MWNTs Mediated Photothermal Therapy Combined With Chemotherapy For The Treatment Of Bone Metastasis Cancer

Various malignant tumors almost invariably metastasizes to bones in patients with advanced disease,approximately 70%of patients with advanced breast cancer and prostate cancer,and 35%of advanced lung cancer patients metastasizes to bones.Bone metastases are responsible for considerable skeletal morbidity,including intractable bone pain,hypercalcemia and pathologic fractures,greatly reducing patients' life quality and life expectancy.Conventional therapies for bone metastatic,like chemotherapy or surgery,all have their own special disadvantages.Chemotherapy,the most widely applied therapy for bone metastases,is highly limited by high toxicity and drug resistance.The rich microenvironment of bone marrow facilitates survival of cancer cells and mediates drug resistance.Surgery is only suitable for removal of the tumors with well-defined and primary lesions but not for treatment of small,poorly defined metastases.Thermal ablation therapies for solid tumors recently emerged as an attractive alternative approach,because it is minimally invasive,extensively indicative and leads to minimal side effects compared with traditional therapies.Currently,several thermal methods have been used for treatment of bone metastases,including focused ultrasound,microwave and radiofrequency ablation,but their effectiveness is limited by nonspecific heating of target tissue and likely risk of healthy tissue injury.Efforts have never ceased to explore novel or alternative approaches which may yield more effective control or even eradication of bone metastatic foci by surmounting the side effects of current therapies.An emerging photothermal therapy(PTT)is advantageous over traditional thermal therapies because its energy source can be adjusted and shaped to provide relatively uniform distribution of heat according to the tumor volume.Therefore,PTT provides better photothermal ablation specific to tumor foci,resulting in a more effective and minimally invasive therapy which leads to few complications.PTT uses near-infrared(NIR)light to activate photothermal agents in a favorable NIR region(700-1,100 nm)to generate high heat which kills malignant cells.In this study,multi-walled carbon nanotubes(MWNTs)were used as a photosensitive material.Carbon nanotubes(CNTs)are made from rolled up graphene with a cylindrical nanostructure in a certain direction,which can absorb the near infrared light to generate high temperature.NIR light at a region of 700-1,100 nm in wavelength is often used in PTT because it penetrates deeply into the tissue and is hardly absorbed by normal tissue.Recently,Multi-Walled Carbon Nanotubes(MWNTs),a class of nanotubes,have been extensively investigated as a sort of promising photothermal agent for PTT of cancer.Previous studies described the therapeutic potentials of PTT using MWNTs plus NIR irradiation for various cancers,but there has been no study into the effects of PTT via MWNTs plus NIR irradiation on bone metastasis of breast cancer.Meantime,the studies mainly focused on the experiment in vitro.Moreover,the study in vivo mostly research the subcutaneous tumor,which simulate normal tumor environment.Therefore,we hypothesized that NIR irradiation combined with MWNTs might be a promising alternative therapy suitable for bone metastatic foci induced by breast cancer.The first part was designed to determine the effectiveness of PTT with MWNTs in an intraosseous model of bone metastasis of breast cancer.Bone metastases treatment methods varied.The traditional treatment methods include surgery,chemotherapy,radiotherapy.New treatment methods include heat therapy,and PTT.It is difficult to completely eradicate the tumor lesions because of the rapid growth of malignant tumors of bone metastases and high incidence of devastating consequences,such as osteolysis,considerable pain,pathological fractures,hypercalcaemia.In addition,treatment methods have different indications.A single treatment is not suitable for a comprehensive therapy of tumors.Combined treatment can significantly reduce the side effects,enhance the curative effect,which attracted more and more attention.PTT in combination with chemotherapy can enhance the therapeutic effect while reducing the side effects of treatment.The mechanism has the following aspects:(1)PTT directly kill tumor cells,while high temperatures generated by hyperthermia can enhance the cytotoxicity of chemotherapy