Purchase this article with an account.
S. A. Barker, Y. I. Chen, J. Sanderson, D. M. R. Georget, U. A. Jayasooriya; Changes in the Secondary Structure of Scleral Collagen Following Application of Model Excipients and Permeants Observed by Fourier Transform Raman Spectroscopy. Invest. Ophthalmol. Vis. Sci. 2007;48(13):5804.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
To assess the potential of Fourier Transform Raman Spectroscopy to detect in-situ changes in the secondary structure of scleral collagen after application of model permeants and pharmaceutical excipients used in ocular formulations.
Fresh (< 4 h post-mortem) porcine eyes were obtained from a local slaughter-house, with the animals having been processed under standard UK regulations for animals for human consumption. The sclera was carefully dissected away from adjoining tissue, sliced and incubated in one of the following aqueous solutions for 2 hours at 35°C prior to analysis: distilled water (control), 2%w/v benzalkonium chloride (BKC), 2%w/v sodium n-dodecyl lauryl sulphate (SDS), 2%w/v phenol and 0.2%w/v sodium fluorescein (NaFl). BKC is used as a preservative in ocular formulations, SDS is used as a surfactant to increase solubility in aqueous solutions and NaFl is used as a model permeant in drug delivery studies. Phenol was used as a positive control as it is known to affect protein behavior. Raman spectra (500 co-added spectra for each sample) were acquired on a Bruker IFS 66 spectrometer equipped with a Bruker FRA 106 accessory. Spectral resolution was 4 cm-1 and laser power was 100 mW at the sample. The spectra were analyzed using OPUS 5.5 software (Bruker Optick GmbH).
Raman bands at 1650 to 1670 cm-1 and 1620 to 1640 cm-1 are characteristic of protein α-helices and ß-sheets, respectively. The ratio of α-helix to ß-sheet conformation in the samples was determined by calculating the ratio of the measured peak heights. The ratios of α-helix to ß-sheet conformation in the samples were: control 2.66 ± 0.56, phenol-treated 1.05 ± 0.20, NaFl-treated 1.54 ± 0.09, BKC-treated 1.48 ± 0.13, SDS-treated 1.94 ± 0.47. There were statistically-significant differences between the control samples and each of the phenol-, NaFl- and BKC-treated samples (P < 0.01 for each), with a shift towards greater ß-sheet contribution. A similar trend was observed in the SDS-treated samples, but this was not statistically significant.
The results indicate that excipients used in ocular formulations have an effect on the secondary structure of scleral collagen. NaFl, used as a model permeant in absorption studies, also showed an effect on the collagen. It is therefore recommended that the use of these materials in pharmaceutical formulations and drug delivery studies be reviewed. Raman spectroscopy allows in-situ examination of the protein secondary structure in sclera.
This PDF is available to Subscribers Only