(J-392) Development of PLG antisense oligonucleotide conjugated nanoparticles to reduce inflammation

Introduction:: Inflammation is commonly initiated due to pathogen exposure. In most cases, inflammation resolves quickly, reduces infection spread, and restores tissue function1,2. At times, inflammation progression becomes non-resolving, which significantly contributes to the pathogenesis of many disease models such as multiple sclerosis, lung fibrosis, and obesity.



Current strategies for reducing inflammation include steroids, but steroids are powerful, act broadly, and have many off-target effects. Conversely, reducing specific inflammatory cytokines has synergistic effects. Nanoparticle strategies, in particular, allow for more targeted approaches and can modify immune responses more directly at sites of inflammation.



The Shea Lab has previously developed polymer NPs that associate with circulating monocytes, neutrophils, and macrophages and influence their phenotype through polymer size, composition, and scavenger receptors3,4,5. Poly(D,L-lactide-co-glycolide) NPs without biological or pharmaceutical agent modifications can influence innate immune cell plasticity, providing a base platform to tune inflammation.



Oligonucleotide-based gene regulators, especially antisense oligonucleotides (ASO) can be designed and modified based on the intended target sequence and have been tested in clinical studies6,7. ASOs are single-stranded and bind to protein in serum, on the cell surface, or intracellularly and enter the nucleus to induce RNAse H and reduce target gene translation8. However, ASOs require optimization for target delivery, safety, and efficacy, which may be addressed using polymer-based nanocarriers.



We investigated reducing inflammation using antiTNFα-ASO conjugated to polymer NPs (PLG-aTNFα-ASO-NPs). We are targeting a specific cell population and looking to precisely target factors associated with inflammation.


Materials and Methods:: PLG NPs were fabricated using the oil in water single emulsion solvent evaporation method. PLG polymer was dissolved in DCM, added to 1% (w/v) PVA solution, and sonicated. The emulsion was poured into a 0.5% (w/v) PVA solution and stirred overnight. Resulting PLG NPs were washed three times in water.



Anti-TNFα-ASO conjugation was done using 10 µM of soluble ASOs and EDC/NHS chemistry9. Particles were washed twice before further analysis or lyophilization with 2% (w/v) sucrose and 3% (w/v) D-mannitol. Prior to lyophilization, particles were frozen at -80°C and freeze-dried for at least 48 hours. Stable, lyophilized particles were rehydrated with water or PBS.



Resuspended PLG-aTNFα-ASO-NPs were placed on a shaker at 300 rpm and 37°C for 7 days. Samples were removed at 0, 2, 4, 8, 12, and 24 hours and for the next 6 days at 24-hour intervals. The supernatant was removed from the particles to determine the amount of ASO still conjugated to the particles.



Particles were washed with PBS and resuspended in macrophage medium before addition to macrophage cultures. PLG-aTNFα-ASO-NPs and blank NPs were added at a concentration of 200 µg/mL. Macrophages were examined with bright-field microscopy after NP treatment.



Gene expression from macrophage cultures was done by lysing cells in Trizol followed by RNA isolation, transcription into cDNA, and qPCR. Gene expression was normalized to the GAPDH housekeeping gene followed by normalization to stimulated M1 macrophages. Cytokines were measured in macrophage media supernatants by ELISA.


Results, Conclusions, and Discussions:: Results

Particle size was not significantly different after ASO conjugation (Figure 1A). Anti-TNFα-ASOs bound to NPs, indicated by a more negative charge than blank NPs (Figure 1B) and a 72% coupling efficiency (Figure 1C). Approximately 7.6% of the total ASO available for conjugation was released from NPs in the first 12 hours of incubation, while the remaining conjugated ASO was stable for up to 7 days (Figure 1D).



NPs were added to macrophages 2 days prior to lipopolysaccharide (LPS) stimulation. After LPS stimulation, cells were harvested after 4 hours, 1 day, and 3 days (Figure 2A). After 4 hours, PLG-aTNFα-ASO-NPs reduced gene expression of TNFα (Figure 2B). PLG-aTNFα-ASO-NPs reduced protein secretion of TNFα for up to 72 hours (Figures 2C, 2D, and 2E).



PLG-aTNFα-ASO-NPs can be freeze-dried and rehydrated. Gene expression of TNFα (Figure 3A), IL-6 (Figure 3B), and IL-1β (Figure 3C) remained knocked down in lyophilized PLG-aTNFα-ASO-NPs. TNFα protein secretion was also reduced at 4 hours (Figure 3D) and 24 hours (Figure 3E).



Conclusions

We successfully fabricated NPs and conjugated ASO to particles with a 72% coupling efficiency. About 7.6% of ASO was released over the first 12 hours, indicating that the majority remained conjugated to the particle.



ASO delivery from the particle could specifically knock down cytokine expression in inflammatory immune cells, as PLG-aTNFα-ASO-NPs knockdown expression was more effective than PLG control. Since TNFα helps orchestrate the cytokine cascade, knocking down its expression could potentially prevent this cascade10.



This nanoparticle platform can be lyophilized and maintain its efficacy, indicating it is shelf-stable.



Discussions

Knocking down TNFα expression as soon as possible is critical to limiting the accumulation of inflammatory immune cells. Our PLG-aTNFα-ASO-NPs led to broad immune cell reprogramming, reducing protein secretion of other inflammatory cytokines. Although IL-1β, IL-6, and TNFα have distinct properties, their functions overlap and interact with each other; all are involved in inflammation and regulation of the immune response11-13. Our NP technology has the potential to be used in disease models with acute inflammation, where rapid reductions in inflammatory markers may change clinical outcomes.


Acknowledgements (Optional): : Thank you to all the Shea Lab Members!

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