Article pubs.acs.org/Biomac
Amphiphilic Graft Copolymers from End-Functionalized Starches: Synthesis, Characterization, Thin Film Preparation, and Small Molecule Loading Lisa M. Ryno, Cassandra Reese, McKenzie Tolan, Jeffrey O’Brien, Gabriel Short, Gerardo Sorriano, Jason Nettleton, Kayleen Fulton, and Peter M. Iovine* Department of Chemistry and Biochemistry, University of San Diego, San Diego, California 92110, United States S Supporting Information *
ABSTRACT: End-functionalized macromolecular starch reagents, prepared by reductive amination, were grafted onto a urethanelinked polyester-based backbone using copper-catalyzed azide− alkyne cycloaddition (CuAAC) chemistry to produce novel amphiphilic hybrid graft copolymers. These copolymers represent the first examples of materials where the pendant chains derived from starch biopolymers have been incorporated into a host polymer by a grafting-to approach. The graft copolymers were prepared in good yields (63−90%) with high grafting efficiencies (66−98%). Rigorous quantitative spectroscopic analyses of both the macromolecular building blocks and the final graft copolymers provide a comprehensive analytical toolbox for deciphering the reaction chemistry. Due to the modular nature of both the urethane-linked polyester synthesis and the postpolymerization modification, the starch content of these novel hybrid graft copolymers was easily tuned from 28−53% (w/w). The uptake of two low molecular weight guest molecules into the hybrid polymer thin films was also studied. It was found that binding of 1-naphthol and pterostilbene correlated linearly with amount of starch present in the hybrid polymer. The newly synthesized graft copolymers were highly processable and thermally stable, therefore, opening up significant opportunities in film and coating applications. These results represent a proof-of-concept system for not only the construction of starch-containing copolymers, but also the loading of these novel polymeric materials with active agents.
1. INTRODUCTION
Our entry point into starch chemistry was focused on delineating fundamental properties of amylose-based inclusion complexes.12−18 Based on these results and all available literature we reasoned that synthetic-natural copolymers containing amylose or high amylose starch could be used as reservoirs for active agents in film or coating applications. Several groups, but especially Borsali and co-workers, have elegantly prepared polysaccharide19−24 and oligosaccharidebased25−27 linear block copolymers from discrete end-modified reagents.28 Grafting-from strategies (where starch is the main chain polymer) have also been heavily pursued.6−8,29−31 Grafting-from approaches most likely dominate the literature because (1) there is a plethora of intrinsic reactive graft sites on the starch itself and (2) the alternative, that is, grafting-to, requires a discrete starch macromolecular reagent. In this context, end-modified starches represent a valuable yet underutilized class of macromolecular reagent. End-modified starches are prepared by site-specifically addressing the unique reducing end group of the polysaccharide. Starch endfunctionalization allows one to conjugate to a wide variety of
Starch represents an abundant, renewable, and low cost hydrophilic biopolymer that exhibits excellent biodegradability and biocompatibility. Beyond these favorable biomaterial properties, starch is an attractive raw material because its structure can easily be manipulated. Molecular weight can be tuned by chemical or biochemical hydrolytic degradation, while structure can be tuned by straightforward chemical reactions or enzymatic methods.1 Starch, therefore, is one of the most versatile polysaccharides for synthesizing new biopolymer or hybrid materials and establishing structure−function relationships. Starch-based materials have some limitations in their physical properties because of their rapid uptake of water, poor mechanical strength, and rather limited processing scope. Standard noncovalent strategies to improve starch-based materials properties include blending and compatibilization.2−4 Covalent modification, in comparison, allows one to broadly tune starch physical properties.5−8 Simply acylating backbone hydroxyl groups on the starch, for example, provides access to a material (starch acetate) that has dramatically improved mechanical and water uptake properties;9,10 chemically modified starches featuring cationic groups are also valuable in terms of their antibacterial properties.11 © 2014 American Chemical Society
Received: April 16, 2014 Revised: July 9, 2014 Published: July 9, 2014 2944
dx.doi.org/10.1021/bm500572v | Biomacromolecules 2014, 15, 2944−2951
Biomacromolecules
Article
Table 1. Summary of Key Characterization Data for Both the (A) ULPE-N3 Backbone Polymers and (B) Hybrid Graft Copolymers 1−3 A name
synthetic yield (%)
N3-loading (μmol N3/g polymer)
Mn (g/mol)
Tg (°C)
theoretical feed ratio (MDI/PCL/N3)
actual feed ratio (MDI/PCL/N3)
ULPE-N3−1 ULPE-N3−2
67 81
182 133
68187 66851
−41.6 −45.6
3:2:1 5:4:1
3:2:1 6:5:1
B
a
name
synthetic yield (%)
starch content (wt %)
grafting efficiency (%)
Tg (°C)
N3/alkyne (mol ratio)
Hybrid-1−53 (from ULPE-N3−1) Hybrid-2−36 (from ULPE-N3−2) Hybrid-3−28 (from ULPE-N3−2)
81 63 >90a
53 36 28
84 74 98
−49.6 −46.7 −46.1
1:2 1:2 1:0.5
