Improved alpha-chymotrypsin stability upon encapsulation in PLGA microspheres by solvent replacement.

Area of Science:

• Biomaterials Science
• Drug Delivery Systems
• Protein Chemistry

Background:

• Dichloromethane (CH2Cl2), an ICH Class 2 solvent, is commonly used for encapsulating proteins in poly (lactic-co-glycolic) acid (PLGA) microspheres.
• CH2Cl2 poses potential toxicity risks, necessitating the exploration of safer, more biocompatible alternatives.
• Protein instability during encapsulation is a significant challenge in developing effective protein-loaded microspheres.

Purpose of the Study:

• To evaluate alternative biocompatible solvents for alpha-chymotrypsin encapsulation in PLGA microspheres.
• To replace CH2Cl2 with a safer solvent without compromising protein stability and encapsulation efficiency.
• To optimize the solid-in-oil-in-water (s/o/w) encapsulation process for improved biocompatibility.

Main Methods:

• Investigated various low-toxicity solvents with differing water solubilities for the s/o/w encapsulation process.
• Optimized the oil-to-water (O:W) ratio during emulsification to maximize protein stability and encapsulation efficiency.
• Assessed protein stability by monitoring insoluble aggregate formation and residual enzyme activity.

Main Results:

• The O:W ratio critically influences protein stability, with hydrophilic solvents generally proving detrimental.
• Optimizing the O:W ratio allowed for successful encapsulation using butyl acetate, a Class 3 solvent.
• Using butyl acetate with poly (ethylene glycol) (PEG) as an emulsifying agent resulted in minimal protein aggregation (approx. 1%) and high residual activity (93 ± 10%).

Conclusions:

• The s/o/w encapsulation technique was successfully modified to replace CH2Cl2 with butyl acetate.
• This modification enhances the biocompatibility of the PLGA microsphere formulation process.
• Alpha-chymotrypsin stability was preserved, demonstrating the feasibility of using safer solvents for protein encapsulation.