resumo
With society’s growing awareness of climate change, novel renewable and naturally sourced materials have received increasing attention as substitutes for petroleum-based products. Laminarin (LAM–OH) is a highly abundant, nontoxic, degradable polysaccharide found in marine organisms and hence is a promising sustainable polymeric candidate. This work reports on a simple, environmentally friendly, and customizable functionalization strategy for producing a toolbox of LAM–OH derivatives under mild conditions. Herein, natural-origin macromolecules exhibiting specific chemical moieties, namely, allyl, amine, carboxylic acid, thiol, aldehyde, and catechol, were prepared and chemically characterized. Furthermore, the obtained polymers were processed into cytocompatible hydrogels, obtained by employing distinct cross-linking mechanisms, to assess their potential for biomedical purposes. The application scope of such polymers could be extended to fields such as catalysis, cosmetics, life sciences, and food packaging, which can also benefit from having sustainable, nontoxic, and degradable materials. Moreover, it is anticipated that the methodology employed to create this library of new natural-based products could be adapted to modify other polysaccharides and biopolymers in general.
autores
Ana M. S. Costa, João M. M. Rodrigues, Maria M. Pérez-Madrigal, Andrew P. Dove, João F. Mano
nossos autores
João M. M. Rodrigues
Investigador AuxiliarProjectos
MUST - Multi-level protection of materials for vehicles by "Smart" nanocontainers (MUST )
Nano-argilas para remoção/captura de fosfatos (P) e sua reutilização como fertilizante (NATURAL)
Collaboratory for Emerging Technologies, CoLab (EMERGING TECHNOLOGIES)
CICECO - Aveiro Institute of Materials (UIDB/50011/2020)
agradecimentos
Introduction ARTICLE SECTIONSJump To Polymers have a tremendous impact on a wide range of modern life activities, spanning from synthetic fibers applied in clothing to biomaterials used in medical devices. (1,2) Despite their obvious usefulness, environmental concerns regarding the raw materials used and their manufacturing processes as well as their end-of-life cycle options are growing. (3,4) Recently, several efforts have been devoted to using more sustainable polymers in science, technology, and industry by, for instance, searching for renewable substitutes of petroleum-based materials with the potential to be recycled. (5−8) Nature is an incomparable source of environmentally friendly and degradable materials. In fact, it is estimated that each year more than 150 billion tons of polysaccharides are produced naturally, with only about 1% of this amount being consumed by humans. (5,9,10) Polysaccharides, such as chitosan, alginate, and laminarin, are among the most abundant macromolecules synthesized in marine organisms. Despite its abundance, laminarin (LAM–OH) has received very little attention, limited to biomedical purposes as a result of its distinct therapeutic functions such as anti-inflammatory, antioxidant, antitumoral, and anticoagulant activities. (11−13) LAM–OH is a low-molecular-weight branched polysaccharide mainly used as an energy storage glucan in brown algae. (14,15) Its structure is composed of glucose units linked by β(1,3) glycosidic bonds as well as by some β(1,6) side-chain branches, which are responsible for its low viscosity, nontoxicity, and high solubility in aqueous and, unusually for polysaccharides, organic solvents. (12,13,16) Furthermore, this natural-origin material is easily degraded into glucose units or oligosaccharide structures by the action of enzymes, some of which are present in the soil or in the oceans. (14,17,18) Such a biodegradability profile is of utmost importance for the end-of-life fate of this sustainable polymer and hence to minimize its effect on existing plastic waste issues. Besides environmental concerns, the potential application of polymers is also highly dependent on the development of competitive candidates against current commercial materials. In other words, they must exhibit not only distinctive and enhanced physicochemical and biological properties, including processability, compatibility, and mechanical strength, but must also be prepared through simple, straightforward, and cost-effective synthesis routes. These aspects remain challenging for many biopolymers, typically owing to their low reactivity and poor solubility. (19) However, by simply functionalizing natural polymers with specific groups, it is possible to process them into multifunctional biomaterials, for example, hydrogels