行染色,激光共聚焦显微镜观察表明,DOX-pGNRs@mSiO2-RGD被细胞内吞后,经溶酶体途径转运；此外,DOX可以在细胞内从载体中释放,作用于细胞核。
以Balb/c小鼠皮下A549瘤为模型,考察了pGNRs@mSiO2-RGD对肿瘤的主动靶向特性。以DiR为活体成像荧光探针,将其载于pGNRs@mSiO2和pGNRs@mSiO2-RGD,活体成像结果显示,pGNRs@mSiO2-RGD对Balb/c荷瘤鼠的肿瘤靶向性优于pGNRs@mSiO2c
第三章为DOX-pGNRs@mSiO2-RGD的热疗-化疗治疗效果评价。细胞实验发现,DOX-pGNRs@mSiO2-RGD加激光照射的热疗-化疗联合治疗对A549细胞的细胞毒性,在材料浓度为40μgmL-1时的毒性最强,优于单纯的DOX化疗和pGNRs@mSiO2-RGD加激光照射热疗。
近红外热成像仪测温结果显示,尾静脉给药2h后,近红外激光照射pGNRs@mSiO2-RGD组裸鼠肿瘤表面的升温幅度高于pGNRs@mSiO2组,30s内从31.5℃升至65.9℃,说明偶联RGD的纳米粒有利于肿瘤热疗。A549荷瘤鼠的药效评价结果显示,DOX-pGNRs@mSiO2-RGD加激光照射对A549肿瘤的抑瘤效果,显著优于单纯的DOX化疗和pGNRs@mSiO2-RGD加激光热疗。
上述的体内药效学评价结果与纳米粒的细胞摄取和靶向性结果相吻合,表明DOX-pGNRs@mSiO2-RGD具有良好的肿瘤靶向性和化疗-热疗联合治疗作用,有望提高抗肿瘤效果。
第四章为pGNRs@mSiO2-RGD的初步毒性评价。A549细胞毒性结果显示,有CTAB残留于孔道内的GNRs@mSiO2,在高浓度时有一定的细胞毒性,经硝酸铵乙醇溶液离子交换去除CTAB后无明显的细胞毒性。单纯激光照射A549细胞,对细胞生存无影响。pGNRs@mSiO2和pGNRs@mSiO2-RGD无明显的细胞毒性。
血常规指标显示,静脉注射pGNRs@mSiO2-RGD不引起各项指标发生明显异常,但pGNRs@mSiO2对血小板和红细胞容积产生一定影响；CD68染色结果显示,pGNRs@mSiO2和pGNRs@mSiO2-RGD不引起心、脾、肺、肾的巨噬细胞增多,仅对肝产生轻微毒性。H&E染色结果显示,DOX-pGNRs@mSiO2和DOX-pGNRs@mSiO2-RGD与对照组相比,组织无明显病理反应,而且可以降低DOX对心脏和肾脏的毒性。表明DOX-pGNRs@mSiO2-RGD具有良好的生物相容性和较低的急性毒性。
Current clinical therapy investigation has shown that thermo-chemotherapy therapy could result in additionally enhanced anticancer efficacy, because hyperthermia can promote drug delivery into tumor and increase the drug toxicity. However, for traditional treatment techniques, the synergistic effects of thermo-chemotherapy are difficult to realize in vivo because co-delivery of chemotherapeutic agents together with hyperthermia sources to the target tissues is still challenging. Fortunately, the rapid development of nanotechnologies provides a new impetus for cancer therapy due to its unique physical properties.
Herin, we developed GNRs@mSiO2nanomaterials as NIR-light-absorbing agents and a drug delivery system for in vivo chemo-photothermal destruction of tumor. In order to target the tumor and increase the therapeutic efficacy, RGD peptides were conjugated on the terminal groups of poly(ethylene glycol)(PEG) on GNRs@mSiO2. DOX, a classic anticancer drug, was also successfully loaded into pGNRs@mSiO2-RGD nanomaterials. The results proved that the synergistic effect of DOX-pGNRs@mSiO2-RGD for the efficacious treatment of A549(Human lung adenocarcinoma epithelial cell line) tumor were better than that of the chemotherapy or photothermal therapy alone.
