English

Poly(ε-caprolactone)-Polypeptide Copolymer Micelles Enhance the Antibacterial Activities of Antibiotics

• Lingshan Chen ^{1, 2, †,} ,
• Yuanxiu Hong ^{1, 2, †,} ,
• Shisheng He ^{1, ,} ,
• Zhen Fan ^{2, 3, ,} ,
• Jianzhong Du ^{1, 2, 4, ,}

• 1.

  Department of Orthopedics, Shanghai Tenth People's Hospital, Shanghai 200072, China

• 2.

  Department of Polymeric Materials, School of Materials Science and Engineering, Tongji University, Shanghai 201804, China

• 3.

  Institute for Advanced Study, Tongji University, Shanghai 200092, China

• 4.

  Key Laboratory of Advanced Civil Engineering Materials of Ministry of Education, Tongji University, Shanghai 201804, China

• ^†These authors contributed equally to this work.

Corresponding author: Shisheng He, tjhss7418@tongji.edu.cn Zhen Fan, fanzhen2018@tongji.edu.cn Jianzhong Du, jzdu@tongji.edu.cn

• Received Date: 28 October 2019
  Accepted Date: 09 December 2019
  Revised Date: 06 December 2019
  Available Online: 15 October 2021
• Fund Project:

Abstract: Bacterial infection is a major threat to human health, and can cause several diseases including gastroenteritis, influenza, tetanus, and tuberculosis. As conventional antibiotic treatment may cause various undesirable effects such as stomach disorder and bacterial resistance, it is necessary to improve the antibacterial efficiency of antibiotics. Here, we synthesized a peptide-based copolymer, poly(ε-caprolactone)-block-poly(glutamic acid)-block-poly(lysine-stat-phenylalanine)[PCL₃₄-b-PGA₃₀-b-P(Lys₁₆-stat-Phe₁₂)] by ring-opening polymerization (ROP) of ε-caprolactone and amino acid N-carboxyanhydride (NCA). Successful synthesis of the copolymer was verified by proton nuclear magnetic resonance and size exclusion chromatography. This copolymer can self-assemble into negatively charged micelles (-26.7 mV) under alkaline conditions by solvent switch method. The micelle structure was confirmed by transmission electron microscopy and dynamic light scattering, and revealed to have a diameter of ~42 nm. Antibiotics were loaded into micelles during the self-assembly process, and cell viability assay was conducted to evaluate its cytotoxicity with and without tobramycin. No obvious cytotoxicity was observed for both micelles when the concentration was lower than 300 μg·mL^-1. The antibiotic-loaded micelles demonstrated very low minimum inhibitory concentrations (MICs) against both Gram-negative Escherichia coli (E. coli) (7.8 μg·mL^-1) and Gram-positive Staphylococcus aureus (S. aureus) (18.2 μg·mL^-1), while the MICs of free tobramycin were 3.9 and 1.0 μg·mL^-1, respectively. The drug-loading content and efficiency of the micelles were 5.2% and 24.3%, respectively. Therefore, the MICs of the loaded tobramycin against E. coli and S. aureus were 0.4 and 0.9 μg·mL^-1, respectively, suggesting that the micelle could enhance the antibacterial activity of antibiotics. Tobramycin-loaded micelles demonstrated a sustained release characteristic, with 85% of the antibiotics released after 8 h. In bacteria-induced acidic microenvironment, the coil conformation of PGA blocks transforms and PGA blocks shrink toward the micelle core. Concomitantly, the carboxyl side chains are protonated in an acidic environment, increasing the hydrophobicity of this micelle. Antibiotics will be captured when reaching the outer core to slow down the releasing process. Furthermore, the poly(lysine-stat-phenylalanine) [P(Lys-stat-Phe)] coronas with broad spectrum intrinsic antibacterial activity can penetrate the bacterial cell membrane, leading to leakage of the cellular contents of the bacteria and ultimately their death. Due to the sustained release property of micelle and the intrinsic activity of the antibacterial peptide segments, this micelle can greatly enhance the antibacterial activity of antibiotics. Overall, this antibiotic-loaded micelle provides a novel approach for significantly reducing the antibiotics dosage and avoiding the associated health risks.

