Study On The Photothermal Properties And Cell Proliferation/Toxicity Of Sodium Humate In Vitro

ObjectiveBy comparing the photothermal properties of six different sodium humate samples under different 808nm laser output power and different concentrations,the best photothermal properties of sodium humate samples and their corresponding parameters were selected.Moreover,through exploring the best samples of sodium humate to human oral keratinocytes（HOK）and oral squamous cancer cells（HSC-4）proliferation/toxic effect,the safety of the samples to normal oral cells and proliferative effect or potential dark toxicity of the samples to cancer cells were preliminarily determined,in order to provide the experimental data for the future research and accelerate the clinical application of humic acid sodium mediated photothermal therapy（PTT）in the treatment of oral cancer.MethodsSix samples of sodium humate were extracted from Baoqing weathered coal,Qitai weathered coal and Tuzuoqi weathered coal by Na₄P₂O₇ solution and Na OH solution,respectively.To investigate the photothermal conversion performance of the samples in vitro,sodium humate samples with different concentrations were irradiated with 808nm laser and the output power of 1W,2W and 3W for 10min,respectively.The safety of the best sodium humate sample to normal oral cells was investigated with HOK as the research object.In addition,the proliferative effect or potential dark toxicity of the best sodium humate sample to cancer cells was investigated with HSC-4 as the research object.The cell relative survival rat of HOK and HSC-4 were detected with CCK-8 method.Results1.Among Baoqing weathered coal,Qitai weathered coal and Tuzuo Qi weathered coal,the amount of humic acid sodium extracted from Qitai weathered coal is the largest.Extraction using Na₄P₂O₇ solution yields higher concentration of sodium humate than that using Na OH solution.Particularly,for Baoqing weathered coal and Tuzuoqi weathered coal samples,the extracted amount is increased by a factor of 3.3 and 4.3 respectively.In addition,the obtained sodium humate sample solution was stored at room temperature for 3 months and not any precipitate was formed or the solution became turbid.Furthermore,for Baoqing weathered coal and Qitai weathered coal,sodium humate extracted by Na OH has better heating performance,whereas Tuzuo Qi weathered coal is opposite.Finally,under 808nm laser irradiation,six kinds of sodium humate samples showed obvious photothermal properties,which increased with the increase of time,concentration and power,the difference was statistically significant（P<0.05）,and the warming effect of Sodium humate sample S5 was the fastest.When the power was 2W and the concentration was 0.5mg/m L,the temperature of Sodium humate sample S5 and S6 was within the ideal tumor temperature sensitive range（43-50℃）.2.For HOK,when the concentration of Sodium humate sample S5 was≤0.5mg/m L,the relative survival rat of cells at different time points remained above 90%.However,when the concentration reached 1mg/m L and 2mg/m L,the relative survival rat of cells decreased significantly.After incubation for 48h,the relative survival rat of cells was 80.42%and64.18%,respectively.After 72h incubation,the relative survival rat of the cells decreased to75.06%and 59.82%respectively,which was significantly different from the control group（P<0.05）.On the other hand,For HSC-4,the cell relative survival rat at different time points remained above 90%when the concentration was≤2.0mg/m L.Conclusion1.Among Baoqing weathered coal,Qitai weathered coal and Tuzuo Banner weathered coal,Qitai weathered coal has the highest humic acid content.Furthermore,extraction using Na₄P₂O₇can improve the utilization rate of ore sources than that using Na OH.Sodium humate sample has not only good dispersion property,but also better stability.Furthermore,for Baoqing weathered coal and Qitai weathered coal,sodium humate extracted by Na OH has better heating performance,whereas Tuzuo Qi weathered coal is opposite.Finally,the photothermal performance of sodium humate sample is positively related to the concentration and laser output power.Overall,Sodium humate sample S5 is the best sodium humate sample with in vitro photothermal properties.The suitable parameters are as follows:laser output power is 2W,and the concentration is 0.5mg/m L.2.When the concentration of Sodium humate sample S5 is between 0.25-0.5mg/m L,it is basically non-toxic to HOK and has good biosafety to normal cells of oral cavity.Within the concentration range of 0.25-2.0mg/m L,S5 has neither obvious proliferation effect nor dark toxicity on HSC-4.Considering the photothermal properties in vitro,sodium humate sample S5 with a concentration of 0.5mg/m L is suitable for the subsequent experimental studies on photothermal killing of cancer cells.