drugs;(2)PTT can enhance tumor local temperature,increasing blood circulation of tumor tissue and permeability of cell membranes,which can increase uptake of chemotherapy drugs;(3)PTT can also increase the expression of the inflammatory factors,enhancing the therapeutic effect.The first part of this study confirmed that MWNTs mediated photothermal therapy can effectively inhibit the growth of bone metastases and bone destruction.However,photothermal therapy alone cannot completely kill the tumor cells.In order to kill tumor cells,we assess the effectiveness and safety of photothermal therapy combined with chemotherapy on a tumor-bearing mouse model.MWNTs mediated photothermal therapy for the treatment of bone metastasis cancerAIM:To evaluate the effectiveness and safety of MWNTs mediated photothermal therapy for the treatment of bone metastasis cancer.Method:MWNTs were mixed with concentrated nitric acids.The oxidized MWNTs were separated from the solution by filtration and dried under vacuum at room temperature.The O-MWNTs were non-covalently functionalized by DSPE-PEG2000.The O-MWNTs-PEG synthesized were systematically characterized by nuclear magnetic resonance(NMR)and transmission electron microscopy(TEM).The damage to tumor cells induced by thermal ablation with MWNTs and NIR irradiation was evaluated in MDA-231 and MCF-7 cells.Cell survival efficiency was measured using the CCK8 assay.To further verify the photothermal effect on cancer cells,the cells were stained with Live-Dead cell staining kit immediately after the photothermal treatment.The therapeutic effect of MWNTs and NIR laser irradiation was evaluated by measuring growth inhibition of the tumor inoculated in mice.Single-cell suspensions of 1.0×106 murine breast cancer EMT6 cells were injected orthotopically into the tibiae of female Balb/c mice.Then,the mice were anesthetized for irradiation on the tumor sites with an NIR laser at 808 nm.We monitored the maximum tactile allodynia and the body weights following treatments in mice.Results:TEM images showed raw MWNTs were aggregated in thick cords PBS at room temperature while PEGylated MWNTs were mostly dissolved in the solution.The average length of MWNTs was estimated to be 1126 nm from 100 counts of MWNTs.The 1H NMR spectra of raw MWNTs only showed the solvent residual peak while PEGylated MWNTs showed PEG resonance peak and the solvent residual peak,indicating successful synthesis of PEGylated MWNTs.The stability test of raw MWNTs and PEGylated MWNTs in PBS showed raw MWNTs gravitated to the bottom of the bottle after 24h,but PEGylated MWNTs were stable even up to 30 days.Evaluation of temperatures of MWNTs solutions clearly showed that the temperatures rose with the period of NIR irradiation(0-240s)and also with the concentration of MWNTs(0-100 ?g/ml).CCK8 assay demonstrated that MWNTs combined with NIR irradiation(808 nm,5W/cm2)induced stronger cytotoxicity compared with either MWNTs or NIR light irradiation alone.The majority of cells treated with either MWNTs alone or NIR irradiation alone(808 nm,5W/cm2)were living ones(green fluorescence),but a large number of cells treated with MWNTs plus NIR laser irradiation(808 nm,5W/cm2)were dead ones(red or yellowish fluorescence).These results suggest that combination of MWNTs and NIR irradiation is necessary to achieve a lethal effect on tumor cells.MWNTs mediated photothermal effect generates significantly temperature elevation in vivo.MWNTs plus NIR irradiation caused dramatic temperature elevation compared with NIR irradiation alone.Additionally,prolonged NIR irradiation duration enhanced the temperature elevation.Moreover,temperature elevation was associated with the dosage of MWNTs.MWNTs mediated photothermal ablation reduces tumor volume and cancer-induced bone destruction.There were no statistically significant differences(P>0.05)in the mean tumor volume among the groups treated with saline,laser alone and 10?g MWNTs,but tumor volume in the group treated with 10?g MWNTs+laser was significantly smaller(P<0.01).The effect of suppression tumor growth was attenuated with decreased dosage of MWNTs of 1?g.1?g MWNTs+laser produced modest suppression in tumor volume,which was significantly larger than that in the group of 10?g MWNTs+laser(P<0.05).In the groups treated with saline,saline+laser and 10?g MWNTs,the bone structure was all severely destroyed and massive osteosclerotic growth was observed.However,in the groups treated with 1?g MWNTs+laser,and 10?g MWNTs+laser,there was only mild