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Blood Clot Initiation by Mesocellular Foams: Dependence on Nanopore Size and Enzyme Immobilization Sarah E. Baker,† April M. Sawvel,† Jie Fan,§ Qihui Shi,† Nicholas Strandwitz,‡ and Galen D. Stucky*,† Department of Chemistry and Biochemistry and Materials Department, UniVersity of California, Santa Barbara, California 93106, and Department of Chemistry, Zhejiang UniVersity, Hangzhou, Zhejiang ProVince 310027, People’s Republic of China ReceiVed August 27, 2008. ReVised Manuscript ReceiVed October 1, 2008 Porous silica materials are attractive for hemorrhage control because of their blood clot promoting surface chemistry, the wide variety of surface topologies and porous structures that can be created, and the potential ability to achieve high loading of therapeutic proteins within the silica support. We show that silica cell-window size variation in the nanometers to tens of nanometers range greatly affects the rate at which blood clots are formed in human plasma, indicating that window sizes in this size range directly impact the accessibility and diffusion of clotting-promoting proteins to and from the interior surfaces and pore volume of mesocellular foams (MCFs). These studies point toward a critical window size at which the clotting speed is minimized and serve as a model for the design of more effective wound-dressing materials. We demonstrate that the clotting times of plasma exposed to MCF materials are dramatically reduced by immobilizing thrombin in the pores of the MCF, validating the utility of enzyme-immobilized mesoporous silicas in biomedical applications.
Introduction The successful inhibition or initiation of blood clotting on material surfaces has major ramifications for the development of biomedical implants and drug release as well as for the treatment of traumatic wounds and a wide variety of surgical procedures that are challenged by potential complications from thrombotic events. The collective mechanistic complexity of the estimated 80 chemical reactions that make up the blood clotting cascade is reflected in the decades of research1-4 that have focused on understanding the cardiovascular system response to the introduction of foreign materials.2,5-10 From a practical point of view, the treatment of traumatic wounds requires readily available, highly efficacious external application of a procoagulant material that gives a high probability for survivability even for arterial wounds with a high-volume blood flow.11,12 Since it is generally agreed that both intrinsic and extrinsic coagulation events are triggered by blood/surface interactions, studies of the various procoagulant, external surface properties that inhibit or promote blood coagulation are of particular interest and can be expected to be important for the † § ‡
Department of Chemistry and Biochemistry, University of California. Zhejiang University. Materials Department, University of California.
(1) Murugesan, S.; Park, T.-J.; Yang, H.; Mousa, S.; Linhardt, R. J. Langmuir 2006, 22, 3461. (2) Vogler, E. A.; Graper, J. C.; Harper, G. R.; Sugg, H. W.; Lander, L. M.; Brittain, W. J. J. Biomed. Mater. Res. 1995, 29, 1005. (3) Garcia, A. J. AdV. Polym. Sci. 2006, 203, 171. (4) Hubbard, D.; Lucas, G. L. J. Appl. Physiol. 1960, 15, 265. (5) Kastrup, C. J.; Runyon, M. K.; Shen, F.; Ismagilov, R. F. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 15747. (6) Evans-Nguyen, K. M.; Schoenfisch, M. H. Langmuir 2005, 21, 1691. (7) Lander, L. M.; Brittain, W. J.; Vogler, E. A. Langmuir 1995, 11, 375. (8) Rosengren, A.; Pavlovic, E.; Oscarsson, S.; Krajewski, A.; Ravaglioli, A.; Piancastelli, A. Biomaterials 2002, 23, 1237. (9) Miller, R.; Guo, Z.; Vogler, E. A.; Siedlecki, C. A. Biomaterials 2006, 27, 208. (10) Kastrup, C. J.; Ismagilov, R. F. J. Phys. Org. Chem. 2007, 20, 711. (11) Hursey, F. X.; Dechene, F. J. Method of Treating Wounds. U.S. Patent 4,822,349, 1989. (12) Ostomel, T. A.; Stoimenov, P. K.; Holden, P. A.; Alam, H. B.; Stucky, G. D. J. Thromb. Thombolysis 2006, 22, 55.
development of new, low-cost materials that are designed to promote or inhibit blood clotting at the foreign material interface. There are many variables that play a significant role in the initiation and continued activation of clotting,12-19 and the autocatalytic1-4 nature of the blood clotting cascade adds to the challenge of determining the dominant variables that accelerate or inhibit clotting at the surface of foreign materials. Heat release, ion exchange capacity, hydration level, particle size, particle morphology, surface area, and surface potential2,4,12-19 are important material properties that can be used to modify the blood clotting response. For example, it is generally accepted that the in vitro blood clotting time decreases as the surface area of a procoagulant material that is exposed to the blood increases.2,20 This relationship is thought to be due to the binding and activation of a group of blood “contact activation proteins” to foreign surfaces, where a higher exposed surface area corresponds to higher levels of protein activation. Factor XII (FXII) is a central contact activation protein, as the surface binding of other contact activating proteins serves to stimulate greater production of activated FXII (FXIIa) in a feedback mechanism. FXIIa then further activates downstream zymogens via the intrinsic pathway of the blood clotting cascade. A promising design strategy for creating wound-dressing materials that quickly stop uncontrolled hemorrhage is to increase the area of the material surface that is accessible to contact activation proteins, such as FXII. We will call this the “protein63.
