Preparation And Application Of High Z Nano-radiosensitizer In Tumor Radiotherapy

Background:Radiation Therapy(RT)is the main treatment option for cancer,but due to toxicity limitations in normal tissues,at a given radiation dose,RT is often used to enhance RT during treatment to improve tumor response.control.At present,there have been several studies involving heavy metals or high-Z materials as radiosensitizers,such as gold nanoparticles.However,these related studies mainly focus on spherical gold nanoparticles.In addition,in addition to gold nanoparticles,there are many related studies on other high-Z materials as radiosensitizers.Another class of materials represented by alkaline earth metal tungstates shows great potential in radiosensitization.Alkaline earth metal tungstates have large X-ray cross sections and relatively low biological toxicity,and are now widely used as scintillation materials.Recently,Ca WO₄nanoparticles have been prepared and their potential as a radiosensitizer has been demonstrated in several studies,but so far,few attempts have been made to explore other alkaline earth metal tungstates in RT.Objective:1.Two different types of new high-Z nano-radiosensitizer ultrathin gold nanowires and alkaline earth metal tungstate BaWO₄ nanoparticles were synthesized and characterized,and their stability,water solubility and safety were evaluated respectively.Create conditions for research and application.2.The prepared and synthesized new high-Z nano-radiosensitizer ultra-thin gold nanowires were coated with polyethylene glycol(PEG)phospholipid layer on the surface to increase its water solubility.Compared with Gold nanoshells(GNSs)particles coated with the same phospholipids,the ability of the two to enhance free radical production under irradiation and to see if oxidative stress can be increased,and then injected into a xenograft model Tumor growth was monitored to assess the potential of ultrathin gold nanowires as radiosensitizers.3.The prepared and synthesized new alkaline earth metal tungstate radiosensitizer BaWO₄ nanoparticles and Ca WO₄ nanoparticles synthesized by a similar method were sequentially coated with polyvinyl pyrrolidone(PVP)to improve their colloidal stability.In an in vitro study,the toxicity of the two nanoparticles was tested,introduced into 4T1 cells to evaluate their ability to generate oxidative stress under radiation conditions,and whether they could enhance radiation-induced cell death,and finally by injection into xenogeneic Tumor cell growth inhibition after transplantation model to study the application value of BaWO₄ nanoparticles in RT.Methods:1.Gold nanowires(GNWs)were synthesized by reducing HAu Cl₄ in hexane.The synthesized GNWs were then coated with a layer of PEGylated phospholipids to make them soluble in water.As a comparison,spherical gold nanoparticles coated with the same phospholipids were also synthesized.The hydrodynamic size and surface charge of both particles were characterized by dynamic light scattering and zeta potential.The morphology and EDS elemental map of the nanoparticles were evaluated by scanning electron microscopy and transmission electron microscopy equipped with an EDAX energy dispersive X-ray spectroscopy system.In vitro and in vivo studies were performed with 4T1 cells,and the cell viability of 4T1 cells was assessed by ATP analysis.4T1 cells were seeded in 96-well plates at an initial density of 5000 cells/well.Twenty-four hours after inoculation,RPMI solutions containing different concentrations of GNWs or GNSs were added.After an additional 24 hours of incubation,the ATPlite 1-step substrate solution was mixed for testing.The luminescence signal was measured using a microplate reader,and the average luminescence intensities were calculated and compared.In order to study the situation of nanoparticles+X-rays,GNWs or GNSs were added to 4T1 cells and incubated for 24 hours,and then 5Gy X-ray radiation was applied.After 24 hours of irradiation,the cells were collected and measured in the same way.Singlet oxygen was measured in the above-mentioned culture medium by singlet oxygen fluorescent probe(Singlet Oxygen Sensor Green,SOSG)method.SOD activity was detected with a free radical detector.Hydroxyl