Physico-chemical evaluation of rationally designed melanins as novel nature-inspired radioprotectors

Abstract

Background: Melanin, a high-molecular weight pigment that is ubiquitous in nature, protects melanized microorganisms against high doses of ionizing radiation. However, the physics of melanin interaction with ionizing radiation is unknown.

Methodology/principal findings: We rationally designed melanins from either 5-S-cysteinyl-DOPA, L-cysteine/L-DOPA, or L-DOPA with diverse structures as shown by elemental analysis and HPLC. Sulfur-containing melanins had higher predicted attenuation coefficients than non-sulfur-containing melanins. All synthetic melanins displayed strong electron paramagnetic resonance (2.14.10(18), 7.09.10(18), and 9.05.10(17) spins/g, respectively), with sulfur-containing melanins demonstrating more complex spectra and higher numbers of stable free radicals. There was no change in the quality or quantity of the stable free radicals after high-dose (30,000 cGy), high-energy ((137)Cs, 661.6 keV) irradiation, indicating a high degree of radical stability as well as a robust resistance to the ionizing effects of gamma irradiation. The rationally designed melanins protected mammalian cells against ionizing radiation of different energies.

Conclusions/significance: We propose that due to melanin's numerous aromatic oligomers containing multiple pi-electron system, a generated Compton recoil electron gradually loses energy while passing through the pigment, until its energy is sufficiently low that it can be trapped by stable free radicals present in the pigment. Controlled dissipation of high-energy recoil electrons by melanin prevents secondary ionizations and the generation of damaging free radical species.

Figure 1. HPLC (a) and EPR spectra of (b) MELex5, (c) MEL1, (d) MEL2, (e) MEL3b, (f) MEL4.

BG, background; PTCA, pyrrole-2,3,5-tricarboxylic acid; PDCA, pyrrole-2,3-dicarboxylic acid; TTCA, 1,3-thiazole-2,4,5-tricarboxylic acid; TDCA, thiazole-4,5-dicarboxylic acid.

Figure 2. Component mass attenuation curves of (a) MELex5, (b) MEL1, (c) MEL2, (d) MEL3b, and (e) MEL4.

Note that both x and y axes are logarithmic scales. Mass attenuation coefficients of interest (energies of 85 and 113 keV) are shown in (f).

Figure 3. X-ray tube and experimental setup for beam calibration and mammalian cell protection studies.

For cell protection studies, 6-well plates with CHO cells were placed in 18×18 cm field where the ionization chamber is located in the figure.

Figure 4. Survival of CHO cells after a 600 cGy dose with synthetic melanins compared to controls (without melanin or with melanin ‘ghosts’ from C. neoformans).

Images of plates irradiated at (a) 85 keV (200 kV_p) and (c) 113 keV (320 kV_p) with each row of 3 wells corresponding to either no melanin, C. neoformans ghosts, MEL1, MEL4, MEL2, or MEL3b (from top left to bottom right). Clonogenic survival plots are shown for (b) 85 keV and (d) 113 keV. The clonogenic survival of irradiated cells was determined by crystal violet staining. Error bars show the standard deviations.

Figure 5. Survival of CHO cells after 661 keV ¹³⁷Cs radiation in presence of 20 or 100 µg/mL MEL2.

The clonogenic survival of irradiated cells was determined by crystal violet staining.