(T1030-06-36) Development of Thermoresponsive Tacrolimus Loaded Nanoemulgel for Localized Immunomodulation in Psoriasis

Purpose: Effective immunosuppression requires targeted drug delivery to the immune cell populations responsible for chronic inflammatory conditions such as psoriasis. The complex pathogenesis of psoriasis involves both innate and adaptive immune systems. The macrophage, dendritic cells, and T-cells create a self-amplifying loop of inflammatory cytokine release, driving the keratinocyte growth and formation of psoriatic lesions^1,2. Existing anti-psoriatic therapeutic approaches are limited to symptomatic relief and often fail to provide targeted action, increasing the risk of severe side effects². The goal of our project is to develop a safe and effective drug delivery platform to control psoriasis by cell-targeted local immunomodulation. We propose to achieve this goal by incorporating an immunosuppressive drug tacrolimus into a nanoemulsion (TAC-NE), which is then homogenously dispersed into a thermo-responsive pluronic-based hydrogel to form a nanoemulgel. The nanoemulgel (TAC-NEG) is designed to be stable, safe, and locally deliver the immunosuppressant tacrolimus to key immune cells (Macrophages and T cells) known to drive psoriasis pathology.  The TAC-NEG acts as a local depot at the injection site, providing a controlled and targeted drug release. The goal of this approach is to minimize systemic exposure to TAC and reduce the dosing frequency for continued control of inflammation in psoriasis, which improves patients' acceptance of local treatment.

Methods: A high percentage of medium-chain triglyceride lipid is used as the hydrophobic internal phase, stabilized with non-ionic surfactants in a sterile phosphate buffer saline as an external phase to formulate a tacrolimus loaded nanoemulsion (TAC-NE). A two-step process is utilized for TAC-NE manufacturing, including the preparation of a homogeneous coarse pre-emulsion, emulsifying the dispersed phase (non-aqueous) with the continuous (aqueous) phase through homogenization. Processing the coarse pre-emulsion under high-pressure microfluidization reduces the droplet size 100-120 nm. Nanoemulsion was manufactured on a microfluidizer LM20 and then incorporated into a pluronic-based thermoresponsive hydrogel via planetary centrifugation³.  The TAC-NE was evaluated for colloidal stability in the presence of biological media and serum, under centrifugation, and filtration using dynamic light scattering (DLS) size measurements. The TAC-NEG was evaluated for rheological behavior⁴, drug content, sterility, and effects on the cell viability using luminescence-based assay³. In vitro drug release profile was established using standard methods³.  The TAC-NEG was in vivo tested in a complete Freund’s adjuvant (CFA) rat inflammation model for anti-inflammatory action and safety⁵. A Near-Infrared Fluorescent (NIRF) dye-loaded NE was injected i.v. to track immune cells in a CFA rat model and animals imaged by a preclinical imager to measure changes in cell infiltration patters as the measure of inflammation in TAC-NEG treated animals and controls.

Results: Our group has previously reported the development of Tacrolimus-loaded microemulsion and nanoemulsion for local and topical immunosuppression^3,6.  In this project, tacrolimus is loaded in a naturally derived lipid-rich nanoemulsion carrier which exhibits a particle size less than 110 nm with a uniform particle size distribution, and narrow dispersity index < 0.1 (Figure 1A-1C). The tacrolimus nanoemulsion (TAC-NE) shows long-term particle size stability. Moreover, stress studies confirm no significant deviation in particle size during filtration and centrifugation or when exposed to cell culture media (Figure 1B-1F). The incorporation of TAC-NE into Pluronic-based hydrogel results in small particle size of 110-120 nm with a narrow monomodal distribution (Figure 2A)⁷. The rheological assessments show thermoresponsive behavior and confirm the sol-to-gel transition of TAC-NEG. The oscillation amplitude demonstrates the linear viscoelastic region below 8% of oscillation strain and the frequency sweep study confirms consistent gel behavior with an increase of angular frequency. The temperature ramp study indicates an increase in viscosity with increasing temperature exhibiting viscoelastic solid behavior of the gel (Figure 2A-2C). Gel shows consistent and reversible behavior under varying strains and temperatures (Figure 2D-2F). Primary in vitro study shows slow drug release profile from TAC-NEG compared to free tacrolimus. Moreover, the TAC-NEG is tested for CellTiter-Glo® luminescent assay for studying the viability of RAW 264.7 macrophages and showed a mean 95% viability with higher concentration (80μl) of TAC-NEG confirming no cytotoxicity (Figure 3A-B). Furthermore, the preliminary in vivo safety profile is also assessed in CFA induced rat model through localized delivery of 450μl TAC-NEG in footpad and was observed till 7 days without showing any toxic effects (Figure 3C). A near-infrared imaging fluorescent (NIRF) dye-loaded nanoemulsion is also injected intravenously for labeling inflammation in rats. The preliminary in vivo assessment confirms the safety and potential use in immunomodulation in a chronic inflammatory rat model.