Calculated yield of this particular hybrid polymer was slightly over 100%, therefore, the yield is reported as >90%. portions spaced 1 h apart. The reaction was left to stir at 40 °C overnight; after the heating period, water (10 mL) was added to the reaction flask and the mixture stirred for 10 min. The reaction mixture was precipitated into ice-cold isopropanol (1 L), filtered, and dried. The crude end-functionalized starch was taken up in warm water (20 mL) and precipitated a second time into cold isopropanol (1 L). The off-white product was isolated by vacuum filtration and dried in a vacuum oven at 45 °C (5.46 g isolated yield). 1H NMR (500 MHz, DMSO-d6): δppm 7.20 (d, J = 5 Hz, 2H aromatic end group), 6.66 (d, J = 5 Hz, 2H aromatic end group), 5.71−5.63, 5.11 (anomeric 1H), 4.75−4.5, 3.66−3.47, 3.6−3.25, 1.65 (s). 1 H NMR Based Determination of Alkyne End Group Density on Starch Biopolymers. The normalized integration values of the 4alkynyl aniline aromatic protons (7.20 and 6.66 ppm) were compared to the anomeric proton (5.10 ppm) integration values. The molar ratio determined from this analysis was converted to an end group density (μmol of end group/g polymer) for a given starch batch using the molecular weights of the anhydroglucose repeat unit and the end group itself. These calculations do not take into account the low level of hydroxypropylation on the starch, and therefore, values obtained reflect slightly higher than actual end group densities. Urethane-Linked Polyester (ULPE-N3-1). A dry 250 mL threeneck flask fitted with an overhead stirrer, reflux condenser, and septum was charged with polycaprolactone diol (PCL diol; 4.017 g, 2.0 mmol) and methylene diphenyl diisocyanate (MDI; 0.759 g, 3.0 mmol) under nitrogen. Toluene (8 mL) was added and the reaction was heated at 80 °C for 4 h. A solution of dibutyltin dilaurate (0.102 g, 2% of solids) and azide-containing chain extender, 11-azidoundecyl 3-hydroxy-2(hydroxymethyl)-2- methylpropanoate (0.327 g, 1.0 mmol) in dry toluene (5.0 mL) was added and stirred at 80 °C for an additional 2 h. The reaction mixture was allowed to concentrate to the consistency of a thick paste during the 2 h chain extension. At the end of the heating period, the reaction mixture was cooled, the residue taken up in a minimum amount of THF, precipitated into ice-cold CH3OH (500 mL), and dried under vacuum at 30 °C for 12 h. The initially isolated precipitate was resuspended in warm THF (15 mL) at 45 °C and again precipitated into ice-cold CH3OH (500 mL). The off-white elastic solid was dried under vacuum at 30 °C for 12 h providing 3.406 g polymer (67% isolated yield). N3-loading density = 182 μmol N3/g polymer. 1H NMR (500 MHz, CDCl3): δ 7.20 and 7.00 (br, 24H, MDI CH2), 6.69 (br, 6H, MDI NH), 3.18 (br, 2H, N3-H2), 2.25 (br, 32H, PCL-H2), 1.58, 1.33. IR: 2900−2700 (C−H), 2097 (N3). Urethane-Linked Polyester (ULPE-N3-2). A dry 250 mL threeneck flask fitted with an overhead stirrer, reflux condenser, and septum was charged with polycaprolactone diol (PCL diol; 4.003 g, 2.0 mmol) and methylene diphenyl diisocyanate (MDI; 0.625 g, 2.5 mmol) under nitrogen. Toluene (8 mL) was added and the reaction was heated at 80 °C for 4 h. A solution of dibutyltin dilaurate (0.048 g, 1% of solids) and 11-azidoundecyl 3-hydroxy-2-(hydroxymethyl)-2-methylpropanoate (0.164 g, 0.5 mmol) in dry toluene (2.5 mL) was added and stirred at 80 °C for an additional 2 h. The reaction mixture was allowed to concentrate to the consistency of a thick paste during the 2 h chain extension. At the end of the heating period the reaction
small molecules, surfaces,32 and polymers while preserving the native hydroxyl-rich structure of the starch backbone. We hypothesized that graft copolymers, featuring starch side chains, may allow for greater control over a material’s final physical properties (e.g., solubility) as compared to block motifs. In the graft-to approach, where starch biopolymers are the grafting agents, one can tune the final concentration of starch in the copolymer by adjusting the macromolecular reagent stoichiometry in the postpolymerization conjugation step. The graft-to approach also allows one to mix and match the type and amount of grafting agent in order to rationally adjust the solubility and physical properties of the final material. We have targeted hybrid graft copolymers featuring urethane-linked polyester (ULPE) backbones and high-amylose starch grafts. There are two main reasons we chose ULPEs as the counterpart to starch. First, ULPEs are widely used in biomedical applications and are known to be biocompatible and biodegradable.33−35 Second, the synthesis of ULPEs is highly modular and allows one to easily tune the polymer structure and, ultimately, the final concentration of starch in the copolymer.36 We also report an initial assessment of film forming properties and the propensity of these materials to retain small hydrophobic molecules. The graft copolymers described herein represent a proof-of-concept system where starch pendant chains can play an active role in sequestering small molecules. The synthesis is versatile and amenable to scaling-up due to the modular nature of polyurethane chemistry and the availability of end-functionalized starches. Lastly, thermal analysis indicates that the newly prepared starch-containing copolymers are thermally robust and may indeed be thermally processable.