radical production was characterized by Aminophenyl fluorescein(APF)assay.The lipid peroxidation product malondialdehyde(MDA)was detected using a TBARs detection kit.The 4T1 tumor model was generated by subcutaneously injecting 2×10⁵ cells into the right skin of female BALB/c mice.GNWs or GNSs were injected intratumorally into mice on day 0 when the mean tumor volume was approximately100 mm3.Tumors were irradiated with three doses(3 Gy×3)on day 0(1 hour after injection),day 1 and day 2.Tumor size and body weight were checked and tumors were measured every 3 days.After 28 days,mice were sacrificed and tumor tissue sections were H&E stained to monitor and compare tumor growth.2.Use the hydrothermal reaction to synthesize BaWO₄ nanoparticles and Ca WO₄ nanoparticles,and coat them with PVP to improve their biocompatibility and colloidal stability.The size of the nanoparticles was characterized by transmission electron microscopy,and the morphology and energy dispersive spectroscopy elemental maps of the nanoparticles were assessed by scanning electron microscopy.The crystallinity of the nanoparticles was assessed by X-ray diffractometer.Nanoparticle coatings were verified by FT-IR.Changes in singlet oxygen levels were analyzed by SOSG analysis.Two different concentrations of PVP-BaWO₄ or PVP-Ca WO₄ nanoparticles were prepared and studied using a mini-X X-ray tube.By using a microplate reader to measure SOSG fluorescence,the ¹O₂ levels of each group before and after 5 Gy X-ray irradiation were evaluated and determined by APF.to characterize the hydroxyl radicals generated in the presence of nanoparticles.In vivo and in vitro studies were performed using 4T1 cells,which were seeded in 96-well plates(10⁴cells per well)and cultured for 12 hours.Fresh medium with PVP-BaWO₄ or PVP-Ca WO₄ nanoparticles was added.Cells after 4 or 24 hours of incubation with nanoparticles were incubated in SOSG.Detection of singlet oxygen levels before and after 5 Gy X-ray irradiation.The PBS solution containing APF was added to the culture plate,the hydroxyl radical level was detected,and the SOD activity was measured by measuring the absorbance at 450 nm.To observe survival in the absence of X-ray radiation,MTT assays were performed after cells were incubated with nanoparticles containing different concentrations of PVP-BaWO₄ or PVP-Ca WO₄ for 24 hours.Afterwards,PVP-BaWO₄ or PVP-Ca WO₄ nanoparticles were incubated with cells for 4 hours,followed by 5 Gy X-ray irradiation for nanoparticle plus radiation assays.Cell viability was measured by MTT assay after20 hours.10⁶ 4T1 cells were inoculated subcutaneously into both flanks of BALB/c mice.When the tumor size reached 100 mm³,each tumor was injected with PVP-BaWO₄or PVP-Ca WO₄ nanoparticles(5 mg/kg).One hour after injection,tumors were irradiated with 10 Gy of radiation.Tumor size was measured every 3 days,mice were sacrificed 18 days later,and tumor changes in mice were analyzed and compared.Results:1.Successfully synthesized two types of new high-Z nano-radiosensitizer ultra-thin gold nanowires and alkaline earth metal tungstate BaWO₄ nanoparticles,both of which have good stability,water solubility and safety.2.The synthesized GNWs were coated with a layer of oleylamine,and their surface was hydrophobic.We coated the GNWs with PEGylated phospholipids,and the resulting DSPE-PEG(2000)amine-coated GNWs were easily dispersed in aqueous solution.For comparison,we also synthesized GNSs and coated them with DSPE-PEG(2000)amine.We incubated GNWs or GNSs with 4T1 cells at different concentrations in the absence of radiation.Both were weakly toxic at lower concentrations,but both caused a significant decrease in cell viability with increasing concentrations,while GNWs showed greater biocompatibility.In the absence of radiation,APF analysis showed that the presence of GNWs increased the production of hydroxyl radicals to a much greater degree than GNSs.However,GNWs plus radiation significantly increased the level of ¹O₂ and the activity of SOD in cells.Thiobarbituric acid response assays and TBARS assays,which were used to measure lipid oxidation levels,also demonstrated increased oxidative stress.Greater lipid oxidation was observed when GNWs were used in