Novel Delivery System for the Bioregulatory Agent Nitric Oxide

5032 Chem. Mater. 2009, 21, 5032–5041 DOI:10.1021/cm901358z

Novel Delivery System for the Bioregulatory Agent Nitric Oxide Harvey A. Liu and Kenneth J. Balkus Jr.* The University of Texas at Dallas, Department of Chemistry and the Alan MacDiarmid NanoTech Institute, 800 West Campbell Road, Richardson, Texas 75080-3021 Received May 17, 2009. Revised Manuscript Received October 2, 2009

The diatomic free radical nitric oxide (NO) plays a crucial role in many physiological functions including vasodilation, angiogenesis, and neurotransmission, as well as some immune responses. NO-mediated processes allude to the possibility that chemical systems capable of generating NO may provide useful therapeutic applications. We present here a method to fabricate an NO-releasing bandage. Zeolite A was embedded within porous polylactic acid fibers of a nonwoven mat generated by electrospinning. These composite fibers were then subjected to mild heat treatment, softening the polymer to encapsulate the embedded zeolite and effectively slowing the nitric oxide release. Introduction Nitric oxide (NO) has long been established as a signaling molecule to promote the relaxation of smooth muscle cells.1 It was not discovered until the late 1980s by Ignarro et al. that nitric oxide and the endothelium-derived relaxing factor (EDRF) was one in the same.2,3 Since the link between EDRF and NO had been established, nitric oxide has been implicated in many other biological processes including wound healing,4,5 inflammation,6 plant disease resistance,7 and even social dysfunction.8 The implication of nitric oxide in so many biological processes has fueled interest in the exogenous delivery of NO. Due to the reactivity of nitric oxide, the conventional storage and delivery of NO as a gas is impractical. This obstacle has fueled research in the synthesis of materials that are able to store and release nitric oxide. Examples of such materials include the diazeniumdiolates,9,10 nitric oxide-releasing gold nanoparticles,11 nitric oxide-releasing polyethyleneimine (PEI) fibers,12 and nitric *Corresponding author. E-mail: [email protected]. Phone: 972-8832659.

(1) Arnold, W. P.; Mittal, C. K.; Katsuki, S.; Murad, F. Proc. Natl. Acad. Sci. U.S.A. 1977, 74(8), 3203–3207. (2) Ignarro, L. J.; Buga, G. M.; Wood, K. S.; Byrns, R. E.; Chaudhuri, G. Proc. Natl. Acad. Sci. U.S.A. 1987, 84(24), 9265–9269. (3) Palmer, R. M. J.; Ferrige, A. G.; Moncada, S. Nature 1987, 327 (6122), 524–526. (4) Weller, R.; Finnen, M. J. Nitric Oxide 2006, 15(4), 395–399. (5) Schaffer, M. R.; Tantry, U.; Gross, S. S.; Wasserkrug, H. L.; Barbul, A. J. Surg. Res. 1996, 63(1), 237–240. (6) Lyons, C. R. Adv. Immunol. 1995, 60, 323–360. (7) Delledonne, M.; Xia, Y.; Dixon, R. A.; Lamb, C. Nature 1998, 394 (6693), 585–588. (8) Wass, C.; Klamer, D.; Fejgin, K.; P^alsson, E. Behav. Brain Res. 2009, 200, 113-116. (9) Keefer, L. K.; Flippen-Anderson, J. L.; George, C.; Shanklin, A. P.; Dunams, T. M.; Christodoulou, D.; Saavedra, J. E.; Sagan, E. S.; Bohle, D. S. Nitric Oxide 2001, 5(4), 377–394. (10) Hrabie, J. A.; Keefer, L. K. Chem. Rev. 2002, 102(4), 1135–1154. (11) Polizzi, M. A.; Stasko, N. A.; Schoenfisch, M. H. Langmuir 2007, 23(9), 4938–4943. (12) Smith, D. J.; Reneker, D. H. Nitric oxide-modified linear poly(ethylenimine) fibers and uses therefor. U.S. Patent 20040131753, 2004.

