Multifunctional Stimuli-Responsive Cellulose Nanocrystals via Dual

Letter Cite This: ACS Macro Lett. 2018, 7, 646−650

pubs.acs.org/macroletters

Multifunctional Stimuli-Responsive Cellulose Nanocrystals via Dual Surface Modification with Genetically Engineered Elastin-Like Polypeptides and Poly(acrylic acid) Jani-Markus Malho,*,⊥,† Jérémie Brand,† Gilles Pecastaings,† Janne Ruokolainen,‡ André Gröschel,§ Gilles Sèbe,† Elisabeth Garanger,*,† and Sébastien Lecommandoux† †

Laboratoire de Chimie des Polymères Organiques (LCPO), CNRS UMR5629, Université de Bordeaux, Bordeaux-INP, Pessac 33607 Cedex, France ‡ Department of Applied Physics, Aalto University School of Science, P.O. Box 15100, 00076 Aalto, Finland § Physical Chemistry and Center for Nanointegration (CENIDE), University of Duisburg-Essen, 45127 Essen, Germany S Supporting Information *

ABSTRACT: Cellulose nanocrystals (CNCs) are promising candidates for a myriad of applications; however, successful utilization of CNCs requires balanced and multifunctional properties, which require ever more applied concepts for supramolecular tailoring. We present here a facile and straightforward route to generate dual functional CNCs using poly(acrylic acid) (PAA) and biosynthetic elastin-like polypeptides (ELPs). We utilize thiol-maleimide chemistry and SI-ATRP to harvest the temperature responsiveness of ELPs and pH sensitivity of PAA to confer multifunctionality to CNCs. Cryo-TEM and light microscopy are used to exhibit reversible temperature response, while atomic force microscopy (AFM) provides detailed information on the particle morphology. The approach is tunable and allows variation of the modifying molecules, inspiring supramolecular engineering beyond the currently presented motifs. The surge of genetically engineered peptides adds further possibilities for future exploitation of the potential of cellulose nanomaterials.

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modification of nanoparticles, including nanocellulosic materials.18−20 Therein, recombinant elastin-like polypeptides (ELPs) are an extremely interesting biopolymer that exhibits, among other properties, lower critical solution phase behavior.21 More importantly, ELPs can be designed and produced with a perfect control of the primary sequence at different scales, which cannot be achieved with conventional synthetic chemistry at the moment.22,23 Despite the promising properties afforded by molecules grafted onto CNCs so far and the diversity of synthetic approaches, the full potential of CNCs has not been reached, and more applied approaches are being sought. Furthermore, chemical modification of CNCs with mutiple functional moieties is still relatively rare, most likely due to the lack of facile conjugation methods, which are also in demand. Toward this goal, we present here dual-functionalized CNCs with poly(acrylic acid) (PAA) chains and biosynthetic elastin-like polypeptides (ELPs) (Scheme 1). The approach is tunable and based on a combination of readily applicable conjugation methods, which should allow more effective utilization of CNCs in the future.

iomimetic studies have revealed the crucial role of proteins, peptides, and biopolymers in Nature’s materials, where unmatched properties stem primarily from hierarchical structures and synergetic functioning.1,2 Bottom-up structuring with nanoparticles as the starting materials has gained a lot of attention among the materials science community. One group of materials of particular interest is cellulose-based nanomaterials, which range from nanofibrils to nanocrystals.3,4 Among these, cellulose nanocrystals (CNCs) are especially interesting due to their promising properties, abundant and renewable character, crystalline structure, and numerous surface hydroxyl groups amenable to chemical modifications.5−7 CNCs are commonly produced via sulfuric acid hydrolysis of cellulose microfibrils, which yields negatively charged rod-like nanoparticles.8,9 However, new routes are constantly being sought, and recently, treatment with hydrochloric acid vapor was described as a new way to access uncharged CNCs with a more homogeneous surface.10 Regardless of the production route, functionalization of CNCs is often achieved via chemical11−13 or physical modifications,14−17 which have led to a wide array of routes and a myriad of novel CNC-based materials in the past decades.8,9 Sustainability is one of the key issues driving the utilization of biosourced polymers to replace synthetic polymers. To this end, a surge of genetically engineered peptides and proteins has emerged as the next generation of precision polymers for the © XXXX American Chemical Society

