MicroRNA (miR) delivery to regulate chronic inflammation hold extraordinary promise, with new therapeutic possibilities emanating from their ability to fine-tune multiple target gene regulation pathways which is an important factor in controlling aberrant inflammatory reactions in complex multifactorial disease. However, several hurdles have prevented advancements in miR-based therapies. These include off-target effects of miRs, limited trafficking, and inefficient delivery. We propose a magnetically guided nanocarrier to transport therapeutically relevant miRs to assist self- resolving inflammation processes at injury sites and reduce the impact of chronic inflammation- related diseases such as tendinopathies. The high prevalence, significant socio-economic burden and increasing recognition of dysregulated immune mediated pathways in tendon disease provide a compelling rationale for exploring inflammation-targeting strategies as novel treatments in this condition. By combining cationic polymers, miR species (e.g., miR 29a, miR155 antagonist), and magnetic nanoparticles in the form of magnetoplexes with highly efficient magnetofection procedures, we developed inexpensive, easy-to-fabricate, and biocompatible systems with competent miR-binding and fast cellular uptake into different types of human cells, namely macrophages and tendon-derived cells. The system was shown to be cell-compatible and to successfully modulate the expression and production of inflammatory markers in tendon cells, with evidence of functional pro-healing changes in immune cell phenotypes. Hence, magnetoplexes represent a simple, safe, and non-viral nanoplatform that enables contactless miR delivery and high- precision control to reprogram cell profiles toward improved pro-regenerative environments.
RES Hub (Norte-01-0145-FEDER-022190).
Chronic inflammatory events have been associated to almost every chronic disease, including cardiovascular-, neurodegenerative- and autoimmune- diseases, cancer, and host-implant rejection. Given the toll of chronic inflammation in healthcare and socioeconomical costs developing strategies to resolve and control chronic states of inflammation remain a priority for the significant benefit of patients. Macrophages (Mφ) hold a central role both in the initiation and resolution of inflammatory events, assuming different functional profiles. The outstanding features of Mφ counting with the easy access to tissues, and the extended networking make Mφ excellent candidates for precision therapy. Moreover, sophisticated macrophage-oriented systems could offer innovative immune-regulatory alternatives to effectively regulate chronic environments that traditional pharmacological agents cannot provide. We propose magnetically assisted systems for balancing Mφ functions at the injury site. This platform combines polymers, inflammatory miRNA antagonists and magnetically responsive nanoparticles to stimulate Mφ functions towards pro-regenerative phenotypes. Strategies with magnetically assisted systems include contactless presentation of immune-modulatory molecules, cell internalization of regulatory agents for functional programming via magnetofection, and multiple payload delivery and release. Overall, Mφ-oriented systems stimulated pro-regenerative functions of Mφ supporting magnetically assisted theranostic nanoplatforms for precision therapies, envisioning safer and more effective control over the distribution of sensitive nanotherapeutics for the treatments of chronical inflammatory conditions.
RES Hub (Norte-01-0145-FEDER-022190).
The aim of the present study was to assess the antibiofilm activity of daptomycin- and vancomycin-loaded poly(methyl methacrylate) (PMMA) and PMMA-Eudragit RL100 (EUD) microparticles against mature biofilms of polysaccharide intercellular adhesin-positive S. epidermidis. The effect of plain, daptomycin- and vancomycin-loaded PMMA and PMMA-EUD microparticles on S. epidermidis biofilms was assessed by isothermal microcalorimetry (IMC) and fluorescence in situ hybridization (FISH). Biofilms were grown for 48h onto poly-urethane pieces of fixed dimensions. Each sample was washed with PBS in order to remove planktonic bacteria and incubated for 24h with different concentrations of acrylic microparticles (20–1.25 mg/mL). The minimal biofilm inhibitory concentration (MBIC) of the antibiotic-loaded particles was defined as the lowest concentration of particles that was able to prevent heat flow associated to the recovery of the biofilms. After incubation with the microparticles, sessile cocci were hybridized with the pan-bacterial EUB338-FITC and the staphylococci-specific STAPHY-FICT probes and stained with DAPI. Biofilm structure and metabolic state were characterized by fluorescence microscopy. According to the IMC results, plain PMMA-particles showed no effect on S. epidermidis biofilms, whereas PMMA-EUD-microparticles negatively influenced the recovery of the biofilm probably due to the highly positive charge of these particles. The MBIC of daptomycin-loaded PMMA-microparticles was 20 mg/mL, whereas vancomycin-loaded PMMA microparticles were not able to inhibit biofilm recovery. Adding EUD to the formulation reduced the MBIC of daptomycin-loaded microparticles to 1.25 mg/mL, corresponding to a 16-fold reduction. Regarding the vancomycin-loaded microparticles, EUD caused a further decrease of their antibiofilm activity. The FISH micrographs corroborated the IMC results and provided additional insights on the antibiofilm effect of these carriers. According to FISH, daptomycin-loaded PMMA-EUD microparticles were responsible for the most pronounced reduction in biofilm mass. In addition, FISH showed that both PMMA and PMMA-EUD microparticles were able to attach to the biofilms. Adding EUD to the formulations proved to be a powerful strategy to improve daptomycin-loaded microparticles antibiofilm activity. In addition, the combination of IMC and FISH was essential in order to fully assess the effect of polymeric microparticles on sessile S. epidermidis. Although the present study enabled gaining further insights on this subject, the nature of these interactions remains unclear. However, this may be a crucial aspect for the enhancement of antibiofilm activity of antibiotic-loaded polymeric microcarriers against mature biofilms. This work was supported by the Portuguese government (Fundação para a Ciência e a Tecnologia) and FEDER (grant SFRH/BD/69260/2010 and research project EXCL/CTM-NAN/0166/2012) and strategic project PEst-OE/SAU/UI4013/2011.
Antibiotic-loaded bone cements are used to decrease occurrence of bone infections in cemented arthroplasties and actually being considered as a more cost-effective procedure when compared to cementless implants [1]. However, considering the challenge of treating device-associated infections there is a reduced number of formulations in the market. Response from the industry to medical need is still slow considering the rapid change in the infecting microbial profile and the emergence of multiresistant strains [2]. In this context, the aim of the work is to evaluate the role of lactose (L), as a porogen, on the antibiotic release from bone cement (BC). Levofloxacin (Lev) and minocycline (M) were the selected antibiotics to be individually loaded into BC due to their low cost and potential application in bone infections [3,4]. Two types of matrices were prepared: 1) Loaded with 2.5% of antibiotics (controls) and 2) Loaded with 10% lactose and 2.5% antibiotic. In vitro drug release and microbiological tests against representative strains causative of bone infections were assessed. Lactose significantly increased the release of both antibiotics. Complete minocycline release after one-week was observed (Fig.1A). Also, lactose increased 3.5-fold the levofloxacin released from BC (Fig.1B). Furthermore, microbiological studies showed that no interaction was observed between lactose and antibiotic as no decrease in drugs antimicrobial activity was observed (Table 1). Considering the results, L-BC matrix appears to be a valuable alternative to available formulations. Future work will include testing other antibiotics as well as mixtures of drugs. Fundação para a Ciência e Tecnologia (Portuguese government) for financial support: EXCL/CTM-NAN/0166/2012 and strategic project PEst-OE/SAU/UI4013/2011.
