Self-Assembly Establishment And Biomedical Application Of Multi-Functional Medical Imaging Probes

Biomedical imaging techniques including optical imaging, magnetic resonance imaging (MRI), ultrasound imaging (US), and CT imaging play an important role in clinical applications. Two or more imaging modalities always combine to abtain more accurate imaging informations in diagnosis. Molecular imaging contrast agents raise medical imaging to the molecular level. In order to obtain more accurate and reliable imaging informations, multi-functional imaging contrast agents can be established by crosslinking imaging probes with high sensitivity (US, optical, or PET imaging) and imaging probes with high resolutions (MRI or CT imaging) by Van der Waals forces. The crosslinking approach is always complex and limited because different imaging probes have various structures and compositions. Self-assembly is a flexible approach to fabricate composites with various compositions by non-covalent bond and provides a broad platform for the establishment of multi-functional medical imaging probes. However, it is rarely reported in the relative fields. In this work, we focused on the self-assembly establishment and biomedical applications of multifunctional medical imaging probes, and the contributions are as follows.(1) We developed a novel self-assembly approach to fabricate polymer/Fe3O4 nanoparticle self-assembled microcapsules (SAMCs). The SAMCs exhibited MRI capabilities caused by the magnetite nanoparticles inside. On this basis, we filled C6Fi4 into the SAMCs to obtain MRI/US dual-modal imaging self-assembled microbubbles (SAMBs) and studied the scheme of the self-assembly approach and the MRI/US imaging capabilities. The SAMC had a unique triple layered shell constructed by "poly(acrylic acid)-IONPs-polyamine" composites. The diameters of the SAMCs could be tuned from 450 nm to 1300 nm corresponding to the molar ratios of their precursors in the self-assembly approach. After filled with C6Fi4, the SAMBs exhibited MR/US dual imaging contrast capabilities in vitro, which related to the diameters of the microbubbles. The larger microbubbles provided better US contrast, but poorer MRI contrast due to lower content of magnetite nanoparticles.(2) The cell cytotoxicity and in vivo MRI/US imaging capabilities of SAMCs and SAMBs were studied systematically. The SAMCs had little cell cytotoxicity in rat NRK kidney cells and BRL 3A liver cells, thus exhibiting good cell viability. The SAMBs exhibited MR/US dual contrast imaging capabilities in the rat liver. The MR imaging disappeared in liver within 45 days, indicating the SAMBs can be cleared from this organ within 45 days. By Prussian Blue staining, the SAMBs showed distribution in liver at first, and a transition to spleen within 15 days, suggesting normal systemic metabolism through the spleen.(3) We fabricated a novel protease sensing near infrared fluorescence (NIRF)/MRI dual-modal imaging probes using self-assembly method and studied the mechanism and the imaging capabilities of the probes. The composite microspheres consisting Cy5.5-poly-L-lysine (PLL) polymers and Fe3O4 nanoparticles and was sensitive to trypsin. The sizes of the microspheres could be controlled by the concentration or the molecular weight of PLL from 100 nm to 300 nm. In the environment without trypsins, the microspheres did not emit light on the basis of Forster Resonance Energy Transfer (FRET), but enhanced MRI contrast imaging intensity. In trypsin activation, PLL polymers within the microspheres were hydrolysis to pull the distances between the fluorescent molecules. The microspheres were thus activated and emitted fluorescence light, while MRI intensity decreased. The protease activated transfer from quenching to de-quenching provided the microspheres more potential applications in diagnosis of trypsin-over-expressed deseases.(4) Based on the trypsin sensitive self-assembled Cy5.5-PLL/Fe3O4 nanopartical composite microspheres, we investigated the cell cytotoxicity and NIRF/MR imaging capabilities in vitro and in vivo. In the cell cytotoxicity and in vitro experiments, we selected trypsin expressed cells (Ges 1, MCF-7 and SW620) and control cells without trypsin expression (BEC, Fb, Hela and A375). The results showed that the microspheres exhibited good biocompatibility. The microspheres had protease sensing NIRF imaging capability in cell experiments. Trypsin expressed cells incubated with microspheres emitted fluorescent light and exhibited MRI capabilities, while cells without trypsin expression didn’t exhibit NIRF imaging capabilities and had lower MRI intensity. We investigated the in vivo trypsin activated NIRF/MR imaging capabilities of microspheres in control nude, trypsin expressed SW620 tumor models and non-trypsin-expressed A375 tumor models. The results showed that the microspheres enabled NIRF imaging in digestive track of nude. In tumor models, the microspheres emit intensive fluorescence and enhanced MRI in SW620 tumors, but quenched fluorescence and slightly enhanced MRI in A375 tumors. Further bio-TEM results indicated that the microspheres were partially taken in SW620 tumor cells and were hydrolysised to IONPs. However few microspheres were taken into A375 tumor cells. The microspheres showed good biocompatibility in vivo, indicating great potential in biomedical application field.In summary, the innovations of our study are as follows:We designed and achieved a novel template-free self-assembly approach drived by electrostatic interaction to fabricate microbubbles for MRI/US dual-modal imaging. We skillfully import enzyme sensitive components into self-assembly approach and firstly fabricated trypsin-activated NIRF/MRI dual-modal imaging probes. The recognition of multiple-modal imaging was developed to the molecular level. The trypsin sensitive microspheres were expected to have important applications in enzyme sensitive tumor detection and gastrointestinal imaging.