for tissue engineering, microparticles for drug delivery, and soft robotics for bioelectronics. (20−22) Chemical grafting is the simplest and most commonly employed strategy for functionalizing polysaccharides while maintaining their intrinsic properties. (23) This methodology can start with an already formed polymeric carrier bearing a large number of reactive chemical groups, for example, amines on chitosan or carboxylic acids on alginate, which are then replaced by the desired functional groups through organic synthesis reactions. (24,25) Herein, we report work aimed at extending the chemistry of LAM–OH to obtain value-added natural-based products and explore the resultant biofunctional materials for innovative applications. To this end, a toolbox of biomaterials was obtained by the chemical derivatization of the LAM–OH polymer via the incorporation of different functional groups, namely, allyl, carboxylic acid, amine, thiol, aldehyde, and catechol, into its backbone, yielding LAM–C═C, LAM–COOH, LAM–NH2, LAM–SH, LAM–C═O, and LAM–DOPA derivatives, respectively. Moreover, their cytotoxicity and their ability to form hydrogels were also evaluated to assess their potential for biomedical purposes. Results and Discussion ARTICLE SECTIONSJump To To develop a modular and flexible functionalization strategy that resulted in the incorporation of defined functional groups into the LAM–OH backbone, two approaches were investigated (Figure 1). First, a LAM–C═C derivative was obtained via a nucleophilic substitution process (reaction i; Figure 1A) that involved the reaction of hydroxyl groups in LAM–OH with an allylation agent, namely, allyl bromide (reaction i; Figure 1B). A strong base such as NaOH is typically required to deprotonate the alcohol group, which allowed its coupling with the allyl halide compound. This allylation method was previously employed to modify other polysaccharides (26−28) and was selected in this work due to the possibility of allyl groups to react with thiol-bearing compounds (represented as HS-R in Figure 1B) via thiol-Michael addition (reaction ii; Figure 1B). Figure 1 Figure 1. (A) Schematic representation of the developed polymeric library containing the synthesized LAM–OH derivatives, namely, LAM–C═C, LAM–COOH, LAM–NH2, LAM–SH, and LAM–C═O. (B) Detailed chemical reactions employed to obtain each LAM–OH functionalization by (i) nucleophilic substitution, (ii) thiol-Michael addition, (iii) oxidation, and (iv) hydrolysis. To simplify, the LAM–OH structure was represented as a glucose monomer. Nevertheless, it is worth stating that LAM–OH is a branched polymer. The LAM–OH structure has previously been chemically characterized through 1H nuclear magnetic resonance (NMR), 13C NMR, and Fourier-transform infrared (FTIR) spectroscopy. Our characterizations are in accordance with those that have been reported (15,29,30) and were further expanded to include and confirm the modification of the produced derivatives (Figure 2). Characteristically, the LAM–C═C derivative displays three new chemical shifts on the corresponding 1H NMR spectrum (Figure 2A; i.e.. a, b, and c at δ = 4.08, 5.30, and 5.97 ppm; substitution degree (SD) of around 47%) that result from the addition of an allyl group during the nucleophilic substitution process, which is corroborated by other works. (31−33) A similar trend is observed in the 13C NMR spectra, specifically, the appearance of the signals at δ = 118.5 and 133.7 ppm that correspond to the new carbons present in the LAM–C═C backbone. Interestingly, different SDs ranging from 5 to 47% can be obtained by tuning the amount of allyl bromide added to the chemical reaction mixture (Figure S1). Figure 2 Figure 2. Chemical characterization of the synthesized biopolymers. (A) 1H NMR and (B) 13C NMR spectra of the native LAM–OH polymer and its derivatives (in D2O) as well as the corresponding peak assignment. The now incorporated allyl group provides a versatile functional handle from which to base further modification to access other functional groups and hence performance. A significant number of studies have shown its versatility for radical thiol–ene addition which allows its implementation to efficiently obtain a wide range of novel materials with distinct functionalities. (34) We took advantage of this versatile reaction to introduce amine, carboxylic acid, and thiol groups into the LAM–OH structure, through the reaction of LAM–C═C with cysteamine hydrochloride, thiolatic acid or thioacetic acid to obtain LAM–NH2, LAM–COOH, or LAM–SH derivatives, respectively, initiated