(13) Alam, H. B.; Burris, D.; DaCorta, J. A.; Rhee, P. Mil. Med. 2005, 170,
(14) Alam, H. B.; Koustova, E.; Rhee, P. World J. Surg. 2005, 29, S7. (15) Marris, E. Nature 2007, 446, 369. (16) Ellis-Behnke, R. G.; Liang, Y.-X.; Tay, D. K. C.; Kau, P. W. F.; Schneider, G. E.; Zhang, S.; Wu, W.; So, K.-F. Nanomedicine: Nanotechnol., Biol., Med. 2006, 2, 207. (17) Ostomel, T. A.; Shi, Q.; Stucky, G. D. J. Am. Chem. Soc. 2006, 128, 8384. (18) Baker, S. E.; Sawvel, A. M.; Zheng, N.; Stucky, G. D. Chem. Mater. 2007, 19, 4390. (19) Ostomel, T. A.; Shi, Q.; Tsung, C.-K.; Liang, H.; Stucky, G. Small 2006, 2, 1261. (20) Zhuo, R.; Siedlecki, C. A.; Vogler, E. A. Biomaterials 2006, 27, 4325.
10.1021/la802804z CCC: $40.75 2008 American Chemical Society Published on Web 11/20/2008
Blood Clot Initiation by Mesocellular Foams Scheme 1. Cross Section of Mesocellular Foam and Associated Geometric Variables
accessible” surface area in the discussion that follows. One route toward increasing the protein-accessible surface area of a blood contacting material is to reduce the size of the material particles.21 However, nanometer-sized particles are not easily removed from a wound and may be toxic.22 In contrast, conventional monolithic wound-dressing materials, such as gauze, are more easily applied to and removed from a wound.23 An alternative approach to maximizing the surface area of a gauze-applicable monolithic material is to introduce porosity. It has been shown that exposure of blood to materials with varying pore sizes in the micrometer regime greatly affects subsequent cellular adhesion and growth.24,25 However, prior to this study, the effects of pore size variation on the length scale relevant to the sizes of the proteins involved in blood clot initiation (nanometers to tens of nanometers) have not been explored. We expect that optimizing the material pore size in this range will improve on current hemostatic agents because both internal and external surface area can be used for contact activation. Mesocellular foams (MCFs) are mesoporous silicate materials with tunable cell diameters in the range of 2-90 nm and cell windows as large as 35 nm.26-29 As shown in the MCF cross section in Scheme 1, the internal surface area of MCFs consists of close-packed, spherical “cells” that have openings, or “windows”, which are smaller than the cell diameter. We will refer to the MCF structures specifically as cells, as opposed to the more general description “pores”, because the latter typically describe structures with continuous channels where the pore diameter and pore-opening diameter are the same. MCFs are excellent materials for size-exclusion chromatography, protein separation,29,30 and enzyme immobilization31,32 due to their uniform cell and window size, their open and accessible structure, and their high pore volume (0.9-3.2 cm3 g-1). The ability to tailor the cell and cell-window sizes of MCFs makes them ideal materials for this study because these properties can be varied independently of the particle size, morphology, and surface chemistry. Furthermore, the large cells in the MCF pore structure (21) Margolis, J. Aust. J. Exp. Biol. 1961, 39, 249. (22) Gwinn, M. R.; Vallyathan, V. EnViron. Health Perspect. 2006, 114, 1818. (23) Z-Medica Corp. http://www.z-medica.com/. (24) Malmberg, P.; Nygren, H. Biomaterials 2002, 23, 247. (25) Roach, P.; Eglin, D.; Rohde, K.; Perry, C. C. J. Mater. Sci.: Mater. Med. 2007, 18, 1263. (26) Schmidt-Winkel, P.; Lukens, W. W., Jr.; Zhao, D.; Yang, P.; Chmelka, B. F.; Stucky, G. D. J. Am. Chem. Soc. 1999, 121, 254. (27) Schmidt-Winkel, P.; Lukens, W. W., Jr.; Yang, P.; Margolese, D. I.; Lettow, J. S.; Ying, J. Y.; Stucky, G. D. Chem. Mater. 2000, 12, 686. (28) Lukens, W. W.,Jr; Yang, P.; Stucky, G. D. Chem. Mater. 2001, 13, 28. (29) Han, Y.; Lee, S. S.; Ying, J. Y. Chem. Mater. 2007, 19, 2292. (30) Han, Y.-J.; Stucky, G. D.; Butler, A. J. Am. Chem. Soc. 1999, 121, 9897. (31) Han, Y.-J.; Watson, J. T.; Stucky, G. D.; Butler, A. J. Mol. Catal. B 2002, 17, 1. (32) Yiu, H. H. P.; Wright, P. A. J. Mater. Chem. 2005, 15, 3690.
