ALPHA INTERFERON LOADED CHITOSAN NANOPARTICLES FOR THE OPTIMIZATION OF ITS THERAPEUTIC USE

CANEPA C; BERINI C; SOSNIK A; BIGLIONE M; IMPERIALE J

Conferencia; 9th Clinical Nanomedicne and Targeted Medicine Conference; 2016

Introduction. Interferon alpha (IFNα) is a protein drug used to treat oncological diseases and viral infections. As it presents a short half-life, it must be administered frequently in order to maintain therapeutic effectiveness. However, its low therapeutic index and fluctuations in drug plasma levels can lead to severe adverse effects 1. In this context, the design of a suitable drug delivery system (DDS) able to protect and control the release of the cargo would permit to reduce the frequency of administration. In this first stage, we designed and characterized a DDS. For this, IFNα2b was encapsulated in chitosan nanoparticles (CHT NPs) which were subjected to physicochemical and biological characterizations.Materials and Methods. CHT NPs were prepared by ionotropic gelification method between the positively charged amino groups of CHT (Low molecular weight CHT, Sigma-Aldrich, USA) and the negative groups of tripolyphosphate (TPP, Sigma-Aldrich, USA) 2. Briefly, a TPP solution was added dropwise to a CHT solution pH 5.5 under constant magnetic stirring. The resulting nanosuspension was then left to gelify for 15 min. To prepare IFNα loaded CHT NPs, a suitable amount of drug was dissolved into de CHT solution. CHT and CHT NPs were characterized by infrared spectroscopy using a Nicolet Spectrometer (Nicolet 380 ATR/FT-IR spectrometer, Avatar Combination Kit). The particle size (Dh), size distribution (PDI) and zeta potential (Z-Pot) of blank and loaded CHT NPs were measured with a Zetasizer Nano-ZS (Malvern Instruments, UK). Unreacted CHT was quantified in the freshly nanosuspension by a colorimetric method reported elsewhere 3 using alizarinsulfonic acid as dye. The encapsulation efficiency (EE) was determined by an indirect method. For this, the concentration of free IFNα was determined with an ELISA commercial kit (Affimetrix eBioSciences). The %EE was calculated according to the following Equation: %EE = [(Do ? Df) / Do)] x 100, where Do is the initial amount of IFNα used and Df is the amount of free IFNα (non-encapsulated drug) (n = 3). CHT NPs stability was physically determined at 4 and 25°C. For this, Dh, PdI and Z-Pot were monitored as a function of time for 30 days, using the technique described above (n = 3). The antiviral activity was determined by measuring the inhibition of the cytopathic effect of Vesicular Stomatitis Virus (VSV) on MDBK cells. MDBK cells (20,000 per well of a 96-well plate) were infected with VSV at a MOI of 0.1. After incubation with soluble IFN or encapsulated IFN, cells were fixed and stained with crystal violet. Absorbance was measured at 590 nm. Absorbance of uninfected control cells was set as 100%. Results and Discussion. Infrared spectra of CHT NPs and CHT evidenced the interaction between CHT and TPP, supporting the formation of CHT NPs. The mean Dh was 381.7 ± 35.2 nm and 353.0 ± 31.2 nm, the PdI 0.472 ± 0.030 and 0.407 ± 0.010 and the Z-Pot 31.4 ± 4.6 mV and 31.8 ± 1.7 mV for blank and IFNα-loaded CHT NPs, respectively. The Z-Pot value suggests not only a net positive surface charge due to an excess of CHT but also physical stability of the DDS. The higher the zeta potential, the stronger the repulsion, the more stable the system becomes. In fact, both blank and IFNα-loaded CHT NPs were stable at 4 and 25°C over a period of 30 days, according to DLS results. The amount of CHT that formed NPs determinated colorimetrically was 95.5% and the encapsulation efficiency was 99.9%. The antiviral activity of encapsulated IFN was significantly higher than that of commercial soluble one