Fluorimetric Cell Analysis with 9-Aryl-Substituted Berberine Derivatives as DNA-Targeting Fluorescent Probes.

DNA-sensitive fluorescent light-up probes based on berberine are presented. This biogenic fluorophore was chosen as central unit to use its potential biocompatibility and its DNA-binding properties. To provide predictable fluorescence quenching in aqueous solution and a fluorescence light-up effect upon DNA binding, aryl substituents were attached at the 9-position by Suzuki-Miyaura coupling reactions. The 9-arylberberine derivatives have a very low fluorescence quantum yield (Φfl =<0.02), which is caused by the radiationless deactivation of the excited state by torsional relaxation about the biaryl axis. In addition, these berberine derivatives intercalate into DNA with high affinity (Kb =2.0-22×104  M-1 ). Except for the nitrophenyl- and hydroxyphenyl-substituted derivatives, all tested compounds exhibited a pronounced fluorescence light-up effect upon association with DNA, because the deactivation of the excited-state by torsional relaxation is suppressed in the DNA binding site. Most notably, it was shown exemplarily with the 9-(4-methoxyphenyl)- and the 9-(6-methoxynaphthyl)-substituted derivatives that these properties are suited for fluorimetric cell analysis. In particular, these probes generated distinct staining patterns in eukaryotic cells (NIH 3T3 mouse fibroblasts), which enabled the identification of nuclear substructures, most likely heterochromatin or nucleoli, respectively.

Photodynamic Eradication of Pseudomonas aeruginosa with Ru-Photosensitizers Encapsulated in Enzyme Degradable Nanocarriers.

The development of new approaches for the treatment of the increasingly antibiotic-resistant pathogen Pseudomonas aeruginosa was targeted by enhancing the effect of local antimicrobial photodynamic therapy (aPDT) using poly(ethylene glycol)-block-poly(lactic acid) (PEG114-block-PLAx) nanocarriers that were loaded with a ruthenium-based photosensitizer (PS). The action of tris(1,10-phenanthroline) ruthenium (II) bis(hexafluorophosphate) (RuPhen3) encapsulated in PEG114-block-PLAx micelles and vesicles was shown to result in an appreciable aPDT inactivation efficiency against planktonic Pseudomonas aeruginosa. In particular, the encapsulation of the PS, its release, and the efficiency of singlet oxygen (1O2) generation upon irradiation with blue light were studied spectroscopically. The antimicrobial effect was analyzed with two strains of Pseudomonas aeruginosa. Compared with PS-loaded micelles, formulations of the PS-loaded vesicles showed 10 times enhanced activity with a strong photodynamic inactivation effect of at least a 4.7 log reduction against both a Pseudomonas aeruginosa lab strain and a clinical isolate collected from the lung of a cystic fibrosis (CF) patient. This work lays the foundation for the targeted eradication of Pseudomonas aeruginosa using aPDT in various medical application areas.

Monitoring Growth and Removal of Pseudomonas Biofilms on Cellulose-Based Fabrics.

Biofilms are often tolerant towards routine cleaning and disinfection processes. As they can grow on fabrics in household or healthcare settings, resulting in odors and serious health problems, it is necessary to contain biofilms through eradication strategies. The current study proposes a novel test model for the growth and removal of biofilms on textiles with Pseudomonas fluorescens and the opportunistic nosocomial pathogen Pseudomonas aeruginosa as model organisms. To assess the biofilm removal on fabrics, (1) a detergent-based, (2) enzyme-based, and (3) combined formulation of both detergent and enzymes (F1/2) were applied. Biofilms were analyzed microscopically (FE-SEM, SEM, 3D laser scanning- and epifluorescence microscopy), via a quartz crystal microbalance with mass dissipation monitoring (QCM-D) as well as plate counting of colonies. This study indicated that Pseudomonas spp. form robust biofilms on woven cellulose that can be efficiently removed via F1/2, proven by a significant reduction (p < 0.001) of viable bacteria in biofilms. Moreover, microscopic analysis indicated a disruption and almost complete removal of the biofilms after F1/2 treatment. QCM-D measurements further confirmed a maximal mass dissipation change after applying F1/2. The combination strategy applying both enzymes and detergent is a promising antibiofilm approach to remove bacteria from fabrics.

