Cascading “Triclick” Functionalization of Poly(caprolactone) Thin Films

Article pubs.acs.org/Biomac

Cascading “Triclick” Functionalization of Poly(caprolactone) Thin Films Quantified via a Quartz Crystal Microbalance Fei Lin,† Jukuan Zheng,† Jiayi Yu,† Jinjun Zhou,† and Matthew L. Becker*,†,‡ †

Department of Polymer Science, The University of Akron, Akron, Ohio 44325, United States Center for Biomaterials in Medicine, Austen Bioinnovation Institute in Akron, Akron, Ohio 44308, United States

‡

S Supporting Information *

ABSTRACT: A series of mono- and multifunctionalized degradable polyesters bearing various “clickable” groups, including ketone, alkyne, azide, and methyl acrylate (MA) are reported. Using this approach, we demonstrate a cascade approach to immobilize and quantitate three separate bioactive groups onto poly(caprolactone) (PCL) thin films. The materials are based on tunable copolymer compositions of εcaprolactone and 2-oxepane-1,5-dione. A quartz crystal microbalance (QCM) was used to quantify the rate and extent of surface conjugation between RGD peptide and polymer thin films using “click” chemistry methods. The results show that alkyne-functionalized polymers have the highest conversion efficiency, followed by MA and azide polymers, while polymer films possessing keto groups are less amenable to surface functionalization. The successful conjugation was further confirmed by static contact angle measurements, with a smaller contact angle correlating directly with lower levels of surface peptide conjugation. QCM results quantify the sequential immobilization of peptides on the PCL thin films and indicate that Michael addition must occur first, followed by azide−alkyne Huisgen cycloadditions.

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INTRODUCTION Synthetic aliphatic polyesters, such as poly(ε-caprolactone) (PCL), poly(lactide)s (PLAs), and poly(glycolide) (PGA), are used widely in biomedical applications such as scaffolds for tissue engineering, drug delivery devices, coatings, and sutures.1−4 However, the expanded use of degradable polyesters into applications requiring functional species to enhance the biointerfacial interactions is limited by the availability of reactive sites for the covalent immobilization of bioactive drugs, peptides, and proteins. In response to this limitation, significant efforts have focused on introducing chemically reactive pendent groups into the monomer species, which afford the placement of functional groups by postpolymerization modification.5−13 Copolymerizing commercial cyclic monomers such as ε-caprolactone (CL), lactide (LA), or glycolide (GA) with analogues possessing protected functional groups is a common strategy to introduce reactive handles. High reaction efficiency and reproducibility are critical when attaching bioactive molecules as they are expensive relative to the polymeric components, and showing efficacy in translational applications is dependent on the ability to correlate structure to function, especially on functional surfaces.14−18 Click chemistry approaches have provided multiple avenues to address these challenges, but optimizing the reaction conditions and quantifying the reaction efficiency remain difficult. The concept of “click chemistry” was introduced by Sharpless and co-workers,19 and the term now represents a wide range of reactions, including copper(I)-catalyzed azide− © 2013 American Chemical Society

alkyne Huisgen cycloaddition (CuAAC), thiol−ene radical additions, Michael additions, Diels−Alder reactions, oxime ligation, etc.20 “Click” reactions are highly selective, highly efficient, and orthogonal to other chemical groups and work in mild reaction conditions.20−23 They are widely used for the postpolymerization functionalization of surfaces, polymers, and nanofibers.6,24−26 In biological applications metal-free strainpromoted azide−alkyne cycloadditions (SPAACs) are attractive in which alkynes are activated using ring strain or incorporating electron-withdrawing groups or both.27−30 Strained alkynes have been shown to react with azide groups directly at room temperature without a metal catalyst.26,31,32 SPAAC has been used to immobilize functional molecules on polymer surfaces using click reactions, though it is still challenging to attach biomolecules on the same surface with different chemical reactions.24,33,34 The combination of ring-opening polymerization (ROP) with highly efficient (click) reactions provides a platform for the construction of degradable polyester materials possessing multiple functional groups.7,35 Emrick and co-workers synthesized alkyne- and azide-derived PCL and further functionalized the polymer with bioactive RGD peptide.35 Alkene sulfonefunctionalized polyester was prepared in the Zhong and Dove group for biomolecule attachment via Michael addition.6,10 Received: May 16, 2013 Revised: June 23, 2013 Published: June 24, 2013 2857