The first part described the construction and characterization of DOX-pGNRs@mSiO2-RGD. CTAB-coated GNRs were firstly synthesized by the seed-mediated method. The TEM image of the obtained CTAB-coated GNRs showed that the average length and width were51.6±2.1and13.2±1.2nm, respectively (about3.9:1aspect ratio). The GNRs@mSiO2were synthesized via a single step coating method, and CTAB molecules on the surface of GNRs served as an organic template for the deposition of a mesoporous silica coating. The GNRs@mSiO2were centrifuged and washed with NH4NO3/ethanol solutions to remove the CTAB residual in the mesoporous channels. The TEM image of the obtained GNRs@mSiO2core-shell nanomaterials indicated that the GNRs were coated with uniform mesoporous silica shells (ca.25nm thickness). The GNRs@mSiO2nanomaterilas have two SPR bands; the longitudinal SPR band is at800nm, whereas the transverse plasmon band is at530nm. The N2adsorption-desorption isotherms and pore size distribution of the GNRs@mSiO2showed the characteristic of mesopores. The pore size distribution exhibited a sharp peak centered at the mean value of2.9nm, indicating a uniform mesopore. The BET surface area and total pore volume were calculated to be348m2/g and0.51cm3/g, respectively. In order to enhance nanomaterials delivery into target cells, RGD peptides were conjugated on the terminal groups of PEG on pGNRs@mSiO2nanomaterials.
Our as-prepared GNRs@mSiO2could be used as effective thermal generators owing to the GNRs ingredients. The photothermal effects depend on GNRs@mSiO2concentration, laser illumination intensity and laser illumination time. DOX was readily loaded into the GNRs@mSiO2, pGNRs@mSiO2and pGNRs@mSiO2-RGD at pH of7.0with a28.1%±1.4%,25.2±1.5%and25.5±1.1%loading capacity. The rates and amounts of DOX released from the nanocarries were strongly dependent on the pH of the medium and the releasing time. The DOX release from nanocarries could be controlled by using NIR laser. After the NIR laser irradiation, the cumulative release of DOX could increase.
The second part described the uptake of pGNRs@mSiO2-RGD and tumor targeting properties. The uptake efficiencies of pGNRs@mSiO2and pGNRs@mSiO2-RGD nanomaterials by A549cells were compared. TEM demonstrated that the uptake amount of pGNRs@mSiO2-RGD in A549cells were significantly more than pGNRs@mSiO2. Meanwhile, DOX-loaded pGNRs@mSiO2and pGNRs@mSiO2-RGD were also employed to investigate cellular uptake characteristics by mean of fluorescent images and flow cytometry of DOX. The fluorescence intensity of A549cells treated with DOX-pGNRs@mSiO2-RGD was considerably higher than that of DOX-pGNRs@mSiO2. Moreover, the cellular uptake of DOX-pGNRs@mSiO2-RGD exhibited time-dependent mode. The quantitative analysis of the cellular uptake by flow cytometry showed that the percent of DOX-positive cells was32.20%after2h incubation with DOX-pGNRs@mSiO2-RGD, but only14.19%with DOX-pGNRs@mSiO2. The results suggested that pGNRs@mSiO2-RGD could act as a transmembrane delivery carrier to increase cell internalization and the DOX intracellular accumulation. With the fluorescent tracking of intracellular pathway, DOX-pGNRs@mSiO2-RGD can enter into the cytoplasm and quickly deliver DOX into the nuclei.
To explore their tumor targeting properties, DiR was employed to load with pGNRs@mSiO2and pGNRs@mSiO2-RGD for in vivo fluorescence imaging. The results showed that pGNRs@mSiO2conjugated with RGD could ensure specific delivery and long-time accumulation in tumor tissues through the active tumor targeting.
The third part described the pharmacodynamic evaluation. Both DOX-pGNRs@mSiO2and DOX-pGNRs@mSiO2-RGD nanocarries were cytotoxic against A549cells in a dose-dependent manner. The treatment efficacy of chemo-photothermal therapy was higher than the separate therapeutic efficacy of chemo-and photothermal therapy.
