Chem. Mater. 2003, 15, 4193-4199
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Preparation of Nitric Oxide (NO)-Releasing Sol-Gels for Biomaterial Applications Stephanie M. Marxer, Aaron R. Rothrock, Brian J. Nablo, Mary E. Robbins, and Mark H. Schoenfisch* Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-3290 Received May 12, 2003. Revised Manuscript Received August 21, 2003
Nitric oxide (NO)-releasing sol-gel materials are synthesized by combining aminefunctionalized alkoxysilanes (aminosilanes) with alkyltrimethoxysilanes (alkylsilanes). Upon hydrolysis and condensation, the amine-functionalized silanes are covalently bound to the alkyltrimethoxysilane backbone and easily converted to diazeniumdiolate NO donors via exposure to high pressures of NO. Immersion of the sol-gel into solution is not required to initiate NO release. The NO-release characteristics of the sol-gels are easily controlled by varying the type and amount of the aminosilane precursor in the sol. The sol-gel coatings release NO for up to 20 d with average fluxes between 8.0 × 10-12 and 5.6 × 10-11 mol‚s-1‚cm-2 (coating thickness of 50 µm) over the first 10 h. These materials exhibit reduced platelet and bacterial adhesion. The results indicate that sol-gel chemistry may be an effective strategy for preparing coatings that release NO both controllably and locally for a range of applications including blood- and tissue-based devices.
I. Introduction Surface-induced thrombosis and biofilm-related infection as a result of platelet and bacterial adhesion, respectively, pose significant health risks to patients receiving in vivo medical devices by altering the environment of the implant and significantly reducing its utility and/or longevity.1,2 The viability of many bloodand tissue-contacting devices is thus highly dependent on materials that resist such biofouling. Numerous strategies have been pursued to address such problems including the synthesis of new polyurethanes, poly(dimethylsiloxane)s (PDMS), polyethylenes, poly(ethylene glycol)s, poly(ethylene oxide)s, and hydrogels.3-6 Other work has focused on the design of materials that actively release anticoagulants such as heparin to reduce platelet adhesion7,8 or antibiotics to reduce bacterial adhesion.3 Despite significant research devoted to the development of materials that resist protein and cell adhesion, biofouling continues to dominate the interfacial chemistry that occurs when biomaterials are utilized in vivo. A promising alternative approach for combating biofouling is based on the use of polymers that controllably * To whom correspondence should be addressed. E-mail: schoenfi@ email.unc.edu. (1) Anderson, J. M. Annu. Rev. Mater. Res. 2001, 31, 81. (2) Wisniewski, N.; Reichert, M. Colloids Surf., B 2000, 18, 197. (3) Hendricks, S. K.; Kwok, C.; Shen, M.; Horbett, T. A.; Ratner, B. D.; Bryers, J. D. J. Biomed. Mater. Res. 2000, 50, 160. (4) Park, J. H.; Park, K. D.; Bae, Y. H. Biomaterials 1999, 20, 943. (5) Desai, N. P.; Hubbell, J. A. J. Biomed. Mater. Res. 1991, 25, 829. (6) Lee, J. H.; Ju, Y. M.; Kim, D. M. Biomaterials 2000, 21, 683. (7) Walpoth, B. H.; Rogulenko, R.; Tikhvinskaia, E.; Gogolweski, S.; Schaffner, T.; Hess, O. M.; Althaus, U. Circulation 1998, 98, II319. (8) Weerwind, P. W.; van der Veen, F. H.; Lindhout, T.; de Jong, D. S.; Cahalan, P. T. Int. J. Artif. Organs 1998, 21, 291.
