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.

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.

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.

Pro-angiogenic near infrared-responsive hydrogels for deliberate transgene expression.

CuS nanoparticles (CuSNP) are degradable, readily prepared, inexpensive to produce and efficiently cleared from the body. In this work, we explored the feasibility of CuSNP to function as degradable near infrared (NIR) nanotransducers within fibrin-based cellular scaffolds. To prepare NIR-responsive CuSNP hydrogels, fibrinogen was dissolved in cell culture medium and supplemented with aqueous dispersions of CuSNP. Fibrinogen polymerization was catalyzed by the addition of thrombin. In some experiments, HUVEC, C3H/10T1/2 or C3H/10T1/2-fLuc cells, that harbor a heat-activated and rapamycin-dependent gene switch for regulating the expression of firefly luciferase transgene, were incorporated to the sol phase of the hydrogel. For in vivo experiments, hydrogels were injected subcutaneously in the back of adult C3H/HeN mice. Upon NIR irradiation, CuSNP hydrogels allowed heat-inducible and rapamycin-dependent transgene expression in cells contained therein, in vitro and in vivo. C3H/10T1/2 cells cultured in CuSNP hydrogels increased metabolic activity, survival rate and fibrinolytic activity, which correlated with changes at the transcriptome level. Media conditioned by CuSNP hydrogels increased viability of HUVEC which formed pseudocapillary structures and remodeled protein matrix when entrapped within these hydrogels. After long-term implantation, the skin patches that covered the CuSNP hydrogels showed increased capillary density which was not detected in mice implanted with matrices lacking CuSNP. In summary, NIR-responsive scaffolds harboring CuSNP offer compelling features in the tissue engineering field, as degradable implants with enhanced integration capacity in host tissues that can provide remote controlled deployment of therapeutic gene products. STATEMENT OF SIGNIFICANCE: Hydrogels composed of fibrin embedding copper sulfide nanoparticles (CuSNP) efficiently convert incident near infrared (NIR) energy into heat and can function as cellular scaffolding. NIR laser irradiation of CuSNP hydrogels can be employed to remotely induce spatiotemporal patterns of transgene expression in genetically engineered multipotent stem cells. CuSNP incorporation in hydrogel architecture accelerates the cell-mediated degradation of the fibrin matrix and induces pro-angiogenic responses that may facilitate the integration of these NIR-responsive scaffolds in host tissues. CuSNP hydrogels that harbor cells capable of controlled expression of therapeutic gene products may be well suited for tissue engineering as they are biodegradable, enhance implant vascularization and can be used to deploy growth factors in a desired spatiotemporal fashion.

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.

Cymantrenyl-Nucleobases: Synthesis, Anticancer, Antitrypanosomal and Antimicrobial Activity Studies.

The synthesis of four cymantrene-5-fluorouracil derivatives (1-4) and two cymantrene-adenine derivatives (5 and 6) is reported. All of the compounds were characterized by spectroscopic methods and the crystal structure of two derivatives (1 and 6), together with the previously described cymantrene-adenine compound C was determined by X-ray crystallography. While the compounds 1 and 6 crystallized in the triclinic P-1 space group, compound C crystallized in the monoclinic P21/m space group. The newly synthesized compounds 1-6 were tested together with the two previously described cymantrene derivatives B and C for their in vitro antiproliferative activity against seven cancer cell lines (MCF-7, MCF-7/DX, MDA-MB-231, SKOV-3, A549, HepG2m and U-87-MG), five bacterial strains Staphylococcus aureus (methicillin-sensitive, methicillin-resistant and vancomycin-intermediate strains), Staphylococcus epidermidis, and Escherichia coli, including clinical isolates of S. aureus and S. epidermidis, as well as against the protozoan parasite Trypanosoma brucei. The most cytotoxic compounds were derivatives 2 and C for A549 and SKOV-3 cancer cell lines, respectively, with 50% growth inhibition (IC50) values of about 7 µM. The anticancer activity of the cymantrene compounds was determined to be due to their ability to induce oxidative stress and to trigger apoptosis and autophagy in cancer cells. Three derivatives (1, 4 and 5) displayed promising antitrypanosomal activity, with GI50 values in the low micromolar range (3-4 µM). The introduction of the 5-fluorouracil moiety in 1 enhanced the trypanocidal activity when compared to the activity previously reported for the corresponding uracil derivative. The antibacterial activity of cymantrene compounds 1 and C was within the range of 8-64 µg/mL and seemed to be the result of induced cell shrinking.