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Scheme 1. Random Copolymers of Poly(ε-caprolactone-co-2-oxepane-1,5-dione) Were Prepared by ROP of CL and 1,4,8Trioxaspiro[4.6]-9-undecanone, Followed by the Deprotection of the Ketone Groups Using Triphenylcarbenium Tetrafluoroboratea

a

The use of multiple functionalized aminooxy reactive groups enables the postpolymerization modification of multiple clickable groups on PCLbased copolymer films. graph. N,N-Dimethylformamide (DMF) (with 0.01 M LiBr) was used as the eluent with a flow rate of 0.8 mL/min at 50 °C. The molecular mass was calculated from universal calibration based on polystyrene standards. Electrospray ionization (ESI) was performed using an HCT Ultra II quadrupole ion trap mass spectrometer (Bruker Daltonics, Billerica, MA) equipped with an electrospray ionization source. MALDI-TOF mass spectra were obtained on a Bruker Ultraflex-III TOF/TOF mass spectrometer (Bruker Daltonics) equipped with a Nd:YAG laser (355 nm). All spectra were measured in positive reflection mode. Sodium trifluoroacetate was used as the cationizing salt. The thermal properties of the materials were recorded using a TA Instruments DSC Q2000. The temperature ramping rate was 10 °C/ min. Data were collected in the second heating cycle. The static contact angles were measured using an advanced goniometer (RaméHart Instrument Co., model 500) at 25 °C using ultrapure water (1 μL) (18 MΩ cm−1) as the probe fluid and analyzed by a drop shape analysis method. Quartz Crystal Microbalance. A Q-sense E4 operator from Biolin Scientific AB was used to study the peptide uptake of the polymer thin film. The SiO2-coated crystal sensor X301(5 MHz resonant frequency) was chosen for spin-coating the substrate. All presented data correspond to the normalized frequency of the seventh overtone. On the basis of the Sauerbrey model,43 the following equation is often utilized to convert the frequency shift into the mass change per area:

PCL with pendent ketone groups has been synthesized by multiple research groups.36−38 The ketone-derived copolymer poly(ε-caprolactone-co-2-oxepane-1,5-dione) (P(CL-co-OPD)) has higher glass transition and melting temperatures, degrades faster, and has stronger mechanical properties than PCL.38−40 The electrophilic ketones in the copolymer backbone can undergo oxime ligation reactions with hydrazine-, semicarbazide-, and aminooxy-functionalized molecules. These reactions have been used for the postpolymerization modification of P(CL-co-OPD) with peptides, drugs, and poly(ethylene oxide).8,12,41,42 Inspired by the “clickable” characteristics of the oxime ligation chemistry, we demonstrate a highly versatile method (Scheme 1) to synthesize PCL possessing single and multiple clickable reactive groups. We show using a cascade approach that only one monomer is required for the preparation of polymers with different reactive sites. Incorporation of additional clickable groups was accomplished via postpolymerization modification through oxime ligation with reactive aminooxy groups. Quartz crystal microbalance (QCM) and static contact angle measurements were used to quantify the conjugation efficiency of the bioactive peptides to a poly(caprolactone) film. 1H NMR and FT-IR also helped to confirm the availability of surface functional groups for chemical reactions.

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m=C

Δf n

(1)

where m is the mass change per area (mg/m2), C is the sensitivity constant, −0.177 (mg/(m2·Hz)), Δf is the change in resonant frequency (Hz), and n is the overtone number. Synthesis of Intermediates R-ONH2 (1, 2, and 3). Synthesis procedures and characterization spectra of the three intermediates with two clickable groups for each are described in detail in the Supporting Information. Synthesis of Monofunctionalized PCL (Scheme 1). Random P(CL-co-OPD) copolymers were prepared by ROP of CL and 1,4,8trioxaspiro[4.6]-9-undecanone (TOSUO), followed by the deprotection of the ketone groups using triphenylcarbenium tetrafluoroborate.37−39 Oxime ligation between keto-PCL and the respective intermediates resulted in three monofunctionalized PCLs: PCLalkyne, PCL-azide, and PCL-MA (MA = methyl acrylate). In a typical experiment, a 20 mL glass vial equipped with a magnetic stir bar was charged with keto-PCL copolymer (0.500 g; 0.4 mmol of the ketone group), intermediate 1 (0.085 g, 0.45 mmol, 1.1 equiv), ptoluenesulfonic acid (catalyst amount 2.0 mg), and THF (5.0 mL).

