Amino acid modified cellulose whiskers

Abstract

L-Leucine amino acid functionalized cellulose whiskers were synthesized via esterification reaction which comprised the reaction between Fmoc–L-leucine and cellulose whiskers and removal of Fmoc-protecting group. This strategy provides a direct, facile way to merge the properties of these biocompatible materials and offers the possibility to introduce biologically active building blocks in cellulose whiskers.

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Cellulose whiskers (CW), which are the fundamental constitutive polymeric motifs of macroscopic cellulosic fibers, have gained increasing attention over the past few years.¹ This trend has developed due to cellulose whiskers unique intrinsic properties such as its nano-scale dimension, high surface area, unique morphology, low density and mechanical strength properties along with their intrinsic renewability, sustainability and biocompatibility.^1,2 Moreover, the hydroxyl groups present at their surface are reactive and offer the possibility for a wide range of chemical modifications including for example esterification, etherification, polymer grafting, oxidation and silylation.^1,3,4,5

Although a series of works dedicated to the chemical modification of cellulose whiskers have been published recently only one study deals with its functionalization with amino acids.⁶ Nevertheless, this work reports the coupling of fluorescent amino acids in oxidized cellulose whiskers and not on native cellulose whiskers. With regard to cellulose, some recent studies have described the successful modification of cellulose fibrous networks and hydroxypropyl and ethyl cellulose with amino acids having as an objective to develop cell-adhesive structures, hydrophobic drug carriers and gas separation membranes, respectively.^7,8,9,10 In fact, amino acids are the building blocks of proteins and peptides and are involved in the development of a wide range of biocompatible applications or architectures.⁹ These include controlled drug delivery systems,¹¹cell-adhesive structures,⁷ chiral recognition materials¹² and polymers¹³ to mention only a few. Therefore, the amino acid esterification of cellulose whiskers is expected to make these materials interesting candidates for drug carriers. The utilization of cellulose whiskers in drug delivery systems has been evaluated and discussed by some authors^14–19 and the obtained results were considered quite encouraging. For example, Mahmoud et al.¹⁵ showed that cellulose whiskers functionalized with two fluorescein isothiocyanates had the ability to penetrate cells with no indication of cytotoxicity. Roman et al.¹⁶ performed the toxicity assessment of cellulose whiskers in human brain microvascular endothelial cells and found that cellulose whiskers were non-toxic and could be used as carriers in targeted drug delivery systems.

In this study, we describe the anchoring of L-leucine moieties to cellulose whiskers that will allow the creation of a binding site to which drugs or targeting molecules can be attached. This strategy will contribute to the development of a cellulose whisker based nano-carrier for drug delivery applications.

The cellulose whiskers were obtained after the sulfuric acid hydrolysis of microcrystalline cellulose according to the methodology described in Bondeson et al.²⁰ with slight modifications. Under these conditions sulfate ester groups will be formed at the surface of cellulose whiskers.

The chemical modification of cellulose whiskers with L-leucine was performed through a two-step process involving the reaction between Fmoc-protected L-leucine and thereafter the removal of Fmoc-protecting group by using a procedure adapted from Kalaskar et al.⁸ and Khan et al.⁹ The presence of sulfate ester groups at the surface of cellulose whiskers was not expected to interfere during the course of the chemical modification and our results were consistent with this expectation. Freeze-dried cellulose whiskers were dispersed in dimethylformamide (DMF). The reaction was conducted at room temperature for 24 h under magnetic stirring using 4-dimethylaminopyridine (DMAP) as a catalyst and N-ethyl-N′-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC.HCl) as a coupling agent. The modified cellulose whiskers were thereafter precipitated in diluted acid solution and washed several times with deionized water and ethanol. The removal of the protecting Fmoc group was performed by stirring the Fmoc–L-leucine CW in a 20% (v/v) piperidine solution in DMF for 30 min. The modified L-leucine CW were washed several times with DMF and left to dry. The reaction schemes are shown in Scheme 1.

Scheme 1 Schematic representation of the chemical reactions involved in the obtaining of modified cellulose whiskers (a) Fmoc–L-leucine modified CW and (b) L-leucine modified CW.

