| The primary transfer pathway of the breast cancer is based on the lymphatic system.Thus to kill or inhibit the metastatic tumor cells effectively is one of decisive factors in prognosis of breast cancer which is of great significance for improving the overall survival of breast cancer.Because there exist multiple routes of metastasis for breast cancer among axillary,interpectoral and internal mammary lymph nodes,and sometimes leaping and micro metastasis will exist.So in traditional lymph node dissection,missing metastatic tumor cells is inevitable,which will lead to the local recurrence of breast cancer and affect the prognosis and survival of patient.Although the current routine use of adjuvant chemotherapy has been applied as clinical conventional method,but the medicine uptake into lymphatic system is extremely low in this method,some tumor cells still remain not to be killed effectively.Therefore,the scientists are still searching for new effective treatment for metastatic lymph nodes in breast cancer.Recent research in tumor therapy,photo thermal therapy(PTT)is a novel treatment.By the laser irradiation upon photo thermal material,the local temperature around photo-thermal material will be raised too high enough to kill tumor cell.Compare to traditional chemotherapy and radiotherapy,PPT has gained intensive attention on the tumor therapy with the advantages of precise locating,minimal invasion and no harm to normal tissue.The key factors in PPT lies on the performance of the photo-thermal material,favorable biocompatibility,high photo-thermal transfer efficiency and good photo stability are general consideration.With the deepening research in the field,the design of photo thermal transfer materials is also towards visualization,multi-functional development,combined with imaging means to achieve visual treatment,has become one of the hot spots of photo thermal treatment.The photoacoustic imaging technology is a novel non-invasive in vivo technology which use the energy transfer from laser to ultrasound.It overcomes the limit of optical imaging(1nm),can detect in vivo deep 5cm of the lesions with higher spatial resolution,significantly improving the detection depth while maintaining higher contrast.The combination of photo thermal imaging and photo thermal therapy not only can be used as a visual means to monitor the distribution of photo thermal material,but also can accurately guide the photo-thermal therapy in precise locating.This will improve precision for photo-thermal therapy of metastatic lymph node in breast cancer.Currently in clinical therapy for metastatic lymph node in breast cancer,due to the existence of clinical stage and individual difference in the situation,traditional single treatment fails to inhibit the transfer thoroughly.The way to cure the metastatic lymph node in breast cancer is towards diversified direction.The photo-thermal therapy,combines with imaging and targeting chemotherapy,should be hope to complete cure the metastatic lymph node in breast cancer.Lymph node targeting chemotherapy,is the use of the phagocyte in lymphatic system.The combination of chemotherapy drugs with carrier,from local injection into the lymphatic system,the drugs can maintain at higher concentration for long time.It can kill tumor cell in the lymphatic system effectively.Generally,it has reduced side effect because of no drugs entering into blood circulation.There is extensive research in gastric cancer and breast cancer treatment.The key of lymphatic targeting chemotherapy is the carrier which should have lymphatic tendency,supper adsorption,position and releasing capacity.As one of a cutting edge technology,the medical nanomaterial catches intensive focus when “precision medical care” era is approaching.In recently years,the research of nanomaterial gradually becomes hot spot for it has specific properties in physics,chemistry and biology.In order to achieve the goal of precision medical care,reducing local recurrence and improving the life quality of patient,we are looking for a new,monomeric material inherent,with good photoacoustic imaging characteristics,not only has a high efficiency of light and heat conversion efficiency,but also can carry the targeted chemotherapy drugs integrated multi-functional nano materials.The carbon nanomaterial is a material with one dimension is less than 100 nm.It has specific properties in optics,electrics,thermology and mechanics.There already many research has been studied about application of carbon nanomaterial in photo-thermal therapy