Fabrication And Application Of Multifunctional Nano-microwave Sensitizers In Tumor Precision Therapy

Objective:Local interventional therapy,including image-guided thermal ablation,has become an important part of non-surgical treatment for tumor patients.Particularly,microwave ablation（MWA）stands out for its high heating efficiency,large ablation area,deep tissue penetration and low susceptibility to the heat-sink,and has gradually evolved as the mainstream choice for tumor thermal ablation.But it still suffers from some drawbacks,such as the rapid diffusion of heat,low curative effect,high tumor recurrence rate and the lack of tissue specificity.Fortunately,the rapid development of nanomedicine has provided new opportunities to combat these limitations.This thesis aims to construct a series of biocompatible nano-microwave sensitizers with low toxicity,excellent microwave heating performance and manifold antitumor capabilities through the design and synthesis of nanomaterials,and to investigate their sensitization effect as well as the mechanism in various tumor models.Chapter One:Targeted delivery of Bi2Se3 Nanoflowers to orthotopic liver tumor via transarterial infusion for enhanced microwave ablation sensibilizationObjective:To prepare a novel non-ionic loaded microwave sensitizer,and then integrate the minimally invasive interventional technology for the microwave sensitization treatment under low power and the microwave protection effect of normal tissues in the rabbit VX2 orthotopic liver tumor model.Methods:1.Synthesis and characterization.The Bi₂Se₃ nanoflowers（Bi₂Se₃ NFs）with multilayered petal morphology were synthesized by adjusting reaction parameters under visible-light irradiation,in which natural amino acid L-Selenocystine and bismuth nitrate pentahydrate were used as the selenium and bismuth sources,respectively.Scanning electron microscopy,transmission electron microscope,energy dispersive spectra,X-ray diffractometer,X-ray photoelectron spectroscopy and Fourier transform infrared spectra were applied to acquire the morphology,size,phase and crystallography,element compositions and surface modification of the samples.2.Evaluation of microwave heating effect.The temperature changes of Bi₂Se₃ NFs with different concentrations(0,0.5,1.0,2.0 mg m L^-1)under microwave irradiation（2450 MHz,5.0W）were monitored in real-time by an infrared thermal imager.Then,the microwave heating effect of Bi₂Se₃ NFs was further verified in fresh lean pork tissues.3.Orthotopic liver tumor model and laparotomy MWA.The rabbit VX2 orthotopic liver tumor models were established by ultrasonic-guided trocar implantation.Next,the VX2-bearing rabbits were randomly divided into four groups（n=3）:control;Bi₂Se₃NFs;MWA;Bi₂Se₃NFs+MWA.Then,the Bi₂Se₃NFs were administrated into the tumor site via the clinical available transcatheter arterial infusion（TAI）interventional operation under the guidance of a digital subtraction angiography device.After TAI interventional procedure,the tumors were then exposed to a microwave probe for thermal ablation after a standard laparotomy.Meanwhile,the whole MWA therapy process was monitored by an infrared thermal imager.4.Therapeutic effect monitoring and mechanism exploration.The changes of tumor volume in each group were dynamically monitored by computed tomography（CT）imaging.Finally,the hematoxylin and eosin（H&E）and immunohistochemistry staining were used to evaluate the levels of coagulative necrosis,apoptosis and heat shock protein 70 in different treatment groups.5.Biosecurity study.Cytotoxicity assay,hemolysis assay,blood routine,blood biochemistry and H&E staining of vital organs were applied to evaluate the biosecurity of Bi₂Se₃NFs.Results:1.The Bi₂Se₃ NFs with an average diameter of 891.17±106.23 nm were produced through a one-step synthesis process under mild conditions.2.The temperature elevation of Bi₂Se₃NFs aqueous dispersions at a concentration of 2.0 mg m L^-1 was significantly higher than that of the deionized water group（38.0±2.6℃vs 54.2±1.6℃,P<0.05）.Moreover,the temperature of the Bi₂Se₃NFs group raised rapidly under microwave irradiation with the increase of concentration,irradiation time and power intensity.All these results indicated the efficient enhancement of microwave thermal effect based on Bi₂Se₃NFs.Similarly,the necrosis diameter and necrosis volume of pork tissues in Bi₂Se₃ NFs group were significantly