Fabrication and Characterization of a Nitric Oxide-Releasing

Jul 11, 2013 - ... Engineering Michigan Technological University Houghton, Michigan 49931, United States ... In addition, the dried matrices stored in...
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Fabrication and Characterization of a Nitric Oxide-Releasing Nanofibrous Gelatin Matrix Caleb Vogt,† Qi Xing,† Weilue He,‡ Bowen Li,§ Megan C. Frost,‡ and Feng Zhao*,† †

Stem Cell and Tissue Engineering Lab, Department of Biomedical Engineering, Michigan Technological University, Houghton, Michigan 49931, United States ‡ Polymer and Biomaterial Lab, Department of Biomedical Engineering, Michigan Technological University, Houghton, Michigan 49931, United States § Department of Material Science and Engineering Michigan Technological University Houghton, Michigan 49931, United States ABSTRACT: Nitric oxide (NO) plays an important role in cardiovascular homeostasis, immune responses, and wound repair. The pro-angiogenic and antimicrobial properties of NO has stimulated the development of NO-releasing materials for wound dressings. Gelatin, an abundant natural biodegradable polymer derived from collagen, is able to promote wound repair. S-Nitroso-N-acetylpenicillamine (SNAP) can release NO under physiological conditions and when exposed to light. The objective of this project was to fabricate a NO-releasing gelatin-based nanofibrous matrix with precise light-controllable ability. Results showed that under controlled phase separation fabrication conditions, the gelatin formed a highly porous matrix with the nanofiber diameter ranging from 50 to 500 nm. Importantly, the removal of the trace amount of divalent metal ions within gelatin generated a more stable nanofibrous structure. N-acetyl-D-penicillamine (NAP) was functionalized onto the matrix and nitrosated with t-butyl nitrite, yielding a SNAPgelatin matrix. Analysis of the photoinitiated NO-release showed that the SNAP-gelatin matrices released NO in a highly controllable manner. Application of increasing light intensities yielded increased NO flux from the matrices. In addition, the dried matrices stored in dark at 4 °C maintained stable NO storage capacity, and the purified (ion-removed) gelatin preserved higher NO-releasing capacity than nonpurified gelatin. The antibacterial effect from the SNAP-gelatin matrices was demonstrated by exposing Staphylococcus aureus (S. aureus) to a light-triggered NO flux. This controllable NO-releasing scaffold provides a potential antibacterial therapeutic approach to combat drug resistant bacteria.



bacterial biofilm that plagues chronic wounds.7 Most NOreleasing materials are based on synthetic materials such as poly(lactic-co-glycolic-co-hydroxymethyl propionic acid),8 silica nanoparticles,9 polyethylene terephthalate and polyurethane.10 The less ideal biocompatibility of these base materials and their degradation byproducts impede their applications in the biomedical field. It is therefore critical to develop natural polymer based NO-releasing materials. S-Nitrosothiols are present in biological systems, where they serve as a reservoir and transporter of NO11 S-Nitroso-Nacetyl-D-penicillamine (SNAP) is one type of S-nitrosothiol that has been used in a wide variety of applications including cell cultures,12 wound healing creams, and NO-releasing polymers.13,14 SNAP also reproduces NO’s known antibacterial property both in vitro15 and in vivo.6 SNAP naturally releases NO under physiological conditions due to interactions with divalent metal ions such as Cu2+, but the release can also be photoinitiated. The NO-donating polymers with SNAP incorporation have demonstrated a capacity for low, control-

INTRODUCTION Nitric oxide (NO) has proven effectiveness in a variety of fields and applications since its debut as “molecule of the year” in 1992. One of the most attractive features of this free radical gas is its antibacterial property. In low doses, NO is a signaling molecule that induces inflammation.1 As the concentration increases, NO is capable of destroying or inhibiting bacteria by forming compounds that react with DNA and inhibit its repair.2,3 The small structure of the gas makes it highly diffusible, enabling its entry into bacteria across the plasma membrane.3 NO can generate reactive molecules that can deaminate and break DNA molecules, and can impede the ability of bacteria to self-repair DNA structure.3,4 Few bacteria are able to escape the antibacterial effect of NO. Therefore, NO is particularly efficient in killing drug-resistant bacteria.5 Various methods of NO delivery have showed a marked inhibitory effect on bacterial cultures, and NO applied to some external wounds can decrease the bacterial load.3,5 By decreasing the risk of infection of wound sites, NO has the potential to reduce wound healing time and repair chronic wounds.6 Compared with common antimicrobial agents such as colloid silver polymyxins or dye component, gaseous NO can easily diffuse into the wound bed and thus is able to destroy the © XXXX American Chemical Society

