Isabel A.C. Ribeiro

Development of novel sophorolipids with improved cytotoxic activity toward MDA-MB-231 breast cancer cells.

Sophorolipids (SLs) are glycolipid biosurfactants, produced as a mixture of several compounds by some nonpathogenic yeast. In the current study, separation of individual SLs from mixtures with further evaluation of their surface properties and biologic activity on MDA‐MB‐321 breast cancer cell line were investigated. SLs were biosynthesized by Starmerella bombicola in a culture media supplemented with borage oil. A reverse‐phase flash chromatography method with an automated system coupled with a prepacked cartridge was used to separate and purify the main SLs. Compositional analysis of SLs was performed by high‐performance liquid chromatography with electrospray ionization mass spectrometry and tandem mass spectrometry. The following diacetylated lactonic SLs were isolated and purified: C18:0, C18:1, C18:2, and C18:3. The critical micelle concentration (CMC) and surface tension at CMC (γCMC) of the purified SLs showed an increase with the number of double bonds. High cytotoxic effect against MDA‐MB‐231 cells was observed with C18:0 and C18:1 lactonic SLs. The cytotoxic effects of C18:3 lactonic SL on cancerous cells were for the first time studied. This cytotoxic effect was considerably higher than the promoted by acidic SLs; however, it induced a lower effect than the previously mentioned SLs, C18:0 and C18:1. To our knowledge, for the first time, C18:1 lactonic SL, in selected concentrations, proved to be able to inhibit MDA‐MB‐231 cell migration without compromising cell viability and to increase intracellular reactive oxygen species. Copyright

Optimization and correlation of HPLC-ELSD and HPLC-MS/MS methods for identification and characterization of sophorolipids.

High-performance liquid chromatography (HPLC) with evaporative light scattering detection (ELSD) and HPLC with electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) methods were implemented and optimized to separate and identify sophorolipids (SLs) produced by Rhodotorula bogoriensis and Starmerella bombicola. SLs are carbohydrate-based amphiphilic biosurfactants with increased interest in pharmaceutical and environmental areas. Rhodotorula bogoriensis and Starmerella bombicola are mainly producers of respectively C22, and C16 and C18 SLs. Mass fragmentation patterns of SLs produced by both yeasts were investigated by HPLC-ESI-MS/MS in the positive mode for [M+Na]+. Based on the established fragmentation pattern, SLs produced by both yeasts were identified and characterized. A correlation between HPLC-ELSD and HPLC- ESI-MS/MS methods was established and made possible the identification of SLs by the HPLC-ELSD technique. TLC is a common tool for the analysis of SLs mixtures. In this work, TLC scrapped bands were analysed by HPLC-ELSD and HPLC-MS allowing the correlation between R(F) values and the identification of sophorolipids by this technique. Identification of monoacetylated and diacetylated C24:0 hydroxy fatty acids sophorolipids produced by Rhodotorula bogoriensis was for the first time accomplished with this study. Although present in lower quantity these longer chain SLs can assume special importance regarding their biological activity and surface active properties.

Novel doped calcium phosphate-PMMA bone cement composites as levofloxacin delivery systems.

Antibiotic-loaded acrylic bone cements (ALABCs) are well-established and cost-effective materials to control the occurrence of bone and joint infections. However, the inexistence of alternative antibiotics other than those already commercially available and the poor ability to bind to bone tissue hampering its biological function are still major drawbacks of ALABCs clinical application. The concept of this research work is to develop a novel bone cement (BC) drug delivery system composed by Mg- and Sr-doped calcium phosphate (CaP) particles as drug carriers loaded into a lactose-modified acrylic BC, which, to the best of our knowledge, has never been reported. CaP particles are known to promote bone ingrowth and current research is focused on using these carriers as antibiotic delivery systems for the treatment of bone infections, like osteomyelitis. Levofloxacin is a fluoroquinolone with anti-staphylococcal activity and adequate penetration into osteoarticular tissues and increasingly being recommended to manage bone-related infections. Also, the lactose-modified BC matrix, with a more porous structure, has already proved to enhance antibiotic release from the BC inner matrix. This novel BC composite biomaterial has shown improved mechanical integrity, biocompatibility maintenance, and sustained release of levofloxacin, with concentrations over the minimum inhibitory concentration values after a 48h while maintaining antibacterial activity over an 8-week period against Staphyloccocus aureus and Staphyloccocus epidermidis, common pathogens associated with bone infections.

