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Colloids and Surfaces B: Biointerfaces xxx (2014) xxx–xxx

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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb

Size- and dose-dependent toxicity of cellulose nanocrystals (CNC) on human fibroblasts and colon adenocarcinoma Zahid Hanif c , Farrukh R. Ahmed c,d , Seung Won Shin a , Young-Kee Kim e , Soong Ho Um a,b,∗ a

School of Chemical Engineering Sungkyunkwan University, Suwon 440-746, South Korea SKKU Advanced Institute of Nanotechnology (SAINT), Sungkyunkwan University, Suwon 440-746, South Korea c Department of Materials Science and Engineering, Gwangju Institute of Science and Technology (GIST), Gwangju 500-712, South Korea d Department of Pharmaceutics, Ziauddin University, Karachi 75600, Pakistan e Department of Chemical Engineering, Hankyong National University, Anseong 456-749, South Korea b

a r t i c l e

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Article history: Received 15 March 2014 Received in revised form 18 April 2014 Accepted 23 April 2014 Available online xxx Keywords: Cellulose nanocrystal Toxicity Cancer cell

a b s t r a c t A controlled preparation of cellulose nanocrystals of different sizes and shapes has been carried out by acid hydrolysis of microcrystalline cellulose. The size- and concentration-dependent toxicity effects of the resulting cellulose nanocrystals were evaluated against two different cell lines, NIH3T3 murine embryo fibroblasts and HCT116 colon adenocarcinoma. It could serve as a therapeutic platform for cancer treatment. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Cellulose, which is a long polymer of ␤ (1–4) linked d-glucose, is one of the most abundant naturally occurring biodegradable and renewable polymers in the world. As an integral part of all plants, it can be derived from a variety of natural resources (e.g., wood, cotton, paper, pulp and microcrystalline cellulose) [1]. Cellulose fibrils are composed of two main parts including both amorphous regions and crystalline regions. The disordered region is easily hydrolyzed by several mineral acids and then changed into an ordered form, appearing as a cellulose nanocrystals [2]. Depending on the preparative method, other types of cellulose nanocrystals in either shape or size can be manufactured. For instance, rod-shaped cellulose nanocrystals are synthesized if the fibrils are exposed to sulfuric acid [3]. The sizes of the cellulose nanocrystals in suspension can be controlled by a mixture of acid components, such as sulfuric acid and hydrochloric acid, once hydrolyzed for a long period of time. The formation mechanism was investigated by studying the liquid crystallinity of the spherical-shaped cellulose nanocrystals [4]. Based on an ecotoxicology evaluation of cellulose nanocrystals on

∗ Corresponding author at: Corresponding author. Tel.: +82 312907348; fax: +82 312907272. E-mail addresses: [email protected], [email protected] (S.H. Um).

different aquatic species such as rain trout, it was determined that different concentrations of cellulose nanocrystals (10–200 mg/l) did not cause any genotoxicity as compared with carboxy methyl cellulose [5]. However, analysis of the potential cytotoxicity of cellulose nanofibers in human lung cells has shown lower cytotoxicity and inflammatory response as compared with both multiwall-carbon nanotubes and asbestos [6]. In particular, further inspection of in vitro cytotoxicity and genotoxicity of nanofibrillar celluloses using the most common cell lines such as human keratinocyte, human cervix carcinoma and mouse hepatoma has demonstrated absence of any cytotoxic or genotoxic behaviors [7]. To date, the use of cellulose nanocrystals has been broadly explored for targeted delivery of therapeutics and bioimaging agents [8–10]. Moreover, they are now utilized as a template for bottom-up hierarchical assembly. After additional chemical modifications, they are able to graft various reactive groups containing nucleic acids or peptides [11]. Inspired by these trends, we assessed the controlled synthesis of cellulose nanocrystals in different sizes and shapes through acid hydrolysis of microcrystalline cellulose. We investigated size- and concentration-dependent toxicity effects against two different cell lines, NIH3T3 murine embryo fibroblasts and HCT116 colon adenocarcinoma, to assess the potential use of cellulose nanocrystals in cancer therapeutics.

http://dx.doi.org/10.1016/j.colsurfb.2014.04.018 0927-7765/© 2014 Elsevier B.V. All rights reserved.

