2.3. Liposomes preparation

Liposomes composed of DOPE, CHEMS e DSPE-PEG₂₀₀₀ and DSPE-PEG₂₀₀₀-DTPA (SpHL-DTPA-PTX) at a molar ratio of 5.7:3.8:0.45:0.05, respectively, were prepared using the standard lipid film hydration method [3]. In brief, pre-determined chloroform aliquots of the lipids and PTX (0.5 mg/ml) were transferred to round bottom flask and a lipid film was obtained by evaporating the organic solvent under reduced pressure. Next, to promote the complete ionization of CHEMS molecules, an aliquot of NaOH solution (0.456 M) was added at a 1:1 molar ratio CHEMS:NaOH. The film was hydrated with NaCl 0.9% (w/v), followed by vigorous shaking in vortex. The vesicles were sonicated (20% amplitude) in an ice bath for 5 min using a high-intensity ultrasonic processor (R2D091109 model; Unique^® Instruments, Indaiatuba, Brazil). The suspension were submitted to a centrifugation process (Sigma 4k-15 centrifuge, Sigma Laborzentrifugen GmbH, Osterode, Germany) at 3000 rpm at 4 °C for 10 min to eliminate non-entrapped PTX. Since the drug shown low water solubility, it precipitate and a drug pellet is formed. The supernatant suspension represent the final and purified formulation. For the folate-coated liposomes, 0.05% of DSPE-PEG₂₀₀₀-folate was added to the lipid film formation.