2. MATERIALS AND METHODS Materials. Molecular weight modified (Mn ∼ 6500, as determined by GPC in DMF 0.01 M LiCl relative to pullulan standards) hydroxypropylated Hylon VII was a generous gift from Ingredion Inc. 1,1,1,3,3,3-Hexafluoro-2-propanol (HFIP), 1-naphthol, polycaprolactone diol (Mn ∼ 2000), dibutyltin dilaurate, sodium triacetoxyborohydride (97%), and ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA) were used as received (Sigma-Aldrich Co.). Pterostilbene (Tokyo Chemical Industry Co., LTD) was also used as received. 11-azidoundecyl 3-hydroxy-2-(hydroxymethyl)-2-methylpropanoate was prepared according to the literature.37 Alkyne End-Functionalized Starch. Molecular weight modified hydroxypropylated Hylon VII (5.0 g) and 4-ethynyl aniline (0.45 g, 3.8 mmol) were added to 100 mL round-bottom flask with stir bar. DMSO (40 mL) was added and the reaction stirred under N2 at 40 °C for 1 h. NaBH(OAc)3 (1.6 g, 7.6 mmol) was added in two equal 2945
dx.doi.org/10.1021/bm500572v | Biomacromolecules 2014, 15, 2944−2951
Biomacromolecules
Article
mixture was cooled, the residue taken up in a minimum amount of THF, precipitated in ice-cold CH3OH (500 mL), and dried under vacuum at 30 °C for 12 h. The initially isolated precipitate was resuspended in warm THF (15 mL) at 45 °C and precipitated again into ice-cold CH3OH (500 mL). The off-white elastic solid was dried under vacuum at 30 °C for 12 h, providing 3.891 g of polymer (81% isolated yield). N3-loading density = 133 μmol N3/g polymer. 1 H NMR Based Determination of N3 Side Chain Density in Urethane-Linked Polyesters. Unique spectroscopic signatures were integrated and normalized for each component of the reaction feed: MDI, azide-containing chain extender, and polycaprolactone. Molar ratios extracted from this analysis were converted to experimental compositions (see Table 1) and compared to theoretical feed ratios. Additionally, the NMR-derived molar ratios were converted to N3 densities (μmol of N3 group/g polymer) for a given composition using the molecular weights of the respective components (Table 1). General Procedure for Synthesizing Hybrid Graft Copolymers. A stock solution containing 1:1 mol/mol Cu(I)Br/ N,N,N′,N″,N″-pentamethyldiethylenetriamine (PMDETA) solution in DMF (0.021 M) was prepared and stored in a glovebox. To a 75 mL pressure vessel was added alkyne end-functionalized starch, the azide-containing urethane-linked polyester, and a stir bar. In the glovebox, the CuBr/PMDETA solution (2× equiv relative to ULPEN3) was added by syringe to the pressure tube, the reaction concentration adjusted to 8−17% (w/w) using supplemental DMF, and the vessel sealed. After removal from the glovebox, the reaction was heated at 40 °C for 18 h. The green-gray solution was cooled to room temperature and precipitated into ice-cold methanol (15X volume of solution) containing 0.5 mM EDTA and isolated by centrifugation. The solid polymer was dissolved in warm DMSO (60 °C, 8% (w/w)) and reprecipitated into ice-cold methanol containing 0.5 mM EDTA. The hybrid polymer was isolated by centrifugation and washed with one portion of CH2Cl2 (20 mL/g polymer) to remove any unreacted urethane-linked polyester, filtered, and subsequently washed with two portions of warm water (35 °C, 15 mL/g polymer) to remove any unreacted starch. The final hybrid graft copolymer was dried by lyophilization and characterized by both thermal and spectroscopic means. Hybrid 1. The target reaction stoichiometry was 1 mol azide on ULPE-N3−1 backbone: 2 mol alkyne from end-functionalized starch. 1 H NMR (500 MHz, DMSO-d6): δ 9.56 (s), 9.48 (s), 8.59 (br, N−H, PCL), 8.30 (br, 1H, triazole-CH), 7.57 and 6.76 (d, J = 4 Hz, anilineCH), 7.34 and 7.08 (br, 4H, MDI-CH), 5.51, 5.41, and 4.57 (br, starch-OH), 5.12 (br, 1H, starch-CH), 3.98 (t, PCL-CH2), 3.67−3.59 (br, starch-CH), 2.27 (br, 2H, PCL-CH2), 1.55−1.51 (br, PCL-CH2), 1.29 (br, PCL-CH). Hybrid 2. The target reaction stoichiometry was 1 mol azide on ULPE-N3−2 backbone: 2 mol alkyne from end-functionalized starch. 1 H NMR (500 MHz, DMSO-d6): δ 9.47 (s), 8.54 (br, N−H, PCL), 8.31 (br, 1H, triazole-CH), 7.57 and 6.76 (d, J = 4 Hz, aniline-CH), 7.34 and 7.08 (br, 4H, MDI-CH), 5.50, 5.40, and 4.58 (br, starchOH), 5.12 (br, 1H, starch-CH), 3.98 (t, PCL-CH2), 3.67−3.59 (br, starch-CH), 2.27 (br, 2H, PCL-CH2), 1.53 (br, PCL-CH2), 1.29 (br, PCL-CH). Hybrid 3. The target reaction stoichiometry was 1 mol azide on ULPE-N3−2 backbone: 0.5 mol alkyne from end-functionalized starch. 1H NMR (500 MHz, DMSO-d6): δ 9.47 (s), 8.54 (br, N−H, PCL), 8.31 (br, 1H, triazole-CH), 7.57 and 6.76 (d, J = 4 Hz, anilineCH), 7.34 and 7.08 (br, 4H, MDI-CH), 5.49, 5.40, and 4.57 (br, starch-OH), 5.11 (br, 1H, starch-CH), 3.98 (t, PCL-CH2), 3.67−3.59 (br, starch-CH), 2.27 (br, 2H, PCL-CH2), 1.53 (br, PCL-CH2), 1.29 (br, PCL-CH). 1 H NMR-Based Determination of Starch Content in Hybrid Polymers. Unique spectroscopic signatures were integrated and normalized for each component of the hybrid polymer: MDI, azidecontaining chain extender, polycaprolactone, and starch (the anomeric proton was used). Using the 1H NMR-derived molar ratios, and knowing the repeat unit molecular weight for each component of the hybrid, the weight percent starch was calculated (see Table 1). For clarity, the starch content of each hybrid polymer is included in the