greater than that of the deionized water group（P<0.05）.However,no obvious microwave heating effect was found under the same condition between the Bi₂Se₃ nanodiscs group and the deionized water group.3.The result from the infrared thermal imager demonstrated that the temperature of the ablated center of Bi₂Se₃ NFs+MWA group（86.7°C）was notably higher than that of the MWA group（51.6°C）.The postoperative CT scans indicated that compared with the MWA group,the Bi₂Se₃NFs+MWA group exhibited a significantly improved tumor growth inhibition,and the tumor doubling time showed a 2.2-fold improvement（P<0.01）.After Bi₂Se₃NFs and MWA treatment,the most severe structural and nuclear damage（karyopyknosis and karyolysis）,as well as cytoplasmic acidophilic degeneration were exhibited in tumor ablation area.Moreover,the Tunel and HSP 70 expression level of tumor tissue in Bi₂Se₃ NFs+MWA group increased more than that of MWA group.4.The CT values increased with the increasing concentration of Bi₂Se₃NFs,and the densities from Bi₂Se₃NFs were higher than that of ioversol at the same concentration.5.No obvious cell and organic toxicity are observed during the experimental period.Conclusion:The as-prepared Bi₂Se₃ nanomaterials with a unique flower-like structure possessed the microwave heating capacity and the auxiliary CT imaging capability.More importantly,minimally invasive interventional technology was utilized for targeted delivery of microwave-sensitive nanoflowers to improve MWA efficacy at the VX2 tumor site and simultaneously reduce side effects in normal tissues.Chapter Two:Hexagonal Nax WO3 nanocrystals with reversible valence states for microwave thermal and chemodynamic combined cancer therapyObjective:To explore the microwave heating ability and Fenton-like catalytic performance of hexagonal Na_xWO₃ nanocrystals and further investigate the combined therapeutic effect of microwave ablation and tumor microenvironment responsive chemodynamic therapy based on nanocrystals on the 4T1-bearing mice.Methods:1.Synthesis and characterization.A general hydrothermal approach of the WO₃ precursor and Na₂SO₄ in the presence of reductant ethylene glycol and polyvinylpyrrolidone（PVP）was used to synthesize hexagonal Na_xWO₃ nanocrystals.Thermogravimetric analysis（TGA）was applied to determine the content of PVP on nanomaterials.The hydrodynamic size and zeta-potential of the as-prepared sample were assessed using Nano Brook Zeta PALS instruments.The other characterization of nanocrystal was the same as that in Chapter One.2.The microwave heating effect of Na_xWO₃ nanocrystals was evaluated by the same method as in Part One.3.Assessment of redox performance in vitro.The reactive oxygen species probe and terephthalic acid were applied to detect the Fenton-like performance of Na_xWO₃ nanocrystals.Ellman’s assay was employed to inspect the glutathione depletion property of oxidized Na_xWO₃nanocrystals.Murine triple-negative breast cancer 4T1 cell lines and human umbilical vein endothelial cells（HUVEC）were selected as models for cancer and normal cells,respectively.Cytotoxicity assay,colony formation assay and mitochondrial membrane potential assay kit JC-1 were performed to detect the selective killing effect and mechanism of Na_xWO₃ nanocrystals on tumor cells.In order to further confirm the contribution of H₂O₂ for·OH generation,an appropriate dose of exogenous H₂O₂（final concentration:100μM）was added to HUVEC cells to construct the H₂O₂ overproducing microenvironment.4.Assessment of synergistic antitumor effect in vivo.4T1 cells were inoculated in the right behind legs of BALB/c normal mice.Then,these mice were randomly divided into four groups until the tumor volume reached about 50 mm³:Control;Na_xWO₃nanocrystals;MWA;Na_xWO₃nanocrystals+MWA.The tumors were treated with microwave irradiation after intratumoral administration of sterile water or Na_xWO₃nanocrystals.After treatment,all mice were fed normally.The body weight and tumor volume in each group were monitored every other day.All mice were sacrificed on the fifteenth day after the treatment.Finally,the excised tumors and organs were stained with hematoxylin and eosin（H&E）or terminal deoxynucleotidyl transferase-mediated d UTP nick-end labeling（Tunel）according to standard procedures.5.The biosecurity of Na_xWO₃ nanocrystals was evaluated in the same way as in Chapter One.Results:1.Hexagonal phase Na_xWO₃ nanocrystals