release nitric oxide (NO).9-12 NO, a diatomic free radical naturally synthesized in the body,13 serves multiple bioregulatory functions in the cardiovascular, respiratory, gastrointestinal, genitourinary, and nervous systems.14-17 NO is produced by endothelial cells in blood arteries to regulate vasodilation and platelet adhesion and activation.18 Similarly, macrophages release NO during the phagocytosis of bacteria.13,19 Thus, several groups have proposed the use of coatings that release NO to improve the biocompatibility of intravascular (blood contacting)- and subcutaneous (tissue)based devices.9,12,20-22 Indeed, plasticized hydrophobic polymers (e.g., silicone rubber, polyurethane, poly(vinyl chloride)) doped with (Z)-1-[N-methyl-N-[6-[(N-methyl(9) Hanson, S. R.; Hutsell, T. C.; Keefer, L. K.; Mooradian, D. L.; Smith, D. J. Adv. Pharmacol. (San Diego) 1995, 6, 383. (10) Smith, D. J.; Chakravarthy, D.; Pulfer, S.; Simmons, M. L.; Hrabie, J. A.; Citro, M. L.; Saavedra, J. E.; Davies, K. M.; Hutsell, T. C.; Mooradian, D.; Hanson, S. R.; Keefer, L. K. J. Med. Chem. 1996, 39, 1148. (11) Espadas-Torre, C.; Oklejas, V.; Mowery, K. A.; Meyerhoff, M., E. J. Am. Chem. Soc. 1997, 119, 2321. (12) Nablo, B. J.; Chen, T.-Y.; Schoenfisch, M. H. J. Am. Chem. Soc. 2001, 123, 9712. (13) Marletta, M. A.; Yoon, P. S.; Iyengar, R.; Leaf, C. D.; Wishnok, J. S. Biochemistry 1988, 27, 8706. (14) Marletta, M. A.; Tayeh, M. A.; Hevel, J. M. BioFactors 1990, 2, 219. (15) Ramamurthi, A.; Lewis, R. S. Ann. Biomed. Eng. 1998, 26, 1036. (16) Ramamurthi, A.; Lewis, R. S. Ann. Biomed. Eng. 2000, 28, 174. (17) Shabani, M.; Pulfer, S. K.; Bulgrin, J. P.; Smith, D. J. Wound Healing Soc. 1996, 4, 353. (18) Radomski, M. W.; Rees, D. D.; Dutra, A.; Moncada, S. J. Pharmacol. 1992, 107, 754. (19) MacMicking, J.; Xie, Q.; Nathan, C. Annu. Rev. Immunol. 1997, 15, 323. (20) Mowery, K. A.; Schoenfisch, M. H.; Saavedra, J. E.; Keefer, L. K.; Meyerhoff, M., E. Biomaterials 2000, 21, 9. (21) Zhang, H.; Annich, G. M.; Miskulin, J.; Osterholzer, K.; Merz, S. I.; Bartlett, R. H.; Meyerhoff, M., E. Biomaterials 2002, 23, 1485. (22) Bohl, K. S.; West, J. L. Biomaterials 2000, 21, 2273.
10.1021/cm034347n CCC: $25.00 © 2003 American Chemical Society Published on Web 10/07/2003
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Scheme 1. Reaction of NO with Amines To Produce Diazeniumdiolate NO Donors
ammoniohexyl)amino]]-diazen-1-ium-1,2-diolate (MAHMA/NO)-NO donor molecules (synthesized by the reaction of secondary amines with NO at elevated pressure23) (Scheme 1, for MAHMA/NO: R and R′ are methyl groups and x ) 6), have been shown to decrease platelet adhesion by releasing low levels of NO at the polymer-solution interface.9-12 The enhanced thromboresistivity using MAHMA/NO was first demonstrated by Hanson et al. for vascular grafts9 and extended by Smith et al. to include several biodegradable polymers.10 Schoenfisch et al. demonstrated improved in vivo performance for intravascular oxygen sensors modified with MAHMA/NO doped into silicone rubber.24 Unfortunately, the NO donor (MAHMA/NO) and its potentially carcinogenic amine decomposition products were found to leach from the hydrophobic polymers.20 To address toxicity concerns, Zhang et al. incorporated amino-alkyl trimethoxysilane cross-linkers into hydroxyl-terminated PDMS polymers.21 In this type of polymer, both diazeniumdiolates and amine decomposition byproducts remained covalently linked to the cured silicone rubber structure and leaching was avoided, while NO release ability was maintained. Notably, small amounts (ca. 2-5 wt %) of both PDMS and the crosslinker were still discovered to leach from the cured silicone rubber films due to incomplete polymerization.21 In addition, a broad range of NO generation rates was unattainable using PDMS-based NO-release polymers. Despite these caveats, NO-releasing