sample name (e.g., Hybrid-1−53, is a hybrid polymer that is 53% (w/ w) starch). Determination of Grafting Efficiencies. Grafting efficiencies were determined by two independent NMR-based methods and reported as averages in Table 1. The first approach compared the theoretical weight % starch in the hybrid polymer (calculated using the reaction stoichiometry and the macromolecular loading values) to the actual weight % starch observed in a given hybrid. The second method quantitated the grafting efficiency based on the relative amount of triazole produced compared to a theoretical triazole value. The aromatic protons of the MDI were used as an internal standard in these calculations as their spectroscopic features are unchanged going from ULPE-N3 base polymer to hybrid. The theoretical triazole value was based on integration of the methylene protons adjacent to the −N3 group in the ULPE-N3 polymer and assuming all azide converts to triazole. Gel Permeation Chromatography. Molecular weights of the ULPE-N3−1 and ULPE-N3−2 were determined on a Polymer Laboratories (now Agilent Technologies) GPC-120 equipped with two PL gel 5 mm MIXED-D columns, refractive index detection, THF mobile phase (1.0 mL/min at 40 °C), and poly(methyl methacrylate) standards (Agilent, PL2020−0101). Sample concentration was 2 mg/ mL. Spectroscopy. 1H NMR (Varian INOVA 500 MHz) spectra were obtained in either DMSO-d6 or CDCl3 at room temperature. Normalized integration values were used to quantify polymer composition and small molecule concentrations in the guest-loaded films. UV−vis measurements were performed on isolated, dried, and digested (DMSO, 2 mg/mL) film pieces. Spectra were recorded on a Carey UV−vis spectrometer in the 260−500 nm spectral region. Guest concentration was determined by comparing the absorbance at λmax (325 nm for 1-naphthol and 327 nm for pterostilbene) to a corresponding standard curve. Attenuated total reflection IR (ATRIR) spectra were obtained using a Jasco FTIR-480 plus. Thermal Properties. DSC data was acquired on a TA Instruments Q20. All samples (except the end-functionalized starch) were heated at 5 °C/min from −70 to 125 °C or 180 °C over 2 full temperature cycles, as indicated in Figure S5. The Tg for each ULPE polymer and hybrid polymer was calculated using the Universal Analysis software (TA Instruments) on the first heating cycle (Table S1). The alkyne end-functionalized starch macromolecular monomer was heated at 10 °C/min from 30 to 180 °C over 3 full cycles. For all samples measured, the thermograms for the final two cycles were, in most cases, identical. It should be noted that for samples containing starch, the first cycle showed a significant loss of water around 100 °C. In all complexes, the thermograms for the last (or only) two cycles were similar, and in most cases, identical. Thermal gravimetric analysis (TGA) was performed on a TA Instruments Q500; samples were heated from 30 to 425 °C at 10 °C/min under nitrogen gas (flow rate = 60 mL/min). Formation of Thin Films. Thin films of the starch-containing graft copolymers were prepared by drop casting colloidal dispersions of the hybrid polymer (0.8−1.6% w/w) in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) (∼1−1.5 mL). Briefly, the suspensions of the hybrid polymers in HFIP were stirred for ∼30 min until uniform. The suspensions were then transferred to 5 mL Teflon beakers and dried overnight under a light stream of N2. The films were further dried in a vacuum oven at 35 °C overnight. Film thickness was estimated by SEM and consistently fell in the range of 25−30 μm (Figure S7). Scanning Electron Microscopy. Samples were prepared by initially coating the sample with gold using an Emitech K550X sputter coater. Both secondary and backscattered electrons were collected from the scanning electron microscope (model: Hitachi S-3400N). Loading of Small Molecule Guests into Hybrid Polymer Films. A 0.85% (w/w) hybrid polymer suspension with 1.6% (w/w) guest relative to solvent was prepared in HFIP (typically approximately 1−1.5 mL) and stirred for 30 min until homogeneous. This solution was transferred to a 5 mL Teflon beaker and the solvent allowed to evaporate under a stream of N2 at room temperature. The film and excess guest were washed with a solvent that solubilized the small 2946
dx.doi.org/10.1021/bm500572v | Biomacromolecules 2014, 15, 2944−2951
Biomacromolecules
Article
Figure 1. 1H NMR overlay of end-functionalized alkyne-starch (bottom spectrum), ULPE-N3−2 backbone (middle spectrum), and Hybrid 3−28 (top spectrum). The 1H NMR spectrum of Hybrid-3 shows unique resonances for each of the key components of the copolymer: the triazole proton (gray star), aromatic protons on the MDI linkers (green diamond), the starch anomeric proton (blue square), and the PCL-alpha protons (red circle). molecule well but not the hybrid polymer (e.g., films containing 1naphthol and pterostilbene were washed with methanol). Multiple film washings were combined and evaporated, and the quantity of small molecule guest not bound in the film quantified. The washed film residue was dried in a vacuum oven at 35 °C for 1 h. This dried film material was resuspended in HFIP, cast again into a Teflon beaker, and the solvent evaporated under a stream of N2 (g). The final washed and transparent thin film was removed from the beaker and further dried in a vacuum oven at 35 °C for 1 h.
elegantly prepared amphiphilic maltoheptose-based block copolymers composed entirely of sugars using copper-catalyzed azide−alkyne cycloaddition (CuAAC) chemistry.39 In this paper, we focus exclusively on the reductive amination of high amylose starches. Using this approach, we installed an alkyne click synthon at the reducing terminus of the biopolymer and later used this synthetic handle to graft starch onto a synthetic backbone polymer. Commercially available 4-alkynyl aniline and sodium acetoxyborohydride were used to prepare alkyne-terminated starches. After precipitation and drying, end group density was assessed by 1D 1H NMR. By integrating the aromatic resonances of the 4-alkynyl aniline end group relative to the anomeric proton of the starch repeat unit, we obtained end group density values (μmol alkyne/g starch) for each batch of starch (Figure 1). Alkyne end group values (as well as azide densities on the ULPE) were used to calculate stoichiometries for subsequent grafting reactions. Variation in the alkyne/azide molar ratio during the grafting reaction represents one approach to rationally controlling starch density in the final amphiphilic polymer. Controlling azide density and, therefore, starch density through feed ratio adjustment in the ULPE itself represents a second mode of compositional control.
