Development of rationally designed affinity-based drug delivery systems

Acta BIOMATERIALIA Acta Biomaterialia 1 (2005) 101–113 www.actamat-journals.com

Development of rationally designed affinity-based drug delivery systems Dustin J. Maxwell, Brandon C. Hicks, Sarah Parsons, Shelly E. Sakiyama-Elbert

*

Department of Biomedical Engineering, Washington University, Campus Box 1097, 1 Brookings Drive, St. Louis, MO 63130-4899, USA Received 13 July 2004; received in revised form 31 August 2004; accepted 1 September 2004

Abstract Many drug delivery systems have been developed to provide sustained release of proteins in vivo. However, the ability to predict and control the rate of release from delivery systems is still a challenge. Toward this goal, we screened a random drug-binding peptide library (12 amino acids) to identify peptides of varying (i.e. low, moderate, and high) affinity for a model polysaccharide drug (heparin). Peptide domains of varying affinity for heparin identified from the library were synthesized using standard solid phase chemistry. A mathematical model of drug release from a biomaterial scaffold containing drug-binding peptide domains identified from the library was developed. This model describes the binding kinetics of drugs to the peptides, the diffusion of free drug, and the kinetics of enzymatic matrix degradation. The effect of the ratio of binding sites to drug, the effect of varying the binding kinetics and the rate of enzymatic matrix degradation on the rate of drug release was examined. The in vitro release of the model drug from scaffold containing the peptide drug-binding domains was measured. The ability of this system to deliver and modulate the biological activity of protein drugs was also assessed using nerve growth factor (NGF) in a chick dorsal root ganglia (DRG) neurite extension model. These studies demonstrate that our rational approach to drug delivery system design can be used to control drug release from tissue-engineered scaffolds and may be useful for promoting tissue regeneration in vivo.
2004 Published by Elsevier Ltd. on behalf of Acta Materialia Inc. Keywords: Combinatorial; Controlled release; Nerve regeneration

1. Introduction The extracellular matrix (ECM) acts as a natural drug delivery system by immobilizing and controlling the release of proteins that promote cell adhesion, migration, proliferation and differentiation [1]. Proteins are sequestered to the ECM based on their affinity for proteoglycans, such as collagen, or glycosaminoglycans, such as heparan sulfate [2,3]. Delivery of proteins from ECM-mimetic materials can be controlled by modifying

*

Corresponding author. Tel.: +1 314 935 7556; fax: +1 314 935 7448. E-mail address: [email protected] (S.E. Sakiyama-Elbert).

the affinity of the material for the protein drug, the number of protein binding sites, or the degradation rate of the material [4,5]. In previous work, we have demonstrated the ability to control the number of proteinbinding sites within an ECM-mimetic material [6,7]; however no systematic approach has been developed to tailor the affinity of the interaction between the protein and the material in order to control the rate of protein release. While the majority of growth factor delivery systems are based on the diffusion of growth factors from degradable polymers, other researchers have also studied affinity-based delivery systems that immobilize and release growth factors based on non-covalent interactions. One example of an affinity-based delivery system is a heparin-based delivery system for heparin-binding

1742-7061/$ - see front matter
2004 Published by Elsevier Ltd. on behalf of Acta Materialia Inc. doi:10.1016/j.actbio.2004.09.002

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growth factors, such as bFGF, developed by Edelman and coworkers [4]. This delivery system consists of heparin-conjugated Sepharose beads that are encapsulated in alginate. Heparin-binding growth factors are immobilized within this delivery system based on electrostatic interactions between basic heparin-binding domains on the growth factors and sulfated groups on heparin. The growth factors are protected from degradation by heparin and are released slowly over time. This delivery system has been tested in a number of animal models and has completed Phase I clinical trials successfully [8]. Heparin-alginate microcapsules containing bFGF were implanted in ischemic, ungraftable myocardial territories in patients undergoing coronary bypass surgery. In the patients with the higher dose of bFGF (100 lg) after 3 months, there was significant improvement in the defect size observed by nuclear magnetic resonance imaging; however, in the placebo group there was a trend toward worsening of the defect size. In addition to using microcapsule-based delivery system, another example of affinity-based delivery systems are matrices functionalized with covalently immobilized growth factor binding sites, as developed by Wissink et al. heparinized collagen matrices were made by cross-linking collagen with EDC/NHS and then immobilizing heparin with a similar scheme [9]. This heparin could in turn serve to immobilize growth factors such as bFGF within the collagen matrices. The bFGF immobilized in these matrices enhanced endothelial cell proliferation on the gels and reduced the minimum cell seeding density required for proliferation by four fold [9,10]. In addition to using affinity-based delivery systems to control the release of growth factors, heparin can also be used as a stabilizing agent for some growth factors. Schroeder-Tefft and coworkers used heparin to stabilize transforming growth factor b2 (TGF-b2) and prevent loss of activity under physiological conditions in vitro and in vivo, when implanted within collagen matrices [5]. They demonstrated that the use of heparin to stabilize TGF-b2 allows retention of growth factor activity for up to 1 month in vivo where as TGF-b2 alone loses its activity within 24 h. These results suggest that heparin can be used to stabilize growth factors with low heparinbinding affinity in vivo and to enhance the delivery of active growth factors from biomaterials. Other affinity-based delivery systems have been developed for growth factor delivery by expressing recombinant fusion proteins of growth factors that contain non-covalent immobilization domains. Tuan and coworkers have developed a TGF-b1 fusion protein that contains an exogenous collagen-binding domain from von Willebrand factor [11]. This TGF-b1 fusion protein has been shown to bind collagen with much higher affinity than recombinant TGF-b1 that lacks the exogenous collagen-binding domain. It was also shown to promote higher levels of migration, growth and differentiation of