A pilot chemo-photothermal therapy study in vivo was performed. A549tumor-bearing mice were randomized into5treatment groups (n=8per group):PBS treated (Group Ⅰ), laser treated (Group Ⅱ), DOX treated (6.25mg kg"1, Group Ⅲ), pGNRs@mSiO2-RGD with laser treated (25mg GNRs@mSiO2-equiv./kg, Group IV), DOX-pGNRs@mSiO2-RGD with laser treated (25mg GNRs@mSiO2-equiv./kg, with an equivalent DOX dosage of6.25mg kg-1, Group V). After2h injection, tumors were illuminated with an808-nm NIR laser for30sec (3W/cm2; spot size,5mm) in Group Ⅱ, Ⅳ Ⅴ. During the irradiation, the temperature rapidly increased from31.5to65.9℃in the focal region in Group Ⅳ mouse. In contrast, the maximum temperature of the tumor surface in Group Ⅱ was only about40.1℃. Among them, the antitumor efficiency of Group Ⅴ was particularly prominent and was superior to all the other groups (P<0.05). At23th days, mice were sacrificed and tumors were excised. The tumor weights of Group Ⅴ were the lowest, showing an inhibition rate of66.45%and52.28%compared with Group Ⅲ, Ⅳ. Compared with chemotherapy or photothermal treatment alone, the combined treatment showed a synergistic effect, resulting in higher therapeutic efficacy for in vivo cancer therapy.
The results of pharmacodynamic evaluation were in agreement with those of cells uptake and tumor targeting properties. pGNRs@mSiO2-RGD could ensure specific delivery and long-time accumulation in tumor tissues through the active tumor targeting, which facilitated the chemo-photothermal therapy.
The fourth part described the biological safety of pGNRs@mSiO2-RGD. Direct irradiation of the cells showed little effect on cell viability. The GNRs@mSiO2, pGNRs@mSiO2and pGNRs@mSiO2-RGD without CTAB residual showed no cells cytotoxicity.
The results of hematology analysis showed that the parameters of platelet and hematocrit in pGNRs@mSiO2group appeared to be significant difference in comparison with saline group. The CD68immunohistochemical staining showd that only light toxicity to liver was observed. The results of H&E staining showed that both DOX-pGNRs@mSiO2group and DOX-pGNRs@mSiO2-RGD group presented no significant differences in tissues including brain, liver, spleen, lung and kidney when compared to DOX group. The heart slice of DOX group exhibited myocardial fiber rupture, which was conduced by the cardiac toxicity of DOX.
本课题设计并构建了一种新型的基于介孔二氧化硅包覆金纳米棒(GNRs@mSiO2)的肿瘤热疗-化疗联合治疗靶向递释系统。该系统可利用mSiO2孔道高效负载药物,通过近红外激光照射,纳米粒发热的同时能加快药物释放；经过PEG和RGD修饰后的纳米粒具有很好的生物相容性和很低的生物毒性,可避免单核巨噬系统的吞噬,并能主动靶向肿瘤细胞和组织。该系统能够同时发挥热疗-化疗的联合治疗效果,相比单纯的化疗或热疗,可显著提高抗肿瘤效果。
