(H-301) Nanobubble Ultrasound Contrast Agents as Delivery Vehicles for Tolerogenic Peptides in Mouse Models of Type 1 Diabetes

Introduction:: Type 1 diabetes (T1D) is an autoimmune disorder characterized by immune-mediated destruction of insulin-producing beta cells, resulting in severe hyperglycemia if untreated. Modulating this autoimmune response to prevent beta cell destruction prior to overt hyperglycemia is essential, yet very few therapeutic approaches have shown reliable and lasting prevention of T1D. Antigen-specific immune therapies that aim to promote the expansion of anti-inflammatory regulatory T cells (Tregs) represent a promising approach to preventing T1D. For instance, a two-week infusion of an insulin B:9-23 peptide mimotope has been shown to expand insulin-reactive Tregs and prevent diabetes onset in non-obese diabetic (NOD) mice. However, this relied on an implanted infusion pump and other insulin-based antigen-specific immune therapeutics have been unsuccessful in clinical trials. Notably, there has been no investigation into alternate and innovative routes of therapeutic delivery. We investigated the usage of ‘nanobubble (NB)’ submicron ultrasound contrast agents as delivery vehicles for insulin peptide therapeutics. NBs are submicron-sized lipid-shelled bubbles that can be visualized and ablated noninvasively using ultrasound. Prior work has shown that islet microvasculature becomes hyperpermeable during presymptomatic T1D, allowing for ‘nanobubble (NB)’ submicron ultrasound contrast agents to accumulate in immune-infiltrated islets. We examine the utility of these lipid-shelled NBs as peptide delivery vehicles to provide islet-targeted delivery and for acoustically driven therapeutic release where therapeutic delivery can be visualized in real-time with ultrasound (Fig. 1A).


Materials and Methods:: NBs were synthesized by forming a lipid precursor solution, mechanically agitating with perfluoropropane gas, and isolating the submicron portion. Peptide-NBs include a NOD insulin B:9-23 peptide mimotope linked to a palmitoyl fatty acid tail with a glycine-rich linker. To assess peptide incorporation in NBs, two methods were employed: First, peptide was FITC-labeled, and the lipid shell was rhodamine-labeled and their colocalization was assessed with confocal microscopy. Second, NOD splenocytes (as a source of antigen-presenting cells) were pulsed with peptide-NBs and co-cultured with an insulin-reactive T cell hybridoma (PCR1-10). IL-2 production, measured via ELISA, was used as a metric of T cell receptor stimulation. NB echogenicity was assessed with contrast enhanced ultrasound (CEUS), both in vitro with a tissue-mimicking phantom and in vivo by intravenous injection in 10w pre-diabetic NOD mice and image-based assessment of pancreatic accumulation. Biodistribution of peptide-NBs was assessed by infusing fluorescently-labeled peptide-NBs intravenously in NOD or SCID control mice and dissecting abdominal organs after 30 min. Tissue was sectioned and imaged with confocal microscopy. Immune infiltration was assessed by scoring H&E-stained sections. Expansion of insulin-reactive Tregs was determined by isolating lymphocytes from pancreatic islets, pancreatic lymph nodes, and spleen of NOD Foxp3-EGFP mice, performing surface antibody stains and insulin-I-A(g7) tetramer stains, and analyzing with flow cytometry.


Results, Conclusions, and Discussions:: Peptide palmitoylation resulted in a 58% increase in peptide/NB colocalization (p < 0.0001). The ability for insulin-peptide from peptide-NBs to stimulate insulin-reactive T cell receptors (TCRs) in vitro was not affected by palmitoylation or incorporation in NBs, with significantly more IL-2 production than blank NB control (p < 0.0001). These results indicate that peptide is being effectively incorporated in the NB lipid shell and peptide palmitoylation is beneficial for more robust and stable incorporation. Additionally, they indicate that peptide modifications and incorporation in NBs do not substantially affect processing and presentation by antigen-presenting cells and ability to stimulate insulin-reactive TCRs. Peptide incorporation did not affect NB echogenicity (in vitro and in vivo), ability to burst with ultrasound, and diameter, but did slightly increase polydispersity (Fig. 1C, p < 0.05). Peptide-NBs accumulated specifically in NOD islets when injected intravenously in NOD mice and not in other abdominal organs or NOD-SCID (control) islets (Fig. 1B, p < 0.0001). NBs effectively delivered palmitoylated peptide to NOD islets compared to controls (Fig. 1B, p < 0.0001), where the extent of peptide delivery correlates with extent of insulitis (p < 0.05). This indicates that NBs can be used to passively target peptide delivery to islets in mice developing T1D. Finally, NOD mice treated with peptide-NBs had a higher proportion of insulin-reactive regulatory T cells amongst insulin-reactive CD4+ T cells in islets, as compared to mice treated with NBs or peptide alone (Fig. 1D, p < 0.05). These results indicate that NBs are a useful tool for modulation of islet-resident T cells towards a more anti-inflammatory state. Antigen-specific therapeutics have great potential, but very few studies have optimized their efficacy using innovative routes of administration. This project serves as an important proof-of-principle for the utility of NBs for peptide delivery and has the potential to improve therapeutic intervention in T1D.


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