Immobilization of LAsparaginase Into Polylactide Coglycolide Nanospheres

3.1.1. Preparation of L-Asparaginase-Loaded Nanospheres

L-Asparaginase loaded nanospheres can be prepared by the water-in-oil-in water solvent evaporation technique (5).

1. Transfer 0.1 mL of 5% L-asparaginase aqueous solution to 0.5 mL ethyl acetate containing 50 mg PLGA placed a 10-mL glass tube (see Note 2).

2. Emulsify the mixture by sonication for 15 s (Branson Sonifier 250 at output 2 [15 W]; see Note 3).

3. Emulsify again by sonication the resulting emulsion (w/o) (same conditions as in step 2) in 2 mL of 1% PVA under cooling in an ice bath to form the second emulsion (wj/o/w2).

4. Dilute this double emulsion into 50 mL of 0.3% PVA and maintain the system under magnetic stirring for 15 min.

5. Transfer the suspension to a 50-mL round bottom flask and evaporate the solvent in a rotary evaporator (Rotavapor Buchi B-480) in order to accelerate the evaporation of the organic solvent (ethyl acetate).

6. Separate the nanoparticles with ultracentrifugation at 30,000g for 15 min (repeat the procedure three times in order to wash the nanoparticles).

7. Resuspend the nanoparticles in 20 mM phosphate buffer, pH 7.4.

3.1.2. Determination of Encapsulation Efficiency

The encapsulation efficiency (EE) indicates the percentage of the enzyme encapsulated with respect to the total amount used for the preparation of the nanoparticles.

1. Weight 20 mg of enzyme-loaded nanoparticles and shake them overnight in 6 mL of 0.1 M NaOH containing 2% sodium dodecyl sulfate (SDS). This allows for the polymer dilution and the release of the encapsulated enzyme.

2. Determine the amount of enzyme by the Lowry-Folin technique (37).

3.1.3. Determination of L-Asparaginase Activity

1. The biological activity of the enzyme is determined by the method described by Jayaram (38). The enzymatic activity of one unit L-asparaginase corresponds to formation of 1 mmol NAD/min at 37°C.

2. The enzyme-loaded nanoparticles are disintegrated by high-speed homogeniza-tion. Fifty-three milligrams of nanoparticles are suspended in 4 mL distilled water and homogenized with an Ultraturrax T-25 (IKA Labortechnik, Staufen, Germany) for 10 min in an ice bath.

3. The tip is rinsed with 1.5 mL distilled water and the homogenized suspension is extracted by end-over-end rotation for 1 h, with 4 mL 66 mM phosphate buffer (pH 7.4) containing Pluronic F68. The enzymatic activity of the supernatant is then assayed.

4. Prepare the reagent from 100 mL anhydrous glycerol, 50 mL 0.5 M Tris buffer, pH 8.45, 50 mg a-ketoglutarate-sodium, 180 U L-glutamic oxaloacetate transaminase, 110 U L-malic dehydrogenase, and 50 mg NADH adjusted to 500 mL with deionized water.

5. Immediately prior to use, 10 mM L-asparagine in 50 mM Tris-HCl buffer, pH 8.45, are added to the solution to a ratio 9:1 (v/v).

6. Incubate 2 mL reagent and 0.5 mL L-asparaginase-containing sample for exactly 1 h at 37°C.

7. Determine the decrease in absorption at 340 nm (Hewlett-Packard UV-VIS spec-trophotometer, model HP8452A).

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