本文第一章为DOX-pGNRs@mSiO2-RGD的构建和表征。采用种子诱导生长法合成均一的长径比为3.92：1的GNRs纳米粒;然后利用GNRs表面CTAB作为模板剂,加入正硅酸乙酯(TEOS)形成Si02包覆GNRs的核壳结构；再用硝酸铵乙醇溶液通过离子交换去除CTAB模板剂,制备GNRs@mSiO2纳米粒。该纳米粒呈棒状结构,粒径长宽为80×50nm,有两个表面等离子共振(Surface plasmon resonance, SPR)峰,横向SPR峰约为530nm,纵向SPR峰约为800nm。纳米粒比表面积为327.42m2/g,孔体积为0.64cm3/g,平均孔道直径2.9nm。为了提高肿瘤靶向效率,对GNRs@mSiO2纳米粒表面进行了PEG和RGD修饰。
GNRs@mSiO2纳米粒可吸收近红外激光并将其转换成大量的热能,发热效果与纳米粒的浓度、激光照射强度、激光照射时间相关。由于合适的介孔孔道和巨大的比表面积,GNRs@mSiO2, pGNRs@mSiO2和pGNRs@mSiO2-RGD纳米粒在pH7.4的PBS溶液中,载DOX药量分别为28.1%士1.4%、25.2±1.5%和25.5±1.1%。DOX释放为pH依赖型,在酸性环境中释放较快；而近红外激光照射也可触发纳米粒快速释放药物。
第二章为pGNRs@mSiO2-RGD的细胞摄取和肿瘤靶向特性考察。生物透射电镜、荧光显微镜直观结果显示,A549细胞对pGNRs@mSiO2-RGD的摄取量大于pGNRs@mSiO2;且孵育时间越长,细胞摄取纳米粒越多DOX-pGNRs@mSiO2-RGD和DOX-pGNRs@mSiO2与A549孵育2h的流式结果表明,修饰RGD的纳米粒其DOX-positive细胞数为32.20%,而未修饰纳米粒仅为14.19%,说明RGD有助于GNRs@mSiO2的细胞内吞。用Lysotracker green和DAPI分别对细胞溶酶体和细胞核进行染色,激光共聚焦显微镜观察表明,DOX-pGNRs@mSiO2-RGD被细胞内吞后,经溶酶体途径转运；此外,DOX可以在细胞内从载体中释放,作用于细胞核。
以Balb/c小鼠皮下A549瘤为模型,考察了pGNRs@mSiO2-RGD对肿瘤的主动靶向特性。以DiR为活体成像荧光探针,将其载于pGNRs@mSiO2和pGNRs@mSiO2-RGD,活体成像结果显示,pGNRs@mSiO2-RGD对Balb/c荷瘤鼠的肿瘤靶向性优于pGNRs@mSiO2c
第三章为DOX-pGNRs@mSiO2-RGD的热疗-化疗治疗效果评价。细胞实验发现,DOX-pGNRs@mSiO2-RGD加激光照射的热疗-化疗联合治疗对A549细胞的细胞毒性,在材料浓度为40μgmL-1时的毒性最强,优于单纯的DOX化疗和pGNRs@mSiO2-RGD加激光照射热疗。
近红外热成像仪测温结果显示,尾静脉给药2h后,近红外激光照射pGNRs@mSiO2-RGD组裸鼠肿瘤表面的升温幅度高于pGNRs@mSiO2组,30s内从31.5℃升至65.9℃,说明偶联RGD的纳米粒有利于肿瘤热疗。A549荷瘤鼠的药效评价结果显示,DOX-pGNRs@mSiO2-RGD加激光照射对A549肿瘤的抑瘤效果,显著优于单纯的DOX化疗和pGNRs@mSiO2-RGD加激光热疗。
上述的体内药效学评价结果与纳米粒的细胞摄取和靶向性结果相吻合,表明DOX-pGNRs@mSiO2-RGD具有良好的肿瘤靶向性和化疗-热疗联合治疗作用,有望提高抗肿瘤效果。
第四章为pGNRs@mSiO2-RGD的初步毒性评价。A549细胞毒性结果显示,有CTAB残留于孔道内的GNRs@mSiO2,在高浓度时有一定的细胞毒性,经硝酸铵乙醇溶液离子交换去除CTAB后无明显的细胞毒性。单纯激光照射A549细胞,对细胞生存无影响。pGNRs@mSiO2和pGNRs@mSiO2-RGD无明显的细胞毒性。
血常规指标显示,静脉注射pGNRs@mSiO2-RGD不引起各项指标发生明显异常,但pGNRs@mSiO2对血小板和红细胞容积产生一定影响；CD68染色结果显示,pGNRs@mSiO2和pGNRs@mSiO2-RGD不引起心、脾、肺、肾的巨噬细胞增多,仅对肝产生轻微毒性。H&E染色结果显示,DOX-pGNRs@mSiO2和DOX-pGNRs@mSiO2-RGD与对照组相比,组织无明显病理反应,而且可以降低DOX对心脏和肾脏的毒性。表明DOX-pGNRs@mSiO2-RGD具有良好的生物相容性和较低的急性毒性。
Current clinical therapy investigation has shown that thermo-chemotherapy therapy could result in additionally enhanced anticancer efficacy, because hyperthermia can promote drug delivery into tumor and increase the drug toxicity. However, for traditional treatment techniques, the synergistic effects of thermo-chemotherapy are difficult to realize in vivo because co-delivery of chemotherapeutic agents together with hyperthermia sources to the target tissues is still challenging. Fortunately, the rapid development of nanotechnologies provides a new impetus for cancer therapy due to its unique physical properties.