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enable a high loading capacity for protein immobilization.26,29 Here, we use mesocellular foams to probe the effect of the cellwindow size on blood clot initiation, as mediated by the activation of the coagulation protein FXII. The pore-opening or cell-window diameter is an important tunable property in wound-dressing materials not only for enabling access of clotting enzymes to the internal surface of the material, but also for enabling the adsorption of clot-promoting enzymes or therapeutic agents within the material that will further accelerate clotting. A wide range of uniform cell-window sizes (to 35 nm) can be selectively created to promote access of thrombin and other enzymes into the very well-defined MCF cell structure. We sought to explore whether the open cell structure of the MCF material would also enable the immobilization of the clotting enzyme thrombin within the MCF cells. Thrombin is a serine protease that is central to the blood coagulation cascade and has the ability to cross-link fibrin even in the absence of other clotting enzymes. As a result, it is likely that thrombin-loaded MCFs would more effectively arrest hemorrhage than would materials that do not deliver thrombin. Furthermore, it has been shown that these mesoporous silica surfaces can be functionalized to optimize utilization of the supported protein so that it is possible to obtain a comparable enzymatic activity and greater stability of the immobilized enzyme compared to those of the same enzyme in the solution phase.32,33 By optimizing the MCF cell-window size and immobilizing a clotting enzyme within these cells, a bifunctional hemostatic agent is created that provides a high surface area for contact activation as well as catalytic, clot-promoting activity. In this study, we have determined that, within a set of procoagulant materials with similar external morphology and surface chemistry, material cell-window size variations in the nanometer range greatly affect the propensity of the material to activate the contact activation proteins involved in the intrinsic pathway of coagulation. Our results confirm that the conventional description of the surface area as defined by gas sorption (BET) measurements is not necessarily relevant to blood-contacting materials. In fact, within the set of materials that we tested, blood clotting was slower in plasma exposed to higher BET surface area materials, contrary to current paradigms. In addition, we found that an enzyme central to several reactions in the bloodclotting cascade, thrombin, can be immobilized within the MCF material at high loading. Our experiments indicated that immobilized thrombin is tightly bound within the cells of the MCF and maintains its catalytic activity, so that low application dosages of these MCF-thrombin agents can significantly reduce the clotting times.
Materials and Methods Synthesis and Characterization of Mesocellular Foams with Varied Pore Sizes. A series of MCF materials with varied cell and cell-window sizes were synthesized according to published procedures,29 on one-quarter scale, using a calcination temperature of 550 °C for all materials. Nitrogen sorption isotherms were obtained using a Micromeritics Tristar 3000 porosimeter. Prior to measurement, the samples were dried at 220 °C under nitrogen for 12 h. The surface areas and pore volumes of the materials from each synthesis were calculated using the BET (Brunauer-Emmett-Teller) analysis, window sizes were determined from the desorption isotherms using the Broekhoff-de Boer (BdB) method, and pore sizes were calculated using the adsorption isotherm data, also using the BdB method. Imaging to (33) Lei, C.; Shin, Y.; Liu, J.; Ackerman, E. J. J. Am. Chem. Soc. 2002, 124, 11242.