bone marrow mesenchymal cells in collagen gels versus native TGF-b1, BMP-2 and bFGF [12]. We have previously developed a heparin-based drug delivery system consisting of four components: (1) fibrin, (2) immobilized heparin-binding peptide, (3) heparin, and (4) heparin-binding growth factor. In this system two pairs of non-covalent interactions serve to immobilize the growth factor in the fibrin matrices. The peptide is crosslinked into the fibrin matrix via a Factor XIIIa substrate and serves to sequester heparin via electrostatic interactions. The bound heparin in turn sequesters growth factor via heparin-binding affinity. We have demonstrated the utility of this approach to promote neurite extension in vitro and sciatic nerve regeneration in vivo [6,7,13]. This approach to affinitybased delivery has proven useful for delivery not only from fibrin matrices, but also for delivery from synthetic hydrogel scaffolds, as well. It was recently shown to improve bone regeneration via controlled BMP-2 delivery in a rat calvarial defect model [14]. In summary, several researchers have demonstrated the feasibility of using affinity-based delivery systems for the sustained delivery of protein both in vitro and in vivo. However, all of the systems developed thus far have required that the researchers identify a binding site (either a polysaccharide or a short peptide) with a high affinity for the matrix or growth factor of interest. This limits the proteins that can be delivered from such system to those known to have moderate to high affinities for previously identified binding sites and provides no mechanism to rationally modulate the affinity of the interaction. The overall goal of this research was to develop improved biomaterials to facilitate tissue regeneration. A combined molecular biological and engineering approach to materials design was used to incorporate drug delivery systems, which can restore key signals that are present in during development, but missing in the adult, that may enhance tissue regeneration. These materials were functionalized with polysaccharide binding sites (in the form of covalently bound peptides) to create an ECM-mimetic that can sequester heparin and/or growth factors and protect them from degradation. Drug release from these materials was controlled by modifying the affinity of the material for the polysaccharide. Currently, no rational methods exist for the selection of domains with varying affinity for polysaccharides or proteins. In this project, combinatorial phage display libraries were used to identify short peptide sequences with varying affinities for polysaccharides, such as heparin. The development of these phage display libraries allows the identification of proteoglycan-binding peptides of varying affinities. Mathematically modeling the binding kinetics and diffusion properties of the drug was then used to enable the rational design of ECM-mimetic materials for drug delivery.

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2. Materials and methods 2.1. Materials All materials were purchased from Sigma (St. Louis, MO) unless otherwise noted.

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on consensus sequences were purchased from Biomolecules Midwest (Waterloo, IL) and made by standard Fmoc chemistry. The crude peptides were purified using a standard C18 reverse phase liquid chromatography (Shimadzu). 2.3. Mathematical modeling

2.2. Heparin binding peptides The peptides used in these experiments were derived by screening the phage display library against a heparin resin. The Ph.D.-12TM Phage Display Library (New England Biolabs Beverly, MA) consists of random peptide 12mers fused to a minor coat protein (gIII) of M13 phage. Heparin Sepharose 6 fast flow resin (Amersham Biosciences Piscataway, NJ) was equilibrated with phosphate buffered saline (PBS, pH 7.4) containing 50 mM NaCl. The phage library (1.5 · 1011 pfu) was added to the column and incubated for 20 min. After washing with 10 column volumes of PBS, the phage were eluted from the column using a stepwise gradient of 0.5, 1.0, 1.5, and 2.0 M NaCl in PBS. The phage collected from each fraction were centrifuged at 11,920 · g for 15 min at 4 C. The resulting pellet was resuspended in 1 mL of 50 mM Tris buffered saline (TBS, pH 7.4), 150 mM NaCl) and amplified in ER2739 bacteria (early logphase growth) for 4.5 h. To collect the phage from the amplification culture, the bacteria were centrifuged at 11,920 · g for 15 min at 4 C. The supernatant was precipitated overnight by adding PEG/NaCl solution (20% (w/v) polyethylene glycol-8000, 2.5 M NaCl) so that its 1/6 the total volume of the solution. Phage were collected by centrifugation at 11,920 · g for 15 min at 4 C. The pellet was re-suspended in 1 mL of TBS and precipitated using PEG/NaCl. After centrifuging the mixture, the supernatant was decanted and precipitate was resuspended in 200 lL TBS to obtain the amplified phage stock solution. The amplified phage (100 lL) were then added to 5 mL of PBS for additional screening against the heparin resin. This process was repeated three times and after each amplification step the phage were tittered. After the third round, the unamplified phage were tittered and the plaques formed were amplified using 1:100 dilution of overnight bacteria in 1 mL of Luria Bertani broth (Fisher Scientific). After incubating for 4.5 h, 500 lL of the supernatant was mixed 200 lL of PEG/NaCl and allowed to sit a room temperature for 10 m. The precipitated phage were collected by centrifugation and the pellet was resuspended in 100 lL of iodide buffer and 250 lL of ethanol was added. The mixture was centrifuged for 10 min and the pellet was washed with 70% ethanol. The pellet (isolated DNA) was resuspended in 30 lL of TBS and sequenced using Big Dye Terminators v3 (Applied Biosystems, Foster City, CA) and -96 sequencing primer for the Ph.D. library (New England Biolabs). Peptides based

A mathematical model was developed to describe release of heparin from fibrin matrices containing covalently immobilized heparin-binding peptides of various affinities. The model was used to estimate the effect of heparin-binding affinity and matrix degradation by cells on the rate of heparin release from the fibrin matrix. The goal was to use the model to predict what range of affinities and drug to binding site ratios would allow us to modulate the rate of release by varying affinity of the binding site in a controlled manner. In order to allow our model to more accurately reflect drug release in the presence of cells, we expanded our previously developed model [6] of the affinity-based drug delivery system to include a term that describes matrix degradation due to cell-activated plasmin. Five species were considered in the model: free heparin (H), immobilized peptide bound to the matrix (PB), free (unbound) peptide (PU), immobilized heparin–peptide complex bound to the matrix (HPB), and free (unbound) heparin–peptide complex (HPU). These species exist in a reaction network (Fig. 1) in which binding and dissociation of heparin and peptide, as well as matrix degradation to liberate degradable

kf

PB + H

HPB

HPU

kr

Fibrin Plasmin kf

PB

PU + H

HPU kr

Plasmin

Fig. 1. Diagram showing the components of the affinity-based delivery system and their binding interactions. Heparin-binding peptide is crosslinked into the fibrin matrix via the transglutaminase activity of Factor XIIIa forming a covalent linkage; heparin can bind to the peptide via electrostatic interactions. Binding (and dissociation) of heparin and peptide can occur for both free and bound peptide. Plasmin can degrade the fibrin matrix releasing bound species from the matrix, as is shown on the right side of the diagram. H denotes heparin, P denotes peptide, U denotes unbound peptide and complex, B denotes matrix-bound peptide and complex. kf and kr denote the forward and reverse rates for the heparin/peptide interaction.

