Elsevier

Acta Biomaterialia

Volume 1, Issue 1, January 2005, Pages 101-113
Acta Biomaterialia

Development of rationally designed affinity-based drug delivery systems

https://doi.org/10.1016/j.actbio.2004.09.002Get rights and content

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.

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 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 protein-binding 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 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 μg) 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 β2 (TGF-β2) 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-β2 allows retention of growth factor activity for up to 1 month in vivo where as TGF-β2 alone loses its activity within 24 h. These results suggest that heparin can be used to stabilize growth factors with low heparin-binding 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-β1 fusion protein that contains an exogenous collagen-binding domain from von Willebrand factor [11]. This TGF-β1 fusion protein has been shown to bind collagen with much higher affinity than recombinant TGF-β1 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-β1, 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 affinity-based 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.

Section snippets

Materials

All materials were purchased from Sigma (St. Louis, MO) unless otherwise noted.

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

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

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

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.

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