Solid-phase synthesis, characterization, and cellular activities of collagen-model nanodiamond-peptide conjugates

Abstract

Nanodiamonds (NDs) have received considerable attention as potential drug delivery vehicles. NDs are small (∼5 nm diameter), can be surface modified in a controllable fashion with a variety of functional groups, and have little observed toxicity in vitro and in vivo. However, most biomedical applications of NDs utilize surface adsorption of biomolecules, as opposed to covalent attachment. Covalent modification provides reliable and reproducible ND-biomolecule ratios, and alleviates concerns over biomolecule desorption prior to delivery. The present study has outlined methods for the efficient solid-phase conjugation of ND to peptides and characterization of ND-peptide conjugates. Utilizing collagen-derived peptides, the ND was found to support or even enhance the cell adhesion and viability activities of the conjugated sequence. Thus, NDs can be incorporated into peptides and proteins in a selective manner, where the presence of the ND could potentially enhance the in vivo activities of the biomolecule it is attached to.

Keywords: cell adhesion; collagen; drug delivery; nanoparticle; triple-helical peptide; wound healing.

FIGURE 1

(Left) High resolution transmission electron microscopic photograph of a single detonation ND particle and (right) 5 nm diameter atomistic model illustrating the ND structure where sp³ diamond carbon is shown in grey, sp³ amorphous carbon in green, sp² carbon in black, oxygen in red, nitrogen in blue, and hydrogen in white. A sector has been cut from the model to show crystalline diamond structure inside the particle.

FIGURE 2

CD properties of α1(I)fTHP and α1(V)THP. (Top) CD spectra of α1(I)fTHP and α1(V)THP. Scans were taken from λ = 180–250 nm. (Bottom) Thermal transition curves of α1(I)fTHP and α1(V)THP. Readings were taken at λ = 225 nm and normalized to fraction folded.

FIGURE 3

Raman spectra of (A) drop-deposited ND-α1(I)fTHP, (B) drop-deposited α1(I)fTHP, (C) 2 mM aqueous solution of α1(I)fTHP, (D) drop-deposited 5/6-Fam, and (E) aqueous suspension of ND. All spectra were measured with 647 nm laser excitation.

FIGURE 4

Fluorescence spectra of (blue) α1(I)fTHP and (red) ND-α1(I)fTHP.

FIGURE 5

Zeta potentials of (top) ND and (bottom) ND-α1(I)fTHP. The three curves represent three replicates.

FIGURE 6

Particle size distribution of (top) ND and (bottom) ND-α1(I)fTHP. The five curves represent five replicates.

FIGURE 7

Adhesion and viability analyses of CHO-K1 cells and BJ fibroblasts. (A) Microscopic images of cell adhesion to peptide and ND–peptide conjugates. Images were taken at 40× magnification under phase contrast microscope. (B) Viability of CHO cells (blue) and BJ fibroblasts (red) following adhesion to PBS, peptide, or ND–peptide conjugates.