by potassium persulfate (KPS). This strategy for modifying laminarin has the added benefit of allowing the pairing under mild conditions and using a water-based solution as a solvent, which further increases the “green” credentials of this approach. (35) The successful modification of LAM–C═C was also proven by 1H NMR spectroscopy by the complete disappearance of the vinyl signals at δ = 5.97 and 5.24–5.36 ppm in combination with the appearance of new signals at δ = 1.88 and 2.68 ppm in all three modifications, also proving that the coupling was complete. (31−33)13C NMR spectroscopic analysis confirmed the disappearance of the allyl resonances and the appearance of two signals at δ = 27.3 and 28.2 ppm that correspond to the alkyl chain adjacent to the newly formed thioether in all three cases (Figure 2). Further resonances attributed to the added functionality were also clearly observed and are in accordance with previously published studies. (36) We therefore conclude that quantitative conversion of the allyl groups was complete within the 1 h reaction time. In all cases, the correct coupling of the distinct moieties on the LAM–OH polymer was further proven by FTIR spectroscopy and colorimetric assays. (See Figure S3 in the Supporting Information for more details.) In order to access the thiol-functional LAM–OH, an additional deprotection step of the water-insoluble intermediate was required. This was undertaken by basic hydrolysis upon adding NaOH. (See Figure S2 for more details.) The complete disappearance of the resonance associated with the acetyl group (δ = 1.73 ppm) confirmed the formation of the LAM–SH compound. Notably, while the SH moieties were protected by an acetyl group, the formation of disulfide bonds due to the oxidation process of the free thiol groups was hampered. Therefore, this intermediate compound can be stored under atmospheric conditions contrary to the LAM–SH derivative. Importantly, other functionalities could be easily inserted into the LAM–OH polymer through the selection of a thiol-bearing compound containing the required chemical group. LAM–NH2 and LAM–COOH biopolymers were targeted due to their possibility to work as substitutes for the most used polysaccharides in biomedical applications, namely, alginate and chitosan. (37−39) LAM–NH2 and LAM–COOH can easily be positively and negatively charged, respectively, and can be employed as polyelectrolytes. (40) Indeed, zeta potential measurements were also performed to confirm the presence of positively charged amine groups in LAM–NH2 and negatively charged carboxylic acid groups in LAM–COOH. The outcomes suggest the successful incorporation of these new functionalities (LAM–NH2, 37.0 ± 0.9 mV; LAM–COOH, −47.6 ± 4.4 mV), highlighting their potential as polyelectrolytes. Such materials have been generically used in the development of new materials, such as multilayer films/coating using the layer-by-layer technique or in the production of coacervates or hydrogels through complexation. (41,42) Chitosan is the only polysaccharide with a positive charge at pH < 6.5, but it has the limitation of being soluble only in dilute acidic solutions. By contrast, the synthesized LAM–NH2 polymer is both positively charged and water-soluble. Moreover, the amount of either amine or carboxylic acid groups on chitosan and alginate, respectively, is not easily controlled. Following the modification strategy described in this work, it is possible to effectively control the SD of the LAM–OH derivatives by tuning the amount of allyl bromide added to the chemical reaction mixture. (See Figure S1 in Supporting Information.) In an alternate approach, a LAM–C═O biopolymer was obtained after an oxidation process (reaction iii; Figure 1B) of the LAM–OH polysaccharide. This oxidized macromolecule is very useful by virtue of its higher reactivity when compared with the hydroxyl groups on the native polymer. (43) Sodium periodate was preferred owing to its selectivity to oxidize vicinal hydroxyl groups at C2 and C3 positions, resulting in the opening of the glucose monomer unit and the formation of two aldehyde groups per each monomer. (16) It is worth noticing that distinct degrees of oxidation could be attained by controlling the addition of NaIO4. (44−46) The resultant oxidized derivative has a larger rotational molecular freedom, enabling its use in further chemical modifications such as reductive amination as well as applications which require higher degradation rates. (47) All of the former reactions occurred under mild conditions, including the use of