Received: May 2, 2018 Accepted: May 14, 2018

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DOI: 10.1021/acsmacrolett.8b00321 ACS Macro Lett. 2018, 7, 646−650

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ACS Macro Letters

Experimental Section, Figure S2 and Table S3).24,26 In contrast, PMPI functionalization was clearly evidenced in the FT-IR spectrum, through the appearance of the maleimide ν(C=O)sym stretching at 1774 cm−1 and aromatic ring ν(=C−H) stretching at 1511 cm−1 (SI, Experimental Section, Figure S2 and Table S4). In addition, a strong band was observed around 1717 cm−1 attributed to the superimposed signals of two carbonyl vibrations: the asymmetric maleimide stretching (ν(C=O)asy) and the CO stretching of the newly formed urethane bond. A recombinant elastin-like polypeptide (ELP) with the primary sequence MW(VPGIG)20C27 was then attached to the surface of the CNCs by thioether linkage formation between the maleimide group on the CNC surface and the free thiol group of the C-terminal Cys of the ELP. For this reaction, CNCs and ELPs were dispersed in DMF and stirred overnight at room temperature, which led to a stable CNC-ELP colloid dispersion (SI, Experimental Section). The reaction was performed in DMF to avoid temperature-induced aggregation of the ELP as observed in aqueous media at room temperature.27 Despite several attempts, unreacted ELPs could not be completely removed from the reaction mixture prior to the ATRP of PtBA. For cryo-TEM imaging the CNC-ELPs were vitrified with liquid ethane-propane mixture once at 10 and 23 °C to follow the temperature-dependent colloidal behavior of CNC-ELPs at the supramolecular level (SI, Materials and Methods). Figure 1

Scheme 1. Functionalization of CNCs with Mixed Brush of PAA and ELPs: (a) Simplified Structure of Unmodified Cellulose Nanocrystals,a (b) CNC Modification with αBromoisobutyryl Bromide (BIBB) via Chemical Vapor Deposition to Introduce the ATRP Initiator (i) and with pMaleidophenylisocyanate (PMPI) to Introduce Maleimide Reactive Groups (ii), and (c) Subsequent Covalent Grafting of ELPs onto CNCs via Thiol-Maleimide Reaction (iii), Followed by SI-ATRP of Poly(tert-butyl acrylate) (PtBA) (iv)

a

The sulfate groups are left out from the scheme for clarity (see SI Figure S1).

The CNCs used in this study were produced via sulfuric acid treatment of softwood and had an average width of 6 nm and length of 110 nm (SI, Materials and Methods). The dual surface functionalization of CNCs with PAA and ELPs was achieved in five main steps (Scheme 1 and SI). An ATRP initiator was first introduced to the surface hydroxyl groups of the CNCs via chemical vapor deposition (CVD) using αbromoisobutyryl bromide (BIBB) as the acylating agent.24 In the second step, p-maleimidophenylisocyanate (PMPI) was reacted in anhydrous DMF with the remaining surface hydroxyl groups to generate dual Br- and maleimide-functionalized CNCs. As evidenced by elemental analysis and Fourier transform infrared spectroscopy (FT-IR) the attachment of PMPI did not affect the previously grafted ATRP initiator (SI, Experimental Section, Figure S2 and Table S1). PMPI is highly sensitive to hydrolysis, thus stringent anhydrous conditions were used to maximize the coupling efficiency. Elemental analyses revealed that approximately 10% of the surface hydroxyl groups were brominated, while only 2.5% were functionalized with PMPI (SI, Experimental Section, Table S1). Such a difference in reactivity can be explained by the larger size of PMPI as compared to BIBB and by the sensitivity of PMPI to hydrolysis that lowers the reaction yield significantly compared to the relatively robust BIBB. Despite the cautious anhydrous conditions used, a small amount of residual water (around 3% wt.) is usually considered to remain adsorbed on the CNCs.25 In addition, the first reaction with BIBB is expected to occur at the most reactive and accessible primary hydroxyl groups (C6), leaving mostly less reactive secondary alcohol groups (C2) available for the reaction with PMPI (Scheme 1 and SI, Experimental Section, Scheme S1). The dual surface modification was also characterized by FT-IR spectroscopy. Although we cannot quantify the amount of ATRP initiator in FT-IR, the small peak at 1730 cm−1 can be assigned to the carbonyl stretching of the grafted BIBB (SI,

