A Thermoresponsive Biodegradable Polymer with ... - ACS Publications

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A Thermoresponsive Biodegradable Polymer with Intrinsic Antioxidant Properties Jian Yang,†,‡ Robert van Lith,†,‡ Kevin Baler,† Ryan A. Hoshi,† and Guillermo A. Ameer*,†,§,∥,⊥ †

Biomedical Engineering Department, Northwestern University, Evanston, Illinois 60208, United States Department of Surgery, Feinberg School of Medicine, Chicago, Illinois 60611, United States ∥ Chemistry of Life Processes Institute, Northwestern University, Evanston, Illinois 60208, United States ⊥ Simpson Querrey Institute for BioNanotechnology in Medicine, Northwestern University, Chicago, Illinois 60611, United States §

S Supporting Information *

ABSTRACT: Oxidative stress in tissue can contribute to chronic inflammation that impairs wound healing and the efficacy of cell-based therapies and medical devices. We describe the synthesis and characterization of a biodegradable, thermoresponsive gel with intrinsic antioxidant properties suitable for the delivery of therapeutics. Citric acid, poly(ethylene glycol) (PEG), and poly-N-isopropylacrylamide (PNIPAAm) were copolymerized by sequential polycondensation and radical polymerization to produce poly(polyethylene glycol citrate-coN-isopropylacrylamide) (PPCN). PPCN was chemically characterized, and the thermoresponsive behavior, antioxidant properties, morphology, potential for protein and cell delivery, and tissue compatibility in vivo were evaluated. The PPCN gel has a lower critical solution temperature (LCST) of 26 °C and exhibits intrinsic antioxidant properties based on its ability to scavenge free radicals, chelate metal ions, and inhibit lipid peroxidation. PPCN displays a hierarchical architecture of micropores and nanofibers, and contrary to typical thermoresponsive polymers, such as PNIPAAm, PPCN gel maintains its volume upon formation. PPCN efficiently entrapped and slowly released the chemokine SDF-1α and supported the viability and proliferation of vascular cells. Subcutaneous injections in rats showed that PPCN gels are resorbed over time and new connective tissue formation takes place without signs of significant inflammation. Ultimately, this intrinsically antioxidant, biodegradable, thermoresponsive gel could potentially be used as an injectable biomaterial for applications where oxidative stress in tissue is a concern.

1. INTRODUCTION In order for new biomaterials to be suitable and widely used in the clinical setting, they should not exacerbate the body’s normal inflammatory response. Although inflammation depends on many factors, oxidative stress is a major one that can significantly contribute to increased acute and chronic inflammation and is often overlooked by the biomaterials community. During the inflammatory response, leukocytes release cytokines, chemokines, and generate excessive reactive oxygen species (ROS) (e.g., superoxide and hydroxyl radicals).1 Pro-oxidant molecules such as ROS can damage DNA, proteins and lipids, impairing normal cell function.2,3 The presence of oxidative stress due to ROS is especially relevant to biodegradable polymers, as accumulation of degradation products may generate excessive ROS, a significant cause of toxicity for many biodegradable materials and medical devices.4−8 Most approaches to deal with this problem focus on conjugating antioxidant molecules to the biomaterial’s surface or adding the antioxidant to the bulk polymer to provide local antioxidant therapy,9−12 but this approach results in materials © XXXX American Chemical Society

with low relative antioxidant mass. Oxidative stress may also be a pathophysiological response due to an imbalance between the production of oxidants and the antioxidant defense mechanism, resulting in a net increase in ROS.13 For example, oxidative stress has been implicated in atherosclerosis,14,15 wound healing,16 and neurodegenerative diseases.17 Therefore, biomaterials that can be delivered through minimally invasive methods and can counter the damaging effects of oxidative stress may be a useful tool for therapies that target these medical problems. Thermoresponsive water-soluble polymers are an important class of biomaterials due to their ability to undergo liquid to solid phase transition under physiologically relevant conditions and the potential for localized drug18−20 or cell21−23 delivery through minimally invasive procedures. Thermoresponsive polymers typically rely on hydrophobic and charge interactions that are sensitive to temperature changes to undergo a phase Received: July 9, 2014 Revised: September 29, 2014

A

dx.doi.org/10.1021/bm5010004 | Biomacromolecules XXXX, XXX, XXX−XXX

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Scheme 1. Synthesis of PPCN Copolymer and Formation of Branched, Antioxidant PPCN Gels