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oxide-releasing zeolites and metal organic frameworks (MOFs).13-23 Zeolites are open stable three-dimensional microporous crystalline aluminosilicates formed by ordered tetrahedras of AlO4 and SiO4.24,25 There is growing interest in zeolites for biomedical applications including their use in drug delivery,26-29 as carriers of low molecular bioactive (13) Boes, A. K.; Wheatley, P. S.; Xiao, B.; Megson, I. L.; Morris, R. E. Chem. Commun. 2008, 46, 6146–6148. (14) Boes, A. K.; Xiao, B.; Megson, I. L.; Morris, R. E. Top Catal. 2008, 1–7. (15) McKinlay, A. C.; Xiao, B.; Wragg, D. S.; Wheatley, P. S.; Megson, I. L.; Morris, R. E. J. Am. Chem. Soc. 2008, 130(31), 10440–10444. (16) Morris, R. E.; Megson, I. L. Bifunctional Material for Nitric Oxide Storage and Production and Use Thereof in Therapy. W.I.P.O. WO2008062160, 2008. (17) Morris, R. E.; Wheatley, P. S. Angew. Chem., Int. Ed. Eng. 2008, 47 (27), 4966–4981. (18) Mowbray, M.; Tan, X.; Wheatley, P. S.; Rossi, A. G.; Morris, R. E.; Weller, R. B. J. Invest. Dermatol. 2008, 128(10), 2546. (19) Wheatley, P. S.; Butler, A. R.; Crane, M. S.; Fox, S.; Xiao, B.; Rossi, A. G.; Megson, I. L.; Morris, R. E. J. Am. Chem. Soc. 2006, 128(2), 502–509. (20) Xiao, B.; Wheatley, P. S.; Morris, R. E. Stud. Surf. Sci. Catal. 2007, 170, 902–909. (21) Xiao, B.; Wheatley, P. S.; Zhao, X.; Fletcher, A. J.; Fox, S.; Rossi, A. G.; Megson, I. L.; Bordiga, S.; Regli, L.; Thomas, K. M.; Morris, R. E. J. Am. Chem. Soc. 2007, 129(5), 1203–1209. (22) Morris, R. E.; Wheatley, P. S.; Butler, A. R. Zeolites for Delivery of Nitric Oxide. W.I.P.O. WO2005003032, 2005. (23) Morris, R. E.; Wheatley, P. S. Adsorption and Release of Nitric Oxide in Metal Organic Frameworks. W.I.P.O. WO2008020218., 2008. (24) Breck, D. W.; Eversole, W. G.; Milton, R. M. J. Am. Chem. Soc. 1956, 78(10), 2338–2339. (25) Breck, D. W.; Eversole, W. G.; Milton, R. M.; Reed, T. B.; Thomas, T. L. J. Am. Chem. Soc. 1956, 78(23), 5963–5971. (26) Horcajada, P.; Marquez-Alvarez, C.; Ramila, A.; Perez-Pariente, J.; Vallet-Regi, M. Solid State Sci. 2006, 8(12), 1459–1465. (27) Rimoli, M. G.; Rabaioli, M. R.; Melisi, D.; Curcio, A.; Mondello, S.; Mirabelli, R.; Abignente, E. J. Biomed. Mater. Res. A. 2008, 87 (1), 156–164. (28) Wong, L. W.; Sun, W. Q.; Chan, N. W.; Lai, W. Y.; Leung, W. K.; Tsang, J. C.; Wong, Y. H.; Yeung, K. L., Stud. Surf. Sci. Catal., 2007; 170, pp 525-530. (29) Yeung, K.-L.; Wong, L.-W.; Sun, W.; Leung, W.-K.; Lai, W.-Y.; Chan, N.-W. Molecular Sieve and Zeolite Microneedles and Preparation Thereof. W.I.P.O. WO2007095859, 2007.

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substances and macromolecules for in-cell chemistry,30 as MRI contrast agents,31-36 hemostatic materials,37,38 and most recently as a vehicle to store and release the NO.13,16,17,19,21,22,39 Zeolite A is produced in excess of 1 M metric tons per year as an ion exchanger in detergent to soften water.40,41 The structure of Zeolite A (LTA) (Figure 1) is cubic and consists of alternating SiO4 and AlO4 tetrahedra that share corners forming a supercage with a pore opening of 4 A˚.25 Exchangeable Naþ cations (not shown) reside in the channels. While the ability of Zeolite A to adsorb nitric oxide was first demonstrated in the late 1970s, it was only recently studied for its antithrombic abilities.16,19,42 Wheatley et al. demonstrated the ability to tailor the release of nitric oxide by altering the type and amount of exchanged metal cations in the zeolite.19 It was shown that the cobalt(II) exchanged zeolite A exhibited the highest adsorption capacity of nitric oxide with the ability to adsorb up to 1.7 mmol/g with approximately 0.7 mmol/g weakly physisorbed.19 The amount of NO adsorbed within the zeolite was shown to be dependent upon the type of transition metal ion that resides in the cages, which followed the trends seen in pressure swing adsorption studies.19,43,44 Zeolite A also presents other characteristics that make it an ideal NO carrier including its ability to complex with NO at atmospheric pressure under dry conditions, thus extending its shelf life for real world applications.19 Recent efforts have focused on the sustained release of the reactive gas using bifunctional zeolites.13,39 In this case, the NO is adsorbed within the pores of a copper(I) exchanged zeolite X (FAU). The secondary function of the Cu(I) species is to reduce NO2- to NO. Upon the addition of a NO2solution, nitric oxide was evolved, resulting in an increased capacity 1 order of magnitude greater and a more sustained release of nitric oxide.13,39 This related to other (30) Dahm, A.; Eriksson, H. J. Biotechnol. 2004, 111(3), 279–290. (31) Balkus, K. J.; Sherry, A. D.; Young, S. W. Zeolite-enclosed transistion and rare earth metal ions as contrast agents for the gastrointestinal tract. U.S. Patent 5,122,363, 1992. (32) Young, S. W.; Qing, F.; Rubin, D.; Balkus, K. J. Jr; Engel, J. S.; Lang, J.; Dow, W. C.; Mutch, J. D.; Miller, R. A. J. Magn. Reson. Imaging 1995, 5(5), 499–508. (33) Bresinska, I.; Balkus, K. J. Jr. J. Phys. Chem. 1994, 98(49), 12989– 12994. (34) Balkus, K. J.; Bresinska, I. J. Alloys Compd. 1994, 207-208(C), 25– 28. (35) Csajbok, E.; Banyai, I.; Elst, L. V.; Muller, R. N.; Zhou, W.; Peters, J. A. Chem.;Eur. J. 2005, 11(16), 4799–4807. (36) Lerouge, F.; Melnyk, O.; Durand, J.-O.; Raehm, L.; Berthault, P.; Huber, G.; Desvaux, H.; Constantinesco, A.; Choquet, P.; Detour, J.; Smaihi, M. J. Mater. Chem. 2009, 19(3), 379–386. (37) Ahuja, N.; Ostomel, T. A.; Rhee, P.; Stucky, G. D.; Conran, R.; Chen, Z.; Al-Mubarak, G. A.; Velmahos, G.; DeMoya, M.; Alam, H. B. J. Trauma 2006, 61(6), 1312–1320. (38) Arnaud, F.; Tomori, T.; Carr, W.; McKeague, A.; Teranishi, K.; Prusaczyk, K.; McCarron, R. Ann. Biomed. Eng. 2008, 36(10), 1708–1713. (39) Boes, A. K.; Xiao, B.; Megson, I. L.; Morris, R. E. Top. Catal. 2008, 1–7. (40) Bajpai, D.; Tyagi, V. K. J. Oleo. Sci. 2007, 56(7), 327–340. (41) Cheetham, A. K.; Eddy, M. M.; Jefferson, D. A.; Thomas, J. M. Nature 1982, 299(5878), 24–26. (42) Cruz, W. V.; Leung, P. C. W.; Seff, K. Inorg. Chem. 1979, 18(6), 1692–1696. (43) Arai, H.; Machida, M. Catal. Today 1994, 22(1), 97–109. (44) Zhang, W. X.; Yahiro, H.; Mizuno, N.; Izumi, J.; Iwamoto, M. Langmuir 1993, 9(9), 2337–2343.