Figure 1. Temperature responsiveness of the CNC-ELPs and pH sensitivity of CNC-ELP-PAAs. The temperature-sensitive behavior of CNC-ELPs was observed macroscopically (inset photos) and by cryoTEM (a) below the transition temperature (20 °C). (c) Zeta-potential values of unmodified CNCs, CNC-PAAs, and CNC-ELP-PAAs at pH 8.4 and 3.0. The drastic change in the zeta potential values demonstrates the pH sensitivity of the binary brush decorated CNCs. Scale bar for the cryo-TEM images of panels (a) and (b) is 50 nm.

exhibits photographs of the macroscopic dispersions (insets) and cryo-TEM images of samples at 10 °C (a) and 23 °C (b). The grafted ELPs are evidenced by darker halos surrounding the CNC rods when the sample is vitrified at 10 °C. The temperature-sensitive nature of CNC-ELPs was clearly observed on the macroscopic sample that turned turbid above 20 °C, while remaining perfectly translucent below 20 °C. The CNC-ELPs agglomerated above the ELP transition temperature confirm the ability of the ELPs to confer temperature responsiveness to the CNCs in aqueous media. 647


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CNCs, CNC-PAAs, and CNC-ELP-PAAs in aqueous dispersions at pH 8.4 and pH 3.0 are provided in Figure 1c. The pKa value of PAA is around 4.5, suggesting that all carboxyl groups of the PAA chains are deprotonated and negatively charged at pH 8.4, while they are protonated (and uncharged) at pH 3.0. Native CNCs presented a negative net charge of −50 mV at pH 8.4 and a slightly higher average value of −34 mV at pH 3.0. The control sample CNC-PAA demonstrated more radical pH sensitivity, with the zeta potential values shifting from −34 mV at pH 8.4 to +36 mV at pH 3.0. In a similar fashion, the CNC-ELP-PAA sample showed pH sensitivity (−41 mV at pH 8.4 to +38 mV at pH 3.0), confirming the pHdependent property of the newly formed CNC-ELP-PAAs afforded by the synthetic polymer chains. The colloidal stability of CNC-ELP-PAAs was not sufficient for cryo-TEM imaging (even at pH 8.4), hence atomic force microscopy (AFM) was used to investigate the dimension of the mixed brush-decorated CNCs. Figure 3 exhibits AFM

CNC-ELPs were then subjected to SI-ATRP in DMF using previously described procedures,24,26 to graft poly(tert-butyl acrylate) (PtBA) chains from the Br- initiating sites. We preferred performing such polymerization after grafting of the ELPs because we expected the PtBA chains to sterically block the maleimide functions and prevent the subsequent grafting of ELPs. An excess amount of tBA monomer was used to facilitate polymerization of relatively long PtBA chains. The reaction was conducted in the presence of ethyl α-bromoisobutyrate (BBIB) as a sacrificial initiator, to estimate the molecular weight of the grafted PtBA polymer from the homopolymer concurrently formed in the reaction medium (SI, Experimental Section).24,26 Utilization of sacrificial initiator allows investigating the degree of polymerization (DP) without cleaving the grafted PtBA chains from the CNCs. The DP was measured in the presence (CNC-BIBB-ELP) and in the absence of ELP chains (CNCBIBB) by size exclusion chromatography (SEC) from the PtBA homopolymer.24,26 The SEC results showed similar DPs, indicating that the grafted ELPs did not affect the polymerization significantly (SI, Experimental Section, Table S5). The polymer chains on the CNC-ELP-PtBA sample were then treated with trifluoroacetic acid (TFA) to yield negatively charged PAA chains on the CNCs. FT-IR confirmed the presence of PtBA and later the presence of hydrolyzed PAA chains (Figure 2), through the observation of the characteristic

Figure 2. FT-IR spectra of the CNC-ELP-PAA, CNC-PAA, CNCPtBA, and pure CNC. The grafting of ELPs and PtBA chains is clearly visible in the FT-IR spectra.