silicone rubbers were shown to have notably improved in vivo blood compatibility (i.e., reduced platelet adhesion).21 Recently, we demonstrated that sol-gel chemistry is another attractive method for synthesizing NO-releasing materials whereby leaching of the residual amine precursors should be avoided because the donor is no longer doped into the polymer, but instead bound to the polymer backbone.12 By exposing sol-gels prepared from amine-functionalized alkoxysilanes (aminosilanes) to high pressures of NO, covalently linked diazeniumdiolates are formed (Scheme 1, where R is a hydrogen and R′ is a silane).23 These materials spontaneously release NO analogous to polymers doped with MAHMA/ NO diazeniumdiolate donors and amino-alkyl trimethoxysilane cross-linked silicone rubber. Herein, a more detailed investigation regarding the synthesis and full characterization of these materials is provided. It will be demonstrated that the NO-release properties (e.g., amount, rate, and duration) of these materials are tunable by varying the structure and/or amount of the aminosilane, allowing for exceptional control over NO release. Furthermore, the flux of NO from sol-gel films is sufficient to reduce both platelet and bacterial adhesion, suggesting that these materials may find utility as potential biomedical coatings. (23) Hrabie, J. A.; Klose, J. R.; Wink, D. A.; Keefer, L. K. J. Org. Chem. 1993, 58, 1472.
Marxer et al. Scheme 2. Structures of (A) Isobutyltrimethoxysilane (BTMOS), (B) (Aminoethylaminomethyl)phenethyltrimethoxysilane (AEMP3), (C) (Aminohexyl)aminopropyltrimethoxysilane), (AHAP3), and (D) N-[3-(Trimethoxysilyl)propyl]diethylenetriamine (DET3)
II. Experimental Section Materials. Ethanol (absolute) and concentrated hydrochloric acid were purchased from Fisher Scientific (Pittsburgh, PA) and used as received. Isobutyltrimethoxysilane (BTMOS) and N-[3-(trimethoxysilyl)propyl]diethylenetriamine (DET3) were purchased from Aldrich (St. Louis, MO). (Aminoethylaminomethyl)phenethyltrimethoxysilane (AEMP3) and N-(6-aminohexyl)aminopropyltrimethoxysilane (AHAP3) were obtained from Gelest (Tullytown, PA). (Structures of BTMOS, AEMP3, AHAP3, and DET3 are given in Scheme 2.) Nitric oxide (99.5%) was obtained from National Welders Supply Co. (Durham, NC) and purified over KOH before use. Whole blood obtained from healthy pigs was provided by the Francis Owen Blood Research Laboratory (Chapel Hill, NC). Pseudomonas aeruginosa (ATCC #19143) was acquired from American Type Culture Collection (Manassas, VA). Distilled water was purified with a Millipore Milli-Q Gradient A-10 system (Bedford, MA) to 18.2 MΩ. All other reagents used were of analytical grade and used as received. Film Preparation. The highest concentration of aminosilane used to prepare sol solutions was 40% (v/v total silane concentration) due to instability of the films upon water immersion at higher aminosilane concentrations. Because aminosilanes hydrolyze considerably faster than non-aminefunctionalized silanes,25,26 sol-gels were prepared using a twostep synthesis. BTMOS (120-190 µL) was mixed with 60 µL of water, 200 µL of ethanol, and 10 µL of 0.5 M HCl for 1 h. By adding 10-80 µL of DET3, AEMP3, or AHAP3 (for a total silane volume of 200 µL), the aminosilane content was varied from 5 to 40%. The sol was mixed for an additional hour before being coated onto glass slides that had been ozone-cleaned for 20 min with a BioForce TipCleaner (Ames, IA). For UV-vis experiments, the sol was spin-cast onto quartz slides at 3000 rpm to obtain thin films. For all other experiments, 30 µL of sol was cast onto 9 × 25 mm glass slides. Sol-gel films were dried at 70 °C for 3 d and then placed in a hydrogenation bomb, which was flushed thoroughly with Ar to remove both water and oxygen, and then exposed to 5 atm NO for 60 h to form the diazeniumdiolate