aqueous-based solvents and low reaction temperatures. As outlined previously, the presence of aldehyde groups on the LAM–C═O derivative was also assessed by 1H NMR, 13C NMR, and FTIR spectroscopy (Figure 2A,B and Figure S4 in the SI). Specifically, the appearance of chemical signals at δ = 4.9–5.2 and 80–100 ppm in the 1H NMR and 13C NMR spectra, respectively, can be ascribed to the formation of hemiacetal groups between aldehydes and neighboring hydroxyls (Figure S4A–C). (48) In fact, the only difference between this derivative and the LAM–OH polymer is the presence of a carbonyl group, which is clearly observed at δ = 200 ppm in LAM–C═O (Figure S4C). This is further supported in the FTIR spectrum of LAM–C═O in which a weak band at 1753 cm–1 appears, supporting the presence of a carbonyl group (Figure S4D). This outcome is in accordance with previously obtained results using other polysaccharides such as alginate, cashew gum, or cellulose (49−53) and can be ascribed to the carbonyl stretching vibration, highlighting the correct incorporation of aldehyde groups in the LAM–OH backbone. In contrast to the allyl substitution and further thiol–ene addition in which no significant degradation was observed by GPC/SEC analysis (SI; Materials and Methods section), the oxidation of LAM–OH resulted in a slight decrease in molecular weight, which suggests that, as may be expected, (54) some degradation of the LAM–OH backbone occurs under these conditions. The modification of laminarin to have a wider range of functional groups now allowed for further selective functionalization at those sites. As an example, a catechol (hereafter referred to as DOPA) moiety, in the form of either 3,4-dihydroxybenzoic acid or dopamine hydrochloride, could be grafted onto the LAM–NH2 or LAM–COOH structure, respectively, using the well-established EDC/NHS coupling (reactions i and ii; Figure 3A). Catechol groups presented in DOPA are known for their adhesive properties under wet conditions. (55) Moreover, catechols and their oxidized form, quinone, are highly reactive toward a wide variety of chemical groups, such as amine, thiol, and metal ions such as iron, offering a large set of possibilities for creating novel materials. (56,57) Figure 3 Figure 3. Synthesis and characterization of the LAM–DOPA structure. (A) The LAM–DOPA derivative was synthesized via the EDC/NHS coupling reaction using either (i) LAM–NH2 or (ii) LAM–COOH as starting polymers. (B) 1H NMR spectra of the (i) LAM–NH2 and LAM–DOPA and (ii) LAM–COOH and LAM–DOPA polymers (in D2O), with the catechol group highlighted in gray. (C) UV–vis spectroscopy profile and (D) FTIR spectra of LAM–OH, DOPA, and LAM–DOPA compounds. The main differences raised from the correct incorporation of the catechol group are assigned with a gray arrow. To demonstrate this possibility, first EDC was reacted with the carboxylic acid groups on either 3,4-dihydroxybenzoic acid (reaction i; Figure 3A) or on LAM–COOH (reaction ii; Figure 3B) to yield an unstable reactive acylisourea ester. Subsequently, NHS was added to stabilize the previously formed intermediate and to convert it to an amine-reactive NHS ester. (58) This ester was then reacted with the amine groups of dopamine hydrochloride (reaction i; Figure 3A) or of the LAM–NH2 biopolymer (reaction ii; Figure 3A) to obtain the final DOPA-modified LAM–OH derivative (LAM–DOPA). To avoid self-polymerization of the catechol group, these reactions were conducted under a N2 atmosphere. By comparing the 1H NMR spectra of LAM–NH2 and LAM–COOH with the LAM–DOPA (Figure 3B; i and ii respectively) resonances at δ = 2.7 and 6.3–6.8 ppm (Figure 3B; resonances a–d), these were ascribed to the methylene hydrogen resonance of the catechol moiety and the phenyl protons’ resonance, respectively, proving that the catechol group was successfully inserted into these biopolymers’ backbones. (59) The reduction of the resonances in the 1H NMR spectrum of the LAM–NH2 and LAM–COOH biopolymers in the spectra of the respective LAM–DOPA (obtained through reaction i or ii; Figure 3A) suggests that the EDC/NHS reaction occurred in these groups and also that the grafting process was incomplete. In fact, the SD was found to be around 10%, which is close to the ones reported in the literature for other polysaccharides, such as hyaluronic acid and chitosan. (60,61) Further characterization of LAM–DOPA was possible by both UV–vis and FTIR spectroscopy. The overlap of the two UV–vis absorption peaks at λ = 270 and 320 nm, which have been associated with the catechol/quinone forms of the