Hybrid organic/metal nanoparticles for photothermal treatment of chronic wounds

Chronic wounds (CWs) are a pathological condition in which an injured tissue fails to restore its anatomic and functional integrity. The normal wound healing process can be affected by several factors, including imbalance of reactive oxygen species, up-regulation of metalloproteinases (MMPs), bacterial infections, and bacteria biofilms, the latter representing a substantial obstacle to wound healing. This thesis aimed at developing lignin (Lig) cobalt-phthalocyanine (CoPc) nanoparticles (NPs) able to restore healing in infected chronic wounds. Lig was conjugated with tannic acid to achieve antioxidant and MMP inhibition properties. CoPc, a complex combining photo-excitable Pc with antimicrobial Co, was selected to provide antibacterial activity and biofilm eradication through photothermal action. Different NPs formulations with varying amounts of CoPc and Lig were synthesized and characterized. UV–vis spectroscopy and Fourier-transform infrared (FTIR) spectroscopy demonstrated the presence of Lig and CoPc in NPs. All NPs had similar size and ζ-potential. DPPH (2,2-Diphenyl-1-picrylhydrazyl) assay ascertained NPs antioxidant properties. NPs obtained with 17.5 mg of Lig and 12 mg of CoPc demonstrated the best homogeneity in terms of size and polydispersity index. Co content of 0.016 mg per mg of NPs was quantified via Inductively Coupled Plasma Mass Spectrometry (ICP). Viability tests on keratinocytes and fibroblasts demonstrated NPs cytocompatibility. TEM images of the NPs showed the particles had irregular rounded morphology, and elemental analysis confirmed the presence of Co. Colony forming unit test showed that NPs inhibited the growth of Staphylococcus aureus at 4 mg/ml concentration. At this concentration, NPs were cytocompatible to fibroblasts and keratinocytes. NPs internalization in S. aureus and Pseudomonas aeruginosa biofilms was observed through Scanning Electron Microscopy coupled with Energy Dispersive X-Ray Analysis. The photothermal properties of the free NPs and of NPs internalized in S. aureus biofilms were studied. When exposed to 660nm laser irradiation for 2 min, the temperature in the biofilm increased by 7°C. Furthermore, when irradiated at 715nm, NPs produced a stable photoacoustic signal over time. To facilitate NPs delivery to wounds, an injectable supra-molecular, UV-cross-linkable hydrogel was used to embed the NPs with the aim of retaining them at the injection site and of controlling their release. Stability tests showed that the hydrogel could maintain its shape in the first 48h and then progressively lost integrity over 7 days. Hydrogels also showed an antioxidant effect and were able to sustain NPs release between 48h and 96h. Overall, the CoPcLig NPs showed promising features for the treatment of CWs, by virtue of their synergistic antioxidant, antibacterial, and photothermal properties. Photoacoustic activity can be also exploited for imaging application, an interesting aspect still poorly investigated. Moreover, NPs could be embedded in injectable hydrogels to achieve a localized delivery and a more controlled release in the wound site. Future tests should be aimed at investigating the therapeutic potential of these platforms and their ability to induce tissue regeneration.