MATERIALS AND METHODS

Materials. All commercial reagents and solvents were purchased from Aldrich or Fisher Scientific and used without additional purification unless specifically noted. CL was dried with CaH2 overnight, distilled in vacuo twice, and stored under argon. All reactions were performed under a blanket of nitrogen unless noted otherwise. Instrumentation. NMR spectra were obtained using a Varian NMRS 300 MHz spectrometer. Chemical shifts are reported in parts per million (δ) and referenced to the chemical shifts of the residual solvent (1H NMR, CDCl3 (7.27 ppm), DMSO-d6 (2.50 ppm), D2O (4.80 ppm); 13C, CDCl3 (77.00 ppm), DMSO-d6 (39.50 ppm)). The following abbreviations were used to explain the multiplicities: s = singlet, d = doublet, t = triplet, br s = broad singlet, m = multiplet. FTIR spectra were recorded on a Shimadzu MIRacle 10 ATR-FTIR spectrometer. Size exclusion chromatography (SEC) analyses were performed using a TOSOH HLC-8320 gel permeation chromato2858

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Figure 1. Individual 1H NMR spectrum of monofunctionalized PCL in CDCl3: (a) PCL-ketone, (b) PCL-alkyne, (c) PCL-azide, (d) PCL-MA. The oxime ligation is quantitative and proceeds under reaction conditions that minimize the degradation of the polymer. Peaks arising from CDCl3 and water are marked with an asterisk. Surface−RGD Conjugation Studies via QCM Measurements. All experiments were performed at 20 ± 0.1 °C using a flow rate of 0.1 mL/min. The concentration of the RGD peptide in buffer solutions was 1 mM. Acetate buffer of CH3OONa/CH3OOH (100 mM, pH 4.5) containing 10 mM aniline catalyst23 was prepared for oxime ligation of keto-PCL, and phosphate-buffered saline (PBS) solution (100 mM, pH 7.4) was used in the Michael addition and SPAAC reactions. In the copper-catalyzed click reaction, sodium ascorbate (2 mM) and CuSO4·5H2O (0.5 mM) were added to the PBS solution as the Cu(I) catalyst supply. In a typical experiment, the polymer-coated QCM sensors were exposed to the respective buffer solution overnight to reach a hydrated equilibrium. The baseline was established over a 10 min interval. The reaction formulation (buffer solution plus peptide) was introduced using a 10 min continuous flow to make sure that the entire chamber was saturated with the reaction formulation. After 1 h, the sensors were rinsed thoroughly with buffer solution until a plateau was reached. Surface Chemistry Studies via 1H NMR. The reaction buffer solutions were identical to those in the QCM experiments, and the concentrations of the small molecules were fixed at 10 mM. Polymer films were prepared via solution-casting of polymer dichloromethane solution (5 mg/mL) onto an aluminum pan and, following drying, were immersed in the reaction formulation. After 1 h, the films were washed extensively with a buffer solution and pure water in sequence and dried in vacuum for further characterization. The o-(prop-2-yn-1yl)hydroxylamine (intermediate 3), 2-hydroxy-1-ethanethiol, 3-azidopropan-1-ol, and propargyl alcohol were chosen as small molecules to investigate the surface reactions of keto-PCL, PCL-MA, PCL-alkyne, and PCL-azide, respectively.

