Real-time in vivo monitoring of the antimicrobial action of combination therapies in the management of infected topical wounds.

The increasing prevalence of non-healing infected wounds has become a serious concern in the clinical practice, being associated to population aging and to the rising prevalence of several chronic conditions such as diabetes. Herein, the evaluation of the bactericidal and antibiofilm effects of the natural antiseptic terpenes thymol and farnesol standing alone or in combination with the standard care antiseptic chlorhexidine was carried out both in vitro and in vivo. The in vitro combinatorial treatment of chlorhexidine associated with those terpenes against Staphylococcus aureus in its planktonic and sessile forms demonstrated a superior antibacterial activity than that of chlorhexidine alone. Real-time in vivo monitoring of infection progression and antimicrobial treatment outcomes were evaluated using the bioluminescent S. aureus strain Xen36. In vivo studies on infected wound splinting murine models corroborated the superior bactericidal effects of the combinatorial treatments here proposed. Moreover, the encapsulation of thymol in electrospun Eudragit® S100 (i.e., a synthetic anionic copolymer of methacrylic acid and ethyl acrylate)-based wound dressings was also carried out in order to design efficient antimicrobial wound dressings.

Iron inactivation by Sporobolomyces ruberrimus and its potential role in plant metal stress protection. An in vitro study.

The endophytic Basidiomycete Sporobolomyces ruberrimus protects its host Arabidopsis arenosa against metal toxicity. Plants inoculated with the fungus yielded more biomass and exhibited significantly fewer stress symptoms in medium mimicking mine dump conditions (medium supplemented with excess of Fe, Zn and Cd). Aside from fine-tuning plant metal homeostasis, the fungus was capable of precipitating Fe in the medium, most likely limiting host exposure to metal toxicity. The precipitated residue was identified by Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), X-Ray Diffraction (XRD) and electron microscopy (SEM/TEM) with energy dispersive X-Ray analysis (EDX/SAED) techniques. The performed analyses revealed that the fungus transforms iron into amorphous (oxy)hydroxides and phosphates and immobilizes them in the form of a precipitate changing Fe behaviour in the MSR medium. Moreover, the complexation of free Fe ions by fungi could be obtained by biomolecules such as lipids, proteins, or biosynthesized redox-active molecules.

Transfer of photothermal nanoparticles using stem cell derived small extracellular vesicles for in vivo treatment of primary and multinodular tumours.

The main current challenges in oncology are (1) avoiding systemic side effects in therapy, and (2) developing alternative treatment strategies for metastatic tumours. Nanomedicine was assumed to provide answers to these issues, but delivering enough therapeutic nanoparticles (NPs) to tumours still remains a huge challenge in nanomaterials-based treatments. Extracellular vesicles (EVs) play a key role in cell communication processes and can be combined with nanomaterials to improve their targeting capabilities. In this work, we leverage the ability of EVs derived from stem cells to reach tumour areas successfully, being used as delivery vehicles for nanoparticles acting as hyperthermia agents. Once small extracellular vesicles (sEVs) loaded with NIR-sensitive hollow gold NPs reached primary subcutaneous solid tumours, they were irradiated with a NIR laser and almost complete tumour remission was obtained. More interestingly, those sEV vehicles were also able to reach multinodular areas similar to those on advanced metastatic phases, eradicating most tumour growth regions in multiple cancerous nodules located in the pancreas region.

Chalcogenide nanoparticles and organic photosensitizers for synergetic antimicrobial photodynamic therapy.