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species were considered. Previously reported kinetic constants for heparin and the antithrombin III (ATIII) heparin-binding site peptide mimetics were used to estimate reasonable theoretical affinity constants and diffusion coefficients for proteins in hydrogels were employed to estimate the various parameters used in the model (given in Table 1). The model consisted of five partial differential equations describing the mass balances for each of the possible species in the delivery system. For those species which are diffusible (free heparin, free peptide, and free heparin–peptide complex) terms describing diffusion and the association and dissociation kinetics were included, whereas in the case of the non-diffusible species (immobilized peptide and immobilized heparin–peptide complex, which are covalently immobilized to the fibrin network), the association and dissociation terms were included, as well as terms that describe cleavage of these species from the matrix to form free peptide or heparin– peptide complex. Michaelis–Menton kinetics were used to describe the degradation of the fibrin matrix by plasmin. A term describing the plasmin activity (Units/mL) in a given volume will be used to describe non-uniform degradation. This term was multiplied by Vmax, which was multiplied by the plasmin concentration. Therefore when there is no plasmin present degradation will not occur. Eq. (1) describes the degradation of a species. The addition of this equation to the unbound species mass bal-

Table 1 Constants used in mathematical modeling Constant

Value

Units

Km Vmax kf Kr r ka DPg DPw DHg DHw DHPUg DHPUw Dplasmin Dplasminogen C0PB C0HPB C0PU C0HPU C0plasmin C0plasminogen C0uPAR

1.25 · 106 5.50 · 105 9.00 · 108 6.80 · 1002 8.00 9.00 · 108 9.05 · 103 1.01 · 102 5.36 · 103 5.95 · 103 5.03 · 103 5.59 · 103 3 · 103 3 · 103 1.84 · 101 4.42 · 106 0.00 0.00 0 2 · 106 22.5 · 109

M M/(min * unit of plasmin) 1/(M * min)[23–25] 1/(M)[26] [18,17] 1/(U/L * min) mm2/min[16] mm2/min mm2/min mm2/min mm2/min mm2/min mm2/min mm2/min M M M M M M M

D is the diffusion coefficient where the subscripts are represented as follows: free heparin (H), free peptide (P), and free heparin–peptide complex (HPU), fibrin gel (g), aqueous release medium (w), and initial condition (0).

ance describes how unbound species increase when a portion of the matrix is degraded. rV max PLASMIN½Species Degraded rK M þ ½Species Degraded

ð1Þ

where r describes the number of peptide binding sites per molecule of fibrinogen, Vmax is the maximum rate of plasmin cleavage, and KM is the Michaelis–Menton constant. This term is added (or subtracted) in the partial differential equation for each of the species that is created or degraded by the plasmin. Values for KM and Vmax were obtained from the literature for fibrin cleavage by plasmin [15]. Modeling was performed assuming that the fibrin matrix was cylindrical with a length of 12 mm and a radius of 3 mm, similar to the geometry of a nerve regeneration conduit. Diffusion was assumed to occur in both the axial (denoted y) and radial (denoted x) directions. At the beginning of the release period, all reactions in the network were assumed to be at equilibrium. The area outside the fibrin cylinder was assumed to contain none of the delivery system components initially, and the release from the cylinder was assumed to be symmetric with respect to both axial and radial diffusion. The model is described by the following equations: oC H ¼ DH rC H  k f C H ðC PB þ C PU Þ þ k r ðC HPB þ C HPU Þ ot ð2Þ oC PB rV max ðPLASMINÞC PB ¼  k f C H C PB þ k r C HPB ot rK m þ C PB ð3Þ oC HPB rV max ðPLASMINÞC HPB ¼ þ k f C H C PB  k r C HPB ot rK m þ C HPB ð4Þ oC PU rV max ðPLASMINÞC PB ¼ DPU rC PU þ ot rK m þ C PB  k f C H C PU þ k r C HPU oC HPU rðPLASMINÞV max C HPB ¼ DHPU rC HPU þ ot rK m þ C HPB þ k f C H C PU  k r C HPU

ð5Þ

ð6Þ

where Ci is the concentration of species i, for i being H, PB, HPB PU, or HPU (the mass balances for which are represented by Eqs. (2)–(6), respectively). The kinetic rate constants are as shown in Fig. 1, namely with kf and kf representing the association and dissociation rate constants for heparin binding to peptide. Di is the diffusion coefficient of species i, for i being H, PU, or HPU (the diffusible species which are not bound to the matrix). The independent variables are x, the distance from

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the axial center of the cylinder, y, the distance from the midline of the cylinder, and t is time from the initiation of release. The system of equations were solved numerically using the FEMLab software package MATLab add-on, Comsol, Burlington, MA), which has been designed to solve coupled systems of non-linear partial differential equations by using a finite element procedure. The initial condition to the model, namely that all reactions are at equilibrium and that no matrix degradation has occurred (CPU and CHPU = 0), are represented by the following equations: k f C H C PB ¼ k r C HPB

ð7Þ

eq ¼ ceq cTOT H þ cHPB H

ð8Þ

eq cTOT ¼ ceq PB þ cHPB P

ð9Þ

This system of coupled non-linear algebraic equations was solved numerically using MatLab and the equilibrium concentrations were simply input to the model as at t = 0, for 3 mm > x P 0, ci ¼ ceq i

for i ¼ H; PB; or HPB

for x > 3 mm, ci ¼ 0

for i ¼ H; PB; or HPB

Table 1 gives the initial conditions used for the system along with a summary of all the constant values used for the numerical solution of the model. In order to determine their diffusion coefficient in water equation (10) was used. A is a constant with a value of 260 cm2/(s · Dalton) and Mw is the molecular weight of the species. Di water ¼ AM w1=3 107

ð10Þ

To determine the diffusion coefficient of the species in the gel Di water was multiplied by 0.9 based on the correlation reported by Saltzman et al. for the diffusion of proteins in porous hydrogels [16]. The values for the diffusion coefficients of each species are in Table 1 and labeled with the appropriate subscript. The units have been converted from cm2/s to mm2/min in order for the modeling program to run properly with our specified geometry. The remaining boundary conditions necessary to complete the model, namely symmetry about x = 0 and y = 0, is represented by for t P 0, at x = 0, oci ¼ 0; ox

for i ¼ H; PU; or HPU

ð11Þ

oci ¼ 0; oy

for i ¼ H; PU; or HPU

ð12Þ

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In the case of localized matrix degradation, plasmin activation from plasminogen by urokinase plasminogen activator (uPA) (while bound to cell surface uPA receptor) was assumed to occur only in the region containing cells (1 mm radius by 1 mm length) within the matrix cylinder (length of 6 mm and radius of 3 mm), and the following two equations were added to the previous model to describe this effect oC PLASMINOGEN ¼ DPLASMINOGEN rC PLASMINOGEN ot  k a C uPAR C PLASMINOGEN