The reaction was allowed to proceed for 4 h at room temperature. The resulting polymer was isolated by precipitation in clod methanol, filtration, and drying in vacuum, giving the product as a white solid in a quantitative yield. Synthesis of Multifunctionalized PCL. Three types of multifunctionalized PCLs were synthesized: PCL-MA-alkyne, PCL-MAazide, and PCL-MA-alkyne-azide. The synthetic process was similar to that of monofunctionalized PCL, except a mixture of intermediates was used in one pot to replace the single functional species. In the initial study, the content of each clickable group was controlled using a defined feed ratio. Synthesis of RGD Peptides. Standard solid-phase (9fluorenylmethoxy)carbonyl (FMOC) methodology was utilized to synthesize RGD possessing different reactive end groups: RGDhydroxylamine, RGD-thiol, RGD-alkyne, RGD-azide, and RGDcyclooctyne. The reactive chemical handles were attached at the Nterminus during the synthesis. Peptides were cleaved from resin using standard conditions (45 min, 95% trifluoroacetic acid (TFA), 2.5% triisopropylsilane (TIPS), 2.5% water (by volume)) and precipitated in cold diethyl ether. The crude solid product was isolated by centrifuge, triturated, washed twice with diethyl ether, and dialyzed in deionized water (molecular weight (MW) cutoff 500 g/mol, cellulose membrane, Pierce), followed by lyophilization. Products were isolated as a white powder. The molecular masses were verified by ESI. Detailed synthesis procedures and characterization are described in the Supporting Information. Polymer-Coated QCM Sensors. The SiO2-coated crystal sensors (X301, 5 MHz resonant frequency) were cleaned thoroughly using standard methods prior to spin-coating. Polymer solutions were prepared by dissolving the functional polyester precursors (30 mg) in chloroform (2 mL) and filtered with a 0.4 μm PTFE filter. The films were spun at 2000 rpm for 1 min, and the acceleration time was 10 s. The thickness of the thin films was measured using spectroscopic ellipsometry as described in Table S1 (Supporting Information).

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RESULTS AND DISCUSSION Synthesis of Functionalized PCL. Scheme 1 shows the synthetic strategy employed in this study. Three bifunctional intermediates were synthesized, which includes an aminooxy

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Three multifunctionalized PCLs were also prepared via a “clickable group conversion” method for evaluation of the reaction efficiency: PCL-MA-alkyne, PCL-MA-azide, and PCLMA-alkyne-azide. The resulting chemical structures are confirmed with 1H NMR as shown in Figure 3. Each of the

group and an additional clickable functional group: alkyne, azide, or MA. Using an oxime ligation, a number of functional intermediates were grafted to keto-PCL, resulting in polyesters containing different clickable groups on the pendant side chains. The conversion reaction was completed in several hours using a stoichiometric feed ratio between the ketone and aminooxy groups at room temperature. Using the keto-PCL precursor, four monofunctionalized polyesters were prepared: keto-PCL for oxime ligation, PCL-azide/alkyne for Huisgen 1,3-dipolar cycloaddition, and PCL-MA for Michael addition. Their chemical structures were first characterized with 1H NMR as shown in Figure 1. The spectrum of keto-PCL is shown in Figure 1a and is identical to spectra reported previously.12,39 The reactions were confirmed using the chemical shifts of H at the “a” position (4.35 ppm) in Figure 1a to peak “i” (4.25 ppm) in Figure 1b, and peaks “c” and “d” shifted to peak “j”. In Figure 1b, the resonances at 4.62 ppm (h) and 2.44 ppm (k) are assigned to the alkyne bond. The chemical shift at 3.20 ppm (m) in Figure 1c was assigned to the methylene bonded to the azide. In Figure 1d three resonance peaks from 5.85 to 6.45 ppm (o, p, and q) come from an alkene group in PCL-MA. MALDI-TOF was utilized to further confirm the chemical composition of PCL-MA. From the mass spectra (Supporting Information, Figure S13), there are only two repeat units: one is m/z 114.1 from the CL unit, and the other is m/z 299.1 exactly from the OPD-MA unit. FT-IR also confirmed the existence of clickable groups in PCL copolymers, as described in Figure 2. In the FT-IR spectra of

Figure 3. 1H NMR spectra of functionalized PCL polymers in CDCl3. Peaks between 5.75 and 6.50 ppm are assigned to the alkene group of MA marked within the red dashed line. Key: (a) PCL-MA-alkyne, characteristic peak of an alkyne (2.45 ppm) marked within the green dashed line; (b) PCL-MA-alkyne-azide, characteristic peak of a methyl group bonded to an azide (3.25 ppm) marked within the blue dashed line; (c) PCL-MA-azide.