Synergistic antimicrobial effects were observed for copper sulfide (CuS) nanoparticles together with indocyanine green (ICG) in the elimination of wild type pathogenic bacteria (Staphylococcus aureus ATCC 29213 and Pseudomonas aeruginosa ATCC 27853) and also opportunistic fungal infective yeast (Candida albicans ATCC 10231). Furthermore, large antibacterial effects were observed for clinical isolates of Methicillin-resistant S. aureus (MRSA) PFGE strain-type USA300. This efficient antimicrobial action was attributed to the combined extra- and intracellular generation of reactive oxygen species upon light irradiation. Instead of the use of visible-light for the activation of common photosensitizers, both ICG and CuS nanoparticles can be activated in the near infrared (NIR)-region of the electromagnetic spectrum and therefore, superior tissue penetration would be expected in a potential elimination of pathogenic microorganisms not only on the skin but also in the soft tissue. In the different bacteria studied a 3-log reduction in the bacterial counts was achieved after only 6 min of NIR irradiation and treatment with ICG or CuS alone at concentrations of 40 and 160 µg mL-1, respectively. A maximum bactericidal effect against S. aureus and USA300 strains was obtained for the combination of both photosensitizers at the same concentration. Regarding P. aeruginosa, a 4-log reduction in the CFU was observed for the combination of CuS and ICG at various concentrations. In Candida albicans the combination of both ICG and CuS and light irradiation showed an antimicrobial dose-dependent effect with the reduction of at least 3-log in the cell counts for the combination of ICG + CuS at reduced concentrations. The observed antimicrobial effect was solely attributed to a photodynamic effect and any photothermal effect was avoided to discard any potential thermal injury in a potential clinical application. The generation of reactive oxygen species upon near infrared-light irradiation for those photosensitizers used was measured either alone or in combination. The cytocompatibility of the proposed materials at the doses used in photodynamic therapy was also demonstrated in human dermal fibroblasts and keratinocytes by cell culturing and flow cytometry studies.

Towards plant-mediated chemistry - Au nanoparticles obtained using aqueous extract of Rosa damascena and their biological activity in vitro.

An eco-friendly, efficient, and controlled synthesis of gold nanoparticles with application of the aqueous extract of Rosa damascena (Au@RD NPs) without using any other reducing agents was studied. Au@RD NPs of narrow size distribution were characterized by UV-vis and FT-IR spectroscopies, transmission electron microscopy, X-ray powder diffraction, X-ray photoelectron spectroscopy, particle size analysis, and zeta potential measurements. In vitro stability experiments revealed that the Au@RD NPs were stable for over a year (pH ~ 3.5), proving a significant stabilizing potential of the aqueous RD extract. The high total content of polyphenols, flavonoids, and reducing sugars along with the powerful antioxidant activity of the RD extract was determined by spectroscopic and analytical methods. Colloids prepared from the purified and lyophilized Au@RD NPs (electrokinetic potential of ca. -33 mV) were stable for at least 24 h under terms similar to physiological conditions (pH = 7.4, PBS). The in vitro cytotoxicity of Au@RD NPs was investigated against peripheral blood mononuclear lymphocytes (PBML), acute promyelocytic leukemia (HL60), and human lung adenocarcinoma (A549). Selective cytotoxicity of Au@RD NPs towards cancer cells (HL60, A549) over normal cells (PBML) in vitro was explicitly demonstrated by viability assays. Comet assay revealed a higher level of DNA damages in cancer cells when compared with normal ones. Apoptotic death in cancer cells was proved by measuring caspases activity. Thus, the developed Au@RD NPs, obtained by the plant-mediated green synthesis, are attractive hybrid materials for the medical applications combining two active components - metal nanoparticles platform and plant-derived metabolites.

Liver Expression of a MiniATP7B Gene Results in Long-Term Restoration of Copper Homeostasis in a Wilson Disease Model in Mice.

Gene therapy with an adeno-associated vector (AAV) serotype 8 encoding the human ATPase copper-transporting beta polypeptide (ATP7B) complementary DNA (cDNA; AAV8-ATP7B) is able to provide long-term copper metabolism correction in 6-week-old male Wilson disease (WD) mice. However, the size of the genome (5.2 kilobases [kb]) surpasses the optimal packaging capacity of the vector, which resulted in low-yield production; in addition, further analyses in WD female mice and in animals with a more advanced disease revealed reduced therapeutic efficacy, as compared to younger males. To improve efficacy of the treatment, an optimized shorter AAV vector was generated, in which four out of six metal-binding domains (MBDs) were deleted from the ATP7B coding sequence, giving rise to the miniATP7B protein (Δ57-486-ATP7B). In contrast to AAV8-ATP7B, AAV8-miniATP7B could be produced at high titers and was able to restore copper homeostasis in 6- and 12-week-old male and female WD mice. In addition, a recently developed synthetic AAV vector, AAVAnc80, carrying the miniATP7B gene was similarly effective at preventing liver damage, restoring copper homeostasis, and improving survival 1 year after treatment. Transduction of approximately 20% of hepatocytes was sufficient to normalize copper homeostasis, suggesting that corrected hepatocytes are acting as a sink to eliminate excess of copper. Importantly, administration of AAVAnc80-miniATP7B was safe in healthy mice and did not result in copper deficiency. Conclusion: In summary, gene therapy using an optimized therapeutic cassette in different AAV systems provides long-term correction of copper metabolism regardless of sex or stage of disease in a clinically relevant WD mouse model. These results pave the way for the implementation of gene therapy in WD patients.

