Letter Cite This: ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX
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Ternary Composite Nanofibers Containing Chondroitin Sulfate Scavenge Inflammatory Chemokines from Solution and Prohibit Squamous Cell Carcinoma Migration William S. Boyle,† Weili Chen,‡ Astrid Rodriguez,§ Samantha Linn,† Jakub Tolar,‡ Karen Lozano,§ and Theresa M. Reineke*,† †
Department of Chemistry, University of Minnesota, Minneapolis, Minnesota 55455, United States Stem Cell Institute and Division of Pediatric Blood and Marrow Transplantation, University of Minnesota, Minneapolis, Minnesota 55455, United States § Department of Mechanical Engineering, University of Texas-Rio Grande Valley, Edinburg, Texas 78539, United States ACS Appl. Bio Mater. Downloaded from pubs.acs.org by TULANE UNIV on 02/10/19. For personal use only.
‡
S Supporting Information *
ABSTRACT: Recessive dystrophic epidermolysis bullosa (RDEB), an inherited disease featuring blistering wounds, causes constant inflammation that leads to the eventual development of an aggressive form of squamous cell carcinoma (RDEB SCC). The persistence of inflammatory chemokines such as MCP-1 and Il-8 in RDEB wounds may foster RDEB SCC carcinogenesis. We report the production of ternary composite nanofibers containing pullulan, chondroitin sulfate, and tannic acid as RDEB wound dressings. The swellable fibers are stable to hydration and absorb ∼500% their weight in water. The fibers remove ∼99% of MCP-1 from solution in 400% for all samples. These mats compare favorably to commercially available gauze pads and the previously fabricated chitosan ternary composites (denoted as “chitosan control”). A positive correlation was found between water absorption and CS%, where the 19% CS mats exhibited 550% water absorption. However, the absorption percentages of the samples were not statistically different from each other based on the Students t-test. The mats maintained their macroscopic structure after submersion (Figure 3b). These water absorption properties indicated that the mats have potential as scaffolds to efficiently absorb and retain wound exudate when used as wound dressings. The ability of the mats to maintain their structure on a microscopic level after saturation with water was also investigated. Mats were submerged in water overnight, rinsed, and lyophilized. These swelled mats were compared to pristine mats by SEM (Figure 3c,d). After treatment with water and lyophilization, the mats exhibit few changes. While some defects can be observed (fused adjacent fibers, flattening) and the fibers appear to have contracted, the fibers do maintain their overall basic structure and do not appear to be degraded. Indeed, these results revealed that these natural product-based ternary composite fibers have promising capabilities to withstand the wet environment of a wound while maintaining their structural integrity in saturated conditions. MCP-1, IL-8, and other chemokines have been implicated in the progression of RDEB-SCC in patients suffering from epidermolysis bullosa.6 To examine the potential antiinflammatory properties of the ternary composite mats in vitro, we investigated the ability of the samples to scavenge MCP-1 from solution. Mats were placed in 96-well plates with 100 μL of RPMI containing 24 μg/mL MCP-1. Samples were removed from the wells at multiple time points, and the MCP1 concentration was quantified compared to an untreated control by ELISA. After 5 min of incubation, the fibers began to scavenge significant amounts (ca. 80%) of MCP-1. This rapid scavenging effect may be the result of the high surface area conferred by the nanostructure of the mats. After 2 h of incubation, 98% of the available MCP-1 had been removed from solution for all tested CS-containing mats. No significant difference was observed in scavenging percentage as a function of CS percentage. Even 6% CS was sufficient to quickly and almost quantitatively remove MCP-1 from solution. The commercially available gauze mats, which are cotton (cellulose) and neutral in charge, exhibited little binding of MCP-1. The control formulation containing chitosan in place of chondroitin sulfate, was fabricated using a process similar to that for the chondroitin sulfate mats. This formulation served as an additional control to understand the MCP affinity for chondroitin sulfate. This formulation, along with the gauze, showed some scavenging of MCP-1; however, the maximum scavenging percentage was 34% compared to nearly complete scavenging with the CS nanofibers. The fibers can quickly remove MCP-1 from solution and have the capacity to hold high concentrations of chemokine over time. After 24 h, no MCP-1 was detected in the solution with the composite mats.