three multifunctionalized polymers contained an MA unit. The characteristic alkene peaks of MA are marked inside the red dashed lines between 5.85 and 6.45 ppm. In Figure 3a,b, PCLMA-alkyne and PCL-MA-alkyne-azide both possess alkyne group signatures as shown by the singlet at 2.45 ppm marked inside the green dashed line. In Figure 3b,c, both PCL-MAalkyne-azide and PCL-MA-azide polymers show azide groups with a chemical shift at 3.20 ppm assigned to the methylene group adjacent to the azide. The synthesis of mono- and multifunctionalized PCLs required one synthesized polymer. A distinct advantage to this method is the oxime ligation was conducted postpolymerization in one pot under mild reaction conditions. Furthermore, the content of each functional group in the final polymer can be tuned using the hydroxylamine feed ratio. This clickable group conversion method avoids the challenges associated with new monomer design and synthesis, which involves the optimization of polymerization conditions, including catalyst, initiator, temperature, solvent conditions, etc. Using a clickable group conversion strategy, a diverse library of functionalized PCLs could be synthesized using a combination of aminooxy intermediates. Conjugation of RGD to a Thin Film of Monofunctionalized PCL. An RGD peptide was chosen as a model peptide model to investigate the reactivity of biomolecule immobilization to polyester surfaces. RGD mediates integrin-based cell adhesion44 in an number of cell types and when tethered appropriately remains bioavailable on surfaces 45,46 and polymers.33,35,47 Using a QCM monitoring experiment, the reaction efficiency of five independent reactive peptide− polymer pairs was investigated. Control experiments were conducted to assess any physical absorption of RGD in each pairing. In Figure 4 the frequency shifts of the experimental pairings (black line) and controls (colored lines) are shown. A decrease in frequency during the experiment indicates an increase in mass on the surface of the QCM sensor. The

Figure 2. FT-IR spectra of monofunctionalized polymers indicate the presence of the oxime-ligated functional groups. (a) PCL-alkyne, −CCH stretch, 3264 cm−1; (b) PCL-azide, −N3 stretch, 2096 cm−1; (c) PCL-MA, CCH stretch, 3077 cm−1, and CC stretch, 1642 cm−1.

PCL-alkyne in Figure 2a, the peak at 3264 cm−1 corresponds to the C−H stretch of the triple bond in PCL-alkyne. The N3 stretch in PCL-azide is reported at 2096 cm−1 in Figure 2b. Characteristic absorption peaks of the alkene groups in PCLMA are present at 3077 and 1642 cm−1 in Figure 2c. Compared to that of keto-PCL, the glass transition temperature of PCLalkyne, PCL-azide, and PCL-MA does not change significantly. However, the melting temperature (Tm) was around 40 °C, much lower than that of keto-PCL, which was 60 °C. The suppression of Tm was attributed to the decrease in chain packing due to the existence of pendant side chains. 2860

