Dual functional polyelectrolyte multilayer coatings for implants: permanent microbicidal base with controlled release of therapeutic agents

Abstract

Here we present a new bifunctional layer-by-layer (LbL) construct made by combining a permanent microbicidal polyelectrolyte multilayered (PEM) base film with a hydrolytically degradable PEM top film that offers controlled and localized delivery of therapeutics. Two degradable film architectures are presented: (1) bolus release of an antibiotic (gentamicin) to eradicate initial infection at the implant site, or (2) sustained delivery of an anti-inflammatory drug (diclofenac) to cope with inflammation at the site of implantation due to tissue injury. Each degradable film was built on top of a permanent base film that imparts the implantable device surface with microbicidal functionality that prevents the formation of biofilms. Controlled-delivery of gentamicin was demonstrated over hours and that of diclofenac over days. Both drugs retained their efficacy upon release. The permanent microbicidal base film was biocompatible with A549 epithelial cancer cells and MC3T3-E1 osteoprogenitor cells, while also preventing bacteria attachment from turbid media for the entire duration of the two weeks studied. The microbicidal base film retains its functionality after the biodegradable films have completely degraded. The versatility of these PEM films and their ability to prevent biofilm formation make them attractive as coatings for implantable devices.

Figure 1

A) Structure of hydrolytically degradable poly(β-amino ester)s, Poly1 and Poly2. B) Structure of microbicidal linear N,N-dodecyl,methyl-PEI (DMLPEI). C) Poly(acrylic acid) (PAA). D) Schematic of poly(β-cyclodextrin) with drug sequestered in the interior of its monomer unit, as well as a close-up structure of a monomeric β-cyclodextrin. E) Structure of diclofenac

Figure 2

Schematic representation of the combination films in this work. A) Gentamicin releasing (Poly1/PAA/GS/PAA)_n, and B) diclofenac releasing (Poly2/PolyCD-DIC)_n combination films, built on top of the microbicidal (DMLPEI/PAA)₁₀.

Figure 3

A, C, and E are figures for diclofenac releasing films, while B, D, and F are for gentamicin releasing films. Thickness and roughness of A) (Poly2/PolyCD-DIC)_n and B) (Poly1/PAA/GS/PAA)_n films (note difference in y axis scale); degradation curves for C) (Poly2/PolyCD-DIC)₂₀ and D) (Poly1/PAA/GS/PAA)₂₀ films (note difference in x axis scale); and drug release curves for E) (Poly2/PolyCD-DIC)₂₀ and F) (Poly1/PAA/GS/PAA)₂₀ films (note difference in x and y axis scales). All films were made on top of base film (DMLPEI/PAA)₁₀.

Figure 4

Percentage of COX enzyme inhibition showing that diclofenac released from (DMLPEI/PAA)₁₀(Poly2/PolyCD-DIC)₁₀ is still active. The released samples from day 1 to day 9 represent non-cumulative drug released from the film.

Figure 5

Kirby-Bauer assays of gentamicin (GS) releasing films eroded for various time periods, ranging from 0 min (as built) to 4 days; Row 1 represents GS films built on (LPEI/SPS)₁₀ base films and Row 2 those built on microbicidal (DMLPEI/PAA)₁₀ base films. A shows decrease in the size of zone of inhibition as time increases. B shows films eroded for 4 days and tested with GS-resistant bacteria to confirm the microbicidal base film functionality; the result shows that the microbicidal (DMLPEI/PAA)₁₀ base film (bottom right sample) is effective in killing the bacteria, while the (LPEI/SPS)₁₀ base film (top right sample) is not. C) Similar results were obtained for (Poly2/PolyCD-DIC)₂₀(DMLPEI/PAA)₁₀ films that had been allowed to undergo complete drug release before testing, showing that microbicidal base film remains active. Note that the dark (black) colored substrate surfaces are bacteria-free, while the lighter beige colored substrate surfaces correspond to contamination by bacteria colonies (each dot corresponds to a colony forming unit).

Figure 6

Media-borne assay with S. aureus with increasing time of incubation in bacterial solution; top row shows bare substrates completely colonized by bacteria (light beige colored dots); bottom row shows (DMLPEI/PAA)₁₀ films with degradable top films completely eroded with no sign of colonization by bacteria (black colored substrate).

Figure 7

Kirby-Bauer assay with S. aureus comparing bare substrate and (DMLPEI/PAA)₁₀ film after incubation in blood plasma for 1 h. The bare substrate shows complete colonization by bacteria (beige colored dots), while film-coated substrate remains uncolonized (black colored substrate).

Figure 8

Cell viability of films relative to bare glass substrates indicating no apparent cytotoxicity of the films. Cells were grown in media with or without serum. Note that the difference in cell viability shown is not statistically significant (t-test p values of 0.36 and 0.84 for data with serum and without serum respectively)

Figure 9

Proliferation (day 1, 3, and 7) of MC3T3-E1 cells on A) bare glass substrates and B) (DMLPEI/PAA)₁₀ films; proliferation of A549 cells on C) bare glass substrates, D) (DMLPEI/PAA)₁₀.