Neuroinflammation is a dynamic immune phenomenon that changes in severity with time after neurotrauma and has a profound impact on neuroregeneration, tissue healing and neuropathic pain, which is a common consequence of peripheral nerve injury (PNI). Macrophages are key cellular mediators of neuroinflammation. Macrophage-targeted nanotherapies, such as complex (perfluorocarbon/hydrocarbon) multimodal nanoemulsions (NEs) provide highly specific imaging signatures of neuroinflammation and hence indirect surrogate metrics of regeneration. We present a novel strategy where these NEs incorporating multiple imaging modalities and biosensors are delivered locally to directly target key cellular players of neuroregeneration. Two representative formulations of a nanotheranostic platform for local delivery of cell targeted NEs are presented: 1) A dual (macrophage and neuronal) targeted nanoparticle laden hydrogel for synergistic modulation of neuroinflammation and analgesia following PNI; and 2) neurotherapeutic loaded nanoparticles with extended release profile for sustained support of neuroregeneration. Each platform is capable of dual imaging payloads (NIRF, MRI and/or PET) and/or cell specific targeting moieties for controlled drug release. In vitro and pilot in vivo results will be presented. Theranostic nanosystem based platforms offer a unique opportunity to sequentially monitor cellular and molecular events at the site of neuronal injury, enabling dynamic, in-vivo mechanistic insights rather than static, ex-vivo histopathologic evaluation. Given their targeted capabilities, these platforms can help achieve personalized treatments that are customized and optimized for patients with PNI.
Proc. SPIE. 9723, Reporters, Markers, Dyes, Nanoparticles, and Molecular Probes for Biomedical Applications VIII
KEYWORDS: Infrared imaging, Near infrared, Proteins, Imaging systems, Magnetic resonance imaging, Luminescence, Inflammation, Flow cytometry, Colon, In vivo imaging, Fluorescent proteins, In vitro testing, Bacteria
Current treatment of inflammatory bowel disease (IBD) is largely symptomatic and consists of anti-inflammatory agents, immune-suppressives or antibiotics, whereby local luminal action is preferred to minimize systemic side-effects. Recently, anti-TNFα therapy has shown considerable success and is now being routinely used. Here we present a novel approach of using perfluorocarbon (PFC) nanoemulsion containing hydrogels (nanoemulgels) as imaging supported delivery systems for anti-TNF alpha probiotic delivery in IBD. To further facilitate image-guided therapy a food-grade lactic acid bacterium Lactococcus lactis capable of TNFα-binding was engineered to incorporate infrared fluorescent protein (IRFP). This modified bacteria was then incorporated into novel PFC nanoemulgels. The nanoemulgels presented here are designed to deliver locally anti-TNFα probiotic in the lower colon and rectum and provide dual imaging signature of gel delivery (MRI) across the rectum and lower colon and bacteria release (NIR). NIR imaging data in vitro demonstrates high IRFP expressing and TNFα-binding bacteria loading in the hydrogel and complete release in 3 hours. Stability tests indicate that gels remain stable for at least 14 days showing no significant change in droplet size, zeta potential and pH. Flow cytometry analyses demonstrate the NIRF expressing bacteria L. lactis binds TNFα in vitro upon release from the gels. Magnetic resonance and near-infrared imaging in vitro demonstrates homogeneity of hydrogels and the imaging capacity of the overall formulation.
Design and development of a new formulation as a unique assembly of distinct fluorescent reporters with nonoverlapping fluorescence spectra and a F 19 magnetic resonance imaging agent into colloidally and optically stable triphasic nanoemulsion are reported. Specifically, a cyanine dye-perfluorocarbon (PFC) conjugate was introduced into the PFC phase of the nanoemulsion and a near-infrared dye was introduced into the hydrocarbon (HC) layer. To the best of our knowledge, this is the first report of a triphasic nanoemulsion system where each oil phase, HC, and PFC are fluorescently labeled and formulated into an optically and colloidally stable nanosystem. Having, each oil phase separately labeled by a fluorescent dye allows for improved correlation between in vivo imaging and histological data. Further, dual fluorescent labeling can improve intracellular tracking of the nanodroplets and help assess the fate of the nanoemulsion in biologically relevant media. The nanoemulsions were produced by high shear processing (microfluidization) and stabilized with biocompatible nonionic surfactants resulting in mono-modal size distribution with average droplet size less than 200 nm. Nanoemulsions demonstrate excellent colloidal stability and only moderate changes in the fluorescence signal for both dyes. Confocal fluorescence microscopy of macrophages exposed to nanoemulsions shows the presence of both fluorescence agents in the cytoplasm.
Highly innovative multimodal perfluorocarbon (PFC) nanoemulsions are presented. They serve
simultaneously as dual-mode imaging reagents (NIR and <sup>19</sup>F MRI), and drug delivery vehicles for water insoluble
cyclooxgenase-2 (COX-2) inhibitors. These features qualify them as theranostic. Cancer progression and metastasis are highly influenced by tumor microenvironment and inflammation. Infiltration of primary tumors with
inflammation-promoting cells (e.g. macrophages) is a negative prognostic factor for cancer patient survival. We
postulate that the suppression of COX-2 enzyme in macrophages by theranostic PFC nanoemulsions will result in
changes in macrophage levels of accumulation in tumors and/or their phenotype, which can suppress tumor-
promoting activity. The presented theranostic nanoemulsions are designed to label immune cells such as macrophages, and deliver celecoxib, a COX-2 inhibitor. The designed theranostic incorporates two fluorescent reporters: a near-infrared (NIR) fluorescent dye for improved optical in vivo imaging, and a distinct fluorescent dye for histological analysis of excised tissues. A high content of PFC in the theranostic allows <sup>19</sup>F MRI to quantitatively assess the distribution of the injected nanomedicine in the peritumoral area, and measure tumor-associated
inflammation, while <sup>1</sup>H MRI provides anatomical context. NIR imaging is used as a complementary in vivo
technique to assess biodistribution of the theranostic. We report preparation and characterization of the nanoemulsions’ colloidal and optical stability, in vitro toxicity, and imaging capabilities. This theranostic offers
flexibility for in vitro and in vivo inflammation imaging and histological analysis using three different imaging
functionalities (fluorescence, NIR and 19F MRI), advancing the monitoring and modulating of tumor-infiltrating
immune cells in vivo.
Conference Committee Involvement (1)
Reporters, Markers, Dyes, Nanoparticles, and Molecular Probes for Biomedical Applications XI
4 February 2019 | San Francisco, California, United States