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increase in mass correlates directly with peptide functionalization and/or protein absorption.48−50 After the introduction of functionalized RGD buffer solution to the functionalized PCL-coated QCM modules, the experimental frequency shifts decreased rapidly, indicating the chemical reactions happened almost immediately. For all five experimental polymer pairings, the extent of the reaction reached ∼80% during the first 20 min. The reactions reached their maximum conversion in about 1 h. This was determined by the establishment of a frequency plateau that corresponds to the complete surface conjugation of the available functional groups. The frequency increased slightly during the buffer rinse after the conjugation plateau and indicated that a small amount of residual physically adsorbed peptide was removed. In the control experiments (colored lines), the small frequency drop (2−5 Hz) due to physical absorption is much smaller than measured in the conjugation reactions. Figure 4a shows the conjugation reaction between keto-PCL and RGD-hydroxylamine. The control experiment uses a sensor coated with plain PCL instead of keto-PCL. Figure 4b shows the reaction of the PCL-MA and RGD-thiol system and two control experiments: unfunctionalized PCL was used to quantify the nonspecific absorption of RGD-thiol peptide, and an RGD peptide with free amine at the N-terminus was used to exclude any reaction between the amine and MA alkene group under the QCM incubation conditions. The frequency drop in each control is much smaller than that in the corresponding reaction experiment, indicating the covalent attachment of the peptide. Figure 4c,d describes the CuAAC conjugation between PCLalkyne and RGD-azide and between PCL-azide and RGDalkyne. In control experiments, there was no copper sulfate in the control buffer formulation. The frequency shift was very small without Cu(I) catalyst. Figure 4e shows the conjugation behavior between PCL-azide and RGD-cyclooctyne. In another control experiment unfunctionalized PCL was used instead of alkyne-functionalized PCL. The small frequency decrease of the control experiment was attributed to physical absorption, and the sharp drop in the experimental pair was due to a covalent reaction between the azide and the strained cyclooctyne via SPAAC. On the basis of eq 1 from the Sauerbrey model, changes in frequency are converted into an adsorbed mass and correspond to a molar amount, as listed in Table 1. While the QCM provides a measure of how much peptide is covalently tethered to the surface, the exact amount of residual surface-available functional groups is unknown, and therefore, the reaction efficiency of surface immobilization cannot be determined. The reported reaction efficiency was calculated using the ratio between the amount of converted functional groups and the total content in the whole film. According to the QCM results, the amounts of surface-conjugated peptides and the respective reaction efficiencies are different for each functionalized polymer and peptide system, though the bulk contents of functional groups are the same in each polymer. Many factors influence the rate and extent of reaction on a surface, especially steric hindrance, reactivity differences, and diffusion limitations of some reagents. In the five pairings we described, keto-PCL reacted with the smallest amount of RGD, and the reaction efficiency, 3.5%, was also the lowest. Steric hindrance can account for this as the side chain of keto-PCL is the smallest and the opportunity for the peptide to be in the correct orientation with the ketone group is reduced. SPACC used to react the PCL-azide system with the peptide derived with

Figure 4. The shift in QCM frequency corresponds to the conjugation efficiency between the peptide and the surface of the polymer thin film. A decrease in frequency indicates a mass increase due to peptide absorption, and the extent of decrease is directly proportional to the amount of increased mass. The data are summarized Table 1. Key: (a) RGD-aminooxy and PCL-ketone; (b) RGD-thiol and PCL-MA; (c) RGD-azide and PCL-alkyne, (d) RGD-alkyne and PCL-azide via CuAAC; (e) RGD-cyclooctyne and PCL-azide via SPAAC. 2861

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Table 1. Summary of the (a) QCM Frequency Shift (Δf) after Peptide Conjugation to the Monofunctionalized Polymer Surface, (b) Amount of Covalently Tethered RGD Calculated from Δf on the Basis of the Sauerbrey Model, (c) Reaction Efficiency Calculated by the Ratio between the Amount of Reacted Functional Groups and the Whole Amount in the Entire Film, and (D) Change of the Contact Angle (Δθ) after Peptide Conjugation PCL-azide PCL-ketone Δf (−Hz) RGD (nmol) efficiency (%) Δθ (−deg)