ð13Þ

oC PLASMIN ¼ DPLASMIN rC PLASMIN ot þ k a C uPAR C PLASMINOGEN

ð14Þ

where Ci is the concentration of species i, for i being plasminogen, plasmin and urokinase plasminogen activator receptor. The activation of plasmin was assumed to be first order with respect to plasminogen and receptor bound uPA with rate, ka and the concentration of occupied uPA receptors (uPAR) was assumed to be 22.5 nM with a local plasminogen concentration of 2 lM and no plasmin present initially. Activation was assumed to occur only in the region containing cells, but both plasminogen and plasmin could diffuse through the entire fibrin matrix and plasminogen was assumed to present at uniform concentration in the matrix initially. The concentration of receptor bound uPA was assumed to remain constant and active throughout simulation. Constant values are given in Table 1. 2.4. Preparation of fibrin matrices Fibrinogen solutions were prepared exactly as described previously, using plasminogen-free fibrinogen from pooled human plasma [17]; this fibrinogen preparation contains factor XIII, the zymogen of the transglutaminase factor XIIIa. Fibrin matrices (400 lL per well, yielding matrices that are approximately 2 mm thick) were made by mixing the components to obtain the following final solution concentrations: 4.0 mg/mL fibrinogen, 2.5 mM Ca++, 2 NIH units/mL of thrombin, 0.25 mM peptide (ATIII peptide or phage consensus peptide, which results in 8 moles of cross-linked peptide per mole fibrinogen), 62.5 lM heparin (sodium salt from porcine intestinal mucosa, 18,000 average MW), and bNGF as indicated (recombinant human, PeproTech Inc., Rocky Hill, NJ). The amount of peptide crosslinked into fibrin matrices during polymerization was previously quantified in the presence of heparin or both heparin and growth factor [18,19,6]. The polymerization mixture was placed into a well (15.6 mm diameter) of a flat 24-well tissue culture plate was incubated for 60 min at 37 C, 95% relative humidity, and 5% CO2.

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2.5. Release studies To monitor the release of heparin from the fibrin matrices, fibrinogen matrices were prepared using known concentrations of fluorescein–heparin (Molecular Probes, Eugene, OR). After polymerization of the fibrin matrix with or without heparin-binding peptide (ATIII peptide or consensus peptides obtained from phage library), the matrices were washed a total of 5 times over 24 h to remove any remaining unbound delivery system components from the matrix. After washing the matrices over 24 h, 400 lL of TBS containing a known fluorescein–heparin concentration was added to each well. These fibrin matrices were incubated at 37 C for 48 h to allow the matrix to reach equilibrium. The fibrin matrices were then washed with 1 ml TBS (5 total washes) and the wash solution was stored at 4 C. The matrices were degraded after collecting the final wash with 0.2 U of plasmin for 2 h at 37 C. The amount of heparin present in the washes and remaining in the degraded matrices was quantified using a fluorometer. The fluorescein–heparin was excited at 490 nm and the emission at 520 nm was measured using a fluorescence multi-well plate reader (CytoFluor, Perseptive Biosystems). Serial dilutions of known fluorescein–heparin concentrations were used to produce a standard curve, which was used to determine the heparin concentration in each wash. 2.6. Dorsal root ganglia culture and analysis Fibrin matrices containing peptide, heparin and NGF were prepared as described above. After polymerization of the fibrin matrix, 1 mL of TBS was added to each well to wash any unbound components of the delivery system from the matrix. The matrices were washed a total of five times over 24 h, four times with TBS and once with 1 mL of modified neural basal medium, consisting of insulin (5 lg/mL), transferrin (100 lg/mL), progesterone (6.4 ng/mL), putrescine (16.11 lg/mL), selenite (5.2 ng/mL) (all from Invitrogen, Carlsbad, CA), 0.1% bovine serum albumin, 20 ng/mL NGF (added only in the case of unmodified fibrin), 0.5 mM L -glutamine, 25 lM L -glutamate, and 1% antibiotic-anti-mycotic solution (Invitrogen) added to neural basal medium (Invitrogen). NGF was not added to the culture media for fibrin matrices containing NGF because we wanted to observe response of neurites to NGF released from the matrix and even low amounts (100 pg/mL) of NGF in the media can stimulate neurite outgrowth in this model (see Fig. 5B of online version) [20]. Neurite survival is poor in absence of any NGF and extension in unmodified gels containing no NGF is about 25% of the optimal level [20]. Dorsal root ganglia (DRGs) were harvested from day 8 White Leghorn chicken embryos and placed in Hanks

buffered salt solution (Invitrogen). One DRG was then implanted inside each fibrin matrix using dissection forceps. Modified neural basal media (1 mL) was then added to each well after 1 h. All measurements of neurite extension were normalized to DRGs cultured in unmodified fibrin matrices (no peptide, heparin, or growth factors present) in a medium of containing 20 ng/mL of NGF. Brightfield images of the DRGs were taken at 48 h with a 2X objective. The images were collected with a CCD camera (Magnifire; Olympus). The images were analyzed using Image-Pro Express software (MediaCybernetics; San Diego, CA) to determine the average length of neurite extension, which was calculated as the radius of an annulus between the DRG body and the outer halo of extending neurites, exactly as described previously [21]. Neurite length for each experiment was normalized by the average length of neurite extension through unmodified fibrin matrices from the same experiment. 2.7. Statistics Statistical analysis was performed using Statistica (version 5.5, Statsoft, Tulsa, OK). Comparative analyses were completed using the ScheffeÕs F post-hoc test by analysis of variance at a 95% confidence level. Mean values and standard deviation are reported, unless otherwise noted. Experiments were performed in triplicate and DRG experiments were performed with 6 DRGs per replicate.