change. In particular, N-isopropylacrylamide (NIPAAm) has been used extensively. The poly(N-isopropylacrylamide) (PNIPAAm) homopolymer exhibits reversible phase separation due to the collapse of extended chains into globular structures that create physical cross-links in aqueous media at a lower critical solution temperature range of 28−32 °C, depending on solvent.24−27 The chain collapse that leads to gelation can cause significant syneresis, the shrinkage of gels, and accompanying water expulsion due to hydrophobic interactions within the system. This phenomenon is a well-known characteristic of PNIPAAm28,29 and can contribute to rapid, uncontrolled release of compounds and can exert detrimental mechanical forces on entrapped cells leading to cell death. Additional problems reported with NIPAAm-based materials include toxicity and nondegradability, which tend to persist30,31 despite the incorporation of several natural and synthetic polymers such as chitosan, gelatin, hyaluronic acid, dextran, poly l-lactide, poly(ethylene)glycol and polycaprolactone to increase biocompatibility.32−46 Moreover, none of these materials are intended to address oxidative stress. Some strategies have been reported to provide antioxidant functionality to thermoresponsive polymers either by conjugating linoleic acid, glutathione peroxidase, or polyaniline.47−49 However, those materials do not directly address an important mechanism for oxidation that involves iron ions in the Fenton reaction and they have not undergone in vivo testing. To address the above challenges, we set out to develop a novel antioxidant degradable nontoxic injectable material that displays low or no syneresis and allows for the easy entrapment of a wide range of therapeutics within its network. Our design strategy involved the synthesis of low molecular weight, hydrophilic polyethylene glycol citrate oligomers that are partially functionalized with acrylate chemical groups for subsequent polymerization with NIPAAm to produce a branched, water-soluble, thermoresponsive polymer with intrinsic antioxidant capacity. Citric acid is an inexpensive multifunctional monomer that can easily form ester bonds in the absence of catalysts,50 and a natural biological compound as a component of the cell’s Krebs cycle. The free carboxyl groups

facilitate further functionalization and form electrostatic interactions with charged biomacromolecules to allow for their slow, controlled release. Importantly, the carboxyl groups of citrate can chelate metals such as iron, a catalyst for the Fenton reaction that produces hydroxyl radicals. Previous attempts to provide thermoresponsive gels with antioxidant functionality have largely ignored metal chelation,47,48,51 despite the fact that transition metals such as iron are heavily implicated in many ROS-related conditions.47,48,51−53 Hence, we expected the inclusion of citric acid to provide significant antioxidative protection. Polyethylene glycol (PEG) is inexpensive, water-soluble, relatively nontoxic, nonimmunogenic, and used in medical implants to resist protein adsorption, platelet adhesion, and bacterial adhesion.54−57 It also forms hydrogen bonds with carboxyl groups, leading to complexation of polymer chains.58 Furthermore, as previously described, the combination of diols with citric acid can lead to intrinsically antioxidant materials.59 Herein, we describe the synthesis of a water-soluble acrylated polydiolcitrate using citric acid, polyethylene glycol, and glycerol 1,3 diglycerolate diacrylate. This water-soluble polydiolcitrate is referred to as poly(polyethylene glycol citrate) acrylate (PPCac) and can be further functionalized with NIPAAm to form poly(polyethylene glycol citrateco-N-isopropylacrylamide) (PPCN) polymer (Scheme 1). We hypothesized that a polyethylene glycol-based thermoresponsive polydiolcitrate would have intrinsic antioxidant properties, provide hydrophilicity and electrostatic repulsion forces to reduce syneresis, and allow for hydrolytic degradation through ester bonds, resulting in a biocompatible, injectable biomaterial suitable for applications where oxidative stress is a concern.

2. MATERIALS AND METHODS 2.1. Materials. N-Isopropylacrylamide (NIPAAm, 98%) was purchased from TCI America, citric acid (99.5%), polyethylene glycol (PEG, Mn = 400), glycerol 1,3-diglycerolate diacrylate, 2,2′-azobis(isobutyronitrile) (AIBN, 98%), 2,2′-azino-bis(3-ethylbenzthiazoline6-sulfonic acid) (ABTS), FeCl2·4H2O, ferrozine, linoleic acid, tween 40, and β-carotene were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). NIPAAm monomer was purified by recrystallization in B