and drug delivery.Carbon nanoparticles,as one morphology of carbon nanomaterial which have very good lymph tendency and supper adsorption of medicine,are now used in the treatment for targeted chemotherapy of metastatic lymph node in breast cancer by some researcher.But application in the field of photo-thermal therapy still remains not to be reported.Therefore,in this article,the performance of photo-acoustic imaging and photo-thermal transfer of three different carbon nanomaterials at specific laser wavelength(808nm)have been investigated.It demonstrates that nanocarbon material has excellent properties in photo-acoustic imaging and photo-thermal transfer.By using PLGA as film forming material,the carbon nanodroplets encapsulating carbon nanoparticles and PFH has been prepared,then the experiment of photo-acoustic imaging and photo-thermal therapy has been conducted simultaneously under the laser irradiation.Finally,for super adsorption of nanocarbon particle,the multifunctional particle carrying anti-tumor drugs(docetaxel)has been prepared.Under the laser irradiation,the PFH takes the phase transformation,leads to the burst of the particles,which will release the drugs encapsulated in it so as to achieve the goal of targeted therapy.This strategy provides a new way of image tracking,targeted guiding,combined treatment for metastatic lymph node in breast cancer.Part one The evaluation of photo-acoustic effect and photo-thermal transfer efficiency of carbon nanomaterial at specific laser irradiationObjective To discover the photo-acoustic effect and photo-thermal transformation of carbon nanomaterial in different structure at specific laser irradiation.Method The different concentration of graphene oxide(GO),multi-wall carbon nanotube(MWNT)and carbon nanoparticles(CNs)suspension were prepared.The photo acoustic imaging effect were observed and photo acoustic value were measured.They are irradiated by laser at 808 nm wavelength and different power,and the temperature were monitored by IR camera.Gold nanorods as control,they are irradiated by laser for 30 s,then turn off the laser,let the samples cool down to the room temperature,then irradiate again.This circle should repeat for 5 times.Results All of three materials were able to photo-acoustic imaging and photo-thermal transfer.The MWNT and CNs shows much stronger photo-acoustic effect than that of GO.After laser irradiation for 30 s,the temperature of MWNT and CNs samples can increase up to 50℃.The laser energy is stronger and the concentration is higher,the higher photo-thermal transfer efficiency will be.The highest temperature of the carbon nano materials was maintained at the highest temperature of the first time after 5 times of irradiation,and the temperature of the gold nanorods increased about 10℃.Conclusion The carbon nanomaterial with different structure are able to generate photo-acoustic effect and photo-thermal transfer.The MWNTs and CNs have stronger photo-acoustic and photo-thermal effect compare to the GO.Carbon nanomaterials have good photo-thermal stability.Part two The visualization photo-thermal therapy and targeted chemotherapy of metastatic lymph node in breast cancer mediated by using carbon nanodropletsObjective To synthetize a kind of carbon nanodroplets(CNPs)carrying CNs and liquid fluorocarbon,to detect physical performance,to observe photo-acoustic imaging and photo-thermal transfer,to investigate the killing effect of laser irradiation upon the MDA-MB-231 cells and treatment efficacy for metastasis lymph nodes.Methods Prepared the CNPs solution by the double emulsification method,the morphology,size,surface and inner structure were observed by optical microscope and TEM.The size distribution of the particles were measured by laser particle size analyzer.The zeta potential was measured by Surface potentiometer,and the concentration of CNs in supernatant and CNPs were measured by UV-vis spectrophotometer,then the encapsulation efficiency and drug loading could be calculated.The photo-acoustic effect and value were observed at a wavelength of 808 nm by photo-acoustic instrument.Irradiate by laser of 808 nm with different power,the temperature change curve was monitored by IR camera,choose the appropriate laser power which can maintain the temperature around 45-50℃ for the following cell experiments.Cultures of the breast cancer MDA-MB-231 cells were added with CNPs carrying fluorescent dye Dil.After incubation for 6h,12 h,the phagocytosis of CNPs was observed by laser confocal.CNPs was introduced into the