Key words:
• Antibiotic
•  /  Self-assembly
•  /  Peptide
•  /  Polymer Micelle
•  /  Antibacterial Activity
•  /  Sustained Release

• 1. [1]

     Morris, C. A.; Conway, H. D.; Everall, P. H. Lancet 1972, 300, 1375. doi: 10.1016/S0140-6736(72)92830-9

  2. [2]

     Thomas, M.; Noah, N. D.; Male, G. E.; Stringer, M. F.; Kendall, M.; Gilbert, R. J.; Jones, P. H.; Phillips, K. D. Lancet 1977, 309, 1046. doi: 10.1016/S0140-6736(77)91272-7

  3. [3]

     Brundage, J. F. Lancet Infect. Dis. 2006, 6, 303. doi: 10.1016/s1473-3099(06)70466-2

  4. [4]

     Khoshnood, S.; Heidary, M.; Haeili, M.; Drancourt, M.; Darban-Sarokhalil, D.; Nasiri, M. J.; Lohrasbi, V. Int. J. Biol. Macromol. 2018, 120, 180. doi: 10.1016/j.ijbiomac.2018.08.037

  5. [5]

     Galagan, J. E. Nat. Rev. Genet. 2014, 15, 307. doi: 10.1038/nrg3664

  6. [6]

     Hersh, D.; Weiss, J.; Zychlinsky, A. Curr. Opin. Microbiol. 1998, 1, 43. doi: 10.1016/s1369-5274(98)80141-0

  7. [7]

     Hostetler, K. Y.; Hall, L. B. Proc. Natl. Acad. Sci. U. S. A. 1982, 79, 1663. doi: 10.1073/pnas.79.5.1663

  8. [8]

     Anne, S.; Reisman, R. E. Ann. Allergy, Asthma, Immunol. 1995, 74, 167.

  9. [9]

     Granowitz, E. V.; Brown, R. B. Crit. Care Clin. 2008, 24, 421. doi: 10.1016/j.ccc.2007.12.011

  10. [10]

      Tan, Y. T.; Tillett, D. J.; McKay, I. A. Mol. Med. Today 2000, 6, 309. doi: 10.1016/s1357-4310(00)01739-1

  11. [11]

      Hancock, R. E. W.; Lehrer, R. Trends Biotechnol. 1998, 16, 82. doi: 10.1016/s0167-7799(97)01156-6

  12. [12]

      Davies, J. Science 1994, 264, 375. doi: 10.1126/science.8153624

  13. [13]

      Jennings, M. C.; Minbiole, K. P. C.; Wuest, W. M. ACS Infect. Dis. 2015, 1, 288. doi: 10.1021/acsinfecdis.5b00047

  14. [14]

      Marambio-Jones, C.; Hoek, E. M. V. J. Nanopart. Res. 2010, 12, 1531. doi: 10.1007/s11051-010-9900-y

  15. [15]

      孙磊, 刘爱心, 黄红莹, 陶小军, 赵彦保, 张治军.物理化学学报, 2011, 27, 722. doi: 10.3866/PKU.WHXB20110235Sun, L.; Liu, A. X.; Huang, H. Y.; Tao, X. J.; Zhao, Y. B.; Zhang, Z. J. Acta Phys. -Chim. Sin. 2011, 27, 722. doi: 10.3866/PKU.WHXB20110235

  16. [16]

      Reiad, N. A.; Salam, O. E. A.; Abadir, E. F.; Harraz, F. A. Chin. J. Polym. Sci. 2013, 31, 984. doi: 10.1007/s10118-013-1263-2

  17. [17]

      Huang, A.; Xu, S.; Wei, G.; Ma, L.; Gao, C. Y. Chin. J. Polym. Sci. 2009, 27, 865. doi: 10.1142/s025676790900459x

  18. [18]

      Ghasemzadeh, H.; Sheikhahmadi, M.; Nasrollah, F. Chin. J. Polym. Sci. 2016, 34, 949. doi: 10.1007/s10118-016-1807-3

