Purpose: To developed PLGA microparticles encapsulating collagenase, elastase and papain, individually, which can be combined for the treatment of keloids, hypertrophic scars and other pathologies experiencing pathological buildup of collagen.
Methods: Collagenase PLGA microparticles are formulated using a double-emulsion method of preparation. Following dissolution in water, collagenase is added to a 5% (wt) PLGA polymer solution in dichloromethane. This emulsion is vortexed for 60 seconds, then sonicated for 60 seconds at 40% amplitude, using a 3mm tip sonicator. This single emulsion is added to a 2% (wt) PVA solution and homogenized for 90 seconds. The resulting emulsion is poured into a beaker containing water and stirred to allow for evaporation of the solvent. After 4 hours of stirring, the microspheres are by centrifugation at 2000 RPM for 3 minutes. Microspheres are washed with water and lyophilized. A similar method was used for fabrication of elastase and papain microsphere fabrication. Loading experiments were conducted by dissolving 10mg microspheres in 4mL DCM. 1 mL water (or buffer at pH 9.0 for elastase and papain) was added to the dissolved microspheres, followed by 5 minutes of vortexing to extract the protease in the aqueous phase. This supernatant was analyzed using a BCA Assay.
Long term protease activity study was conducted, Porcine tendon (~100g) was placed in a 300kD MWCO membrane with protease in buffered solution or protease microspheres in suspension (buffered) (1mL). The sealed membrane was placed in 10mL buffer in a 50mL conical tube, and tubes were stored and shaken at 37˚C. The buffer was sampled and assayed for soluble protein using a BCA kit. 5mL buffer was replaced at each sampling.
Results: The study showed that the protease were release from the particles into the solution and where able to degrade the tendon. The amount of collagen degraded was measured as the amount of soluble protein present in the solution, incase of protease solution 2% of the soluble protein was measured but incase of collagenase particles the amount of soluble protein estimated was higher about 6%. Similar observation was seen for the protease, collagenase and papain being released from PLGA particles. This was observed due to improved activity of the protease from the microparticles compared to the protease in solution.
Conclusion: PLGA microspheres encapsulating collagenase, elastase and papain, can be fabricated using the double emulsion/solvent extraction method, resulting in increased protease activity.
Pardeep Gupta– Scientific Advisor, Hyalo Technologies, Pennsylvania
Shalabh Jain– Chairman, CEO and Founder, Hyalo Technologies, Philadelphia, Pennsylvania
Buu Tu– Research and Development Scientist, Hyalo Technologies