대한핵의학회지 (The Korean Journal of Nuclear Medicine)

대한핵의학회 (The Korean Society of Nuclear Medicine)
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간세포 지향성 Galactosylated Chitosan 화합물의 표지 수율 향상 및 세포 독성에 대한 연구

A Study on the Labeling Efficiency and Cytotoxicity of Hepatocyte-targeting Galactosylated Chitosan Compounds

  • 김대응 (원광대학교 의과대학 핵의학교실) ;
  • 정환정 (전북대학교 의과대학 핵의학교실) ;
  • 김은미 (전북대학교 의과대학 핵의학교실) ;
  • 김세림 (전북대학교 의과대학 핵의학교실) ;
  • 강윤희 (전북대학교 의과대학 핵의학교실) ;
  • 김민우 (전북대학교 의과대학 핵의학교실) ;
  • 김창근 (원광대학교 의과대학 핵의학교실) ;
  • 손명희 (전북대학교 의과대학 핵의학교실)
  • Kim, Dae-Weung (Department of Nuclear Medicine, Wonkwang University School of Medicine) ;
  • Jeong, Hwan-Jeong (Department of Nuclear Medicine, Chonbuk National University Medical School and the Hospital) ;
  • Kim, Eun-Mi (Department of Nuclear Medicine, Chonbuk National University Medical School and the Hospital) ;
  • Kim, Se-Lim (Department of Nuclear Medicine, Chonbuk National University Medical School and the Hospital) ;
  • Kang, Yun-Hee (Department of Nuclear Medicine, Chonbuk National University Medical School and the Hospital) ;
  • Kim, Min-Woo (Department of Nuclear Medicine, Chonbuk National University Medical School and the Hospital) ;
  • Kim, Chang-Guhn (Department of Nuclear Medicine, Wonkwang University School of Medicine) ;
  • Sohn, Myung-Hee (Department of Nuclear Medicine, Chonbuk National University Medical School and the Hospital)
  • 발행 : 2005.10.31
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초록

목적 : 저자들은 이전 연구에서 $^{99m}Tc$-GC을 합성해 생체내 분포를 확인했다. 그 결과 GC은 고도의 간 특이성을 보였으며, 간 특이 영상을 위한 방사성 의약품으로서의 가능성을 확인할 수 있었다. 그러나 $^{99m}Tc$-GC는 표지 수율과 안정성 면에서 불만족스러운 결과를 보였다. 이번 연구는 GC를 메틸화하는 방법과 HYNIC의 도입을 통해 표지수율과 안정성을 향상시킬 수 있는지 알아보고, 그와 함께 이 화합물들의 세포 독성에 대해 연구해 보고자 하였다. 대상 및 방법 : 합성한 GC, GMC, GCH를 $^{99m}Tc$로 표지하고 상온과 사람 혈청에서 표지 안정성을 측정하였으며, 6시간까지 시행하였다. 각 화합물을 0, 0.5, 1, 1.25, 5, 10, 25, 100 ${\mu}g/ml$ 농도로 첨가한 HeLa와 HepG2 세포를 18시간동안 배양한 뒤 세포 생존능을 MTT를 이용해 측정하였다. 결과 : 표지 후 실온에서의 안정성 실험을 한 결과 이동상으로 아세톤을 사용한 경우 $^{99m}Tc$-GC는 15분에 96%, 1시간에 88%였고, $^{99m}Tc$-GMC는 15분, 1시간. 6시간에서 각각 100%, 97%, 89%의 향상된 표지율을 보였고 $^{99m}Tc$-GCH는 6시간까지 95% 이상을 보여 안정성이 보다 더 향상됨을 확인하였다. 또한 이동상으로 생리 식염수를 사용한 경우 $^{99m}Tc$-GC는 15분, 1시간에 73.9%, 72%를 보였고, $^{99m}Tc$-GMC의 경우 15분, 1시간, 6시간에서 96.3%, 95.8%, 75.6%의 표지율을 보였다. $^{99m}Tc$-GCH는 90% 이상의 표지 안정성을 보였고, 사람 혈청에서 6시간까지 95.7%의 표지율을 보였다. MTT를 시행한 결과. 각 화합물을 처리한 세포주의 생존능은 대조군과 비교할 때 통계적으로 유의한 차이가 없었다. 결론 : 저자들은 GC를 메틸화하는 방법과 HYNIC의 도입을 통해 새로운 간세포 특이적 지향성을 갖는 키토산 화합물을 합성하였고, 이 화합물들은 $^{99m}Tc$으로 표지할 때 향상된 표지 수율과 뛰어난 표지 안정성을 보였다. 그리고 각 화합물들의 세포 독성 역시 원래 키토산과 의의있는 차이가 없이 매우 낮은 정도임을 증명하였다.

