Biopolym. Cell. 2022; 38(1):58-68.
Bioorganic Chemistry
Transformation of the moss (Ceratodon purpureus) with plasmid DNA delivered by novel block-copolymers of the dimethylaminoethyl methacrylate
1, 2Finiuk N. S., 3Mitina N. E., 4Lobachevska O. V., 3Zaichenko A. S., 1, 2Stoika R. S.
  1. Institute of Cell Biology, NAS of Ukraine
    14/16, Drahomanov Str., Lviv, Ukraine, 79005
  2. Ivan Franko National University of Lviv
    4, Hrushevskoho Str., Lviv, Ukraine, 79005
  3. Lviv Polytechnic National University
    12, S. Bandery Str., Lviv, Ukraine, 79013
  4. Institute of Ecology of the Carpathians of National Academy of Sciences of Ukraine
    4, Kozelnytska Str., Lviv, Ukraine, 79026


Aim. To investigate the potential of poly(2-dimethylamino)ethyl methacrylate (DMAEMA)-based block-like polymers to serve as gene delivery carriers in moss Ceratodon purpureus (Hedw.) Brid. protoplasts, and to evaluate the level of their phytotoxicity. Methods. Organic synthesis; DNA gel retardation assay; adapted PEG-mediated transformation protocol; PCR; light microscopy. Results. The formation of pDNA complex with DMAEMA-based carriers took place at 0.01-0.1 % concentrations of the polymer. The poly-DMAEMA carriers F8-DM1, F8-DM2 (fluorine-containing), LAcr-DM1, LAcr-DM2 (lauryl acrylate-containing), BAcr-DM1, and BAcr-DM2 (butyl acrylate-containing) were effective as carriers of plasmid DNA pSF3 at C. purpureus transformation. PCR analysis confirmed that the transformants of C. purpureus moss contain GFP as a gene of interest after the protoplast transformation by polymers LAcr-DM2, LAcr-DM1, BAcr-DM2, BAcr-DM1 and F8-DM2. The poly-DMAEMA carriers at working concentration (0.0025 %) were relatively non-toxic for protoplasts of C. purpureus moss. 83.1-93.9 % of viable protoplasts of C. purpureus moss were found after the treatment with studied carriers at that dose. However, at 0.25 % i.e. 100 times higher concentration than that used for moss transformation, the poly-DMAEMA carriers reached their IC50 level. Conclusion. The novel block-like poly-DMAEMA carriers were effective in transformation of C. purpureus moss protoplasts and demonstrated low toxicity.
Keywords: polymeric carrier, poly(2-dimethylamino)ethyl methacrylate, moss Ceratodon purpureus, protoplasts transformation, toxicity.


[1] Kumar S, Nehra M, Dilbaghi N, Marrazza G, Tuteja SK, Kim KH. Nanovehicles for plant modifications towards pest- and disease-resistance traits. Trends Plant Sci. 2020; 25(2):198-212.
[2] Liu Y, Wu H, Chen H, Liu Y, He J, Kang H, Sun Z, Pan G, Wang Q, Hu J, Zhou F, Zhou K, Zheng X, Ren Y, Chen L, Wang Y, Zhao Z, Lin Q, Wu F, Zhang X, Guo X, Cheng X, Jiang L, Wu C, Wang H, Wan J. A gene cluster encoding lectin receptor kinases confers broad-spectrum and durable insect resistance in rice. Nat Biotechnol. 2015; 33(3):301-5.
[3] Himmel ME, Ding SY, Johnson DK, Adney WS, Nimlos MR, Brady JW, Foust TD. Biomass recalcitrance: engineering plants and enzymes for biofuels production. Science. 2007; 315(5813):804-7.
[4] Chen Q, Lai H. Gene delivery into plant cells for recombinant protein production. Biomed Res Int. 2015; 2015:932161.
[5] Demirer GS, Zhang H, Matos JL, Goh NS, Cunningham FJ, Sung Y, Chang R, Aditham AJ, Chio L, Cho MJ, Staskawicz B, Landry MP. High aspect ratio nanomaterials enable delivery of functional genetic material without DNA integration in mature plants. Nat Nanotechnol. 2019; 14(5):456-64.
[6] Baltes NJ, Gil-Humanes J, Voytas DF. Genome engineering and agriculture: opportunities and challenges. Prog Mol Biol Transl Sci. 2017; 149:1-26.
[7] Altpeter F, Springer NM, Bartley LE, Blechl AE, Brutnell TP, Citovsky V, Conrad LJ, Gelvin SB, Jackson DP, Kausch AP, Lemaux PG, Medford JI, Orozco-Cárdenas ML, Tricoli DM, Van Eck J, Voytas DF, Walbot V, Wang K, Zhang ZJ, Stewart CN Jr. Advancing crop transformation in the era of genome editing. Plant Cell. 2016; 28(7):1510-20.
