The use of a drug delivery platform composed of naturally self-assembled polypeptides is often attractive due to its inherent biocompatibility and biodegradability. Amelogenin is a non-toxic and affordable protein that can easily be produced in large-scales. It has been used clinically for wound healing and dental applications for several years with no side effects reported [18
]. Even if amelogenin often is viewed largely as an enamel associated protein, it has recently been found to be expressed in several tissues including brain, eye, cartilage and bone [21
In order to target the release of a drug to a specific location in the body, differences in pH between tissues or cellular compartments can be utilized. For example, increased drug release at acidic pH can target a drug to a cancer tumor where the pH often is more acidic. In order to be functional the drug delivery system must be responsive to the pH change at the target site. The enamel matrix protein amelogenin has a solubility profile that is greatly affected by pH (Figure ), which can be potentially useful in creating a pH response in drug delivery systems composed at least partly of amelogenin. In this study the use of amelogenin as a pH responsive component was assessed by testing its ability to entrap/release other proteins upon changes in pH, and by studying the effect of pH change on the structure of gelatin microparticles containing amelogenin.
The results in this study demonstrate that amelogenin can be used to entrap other proteins, in a reversible manner. BSA and insulin were found to aggregate together with amelogenin (Figure ), suggesting formation of composite particles, containing both amelogenin and the target proteins. No BSA or insulin were found in the pellet of the control samples, indicating that the aggregates, formed together with amelogenin, were indeed composite aggregates, and not simply a mixture of two different types of aggregates. Interestingly, the presence of BSA also seemed to increase the amount of amelogenin in the soluble fraction, compared to the amelogenin control. Both amelogenin and BSA have a hydrophobic character and they may interact with each other both in solution and during aggregation, leading to increased solubility of the amelogenin part. All the amelogenin composite aggregates could be completely solubilized at acidic conditions. The reversibility of the aggregation process makes it possible to release the protein entrapped with amelogenin, following a decrease in pH. Most proteins were susceptible for entrapment in an amelogenin matrix and the amelogenin particles could carry substantial amounts of an entrapped target protein, up to 30%. Out of the proteins tested, only lysozyme encapsulation was not achieved in this study, probably due to strong protein-protein intermolecular interactions affecting amelogenin aggregation. As opposed to BSA (pI 4.8) and insulin (pI 5.3) lysozyme has an isolectric point (pI 10.7) in the far alkaline range. Although hydrophobic interactions are essential for the initial amelogenin nanosphere formation, the charged and hydrophilic C- terminus has a pronounced effect on the amelogenin aggregation. At neutral pH, lysozyme is positively charged and can thereby perturb and interfere with the native intermolecular amelogenin interactions. The results from this study therefore point to a potential use of amelogenin in targeted release of pharmaceutical proteins, induced by acidification, but the charge of the entrapped protein may be critical for proper particle formation.
To test if the pH dependent aggregation properties of amelogenin could be used further to confer pH sensitivity also to other drug delivery systems, without any inherent pH responsiveness, gelatin/amelogenin microparticles were prepared using an emulsion-based method. The purpose of mixing amelogenin with gelatin was to determine if only a minor fraction of amelogenin could provide a response to pH changes in the gelatin particles, potentially giving the composite particles beneficial properties from both components. The gelatin microparticles containing amelogenin displayed a modulated structure compared to control particles without amelogenin. The difference was most pronounced at pH 7 where the amelogenin containing particles had a porous surface not observed in the control particles (Figure ). At pH 4, a difference in the surface structure was also observed between the amelogenin and control particles, indicating that the presence of amelogenin may have effect on the particles at both acidic and neutral pH. However, the porous structure was confined to the pH 7 particles, suggesting a more pronounced effect of amelogenin on the particle surface structure at neutral pH. At pH 4, the gelatin particles appeared to lose some of their structural integrity and adapt a more flat morphology. This effect was less pronounced in the amelogenin containing particles, suggesting a stabilizing effect of amelogenin. The results clearly indicate that the amelogenin containing gelatin particles change from having a porous surface at pH 7, to having a non-porous surface when pH becomes acidic, or vice versa. The analysis carried out in this study cannot distinguish if the pores extend deeper into the particles, or if they are isolated to the surface layer. However, we propose that the change in microparticle surface porosity at pH 7 is an effect of the amelogenin solubility profile and the tendency of amelogenin to self-assemble at neutral pH. A change in amelogenin protein structure, due to changes in pH, could modulate the interaction between both amelogenin molecules and components in the gelatin, leading to a modified structure of the microparticles. Crosslinking of amelogenin with glutaraldehyde under the conditions used does not covalently attach amelogenin to gelatin (Figure ), making it possible for amelogenin to act independently of the gelatin molecules inside the particles. The observed changes in porosity are likely to have a large effect on particle release, degradation, tissue penetration, and other pharmacologically important properties. If a porous or non-porous surface is most beneficial at a given condition depends on the application, but having the ability to change between these two states can be helpful to achieve a pH mediated delivery of substances from the particle. In addition, the results obtained in this study are supported by previous observations that an amelogenin gel, formed at pH 6.8, develops voids in response to temperature, turning the gel opaque when the temperature is changed from 4°C to 24°C [22
]. The temperatures used in our study correspond with those where the voids are formed. In our study, amelogenin comprises only a small fraction (~2.5%) of the solid material in the microparticles, indicating that amelogenin confers a potent response to pH changes, even after crosslinking of the particle with glutaraldehyde. This could be a useful property when designing new drug delivery systems, or in other processes where pH responsiveness is desirable.
The amelogenin containing microparticles proved to be more resistant to trypsin degradation compared to the control particles without amelogenin (Figure ). Since amelogenin is insoluble at pH 7, at which the proteolysis was performed, it may be a poor trypsin substrate and therefore shield the particle from degradation. Decreased trypsin sensitivity can be an advantage if an extended release in the small intestine is desired, or release further down the gastrointestinal tract. The data from this study suggest that drug delivery systems composed partly of amelogenin could have an increased tolerance towards trypsin degradation.
In our current study, we utilized native amelogenin which is insoluble between pH 6 and 7.5. By modifying the surface properties of amelogenin by for instance site-directed mutagenesis, this pH range may be modified to cover a desired pH optimum or shifting it to a wider or more narrow range. Particularly, the charged residues in the C-terminal end of amelogenin have proved to be critical in the amelogenin self-assembly [9
]. By dissecting the influence of these residues by protein engineering methods novel amelogenins can be obtained.