Please cite this article in press as: Z. Hanif, et al., Size- and dose-dependent toxicity of cellulose nanocrystals (CNC) on human fibroblasts and colon adenocarcinoma, Colloids Surf. B: Biointerfaces (2014), http://dx.doi.org/10.1016/j.colsurfb.2014.04.018

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2. Experimental 2.1. Materials Microcrystalline cellulose (Avicel PH-101) was purchased from Fluka. Sulphuric acid and hydrochloric acid were purchased from Daejung Chemical Company, South Korea. Dialysis tubes with a molecular cut-off size of 10,000 were purchased from Serva Electrophoresis, Germany. Deionized water was used to carry out all the experimental procedures. 2.2. Preparation of cellulose nanocrystals Four cellulose nanocrystals in different sizes, labeled CNC1, CNC2, CNC3 and CNC4, were prepared from microcrystalline cellulose (MCC) (Fluka, Avicel PH-101) via a controlled acid hydrolysis as described previously by our research group [12]. Briefly, CNC1 was prepared by acid hydrolysis of microcrystalline cellulose with 9 M sulphuric acid once exposed at 50 ◦ C for 180 min. CNC2 was prepared by the same method as that used for CNC1 except that it was hydrolyzed with a mixture of acids (mixture of 18 M H2 SO4 and 12 M HCl) at the volume ratio of 3:1). CNC3 was also prepared by the same method as that for CNC1 with one modification that it was first treated with 5 M of sodium hydroxide for 4 h, followed by a mixture of strong acids (18 M H2 SO4 and 12 M HCl) at a volume ratio of 3:1 for hydrolysis at 50 ◦ C for 140 min. CNC4 was similarly prepared by strong hydrochloric acid (12 M) hydrolysis at 50 ◦ C for 180 min. In each case, the reaction was quenched by adding a10-fold volume of deionized water, followed by complete removal of acids by a repeated washing-off, centrifugation and then dialysis. The cleanup process involved first centrifuging the suspension at 10,000 rpm for 10 min, decanting the sample, and then adding fresh deionized water and centrifuging again. The centrifugation/washing cycles were repeated five times until the pH of the solutions reached in the range of 3 to 5. The supernatant became turbid and was collected by dialysis and poured into deionized water for 4 days in order to make sure that it was free of any acid residues. It was finally lyophilized for 3 days in order to obtain a dry cellulose nanocrystalline in a white powder form. To evaluate the nanocrystals, a variety of imaging tools were used including scanning electron microscopy (Hitachi Model S4700H) and a transmission electron microscopy to study the external morphology of the cellulose crystals, and X-ray diffractometery (XR-Rigaku) was operated at 40 kV and 30 mA to record the crystalline patterns of all the samples.

Fig. 1. Crystalline analysis of MCC and CNCs (top view) and SEM images of MCC (Bottom view). As labeled in the inset, black line indicates the diffraction spectra of commercial MCC. The rest corresponds to the crystalline patterns of CNCs:CNC1 (red), CNC2 (green) and CNC3 (violet) and CNC4 (magenta), respectively. The scale bar in the SEM image represents 100 ␮m. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

with PBS buffer, followed by the addition of 100 ␮L of culture media containing various concentrations (e.g., 1000, 500, 250, 100, and 10 ␮g/mL) of cellulose nanocrystals. They were added into each cell-containing well. The cells were also treated with cellulose

2.3. Cell culture and reagents NIH3T3 murine embryo fibroblast cells and HCT116 colon adenocarcinomas, which were gifts from Prof. Sangyong Jon in KAIST, were maintained as adherent cultures and then grown as monolayers in a humidified incubator operated under 95% air and 5% CO2 at 37 ◦ C in Dulbecco’s Modified Eagle’s Medium (DMEM) containing high glucose media (GIBCO-Invitrogen, Grand Island, NY) supplemented with 10% (v/v) fetal bovine serum (FBS, GIBCO) and 100 IU/mL Anti-AntiR (GIBCO). 2.4. In-vitro cell viability assay The in-vitro cell viabilities of both HCT116 and NIH3T3 against cellulose nanocrystals (CNCs) were assessed using a WST-1 assay (Biovision, CA, USA). Cells were seeded into a 96-well plate (Flat Bottom Costar, Corning, NY, USA) at a density of 2.5 × 104 cells per a well for HCT116 and 3.0 × 104 cells per a well for NIH3T3. The cells were incubated at 37 ◦ C, 5% CO2 and 95% humidified air for 24 h. After 24 h incubation, the cells were washed out completely

Fig. 2. External morphologic observation of cellulose nanocrystals under TEM: (a) CNC1, (b) CNC2, (c) CNC3, (d) CNC4. All scale bars represent 200 nm except that of (d) is in 500 nm.