3. Results and discussion The goal of this research was to develop a drug delivery system to enable controlled release during wound healing, such that heparin-binding growth factor is released in response to cellular activity during healing. Fibrin was selected as the base material for this delivery system, because it provides a three-dimensional scaffold for wound repair, and the adhesive and drug delivery characteristics of fibrin could be tailored for the wound healing model of interest. The drug delivery system developed in these studies consisted of heparin-binding peptides covalently immobilized to fibrin, and heparin bound to these immobilized peptides. Release from such a system can occur through two different mechanisms: the dissociation of heparin from matrix-bound peptide and the subsequent diffusion of free heparin-binding growth factor from the matrix, or proteolytic degradation of the fibrin matrix. The first mechanism of release described above is passive and occurs in the presence or absence of cells, while the second two mechanisms are active and occur only in the presence of cells. To determine the effect of the drug delivery system parameters (such as the relative ratios of heparin to peptide and

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affinity of the peptide for heparin) on release, mathematical modeling was performed. The effect of cell-mediated matrix degradation on release was also examined in the model by incorporating a term that described matrix degradation. The effect of modulating the affinity of the peptide for heparin was also examined in using in vitro release studies and using chick DRGs, an in vitro model of nerve regeneration to examine the effect of affinity on the activity of growth factors delivered from affinity-based drug delivery systems. 3.1. Phage library screening A random 12 amino acid combinatorial phage display library was screened using heparin affinity chromatography with a step gradient of NaCl concentration to determine sequences of random peptides with low (1.0 M NaCl fraction), medium (1.5 M NaCl fraction) and high (2.0 M NaCl fraction) affinities for heparin. Screening was repeated three times and the phage isolated from the third round of screening were amplified for sequencing. Eight to 10 sequences were isolated for each fraction (low, medium and high affinity) and are given in Table 2. For each fraction a consensus sequence (in italics) was identified and synthesized by solid phase peptide chemistry (Table 3). A Factor XIIIa substrate (NQEQVSP) from a2-plasmin inhibitor was added to the N-terminus of each peptide to allow crosslinking in fibrin matrices during polymerization [22].

3.2. Mathematical modeling of release In order to better understand the effect of peptide heparin-binding affinity on release, a mathematical model of the delivery system was developed. The equations were then solved numerically for each set of delivery system parameters, as described above. Previously we developed a model that describes only passive, diffusion-based release of drug and not cell-mediated active release [6]. In this paper, we describe a method for incorporating enzyme-mediated degradation of the fibrin matrix into the model. This improved model allows us to more closely approximate the rate of release that occurs in the presence of global matrix degradation or in the case of a point source of degradation (approximating

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Table 2 Phage library peptide sequences for heparin 12mer library CLONE #

Sequence

Affinity fraction

1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 Consensus Consensus Consensus

LLADTTHHRPWT STVSRTPQWALA SPLLSTRAVQLS NSAHYMTSGVQAS AVPHRVGGLHSL SHNLVHGPLPPH ASMTSPTTLPPRT NSAHYMSGVQAS SPLTVPYERKLL THHPQSRLAYMA NSAHYMSGVQAS QTHYTPSKSFLR ILANDLTAPGPR ILANDLTAPGPR VSYPHKDYAPKL DLHPRRPPTIHD SVSVGMKPSPRP QQQITTSSMHFD ALPELSSLPESA ALPELSSLPESA YTLASVPHNDTP ALHSPHAFRPTH QAENPNLLAATR ALPELSSLPESA QAQHSFGQVFSR KQATELPALPHP SVSVSMKPSPRP NSAHRTRGRQRS SSANGKKPSTRR ALPNSSKLAPSR

High High High High High High High High Medium Medium Medium Medium Medium Medium Medium Medium Medium Medium Low Low Low Low Low Low Low Low Low High Medium Low

a cell) and can examine the effects of matrix degradation rate on drug release. Model results showed that matrix degradation can have a major impact on release resulting in more rapid diffusion of the free heparin (drug) from the matrix. The wave front of heparin release propagates much further into the matrix in 1 h in the presence of global matrix degradation (Fig. 2), when modeled as described above. The model is able to determine the concentration each of the five system components throughout the matrix and release media. This model also demonstrates that a localized cell source of plasmin activation leads to localized release of heparin from the matrix (Fig. 2C), but the release is much smaller than in the case of global matrix degradation (Fig. 2B). Our results also demonstrate that the affinity of the peptide for heparin plays an important role in the rate

Table 3 Peptide sequences used for release studies Resin

Length

Sequence

Column wash

Heparin Heparin Heparin Control Control

12 12 12 ATIII CATIII

NQEQVSPGALPNSSKLAPSR NQEQVSPGSSANGKKPSTRR NQEQVSPGNSAHRTRGRQRS NQEQVSPK(bA)FAKLAARLYRKA-NH2 AcGCGK(bA)FAKLAARLYRKA-NH2

Low Med High – –

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Fig. 2. Effect of matrix degradation on the rate of heparin release. Heparin (free species only) concentration at 60 min is plotted versus position in the matrix (denotes by smaller rectangle, 3 · 6 mm) or release medium (larger square, 10 · 10 mm). X- and Y-axis represent millimeters. (A) Release of heparin from the matrix in the absence of degradation by plasmin. (B) Release in the presence of global matrix degradation (0.2 U of plasmin/matrix). As expected, release occurs more quickly in the presence of degradation. (C) Release from a cell source located in the corner of the matrix (1 · 3 mm) representing cells migrating into a small region of the matrix, which are activating plasmin from plasminogen via cell surface receptor-bound uPA.

Fig. 3. Effect of peptide affinity on simulated heparin release from fibrin matrices. Heparin (free species only) concentration (M) at 60 min (0.02 U of plasmin/matrix) is plotted versus position in the matrix (smaller rectangle) or release medium (larger square) in mm. (A) No reaction (no binding to matrix), (B) KD = 8.1 · 101, (C) KD = 8.1 · 102, (D) KD = 8.1 · 104, (E) KD = 8.1 · 106, (F) KD = 8.1 · 108. X- and Y-axis represent millimeters. The results show that as the affinity of the peptide for the heparin increases, the amount of heparin released decreases over time. There appears to be little effect of increasing the peptide affinity beyond KD = 1 · 104 for a peptide to heparin ratio of (100:1).

