Fabrication of biocatalytic hybrid particles. Our approach to fabricate multifunctional Pickering emulsion polymerization stabilizers started with production of biocatalyst loaded LNPs (GOx-chi-LNPs) via two-step adsorption immobilization strategy (Fig. 1).
First, LNPs (ζ-potential − 40 mV) were produced by solvent-exchange methodology from previously characterized pine kraft lignin (Table S1). The resulting LNPs (diameter 97 nm) were used to adsorb chitosan (10 wt% relative to LNPs) as a cationic polyelectrolyte. As previously reported, we obtained colloidally stable cationic chitosan-coated LNPs (chi-LNPs) (ζ-potential + 32 mV and diameter 190 nm) that were here used to adsorb GOx (10 mg g-1 chi-LNPs) to yield the enzyme-immobilized LNPs (GOx-chi-LNPs) (Figure S1 and S2). The increase in size and surface charge as determined by dynamic light scattering (DLS) analysis of the resulting particles after the two-step adsorption procedure (215 nm and + 41 mV) verified an effective electrostatic interaction between GOx and chi-LNPs (Fig. 2a-b and table S2).
Scanning electron microscopy (SEM) images confirmed the formation of spherical and uniformly shaped biocatalyst-loaded particles (GOx-chi-LNPs) that exhibited a high tendency to agglomerate upon drying (Fig. 2c). A protein mass balance based on Bradford protein assay (BSA standards), showed that 88% of GOx adsorbed on chi-CLPs, while the remaining 12% was present in solution or adsorbed on the smallest particles that did not sediment during the centrifugation step used in the sample preparation for the protein assay.
Since polymerization reactions are carried out in a broad range of temperatures, we first studied thermal stability of the immobilized GOx in comparison to the free enzyme. It turned out that the immobilized GOx remained active until 80 ºC, with a temperature optimum at 65 ºC compared to about 50 ºC of the free enzyme (Fig. 2d). One possible explanation for this increased stability is the ability of the chi-LNPs to scavenge hydrogen peroxide produced by GOx (Fig. 1, and vide infra for more details). Comparison before and after the immobilization step of enzyme activity also confirmed a high retention of activity, which could be associated to a low kinetic constraint from the embedment of the enzyme on the chitosan hydrogel layer over the LNPs (Figure S3 and Table S3).
Enzyme-degassed SET-LRP-mediated Pickering emulsion using GOx-chi-LNPs as emulsifiers. With the biocatalyst-loaded particles available as a colloidally stable dispersion, we assessed whether or not the biocatalytic emulsifier system is suitable for conducting SET-LRP reactions. All the polymerization were conducted in aqueous buffer solutions (pH = 6) at 50 ºC in Pickering emulsions produced by mixing the monomer, ligand and initiator (oil phase) with an aqueous dispersion containing GOx-chi-LNPs (20 g per L of monomer) and glucose (0.1 M) by ultrasonication (See methods section and Table S4 for more details of polymerization conditions). The emulsion droplets were efficiently covered by GOx-chi-LNPs (Figure S4 and Table S5) regardless of the monomer employed, and polymerization was initiated by adding a small volume of aqueous Cu(0) powder dispersion into the Pickering emulsion. Nanosized Cu(0) particles (40–60 nm) were used to ensure a high active surface area and provide a fast initiation step, crucial to the domain propagation step. The polymerization reactions were allowed to run in either open or closed vials.
The feasibility of GOx-chi-LNPs to provide an oxygen tolerant and controlled polymerization process was evaluated by the polymerization of butyl methacrylate (BMA) as a model monomer in closed vials to avoid the loss of volatile BMA from the reaction mixture (Fig. 3a). A kinetic polymerization study revealed a pseudo-first-order polymerization kinetics and an excellent linearity between ln([M0]/[M]) vs time (Fig. 3b, circles), indicating an equal and constant propagation of the growing polymeric chains, the principal feature of a living radical polymerization process. In addition, any appreciable induction period could not be observed, which confirms an efficient degassing process catalyzed from GOx-chi-LNPs. As a visual observation it was noted that the appearance of the emulsion became more opaque as a result of the polymerization (Fig. 3c-d). The high tolerance to air atmosphere was additionally assed by temporally opening and closing the reaction vial in 20 minutes intervals during 4 hours, which essentially confirmed that exposure to air had no effect on the polymerization rate or extent of control even at the most oxygen-exposed periods (open vial times) (Figure S5). For comparison, chi-LNPs without GOx were used by applying nitrogen gas purging as a degassing methodology (Fig. 3b, triangles) and without any degassing protocol (Fig. 3b, squares). It can be very clearly observed that the system of chi-LNPs without degassing measures showed an induction period of more than 3 h before the polymerization began to proceed, which confirms previous findings on the inhibition of the reaction by dissolved oxygen. In the case of chi-LNPs, applying N2 bubbling for degassing, a similar kinetic profile than that observed for GOx-chi-LNPs could be obtained, albeit at a slightly slower polymerization rate, which could be associated to the inevitable oxygen contamination resulting from the sampling process despite applying concurrent purging with N2.