These results demonstrate the ability of the nonwoven nanofiber mats to successfully scavenge biologically relevant amounts of protein from solution. In a wound environment, the fibers could act as a sink for MCP-1 and other GAGbinding chemokines, leading to reduced inflammation and improved wound healing. To further test the utility of the nanofibers as chemokine scavengers, the fibers were exposed to conditioned media derived from RDEB fibroblast cells. In order to use the conditioned media for supporting the growth of RDEB-SCC cells, RDEB fibroblasts at confluency were cultured with EpiLife media supplemented with EpiLife Defined Growth Supplement (EDGS) and 50 μg/mL L-ascorbic acid.30 Media were collected 48 h later and centrifuged to remove debris to yield conditioned media. Conditioned media were placed in a tube containing a section of the 19% CS mat. The media were sampled prior to scavenging to determine the initial chemokine concentration. The media were incubated with the nanofibers for 24 h at 4 °C and agitated periodically. After incubation, the fibers were removed, and the chemokine concentration was quantified by ELISA.22 As can be seen in Figure 4b, scavenging reduced the concentration of MCP-1 by 64% (from 760 to 273 pg/mL). The concentration of Il-8 was also reduced by 67% (from 417 to 134 pg/mL). These results differ from the time course scavenging experiment where quantitative reduction of the chemokine concentration was achieved after 2 h. Since the starting concentration of the conditioned media was orders of magnitude lower, it is possible that an equilibrium state was being reached with the conditioned media that resulted in a reduced scavenging percentage. It is also possible that excreted molecules from the cells may be interfering with the ability of the nanofibers to completely remove chemokines. The negative charge associated with chondroitin sulfate may lead to nonspecific accumulation of anionic species in complex conditions such as a wound environment. These effects would need to be characterized should these materials be deployed in a clinical context. Regardless, these results revealed that the nanofibers significantly reduced the concentration of inflammatory chemokines associated with RDEB-SCC from complex conditioned media. To determine whether the chemokine scavenging was sufficient to modify the behavior of RDEB-SCC cells, the scavenged conditioned media were used as a chemoattractant in a cell migration assay. RDEB-SCC cells (5 × 104) were plated on permeable membranes within a transwell 24-well insert. The lower chamber was then filled with RDEB-SCC conditioned media, control media, or scavenged conditioned media, and the cells were allowed to migrate for 24 h. The cells on the top of the inset were gently removed with a cotton swab after 24 h. The migrated cells on the bottom of the inset were stained with Crystal Violet stain and quantified by counting under a microscope. All results were normalized to RDEB-SCC conditioned media migration as seen in Figure 4c. More than twice as many cells migrated toward the conditioned media when compared with control media. In contrast, cell migration halts almost entirely in scavenged media. It should be noted that a decrease in migration compared to control media could also be in part due to nonspecific scavenging of essential nutrients from the media. While this effect is difficult to characterize and distinguish, scavenging of conditioned media clearly reverses the migration normally triggered by RDEBSCC conditioned media. The reduction of RDEB-SCC D
DOI: 10.1021/acsabm.8b00690 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX
Letter
ACS Applied Bio Materials
and maintain their structure when hydrated. These fibers efficiently scavenge pro-inflammatory chemokines (MCP-1 and Il-8) from culture media that model wound exudate. These materials may provide an anti-inflammatory environment for wound healing in RDEB patients and may slow the development of RDEB-SCC as shown by our data, indicating that cultured SCC cell migration was inhibited. To this end, these mats could have broad utility for improving healing of a variety of chronic wounds. Furthermore, the chemical functionality of pullulan and CS may provide convenient handles to produce protein-conjugated materials that can further promote a positive interaction with wound environments and other therapeutic molecules that could further promote wound healing.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsabm.8b00690. Detailed materials and methods: general, development of ternary composite nanofibers, water absorption coefficient, scanning electron microscopy, chemokine scavenging, scavenging of conditioned media, RDEBSCC migration (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Karen Lozano: 0000-0002-6676-8632 Theresa M. Reineke: 0000-0001-7020-3450 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The work was supported by the National Science Foundation through the NSF PREM Award DMR 1523577. Part of this work was done at the College of Science and Engineering Characterization Facility, University of Minnesota, which has received capital equipment funding from the NSF through the UMN MRSEC program under Award Number DMR1420013. This work was also funded by the NIH National Heart Lung, and Blood Institute (R01 AR063070, and R01 HL108627) and the NIH National Institute of Arthritis and Musculoskeletal and Skin Diseases (R01 AR059947-01A1). We also acknowledge Cristobal Rodriguez for his contribution to work leading up to this project.