Smart Dressings Based on Nanostructured Fibers Containing Natural Origin Antimicrobial, Anti-Inflammatory, and Regenerative Compounds.

A fast and effective wound healing process would substantially decrease medical costs, wound care supplies, and hospitalization significantly improving the patients' quality of life. The search for effective therapeutic approaches seems to be imperative in order to avoid the aggravation of chronic wounds. In spite of all the efforts that have been made during the recent years towards the development of artificial wound dressings, none of the currently available options combine all the requirements necessary for quick and optimal cutaneous regeneration. Therefore, technological advances in the area of temporary and permanent smart dressings for wound care are required. The development of nanoscience and nanotechnology can improve the materials and designs used in topical wound care in order to efficiently release antimicrobial, anti-inflammatory and regenerative compounds speeding up the endogenous healing process. Nanostructured dressings can overcome the limitations of the current coverings and, separately, natural origin components can also overcome the drawbacks of current antibiotics and antiseptics (mainly cytotoxicity, antibiotic resistance, and allergies). The combination of natural origin components with demonstrated antibiotic, regenerative, or anti-inflammatory properties together with nanostructured materials is a promising approach to fulfil all the requirements needed for the next generation of bioactive wound dressings. Microbially compromised wounds have been treated with different essential oils, honey, cationic peptides, aloe vera, plant extracts, and other natural origin occurring antimicrobial, anti-inflammatory, and regenerative components but the available evidence is limited and insufficient to be able to draw reliable conclusions and to extrapolate those findings to the clinical practice. The evidence and some promising preliminary results indicate that future comparative studies are justified but instead of talking about the beneficial or inert effects of those natural origin occurring materials, the scientific community leads towards the identification of the main active components involved and their mechanism of action during the corresponding healing, antimicrobial, or regenerative processes and in carrying out systematic and comparative controlled tests. Once those natural origin components have been identified and their efficacy validated through solid clinical trials, their combination within nanostructured dressings can open up new avenues in the fabrication of bioactive dressings with outstanding characteristics for wound care. The motivation of this work is to analyze the state of the art in the use of different essential oils, honey, cationic peptides, aloe vera, plant extracts, and other natural origin occurring materials as antimicrobial, anti-inflammatory and regenerative components with the aim of clarifying their potential clinical use in bioactive dressings. We conclude that, for those natural occurring materials, more clinical trials are needed to reach a sufficient level of evidence as therapeutic agents for wound healing management.

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).

Enhancing of plasmonic photothermal therapy through heat-inducible transgene activity.

We explore the synergistic effect of photothermal therapy and gene therapy, simultaneously triggered by silica-gold nanoshells (NS) or hollow gold nanoparticles (HGNPs) in human HeLa cells following near-infrared (NIR) light irradiation. Thermal transfer from NS was higher than that displayed by HGNPs, owing to a differential interaction of the nanomaterial with the biological environment. Under sublethal photothermal conditions, NS and HGNPs effectively modulated the expression levels of a DsRed-monomer reporter gene controlled by the highly heat-inducible human HSP70B promoter, as a function of nanomaterial concentration and length of laser exposure. Hyperthermia treatments at doses that do not promote cell death generated a lethal outcome in HeLa cells harboring the fusogenic GALV-FMG transgene under the control of the HSP70B promoter. Combination of lethal photothermia with the triggering of the cytotoxic transgene resulted in a dramatic increase of the cell-ablation area as a result of the synergistic activity established. FROM THE CLINICAL EDITOR: In this study photothermal therapy and gene therapy, simultaneously triggered by silica-gold nanoshells or hollow gold nanoparticles, was investigated in human HeLa cells following near-infrared (NIR) light irradiation. It is shown that the combination of lethal photothermia with the triggering of the cytotoxic transgene at sublethal levels results in a synergistic cytotoxic effect in vitro.