14256 Langmuir, Vol. 24, No. 24, 2008 determine the particle size and morphology of the MCF materials was performed using an FEI XL30 electron microscope. Transmission electron microscopy (TEM) micrographs were collected using an FEI Tecnai G2 Sphera microscope operating at 200 kV. TEM samples were prepared by immersing a lacey carbon grid in an ethanolic solution of the MCF-33 material. Determination of Clotting Activity of MCF Materials. Frozen pooled human plasma (PHP) and Coumadin plasma (with an international normalized ratio of 2.4) were purchased from George King Biomedical (Overland Park, KS) and handled according to the package inserts. Factor XII-deficient plasma was purchased from George King Biomedical and handled according to the directions provided in the package insert. A Hemoscope Thrombelastograph (TEG) was used to compare the clotting properties of the MCF and QuikClot hemostatic agents. The TEG measures several parameters likely to be relevant to the effectiveness of a hemostatic material, including time until initial clot formation and the rate of clot formation, by measuring the torsion of a small sample of blood around a wire during coagulation. The TEG was operated as follows. First, 20 µL of 0.2 M CaCl2 was added to the TEG cup, which was heated to 37.0 °C. Then 340 µL of plasma was added to the cup, followed immediately by addition of the clotting agent from a 1 dram glass vial. The sample cup was then loaded into position for commencement of the measurement. To determine the effects of pore size alone on the clotting parameters of human plasma, we attempted to keep the total external volume of MCF spheres added to the plasma similar by keeping the total pore volume constant at 0.006 cm3. Therefore, a different mass of each MCF was added to the plasma, according to the pore volume. These masses were 6.4 mg of MCF 6, 4 mg of MCF 11, 4.3 mg of MCF 20, 2.6 mg of MCF 26, and 2.6 mg of MCF 33. The clotting activity of QuikClot13,14 was maintained by handling and storing the material in an argon drybox. Sealed QuikClot packages (Z-Medica Inc.) were transferred into the argon dry box before opening, and 20 mg of QuikClot was measured into 1 dram glass vials. The vials were sealed and removed from the drybox within 30 min of use. Thrombin Loading and Determination of the Enzymatic Activity of MCF-Thrombin. The mass of bovine thrombin (Aldrich, 236 NIH units/mg) adsorbed to each MCF sample was determined by measuring the background-corrected absorbance of a 2 mg/mL thrombin solution in quartz cuvettes prior to and after exposure to the solid. The MCF material was removed from the thrombin solution by centrifugation, and the optical absorbance of only the solution phase was measured. Typically 0.6-1 mL of 2 mg/mL thrombin in HEPES buffer (20 mM, pH 5.3) was added to 2-4 mg of MCF in a plastic 1.5 mL centrifuge tube and incubated at 4 °C overnight. Thrombin adsorption to the sides of the plastic centrifuge tubes in which the thrombin-immobilization studies were carried out was found to be negligible. The quantities used in Figure 3a were 0.8 mL of 2 mg/mL thrombin solution (shown in Figure 3a at 50% concentration) and 4 mg of MCF-33. The MCF loading capacity for thrombin was calculated as follows: the absorbance value of 0.75 corresponded to 1.6 mg of thrombin in this experiment. After incubation with 4 mg of MCF, the absorbance of the solution decreased by 0.25 unit, or 33%, indicating that 33% of the initial 1.6 mg present in the solution, or 0.53 mg of thrombin, adsorbed to 4 mg of MCF material, corresponding to a loading of 13%, w/w. Because some fraction of thrombin initially determined to be absorbed to the MCF may have been only loosely bound and removed by subsequent washing steps, the calculation described may overestimate the extent of thrombin loading. The resulting MCF-thrombin (MCF-T) material was then washed four times with HEPES buffer using centrifugation and the supernatant removed. In all cases, after two centrifugation steps thrombin was not detectable in the supernatant by UV/vis. To determine whether the thrombin was irreversibly bound to the MCF and retained its activity, we used a chromogenic substrate for
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Figure 1. (a) Morphology of MCFs. MCF-33 is shown. The scale bar represents 20 µm. (b) Higher resolution SEM image showing the morphology of the MCF-33 surface. The scale bar represents 1 µm; the inset scale bar represents 250 nm. (c) Representative high-resolution TEM micrograph of MCF-33.
thrombin, β-Ala-Gly-Arg-p-nitroanalide diacetate34 (Sigma-Aldrich). We used a stock solution of this substrate [3 mg/mL in HEPES buffer (20 mM, 0.15 M NaCl), pH 7.4] and added 100 µL of substrate solution and 800 µL of HEPES pH 7.4 buffer to 4 mg of MCF-T pellet (prepared as described above) and inverted the resulting suspension several times. After 8 min, the MCF-T suspension was visibly yellow and was centrifuged for 2 min and the supernatant removed to determine the amount of substrate cleaved by thrombin after 10 min. To determine the 15 min time point, the MCF-T suspension was immersed in the substrate solution for 13 min prior to 2 min centrifugation. To compare the activity of the immobilized thrombin to that in solution, we added the volume of 2 mg/mL thrombin solution necessary to equal the mass of thrombin adsorbed to the MCF material and used the same assay conditions. This volume (265 µL for 13% thrombin loading on 4 mg of MCF) was added to 100 µL of chromogenic substrate and 800 µL of HEPES pH 7.4 (34) Prasa, D.; Svendsen, L.; Sturzebecher, J. Thromb. Haemostasis 1997, 78, 1215.
