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Poly(DL-lactic acid) scaffolds as a bone targeting platform for the co-delivery of antimicrobial agents against S. aureus-C.albicans mixed biofilms

Publication . Zegre, M.; Barros, J.; Ribeiro, Isabel A. C.; Santos, C; Caetano, L. A.; Gonçalves, Lídia; Monteiro, F. J.; Ferraz, M. P.; Bettencourt, Ana

2022-06Journal article

Open access

Novel particulate antibiotic-loaded platforms as sustained drug delivery systems for bone infection treatment

Publication . Ferreira, Inês da Fonseca Santos, 1985-1; Almeida, António José Leitão das Neves, 1963-; Bettencourt, Ana Francisca Campos Simão; Trampuz, Andrej

The most common infecting microorganisms in bone infections are staphylococci, namely Staphylococcus aureus and Staphylococcus epidermidis. Conventionally, complicated bone infections caused by Gram-positive bacteria are treated with vancomycin. However, emergence of resistant staphylococci to vancomycin led to the increased use of daptomycin, which is bactericidal against resistant staphylococci. However, in severe bone infections, daptomycin efficacy is often limited, due to insufficient drug bioavailability at the infected site and biofilm tolerance; hence novel approaches are needed to enhance daptomycin antibiofilm effect. Over the last decades, polymeric nano-/microparticles have emerged as a worthy strategy to enhance the antibiofilm effect of clinically available antibiotics. In this context, daptomycin was encapsulated into polymeric microparticles composed by poly(methyl methacrylate (PMMA), PMMA-Eudragit RL 100 (EUD) and poly-caprolactone (PCL). Vancomycin-loaded microparticles were also prepared as controls. All particles were obtained by an optimised double emulsion-solvent evaporation method and subsequently freeze-died. The final particles presented a spherical morphology within the micrometre size range, high drug encapsulation and yield of preparation. Additionally, the effect of the microparticles on cell viability of ISO-compliant cells, fibroblasts, and osteoblasts was tested. Although some formulations induced a slight decrease in cell viability, none of them was considered cytotoxic. Bearing in mind the main objective of this work, daptomycin-loaded PMMA-EUD and PCL microparticles presented the highest in vitro drug release, with concentrations above the minimal inhibitory and bactericidal concentration of the tested strains. Nevertheless, the antibacterial activity of all formulations was assessed against planktonic and sessile clinically relevant staphylococci by isothermal microcalorimetry (IMC). Further characterization of microparticles-biofilm interaction, as well as assessment of their effect on biofilm size, structure and metabolic state, was performed by fluorescence in situ hybridization (FISH). Daptomycin-loaded PMMA-EUD and PCL microparticles proved to be the most effective against the tested strains with high antibiofilm activity. Finally, the microencapsulation of daptomycin into polymeric carriers proved to be an advantageous approach, thus making them potential candidates as sustained drug delivery systems for bone infections treatment. In addition, the innovative combination of IMC with FISH was essential in order to gain further insights on the antibiofilm effects of microparticulate systems, as well as on their interaction with sessile bacteria.

2015Doctoral thesis

Open access

Investigation of new formulations of acrylic bone cement containing antibiotics

Publication . Matos, Ana Cristina Mendes Correia de, 1968-; Bettencourt, Ana Francisca de Campos Simão; Almeida, António José Leitão das Neves, 1963-; Vaz, Mário Augusto Pires

