Polymeric micelles in drug delivery: An insight of the techniques for their characterization and assessment in biorelevant conditions.

Polymeric micelles, i.e. aggregation colloids formed in solution by self-assembling of amphiphilic polymers, represent an innovative tool to overcome several issues related to drug administration, from the low water-solubility to the poor drug permeability across biological barriers. With respect to other nanocarriers, polymeric micelles generally display smaller size, easier preparation and sterilization processes, and good solubilization properties, unfortunately associated with a lower stability in biological fluids and a more complicated characterization. Particularly challenging is the study of their interaction with the biological environment, essential to predict the real in vivo behavior after administration. In this review, after a general presentation on micelles features and properties, different characterization techniques are discussed, from the ones used for the determination of micelles basic characteristics (critical micellar concentration, size, surface charge, morphology) to the more complex approaches used to figure out micelles kinetic stability, drug release and behavior in the presence of biological substrates (fluids, cells and tissues). The techniques presented (such as dynamic light scattering, AFM, cryo-TEM, X-ray scattering, FRET, symmetrical flow field-flow fractionation (AF4) and density ultracentrifugation), each one with their own advantages and limitations, can be combined to achieve a deeper comprehension of polymeric micelles in vivo behavior. The set-up and validation of adequate methods for micelles description represent the essential starting point for their development and clinical success.

Cell penetrating peptides in ocular drug delivery: State of the art.

Despite the increasing number of effective therapeutics for eye diseases, their treatment is still challenging due to the presence of effective barriers protecting eye tissues. Cell Penetrating Peptides (CPPs), synthetic and natural short amino acid sequences able to cross cellular membrane thanks to a transduction domain, have been proposed as possible enhancing strategies for ophthalmic delivery. In this review, a general description of CPPs classes, design approaches and proposed cellular uptake mechanisms will be provided to the reader as an introduction to ocular CPPs application, together with an overview of the main problems related to ocular administration. The results obtained with CPPs for the treatment of anterior and posterior segment eye diseases will be then introduced, with a focus on non-invasive or minimally invasive administration, shifting from CPPs capability to obtain intracellular delivery to their ability to cross biological barriers. The problems related to in vitro, ex vivo and in vivo models used to investigate CPPs mediated ocular delivery will be also addressed together with potential ocular toxicity issues.

Microemulsion containing triamcinolone acetonide for buccal administration.

The aim of the present work was to investigate the potential of microemulsions for the buccal administration of triamcinolone acetonide. Microemulsions were developed by the construction of pseudoternary phase diagrams, using the aqueous titration method. Among all microemulsions prepared and tested for stability, three were selected and submitted to characterization and in vitro permeation/retention experiments, using pig esophageal epithelium, an accepted model of the buccal mucosa. Furthermore, one microemulsion was added of excipients (stearylamine, CTAB and chitosan) able to alter the charge of droplets. The results obtained show that the permeation of triamcinolone acetonide across pig esophageal epithelium was not influenced by the droplet size nor by the composition, but only by the presence of chitosan, polysaccharide able to increase the transport across mono and stratified epithelia. The determination of the permeation parameters allowed us to show that chitosan acts on the diffusion parameter across the tissue and not on the partitioning parameter; for the same reason the tissue retention of triamcinolone acetonide was not modified. Triamcinolone flux (2.6 µg cm-2 h-1) was too low to make systemic administration feasible (dose required 2.5 to 60 mg/day). The amount of triamcinolone acetonide recovered in the mucosa after only 10 min. of microemulsion application was much higher than after overnight application of the commercial paste Omicilon® A. This suggests that triamcinolone acetonide microemulsions can be an interesting alternative to the commercial formulation to treat diseases of the buccal mucosa. Owing to the fast uptake by the tissue, the formulation can be used as a mouthwash.

In vitro permeability of a model protein across ocular tissues and effect of iontophoresis on the transscleral delivery.

