Self-assembling nanocomposites for protein delivery: Supramolecular interactions between PEG-cholane and rh-G-CSF.

PEG(5kDa)-cholane, PEG(10kDa)-cholane and PEG(20kDa)-cholane self-assembling polymers have been synthesised by the end-functionalisati`on of 5, 10 and 20kDa linear amino-terminating monomethoxy-poly(ethylene glycol) (PEG-NH(2)) with 5β-cholanic acid. Spectroscopic studies and isothermal titration calorimetry showed that the CMC of the PEG-cholane derivatives increased from 23.5±1.8 to 60.2±2.4μM as the PEG molecular weight increased. Similarly, light scattering analysis showed that the micelle size increased from 15.8±4.9 to 23.2±11.1nm with the PEG molecular weight. Gel permeation studies showed that the polymer bioconjugates associate with recombinant human granulocyte colony stimulating factor (rh-G-CSF) to form supramolecular nanocomposites according to multi-modal association profiles. The protein loadings obtained with PEG(5kDa)-cholane, PEG(10kDa)-cholane and PEG(20kDa)-cholane were 7.4±1.1, 2.7±0.3 and 2.1±0.4% (protein/polymer, w/w %), respectively. Scatchard and Klotz analyses showed that the protein/polymer affinity constant increased and that the number of PEG-cholane molecules associated to rh-G-CSF decreased as the PEG molecular weight increased. Isothermal titration calorimetry confirmed the protein/polymer multi-modal association. Circular dichroism analyses showed that the polymer association alters the secondary structure of the protein. Nevertheless, in vitro studies performed with NFS-60 cells showed that the polymer interaction does not impair the biological activity of the cytokine. In vivo studies performed by intravenous and subcutaneous administrations of rh-G-CSF to rats showed that the association with PEG(5kDa)-cholane prolongs the body exposure of the protein. After subcutaneous administration, the protein t(max) values obtained with rh-G-CSF and 1:14 and 1:21 rh-G-CSF/PEG(5kDa)-cholane (w/w ratio) nanocomplexes were 2, 8 and 24h, respectively. The 1:21 (w/w) rh-G-CSF/PEG(5kDa)-cholane formulation resulted in 149% relative bioavailability, and the pharmacokinetic behaviour was similar to that obtained with an equivalent protein dose of rh-G-CSF chemically conjugated with one linear 20-kDa PEG. A single administration of a 1.5mg/kg dose of a 1:21 (w/w) rh-G-CSF/PEG(5kDa)-cholane formulation induced a high production of white blood cells for 96h.