## 5 Coating Materials with Embedded Antibiotics and Other Antibofilm Agents
### Usnic Acid  
Usnic acid (UA) is a secondary metabolite found in lichens with strong antimicrobial activity against _S. epidermidis_, _S. aureus_, _E. faecalis_, _Mycobacterium tuberculosis_, and some pathogenic fungi [46]. In spite of these interesting biological properties, the utilization of this dibenzofuran derivative (Figure 4) was limited by its low water solubility and high hepatotoxicity.  
The aforesaid properties of UA prompted our group to start a series of research studies aiming to develop novel dosage forms of UA with increased biocompatibility and bioavailability and decreased toxicity.  
In a first approach, we developed a novel \(\text{Fe}_{3}\text{O}_{4}\) nano-ferrofluid formulation for the magnetically targeted delivery of UA [47]. Briefly, superparamagnetic \(\text{Fe}_{3}\text{O}_{4}\) nanoparticles stabilized by an oleic acid (OA) shell were prepared by microwave-assisted synthesis, subjecting a mixture of ferrite nanoparticles and OA to a microwave field for 10 min [48]. \(\text{Fe}_{3}\text{O}_{4}\) nanoparticles were prepared by the coprecipitation method following the [PERSON]'s procedure. An extra-shell of the bioactive UA was adsorbed onto the OA-stabilized \(\text{Fe}_{3}\text{O}_{4}\) nanoparticles by adding UA into a chloroform dispersion of the prepared  
Figure 3: Chemical structure of chlorhexiidine.  
Fe\({}_{3}\)O\({}_{4}\)-OA core-shell magnetic nanoparticles and mixing until the complete evaporation of the solvent. The nanoparticles were separated magnetically, air-dried, re-dispersed in chloroform, and the procedure was repeated once more. The antibiofilm activity against _S. aureus_ was assessed by VCC assay and by CLSM visualization of the _S. aureus_ biofilm. Both positive (UA-coated coverslips) and negative (coverslips coated with bare Fe\({}_{3}\)O\({}_{4}\) core-shell nanoparticles) controls were used. Both the qualitative (CLSM) and the quantitative (VCC) assay revealed a great improvement of the antibiofilm activity by incorporation of UA into the nanofluid.  
In another work [49], we used the advanced laser deposition technique MAPLE to easily fabricate thin film coatings of polylactic acid (PLA)-polyvinyl alcohol (PVA) microspheres loaded with UA on Ti substrate. The UA-loaded PLA-PVA microspheres were prepared by the emulsion solvent evaporation method. The best films were obtained working with a laser fluence of 300 mJ/cm\({}^{2}\). The cytotoxicity of the UA-loaded coatings was assessed comparatively with uncoated control substrates using mesenchymal stem cells (MSC) that were stained with fluorescein diacetate (FDA) and subsequently examined by fluorescence microscopy to reveal the potential morphological changes. The outcomes showed that the prepared thin coatings have no adverse effect on the normal morphology and growth of the MSC. VCC assays demonstrated that the newly fabricated UA-loaded coatings inhibit _S. aureus_ biofilms at all stages from initiation to maturation.  
We also succeeded in the MAPLE deposition of thin films consisting of inclusion complexes of UA with \(\gamma\)-cyclodextrintrin (\(\gamma\)-CD) on double-side polished silicon and glass slides [50]. The optimal compromise between the amount and the structural integrity of the laser transferred material was obtained at the laser fluence of 500 mJ/cm\({}^{2}\). The biocompatibility of the \(\gamma\)-CD-UA films comparatively with uncoated controls was assessed in vitro on endothelial cells by fluorescence microscopy using the RED CMTPX dye. No cytotoxic effect was observed, a result that was also confirmed by an additional MTT assay. _S. aureus_ biofilm development on \(\gamma\)-CD-UA-coated silicon and glass materials, and on uncoated controls, respectively, was assessed by the VCC method. The results proved the capacity of the \(\gamma\)-CD-UA coatings to hinder both bacterial attachment and biofilm formation, with the antibiofilm activity being maintained for at least 72 h.