phenolic group on the LAM–DOPA derivative, was clear in the UV–vis profile of LAM–DOPA. These two peaks are not present on the UV–vis profile of the LAM–OH polymer, which also confirms the successful incorporation of the catechol groups on the LAM–DOPA biopolymer. (60) The FTIR spectra of LAM–DOPA also exhibited additional peaks consistent with the main vibrational modes of dopamine, namely, the C–H vibration (2850 cm–1), the C═C aromatic stretch (1519 cm–1), the C═O stretch (1720 cm–1), the amide bond C–N vibration (1646 cm–1), the C–O stretching vibrations (1408 cm–1), and the phenol OH bend (1353 cm–1). All of these characterizations proved the correct incorporation of catechol groups on the LAM–DOPA derivative. (60,62,63) To demonstrate the potential utility of the modified laminarin reported herein, we chose to investigate their potential in the formation of hydrogels. Hydrogels are highly hydrated 3D cross-linked polymeric networks that exhibit outstanding physicochemical properties, including excellent biocompatibility, high permeability to various solutes, and high ionic conductivity, that render them appealing as scaffolds for tissue engineering, microparticles for drug delivery, and soft actuators for biosensors. (64−66) One class of such systems is injectable hydrogels, whose successful translation to clinics is dependent on meeting application-specific design criteria, such as injectability, mechanical properties, and cytocompatibility. (67) An attractive feature of the LAM–OH polysaccharide is its low viscosity, which facilitates its implementation through a minimally invasive strategy. (68,69) A cross-linking mechanism, which is determined by the chemical functionality of the polymer or/and the cross-linking agents, is typically required to confer mechanical performance to the final material structure. This step can occur in situ by the establishment of, for example, ionic interactions, hydrogen bonds, and/or covalent bonds within the mixing tip or under physiological conditions. (70,71) Herein, it was hypothesized that the previously developed functional biopolymers could be used to produce injectable hydrogels. To this end, LAM–NH2 was combined with the LAM–C═O polysaccharide to produce hydrogels via the formation of dynamic covalent bonds between their amine and aldehyde groups, respectively (i.e., Schiff base; right corner of Figure 4A). Figure 4 Figure 4. Hydrogel production through an (A) chemical or (B) photo-cross-linking process under mild conditions. Oscillatory time-sweep assays were performed to determine the hydrogels’ gelling point, which occurs when the storage modulus (G′) is equal to the loss modulus (G′′). The insets correspond to the (left corner) schematic representation of the obtained cross-links formed when combining the amine groups in LAM–NH2 with the aldehyde moieties in LAM–C═O (Chem condition: Schiff base) or between the thiol moieties in LAM–SH with the allyl groups in LAM–C═C after UV radiation in the presence of a photoinitiator (Photo condition, thiol–ene reaction) and (right corner) pictures of the obtained hydrogels. (C) Cell viability results presented as a % of the positive control. Cells were placed in contact with leachables of the former two cross-linked networks (Chem and Photo), obtained after 1 or 3 days of extraction in complete culture medium. (D) LAM–DOPA/FeIII hydrogel formation. In the photograph (i), a droplet of LAM–DOPA polymer was deposited above a glass slide. Afterward, FeIII solution was added to the LAM–DOPA solution (ii). The last photograph (iii) displays the formation of a hydrogel through coordination bonds by adding NaOH. (iv) As the pH increases, the hydrogel structure becomes more cross-linked due to the formation of bis- and tris-DOPA/Fe complexes. Hydrogels (Figure 4A, right corner) were formed under physiological conditions (pH 7.4 and 37 °C) without any initiator or catalyst. Oscillatory rheology assays were performed to determine the gelation time for these materials, characterized by the crossover between the storage (G′) and loss (G′′) moduli (Figure 4A). In this system, the gelation time was approximately 450 s, proving the conversion from a fluid-like state to a hydrogel. The obtained values are of the same magnitude as those found in the literature for the same cross-linking mechanism using different polysaccharides. (69,72) The thiol and alkene functional laminarin derivatives are ideally placed to enable gelation by photo-cross-linking (Figure 4B, left corner). Light-mediated thiol–ene reactions are a simple and cost-effective