10.0 0.5 3.5 4.1

± ± ± ±

1.1 0.1 0.4 1.0

PCL-MA 29.8 1.4 9.5 10.9

± ± ± ±

PCL-alkyne

3.5 0.2 1.1 2.9

47.8 2.4 16.4 15.8

± ± ± ±

1.9 0.1 0.6 2.1

CuAAC 24.5 1.2 7.9 9.5

± ± ± ±

1.9 0.1 0.6 0.9

SPAAC 42.7 1.6 11.1 10.2

± ± ± ±

5.7 0.2 1.5 1.8

two spectra in Figure 6a shows a new resonance at 4.62 ppm that is assigned to the alkyne bond and a small wide shoulder between 4.2 and 4.3 ppm that proves the occurrence of the oxime ligation. In Figure 6b, which shows the PCL-alkyne precursor, the evidence for a surface chemical reaction is obvious. In addition to peaks labeled “b”, “c”, and “d” as noted in Figure 6b, small peak “a” at 7.8 ppm is assigned to the H of the triazole ring following the CuAAC cycloaddition. The decrease of the stretch associated with the alkyne was also observed via FT-IR (Supporting Information, Figure S5). Similarly, in Figure 6c for PCL-azide, the small peak at 7.8 ppm following the reaction corresponds to the triazole H. Peak “c” at 4.45 ppm is assigned to the methyl groups after reaction, which shifts downfield from 3.2 ppm in the original PCL-azide polymer. Meanwhile, the corresponding decrease of the azide stretch in the FT-IR (Figure S5) confirms the CuAAC reaction. In Figure 6d for the PCL-MA system, there is a new wide multipeak resonance between 2.75 and 3.75 ppm after the reaction, which is assigned to the product of Michael addition between 2-hydroxy-1-ethanethiol and MA. The peak at 3.5 ppm is assigned to the methylene group adjacent to the hydroxyl. The surface conjugation reactions of small molecules to polymer films occur in a manner similar to that of peptide− polymer film conjugation. In the QCM experiment, only peptides derivatized with the complementary reactive sites are tethered to the surfaces, resulting in a proportional decrease of frequency. Using small-molecule model reactions, the NMR signal shifts after conjugation demonstrate the occurrence of the click reactions. Cascade Conjugation of RGD to Multifunctionalized PCL Thin Films. The QCM was used to monitor the peptide conjugation via orthogonal click chemistry to the thin film surface of a multifunctionalized polymer in a series of cascading sequential reactions. In Figure 7a for the PCL-MA-alkyne system, a Michael addition was designed to perform the initial reaction followed by CuAAC reaction. After introduction of the RGD-thiol, the frequency decreased quickly by 15 Hz due to peptide conjugation to the MA units. Compared to the frequency shift of the monofunctionalized PCL-MA described above, this shift is smaller because the MA content in PCL-MAalkyne is only 5%, which is half that in the PCL-MA polymer. Following the Michael addition, the sensors were rinsed thoroughly with buffer solution until equilibrium, and then reaction formulation of RGD-azide for the second conjugation was loaded into the QCM chamber. Following the addition of aRGD-azide solution, the frequency again dropped quickly, indicating the covalent CuAAC addition of RGD to the polymer surface. Figure 7b shows the frequency behavior in the PCL-MA-azide system, which is very similar to that in the PCLMA-alkyne system. The initial Michael addition again showed a sharp frequency drop followed by the SPAAC reaction. In the

dibenzyl ring fused cyclooctyne (DIBO) resulted in higher peptide conversion than traditional CuAAC, even though the N-terminus of RGD-cyclooctyne is more bulky than that of RGD-alkyne. The copper-mediated cycloaddition was recently shown to involve two copper atoms,51 and the resulting CuAAC intermediate is a bulky complex between copper(I) and the alkyne, which results in a smaller amount of peptide reaction. In addition, the DIBO group is very lipophilic and as a result may be driven to the surface of the polymer film, resulting in more opportunities for the chemical reaction to occur.52 The PCL-MA-derivatized film reacted with more peptide through the Michael addition than PCL-azide via CuAAC due primarily to steric hindrance from the Cu(I)− alkyne complex. Interestingly, the PCL-alkyne resulted in the highest RGD immobilization, double the amount reported in the reverse of PCL-azide pairing, even though CuAAC intermediates were necessary in both systems. It is possible that more alkyne groups are present on the surface of the PCLalkyne film, but it is very difficult to confirm this. Another possible explanation is related to the steric hindrance as described above and associated diffusion. Together with the QCM data, contact angle (CA) measurements were used to confirm the changes in hydrophobicity of the polymer surfaces. The PCL film surfaces are hydrophobic with a contact angel around 78°. Following the peptide conjugation, the polymer surfaces are more hydrophilic due to the peptide attachment, which is confirmed with a smaller contact angle (Table 1, Figure 5). For example, the contact

Figure 5. Static contact angle of the PCL-alkyne thin film before (a) and after (b) RGD-azide peptide conjugation. A contact angle drop after reaction indicates an increase of the surface hydrophylicity due to water-soluble RGD attachment to the film.

angle of the PCL-alkyne film dropped from 78° to 63° following the peptide reaction. The magnitude of the contact angle decrease correlates directly with the amount of peptides tethered to the surface and agrees with the data acquired by QCM measurements. Conjugation of Small-Molecule Probes to PCL Films. To further confirm the availability of functional groups on the polymer surface for chemical reactions, a series of small molecules were chosen to react with the polymer films as model reactions. The 1H NMR spectra are shown in Figure 6, where the red line denotes the labeled chemical shifts following the reactions. O-(Prop-2-yn-1-yl)hydroxylamine (intermediate 3) was used to react with keto-PCL. A close comparison of the 2862

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Figure 6. 1H NMR spectra of solution-cast films in CDCl3 before (red) and after (black) surface reactions: (a) keto-PCL with O-(prop-2-yn-1yl)hydroxylamine (intermediate 3); (b) PCL-alkyne with 3-azidopropan-1-ol; (c) PCL-azide with propargyl alcohol; (4) PCL-MA with 2-hydroxyl1-ethanethiol.

