Targeted molecular imaging offers a major advantage for medical diagnosis by its ability to provide three-dimensional mapping of the target tissues compared to other techniques, such as biopsy, by utilizing specific targeted imaging probes. Despite the rapid growth in molecular imaging and many successful clinical translations of targeted imaging probes, there are still many medical needs, including assessing macrophage localization and polarization, that have not been satisfied. Macrophages play an important role in the immune system and are associated with a variety of diseases, including cancer and atrial fibrillation. In the tumor microenvironment, tumor associated macrophages (TAMs) represent a predominant component of the total tumor mass, and TAMs play a complex and diverse role in cancer pathogenesis with potential for either tumor suppressive, or tumor promoting phenotype. In atrial fibrillation, macrophages are linked with chronic inflammation and thrombosis in the vulnerable left atrial appendage, which makes a perfect target for developing the site-specific anticoagulant. In this dissertation work, we developed several novel imaging agents for magnetic resonance imaging (MRI) and fluorine-19 magnetic resonance imaging (19F-MRI) to target macrophages and macrophage subtypes in cancer. Also, a macrophage-targeted site-specific anticoagulant has been discussed. We have previously found that sulfated-dextran coated iron oxide nanoparticles (SDIO) can target macrophage scavenger receptor A (SR-A, also known as CD204). Since CD204 (SR-A) is considered a biomarker for the M2 macrophage polarization, these SDIO might provide M2-specific imaging probes for MRI. In the first project described in this dissertation, we investigate whether SDIO can label M2- polarized cells in vitro. We evaluate the effect of degree of sulfation on uptake by primary cultured bone marrow derived macrophages (BMDM) and found that a higher degree of sulfation led to higher uptake, but there were no differences across the subtypes. We further examine the localization of SDIO in TAMs in vivo, in the mammary fat pad mouse model of breast cancer. We demonstrate that uptake by TAMs expressing SR-A scales with degree of sulfation, consistent with the in vitro studies. The TAMs demonstrate M2-like function and secrete Arg-1 but not iNOS. Uptake by these M2-like TAMs is validated by immunohistochemistry. SDIO shows promise as a valuable addition to the toolkit of imaging probes targeted to different biomarkers for TAMs, even though the analysis of the BMDM showed similar SR-A expression across stimulation conditions, suggesting that this classic model for macrophage subtypes may not be ideal for definitive M2 subtype marker expression, especially SR-A.
Given the in vivo labeling potential of SDIO, we aimed to provide increased specificity by appending targeting moieties to imaging platforms to allow markers like CD204 to be used alongside others. Here, we describe an in vivo imaging probe platform that is readily modifiable to accommodate binding of different molecular targeting moieties and payloads for multimodal image generation. In this work, we demonstrate the utility of perfluorocarbon (PFC) nanoemulsions incorporating dibenzocyclooctyne (DBCO) by enabling post-emulsification functionalization via a click reaction with azide-containing ligands. Further, the DBCO-PFC NE has been modified by adding two different lengths of polyethylene glycol (PEG) group, PEG350 and PEG600, in the nanoemulsion formula to minimize the non-specific uptake by phagocytes in vitro and in vivo as well as help DBCO stand out from the lipid shell of the nanoemulsions. Here, we proposed 4 targeting ligands to specifically target 4 biomarkers for M1/M2, which would allow us to do multi-target imaging using 19F-NMR in the future, specifically, CD40 and CD86 for M1 targeting, while CD206 and CD204 for M2 targeting. In this dissertation work, we have determined the optimal polarization condition that fits our needs for polarized THP-1 derived macrophages and RAW 264.7 macrophages. We have also reported the expression of these biomarkers in the 4T1 mouse breast cancer model in vivo by immunohistochemistry. Further, we have reported the synthesis and development of CD40 (for M1) and CD204 (for M2) targeted 19F nanoprobes. In particular, we evaluated the uptake efficiency of these two nanoprobes on in vitro polarized THP-1 derived macrophages and/ or RAW 264.7 macrophages, which provides promising results that support the possibility of in vivo imaging.
We also reported the development of a site-specific anticoagulant for atrial fibrillation (AF) patients. AF patients normally will receive anticoagulant treatment to prevent thrombosis events, but current anticoagulants, such as Warfarin, suffer a serious hemorrhage risk. Therefore, developing a safer and localized anticoagulant attracts our interest. SDIO, which we developed and reported previously, has a sulfated dextran coating on its surface, and free dextran sulfate has similar anticoagulant properties as heparin. Confirming that SDIO has a similar anticoagulant property as free dextran sulfate, we labeled SDIO with 64Cu as the radiotracer to assess the biodistribution of SDIO by PET-CT and validate SDIO accumulation in the inflamed heart. We also evaluated the CD204 expression in the left and right atrial appendages which is related to inflammation. Moreover, the immunohistochemistry for CD204 on human AF and NSR specimens supports the translational potential for SDIO as an anticoagulation drug for the clinic. We further conjugated SDIO on a PDMS surface to assess the anticoagulant activities of immobilized SDIO, which serves as a preliminary study to demonstrate that SDIO would retain anticoagulant activity even when surface bound. These results suggested that SDIO has the potential to be used as a site-specific anticoagulant. Future directions which include in vivo anticoagulant assays and SDIO treatment studies are discussed.
