Poly(ε-lysine) dendrons as modulators of quorum sensing in Pseudomonas aeruginosa.

The opportunistic pathogen Pseudomonas aeruginosa is a significant contributor to recalcitrant multi-drug resistant infections. In a vigorous search for alternative therapeutic approaches, the communication system used by this bacterium to synchronise the expression of genes involved in pathogenicity has been identified as a potential target. Poly(ε-lysine) dendrons, composed of three branching generations, were examined herein for their anti-virulence potential and ability to disperse within P. a eruginosa biofilms. These hyperbranched macromolecules reduced attachment and biomass production under different nutrient growth conditions, and at concentrations that were not lethal to planktonic cells (0.2, 0.4 and 0.8 mg/mL). Fluorescent labelling revealed the intracellular localisation and cell-penetrating capacity of the dendron, and showed the rapid uptake and release of unexploited dendron from pre-established P. a eruginosa biofilms. Additionally, the dendron induced complete attenuation of LasA protease, a marker of quorum sensing inactivation, by preventing its accumulation in the external environment. This study thus demonstrates the anti-virulence potential of this class of macromolecules, and could represent a novel therapeutic approach for the treatment of antibiotic-resistant P. a eruginosa infections.

Osseointegration of titanium implants functionalised with phosphoserine-tethered poly(epsilon-lysine) dendrons: a comparative study with traditional surface treatments in sheep.

The aim of this study was to analyse the osseointegrative potential of phosphoserine-tethered dendrons when applied as surface functionalisation molecules on titanium implants in a sheep model after 2 and 8 weeks of implantation. Uncoated and dendron-coated implants were implanted in six sheep. Sandblasted and etched (SE) or porous additive manufactured (AM) implants with and without additional dendron functionalisation (SE-PSD; AM-PSD) were placed in the pelvic bone. Three implants per group were examined histologically and six implants were tested biomechanically. After 2 and 8 weeks the bone-to-implant contact (BIC) total values of SE implants (43.7±12.2; 53.3±9.0%) and SE-PSD (46.7±4.5; 61.7±4.9%) as well as AM implants (20.49±5.1; 43.9±9.7%) and AM-PSD implants (19.7±3.5; 48.3±15.6%) showed no statistically significant differences. For SE-PSD and AM-PSD a separate analysis of only the cancellous BIC demonstrated a statistically significant difference after 2 and 8 weeks. Biomechanical findings proved the overall increased stability of the porous implants after 8 weeks. Overall, the great effect of implant macro design on osseointegration was further supported by additional phosphoserine-tethered dendrons for SE and AM implants.

Gadolinium Tagged Osteoprotegerin-Mimicking Peptide: A Novel Magnetic Resonance Imaging Biospecific Contrast Agent for the Inhibition of Osteoclastogenesis and Osteoclast Activity.

The control of osteoblast/osteoclast cross-talk is crucial in the bone remodelling process and provides a target mechanism in the development of drugs for bone metabolic diseases. Osteoprotegerin is a key molecule in this biosignalling pathway as it inhibits osteoclastogenesis and osteoclast activation to prevent run-away bone resorption. This work reports the synthesis of a known osteoprotegerin peptide analogue, YCEIEFCYLIR (OP3-4), and its tagging with a gadolinium chelate, a standard contrast agent for magnetic resonance imaging. The resulting contrast agent allows the simultaneous imaging and treatment of metabolic bone diseases. The gadolinium-tagged peptide was successfully synthesised, showing unaltered magnetic resonance imaging contrast agent properties, a lack of cytotoxicity, and dose-dependent inhibition of osteoclastogenesis in vitro. These findings pave the way toward the development of biospecific and bioactive contrast agents for the early diagnosis, treatment, and follow up of metabolic bone diseases such as osteoporosis and osteosarcoma.

Hyperbranched poly(ϵ-lysine) substrate presenting the laminin sequence YIGSR induces the formation of spheroids in adult bone marrow stem cells.

Unlike the fibroblast-like cells formed upon monolayer culture of human mesenchymal stem cells, the natural stem cell niche of the bone marrow and other types of tissues favours the formation of 3-dimensional (3D) cell clusters. The structuring and biological activity of these clusters are regulated by the contacts established by cells with both the basement membrane and neighbour cells and results in their asymmetric division and the consequent maintenance of both a stem population and a committed progeny. The present work demonstrates the potential of a synthetic substrate to mimic the stem cell niche in vitro. The side amino groups of a linear Poly-L-lysine were modified with hyperbranched poly-(ϵ-lysine) peptides, named as dendrons, tethered with the laminin-mimicking sequence, YIGSR. These dendrons presented the YIGSR sequence at the uppermost molecular branching ensuring a controlled spacing of the bioligand. When used to coat the surface of tissue culture plates in a serum-free in vitro cell culture system, the substrate was able to mimic the most relevant features of the basement membrane of the stem cell niche, i.e. the mesh structure of Collagen Type IV and the availability of laminin bioligands relevant to integrin biorecognition. The substrate biomimetic properties were tested for their ability to support the formation of human bone marrow mesenchymal stem cells (hMSCs) 3D spheroids similar to those observed in the natural stem cell niches and their ability to maintain stem cell pluripotency markers. These features were related to the substrate-specific expression and localisation of (i) cell adhesion receptors (i.e. ß-integrin and N-cadherin), (ii) transcription factors of pluripotency markers and cytoskeleton protein and (iii) regulators of cell migration throughout cell culture passages 2 to 4. The results clearly demonstrate the formation of 3D spheroids starting from the asymmetric division of substrate-adhering spread cells, the clustering of relevant integrins and the expression of specific intracellular pathways controlling cytoskeleton formation suggesting their potential use as a substrate for the handling of stem cells prior to transplantation procedures.