3. RESULTS AND DISCUSSION Synthesis and Characterization of Synthetic and Biopolymer Building Blocks. There are several published synthetic routes to end-functionalized oligosaccharides and starches. First, oxidatively prepared terminal lactones are accessible and can be opened, and therefore conjugated, with an assortment of nucleophiles. Loos et al. has used this approach to prepare amylose-b-polystyrene block copolymers.23,38 Oxime end group chemistry has also been used to prepare polysaccharide-b-PEG block copolymers.24 Alkyne incorporation at the oligo- or polysaccharide reducing end has also been used to generate block copolymer structures (but not graft copolymers).20−22,26,27 Lastly, Borsali et al. have also 2947
dx.doi.org/10.1021/bm500572v | Biomacromolecules 2014, 15, 2944−2951
Biomacromolecules
Article
Scheme 1. Shown is the Key Synthetic Grafting Step that Covalently Links End-Functionalized High Amylose Starch to an Azide-Pendant Urethane-Linked Polyester (ULPE) Using Copper-Catalyzed Azide−Alkyne Cycloaddition (CuAAC) Chemistrya
a
Although a strictly linear amylose macromolecular reagent is shown for clarity, the actual macromolecular reagent was hydroxypropylated high amylose starch.
Polycaprolactone (PCL) was the main component of the ULPE. We employed a prepolymer approach where, initially, PCL diol (Mn = 2000 g/mol) was condensed with MDI in toluene to prepare an isocyanate-terminated prepolymer. The prepolymer was then chain extended using an azide-containing diol ester of bis-MPA originally prepared by Altintas et al.37 Two different feed ratios gave rise to two different ULPEs: ULPE-N3−1 and ULPE-N3−2. Physical properties and molar compositions of these two polymers are summarized in Table 1. Azide loadings ranged from 182 μmol/g in ULPE-N3−1 to 133 μmol/g in ULPE-N3−2. Molecular weights (Mn) of the two chain extended ULPE polymers relative to poly(methyl methacrylate) standards were approximately 67000 g/mol (see Table 1). Synthesis of Hybrid Amphiphilic Polymers Using CuAAC Chemistry. The synthesis of starch-containing hybrid graft copolymers is shown in Scheme 1. DMF was chosen as the reaction solvent due to its ability to solvate the polymeric building blocks and its compatibility with the CuAAC methodology. Copper(I) conditions were used, as compared to aqueous copper(II), to avoid a potential oxidation of starch by Cu(II).40 Three hybrid graft copolymers were synthesized from two different ULPE-N3 base polymers. Hybrid-1−53 has the highest starch content (53% w/w), whereas Hybrid-3−28 has the lowest (28% w/w). In the case of Hybrid-1−53 and Hybrid-2−36, a 2-fold molar excess of alkyne end-functionalized starch was used relative to each azide site. In Hybrid-3− 28, the alkynyl starch was used as the limiting reagent relative to azide sites in order to achieve the lowest amount of starch in the final composition. Overall, the synthetic approach was highly versatile and allowed for straightforward adjustment of polysaccharide content. The purification of Hybrids 1−3 involved several important washing steps designed to remove copper catalyst, unreacted starch (or starch that did not contain an end group in the first place), and unfunctionalized ULPE-N3. The bulk of copper catalyst was removed by an initial precipitation of the reaction mixture into methanol containing 0.5 mM EDTA; a second
precipitation was required, however, to more fully sequester trace copper. A warm water wash was used to remove any unreacted starch from the hybrid while dichloromethane was used to remove unreacted or lightly substituted ULPE-N3. All washes were quantified and analyzed spectroscopically; in each case, the mass balance for the entire synthesis and purification (multiple wash steps) was in the range of 85−90%. Characterization of Hybrid Graft Copolymers. Spectroscopy. Purified starch-containing Hybrids 1−3 were analyzed spectroscopically to determine (1) the composition of the final hybrid polymer (i.e., wt % starch in the graft copolymer) and (2) the presence or absence of unreacted azide functional groups. Figure 1 shows the 1H NMR spectra of Hybrid-3−28 and its corresponding polymeric building blocks. Importantly, the spectrum of Hybrid-3−28 is essentially the sum of the end-functionalized starch and ULPE-N3−2 spectra. Triazole formation and, therefore, proof of grafting to the ULPE are confirmed based on the diagnostic peak found at 8.3 ppm (top spectrum, Figure 1). 1H NMR spectra for the other hybrid polymers, as well as the ULPE-N3−1 backbone can be found in the Supporting Information (Figures S1−S3). The grafting efficiencies of starch to the ULPE backbone were studied by 1H NMR (see Table 1 and the Materials and Methods). Hybrids 1 and 3 were synthesized with grafting efficiencies of approximately 84 and 98%, respectively, while Hybrid-2−36’s grafting efficiency was approximately 74%. Hybrid-3−28’s grafting efficiency was 98%, but it is important to note that this value is in relation to the 33% of the total azides targeted in the reaction stoichiometry. Essentially, for Hybrid-3−28 we purposely under-functionalized the backbone polymer. Unlike the syntheses of Hybrid-1−53 and Hybrid-2− 36 where azide was limiting, alkyne-functionalized starch limited the production of Hybrid-3−28. Expressing the grafting data in another way, Hybrid 1 features, on average, 12 polysaccharide grafts per ULPE backbone polymer. Hybrids 2 and 3 have approximately 6 and 4 grafts per main chain, respectively. 2948
dx.doi.org/10.1021/bm500572v | Biomacromolecules 2014, 15, 2944−2951
Biomacromolecules
Article
thermal processing techniques. The hybrid polymer with the least amount of starch (Hybrid-3−28) has the greatest resistance to degradation, with the temperature at 50% loss (T50%) approximately 90 °C higher than the ULPE backbone and over 140 °C higher than the alkyne end-functionalized starch itself. Noncovalent Loading of Small Molecule Guests into Graft Copolymers. It is well established that small molecule hydrophobic guests can induce the formation of and bind inside (or between) amylose helices.13−16 Our laboratory12 and others4,18,35,41−43 have studied the impact of amylose molecular weight and chemical structure, as well as the nature of the hydrophobic guest, on the propensity to form inclusion complexes. Based on our prior experience with amylose inclusion complexes and all available literature, we hypothesized that manipulating the grafting density of high amylose starch in the graft copolymer composition would directly impact small molecule loading profiles. The impact of starch content in Hybrids 1−3 on the loading density of small molecule guests in a film was assessed by UV− visible spectroscopy (Figure 3). Herein we present the results
Hybrid graft copolymers were also studied by FT-IR. Figure 2 shows an overlay plot of ULPE-N3−2 and its offspring hybrid
Figure 2. FT-IR spectra of the azide pendant polyurethane-linked polyester backbone ULPE-N3−2 and hybrid graft copolymer progeny.
