Effect of gamma radiation dose on the fabrication of alpha-elastin nanoparticles
by gamma-ray crosslinking
Mari Fujimoto1, Mayuko Takeda1, Kouji Okamoto2, and Masakazu Furuta1*
1

Department of Biological Science, Graduate school of Science,

Osaka Prefecture University, 1-2 Gakuen-cho, Naka-ku, Sakai, Osaka, 599-8570, Japan2

Department of Bioscience and Bioinformatics, Kyushu Institute of Technology,
680-4 Kawazu, Iizuka, Fukuoka 820-8502, Japan*E-mail: mfuruta@b.s.osakafu-u.ac.jp

Introduction:
Elastin is an essential component in animal tissues as well as collagen. Elastin molecules are formed
from relatively loose and unstructured polypeptide chains that are covalently cross-linked into arubberilike elastic meshwork 1. A unique feature of elastin is that it undergoes self-assembly called
coacervation under selected conditions of concentration and temperature called cloudy point (CP).
Taking advantage of this thermosensitive feature, we employed alpha-elastin and examined theformation of nano sized droplets above its CP using various heating procedures. We obtained
nanoparticles from the droplets of by gamma-ray crosslinking under a temperature above CP 2.
In this work, we investigated that effect of gamma-irradiation to form crosslinked nanoparticles.

Materials and Methods:
Materials
Alpha-elastin (extracted from bovine neck ligaments) was purchased from Elastin Product Co., Inc.
Preparation of alpha-elastin aggregates in solutions by heating
Ten mg/ml of alpha-elastin dissolved in aqueous solution. The solution of alpha-elastin was slowly
heated from 4 oC to 60 oC in 30 min.
Dynamic Light Scattering (DLS)DLS measurements were done to determine the size of the aggregated alpha-elastin, using a NICOMPMODEL 370 system (USA). The size distributions of the aggregated alpha-elastin before and afterirradiation were measured by DLS at 60 oC.
Gamma-irradiation
The heated alpha-elastin sample was put into thermos bottle and it irradiated at 1, 3, 5, 7.5, 15 and 30
kGy with 60Co gamma-rays using 60Co-gamma irradiation pool at Osaka Pref. University (dose rate: 7.9
kGy / h). The temperature of the sample was kept at 60 oC during irradiation. We prepared thetemperature in the thermos bottle every 1 h.
Sodium Dodecyl Sulfate-Poly-Acrylamide Gel Electrophoresis (SDS-PAGE)
In order to analyze molecular weight of the crosslinked polypeptides after gamma-irradiation, wecarried out SDS-PAGE. SDS-PAGE was performed using 10 % polyacrylamide gel and 0.5 mg of thepolypeptide sample was loaded onto each lane of the gel (Thickness: 1mm) and run in a Mini-PROTEIN
3 cell (Bio-Rad) at constant voltage (200V) for 45 min. Separated polypeptides were visualized by cupper
staining kit (Bio-Rad).
Transmission Electron Microscope (TEM)
The morphology and particle size distribution of the aggregated polypeptide particles were examinedusing a HITACHI, EF2000 TEM operated at 200 keV. Samples for the TEM were dissolved in deionizedwater to a final concentration of 10 mg/ml. The sample solution was added to carbon-coated 400-meshcopper grids (VECO) and excess sample was absorbed in filter paper and the grid was dried at room
temperature before observing under TEM.

Results and Discussion:
Alpha-elastin was aggregated in aqueous solution with heating to 60oC (above CP) and the size of theaggregates was distributed about 190 nm before irradiation. After irradiation, we did not obtain the stable
aggregated alpha-elastin at the doses of 1 and 3 kGy. However after 5, 7.5, 15, and 30-kGy irradiation,aggregated alpha-elastin with the distribution ranging 150 – 220 nm was detected at 60oC (above CP).
After cooled to 5oC (below CP), the size of aggregated alpha-elastin were distributed around 60 nm – 70
nm for 7.5 and 15 kGy and 160 nm for 30 kGy, suggesting that we could obtain the stable aggregatedalpha-elastin above 7.5 kGy. Yield of the aggregatedalpha-elastin collected at 4oC after irradiation was about 10 %irrespective of the dose of 7.5, 15 and 30 kGy. We analyzed
the molecular weight of the aggregated alpha-elastin
molecules using SDS-PAGE. The molecular weight increased
with increasing doses, indicating that the alpha-elastin wascrosslinked

bygamma

irradiation.

Weobserved

themorphology of the aggregated alpha-elastin crosslinked by 30
kGy gamma irradiation. Figure showed that almost all of theaggregates were spherical and sizes were distributed within
about 60-220 nm at room temperature.
References:1

Bruce, A., Alexander, J., Julian, L., Martin, R., Keith, R., Peter, W. In MOLECULAR BIOLOGY OF

THE CELL, 4th ed.; Bruce, A., Alexander, J., Julian, L., Martin, R., Keith, R., Peter, W., Eds.; Garland
Science: New York, 2002; p152.
2Mari, Fujimoto; Kouji, Okamoto; Masakazu, Furuta Radiation Physics and Chemistry (in press).

3

Neradovic, D.; Soga, O.; Van Nostrum, C.F.; Hennink, W.E. Biomaterials, 2004, 25, 2409-2418.