of release from our system. As the affinity for the peptide for the heparin increases, the rate of release from the delivery system decreases at similar heparin to peptide ratios (Fig. 3). In this simulation we observed that initially increasing affinity of the heparin binding peptide showed a dramatic decrease in the amount of heparin released, but as the affinity was increased to KD = 1 · 106 and above, few changes were observed. This plateau was dependent upon the ratio of heparin to peptide, thus we would expect that if the peptide to heparin ratio were decreased (100:1 was used for these simulations) that the plateau would occur at the higher affinity (KD < 1 · 106). This result suggests that affinity of the binding site becomes more important when a large

amount of growth factor is required and the number of binding sites is constrained within the delivery system. 3.3. Experimental release characterization Release rates were measured experimentally in the absence of cells and the results of these studies were compared with the mathematical modeling to determine whether the model is a valid predictive tool. Fibrin matrices were prepared containing peptides (listed in Table 3) identified from the phage display library. Briefly, the matrices were washed thoroughly over 24 h to remove any unbound peptide and then incubated with solutions containing known concentrations of heparin–

D.J. Maxwell et al. / Acta Biomaterialia 1 (2005) 101–113

Table 4 Dissociation equilibrium constants measured for heparin-binding peptides Peptide

KD (105 M)

Low Medium High Fibrin

9.0 ± 0.9 6.1 ± 1.2 3.8 ± 0.7 10.6 ± 2.2

heparin to come to equilibrium for 48 h (Table 4). The constants were measured for three heparin-to-peptide ratios and little variation was observed. The dissociation constants increase with decreasing affinity, as expected and all are lower than the constant measured for fibrin alone, which has weak heparin-binding affinity. This estimated dissociation constants were evaluated in our mathematical model (in the absence of matrix degradation) with a release geometry matching that of the 24

0.8 0.7

Fraction of Heparin Released

fluorescein for 48 h. The matrices were then degraded immediately or washed thoroughly for 24 h to remove any unbound heparin. The heparin released into the washes and retained in the matrices was measured using a fluorometer and the concentration was determined using a standard curve. The results in Fig. 4 demonstrate varying rates of heparin release from the matrix for the low, medium and high affinity peptides prepared from the 12mer heparin phage display library. A high affinity control peptide, containing the heparin-binding domain from ATIII, and matrices lacking peptide were used as controls to demonstrate the clear binding of each peptide sequence. The high and medium affinity peptide sequences retained approximately 46% and 43% of the heparin bound at 24 h, respectively, whereas approximately 35% was retained by the low affinity peptide. The results demonstrate that the low, medium, and high affinity heparin binding peptides release heparin from the fibrin matrices at different rates based on their binding affinities toward the heparin. The ATIII peptide (high affinity control) retained approximately 60% of heparin, which was significantly different from the low and medium affinity peptides, but not the high affinity peptide. In the absence of peptide 21% of the heparin was retained, which was significantly less than that retained by the medium and high affinity peptides, but not the low affinity peptide. These results demonstrate the all of the peptides have a moderate affinity for heparin and that the release rate decreases with increasing affinity (phage screening condition stringency) as expected. The equilibrium dissociation constants for the three peptides were estimated by allowing free and bound

0.6 0.5 0.4 0.3 0.2

H:P 1:25 H:P 1:50 H:P 1:100 H:P 1:500

0.1 0.0 0

ATIII High Medium Low No peptide

0.9 0.8 0.7

10

15

0.5 0.4 0.3 0.2 0.1

Time (hr)

0.6 0.5 0.4 0.3 '25 '50 '100 '500

0.2 0.1

0.0 5

10

25

0.7

0.6

0

20

0.8

Fraction of Heparin Released

F r action Total Heparin R e leased

5

(A)

1.0

109

15

20

25

30

0.0 0

Time (hr)

Fig. 4. Heparin release from fibrin matrices containing 12mer peptides from the heparin phage display library. Fibrin matrices containing the 12mer heparin-binding or ATIII peptides were equilibrated with fluoroscein–heparin for 48 h and then washed thoroughly to examine heparin release for a 100:1 molar ratio of peptide to heparin. The release rate of heparin increased as the affinity for the peptide decreased, as expected.

(B)

5

10

15

20

25

Time (hr)

Fig. 5. Effect of peptide to heparin ratio on the rate of heparin release. Release of heparin was measured over 24 h for four ratios of peptide to heparin (25:1, 50:1, 100:1, and 500:1). (A) Medium affinity heparinbinding peptide, (B) high affinity heparin-binding peptide. The results demonstrate that higher ratios of peptide to heparin have slower release rates than lower ratios of peptide to heparin.

D.J. Maxwell et al. / Acta Biomaterialia 1 (2005) 101–113

well plates used for experimental release studies. The percent heparin released from the matrix at 24 h in our model was similar to the in vitro release data shown in Fig. 4, suggesting that our model can predict heparin release and can be used for rational delivery system design. The effect of the ratio of peptide to heparin on heparin release was examined for the high and medium affinity peptides. The ratio of heparin to peptide was varied from (1:25 to 1:500) and release from fibrin matrices was measured as above. The results (Fig. 5) demonstrate that as the ratio of peptide to heparin decreases (thus decreasing the concentration of free heparin-binding sites) the rate of heparin release increases. This result was observed for both high and medium affinity peptides. This demonstrates that both affinity and the relative concentration of heparin to peptide can be used to modulate the rate heparin release from affinity-based drug delivery systems. 3.4. In vitro activity assay—DRG cultures We have previously demonstrated that an affinitybased delivery system consisting of heparin-binding peptide, heparin and NGF can be used to promote neurite extension in vitro and nerve regeneration in vivo [7,13]. In this study, the effect of heparin-binding peptide affinity on NGF release and biological activity was assessed using the low, medium and high affinity heparin-binding peptides identified in this study. A dose– response study was performed to determine the optimal concentration of NGF in the matrix to stimulate neurite extension similar to that observed for a previously deter-

1.4

Normalized Neurite Extension

110

1.2

Low Affinity Peptide Medium Affinity Peptide High Affinity Peptide NGF in Gel

1.0

* *

0.8 0.6 0.4

*

0.2 0.0 1 ng/ml

10 ng/ml

25 ng/ml

100 ng/ml

200 ng/ml

NGF Concentration

Fig. 6. Dose–response study to determine the optimal concentration of NGF for fibrin matrices containing heparin-binding peptides of varying affinities. * denotes p < 0.05 compared to NGF polymerized in unmodified fibrin matrices. These results show that high affinity heparin-binding delivery systems promote neurite extension at lower NGF concentrations then in the absence of the delivery system.

mined optimal concentration of NGF (20 ng/mL) in the media [20]. Varying concentrations of NGF were incorporated in the delivery system with heparin and the low, medium, and high affinity heparin-binding peptides. NGF polymerized in fibrin matrices with no delivery system was used as the control. Fibrin matrices containing the delivery system and NGF was washed thoroughly and DRGs were implanted within fibrin matrices. To measure the effect of the different peptides, the neurite extension from the cell body of the DRG was measured (Fig. 6).

