(T1230-09-56) Challenges in the Development of Polycaprolactone Microparticles for H₂S Donor Encapsulation Using Coacervation

Purpose: Glaucoma is an eye disorder characterized by increased intraocular pressure (IOP), leading to progressive vision loss due to the degeneration of retinal ganglion cells (RGCs) and optic nerve damage[1,2]. Hydrogen sulfide (H₂S) has emerged as a promising therapeutic agent for glaucoma due to its ability to lower intraocular pressure and provide neuroprotection [3]. However, its gaseous nature, inherent toxicity, and instability in aqueous environments pose significant hurdles for its safe and efficient controlled delivery [4]. Organic coacervation using biodegradable polymers such as polycaprolactone (PCL) offers a non-aqueous approach ideal for encapsulating H₂S donors. While this technique is widely applied for encapsulating proteins and peptides[5], its non-aqueous nature also makes it ideal for encapsulating small, unstable molecules like H₂S donors. Optimizing the coacervation process is essential for maximizing drug encapsulation, as small changes in formulation parameters (stirring speed, silicone oil concentration, and surfactant type and concentration) can significantly affect particle morphology, size, and uniformity. This study focuses on the formulation of blank PCL microparticles via coacervation–solvent evaporation and solvent extraction methods, systematically evaluating key parameters to overcome challenges for maximizing H₂S donor encapsulation.

Methods: Polycaprolactone (PCL, MW ~14,000) solution [5.7% (w/w)] was prepared by dissolving PCL in 10 mL dichloromethane (DCM), with surfactants (0.1%-2.26%) Two methods were used for preparing the PCL particles loaded with H₂S donor. For the coacervation–solvent evaporation method, PCL solution containing 1.13% glyceryl monooleate (GMO, HLB 3.4) was stirred at 376 rpm with an overhead stirrer. Silicone oil (1000 cSt, 36% (w/w)) was added all at once, followed by solvent (DCM) evaporation under a fume hood. Coacervate droplets were solidified by pouring the mixture into n-hexane (15× silicone oil volume) and stirring at 376 rpm for 15 minutes. For the solvent extraction method, the optimal quantity of silicone oil (1000 cSt) required to obtain stable coacervates was determined by adding silicone oil at a rate of 0.5 mL/min to the 5.7% (w/w) PCL solution and subsequent microscopic observations. Various amounts of silicon oil (45–60% (w/w)) were added with the help of a glass syringe to the PCL solution (with or without surfactant) in a closed glass jar with overhead stirring at 376 rpm. Following coacervation, the mixture was rapidly transferred to n-hexane (15× the combined volume of silicone oil and DCM) for particle solidification under controlled stirring (472 rpm or 1048 rpm) for 1 hour to ensure effective solvent extraction. The resulting microparticles were separated by centrifugation (1500 rpm for 2 minutes), washed with n-hexane, and dried overnight under a fume hood. Particle morphology was examined using scanning electron microscopy (SEM), while particle size and size distribution were analyzed with a Bettersizer 2600 particle size analyzer.

Results: The coacervation–solvent evaporation method yielded smooth, spherical microparticles when the coacervation step was stirred for 45 minutes [Table 1(F1), D50 = 59.68 ± 6.95 µm]. However, increasing the stirring time to 75 minutes [Table 1 (F2)] led to a larger particle size (125.33 ± 31.99 µm) and loss of spherical morphology, while reproducibility of this method was limited, likely due to uneven solvent evaporation. For the coacervation–solvent extraction method, the stability window study revealed that stable coacervates were obtained when the silicone oil concentration ranged between 45–60%(w/w). An increase in silicon oil content led to a reduction in particle size, decreasing from 379.6 ± 28.96 µm [Table 2 (F3)] at 45%(w/w) to 113.9 ± 6.32 µm [Table 2 (F10)] at 60.3%(w/w). Furthermore, increasing the solidification speed from 472 to 1048 rpm further reduced the particle size of (F10) to 70.11 ± 8.16 µm [Table 2 (F11)], although particle aggregation was observed. To reduce aggregation, surfactants were added to either the PCL solution or n-hexane. Adding 0.1% SPAN-85 [Table 2 (F5)] or GMO [Table 2 (F6)] to n-hexane had minimal effect, while incorporating 2.26% GMO [Table 2 (F8)] into the PCL solution significantly improved particle separation compared to 1.13% GMO [Table 2 (F7)]. The addition of SPAN-85 [Table 2 (F9)] into the PCL solution was not effective, as it destroyed the smooth surface of the particles.

Conclusion: PCL microparticles were successfully prepared using both coacervation solvent evaporation and solvent extraction methods. In solvent evaporation, excessive stirring increased particle size and disrupted smooth, spherical morphology. For solvent extraction, stable coacervates formed within a silicone oil concentration range of 45–60%(w/w). Incorporation of 2.26% GMO surfactant effectively improved particle separation. The optimized solvent extraction method with 52.49%(w/w) silicone oil, a solidification speed of 1048 rpm, and 2.26% GMO (F9) yielded the most uniform, minimally aggregated particles. The delivery method developed can be used for loading H₂S donor in future study.

References: [1] Mhatre S, Opere C A and Singh S 2022 Unmet needs in glaucoma therapy: The potential role of hydrogen sulfide and its delivery strategies J. Controlled Release 347 256–69
[2] Agarwal R, Gupta S, Agarwal P, Saxena R and Agrawal S 2009 Current concepts in the pathophysiology of glaucoma Indian J. Ophthalmol. 57 257
[3] Liu H, Perumal N, Manicam C, Mercieca K and Prokosch V 2020 Proteomics Reveals the Potential Protective Mechanism of Hydrogen Sulfide on Retinal Ganglion Cells in an Ischemia/Reperfusion Injury Animal Model Pharmaceuticals 13 213
[4] Rai A, Mhatre S, Chandler C, Opere C and Singh S 2024 Application of Quality by Design in the Development of Hydrogen Sulfide Donor Loaded Polymeric Microparticles AAPS PharmSciTech 25 132
[5] Aftabroushad S and Doelker E Factors Influencing the Entrapment of a Water Soluble Model Drug into Injectable Microparticles Prepared Using Solvent Evaporation and Phase Separation Techniques

Acknowledgements: Financial support from School of Pharmacy and Health Professions, Creighton University.

Funding: This study was funded by NIH NEI 1R15EY032275-01A1. 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: Effect of process parameters in solvent evaporation on particle size and span value

Table 2: Effect of process parameters in solvent extraction on particle size and span value