NO donor. After NO was purged from the hydrogenation bomb by flushing six times with Ar, the solgels were removed from the vessel and stored in a sealed container at -20 °C prior to use. Material Characterization. Diazeniumdiolate formation was confirmed with UV-vis spectroscopy. Absorption measurements were obtained using a Perkin-Elmer Lamda 40 UV/ vis spectrometer (Norwalk, CT). To measure pore surface area, the sol-gel was removed from the glass slide and ground into a fine powder. A Micromeritics ASAP 2405N surface area and pore size analyzer (Norcross, (24) Schoenfisch, M. H.; Mowery, K. A.; Rader, M. V.; Baliga, N.; Wahr, J. A.; Meyerhoff, M., E. Anal. Chem. 2000, 72, 1119. (25) Charbouillot, Y.; Ravaine, D.; Armand, M.; Poinsignon, C. J. Non.-Cryst. Solids 1988, 103, 325. (26) Cao, W.; Hunt, A. J. Mater. Res. Soc. Symp. Proc. 1994, 346, 631.
Nitric Oxide (NO)-Releasing Sol-Gels GA) was used to characterize N2 and Kr sorption. Surface area was calculated using the Brunauer-Emmett-Teller (BET) equation.27 The detection limit of the instrument was ∼0.5 m2/ g. Sol-gel stability in aqueous solution was evaluated by soaking the films in phosphate-buffered saline (PBS) (pH 7.4) for various times (1-14 d). The amount of Si in the soak solutions, an indicator of sol-gel fragmentation, was measured with an ARL-Fisons Spectraspan 7 direct current plasma optical emission spectrometer (DCP-OES; Beverly, MA). This instrument has a detection limit of 0.5 ppm and was calibrated using standards of 0.0, 1.0, 2.0, 5.0, and 10.0 ppm Si in PBS. The level of fragmentation (% fragmentation) was calculated as a mole percent using the following equation:
Volume Soak Solution (mL) MW Si(µg/µmol) × 100% Amount Si in film (µmol)
Measured Si(ppm) ×
The number of moles of Si in the sol-gel film was calculated based on the volumes of silane solutions used to prepare the starting sol and both the density and MW of the materials provided by the manufacturer. The effect of sol-gel composition on surface wettability was evaluated with a KSV Instruments Cam 200 Optical Contact Angle Meter (Helsinki, Finland). Static water contact angles were obtained before and after diazeniumdiolate formation. Characterization of NO Release Properties. NO release was determined using a Sievers NOA 280 Chemiluminescence Nitric Oxide Analyzer (Boulder, CO). The instrument was calibrated before each experiment with air passed through a zero filter (purchased from Sievers) and 43.5 ppm NO gas (balance nitrogen). To measure NO release, sol-gel-modified glass slides were immersed in PBS. The NO generated was carried to the analyzer by a stream of nitrogen bubbled into the PBS, where it was detected directly through reaction with ozone. For a given sol-gel film, several real-time data points were obtained and averaged at various intervals (e.g., t ) 0, 5, 10, and 24 h) to determine the flux of NO from the sol-gel film. To evaluate the effect of temperature on NO release, solgels were analyzed and stored at 0, 25, and 37 °C. The efficiency of converting amine groups to diazeniumdiolates was estimated by curve-fitting the NO release profiles to approximate the total amount of NO, assuming 100% of the bound NO would be released from the sol-gel. The following equation was then used to determine conversion efficiency (C.E.):