These results not only validate our original hypothesis and confirm GOx-chi-LNPs as efficient degassing stabilizers for Pickering emulsions polymerizations, but also demonstrate that GOx-chi-LNPs can provide even a faster polymerization rate than chi-LNPs after applying nitrogen purging, which could be of potential interest for industrial applications. Moreover, molecular weight analysis at different polymerization times by GPC revealed a linear increase of molar mass with an excellent agreement with the theoretical molecular weight values, leading to well defined polymers (Mw/Mn = 1.30 − 1.15) during the whole polymerization process regardless of the type of LNPs (chi-LNPs or GOx-chi-LNPs) used as emulsifiers (Fig. 3e and S6). Therefore, these results validated the potential of lignin nanoparticles as functional emulsifiers for SET-LRP.
Self-scavenger ability of GOx-chi-LNPs towards hydrogen peroxide. Inactivation by hydrogen peroxide of enzymes in general and GOx in particular is a problem that has been alleviated by additives such as the electron acceptor system benzoquinone-hydroquinone. Moreover, as already mentioned, H2O2 may initiate Fenton-like oxidation processes and promote the generation of new chains, leading to uncontrolled growing of the polymeric chains and thus affecting the final molecular weight distribution. It is thus important to note that our system was able to generate well-defined polymers without the need of any extraneous reducing agents (e.g. sodium pyruvate) to eliminate the H2O2 formed during the degassing step. This fact could be attributed to the inherent nature of both chitosan and lignin to consume H2O2 in situ. To test this hypothesis, we evaluated the ability of GOx-chi-LNPs to consume H2O2. First, H2O2 was dissolved in an aqueous solution (pH = 6) and mixed with horseradish peroxidase (HRP, EC 22.214.171.124) in opened vials with and without LNPs or GOx-chi-LNPs, and then o-dianisidine was added as a substrate for HRP (Figure S7a). In the absence of LNPs or GOx-chi-LNPs, a significant increase in absorption at 500 nm was observed by UV-vis spectroscopy due to the formation of the oxidized dimer of o-dianisidine (ε[500 nm] = 7.5 mM in water) catalyzed by HRP in the presence of H2O2 (Figure S7b). In contrast, when the reaction was performed in the presence of LNPs, a noteworthy decrease in the UV absorption of oxidized o-dianisidine indicated an effective consumption of H2O2 by LNPs (Figure S7c and S7e). Additionally, it was interesting to observe that when GOx-chi-LNPs were added to the reaction mixture, only trace levels of the oxidized product could be determined, suggesting an excellent combined effect of lignin and chitosan to scavenge the H2O2 present in the system. (Figure S7d). Additionally, to mimic the polymerization conditions, the same amount of BMA, glucose and GOx-chi-LNPs were dissolved in an aqueous solution (pH = 6) and exposed to air. Then, o-dianisidine and HRP solution were added, and the analysis of the resulting solution confirmed that the mere presence of GOx-chi-LNPs suppressed the H2O2 formed in the reaction media (Figure S8). These results confirm the scavenger ability of our system towards H2O2, which is beneficial not only for the polymerization process but also to protect the enzyme against the oxidative denaturation, which could also explain the high retention of enzyme activity that the immobilized GOx exhibited at high temperatures (Fig. 2d).