Lipid nanocapsules decorated and loaded with cannabidiol as targeted prolonged release carriers for glioma therapy: In vitro screening of critical parameters.

The therapeutic potential of cannabinoids has been truly constrained heretofore due to their strong psychoactive effects and their high lipophilicity. In this context, precisely due to the lack of psychoactive properties, cannabidiol (CBD), the second major component of Cannabis sativa, arises as the phytocannabinoid with the most auspicious therapeutic potential. Hence, the incorporation of CBD in lipid nanocapsules (LNCs) will contribute to overcome the dosing problems associated with cannabinoids. Herein, we have prepared LNCs decorated and loaded with CBD for glioma therapy and screened in vitro their critical parameters. On the one hand, we have encapsulated CBD into the oily core of LNCs to test their in vitro efficacy as extended-release carriers against the human glioblastoma cell line U373MG. The in vitro antitumor effect was highly dependent on the size of LNCs due to its pivotal role in the extent of CBD release. Effectively, a comparison between two differently-sized LNCs (namely, 20-nm and 50-nm sized carriers) showed that the smaller LNCs reduced by 3.0-fold the IC50 value of their 50-nm sized counterparts. On the other hand, to explore the potential of this phytocannabinoid to target any of the cannabinoid receptors overexpressed in glioma cells, we decorated the LNCs with CBD. This functionalization strategy enhanced the in vitro glioma targeting by 3.4-fold in comparison with their equally-sized undecorated counterparts. Lastly, the combination of CBD-loading with CBD-functionalization further reduced the IC50 values. Hence, the potential of these two strategies of CBD incorporation into LNCs deserves subsequent in vivo evaluation.

High-Precision Photothermal Ablation Using Biocompatible Palladium Nanoparticles and Laser Scanning Microscopy.

Herein, we report a straightforward method for the scalable preparation of Pd nanoparticles (Pd-NPs) with reduced inherent cytotoxicity and high photothermal conversion capacity. These Pd-NPs are rapidly taken up by cells and able to kill labeled cancer cells upon short exposure to near-infrared (NIR) light. Following cell treatment with Pd-NPs, ablated areas were patterned with high precision by laser scanning microscopy, allowing one to perform cell migration assays with unprecedented accuracy. Using coherent Raman microscopy, cells containing Pd-NPs were simultaneously ablated and imaged. This novel methodology was combined with intravital imaging to mediate microablation of cancerous tissue in tumor xenografts in mice.

Magneto-plasmonic nanoparticles as theranostic platforms for magnetic resonance imaging, drug delivery and NIR hyperthermia applications.

PEGylated magneto-plasmonic nanoparticles with a hollow or semi-hollow interior have been successfully synthesized and their physico-chemical characteristics have been investigated. The hollow interior space can be used to store drugs or other molecules of interest whereas magnetic characterization shows their potential as contrast agents in magnetic resonance imaging (MRI) applications. In addition, their plasmonic characteristics in the near infrared (NIR) region make them efficient in photothermal applications producing high temperature gradients after short irradiation times. We show that by controlling the etching conditions the inner silica shell can be selectively dissolved to achieve a hollow or semi-hollow interior without compromising the magnetic or plasmonic characteristics of the resulting nanoparticles. Magnetic measurements and transmission electron microscopy observations have been used to demonstrate the precise control during the etching process and to select an optimal concentration of the etching reagent and contact time to preserve the inner superparamagnetic iron oxide-based nanoparticles and the plasmonic properties of the constructs. Drug loading capabilities were also evaluated for both semi-hollow and as-synthesized nanoparticles using Rhodamine B isothiocyanate as a model compound. The nanoparticles produced could be potentially used as "theranostic" nanoparticles with both imaging capabilities and a dual therapeutic function (drug delivery and hyperthermia).