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Figure 1. Cubic structure of Zeolite A (LTA).

studies in the delivery of nitric oxide that have focused on the development of NO releasing creams based on the acidification of sodium nitrite by citric acid.4 This acidified cream has been demonstrated to exhibit antifungal ability and be effective in wound healing in normal and diabetic mice.45 While such topical creams could be used for wound healing or antifungal treatment, the application in areas such donor organ preservation during procurement and transplantation is not practical. For example, one of the major factors in a successful heart transplant is adequate organ preservation during the procurement, storage, as well as the implantation stages. Currently, cardiac preservation is based on a single flush induction of cardioplegia and hypothermic storage in a solution.46 While this method is effective in the preservation of cardiac muscles, time is still the major factor in healing because the heart can only tolerate 4-5 h of ischemia. Prolonged periods of ischemia lead to increased acute and chronic organ failure due to endothelium damage. It is well documented that damage to the endothelium, the innermost lining of the arteries, is one of the major limiting factors in heart preservation.46 Under normal conditions, the endothelium synthesizes compounds that induce vascular smooth muscle relaxation, the endothelium dependent nitric oxide (EDNO), and endothelium dependent hyperpolarization factor (EDHF), which are not synthesized during transplantation. The ability to deliver regulated amounts of nitric oxide may provide a means to better preserve the endothelium and increase the success rate of organs transplants. The implementation of a NO-releasing cream in this instance would not satisfy the needs for (45) Anyim, M.; Benjamin, N.; Wilks, M. Int. J. Antimicrob. Agents 2005, 26(1), 85–87. (46) Podesser, B. K.; Gottardi, R.; Steitelberger, R.; Hallstrom, S. Vasc. Dis. Prev. 2005, 2, 77–85.