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2.4. Morphology Assessment. Morphology of the PPCN gel was visualized using a quick-freeze deep etch (QFDE) method (detailed protocol in Supporting Information). PPCN solution was placed directly on the QFDE specimen disks, heated above its LCST until solid, and slam frozen. After etching, an exact replica of exposed gel structure was made and examined using scanning electron microscopy (SEM). 2.5. Antioxidant Properties. 2.5.1. Free Radical Scavenging. The ability of PPCN gels to scavenge the free radical cation, 2,2′azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) was assessed. A stock solution of 7 mM ABTS and 2.45 mM sodium persulfate in MQ water was prepared and left for 16 h in the dark at room temperature, after which the solution was sequentially filtered with 5, 1, and 0.45 um filters. This working solution was then exposed to samples (50 mg/mL) and incubated at 37 °C. At each time point, ABTS solution was sampled, diluted with MQ water 1:1 and the absorbance measured at 734 nm. All measurements were performed in triplicate. The antiradical activity was measured as % inhibition of free radicals by measuring the decrease in absorbance compared to control solutions. ePTFE and PLLA were used as negative controls because they are commonly used biomaterials that do not have antioxidant properties. Furthermore, in the case of PLLA, it has been shown to induce oxidative stress.61−63 2.5.2. Iron Chelation. The iron chelation activity was assessed by incubating samples (1 mL gelled, 100 mg/mL for PNIPAAm and PPCN, or 100 mg for solid polymers ePTFE and PLLA) with 0.25 mM FeCl2·4H2O solution (50 mg/mL) at 37 °C. ePTFE and PLLA were used as negative controls because they are not known to chelate metals. Supernatants were collected at each time point and reacted with 5 mM ferrozine indicator solution in a 5:1 ratio. Ferrous ions chelated by polymers will not be available for ferrozine reaction, resulting in lower color development. Absorbance was measured at 562 nm and the percentage of iron chelated calculated. 2.5.3. Lipid Peroxidation Inhibition. The antioxidant activity of polymers was also evaluated using the β-carotene-linoleic acid assay with a few modifications as described below.64 Briefly, tween 40 (4 g), β-carotene (4 mg), and 0.5 mL of linoleic acid were mixed in 20 mL of chloroform. After removing chloroform in a rotary evaporator, 30 mL of prewarmed Britton buffer (100 mM, pH 6.5) was added to 1 mL of the oily residue with vigorous stirring. Aliquots (1 mL) of the obtained emulsion were added to the samples (1 mL gelled, 100 mg/mL for PNIPAAm and PPCN, or 100 mg solid polymer for ePTFE or PLLA). ePTFE and PLLA were used as controls as they are not known to inhibit lipid peroxidation. Reaction mixtures were incubated at 45 °C for 100 min. Spontaneous oxidation of linoleic acid at 45 °C leads to β-carotene discoloration, which was monitored by the decrease in absorbance at 470 nm, starting immediately after sample preparation (t = 0 min). 2.6. Protein Entrapment and Release. A biologically relevant model chemokine, stromal cell derived factor 1α (SDF-1α; R&D Systems, 500 ng/mL doped with 1:100 iodinated SDF-1α65) was added to PPCN and PNIPAAm solutions (100 mg/mL, pH 7.4 in PBS), which were allowed to solidify at 37 °C for 30 min and gently rinsed with 37 °C PBS. Samples were incubated in PBS, kept at 37 °C, and at predetermined intervals the PBS supernatant from the gels was sampled and replaced by warm PBS. Total protein in the gel was quantified using a gamma counter by measuring the samples directly. A standard curve of iodinated SDF-1α protein was used to quantify released protein content. In vitro bioactivity of released SDF-1α was assessed using a modified transwell migration assay to assess cell migration toward a SDF-1α gradient (full protocol in Supporting Information). 2.7. In Vitro Cell Entrapment, Viability, and Proliferation in PPCN Gels. Human aortic smooth muscle cells (HaSMCs) and umbilical vein endothelial cells (HUVECs; Lonza, Walkersville, MD) were cultured in SmGM-2 and EGM-2, respectively, in a humidified incubator equilibrated with 5% CO2 at 37 °C. For all experiments, PPCN and PNIPAAm (pH 7.4) were sterilized with ethylene oxide gas. For cell compatibility assessment, PPCN and PNIPAAm were reconstituted in the respective cell type’s growth media (100 mg/mL),

hexane and dried under vacuum. All other chemicals used were of analytical grade without further purification unless stated. 2.2. Polymer Preparation and Characterization. PPCN was synthesized according to a two-step procedure that included a polycondensation reaction and free radical polymerization (Scheme 1). First, poly(polyethylene glycol citrate) acrylate prepolymer (PPCac) was prepared by a polycondensation reaction at 140 °C for 45 min by melting citric acid, PEG, and glycerol 1,3-diglycerolate diacrylate at a 5:9:1 molar ratio under constant stirring. PPCac and NIPAAm were added to a three-necked flask in a 1:1 mass ratio and dissolved in 1,4-dioxane (equimolar to NIPAAm). Azobis(isobutyronitrile) (AIBN) radical initiator was added to the PPCac and NIPAAm mixture (final concentration: 6.5 × 10−3 M) and reacted for 8 h at 65 °C in a nitrogen atmosphere without prior deoxygenation of the polymer solution. The reaction products were dissolved in 1,4dioxane and purified by precipitation in diethyl ether and vacuumdried. As a reference, poly(N-isopropylacrylamide) (PNIPAAm) homopolymer was also synthesized by free radical polymerization using a similar procedure.60 The pH was adjusted to 7.4 using NaOH and HCl and the typical polymer concentration in solution was 100 mg/mL in PBS or culture media (DMEM). The 1H NMR spectrum of PPCN was recorded using a Bruker Ag500 NMR spectrometer at ambient temperature, using DMSO-d6 as solvent, and tetramethylsilane (TMS) as the internal reference. Samples were incorporated in KBr pellets and Fourier transform IR transmission spectra were recorded on a Thermo Nicolet Nexus 870 spectrometer by accumulation of 32 scans, with a resolution of 8 cm−1. The number-average molecular weight Mn was determined using gel permeation chromatography (GPC; Agilent 1100 series equipped with Brookhaven BI-DNDC differential refractometer and BI-MwA Light scattering detector). GPC consisted of an Agilent G1310A pump and a PLgel 10 um Mixed-B LS column. Monodisperse polyethylene glycol (PEG) standards with four number-averaged molecular weights (395, 1425, 5400, 13200 Da, American Polymer Standards Corporation, Mentor, OH) were used to obtain an internal calibration curve, as they are most representative for our water-soluble polymers. The dn/dc value used for PEG in DMF was 0.07815 mL/g, and the dn/dc of DMF was 1.431 mL/g. All the standards and samples were dissolved to 10 mg/mL in DMF with 10 mM LiBr as mobile phase at a rate of 1.0 mL/min, injection volume of 100 μL, and measurements were taken at 25 °C (620 nm). 2.3. Lower Critical Solution Temperature, Syneresis, and Degradation. Cloud point measurements were performed in a Jasco815 circular dichroism (CD) spectrophotometer by monitoring the transmittance. The absorbance at 450 nm was measured at 1 °C/min from 15 to 45 °C. The temperature at 50% transmittance was defined as the LCST. To assess syneresis, samples (100 mg/mL, pH 7.4 in phosphate buffered saline (PBS)) were added to glass vials and solidified by incubation at 37 °C for 30 min. Excess water not contained within solidified gels was removed by aspiration and weighed. To calculate percent water loss, the following equation was used:

