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. 2022 Dec 7;14(3):303-317.
doi: 10.1039/d2py01012g. eCollection 2023 Jan 17.

How does the polymer architecture and position of cationic charges affect cell viability?

Joana S Correia  1 , Sofía Mirón-Barroso  1 , Charlotte Hutchings  1 , Silvia Ottaviani  2   3 , Birsen Somuncuoğlu  1 , Leandro Castellano  3   4 , Alexandra E Porter  1 , Jonathan Krell  5 , Theoni K Georgiou  1
Affiliations

How does the polymer architecture and position of cationic charges affect cell viability?

Joana S Correia et al. Polym Chem. .

Abstract

Polymer chemistry, composition and molar mass are factors that are known to affect cytotoxicity, however the influence of polymer architecture has not been investigated systematically. In this study the influence of the position of the cationic charges along the polymer chain on cytotoxicity was investigated while keeping constant the other polymer characteristics. Specifically, copolymers of various architectures, based on a cationic pH responsive monomer, 2-(dimethylamino)ethyl methacrylate (DMAEMA) and a non-ionic hydrophilic monomer, oligo(ethylene glycol)methyl ether methacrylate (OEGMA) were engineered and their toxicity towards a panel of cell lines investigated. Of the seven different polymer architectures examined, the block-like structures were less cytotoxic than statistical or gradient/tapered architectures. These findings will assist in developing future vectors for nucleic acid delivery.

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Conflict of interest statement

There are no conflicts to declare.

Figures

Fig. 1
Fig. 1. Chemical structures and names of the monomers used in this study and a schematic representation of the architectures synthesised. OEGMA300 and DMAEMA units are represented in green and blue, respectively.
Fig. 2
Fig. 2. GPC chromatogram of the AB diblock copolymer, OEGMA30016-b-DMAEMA32 (P1), and its precursor. OEGMA30014 (abbreviated OEG14 on inset legend) and the final polymer (abbreviated OEG16-b-DMA32 on inset legend) are represented by the green dashed line and blue solid line, respectively.
Fig. 3
Fig. 3. H1 NMR spectra of the AB diblock copolymer, OEGMA30016-b-DMAEMA32 (P1), and its precursor. The first block of the copolymer, OEGMA300, is represented in green (chemical structure and spectra), the second block, DMAEMA, is represented in bright blue (chemical structure), and the final spectra in bright blue.
Fig. 4
Fig. 4. Zeta potential (ζ, mV) of each copolymer and PEI at physiological pH (at 1 wt% in PBS). Results are shows as mean ± SEM (n = 9). Dotted line represents neutral surface charge (ζ = 0 mV). Significances denoted were investigated using a one-way ANOVA and Tukey's multiple comparisons test, where ****P < 0.0001, ***P < 0.001, **P < 0.01 and *P < 0.05. P-Values in bracket compare each architecture to copolymer AB (P1). Significance in arrow (top) was found to be the same for all copolymers, when compared to PEI.
Fig. 5
Fig. 5. Effect of monomer distribution on non-malignant cell lines. HEK-293 (A) and HPNE (B) were treated with increasing concentrations of polymer solutions for 24 hours. Polymer concentrations, for all copolymers, are: 25 μg mL−1 (lilac), 50 μg mL−1 (green), 125 μg mL−1 (orange), 250 μg mL−1 (blue) and 500 μg mL−1 (pink). Polymer abbreviations for P1 to P7 are AB, ABA, BAB, ABAB, AcoB, A(AcoB)B and gradient, respectively. PEI was used a positive control (cytotoxic). Cell viability is reported as percentage relative to untreated control cells (0 μg mL−1, not plotted). Black dotted line indicates 100% cell viability (control). Data shown as mean ± SEM from triplicates. Differences in mean cell viability between polymers was investigated using Two-way ANOVA and Tukey's multiple comparison tests. Significance denoted ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05.
Fig. 6
Fig. 6. Effect of monomer distribution on PDAC cell lines. BxPC-3 (A), PANC-1 (B) and S2-007 (C) cells were treated with increasing concentrations of polymer solutions for 24 hours. Polymer concentrations, for all copolymers, are: 25 μg mL−1 (lilac), 50 μg mL−1 (green), 125 μg mL−1 (orange), 250 μg mL−1 (blue) and 500 μg mL−1 (pink). Polymer abbreviations for P1 to P7 are AB, ABA, BAB, ABAB, AcoB, A(AcoB)B and gradient, respectively. PEI was used a positive control (cytotoxic). Cell viability is reported as percentage relative to untreated control cells (0 μg mL−1, not plotted). Black dotted line indicates 100% cell viability (control). Data shown as mean ± SEM from triplicates. Differences in mean cell viability between polymers was investigated using Kruskal–Wallis tests and Dunn's multiple comparison testes. Significance denoted ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05.
Fig. 7
Fig. 7. Conceptual illustration of the effect of monomer distribution on cell membrane interaction. Blue sections represent partially protonated DMAEMA units, green sections represent other monomer units (in the case of this schematic, OEGMA300). A wider distribution of cationic charges may lead to a higher number of interactions with the negatively charged cellular membrane, disturbing the membrane across more locations, leading to increased toxicity.
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