• cyclosporine A
• drug delivery
• eye
• in situ gel
• nanomicelles

The aim of the present study was the development and characterization of new nanomicellar formulations containing cyclosporine A (CyA) for the treatment of ocular diseases, such as dry eye syndrome and chronic uveitis.
In the first part of this thesis, starting from a previous study, a promising nanomicellar formulation, prepared by taking advantage from the surfactants ability to self-assembling in an aqueous medium at 50°C, was evaluated. The selected formulation (Nano1HAB-CyA), containing 1.0%w/w of a mixture of surfactants (Vitamin E-TPGS and OPEE in a ratio of 2.25:1), 0.01% w/w of hyaluronic acid (HA), and CyA (0.1%w/w) was characterized and its ability to transport the drug across the scleral tissue was evaluated.
First of all, the technological characterization of Nano1HAB-CyA formulation was performed by measuring pH, osmolality and size distribution immediately after preparation and filtration through a 0.22 μm cellulose acetate membrane filter to assess: a) the ocular biocompatibility and b) the nanomicellar dimensions suitability to deliver the drug in the anterior and/or posterior segment of the eye. The pH, osmolality and size (10-12 nm) of the formulation were in the physiological range and suitable for ocular administration. The amount of drug loaded into the nanomicelles was detected by RP-HPLC analysis after dilution of the formulation with an organic solvent able to disrupt the nanostructure and promote the drug solubilization in the surrounding environment. Further characterizations were performed to evaluate the stability of the nanomicelles. In particular, the temperature at which nanomicelles dissociate and lose their structure was evaluated by a turbidimetric analysis detecting the cloud point in both unloaded and drug-loaded nanomicelles, and the time required to the micelles to self-regenerate at room temperature (regeneration time, RT) was measured. The thermal dissociation of the Nano1HAB-CyA occurred at enough high temperature (32°C) to provide stability of the formulation at room temperature and the RT resulted approximately 11 minutes. Short-term stability of the CyA-loaded nanomicellar solution was also evaluated at 4 and 20°C by measuring the percentage of drug recovered at predetermined time intervals (1,2,7,14, and 30 days) by HPLC analysis. The Nano1HAB–CyA resulted stable at 4 and 20°C up to 30 days with a recovery of CyA of about 95%.
Moreover, an ex vivo permeation study was carried out on excised scleral tissue of rabbit eyes by using horizontal diffusion cells to evaluate the ability of the nanomicelles to penetrate the scleral barrier.
To investigate Nano1HAB–CyA ability to interact with the external ocular tissues (cornea and sclera) fluorescein-labelled hyaluronic acid (HA-FITC), obtained by a conjugate addition reaction and characterized by a differential scanning calorimetry (DSC) analysis, was used to produce labelled nanomicelles (Nano1HABFITC-CyA). This study was performed with a microscopic imaging analysis on fixed tissues at the end of ex-vivo permeation study.
In the second part of the thesis, a new combination of drug-loaded non-ionic surfactant nanomicelles and in situ gelling system was developed to take advantage of: a) nanomicelles ability to incorporate insoluble drugs and carry it through biological barriers and b) improvement of the residence time of the formulation in the precorneal area by the formation of a viscoelastic gel due to the presence of specific amount of ions, starting from a sol-gel dispersion. Three different calcium ion concentrations were added to a previously
selected dispersion of 0.1%w/w gellan gum without any variation in the concentration of the
monovalent cations (Na+ and K*). The nanomicelles having the selected surfactants
composition (Vitamin E-TPGS and OPEE in a ratio of 2.25:1, 1%w/w) used in the previous
study, (without HA) were prepared by using the polymeric gellan gum dispersion as solvent.
The preparation of the formulation was completed with the centrifugation of the
surfactant/polymeric dispersion and leaving the gel to stabilize for 12 hours before any use.
The formulations (NanoG1-3) were characterized measuring pH, osmolality, size
distribution and clarity, to assess the presence of the nanomicelles into the gel system,
analyse their size in view of ocular administration, and their stability. The technological
parameters of the formulations resulted suitable for ocular administration and the
nanomicelles diameter was comparable to the original Nano1HAB–CyA (10-12 nm).
The rheological behaviour of the polymeric dispersions in presence or absence (reference)
of nanomicelles were measured at 32°C immediately after preparation to verify any possible
surfactant influence and to evaluate the increase in viscosity after dilution of the polymeric
dispersions with Artificial Tear Fluid (ATF, ratio 3:0.7).
On the basis of the obtained data, one of the three developed formulations (NanoG-2) was
selected to load CyA (NanoG-2CyA). The effective successful drug encapsulation was
checked by dynamic light scattering and the amount of drug encapsulated measured on
three different batches of NanoG-2CyA by HPLC analysis to verify the reproducibility of the
preparation method.
Finally, to assess the capacity of the studied formulations (Nano1HAB and NanoG-2) to
produce a polymeric film on the ocular surface, a fluorescent probe (Coumarin-6), was
encapsulated into nanomicelles and their residence time in vivo evaluated on New Zealand
rabbits under a wavelength excitation of 366nm.