Fig. 7. Photomicrographs of DRG cultures containing different heparin-binding peptides and heparin at optimal NGF concentrations. (A) High affinity peptide with 25 ng/mL NGF, (B) medium affinity peptide with 25 ng/mL NGF, (C) low affinity peptide with 100 ng/mL NGF, (D) NGF (100 ng/mL) polymerized in unmodified fibrin matrices, (E) unmodified matrices with NGF (20 ng/mL) in media, (F) NGF (25 ng/mL) polymerized in unmodified fibrin matrices. These results demonstrate that the higher affinity peptides retain NGF at lower concentrations and promote neurite extension.

D.J. Maxwell et al. / Acta Biomaterialia 1 (2005) 101–113 1.25

Normalized Neurite Extension

(A) 1.00

0.75

0.50

* *

0.25

* H+NGF

P+NGF

P+H

P

P+H+NGF

0.00

1.25

(B) Normalized Neurite Extension

1.00

0.75

*

0.50

0.25

P+H+NGF in media

P+NGF in Media

NGF in Gel

0.00 NGF in Media

The results show that the fibrin matrices containing the medium and high affinity peptides can promote neurite extension at lower NGF concentrations than delivery systems containing the low affinity peptide. Although the optimal dose of both the high and medium affinity peptides is approximately 25 ng/mL, the high affinity peptide starts to promote neurite extension similar to the optimal media concentration at a concentration of 10 ng/mL. We attribute this difference to the ability of the high affinity peptide to retain the more growth factor in the delivery system at lower NGF concentrations. At 10 and 25 ng/mL of NGF with the high affinity peptide, the neurite extension was statistically different from NGF polymerized in unmodified fibrin matrices. Similarly, the low affinity heparin-binding peptides had no effect in promoting nerve regeneration at lower concentrations. Neurite extension for matrices containing 100 ng/mL NGF was similar to that observed with an optimal media concentration of NGF (20 ng/ mL) for all three peptides. The results indicate that the low affinity sequence binds heparin weakly since it cannot be distinguished from the NGF polymerized in the matrix without the delivery system. In each case, an increase in neurite extension beyond the level obtained with an optimal dose of NGF in the media could not be observed for each peptide sequence. For each group, the density of neurites extending from the cell body was similar for optimal concentrations of NGF in the fibrin matrix (Fig. 7). A series of control experiments were performed to determine if the increased neurite extension at low doses (less than or equal to 25 ng/mL of NGF) for the medium and high affinity peptides could be attributed to the delivery systemÕs ability to retain NGF (Fig. 8). Fibrin matrices containing delivery system and high affinity peptide with NGF (25 ng/ml) were compared to fibrin matrices lacking all of the delivery system components. A systematic approach of removing one or more components from the fibrin matrices, allowed the role of each component to be explored and a significant reduction in neurite extension was observed in all cases. As expected, fibrin matrices containing the high affinity binding peptide and/or heparin shows little neurite extension without NGF present in the delivery system. Furthermore when the peptide is removed from the delivery system, neurite extension in matrices containing heparin and NGF was greatly reduced. This result indicates that the peptide interacts with the heparin to retain NGF in the fibrin matrix. By removing the peptide, the NGF and heparin are no longer bound to the matrix and diffuse out during the washing protocol prior to DRG implantation. Interestingly, matrices containing no heparin (only peptide and NGF polymerized within the matrix) show only a moderate decrease in neurite extension. This effect may be attributed to a combination of factors,

111

Fig. 8. Effect of delivery system components on DRG neurite extension with high affinity heparin-binding peptide. (A) Effect of the removal of delivery system components on neurite extension. P = high affinity peptide; H = heparin; NGF = nerve growth factor. All experiments were normalized to unmodified fibrin matrices containing NGF (20 ng/mL) in the media. Removal of one or more delivery system components resulted in a decrease in neurite extension. (B) Control experiments comparing NGF polymerized in fibrin matrices to matrices with NGF in the media. NGF polymerized in unmodified fibrin matrices shows a significant decrease in neurite extension suggesting that most NGF diffuses out of the delivery system during the washing protocol, prior to DRG implantation. * denotes p < 0.05 versus 20 ng/mL of NGF in the culture media and p < 0.05 versus P + H + NGF (25 ng/mL) with high affinity heparin-binding peptide.

NGF (25 ng/mL) in unmodified matrices can promote approximately 50% of the level of normal neurite extension observed with NGF in culture medium (Fig. 8B)

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and heparin binding peptides have been shown to enhance neurite extension by 35–75% versus unmodified fibrin in the presence of NGF in the culture media [18], so these two factors may additively increase the level of neurite extension to approximately 75% that observed with NGF in culture media. Overall, these results demonstrate that in order to obtain the maximal level of neurite extension, all three delivery system components, peptide, heparin and NGF are required.

Acknowledgments Support for this research was provided by a grant from the Whitaker Foundation. The author would like to thank Sara Taylor, Kelly Foyil, Shannon Hughes, and Shadatal Ghosh (Washington University) for technical assistance.

References 4. Conclusions A great number of in vivo studies have demonstrated the ability of growth factors to enhance regeneration of tissues including bone and nerve. However, the researchers acknowledge that they may not be using an optimal delivery system and in the case of affinity-based delivery systems, they often lack the ability to tailor the rate of release to the rate of the tissue regeneration. Developing a system for controlled, prolonged release could clearly impact the field of tissue engineering and aid the in the design of biomaterial scaffold that are better able to interact with their biological environment during the regeneration process. The combinatorial approach to drug delivery system design described here can be used to develop delivery systems with varying affinities for growth factor binding molecules, such as heparin or directly for the growth factor of interest, such as NGF. These affinity-based delivery systems could provide sustained release of growth factors to enhance regeneration allow the temporal role of growth factor delivery to explored in further detail, allowing researchers to potentially isolate the most important time frame for growth factor delivery during tissue regeneration. In some models such as nerve regeneration, the conventional wisdom suggests that longer periods of delivery would be desirable for regeneration, based on the 1–2 month time frame for nerve regeneration [13]. Conversely, in other wound healing models, such as bone regeneration, it is believed to be beneficial to have more rapid protein release to stimulate the migration and differentiation of precursor cells from the host tissue during the early phases of tissue regeneration (H Bentz, personal communication). In this case, a material containing low to moderate affinity binding sites might be more useful to provide more rapid release. The rational design of materials allows the release profile to be predicted prior to scaffold synthesis and allows the design of materials with rapid release profiles, slow release profile, or intermediate release profiles. Thus, rational design of drug delivery systems can be used to test hypothesis about the best time scale for growth factor delivery during tissue regeneration and provide insight into the design of biologically active materials for tissue regeneration.