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transplant organs due to the low amounts of nitric oxide evolved, the risk of washing away the cream during storage in the transport solution, and the slow rate of release. Additionally, the application of a cream directly to the transplant organ may introduce other health issues to the donor organ. While the ability of zeolites to store large amounts of nitric oxide and release viable amounts in a regulated rate is promising, the application of the free powder to transplant organs is also not practical. The goal of the present work is to fabricate a composite fiber material consisting of nitric oxide infused Zeolite A and a biocompatible, biodegradable, hydrophobic polymeric material, polylactic acid (PLA). The use of polylactic acid affords many advantages including its biocompatibility due to its hydrolysis product, lactic acid, which is commonly found in our bodies as well as its implications in drug delivery.47,48 Previous studies have used polylactic acid to encapsulate drugs for delivery and have not demonstrated any complications with its application.49-52 Additionally, polylactic acid has also been implicated as an aid in the delivery of drugs across the epidermis of rabbits.53 A free-standing nonwoven bandage was fabricated by electrospinning which could be easily handled and manipulated. Additionally, by controlling the PLA fiber porosity the rate of nitric oxide release can be tuned. In contrast to other polymer-based NO delivery systems, only moisture is required to release the NO from the encapsulated zeolites. The zeolite polymer composite bandage could be used to wrap a donor organ ensuring intimate contact and direct the delivery of nitric oxide. Additionally, these nonwoven fabrics could also find applications in smart textiles such as NO-releasing socks for diabetic patients, who have been shown to produce less nitric oxide than healthy patients.4,54,55 Experimental Section Materials. All reagents were used without further purification. Lactic acid polymer (∼100 kDa) was purchased from Dajac Laboratories. Sodium hydroxide, sodium aluminate, and sodium metasilicate were purchased from Sigma-Aldrich. Nitric oxide was purchased from Airgas (c.p. grade). All solvents were used as received and were obtained from VWR. (47) Von Recum, H. A.; Cleek, R. L.; Eskin, S. G.; Mikos, A. G. Biomaterials 1995, 16(6), 441–447. (48) Zhang, X.; Wyss, U. P.; Pichora, D.; Goosen, M. F. A. J. Bioact. Compat. Polym. 1994, 9(1), 80–100. (49) Anderson, J. M.; Shive, M. S. Adv. Drug Delivery Rev. 1997, 28(1), 5–24. (50) Grizzi, I.; Garreau, H.; Li, S.; Vert, M. Biomaterials 1995, 16(4), 305–311. (51) Jalil, R.; Nixon, J. R. J. Microencapsul. 1989, 6(4), 473–484. (52) Kwon, G. S.; Okano, T. Adv. Drug Delivery Rev. 1996, 21(2), 107– 116. (53) Sebastiani, P.; Nicoli, S.; Santi, P. Int. J. Pharm. 2005, 292(1-2), 119–126. (54) De Vriese, A. S.; Verbeuren, T. J.; Van De Voorde, J.; Lameire, N. H.; Vanhoutte, P. M. Br. J. Pharmacol. 2000, 130(5), 963–974. (55) Lee, P. C.; Salyapongse, A. N.; Bragdon, G. A.; Shears Ii, L. L.; Watkins, S. C.; Edington, H. D. J.; Billiar, T. R. Am. J. Physiol. Heart Circ. Physiol. 1999, 277 (446-4), H1600-H1608. (56) Robson, H.; Lillerud, K. P. In Verified Synthesis of Zeolitic Materials, 2nd revised ed.; International Zeolite Association: Amsterdam, Netherlands, 2001.

Liu and Balkus Synthesis of Zeolite A. Zeolite A was synthesized according to a verified procedure.56 Sodium hydroxide (0.723 g) was first dissolved in 80 mL of deionized (DI) water then divided into two equal proportions. One half of the sodium hydroxide solution was combined with sodium aluminate (8.258 g) and stirred at room temperature (RT) until clear. The second half was mixed with sodium metasilicate (15.48 g) at RT until a clear solution was achieved. These clear solutions were then combined with stirring at RT, resulting in a thick white gel. This gel was further stirred until homogeneous, transferred to a 150 mL polypropylene bottle, and subsequently heated for 24 h at 90 °C. The resulting white precipitate was washed repeatedly with 10 mL aliquots of deionized water and isolated through centrifugation. Synthesis of Nanosized Zeolite A. Nanosized LTA was synthesized according to a previously published method.57 Aluminum isopropoxide (0.75 g), trimethyl ammonium hydroxide (5.0 g), 1 M sodium hydroxide solution (0.58 mL), and 7 mL of DI water was stirred in a polypropylene bottle with a warm water bath regulated to 40 °C until clear. In a separate propylene bottle, 30% silica sol (2.25 g) was combined with 2.0 mL of DI water and stirred under a warm water bath regulated to 40 °C until clear. Then both solutions were combined and placed in an oven for 24 h at 80 °C. The resulting solution was a suspension of zeolite nanoparticles, which was washed repeatedly with a water/ethanol mixture, followed by centrifugation. After repeated washings and centrifugation of the solution, the zeolites start becoming visible as a white precipitate. When the white precipitate formed, several more washes were made and again centrifuged to separate the zeolite from the supernatant followed by drying overnight at 90 °C under reduced pressure. Ion Exchange of Zeolites. The cobalt(II) exchanged zeolite A was prepared by combining Zeolite A (1 g) in a 0.05 M solution of cobalt acetate (100 mL) with stirring at room temperature (RT) at least 24 h. The zeolite was isolated through centrifugation followed by two more washing and centrifugation steps before the zeolites were dried overnight at 80-90 °C under reduced pressure. Elemental analysis of the blue Co-Zeolite A was performed and certified by Galbraith Laboratories, Inc. by ICP (9.27 wt %). Electrospinning of Zeolites in Polylactic Acid. The electrospinning of polylactic acid (PLA) polymer fibers was carried out by first dispersing 1.50 g of polylactic acid in 10 mL of dry chloroform. Then, dry Co-Zeolite A (0.2 g) was dispersed in 500 mL of chloroform and sonicated until the zeolite crystals were well-dispersed. This suspension was then mixed with the 10% w/w PLA solution with stirring, resulting in a blue viscous polymer solution. A syringe charged with the composite polymer melt was then fed through a 20-gauge needle at a rate of 0.20-0.25 mL/h. To initiate electrospinning, a voltage of 10 kV was applied to the needle using a variable high voltage power supply (ES50P-5W, Gamma High Voltage Research). Aligned fibers were then collected using a grounded rotating drum covered with aluminum foil positioned at a distance of 20 cm from the needle tip. Nitric Oxide Loading and Release Experiments. The zeolite absorption of nitric oxide was conducted according to the method of Wheatley et al.19 The cobalt ion-exchanged zeolite A (0.2 g) was placed in a pressure vessel and dehydrated overnight at 90 °C under vacuum and subsequently exposed to dry nitric oxide under pressure (4 atm) for 1 h, evacuated, and then (57) Mintova, S.; Olson, N. H.; Valtchev, V.; Bein, T. Science 1999, 283 (5404), 958–960.

