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It was found that polypeptide I aggregates above 28 oC in deionized solution.
However, polypeptide I yield hydrogels when prepared and irradiated at the same heating rate used for α-elastin. The size of the hydrogels was found to decrease with increased heating rates. However, stable cross-linked nanoparticles could not be obtained. Therefore, 4oC of polypeptide I solution was dropped into deionized water at 42oC to generate a final concentration of polypeptide I of 5 mg/ml. The polypeptide rapidly aggregated when subjected to a “Heat shock (40oC/min)” process and was subsequently irradiated to yield stable cross-linked nanoparticles.
On the other hand, polypeptide II and III did not undergo aggregation in deionized solution over the range of temperatures investigated and stable cross-linked nanoparticles could not be obtained over the usual range of concentrations and heating rates. Therefore, polypeptide II and III were dissolved in 10 mM phosphate buffer (pH 6.2) with the expectation that aggregation would be improved. As a result, only polypeptide II could aggregate. The polypeptide II solution was heated from 4oC to 50oC by various heating processes and was subsequently irradiated. Stable cross-linked nanoparticles were obtained using the same “Slow heating (1.5oC/min)” and irradiation conditions used for producing cross-linked nanoparticles of α-elastin. The size of the polypeptide II nanoparticles was ca. 60 nm and the optimal concentration was found to
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be 7 mg/ml. Stable polypeptide III cross-linked nanoparticles could not be obtained using any of the typical buffer solutions, concentrations and heating rates.
These result, reconfirmed the finding that the repeating GVGVP motif is an important characteristic for obtaining aggregation of α-elastin and the cross-linked structure. Charged amino acid residues prevent the polypeptide from dissolving in deionized water. As a result, formation of stable cross-linked nanoparticles and aggregation of the polypeptide does not occur. Moreover, in case of α-elastin and other polypeptides including charged amino acid side chains, “Slow heating” is required to efficiently obtain cross-linked nanoparticles. It is necessary for α-elastin and polypeptide chains to come into close proximity to obtain efficient cross-linking. “Slow heating” might contribute to efficient aggregation to improve the cross-linking process by enhancing the interactions of hydrophobic side-chains. Since α-elastin has many charged amino acid side chains within the polypeptides, charged repulsions have the effect of counteracting the interactions of hydrophobic side-chains.
In order to characterize the aggregation of the GVGVP hydrophobic motif within α-elastin, “Slow heating (1.3oC/min)”, “Fast heating (3.8oC/min)”, and “Heat shock (40oC/min)” methods were employed for polypeptide I consisting of only the GVGVP motif. The CD spectrum of the sample prepared using the “Heat shock” process has a
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negative band near 225 nm which represents a characteristic band of a type-II β-turn.
This spectrum is distinct from the spectra of other samples prepared by slower heating methods. The “Heat shock” process also had a greater effect on the hydrophobic interactions than the “Fast heating” and “Slow heating” processes according to fluorescence spectrometry using ANS-Mg as a probe. Consequently, it is expected that the “Heat shock” process might encourage high levels of close contact hydrophobic interactions among the amino acid side chains of the polypeptides.
Molecular dynamics (MD) simulation of the three molecules of (VPGVG)18 also provided an encouraging information on the aggregation process. A type-II β-turn structure defined initially for the simulation was remained within the aggregated polypeptide. Moreover, hydrophobic amino acids such as Val and Pro were arranged in close proximity within the assembly of the polypeptides within the simulation periods for 10000 ps.
The intensity of the band representing the type-II β-turn of the irradiated polypeptide I prepared by “Heat shock” was reduced by 25% after irradiation, suggesting that the type-II β-turn might be involved in cross-linking among polypeptides.
A single Val, Pro and Gly mixture solution was investigated to characterize the
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cross-linking induced by gamma irradiation. Val was found to be more likely to be reduced. This suggests that the Val residue of the (VPGVG)n motif is cross-linked as a result of gamma irradiation.
A schematic model of the efficient nanoparticle formation process is illustrated in Fig. 6-1. Polypeptide I dissolve in water at 4°C (below CP). Increasing the temperature to 42°C (above CP) using the “Heat shock” process causes the polypeptides to form a type-II β-turn structure during the course of aggregation. The aggregation of polypeptide I has cross-links between Val residues within the polypeptide I which are initiated by gamma irradiation below 42°C (above CP). This produces stable nanoparticle formation even below CP.
