Protein release from gelatin matrices

Abstract

Gelatin is a denatured, biodegradable protein obtained by acid and alkaline processing of collagen. This processing affects the electrical nature of collagen, yielding gelatin with different isoelectric points (IEPs). When mixed with positively or negatively charged gelatin, an oppositely charged protein will ionically interact to form a polyion complex. This review article describes protein release from charged gelatin matrices on the basis of this polyion complexation. The biodegradable hydrogel matrices are prepared by chemical crosslinking of acidic or basic gelatin and are enzymatically degraded in the body with time. The degradation is controllable by changing the extent of crosslinking, which, in turn, produces hydrogels with different water contents. The time course of protein release is in good accordance with the rate of hydrogel degradation. It is very likely that the protein drug complexed with gelatin hydrogel is released as a result of its biodegradation. This gelatin hydrogel system releases the protein drug under maintenance of biological activity. This article will focus on experimental data that sustained release of growth factor from the gelatin hydrogels is very effective in exerting the biological functions of the growth factor.

Introduction

Recent advances in biotechnology has made it possible to produce various clinically useful peptides and proteins. While this technology has brought about the discovery and mass production of these bioactive macromolecules, several challenges need to be addressed with regard to their sustained delivery in a convenient, controlled manner, and targeting formulations. In contrast to conventional synthetic pharmaceuticals, proteins are susceptible to proteolysis, chemical change and denaturation during storage and administration in the body 1, 2. Significant efforts have been made to improve formulations for better stabilization of proteins over a sufficiently long storage time. Additional research has focused on the development of dosage forms that either prolong the biological activity of protein in the body or assist in targeting the protein to a specific tissue. One possible way to prolong activity is to incorporate a protein drug into an appropriate matrix for achieving sustained release of the drug at the site of action over a long period of time. It is highly possible that protein is protected against proteolysis and antibody neutralization, as far as it is, at least, incorporated in a release matrix for prolonged retention of the protein activity in vivo. There have been a number of research reports on protein release from polymer matrices: poly(l-lactic acid) (PLLA) and its copolymers with glycolic acid (PLGA) 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, PLGA polymer blends 18, 32, 33, PLLA–polyethylene glycol (PEG) copolymers 34, 35, poly(cyanoacrylates) 36, 37, poly(anhydrides) 38, 39, 40, poly(ortho esters) 41, 42, polyphosphazene [43], poly(vinyl alcohol) [44], poly(vinyl pyrrolidone) [45], poly(acrylic acid) [46], poly(ethylene–co-vinyl acetate) 47, 48, cellulose derivatives 49, 50, 51, hyaluronic acid derivatives 52, 53, alginate 54, 55, 56, 57, 58, collagen 59, 60, 61, gelatin 60, 61, 62, 63, 64, 65, 66, 67, starch 68, 69, dextran [70]and fibrin [71]. As is stated in other chapters of this special issue, the largest problem of protein release technology is the loss of biological activity of the protein released from a protein–polymer formulation. Thus, unless this problem is solved by a breakthrough, it seems difficult to expect a further research development in the area of protein release. It has been demonstrated that this activity loss results from denaturation and deactivation of protein during the formulation process with a polymer matrix. When exposed to harsh environmental changes, such as heating and exposure to sonication and organic solutions, protein is generally denatured, losing its biological activity 9, 14, 20. Therefore, it is important to exploit a new formulation method of protein carrier with polymers under mild conditions to minimize protein denaturation. From this viewpoint, polymer hydrogel may be a preferable candidate as a protein release matrix because of its biosafety and its high inertness towards protein drugs [72]. However, sustained release of protein over a long time period will not be expected from hydrogels, since the release rate of protein from hydrogels is generally diffusion-controlled through aqueous channels in the hydrogels. Thus, for achieving effective protein release, it will be a key strategy to immobilize the protein drug to polymer carrier molecules constituting the hydrogel through some molecular interactions. For one trial, we have been attempting to take advantage of electrostatic interactions between protein and polymer molecules for the sustained release of protein from the polymer hydrogel.

