TSitologiya i Genetika 2020, vol. 54, no. 5, 75-88
Cytology and Genetics 2020, vol. 54, no. 5, 437–448, doi: https://www.doi.org/10.3103/S0095452720050096

Evaluation of phytotoxicity and mutagenicity of novel DMAEMA-containing gene carriers

Finiuk N., Romanyuk N., Mitina N., Lobachevska O., Zaichenko A., Terek O., Stoika R.

  1. Institute of Cell Biology of National Academy of Sciences of Ukraine, Drahomanov st., 14/16, Lviv, 79005, Ukraine
  2. Ivan Franko National University of Lviv, Hrushevskyy st., 4, Lviv, 79005, Ukraine
  3. Lviv Polytechnic National University, Bandera st., 12, Lviv, 79013, Ukraine
  4. Institute of Ecology of the Carpathians of National Academy of Sciences of Ukraine, Kozelnytska st., 4, Lviv, 79026, Ukraine

SUMMARY. A use of novel carriers for gene delivery is rapidly gro-wing, thus, investigation of potential phytotoxic and mutagenic action of gene delivery carriers is important for excluding their negative side effects. We found that poly-DMAEMA carriers used in 0,0025 % dose exhibited weak cytotoxic effect towards Allium cepa plant. In higher dose (0,025 %), they slightly (by 26–55 %) increased the level of catalase activity, but they did not affect the level of superoxide dismutase activity and malonic dialdehyde content in roots of A. cepa. The results of ana-telophase test in A. cepa did not demonstrate a genotoxic activity of the polymeric carriers used in 0.0025 % concentration and its higher dose (0,025 %). Slight genotoxic activity was detected only for BGP24 and BGP26, PEG-containing poly-DMAEMA carriers used in 0,025 %. The DMAEMA-based polymers did not possess muta-genic potential estimated in Ames test (–S9 and +S9). Thus, low phytotoxicity and absence of mutagenic action of novel polymeric carriers suggest their potential as promising nanocarriers for gene delivery into plant cells.

Keywords: poly(2-dimethylamino)ethyl methacrylate, polymeric carrier, ana-telophase assay, Ames test, catalase, superoxide dismutase, malonic dialdehyde

TSitologiya i Genetika
2020, vol. 54, no. 5, 75-88

Current Issue
Cytology and Genetics
2020, vol. 54, no. 5, 437–448,
doi: 10.3103/S0095452720050096

Full text and supplemented materials


1. Cunningham, F.J., Goh, N.S., Demirer, G.S., Matos, J.L., and Landry, M.P., Nanoparticle-mediated delivery towards advancing plant genetic engineering, Trends Biotechnol., 2018, vol. 36, no. 9, pp. 882–897. https://doi.org/10.1016/j.tibtech.2018.03.009

2. Demirer, G.S., Zhang, H., Matos, J.L., Goh, N.S., Cunningham, F.J., Sung, Y., Chang, R., Aditham, A.J., Chio, L., Cho, M.J., Staskawicz, B., and Landry, M.P., High-aspect ratio nanomaterials enable delivery of functional genetic material without DNA integration in mature plants, Nat. Nanotechnol., 2019, vol. 14, no. 5, pp. 456–464. https://doi.org/10.1038/s41565-019-0382-5

3. Tomlinson and Rolland, A.P., Controllable gene therapy: pharmaceutics of non-viral gene delivery systems, J. Control. Release, 1996, vol. 39, nos. 2–3, pp. 357–372. https://doi.org/10.1016/0168-3659(95)00166-2

4. Lv, H., Zhang S., Wang B., Cui S., and Yan J. Toxicity of cationic lipids and cationic polymers in gene delivery. J. Control. Release, 2006, vol. 114, no. 1, pp. 100–109. https://doi.org/10.1016/j.jconrel.006.04.014

5. Cerda-Cristerna B.I., Flores H., Pozos-Guillén A., Pérez E., Sevrin C., and Grandfils C. Hemocompatibility assessment of poly(2-dimethylaminoethylmethacrylate) (PDMAEMA)-based polymers, J. Control. Release, 2011, vol. 153, no. 3, pp. 269–277. https://doi.org/10.1016/j.jconrel.2011.04.016