Herin, we developed GNRs@mSiO2nanomaterials as NIR-light-absorbing agents and a drug delivery system for in vivo chemo-photothermal destruction of tumor. In order to target the tumor and increase the therapeutic efficacy, RGD peptides were conjugated on the terminal groups of poly(ethylene glycol)(PEG) on GNRs@mSiO2. DOX, a classic anticancer drug, was also successfully loaded into pGNRs@mSiO2-RGD nanomaterials. The results proved that the synergistic effect of DOX-pGNRs@mSiO2-RGD for the efficacious treatment of A549(Human lung adenocarcinoma epithelial cell line) tumor were better than that of the chemotherapy or photothermal therapy alone.
The first part described the construction and characterization of DOX-pGNRs@mSiO2-RGD. CTAB-coated GNRs were firstly synthesized by the seed-mediated method. The TEM image of the obtained CTAB-coated GNRs showed that the average length and width were51.6±2.1and13.2±1.2nm, respectively (about3.9:1aspect ratio). The GNRs@mSiO2were synthesized via a single step coating method, and CTAB molecules on the surface of GNRs served as an organic template for the deposition of a mesoporous silica coating. The GNRs@mSiO2were centrifuged and washed with NH4NO3/ethanol solutions to remove the CTAB residual in the mesoporous channels. The TEM image of the obtained GNRs@mSiO2core-shell nanomaterials indicated that the GNRs were coated with uniform mesoporous silica shells (ca.25nm thickness). The GNRs@mSiO2nanomaterilas have two SPR bands; the longitudinal SPR band is at800nm, whereas the transverse plasmon band is at530nm. The N2adsorption-desorption isotherms and pore size distribution of the GNRs@mSiO2showed the characteristic of mesopores. The pore size distribution exhibited a sharp peak centered at the mean value of2.9nm, indicating a uniform mesopore. The BET surface area and total pore volume were calculated to be348m2/g and0.51cm3/g, respectively. In order to enhance nanomaterials delivery into target cells, RGD peptides were conjugated on the terminal groups of PEG on pGNRs@mSiO2nanomaterials.
Our as-prepared GNRs@mSiO2could be used as effective thermal generators owing to the GNRs ingredients. The photothermal effects depend on GNRs@mSiO2concentration, laser illumination intensity and laser illumination time. DOX was readily loaded into the GNRs@mSiO2, pGNRs@mSiO2and pGNRs@mSiO2-RGD at pH of7.0with a28.1%±1.4%,25.2±1.5%and25.5±1.1%loading capacity. The rates and amounts of DOX released from the nanocarries were strongly dependent on the pH of the medium and the releasing time. The DOX release from nanocarries could be controlled by using NIR laser. After the NIR laser irradiation, the cumulative release of DOX could increase.
