Immobilization of bioactive moieties on polycaprolactone scaffold surface: RGD sequence bioconjugation after aminolysis

Filippo Causa 1Edmondo Battista 1Raffaella Della Moglie 3Paolo A. Netti 2,3

1. Dipartimento di Medicina Sperimentale e Clinica (UMG), Viale Europa, Catanzaro 88100, Italy
2. Department of Materials & Production Engineering, University of Naples "Federico II" (DIMP-CRIB), Piazzale Tecchio, 80, Napoli 80125, Italy
3. Interdisciplinary Centre on Biomaterials (CRIB), Napoli 80125, Italy

Abstract

PCL is a semicrystalline thermoplastic polyester widely used in biomedical field. This popular biomaterials for tissue engineering scaffold often has suboptimal properties when analyzed for cell attachment and growth, thus, it should be desirable to improve its performance by immobilization of bioactive moieties on its surface.

Moreover, short amino acid sequence such as arginine-glycine-aspartic acid (RGD) has been widely used as recognition motif to mediate cell attachment.

The aim of this work is to set-up a procedure to covalently immobilize RGD-based peptide sequences onto functionalized PCL surface. The procedure consists of three steps: aminolysis of polymeric scaffold, grafting of a tether and peptide immobilization.

The aminolysis of PCL substrates with 1,6-hexanediamine has been investigated and the yield of the reaction evaluated. In particular, the amount of amino group introduced, measured by Ninhidryn assay reached a maximum NH2 density of ~1*10-7mol/cm2 (30 minutes, 37°C)(fig 1).

Contact angles decrease with increasing time of treatment, ranging from 76° (pure) to 67° (90 min at 37°C). The rates of NH2 grafting at different temperatures (from 24 to 37°C) ranged from 0.92 to 3.2*10-8 mol/cm2min, and, an activation energy of 59 kJ/mol have been evaluated.

Moreover, ATR-FTIR spectroscopy characterization evidenced the presence of amine group (3337cm-1 NH stretch, 1639cm-1 and 1556cm-1 -CONHR- absorption) (fig 2). Afterwards, indeed, glutaraldeide solutions were used to graft GRGDY-Rho sequences on scaffold surface after reaction in mild aqueous conditions by reductive amination.

CLSM observations were also preformed on cross-sections of 3D PCL scaffolds demonstrating the capability to control in space and amount the peptide immobilization also in the case of 3D PCL substrate to be used as scaffold for tissue engineering.

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Fig 1


Fig 2
 

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Presentation: Poster at E-MRS Fall Meeting 2008, Symposium L, by Filippo Causa
See On-line Journal of E-MRS Fall Meeting 2008

Submitted: 2008-05-12 11:41
Revised:   2009-06-07 00:48