water loss(%) =

M H2O(mg) M i(mg)

·100

MH2O is the water mass expelled from the polymer during gelation and Mi is the initial water mass of the polymer solution. Hydrolytic degradation of PPCN and PNIPAAm gels (pH 7.4, 100 mg/mL) was examined by measuring mass loss after incubation in PBS at 37 °C. Samples were placed in a 37 °C incubator for 30 min, and warm PBS was added. After PBS was removed at specified time intervals, the polymers were snap-frozen, lyophilized, and weighed (Wt). The mass loss percentage was calculated as 100 × (Wo − Wt)/ Wo, with Wo the initial weight of lyophilized gels. Samples were prepared in triplicate for each data point. A thermostat mixer was used to keep all samples at 37 °C during handling to prevent loss of material into the PBS phase due to gel instability as a result of inadvertent cooling. The characterization of the thermal and rheological properties of the gels is described in the Supporting Information. C


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Figure 1. 1H NMR spectra confirm formation of poly(polyethylene glycol citrate-co-N-isopropylacrylamide) (PPCN) polymer and poly(polyethylene glycol citrate) acrylate prepolymer (PPCac; inset) in DMSO-d. PNIPAAm spectrum in Supporting Information, Figure S1.

Figure 2. FTIR spectra of PNIPAAm, PPCac prepolymer, and PPCN confirm copolymerization of PNIPAAm with PPCac. Samples were prepared in KBr pellets.

D


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cells were added (5.0 × 104 cells/mL), and the cell-polymer suspensions were added to 48 well ultralow attachment plates (Corning, NY). After solidification for 30 min at 37 °C, warm media was added. Cell viability was assessed after 3 days of culture using the Live/Dead assay (Life Technologies, Carlsbad, CA). For cell proliferation assessment, at each time point gels were allowed to reliquefy at room temperature, centrifuged to remove oligomer, and DNA content was measured using picogreen assay (Life Technologies, Carlsbad, CA) according to manufacturer’s instructions. 2.8. In Vivo Biocompatibility and SDF-1α Bioactivity Assessment. A rat subcutaneous injection model was used to assess the in vivo biocompatibility of gels (detailed protocol in Supporting Information). All animal experiments were approved by Northwestern University’s Animal Care and Use Committee. Each rat received four 150 μL injections of PNIPAAm and PPCN (100 mg/mL, pH = 7.4) and animals were euthanized at 3 and 30 days after injection. Implants and surrounding tissue were snap-frozen, sections of 10 μm cut, and three sections per slide stained with hematoxylin and eosin, as well as masson trichrome. Sections were also stained with mouse antirat CD68 and counterstained with 6-diamino-2-phenylindole hydrochloride (DAPI; Sigma-Aldrich, St. Louis, MO) to assess macrophage infiltration. To assess SDF-1α bioactivity in vivo, PPCN with SDF-1α (500 ng/mL) was injected in select animals. After 30 days, sections were stained with rabbit antirat CXCR4 with Alexa Fluor594 as secondary antibody. The delimitation between gel and native tissue was determined by differential interference contrast (DIC) imaging. 2.9. Statistical Methods. Statistical analysis was performed using Microsoft Excel software and Graphpad Prism 5.0 (Graphpad Software Inc., La Jolla, CA). Data from independent experiments were quantified and analyzed for each variable. Comparisons between two treatments were made using student’s t test (two tail, unequal variance) and comparisons between multiple treatments were made with analysis of variance (ANOVA) with Bonferroni posthoc analysis. A value of p < 0.05 was considered to be statistically significant.

Table 1. Characterization of PNIPAAm, PPCac, and PPCN PNIPAAm monomer molar ratioa feed ratio actual ratio Molecular Weightb Mn (kDa) PDI

9.74 1.93

PPCac

PPCN

CA/PEG/GDD

CA/PEG/GDD/NIPAAm

5:9:1 1.9:2.5:1

5:9:1:43.2 2.6:3.1:1:26.5

2.47 1.44

9.95 3.46

a

Calculated by 1H NMR by peaks 1, 2, 5, and 11 assigned to CA, PEG, GDD, and NIPAAm. bDetermined by GPC using polyethyeneglycol (PEG) standards in DMF.