Purpose: In prior study, we synthesized $^{99m}Tc$-galactosylated chitosan (GC) and performed in vivo biodistribution study, showed specific targeting to hepatocyte. The aim of this study is to evaluate the labeling efficiency and cytotoxicity of modified galactosylated chitosan compounds, galactosyl methylated chitosan (GMC) and HYNIC-galactosylated chitosan (GCH). Materials and Methods: GC, GMC and GCH were synthesized and radiolabeled with $^{99m}Tc$. Then, they were incubated for 6 hours at room temperature and human serum at $37^{\circ}C$. Labeling efficiencies were determined at 15, 30 m, 1, 2, 3 and 6 h after radiolabeling. To evaluate cytotoxicity, MTT assay was performed in HeLa and HepG2 cells. Results: In comparison with them of $^{99m}Tc$-GC labeling efficiencies of $^{99m}Tc$-GMC were significantly improved (100, 97 and 89%) in acetone and 96.3, 95.8 and 75.6% in saline at 15 m, 1 and 6 h, respectively). Moreover, $^{99m}Tc$-GCH showed more improved labeling efficiencies (>95% in acetone and human serum and >90% in saline at 6 h). In MTT assay, cytotoxicity was very low and not different from that of controls. Conclusion: These results represent that these compounds are radiochemically compatible radiopharmaceuticals, can be used in hepatocyte specific imaging study and in vivo gene or drug delivery monitoring.