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Fig. 6-1 Postulated reaction scheme for the formation of the cross-linked nanoparticles.
Cross-linked polypeptide nanoparticles were tested for applications in drug delivery systems (DDS). Methotrexate was used to load polypeptide I and release was effected with saline 10 mM phosphate buffer, pH 7.2 - 7.4, at 37oC. Eighty percent of the methotrexate was released within 2 hours with the remaining twenty percent
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gradually released over the following week. Cisplatin was employed to load polypeptide II under various pH conditions and release was initiated with saline at 37oC. Cisplatin was gradually released over the period of at least one week when cisplatin loading was carried out at pH 9. Cisplatin is a platinum chelate complex with four ligands (two ammonia ligands and two chloride ions). These ligands form a complex with carboxylate groups of cross-linked polypeptide II nanoparticles with release of cisplatin from the complex under saline conditions. Therefore, the amount of cisplatin loaded and its initial release can be controlled by altering the pH. These results are promising for development of nanoparticles in DDS applications.
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Acknowledgements
The present study has been carried out under the conduct of Associ. Prof.
Masakazu Furuta, in the graduate school of science, Osaka Prefecture University. The author wishes to express her deep gratitude to him for his helpful advice and warm encouragement throughout this work.
The author wishes to express her deep gratitude to Prof. Masahito Oka, in the graduate school of science, faculty of liberal arts and sciences, Osaka Prefecture University, for his skilled technical assistance and discussions related to CD spectra, MD simulation and valuable suggestion.
The author wishes to express her deep gratitude to Prof. Masayuki Hara, in the graduate school of science, Osaka Prefecture University, for valuable suggestion and constant discussion.
The author wishes to express her deep gratitude to Prof. Toshiji Tada, in the graduate school of science, Osaka Prefecture University, for valuable suggestion.
The authors would like to express their sincere gratitude to Prof. Dan W. Urry, in the Minnesota University for supplying the elastin protein-based polymers and valuable discussions.
The author wishes to express her deep gratitude to Dr. Masamichi Iwama in the
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Bioelastic Japan Co. and Dr. Mituhiro Murata in the JSR Co. for their technical support and valuable discussions since the early days of this work.
The author wishes to express her deep gratitude to Dr. Koji Inai, in the graduate school of science, Osaka Prefecture University, for molecular dynamics simulation and valuable suggestion.
The author wishes to express her deep gratitude to Prof. Kouji Okamoto, Ms. Eri Shiratuchi and Mr. Suguru Taniguchi in the graduate school of life science and systems engineering, Kyushu Institute of Technology, for his or her skilled technical assistance and discussions related to TEM, CD spectra, UV spectra, MALDY TOFF MASS and valuable suggestion.
The author wishes to express her deep gratitude to Associ. Prof. Hiroshi Tsuda in the graduate school of materials science, Osaka Prefecture University, for his skilled technical assistance and discussions related to TEM.
The author wishes to express her deep gratitude to Tenure Track Instructor Tomoaki Nishino and technical assistant Ms. Taeko Yuki in the Nanoscience and nanotechnology research center, research institute for the twenty-first century, Osaka Prefecture University, for his or her skilled technical assistance and discussions related to AFM.
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The author wishes to express her deep gratitude to Assist. Prof. Shigenori Nishimura in the graduate school of life and environmental sciences, Osaka Prefecture University, for his skilled technical assistance and discussions related to CD.
The author wishes to express her deep gratitude to Associ. Prof. Harumi Fukada in the graduate school of life and environmental sciences, Osaka Prefecture University, for her skilled technical assistance and discussions related to DSC.
The author wishes to express her deep gratitude to Prof. Yoshiaki Hirano in the faculty of chemistry, materials and bioengineering, high technology research center, Kansai University, for his skilled technical assistance and discussions related to synthesis VPGVG.
The author wishes to express her heartfelt gratitude to Professor Takao Yamamoto and Instructor Satoshi Seino, in the graduate school of engineering, Osaka University, for useful suggestion and experiment on amino acid analysis.
The author would like to express much appreciation to Ms. Mayuko Takeda in the school of science, Osaka Prefecture University, for her assistance with this study.
Finally, the author would like to express much appreciation to members of the laboratory of the cellular regulatory science, in the graduate school of science, Osaka Prefecture University.