Bare Eye Detection of Bacterial Enzymes of Pseudomonas aeruginosa with Polymer Modified Nanoporous Silicon Rugate Filters.

The fabrication, characterization and application of a nanoporous Silicon Rugate Filter (pSiRF) loaded with an enzymatically degradable polymer is reported as a bare eye detection optical sensor for enzymes of pathogenic bacteria, which is devoid of any dyes. The nanopores of pSiRF were filled with poly(lactic acid) (PLA), which, upon enzymatic degradation, resulted in a change in the effective refractive index of the pSiRF film, leading to a readily discernible color change of the sensor. The shifts in the characteristic fringe patterns before and after the enzymatic reaction were analyzed quantitatively by Reflectometric Interference Spectroscopy (RIfS) to estimate the apparent kinetics and its dependence on enzyme concentration. A clear color change from green to blue was observed by the bare eye after PLA degradation by proteinase K. Moreover, the color change was further confirmed in measurements in bacterial suspensions of the pathogen Pseudomonas aeruginosa (PAO1) as well as in situ in the corresponding bacterial supernatants. This study highlights the potential of the approach in point of care bacteria detection.

Effects of the Sex Steroid Hormone Estradiol on Biofilm Growth of Cystic Fibrosis Pseudomonas aeruginosa Isolates.

Women with cystic fibrosis (CF) have a significantly lower life expectancy compared to men, which is indicated by an earlier impairment of lung function due to chronic colonization with biofilm formed by Pseudomonas aeruginosa. There is growing evidence that blood serum concentrations of the steroid sex hormone estradiol (E2) correlate with the occurrence of pulmonary exacerbations in CF but also play a role in the mucoid switch of P. aeruginosa. This study aims to shed light on possible microbiological reasons for sexual dimorphism in CF by investigating the influence of E2 on biofilm formation of P. aeruginosa CF isolates. For this purpose, 10 CF isolates of the respiratory tract derived from different CF patients have been treated with E2 in a microtiter plate biofilm model. Biofilms have been examined by crystal violet assays, field emission scanning electron microscopy (FE-SEM), 3D laser scanning microscopy (LSM), and quorum sensing (QS) reporter assays of the supernatants taken from biofilms. This allowed us to simultaneously investigate the effects of E2 on attached biofilm mass, biofilm ultrastructure, and QS activity. Upon E2 treatment, six out of 10 investigated CF isolates showed an increase of attached biofilm mass, whereas biofilms from two tested non-CF laboratory strains (PAO1 and ATCC19660) did not. Moreover, FE-SEM and 3D LSM analyses of the E2 responsive CF biofilms revealed ultrastructural remodeling of biofilm structure at different scales with increased formation of prominent biofilm spots, enhanced coverage with extracellular polymeric substance (EPS), and extended average surface roughness. QS activity measurements performed in biofilm supernatants via luminescence acyl homoserine lactone (AHL) reporter assays further showed that E2 treatment may also modulate QS signaling, as shown in an E2 sensitive CF isolate. Together, our results suggest the biofilm modulating effects of E2 on various clinical CF isolates that are documented by both biomass and ultrastructural changes of biofilms. The gained new insight into the influence of steroid hormones on P. aeruginosa biofilm phenotypes might pave the way for novel future approaches in personalized medicine based on the patients' sex and hormonal status.

Ruthenium(II) Polypyridyl Complexes for Antimicrobial Photodynamic Therapy: Prospects for Application in Cystic Fibrosis Lung Airways.