tool to fabricate 3D hydrogel networks with controlled shape. This strategy is mediated by a radical-based mechanism promoted by the irradiation of an initiator. It is worth noting that the LAM–C═C polymer was incapable of homopolymerization since it is a vinyl-ether, ensuring that the only reaction that took place was the thiol–ene click reaction. (73,74) The gels were successfully formed (Figure 4B, right corner) and displayed robust mechanical properties that were an order of magnitude higher than for the LAM–C═O/LAM–NH2 materials. Given the potential of these materials to be used as a biomaterial platform, consideration was given to their compatibility with cells. (75) The previous two hydrogels obtained through chemical reaction and photocuring were tested for their cytocompatibility, following ISO guidelines, in assessing the ability of the developed biomaterials to support living cell encapsulation. To this end, hydrogel leachables, collected after either 1 or 3 days of extraction in a complete culture medium, were placed in contact with a standard cell line and their metabolic activity was measured. The values represented are relative to the positive control values, which were considered to be 100%. The developed hydrogels do not present any significant differences compared with the positive control, meaning that the produced hydrogels are cytocompatible and that cells remained alive after contact with the leachables (Figure 4C). To prompt the cell adhesion of the developed hydrogel devices, other functionalization strategies could be employed such as the incorporation of cell-adhesive RGD tripeptide (Arg-Gly-Asp). Besides the potential to be injected, the possibility to stabilize the polymer networks by applying a cross-linking mechanism, and cytocompatibility, it is also required that the biomaterials are adhesive to ensure their correct fixation to the place to be treated. (76) DOPA moieties are present in the mussel byssus attachment system and are known to promote this marine organism underwater adhesion. (77) In this work, another attempt to form hydrogels was made by employing a coordination chemistry mechanism between the DOPA-functionalized biopolymer and iron ions. To this end, the LAM–DOPA polymer was mixed with FeIII ions in a 3:1 catechol/FeIII molar ratio (Figure 4D, left panel). Afterward, the pH was increased by the addition of NaOH. It is known that the number of DOPA/FeIII coordination bonds, from mono to tris complexes, is controlled by the pH value (Figure 4D, right panel). Interestingly, after adding EDTA, the previous hydrogel structure is decomposed, recovering its liquid-like state and consequently suggesting that covalent cross-linking does not play a significant role in the overall cross-links in the hydrogel. This reversible mechanism was also already used to produce hydrogels with adhesive properties and a self-healing ability. (78) Moreover, as already stated the amine conversion rate was not complete, meaning that there are still some amine groups available on the LAM–DOPA derivative. This is very important as the presence of charged groups close to DOPA moieties is known to promote higher adhesion rates. Conclusions ARTICLE SECTIONSJump To The structure of polysaccharides offers a wide variety of chemical groups with the potential to be functionalized and to incorporate desired physicochemical and biological features. Herein, a new collection of non-petroleum-based materials was developed through the derivatization of the LAM–OH macromolecule under mild conditions. Indeed, the employed simple, cost-effective, modular methodology enabled the successful design and production of a wide range of biopolymers exhibiting distinct chemical groups, namely, allyl, amine, carboxylic acid, thiol, aldehyde, and catechol, without significant effects on the bulk properties of LAM–OH. Likewise, the environmentally friendly strategy uses aqueous-based solutions as solvents, which has the advantage of removing additional and labor-intensive steps to make the polymers soluble. This methodology is envisioned to be easily translated to the manufacture of these or other LAM–OH derivatives and to enable the exploration of its potential to modify other materials bearing large numbers of hydroxyl groups, such as the highly abundant cellulose polymer. The potential of these materials for biomedical purposes was shown by their noncytotoxicity as well as their ability to be processed into hydrogels on the basis of (i) chemical dynamic cross-links via the formation of Schiff bases, (ii) a