[8] Liu YC, Vidali L. Efficient polyethylene glycol (PEG) mediated transformation of the moss Physcomitrella patens. J Vis Exp. 2011; (50):2560.
[9] King BC, Vavitsas K, Ikram NK, Schrøder J, Scharff LB, Bassard JÉ, Hamberger B, Jensen PE, Simonsen HT. In vivo assembly of DNA-fragments in the moss, Physcomitrella patens. Sci Rep. 2016; 6:25030.
[10] Jing L, Wenjing Q, Dan S, Zhengquan H. Genetic transformationof moss plant. Afr J biotechnol. 2013; 12(3):227-32,
[11] Gleba Y, Klimyuk V, Marillonnet S. Viral vectors for the expression of proteins in plants. Curr Opin Biotechnol. 2007; 18(2):134-41.
[12] Mizrachi A, Shamay Y, Shah J, Brook S, Soong J, Rajasekhar VK, Humm JL, Healey JH, Powell SN, Baselga J, Heller DA, Haimovitz-Friedman A, Scaltriti M. Tumour-specific PI3K inhibition via nanoparticle-targeted delivery in head and neck squamous cell carcinoma. Nat Commun. 2017; 8:14292.
[13] Pamornpathomkul B, Wongkajornsilp A, Laiwattanapaisal W, Rojanarata T, Opanasopit P, Ngawhirunpat T. A combined approach of hollow microneedles and nanocarriers for skin immunization with plasmid DNA encoding ovalbumin. Int J Nanomedicine. 2017; 12:885-98.
[14] Sung YK, Kim SW. Recent advances in the development of bio-reducible polymers for efficient cancer gene delivery systems. Cancer Med J. 2019; 2(1):6-13.
[15] Lee HJ, Park J, Lee GJ, Oh JM, Kim TI. Polyethylenimine-functionalized cationic barley β-glucan derivatives for macrophage RAW264.7 cell-targeted gene delivery systems. Carbohydr Polym. 2019; 226:115324.
[16] Chang FP, Kuang LY, Huang CA, Jane WN, Hung Y, Hsing YC, Mou CY. A simple plant gene delivery system using mesoporous silica nanoparticles as carriers. J Mater Chem B. 2013; 1(39):5279-87.
[17] Demirer GS, Zhang H, Goh NS, Pinals RL, Chang R, Landry MP. Carbon nanocarriers deliver siRNA to intact plant cells for efficient gene knockdown. Sci Adv. 2020; 6(26):eaaz0495.
[18] Zhang H, Cao Y, Xu D, Goh NS, Demirer GS, Cestellos-Blanco S, Chen Y, Landry MP, Yang P. Gold-nanocluster-mediated delivery of sirna to intact plant cells for efficient gene knockdown. Nano Lett. 2021; 21(13):5859-66.
[19] Mitter N, Worrall EA, Robinson KE, Li P, Jain RG, Taochy C, Fletcher SJ, Carroll BJ, Lu GQ, Xu ZP. Clay nanosheets for topical delivery of RNAi for sustained protection against plant viruses. Nat Plants. 2017; 3:16207.
[20] Wong MH, Misra RP, Giraldo JP, Kwak SY, Son Y, Landry MP, Swan JW, Blankschtein D, Strano MS. Lipid exchange envelope penetration (LEEP) of nanoparticles for plant engineering: a universal localization mechanism. Nano Lett. 2016; 16(2):1161-72.
[21] Loczenski Rose V, Shubber S, Sajeesh S, Spain SG, Puri S, Allen S, Lee DK, Winkler GS, Mantovani G. Phosphonium polymethacrylates for short interfering RNA delivery: effect of polymer and RNA structural parameters on polyplex assembly and gene knockdown. Biomacromolecules. 2015; 16(11):3480-90.
[22] Li L, Tian H, He J, Zhang M, Li Z, Ni P. Fabrication of aminated poly(glycidyl methacrylate)-based polymers for co-delivery of anticancer drugs and the p53 gene. J Mater Chem B. 2020; 8(41):9555-65.
[23] Haladjova E, Chrysostomou V, Petrova M, Ugrinova I, Pispas S, Rangelov S. Physicochemical properties and biological performance of polymethacrylate based gene delivery vector systems: Influence of amino functionalities. Macromol Biosci. 2021; 21(2):e2000352.