Design of selective production of sophorolipids by Rhodotorula bogoriensis through nutritional requirements

Rhodotorula bogoriensis is known as the producer of longer chain acidic sophorolipids (SLs) with a unique hydroxylation position where the sophorose unit is linked to the 13‐hydroxydocosanoic acid. The influence of initial inoculum concentration, hydrophilic and hydrophobic carbon, and nitrogen sources on R. bogoriensis growth and SL production was evaluated to obtain a selective SL production. Experiments took place in microtiter plates, used as minireactors, after the verification of its suitability compared with shake flasks.

Key-properties outlook of a levofloxacin-loaded acrylic bone cement with improved antibiotic delivery.

Antibiotic-loaded acrylic bone cements (ALABCs) are widely used to decrease the occurrence of bone infections in cemented arthroplasties and actually being considered as a more cost-effective procedure when compared to cementless implants. However, ALABCs have a major drawback, which is the incomplete release of the antibiotics and, as a result, pathogens that commonly are responsible for those infections are becoming resistant. Consequently, it is of most relevance to find new antibacterial agents to load into BC with an effective mechanism against those microorganisms. This research work intended to load levofloxacin, a fluoroquinolone with anti-staphylococcal activity and adequate penetration into osteoarticular tissues, on lactose-modified commercial bone cement (BC). This modified BC matrix exhibited increased levofloxacin release and delayed Staphylococcus aureus biofilm formation. Further insights on material-drug interaction during BC setting were investigated by density functional theory calculations. The obtained results suggested that favorable covalent and non-covalent interactions could be established between levofloxacin and the BC. Moreover, BC mechanical and biocompatibility properties were maintained. These features justify the potential of levofloxacin-loaded modified-BC as a valuable approach for local antibiotic delivery in bone infections management.

Can Sophorolipids prevent biofilm formation on silicone catheter tubes

Given the impact of biofilms in health care environment and the increasing antibiotic resistance and/or tolerance, new strategies for preventing that occurrence in medical devices are obligatory. Thus, biomaterials surface functionalization with active compounds can be a valuable approach. In the present study the ability of the biosurfactants sophorolipids to prevent biofilms formation on silicone rubber aimed for medical catheters was investigated. Sophorolipids produced by Starmerella bombicola, identified by HPLC-MS/MS were used to cover silicone and surface characterization was evaluated through contact angle measurements and FTIR-ATR. Results revealed that sophorolipids presence on silicone surface decreased the hydrophobicity of the material and biofilm formation of Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 25922. Antibiofilm activity was evaluated through different methods and was more pronounced against S. aureus. Furthermore, biocompatibility of silicone specimens with HaCaT cells was also obtained. From this study it was possible to conclude that sophorolipids seem to be a favourable approach for coating silicone catheters. Such compounds may represent a novel source of antibiofilm agents for technological development passing through strategies of permanent functionalization of surfaces.

Levofloxacin-loaded bone cement delivery system : highly effective against intracellular bacteria and Staphylococcus aureus biofilms

Staphylococcus aureus is a major pathogen in bone associated infections due to its ability to adhere and form biofilms on bone and/or implants. Moreover, recrudescent and chronic infections have been associated with S. aureus capacity to invade and persist within osteoblast cells. With the growing need of novel therapeutic tools, this research aimed to evaluate some important key biological properties of a novel carrier system composed of acrylic bone cement (polymethylmethacrylate - PMMA), loaded with a release modulator (lactose) and an antibiotic (levofloxacin). Levofloxacin-loaded bone cement (BC) exhibited antimicrobial effects against planktonic and biofilm forms of S. aureus (evaluated by a flow chamber system). Moreover, novel BC formulation showed high anti-bacterial intraosteoblast activity. This fact led to the conclusion that levofloxacin released from BC matrices could penetrate the cell membrane of osteoblasts and be active against S. aureus strains in the intracellular environment. Furthermore, levofloxacin-BC formulations showed no significant in vitro cytotoxicity and no allergic potential (measured by the in vivo chorioallantoic membrane assay). Our results indicate that levofloxacin-loaded BC has potential as a local antibiotic delivery system for treating S. aureus associated bone infections.

