Self-assembling nanocomposites for protein delivery: supramolecular interactions between PEG-cholane and rh-G-CSF.
PEG(5 kDa)-cholane, PEG(10 kDa)-cholane and PEG(20 kDa)-cholane self-assembling polymers have been synthesised by the end-functionalisation of 5, 10 and 20 kDa 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 ± 2.4 μM as the PEG molecular weight increased. Similarly, light scattering analysis showed that the micelle size increased from 15.8 ± 4.9 to 23.2 ± 11.1 nm 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(5 kDa)-cholane, PEG(10 kDa)-cholane and PEG(20 kDa)-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(5 kDa)-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(5 kDa)-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(5 kDa)-cholane formulation induced a high production of white blood cells for 96 h.
Department of Pharmaceutical and Pharmacological Sciences, University of Padua, Via F. Marzolo, 5, 35131 Padova, Italy.
Stefano
S
Salmaso
Sara
S
Bersani
Francesca
F
Mastrotto
Giancarlo
G
Tonon
Rodolfo
R
Schrepfer
Stefano
S
Genovese
Paolo
P
Caliceti
D061605:Administration, Intravenous; D000818:Animals; D002757:Cholanes / Q000737:chemistry*; D002942:Circular Dichroism; D003692:Delayed-Action Preparations / Q000737:chemistry*; D016179:Granulocyte Colony-Stimulating Factor / Q000008:administration & dosage* / Q000493:pharmacokinetics* / Q000494:pharmacology; D006801:Humans; D007279:Injections, Subcutaneous; D007958:Leukocyte Count; D008297:Male; D053761:Nanocomposites / Q000737:chemistry*; D011092:Polyethylene Glycols / Q000737:chemistry*; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000008:administration & dosage / Q000493:pharmacokinetics / Q000494:pharmacology
D002757:Cholanes; D003692:Delayed-Action Preparations; D011994:Recombinant Proteins; D016179:Granulocyte Colony-Stimulating Factor; D011092:Polyethylene Glycols