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Figure 2. (a) SEM micrograph of as-synthesized Zeolite A and (b) X-ray diffraction spectra of as-synthesized Zeolite A.

the excess nitric oxide was flushed out of the vessel by purging with argon for 30 min. In order to adsorb the nitric oxide in the zeolites encapsulated within the polymer fibers, the fibers were first placed in the pressure vessel then dried in a vacuum oven at approximately 40 °C overnight to dry the samples, resulting in bluish fibers. The vessel was then purged with argon before the induction of nitric oxide for 15-20 min, followed by the exposure of the fibers to dry nitric oxide under pressure (4 atm) for 1 h. The nitric oxide was then evacuated and the vessel was purged with argon to expel the excess nitric oxide. For the annealed fibers, the NO charged fibers were immediately placed in an oven regulated at 65 °C for 30 min followed by 10 min to cool to room temperature. This process was repeated three times. After the last annealing cycle, the vessel was again purged with argon to expel any excess or desorbed nitric oxide resulting from the heat treatment. All samples including free zeolites, embedded zeolites, as well as heat treated zeolites were quantified for nitric oxide release immediately upon infusion of nitric oxide or upon heat treatment. Quantification of NO Release. During the quantification of NO release, the samples were kept in the infusion vessel to ensure that nitric oxide was not released in the transfer process by the contamination from atmospheric moisture. In order to quantify the loading and release of nitric oxide from both the free-zeolites as well as the zeolites embedded in the polymer fibers, a flow of argon was passed through a vessel containing a saturated salt solution that regulated the relative humidity (magnesium chloride, 33% RH). The argon carried the moisture rich gas to the sample to induce NO release. The evolved NO was carried by the argon and subsequently bubbled through a vessel containing 10 mL of deionized water where the nitric oxide would ultimately react to form nitrite ions. From this solution, 100 μL aliquots were taken at predetermined intervals and the concentration of nitrite determined by UV-vis using the Griess Reagent.58,59 All experiments were done in triplicate. In a typical Griess Assay, equal volumes of N-(1naphthyl)ethylenediamine (component A) and sulfanilic acid (component B) were mixed to form the Griess reagent. The reagent was prepared as needed and used immediately. In a 30 mL scintillation vial, 100 μL of Griess reagent, 100 μL of the nitrite-containing sample, and 2.8 mL of deionized water was mixed. The mixture was then allowed to incubate for 30 min, which allowed ample time for the pink color to form. The samples were tested at a wavelength of 548 nm against a (58) Heines, S. V. J. Chem. Educ. 1958, 35(4), 187–191. (59) Smith, M. A.; Gower, W. R. J. Chem. Educ. 1952, 29, 176–177.

reference sample consisting of 100 μL of Griess reagent with 2.9 mL of DI water. Initially, samples were combined with granules of cadmium metal to reduce any nitrate to nitrite, but further testing of samples did not reveal significant differences in the nitrite detected in samples with or without the cadmium granules, and thus, cadmium was later omitted. Instrumentation. The composite fibers were evaluated by scanning electron microscopy and energy dispersive absorption X-ray (EDAX) using a Leo 1530 VP field emission electron microscopy from Au/Pd coated samples. Fourier transform infrared (FT-IR) spectra were recorded from a Nicolet Avatar 360 FT-IR spectrophotometer. UV-vis spectrophotometry (Shimadzu UV-1601PC) was used to determine the amount of NO released. Powder X-ray diffraction (Rigaku Ultima III, Cu KR radiation) was used to characterize Zeolite A.

Results and Discussion Characterization of Cobalt-Zeolite A. A scanning electron micrograph (SEM) of the as synthesized LTA zeolite is shown in Figure 2a, and its corresponding X-ray diffraction (XRD) pattern is depicted in Figure 2b. Both the image and XRD patterns are in good agreement with those of previously verified results.56 The SEM micrograph reveals the cubic zeolite crystals with sizes ranging from 1 to 2 μm. It is important to note here that upon the influx of nitric oxide, the blue color of the zeolite begins to fade into a darker blue-gray, which subsequently changes to pink upon exposure to moisture, which has been previously attributed to the interaction of NO with the cobalt ions.19 The cobalt exchanged zeolite A was utilized for all experiments in this study because it had been previously demonstrated to adsorb the highest amount of nitric oxide.19 Characterization of Nanosized Cobalt-Zeolite A. A scanning electron micrograph (SEM) of the as-synthesized LTA nanozeolites are shown in Figure 3a, and its corresponding X-ray diffraction (XRD) pattern is depicted in Figure 3b. Both the image and XRD patterns are in good agreement with those of previously verified results.56 The SEM micrograph reveals the cubic zeolite crystalline structures with sizes of ∼100 nm. Similar to the micrometer-sized zeolites, the nanozeolites were exchanged with cobalt(II). Upon the influx of nitric oxide, the blue color of the zeolite begins to slightly fade into a

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Figure 3. (a) SEM micrograph of as-synthesized nanozeolite A and (b) X-ray diffraction pattern of nanozeolite A.

