The Experimental Study Of Dual-mode Nanobubbles As Targeted Contrast Agents For Cancer Molecular Imaging And Photothermal Therapy

PurposePrepare a dual-mode nanobubbles,FA-NBs-IR780,as targeted contrast agents for cancer molecular imaging and accurate therapy.First,through the in vitro tests to detect the basic characteristics of FA-NBs-IR780 sufficiently.Then,through the in vivo and in vitro tests to verify the enhanced cancer target ability of FA-NBs-IR780.Through the ultrasound contrast enhanced imaging and near-infrared fluorescence of FA-NBs-IR780 in vivo to test its cancer molecular imaging.In addition,through the in vitro and in vivo experiments to test the photothermal therapy(PTT)of cancer when FA-NBs-IR780 upon near-infrared(NIR)irradiation.The ultimately destination is to provide a noval dual-mode nanobubbles as targeted contrast agents for cancer accurate diagnosis and treatment.Methods1.10 mg DPPC,4 mg DSPE-PEG2000-FA and 0.3 mg IR-780 were mixed together.Using the thin-film hydration and mechanical vibration to fabricate a dual-mode targeted nanobubbles,named as FA-NBs-IR780.The morphology of the FA-NBs-IR780 were characterized by transmission electron microscopy(TEM).The particle size and Zeta potential size of the FA-NBs-IR780 was further assessed with a nanometer particle size/zeta analyzer.The toxicity to cells of IR-780s was tested by CCK-8 method.The echogenicity of FA-NBs-IR780 was determined by ultrasound contrast imaging via a self-made device in vitro.The optical characteristic of FA-NBs-IR780 was assessed via confocal laser scanning microscopy(CLSM)in vitro.2.The cancer cellular target efficiency of FA-NBs-IR780 was estimated in vitro using human glioblastoma cell line U87 and human breast cancer cell line MDA-MB-231.The cancer cellular target ability was first evaluated by CLSM and compared with human renal epithelial cell line 293T.Then we used flow cytometry to quantify appraise cancer cellular target efficiency of FA-NBs-IR780 in vitro.3.We established the U87 cancer-bearing nude mouse model.The effect of ultrasound cancer contrast-enhanced imaging of FA-NBs-IR780 was investigated using a clinical ultrasound contrast system in vivo.To further confirm that FA-NBs-IR780 could be used as an NIRF imaging agents,whole-body NIR images were observed using an in vivo fluorescence imaging system(IVIS).The ability of FA-NBs-IR780 passing through the tumor vessels was observed by CLSM using the pathological sections of cancer.4.The CLSM was used to visually estimate the photothermal therapy(PTT)of FA-NBs-IR780 upon NIR irradiation in vitro using U87 cells,and the temperatures were measured.Then the antitumor ability of FA-NBs-IR780 upon NIR irradiation was quantify evaluated by cell counting kit(CCK-8)in vitro.5.The cancer-bearing nude mice were used to observe the antitumor ability of FA-NBs-IR780 upon NIR laser irradiation.First,the distribution and metabolic rate of FA-NBs-IR780 in cancer and the major organs were observed by IVIS.Then the FA-NBs-IR780 was injected by tail vein.After 12 h,the cancers were exposed to 808 nm laser at a power density of 1 W/cm~2 for 2 min.The PTT efficacy of FA-NBs-IR780 upon NIR laser irradiation was evaluated by measure the temperatures at the cancer site,observe the morphology and surface changes of the cancers,record the cancer volumes and nude mice body weights every 3 days.The anticancer efficacy was further evaluated by immunohistochemical analyses,including terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL staining)to evaluate cell apoptosis and proliferating cell nuclear antigen(PCNA)immunolocalization to evaluate cell proliferation.Results1.We fabricated the dual-mode nanobubbles,FA-NBs-IR780,as targeted contrast agents for cancer successfully.The average diameter of FA-NBs-IR780 was 591.6±52.3nm.The results of TEM image revealed that FA-NBs-IR780 were approximately spherical in morphology and were well dispersed with a small size of approximately 500 nm.FA-NBs-IR780 also exhibited optimal ultrasound contrast-enhanced imaging and fluorescence imaging in vitro.Additionally,FA-NBs-IR780 had good stability and non-toxic to cells.2.The results of CLSM and flow cytometry proved that FA-NBs-IR780 could specific target the cancer cells.At the same time,no target ability was observed between FA-NBs-IR780 and renal epithelial cell 293T.Notably,the cancer target ability of FA-NBs-IR780 is stronger than that of NBs-IR780,FA-NBs and free IR-780.The result of CLSM also proved that FA-NBs-IR780 could through the cancer vessels due to its nano-size,and accumulate massively at the cancer tissue space using the pathological sections.3.The obvious ultrasound contrast-enhanced imaging of FA-NBs-IR780 was observed at the tumor site of U87 tumor-bearing mice.The ultrasound contrast-enhanced imaging of FA-NBs-IR780 have higher peak intensity and longer duration time.Tumor-bearing mice treated with FA-NBs-IR780 also presented excellent fluorescence imaging at the tumor site which was confirmed by IVIS.4.The FA-NBs-IR780 could obviously increase the local temperature of the therapy site and exhibited superior PTT efficacy to cancer cells when upon NIR laser irradiation in vitro,which was confirmed by CLSM and CCK-8.5.The results of IVIS showed that FA-NBs-IR780 accumulated at the tumor site massively and the peak intensity was at 12 h after injection.The in vivo experiments confirmed that FA-NBs-IR780 upon NIR laser irradiation could obviously increase the local temperature of the tumor site and strikingly inhibit the tumor’s growth.The immunohistochemical results also confirmed that FA-NBs-IR780 upon NIR laser irradiation could induce cell apoptosis and decrease cell proliferation.And as the results showed,the PTT therapy could distinguish cancer locally without other systemic toxicities.ConclusionsUsing the modified thin-film hydration,we successfully produced a noval dual-mode nanobubbles,FA-NBs-IR780,as target contrast agents for cancer.FA-NBs-IR780 displayed relatively small sizes and good stability.FA-NBs-IR780 have excellent cancer targeted ability which were confirmed in vitro and in vivo.FA-NBs-IR780 also exhibited excellent ultrasound contrast enhanced imaging and NIR imaging.Thus,FA-NBs-IR780 have excellent cancer targeting specificity and dual-mode cancer target imaging.At the same time,FA-NBs-IR780 could improve IR-780 iodide hydrophobicity,promote IR-780 iodide accumulate at the cancer site.FA-NBs-IR780 upon NIR laser irradiation could obviously increase the local temperature of the tumor site and strikingly inhibit the tumor’s growth.All these results indicated that FA-NBs-IR780 would be an ideal cancer targeted agent for accurate diagnosis and photothermal therapy of cancer.