Received: December 22, 2012 Revised: July 10, 2013

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dx.doi.org/10.1021/bm301984w | Biomacromolecules XXXX, XXX, XXX−XXX

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samples were immersed in 1,4-dioxane for solvent exchanging for 24 h. Then the samples were frozen for 4 h at −20 °C, and then lyophilized for 2 days at −45 °C. The chemical cross-linking of the nanofiber matrix was done for 24 h at room temperature with EDC (5 mM) dissolved in a solution of acetone/water (90/10). Thiolactone Synthesis. The thiolactone was synthesized following previous publications.13,29 Briefly, 5 g of N-acetyl-Dpenicillamine and 10 mL of acetic anhydride were each dissolved separately in 10 mL of pyridine and chilled in ice for 1 h. The two solutions were covered with foil and mixed together at room temperature for 2 days. The resulting red solution was rotary evaporated until it became highly viscous, then it was dissolved in 20 mL of chloroform. The solution was extracted three times with 1 M HCl, and then dried with MgSO4. The solution was then vacuumfiltered to remove the MgSO4 and rotary evaporated at room temperature to remove the chloroform. The resulting solid was rinsed with hexanes and air-dried. Chemical Functionalization of Nanofibrous Gelatin Matrix. The gelatin nanofibrous matrices were immersed in a 4.5 mM thiolactone solution of toluene/ethanol (2/1, v/v), and shaken for 24 h at 37 °C. The samples were then removed from the thiolactone solution and rinsed three times with toluene. t-Butyl nitrite was cleaned by washing three times with equal volumes of 15 mM cyclam. In the absence of light, the nanofibers were immersed in a solution of 4.5 mL cleaned t-butyl nitrite and 10 mL toluene for 30 min, and then rinsed three times with toluene and three times with water. The matrices were stored in DIH2O in the dark within a 4 °C refrigerator, and then used in the second day. Morphology Observation. The structure and size of the nanofibrous gelatin matrices before and after the chemical functionalization were observed with a Hitachi S-4700 FE-SEM scanning electron microscope (SEM). The accelerating voltage was set to 10 kV and a current 5 mA. The samples were sputter coated with gold/palladium to a thickness of 10 nm before the observation with SEM. Chemical Structure Characterization. The effect of SNAP functionalization of the gelatin chemical structure was observed with an attenuated total reflectance-infrared spectroscopy (FTIR-ATR) (Perkin-Elmer, Waltham, MA). The samples were frozen for 4 h at −20 °C, and then lyophilized in the dark for 2 days at −45 °C. The dried samples were frozen in liquid nitrogen for 10 s, crushed into small pieces with a mortar and pestle, and then analyzed according to the instrument’s standard operating procedures. The nitrogen components of the gelatin matrices prior to and after the SNAP conjugation were also measured by a carbon, hydrogen, and nitrogen (CHN) elemental analyzer (Model 240 XA, Perkin-Elmer) via the combustion approach. The dried samples were weighed and analyzed according to the instrument’s standard operating procedures. Ion Content Measurement. The ion content of the gelatin was determined with inductively coupled plasma (ICP) mass spectroscopy (Leeman Laboratories, Lowell, MA). Briefly, the gelatin solution was allowed to gel and dry in a vacuum desiccator for 2 days and burned at 450 °C for 30 min. The resulting charred substance was then crushed and burned again into ash. The ash was dissolved in 0.1 M HCl and filtered for ICP test of the ion content. Three repeats were performed for each experimental condition. The results were expressed as means ± standard deviations. The statistical analysis was analyzed by Student’s t test, and p < 0.05 was used to determine whether a significant difference existed in the samples. Light-Activated NO Release. The photoinitiated NO release was accomplished with a 527 nm wavelength light-emitting diode (LED; C503B-BAN-CY0C0461, Mouser Electronics, Mansfield, TX) and a multiple output voltage controller. An additional 138 Ω resistor was in series with the LED. The NO flux was measured with a Sievers 280i Nitric Oxide Analyzer (GE Instruments, Boulder, CO). Samples with an average weight of 0.03 g were placed in a foil-covered glass container and illuminated with the LED from a distance of 5 cm. The nitric oxide flux was carried away by continuous N2 flow and blowed into the analyzer. Samples were subjected to various light intensities by varying the applied voltage to the LED (0, 3, 4.5, 6 V). The

