Synthesis And Evaluation Of Indocyanine Green-Loaded Nanoprobes For Tumor Imaging

Objective:Breast cancer is one of the familiar diseases in women. According to World Health Organization 2014 reports, the breast cancer incidence of women ranks the first among all over the world; breast cancer is the leading cause of death in women. Cause early detection of breast cancer can obtain 90% survival. The development of nano theranostic system that are capable of many functions, including detecting cancer, targeted therapy and monitoring of therapeutic efficiencies, is a hot spot in current research. Many imaging modalities such as magnetic resonance imaging, positron-emission tomography, and computed tomography have been developed and used in biomedical fields. However, they could result in severe radiation risk that patients often experience. To build a non-toxic nanoparticulate drug-delivery system which could smartly integrate diagnostic and therapeutic functions is an urgent need for the clinical applications. Indocyanine green(ICG) has been the only NIR fluorescent dye so far approved by the FDA to be used in clinics. ICG can absorb NIR light and convert it into cytotocix heat for tumor treatment, so it is also used as a therapeutic molecule for phototheral therapy.In this article, we considered two nanocarriers to carry ICG, and exhibits great performances in both imaging and therapy. Methods:Section 1: A fluorescent nanoprobe based on liposome was synthesized by the hydrophobic interaction of phosphatidyl ethanolamine and ICG, the resulting nanoprobe was called LipoICG. In order to enhance the stability of liposome, the LipoICG was coated with HSA, the resulting novel fluorescent nanoprobe, namely H-LipoICG, was synthesized for tumor imaging. Size and zeta potential of H-LipoICG were measured using a zetasizer system, the morphology and structure of nanoparticles were obtained through TEM, the kinetics of ICG release in vitro were quantified by dialysis method. The in vitro cellular uptake assay was produced by CLSM and cell viability was produced by CCK-8 assay. Finally, the biodistribution analysis of ICG was obtained by using the IVIS in vivo imaging system with xenograft tumor model.Section 2: In this work, the pH of human serum albumin(HSA) was adjusted to 3.0; ICG was fully encapsulated in positively charged HSA. After protein desolvation by acetone, glutaraldhyde was added to induce particle cross-linking, the resulting nanoparticl——HIP NPs was synthesized. Size and zeta potential of nanoparticles were measured using a zetasizer system, the morphology and structure of nanoparticles were obtained through transmission electron microscope(TEM), the kinetics of ICG release in vitro were quantified by dialysis method. The in vitro cellular uptake assay was produced by laser scanning confocal microscope(LSCM) and cell viability was produced by CCK-8. The biodistribution analysis of ICG was obtained by IVIS in vivo imaging system. Furthermore, the anticancer effect of photothermal therapy was investigated in nude mice bearing MCF-7 subcutaneous tumor model. Conclusions:Section 1: In this study, the LipoICG was synthesized by the interaction of indocyanine green and ethanolamine phosphatidyl. HSA coating was prepared to enhance the stability of LipoICG, the resulting fluorescent probe was called H-LipoICG. The LipoICG was spherical shape with particle size of 94.47±0.13 nm, the encapsulation efficiency of ICG was 81.5%. The particle size of H-LipoICG was increased to 121.5±0.85 nm as a result of HSA coating. However, the encapsulation efficiency of ICG was increased to 98.2%, which demonstrated that the encapsulation of HSA could significantly improve the loading capacity of liposome, thereby avoiding the leakage of ICG. The drug release experiment showed that the release of ICG in H-LipoICG was less than 10% within 144 h, while it’s more than 60% in LipoICG. The encapsulation of HSA could avoid the ICG leakage from liposome, the fluorescence intensity could further be kept in the nanoprobe. Our in vitro experimental results demonstrated that H-LipoICG could efficiently internalize into cells and was nontoxic. We also observed tumor-specific biodistribution of nanoparticles of in vivo xenografts. An optimized tumor contrast signal was observed after 8 h intravenous administration of H-LipoICG, and the fluorescent signal was lasted up to 24 h. While LipoICG mostly distributed in liver and intestine after administration. Accordingly, the resulting H-LipoICG exhibited some advantages as being a fluorescence nanoprobe. Such as good biocompatibility, excellent fluorescent stability, strengthen physiochemical property, superior photothermal effect and high tumor-targeting ability. Therefore, H-LipoICG could be considered as an ideal contrast agent for diagnosis and therapy of breast cancer.Section 2: The resulting HIP NPs presented spherical shape with the particle size of 121.4±1.9 nm. The encapsulation efficiency of ICG was as high as 88.8%, and the drug release experiment showed that HIP NPs was stable because the leakage of ICG was less than 20% within 144 h. The intracellular uptake experiment proved that the HIP NPs could be easily internalized into human breast cancer cells and exhibited good biocompatibility. However, when treated with NIR irradiation, cells was fastly be killed by the photothermal effect of HIP NPs. The in vivo experiments showed that HIP NPs was preferentially accumulated in tumor via EPR effect after 8h administration, and its circulation time was much longer than ICG solution, the fluorescence signal could maintain for at lest 24 h. Furthermore, highly effectively photothermal therapy under the guidance of NIR imaging was conducted. Administration of HIP NPs along with NIR irradiation treatment could result in complete inhibition of subcutaneous tumors. Moreover, the body weight and health condition of HIP NPs group had no significant difference compared with control group. Therefore, the biocompatible and biodegradable nanoparticulate system proved a potential application in diagnosis and therapy of breast cancer.