Preparation of Drug-Loaded Polymeric Nanoparticles and Evaluation of the Antioxidant Activity Against Lipid
3.9. In vitro Lipid Peroxidation
1. The lipid peroxidation is induced by ascorbyl radical (iron–
ascorbate) (30).
2. Buffer is placed in test tubes: 20L (v1) of 1 M Tris–HCl, pH 7.4, is added to reach 0.1 M Tris–HCl, pH 7.4, in the final volume of 200L (vtotalof the reactional medium).
3. Distilled water (v2) is added to the test tubes after its cal-culation (see Note 10).
4. The volume (v3) of drug solution (see Note 11) or melatonin-loaded formulations (nanocapsule or nanosphere suspensions) corresponding to the antioxidant dose of 0.2 mM (18.6 L) or 0.4 mM (37.2 L) is added in the test tubes. The controls for the experiments correspond to the respective formulations prepared in the absence of the drug (unloaded formulations) (see Note 12).
5. In the test tubes, 10L (v4) of 500M FeSO4solution is added to reach 25M FeSO4.
6. In the test tubes, 10 L (v5) of 10 mM ascorbic acid is added to reach 500M ascorbic acid.
7. The membrane substrates, microsomes or liposomes, are added (v6) to the reactional medium in the test tubes at 1 mg/mL microsomal protein/mL (25L) or lipids (lipo-somes) at 12.5 mg/mL (53.6L) (see Note 13).
8. The samples are then incubated for 30 min at 37◦C.
9. After the incubation period, 200L of 12% trichloroacetic acid and 200L of 0.73% thiobarbituric acid are added to stop the reaction.
10. The mixture is then maintained at 100◦C for 30 min (see Note 14).
11. The mixture is cooled and centrifuged (10,000×g for 5 min) (see Note 15).
12. The supernatants are monitored at 535 nm.
13. The amount of lipid peroxidation is determined using the molar extinction coefficient of 1.56 × 105/M/cm and expressed as thiobarbituric acid reactive substances
118 Pohlmann et al.
LIPOSOMES
0 10 20 30 40 50 60 70
SOL NC NS
Protection of melatonin against lipid peroxidation (%)
0.2 mM 0.4 mM
*
#*
* *
*
*
MICROSOMES
0 10 20 30 40 50 60 70
Protection of melatonin against lipid peroxidation (%)
0.2 mM 0.4 mM
*
*
#*
#SOL NC NS
#
Fig. 7.3. Effect of melatonin encapsulation on lipid peroxidation induced by the ascorbyl-free radical (SOL= melatonin solution containing 1% ethanol; NC = Eudragit S100R nanocapsule suspension; NS= Eudragit S100R nanosphere suspension). This protec-tion is relative to the formulaprotec-tions prepared without melatonin (controls).∗Statistical differences between each formulation and its respective control established usingt test (p< 0.05); #statistical differences between the solution (0.4 mM of melatonin) and the formulations established usingt test (p< 0.05). (Data published in 19).
(TBARS). The calculated protection against lipid peroxida-tion is relative to the formulaperoxida-tions prepared without mela-tonin (controls). An example of the results is shown in Fig. 7.3.
4. Notes
1. The solution is prepared under exhaustion (fumed hood) and it is filtered (pore size: 0.22 or 0.45m) before use.
2. The solution is prepared just before the experiment.
3. In the case of the antioxidant drug insolubility in water-miscible solvents (acetone or ethanol), this method can-not be employed. Other methods can be used, as the emulsification–diffusion, which employs ethyl acetate, ben-zyl alcohol, or propylene carbonate as solvents (3, 22).
4. The melatonin-loaded nanoparticle suspensions must be stored to protect from the light. In this way, evaporation is carried out in an amber flask and the suspension is stored in an amber recipient.
5. The usual dilution is 500-fold, but changing the qualitative composition of the suspension a study of dilution is nec-essary. In general, the dilution ranges between 500- and 2,000-folds.
Preparation of Drug-Loaded Polymeric Nanoparticles 119
6. The diameters usually observed for nanoparticles prepared using preformed polymers range between 100 and 500 nm (3, 4). Polydispersion indexes lower than 0.2 indicate homogeneous systems presenting a satisfactory narrow par-ticle distribution.
7. Generally, zeta potential values higher than 30 mV (positive or negative) indicate relatively stable nanoparticle suspen-sions due to the repulsion between particles reducing their aggregation (4).
8. Generally, the suspension is diluted 10-fold in water, but depending on the composition a study should be carried out to optimize the dilution.
9. The melatonin calibration curve is prepared between 2.5 and 17.5 g/mL in acetonitrile. The drug analytical method is validated following the ICH or the USP 26 (31, 32).
10. Volume v2 is calculated by the following: v2 = 200 – v1 – v3– v4– v5– v6, where v1, v3, v4, v5, and v6correspond to the volumes of the solution: Tris–HCl, antioxidant, FeSO4, ascorbic acid, and substrates (liposomes or microsomes), respectively.
11. The 0.5 mg/mL melatonin solution is prepared using dis-tilled water containing 1% ethanol (v/v).
12. Each test tube must have a respective control prepared in the same condition omitting the drug.
13. Volume v6 can change depending on the total volume of the resulting liposome suspension or microsomal protein content.
14. Using a hot syringe needle, a small orifice in the test tubes must be performed for pressure compensation.
15. The supernatants must be transparent. The use of an ultra-centrifuge or a solvent can be necessary.
Acknowledgments
The authors acknowledge the financial support from Conselho Nacional de Desenvolvimento Cient´ıfico e Tecnol ´ogico (CNPq), Fundac¸˜ao de Amparo `a Pesquisa do Estado do Rio Grande do Sul (FAPERGS), Fundac¸˜ao de Amparo `a Pesquisa do Estado de Santa Catarina (FAPESC), Coordenac¸˜ao de Aperfeic¸oamento de Pessoal de N´ıvel Superior (CAPES), Rede Nanocosm´eticos CNPq/MCT-Brazil.
120 Pohlmann et al.
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