Antibiotic-loaded bone cement (ALABC), is the common designation for polymethylmethacrylate bone cement (BC), used as drug-delivery system to prevent or to treat bone related-infections. Although presenting some disadvantages, the use of ALABC is still considered the standard of care for patients with chronic bone and joint infection, providing local delivery of high levels of antibiotics for an extended period without exceeding systemic toxicity, while being a more costeffective procedure when compared to cementless implants. Described and reported ALABCs drawbacks include the inadequate release of the loaded antibiotic, the lack of bioactivity and the poor diversity of antibiotics available in commercial premixed formulations. The base-concept of this study was to develop a novel ALABC with improved antibiotic release through the inclusion of particulate drug delivery systems and a release modulator without hampering the antibacterial activity of antibiotics or the BC mechanical and biocompatibility properties. Levofloxacin, a 3rd generation fluoroquinolone, and minocycline, a tetracycline, were the elected antibiotics to load into BC. The rationale behind this choice was related to their adequate microbiological and physicochemical characteristics. Both antibiotics present a broad-spectrum of activity against the main organisms responsible for bone and joint infections, namely Staphylococcus spp. Physicochemically, both are molecules with amphiphilic characteristics - greater for levofloxacin; soluble in acidic aqueous media; with high melting points (over 200ºC) and available in powder form; the latter two characteristics being restrictive when choosing for antibiotics to load into BC. Two main strategies were explored for the inclusion of antibiotics into particulate systems previous to incorporation into BC: 1) by encapsulation into PMMA; 2) by adsorption into calciumphosphate particles (CaPs). To improve drug release from the matrix, a pharmaceutical excipient was used as release modulator, lactose, and loaded into the BC powder component. A step-by-step approach was pursued: 1st. Assessment of antibiotics encapsulation into PMMA particles and of antibiotics in vitro release followed by loading antibiotic-loaded-particles into BC; The PMMA biopolymer was chosen to prepare PMMA-particles (PMMAp) foreseeing a mechanical reinforcement of the final ALABC matrix, because PMMA is the base-polymer of both systems. Plain, levofloxacin- and minocycline-loaded particles were successfully prepared using the double-emulsion solvent evaporation method. Since only minocycline-PMMAp registered an interesting in vitro release profile, studies proceeded with these particles and 15% (wparticles/wBC) were loaded into BC, which, on the other hand, hindered BC setting. 2nd. Effect of the inclusion of lactose into BC, monitoring antibiotics in vitro release, quasi-static mechanical properties and biocompatibility of the resultant matrices; Each powdered antibiotic was directly loaded into BC, and lactose, was added to each formulation. The amount of antibiotic loaded corresponded to the low-dose currently used in commercial ALABCs formulations - 2.5% (w/wBC) - in order to provide an effective antimicrobial activity and preserve the mechanical properties. As to lactose, 10% (wL/wBC) resulted in the optimised amount to be loaded into BC. This lactose-modified BC matrix allowed total release of the minocycline after a one-week period, and a 3.5-fold increase of levofloxacin release compared to control without lactose, over a 7-week period. 3rd. Inclusion of levofloxacin-adsorbed doped CaPs into BC and monitoring of the antibiotics in vitro release, quasi-static mechanical properties and biocompatibility of the resultant matrices; Intending to improve antibiotic release and bioactivity calcium-phosphate particles (CaPs) were tested as drug delivery system. Mg- and Sr-doped CaPs were prepared as levofloxacin carriers and were loaded into the 10% (wL/wBC) lactose-modified acrylic BC at 2.5% (wCaPs/wBC). This novel BC composite revealed a sustained release of levofloxacin over an 8-week period, with concentrations over the Staphylococcus spp. minimum inhibitory concentration values after 48 h. The novel 10% (wL/wBC) lactose-loaded ALABC, independently of the antibiotic or CaPs loaded, followed the same release mechanistic based on dissolution and subsequent diffusion of the antibiotic from the matrix. Both minocycline and levofloxacin maintained antibacterial activity against the Staphylococcus spp. after being released from ALABC matrix. Though this result suggest that polymerization setting did not affect these antibiotics, a novel in silico approach revealed the existence of covalent and non-covalent interactions between the levofloxacin and the BC matrix. Evaluation of the antibiotic-lactose-modified BC matrices regarding the quasi-static mechanical properties according to standard ISO 5833, clearly demonstrated that the mechanical performance was not compromised. Biocompatibility was also successfully evaluated following standard ISO 10993-5 with fibroblasts and osteoblasts cell lines incubated with extracts or in direct contact with BC composites, respectively. Results have shown that neither lactose nor the loaded antibiotics compromised the biocompatibility of the BC. All considered, these features justify the potential of lactose-loaded BC as a valuable step forward on the development of novel BC composites, namely with lactose, as release modulator, and doped CaP particles, as antibiotic carriers, for the control of bone and joint infections.

