(T1530-01-02) Exploring the Properties of Hexagonal Liquid Crystalline Nanoparticles for Co-delivery of Dexamethasone Acetate and siRNA TNF-α

Purpose: Non-lamellar liquid-crystalline nanoparticles (LCN) are colloidal platforms with excellent properties for cutaneous drug delivery, such as high ordering and internal symmetry, entrapment capacity and sustained release of hydrophilic, lipophilic or amphiphilic molecules and biocompatibility with different biological interfaces [1]. Considering the co-delivery capability of LCN, and aiming additive or synergistic therapeutic effects, we explored the properties of reverse hexagonal LCN as platforms loaded with dexamethasone acetate (DMA) and small interfering RNA (siRNA) molecules directed to TNF-α for the treatment of inflammatory skin diseases. DMA is a potent synthetic lipophilic glucocorticoid often used in the treatment of inflammatory and allergic skin diseases, exerting its anti-inflammatory and anti-proliferative effects via genomic and non-genomic pathways [2]. Associated with this, siRNA molecules targeting specific targets of the inflammatory cascade (e.g. cytokines and transcription factors) have been considered ideal candidates to treat a range of diseases, including skin diseases [3].

Methods: The LCN were obtained from the ultrasonic dispersion of the bulk gel composed of monoolein, oleic acid, DMA, cationic polymer and aqueous phase (8:2:0.1:0.5:89.5, w/w/w/ p/p). The physicochemical characteristics of the LCN were obtained by means of small angle X-ray scattering (SAXS), dynamic and electrophoretic light scattering, nanoparticle concentration, DMA encapsulation efficiency and electrophoretic assays with siRNA. In vitro studies in Franz diffusion cells were carried out to evaluate the DMA release and its cutaneous distribution in the biological membrane. Cellular studies were performed to assess the cytocompatibility and internalization rates of LCN in Raw264.7 macrophage cell line. TNF-α gene silencing was determined in Raw264.7 cells stimulated with lipopolysaccharide (LPS).

Results: LCN exhibited unimodal, symmetric size distribution curves with mean size between 144 and 166 nm, low polydispersion (0.11 to 0.18), DMA encapsulation greater 90% and high siRNA complexation (Table 1 and Fig. 1 C). SAXS analysis confirmed an internal mesophase of reverse hexagonal symmetry (Fig 1. B). In vitro skin permeation and retention results showed that LCN promoted greater skin penetration and retention of DMA compared to free drug (Fig 2-A). Still, complementary to these data, CLSM micrographs obtained from skin fragments treated with LCN-DMA complexed with siRNA labeled AF647 dye demonstrated a predominance of fluorescence throughout the SC and EP+D region (Fig 2-B). In cellular studies, LCN demonstrated better cytocompatibility and a greater ability to deliver siRNA (Fig 3-A,B). Furthermore, the co-delivery of DMA and siRNA targeted TNF-α by LCN promoted a marked reduction of TNF-α levels in inflammatory macrophages. In all protocols examined, we showed that siRNA TNF-α presented silencing activity only when transported by LCN. In addition these nanoparticles also improved the effect of ADM (Fig 3-C).

Conclusion: Taking together, these results show that LCN holds the merits of successful delivery of siTNF-α to machophages and its combination with DMA leads to a significant reduction in secreted TNF-α levels under different experimental conditions of inflammation, being a potential strategy for the therapy of inflammatory skin diseases involving a complex network of cytokines and other pro-inflammatory mediators.

References: [1] A.V.P. Silvestrini, B.W. Debiasi, F.G. Praça, M.V.L.B. Bentley, Progress and challenges of lyotropic liquid crystalline nanoparticles for innovative therapies, Int. J. Pharm. 628 (2022) 122299. https://doi.org/10.1016/j.ijpharm.2022.122299.
[2] G. Bor, S. Salentinig, E. Şahin, B. Nur Ödevci, M. Roursgaard, L. Liccardo, P. Hamerlik, S.M. Moghimi, A. Yaghmur, Cell medium-dependent dynamic modulation of size and structural transformations of binary phospholipid/ω-3 fatty acid liquid crystalline nano-self-assemblies: Implications in interpretation of cell uptake studies, J. Colloid Interface Sci. 606 (2022) 464–479. https://doi.org/10.1016/j.jcis.2021.07.149.
[3] A.V.P. Silvestrini, A.L. Caron, J. Viegas, F.G. Praça, M.V.L.B. Bentley, Advances in lyotropic liquid crystal systems for skin drug delivery, Expert Opin. Drug Deliv. 00 (2020) 1–25. https://doi.org/10.1080/17425247.2020.1819979.

Acknowledgements: This work was developed within the framework of National Institute of Science and Technology of Pharmaceutical Nanotechnology (INCT-Nanofarma), which is supported by São Paulo Research Foundation (FAPESP, grant #2018/08253-9 and #2022/01969-4) and National Council for Scientific and Technological Development (CNPq, Brazil, grant #465687/2014-8).

Fig. Characterization of NLCs.

Fig. 2. (A) In vitro permeation and skin retention of DMA and (B) In vitro skin delivery of siRNA labeled AF647 dye.

Fig 3. (A) Cytocompatibility, (B) cellular uptake and (C) TNF-α levels in inflammatory macrophages.