culture medium and incubated for 12 h,irradiated the samples by laser with power of 1.0 W/cm2 for 0,1,2,4min separately,then incubated for another 12 h,the apoptosis rate was measured by Annexin V /FITC flow cytometry.The experimental model in vitro was established.Irradiated the spots of subcutaneous at depth of 1.0,2.0,3.0,4.0cm respectively by different laser power,the temperature change was monitored by IR camera and temperature curve was plot,choose the laser power which can maintain temperature around 45-50℃ for the following animal experiment.The rabbit VX2 cancer lymph node metastasis model was built in 3 groups: CNs group,NPs group and CNPs group.Each group would receive local injection with CNs,NPs and CNPs separately by foot.After 24 h,the distribution of particles in cancer lymph node were observed.Irradiated the samples by 808 nm laser with power 1.5W/cm2,monitored the temperature change by IR camera.Each group would conduct the following experiments before treatment and after treatment for 4 weeks: the size of lymph node was measured by 2D ultrasonography and the volume was calculated.The blood flow signal was measured and classified by the Color Doppler flow imaging.The hardness of lymph node will be observed and measured by elasticity imaging mode.After treatment for 4 weeks,the lymph nodes were dissected,the tumor metastasis and cell necrosis were observed by HE staining,the apoptotic index was detected by TUNEL method,the expression of Ki-67,CD44v6 and E-cadherin were detected by immunohistochemistry.Results The appearance of CNPs prepared in this study was black emulsion.The characters of spherical shape,unique diameter,smooth surface,uniform dispersion of CNPs were observed by optical microscope and TEM.The diameter of CNPs was(452.7±23.6)nm,zeta potential was-(20.8±1.6)m V,the encapsulation efficiency was(51.98±2.86)%,(52.50±3.22)% in supernatant and CNPs separately.And the drug loading were(2.95±0.12)%,(2.83±0.16)% respectively.The photo-acoustic imaging performs well,its value was 6.92±1.45.The photo-thermal transfer efficiency was high,irradiated by laser at power 1.0W/cm2,the temperature increased rapidly and maintained around 45-50℃.Laser confocal microscopy observed that CNPs could be phagocytosed by MDA-MB-231 cells and had a time-dependent manner.The flow cytometry showed that CNPs could kill MDA-MB-231 cell after laser irradiation,the longer duration irradiation lasted,the stronger killing effect could be obtained.In vitro experiment showed that the depth of CNPs in the subcutaneous could have great influence on the photo-thermal transfer efficiency.The depth ≤2.0cm,the CNPs had higher photo-thermal transfer efficiency,the temperature could increase rapidly to 45-50℃ and lasted for long time at laser irradiation of 1.5W/cm2.The depth >2.0cm,the significant decrease of photo-thermal transfer efficiency would be observed.The CNPs could be used for photo-acoustic imaging in metastatic lymph node and had certian therapeutic effects.After 4 weeks of treatment,the NPs group showed increases in node diameter compare to that of before treatment.The CNs group and CNPs group showed slightly decreases in node diameter,but blood flow significantly reduced and elasticity value decreased.HE staining observed that there exists significant necrosis in lymph nodes of CNPs and CNs group,but residual cancer cells remained to be visible in some lymph nodes.TUNEL method showed the apoptosis index in CNPs and CNs groups were much higher than NPs group.In CNPs and CNs groups the necrotic tumor cells showed homogeneous brown staining,the surviving tumor cells showed low expression of Ki-67,negative expression of CD44v6,and positive expression of E-Cadherin.But in NPs group the tumor cells showed high expression of Ki-67,positive expression of CD44v6,and negative expression of E-Cadherin.Conclusion CNPs could be synthesized by the double emulsification method successfully.It had excellent performance in photo-acoustic effect and photo-thermal transfer in vitro.It could kill the breast cancer MDA-MB-231 cells significantly after laser irradiation.The photo-acoustic effect of CNPs in vivo was also good,and after laser irradiation upon the metastatic lymph node,the CNPs still had certain therapeutic effect.But the photo-thermal performance would decrease with increases of the depth to subcutaneous.Part 3 The visualization photo-thermal therapy and targeted chemotherapy of metastatic lymph node in breast cancer mediated by using multifunctional nanodropletsObjective To prepare a