Biofunctionalization of biomaterials for nitric oxide delivery: potential applications in regenerative medicine

Figures & data

Figure 1 NO can influence a great many physiological systems through a range of bio-physiological mechanisms.

Abbreviation: NO, nitric oxide.

Figure 2 Overview of globule formation and SNAP conjugation.

Notes: (A) Chemical diagram of conjugation of long chain aliphatic hydrocarbons (OD/DD) with SNAP and formation of OD–SNAP and DD–SNAP. (B) Incorporation of DD–SNAP or OD–SNAP with a hydrophobic polymeric scaffold and the proposed distribution of SNAP-conjugated long-chain aliphatic hydrocarbons and the diagrammatic illustration of NO release. (C) Negatively stained TEM imaging of aliphatic hydrocarbon globules; the red arrow indicates a fatty acid globule.
Abbreviations: DD, dodecylamine; HBTU, O-(1H-benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexoflurophosphate; HOBt, Hydroxybenzotriazole; NO, nitric oxide; OD, octadecylamine; SNAP, S-nitro-N-acetylpenicillamine.

Table 1 Summary of the peaks obtained by LC–MS of SNAP, DD–SNAP, and OD–SNAP

Molecule	Expected peak at mass (u)	Measured peak at mass (u)	Positive ion
SNAP	221.25	221.1	C7H13N2O4S^+
DD	186.35	186.3	C12H28N^+
DD–SNAP	388.58	388.6	C19H38N3O3S^+
OD	270.51	270.4	C18H40N^+
OD–SNAP	472.74	472.7	C25H50N3O3S^+
DD–SNAP without NO	358.58	358.5	C19H38N2O2S^+
OD–SNAP without NO	442.74	442.7	C25H50N2O2S^+

Abbreviations: DD, dodecylamine; LC–MS, liquid chromatography–mass spectrometry; NO, nitric oxide; OD, octadecylamine; PCU, poly(carbonate-urea); SNAP, S-nitro-N-acetylpenicillamine.

Figure 3 Characterization of LCAH–SNAP–PCU.

Notes: (A) SEM images indicating porous structures of PCU and DD/OD–SNAP globular structures (pseudo colored green). (B) Mean contact angle measurements (θ) in mean ± SD and images of water drop on the surface of PCU (control) =107.8±1.9, DD–SNAP-P (2%) =109.0±5.1, and OD–SNAP-P (2%) =135.8±14.5.
Abbreviations: DD, dodecylamine; LCAH, long-chain aliphatic hydrocarbon; OD, octadecylamine; PCU, poly(carbonate-urea); SEM, scanning electron microscopy; SNAP, S-nitro-N-acetylpenicillamine.

Figure 4 Details of chemiluminescence studies.

Notes: (A) Schematic representation of chemiluminescence set-up for NO release measurement. (B) Four distinct phases of average NO release catalyzed with 1 M copper sulfate over a 5 h period for DD–SNAP, OD–SNAP, and SNAP–PCU (control). Values are shown as mean ± SD. (C) Rates of NO release in parts per billion per minute illustrating relative NO release during the four distinct phases. Values are shown as mean ± SD. Statistical significance is indicated with an *.
Abbreviations: DD, dodecylamine; NO, nitric oxide; NO₂, nitrogen dioxide; OD, octadecylamine; O₃, ozone; PCU, poly(carbonate-urea); PMT, photomultiplier tube; SNAP, S-nitro-N-acetylpenicillamine.

Figure S1 FTIR spectra of DMF, OD–SNAP, and OD–SNAP.

Notes: Arrows indicate the peaks of interest. FTIR was used to characterize the amide bond formation in both the DD and OD mixtures. In the DD–SNAP sample, DMF that has amide bonds has masked the amide bond formation of the DD–SNAP despite vacuum drying the traces of DMF. In the case of OD–SNAP, the same masking effect is can be seen. As OD is insoluble, it was easier to separate out. Peaks at 2850 and 2927 that correspond to the alkane groups (-CH₂) of the long hydrocarbon chain. A peak at 1690 corresponds to the C=O of the amide bond. There is a relatively small but very broad peak ~3450, which could be attributed to the N–H of the amide bond.
Abbreviations: DD, dodecylamine; DMF, dimethylformamide; FTIR, Fourier transform infrared; OD, octadecylamine; SNAP, S-nitro-N-acetylpenicillamine.

Figure S2 Characterization of LCAH–SNAP–PCU: AFM images showing changes in topography after the addition of OD/DD–SNAP.

Abbreviations: AFM, atomic force microscopy; DD, dodecylamine; LCAH, long-chain aliphatic hydrocarbon; OD, octadecylamine; PCU, poly(carbonate-urea); SEM, scanning electron microscopy; SNAP, S-nitro-N-acetylpenicillamine.

Figure S3 Negatively stained TEM imaging of aliphatic hydrocarbon globules in solvent (1% uranyl acetate). Red arrows indicate the presence of hydrocarbon globules.

Abbrevaition: TEM, transmission electron microscope.

Figure S4 Values for electrical conductivity (pFm^-1) at 80% RH measured after subjecting polymers to increase in RH at 1%/min from 20 to 80% RH and then leaving polymer at 80% RH for 30 min.

Notes: Image presents a comparison of dielectric values for polyurethanes with modifications with DD/OD–SNAP and native SNAP compared to polyurethane graft in its natural state. Lowest value for OD–SNAP–PCU indicates most hydrophobic material.
Abbreviations: DD, dodecylamine; OD, octadecylamine; PCU, poly(carbonate-urea); SNAP, S-nitro-N-acetylpenicillamine.