  19. [19]

      Sun, H.; Hong, Y. X.; Xi, Y. J.; Zou, Y. J.; Gao, J. Y.; Du, J. Z. Biomacromolecules 2018, 19, 1701. doi: 10.1021/acs.biomac.8b00208

  20. [20]

      Zhou, C. C.; Wang, M. Z.; Zou, K. D.; Chen, J.; Zhu, Y. Q.; Du, J. Z. ACS Macro Lett. 2013, 2, 1021. doi: 10.1021/mz400480z

  21. [21]

      袁康, 周雪, 杜建忠.物理化学学报, 2017, 33, 656. doi: 10.3866/PKU.WHXb201701162Yuan, K.; Zhou, X.; Du, J. Z. Acta Phys. -Chim. Sin. 2017, 33, 656. doi: 10.3866/PKU.WHXb201701162

  22. [22]

      Wang, M. Z.; Wang, T.; Yuan, K.; Du, J. Z. Chin. J. Polym. Sci. 2016, 34, 44. doi: 10.1007/s10118-016-1725-4

  23. [23]

      李婷, 向双飞, 东为富, 马丕明, 施冬健, 陈明清.物理化学学报, 2016, 32, 2761. doi: 10.3866/PKU.WHXB201608261Li, T.; Xiang, S. F.; Dong, W. F.; Ma, P. M.; Shi, D. J.; Chen, M. Q. Acta Phys. -Chim. Sin. 2016, 32, 2761. doi: 10.3866/PKU.WHXB201608261

  24. [24]

      Zou, Y. J.; He, S. S.; Du, J. Z. Chin. J. Polym. Sci. 2018, 36, 1239. doi: 10.1007/s10118-018-2156-1

  25. [25]

      余康, 田翠翠, 李霞, 廖学品, 石碧.物理化学学报, 2018, 34, 543. doi: 10.3866/PKU.WHXB201709291Yu, K.; Tian, C.; Li, X.; Liao, X.; Shi, B. Acta Phys. -Chim. Sin. 2018, 34, 543. doi: 10.3866/PKU.WHXB201709291

  26. [26]

      Xu, D. F.; Cai, J.; Zhang, L. N. Chin. J. Polym. Sci. 2016, 34, 1281. doi: 10.1007/s10118-016-1840-2

  27. [27]

      Wade, R. J.; Burdick, J. A. Nano Today 2014, 9, 722. doi: 10.1016/j.nantod.2014.10.002

  28. [28]

      Smith, K. H.; Tejeda-Montes, E.; Poch, M.; Mata, A. Chem. Soc. Rev. 2011, 40, 4563. doi: 10.1039/c1cs15064b

  29. [29]

      Oh, J. K. Soft Matter 2011, 7, 5096. doi: 10.1039/c0sm01539c

  30. [30]

      Zhou, Y.; Huang, W.; Liu, J.; Zhu, X.; Yan, D. Adv. Mater. 2010, 22, 4567. doi: 10.1002/adma.201000369

  31. [31]

      Mai, Y.; Eisenberg, A. Chem. Soc. Rev. 2012, 41, 5969. doi: 10.1039/c2cs35115c

  32. [32]

      Philp, D.; Stoddart, J. F. Angew. Chem., Int. Ed. 1996, 35, 1154. doi: 10.1002/anie.199611541

  33. [33]

      Li, H.; Li, J.; He, X.; Zhang, B.; Liu, C.; Li, Q.; Zhu, Y.; Huang, W.; Zhang, W.; Qian, H.; et al. Chin. Chem. Lett. 2019, 30, 1083. doi: 10.1016/j.cclet.2019.01.003

  34. [34]

      Yang, Z.; Peng, Y.; Qiu, L. Chin. Chem. Lett. 2018, 29, 1839. doi: 10.1016/j.cclet.2018.11.009

  35. [35]

      Antonietti, M.; Forster, S. Adv. Mater. 2003, 15, 1323. doi: 10.1002/adma.200300010