with a length of 100-150 nm and a diameter of about 5 nm were synthesized through an eco-friendly hydrothermal approach.The content of PVP on nanomaterials was determined to be 7.86%by TGA.The dynamic light scattering analysis revealed that no significant changes were observed in the hydrodynamic size of Na_xWO₃ nanocrystals after being maintained in physiological solutions for 7 days.2.Under the same microwave irradiation,the Na_xWO₃aqueous solution with the concentration of 30 mg m L^-1 showed a considerable temperature change of 53.3±1.85℃within 6 min,while the temperature of deionized water only rose by 37.7±1.11℃within the same time（P<0.001）.3.The high-resolution scans of tungsten core-level（W 4f）illustrated that the as-prepared Na_xWO₃nanocrystals possessed reversible redox valence states(W⁵⁺/W⁶⁺).The fluorescence spectra of DCF and TAOH in an external simulated fluid showed that Na_xWO₃nanocrystals had the potential as chemical catalytic nanomedicine to boost reactive oxygen species generation via decomposing H₂O₂ into highly toxic·OH.Moreover,the catalytic activity enhanced rapidly along with the increase of the concentration of substrate,reaction temperature and reaction time.Ellman’s assay demonstrated that the cells treated with oxidized Na_xWO₃ nanocrystals displayed a significant reduction（about57.6%）in the ratio of reduced GSH and oxidized GSSG compared with the control group（P<0.05）.Cytotoxicity assay indicated that the viability of 4T1 cells decreased to67.8±2.6%when the concentration of Na_xWO₃ nanocrystals was up to 400μg m L^-1.Conversely,the viability of Na_xWO₃ nanocrystals-incubated HUVEC cells was still close to 100%.Whereas,an obvious decrease of cell viability（77.2±5.7%）in HUVEC cells was observed after the addition of exogenous H₂O₂together with Na_xWO₃ nanocrystals（taking the cell viability of HUVEC cells treated with H₂O₂ alone as control）.The consequences of the colony formation assay were in good agreement with the cytotoxicity assay.JC-1 assay demonstrated the change of mitochondrial membrane potential in Na_xWO₃ nanocrystals-incubated 4T1 cells.All the above results showed that Na_xWO₃ nanocrystals could selectively inhibit tumor cell growth by reactive oxygen species-induced mitochondrial dysfunction,but less injury to normal cells.4.Compared with the control group,the tumor volume of the Na_xWO₃nanocrystals group was slightly reduced（P<0.05）,and the tumor growth was prominently depressed in mice treated with MWA alone.The most encouraging result was that the strongest tumor volume inhibition（P<0.001）,the most severe cancer cell apoptosis and necrosis were accomplished in the Na_xWO₃nanocrystals+MWA treatment group.5.No significant changes in body weights and hematological indicators were observed during the follow-up period.Conclusion:Hexagonal Na_xWO₃ nanocrystals with the excellent catalytic activity could be utilized to generate additional heat under microwave irradiation and concurrently produce highly toxic·OH in tumor sites,ultimately attaining a collaborative antitumor effect of external MWA and internal CDT.Chapter Three:Hyperthermia and chemotherapy based on carbon nanoparticles for synergic inhibition of primary breast cancer growth and lymph node metastasisObjective:To explore the synergic therapeutic effect of hyperthermia therapy and chemotherapy based on clinically approved carbon nanoparticles on the metastatic 4T1-bearing mice.Methods:1.Characterization.The absorption ability of carbon nanoparticles suspension injection（CNSI）in a specific spectral range was detected by UV-Vis-NIR spectrophotometer.The nanomaterial was characterized in the same way as in Chapter One.2.The heating effect of CNSI under microwave irradiation（2450 MHz）or NIR-II laser irradiation(0.8 W cm^-2)was evaluated using the same method as in Chapter One.3.Drug loading and releasing.CNSI(0.2 mg m L^-1)and pirarubicin hydrochloride for injection(THP,0.2 mg m L^-1),a first-line chemotherapy drug for breast cancer,were placed in a beaker.After stirring at room temperature for different times（0,3,6,9,12,15,19,24h）,the supernatant was collected by centrifugation.UV-vis spectrophotometer was used to measure the absorbance of the supernatant at 480nm.Finally,the pirarubicin-loading ability was calculated as follows:drug loading（w/w）=the mass of THP adsorbed/the mass of THP added during the loading process.In order to evaluate the