6. Plamper, F.A., Synatschke, C.V., Majewski, A.P., Schmalz, A., Schmalz, H., and Müller, A.H.E., Star-shaped poly[2-(dimethylamino)ethyl methacrylate] and its derivatives: toward new properties and applications, Polimery, 2014, vol. 59, no. 1, pp. 66–73. https://doi.org/10.14314/polimery.2014.066

7. Zhang, S., Xu, Y., Wang, B., Qiao, W., Liu, D., and Li, Z., Cationic compounds used in lipoplexes and polyplexes for gene delivery, J. Control. Release, 2004, vol. 100, no. 2, pp. 165–180. https://doi.org/10.1016/j.jconrel.2004.08.019

8. Agarwal, S., Zhang, Y., Maji, S., and Greiner, A., PDMAEMA based gene delivery materials, Materials Today, 2012, vol. 15, no. 9, pp. 388–393. https://doi.org/10.1016/S1369-7021(12)70165-7

9. Arnold, A.E, Czupiel, P., and Shoichet, M., Engineered polymeric nanoparticles to guide the cellular internalization and trafficking of small interfering ribonucleic acids, J. Control. Release, 2017, vol. 259, pp. 3–15. https://doi.org/10.1016/j.jconrel.2017.02.019

10. Cheng, Q., Du, L.L., Meng, L.W., Han, S.C., Wei, T., Wang, X.X., Wu, Y.D., Song, X.Y., Zhou, J.H., Zheng, S.Q., Huang, Y.Y., Liang, X.J., Cao, H.Q., Dong, A.J., and Liang, Z.C., The promising nanocarrier for doxorubicin and siRNA co-delivery by PDMAEMA-based amphiphilic nanomicelles, ACS Appl. Mater. Interfaces, 2016, vol. 8, no. 7, pp. 4347–4356. https://doi.org/10.1021/acsami.5b11789

11. Ficen, S.Z., Guler, Z., Mitina, N., Finuk, N., Stoika, R., Zaichenko, A., and Ceylan, S.E., Biophysical study of novel oligoelectrolyte based non-viral gene delivery systems to mammalian cells, J. Gene Med., 2013, vol. 15, no. 5, pp. 193–204. https://doi.org/10.1002/jgm.2710

12. Filyak, Ye., Finiuk, N., Mitina, N., Bilyk, O., Titorenko, V., Hrydzhuk, O., Zaichenko, A., and Stoika, R., A novel method for genetic transformation of yeast cells using oligoelectrolyte polymeric nanoscale carriers, BioTechniques, 2013, vol. 54, no. 1, pp. 35–43. https://doi.org/10.2144/000113980

13. Finiuk, N., Chaplya, A., Mitina, N., Boiko, N., Lobachevska, O., Miahkota, O., Yemets, A., Blume, Ya., and Stoika, R., Genetic transformation of moss Ceratodon purpureus by means of polycationic carriers of DNA, Cytol. Genet., 2014, vol. 48, no. 6, pp. 345–351. https://doi.org/10.3103/S0095452714060048

14. Finiuk, N., Buziashvili, A., Burlaka, O., Zaichenko, A., Mitina, N., Miagkota, O., Lobachevska, O., Stoika, R., Blume, Ya., and Yemets, A., Investigation of novel oligoelectrolyte polymer carriers for their capacity of DNA delivery into plant cells, Plant Cell Tiss. Organ Cult., 2017, vol. 131, pp. 27–39. https://doi.org/10.1007/s11240-017-1259-7

15. von Gersdorff, K., Sanders, N.N., Vandenbroucke, R., De Smedt, S.C., Wagner, E., and Ogris, M., The internalization route resulting in successful gene expression depends on both cell line and polyethylene-minepolyplex type, Mol. Ther., 2006, vol. 14, no. 5, pp. 745–753. https://doi.org/10.1016/j.ymthe.2006.07.006

16. You, Y.Z., Manickam, D.S., Zhou, Q.H., and Oupický, D., Reducible poly(2-dimethylaminoethyl methacrylate): synthesis, cytotoxicity, and gene delivery activity, J. Control. Release, 2007, vol. 122, no. 3, pp. 217–225. https://doi.org/10.1016/j.jconrel.2007.04.020

17. Marslin, G., Sheeba, C.J., and Franklin, G., Nanoparticles alter secondary metabolism in plants via ROS burst, Front. Plant Sci., 2017, vol. 8, p. 832. https://doi.org/10.3389/fpls.2017.00832

