(T1230-06-36) Scalable Theranostic Nanoemulsion for Sustained Non-opioid Analgesia: Modulating Macrophage Dynamics in Peripheral Nerve Injury

Purpose: Peripheral nerve injuries (PNIs) refer to the damage of the peripheral nerves, which leads to sensorimotor impairments. PNIs can result from various nerve traumas, including crushing, contusions, or penetrating injuries ¹. Therefore, the incidence of PNIs is common in both civilians and warfighters. Neuroinflammation and neuropathic pain are common consequences following PNI. Approximately 73% of patients diagnosed with PNI experience neuropathic pain due to underlying neuroinflammation ². Despite the significant progress in understanding the mechanisms of PNI-associated neuroinflammation, 69.4% of patients suffering from PNIs receive prescription opioids early on for their pain management ³. This is because the recommended non-opioid analgesics, such as calcium and sodium channel blockers or NSAIDs, provide suboptimal pain relief ⁴. In this project, we argue that the current non-opioid analgesics fail to provide long-term pain relief because of their inability to be directly delivered to immune cells involved in the pathophysiology of neuroinflammation, which triggers neuropathic pain. The temporal specificity of macrophage phenotypes and the differential cytokine profiles observed in PNI suggest that immunomodulating macrophage phenotypes from pro-inflammatory (M₁-like) to anti-inflammatory (M₂-like) and decreasing their infiltration at the site of nerve injury would create a nerve microenvironment that promotes resolution of neuroinflammation, ultimately preventing development of neuropathic pain ^5,6. Thus, we have developed a scalable theranostic nanoemulsion (NE), a colloidal dispersion of immiscible liquids stabilized with surfactants, to facilitate sustained release of non-opioid analgesics directly to infiltrating macrophages. This approach will reprogram their pro-inflammatory phenotype to an anti-inflammatory pro-regenerative phenotype.

Methods: Theranostic NE was formulated using microfluidizers M110S (25 mL) and LM20 (175 mL). Celecoxib (CXB, selective inhibitor of cyclooxygenase 2) and DiR (near-infrared fluorescent imaging (NIRF) dye) were first dissolved in hydrocarbon oil and then vortexed with liquid perfluorocarbon (¹⁹F MRI tracer). The blend was emulsified using a non-ionic surfactant solution. Orthogonal techniques such as dynamic light scattering and nanoparticle tracking analysis were used to determine the droplet diameter and concentration. Routine follow-ups were performed to evaluate the long-term colloidal and fluorescent stability. Celecoxib percent encapsulation efficiency (%EE) was measured using HPLC. The release of celecoxib was studied using a dialysis bag release set-up at 37 under sink conditions. CellTiter-Glo^® ATP assay was used to study the effect of theranostic NE on macrophage viability. The ability of theranostic NE to skew macrophage phenotype was assessed using multicolor flow cytometry and ELISAs. To demonstrate the ability of CXB-loaded theranostic NE to decrease M₁-like macrophage infiltration at the nerve injury site, we used the sciatic nerve transection and repair (SN-TSR) rodent model of neuroinflammation and neuropathic pain. Macrophage infiltration at the site of nerve injury was tracked using an in vivo Pearl Trilogy Imager. Sciatic nerve hematoxylin and eosin (H&E) staining was performed to demonstrate resolution of neuroinflammation due to treatment with CXB-loaded theranostic NE compared to drug-free NE. Immunofluorescence assay was performed to confirm the co-localization of theranostic NE droplets in COX-2+CD68+ macrophages.

Results: A theranostic NE encapsulating CXB (model drug) with two diagnostic payloads (a NIRF dye and a ¹⁹F MRI tracer) was formulated using a microfluidizer (Figure 1A). The average droplet diameter determined using orthogonal techniques was around 140 nm (Figure 1B). The colloidal and fluorescent stability of theranostic NE was maintained up to three months. The %EE of celecoxib was 79.5 0.5 %. Sustained release for two weeks with no initial burst was observed from the CXB-loaded theranostic NE compared to the free CXB solution (Figure 1C). The developed theranostic NE decreased the percentage of M₁-like CD86+ macrophages compared to other groups (Figure 1D). Consequently, we also observed a decrease in the release of pro-inflammatory mediators and cytokines. In a clinically relevant SN-TSR model, treatment with CXB-loaded theranostic NE decreased macrophage infiltration significantly as compared to drug-free NE, surgical sham control, and naïve rodents (Figure 2A and 2B). Sciatic nerve H&E staining showed that treatment with CXB-loaded theranostic NE promoted axon survival and myelin preservation, while was associated with extensive myelin disruption and axonal loss. The infiltration of COX-2+CD68+ pro-inflammatory macrophages was significantly higher in drug-free NE group compared to the CXB-loaded theranostic NE group. The theranostic NE was scaled to a 175 mL batch using a robust microfluization manufacturing technique without compromising the physicochemical characteristics and functional efficacy (Figure 3A-C).