lable release through the application of varying light intensities.13 Thus, the application of SNAP in antibacterial wound dressing becomes very attractive because of the lighttriggered delivery system and its precisely controllable NOreleasing property. Gelatin is a nonimmunogenic biopolymer obtained from collagen. Due to the triple helix conformation of native collagen, gelatin has a unique gelation property. It can be processed into hydrogels,16 three-dimensional (3D) nanofibrous meshes,17 or electrospun into nanofiber mats.18 Gelatin has been widely used in wound dressing applications because of its high water absorption capacity and ability to activate macrophages and homeostasis in bleeding wounds.19 Antibacterial wound dressings fabricated with silver nanoparticle incorporated gelatin was also reported.20 The amino acids of gelatin contain many pendant free amine groups that are capable of functionalization with different molecules21 or with different polymer substrates.22 It is attractive to use a gelatin nanofiber matrix to deliver therapeutic or antimicrobial agents due to its high surface area to volume ratio.23 Gelatin contains trace amount of divalent metal ions such as Cu2+, which have important functions in promoting self-assembly of collagenrelated peptides,24,25 increasing the cross-linking level of collagen26 and initiating NO release from SNAP. This work describes the preparation and SNAP functionalization of a NO-releasing gelatin nanofibrous matrix, created by a phase separation fabrication method. The effect of purifying the divalent metal ions from the gelatin as well as the NO release from the nanofibrous gelatin matrix in response to light intensity were examined. Staphylococcus aureus (S. aureus) is a common cause of skin infections such as boils and is frequently used as a model bacterial for wound healing study. It is also a major human pathogen interfering with host-cell functions. Impaired wound healing is often observed in S. aureus-infected wounds.27,28 As a material for potential antibacterial wound healing application, the NO-releasing gelatin matrices’ lighttriggered antibacterial effect on S. aureus was demonstrated.



MATERIALS AND METHODS

Materials. Gelatin (type B, bovine skin), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), 1,4-dioxane, and acetic anhydride (ACS grade) were purchased from Sigma-Aldrich (St. Louis, MO). Ethyl alcohol, 200 proof, was purchased from Decon Laboratories (King of Prussia, PA). Chelex 100 resin was purchased from Bio-Rad (Hercules, CA). Toluene, ACS grade, was purchased from Mallinckrodt Baker, Inc. (Phillipsburg, NJ). N-acetyl-D-penicillamine (NAP) was purchased from Aldrich Chemicals (Allentown, PA). Pyridine and concentrated hydrochloric acid was purchased from EMD Chemicals (Gibbstown, NJ). Magnesium sulfate (anhydrous, 99.5%) was purchased from Alfa Aesar (Ward Hill, MA). 1,4,8,11tetraazacyclotetradecane (cyclam) and t-butyl nitrite (90% pure) were obtained from Acros Organics (Ward Hill, MI). Agar was purchased from Becton Dickinson and Company (Sparks, MD). The Griess assay kit was purchased from Invitrogen (Rockville, MD). Nanofibrous Gelatin Matrix Preparation. The method for the preparation of nanofibrous gelatin matrix via temperature-induced phase separation is described in the literature.17 An additional step of treatment with Chelex resin to remove metal ions was included. Briefly, gelatin was dissolved in a mixture of ethanol and water at 60 °C. The ratio of ethanol/water ranged from 10/90, 30/70, to 50/50 (v/v). Chelex 100 resin (5 g/100 mL) was added to the solution and allowed to react for 18 h at 60 °C. The resulting supernatant was pipetted into 5 mL plastic tubes and sealed. The solution was transferred to −80 °C to induce phase separation. After 4 h, the dish was inserted into an ethanol bath at −20 °C for 24 h. The gelatin B

dx.doi.org/10.1021/bm301984w | Biomacromolecules XXXX, XXX, XXX−XXX

Biomacromolecules

Article

approximate luminous intensity corresponding to 3, 4.5, 6 V were 16.1, 24.1, 53.6 cd, respectively. The long-term NO release under physiological conditions was measured by using the Griess assay. To do this, the samples were immersed in pH 7.4 phosphate buffer saline (PBS) at 37 °C and illuminated by the LED (0, 3, 4.5, and 6 V) using a 300 Ω resistor for up to 24 h. The corresponding luminous intensities were 0, 9.1, 13.4, and 29.5 cd, repectively. The NO storage capacity of the samples stored under different conditions was also examined by the Griess assay. Wet samples were stored in DIH2O in the dark at 4 °C for 1, 2, and 3 days, and dried samples were stored in dry conditions in the dark at 4 °C for 1, 2, and 3 days. Then the samples were immersed in pH 7.4 PBS solution at room temperature and illuminated by the LED (300 Ω, 3 V and 9.1 cd). The nitric oxide generation from samples over 24 h was monitored at specified time points. Antimicrobial Property Examination. The antibacterial efficacy of gelatin nanofibrous matrix was examined against S. aureus. Thirty microliters of S. aureus suspension containing approximately 3 × 105 CFU/mL was added onto an agar gel in 6-well plate. Four sterile glass beads were placed on the top of agar gel and shaken for 30 s to spread the inoculums uniformly. The test sample with an average weight of 0.02 g was located at the center of the medium plate, covered and placed into a 37 °C incubator, and illuminated with the same LED at 0, 3, 4.5, and 6 V using 300 Ω resistor (the corresponding luminous intensity were 0, 9.1, 13.4, and 29.5 cd approximately). The bacteria growth in the plate was investigated after 24 h incubation. The zone of inhibition was found by taking 16 measurements from each well. The samples were estimated as circles, and measurements were made perpendicularly from the edge of the sample to the nearest bacterial colony.