Figure 4. (a) Total release profile of Co-Zeolite A (0.2 g) exposed to an RH of 33% and (b) total release profile of nanoparticles of Co-Zeolite A (0.2 g) exposed to an RH of 33%.

darker hue, which subsequently changes to pink upon exposure to moisture.19 Pure Co-Zeolite A Release Studies. A typical nitric oxide release profile from 0.2 g of Zeolite A is shown in Figure 4a. In this study, a flow rate of 180 cm3/min was used to carry an atmosphere of approximately 33% relative humidity to the powdered zeolite to induce nitric oxide release. This is in agreement with the NO release profile reported by Wheatley et al. where an initial burst of nitric oxide is observed, followed by a rapid drop in the rate of release. This abrupt release is attributed to the strong affinity of the zeolite for atmospheric moisture, indicating the importance of flow rate and moisture present in controlling the nitric oxide release. In our storage and release studies of the nitric oxide from Co-Zeolite A, a maximum storage capacity of ∼0.774 mmol/g was observed, a slightly lower loading than that seen by Wheatley et al. (∼1 mmol NO/g).19 This decreased loading of nitric oxide could be due to the level of Co2þ exchanged within the zeolite, which has been implicated to have a direct relationship with the amount of NO adsorbed. The zeolite A used in our study was measured to contain 9.27 wt % cobalt, in contrast to the 19.8 wt % reported by Wheatley et al.19 While zeolites have demonstrated a high capacity of nitric oxide storage, its sensitivity toward moisture requires encapsulation. Embedding the zeolite within a biocompatible

water-degradable polymer matrix may serve to temporarily shield the zeolite from atmospheric moisture and ultimately slow nitric oxide release. Nitric oxide storage and release from cobalt-exchanged nanozeolites were also studied and are shown in Figure 4b. The maximum loading of nitric oxide observed in the 0.2 g sample of the nanoparticles was approximately ∼0.90 mmol/g with an average of ∼0.85 mmol/g of nanozeolites. The loading of the nitric oxide in the nanoparticles were slightly higher than that of the micrometer sized zeolites, though the difference was not statistically significant. The initial rate of release in the nanosized zeolites was also observed to be slightly higher than that of the micrometer sized particles of LTA, which may be attributed to the decreased size of the zeolites. Electrospinning of Composite Fibers. PLA fibers were fabricated using the technique of electrospinning. Electrospinning involves the introduction of an external electrostatic field, which is applied to a conducting fluid. When the electric field is in equilibrium with the droplet, a suspended conical droplet, the Taylor cone, is formed.26 Additional voltage causes the electric field to exceed the surface tension causing the droplet to become unstable resulting in the formation of a tiny jet, which is ejected from the surface of the droplet as an elongating polymer melt. This elongating melt experiences a whipping action

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Figure 5. Digital image of composite polylactic acid fibers containing metal cation exchanged zeolite. (inset) Bandage exposed to atmospheric moisture for 24 h.

where the polymer stretches resulting in polymer fibers, which are collected on a grounded target.22,26,32,33 This technique has been utilized to produce a wide range of fibrous materials with their diameter ranging from the micrometer to the submicrometer range. Many materials have exploited this technique including polymers,60-62 composites,63,64 and ceramic materials.65 In our study, we have electrospun Zeolite A as a suspension within the polymer melt, resulting in the encapsulation of the zeolite within the polymer fiber matrix. Porous Fibers with Micrometer-Sized Zeolite A. The electrospun fibers as a free-standing paper exhibited a bluish color, due to the presence of the cobalt exchanged zeolite (Figure 5). The uniform blue color seen throughout the bandage indicates a homogeneous dispersion of the zeolite within the fibers. The electrospinning of polylactic acid, which is commonly used in biological drug delivery,66-68 resulted in a free-standing bandage that was robust and very easily handled. Upon exposure to atmospheric moisture for an extended period of time, these fibers took upon a pinkish hue, similar to that seen in the free hydrated zeolite particles (Figure 5 inset). The electrospun composite fibers are shown in Figure 6a and b. The zeolites were well-dispersed; however, some crystals are located at the outer surface of the fiber as shown in Figure 6b. The average fiber diameters are approximately 10 μm, though fibers as large as 25 μm are seen. We have previously demonstrated the formation of porous PLA fibers electrospun from a 10% PLA (60) Reneker, D. H.; Chun, I. Nanotechnology 1996, 7(3), 216–223. (61) You, Y.; Min, B. M.; Lee, S. J.; Lee, T. S.; Park, W. H. J. Appl. Polym. Sci. 2005, 95(2), 193–200. (62) Reneker, D. H.; Yarin, A. L. Polymer 2008, 49(10), 2387–2425. (63) Li, D.; Xia, Y. Nano Lett. 2004, 4(5), 933–938. (64) Xu, X.; Zhuang, X.; Chen, X.; Wang, X.; Yang, L.; Jing, X. Macromol. Rapid Commun. 2006, 27(19), 1637–1642. (65) Madhugiri, S.; Sun, B.; Smirniotis, P. G.; Ferraris, J. P.; Balkus, K. J. Jr. Microporous Mesoporous Mater. 2004, 69(1-2), 77–83. (66) Agarwal, S.; Wendorff, J. H.; Greiner, A. Polymer 2008, 49(26), 5603–5621. (67) Sill, T. J.; von Recum, H. A. Biomaterials 2008, 29(13), 1989–2006. (68) Liang, D.; Hsiao, B. S.; Chu, B. Adv. Drug Delivery Rev. 2007, 59 (14), 1392–1412.

