Use of human amelogenin in molecular encapsulation for the design of pH responsive microparticles

Abstract

Background: Proteins can be used in drug delivery systems to improve pharmacological properties of an active substance. Differences in pH between tissues can be utilized in order to achieve a targeted drug release at a specific location or tissue, such as a tumor. The enamel matrix protein amelogenin has a pH dependent solubility profile and self-assemble to form aggregates at neutral pH. This could make amelogenin useful in the design of pH responsive drug delivery systems.

Results: In this study amelogenin was evaluated as a pH responsive component in drug delivery applications. This was achieved by testing the ability of amelogenin to entrap/release other proteins upon changes in pH, and by testing if amelogenin could confer pH responsiveness to an existing and versatile drug delivery system, such as gelatin microparticles. Amelogenin was able to encapsulate bovine serum albumin and insulin, whichwere used as model target proteins. The composite aggregates of amelogenin and target protein were formed at neutral pH and could be reversibly solubilized at weakly acidic pH. Gelatin microparticles prepared in the presence of amelogenin, showed a modulated structure in response to pH change, when studied by scanning electron microscopy, compared to particles without amelogenin. At neutral pH amelogenin induced formation of pores in the particle surface, which were not present at acidic pH, or in particles lacking amelogenin.

Conclusions: The results from this study demonstrate that amelogenin can be a useful component in drug delivery systems in order to achieve a pH dependent response.

Figure 1

Amelogenin samples at pH 4 (left tube) and at pH 7 (right tube). At pH 7 amelogenin spontaneously self assembles to form insoluble aggregates.

Figure 2

SDS-PAGE analysis of encapsulation experiments with amelogenin (AMG) mixed with BSA (Panel A) or insulin (Ins.) (Panel B). By changing the pH from weakly acidic to neutral the aggregation of amelogenin is induced, which entraps the target molecule. Proteins captured in the aggregates are released upon acidification will end up in the solubilized pellet sample (P). The soluble fraction (S) contains proteins not precipitated or captured by the neutralization. The three subpanels are taken from the same gel. Blue arrowsindicate amelogenin, green arrow indicates BSA, and red arrow indicates insulin.

Figure 3

SDS-PAGE analysis of amelogenin (AMG) and gelatin samples crosslinked with glutaraldehyde. The samples have been crosslinked for 0, 5, 30 minutes, and 2 h. Crosslinking of amelogenin in the presence of gelatin does not alter the size of the amelogenin multimers, compared to crosslinking in the absence of gelatin. This indicates that only a minor portion of the amelogenin molecules are covalently linked with gelatin during the glutaraldehyde crosslinking reaction.

Figure 4

SEM analysis of microparticles made from gelatin, with (B, D, F, H, J, L) and without (A, C, E, G, I, K) amelogenin (AMG). The particles were incubated at pH 7 (A-F) or at pH 4 (G-L) before preparation for SEM. The presence of amelogenin affected the surface structure of the particles at pH 7 and small pores were observed in the amelogenin containing particles (panel F), but not in the control particles without amelogenin (panel E). A difference in the structure between amelogenin and control particles was also observed at pH 4 (panel G-L), but no pores seemed to form at acidic pH.

Figure 5

Analysis of gelatin and gelatin/amelogenin (AMG) microparticle degradation at pH 7.0 in presence of trypsin. The bars indicate the accumulated release of aniline blue from the particles upon proteolytic degradation, as measured by aborbance at 600 nm. The error bars represent the standard deviation based on four replicates. The amelogenin containing microparticles showed an improved resistance to trypsin degradation after 1 h incubation.