The aim of this work was to study the penetration of cytochrome c, a positively charged model protein (MW 12.4 kDa, charge at pH 8.2: +9), across different ocular tissues, and to evaluate the potential of iontophoresis to enhance and control the transscleral transport. The passive transport of cytochrome c across the sclera and across the bilayer choroid-Bruch's membrane was evaluated using Franz diffusion cells and porcine tissues. The affinity of cytochrome c for melanin was measured by means of in vitro binding experiments. The iontophoretic (anodal) permeation was studied as a function of donor concentration (from 5 to 70 mg/ml) and current intensity (from 0.9 to 3.5 mA; density from 1.5 to 5.8 mA/cm(2)), and the contribution of electroosmosis on cytochrome c transport was evaluated by using a high molecular weight fluorescent dextran (FD-150, 149 kDa) as neutral marker. Finally, the possibility of tuning cytochrome c permeation rate was investigated on a 70 mg/ml cytochrome c solution, by alternating passive permeation and iontophoresis at different intensities. Cytochrome c permeated the sclera with a passive permeability coefficient of about 2.5 × 10(-6)cm/s, comparable to molecules of similar molecular radius. The choroid-Bruch's layer was an important barrier to penetration, since its presence reduced 5-7 times the amount permeated after 5h, also because of the presence of melanin that binds cytochrome. Iontophoresis (2.9 mA/cm(2)) enhanced cytochrome c penetration across the sclera at all the concentrations tested, increasing about ten times the amount permeated after 2h. The effect was proportional to current density: the enhancement factor (measured on a 10mg/ml solution), resulted 6.0 ± 4.3 (i=0.9 mA; density=1.5 mA/cm(2)), 10.6 ± 4.1 (i=1.75 mA; density=2.9 mA/cm(2)), 33.2 ± 8.3 (i=1.75 mA; density=5.8 mA/cm(2)). Iontophoretic (density=2.9 mA/cm(2)) experiments performed with FD-150, an electroosmotic flow (EO) marker, demonstrated that cytochrome c, at concentration higher that 1mg/ml, dramatically reduced the EO flow and that, despite the high MW, the main mechanism for cytochrome c iontophoretic permeation is electrorepulsion. Finally, by alternating in the same experiment passive permeation and iontophoresis at different current intensities, a precise modulation of cytochrome c release was obtained, thus indicating the possibility of tuning the release as a function of specific therapeutic needs.

In vitro trans-scleral iontophoresis of methylprednisolone hemisuccinate with short application time and high drug concentration.

Trans-scleral iontophoresis, i.e. the application of small electric current to enhance drug transport across sclera is an option for non-invasive delivery of corticosteroids to the posterior segment of the eye. In this paper, in vitro trans-scleral iontophoresis of methylprednisolone hemisuccinate was investigated using concentrated drug solutions and short application times to mimic the iontophoretic conditions of in vivo studies. The drug at the donor concentration of 45 mg/ml was delivered through isolated porcine sclera under passive and iontophoretic conditions (cathodal, 2.4 mA) for 2-15 min. In a second set of experiments, the drug was delivered for 5 min at current intensities of 0.9-7.2 mA. After donor removal, drug release was followed up to 24 h. The exposure of concentrated solutions to sclera for 2-15 min under passive conditions caused a notable accumulation of drug up to 0.8 mg/cm², the release of which was successively followed for 24 h. In cathodal iontophoresis, the amount of accumulated drug increased proportionally to the charge between 0.3 and 1.44 Coulomb. When the charge was increased to 2.16 Coulomb by increasing the application time or current intensity, no further enhancement was recorded. This behaviour can be ascribed to substantial drug adsorption on the scleral tissue, as demonstrated through streaming potential studies, with the consequent increase of the electroosmotic flow that opposes drug transport. The set up suggested here could help in defining the optimal conditions for in vivo studies with animal models and reducing the number of in vivo experiments.