Figure 6. SEM image of (a, b) electrospun PLA fibers containing ∼13% Zeolite A.

solution in chloroform.69 The formation of pores throughout the fiber is caused by the phase separation of the polylactic acid in chloroform, which coupled with the rapid solvent evaporation during electrospinning results in a porous morphology. The loading of Zeolite A within the nonwoven mat was measured as a weight ratio with respect to the amount of polylactic acid in each sample. In a typical bandage fabrication, ∼0.2 g of zeolite A was suspended with ∼1.5 g of polylactic acid. In our study, this ratio was the highest loading of Zeolite A that could be electrospun to form uniform fibers. Increasing the amount of zeolite suspended within the polymer melt to 0.4 g resulted in larger nonuniform beaded fibers up to 40 μm in diameter with smaller fibers approximately 1-2 μm scattered throughout (Figure 7a). Further increasing the amount of zeolites to 0.6 g resulted the large aggregates of the polymer melt resembling sheets and films, again with smaller fibers scattered throughout (Figure 7b). This effect was attributed to large aggregates of the zeolite inside the fibers resulting in the beaded morphology. Silicon EDAX mapping of the electrospun PLA composite fibers with the optimized loading of zeolites is shown in Figures 8a and b. Each of the circular groupings of silica measuring ∼1 μm in diameter is indicative of a zeolite crystalline particle. It can be seen in the mapping of silica that the zeolites are well-distributed within the polymer matrix and that little to no aggregation of the zeolite particles were observed at this loading. A representative freeze-fractured cross-section of the porous fiber is shown in Figure 9. The crimped fiber (Figure 9) results from the collapse of the inner pores of (69) Liu, H. A.; Gnade, B. E.; Balkus, K. J. Jr. Adv. Funct. Mater. 2008, 18(22), 3620–3629.

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Figure 9. Freeze-fractured cross-section of porous electrospun fiber without zeolites.

Figure 7. SEM micrograph of (a) sample containing 0.4 g zeolite and (b) sample containing 0.6 g zeolite.

Figure 10. SEM micrograph of (a) electrospun nanozeolite composite fiber and (b) fiber showing aggregation of nanozeolites.

Figure 8. Silicon EDAX mapping of electrospun PLA fibers (a) SEM image of composite fiber and (b) corresponding Si EDAX mapping.

the fiber. While this does not reveal whether the pores are interconnecting, it does demonstrate that the pores of the fiber are not only seen on the outer surface of the fiber, but within the core of the fiber as well. The presence of porous fibers is integral in the fabrication of the bandage, in that it allows for the infusion of nitric oxide to the embedded zeolites. Due to the reactivity of the NO containing zeolites to moisture, the charging of the zeolites with nitric oxide could not be performed before electrospinning.

Porous Fibers with Nanozeolite A. As with the micrometer-sized zeolites A, the amount of nanozeolites in the bandage was measured with respect to the amount of polylactic acid. A typical bandage contained ∼0.2 g of the nanozeolite. A scanning electron micrograph of the electrospun nanozeolite composite fiber is shown in Figure 10a and b. The fibers with the nanozeolites typically had diameters ranging from 1 to 3 μm, though occasionally, fibers as large as 5-8 μm were seen as well. Another feature seen in the nanozeolite composite bandages were aggregates that caused slight beading in the electrospun fibers (Figure 10b). These aggregates of nanozeolites within the polymer matrix, which is more apparent in the EDAX map of Si (Figure 11). In this instance, the fiber was not beaded, but due to the electron beam damage caused by the long exposure of the polylactic acid to the electron gun the fiber softened, resulting in what appears to be a beaded fiber. The inset of Figure 11a shows an image of the fiber before EDAX mapping and e-beam damage revealing a uniform fiber with a diameter of ∼1 μm. EDAX mapping revealed that the

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Figure 12. Total release profile of nitric oxide from (9) micrometer-sized zeolite composite fibers and (() nano-sized zeolite composite fibers.

Figure 11. (a) SEM image of composite fiber with (inset) composite fiber before e-beam damage and (b) corresponding Si EDAX mapping of nanozeolite composite fibers.

nanozeolites within a uniform polymer fiber were embedded as larger aggregates, approximately 400 nm in width. Release of Nitric Oxide from Porous Composite Fibers. The release profile of nitric oxide from porous fibers was similar to that of the free zeolite (Figure 12). The fibers first experienced an initial burst in nitric oxide release followed by a rapid decline and subsequent exhaustion of the nitric oxide. Comparison between the NO stored in the free zeolites (0.775 mmol/g) with the zeolites embedded in the polymer fiber matrix (0.55 mmol/g) revealed that the bandage exhibited less nitric oxide storage capacity. This deficiency in the nitric oxide measured could be due to the electrospinning process where some of the fibers are not deposited upon the grounded target but are collected in the electrospinning setup area, resulting in a loss of zeolite sample and a reduction in the NO detected. Another source of reduced NO storage could be attributed to the inaccessibility of a small amount of zeolites that were embedded within the polymer fiber. While most of the pores observed are interconnected, a small portion of the pores are isolated, thus shielding the zeolite from the NO gas. The release of nitric oxide from the nanozeolite composite is also shown in Figure 12 and reveals the same release characteristic as that of the micrometer sized zeolites and the free zeolites. Though, as seen with the free zeolites, the rate of release again was observed at a slightly faster rate. This faster release of nitric oxide was attributed to the nanoparticles as well as the smaller diameter fibers (∼1 μm), which exposed more surface area to moisture. The nanozeolites in the porous fibers also showed a slight decrease in the amount of NO storage (∼0.73 mmol/g) in comparison to the free nanozeolites (∼0.85 mmol/g), though the decrease in NO storage was not as significant as that seen in the micrometer-sized