Chem. Mater., Vol. 15, No. 22, 2003 4195 Plain glass slides, 40% AHAP (balance BTMOS) sol-gel control films, and identical sol-gel films capable of NO release (i.e., exposed to 5 atm NO) were immersed in PRP solutions for 30 min. The slides were then rinsed with Tyrode’s buffer (pH 7.35) to remove loosely adhered platelets. Attached platelets were fixed with a 1% glutaraldehyde solution (Tyrode’s buffer) to ensure preservation of cell morphology. Finally, the surfaces were rinsed with Tyrode’s buffer and water before being chemically dried using 50% EtOH (v/v, water) for 5 min, 75% (v/v, water) for 5 min, 95% (v/v, water) for 5 min, and 100% for 10 min and hexamethyldisilazane (HMDS) (overnight). Phase contrast images of these surfaces were obtained using a Zeiss Axiovert 200 inverted microscope (Chester, VA). P. aeruginosa cultures were grown from a -80 °C stock in tryptic soy broth (TSB) for 12 h at 37 °C. A 2 mL aliquot of the cell culture was inoculated into 200 mL of TSB and incubated for ca. 6 h at 37 °C. Cells were centrifuged and resuspended in PBS. Viable cell counts on tryptic soy agar were performed to obtain the viable cell concentration. 40% AHAP (balance BTMOS) sol-gels were incubated in 5 mL of PBS for 30 min to initiate steady NO release. Sol-gel films were immersed and gently stirred in 5 mL of the cell suspension for 30 min at 37 °C. Surfaces were then rinsed with water and fixed in a 2% glutaraldehyde solution for 15 min. Images of these surfaces were obtained using phase contrast microscopy.
III. Results and Discussion
The theoretical NO-release capability of the sol-gel is based on the initial concentration of aminosilane (diazeniumdiolate precursor) in the starting sol and the amount of sol cast onto the glass slide. Of note, although DET3 is a triamine, the number of diazeniumdiolates that form on its side chain was assumed to be 1 based on the structure of the diazeniumdiolate form of diethylenetriamine (DETA/NO) proposed by Keefer et al.28 Biocompatibility Studies. Platelet-rich plasma (PRP) was obtained from acid citrate dextrose (ACD)-anticoagulated porcine blood (1 part ACD to 9 parts whole blood) by centrifugation at 200g for 15 min at room temperature.29 Calcium chloride (CaCl2) was added to the PRP at a concentration of 0.25-0.50 mM Ca2+ to maintain normal platelet activity.29
With control of a range of processing conditions including pH, solvent, water content, dry/cure time and temperature, and the type and concentration of the silane precursor (or precursors), sol-gel chemistry has been widely employed to synthesize materials with an assortment of physical properties (e.g., wettability and micropore structures) and chemical characteristics.30-32 Herein, experiments were conducted to determine whether sol-gel chemistry might be useful for preparing materials capable of generating NO at variable rates and amounts without concomitant leaching of the amine precursor. Sol-Gel Synthesis. Isobutyltrimethoxysilane (BTMOS) was chosen as the initial backbone alkoxysilane because the most stable and smooth films with the largest variety of aminosilanes were formed with BTMOS over other commonly used silanes (e.g., methyltrimethoxysilane). Because aminosilanes hydrolyze considerably faster than the above alkoxysilanes,26,33 sol-gels were prepared in a two-step synthesis involving (1) the prehydrolysis of the alkoxysilane and (2) the addition of the aminosilane. Cast films were dried/baked in an oven at 70 °C for 3 d. This temperature was chosen to accelerate the drying process. Smooth, optically transparent films were thus formed for combinations of DET3, AEMP3, or AHAP3 and BTMOS for aminosilane concentrations up to 40%. Sol-gel films prepared using either a one-step synthesis, greater aminosilane concentrations (> 45%), and bake temperatures >70 °C were considered inadequate as biomaterials because they were either opaque, brittle, or nonhomogeneous. A property of sol-gels that have made these materials attractive for numerous applications including coatings
(27) Brunauer, S.; Emmett, P. H.; Teller, E. J. Am. Chem. Soc. 1938, 60, 309. (28) Keefer, L. K.; Nims, R.; Davies, K. M.; Wink, D. A. Methods in Enzymology; Academic Press: New York, 1996; Vol. 268, p 281. (29) Cazenave, J.; Mulvihill, J. The role of platelets in bloodbiomaterial interactions; Missirlis, Y., Wautier, J.-L., Eds.; Kluwer: Dordrecht, 1993; p 69.
