(T1430-04-24) Method Optimization to Reduce Leakage of Encapsulated Protein from Ethylcellulose Microcapsules Using Bovine Serum Albumin as a Model Protein

Purpose: Phenylalanine Ammonia Lyase (PAL) is an enzyme that metabolizes phenylalanine (Phe). We propose to encapsulate PAL in non-digestible ethylcellulose (EC) microcapsules as a potential treatment for Phenylketonuria (PKU). Microcapsules will be administered with food. Dietary Phe will diffuse into the microcapsules and be metabolized by PAL thereby reducing Phe absorption in PKU patients. However, there is extensive leakage of PAL out of the microcapsules. Leaked PAL will lose its activity in the gastrointestinal tract and compromise the effectiveness of our therapeutic strategy. The overall purpose of our work is to devise formulation strategies to reduce leakage of PAL from microcapsules. Microcapsules were manufactured by the solvent evaporation technique and Bovine Serum Albumin (BSA) was used as a model protein. Various formulation parameters were evaluated for their effects on microcapsule particle size, protein encapsulation efficiency (EE), protein leakage (PL), and surface morphology.

Methods: Manufacture of micronized BSA-trehalose particles: An aqueous solution of BSA and trehalose, a thermal protectant, was spray dried to form solid, spherical particles. Manufacture of microcapsules: 20mg of the BSA-trehalose particles were homogenized in an EC solution in an organic solvent to create a s/o suspension. This suspension was added to a 1% w/v aqueous PVA solution under homogenization to form a s/o/w dispersion. The dispersion was placed under high speed magnetic stirring overnight to allow for complete organic solvent extraction and subsequent EC precipitation around the particles to form microcapsules. The filtered microcapsules were collected and lyophilized for 24 – 48 hrs. The following parameters were assessed for their impact on microcapsule properties: organic solvent for dissolving EC and forming the organic phase; concentration of EC solution; phase:volume ratios of the organic phase to the external aqueous phase; homogenization speed for manufacturing the final s/o/w dispersion. EE of BSA was determined by shaking aliquots of microcapsules in dichloromethane (DCM): ethanol (1:1) solution to dissolve the polymer shell and simultaneously precipitate the encapsulated protein. The protein pellet was resolubilized in an alkaline SDS solution and protein content was determined by the Lowry assay. Protein leakage was determined by suspending aliquots of microcapsules in water and incubating them in a rotating shaker at 37°C for 15 minutes, 1 hour, and 3 hours. At each time point, protein content in the release medium was determined by the Lowry assay. Microcapsule sizes were determined by a Malvern Mastersizer 3000 and the D₅₀ value and Span are reported. Surface morphology was observed using a Scanning Electron Microscope (SEM).

Results: Microcapsules manufactured with EC dissolved in DCM had an EE of BSA of 40.0±5.2% and cumulative 3-hour leakage of 40.3±3.7%. When EC was dissolved in ethyl acetate (EA), EE increased to 61.7±5.8% with little change in leakage. Increasing the EC concentration from 4% to 6% markedly increased EE to 84.3±0.9% but did not affect leakage. Decreasing the phase:volume ratio of organic to aqueous phase from 5:10mL to 5:60mL decreased EE to 45.5±11.5% but reduced cumulative leakage to 18.7±11.6%. Reducing the homogenization speed in the formation of the s/o/w dispersion from 20000 RPM to 8000 RPM decreased EE to 36.2±7.1 % and markedly reduced cumulative leakage to 4.6±6.1%. Table 1 reports the average particle sizes and span of each manufactured batch. At each step in the formulation optimization process, microcapsule sizes increased while span values remained relatively constant. Figure 1 presents the protein leakage profile of microcapsules over a 3-hour incubation. As formulation conditions were optimized, both burst release and cumulative leakage progressively decreased. Most of the protein leakage occurred as burst release within 15 mins, with little additional leakage observed over 3 hours. Figure 2 shows an SEM image of microcapsules using the optimized formulation conditions. Microcapsules appear relatively round, and semi-porous.

Conclusion: Dissolving EC in EA instead of DCM increased the EE of BSA. EC has lower solubility in EA compared to DCM and EA has greater solubility in the external aqueous phase. This results in rapid extraction of EA into the external aqueous phase, precipitation of EC around the solid BSA core, and accounts for the increased entrapment of protein. The increase in EC concentration increased EE and microcapsule size, but surprisingly did not affect leakage. A higher concentration of EC presents more EC for entrapment of protein and likely accounts for the larger microcapsule size. Decreasing the organic to aqueous phase:volume ratio along with reduction of homogenization speed of the final s/o/w dispersion from 20000 RPM to 8000 RPM dramatically increased microcapsule size and markedly reduced leakage. The larger particle size and the consequent greater diffusional distance needed for protein leakage likely accounted for the reduced BSA leakage. The thicker EC coat of these larger particles likely contributed to the reduced BSA leakage. Our studies identified microcapsule formulation conditions that markedly reduced the leakage of BSA. Future experiments will replicate these results using the larger molecular weight PAL.

Acknowledgements: The authors acknowledge the St.Joseph's University Department of Pharmaceutical Sciences


Table 1 Particle sizes of microcapsule batches manufactured under varied formulation conditions

a mean ± S.D. of 40 measurements


Figure 1: Protein leakage from microcapsules manufactured under varied formulation conditions
Each value is mean ± S.D.; a n=2, b n=3, c n=6
In order to improve visual clarity, error bars have been omitted and average/mean leakage values are reported for each batch



Figure 2: SEM image of microcapsules manufactured using final formulation conditions