bone destruction.Especially in the groups treated with 10?g MWNTs+ laser,the bone structure was significantly protected.Tumor-bearing tibiae treated with 10?g MWNTs+laser showed a statistically significant decrease in bone volume compared with those treated with saline,laser alone and 10?g MWNTs(P<0.05),but 1?g MWNTs+laser led to only a slight reduction in bone volume compared with the other three groups(P>0.05).MWNTs plus NIR laser irradiation caused a remarkably greater suppression of tumor growth compared with treatment with either MWNTs injection or NIR irradiation alone,significantly reducing the amount of tumor-induced bone destruction.MWNTs mediated photothermal ablation does not affect tactile allodynia and body weight in mice.There were no significant between-group differences in the maximum tactile allodynia and body weights after photothermal therapy.These results clearly indicated that MWNTs combined with NIR irradiation did not affect the withdrawal threshold or body weight,thus implying the PTT might be a safe therapy used in tumor-bearing mice.Conclusion:Prolonged duration of NIR irradiation and increased dosage of MWNTs enhanced photothermal effect of PTT.MWNTs plus NIR irradiation reduced the tumor size in the mice,and protected the bone from cancer-induced destruction in an intraosseous model of bone metastasis of breast cancer.Significantly,our therapy for the bone metastasis of breast cancer,MWNTs plus NIR irradiation,achieved high antitumor efficacy without significant side effects,as proved by nociceptive tests and body weight measurements.MWNTs/Dox mediated photothermal therapy combined with chemotherapy for the treatment of bone metastasis cancerAIM:To evaluated the effectiveness and safety of MWNTs/Dox mediated photothermal therapy combined with chemotherapy for the treatment of bone metastasis cancer.Method:MWNTs were mixed with concentrated sulfuric and nitric acids.The oxidized MWNTs were separated from the solution by filtration and dried under vacuum at room temperature.The O-MWNTs were non-covalently functionalized by DSPE-PEG2000.The O-MWNTs-PEG synthesized were systematically characterized by Raman spectrometer,fourier transform infrared spectroscopy(FT-IR)nuclear magnetic resonance(NMR)and transmission electron microscopy(TEM).DOX-loaded O-MWNTs-PEG(MWNTs/Dox)were prepared through ?-? action.The drug release rate at different PH value of the environment and release rate of the drug under laser irradiation were evaluated.The damage to tumor cells induced by thermal ablation with MWNTs and NIR irradiation was evaluated in MDA-231 cells.The therapeutic effect of MWNTs/Dox-mediated photothermal therapy combined with chemotherapy was evaluated by measuring growth inhibition of the tumor inoculated in mice.We monitored the H&E staining tissue sections and body weights following treatments in mice.Results:TEM images showed average length of MWNTs was estimated to be 99.57±61.20 nm from 100 counts of MWNTs.The bands at 1210 cm-1 are attributed to the vibration of C-O bonds,confirming the introduction of carboxyl to the carbon skeleton.The Raman spectroscopic analysis showed the higher intensity of D bands of O-MWNTs in comparison to MWNTs,which is due to the more defects at the surface of O-MWNTs.Characteristic resonance peaks of PEG was observed in the spectrum of O-MWNTs-PEG,demonstrating the successful synthesis of O-MWNTs-PEG.The MWNTs solution has good dispersibility and stability after decorating with PEG2000.O-MWNTs-PEG dispersed well in PBS solution or DMEM cell culture medium and remained stable for four weeks without any detectable agglomeration.With the increase of the laser power(i.e.,from 1 to 1.2 and 1.5w/cm2),the magnitude of the temperature elevation can be increased by 42.6±0.26,49.2±0.89,58.1±1.10?,respectively,in the same irradiation time of 60s.These demonstrated that MWNTs is a good platform for PTT.The loading of Dox was testified by reddish color of MWNTs/Dox solution.Moreover,after loaded with Dox,the characteristic of peak of Dox was found in the composites of MWNTs/Dox,demonstrating the successful loading of Dox on the surface of MWNTs.The loading efficiency of Dox was as high as 88.57%,showing the MWNTs is a promising candidate for drug carrier materials.The accumulative release of Dox were only 8.4%and 40.7%in 48h in PH 9.0 and PH 7.4,respectively.However,when the PH decreased to PH 5.5,the Dox was rapidly released in the early stage,and finally the accumulative release of Dox was up to 77.4%.The