It has been well recognized in polymer science that a positively or negatively charged polyelectrolyte electrostatically interacts with an oppositely charged partner to form a polyion complex 73, 74. It seems unlikely that all of the ionic interactions between the two polyelectrolytes with many charged groups are dissociated at the same time. As a result, in contrast to low-molecular-weight electrolytes, stable bonding will occur between the oppositely charged polyelectrolytes, which will not be dissociated easily. In the research field of pharmaceutical science, this polyion complexation is not a new technology but has been extensively explored for drug coating and encapsulation. The application of this polyion complexation, which we will describe here, is “Drug complexation with polymer carriers”. This is a new trial that will allow us to pharmaceutically modify a charged polymeric drug to increase its stability, targeting and sustained release, leading to enhanced therapeutic efficacy. Charged drugs available for this trial include proteins and oligo- and polynucleotides, while biodegradable polymers, such as proteins, polysaccharides and poly(amino acid)s, are applicable as the polymer carriers. Another representative research field of “Drug complexation with polymer carriers” that has been reported is gene therapy. It has been demonstrated that complexation with positively charged polymers enabled negatively charged DNA to have an enhanced stability and transfection efficiency to cells 75, 76, 77. However, it is unclear whether or not such a formulation also functions as a matrix for sustained release of polynucleotides. On the other hand, few applications have been reported on polyion complexation for sustained release of macromolecular drugs from polymer matrices. Although low-molecular-weight pharmaceuticals have been shown to release from polymer matrices on the basis of their ionic interaction 78, 79, 80, this is, however, different from polyion complexation.

Fig. 1 shows a conceptual scheme of protein drug release from a biodegradable polymer carrier on the basis of polyion complexation. A positively charged protein drug is electrostatically complexed with negatively charged polymer chains, constituting a carrier matrix. If an environmental change, such as increased ionic strength, occurs, the complexed drug will be released from the drug–carrier complex. Even if such an environmental change does not take place, degradation of the polymer carrier itself will also lead to drug release. Because the latter is more likely to happen in vivo than the former, it is preferable that the drug carrier is prepared from biodegradable polymers. The profile of drug release in this drug–carrier system is regulated by the change of carrier biodegradation.

When we make use of polyion complexation for sustained release of a protein drug, it is absolutely necessary to employ a highly bio-safe polyelectrolyte as the carrier matrix. In addition, if biodegradability is required for the carrier, the material to be used will be restricted to natural polymers with charged groups, such as proteins and polysaccharides. Therefore, as the carrier polymer, we have selected biodegradable gelatin, which is extensively used for industrial, pharmaceutical and medical purposes. The biosafety of gelatin has been proved through its long clinical usage as a plasma expander, in surgical biomaterials and as an ingredient in drugs [81]. Another unique advantage of gelatin as a drug carrier is the electrical nature of gelatin, which can be changed by the collagen processing method [82]. For example, the alkaline process, through hydrolysis of amide groups of collagen, yields gelatin with a high density of carboxyl groups, which makes the gelatin negatively charged. This reduces the isoelectric point (IEP) of gelatin. In contrast, the electrostatic nature of collagen is hardly modified through the acid process because of a less invasive reaction to amide groups of collagen. As a result, the IEP of the gelatin that is obtained will remain similar to that of collagen. In other words, a variety of gelatin samples with different IEP values are available (Fig. 2).

If a protein to be released is acidic, basic gelatin with an IEP of 9.0 is preferable as the carrier material, while acidic gelatin, with an IEP of 5.0, will be applicable to the sustained release of a basic protein. Both gelatins are insolubilized in water to prepare a hydrogel through chemical crosslinking, for instance, with water-soluble carbodiimides and glutaraldehyde. It was reported that a model protein could be immobilized into albumin–heparin microspheres [83]or into a carrier of non-biodegradable synthetic polymer [84], through polyion complexation, and that this protein was released from the carriers upon environmental change. However, these experiments were conducted under in vitro conditions and the biological activity of the protein released was not determined. Edelman et al. [54]reported one trial of sustained release of basic growth factor by using heparin incorporated into alginate beads. The sustained release of various bioactive proteins from a collagen matrix has also been investigated 61, 85, 86and it has been shown that protein release was regulated by collagen swelling, but the contribution of ionic interactions between the proteins and collagen was not studied. Protein release from charged polysaccharides is discussed in another chapter in this issue. Since research on protein release based on polyion complexation has just started, this article mainly describes the preparation of biodegradable hydrogels from gelatin with two different IEP values and their efficacy as a sustained release carrier of a bioactive protein, together with our current findings on hydrogel degradation and protein release.