Antimicrobial photodynamic therapy (aPDT) depends on a variety of parameters notably related to the photosensitizers used, the pathogens to target and the environment to operate. In a previous study using a series of Ruthenium(II) polypyridyl ([Ru(II)]) complexes, we reported the importance of the chemical structure on both their photo-physical/physico-chemical properties and their efficacy for aPDT. By employing standard in vitro conditions, effective [Ru(II)]-mediated aPDT was demonstrated against planktonic cultures of Pseudomonas aeruginosa and Staphylococcus aureus strains notably isolated from the airways of Cystic Fibrosis (CF) patients. CF lung disease is characterized with many pathophysiological disorders that can compromise the effectiveness of antimicrobials. Taking this into account, the present study is an extension of our previous work, with the aim of further investigating [Ru(II)]-mediated aPDT under in vitro experimental settings approaching the conditions of infected airways in CF patients. Thus, we herein studied the isolated influence of a series of parameters (including increased osmotic strength, acidic pH, lower oxygen availability, artificial sputum medium and biofilm formation) on the properties of two selected [Ru(II)] complexes. Furthermore, these compounds were used to evaluate the possibility to photoinactivate P. aeruginosa while preserving an underlying epithelium of human bronchial epithelial cells. Altogether, our results provide substantial evidence for the relevance of [Ru(II)]-based aPDT in CF lung airways. Besides optimized nano-complexes, this study also highlights the various needs for translating such a challenging perspective into clinical practice.

Nanomedicine-based strategies to improve treatment of cutaneous leishmaniasis.

Nanomedicine strategies were first adapted and successfully translated to clinical application for diseases, such as cancer and diabetes. These strategies would no doubt benefit unmet diseases needs as in the case of leishmaniasis. The latter causes skin sores in the cutaneous form and affects internal organs in the visceral form. Treatment of cutaneous leishmaniasis (CL) aims at accelerating wound healing, reducing scarring and cosmetic morbidity, preventing parasite transmission and relapse. Unfortunately, available treatments show only suboptimal effectiveness and none of them were designed specifically for this disease condition. Tissue regeneration using nano-based devices coupled with drug delivery are currently being used in clinic to address diabetic wounds. Thus, in this review, we analyse the current treatment options and attempt to critically analyse the use of nanomedicine-based strategies to address CL wounds in view of achieving scarless wound healing, targeting secondary bacterial infection and lowering drug toxicity.

Antimicrobial Photodynamic Therapy: Latest Developments with a Focus on Combinatory Strategies.

Antimicrobial photodynamic therapy (aPDT) has become a fundamental tool in modern therapeutics, notably due to the expanding versatility of photosensitizers (PSs) and the numerous possibilities to combine aPDT with other antimicrobial treatments to combat localized infections. After revisiting the basic principles of aPDT, this review first highlights the current state of the art of curative or preventive aPDT applications with relevant clinical trials. In addition, the most recent developments in photochemistry and photophysics as well as advanced carrier systems in the context of aPDT are provided, with a focus on the latest generations of efficient and versatile PSs and the progress towards hybrid-multicomponent systems. In particular, deeper insight into combinatory aPDT approaches is afforded, involving non-radiative or other light-based modalities. Selected aPDT perspectives are outlined, pointing out new strategies to target and treat microorganisms. Finally, the review works out the evolution of the conceptually simple PDT methodology towards a much more sophisticated, integrated, and innovative technology as an important element of potent antimicrobial strategies.

Drug Release from Thermo-Responsive Polymer Brush Coatings to Control Bacterial Colonization and Biofilm Growth on Titanium Implants.