키워드

참고문헌

  1. Pricer WE, Ashwell G. The binding of desialylated glycoproteins by plasma membranes of liver. J Biol Chem 1971;246:4825-33
  2. Morell AG, Irvine RA, Sternlieb I, Scheinberg IH, Ashwell G. Physical and chemical studies on ceruloplasmin. V. Metabolic studies on sialic acid-free ceruloplasmin in vivo. J Biol Chem 1968;243: 155-9
  3. Marshall JS, Green AM, Pensky J, Williams S, Zinn A, Carlson DM. Measurement of circulating desialylated glycoproteins and correlation with hepatocellular damage. J Clin Invest 1974;54:555-62 https://doi.org/10.1172/JCI107792
  4. Sawamura T, Nakada H, Hazama H, Shiozaki Y, Sameshima Y, Tashiro Y. Hyperasialoglycoproteinemia in patients with chronic liver diseases and/or liver cell carcinoma. Asialoglycoprotein receptor in cirrhosis and liver cell carcinoma. Gastroenterology 1984;87:1217-21
  5. Spiess M. The asialoglycoprotein receptor: a model for endocytic transport receptors. Biochemistry 1990;29:10009-18 https://doi.org/10.1021/bi00495a001
  6. Bettinger T, Remy JS, Erbacher P. Size reduction of galactosylated PEI/DNA complexes improves lectin-mediated gene transfer into hepatocytes. Bioconjug Chem 1999;10:558-61 https://doi.org/10.1021/bc990006h
  7. Niidome T, Urakawa M, Sato H, Takahara Y, Anai T, Hatakayama T, et al. Gene transfer into hepatoma cells mediated by galactosemodified alpha-helical peptides. Biomaterials 2000;21:1811-19 https://doi.org/10.1016/S0142-9612(00)00076-4
  8. Stadalnik RC, Vera DR, Woodle ES, Trudeau WL, Porter BA, Ward RE, et al. Technetium-99m NGA functional hepatic imaging: preliminary clinical experience. J Nucl Med 1985;26:1233-42
  9. Sasaki N, Shiomi S, Iwata Y, Nishiguchi S, Kuroki T, Kawabe J, et al. Clinical usefulness of scintigraphy with 99mTc-galactosyl- human serum albumin for prognosis of cirrhosis of the liver. J Nucl Med 1999;40:1652-6
  10. Jeong JM, Hong MK, Lee J, Son M, So Y, Lee DS, et al. $^{99m}Tc$-neolactosylated human serum albumin for imaging the hepatic asialoglycoprotein receptor. Bioconjug Chem 2004;15:850-5 https://doi.org/10.1021/bc0342074
  11. Park IK, Kim TH, Park YH, Shin BA, Choi ES, Chowdhury EH, et al. Galactosylated chitosan-graft-poly(ethylene glycol) as hepatocytetargeting DNA carrier. J Control Release 2001;76:349-62 https://doi.org/10.1016/S0168-3659(01)00448-5
  12. Park IK, Yang J, Jeong HJ, Bom HS, Harada I, Akaike T, et al. Galactosylated chitosan as a synthetic extracellular matrix forhepato cytes attachment. Biomaterials 2003;24:2331-7 https://doi.org/10.1016/S0142-9612(03)00108-X
  13. Sandford P. Chitosan: commercial uses and potential applications. In: Skjakbraek G, Anthonsen T, Sandford P, editors. Chitin and Chitosan. London, UK:Elsevier Applied Science; 1989. p. 51-69
  14. Muzzarelli RAA, Rocchetti R. Enhanced capacity of chitosan for transition-metal ions in sulphate-sulphuric acid solutions. Talanta 1974;21:1137-43 https://doi.org/10.1016/0039-9140(74)80097-4
  15. Rao SB, Sharma CP. Use of chitosan as a biomaterial: studies on its safety and hemostatic potential. J Biomed Mater Res 1997; 34:21-8 https://doi.org/10.1002/(SICI)1097-4636(199701)34:1<21::AID-JBM4>3.0.CO;2-P
  16. Ngah WS, Ab Ghani S, Kamari A. Adsorption behaviour of Fe(II) and Fe(III) ions in aqueous solution on chitosan and cross-linked chitosan beads. Bioresour Technol 2005;96:443-50 https://doi.org/10.1016/j.biortech.2004.05.022
  17. Skorik YA, Gomes CA, Podberezskaya NV, Romanenko GV, Pinto LF, Yatluk YG. Complexation models of N-(2-carboxyethyl) chitosans with copper(II) ions. Biomacromolecules 2005;6:189-95 https://doi.org/10.1021/bm049597r
  18. Kim EM, Jeong HJ, Park IK, Cho CS, Kim CG, Bom HS. Hepatocyte-targeted nuclear imaging using $^{99m}Tc$-galactosylated chitosan: conjugation, targeting, and biodistribution. J Nucl Med 2005;46:141-5
  19. Edwards DS, Liu S, Ziegler MC, Harris AR, Crocker AC, Heminway SJ, et al. RP463: a stabilized technetium-99m complex of a hydrazino nicotinamide derivatized chemotactic peptide for infection imaging. Bioconjug Chem 1999;10:884-91 https://doi.org/10.1021/bc990049y
  20. Decristoforo C, Mather SJ. Technetium-99m somatostatin analogues: effect of labelling methods and peptide sequence. Eur J Nucl Med 1999;26(8):869-76 https://doi.org/10.1007/s002590050461
  21. Siecal AB, Thanou M, Kortze AF, Verhoel JC, Brussee J, Junginger HE. Preparation and NMR characterization of highly substituted N-trimethyl chitosan chloride. Carbohydrate Polymers 1998;36: 157-65 https://doi.org/10.1016/S0144-8617(98)00009-5
  22. Saha GB. Fundamentals of nuclear pharmacy. 4th ed. New York: Springer-Verlag; 1998. p. 98-9
  23. Arano Y. Delivery of diagnostic agents for gamma-imaging. Adv Drug Deliv Rev 1999;37:103-20 https://doi.org/10.1016/S0169-409X(98)00102-1
  24. Domingo JL. Developmental toxicity of metal chelating agents. Reprod Toxicol 1998;12:499-510 https://doi.org/10.1016/S0890-6238(98)00036-7
  25. Rhazi M, Desbrieres J, Tolaimate A, Rinaudo M, Vottero P, Alagui A. Contribution to the study of the complexation of copper by chitosan and oligomer. Polymer 2002;43:1267-76 https://doi.org/10.1016/S0032-3861(01)00685-1