polymers. As expected, the N3 spectroscopic signature at 2100 cm−1 is clearly evident in the ULPE-N3−2 (Figure 2, black line) and Hybrid-3−28 spectra. Hybrid-3−28 was synthesized in a manner that left, unreacted, N3 sites on the ULPE backbone. Interestingly, the spectra of Hybrid-2−36 (Figure 2, red line) and Hybrid-1−53 (Figure S4) do not show the presence of free azide despite an associated grafting efficiency under 100%. The reaction stoichiometry in each case featured excess starch so it was anticipated that the grafting would be exhaustive, however, our reported grafting efficiencies indicate that not all sites have been functionalized. One possible explanation for not observing free azide in Hybrid-1−53 and Hybrid-2−36 is simply that the density of free azide falls below our detection limit. In fact, when the spectra for Hybrids 1 and 2 are significantly intensified in the 2100 cm−1 region, one can potentially interpret a very weak and broad peak attributable to azide. Thermal Analysis. The thermal properties of the hybrid graft copolymers were studied by differential scanning calorimetry (DSC) (Table S1). Individual DSC traces for each ULPE backbone, the alkyne end-functionalized starch, and hybrid graft copolymers can be found in the Supporting Information (Figure S5). The Tg for each ULPE polymer and hybrid polymer was calculated using the Universal Analysis software (TA Instruments) on the first heating cycle (Table 1). Compared to polycaprolactone (Tg ∼ −60 °C), both the ULPE and hybrid polymer samples show an approximately 20 °C higher Tg. The addition of starch decreased the Tg of the hybrid polymer relative to its parent ULPE base polymer. This is most dramatically observed between the ULPE-N3−1 polymer and its offspring Hybrid-1−53 (containing the largest quantity of starch). Hybrid-1−53 has a Tg value approximately 8 °C lower than the ULPE-N3−1 base polymer. Thermogravimetric analysis (TGA) was also used to study the decomposition profiles of the ULPE base polymers, alkyneend functionalized starch, and the hybrid graft copolymers (Figure S6). The end-functionalized starch macromolecular building block shows an initial loss of water beginning around 70 °C and another significant weight loss beginning around 200 °C. Hybrid polymers 1−3 retain comparatively small amounts of water relative to the end-functionalized starch alone and are more resistant to thermal degradation than their individual macromolecular building blocks (Figure S6, Table S2). In general, the high temperature degradation for Hybrids 1−3 begins at approximately 280 °C and advocates well for future
Figure 3. Plot showing the wt % guest in complex, as determined by UV−visible spectroscopy, vs wt % starch in the hybrid polymer. It should be pointed out that the 0 wt % starch data point for 1-naphthol and pterostilbene overlap.