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Langmuir, Vol. 24, No. 24, 2008 14257 Table 1. Nitrogen Sorption Surface Area Characteristics for the MCF Series mass of material BET pore surface volume used in the BJH window BJH cell material size (nm) size (nm) area (m2/g) (cm3/g) TEG (mg) MCF-6 MCF-11 MCF-20 MCF-26 MCF-33
5.9 11.2 20.7 26.4 33.1
10.0 24.5 42.7 41.9 52.1
679 739 375 364 294
0.95 1.53 1.4 2.3 2.4
6.4 4 4.3 2.6 2.6
Results and Discussion
Figure 2. Effects of the MCF window size on the (a) time to clot formation and (b) rate of clot formation. The figure was generated from four replicate measurements of each sample; the error bars represent the standard deviation among these replicates.
buffer, and the absorbance was measured at 405 nm for 15 min. To control the effects of dilution that occurred by adding this additional 265 µL solution to the substrate solution, we also compared the activity of the solution-phase thrombin to that of MCF-T that was diluted by an additional 265 µL of HEPES pH 7.4 buffer. In all cases, the activity of the MCF-T was greater (20-30% higher absorbance values due to MCF-T than solution-phase thrombin after 15 min) than that of the same mass of thrombin in solution. To test for long-term leaching of thrombin from MCF, we measured the activity of the MCF-T supernatant after 12 days and found negligible activity, while the MCF-T material retained 1/3 of the initial activity after 12 days. To determine whether the high ionic strength of blood might cause thrombin to be released from the MCF pores, we immersed MCF-T in simulated body fluid (composition i35) for 30 min, centrifuged the resulting mixture to remove the MCF-T, and measured the activity of thrombin in the supernatant. We found negligible activity. Characterization of Thrombin-Loaded MCFs in Human Plasma. We compared the clotting performance of 20 mg of dehydrated QuikClot, 2 mg of MCF-33 (a control), and 2 mg of MCF-33 immobilized with thrombin in both pooled human plasma and in Coumadin plasma using the TEG. The MCF-T was prepared as described above. To ensure that the MCF control had surface chemistry identical to that of MCF-T, the MCF control was also immersed in HEPES pH 5.3 buffer overnight and washed extensively using centrifugation with HEPES pH 7.4 buffer. After removal of the supernatant, the MCF samples (2 mg) were resuspended in 50 µL of HEPES pH 7.4 buffer and were added to the plasma as a slurry using a 200 µL pipet. Control experiments verified that the addition of 50 µL of the HEPES pH 7.4 buffer alone did not significantly change the plasma-clotting times. (35) Oyane, A.; Kim, H.-M.; Furuya, T.; Kokubo, T.; Miyazaki, T.; Nakamura, T. J. Biomed. Mater. Res. 2003, 65A, 188.
Clotting Time Dependence on the Window Size. Porous inorganic materials offer extremely high material surface area and have a myriad of applications such as in heterogeneous catalysis,36,37 photovoltaic systems,38 size exclusion chromatography,29,30 and protein immobilization31 and as fast-acting hemostatic agents.12 While these materials can be synthesized with a wide range of pore diameters and pore-opening diameters, for porous materials to be an effective medium for protein immobilization or rapid blood clot initiation, the pore-opening diameter must be large enough to accommodate macromolecules in the tens of nanometers size regime. Previous investigations into the use of high surface area, porous aluminosilicates with cage structures as procoagulant materials showed that the time to clot initiation could be correlated to the material surface area, as measured by nitrogen adsorption.12 However, the materials used in that study had cage-diameter openings that were e10 Å, resulting in a large internal surface area that was not accessible to contact-activating coagulation proteins. The data we present here clearly demonstrate that tuning the pore-opening diameter of porous silicate materials modulates the protein-accessible surface area of the material and, therefore, the procoagulant activity of porous inorganic surfaces. To probe the effects of protein-accessible surface area on surface-activated blood clotting proteins, we created a model set of materials that would allow us to vary the cell-window diameter while keeping other properties, such as the surface chemistry, particle size, and morphology constant. Silica-based MCFs with a range of cell-window and cell diameters were created by varying the synthesis conditions during the supramolecular templating process. Nitrogen sorption data (Table 1) verified that the materials, designated by window size as MCF-6, MCF-11, MCF21, MCF-26, and MCF-33, had a range of window and cell size distributions, pore volumes, and BET surface areas. These parameters are illustrated in Scheme 1 for clarification. The BET surface area decreased and the pore volume increased within the series of samples, confirming the increased amount of void space present in the samples with larger cell-window openings and larger cells. While both the cell-window opening and the cell diameter increased within this series of samples (MCF-6 to MCF33), it was assumed that the cell-window diameter was the primary constraint on the ability of proteins to access the internal surface area of the material. Scanning electron microscopy (SEM) was used to confirm that the particle size and morphology remained constant within the MCF series, despite the varied window and cell size distributions among the samples (Figure S1, Supporting information). A representative SEM image of the MCF particle (36) Andersson, M.; Birkedal, H.; Franklin, N. R.; Ostomel, T. A.; Boettcher, S. W.; Palmqvist, A. E. C.; Stucky, G. D. Chem. Mater. 2005, 17, 1409. (37) Zheng, N.; Stucky, G. D. J. Am. Chem. Soc. 2006, 128, 14278. (38) Coakley, K. M.; Liu, Y.; McGehee, M. D.; Frindell, K. L.; Stucky, G. D. AdV. Funct. Mater. 2003, 13, 301.