Figure 4. Scavenging of MCP-1 from solution by the composite nanofiber mats. (A) Scavenging percentage was determined by ELISA assays by quantification of MCP-1 concentration in solutions treated with nanofiber mats compared to the control solution (no mat) over time. Error bars represent the standard deviation of three replicates. (B) Concentration of MCP-1 and IL-8 in RDEB-SCC conditioned media before and after scavenging with 19% CS nanofibers as determined by ELISA. Error bars represent the average of three replicates. The asterisk (*) denotes a significant (P < 0.05) decrease compared to unscavenged conditioned media by the Student’s t-test. (C) Migration of RDEB-SCC triggered by conditioned media, control media, and scavenged conditioned media. Error bars represent the average of three replicates. * denotes a significant decrease compared to the conditioned media. ** denotes a significant decrease compared to fresh media. Significance was determined using the Student’s t-test.
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REFERENCES
(1) Hovnanian, A.; Hilal, L.; Blanchet-Bardon, C.; De Prost, Y.; Christiano, A. M.; Uitto, J.; Goossens, M. Recurrent Nonsense Mutations within the Type Vii Collagen Gene in Patients with Severe Recessive Dystrophic Epidermolysis Bullosa. Am. J. Hum. Genet. 1994, 55, 289−296. (2) Wagner, J. E.; Ishida-Yamamoto, A.; Mcgrath, J. A.; Hordinsky, M.; Keene, D. R.; Riddle, M. J.; Osborn, M. J.; Lund, T.; Dolan, M.; Blazar, B. R.; Tolar, J. Bone Marrow Transplantation for Recessive Dystrophic Epidermolysis Bullosa. N. Engl. J. Med. 2010, 363, 629− 639. (3) Krupiczojc, M. A.; O’toole, E. A. Management of Cutaneous Squamous Cell Carcinoma in Patients with Epidermolysis Bullosa. Br. J. Dermatol. 2016, 174, 56−67.