Figure 7. Shift in the QCM frequency for the cascading conjugation between the peptide and the surface of the multifunctionalized polymer thin film: (a) PCL-MA-alkyne, Michael addition between RGD-thiol and MA units first, CuAAC between RGD-azide and alkyne units second; (b) PCLMA-azide, Michael addition between RGD-thiol and MA units first, SPAAC between RGD-cyclooctyne and azide units second; (c) PCL-MA-alkyneazide, Michael addition between RGD-thiol and MA units first, SPAAC between RGD-cyclooctyne and azide units second, CuAAC between RGDazide and alkyne units third.

the MA double bond and thiol group was active only when it was conducted at the first step. If CuAAC or SPAAC was performed first, there was no frequency shift attributed to the Michael addition in subsequent reactions. Figure 8 shows a small frequency shift in the second step after RGD-thiol, following the initial SPAAC reaction. This distinction could be due to a number of factors, such as a change in the surface microenvironment, since MA-thiol conjugation is pH influenced,53,54 or the interaction between triazole and MA

two systems, both the copper-catalyzed and the strainpromoted click reactions are available for reaction following the Michael addition. In the trifunctionalized polymer PCLMA-alkyne-azide, the reactions follow a predetermined sequence: Michael addition, SPAAC, and CuAAC, respectively. Figure 7c shows the obvious irreversible frequency shift at each step following the peptide addition, indicating the successful conjugation of RGD to the film surface. In all three multifunctionalized PCL systems, the Michal addition between 2863

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Functional Materials Center. We also acknowledge the use of a quartz crystal microbalance which was acquired as part of an NSF-MRI (National Science Foundation Major Research Instrumentation) award (Grant DMR-1126544). We gratefully acknowledge mass spectrometry confirmation of our peptide precursors from Ms. Kai Guo and Professor Chrys Wesdemiotis at The University of Akron.

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Figure 8. Shift in the QCM frequency in the PCL-MA-azide system. In the first step (black line) RGD-cyclooctyne for SPAAC was introduced. A significant decrease of frequency indicates the extent of a chemical reaction. In the second step (red line) RGD-thiol for Michael addition was introduced. A slight drop of frequency indicates that little to no chemical reaction occurred.

deactivating MA due to the electron density increase of the MA double bond due to triazole electron donation.55 The precise reason remains unknown at this time.

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CONCLUSIONS In summary, a series of mono- and multifunctionalized polyesters were synthesized by grafting clickable groups to PCL-co-OPD (keto-PCL) using an oxime ligation at room temperature. The materials demonstrated the ability to immobilize bioactive peptides to the polymer surface using several highly efficient (click) reactions. The extent of the individual reactions was confirmed by QCM and contact angle experiments. 1H NMR and FT-IR were used to prove the availability of clickable groups on the surface of the polymer film. Using the reactivity differences of the pendent side chain groups, a cascade of orthogonal reactions was also demonstrated. Three multifunctional polymer precursors were prepared for the sequential immobilization of peptides on the polymer surface. We further determined that the Michael addition must occur first in the cascading approach. Changing the order of the reaction sequence was not possible in using these reactions. In general, the described methodology provides a tool to synthesize functional PCLs bearing different clickable groups. This new system is likely to find applications in the biomolecular conjugation of peptides and drugs.24,34

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ASSOCIATED CONTENT

S Supporting Information *

Synthesis and characterization details of all molecules and QCM procedures. This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: 330-972-2834. Fax: 330972-5290. Notes

The authors declare no competing financial interests.

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ACKNOWLEDGMENTS We gratefully acknowledge financial support from the National Science Foundation (Grant DMR-1105329) and the Akron 2864

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Biomacromolecules

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