Ultrasound-assisted Cu(I)-catalyzed azide-alkyne click cycloaddition as polymer-analogous transformation in chitosan chemistry. High antibacterial and transfection activity of novel triazol betaine chitosan derivatives and their nanoparticles
Authors: Artem P Dysin
Aleh V Kurliuk
Andreii S Kritchenkov
Elena P Suchkova
Tatsiana V Shakola
Ludmila A Zabodalova
Olga V Volkova
Anton R Egorov
Journal: International Journal of Biological Macromolecules (2019)
Discipline: BIOLOGY & BIOCHEMISTRY
Keywords: Ultrasound-assisted click CuAAC; Chitosan chemistry; Nanoparticles; Antibacterial activity; Transfection activity
Affiliations: Peoples' Friendship University of Russia (RUDN University), 6 Miklukho-Maklaya Street, Moscow 117198, Russian Federation; Institute of Macromolecular Compounds of the Russian Academy of Sciences, Bolshoi pr. VO 31, St. Petersburg 199004, Russian Federation; Saint-Petersburg National Research University of Information Technologies, Mechanics, and Optics, Kronverkskii pr. 49, 197101 St. Petersburg, Russian Federation. Electronic address: ., Institute of Macromolecular Compounds of the Russian Academy of Sciences, Bolshoi pr. VO 31, St. Petersburg 199004, Russian Federation; Saint-Petersburg National Research University of Information Technologies, Mechanics, and Optics, Kronverkskii pr. 49, 197101 St. Petersburg, Russian Federation., Saint-Petersburg National Research University of Information Technologies, Mechanics, and Optics, Kronverkskii pr. 49, 197101 St. Petersburg, Russian Federation., Vitebsk State Medical University, Frunze av. 27, Vitebsk 210009, Belarus.

Abstract:  In this work, we involved ultrasound-assisted click CuAAC in chitosan chemistry. Ultrasound-mediated CuAAC between propargylic ester of betaine and azido chitosan derivative proceeds fast in water under aerobic conditions and gives rise novel water-soluble triazole betaine chitosan derivatives. Using ionic gelation technique, we prepared and characterized nanoparticles from the synthesized chitosan derivatives. We studied antibacterial and transfection activity of the novel chitosan derivatives and their nanoparticles. The nanoparticles with size ca. 100 nm and ζ-potential ca. +65 mV proved to possess outstanding antibacterial activity, which is much more than that of the triazole betaine derivatives in their native form, and it is equal to the activity of ampicillin and gentamicin. Opposite, triazole betaine chitosan derivatives in their native form are characterized by remarkable transfection activity as compared with their nanoparticles. The most active triazole betaine chitosan derivatives are derivatives of moderate molecular weight with moderate degree of substitution. Their transfection activity is extremely high for chitosan species and it is comparable (values of the same order) with activity of Lipofectin - commercially available gene delivery vector. 

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PubPeer comments:
 ... 2. Section 2.11  The gel retardation assay was performed as described in [10]. No gel electrophoresis data is presented in the article. 3. Section 2.13 and 2.10 are from https: doi.org 10.1016 j.carbpol.2017.11.093 4. Section 3.1 For 18 chitosan derivatives bearing azide groups, not a single NMR spectrum is presented in the article. 5. Section 3.2. NMR and MS data are missing. 6. Section 3.3. For 18 samples containing triazol-betaine substituents (according to table 2) only one NMR spectrum is presented. Moreover 1H NMR spectrum presented on Fig. 1 does not correspond to structure mentioned. According to HMDB (human metabolome database) betaine methyl protons signal in water solution has a chemical shift of 3.25 ppm. In Fig. 1 there are no signals in range of 3.0 - 3.5 ppm. The only signal close to that range is wide signal at 3.5 ppm corresponding to H-2 proton of chitosan itself. Betaine methyl protons signal would be a singlet with integral intensity of 9xDS. If TB-CS-A-II-M had a DS of 0.41 (as a result of full conversion of AZ-CS-A-II-M from Table 1), it s intensity would be 3.69 which is much higher than acetyl protons signal at 2.4 ppm and H-2 at 3.5 ppm, which integral intensity is roughly around 1. ![file] 7. Section 3.5 In experimental section AFM and SEM are not described at all. 8. Section 3.6. From this section it is clear that the author mixes up two different methods: gel electrophoresis or electrophoretic mobility shift assay and EtBr fluorescence quenching. First authors describe gel electrophoresis (but do not present gel photos):  Formation of polyplexes u201cchitosan derivative DNAu201d significantly reduces electrophoretic mobility of DNA due to neutralization of negative charge of its phosphate groups and the formation of large spherical particles that are not capable of diffusion into the gel. Because of gel electrophoresis experiments, we found that DNA migration stops at mass ratio of polysaccharide: DNA 0.5:1. Then EtBr fluorescence quenching is described, but authors continue calling in gel electrophoresis with no raw data at all.  For the quantitative assessment of efficiency of DNA binding to cationic polymers, we used method of agarose gel electrophoresis of polyplexes in the presence of ethidium bromide. This method is based on the competitive replacement of a cationic dye by cationic polysaccharides, which leads to the quenching of fluorescence. We obtained polyplexes solutions with a chitosan derivative: DNA or starting chitosan:DNA mass ratio from 0.05:1 to 2:1. An increase for chitosan derivative in the samples led to a decrease in the fluorescence intensity of the bands on the electrophoregram as compared to the starting DNA. 9. Section 3.7. Transfection efficiency of control agents is the same by numbers as in previously published article ![file] ![file] ,