FTIR analysis of the PNIPAAm homopolymer shows its characteristic peaks due to NH stretching at 3430 cm−1, amide I and amide II band CO stretching at 1660 and 1550 cm−1, and CH2 and CH3 specific peaks at 2875−2970, 1460, 1385, 1370, 1170, and 1125 cm−1. The CH2 and CH3 specific peaks are obscured in the PPCN spectrum by the alkane peak from PPCac. There is also a peak at 3075 cm−1 from the amide B band. The PPCac prepolymer as expected has particularly strong bands at 3410 cm−1 due to OH stretching from the carboxylic groups, at 2880−2910 cm−1 from the alkane CH groups, at 1740 cm−1 from ester CO stretching, and at 1105 cm−1 from alcohol C−O stretching. We can also identify a peak at 1640 cm−1 from the diacrylate CC bond available for radical polymerization with NIPAAm. In the PPCN spectrum, the broad OH band in PPCac has been replaced by the narrower NH peak of NIPAAm amide. The peak at 3410 cm−1 from PPCac is diminished due to further esterification and obscured by the −NH stretch from PNIPAAm incorporation. Amide II CO stretching is apparent at 1550 cm−1. Likewise, the ester CO peak at 1740 cm−1 and alcohol C−O peak at 1105 cm−1, as well as the alkane peaks from 2880 to 2910 cm−1 from PPCac are identified. The latter are reduced in intensity. A band around 1640−1660 cm−1 in PPCN is likely due to the amide II in NIPAAm, although overlap with the CC 1640 cm−1 peak means it cannot be excluded that some unreacted double bonds remain (Figure 2, Table 2). Using gel permeation chromatography (GPC), the molecular weight of PPCac and PPCN were determined to be approximately 2.47 and 9.95 kDA, with PNIPAAm homopolymer’s Mn determined to be 9.74 kDa (Table 1). The polydispersity of PPCN was higher than PNIPAAm at 3.46 versus 1.93, which may account for the broader curves in the cloud point measurements to obtain the LCST. The product of the radical polymerization reaction of PPCac with NIPAAm was of lower molecular weight than expected, which could be due to the intrinsic radical scavenging properties of PPCac (see also section 3.4 and Supporting Information, S2.7), which may be inhibiting the radical polymerization process by removing formed radicals, limiting the chain growth. This phenomenon of radical scavenging by polydiolcitrates has been previously reported.59 3.2. Lower Critical Solution Temperature, Syneresis, and Degradation. After adjusting the pH of the polymer solutions to 7.4, the transmittance versus temperature data from PPCN and PNIPAAm show phase transition profiles with LCSTs of 26.3 ± 0.2 and 28.9 ± 0.8 °C, respectively (Figure 3A). The incorporation of hydrophilic segments such as PEG and citric acid may cause ordering of water molecules, making

3. RESULTS AND DISCUSSION 3.1. Polymer Preparation and Characterization. The radical polymerization reaction between PPCac and NIPAAm results in the synthesis of poly(polyethylene glycol citrate-co-Nisopropylacrylamide) (PPCN), as verified by the 1H NMR and FTIR spectra (Figures 1 and 2, respectively). To provide insight into the effects of copolymerizing a polydiolcitrate with NIPAAm, PNIPAAm homopolymer was synthesized as a reference material. As determined by the 1H NMR, peaks labeled 3 around 3.5 ppm indicate PEG incorporation in both PPCac and PPCN, with the −CH2 group near ester bond after esterification shifted to 4.2 ppm (peak 2, see also Figure 1, inset). The multiple peaks at 2.79 ppm were assigned to the protons in −CH2− from citric acid, and the peak at 1.94 ppm was assigned to −CH2− in the NIPAAm unit (Figure 1). The characteristic peaks of the NIPAAm segments ranging from 0.8 to 2.2 ppm are found in the PPCN, denoted by the numbers 8, 9, and 10. The molar ratio of citric acid/NIPAAm in PPCN was approximately 1:10, as calculated from the signal intensities of peak 10 from PNIPAAm and peak 1 from citric acid, reasonable based on the molar feed ratio of 1:8.64. Vinyl protons were identified around 6.0 and 5.7 ppm in PPCac (Figure 1, red arrows, peaks 13 and 14 in inset), indicating the presence of acrylate groups after polycondensation. Disappearance of these peaks after free radical polymerization indicates successful addition of NIPAAm residues. Results were corroborated with the 1H NMR spectrum of PNIPAAm homopolymer (Figure S1), with identical peaks as assigned to NIPAAm units in PPCN. The ratios of citric acid, PEG, GDD, and NIPAAm were determined to be 2.6:3.1:1:26.5 from NMR (Table 1). E


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Table 2. Observed FTIR Bands (4000−500 cm−1) in PNIPAAm, PPCac, and PPCN wavenumber (cm−1)

