Investigation Of Ucnps-based Composite As An Agent For Multimodal Cancer Imaging And Photothermal Therapy

Part ⅠPreparation and initial application of PEDOT:UCNP:Fe₃O₄:PEG for multimodal cancer imagingObjective: To study the feasibility and value of PEDOT:UCNP:Fe₃O₄:PEG as an agent for multimodal cancer imaging.Methods: 1) The high temperature thermal decomposition method was selected to synthesize Na YF4（Y : Yb : Er =78% : 20% : 2%） UCNPs. Then, OA-PAA was coated to increase the water soluble of UCNPs. Next, the PAH coated PEDOT:PSS was the core, UCNP-OA-PAA and DMSA modified Fe₃O₄ were adsorbed on the surface of PEDOT:PAH by electrostatic adsorption. A layer-by-layer（LBL） polymer coating approach is developed to functionalize PEDOT:UCNP:Fe₃O₄ with PAA, PAH and PEG introduced to improve the physiological stability of those nanoparticles. Next, the morphology of UCNPs-based composites and zeta potentials of different layers of PEDOT:UCNP:Fe₃O₄:PEG were assessed. 2) The MR T2-weighted images were performed using a series of PEDOT:UCNP:Fe₃O₄:PEG solutions with different concentrations. 3) A series of PEDOT:UCNP:Fe₃O₄:PEG solutions with different concentrations and deionized water were exposed to the 808 nm laser irradiation for 5 min, IR thermal camera（IRS E50） was used to monitor the temperature of the solutions. 4) The cytotoxicity of PEDOT:UCNP:Fe₃O₄:PEG was evaluated by MTT assay. 5) 4T1 tumorbearing mice were intratumor injected with PEDOT:UCNP:Fe₃O₄:PEG prior to imaging. T2-weighted MR imaging studies were conducted on a MRI scanner equipped with a special coil designed for small animal imaging. 6) PEDOT:UCNP:Fe₃O₄:PEG solutions incubated with 4T1 cells for 24 h. Using DAPI stain nuclei, the fluorescent of PEDOT:UCNP:Fe₃O₄:PEG was observed by fluorescent microscope. 7) In vivo UCL imaging was carried out using a modified Maestro EX in vivo imaging system（CRi. Inc.） with an external 980 nm laser as the excitation source.Results: 1) The transmission electron microscopy（TEM） imaging showed that the synthesized UCNPs nanoparticles were monodispersed nanocrystals with a uniform average diameter of 30 nm. PEDOT:UCNP:Fe₃O₄:PEG exhibited well stability in different solutions after different layers of modification. The zeta potential of PEDOT:UCNP:Fe₃O₄:PEG solution was close to zero. 2) MR images showed PEDOT:UCNP:Fe₃O₄:PEG could serve as an agent for MR imaging and showed a concentration-dependent darkening effect. 3) PEDOT:UCNP:Fe₃O₄:PEG was a powerful agent under 808 nm laser irradiation. 4) In vitro cytotoxicity assay showed no significantcy totoxicity of PEDOT:UCNP:Fe₃O₄:PEG was observed even at high concentrations. 5) After intratumor injection of PEDOT:UCNP:Fe₃O₄:PEG,remarkably reduced T2-weighted MR signals were observed in the tumor. The tumor capsule was complete, and the PEDOT:UCNP:Fe₃O₄:PEG solution was evenly distributed in the tumor. 6) With the help of fluorescent microscope, the green fluorescent emissioned by PEDOT:UCNP:Fe₃O₄:PEG was observed in the cells after incubated with PEDOT:UCNP:Fe₃O₄:PEG solutions for 24 h. 7) After intratumor injection of PEDOT:UCNP:Fe₃O₄:PEG, remarkably enhanced UCL signals were observed in the tumor under NIR.Conclusion: PEDOT:UCNP:Fe₃O₄:PEG exhibit excellent compatibility in physiological solutions, little in vitro toxicity. Besides, PEDOT:UCNP:Fe₃O₄:PEG could serve as an agent for MR imaging. Under near-infrared（NIR） light, PEDOT:UCNP:Fe₃O₄:PEG could serve as an imaging probe due to the unique upconversion photoluminescence, as well as a high photothermal conversion agent for photothermal therapy. Therefore, PEDOT:UCNP:Fe₃O₄:PEG could serve as an agent for multimodal cancer imaging and it may have great potential in photothermal therapy.Part ⅡStudy of PEDOT:UCNP:Fe₃O₄:PEG as an agent for photothermal therapyObjective Investigation of PEDOT:UCNP:Fe₃O₄:PEG as an agent for photothermal therapy.Methods 1) 4T1 cells were exposed to the laser irradiation with different power density for 5 min after incubated with PEDOT:UCNP:Fe₃O₄:PEG solutions for 6 h, Relative cell viabilities were determined by the standard MTT assay after 24 hours. 2) 4T1 subcutaneous tumors were generated by subcutaneous injection of 4T1 cell suspension onto the back of each mice. 3) Mice bearing 4T1 tumors were randomized into four groups,the tumor sizes were measured before treatment. The experimental group（group d） were intratumor injected with PEDOT:UCNP:Fe₃O₄:PEG and exposed to the 808-nm NIR laser at the power density of 0.8W/cm2 for 5 min. For control groups, mice were either injected with the same volume of saline only（group a）, or injected with PEDOT:UCNP:Fe₃O₄:PEG but without laser exposure（group b）. Group c were injected with the same volume of saline before laser irradiation. The tumor surface temperatures were recorded by an IR thermal camera. The tumor sizes were measured by a caliper every the other day. 5) During the test, the behavior of each mice were observed every day. After the test, major organs from those mice were harvested, stained with H&E and examined by a digital microscope.Results 1) Upon 808 nm laser irradiation, the cell viabilities gradually decreased with the rise of laser power density, indicating the effectiveness of NIR-induced PTT mediated by PEDOT:UCNP:Fe₃O₄:PEG. 2) Tumor bearing model of mice was successively prepare in one week. 3) After laser irradiation, tumors on the experimental group mice were completely eliminated one day post irradiation. Not only that, mice in the experimental group were tumor-free after treatment and survived over 60 days without a single case of animal death or tumor re-growth. Tumors on the control group mice continues to grow and all mice died within 60 days. 4) Neither mouse death nor noticeable abnormal behavior was observed during the test. Histological examination by H&E staining also uncovered no appreciable adverse effect of PEDOT:UCNP:Fe₃O₄:PEG to the examined major organs.Conclusion PEDOT:UCNP:Fe₃O₄:PEG show a good therapeutic effect in the cellular level of photothermal therapy. While being a potent PTT agent for in vivo tumor ablation with excellent therapeutic efficacy, PEDOT:UCNP:Fe₃O₄:PEG exhibit no appreciable toxicity to the treated animals. Therefore,PEDOT:UCNP:Fe₃O₄:PEG could serve as an agent for multimodal cancer imaging and photothermal therapy.