  36. [36]

      Sun, T.; Yang, X.; Zhu, C.; Zhao, N.; Dong, H.; Xu, J. Chin. Chem. Lett. 2019, 30, 477. doi: 10.1016/j.cclet.2018.07.014

  37. [37]

      Zhao, X.; Pan, F.; Xu, H.; Yaseen, M.; Shan, H.; Hauser, C. A. E.; Zhang, S.; Lu, J. R. Chem. Soc. Rev. 2010, 39, 3480. doi: 10.1039/b915923c

  38. [38]

      Bryaskova, R.; Pencheva, D.; Kyulavska, M.; Bozukova, D.; Debuigne, A.; Detrembleur, C. J. Colloid Interface Sci. 2010, 344, 424. doi: 10.1016/j.jcis.2009.12.040

  39. [39]

      Yuan, W. Z.; Wei, J. R.; Lu, H.; Fan, L.; Du, J. Z. Chem. Commun. 2012, 48, 6857. doi: 10.1039/c2cc31529g

  40. [40]

      Xi, Y. J.; Wang, Y.; Gao, J. Y.; Xiao, Y. F.; Du, J. Z. ACS Nano 2019. doi: 10.1021/acsnano.9b03237

  41. [41]

      Benson, J. R.; Hare, P. E. Proc. Natl. Acad. Sci. U. S. A. 1975, 72, 619. doi: 10.1073/pnas.72.2.619

  42. [42]

      Deacon, J.; Abdelghany, S. M.; Quinn, D. J.; Schmid, D.; Megaw, J.; Donnelly, R. F.; Jones, D. S.; Kissenpfennig, A.; Elborn, J. S.; Gilmore, B. F.; et al. J. Controlled Release 2015, 198, 55. doi: 10.1016/j.jconrel.2014.11.022

  43. [43]

      Lilja, M.; Soerensen, J. H.; Brohede, U.; Astrand, M.; Procter, P.; Arnoldi, J.; Steckel, H.; Stromme, M. J. Mater. Sci.: Mater. Med. 2013, 24, 2265. doi: 10.1007/s10856-013-4979-1

  44. [44]

      Zhou, C. C.; Yuan, Y.; Zhou, P. Y.; Wang, F. Y. K.; Hong, Y. X.; Wang, N. S.; Xu, S. G.; Du, J. Z. Biomacromolecules 2017, 18, 4154. doi: 10.1021/acs.biomac.7b01209

  45. [45]

      Wise, J. P.; Goodale, B. C.; Wise, S. S.; Craig, G. A.; Pongan, A. F.; Walter, R. B.; Thompson, W. D.; Ng, A. K.; Aboueissa, A. M.; Mitani, H.; et al. Aquat. Toxicol. 2010, 97, 34. doi: 10.1016/j.aquatox.2009.11.016

  46. [46]

      Wei, L.; Cai, C.; Lin, J.; Chen, T. Biomaterials 2009, 30, 2606. doi: 10.1016/j.biomaterials.2009.01.006

  47. [47]

      Johnson, W. C., Jr.; Tinoco, I., Jr. J. Am. Chem. Soc. 1972, 94, 4389. doi: 10.1021/ja00767a084

  48. [48]

      Sun, J.; Deng, C.; Chen, X.; Yu, H.; Tian, H.; Sun, J.; Jing, X. Biomacromolecules 2007, 8, 1013. doi: 10.1021/bm0609792

  49. [49]

      Chung, J. E.; Yokoyama, M.; Okano, T. J. Controlled Release 2000, 65, 93. doi: 10.1016/s0168-3659(99)00242-4

  50. [50]

      Chan, A. S.; Chen, C. H.; Huang, C. M.; Hsieh, M. F. J. Nanosci. Nanotechnol. 2010, 10, 6283. doi: 10.1166/jnn.2010.2536

  51. [51]

      Hang, Z.; Ni, R.; Zhou, J.; Mao, S. Drug Discovery Today 2015, 20, 380. doi: 10.1016/j.drudis.2014.09.020

•