The second part described the uptake of pGNRs@mSiO2-RGD and tumor targeting properties. The uptake efficiencies of pGNRs@mSiO2and pGNRs@mSiO2-RGD nanomaterials by A549cells were compared. TEM demonstrated that the uptake amount of pGNRs@mSiO2-RGD in A549cells were significantly more than pGNRs@mSiO2. Meanwhile, DOX-loaded pGNRs@mSiO2and pGNRs@mSiO2-RGD were also employed to investigate cellular uptake characteristics by mean of fluorescent images and flow cytometry of DOX. The fluorescence intensity of A549cells treated with DOX-pGNRs@mSiO2-RGD was considerably higher than that of DOX-pGNRs@mSiO2. Moreover, the cellular uptake of DOX-pGNRs@mSiO2-RGD exhibited time-dependent mode. The quantitative analysis of the cellular uptake by flow cytometry showed that the percent of DOX-positive cells was32.20%after2h incubation with DOX-pGNRs@mSiO2-RGD, but only14.19%with DOX-pGNRs@mSiO2. The results suggested that pGNRs@mSiO2-RGD could act as a transmembrane delivery carrier to increase cell internalization and the DOX intracellular accumulation. With the fluorescent tracking of intracellular pathway, DOX-pGNRs@mSiO2-RGD can enter into the cytoplasm and quickly deliver DOX into the nuclei.
To explore their tumor targeting properties, DiR was employed to load with pGNRs@mSiO2and pGNRs@mSiO2-RGD for in vivo fluorescence imaging. The results showed that pGNRs@mSiO2conjugated with RGD could ensure specific delivery and long-time accumulation in tumor tissues through the active tumor targeting.
The third part described the pharmacodynamic evaluation. Both DOX-pGNRs@mSiO2and DOX-pGNRs@mSiO2-RGD nanocarries were cytotoxic against A549cells in a dose-dependent manner. The treatment efficacy of chemo-photothermal therapy was higher than the separate therapeutic efficacy of chemo-and photothermal therapy.
A pilot chemo-photothermal therapy study in vivo was performed. A549tumor-bearing mice were randomized into5treatment groups (n=8per group):PBS treated (Group Ⅰ), laser treated (Group Ⅱ), DOX treated (6.25mg kg"1, Group Ⅲ), pGNRs@mSiO2-RGD with laser treated (25mg GNRs@mSiO2-equiv./kg, Group IV), DOX-pGNRs@mSiO2-RGD with laser treated (25mg GNRs@mSiO2-equiv./kg, with an equivalent DOX dosage of6.25mg kg-1, Group V). After2h injection, tumors were illuminated with an808-nm NIR laser for30sec (3W/cm2; spot size,5mm) in Group Ⅱ, Ⅳ Ⅴ. During the irradiation, the temperature rapidly increased from31.5to65.9℃in the focal region in Group Ⅳ mouse. In contrast, the maximum temperature of the tumor surface in Group Ⅱ was only about40.1℃. Among them, the antitumor efficiency of Group Ⅴ was particularly prominent and was superior to all the other groups (P<0.05). At23th days, mice were sacrificed and tumors were excised. The tumor weights of Group Ⅴ were the lowest, showing an inhibition rate of66.45%and52.28%compared with Group Ⅲ, Ⅳ. Compared with chemotherapy or photothermal treatment alone, the combined treatment showed a synergistic effect, resulting in higher therapeutic efficacy for in vivo cancer therapy.
The results of pharmacodynamic evaluation were in agreement with those of cells uptake and tumor targeting properties. pGNRs@mSiO2-RGD could ensure specific delivery and long-time accumulation in tumor tissues through the active tumor targeting, which facilitated the chemo-photothermal therapy.
The fourth part described the biological safety of pGNRs@mSiO2-RGD. Direct irradiation of the cells showed little effect on cell viability. The GNRs@mSiO2, pGNRs@mSiO2and pGNRs@mSiO2-RGD without CTAB residual showed no cells cytotoxicity.
The results of hematology analysis showed that the parameters of platelet and hematocrit in pGNRs@mSiO2group appeared to be significant difference in comparison with saline group. The CD68immunohistochemical staining showd that only light toxicity to liver was observed. The results of H&E staining showed that both DOX-pGNRs@mSiO2group and DOX-pGNRs@mSiO2-RGD group presented no significant differences in tissues including brain, liver, spleen, lung and kidney when compared to DOX group. The heart slice of DOX group exhibited myocardial fiber rupture, which was conduced by the cardiac toxicity of DOX.
引文
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