Conclusion: We successfully formulated a scalable theranostic NE that provides sustained release of non-opioid analgesic to treat neuroinflammation in PNI. We demonstrated that the sustained release of CXB promotes macrophage immunomodulation towards an anti-inflammatory phenotype at the site of nerve injury. Consequently, decreasing further infiltration of M₁-like macrophages at the neuroinflammatory foci. This theranostic NE platform is designed to treat neuroinflammation for long-term analgesia rather than symptomatic relief, ultimately avoiding the need for prescription opioids in patients with PNIs.

References: 1 Gaudet, A. D., Popovich, P. G. & Ramer, M. S. Wallerian degeneration: gaining perspective on inflammatory events after peripheral nerve injury. Journal of neuroinflammation 8, 1-13 (2011).
2 Miclescu, A. et al. Chronic neuropathic pain after traumatic peripheral nerve injuries in the upper extremity: prevalence, demographic and surgical determinants, impact on health and on pain medication. Scandinavian Journal of Pain 20, 95-108 (2019).
3 Callaghan, B. C. et al. Longitudinal pattern of pain medication utilization in peripheral neuropathy patients. Pain 160, 592-599 (2019).
4 Lopes, B. et al. Peripheral nerve injury treatments and advances: one health perspective. International journal of molecular sciences 23, 918 (2022).
5 Domoto, R., Sekiguchi, F., Tsubota, M. & Kawabata, A. Macrophage as a peripheral pain regulator. Cells 10, 1881 (2021).
6 Janjic, J. M. et al. Low-dose NSAIDs reduce pain via macrophage targeted nanoemulsion delivery to neuroinflammation of the sciatic nerve in rat. Journal of neuroimmunology 318, 72-79 (2018).
7 Vichare, R. et al. Folate-conjugated near-infrared fluorescent perfluorocarbon nanoemulsions as theranostics for activated macrophage COX-2 inhibition. Scientific Reports 13, 15229 (2023).

Acknowledgements: We acknowledge Dr. Vijay S. Gorantla and his team for providing surgical training and development of SNTSR model.
This work was supported by CDMRP awards W81XWH-20-1-0854 and W81XWH-20-1-0276.


Figure 1. A) Schematic representation of CXB-loaded theranostic nanoemulsion with two diagnostic payloads. B) Overlay of average size distribution by intensity measured for CXB-NE and drug-free (DF-NE) on Day 0 and TEM of CXB-loaded theranostic NE. C) In vitro sustained release of CXB from CXB-NE, whereas CXB-free drug (CXB-FD) shows initial burst release at pH = 7.4 at 37oC. D) Quantitative changes in CD86+ macrophage expression in the presence of varying doses of CXB-NE, CXB FD (40 𝜇M) and DF-NE (volume-matched to the highest concentration). The data is shown as the mean ± SD. n = 3/group independent cell culture, and 40,000 cells were counted. One-way ANOVA with Tukey’s multiple comparisons test. ns not significant, ****p < 0.00005.


Figure 2. A) Timeline of sciatic nerve transection and repair surgery (clinically relevant rodent model of PNI) and representative whole-body NIRF images showing the accumulation of macrophages at the site of neuroinflammation (sciatic nerve). Quantification of the NIRF signal intensity from the ROI after administration of a single dose (300 𝜇L) of CXB-NE or DF-NE to track infiltrating macrophages at the site of nerve injury in B) male rodents and C) Female rodents. Sham surgical control received single dose of DF-NE to track macrophages post muscle injury. Naïve rodent were administered DF-NF to establish baseline fluorescence. Two-way, repeated measures ANOVA, Šídák’s multiple comparisons test, ns not significant, ****p < 0.0005


Figure 3. A) Demonstration of the manufacturing process on LM20 microfluidizer. B) A size versus microfluidization pass relationship on LM20 microfluidizer. C) Overlay of average size distribution comparing the size of a small-scale theranostic NE against a large-scale theranostic NE.