Notes

1. Although different types of the polymer PLGA may be used, a higher encapsulation efficiency of the enzyme is obtained with the PLGA Resomer 503H (with free carboxyl-end groups).

2. The organic solvent used to dissolve the polymer, ethyl acetate, has been reported to maintain L-asparaginase activity. Moreover, the loss of enzyme activity has been shown to be dependent on the initial concentration of the enzyme. At higher concentrations the activity is better preserved.

3. The sonication energy used in this step is gentle enough to preserve enzyme activity.

4. Different lipids and methods can be used for the preparation of enzyme-containing liposomes. However, the encapsulation efficiency, enzymatic activity, and protein-to-lipid ratio are found to be highly dependent on the lipid composition used.

a. If dicetyl phosphate is used, a loss of enzymatic activity is observed.

b. The addition of stearylamine increases the encapsulation efficiency up to 100%, with only a mild loss of enzymatic activity.

c. Low-encapsulation efficiencies are observed with lipid mixtures of dimyristoyl-phosphatidylcholine (DMPC) with dioleoyl-phosphatidylcholine (DOPC).

5. The temperature of the aqueous phase must be higher than the main lamellar chain-melting phase transition temperature (Tm) of the phospholipid mixture.

6. The use of 0.3 M mannitol instead of deionized water in the rehydration step leads to an improved recovery, since it maintains a physiological intraliposomal osmolarity.

7. As an alternative of DRV liposomes, VET (extruded vesicles) liposomes can be made, although the encapsulation efficiency of the latter is much lower.

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