Multilayer films assembled from naturally-derived materials for controlled protein release

Abstract

Herein we designed and characterized films composed of naturally derived materials for controlled release of proteins. Traditional drug delivery strategies rely on synthetic or semisynthetic materials or utilize potentially denaturing assembly conditions that are not optimal for sensitive biologics. Layer-by-layer (LbL) assembly of films uses benign conditions and can generate films with various release mechanisms including hydrolysis-facilitated degradation. These use components such as synthetic polycations that degrade into non-natural products. Herein we report the use of a naturally derived, biocompatible and degradable polyanion, poly(β-l-malic acid), alone and in combination with chitosan in an LbL film, whose degradation products of malic acid and chitosan are both generally recognized as safe (GRAS) by the FDA. We have found that films based on this polyanion have shown sustained release of a model protein, lysozyme that can be timed from tens of minutes to multiple days through different film architectures. We also report the incorporation and release of a clinically used biologic, basic fibroblast growth factor (bFGF), which demonstrates the use of this strategy as a platform for controlled release of various biologics.

Figure 1

Potentiometric titration of PMLA in water at room temperature. The symbols represent measured values of PMLA (inset) and the solid line is a four-parameter fit, giving a measured pK_a of 4.58 ± 0.02.

Figure 2

Film growth of (polycation/PMLA)_n films. Growth curves represent film assembly with polycations of PAH (a), chitosan (b), PLL (c), and LPEI (d).

Figure 3

PLL^Fluor release profile from (PLL^Fluor/PMLA)₂₀ films incubated in PBS, pH 7.4 at 37°C.

Figure 4

Film growth curves (a,c) and lysozyme release profiles in PBS, pH 7.4 at 37°C (b,d) for (lysozyme/PMLA)_n films (a,b) and (chitosan/PMLA/lysozyme/PMLA)_n films (c,d). Release data were from 120 bilayer films (b), and 20 (circles) and 60 (squares) tetralayer films (d).

Figure 5

Release of lysozyme into PBS, pH 7.4 at 37°C from (chitosan/polyanion/lysozyme/polyanion)₂₀ films of 100:0 (red circles), 75:25 (green squares), 50:50 (blue triangles), 25:75 (purple inverted triangles), and 0:100 (black diamonds) PMLA:PAA molar ratios. Symbols represent measured data and solid lines are their first-order fits.

Figure 6

Film characteristics of (chitosan/PMLA/bFGF/PMLA)_n films including its growth curve (a), release profiles in PBS, pH 7.4 at 37°C for 20 (circles) and 60 tetralayers (squares) (b), and proliferative activity of PBS, 10 ng/mL of as-received bFGF and film-released bFGF on NIH3T3 cells (c).