photo-cross-linking process between thiol and allyl moieties in the presence of a photoinitiator and UV light, and (iii) coordination bonds between catechol groups and iron ions. These multifunctional biomaterials are just one example of a possible application of the developed materials, but the possibilities are endless. For instance, taking into consideration the zeta potential values obtained for LAM–NH2 and LAM–COOH, these two derivatives could be complementarily assembled into multilayered substrates using a layer-by-layer strategy. Moreover, these materials could be developed into a sustainable materials platform to replace petrochemically derived plastics, and work to investigate their bulk material properties is ongoing. These advances in natural-based materials are envisioned to inspire and lead to commercially competitive applications, in the near future, for such multifunctional materials made from natural waste with the possibility to be recycled. Supporting Information ARTICLE SECTIONSJump To The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jacs.0c09489. Description of materials/reagents used; synthesis procedures and characterization of the obtained materials by NMR, FTIR, UV–vis, and colorimetric assays; hydrogel preparation; rheological measurements; and statistical analysis (PDF) Modular Functionalization of Laminarin to Create Value-Added Naturally Derived Macromolecules 45 views 38 shares 0 downloads Skip to figshare navigation S1Supporting InformationModular Functionalization of Laminarin to Create Value-Added Naturally-Derived macromoleculesAna M.S. Costaa, João M.M. Rodriguesa, Maria M. Pérez-Madrigalb, Andrew P. Doveb*, João F. Manoa*a CICECO – Aveiro Institute of Materials, Department of Chemistry, University of Aveiro, 3810-193, Aveiro, Portugalb School of Chemistry, University of Birmingham, Edgbaston, B15 2TT, Birmingham, UKCorresponding authors: Prof. Andrew P. Dove (a.dove@bham.ac.uk) and Prof. João F. Mano (jmano@ua.pt)TABLE OF CONTENTS1. Materials and Methods......................................................................................................S21.1. Materials:...................................................................................................................S21.2. 1H and 13C Nuclear Magnetic (NMR) spectroscopy: .................................................S21.3. Fourier Transform Infrared (FTIR) spectroscopy: .....................................................S21.4. Zeta potential: ...........................................................................................................S21.5. Gel Permeation Chromatography (GPC)/Size-exclusion chromatography (SEC): ...S21.6. Ultraviolet–visible (UV-Vis) spectroscopy: ................................................................S31.7. Synthesis of LAM-OH functionalized with allyl groups (LAM-C=C) via nucleophilic substitution: ......................................................................................................................S31.8. Synthesis of LAM-C=C derivatives, namely LAM-NH2, LAM-COOH and LAM-SH, by thiol Michael addition:.......................................................................................................S31.9. Synthesis of LAM-C=O derivative through an oxidation process: .............................S41.10. Synthesis of LAM-DOPA polymer via EDC/NHS coupling using either LAMCOOH or LAM-NH2 derivatives as starting materials: .................................................................S41.11. Hydrogel Preparation through: ................................................................................S51.11.1. Chemical crosslinking between LAM-NH2 and LAM-C=O derivatives: .........S51.11.2. Photocrosslinking between LAM-SH and LAM-C=C derivatives: .................S51.11.3. Crosslinking promoted by coordination bonds between Fe3+ ions and the catechol groups on LAM-DOPA derivative: ..............................................................S51.12. Rheological measurements:....................................................................................S51.13. Hydrogel cytotoxicity: ..............................................................................................S61.14. Statistical Analysis: .................................................................................................S62. Supplementary Data..........................................................................................................S82.1. Tuning the SD of LAM-(C=C) derivative ...................................................................S82.2. Removal