Abstract
Enzymes, as natural and potentially long-term treatment options, have become one of the most sought-after pharmaceutical molecules to be delivered with nanoparticles (NPs); however, their instability during formulation often leads to underwhelming results. Various molecules, including the Tween® polysorbate series, have demonstrated enzyme activity protection but are often used uncontrolled without optimization. Here, poly(lactic-co-glycolic) acid (PLGA) NPs loaded with β-glucosidase (β-Glu) solutions containing Tween® 20, 60, or 80 were compared. Mixing the enzyme with Tween® pre-formulation had no effect on particle size or physical characteristics, but increased the amount of enzyme loaded. More importantly, NPs made with Tween® 20:enzyme solutions maintained significantly higher enzyme activity. Therefore, Tween® 20:enzyme solutions ranging from 60:1 to 2419:1 mol:mol were further analyzed. Isothermal titration calorimetry analysis demonstrated low affinity and unquantifiable binding between Tween® 20 and β-Glu. Incorporating these solutions in NPs showed no effect on size, zeta potential, or morphology. The amount of enzyme and Tween® 20 in the NPs was constant for all samples, but a trend towards higher activity with higher molar rapports of Tween® 20:β-Glu was observed. Finally, a burst release from NPs in the first hour with Tween®:β-Glu solutions was the same as free enzyme, but the enzyme remained active longer in solution. These results highlight the importance of stabilizers during NP formulation and how optimizing their use to stabilize an enzyme can help researchers design more efficient and effective enzyme loaded NPs.
Highlights
Enzyme replacement therapy (ERT), which is based on the periodic administration of specific enzymes, is currently the most suitable therapy for difficult-to-treat diseases caused by a deficit in enzymes, such as lysosomal storage disorders (LSDs) [6,7,8,9]
The aim was to determine which variant poses the highest potential for formulating and preserving enzyme activity in poly(lactic-co-glycolic) acid (PLGA) NPs. β-Glu mixed in solution with each different Tween® in the aqueous phase before formulation with the double emulsion method did not produce any difference in size or ζ-pot of the
NPs compared to empty PLGA NPs or those containing free β-Glu, resulting unanimously with monodisperse particles ranging from 177 to 208 nm with low polydispersity index (PDI) < 0.2 and a negative surface charge of the NPs ranging from −16 to −23 mV (Table 2)
Summary
Therapeutic application of enzymes is still hampered by several obstacles that limit their clinical benefits because these macromolecules frequently fail in crossing biological barriers such as the blood brain barrier (BBB), and do not reach therapeutic concentrations in the target tissues [1,4,5,10,11,12,13]. They show immunogenicity, short half-lives in blood
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