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Figure 3. (a) Optical absorbance measurement indicating the degree of thrombin immobilization on MCF-33. (b) Comparison of the enzymatic activity of 0.4 mg of thrombin in solution vs that adsorbed to MCF as indicated by absorbance at 405 nm. (c) Time-dependent absorbance due to cleavage of the chromogenic substrate for thrombin. The lack of absorbance change in the MCF-T supernatant (black) at both 10 and 15 min time points indicates that the thrombin was confined to the surface of the MCF-T.
morphology, in this case MCF-33, is shown in Figure 1a. The image indicates that the MCF particles consist primarily of 5-10 µm spheres with some fused spheres. The higher resolution SEM images (Figure 1b and inset) show the porous surface of MCF33. High-resolution TEM was used to confirm the presence of a uniform and ordered porous network. A representative image of MCF-33 (Figure 1c) shows that the material has highly ordered cells with a uniform diameter of 25 nm. These results show that the MCF materials synthesized are suitable for use as model materials to investigate the effects of pore window size on clot initiation. TEG measurements in PHP were used to determine the impact of the MCF window size on the activation of blood coagulation proteins. The mass of material used in the analysis was adjusted for each MCF sample to account for variations in pore volume
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within the MCF series and to ensure that differences in clotting activity were due entirely to changes in the cell-window diameter. As shown in Table 1, to keep the pore volume constant among the series of MCF samples, generally a greater mass of material was used as the cell-window diameter decreased. Figure 2a shows the time to clot initiation of pooled human plasma after exposure to the MCF samples of each window size. As the window size increased from 6 to 33 nm, the average time to blood clot initiation decreased from 9.8 to 6 min. Furthermore, the TEG parameter designated as the rate of clot formation (Figure 2b), which is related to the time required for the blood clot to reach maximum clot strength, increased significantly when the average window size of the material was greater than 20 nm. An illustration depicting how the clotting time and rate were derived from the raw TEG data is shown in the Supporting Information (Figure S2). The dramatic effect of the MCF cell-window size on blood clot initiation and the rate of blood clot formation in human plasma is likely to be the result of increased protein-accessible surface area that is available with the larger cell-window diameters. We focus primarily on the surface area accessible to coagulation protein factor XII (FXII) in this discussion because clotting times of FXII-deficient plasma in the presence of MCF were greatly prolonged compared with the clotting times of normal plasma in the presence of MCF. These data indicate that the MCF materials accelerate clotting in an FXII-dependent mechanism (Supporting Information, Figure S3). FXII has an estimated hydrodynamic radius of 7.5 nm, on the basis of the molecular weight (78 000),39 and is known to be rapidly converted to the active form (FXIIa) when exposed to negatively charged surfaces (such as silica) in the presence of other plasma proteins.40 Given the diffusion coefficient approximation used for activating proteins involved in the blood clotting cascade of 5 × 10-11 m2/s,5 the diffusion length of FXII is ∼100 µm on the time scale of clot initiation (6 min) in these experiments. Therefore, when not physically blocked by prohibitively small cell-window diameters, it is likely that FXII and other contact activation proteins of similar size are able to diffuse into MCF cells, adhere, activate, and diffuse out of the material during the clot initation phase of the clotting cascade. The extent of FXII activation has been shown to scale with the silica surface area exposed to the plasma when the particles are nonporous silica beads.2 Our results, which indicate a sharp decrease in clotting times with window sizes greater than 11 nm, are consistent with the hypothesis that the increased clotting activity of larger pore silicates corresponds to increased access of FXII, and other surface-active enzymes that participate in activation of FXII, to the internal surface area of the MCF. These results are of particular importance considering that less of the MCF material was used in TEG experiments performed on samples with larger window diameters, but faster clotting times were observed. Tuning the cell-window diameter to accommodate contact-activating proteins, such as FXII, allowed us to conserve sample mass by creating a more efficient procoagulant material where both the internal and the external surfaces could be utilized for blood clot initiation. Within the series of MCF samples studied here, the lower surface area materials (as determined by nitrogen sorption isotherms, Table 1) resulted in faster clot formation, which is contrary to the paradigm that the clotting time decreases with increasing procoagulant material surface area.2,41 Therefore, the surface area as defined by nitrogen sorption isotherms does not (39) Zhuo, R.; Siedlecki, C. A.; Vogler, E. A. Biomaterials 2007, 28, 4355. (40) Griffin, J. H. Proc. Natl. Acad. Sci. U.S.A. 1978, 75, 1998.