Despite decades of biomedical advances, the colonization of implant devices with bacterial biofilms is still a leading cause of implant failure. Clearly, new strategies and materials that suppress both initial and later stage bacterial colonization are required in this context. Ideal would be the implementation of a bactericidal functionality in the implants that is temporally and spatially triggered in an autonomous fashion at the infection site. Herein, the fabrication and validation of functional titanium-based implants with triggered antibiotic release function afforded via an intelligent polymer coating is reported. In particular, thermo-responsive poly(di(ethylene glycol) methyl ether methacrylate) (PDEGMA) brushes on titanium implants synthesized via a surface-initiated atom transfer radical polymerization with activators regenerated through the electron transfer technique (ARGET ATRP) allows for a controlled and thermally triggered release of the antibiotic levofloxacin at the wound site. Antibiotic loaded brushes are investigated as a function of thickness, loading capacity for antibiotics, and temperature. At temperatures of the infection site >37 °C the lower critical solution temperature behavior of the brushes afforded the triggered release. Hence, in addition to the known antifouling effects, the PDEGMA coating ensured enhanced bactericidal effects, as demonstrated in initial in vivo tests with rodents infected with Staphylococcus aureus.

Multiplexed detection and differentiation of bacterial enzymes and bacteria by color-encoded sensor hydrogels.

We report on the fabrication and characterization of color-encoded chitosan hydrogels for the rapid, sensitive and specific detection of bacterial enzymes as well as the selective detection of a set of tested bacteria through characteristic enzyme reactions. These patterned sensor hydrogels are functionalized with three different colorimetric enzyme substrates affording the multiplexed detection and differentiation of α-glucosidase, ß-galactosidase and ß-glucuronidase. The limits of detection of the hydrogels for an observation time of 60 min using a conventional microplate reader correspond to concentrations of 0.2, 3.4 and 4.5 nM of these enzymes, respectively. Based on their different enzyme expression patterns, Staphylococcus aureus strain RN4220, methicillin-resistant S. aureus (MRSA) strain N315, both producing α-glucosidase, but not ß-glucuronidase and ß-galactosidase, Escherichia coli strain DH5α, producing ß-glucuronidase and α-glucosidase, but not ß-galactosidase, and the enterohemorrhagic E. coli (EHEC) strain E32511, producing ß-galactosidase, but none of the other two enzymes, can be reliably and rapidly distinguished from each other. These results confirm the applicability of enzyme sensing hydrogels for the detection and discrimination of specific enzymes to facilitate differentiation of bacterial strains. Patterned hydrogels thus possess the potential to be further refined as detection units of a multiplexed format to identify certain bacteria for future application in point-of-care microbiological diagnostics in food safety and medical settings.

Quantitative E. coli Enzyme Detection in Reporter Hydrogel-Coated Paper Using a Smartphone Camera.

There is a growing demand for rapid and sensitive detection approaches for pathogenic bacteria that can be applied by non-specialists in non-laboratory field settings. Here, the detection of the typical E. coli enzyme ß-glucuronidase using a chitosan-based sensing hydrogel-coated paper sensor and the detailed analysis of the reaction kinetics, as detected by a smartphone camera, is reported. The chromogenic reporter unit affords an intense blue color in a two-step reaction, which was analyzed using a modified Michaelis-Menten approach. This generalizable approach can be used to determine the limit of detection and comprises an invaluable tool to characterize the performance of lab-in-a-phone type approaches. For the particular system analyzed, the ratio of reaction rate and equilibrium constants of the enzyme-substrate complex are 0.3 and 0.9 pM-1h-1 for ß-glucuronidase in phosphate buffered saline and lysogeny broth, respectively. The minimal degree of substrate conversion for detection of the indigo pigment formed during the reaction is 0.15, while the minimal time required for detection in this particular system is ~2 h at an enzyme concentration of 100 nM. Therefore, this approach is applicable for quantitative lab-in-a-phone based point of care detection systems that are based on enzymatic substrate conversion via bacterial enzymes.

Targeting non-apoptotic cell death in cancer treatment by nanomaterials: Recent advances and future outlook.