Conclusion: Therefore, this study shows a thermoresponsive and biocompatible TAC-NEG which offers a promising platform for the development of localized immunosuppression strategies in psoriasis. TAC-NEG shows reversible behavior and undergoes complete gelation when injected into body temperature (37˚C) making it suitable for sustained drug release. The presented pluronic-based TAC-NEG confirms its physical stability and viability both in vitro and in vivo. The CFA-induced rat model treated with TAC-NEG provides tracking inflammation and evaluating potential in immunomodulation for future experiments.

References: 1. Sieminska, I., Pieniawska, M. & Grzywa, T.M. The Immunology of Psoriasis-Current Concepts in Pathogenesis. Clin Rev Allergy Immunol 66, 164-191 (2024).
2. Yadav, K., et al. A complex molecular landscape to drug delivery concept for achieving precise therapy in psoriasis. Medicine in Drug Discovery 22(2024).
3. Vichare, R., et al. A Reversibly Thermoresponsive, Theranostic Nanoemulgel for Tacrolimus Delivery to Activated Macrophages: Formulation and In Vitro Validation. Pharmaceutics 15(2023).
4. Das, A.C., et al. Injectable, reversibly thermoresponsive captopril-laden hydrogel for the local treatment of sensory loss in diabetic neuropathy. Sci Rep 14, 18978 (2024).
5. Vichare, R., et al. Theranostic nanoemulsions suppress macrophage-mediated acute inflammation in rats. J Nanobiotechnology 23, 80 (2025).
6. Srishti, S.A., et al. Quality by Design (QbD)-Driven Development and Optimization of Tacrolimus-Loaded Microemulsion for the Treatment of Skin Inflammation. Pharmaceutics 16(2024).
7. Jelena M. Janjic, Rebecca McCallin, Lu Liu, Caitlin Crelli, Amit Chandra Das and Anneliese Troidle. In vitro Quality Assessments of Perfluorocarbon Nanoemulsions for Near-infrared Fluorescence Imaging of Inflammation in Preclinical Models. J Neuroinflammation 18, 299 (2021).

Acknowledgements: This work was funded in part by the following CDMRP awards: W81XWH-19-1-0828, W81XWH-19-1-0389, W81XWH-20-1-0730, and W81XWH-20-1-0769 awarded to J. M. Janjic.


Figure 1: A. The structure of oil in water (O\W) nanoemulsion having the hydrobhobic drug tacrolimus in the core oil droplet stabilized by the surfactants in the interface and surrounded by the aqueous phase. B. The particle size assessment till day 90, B. The particle size distribution C. Impact of incubation in 20% FBS in DMEM containing culture media and water in 37C in particle size. E. Impact of centrifugation on particle size. F. Impact of filtration on particle size.


Figure 2: A. Particle size distribution of TAC-NEG, Rheological assessments including B. Oscillation amplitude sweep test (OA) and C. Oscillation frequency (OF) sweep test. D. Viscosity with temperature ramp up test E & F. Alternate step strain tests for assessing injectability and thermoresponsiveness varying strains and temperature.


Figure 3: A. Primary in vitro drug release profile from TAC-NEG B. Assessment of percentage viability in raw macrophage 264.7 with TAC-NEG treatment varying concentrations C. Preliminary evaluation of in vivo toxicity assessment of TAC-NEG with NIRF dye loaded nanoemulsion in CFA induced rat model for tracking inflammation.