(30) Brinker, C. J.; Scherer, G. Sol-Gel Science; Academic Press: New York, 1989. (31) Collinson, M., M. Trends Anal. Chem. 2002, 21, 30. (32) Elferink, W. J.; Nair, B. N.; de Vos, R. M.; Keizer, K.; Verweij, H. J. Colloid Interface Sci. 1996, 180, 127. (33) Rousseau, F.; Poinsignon, C.; Garcia, J.; Popall, M. Chem. Mater. 1995, 7, 828.
NO release (µmol) × 100 2 mol of NO µmol of aminosilane cast × mol of aminosilane
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Figure 1. UV-vis spectra of 20% DET3 (balance BTMOS) sol-gel film before exposure to NO (+), and 40% (×), 30% (O), and 20% (- - -) DET3 (balance BTMOS) sol-gel films after exposure to NO. (Note: 30% and 40% DET3 films prior to NO exposure overlay the 20% DET3 spectrum and are not shown for simplicity.)
for sieve columns and sensors is their high porosity.30 Surface areas of dried sol-gels (xerogels) typically range from 500 to 900 m2/g.30 Sol-gels prepared with aminosilanes, however, have previously been reported as significantly less porous.25 Indeed, the aminosilane-solgel systems studied (AEMP3/BTMOS, AHAP3/BTMOS, and DET3/BTMOS) are each characterized by surface areas of less than ∼0.5 m2/g (BET surface analysis) at aminosilane concentrations >5%. Due to the low surface area, pore volume data were not acquired. The high density of these sol-gel systems is attributed to the basic environment that exists within the sol-gel network due to the presence of the amines, promoting accelerated condensation. NO Release Characteristics. Characterization of the NO-releasing sol-gel films after reaction of the amines with NO involved confirming the formation of the diazeniumdiolate-NO donor within the sol-gel matrix by both acquiring UV absorbance spectra of thin sol-gel films and measuring actual NO release with chemiluminescence-based detection.34 Typical UV-vis spectra of sol-gel films before and after exposure to NO are shown in Figure 1. The appearance of a peak at 250 nm after NO exposure is consistent with the findings of Hrabie et al., who reported absorption maxima at ca. 250 nm for a variety of diazeniumdiolates.23 As expected, the absorbance at 250 nm increased with greater amounts of aminosilane since more diazeniumdiolates were formed. Diazeniumdiolate dissociation to NO from the solgel materials is assumed to be the reverse pathway to its formation.35 This reaction is assumed to be accelerated when the diazeniumdiolate molecules encounter water or another proton source (protonation of the amino nitrogen)36 or when the temperature is increased,37 shifting the equilibrium between the aminosilane precursor and the diazeniumdiolate to the left (Scheme 1). Diazeniumdiolate formation was also confirmed by measuring the amount of NO released from (a) aminosilane-based sol-gels, (b) sol-gels prepared (34) Kelm, M.; Yoshida, K. Methods of Nitric Oxide Research; John Wiley: New York, 1996. (35) Drago, R. S.; Ragsdale, R. O.; Eyman, D. P. J. Am. Chem. Soc. 1961, 83, 4337. (36) Maragos, C. M.; Morley, D.; Wink, D. A.; Dunams, T. M.; Saavedra, J. E.; Hoffman, A.; Bove, A. A.; Isaac, L.; Hrabie, J. A.; Keefer, L. K. J. Med. Chem. 1991, 34, 3242. (37) Ragsdale, R. D.; Karstetter, B. R.; Drago, R. S. Inorg. Chem. 1965, 4, 420.
Marxer et al.
Figure 2. NO flux from 40% DET3 (9), AHAP3 (2), and AEMP3 (b) (balance BTMOS) sol-gel films at 25 °C.
with 100% BTMOS precursor (controls), and (c) plain glass slides (blanks) freshly reacted with NO at high pressure. NO is generated steadily from the diazeniumdiolate-sol-gel films while only slight release (