associated with two guests: 1-naphthol and pterostilbene. Naphthol is known to form V8 inclusion complexes with amylose.44−46 Pterostilbene was chosen as the second guest due to its stilbene-like structure and biomedical relevance (it is found in the skin of blueberries and has been reported to have antioxidant and anticarcinogenic properties beneficial to human health47). Guests were loaded into the thin films using a dropcasting/coevaporation method. Increasing the amount of guest relative to the host polymer in the coevaporation procedure was found to make little difference in the final concentration of guest included in the film. It is important to note that the loading protocol also includes several rigorous washing steps to remove unbound or loosely bound guest (Figure S8). Figure 3 shows the results of our loading studies with Hybrids 1−3 and two different guests. We observe a dramatic difference in the slope (and therefore relationship between amount of guest bound and the amount of starch contained in the hybrid polymer) for the two guests studied. 1-Naphthol, which is known to bind to amylose both inside the α-helix and in between helices,44,46 shows a correlation between the guest concentration in the film and the amount of starch contained in the hybrid polymer (slope = 0.732 ± 0.029, R2 = 0.999). The amount of pterostilbene observed in complex also increased linearly with increasing amounts of starch present in the hybrid 2949
dx.doi.org/10.1021/bm500572v | Biomacromolecules 2014, 15, 2944−2951
Biomacromolecules
■
polymers (slope = 0.168 ± 0.013, R2 = 0.994), albeit to a more modest extent than 1-naphthol. Interestingly, when the ULPE-N3−2 backbone was used as a control (0% starch) in the loading studies, a greater amount of guest (both 1-naphthol and pterostilbene) was observed bound than observed for the hybrid polymer with the least amount of starch (Hybrid-3−28). We believe the lack of swelling in the ULPE-N3 films during the methanol washing steps, as well as the physical properties of the film itself, are leading to some finite guest entrapment. To probe this issue further, we conducted a rudimentary swelling study with several of our polymeric materials (see Figure S9). All the starch-containing films swell substantially (e.g., a film of Hybrid-1−53 swells to 150% of original mass) in the presence of the wash solvent methanol and the extent of swelling correlates with the weight % of starch in the film. As expected, the ULPE-N3 backbone polymers do not swell to any significant extent (Figure S9). It would seem reasonable that more extensive swelling would lead to more efficient washing of guest out of the film. This is not the observed trend. Instead, as the swelling capacity increases (i.e., more starch is added to the hybrid polymer) more guest is found in the film. This suggests that the polysaccharide grafts are important in loading guest and that the guest is retained more effectively in the hybrid polymers with higher starch content. We cannot conclude what the exact nature of the interaction is between the guest molecules and the polysaccharide side chains, although this is an area we are currently investigating. Lastly, we conducted control guest loading experiments with the starch macromolecular building block in the absence of the ULPE backbone. It should be pointed out that these experiments were conducted on powders, not films, and the solubility of the end-functionalized starch (as well as the 1-naphthol and pterostilbene) in the loading solvent (HFIP) was poor. Nonetheless, when prepared in an identical manner as the actual hybrid copolymer film samples, isolated starch powders were found to contain the lowest loadings of guests. Loading of starch with 1-naphthol or pterostilbene resulted in complexes that contained 6.86 ± 3.46 and 0.98 ± 0.14% (w/w), respectively. The lack of solubility of both the starch host and the small molecule guests in the HFIP most likely limits loading in the starch-alone controls.
Article
ASSOCIATED CONTENT
S Supporting Information *
Raw and summarized thermal characterization data for newly synthesized polymers, spectroscopic data (NMR and IR), and SEM images of films described in the text. Two figures related to the small molecule loading study (Figures S8 and S9). This material is available free of charge via the Internet at http:// pubs.acs.org.
■
AUTHOR INFORMATION
Corresponding Author
*Phone: (619) 260-4028. Fax: (619) 260-2211. E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS The authors acknowledge Carmine P. Iovine for helpful discussions. Financial support was also made possible by the National Science Foundation under Grant Nos. CHE 1305117 and CHE 0746309. Lastly, the authors would also like to acknowledge the Dreyfus Foundation for financial support and O’Reilly Science Art for the paper’s table of contents artwork.
■
REFERENCES
(1) Liu, G.; Hong, Y.; Gu, Z. Carbohydr. Polym. 2013, 98 (1), 995− 1001. (2) Lu, D. R.; Xiao, C. M.; Xu, S. J. eXPRESS Polym. Lett. 2009, 3 (6), 366−375. (3) Brandelero, R. P.; Grossmann, M. V.; Yamashita, F. Carbohydr. Polym. 2012, 90 (4), 1452−60. (4) Wang, X. L.; Yang, K. K.; Wang, Y. Z. J. Macromol. Sci., Polym. Rev. 2003, C43 (3), 385−409. (5) Cyras, V. P.; Zenklusen, M. C. T.; Vazquez, A. J. Appl. Polym. Sci. 2006, 101 (6), 4313−4319. (6) Sugih, A. K.; Picchioni, F.; Janssen, L. P. B. M.; Heeres, H. J. Carbohydr. Polym. 2009, 77 (2), 267−275. (7) Najemi, L.; Jeanmaire, T.; Zerroukhi, A.; Raihane, M. Starch/ Staerke 2010, 62 (2), 90−101. (8) Bertoldo, M.; Zampano, G.; La Terra, F.; Villari, V.; Castelvetro, V. Biomacromolecules 2011, 12 (2), 388−398. (9) Fringant, C.; Desbrieres, J.; Rinaudo, M. Polymer 1996, 37 (13), 2663−2673. (10) Parandoosh, S.; Hudson, S. M. J. Appl. Polym. Sci. 2003, 48, 787. (11) Guan, Y.; Qian, L.; Xiao, H.; Zheng, A. Cellulose 2008, 15 (4), 609−618. (12) Ryno, L. M.; Levine, Y.; Iovine, P. M. Carbohydr. Res. 2014, 383, 82−8. (13) Godet, M. C.; Tran, V.; Colonna, P.; Buleon, A. Int. J. Biol. Macromol. 1995, 17 (6), 405−8. (14) Rappenecker, G.; Z, P. Carbohydr. Res. 1981, 89 (1), 11−19. (15) Putaux, J. L.; Nishiyama, Y.; Mazeau, K.; Morin, M.; Cardoso, M. B.; Chanzy, H. Macromol. Symp. 2011, 303 (1), 1−9. (16) Putseys, J. A.; Lamberts, L.; Delcour, J. A. J. Cereal Sci. 2010, 51 (3), 238−247. (17) Nishiyama, Y.; Mazeau, K.; Morin, M.; Cardoso, M. B.; Chanzy, H.; Putaux, J. L. Macromolecules 2010, 43 (20), 8628−8636. (18) Zaslow, B. Biopolymers 1963, 1 (2), 165−169. (19) Houga, C.; Giermanska, J.; Lecommandoux, S.; Borsali, R.; Taton, D.; Gnanou, Y.; Le Meins, J. F. Biomacromolecules 2009, 10 (1), 32−40. (20) Aissou, K.; Otsuka, I.; Rochas, C.; Fort, S.; Halila, S.; Borsali, R. Langmuir 2011, 27 (7), 4098−4103. (21) Bosker, W. T. E.; Agoston, K.; Stuart, M. A. C.; Norde, W.; Timmermans, J. W.; Slaghek, T. M. Macromolecules 2003, 36 (6), 1982−1987.