Figure 13. Cross section of heat-treated fibers.

zeolite composite fibers. This is likely due to the smaller diameter of the fibers, which allowed more access of the infused nitric oxide to the zeolites embedded within the fibers. Sealing/Softening Porous Polymer Fibers. The ability to incorporate nitric oxide-releasing zeolites within the fibers of a free-standing bandage expands the utility of these materials. The abrupt release of NO may be useful for the preservation of organs and tissues. However, other applications may require slower release rates. The ability to slow the initial rate of release of NO from these fibers might be achieved by changing the fiber porosity. Heating the PLA polymer fibers slightly above the glass transition temperature (Tg = 55-60 °C) allows the polymer chains to flow and effectively protect the zeolite from external moisture. A scanning electron micrograph of the heattreated fibers is shown in Figure 13. While cross sections of the polymer fiber revealed a continuous polymer matrix devoid of pores in the core, the outer surface of the fibers still exhibited a porous morphology. Additionally, after heating the PLA fibers, the bandage was not as flexible as the porous PLA fibers. The onset of brittleness in PLA fibers has been observed after repeated heating and cooling of the PLA fibers.70 The temperature cycles resulted in the annealing of the polylactic acid, which has been shown to increase the crystallinity and thus the brittleness of the polymer.71 (70) Lim, L. T.; Auras, R.; Rubino, M. Prog. Polym. Sci. 2008, 33(8), 820–852. (71) Park, S. D.; Todo, M.; Arakawa, K.; Koganemaru, M. Polymer 2006, 47(4), 1357–1363.

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Figure 16. Effect of heat treatment on nitric oxide stored in CoZeolite A.

Figure 14. (a) SEM image of heat-treated cross section and (b) corresponding Si EDAX mapping of the cross section of the heat-treated fiber.

Figure 17. Release profile of nanozeolite (b) porous and (0) heat-treated composite fibers.

Figure 15. Release profile of microzeolite composite fibers: (0) porous fibers and (2) heat-treated fibers.

The silicon EDAX mapping of a cross section of the heat-treated fibers is shown in Figure 14 and provides further evidence of a homogeneous distribution of micrometer-sized zeolites within the PLA fibers. The Si mapping of the cross section reveals four particles approximately 1 μm in diameter attributed to an individual particle of the LTA, which was evenly dispersed and showed no signs of the aggregations as seen with the nanozeolite composite fibers. The release profile of the heat-treated samples is represented in Figure 15 and reveals a more prolonged release as compared to the porous composite fibers. Additionally, the initial rate of NO release is much less pronounced than the porous fibers (Figure 15). However, the samples that were subjected to the heat treatment exhibited a lower total release of nitric oxide. Upon further investigation of the free zeolites, it was observed that the heat treatment used to seal the polymer fibers also results in a 40% reduction in nitric oxide stored within the

zeolites (Figure 16). This trend was also seen in the nanozeolite heat-treated fiber composites (Figure 17) where the amount of NO stored decreased from ∼0.7 to ∼4 mmol/g. While the rate of the heat-treated fibers exhibits a slowed release profile as compared to the release from porous fibers, the reduction in rate is not attributed to the hydrolysis of the polylactic acid, which possesses a long half-life lasting several days.47,49,72 As can be seen from the SEM images of the heat-treated polylactic acid, pores still remained on the outer surface of the fiber. Thus, the reduction in rate was attributed mainly to partial encapsulation of the zeolites. The heat creates tortuous pathways for the atmospheric moisture to reach the zeolites. If the zeolites were completely encapsulated by the polylactic acid, then the release would be expected to depend more on the hydrolysis rate of the polymer and the rate of nitric oxide release would have been much longer. Conclusion We have demonstrated the ability to incorporate nitric oxide releasing compounds and materials within electro(72) Andreopoulos, A. G.; Hatzi, E.; Doxastakis, M. J. Mater. Sci. Mater. Med. 1999, 10(1), 29–33.

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spun fibers that exhibit the release of nitric oxide comparable to past studies. This study has also demonstrated the ability to slow the rate of nitric oxide release through the shielding of the zeolites within the polymer matrix of fiber. Preliminary studies have also been conducted on isolated rat hearts that demonstrate an increase


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in coronary flow upon exposure to the NO releasing bandage. Acknowledgment. We would like to thank the Welch foundation (grant no. AT1153) and SPRING for funding this research.

Novel Delivery System for the Bioregulatory Agent Nitric Oxide

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