Blood Clot Initiation by Mesocellular Foams
appear to correlate with clot initiation by biomaterials with the window/pore sizes evaluated in our study. More relevant is the protein-accessible surface area and pore volume, which are dependent on the pore sizes in the tens of nanometers range. The results indicate that the material window size, rather than the BET surface area, is a more relevant parameter to consider in the design of porous wound-dressing materials for hemorrhage control. In particular, we show that window sizes of 30 nm or greater may be beneficial for fast blood clot activation. We were not able to test larger window sizes because 35 nm is the upper size limit for current MCF synthesis methods. Synthesis routes to achieve larger window and cell sizes are under investigation. Thrombin Immobilization on MCF-33. Thrombin is a 36 kDa serine protease with dimensions42 of 3 × 8 nm that affects several enzymatic reactions in the blood clotting cascade and has a critical function in the wound-healing process.43,44 Within the coagulation cascade, thrombin facilitates the activation of FXI in the intrinsic pathway, activates factors V and VIII, stimulates platelet activation, converts soluble fibrinogen to the insoluble fibrin monomer, and converts FXIII to FXIIIa, which cross-links insoluble fibrin polymers to form a robust blood clot. Due to the ability of thrombin to activate numerous clotting factors and to directly produce insoluble fibrin, exogenous thrombin can generate a blood clot at the wound site despite clotting factor deficiencies, such as hemophilia,45 and is used to stop bleeding in more than 500 000 surgical procedures annually in the United States.45 Thrombin is typically applied to the wound site either alone in solution form or in a gelatin sponge saturated with a thrombin solution, such as the Gelfoam sponge.46 Since thrombin can be fatal if introduced directly into the blood stream, immobilizing it within a microporous scaffold may improve the safety of topically administered thrombin by localizing enzymatic activity at the wound site and preventing thrombin entry into the blood stream. The large pore volumes as well as the tunable cell sizes and cell-window diameters of MCFs make them an ideal platform for immobilizing a high weight percentage of enzymes.31 Of the cell-window diameters studied here, MCF-33 was determined to have the highest clotting activity, due to the increased amount of protein-accessible surface area that is present in MCF materials with window diameters above 20 nm. Therefore, it is likely that immobilizing thrombin on MCF-33 will have a dramatic impact on the clotting activity of the material due to the combined effect of adding exogenous thrombin to the wound site and providing a large protein-accessible surface area for contact activation to occur. Furthermore, it is expected that the large pore volume (2.4 cm3/g) of MCF-33 will accommodate a significantly larger quantity of immobilized thrombin than the gelatin sponge technologies that are currently in place. The loading efficiency of bovine thrombin (pI 7.1) on equal masses (1 mg) of MCF-33 and Gelfoam, a commercial gelatin sponge, was determined at pH values between 9.4 and 5.4 by comparing the optical absorption at 280 nm of a thrombin solution before and after exposure to each material. We found that the (41) Chatterjee, K.; Vogler, E. A.; Siedlecki, C. A. Biomaterials 2006, 27, 5643. (42) Harmison, C. R.; Landaburu, R. H.; Seegers, W. H. J. Biol. Chem. 1961, 236, 1693. (43) Lundblad, R. L.; Bradshaw, R. A.; Gabriel, D.; Ortel, T. L.; Lawson, J.; Mann, K. G. Thromb. Haemostasis 2004, 91, 851. (44) Hoffman, M.; Harger, A.; Lenkowski, A.; Hedner, U.; Roberts, H. R.; Monroe, D. M. Blood 2006, 108, 3053. (45) Bishop, P.; Lawson, J. Nat. Drug DiscoVery 2004, 3, 684. (46) Cantor, M. O.; Kennedy, C. S.; Reynolds, R. P. Am. J. Surg. 1951, 82, 230.