drug-releasing capacity,CNSI/THP was dispersed in 2 m L p H=7.4 PBS and transferred to a dialysis bag with a molecular weight cutoff of 8000-14000 Da.Then,the dialysis bag was placed in a beaker containing 100 ml of p H=7.4 PBS（external dialysis solution）,and put on a shaking table at 37℃and 120 rpm.Similarly,UV-vis spectrophotometer was used to measure the absorbance of dialysate at 480nm.4.Assessment of synergistic antitumor effect in vitro.Cytotoxicity assay,wound healing and colony formation assay were performed to detect the killing effect of CNSI/THP on 4T1 tumor cells.Cytotoxicity assay and live/dead staining were used to assess the killing effect of CNSI/THP combined with NIR-II photothermal therapy on 4T1 tumor cells.5.Evaluation of sentinel lymph node staining and photoacoustic imaging performance in vivo.CNSI was directly injected into the subiliac lymph node of mice and then the direction of lymphatic drainage and the blackening of lymph nodes were observed and recorded carefully.The photoacoustic imaging performance of CNSI for tumor and sentinel lymph nodes was examined using a multispectral optoacoustic tomography system.Results:1.CNSI was composed of spherical carbon particles with a mean size of10-50 nm.These particles were slightly aggregated in physiological solution,forming large aggregates with a hydrodynamic diameter of 150 nm.The Vis-NIR spectrum indicated that CNSI had a broad absorption from the visible to NIR region.2.The temperature of CNSI aqueous solution raised rapidly under microwave irradiation with the increase of concentration,irradiation time and power intensity,which was all higher than the temperature change of the deionized water.Moreover,the excellent NIR absorption ability of CNSI endowed it with notable NIR-II photothermal productivity.The photothermal conversion efficiency of CNSI was calculated to be 37.7%.3.After stirring at room temperature for 20 h,the THP loading ratio of CNSI was measured to be0.7 mg/mg.After stirring at 37℃for 12 h,a slow release was observed in CNSI/THP group,with an accumulative release rate of 22.9%.These results indicated that CNSI possessed high-efficiency drug loading capability and drug sustained-release behavior.4.The result of wound healing assay and colony formation assay demonstrated that CNSI/THP could effectively inhibit the migration（34.9±1.1%vs 25.3±1.8%,P<0.05）and proliferation（100±4.3%vs 24.8±4.6%）of tumor cells.Both cytotoxicity assay and live/dead staining experiment verified that CNSI had excellent NIR-II photothermal conversion efficiency,and combined with THP could induce the strongest tumor cells apoptosis and necrosis.5.A mouse model was applied to confirm the lymphatic system tracing capacity of CNSI and simultaneously identify the drainage pathways from the subiliac lymph node to the proper axillary lymph node.Furthermore,CNSI had excellent photoacoustic imaging characteristics which could be used to identify the tumor-associated sentinel lymph node.Conclusion:Based on the clinical approved CNSI lymphatic tracer,a novel multifunctional CNSI/THP nanocomposite with good microwave heating ability,efficient NIR-II photothermal conversion efficiency,high drug loading capacity and photoacoustic imaging performance was proposed.Moreover,the CNSI/THP nanocomposites combined with external laser irradiation could effectively inhibit the proliferation and migration of 4T1 breast cancer cells.Summary:Collectively,to overcome the limitations of MWA in clinical,such as the lack of tissue specificity and high tumor recurrence rates,three novel nano-microwave sensitizers,including Bi₂Se₃ NFs,hexagonal Na_xWO₃ nanocrystals and multifunctional CNSI/THP nanocomposite,were successfully fabricated,and their excellent therapeutic effects in several tumor models were also achieved.Three works of this paper have gradually achieved the anti-tumor efficacy from single suppression of local primary lesion to synergistic inhibition of lymphatic metastasis.This thesis not only breaks the exclusion zone of MWA for the treatment of HCC in high-risk sites,but also deepens the research on the microwave sensitization effect of nanomaterials with a unique morphology.To sum up,the above work offers a firm theoretical and experimental ground to further enhance the efficacy of MWA in cancer therapy,and also provides an innovative strategy for the development of efficient microwave sensitizers.