multifunctional nanodroplets DOC-CNPs and physical properties were measured.The photo-acoustic imaging,photo-thermal conversion and drug release were observed.After the laser irradiation upon the breast cancer MDA-MB-231 cells,the killing effect and therapeutic efficacy for metastatic lymph node was investigated.Methods To prepare the DOC-CNPs by the double emulsification method.The morphology,size,surface and inner structure were observed by optical microscope and TEM.The size distribution was measured by laser particle size analyzer.The zeta potential was measured by Surface potentiometer.The photo-acoustic effect was observed by photo-acoustic instrument.Irradiate by laser,the temperature was monitored by IR camera.The concentration of docetaxel in supernatant and DOC-CNPs were measured by HPLC,the encapsulation efficiency and drug loading were calculated.The DOC-CNPs were loaded into dialysis bag and irradiated by laser at wavelength of 808 nm,power of 1.0W/cm2,sampled at different time point,the concentration of docetaxel was measured by HPLC,the cumulative drug release rate at each time point was calculated,then the drug release time curve could be established.The MDA-MB-231 cells were cultured and divided into 4 groups: contrast group(CT),DOC-CNPs group(DC),laser irradiation group(LA),DOC-CNPsand laser irradiation group(DC+LA).The CT group should not receive any treatment.DOC-CNPs solution should be introduced into each well of the DC group and DC+LA group.The LA group and DC+LA group would be irradiated by laser with the power of 1.0W/cm2 for 4min.After appropriate treatment,the apoptosis rate of all of the cells would be detected by Annexin V /FITC flow cytometry.The rabbit VX2 metastatic lymph node transfer model was built and divided into 4 groups: CT group will receive no any treatment.DC group and DC+LA group would receive local injection DOC-CNPs solution by foot,after 24 h,the distribution of DOC-CNPs in metastatic lymph node would be observed by photo-acoustic imaging.The LA group and DC+LA group would receive laser irradiation of power 1.5 W/cm2 each day for 10 min.The size of node,blood flow and hardness in each group will be monitored by ultrasound imaging before and after treatment for 4weeks.After treatment for 4 weeks,the lymph nodes were dissected,the tumor metastasis and cell necrosis were observed by HE staining,the apoptotic index was detected by TUNEL method,the expression of Ki-67,CD44v6 and E-cadherin were detected by immunohistochemistry.Results The appearance of DOC-CNPs prepared in this study was black emulsion.Observation under optical microscope and TEM showed spherical shape with unique size,evenly dispersion and smooth surface.The size was(463.7±25.2)nm,the zeta potential was-(22.6±1.2)m V,the photo-acoustic imaging performed well,the photo-thermal transfer efficiency was high,the encapsulation efficiency of docetaxel were(63.85±2.41)%,(64.16±1.87)%respectively in supernatant sand in DOC-CNPs calculated by the measurement of HPLC,and the drug loading were(10.08±0.39)%,(9.83±0.42)% respectively.Under the laser irradiation the drug release rate increased with the time prolonging,while it was lower in the control group.The flow cytometry showed the apoptosis rate in DC+LA group is greater than that of other group significantly.DOC-CNPs could effectively generate photo-acoustic imaging for metastatic lymph node,and had excellent therapeutic effect for tumor metastatic lymph node after laser irradiation.After treatment for 4weeks,the volume of lymph node in CT group and LA group increased significantly compare to those before treatment,but the volume of lymph node in DC+LA group decreased significantly,the blood flow and elasticity also reduced obviously.The massive necrosis happened in lymph node of DC+LA group shown by HE staining.The higher apoptosis rate of DC+LA group than that of other groups.In CT and LA group the tumor cells showed high expression of Ki-67,positive expression of CD44v6,and negative expression of E-Cadherin,in DC group some of the necrotic tumor cells showed homogeneous brown staining,in DC+LA group almost all of the tumor cells showed homogeneous brown staining.Conlusion The double emulsification method can prepare DOC-CNPs successfully,the properties of photo-acoustic effect,photo-thermal conversion and drug loading were excellent.It had strong killing effect for breast cancer cell after irradiated by laser.It demonstrates significant therapeutic effect for metastatic lymph node. |
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