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optimal pH for immobilizing thrombin on MCF was in the range between 5.4 and 6.4, which enabled an enzyme loading capacity of 13%, w/w (Figure 3a). However, the maximum loading capacity determined for the Gelfoam sponge was only 2%, w/w, at pH 6.4 (data not shown). Though it was not confirmed, it is likely that the higher thrombin loading capacity of the MCF-33 material is due to the increased pore volume of the MCF-33 material compared to the Gelfoam sponge. These results imply that the porous network of MCF-33 yields a much higher thrombin loading capacity than the same mass of the gelatin sponge. The large cell-window diameter of MCF-33 allows for maximum transport of thrombin into the interior of the material, and the large pore volume allows for considerable retention of the enzyme within the porous network of the material. By optimizing the material properties of MCF, we were able to create an enzyme support scaffold with a thrombin loading capacity superior to that of the currently available Gelfoam product. To evaluate whether the immobilized thrombin was active and tightly bound within the mesocellular structure of the MCF material, MCF-T was exposed to β-Ala-Gly-Arg-p-nitroanalide diacetate, which is a colorimetric substrate specific to thrombin. The activity of thrombin in the presence of the colorimetric substrate in this assay corresponds to the rate of change in optical absorbance of the substrate solution at 405 nm. To prevent the MCF material from interfering with absorbance measurement, each sample was centrifuged and the supernatant was analyzed in all cases. Representative results from these experiments are shown in Figure 3b. As shown in the figure, the MCF-T supernatant solutions (resulting from exposure to approximately 400 µg of thrombin adsorbed to 4 mg of MCF) led to higher absorbance values at 405 nm (typically 20-30% greater) after 10 and 15 min time intervals than 400 µg of solution-phase thrombin (in 200 µL of buffer), indicating that immobilized thrombin catalyzed the cleavage of the chromogenic substrate at a higher rate than solution-phase thrombin. Figure 3c shows the time dependence of the data from which Figure 3b was derived. The blue trace with a positive slope in Figure 3c shows the absorbance change due to cleavage of the substrate by solutionphase thrombin during the initial 15 min. The black traces in Figure 3c represent the time-dependent absorbance of chromogenic substrate in the MCF-T supernatant which was removed from the MCF-T by centrifugation after 10 and 15 min. As can be seen in the figure, the absorbance of the MCF-T supernatant at 405 nm did not change significantly with time after the removal of the MCF-T, indicating that the thrombin activity was localized to the MCF-T. These data are consistent with highly active, irreversibly adsorbed thrombin present in the pores of the MCF materials. Blood Clotting Activity of Thrombin-MCF-33. To ascertain whether MCF-T could catalyze the formation of fibrin in human plasma in vitro, including human plasma with a clotting deficiency, we evaluated the MCF-T and other clotting agents using the TEG in both normal PHP and plasma from an individual taking Coumadin. Coumadin therapy decreases vitamin Kdependent synthesis of several proteins necessary for propagation of blood clot formation, including thrombin, and factors VII, IX, and X and is prescribed to about 2 million patients annually in the United States. Because of difficulties in Coumadin dosing, Coumadin can lead to hemorrhage and is the second most common drug leading to adverse events that require hospitalization.47 Thrombin is able to bypass reactions involving these proteins by (47) Bloom, M. FDA alters warfarin label to reflect gene tests for bleeding risks. Medpage Today, Aug 16, 2007.
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Figure 4. Clotting times of pooled human plasma (PHP) and Coumadin plasma alone (left) after exposure to MCF-33, MCF-thrombin, and QuikClot clotting agents. The figure was generated from three replicate measurements of clotting times; the error bars represent the standard deviation among these replicates.
stimulating the activation of other clotting factors in the coagulation cascade, and it is likely that MCF-T will work equally well in normal PHP and in Coumadin plasma. In addition to evaluating the effectiveness of MCF-T compared with MCF to determine the effects of thrombin immobilization, we also compared the clotting effectiveness of the MCF-T to that of QuikClot, a zeolite composite material which has been shown to be the most effective of commercially available hemostatic agents in animal studies.13,14 The results of the in vitro evaluation of MCF-T are shown in Figure 4. The left side of Figure 4 shows that PHP clots significantly faster than the Coumadin plasma, which is expected due to the lower concentrations of vitamin K-dependent clotting factors in the Coumadin plasma. Figure 4 (inset) shows that adding 20 mg of dehydrated QuikClot dramatically reduced the clotting times of both PHP and Coumadin plasma. Surprisingly, only 2 mg of MCF-33 (suspended in 50 µL of buffer) accelerated blood clot formation similarly to 20 mg of QuikClot. The fact that an order of magnitude lower mass of MCF-33 was required to demonstrate in vitro effectiveness similar to that of QuikClot may be due to the comparatively higher degree of proteinaccessible surface area of MCF-33. The cage-opening diameter of the zeolite in QuikClot is