Please cite this article in press as: Z. Hanif, et al., Size- and dose-dependent toxicity of cellulose nanocrystals (CNC) on human fibroblasts and colon adenocarcinoma, Colloids Surf. B: Biointerfaces (2014), http://dx.doi.org/10.1016/j.colsurfb.2014.04.018

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Fig. 3. Size evaluation of synthetic cellulose nanocrystals. Summarized is the particle size of each cellulose nanocrystals (left) and the corresponding table shows the particular size values (nm) for each cellulose nanocrystal (right).

nanocrystals in various sizes. Non-treated cells of only culture media served as the control. Sample media with different concentrations of cellulose nanocrystals were poured into wells with no cells to serve as blank. The 96-well plates were then incubated for 24 h for the treatment of cells under the same conditions described above. Following the treatment with cellulose nanocrystals, the cells were gently washed three times with PBS and then 100 ␮L of fresh culture media was added to each well in the plate. Then, 10 ␮L of WST-1 solution, prepared according to the manufacturer’s protocol, was added. The plates were allowed to incubate for 1 h in the dark and then the absorbance (bottom-read) at 9 different points in each well was taken at scan mode with the wavelength

of 460 nm (note that 620 nm was used as a reference wavelength) using SpectraMax M5e (Molecular Devices) 96-well plate reader. For mathematical analysis, values from media were subtracted from corresponding values from treated cells in each well. The percent value indicating the cell viability was obtained by dividing values of treated cells by those of untreated cells as a control. 3. Results and discussion Fig. 1 shows the XRD diffraction patterns of both microcrystalline cellulose and cellulose nanocrystals as prepared by controlled acid hydrolysis with different aspect ratios, which are

Fig. 4. Cytotoxic assay of HCT116 and NIH3T3 cells treated with cellulose nanocrystals. According to a 24 h time period, cell viability was further studied at different concentrations of each CNC. Here, we note that WST-1 assay was used (* = p < 0.05 compared to the control; ˛ = p < 0.05 compared to cell viability at 500 ␮g/mL; n = 3).

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labeled as CNC1, CNC2, CNC3 and CNC4. XRD diffraction can be utilized to investigate the crystalline structure of pristine microcrystalline and also to confirm the subsequent acid-treatment of the microcrystalline for the formation of cellulose nanocrystals. The XRD spectra clearly showed that the acid treatment of microcrystalline cellulose with different acid compositions and concentrations at determined time periods did not significantly alter crystalline structures in the four samples, CNC1, CNC2, CNC3 and CNC4. Interestingly, the acid hydrolysis procedures did not disturb the crystallinity, but did affect the amorphous regions of microcrystalline cellulose [13,14]. As described in a previously published article from our group [12], all cellulose nanocrystals, even under different conditions, were observed to be rod-shaped, but with different mean sizes (Figs. 2 and 3): CNC1, 256 nm; CNC2, 140.5 nm; CNC3, 108.4 nm; and CNC4, 1174 nm. CNCs of different sizes were evaluated at various concentrations for in-vitro cell cytotoxicity against HCT116 colon carcinoma and NIH3T3 fibroblast cell lines by WST-1 assay. Our results showed that none of the four CNCs of varying size presented any substantial cytotoxicity against either cell line at various concentrations ranging from 10 ␮g/mL to 250 ␮g/mL (and even 500 ␮g/mL in the case of CNC2). These results are consistent with the previous study reporting apparently no cytotoxicity at lower concentration against various cell lines [5,15]. However, in our study, CNC1, CNC3 and CNC4, at much higher concentrations of 500 ␮g/mL and 1000 ␮g/mL, did induce significant cytotoxic effects, as shown in Fig. 4. Substantial cytotoxicity was also observed with CNC3 at 1000 ␮g/mL as compared to 500 ␮g/mL. The possible explanation to this phenomena could be that a substantial proportion of smaller particle sizes (below ∼50 nm) of CNC 3 might get uptaken in higher concentrations owing to clathrin and caveolin independent pinocytosis which favors this phenomena and subsequent cytotoxicity due to the interference of elongated nanocrystals with cellular organelles that can render structural and chemical damage [16–18]. In comparison with CNC1 and CNC2 in the smaller sizes, CNC4 was usually much more cytotoxic because of its tendency to form gel in a suspension when it was incubated at higher concentrations, which might have blocked the passage for gases through cell membranes. We clearly observed that the color of phenol red in the cell culture media was significantly more acidic in the case of CNC4 at 1000 ␮g/mL. Overall, the CNCs did not reveal cytotoxicity at various concentrations up to 250 ␮g/mL, demonstrating that CNCs of various sizes, as prepared in our laboratory exhibit remarkably great potential for future biomedical and biotechnological applications.