purified gelatin (Figure 1 C, F). However, the purified sample had finer fibers and a more porous structure than the nonpurified sample. The results clearly showed that the ion removal facilitated the formation of a gelatin nanofibrous matrix. The morphological change of gelatin matrices during crosslinking and SNAP conjugation is shown in Figure 2. Compared with samples before cross-linking, the treatment caused a considerable amount of volume shrinkage and a slight change in the shape of the fibers, but the overall structure was preserved (Figure 2B). The SNAP functionalization did not cause noticeable change in the shape of the nanofibers (Figure 2C). Both cross-linking and SNAP functionalization procedures caused the size of the fibers and interstitial space to increase somewhat, but the fiber diameters remained less than 1 μm. The average fiber diameter of purified gelatin matrix changes from 42 ± 14 nm to 68 ± 19 nm during cross-linking and again to 162 ± 53 nm following SNAP functionalization. Figure 2D showed the structural uniformity of the finished product. Chemical Structure. Scheme 1 illustrated the synthetic scheme for preparing the SNAP-gelatin matrices. The carboxyl group of thiolactone reacted with the primary amine group on gelatin, resulting in a free thiol group that was available to form the corresponding S-nitrosothiol. The reaction of thiolactone with the gelatin was the first step for the two-step conjugation of SNAP. One of the functions of thiolactone was to react with primary amine groups on gelatin; the other function of thiolactone was to provide a “−SH” bond to react with tbutyl nitrite in the second step forming S−NO.13,29 Figure 3 showed the FTIR-ATR spectra of the control and purified gelatin matrices before and after SNAP functionalization. The characteristic bands of the coiled gelatin structure were the C O stretching for amide I at around 1628 cm−1, N−H deformation for amide II at around 1540 cm−1, C−N stretching for amide II at around 1400 cm−1, and N−H bending for amide III at around 1230 cm−1. The band position was determined by the backbone conformation and hydrogen bonding. Compared with SNAP-conjugated samples, untreated nonpurified and purified samples exhibited higher intensity peaks at around 1400 cm−1. The phenomenon could be explained by the incorporation of SNAP decreasing the intermolecular association in gelatin. The N−H bending band shifted to lower frequency after SNAP treatment, which also indicated the change of the molecular structure. The SNAP conjugation was further proved by CHN elemental analysis, which showed that both nitrogen and carbon contents increased after the reaction. For each milligram of gelatin, the newly incorporated nitrogen and carbon were 0.69 ± 0.14 and 3.45 ± 0.12 μmol, respectively. Although a part of newly incorporated nitrogen might come from the physically entrapped thiolactone, according to the SNAP−gelatin reaction mechanism, when thiolactone reacted with an amine group, the sulfur ring structure opened and provided a free thiol group to react with t-butyl nitrite. Therefore, only the thiolactone that was covalently bonded to the primary amine could be S-nitrosated and therefore NO-releasing.13,30 Taken together, the FT-IR spectra, the NO release pattern as well as the CHN element analysis synergistically prove that the SNAP groups were covalently bonded in the gelatin molecules. Light-Activated NO Release. The nanofibrous matrices demonstrated a stable, controllable NO release when exposed to light. Figure 4 showed a comparison between nonpurified and purified gelatin. The light responsive NO release of both



RESULTS An interesting development occurred during the phase separation step of the gelatin nanofibrous matrix preparation. The top surface of the gelatin, which was in immediate contact with the 1,4-dioxane, became a yellow-brown gel-like layer with a swelling behavior similar to a gelatin hydrogel. Underneath this layer, a white substance made up of nanofibers was found. This occurrence necessitated the separation of the two layers immediately following solvent exchange with 1,4-dioxane. All of the tests were conducted with the white nanofibrous material. Ion Content. Table 1 showed the effect of the purification process on the concentrations of select metal ions in the Table 1. Divalent Metal Ion Concentrations in Purified and Nonpurified Gelatin metal ions

Fe2+

Cu2+

Ca2+

nonpurified (ppm) purified (ppm) p-value

29.78 ± 1.16 4.14 ± 0.15