PNIPAAm

A B C

3430 3075 2875−2970

X X X

X X X

D E F G H I

1650 1550 1460 1370, 1385 1125,1170 3410

X X X X X

X X

J K L M N

PPCac

PPCN

X

X

2880−2910 1740 1640 1105

X X X X

X X (X) X

950

X

X

unique nano- and microscale hierarchical features (Figure 3). Below 40 mg/mL, PPCN forms a white suspension of nanoparticles above the LCST. Resistance to compaction is important for tissue engineering or cell delivery applications because scaffolds that experience significant compaction will reduce cell viability and function. Mechanical forces applied by the scaffold to the cells likely damage them or negatively affect their differentiation.73 Moreover, gel shrinkage upon injection hinders tissue repair.74 In this regard, the percent water mass loss due to compaction upon gelation, or syneresis, of PNIPAAm and PPCN gels at 37 °C was assessed (Figure 3C,F). PNIPAAm exhibited significant water mass loss and compaction upon gelation, with a water loss of 61.5 ± 3.9%. In contrast, the PPCN gel had a water loss of only 16.0 ± 3.4% and retained most of its original volume (low syneresis). Syneresis is further reduced by increasing concentration of PPCN (Figure S3). PEG citrate segments within PPCN gels may help to prevent water expulsion from the network caused by the hydrophobic interactions between NIPAAm segments above their LCST. Watson et al. recently addressed the important issue of syneresis in thermoresponsive gels by synthesizing a copolymer that allowed chemical cross-linking of the gel, rendering the phase change above the LCST as irreversible.75 Unlike the latter strategy, the PPCN described here is relatively simple to synthesize and scale up, maintains the reversibility of the phase change, and has intrinsic antioxidant properties. (see section 3.4) Also, polyester segments within the PPCN allow for hydrolytic degradation over time. In vitro degradation studies show a mass loss of 24.7 ± 5.3% at two months versus no degradation for PNIPAAm gels (Figure 3B). Differential scanning calorimetry data show that the transition temperature present in PNIPAAm homopolymer is absent in PPCN likely due to the presence

assignment −NH stretch66 amide −NH stretch66 CH2, CH3 vibrations66 amide I66 amide II66 CH2 bend66 CH3, CH2 bend66 CH3, skeletal66 carboxyl −OH stretch67 alkane −CH stretch68 ester CO stretch68 CC stretch69 alcohol C−O stretch67 PEG400 ether C−O vibration (Sigma)

entropy of mixing more negative, thereby lowering the LCST.70 A similar reduction in LCST was previously observed when PEG was combined with PNIPAAm.46,70,71 Moreover, the phase transition temperature range was broadened for PPCN, a previously reported phenomenon when PNIPAAm was copolymerized with hydrophilic monomers.72 This broadening effect can also be ascribed to increased variation in branch points or the wide molecular weight distribution of the PPCN. Above the LCST and at solution concentrations above 40 mg/ mL, PPCN self-assembles into a hyperbranched network that forms the basis for a soft porous gel-like biomaterial with

Figure 3. PPCN forms biodegradable fibrillar gels with unique microscale and nanoscale hierarchical features. (A) Phase transition behavior of PPCN and PNIPAAm in phosphate buffered saline (PBS) and cell culture media; N = 3, mean ± SD. (B) Hydrolytic degradation over time; N = 3, mean ± SD; P < 0.05 for all time points after t = 0 days. (C) Quantitation of water loss upon gelation; N = 6, mean ± SD; P < 0.05. (D, E) SEM image of PPCN processed via QFDE showing the hierarchical micro- and nanoscale features. (F) Digital picture showing reduced PPCN gel compaction relative to PNIPAAm. All samples were prepared in PBS at a concentration of 100 mg/mL, neutralized to pH 7.4. Image was taken 30 min after gelation at 37 °C. F


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Figure 4. PPCN has intrinsic antioxidant properties: (A) ABTS radical scavenging capacity; (B) Iron chelation capacity; (C) Inhibition of lipid peroxidation. All samples were prepared in PBS at a concentration of 100 mg/mL, neutralized to pH 7.4; N = 3, mean ± S.D., *P < 0.05, **P < 0.01, ***P < 0.001 for all experiments.

Figure 5. PPCN entraps and slowly releases bioactive SDF-1α. (A) SDF-1α encapsulation efficiency in PNIPAAm and PPCN; N ≥ 3, mean ± S.D.; (B) SDF-1α cumulative release; N = 3, mean ± S.D.; *P < 0.05; (C) Transmigration of HUVECs in response to released SDF-1α relative to negative control. “Released SDF-1α” concentration: 63.99 ± 5.4 ng/mL; *P < 0.05, N = 4, mean ± SD. All gels were prepared at concentration of 100 mg/ mL, neutralized to pH 7.4. SDF-1α loading was 500 ng/mL.

