Nano and microparticulate chitosan-based systems for antiviral topical delivery.

Acyclovir (ACV) is one of the drugs of choice for the treatment of epidermal, ocular or systemic herpetic infections. Nevertheless, its trans-mucosal limited absorption and the scarce contact time of the formulation with the mucosal surface - especially in the ocular mucosa - constitute a big limitation of the antiviral efficiency. The most effective way to solve these problems is to increase the quantity and the residence time of the drug over the ocular surface. In order to cope with all these requirements, micro-particles (MPs) and nano-particles (NPs) containing ACV have been developed using cross-linked chitosan with tripolyphosphate (TPP) due to the biocompatibility, bio-adhesion ability and the potential power as penetration enhancer of this polymer. Particles were characterized by Fourier-transformed infrared (FTIR) spectroscopy, X-ray diffraction, SEM, Zeta potential and particle size. Encapsulation efficiency and release profiles in flow through diffusion cells were also determined. Besides the Slug Mucosal Irritation (SMI) assay has been applied as an alternative to the Draize test to predict the mucosal irritation of the selected formulation. FTIR and X-ray results suggested an electrostatic interaction ACV-Chitosan that made ACV be molecularly dispersed within the polymer matrix. Encapsulation efficiency was 75% for MP and 16% for NP. Release profiles in flow through diffusion cells were also determined. From the diffusion profiles, it was found that the amounts of ACV effectively diffused in 24h were 30, 430 and 80 µg for the ACV solution, MP and NP respectively. SMI results showed that chitosan-based particles induced moderate irritation and mild tissue damage, what supposes that ACV-MP constitute a promising alternative for further development of an antiviral formulation.

Validation protocol of an automated in-line flow-through diffusion equipment for in vitro permeation studies.

Transdermal drug delivery experiments are often tedious and time consuming in terms of sampling, labor, etc. In this way, the automation of such experiments has increased in the last few years. A protocol suitable to validate an automated diffusion equipment with seven in-line flow-through cells is described. The proposed protocol was divided into two parts. First, validation of each component which makes up the whole equipment, including the study of the statistical variability of the internal volumes between the cells, the temperature into the different chambers, the time and sample volumes, etc. In the second part, a series of permeation studies were carried out comparing the performance of the system against a classical Franz-type diffusion cell. Ketoprofen was used as a model drug. It was proved the low variability of the replicates obtained with the automated flow-through diffusion cells. The best work conditions as flow rate into the receptor chamber, temperature, etc., as well as the best mathematical approach for the diffusion data, were determined. The advantages in terms of time saved and easiness of validation of the flow-through cell design in comparison to the Franz-type cell were evidenced.

Improved RPLC determination of acyclovir using hexylamine as silanol masking agent.

The aim of the present work is to improve the sensitivity in the RPLC determination of acyclovir [9-(2-hydroxy ethoxymethyl) guanine] (ACV) and guanine, the major impurity of the drug synthesis and one of the compounds found in the chemical degradation process of ACV. The method was applied to the quantification of drug in liposomal formulations. The most important problem for RPLC analysis of both compounds are their high pKa values, mainly guanine, and the interaction with reactive silanol groups in the stationary phase. In order to avoid these problems there are four basic strategies: (i) ionic pair reagents, (ii) deactivated silica columns, (iii) polymeric based columns and (iv) silanol masking agents. A validation protocol was followed to develop the analytical method, using a Spherisorb ODS (250 x 4.6 mm i.d.) analytical column, with a mobile phase of 95% aqueous phosphate buffer (pH 3.0) and 5% HPLC methanol pumped isocratically at 1.3 ml/min(-1), with ultraviolet detection at 254 nm. The results showed a high reproducibility in retention time value, with R.S.D. of 2.37% for ACV and 0.32% for guanine. The lowest concentration levels assayed, 0.15 microg/ml(-1) for guanine and 1 microg/ml(-1) for ACV, showed good R.S.D. in the quantification parameter (peak area) 11.0% (guanine) and 9.64% (ACV)

Characterization of 5-fluorouracil loaded liposomes prepared by reverse-phase evaporation or freezing-thawing extrusion methods: study of drug release.

