| Objective:Osteosarcoma（OS）is a common primary malignant bone tumor originated from bone mesenchymal cells,which has the characteristics of poor prognosis,high metastasis rate and high recurrence rate.Surgical intervention combined with adjuvant chemotherapy/radiotherapy is a common method for the osteosarcoma treatment,but surgical resection inevitably makes the residual tumor difficult to remove,easily leads to tumor metastasis and recurrence,and large bone defects at the resection site.Chemotherapy/radiotherapy also has disadvantages such as many side effects and serious drug resistance.In recent years,Au-based nanomaterials have received extensive attention,especially gold nanorod（Au NR）have been intensively studied and applied in the field of photothermal therapy.In this study,we prepared a multifunctional Au NR@CA nanohybrids by surface modification of Au NR via Au-catechol bond using Chlorogenic acid（CA）as catechol anchor for the synergistic treatment of OS and bone defects.Methods:1.Preparation and characterization of Au NR@CA nanohybridsThe maximum binding amount of CA to Au NR was calculated by Ultraviolet-visible spectroscopy（UV-Vis）.Fourier transform infrared（FTIR）spectrometer was used to study characteristic functional groups.The morphology of Au NR@CA nanohybrids was observed by transmission electron microscope（TEM）.Zeta potential was measured by dynamic light scattering particle size analyzer,and water solubility was investigated.2.Photothermal effect of Au NR@CA nanohybridsThe UV absorption properties of Au NR@CA nanohybrids were studied by UV-Vis,and the photothermal effect and photothermal stability were investigated.3.Antitumor effect of Au NR@CA nanohybridsCell counting kit 8（CCK-8）was used to determine the effect of Au NR@CA nanohybrids on the survival rate of human osteosarcoma cells（Saos-2 cells）before and after 808 nm near infrared（NIR）laser irradiation.The content of adenosine triphosphate（ATP）in cells was detected by ATP content determination kit.The effect of Au NR@CA on cell apoptosis before and after NIR laser irradiation was investigated by flow cytometry.The m RNA expression levels of apoptosis-related genes in cells were examined by quantitative real-time PCR（q PCR）.The tumor-bearing nude mouse model was established to study the photothermal effect of Au NR@CA nanohybrids in the tumor site of mice,and to investigate the antitumor effect in vivo and the toxic and side effects during the treatment.4.Bone regeneration induced by Au NR@CA nanohybridsThe alamar Blue?HS cell viability assay kit was used to determine the activity of Au NR@CA nanohybrids on mouse embryonic osteoblast precursor cells（MC3T3-E1cells）before and after 808 nm NIR laser irradiation.Alkaline phosphatase（ALP）staining and activity detection and Alizarin Red S（ARS）staining were used to determine the degree of cell differentiation and mineralization.The m RNA expression levels of related genes in the cells were examined by q PCR.The mouse model of calvarial defect was established to study the photothermal effect of Au NR@CA nanohybrids in the calvarial defect of mice.The effect of bone regeneration in vivo was investigated by micro-computed tomography（micro-CT）,H&E staining and Masson staining.Results:1.Preparation and characterization of Au NR@CA nanohybridsAu NR@CA nanohybrids were synthesized by one-step assembly via Au-catechol bonds.The maximum binding amount of CA was reached 4.5 g per 1 g Au NR by UV-Vis.The FTIR spectra demonstrated the successful synthesis of CA-modified Au NR.The results of TEM and dynamic light scattering particle size analyzer showed that Au NR@CA had a rod-like structure with a particle size of about 64 nm,an aspect ratio of about 3.4,and a negatively charged surface.In addition,compared with Au NR@CTAB and free CA,the water solubility of Au NR@CA was greatly improved.2.Photothermal effect of Au NR@CA nanohybridsThe UV absorption spectrum results showed that the Au NR@CA has a strong absorption peak at 814 nm,suggesting that Au NR@CA is a potential photothermal agent.The photothermal properties of Au NR@CA were further evaluated and the results showed that the temperature of Au NR@CA could be increased to 68.3°C after being irradiated by an 808 nm NIR laser with a laser power density of 2.0 W·cm-2 for 10 min,and the photothermal effect was positively correlated with the laser power density.In addition,the absorbance at the maximum absorption wavelength of Au NR@CA after stored for 0 h,1 h,2 h,3 h,4 h and 5 h was relatively consistent,and the maximum temperature has no obvious