4. Conclusions We successfully constructed a cellulose nanocrystal using a facile acidic dissolution method of larger cellulose microcrystallines. It was proven that its cytotoxicity may be irrespective of different sizes as well as doses less than 250 ␮g/mL. We speculate that the biological compatibility of the CNCs may hold a promise for the future of biotechnological applications [7]. Acknowledgments This work was supported by grants from the National Research Foundation of Korea (NRF) funded by the Ministry of Future Creation and Science (grant nos. 2013R1A1A1058670 and 2013R1A1A2016781). Support was also provided by the Korea Health Technology R&D Project of the Ministry of Health and Welfare of the Republic of Korea (grant no. A110552). References [1] Y. Habibi, L.A. Lucia, O. Rojas, Chem. Rev. 110 (2010) 3479–3500. [2] S. Beck-Candanedo, M. Roman, D.G. Gray, Biomacromolecules 6 (2005) 1048–1054. [3] R.H. Marchessault, F.F. Morehead, N.M. Walter, Nature 184 (1959) 632–633. [4] N. Wang, E. Ding, R. Cheng, Langmuir 24 (2008) 5–8. [5] T. Kovacs, V. Naish, B. O’Connor, C. Blaise, F. Gagne, L. Hall, V. Trudeau, P. Martel, Nanotoxicology 4 (3) (2010) 255–270. [6] J.D. Martin, E. Clift, J. Foster, D. Vanhecke, D. Studer, P. Wick, P. Gehr, B. RothenRutishauser, C. Weder, Biomacromolecules 12 (2011) 3666–3673. [7] M. Pitkänen, U. Honkalampi, A. Von Wright, A. Sneck, H.P. Hentze, J. Sievanen, J. Hiltunen, E.K.O. Hellen, International Conference on Nanotechnology for Forest Products Industry, Otaneimi, Espoo, Finland, 27–29 September 2010, Curran, Finland, 2010. [8] K. Fleming, D. Gray, S. Prasannan, S. Matthews, J. Am. Chem. Soc. 122 (2000) 5224. [9] S.P. Dong, M. Roman, J. Am. Chem. Soc. 129 (2007) 13810. [10] Y. Habibi, A. Dufresne, Biomacromolecules 9 (2008) 1974. [11] A.P. Mangalam, J. Simonsen, A.S. Benight, Biomacromolecules 10 (2009) 497–504. [12] C. Baek, Z. Hanif, S-W. Cho, D-I. Kim, S.H. Um, J. Biomed. Nanotech. 9 (7) (2013) 1293–1298. [13] N. Wang, E. Ding, R. Cheng, Polymer 48 (12) (2007) 3486–3493. [14] L.D. Majdanac, D. Poleti, M.J. Teodorovic, Acta Polym. 42 (8) (1991) 351–357. [15] A. Hirani, University Libraries, Virginia Polytechnic Institute and State University, Blacksburg, VA, 2009. [16] J. Zhu, L. Liao, L. Zhu, P. Zhang, K. Guo, J. Kong, C. Ji, B. Liu, Talanta 107 (2013) 408–415. [17] Keiji Hirota, Hiroshi Terada, in: Brian Ceresa (Ed.), Endocytosis of Particle Formulations by Macrophages and Its Application to Clinical Treatment, Molecular Regulation of Endocytosis, InTech, Rijeka, 2012, http://dx.doi.org/10.5772/45820. [18] A. Arnida, H. Malugin, Ghandehari, J. Appl. Tox 30 (3) (2010) 212–217.

Please cite this article in press as: Z. Hanif, et al., Size- and dose-dependent toxicity of cellulose nanocrystals (CNC) on human fibroblasts and colon adenocarcinoma, Colloids Surf. B: Biointerfaces (2014), http://dx.doi.org/10.1016/j.colsurfb.2014.04.018

Size- and dose-dependent toxicity of cellulose nanocrystals (CNC) on human fibroblasts and colon adenocarcinoma.

A controlled preparation of cellulose nanocrystals of different sizes and shapes has been carried out by acid hydrolysis of microcrystalline cellulose...
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