Liposomes characterizations

3.1 Particle size distribution and zeta potential measurement

The particle size, polydispersity index (PDI) and zeta potential of all lipid vesicles were determined by photon correlation spectroscopy (PCS, Zetasizer Nano series, Nano-ZS, Malvern Instruments Ltd., Worcestershire, UK). Before size and zeta potential measurements, different liposome samples were diluted 20-times with filtered water prepared by using polycarbonate membrane (Minisart^® syringe end filters 0.2 µm, Sartorious AG, Göttingen, Germany). Measurements were made at 25 ˚C with a fixed angle of 137°. Sizes quoted are the z-average mean for the liposomal hydrodynamic diameter.

The polydispersity index (PDI) was determined as a measure of homogeneity.

Small values of PDI ( < 0. 3) indicate a homogenous population, while PDI  0. 3

indicate high heterogeneity [ 102] . For the measurement of zeta potential, the electrophoretic mobility of the dispersion was determined by laser Doppler velocimetry. The zeta potential values were calculated by the Smoluchowski approximation of Henry’ s equation. Calculation of zeta potential ( mV) was done by the instrument from electrophoretic mobility.

3.2 Deformability study

The vesicle suspension was driven through a microporous filter by an external constant pressure of high pressure. The size of the vesicles was monitored by dynamic light scattering measurement before and after the filtration through a microporous filter with pore diameter 50 nm using a stainless steel pressure filter holder for 47 mm diameter filters, with 200 ml capacity barrel. Deformability value can be followed by the equation 2, which was developed by Bergh et al. (2001) [103].

D = J × (rv/rp)² Equation 2

Where J is the amount of vesicle suspension extruded (g) for 10 min, rv the size of vesicles after extrusion (nm) and rp the pore size of the barrier (nm). To measure J, the vesicles were extruded through a polycarbonate membrane (Nuclepore, Whatman Inc., MA, USA) with a pore diameter of 50 nm (rp), at a pressure of 2.5 bar. After 10 min of extrusion, the extrudate was weighed (J), and the average vesicle diameter after extrusion (rv) was measured by dynamic light scattering (DLS).

3.3 Percentage of Entrapment efficiency (%EE)

Free carboxyfluorescein was separated from entrapped CF using an ultracentrifugation method. The liposomal suspension (0.5 ml) was added to an Eppendorf tube (1 ml) and centrifuged at 4 °C at 14,000 g for 30 min. The non-entrapped CF in the filtrate was quantified the concentration by fluorescence spectrophotometry. Entrapment efficiency of CF was calculated indirectly from the amount of free CF, according to the following equation 3:

Entrapment efficiency (%) = (1 – Cf/ Ct)  100 Equation 3

with Cf as the amount of free CF and Ct as the total amount of CF.

3.4 Storage-stability studies

Samples were stored in tightly capped tubes at room temperature for 90 days.

Physical stability studies of liposome formulations were carried out to investigate the particle size and percentage of CF remained in liposomes during storage.

Samples from each were taken at time intervals of 0, 30, 60, and 90 days.

3.5 Skin permeability study

3.5.1 Ex vivo permeability of carboxyfluorescein

The skin samples were mounted in Franz diffusion cells under non-occlusive condition with the effective surface area of 0.5 cm² and a receptor volume of 5.7 ml. The dermal side of the skin was exposed to the receptor fluid and the SC remained in contact with the donor compartment left dry and open to the atmosphere. The temperature was maintained at 37 ± 0.1 °C, in order to maintain the skin surface at 32 °C, and the receptor solution was continuously stirred at 700 rpm with a small bar magnet placed inside the cell. Five µl of each liposomal formulation were applied onto the skin’s surface and uniformly spread. Samples (1 ml) were taken from the receptor fluid at 3, 6, 12 and 24 h after application. At each point, the receptor cell content was replaced by 1 ml of fresh solution. The samples were diluted to proper concentration before measuring the amount of CF by spectrofluorometer. Three replicates were used for the study.

3.5.2 Ex vivo and in vivo tape-stripping of the stratum corneum

The ex vivo and in vivo skin penetration studies were carried out by the tape stripping technique in a modified form after Coderch et al. (1996) [104]. Before this ex vivo measurement, the excised human skin was stretched and mounted with pins on cork discs and covered with aluminium mask with a central hole of 8 mm in diameter. In case of in vivo experiments, two volunteers were tested on their own both (left and right) sides of forearm and were applied the tested formulations with a circle-shaped of 8 mm. The applied volume of liposomal suspension for each experiment was of 5 µl.

After 2 hours, the SC was successively removed by stripping with an adhesive tape (CristallKlarTesa^®, Beiersdorf AG, Hamburg, Germany). Each tape was put onto the skin and a weight of 2 kg was placed on the tape for 10 s. Afterward the tape was rapidly removed with forceps and transferred into a glass vial of suitable size. Fifteen stripping procedures were performed consecutively. One ml of ethanol/ PBS pH7.4 (1:1, v/v) was added to each vial to extract the CF from the strip. Each vial was vortexed for 2 min and then sonicated for 2 min, after which they were kept overnight at room temperature under light protection. The strips were analysed for CF content by fluorescence spectrophotometry. The results were expressed as percentage of the applied CF dose penetrated into the skin.

3.5.3 Fluorescence assay of carboxyfluorescein

The concentration of CF was determined by fluorescence spectroscopy.

Fluorescence detection was performed at an excitation of 490 nm and an emission of 520 nm in the case of CF. The method was validated for linearity, accuracy and precision. The linear range during the measurements for CF was from 0.1 to 4.0 µM (r² = 0.99). The software used was Optima, version 2.10, BMG Lab Tech.

3.6 Data analysis

All reported data are mean ± S.D. Statistical significance was interpreted by Student’s t-test using Graph Pad Prism^® Ver.6 software and considered to be significant at P < 0.05.

References

1 Muller RH, Keck CM: Challenges and solutions for the delivery of biotech drugs--a review of drug nanocrystal technology and lipid nanoparticles. Journal of biotechnology 2004;113:151-170.

2 Šentjurc M, Vrhovnik K, Kristl J: Liposomes as a topical delivery system: The role of size on transport studied by the epr imaging method. Journal of Controlled Release 1999;59:87-97.

3 van den Bergh BA, Wertz PW, Junginger HE, Bouwstra JA: Elasticity of vesicles assessed by electron spin resonance, electron microscopy and extrusion measurements. Int J Pharm 2001;217:13-24.

4 Coderch L, Fonollosa J, De Pera M, Estelrich J, De La Maza A, Parra JL: Influence of cholesterol on liposome fluidity by epr: Relationship with percutaneous absorption. Journal of Controlled Release 2000;68:85-95.