pro-oxidant properties.61−63 Although citric acid has previously been shown to scavenge the radical cation N,N-dimethyl-pphenylenediamine,77 it is not a typically reported property for the monomer. Nonetheless, polydiolcitrate oligomers have previously been reported to scavenge radicals59 and PPCac, as the polydiolcitrate component in PPCN, has ABTS scavenging properties (Figure S6). The observed radical scavenging activity of PNIPAAm suggests, however, that there may be a third factor at play. Since superoxide radical scavenging by PNIPAAm has been observed before,48 this may be an inherent property of its structure. Nevertheless, our results show additional radical scavenging capacity for PPCN as expected. The radical scavenging activity could potentially be further increased by incorporating ascorbic acid in the PPCN backbone, as reported previously by us.59 PPCN provides strong iron chelation due to citric acid content (Figure 4B). PPCN is able to completely chelate iron ions present, while PNIPAAm, ePTFE, and PLLA do not. Lipid peroxidation was selectively inhibited by PPCN as assessed by the β-carotene bleaching assay (Figure 4C). After 100 min of heating at 45 °C, exposure to PPCN led to almost complete abolishment of lipid peroxidation. PNIPAAm, ePTFE, and PLLA, in contrast, did not inhibit lipid peroxidation (Figure 4C). The known role of iron in lipid peroxidation64 explains partially why PPCN, as an iron chelator, inhibits lipid peroxidation. 3.5. Protein Entrapment and Release. Gelled PPCN efficiently binds and slowly releases bioactive proteins.

of the hydrophilic polyethylene glycolcitrate repeat units (Supporting Information). Rheology data show an LCST as expected and demonstrate the capacity of PPCN to maintain its mechanical properties at temperatures above its LSCT, consistent with reduced syneresis, whereas PNIPAAm undergoes significant change in moduli above LCST likely due to compaction (Supporting Information). 3.3. Morphology. Scanning electron microscopy of PPCN gel samples obtained via quick-freeze deep-etch (QFDE) processing reveals a fibrilar nanoarchitecture that consists of branches and nodules (Figure 3D,E). PPCN self-assembles to form a biomaterial with pore sizes of 10.60 ± 2.79 μm, while PNIPAAm has a significantly smaller pore size of 1.77 ± 0.92 μm, likely due to PNIPAAm aggregation and compaction upon gelation. The unique hierarchical porosity of PPCN gels allows entrapment of cells with high viability, and also the entrapment and slow release of biomacromolecules and nanoparticles. It also facilitates the diffusion of nutrients and waste products from cells within the network.76 Due to the ability of PPCN to self-assemble and entrap targets that are within a wide size range (from nano to micro), we refer to them as nanonets. 3.4. Antioxidant Properties. PPCN uniquely exhibits significant direct and indirect antioxidant capacity. Both PPCN and PNIPAAm showed rapid free radical scavenging activity as per ABTS assay with over 70 and 45% of radicals scavenged within 48 h, respectively (Figure 4A). ePTFE, on the other hand, does not present any radical scavenging effect and PLLA seems to stabilize the radical, which is consistent with reported G


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Figure 6. PPCN supports cell entrapment and proliferation. (A−C) Human aortic smooth muscle cell viability and proliferation in the gel after entrapment; red: ethidium homodimer staining of dead/dying cells; green: calcein AM staining of live cells; N ≥ 4, mean ± S.D., P < 0.001. (D−F) Human umbilical vein endothelial cell viability and proliferation in the gel after entrapment; red: ethidium homodimer staining of dead/dying cells; green: calcein AM staining of live cells; N ≥ 3, mean ± S.D., P < 0.001. All gels were prepared at a concentration of 100 mg/mL, neutralized to pH 7.4. Scale bars: 200 μm.

when cells are entrapped significant loss of viability occurs in PNIPAAm after only 3 days of culture, whereas the majority of cells are viable within PPCN (Figure 6). Moreover, cells have remained viable within the PPCN gel for at least 72 days (Figure S7). Additionally, both HUVECs and HaSMCs proliferated well inside PPCN gels, indicating effective nutrient diffusion through the gels. These results confirm that PPCN provides a favorable environment for cell entrapment likely due to the hierarchical porosity, hydrophilicity, and low-compaction properties of the network.90 3.7. In Vivo Biocompatibility and SDF-1α Bioactivity. PPCN immediately forms a macroscopic gel in situ when injected into rat subcutaneous tissue, facilitates new connective tissue formation with a minimal foreign body response, and is significantly resorbed by the body at 30 days postimplantation. Solutions of PPCN and PNIPAAm were injected into the dorsal subcutaneous region of rats and tissue response was evaluated at 3 days and 30 days after implantation. No infections or abscesses at the sites of injection were observed. A palpable lump was formed at the site of injection for both materials (Figure S8). Cellular infiltration was observed at the interface of PPCN gels and tissue as early as 3 days, but not in PNIPAAm gels (Figure 7). Cells migrated along the PPCN pore walls (Figure S9). At 30 days, there was significant new tissue formation throughout the PPCN injection site including some fibrous tissue that formed at the interface between native tissue and implant, whereas the PNIPAAm injection site showed evidence of the PNIPAAm gel surrounded by a fibrous capsule. Staining for monocytes/macrophages revealed few CD68-positive cells (Figure 7), while Masson Trichrome staining of cross sections confirmed the presence of collagen within the new connective tissue that formed at the PPCN injection site at 30 days (Figure S9). Regarding the PPCN samples that contained SDF-1α, tissue samples analyzed after

