| Objective:The inherent radioresistance and inaccuracy of localization of tumors weaken the clinical implementation effectiveness of radiotherapy.To overcome these limitations,bifunctional bismuth oxide nanoparticles modified by hyaluronic acid?HA-Bi2O3NPs?was synthesized by one-pot hydrothermal method for target-specific computed tomography?CT?imaging and radiosensitization of tumor.After functionalization of hyaluronic acid?HA?,the HA-Bi2O3 NPs possessed favorable solubility in water,excellent biocompatibility and were uptaken specifically by cancer cells over expressing CD44 receptors.The as-prepared HA-Bi2O3 NPs exhibited highly X-ray attenuation efficiency and ideal radiosensitivity via synergizing X-ray to induce cell apoptosis and arrest the cell cycle in a dose-dependent manner in vitro.Remarkably,these properties offered excellent performance in active-tartgeting CT imaging and enhancement of radiosensitivity for inhibition of tumor growth.These findings demonstrated that HA-Bi2O3 NPs as theranostic agents exhibited great promise for CT imaging-guided radiotherapy in tumors diagnosis and treatment.Methods:HA-Bi2O3 NPs were prepared using the bismuth source material and sodium hyaluronate via a slightly modified procedure of the polyol method.HA-Bi2O3 NPs was characterized by transmission electron microscopy/High Resolution Transmission Electron Microscopy?TEM/HR-TEM?,NanoDLS particle size analyzer,X-ray diffraction?XRD?,Fourier transform infrared spectrometry?FT-IR?and X-ray photoelectron spectroscopy?XPS?.In order to verify the HA receptor-mediated cellular uptake behavior,we used real-time PCR,flow cytometry assay and fluorescence microscopy images by Confocal Laser Scanning Microscope?LSCM?.The biocompatibility characterization of HA-Bi2O3 NPs was characterized by CCK-8 assay,hemolysis assay,histologicalanalysis and Inductively coupled plasma mass spectrometry?ICP-MS?.The CT imaging for tumor of HA-Bi2O3 NPs was demonstrated by ICR mice,the subcutaneous tumor models.The radiosensitization of HA-Bi2O3 was conducted by CCK-8 assay,Live-dead staining assay,flow cytometry assay and clonogenic assay in vitro and subcutaneous tumor models in vivo.Results:1.Synthesis and characterization of HA-Bi2O3 NPsWe successfully synthesized the HA-Bi2O3 NPs.As shown in the NanoDLS particle size analyzer,HA-Bi2O3 NPs had a well-defined structure of 45 ± 0.6 nm diameter,the average diameter of HA-Bi2O3 NPs in aqueous solution kept stable for 8days.TEM/HRTEM showed that the HA-Bi2O3 NPs all exhibited uniform dispersion and discrete quasi-spherical shape without apparent aggregation,possessed a uniform lattice structure with the lattice fringe of d = 3.3 ± 0.2 ?.X-ray diffraction?XRD?pattern further investigated the phase structure of HA-Bi2O3 NPs were heterostructure,and these diffraction peaks of HA-Bi2O3 NPs matched well with the characteristic peaks of cubic Bi2O3?JCPDS 2004 06-0312?.FT-IR spectroscopy showed the HA-Bi2O3 NPs composed of C=O,-COOH and-OH.The XPS survey spectrum of the as-produced HA-Bi2O3 NPs showed Bi atoms incorporated into the nanoparticles and mainly composed of bismuth,carbon and oxygen elements.2.HA receptor-mediated cellular uptake behavior of HA-Bi2O3 NPsAs previous reported,relative expression of CD44 in SMMC-7721 was higher than in MCF7 according to real-time PCR results.From flow cytometry assay,it was also worthy of note that SMMC-7721 cells exhibited much higher uptake of HA-Bi2O3 NPs than MCF7,implying the synergistic effect of CD44 on cellular internalization of HA-Bi2O3 NPs.3.The biodistribution and histocompatibility of the HA-Bi2O3 NPsThe inherent cytotoxicity of HA-Bi2O3 NPs was assessed in MCF7,SMMC-7721 and VSMC cells via CCK-8 assay,all the HA-Bi2O3 NPs at different concentration exhibited negligible cytotoxicity.Even at the concentration of 400 g/m L and a 72-hours exposure time,the cell inhibition rate were all less than 10%.Asshown in hemolysis assay,no obvious hemolysis phenomenon was observed after incubation with different concentrations from 12.5 to 400 ?g/mL for 2 hours administraton,which was similar to the negative normal saline.The ICP-MS results reported that the NRs presented more appreciable accumulation in the liver?27.80mg/g?and kidney?7.40 mg/g?.Histological assessment of the harvested susceptible organs?liver,heart,spleen,lung,and kidney?showed that the HA-Bi2O3 NPs did not cause obvious adverse effects,such as tissue damage or inflammation,when compared to the control group,showing good organ compatibility.4.Experimental study on the CT imaging for tumor using HA-Bi2O3 NPsWe investigated the X-ray attenuation capacity of the HA-Bi2O3 NPs compared with the Omnipaque?commercial CT contrast agents?,the CT image brightness increased with the HA-Bi2O3 NPs