A Thermosensitive Carrier For The Controlled Delivery Of Biologically Active Molecules To Potentiate Bone Repair

Introduction: Delivery of functionally active molecules to a variety of musculoskeletal tissues has been a major focus of investigation. However, use of biologically active proteins is hindered by high cost and difficulty with long term storage. Peptides are an attractive alternative to active proteins because of their relatively simple structure and greater stability. These traits reduce the cost and complexity of delivering bioactive molecules for tissue engineering. In vivo biopanning studies have revealed bone targeting peptides that promote bone regeneration. We have previously shown that bone targeting peptides potentiate the differentiation of marrow mesenchymal cells in vitro [1]. The peptides were also delivered to unicortical defects in the femur with gelfoam, a porous material consisting of reconstituted type I collagen, potentiating the regeneration of bone and marrow in vivo [2]. Local delivery of the peptide in a controlled and sustained manner using appropriate carriers is important to healthy bone growth. Injectable in situ gelling hydrogels have great potential as carrier matrices for a wide range of applications such as drug delivery and tissue engineering [3]. The advantages of gelling systems over preformed matrices include the ability to conform to complex shapes, delivery of bioactive molecules or cells to a defect site under mild conditions, and minimally invasive surgery. For effective clinical application, injectable systems should gel at physiological conditions in a short time period. An injectable thermosensitive system based on the biocompatible natural polymer chitosan has been developed as a delivery vehicle for the peptide [4]. This novel thermosetting injectable system will serve as a viable carrier vehicle to deliver osteogenic peptides locally to a defect site to enhance bone repair. Materials and Methods: Chitosan with an 85% minimum degree of deacetylation (DDA) in solution with ammonium hydrogen phosphate (AHP) exhibits thermosensitive behavior and is transformed into a gel in 10 minutes at 37 °C. The thermogelling solution is prepared by mixing the AHP salt into a liquid solution of chitosan dissolved in 0.5% acetic acid. Release of the osteogenic peptide, R1, and bone morphogenetic protein 2 (BMP-2) was studied. The thermogelling solution was mixed with AHP and the desired amount of peptide or protein was added. The thermogel solution containing 300 nanomoles of the R1 peptide was submerged in PBS solution at 37 °C. Samples were collected by completely removing and replacing the release media. Peptide content was analyzed by HPLC. For comparison with a larger polypeptide with osteogenic properties, the release of 0.3 nanomoles of BMP-2 was studied under the same conditions. BMP-2 release was measured using the R&D Systems Quantikine BMP-2 Immunoassay. In addition, degradation of the chitosan thermogel was studied. Samples of chitosan thermogel were submerged in PBS solution at 37 °C or in lysozyme solution with concentration of 1 mg/mL water at 37 °C to explore the effect of enzymatic degradation on the gel and approximate in vivo behavior. Results: In vitro experiments with peptide R1 showed maximum osteogenic activity at a 5 nanomolar concentration [1]. Controlled release of the peptide was attempted in order to release at least this concentration of R1 into the site of a bone defect. To assess the release characteristics from the chitosan thermogel, R1 was added to a thermogelling solution prepared from 85% DDA chitosan. Samples were taken over the course of seven days to measure a burst release of the peptide. More than 50% of the peptide was released in the first 24 hours and almost 70% was released by day five (Figure 1). The rate of release far exceeded the desired 5 nanomolar per day with almost 5 nanomoles of peptide being released even on the fifth day. This release profile demonstrates that although the peptide eluted easily from the polymer matrix, it is possible to prepare a peptide-thermogel mixture that allows sustained release over several days. The sustained release of high levels of peptide indicates that a smaller amount of peptide can be used, and therefore reduce the cost of the delivery system further. The rate of release of recombinant BMP-2 from the same thermogelling system was investigated. Only 4.5% of the BMP-2 was released, all during the first 24 hours. Effective release of this recombinant BMP-2 protein, which is considerably larger than the R1 peptide, would be controlled by enzymatic degradation of the chitosan carrier. Degradation of the chitosan thermogel was determined by measuring loss of mass over twelve days. In order to avoid impeding the healing of critical sized bone defects in a rat animal model, the delivery vehicle must be completely degraded and removed from the site of injury within 5 weeks. The thermogel incubated in a solution of lysozyme at 37 °C completely degraded in 12 days. By contrast, chitosan incubated in PBS without lysozyme showed only a 20% mass loss in the same time period. These results suggest that enzymatic degradation of the thermogels will promote the complete release of peptide or protein. Moreover, changes in the degradation rate can be modified by changing the degree of deacetylation, thereby tailoring the final release characteristics of biologically active components such as osteogenic peptides from the mixture. Discussion: An injectable chitosan thermogel has been shown to possess properties that could be favorable for the delivery of a biologically active peptide to promote healing of defects in bone. The release rate is sufficient to deliver therapeutic levels of peptide over several days. The degradation rate of the thermogel should not impede the healing process and can be tailored for effective biological activity and clinical uses by modifying the deacetylation of chitosan. The release of a biologically active peptide from an injectable solution that gels at physiological temperature suggests that further applications may be possible for tissue repair and regeneration. References: 1) Vedavathi M, Beck G, Huang D & Balian G. ORS Trans Vol.32, 125, 2007. 2) Huang D, Beck G, Vedavathi M & Balian G. ORS Trans Vol.31, 152, 2006. 3) Ruel-Gariepy E, Leroux JC. Euro J Pharma Biopharma 58: 409, 2004. 4) Nair LS & Laurencin CT. US Patent Appl. 60/705, 812. Acknowledgements: Supported by NIH grants T32AR050960 and R21AR53579.