18. Rao, S. and Shekhawat, G.S., Phytotoxicity and oxidative stress perspective of two selected nanoparticles in Brassica juncea, 3 Biotech, 2016, vol. 6, no. 2, p. 244. https://doi.org/10.1007/s13205-016-0550-3

19. Schallon, A., Jerome, V., Walther, A., Synatschke, C.V., Muller, A.H.E., and Freitag, R., Performance of three PDMAEMA-based polycation architectures as gene delivery agents in comparison to linear and branched PEI, React. Funct. Polym., 2010, vol. 70, no. 1, pp. 1–10. https://doi.org/10.1016/j.reactfunctpolym.2009.09.006

20. Voronov, S.A., Kiselyov, E.M., Minko, S.S., Budishevska, O.G., and Roiter, Y.V., Structure and reactivity of peroxide monomers, J. Polym. Sci. Pol. Chem., 1996, vol. 34, no. 12, pp. 2507–2511. https://doi.org/10.1002/(SICI)1099-0518(19960915)34:12<2507::AID-POLA24>3.0.CO;2-B

21. Paiuk, O., Mitina, N., Slouf, M., Pavlova, E., Finiuk, N., Kinash, N., Karkhut, A., Manko, N., Gromovoy, T., Hevus, O., Shermolovich, Y., Stoika, R., and Zaichenko, A., Fluorine-containing block/branched polyamphiphiles forming bioinspired complexes with biopolymers, Colloids Surf. B Biointerfaces, 2019, vol. 174, pp. 393–400. https://doi.org/10.1016/j.colsurfb.2018.11.047

22. Zaichenko A., Mitina, N., Shevchuk, O., Rayevska, K., Lobaz, V., Skorokhoda, T., and Stoika, R., Development of novel linear, block and branched oligoelectrolytes and functionally targeting nanoparticles, Pure Appl. Chem., 2008, vol. 80, no. 11, pp. 2309–2326. https://doi.org/10.1351/pac200880112309

23. Kirmse, W., Organic Elemental Analysis: Ultramicro, Micro, and Trace Methods, New York: Academic, 1983.

24. Critchfield, F.E., Organic Functional Group Analysis—International Series of Monographs on Analytical Chemistry, Pergamon Press, 1963.

25. Fiskesjo, G., Allium test, Methods Mol. Biol., 1995, vol. 43, pp. 19–127.

26. Cove, D., Perroud, P.F., Charron, A., McDaniel, S., Khandelwal, A., and Quatrano, R., The moss Physcomitrella patens. A novel model system for plant development and genomic studies, in Emerging Model Organisms, A Laboratory Manual, New York: Cold Spring Harbor Laboratory Press, 2009. https://doi.org/10.1101/pdb.emo115

27. Goth, L., A simple method for determination of serum catalase activity and revision of reference range, Clin. Chim., 1991, vol. 196, nos. 2–3, pp. 143–151. https://doi.org/10.1016/0009-8981(91)90067-M

28. Kumar, G. and Knowles, N.R., Changes in lipid peroxidation and lipolytic and free-radical scavenging enzyme activities during aging and sprouting of potato (Solanum tuberosum) seed-tubers, Plant Physiol., 1993, vol. 102, no. 1, pp. 115–124. https://doi.org/10.1104/pp.102.1.115

29. Rank, J. and Nielsen, M.H., A modified Allium test as a tool in the screening of the genotoxicity of complex mixtures, Hereditas, vol. 118, no. 1, pp. 49–53. https://doi.org/10.1111/j.1601-5223.1993.t01-3-00049.x

30. Kiełkowska, A., Allium cepa root meristem cells under osmotic (sorbitol) and salt (NaCl) stress in vitro,Acta Bot. Croat., 1993, vol. 76, no. 2, pp. 146–153. https://doi.org/10.1515/botcro-2017-0009

31. Mortelmans, K. and Zeiger, E., The Ames Salmonella/microsome mutagenicity assay, Mutat. Res., 2000, vol. 455, nos. 1–2, pp. 29–60. https://doi.org/10.1016/s0027-5107(00)00064-6

32. OECD Guideline for Testing of Chemicals: Bacterial Reverse Mutation Test, TG 471. Adopted July 1997. Available at chrome-extension://oemmndcbldboiebfnladdacbdfmadadm/https://www.oecd.org/chemicalsafety/risk-assessment/1948418.pdf.