2015Doctoral thesis

Open access

Acrylic microparticles increase daptomycin intracellular and in vivo anti-biofilm activity against Staphylococcus aureus

Publication . Woischnig, Anne Kathrin; Gonçalves, Lidia M.; Ferreira, Maxime; Kuehl, Richard; Kikhney, Judith; Moter, Annette; Ribeiro, Isabel A. C.; Almeida, António J.; Khanna, Nina; Bettencourt, Ana Francisca

Daptomycin (DAP) is a cyclic lipopeptide antibiotic with potential clinical application in orthopedic infections caused by staphylococci. However, it failed to eradicate Staphylococcus aureus in vitro, in intracellular infection studies, as well as in vivo in an experimental model of implant-associated biofilm infections. In this study, the antimicrobial effect of DAP encapsulated in poly(methyl methacrylate)-Eudragit (PMMA-EUD) microparticles (DAP-MPs) on intracellular S. aureus was evaluated in human osteoblast cells using fluorescence in situ hybridization (FISH) analysis. Encapsulated DAP was able to reduce the amount of intracellular S. aureus by 73% compared to blank microparticles (MPs). Then, the advantage of treating with DAP-MPs versus free DAP was evaluated in a murine model of implant-associated biofilm infection. Free DAP showed a >3 log10 decrease in planktonic and adherent bacteria but failed to eradicate adherent methicillin-resistant S. aureus (MRSA), whereas DAP-MPs showed a clearance of planktonic MRSA, significantly reduced adherent MRSA by more than 3 log10 and cured the infection in 60%. This was linked to the prolonged higher DAP concentration within the tissue cage fluid compared to free DAP. To our knowledge, this study provides the first evidence for the high intracellular and in vivo anti-biofilm efficacy of DAP-MPs to target staphylococcal infections.

2018Journal article

Restricted access

Engineering a multifunctional 3D-printed PLA-collagen-minocycline-nanoHydroxyapatite scaffold with combined antimicrobial and osteogenic effects for bone regeneration

Publication . Martin, Victor; Ribeiro, Isabel A. C.; Alves, Marta M.; Gonçalves, Lídia; Claudio, Ricardo A.; Grenho, Liliana; Fernandes, Maria H.; Gomes, Pedro; Santos, Catarina F.; Bettencourt, Ana

3D-printing and additive manufacturing can be powerful techniques to design customized structures and produce synthetic bone grafts with multifunctional effects suitable for bone repair. In our work we aimed the development of novel multifunctionalized 3D printed poly(lactic acid) (PLA) scaffolds with bioinspired surface coatings able to reduce bacterial biofilm formation while favoring human bone marrow-derived mesenchymal stem cells (hMSCs) activity. For that purpose, 3D printing was used to prepare PLA scaffolds that were further multifunctionalized with collagen (Col), minocycline (MH) and bioinspired citrate- hydroxyapatite nanoparticles (cHA). PLA-Col-MH-cHA scaffolds provide a closer structural support approximation to native bone architecture with uniform macroporous, adequate wettability and an excellent compressive strength. The addition of MH resulted in an adequate antibiotic release profile that by being compatible with local drug delivery therapy was translated into antibacterial activities against Staphylococcus aureus, a main pathogen associated to bone-related infections. Subsequently, the hMSCs response to these scaffolds revealed that the incorporation of cHA significantly stimulated the adhesion, proliferation and osteogenesis-related gene expression (RUNX2, OCN and OPN) of hMSCs. Furthermore, the association of a bioinspired material (cHA) with the antibiotic MH resulted in a combined effect of an enhanced osteogenic activity. These findings, together with the antibiofilm activity depicted strengthen the appropriateness of this 3D-printed PLA-Col-MH-cHA scaffold for future use in bone repair. By targeting bone repair while mitigating the typical infections associated to bone implants, our 3D scaffolds deliver an integrated strategy with the combined effects further envisaging an increase in the success rate of bone-implanted devices.

2019Journal article

Restricted access