Novel Antibacterial Agents: An Emergent Need to Win the Battle Against Infections

BACKGROUND Multiple strategies have been recommended for prevention and control of antibacterial resistance. Solutions will need to be found soon if we are not to run the serious risk of losing the ability to treat bacterial infections, especially the ones arising from multi-resistant strains. Deep knowledge of the resistance mechanisms followed by novel therapeutic drugs and vaccines are needed. A consolidated, multidisciplinary and regulated strategy is required by this challenge. OBJECTIVE This review will be focused on new strategies to control infections. Among strategies to tackle antibiotic resistance that have been under investigation, are the use of antimicrobial peptides, phage therapy and phage enzymes, therapeutic antibodies, quorum sensing inhibitors and, finally, the antibacterial nanomedicines. Although all of the approaches seem to be effective, and at least one of them has been in use for relatively a long time (phage therapy), antibacterial nanomedicines show the most diverse range of different approaches regarding potential translation to clinics. RESULTS & CONCLUSION Several advances have been made but a great effort is still mandatory in order to reach feasible, effective and marketable novel antimicrobial products.

Biopolymeric coatings for delivery of antibiotic and controlled degradation of bioresorbable Mg AZ31 alloys

ABSTRACT Magnesium and magnesium alloys are attracting considerable interest as biodegradable materials with high potential for application as temporary implants. The high corrosion rate of Mg-based implants is considered a serious drawback, and it is crucial to design novel surface protection strategies that minimize the detrimental effects of corrosion, while contributing for introducing additional functionalities on the material surface. In this work, a layer-by-layer coating architecture composed of an inner poly(lactic-co-glycolic) acid layer, working as adhesion promoter, and additional polycaprolactone (PCL) layers working as reservoirs for antibiotic (levofloxacin) and for nanohydroxyapatite (nanoHA) particles was applied on the Mg alloy AZ31. The results demonstrate that the composition and number of PCL layers can tailor the biodegradation of the bare magnesium alloy, surface wettability, and the kinetics of release of antibiotic (levofloxacin). The distribution of nanoHA in the coating architecture plays a crucial role on tailoring the desired biocompatible functionalities and corrosion protection of the bare alloy. GRAPHICAL ABSTRACT

Novel sintering-free scaffolds obtained by additive manufacturing for concurrent bone regeneration and drug delivery: Proof of concept

Advances on the fabrication of sintering-free biphasic calcium phosphate (BCP)/natural polymer composite scaffolds using robocasting as additive manufacturing technique are presented in the present work. Inks with high amounts of BCP powders (45 vol%) containing different HA/β-TCP ratios, in presence of crosslinked polymer, were successfully fine-tuned for extrusion by robocasting. The non-existence of sintering step opened the possibility to obtain drug loaded scaffolds by adding levofloxacin to the extrudable inks. The drug presence induced slightly changes on the rheological behaviour of the inks, more emphasized for the BCP compositions with higher amounts of β-TCP, and consequently, on the microstructure and on the mechanical properties of the final scaffolds. The strong interaction of β-TCP with chitosan difficult the preparation of suitable rheological inks for printing. Drug delivery studies revealed a fast release of levofloxacin with a high burst of drug within the first 30 min. Levofloxacin loaded samples also presented bacteria growth inhibition ability, proving that antibiotic was not degraded during the fabrication process and its bactericidal efficacy was preserved. From the results obtained, the composite scaffolds containing higher amounts of HA (around 80% HA/20% β-TCP) constitute a promising bi-functional synthetic bone substitute for simultaneous local bone regeneration and infection treatments.