Many tumors develop resistance to most of the apoptosis-based cancer therapies. In this sense targeting non-apoptotic forms of cell death including necroptosis, autophagy and ferroptosis may have therapeutic benefits in apoptosis-defective cancer cells. Nanomaterials have shown great advantages in cancer treatment owing to their unique characteristics. Besides, the capability of nanomaterials to induce different forms of cell death has gained widespread attention in cancer treatment. Reports in this field reflect the therapeutic potential of necroptotic cell death induced by nanomaterials in cancer. Also, autophagic cell death induced by nanomaterials alone and as a part of chemo-, radio- and photothermal therapy holds great promise as anticancer therapeutic option. Besides, ferroptosis induction by iron-based nanomaterials in drug delivery, immunotherapy, hyperthermia and imaging systems shows promising results in malignancies. Hence, this review is devoted to the latest efforts and the challenges in this field of research and its clinical merits.

Alterations in inflammatory markers and clinical outcome after spontaneous intracerebral hemorrhage - Preliminary results.

OBJECTIVE: After an intracerebral hemorrhage, there is an immunological reaction, the specific mechanism of which is not fully understood, that seems to contribute to secondary brain injury. In this study, we investigated alterations of inflammatory markers in the blood and clinical outcome after an intracerebral hemorrhage. METHODS: Between July 2013 and February 2016, we performed a prospective study for which we recruited patients who had suffered an intracerebral hemorrhage. Using various scoring scales we evaluated the neurological state upon admission and discharge, and at one and three months following the ICH. During the hospital stay, various inflammatory markers were examined in blood samples. RESULTS: Out of 132 screened patients, 27 were included (48.2% male, mean age 68 years). We found significantly elevated serum concentrations of interleukin-6 (p=0.006) at the time of admission and throughout days three and five. There were also elevated c-reactive protein and granulocyte-colony stimulating factor concentrations found. The concentrations of these immune parameters showed significant monotonic relationships. The ROC analyses showed a better discrimination for mortality with regard to the percentage of T helper cells than with regard to the ICH volume alone. CONCLUSION: Our results may be regarded as preliminary evidence of the occurrence of inflammation after intracerebral hemorrhage. If there is a relationship between inflammation and clinical outcome remains speculative.

Novel therapeutic strategies for Alzheimer's disease: Implications from cell-based therapy and nanotherapy.

Alzheimer's disease (AD) is a multifactorial neurodegenerative disease which leads to progressive dysfunction of cognition, memory and learning in elderly people. Common therapeutic agents are not only inadequate to suppress the progression of AD pathogenesis but also produce deleterious side effects; hence, development of alternative therapies is required to specifically suppress complications of AD. The current review provides a commentary on conventional as well as novel therapeutic approaches with an emphasis on stem cell and nano-based therapies for improvement and management of AD pathogenesis. According to our overview of the current literature, AD is a multi-factorial disorder with various pathogenic trajectories; hence, a multifunctional strategy to create effective neuroprotective agents is required to treat this disorder.

Enhanced Colorimetric Differentiation between Staphylococcus aureus and Pseudomonas aeruginosa Using a Shape-Encoded Sensor Hydrogel.

Herein, we demonstrate a combined fluorescent probe/shape-encoded hydrogel strategy for the fast, sensitive, and selective detection of bacterial species via their characteristic enzymes. A poly(vinyl alcohol) (PVA) hydrogel loaded with the fluorescent probe N,N'-(3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-3',6'-diyl)bis(2,2,3,3,3-pentafluoropropanamide) (ACS-HNE) was designed for the detection of elastase, an enzyme produced by Pseudomonas aeruginosa. Likewise, a chitosan-derived hydrogel was loaded with the fluorescent probe 4-methylumbelliferyl-α-d-glucopyranoside (MUD) by entrapment for the selective detection of α-glucosidase, an enzyme produced by Staphylococcus aureus. For an observation time of 60 min, limits of detection (LODs) of ≤20 nM for elastase and ≤30 pM for α-glucosidase were obtained, which in the latter case is 3 orders of magnitude better than related chitosan systems with covalently coupled substrate. To illustrate the potential utility of these highly sensitive sensor hydrogels as a simple point-of-care test system, shaped hydrogel slabs representing the letters P and S were manufactured to detect P. aeruginosa and S. aureus, respectively. These shapes were shown to provide an additional unique color code under UV illumination corresponding to the characteristic enzyme produced by the corresponding bacteria. This study shows potential for the future development of an effective and simple point-of-care test for the rapid identification of bacterial species that can be operated by nonspecialists.

