Enzyme Stability in Nanoparticle Preparations Part 1: Bovine Serum Albumin Improves Enzyme Function

Abstract

Enzymes have gained attention for their role in numerous disease states, calling for research for their efficient delivery. Loading enzymes into polymeric nanoparticles to improve biodistribution, stability, and targeting in vivo has led the field with promising results, but these enzymes still suffer from a degradation effect during the formulation process that leads to lower kinetics and specific activity leading to a loss of therapeutic potential. Stabilizers, such as bovine serum albumin (BSA), can be beneficial, but the knowledge and understanding of their interaction with enzymes are not fully elucidated. To this end, the interaction of BSA with a model enzyme B-Glu, part of the hydrolase class and linked to Gaucher disease, was analyzed. To quantify the natural interaction of beta-glucosidase (B-Glu,) and BSA in solution, isothermal titration calorimetry (ITC) analysis was performed. Afterwards, polymeric nanoparticles encapsulating these complexes were fully characterized, and the encapsulation efficiency, activity of the encapsulated enzyme, and release kinetics of the enzyme were compared. ITC results showed that a natural binding of 1:1 was seen between B-Glu and BSA. Complex concentrations did not affect nanoparticle characteristics which maintained a size between 250 and 350 nm, but increased loading capacity (from 6% to 30%), enzyme activity, and extended-release kinetics (from less than one day to six days) were observed for particles containing higher B-Glu:BSA ratios. These results highlight the importance of understanding enzyme:stabilizer interactions in various nanoparticle systems to improve not only enzyme activity but also biodistribution and release kinetics for improved therapeutic effects. These results will be critical to fully characterize and compare the effect of stabilizers, such as BSA with other, more relevant therapeutic enzymes for central nervous system (CNS) disease treatments.

Nerve Growth Factor Biodelivery: A Limiting Step in Moving Toward Extensive Clinical Application?

“…The greatest drawback concerns the requirements of nanoproduction (such as stirring, heating, sonication, organic solvents, etc. ), which severely impact the stability and maintenance of the biological drug's pharmacological activity (Duskey et al, 2020). Some of these requirements relate to the polymer/lipid used in the formulation, and should be carefully designed and always adapted to the "stability features" of the embedded drug.…”

Tween® Preserves Enzyme Activity and Stability in PLGA Nanoparticles

“…One possible approach to prevent the loss of enzyme activity in solution or during formulative steps lies in the addition of stabilizers such as sugars [50][51][52][53], emulsifiers [54,55], and serum albumins [56][57][58]. Specific examples include: (1) Yun et al used rabbit serum albumin in PLGA NPs encapsulating superoxide dismutase showing acceptable enzyme activity in vitro and in vivo [46]; (2) Osman et al who tested PLGA NPs encapsulating DNaseI with the addition of hydroxypropyl-β-cyclodextrin (shown in other literature as an enzyme stabilizer [59]) [60]; (3) Atkins et al that covalently linked the surfactant alkyl-glycolic acid ethoxylate to the surface of hen egg-white lysozyme to form a single enzyme NP with a surfactant shell which led to an up to 7-fold increase of enzyme activity in solution [61]; however, while offering protective features, these stabilizers can impact the physicochemical characteristics (size, morphology, and zeta potential), encapsulation efficiency of the pharmaceutic, and the pharmaceutical properties (release kinetics, biodistribution, cell uptake, etc.)…”

“…Specific examples include: (1) Yun et al used rabbit serum albumin in PLGA NPs encapsulating superoxide dismutase showing acceptable enzyme activity in vitro and in vivo [46]; (2) Osman et al who tested PLGA NPs encapsulating DNaseI with the addition of hydroxypropyl-β-cyclodextrin (shown in other literature as an enzyme stabilizer [59]) [60]; (3) Atkins et al that covalently linked the surfactant alkyl-glycolic acid ethoxylate to the surface of hen egg-white lysozyme to form a single enzyme NP with a surfactant shell which led to an up to 7-fold increase of enzyme activity in solution [61]; however, while offering protective features, these stabilizers can impact the physicochemical characteristics (size, morphology, and zeta potential), encapsulation efficiency of the pharmaceutic, and the pharmaceutical properties (release kinetics, biodistribution, cell uptake, etc.) of the NPs [62,63]; 4) Another work used bovine serum albumin (BSA) and its ability to stabilize β-Glu during the formulation process [56]. BSA significantly enhanced loading capacity from 6% to 30%, and was shown to quantifiably directly bind to the enzyme (studied by isothermal titration calorimetry (ITC)) forming complexes in solution.…”

“…This translated to an equal stabilization of the enzyme activity due to a BSA:β-Glu enzyme complex and a higher loading content of enzyme in the NPs. Moreover, the direct complexation of the two affected release kinetics from the PLGA matrix, shifting the release from an immediate burst release over 3 h (enzyme alone) to an extended release over 6 days [56].…”

“…This work focused on elucidating if Tween ® can have a stabilizing effect on a model enzyme during the formulation process into safe and biodegradable NPs. To this end, we combined β-Glu, the same model enzyme linked to the LSD Gaucher [65] used in our previous work with the stabilizer BSA [56], with Tween ® stabilizers and loaded them into PLGA NPs. It was determined if the type and amount of Tween ® (Tween ® 20, 60 and 80) in the enzyme solution affects the enzyme stability during NP formation.…”