(M1330-03-18) Crafting Lipid-Polymer Nanoparticles of HDAC Inhibitors for the Treatment of Retinitis Pigmentosa

Purpose: Retinitis Pigmentosa (RP) is an inherited retinal disease-causing color and peripheral vision loss, leading to blindness. Current treatments aim to slow disease progression but with no cure. New therapeutic approaches such as retinal prostheses, gene therapy, and neuroprotection have gained interest. Histone deacetylase inhibitors (HDACi) show promise for neuroprotection and neuronal regeneration in RP and other retinal and neuronal cells. Clinical trials have demonstrated the neuroprotective effects of HDAC inhibitors in RP. Injecting HDACi like Trichostatin A (TSA) and Vorinostat into the eye has shown slow photoreceptor cell degeneration in advanced RP mouse models. However, the rapid metabolic degradation of these HDAC inhibitors limits their efficacy, necessitating frequent injections. To overcome this limitation, lipid-polymer hybrid nanoparticles for sustained delivery of HDAC inhibitors have been investigated in this study. These nanoparticles prevent rapid degradation and eliminate the need for repeated intravitreal injections of HDAC inhibitors. Our hypothesis is that encapsulating HDACi in lipid-polymer hybrid nanoparticles will prevent its rapid metabolic degradation and lead to sustained drug delivery of HDAC inhibitors through the protective advantages of liposomes and the sustained drug release properties of polymeric nanoparticles. The goal of this study is to develop and characterize this hybrid drug delivery system, enhancing the therapeutic efficacy of HDAC inhibitors for RP treatment.

Methods: The nanoparticles (NPs) were formed through a combination of nanoprecipitation and self-assembly methods. Briefly, PLGA (poly lactic-co-glycolic acid, 85:15), a hydrophobic polymer, was dissolved in acetonitrile (ACN) at a concentration of 2 mg/mL. Vorinostat and Trichostatin A were added to the dissolved polymer solution at a concentration of 10% w/w. Soya lecithin (90 %) and DSPE-PEG (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[polyethylene glycol-2000]) (8.5/1.5 molar ratio) were dissolved in 4% ethanol, heated to 65°C, and allowed to cool at room temperature. The PLGA solution was added dropwise to the preheated lipid solution under gentle stirring and then vortexed for 3 minutes to allow the lipids to disperse and self-assemble. The addition of PLGA solution in the lipid system resulted in a 14% lipid-to-polymer ratio. The lipid-polymer solution was continuously stirred for 2 hours at room temperature to allow the nanoparticles to self-assemble and the organic solvent to evaporate. The lipid-polymer solution was washed with deionized water (DI water) three times employing centrifugation at 9500 g using an Amicon Ultra-4 filter with a molecular weight cutoff of 30 kDa. The nanoparticles were tested for particle size, polydispersity index, and zeta potential using a particle size analyzer (Nanobrook 90 Plus PALS, Brookhaven). To further analyze the morphology and particle size, scanning electron microscopy (SEM) was utilized. Samples for SEM were prepared by dropping 10 μL of NP solution onto a carbon tape. After allowing the droplet to dry naturally overnight at room temperature, the sample was subjected to SEM imaging. An HPLC method was developed to quantify both drugs for in vitro drug release study. Separation was achieved using a C-18 column (Phenomenex, 250x4.60 mm, 5 microns), with an isocratic mobile phase that consisted of 40.0% (v/v) acetonitrile and 60% (v/v) water (pH 2.5) for Vorinostat, and 70.0% (v/v) acetonitrile and 30% (v/v) water (pH 2.5) for Trichostatin A. The mobile phase flow rate was maintained at 1.0 mL/min. A PDA detector at 241 nm and 231 nm was used to detect Vorinostat and Trichostatin A, respectively.

Results: The particle size for blank and drug-loaded nanoparticles ranges from 81.23 nm to 85.43 nm. Similarly, PDI and zeta potential varied from 0.13 to 0.19 and -114.91 mV to -68.54 mV, respectively (Table 1). SEM images also confirmed the formation of nanoparticles and their spherical shape (Figure 1). Both blank and drug-loaded particles were physically stable for 7 days when stored at both room temperature and 4⁰C and they had similar diameters and surface charges as compared to day 0 formulations. The retention time of Vorinostat was 4.13 minutes (Figure 2a). TSA was eluted at 3.21 minutes (Figure 2b). Standard curves for both drugs were linear over a concentration range from 0.19 µg/mL to 100µg/mL.

Conclusion: The study demonstrated successful formulation and characterization of polymer-lipid hybrid HDAC inhibitor particles in nano-size ranges. The drug-loaded nanoparticles had a zeta potential of about -70.00 mV to -115.00 mV and were polydisperse. These nanoparticles were also physically stable for one week, which could be attributed to their significant zeta potential value. Additionally, the HPLC methods for linearity were found to be within the acceptable range for both drugs.

References: 1. L. Zhang et al., “Self-assembled lipid--polymer hybrid nanoparticles: a robust drug delivery platform,” ACS Nano, vol. 2, no. 8, pp. 1696–1702, Aug. 2008, doi: 10.1021/nn800275r.
2. M. Samardzija et al., “HDAC inhibition ameliorates cone survival in retinitis pigmentosa mice,” Cell Death Differ., vol. 28, no. 4, pp. 1317–1332, Apr. 2021, doi: 10.1038/s41418-020-00653-3.

Acknowledgements: We would like to acknowledge the School of Pharmacy and Health Professions, Department of Pharmacy Sciences, Creighton University and George F. Haddix President’s faculty Research fund for funding. Conflict of Interest Statements: We confirm that there are no conflicts of interest associated with this work, and there has been no significant financial support for this work that could have influenced its outcome.

Table 1: Characterization of lipid-polymer nanoparticles.

Figure 1: Scanning Electron Microscopy (SEM) image depicting the hybrid nanoparticles.

Figure 2: HPLC peaks. A) Vorinostat (4.13 min), and B) Trichostatin A (3.21 min).