Figure 3. AFM height images (a), height profiles (b), and phase images (c) of unmodified CNCs, CNC-PAAs, and CNC-ELP-PAAs. The average widths (with standard deviations) were calculated by measuring a minimum of eight different CNCs at three distinct positions.

carbonyl stretching vibrations at 1732 and 1680 cm−1, respectively (Figure 2 and SI, Experimental Section, Table S3). The persistence of ELP chains during polymerization and hydrolysis was confirmed by the stretching band at 1543 and 1640 cm−1 (Figure 2 and SI, Experimental Section, Figure S3). While the removal of the free ELP chains was not possible prior to the ATRP, the free ELPs were efficiently removed by washing after polymerization of the PtBA (and prior to the acid hydrolysis step) due to increased hydrophobicity and reduced solvation of the CNC-ELP-PtBA compared to the free ELP chains. The amount of grafted ELP was quantified by UV−vis spectroscopy using the absorbance at 280 nm of the tryptophan at the N-terminal end of the ELPs (SI, Experimental Section, Figure S4).27 UV−vis results and calculations showed a similar amount of ELPs in the CNC-ELP-PAA than the amount of maleimide groups on PMPI-modified CNCs (SI, Experimental Section). The pH sensitivity of the CNC-ELP-PAA was investigated by zeta potential measurements. Zeta potentials for unmodified

images of unmodified CNCs, CNC-PAAs, and CNC-ELPPAAs. Samples were dried for the AFM imaging, which causes shrinking and collapsing of PAA and ELP chains and thus affects the appearance of the surface-modified CNCs. Nevertheless, significant differences between samples with different surface modification were observed. The AFM height images revealed clear differences in the average width of unmodified CNCs (27 nm), CNC-PAAs (47 nm), and CNC-ELP-PAAs (79 nm) (Figure 3). Furthermore, the height profiles of CNCPAAs and CNC-ELP-PAAs differed significantly. Both the width and the height profiles can be explained by more crowded surfaces of the CNC-ELP-PAAs. The ELPs also very likely force the PAA chains to extend themselves away from the CNC surface, which leads to a larger overall width and lower average height because the PAA chains are unable to fold on the top of the CNCs during drying. The amount of the grafted 648


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for funding. This work made use of Aalto University Nanomicroscopy Center facilities (Aalto-NMC).

PtBA at the CNC surface was estimated based on the DP of the polymer, the quantity of the ATRP initiator at the surface prior to the polymerization, and the TGA thermograms of the samples, all of which led to an estimated width of approximately 85 nm for CNC-ELP-PAA (SI, Experimental Section). The estimated value fits relatively well with the measured width of 79 nm of the CNC-ELP-PAAs from the AFM images (Figure 3). The phase images display also differences between CNCPAAs and CNC-ELP-PAAs, where the softer area surrounding the CNC appears larger, agreeing well with the extended brush morphology due to crowding. Similar extension of the PAA chains has already been observed before, when PAA-grafted CNCs were complexed with diblock polymers.26 Finally, the CNC-ELP-PAAs were subjected to temperaturecontrolled light microscopy imaging to confirm their temperature responsiveness in an aqueous environment. The presence of agglomerates was observed above 20 °C, but when the sample was cooled below 20 °C, the agglomerates redispersed rapidly and easily to form a homogeneous dispersion. The temperature-dependent transition from agglomerates to the dispersed state was reversible as the agglomerates reappeared upon heating above 20 °C (SI, Experimental Section, Figure S6). In summary, we present the first study of mixed brushdecorated CNCs, combining both synthetic and biosynthetic polymers on biopolymer nanocrystals. Our approach combines two relatively facile synthetic routes for the generation of multifunctional cellulose nanocrystals. The resulting CNCELP-PAAs display pH sensitivity stemming from the PAA chains and temperature responsiveness resulting from the thermal behavior of the ELPs. Bridging the gap between multifunctionality and supramolecular engineering paves the way for more evolved supracolloidal engineering strategies, which should enable more efficient utilization of biopolymers in the future.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsmacrolett.8b00321. Materials, methods, characterization, and experimental details (PDF).

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REFERENCES

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

André Gröschel: 0000-0002-2576-394X Elisabeth Garanger: 0000-0001-9130-8286 Sébastien Lecommandoux: 0000-0003-0465-8603 Present Address ⊥

Nolla Antimicrobial Oy, Viikinkaari 4, 00790 Helsinki, Finland.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Emil Aaltonen Säät iö and Tutkijat Maailmalle Säät iö (Teknologiateollisuuden100-vuotissäaẗ iö) are acknowledged 649


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Multifunctional Stimuli-Responsive Cellulose Nanocrystals via Dual

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