[1] Roghani M, Moscatelli D. Basic fibroblast growth factor is internalized through both receptor-mediated and heparan sulfatemediated mechanisms. J Biol Chem 1992;267:22156–62. [2] Rifkin D, Moscatelli D. Recent developments in the cell biology of basic fibroblast growth factor. J Cell Biol 1989;109:1–6. [3] Vlodavsky I, Fuks Z, Ishai-Nichaeli R, Bashkin P, Levi E, Korner GB-SR, et al. Extracellular matrix-resident basic fibroblast growth factor: implication for the control of angiogenesis. J Cell Biochem 1991;45:167–76. [4] Edelman E, Mathiowitz E, Langer R, Klagsbrun M. Controlled and modulated release of basic fibroblast growth factor. Biomaterials 1991;12:612–26. [5] Schroeder-Tefft J, Bentz H, Estridge T. Collagen and heparin matrices for growth factor delivery. J Control Release 1997;49: 291–8. [6] Sakiyama-Elbert S, Hubbell J. Development of fibrin derivatives for controlled release of heparin-binding growth factors. J Control Release 2000;65:389–402. [7] Sakiyama-Elbert S, Hubbell J. Controlled release of nerve growth factor from a heparin-containing fibrin-based cell ingrowth matrix. J Control Release 2000;69:149–58. [8] Laham R, Sellke F, Edelman E, Pearlman J, Ware J, Brown D, et al. Local perivascular delivery of basic fibroblast growth factor in patients undergoing coronary bypass surgery. Circulation 1999;100:1865–71. [9] Wissink MJ, Beernink R, Poot AA, Engbers GH, Beugeling T, van Aken WG, et al. Improved endothelialization of vascular grafts by local release of growth factor from heparinized collagen matrices. J Control Release 2000;64:103–14. [10] Wissink MJ, Beernink R, Scharenborg NM, Poot AA, Engbers GH, Beugeling T, et al. Endothelial cell seeding of (heparinized) collagen matrices: effects of bFGF pre-loading on proliferation (after low density seeding) and pro-coagulant factors [in process citation]. J Control Release 2000;67:141–55. [11] Tuan TL, Cheung DT, Wu LT, Yee A, Gabriel S, Han B, et al. Engineering, expression and renaturation of targeted tgf-beta fusion proteins. Connect Tissue Res 1996;34:1–9. [12] Andrades JA, Han B, Becerra J, Sorgente N, Hall FL, Nimni ME. A recombinant human TGF-beta1 fusion protein with collagenbinding domain promotes migration, growth, and differentiation of bone marrow mesenchymal cells. Exp Cell Res 1999;250:485–98. [13] Lee AC, Yu VM, Lowe 3rd JB, Brenner MJ, Hunter DA, Mackinnon SE, et al. Controlled release of nerve growth factor enhances sciatic nerve regeneration. Exp Neurol 2003;184: 295–303. [14] Pratt AB, Weber FE, Schmoekel HG, Muller R, Hubbell JA. Synthetic extracellular matrices for in situ tissue engineering. Biotechnol Bioeng 2004;86:27–36. [15] Hurlet-Jensen A, Koehn JA, Nossel HL. The release of b beta 142 from fibrinogen and fibrin by plasmin. Thromb Res 1983;29:609–17. [16] Saltzman W, Radomxky M, Whaley K, Cone R. Antibody diffusion in human cervical mucus. Biophys J 1994;66:508–15.

D.J. Maxwell et al. / Acta Biomaterialia 1 (2005) 101–113 [17] Schense J, Hubbell J. Cross-linking exogenous bifunctional peptides into fibrin gels with factor XIIIa. Bioconjug Chem 1999;10:75–81. [18] Sakiyama SE, Schense JC, Hubbell JA. Incorporation of heparinbinding peptides into fibrin gels enhances neurite extension: an example of designer matrices in tissue engineering. FASEB J 1999;13:2214–24. [19] Schense JC, Hubbell JA. Cross-linking exogenous bifunctional peptides into fibrin gels with factor XIIIa. Bioconjug Chem 1999;10:75–81. [20] Sakiyama-Elbert SE, Panitch A, Hubbell JA. Development of growth factor fusion proteins for cell-triggered drug delivery. FASEB J 2001;15:1300–2. [21] Herbert C, Bittner G, Hubbell J. Effect of fibrinolysis on neurite growth from dorsal root ganglia cultured in two- and threedimensional fibrin gels. J Comp Neurol 1996;365:380–91. [22] Ichinose A, Tamaki T, Aoki N. Factor XIIIa-mediated crosslinking of NH2-terminal peptide of a2-plasmin inhibitor to fibrin. FEBS Lett 1983;152:369–71.

113

[23] Tyler-Cross R, Sobel M, Marques D, Harris R. Heparin binding domain peptides of antithrombin III: analysis by isothermal titration calorimetry and circular dichroism spectroscopy. Protein Sci 1994;3:620–7. [24] Kridel SJ, Chan WW, Knauer DJ. Requirement of lysine residues outside of the proposed pentasaccharide binding region for high affinity heparin binding and activation of human antithrombin iii. J Biol Chem 1996;271:20935–41. [25] Tyler-Cross R, Sobel M, McAdory L, Harris R. Structure– function relations of antithrombin III–heparin interactions as assessed by biophysical and biological assays and molecular modeling of peptide–pentasaccharide-docked complexes. Arch Biochem Biophys 1996;334:206–13. [26] Olson S, Srinivasan D, Bjork I, Shore J. Binding of high affinity heparin to antithrombin III. Stopped flow kinetic studies of the binding interaction. J Biol Chem 1981;256: 11073–9.