The FT-IR spectra of Fmoc-protected and unprotected L-leucine modified cellulose whiskers presented some main features which demonstrate the occurrence of an esterification reaction between the acid groups of Fmoc–L-leucine and the hydroxyl groups of cellulose whiskers (Fig. 1). In the spectra of Fmoc-protected modified cellulose whiskers it was possible to observe the appearing of: (i) a shoulder at 1749 cm⁻¹ and a peak at 1705 cm⁻¹ assigned to carbonyl stretching in the ester and carbamate groups, respectively, and (ii) a peak at 1543 cm⁻¹ assigned to N–H bending of the carbamate group and to aromatic skeletal vibrations. After the removal of the Fmoc protecting group the carbonyl stretching assigned to the ester group (1749 cm⁻¹) became more evident while that ascribed to carbamate group disappeared (1705 cm⁻¹). Additionally, the peak at 1543 cm⁻¹ also disappeared. These features indicate that Fmoc deprotection was successful. Nevertheless, after the removal of the Fmoc protecting group no bands assigned to N–H stretching and bending in the range of 3500–3000 cm⁻¹ and 1640–1560 cm⁻¹, respectively, were noticeable. This can be caused by the overlapping of OH stretching band of unreacted hydroxyl groups, in the range of 3000–3600 cm⁻¹, and the bending of adsorbed water molecules at approximately 1650 cm⁻¹.

Fig. 1 FT-IR comparative spectra of (a) non-modified CW (b) Fmoc–L-leucine modified CW and (c) L-leucine modified CW.

Further confirmation of the introduction of Fmoc–L-leucine moieties at the surface of CW was acquired by XPS analysis. The XPS spectra obtained for non-modified CW and Fmoc-protected and unprotected L-leucine modified CW are presented in Fig. 2. The XPS spectra of non-modified CW exhibit the presence of two peaks at around 533 and 285 eV which correspond, respectively, to oxygen (O) and carbon (C). After chemical modification the appearance of a new peak at approximately 400 eV was observed and assigned to the presence of nitrogen at the surface of the modified CW samples. The quantification of the elements at the surface revealed in fact that after chemical modification the content of nitrogen increased from 0% for non-modified whiskers, to 1.77% and 1.48% for Fmoc-protected and unprotected L-leucine modified cellulose whiskers, respectively.

Fig. 2 XPS comparative spectra of (a) non-modified CW (b) Fmoc–leucine modified CW and (c) L-leucine modified CW.

The crystallinity index was determined by ¹³C CP/MAS NMR spectroscopy by using the peak areas of the C-(4) atoms of crystalline and amorphous cellulose fractions according to a literature method.²¹ The crystallinity index determined for the non-modified and modified cellulose whiskers was approximately 60% which indicates that the crystalline structure of cellulose whiskers remains unchanged through the course of the chemical modification. This crystallinity value is in agreement to that found by Goetz et al.²² for cellulose whiskers isolated from microcrystalline cellulose.

TEM morphological analysis revealed that the size of cellulose whiskers does not change significantly after chemical modification. Both non-modified and modified cellulose whiskers presented an average length and diameter of approximately, 174 ± 28 nm and 6.1 ± 1.4 nm, respectively (Fig. 3).

Fig. 3 TEM images of (a) non-modified CW and (b) L-leucine modified CW.

In conclusion, in this work we have demonstrated the possibility to modify cellulose whiskers with L-leucine amino acid by esterification reaction. The occurrence of esterification reaction was confirmed by FT-IR and XPS and the successful Fmoc removal was identified by FT-IR spectroscopy. The structural integrity of the cellulose whiskers was keep intact along the course of the chemical modification as demonstrated by TEM and ¹³C CP/MAS NMR spectroscopy. The covalent conjugation of amino acids and cellulose whiskers represents the first step in the development of a new range of biocompatible and biologically active nanomaterials.

Acknowledgements

The authors gratefully acknowledge Solvay S.A. for financial support, Marcus Foston for solid-state NMR measurements and Rajalaxmi Dash for XPS analysis.

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Footnote

† Electronic supplementary information (ESI) available: Experimental details. See DOI: 10.1039/c1ra00647a

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