change after irradiation with 808 nm NIR laser for 10 min.Moreover,Au NR@CA maintained basically unchanged photothermal properties after four laser on/off cycles,indicating that it had good photothermal stability and can be used as an excellent photothermal agent.3.Antitumor effect of Au NR@CA nanohybridsThe antitumor effects of Au NR@CA nanohybrids were studied at the cellular and animal levels.The results of CCK-8 showed that Au NR@CA significantly inhibited the proliferation of Saos-2 cells after irradiation with an 808 nm NIR laser at 2.0 W·cm-2 for10 min.The results of ATP content determination showed that Au NR@CA nanohybrids after NIR laser irradiation have significantly reduced the ATP level in Saos-2 cells.The results of apoptosis assay showed that Au NR@CA combined with photothermal effect killed tumor cells mainly by promoting the late apoptosis of Saos-2 cells.The expression levels of apoptosis-related genes Bcl-2,Caspase-3 and p21 m RNA in cells were detected by q PCR.The results showed that Au NR@CA irradiated by NIR laser promoted the apoptosis of Saos-2 cells by down-regulating the expression of Bcl-2 m RNA and up-regulating the m RNA expression of Caspase-3 and p21.The results of in vivo photothermal effect showed that the tumor site temperature of the nude mice in the Au NR@CA group could be increased to 54.9°C after irradiation with NIR laser at 2.0W·cm-2 for 10 min,indicating that Au NR@CA had a high photothermal conversion efficiency.The results of in vivo antitumor experiments showed that intratumor injection of Au NR@CA under NIR laser irradiation could inhibit tumor growth,inhibit angiogenesis,inhibit tumor cell proliferation and promote tumor cell apoptosis.The body weight of the mice was normal during the treatment and no obvious histopathological abnormalities were observed after the treatment,indicating that Au NR@CA nanohybrids irradiated by NIR laser had good biosafety.4.Bone regeneration induced by Au NR@CA nanohybridsThe effects of Au NR@CA nanohybrids on bone regeneration were studied at the cellular and animal levels.Au NR@CA had no effect on the viability of MC3T3-E1 cells after being irradiated with a mild NIR laser at 808 nm with the density of 1.0 W·cm-2 for1 min,indicating that it had good biocompatibility.The cell viability assay results at day1,3 and 7 showed that Au NR@CA could promote the proliferation of MC3T3-E1 cells after irradiation with 1.0 W·cm-2 NIR laser at 808 nm for 1 min.Au NR@CA significantly increased ALP activity after NIR laser irradiation,which indicated that the degree of osteogenic differentiation was high.Meanwhile,ARS staining experiment showed that Au NR@CA irradiated by NIR laser significantly promoted the mineralization of MC3T3-E1 cells.The m RNA expression levels of osteogenesis-related genes ALP,Runx2,OPN and OCN in cells were detected by q PCR.The results showed that Au NR@CA after NIR laser irradiation promoted the osteogenic differentiation of MC3T3-E1 cells by up-regulating the m RNA expression of ALP,Runx2,OPN and OCN.In addition,Au NR@CA irradiated by NIR laser up-regulated the m RNA expression of HSP47 and HSP70,which indicated that the mechanism of osteogenesis induced by Au NR@CA by photothermal stimulation may be related to the increase of HSP expression in MC3T3-E1 cells.The results of in vivo photothermal effect showed that the temperature of the calvarial defect site of the mice in the Au NR@CA group could be increased to 42.3℃after irradiation with NIR laser at 1.0 W·cm-2 for 1 min,resulting in a mild photothermal effect suitable for bone regeneration.The results of in vivo study showed that Au NR@CA nanohybrids irradiated by NIR laser could significantly promote the formation of new bone in the calvarial defect area of mice and promote bone regeneration.Conclusion:In conclusion,Au NR@CA nanohybrids were assembled by one-step method via Au-catechol bond for tumor inhibition and bone regeneration.The Au NR@CA nanohybrids possess precise nanostructures,good water solubility and excellent photothermal properties.Under controllable NIR laser irradiation,on the one hand,Au NR@CA exhibited excellent antitumor activity,which can promote cell apoptosis and inhibit tumor growth;on the other hand,Au NR@CA showed outstanding osteogenic ability,which can induce osteogenic differentiation of MC3T3-E1 cells and promote bone regeneration.In a word,the study proposes a novel biomimetic strategy for the synergistic treatment of OS and bone defects through controlled hyperthermia. |
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