of acetyl groups from the intermediate compound of the LAM-SH ............S92.3. FTIR spectroscopy of LAM-OH, LAM-C=C, LAM-NH2 and LAM-COOH and colorimetric assays.........................................................................................................S102.4. Detailed characterization of LAM-C=O derivative by 1H-NMR, 13C-NMR and FTIR spectroscopy ..................................................................................................................S113. References:......................................................................................................................S12 S21. Materials and Methods1.1. Materials:Laminarin from Eisenia Bicyclis (LAM, CAS: 9008-22-4, Mw≈6762 g/mol, PDI=1.7 determined by GPC against PEG standards) was acquired from Carbosynth. Allyl bromide (99%, stabilized), sodium hydroxide (NaOH) pellets, glacial acetic acid (>99%) and hydrochloric acid (HCl, ≈37% w/w) were obtained from Thermo Fisher Scientific, while ethanol (≥99.8%) was purchased from VWR. Cysteamine hydrochloride (≥97.0%), 2-hydroxy-4’-(2-hydroxyethoxy)-2-methylpropiophenone (I2959, 98%), tris(2-carboxyethyl) phosphine hydrochloride (TCEP, ≥99.9%), potassium persulfate (KPS, 99%), sodium periodate (NaIO4, ≥98%), thioacetic acid (96%), ethylene glycol (≥99%), dopamine hydrochloride and thiolactic acid (95%) were purchased from Sigma-Aldrich. 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC, >98%), N-hydroxysuccinimide (NHS, >98%), 3,4-dihydroxybenzoic acid (>98%) were obtained from TCI Chemicals. Spectra/Por dialysis membranes with a molecular weight cut-off of 3.5 kDa were supplied from Spectrum Laboratories. All reagents used were of analytical grade and, hence, no further purification was performed. Unless otherwise stated, water purified in an Elix® Advantage 3 system (Merck) was used throughout.1.2. 1H and 13C Nuclear Magnetic (NMR) spectroscopy:1H and 13C NMR spectra were recorded on a BrukerAvance-300 spectrometer at 300.13 MHz and 75.47 MHz, respectively, in D2O and at 298 K. Chemical shifts are reported as parts per million (ppm) downfield from the internal standard trimethylsilane (TMS).1.3. Fourier Transform Infrared (FTIR) spectroscopy:IR spectra of the freeze-dried samples were recorded with an attenuated total reflectance (ATR) accessory on a Mattsson 7000 galaxy series spectrometer at room temperature (RT). For each sample, 256 scans were collected with a resolution of 4 cm-1 in the spectral width region of 4000–400 cm−1.1.4. Zeta potential:The zeta potentials of the synthesized compounds in water were obtained using a Malvern Zetasizer Nano-ZS (Malvern Instruments Ltd.) under an applied electric field and at 25 °C. All determinations were repeated five times.1.5. Gel Permeation Chromatography (GPC)/Size-exclusion chromatography (SEC):GPC/SEC measurements were performed on a 390-MDS Multi-Detector GPC/SEC System from Agilent Technologies fitted with RI and UV detectors (λ = 280 nm), 0.2 M NaNO3 with 0.01 % NaN3 solution was used as eluent at a flow rate of 1.0 mL min−1 and at 40 °C. SEC data were calibrated against PEG standards. Share Download figshare Terms & Conditions Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html. Author Information ARTICLE SECTIONSJump To Corresponding Authors Andrew P. Dove - School of Chemistry, University of Birmingham, Edgbaston B15 2TT, Birmingham, U.K.; Orcidhttp://orcid.org/0000-0001-8208-9309; Email: a.dove@bham.ac.uk João F. Mano - CICECO − Aveiro Institute of Materials, Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal; Orcidhttp://orcid.org/0000-0002-2342-3765; Email: jmano@ua.pt Authors Ana M. S. Costa - CICECO − Aveiro Institute of Materials, Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal João M. M. Rodrigues - CICECO − Aveiro Institute of Materials, Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal; Orcidhttp://orcid.org/0000-0002-4031-8000 Maria M. Pérez-Madrigal - School of Chemistry, University of Birmingham, Edgbaston B15 2TT, Birmingham, U.K.; Orcidhttp://orcid.org/0000-0002-2498-8485 Notes The authors declare no competing financial interest. Acknowledgments ARTICLE SECTIONSJump To This work was developed within the scope of the CICECO-Aveiro Institute of Materials project (UIDB/50011/2020 and UIDP/50011/2020) and projects COP2P (PTDC/QUI-QOR/30771/2017) and MARGEL (PTDC/BTM-MAT/31498/2017), financed by national funds through the FCT/MEC and, when appropriate, co-financed by FEDER under the PT2020 Partnership Agreement. A.M.S.C. also acknowledges FCT for her Ph.D. grant (SFRH/BD/101748/2014).