33. Yang, J., Cao, W., and Rui, Y., Interactions between nanoparticles and plants: phytotoxicity and defense mechanisms, J. Plant Interact., 2017, vol. 12, no. 1, pp. 158–169. https://doi.org/10.1080/17429145.2017.1310944

34. Rajeshwari, A., Roy, B., Chandrasekaran, N., and Mukherjee, A., Cytogenetic evaluation of gold nanorods using Allium cepa test, Plant Physiol. Biochem., 2016, vol. 109, pp. 209–219. https://doi.org/10.1016/j.plaphy.2016.10.003

35. Shetty, A., Venkatesh, T., Suresh, P.S., and Tsutsumi, R., Exploration of acute genotoxic effects and antigenotoxic potential of gambogic acid using Allium cepa assay, Plant Physiol. Biochem., 2017, vol. 118, pp. 643–652. https://doi.org/10.1016/j.plaphy. 2017.08.005

36. Ahmed, B., Dwivedi, S., Abdin, M.Z., Azam, A., Al-Shaeri, M., Khan, M.S., Saquib, Q., Al-Khedhairy, A.A., and Musarrat, J., Mitochondrial and chromosomal damage induced by oxidative stress in Zn2+ ions, ZnO-bulk and ZnO-NPs treated Alliumcepa roots, Sci. Rep., 2017, vol. 7, p. 40685. https://doi.org/10.1038/srep40685

37. Lah, B., Zinko, B., Tisler, T., and Marinsek-Logara, R., Genotoxicity detection in drinking water by Ames test, Zimmermann test and Comet assay, Acta Chim. Slov., 2005, vol. 52, pp. 341–348.

38. McCarren, P., Springer, C., and Whitehead, L., An investigation into pharmaceutically relevant mutagenicity data and the influence on Ames predictive potential, J. Cheminform., 2011, vol. 3, p. 51. https://doi.org/10.1186/1758-2946-3-51

39. Lin, S., Du, F., Wang, Y., Li, S., Liang, D., Yu, L., and Li, Z., An acid-labile block copolymer of PDMAEMA and PEG as potential carrier for intelligent gene delivery systems, Biomacromolecules, 2008, vol. 9, no. 1, pp. 109–115. https://doi.org/10.1021/bm7008747

40. Sharma, R., Lee, J.-S., Bettencourt, R.C., Xiao, Ch., Konieczny, S.F., and Won, Y.-Y., Effects of the incorporation of a hydrophobic middle block into a PEG-polycation diblock copolymer on the physicochemical and cell interaction properties of the polyer-DNA complexes, Biomacromolecules, 2008, vol. 9, no. 1, pp. 3294–3297. https://doi.org/10.1021/bm800876v

41. Pirotton, S., Muller, C., Pantoustier, N., Botteman, F., Collinet, S., Grandfils, C., Dandrifosse, G., Degée, P., Dubois, P., and Raes, M., Enhancement of transfection efficiency through rapid and noncovalent post-PEGylation of poly(dimethylaminehtylmethacrlyate)/DNA complex, Pharm. Res., 2004, vol. 21, no. 8, pp. 1471–1479. https://doi.org/10.1023/b:pham.0000036923.25772.97

42. Hong, J., Peralta-Videa, J.R., Rico, C., Sahi, S., Viveros, M.N., Bartonjo, J., Zhao, L., and Gardea-Torresdey, J.L., Evidence of translocation and physiological impacts of foliar applied CeO2 nanoparticles on cucumber (Cucumis sativus) plants, Environ. Sci. Technol., 2014, vol. 48, no. 8, pp. 4376–4385. https://doi.org/10.1021/es404931g

43. Song, U. and Lee, E.J., Ecophysiological responses of plants after sewage sludge compost applications, J. Plant Biol., 2010, vol. 53, pp. 259–267. https://doi.org/10.1007/s12374-010-9112

44. Garg, N., and Manchanda, G., ROS generation in plants: boon or bane? Plant Biosyst., 2009, vol. 143, pp. 81–96. https://doi.org/10.1080/11263500802633626

45. Kenneth, W.A., Advanced Techniques in Chromosome Research, CRC Press, 1991.