Applicability to other monomers and synthesis of well-defined block copolymers. Having demonstrated the robust nature of GOx-chi-LNPs to act as functional surfactant to obtain well-defined hydrophobic polymers from oxygen-tolerant SET-LRP, we explored versatility of the system by targeting the polymerization of different vinyl monomer of relevant industrial interest. For this propose, we conducted polymerizations under identical degree of polymerization (DP = 200) to those applied for BMA for two additional hydrophobic monomers, namely methyl acrylate (MA) and styrene (S). In both cases, near to quantitative monomer conversion (90%) was achieved, and molecular weight analysis of the resulting polymers revealed symmetrical monomodal peaks (Mw/Mn = 1.16 for MA and 1.26 for S) with an excellent agreement between the experimental and theoretical molecular weights values (Fig. 4a), proving the versatility of this system to polymerize, in a controlled manner, different families of hydrophobic vinyl monomers in aqueous phase. We also decided to push the limits of our system by targeting different degrees of polymerization (DPn), ranging from 50 to 500 for BMA. The results are summarized in Table S6. In all cases, well-defined polymers with narrow dispersities (Mw/Mn = 1.29 − 1.16) and experimental molecular weight values close to the theoretical ones could be obtained, indicating a high versatility of the system to deliver well defined polymers in a wide range of molecular weights (Fig. 4b and Figure S9).
The high chain-end fidelity of the obtained polymers, a critical parameter to synthetize more complex architectures (e.g. block and multiblock copolymers)[36–38] was also assed for our system. Low molecular weight PMA (M/I = 50) was synthetized, isolated and analyzed by 1H NMR spectroscopy, which revealed virtually complete chain-end functionality (96%) based on the retention of the bromine chain-ends (Figure S10). Indeed, chain extension experiments by stopping the reaction, purification of the macromonomer through precipitation and re-dispersion to continue the polymerization of a second block also confirmed a nearly perfect chain-end functionality regardless of the monomer employed (Fig. 4c and S11). The successful formation of quasi real AB block copolymers was confirmed by a clear shift in the GPC curves towards high molecular weight values without noticeable shoulders or tailing (Fig. 4c and S11), indicating the absence of unreactive polymeric chain produced in recombination processes of growing polymeric chains (e.g. bimolecular termination processes). Overall, these results not only validate GOx-chi-LNPs as an efficient stabilizers for more sophisticated controlled polymerization techniques under the presence of oxygen, but also revealed their high versatility to stabilize and enable SET-LRP of different vinyl monomers leading to well-defined polymers as building units for more complex and sophisticated architectures.
Analysis of latex dispersion and production of lignin-based composites. Finally, the quality of the latex dispersions obtained after the SET-LRP process was also evaluated by SEM and optical light microscopy. Regardless of the monomer employed, the latex dispersions were in all cases composed of uniform spherical polymeric microbeads (Fig. 5 and Figure S12), with a similar size to that of the initial monomer droplets (Compare Table S5 and S7). As a representative example, BMA latex dispersion was selected for a more detailed analysis (Fig. 5). Lignin-coated PBMA microparticles could be obtained by isolation of the particles by a simple centrifugation/re-dispersion step. Based on the microscopic images, it can be clearly observed that the PBMA microspheres contained a thin amorphous layer (Fig. 5b and c), which demonstrate the high emulsifier capacity of GOx-chi-LNPs system. Such a uniform distribution of the nanoparticles in the polymeric matrix inspired us to envision that the polymerization system could also provide an effective and direct method to prepare lignin-based composites with homogeneously dispersed lignin particles in the polymer matrix. With this objective in mind, we evaluated the preparation of composites by simple melting at 160 ºC of the PBMA latex dispersion stabilized with a higher content of GOx-chi-LNPs (6 wt % relative to BMA) (Figure S13a). SEM analysis of the surface and cross sections of PBMA-GOx-chi-LNPs film composites (Figure S13b-c) confirmed the uniform distribution of GOx-chi-CLPs without agglomeration within the PBMA matrix, which indicates an unprecedented and effective interaction of the particles even within such hydrophobic polymeric matrixes. These results make it possible to form lignin-based polymer composites in a simple way, avoiding chemical functionalization of lignin to improve the dispersability within the polymeric matrix. Last but not least, it is also important to note that highly purified PBMA microparticles without the presence of lignin could also be obtained by simple aqueous basic extraction of the latex dispersions under alkaline conditions (Fig. 5d-f).