migration could result in reduced progression of RDEB-SCC in patients treated with nanofiber wound dressings. In conclusion, this work demonstrates a fast and scalable synthesis of hydrophilic nanofibers derived from natural feedstocks. The fibers are uniform in size regardless of CS content, absorb large volumes of water relative to their mass, E
DOI: 10.1021/acsabm.8b00690 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX
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ACS Applied Bio Materials (4) Knaup, J.; Gruber, C.; Krammer, B.; Ziegler, V.; Bauer, J.; Verwanger, T. Tgfβ-Signaling in Squamous Cell Carcinoma Occurring in Recessive Dystrophic Epidermolysis Bullosa. Anal. Cell. Pathol. 2011, 34, 339−353. (5) Martins, V. L.; Vyas, J. J.; Chen, M.; Purdie, K.; Mein, C. A.; South, A. P.; Storey, A.; Mcgrath, J. A.; O’toole, E. A. Increased Invasive Behaviour in Cutaneous Squamous Cell Carcinoma with Loss of Basement-Membrane Type Vii Collagen. J. Cell Sci. 2009, 122, 1788−1799. (6) Breitenbach, J. S.; Rinnerthaler, M.; Trost, A.; Weber, M.; Klausegger, A.; Gruber, C.; Bruckner, D.; Reitsamer, H. A.; Bauer, J. W.; Breitenbach, M. Transcriptome and Ultrastructural Changes in Dystrophic Epidermolysis Bullosa Resemble Skin Aging. Aging 2015, 7, 389−411. (7) Sindrilaru, A.; Peters, T.; Wieschalka, S.; Baican, C.; Baican, A.; Peter, H.; Hainzl, A.; Schatz, S.; Qi, Y.; Schlecht, A.; Weiss, J. M.; Wlaschek, M.; Sunderkö tter, C.; Scharffetter-Kochanek, K. An Unrestrained Proinflammatory M1Macrophage Population Induced by Iron Impairs Wound Healing in Humans and Mice. J. Clin. Invest. 2011, 121, 985−997. (8) Waugh, D. J. J.; Wilson, C. The Interleukin-8 Pathway in Cancer. Clin. Cancer Res. 2008, 14, 6735. (9) Steen, A.; Larsen, O.; Thiele, S.; Rosenkilde, M. M. Biased and G Protein-Independent Signaling of Chemokine Receptors. Front. Immunol. 2014, 5, 277−290. (10) Koide, N.; Nishio, A.; Sato, T.; Sugiyama, A.; Miyagawa, S.-I. Significance of Macrophage Chemoattractant Protein-1 Expression and Macrophage Infiltration in Squamous Cell Carcinoma of the Esophagusa. Am. J. Gastroenterol. 2004, 99, 1667−1674. (11) Martins, V. L.; Caley, M. P.; Moore, K.; Szentpetery, Z.; Marsh, S. T.; Murrell, D. F.; Kim, M. H.; Avari, M.; Mcgrath, J. A.; Cerio, R.; Kivisaari, A.; Kähäri, V. M.; Hodivala-Dilke, K.; Brennan, C. H.; Chen, M.; Marshall, J. F.; O’Toole, E. A. Suppression of Tgfβ and Angiogenesis by Type Vii Collagen in Cutaneous Scc. JNCI, J. Natl. Cancer Inst. 2016, 108, djv293. (12) Guerra, L.; Odorisio, T.; Zambruno, G.; Castiglia, D. Stromal Microenvironment in Type Vii Collagen-Deficient Skin: The Ground for Squamous Cell Carcinoma Development. Matrix Biol. 2017, 63, 1−10. (13) Küttner, V.; Mack, C.; Rigbolt, K. T. G.; Kern, J. S.; Schilling, O.; Busch, H.; Bruckner-Tuderman, L.; Dengjel, J. Global Remodelling of Cellular Microenvironment Due to Loss of Collagen Vii. Mol. Syst. Biol. 2013, 9, 657−657. (14) Guo, J.; Zhou, H.; Akram, M. Y.; Mu, X.; Nie, J.; Ma, G. Characterization and Application of Chondroitin Sulfate/Polyvinyl Alcohol Nanofibres Prepared by Electrospinning. Carbohydr. Polym. 2016, 143, 239−245. (15) Liu, Y.; Cai, S.; Shu, X. Z.; Shelby, J.; Prestwich, G. D. Release of Basic Fibroblast Growth Factor from a Crosslinked Glycosaminoglycan Hydrogel Promotes Wound Healing. Wound Repair Regen. 