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

Stefano Salmaso, Sara Bersani, Francesca Mastrotto, Giancarlo Tonon, Rodolfo Schrepfer, Stefano Genovese, Paolo Caliceti

Research output: Contribution to journalArticlepeer-review

Abstract

PEG5 kDa-cholane, PEG10 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-NH2) 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 PEG5 kDa-cholane, PEG10 kDa-cholane and PEG20 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 PEG5 kDa-cholane prolongs the body exposure of the protein. After subcutaneous administration, the protein tmax values obtained with rh-G-CSF and 1:14 and 1:21 rh-G-CSF/PEG5 kDa-cholane (w/w ratio) nanocomplexes were 2, 8 and 24 h, respectively. The 1:21 (w/w) rh-G-CSF/PEG5 kDa-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 1.5 mg/kg dose of a 1:21 (w/w) rh-G-CSF/PEG5 kDa-cholane formulation induced a high production of white blood cells for 96 h.

Original languageEnglish
Pages (from-to)176-184
Number of pages9
JournalJournal of Controlled Release
Volume162
Issue number1
DOIs
Publication statusPublished - Aug 20 2012

Keywords

  • Amphiphilic polymers
  • Granulocyte colony stimulating factor
  • Nanocomposites
  • Poly(ethylene glycol)
  • Protein delivery
  • Supramolecular association

ASJC Scopus subject areas

  • Pharmaceutical Science

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