Poster J5 - Development of an in situ forming microparticle system for sustained delivery of punicalagin

Thursday, October 24, 2024

2:45 PM - 3:45 PM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Steve Elder, PhD (he/him/his)

Professor
Mississippi State University
Mississippi State, Mississippi, United States

Co-Author(s)

KT

Kenna Turner

Lab Assistant
Mississippi State University
meridian, Mississippi, United States

Co-Author(s)

AB

Austen Breland

Lab Assistant
Mississippi State University
ellisville, Mississippi, United States

Introduction: Intra-articular drug delivery is appealing for the treatment of osteoarthritis (OA) because it minimizes the dose and reduces off-target effects, but short intra-articular dwell time due to efficient lymphatic drainage is problematic. Our innovative approach to overcome this problem is to use in situ forming microparticles (ISM). ISM systems consist of an internal drug-containing polymer phase emulsified with a continuous oil phase. When injected into an aqueous environment, the polymeric phase hardens, forming microparticles in situ. ISMs could be used to boost the effectiveness of many experimental drugs that rely on long-term exposure. Unfortunately, an arsenal of safe and effective disease-modifying OA drugs (DMOADs) has yet to be developed. Current treatments are palliative and do not slow the rate of cartilage degeneration.
We advance punicalagin (PCG), a polyphenol from pomegranate fruit, as a potential DMOAD with multiple chondroprotective mechanisms. For example, it inhibits the activity of MMP-13 and ADAMTS-5, the catabolic enzymes primarily responsible for ECM degradation in OA. The long-term objective of this research is to create punicalagin-releasing in situ forming microparticles which could be administered through intra-articular injection. It could be given prophylactically to patients at high risk of developing OA or to slow disease progression in patients with early-stage OA.
We hypothesized that PCG release kinetics are highly tunable through varying the proportions of polycaprolactone (PCL) and poly(lactic-co-glycolic acid) (PLGA). The purpose of this study was to determine the effect of the PCL:PLGA ratio on PCG loading efficiency, microparticle size, and the kinetics of PCG release.

Materials and

Methods: Microparticles were formed by first dissolving PCG (0.15 mg/mg polymer), PCL, PLGA and poloxamer 188 (2.5% w/w) in 1,4-dioxane. Total polymer concentration was held constant at 15% w/w. An external oily phase was comprised of sesame oil containing 5% Span 80. The polymer phase and the oil phase were mixed at a 4:1 oil:polymer ratio and emulsified using a handheld homogenizer. Drug release experiments were performed in triplicate using 2 ml aliquots that were transferred to dialysis tubing (MWCO 12-14 kD) containing 2 ml PBS. The bags were then placed into 9 ml PBS and maintained in an incubated shaker (37 °C, 100 rpm). The external PBS was periodically collected and replaced in its entirety. The optical absorbance at 378 nm (A378) was measured to determine the amount of PCG released during each interval. Separate batches were prepared for determining encapsulation efficiency. After 60 minutes of shaking, the amount of unencapsulated PCG in the PBS within the tubing was determined from A378. Encapsulation efficiency was calculated as ((total PCG added-unencapsulated PCG))/(total PCG added)×100.
A third emulsion was prepared as above with the addition of rhodamine to the polymer solution. Approximately 4 ml of the rhodamine-containing emulsion was pipetted into 10 ml PBS. After stirring gently for 2 h, suspended microparticles were imaged on a fluorescence microscope and particle size determined using digital image analysis. Remaining particles were stirred for and additional 48 h, isolated by filtration, vacuum dried and imaged on a scanning electron microscope.

Results, Conclusions, and Discussions: As shown in Figure 1, all formulations resulted in roughly spherical as well as irregularly shaped microparticles. For all groups, most spherical particles were around 3 µm in diameter. Ideally, the microsphere size would be close to 10 μm diameter. Microspheres of that size are well tolerated in joints [1–3]. Particles larger than 10 μm tend to elicit a foreign body reaction but are not phagocytosed, whereas those under 5 μm may be phagocytosed by macrophages and foreign body giant cells [4], or they may be eliminated from the joint by the lymphatic pathway [3]. We speculate that microparticle size can be increased through less vigorous mixing during emulsification and lowering the oil:polymer ratio.
For all groups except 100% PLGA, the encapsulation efficiency was in the range of 58 – 71% (Table 1); encapsulation efficiency for 100% PLGA was much lower. Encapsulation efficiency could be affected by the type of polymer, the polymer concentration, and solvent’s miscibility with water. These factors will be explored in future work. The pattern of sustained PCG release was highly tunable based on the PCL:PLGA ratio (Figure 2). There was a statistically significant difference among all groups by two-way ANOVA. The highest and lowest rates of release were observed for 100% PLGA and 100% PCL, respectively. The other formulations were intermediate, exhibiting the general trend of slower rate of release with increasing PCL proportion. Most of the PCG added to 100% PLGA was not encapsulated. Therefore, it was “released” immediately in burst fashion. There was much lower burst release of PCG from formulations that included PCL. PCL could slow the rate of release due to forming more dense microparticles and hydrolyzing more slowly than PLGA. Overall, the data show that sustained and tunable release of PCG can be achieved from in situ forming microparticles comprised of PCL and PLGA. Future research will be aimed at increasing microparticle size and encapsulation efficiency.

References
1. Chen et al. Drug Deliv. 2014;21(5):342-350
2. Zhang and Huang. Drug Deliv. 2012;19(5):255-263
3. Pradal et al. Int J Pharm. 2016;498(1-2):119-129
4. Anderson and Shive. Adv Drug Deliv Rev. 2012;64(SUPPL.):72-82

Acknowledgements (Optional): Research reported in this publication was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health under award number R15AR083143 and by the National Institute of General Medical Sciences of the National Institutes of Health under award number GM123920.