4. CONCLUSIONS Here we describe, for the first time, the synthesis and characterization of amphiphilic graft copolymers featuring starch-derived biopolymer pendant chains prepared using high yielding CuAAC chemistry. The availability of a wide array of alkyne end functionalized starch biopolymer reagents and azide functional host polymers paves the way for a selection of graft copolymers that can be customized for a wide array of applications. Three different hybrid polymers were prepared in excellent yield with starch content as high as 53 wt %. Thin films of these materials were prepared by solvent casting and loaded with small hydrophobic molecules. Guest loading was directly dependent on the structure of the guest and the amount of starch in the hybrid. These natural-synthetic graft copolymers are amenable to solution-phase processing, thermally robust and capable of sequestering small molecules due to the presence of the polysaccharide grafts. Future work will concentrate on defining the surface characteristics, biodegradability and release profiles of these materials. 2950
dx.doi.org/10.1021/bm500572v | Biomacromolecules 2014, 15, 2944−2951
Biomacromolecules
Article
(22) Loos, K.; Muller, A. H. Biomacromolecules 2002, 3 (2), 368−73. (23) Loos, K.; Stadler, R. Macromolecules 1997, 30 (24), 7641−7643. (24) Novoa-Carballal, R.; Muller, A. H. E. Chem. Commun. 2012, 48 (31), 3781−3783. (25) Haddleton, D. M.; Ohno, K. Biomacromolecules 2000, 1 (2), 152−6. (26) Otsuka, I.; Fuchise, K.; Halila, S.; Fort, S.; Aissou, K.; PignotPaintrand, I.; Chen, Y.; Narumi, A.; Kakuchi, T.; Borsali, R. Langmuir 2010, 26 (4), 2325−2332. (27) Otsuka, I.; Osaka, M.; Sakai, Y.; Travelet, C.; Putaux, J. L.; Borsali, R. Langmuir 2013, 29 (49), 15224−30. (28) Schatz, C.; Lecommandoux, S. Macromol. Rapid Commun. 2010, 31 (19), 1664−84. (29) Barikani, M.; Mohammadi, M. Carbohydr. Polym. 2007, 68 (4), 773−780. (30) Kweon, D. K.; Cha, D. S.; Park, H. J.; Lim, S. T. J. Appl. Polym. Sci. 2000, 78 (5), 986−993. (31) Maruyama, A.; Ishihara, T.; Kim, J. S.; Kim, S. W.; Akaike, T. Bioconjugate Chem. 1997, 8 (5), 735−742. (32) Cao, Z.; Tsoufis, T.; Svaldo-Lanero, T.; Duwez, A. S.; Rudolf, P.; Loos, K. Biomacromolecules 2013, 14 (10), 3713−22. (33) Lamba, N. M. K.; Woodhouse, K. A.; Cooper, S. L. Polyurethanes in Biomedical Applications; CRC Press: Boca Raton, FL, 1998. (34) Lendlein, A.; Langer, R. Science 2002, 296 (5573), 1673−1676. (35) Yang, L. X.; Wei, J. Z.; Yan, L. S.; Huang, Y. B.; Jing, X. B. Biomacromolecules 2011, 12 (6), 2032−2038. (36) Sardon, H.; Chan, J. M. W.; Ono, R. J.; Mecerreyes, D.; Hedrick, J. L. Polym. Chem. 2014, 5, 3547−3550. (37) Altintas, O.; Tunca, U.; Barner-Kowollik, C. Polym. Chem. 2011, 2 (5), 1146−1155. (38) Loos, K.; Boker, A.; Zettl, H.; Zhang, A. F.; Krausch, G.; Muller, A. H. E. Macromolecules 2005, 38 (3), 873−879. (39) Modolon, S. D.; Otsuka, I.; Fort, S.; Minatti, E.; Borsali, R.; Halila, S. Biomacromolecules 2012, 13 (4), 1129−1135. (40) Achremowicz, B.; Gumul, D.; Bala-Piasek, A.; Tomasik, P.; Haberko, K. Carbohydr. Polym. 2000, 42 (1), 45−50. (41) Tusch, M.; Kruger, J.; Fels, G. J. Chem. Theory Comput. 2011, 7 (9), 2919−2928. (42) Yang, L. Q.; Zhang, B. F.; Yi, J. Z.; Liang, J. B.; Liu, Y. L.; Zhang, L. M. Starch/Staerke 2013, 65 (7−8), 593−602. (43) Zabar, S.; Lesmes, U.; Katz, I.; Shimoni, E.; Bianco-Peled, H. Food Hydrocolloids 2009, 23 (7), 1918−1925. (44) Cardoso, M. B.; Putaux, J. L.; Nishiyama, Y.; Helbert, W.; Hytch, M.; Silveira, N. P.; Chanzy, H. Biomacromolecules 2007, 8 (4), 1319−26. (45) Le Bail, P.; Rondeau, C.; Buleon, A. Int. J. Biol. Macromol. 2005, 35 (1−2), 1−7. (46) Zhu, F.; Wang, Y. J. Food Chem. 2013, 138 (1), 256−62. (47) McCormack, D.; McFadden, D. Oxid. Med. Cell. Longevity 2013, 2013, 575482.
2951
dx.doi.org/10.1021/bm500572v | Biomacromolecules 2014, 15, 2944−2951