Injectable thermoresponsive biomaterials, although potentially useful for protein delivery, often have low protein loading efficiencies,78 do not maintain protein activity over time,79 and have a large initial burst release.80 The ability of PPCN to overcome those issues was evaluated with the model chemokine stromal cell-derived factor 1α (SDF-1α), an important homing factor.81 SDF-1α has a net charge of +8 and isoelectric point of 9.9 at pH 7.4.82,83 The loading efficiency of SDF-1α in PPCN gels was 102.8 ± 19.7%, while PNIPAAm had a loading efficiency of only 16.1 ± 11.5% (Figure 5A). The lower loading efficiency for PNIPAAm is likely due to its compaction, as well as the lack of negative charges for electrostatic interactions with proteins. PPCN gels released SDF-1α for at least 2 weeks with minimal burst release (Figure 5B). An ideal protein release system should not only deliver proteins in a controlled manner, but also maintain their bioactivity. Protein aggregation and detrimental protein-gel interactions can occur during gel formation, storage, and over time, resulting in protein denaturation.84 In PPCN gels, released SDF-1α is bioactive as evident from the promotion of HUVEC migration, assessed via transwell migration assay (Figure 5C). Similar results have been obtained with lysozyme and confirm that PPCN gels can entrap and release bioactive proteins with high efficiency. Complexation between the proteins and the carboxyl groups in PPCN likely plays an important role in these findings.85,86 3.6. In Vitro Cell Entrapment, Viability, and Proliferation. PPCN gels support three-dimensional cell proliferation whereas PNIPAAm gels do not (Figures 6 and S7). Human aortic smooth muscle and umbilical vein endothelial cells were entrapped within the gels (3D culture) to evaluate in vitro cytocompatibility. Although 2D cell culture on PNIPAAm grafted surfaces has been widely reported as a basis for cellsheet tissue engineering applications,87−89 use of PNIPAAm for 3D in vivo applications is limited due to its toxicity. Indeed, H


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lipid peroxidation. Further, PPCN nanonets exhibit very low syneresis, can deliver chemokines in a controlled manner while retaining their bioactivity in vitro, as well as in vivo, and support cell entrapment, viability, and proliferation. Finally, the PPCN nanonets were shown to be biocompatible and bioresorbable when evaluated in vivo. This antioxidant injectable polymer represents a new biomaterials platform that may be useful in regenerative engineering applications.

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ASSOCIATED CONTENT

S Supporting Information *

Additional NMR, differential scanning calorimetry, rheology, and LCST characterization data, as well as long-term cell encapsulation data. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel.: +1-847-467-2992. Fax +1-847-491-4928. Author Contributions ‡

These authors contributed equally (J.Y. and R.v.L.).

Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This research was supported in part by the National Institutes of Health under Ruth L. Kirschtein National Research Service Award 1F31EB014698-01 (KB) and grant 5R01EB017129-02 (GAA) both from the National Institute of Biomedical Imaging and Bioengineering, and made use of the NUANCE and IMSERC Centers at Northwestern University, which have received support from the MRSEC program (NSF DMR0520513) at the Materials Research Center, Nanonascale Science and Engineering Center (EEC-0118025/003), both programs of the National Science Foundation; the State of Illinois; and Northwestern University. This work was also supported by the National Center for Research Resources, Grant 5UL1RR025741, and is now at the National Center for Advancing Translational Sciences, Grant 8UL1TR000150. The authors thank Xue Song Wang for help with histology. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.

Figure 7. PPCN is resorbed and supports cell infiltration and new tissue formation with minimal inflammatory response. (A−D) Haematoxylin-Eosin (H&E) staining of subcutaneous tissue injected with PPCN or PNIPAAm (100 mg/mL, pH = 7.4) that was explanted at 3 and 30 days. (E−H) Immunofluorescence staining of subcutaneous tissue injected with PPCN or PNIPAAm. Blue and green correspond to DAPI nuclear counterstain and CD68 immunohistochemical staining of macrophages, respectively. Dotted white line indicates the border of the polymer with native tissue; T = native tissue, P = polymer location. Scale bars: 100 μm.

30 days show significant presence of CXCR4+ cells, suggesting preservation of the chemokine’s bioactivity by PPCN in vivo (Figure 8).

4. CONCLUSIONS We have synthesized and characterized a novel intrinsically antioxidant citric acid-based thermoresponsive gel referred to as nanonets for the delivery of nano- to macrotherapeutics where oxidative stress in tissue is a concern. PPCN nanonets show free radical scavenging activity, can chelate iron ions and inhibit

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REFERENCES

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Figure 8. Subcutaneous tissue that received PPCN + SDF-1α gels shows CXCR4 positive cells at 30 days postinjection. Immunofluorescence staining of subcutaneous tissue injected with PPCN (A) or PPCN + SDF-1α (B) explanted at 30 days. Blue and red correspond to DAPI nuclear counterstain and CXCR4 immunohistochemical staining, respectively. Scale bars: 50 μm. I


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