Effect of surface organic coatings of cellulose nanocrystals on the viability of mammalian cell lines

Abstract

Cellulose nanocrystals (CNCs) have emerged as promising candidates for a number of bio-applications. Surface modification of CNCs continues to gain significant research interest as it imparts new properties to the surface of the nanocrystals for the design of multifunctional CNCs-based materials. A small chemical surface modification can potentially lead to drastic behavioral changes of cell-material interactions thereby affecting the intended bio-application. In this work, unmodified CNCs were covalently decorated with four different organic moieties such as a diaminobutane fragment, a cyclic oligosaccharide (β-cyclodextrin), a thermoresponsive polymer (poly[N-isopropylacrylamide]), and a cationic aminomethacrylamide-based polymer using different synthetic covalent methods. The effect of surface coatings of CNCs and the respective dose-response of the above organic moieties on the cell viability were evaluated on mammalian cell cultures (J774A.1 and MFC-7), using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-nyltetrazolium bromide and lactate dehydrogenase assays. Overall, the results indicated that cells exposed to surface-coated CNCs for 24 h did not display major changes in cell viability, membrane permeability as well as cell morphology. However, with longer exposure, all these parameters were somewhat affected, which appears not to be correlated with either anionic or cationic surface coatings of CNCs used in this study.

Keywords: LDH; MTT; cell viability; cellulose nanocrystals; surface coating.

Figure 1

FT-IR of (A) unmodified CNCs, (B) CNCs-poly(APMA), (C) CNCs-β-CD. Abbreviations: CNCs, cellulose nanocrystals; CNCs-β-CD, CNCs grafted with β-cyclodextrin; FT-IR, Fourier transform infrared spectroscopy; poly(APMA), poly(N-3-aminopropyl)methacrylamide; poly(NIPAAm), poly(N-isopropylacrylamide).

Figure 2

STEM images of unmodified and modified CNCs: (A) unmodified CNCs, (B) CNCs-AMINE, (C) CNCs-poly(NIPAAm), and (D) CNCs-poly(APMA). Note: Scale bar=100 nm. Abbreviations: AMINE, amino-functionalized; CNCs, cellulose nanocrystals; CNCs-β-CD, CNCs grafted with β-cyclodextrin; poly(APMA), poly(N-3-aminopropyl)methacrylamide; poly(NIPAAm), poly(N-isopropylacrylamide); STEM, scanning transmission electron microscopy.

Figure 3

Cytotoxicity of unmodified and modified CNCs on J774A.1 (A) and MCF-7 (B) cells. Notes: After 24 h of treatment, cell viability was determined by MTT assay. Data are expressed as percentage of control (non-treated cells, 100% viability) using the mean value and SD from triplicate experiments. *p<0.01 compared to control. Abbreviations: AMINE, amino-functionalized; CNCs, cellulose nanocrystals; CNCs-β-CD, CNCs grafted with β-cyclodextrin; MTT, 4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide; poly(APMA), poly(N-3-aminopropyl)methacrylamide; poly(NIPAAm), poly(N-isopropylacrylamide).

Figure 4

Cytotoxicity of unmodified and modified CNCs on J774A.1 (A) and MCF-7 (B) cells. Notes: After 48 h of treatment, cell viability was determined by MTT assay. Data are expressed as percentage of control (non-treated cells, 100% viability) using the mean value and SD from triplicate experiments. *p<0.01 compared to control. Abbreviations: AMINE, amino-functionalized; CNCs, cellulose nanocrystals; CNCs-β-CD, CNCs grafted with β-cyclodextrin; MTT, 4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide; poly-(APMA), poly-(N-3-aminopropyl) methacrylamide; poly-(NIPAAm), poly(N-isopropylacrylamide).

Figure 5

Cytotoxicity of unmodified and modified CNCs on J774A.1 cells. Notes: After 48 h of treatment, cell viability was determined by LDH assay. Data are expressed as percentage of cytotoxicity over the control using the mean value and SD from triplicate experiments. *p<0.01 compared to control. Abbreviations: CNCs, cellulose nanocrystals; CNCs-β-CD, CNCs grafted with β-cyclodextrin; LDH, lactate dehydrogenase; poly-(APMA), poly-(N-3-aminopropyl) methacrylamide; poly-(NIPAAm), poly(N-isopropylacrylamide).

Figure 6

Microscope images of J774A.1 cells treated with unmodified and modified CNCs (50 μg/mL). Notes: After indicated times of treatment, images of live cells were taken in bright field with inverted microscope at 400× magnification. White arrows indicate nanomaterial-cell aggregates, black arrows indicate membrane-nanomaterial interactions and red arrows indicate cell enlargement and elongation. Abbreviations: AMINE, amino-functionalized; CNCs, cellulose nanocrystals; CNCs-β-CD, CNCs grafted with β-cyclodextrin; APMA, (N-3-aminopropyl) methacrylamide; NIPAAm, N-isopropylacrylamide.

Scheme 1

Synthesis of surface-modified CNCs (A) anionic CNCs-AMINE, (B) anionic CNCs-β-CD, (C) anionic CNCs-poly(NIPAAm) and cationic CNCs-poly(APMA). Abbreviations: AMINE, amino-functionalized; CNCs, cellulose nanocrystals; CNCs-β-CD, CNCs grafted with β-cyclodextrin; poly-(APMA), poly-(N-3-aminopropyl) methacrylamide; poly-(NIPAAm), poly(N-isopropylacrylamide); rt, room temperature; EDC, N-Ethyl-N′-(3-dimethylaminopropyl)carbodiimide; NHS, N-hydroxysuccinimide; MES, 2-(N-Morpholino)ethanesulfonic acid; THF, tetrahydrofuran; DMAP, 4-(Dimethyamino)pyridine; PMDETA, N,N,N′,N″,N″-pentamethyldiethylenetriamine.