• characterization
• cyclosporine A
• nanomicelles
• ocular drug delivery

The aim of the present study was to evaluate a combined strategy involving nanomicelles based on non ionic surfactants and hyaluronic acid (HA) as mucoadhesive polymer to improve ocular bioavailability of cyclosporine A, a poorly soluble drug.
From a previous study, a nanomicellar formulation, Nano1HAB-CyA, containing a total surfactant amount of 1% w/w (Vitamin E-TPGS and IGEPAL® in a ratio of 2.25:1), hyaluronic acid 0.01%w/w, and loaded with CyA (0.1%w/w) was selected by a statistical design of experiment (DoE). Nanomicelles were prepared by the melting method and using an isotonic phosphate buffer solution (PBS) as solvent.
First of all, the technological characterization of the prepared formulation was performed by measuring the pH, osmolarity and size distribution immediately after preparation and filtration through a 0.22 m cellulose acetate membrane filter to assess the ocular biocompatibility and the appropriate dimension to reach the anterior chamber of the eye, respectively. pH and osmolality of the formulation resulted suitable for a topical ocular administration and the particle diameter was in a size range of 10-18 nm, allowing to penetrate the ocular tissues. Afterwards, the drug content (entrapment) and the loading capacity of the formulation was determined by reverting the nanomicelles in organic solvent to extract the drug from the nanomicelles core and the CyA concentration was then analysed by RP-HPLC. The drug entrapment and loading efficiency was in a range of 75 – 85% and 10 – 12%, respectively, with 0.1%w/w final CyA concentration. Subsequently, short-term stability of nanomicelles at 4, 20 and 32°C (storage, room and ocular surface temperature) was also evaluated measuring the amount of drug remained in the formulation at predetermined time interval (1,2,7,14 and 30 days) by HPLC, and assessing drug possible degradation and checking nanomicelles structural preservation by dimensional and turbidity analysis. CyA was stable at 4°C and 20°C up to 30 days with no sign of degradation; the nanomicelles seem to maintain their structure over the time at 4°C and 20°C until at least 48 hours. At 32°C the nanomicelles lose their structure and regenerate in approximately 11 min when allowed to cool at room temperature. Moreover, a comparison between the formulation under study and the same formulation without HA (Nano1-CyA) was done to evaluate the HA role as a stabilizer in addition to its mucoadhesive properties. No statistical differences between the two nanomicellar formulations were observed in term of size (Nano1HAB-CyA vs Nano1-CyA ), even if Nano1-CyA showed a higher coefficient of variation (CV%) than Nano1HAB-CyA.
Secondly, in vitro release studies of CyA from the nanomicellar formulation Nano1HAB-CyA were performed at 32°C in water by using the dialysis method following the process for 30 hours. An ethanolic solution of the drug at the same concentration (0.1% w/w) was used as reference solution. Before the in vitro release experiments, the possible interactions between the polypeptide and the materials used in this test have been determined. On the basis of the obtained results, all materials used for the release studies were subjected to treatment with a Tween® 20 (0,05%) solution. The Nano1HAB-CyA formulation showed a diffusive release profile, releasing in 6 hours twice the amount of CyA (50 mcg) compared to the reference solution (25 mcg) suggesting a positive influence of the nanomicellar structure on the release of the drug. Moreover, the release of Nano1HAB-CyA was followed up to 30 hours showing that the formulation under study was able to release a total amount of drug of about 114 mcg.
Furthermore, an ATR-FITR analysis on raw materials (CyA, hyaluronic acid, vitamin E-TPGS and IGEPAL®) and on the freeze-dried Nano1HAB-CyA formulation was carried out to evaluate probable structural changes and interactions among the formulation components. In this study, it is to be highlighted that the Nano1HAB-CyA formulation ATR-IR spectrum exhibits CyA characteristic peak, which could demonstrate the successful loading of the drug in the nanoparticles, and a C=O vibration signal shift of Vit E-TPGS, suggesting an interaction between the drug and the surfactant.
Finally, the in vivo pharmacokinetics of the formulation under study in the tear fluid of New Zealand rabbits (animal model) was evaluated in order to investigate if the nanomicellar formulation produced a sustained release in the precorneal area, avoiding daily repeated administrations. As reference, a formulation with the same composition of an US commercial product not available on the European market (Restasis®), and loaded with the same amount of CyA of the nanomicelles formulation, was used. Pharmacokinetics studies have shown that Nano1HAB-CyA and the reference emulsion were comparable concerning the pharmacokinetics parameters: elimination rate constant (Ke), half-life (t1/2) and medium tear fluid retention time (MRT).