PH-dependent drug release system would be superior to drug application,because the micro-environment of the extracellular tissues of tumor are acidic,which is benefit for the cancer therapy.Moreover,the drug release rate was accelerated with irradiation.28.08%of Dox was released form MWNTs/Dox composites in the first 10min,however,only 1.78%of drug was released after 10min.These results indicated that the hyperthermia could increase the drug release,which is benefit to the cancer therapy.Cell survival efficiency was measured using the CCK8 assay.MWNTs/Dox-mediated photothermal therapy combined with chemotherapy can significantly inhibit tumor cell viability.Cell viability of MWNTs/Dox plus laser irradiation was significantly lower than the cells treated with free Dox or MWNTs plus laser irradiation.cell viability did not change significantly under 808nm laser irradiation alone 2min,Cell viability significantly reduced to 49.5%when combined with 100 ?g/ml of the MWNTs,indicating that MWNTs mediated photothermal therapy can effectively kill tumor cells.Cell viability was 78.1%when cultured with 10 ?g/ml DOX incubation,and cell viability was 75.8%when combined with laser irradiation.Cell viability was 75.8%when cultured with MWNTs/Dox incubation,however,cell viability significantly reduced to 35.7%when combined with laser irradiation.All these demonstrated that photothermal therapy combined with chemotherapy can achieve better results.After irradiated with a 808nm laser(1.5 w/cm2),only minimal temperature elevation was recorded in the PBS group,in contrast with significant temperature increase in the MWNTs group and MWNTs/Dox group.The temperature in the tumor surface injected with MWNTs and MWNTs/Dox composites were 48.4 and 48.9? under 60s NIR irradiation,respectively.The effect of suppression tumor growth of MWNTs/Dox-mediated photothermal therapy combined with chemotherapy was significantly higher than that of MWNTs-mediated photothermal therapy alone or chemotherapy alone.apparent osteolytic destruction was observed in the saline and Dox groups,only partial bone destruction was found in the MWNTs plus laser irradiation group.In comparison,the mice treated with MWNTs/Dox plus laser irradiation showed the lest osteolytic destructions.The combination of chemo-and photothermo-therapy could not only inhibit the growth of the metastasis tumors but also prevent the osteolytic destructions in an bone metastases model.MWNTs/Dox-mediated photothermal therapy combined with chemotherapy does not affect H&E staining tissue sections and body weights in mice.There were no significant between-group differences in the H&E staining tissue sections and body weights after photothermal therapy.These results clearly indicated that MWNTs/Dox-mediated photothermal therapy combined with chemotherapy did not affect the H&E staining tissue section or body weight,thus implying the MWNTs/Dox-mediated photothermal therapy combined with chemotherapy might be a safe therapy used in tumor-bearing mice.Conclusion:MWNTs/Dox-mediated photothermal therapy combined with chemotherapy achieved a better effect in reduction of tumor size and protection the bone from cancer-induced destruction in an intraosseous model of bone metastasis of breast cancer.Significantly,our therapy for the bone metastasis of breast cancer,MWNTs/Dox-mediated photothermal therapy combined with chemotherapy,achieved high antitumor efficacy without significant side effects,as proved by HE and body weight measurements.This paper evaluated the efficacy and safety of MWNTs mediated photothermal therapy and MWNTs/Dox-mediated photothermal therapy combined with chemotherapy for the treatment of bone metastases.In the first part,MWNTs mediated photothermal therapy caused a remarkably greater suppression of tumor growth compared with treatment with either MWNTs injection or NIR irradiation alone,significantly reducing the amount of tumor-induced bone destruction.Meantime,MWNTs mediated photothermal therapy achieved high antitumor efficacy without significant side effects.In the second part,MWNTs/Dox-mediated photothermal therapy combined with chemotherapy could not only inhibit the growth of the metastasis tumors but also prevent the osteolytic destructions in an bone metastases model,which is better than photothermal therapy alone and chemotherapy alone.MWNTs/Dox-mediated photothermal therapy combined with chemotherapy is a promising therapeutic alternative because it is safe and effective for bone metastatic foci.