Nanosized systems for efficient delivery of antitumoral and anti-inflammatory drugs

The purpose of the present PhD thesis was to study two different nanosized systems, liposomes and nanosuspensions. Liposomes were develop with the aim to improve the Triiodothyronine (T3) delivery to hepatic cancer cells and nanosuspensions were proposed to enhance dermal bioavailability of Diclofenac acid, a potent nonsteroidal anti-inflammatory drug (NSAID) with a very low aqueous solubility. T3, is a thyroid hormone normally synthesized and secreted by the thyroid gland and through interaction with its nuclear receptors (TRs) plays an essential role in morphogenesis and differentiation. However, there is increasing evidence for its role in hepatocellular carcinoma (HCC) suppression. Three different liposome systems, such as, conventional, Stealth (PEGylated) and Lf-modified-Stealth liposomes were successfully prepared, by the film hydration method, and characterized. Liposomes showed a mean diameter ranging between 86 and 126 nm and a PI lower than 0.28, as shown by PCS measurements. Liposomes cell interactions and cellular uptake were evaluated in three different HCC target cells (FaO, HepG2 and SKHep) by confocal microscopy using liposomes labeled with the lipophilic marker Rho-PE and loaded with the hydrophilic probe CF. Finally, in vitro cytotoxicity studies were carried out by using MTT assay to evaluate the toxicity of the liposome delivery system and to test the effect of T3 when incorporated into liposomes. These studies provides the great potential of liposomes as suitable carrier for T3 delivery to hepatoma cells. Internalization studies performed using Lf-modified-liposomes labeled with the lipophilic marker Rho-PE and loaded with the hydrophilic probe CF clearly demonstrated the effective internalization of both hydrophilic and lypophilic markers. Lf-liposome might markedly enhance the specific cell binding and cellular uptake in hepatoma cells due to the mediating of Lf that could bind to multiple receptors on cell surface such as ASGP-R with high affinity. From our data, emerges that liposome delivery system may insure a specific and sustained drug delivery, a reduced therapeutic dose and, in particular, should avoid deleterious side effects of T3 treatment. Overall, our results showed that liposomes are good candidates as liver delivery system of T3 since cell viability tests performed by using hepatoma cell lines demonstrated a very low toxicity of all three lyposome formulations. On the other hand, nanocrystal formulations, containing two different diclofenac acid crystal forms, were developed with the aim to improve dermal drug bioavailability. Nanosuspensions were obtaining using wet media milling technique and were characterized in terms of size distribution, morphology, zeta potential, differential scanning calorimetry and X-ray diffraction powder. The ability of the nanocrystals to improve dermal drug bioavailability was investigated in vitro using Franz diffusion vertical cells and newborn pig skin, in comparison with diclofenac acid coarse suspensions and a commercial topical formulation containing diclofenac sodium. Nanocrystals exhibited a mean diameter ranging between 279 and 315 nm and a PI lower than 0.25, as shown by PCS measurements. The XRDP and DSC analysis clearly indicated that the preparation process did not modify the diclofenac polymorphic forms. In vitro transdermal delivery experiments showed an improved skin deposition and permeation of the nanocrystals compared to coarse suspensions and diclofenac sodium commercial topical formulation. These results highlight the fundamental role of the crystal size on drug solubility and, thus, on the ability of a poorly soluble drug to cross the skin and accumulate in the deeper skin layers. Afterwards, the aims of the work were to develop Diclofenac acid (DCF) nanosuspensions obtained by the addition of a permeation enhancer, Transcutol (TRC), in the aqueous stabilizer solution and to study the effect of Transcutol on the transdermal permeation and skin accumulation of DCF nanocrystals. DCF-TRC-nanosuspensions, prepared using the wet media milling technique, were characterized in terms of size distribution, morphology, zeta potential, differential scanning calorimetry, X-ray diffraction powder and Fourier transform infrared spectroscopy.The influence of Transcutol concentration on skin penetration ability of DCF nanosuspensions was evaluated by in vitro skin penetration and permeation studies using Franz diffusion vertical cells and newborn pig skin, in comparison with DCF nanosuspension without TRC, DCF coarse suspension and a commercial topical formulation containing diclofenac sodium. Nanosuspensions with Transcutol exhibited a mean diameter of ∼350 nm and a polydispersity index ranging between 0.21 and 0.26, values greater than those of the nanosuspension without TRC. In vitro permeations studies showed that the increase of TRC in the nanosuspension formulations decreased the DCF skin delivery probably because the higher average diameter values of TRC nanosuspensions could decrease the DCF dissolution velocity with respect to nanosuspension without TRC. Moreover, although TRC is considered a powerful solubilizing agent, the increase of its concentration in the nanosuspension formulations determined the DCF solubility decrease. In light of these results, in the last part of this thesis nanocrystals of diclofenac (DCF) are proposed as a novel approach to treat skin inflammation, and their efficacy was validated in an animal model. Diffusion of DCF through mouse skin was investigated ex vivo, and the topical anti-inflammatory potential of the DCF nanosuspension was assessed in vivo by testing their activity against common inflammatory endpoints: inhibition of chemically induced oedema and leucocyte infiltration (reflected in myeloperoxidase–MPO-activity). TPA (12-O-tetradecanoylphorbol-13-acetate) is commonly used to induce inflammation in animal models. The therapeutic efficacy of the nanosuspension was compared with those of a DCF coarse suspension and a commercial topical preparation containing diclofenac diethylamine, Voltaren Emulgel®. In vivo and ex vivo results have proven the superior anti-inflammatory efficacy of the nanocrystal suspension and its actual ability to localize the drug in the site of inflammation, compared to a commercial product (Voltaren). In conclusion, this study highlights the great potential of using nanocrystal suspensions as an effective strategy to improve topical bioavailability of poorly water-soluble drugs as well as a valid therapeutic approach.