[24] Paiuk O, Mitina N, Slouf M, Pavlova E, Finiuk N, Kinash N, Karkhut A, Manko N, Gromovoy T, Hevus O, Shermolovich Y, Stoika R, Zaichenko A. Fluorine-containing block/branched polyamphiphiles forming bioinspired complexes with biopolymers. Colloids Surf B Biointerfaces. 2019; 174:393-400.
[25] Ficen SZ, Guler Z, Mitina N, Finiuk N, Stoika R, Zaichenko A, Ceylan SE. Biophysical study of novel oligoelectrolyte-based nonviral gene delivery systems for mammalian cells. J Gene Med. 2013; 15(5):193-204.
[26] Cheng H, Wu Z, Wu C, Wang X, Liow SS, Li Z, Wu YL. Overcoming STC2 mediated drug resistance through drug and gene co-delivery by PHB-PDMAEMA cationic polyester in liver cancer cells. Mater Sci Eng C Mater Biol Appl. 2018; 83:210-7.
[27] Filyak Y, Finiuk N, Mitina N, Bilyk O, Titorenko V, Hrydzhuk O, Zaichenko A, Stoika R. A novel method for genetic transformation of yeast cells using oligoelectrolyte polymeric nanoscale carriers. Biotechniques. 2013; 54(1):35-43.
[28] Finiuk N, Buziashvili A, Burlaka O, Zaichenko A, Mitina N, Miagkota O, Lobachevska O, Stoika R, Blume Ya, Yemets A. Investigation of novel oligoelectrolyte polymer carriers for their capacity of DNA delivery into plant cells. Plant Cell Tiss Organ Cult. 2017; 131:27-39.
[29] Wagner TA, Cove DJ, Sack FD. A positively gravitropic mutant mirrors the wild-type protonemal response in the moss Ceratodon purpureus. Planta. 1997; 202(2):149-54.
[30] Byun MY, Seo S, Lee J, Yoo Y-H, Lee H. Transfection of arctic Bryum sp. KMR5045 as a model for genetic engineering of cold-tolerant mosses. Front Plant Sci. 2021; 11:609847.
[31] Zaichenko A, Mitina N, Shevchuk O, Rayevska K, Lobaz V, Skorokhoda T, Stoika R. Development of novel linear, block and branched oligoelectrolytes and functionally targeting nanoparticles. Pure Appl Chem. 2008; 80(11):2309-26.
[32] Cove DJ, Perroud PF, Charron AJ, McDaniel SF, Khandelwal A, Quatrano RS. The moss Physcomitrella patens: a novel model system for plant development and genomic studies. Cold Spring Harb Protoc. 2009; 2009(2):pdb.emo115.
[33] Zeidler M, Hartmann E, Hughes J. Transgene expression in the moss Ceratodon purpureus. J Plant Physiol. 1999; 154(5-6):641-50.
[34] Cunningham FJ, Goh NS, Demirer GS, Matos JL, Landry MP. Nanoparticle-mediated delivery towards advancing plant genetic engineering. Trends Biotechnol. 2018; 36(9):882-97.
[35] Hayes M, Smith A, Arrasmith C, Davis W, Daniels CR. Initial characterization of PDMAEMA: Styrene porous polymer monolithic morphologies. Appl Sci. 2021; 11(15):7097.
[36] Chountoulesi M, Pippa N, Chrysostomou V, Pispas S, Chrysina ED, Forys A, Otulakowski L, Trzebicka B, Demetzos C. Stimuli-responsive lyotropic liquid crystalline nanosystems with incorporated poly(2-dimethylamino ethyl methacrylate)-b-poly(lauryl methacrylate) amphiphilic block copolymer. Polymers (Basel). 2019; 11(9):1400.
[37] Synatschke CV, Schallon A, Jérôme V, Freitag R, Müller AH. Influence of polymer architecture and molecular weight of poly(2-(dimethylamino)ethyl methacrylate) polycations on transfection efficiency and cell viability in gene delivery. Biomacromolecules. 2011; 12(12):4247-55.
[38] Lv J, Cheng Y. Fluoropolymers in biomedical applications: state-of-the-art and future perspectives. Chem Soc Rev. 2021; 50(9):5435-67.
[39] Tan E, Lv J, Hu J, Shen W, Wang H, Cheng Y. Statistical versus block fluoropolymers in gene delivery. J Mater Chem B. 2018; 6(44):7230-8.
[40] Yuan Z, Guo X, Wei M, Xu Y, Fang Z, Feng Y, Yuan W. Novel fluorinated polycationic delivery of anti-VEGF siRNA for tumor therapy. NPG Asia Materials. 2020; 12:1-11.
[41] Liu H, Wang Y, Wang M, Xiao J, Cheng Y. Fluorinated poly(propylenimine) dendrimers as gene vectors. Biomaterials. 2014; 35(20):5407-13.