concentration,which was similar to the behavior of the Omnipaque solution.After intravenous injection of HA-Bi2O3 NPs,the CT images were acquired through different scans time,HA-Bi2O3 NPs exhibited gradual increases of CT signal intensities at tumor within 10 min.Compared with pre-injection,a great contrast enhancement in tumor was observed clearly,indicating that HA-Bi2O3 have a time-dependent increase of signals and exhibited a strong CT imaging capability at 10 min post-injection,the signal both in the kidney and bladder strongly increased at 30 minutes post-injection,which indicated effective clearance of HA-Bi2O3 NPs via renal matabolismout of body.5.Study on the radiosensition of HA-Bi2O3 NPs in vitro and in vivoUsing CCK-8 assay,no significant difference in inhibition was found in HA-Bi2O3 NPs group,while the combination of X-ray radiation and HA-Bi2O3 NPs significantly inhibited proliferation of cells?P<0.05?.The as-prepared HA-Bi2O3 NPs have radiosensitivity effect.With increasing the concentrations of HA-Bi2O3 NPs and the doses of radiation,the inhibition rate of SMMC-7721 cells dramatically increased to 76.5% during treatment with 9Gy radiation and 400g/m L of HA-Bi2O3 NPs.clonogenic assay showed that single HA-Bi2O3 NPs has no dramatically influence on the SMMC-7721 cells colony formation.With 6Gy radiationthe,survival fraction of cells was 46.7%.The survival fraction of SMMC-7721 cells dramatically decreased to29.8% during treatment with 6Gy radiation and 200?g/mL of HA-Bi2O3 NPs?P<0.05?.As shown in live-dead staining assay,intensive green fluorescence without red was observed in the SMMC-7721 cells treated with single HA-Bi2O3 NPs which verified high cell viability,but a small amount of red fluorescence can be seen in the X-ray radiation treatments which indicated slightly cell apoptosis.However,bright red fluorescence could be seen in the combination treatment of HA-Bi2O3 NPs?200 g/ml?and radiation?6 Gy?.As shown in flow cytometry assay,almost no apoptosis or necrosis cells were observed in the group of no radiation treatment with the concentration up to 200 ?g/m L.When the concentration was is 200 ?g/mL,after radiation,the early apoptosis rate of cells was 41.2%;the late apoptosis/necrosis rate reached 14.1%.As analyzed by flow cytometry,radiation?6 Gy?alone slightly caused 19.87% of G2/M phase arrest,and HA-Bi2O3 NPs?from 0 to 200g/ml?activated apoptotic cell death from 0.446% to 2.34%,as reflected by the Sub-G1 proportions.However,the combination of HA-Bi2O3 NPs and radiation dose-dependently increased the extent of G2/M phase arrest and cell apoptosis.For instance,combination of HA-Bi2O3 NPs?200g/ml?and radiation at 6 Gy enhanced the proportions of G2/M phase arrest and Sub-G1 peak to 33% and 21.59%respectively.As show in tumor growth curve,The control group?PBS?and single HA-Bi2O3 NPs,respectively,resulted in 270% increases of tumor volumes compared to their original volumes,from the survival curves,the median survival time was 17 days and 20 days.This data indicated that the HA-Bi2O3 NPs has no influence on the tumor growth after 10 days.In the group treated with X-ray irradiation alone,tumor growth was inhibited during 4-10 days,as seen by a decrease in tumor volume of approximately 14.6%,the median survival time was 24 days.In contrast,the HA-Bi2O3 NPs treatment combined with radiation group showed that the tumors shrank by a maximum volume percentage of 33.9%?P<0.05?,the median survival time was 35 days.Conclusion: 1.We have developed a bifunctional HA-Bi2O3 by simple BiCl3·6H2O as the bismuth source material and sodium hyaluronate as the targeting molecules.2.The HA-Bi2O3 NPs revealed favorable solubility,stability and dispersion in water.HA-Bi2O3 NPs had a well-defined structure of diameter and possessed a uniform lattice structure with the lattice fringe,the diffraction peaks of HA-Bi2O3 NPs matched well with the characteristic peaks of cubic Bi2O3.The functional groups of HA-Bi2O3 NPs mainly contained certain plentiful C=O,-COOH,and-OH groups.The as-produced HA-Bi2O3 NPs mainly composed of bismuth,carbon and oxygen atoms.HA-Bi2O3 NPs were discrete excellent biocompatibility and no obvious toxicity.3.The X-ray attenuation capacity of the HA-Bi2O3 NPs compared with the Omnipaque?commercial CT contrast agents?,the CT image brightness increased with the HA-Bi2O3 NPs concentration,which was similar to the behavior of the Omnipaque solution.By the enhanced permeability and retention?EPR?effect,HA-Bi2O3 NPs have great promising applications in tumors diagnosis.4.HA-Bi2O3 NPs could be an efficient alternative to improve radiosensitization enhancements for radiotherapy and had great promising applications in tumors radiotherapy. |
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