2007, 15, 245−251. (16) Arslan, E.; Guler, M. O.; Tekinay, A. B. GlycosaminoglycanMimetic Signals Direct the Osteo/Chondrogenic Differentiation of Mesenchymal Stem Cells in a Three-Dimensional Peptide Nanofiber Extracellular Matrix Mimetic Environment. Biomacromolecules 2016, 17, 1280−1291. (17) Yergoz, F.; Hastar, N.; Cimenci, C. E.; Ozkan, A. D.; Tekinay, T.; Guler, M. O.; Tekinay, A. B. Heparin Mimetic Peptide Nanofiber Gel Promotes Regeneration of Full Thickness Burn Injury. Biomaterials 2017, 134, 117−127. (18) Witt, D. P.; Lander, A. D. Differential Binding of Chemokines to Glycosaminoglycan Subpopulations. Curr. Biol. 1994, 4, 394−400. (19) Middleton, J.; Neil, S.; Wintle, J.; Clark-Lewis, I.; Moore, H.; Lam, C.; Auer, M.; Hub, E.; Rot, A. Transcytosis and Surface Presentation of Il-8 by Venular Endothelial Cells. Cell 1997, 91, 385− 395. (20) Kuschert, G. S. V.; Coulin, F.; Power, C. A.; Proudfoot, A. E. I.; Hubbard, R. E.; Hoogewerf, A. J.; Wells, T. N. C. Glycosaminoglycans
Interact Selectively with Chemokines and Modulate Receptor Binding and Cellular Responses. Biochemistry 1999, 38, 12959−12968. (21) Johnson, Z.; Proudfoot, A. E.; Handel, T. M. Interaction of Chemokines and Glycosaminoglycans: A New Twist in the Regulation of Chemokine Function with Opportunities for Therapeutic Intervention. Cytokine Growth Factor Rev. 2005, 16, 625−636. (22) Lohmann, N.; Schirmer, L.; Atallah, P.; Wandel, E.; Ferrer, R. A.; Werner, C.; Simon, J. C.; Franz, S.; Freudenberg, U. Glycosaminoglycan-Based Hydrogels Capture Inflammatory Chemokines and Rescue Defective Wound Healing in Mice. Sci. Transl. Med. 2017, 9, eaai9044. (23) Xu, F.; Weng, B.; Gilkerson, R.; Materon, L. A.; Lozano, K. Development of Tannic Acid/Chitosan/Pullulan Composite Nanofibers from Aqueous Solution for Potential Applications as Wound Dressing. Carbohydr. Polym. 2015, 115, 16−24. (24) Padron, S.; Fuentes, A.; Caruntu, D.; Lozano, K. Experimental Study of Nanofiber Production through Forcespinning. J. Appl. Phys. 2013, 113, No. 024318. (25) Xu, F.; Weng, B.; Materon, L. A.; Gilkerson, R.; Lozano, K. Large-Scale Production of a Ternary Composite Nanofiber Membrane for Wound Dressing Applications. J. Bioact. Compat. Polym. 2014, 29, 646−660. (26) El Hachem, M.; Zambruno, G.; Bourdon-Lanoy, E.; Ciasulli, A.; Buisson, C.; Hadj-Rabia, S.; Diociaiuti, A.; Gouveia, C. F.; Hernández-Martín, A.; De Lucas Laguna, R.; et al. Multicentre Consensus Recommendations for Skin Care in Inherited Epidermolysis Bullosa. Orphanet J. Rare Dis. 2014, 9, 76−96. (27) Kim, T. J.; Silva, J. L.; Kim, M. K.; Jung, Y. S. Enhanced Antioxidant Capacity and Antimicrobial Activity of Tannic Acid by Thermal Processing. Food Chem. 2010, 118, 740−746. (28) Jones, V.; Grey, J. E.; Harding, K. G. Wound Dressings. BMJ. [Br. Med. J.] 2006, 332, 777−780. (29) Boateng, J. S.; Matthews, K. H.; Stevens, H. N. E.; Eccleston, G. M. Wound Healing Dressings and Drug Delivery Systems: A Review. J. Pharm. Sci. 2008, 97, 2892−2923. (30) Vanden Oever, M.; Muldoon, D.; Mathews, W.; Mcelmurry, R.; Tolar, J. Mir-29 Regulates Type Vii Collagen in Recessive Dystrophic Epidermolysis Bullosa. J. Invest. Dermatol. 2016, 136, 2013−2021.
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DOI: 10.1021/acsabm.8b00690 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX
