Release of a model protein from biodegradable self assembled films for surface delivery applications

Abstract

Layer-by-layer (LbL) films have multiple features that make them attractive for drug delivery, including the potential to sequentially deliver growth factors from implantable medical devices or tissue engineering scaffolds. To date, however, characterization has been lacking for protein delivery from such films. Here, LbL polyelectrolyte films constructed with the model protein lysozyme and a hydrolytically degradable and biocompatible synthetic polycation are characterized. Milligram/cm(2) scale linear or power law release profiles can be achieved over 2 to 34 days, and control over loading and release are demonstrated through parameters such as tuning the degradability of the synthetic polycation, changing the number of layers used, or changing the polysaccharide polyanion. Functionality is maintained at nearly 100%, underscoring mild processing conditions apt to preserve fragile protein function. LbL films thus have promise as a tool for exploring protein modulation of the interaction between implanted surfaces and the cells they contact.

Figure 1

Film characterization of (Poly1/heparin/lysozyme/heparin) tetralayers by UV-Vis spectroscopy, profilometry and instant dissolution methods. Percent of value at 80 tetralayers is plotted vs. number of tetralayers to allow comparison of curves. Agreement is seen in the three methods of detecting film building indicating that the films are growing in thickness and incorporating protein.

Figure 2

Total release and release time span are affected by number of tetralayers. 2A) Replicate samples were dipped with the architecture (Poly1/heparin/lysozyme/heparin)n and released at 37°C. Total release in μg/cm² is plotted vs. time in days. Note that both the amount of protein incorporated and the time to total release are increased with increasing numbers of tetralayers, suggesting surface erosion as a mechanism of release. 2B) The signal value for each timepoint was taken as a percentage of the signal at the last timepoint to allow comparison of release kinetics. Percent of total value is plotted vs. time in days. 2C) Fractional release of lysozyme is 85–95%. Films of various tetralayers of (Poly1/heparin/lysozyme/heparin) films were either instantaneously released as described in materials and methods or allowed to elute into PBS at 37°C. Total protein loaded or released protein are plotted versus number of tetralayers.

Figure 3

Poly2 Film Characterization 3A) The signal recorded at each point of construction is taken as a percent of the signal at 50 tetralayers and plotted against the number of (Poly2/heparin/lysozyme/heparin) tetralayers to allow comparison of curves. By comparison of curves, it is possible to see the transition from exponential to linear building regimes. 3B) When Poly2 is layered in the architecture [(Poly2/heparin/lysozyme/heparin)₅₀], and released at 37°C, release of over 34 days is achieved, showing the tunability of this system in response to a designed synthetic polymer. Total release in μg/cm² is plotted vs. time in days.

Figure 4

Chondroitin film characterization. 4A) The signal recorded at each point of construction is taken as a percentage of the signal at 50 tetralayers and is plotted against the number of (Poly1/chondroitin/lysozyme/chondroitin) tetralayers to allow comparison of the curves. It is possible to see agreement of different measurement techniques and an exponential pattern of growth. 4B) Release of films constructed with lysozyme and chondroitin. Total release in μg/cm² is plotted vs. time in days. Note increased loading with increased numbers of tetralayers. 4C) The signal value for each timepoint was taken as a percentage of the signal at the last timepoint to allow comparison of release kinetics between films with different numbers of tetralayers. Percentage of total release is plotted vs. time in days. Results at 12 and 20 tetralayers included large error in measurements corresponding to very low release rates and were therefore omitted from the plot.

Figure 5

The total amount of protein detected using the micro-BCA kit (total protein) was plotted with the total amount of protein detected using the kinetic functional lysozyme assay (functional protein) to elucidate the functionality of the released protein. The amount of total protein released (ug) and functional protein released (ug) is plotted against time in days. This [(Poly1/heparin/lysozyme/heparin)₄₃] film shows nearly 100% maintenance of enzyme activity.

Scheme 1

Structural characteristics of the synthetic polymers used. Note that Poly1 and Poly2 differ only by two methylene units in the backbone, increasing the hydrophobicity of the region next to the ester bond.