Tween® Preserves Enzyme Activity and Stability in PLGA Nanoparticles

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.

Keywords: Tween® stabilization; enzyme delivery; enzyme stabilization; nanomedicine; polymeric nanoparticles.

Figure 1

Activity Comparison. Activity of β-Glu mixed with Tween^® 20, 60, 80 and encapsulated into PLGA NPs. Statistical analysis was performed using the Student t test where * p < 0.05, ** p < 0.01 and N = 3 individual NP formulations for each type.

Figure 2

Isothermal titration calorimetry analysis of Tween^® 20 and β-glucosidase. (A) Raw titration data of Tween^® 20 (0.5 mM) titrated over β-Glu (37 µM) in PBS buffer, pH 6. (B) Binding isotherm of Tween^® 20 titration over β-Glu, obtained by integrating raw data for the protein titration. The blue dots represent the experimental data and the red curve represents the fit of the data using a single set of sites model.

Figure 3

SEM-FEG Nanoparticle images; (A) NPs PLGA:Solution0; (B) NPs PLGA:Solution1, (C) NPs PLGA:Solution2; (D) NPs PLGA:Solution3; (E) NPs PLGA:Solution4; (F) Solution Tween^® 20: β-Glu non-formulated. Note: all formulations contain 5 mg β-Glu.

Figure 4

Activity Comparison. Activity of Tween^® 20:β-Glu solutions at different molar ratios (Solutions 1-4) and encapsulated into PLGA NPs. Statistical analysis was performed using the Student t test where ** p < 0.01 and N = 3 individual NP formulations of each type.

Figure 5

Quantification of the release and enzyme activity of β-glucosidase from the nanoparticles. Grey: control Solution 0 NPs (no Tween^® 20). Black: Solution 4 NPs (with Tween^® 20). (A) %Release of β-Glu at pH 7.4, (B) %Release of β-Glu at pH 4.5, (C) Activity of released β-Glu at pH 7.4, (D) Activity of released β-Glu at pH 4.5. Analysis of N = 3 separate NP formulations.

Figure 6

Combination Stabilization. Measurement of the β-Glu Activity in NPs formulated with: BSA:Enzyme, Tween^® 20:enzyme, or BSA:Tween^® 20:enzyme solution.. Statistical analysis was performed using the Student t test where, ** p < 0.01 and measured for N = 3 individual NP formulations of each type.