combination with RT.This reflects the effect of increased free radical production on cellular oxidative stress.For assessing whether GNWs contribute to RT-induced toxicity.Neither GNWs nor GNSs caused severe toxicity in the absence of radiation.When a 5 Gy dose of radiation was applied to the cells for 24 hours,the ATPlite assay showed that the toxicity of both nanomaterials was enhanced to a certain extent.In the 4T1subcutaneous tumor model assay,GNWs showed a more pronounced reduction in tumor cell numbers relative to GNSs.At the same time,we did not observe signs of acute toxicity in either,nor did we observe weight loss in mice throughout the experiment.3.Unmodified BaWO₄ nanoparticles are easily aggregated in aqueous solution.To improve colloidal stability,PVP-coated BaWO₄ and Ca WO₄ nanoparticles were synthesized by incubating PVP and BaWO₄ or Ca WO₄ nanoparticles in DMF/H₂O solution.We then irradiated the PVP-BaWO₄ or PVP-Ca WO₄ nanoparticles with X-rays at a dose of 5 Gy.Singlet oxygen radicals and hydroxyl radicals were detected by SOSG method and APF method,respectively.The singlet oxygen content hardly changes in the presence of nano-barium oxide or nano-calcium oxide,but the number of hydroxyl radicals increases sharply in the presence of tungstate,which is more obvious with the presence of nano-barium oxide.In the absence of X-ray irradiation,the toxicity of PVP-BaWO₄ nanoparticles was found to be low.In contrast,the decrease in cell viability of Ca WO₄ nanoparticles after incubation for 24hours was much higher than that of BaWO₄ nanoparticles.PVP-BaWO₄ or PVP-Ca WO₄ nanoparticles were incubated with 4T1 cells for 24 hours,and then irradiated with 5 Gy dose of X-rays.SOSG detection found that the levels of ¹O₂produced by PVP-BaWO₄ and Ca WO₄ nanoparticles in the cells increased after 4hours,and hydroxyl radicals The effect is greater,the APF fluorescence of PVP-BaWO₄ and PVP-Ca WO₄ nanoparticles increased by 9.8 and 6.4 times,respectively.But for Ca WO₄ nanoparticles,the increasing trend of free radical concentration gradually weakened with time.For BaWO₄ nanoparticles,the enhancement of free radicals is still very significant even after 24 hours.The effect of PVP-BaWO₄ and PVP-Ca WO₄ nanoparticles on cell viability under 5 Gy dose irradiation was evaluated by MTT method.Comparable levels of decline due to the cytotoxicity of these two nanoparticles were observed.But in the absence of radiation,Ca WO₄ nanoparticles are relatively toxic.The radiotherapy effects of BaWO₄ and Ca WO₄ were evaluated in a mouse model.The tumor growth inhibition rate in the Ca WO₄+RT group was significantly lower than that in the BaWO₄+RT group.Meanwhile,no signs of acute or long-term toxicity were observed in animals treated with BaWO₄+RT.Conclusion:1.In this study,two novel radiosensitizers of high-Z nanomaterials have been successfully prepared.Both of them have stable properties,good water solubility and biocompatibility.They can be used as radiosensitizers to enhance RT treatment and have lower performance.toxicity.2.Ultrathin gold nanowires are promising radiosensitizers.Under X-ray irradiation,GNWs are significantly better than GNSs in enhancing radical generation capacity.In vitro analysis found that GNWs induced lipid peroxidation and increased intracellular oxidative stress under irradiation.When tested in vivo,GNWs inhibited tumors better than GNSs in the presence of radiation.At the same time,it is less toxic and can be safely injected into tumors to enhance RT.Although this study is currently performed in a breast cancer model,the approach could be attempted to be extended to the treatment of other types of cancer.3.BaWO₄ nanoparticles can effectively enhance RT and have great potential as novel radiosensitizers.When tested in vitro,BaWO₄ nanoparticles were less toxic than Ca WO₄ nanoparticles in the absence of irradiation,while Ba WO4 nanoparticles increased hydroxyl radical production more effectively than Ca WO₄ nanoparticles under irradiation conditions,with a significant induction of Oxidative stress can significantly improve the efficacy of RT.When tested in vivo,BaWO₄ nanoparticles enabled more effective tumor inhibition without causing significant systemic toxicity.