Hydrolyzed galactomannan to actively target rifampicin-loaded nanocarriers to macrophages

MORETTON M.A.; CHIAPPETTA D.A.; ANDRADE F.; DAS NEVES J.A.; FERREIRA D.; SARMENTO B.; SOSNIK A.

Rosario

Encuentro; RICIFA 2012. The 2nd International Meeting on Pharmaceutical Sciences; 2012

Introduction Rifampicin (RIF) is one of the most effective first-line antituberculosis drugs [1]. The inhalatory route has emerged as an attractive therapeutic approach to diminish systemic adverse affects. Alveolar macrophages are the reservoir of the non-replicant form of M. Tuberculosis. Macrophages recognize surface mannose residues through lectin-like receptors [2]. Polymers containing mannose residues (e.g., galactomannan, GalM) could be used to modify the surface of drug-loaded nanocarriers, enhancing the uptake by macrophages. At the same time, drug-loaded nanocarriers must be deposited in the lower respiratory tract after inhalation [3]. Muco-adhesive polymers like chitosan (Chit) could enhance the retention of the nanocarriers in the respiratory system. The present study explored the development of two types of RIF-loaded nanocarriers modified with hydrolyzed galactomannan (GalM-h). The systems were fully characterized and the cellular uptake by macrophages in vitro was assessed. 2. Materials and Methods Epsilon-caprolactone (CL), tin(II) 2-ethylhexanoate (SnOct), chitosan (Chit, ~50 kg/mol), tripolyphosphate pentasodium salt (TPP), fluorescein 5(6)-isothiocyanate, concanavalin A (Con A), galactomannan (GalM), poly(ethyleneglycol) (PEG10000, 10 kg/mol), RIF and solvents were used as received. GalM-h was produced by hydrolysis in HCl (80°C, 1h). Chit nanoparticles without (Chit-NP) and with GalM-h (GalM-h/Chit-NP) were prepared by ionotropic gelation with TPP. A PCL-PEG-PCL copolymer was synthesized by the ring opening polymerization of CL by PEG10000 in presence of SnOct. PCL-PEG-PCL polymeric micelles (PMs) (1% w/v) coated with Chit (Chit-PM) and GalM-h/Chit (GalM-h/Chit-PM) were obtained by the acetone diffusion technique [4]. The size, size distribution, and zeta potential of the systems were measured with a Zetasizer Nano-Zs. The binding of the nanocarriers to a lectin (Con A) in vitro at 37 °C was monitored by Dynamic Light Scattering. Qualitative cellular uptake of fluorescein-labelled nanocarriers was assessed by fluorescence microscopy. Quantitative uptake was assessed by measuring intracellular RIF concentrations by HPLC-UV. Results The size of Chit-NP and GalM-h/Chit-NP ranged between 263 and 340 nm and the zeta-potential between +18.0 and +24.5 mV. However, the theoretical RIF payload was 1.0% w/w. PMs displayed a bimodal size distribution with a less positive zeta potential. More importantly, RIF encapsulation increased 12.9-fold with respect to NPs. Con A agglutination results confirmed the presence of GalM-h on the surface of both systems. Qualitative uptake studies by fluorescence microscopy indicated that GalM-h-modified NPs and PMs, but not Chit-NPs, were successfully taken-up by RAW 264.7 cells. The intracellular/cell associated levels of RIF for GalM-h modified-systems (1.1 μg/mg protein) were significant greater in comparison with systems containing only Chit (0.36 μg/mg protein) and RIF control solution (0.59 μg/mg protein) after 6h. Discussion and Conclusion The cationic nature of Chit led to a positively-charged surface in both types of nanocarriers. However, Chit-NPs were not taken up by macrophages probably due to the Chit hydrophilic nature [5]. Incorporation of GalM-h promoted the uptake and RIF was accumulated to a greater extent. Therefore, these modified systems exhibit potential as RIF carriers to promote macrophage-targeting in alternative TB inhalatory therapy. Future studies will be focused on the development of powders for inhalation and aerodynamic diameter measurements. Acknowledgements Cooperation program of Ministerio de Ciencia, Tecnología e Innovación Productiva (Argentina) and Fundação para a Ciencia e Tecnología (Portugal) for the period 2010-2011. References 1. Sosnik A. et al. Adv. Drug Deliv. Rev., 2010, 62, 547-559. 2. Pandey R. et al. J. Antimicrob. Chemother., 2005, 55, 430-435. 3. Andrade F. et al. Emerging Topics in Nanotechnology, John Wiley & Sons: New York, 2012. 4. Moretton M.A. et al. Colloids Surf B: Biointerfaces., 2010, 79, 467-479. 5. Sarmento et al. Carbohyd. Polym., 2011, 84, 919-925.