Entrapment of the anti-tumoral drug 5-fluorouracil (5-FU) in unilamellar liposomes prepared by freeze-thawing extrusion technique (FATVET) and the reverse-phase evaporation method (REV) from natural (bovine brain) sphingomyelin (SM) and synthetic distearoylphosphatidylcholine (DSPC) phospholipids was studied. Reverse-phase evaporation vesicles obtained from DSPC sized through polycarbonate membranes of 0.2 micron pore size were found to entrap roughly double amounts of drug than did extruded liposomes (0.1 micron pore size); however, s-REV in these preparations were more heterogenous in vesicle size than FATVET. The rate of in vitro drug release from the liposomes was found to be dependent of the bilayer composition and the method used to prepare the vesicles. The permeability coefficient P obtained was approx. 10(-11) m/s. The results suggest that 5-FU release is kinetically controlled by an interfacial process seemingly dependent on the surface activity of the drug. Also, the physical state of the bilayer determines the retention capacity of the vesicles. Thus, liposomes consisting of distearoylphosphatidylcholine whose acyl chains were in a gel state at the working temperature (37 degrees C) retained 70% of encapsulated 5-FU after 1 h, whereas liposomes composed of natural bovine brain sphingomyelin retained only 15% over the same period.

Analysis of the particle size distribution and internal volume of liposomal preparations.

In this work we studied the particle size distribution of three liposomal preparations by quasi-elastic light scattering spectroscopy. Sized unilamellar vesicles of small diameter (s-SUV) were prepared by ultrasonication and subsequent centrifugation followed by extrusion through polycarbonate membranes of 0.2-micron pore size. Large unilamellar vesicles were obtained by reversed-phase evaporation (REV) and extrusion through polycarbonate filters with or without preliminary freezing-thawing cycles (VETI and VETII, respectively). After preparation, REV were sized to small diameter REV (s-REV) by extrusion through 0.4- and 0.2-micron polycarbonate membranes. According to the results, the s-SUV preparations were made up of two subpopulations, the major of which consisted of vesicles that were 26 nm in mean diameter and accounted for 95% of the overall s-SUV population. The s-REV dispersions always resolved into two populations centered at 120 and 380 nm, the relative proportions of which depended on the pore size of the filters used. VET structures were composed of a single population of vesicles that were approximately 100 nm in mean diameter. Cholesterol inclusion into the bilayer composition extended the distribution without altering its mean value. On the other hand, the internal volumes calculated from mean diameters or assuming a Gaussian distribution were inconsistent with experimental data obtained by usual techniques.

Comparison of particle size and encapsulation parameters of three liposomal preparations.

Liposomes can be produced by using various techniques such as mechanical agitation, ultrasonication, ether injection, detergent dialysis, reversed-phase evaporation, extrusion through polycarbonate filters of appropriate pore size, etc. Each procedure provides liposomes with their own perculiar features so the choice of a given one to obtain suitable vesicles for use as drug delivery systems should be based on careful experimentation. In this work we tested three liposome preparation methods that provide unilamellar structures with high yields. In fact, unilamellar vesicles of small diameters (SUV) were prepared by ultrasonication and large unilamellar vesicles (LUV) were obtained by (a) reversed-phase evaporation (REV) and (b) extrusion through polycarbonate filters--with or without preliminary freezing-thawing (VETI and VETII, respectively). According to the results obtained, subsequent filtration of the SUV and REV liposomal preparations results in dramatically decreased average diameters and polydispersity indices, as well as in marked changes in their encapsulation parameters and in lipid losses. Among extruded liposomes, those obtained with preliminary freezing-thawing (VETII) resulted in a higher lipid recovery, even though their average hydrodynamic diameter and polydispersity index as measured by quasi-elastic light-scattering spectroscopy (QELSS) were quite similar to those of the VETI counterparts.