The Role of Stabilization-Free Microsurgical Decompression in the Surgical Treatment of Spinal Metastases.

OBJECTIVE: Symptomatic spine metastases are found in about 10% of patients with cancer. As the long-term survival of patients with carcinoma rises, the number of patients with symptomatic spine metastases is also increasing. In our tertiary referral center, patients usually present rapidly progressive neurologic disorders, which require an urgent treatment decision. Treatment options include extensive 360° stabilizations. These complex interventions are not always readily available. We examined the extent to which the patient population benefited from decompressive surgery without stabilization. We hypothesize that patients benefit from merely dorsal decompression, which preserves stability when they experience symptomatic spine metastases. METHODS: We performed a retrospective analysis of electronic patient data from 19 patients, who were treated for symptomatic spine metastases by hemilaminectomy between 2009 and 2017. We evaluated the preoperative and postoperative neurologic functions using the American Spinal Injury Association (ASIA) Impairment Scale. A comparative literature analysis was carried out to assess the Spinal Neoplastic Instability Score, Tokuhashi score, and Tomita score. RESULTS: Nine participants had prostate cancer, 4 had mammary carcinoma, 3 had bronchial carcinoma, and 3 had other cancers. The median preoperative ASIA score was C, postoperatively, the score significantly improved to D (sign test P = 0.002). None of the patients needed stabilization within the follow-up period of up to 56 months. CONCLUSIONS: In our patient population, minimal intervention could significantly improve neurologic disorders. This outcome was seen over the whole study period. Even though different scoring systems suggest stabilization, our results show that spinal decompression alone might be indicated as well.

Colorimetric and Fluorimetric DNA Detection with a Hydroxystyryl-Quinolizinium Photoacid and Its Application for Cell Imaging.

The combination of styryl dye properties with the acidity and strong photoacidity of the 2,2'-[(1''-hydroxy-4''-methyl-(E)-2'',6''-phenylene)]-bisquinolizinium enables the detection of DNA by distinct absorption and emission color changes and the fluorimetric detection of DNA in cells with epifluorescence and confocal fluorescence microscopy.

Propagation and Purification of Human Induced Pluripotent Stem Cells with Selective Homopolymer Release Surfaces.

The selective detachment of undifferentiated human induced pluripotent stem (iPS) cells from a thermal release coating, fabricated from a tailored poly(di(ethylene glycol) methyl ether methacrylate) (PDEGMA) homopolymer layer on gold, is reported. By exploiting the mild, thermally triggered release of iPS cell colonies in the absence of any releasing reagent, pluripotent iPS cells are shown to be selectively separated from spontaneously differentiated cells. The maintained pluripotency and high cell viability of detached and reseeded iPS cell colonies were confirmed and suggest the feasibility of a generally applicable platform approach for cell separation and purification in the context of iPS cell culture, differentiation of pathologically altered cells and normal cells, as well as isolation of different cell types derived from certain tissues, for example, from biopsies.

Toward Label-Free Selective Cell Separation of Different Eukaryotic Cell Lines Using Thermoresponsive Homopolymer Layers.

Ultrathin thermoresponsive poly(di(ethylene glycol) methyl ether methacrylate) (PDEGMA) homopolymer layers are reported as a novel platform for label-free temperature-stimulated cell separation from a coculture of eukaryotic cell lines. Pancreatic tumor cells (PaTu 8988t) and fibroblasts (NIH 3T3) were shown to attach and proliferate on PDEGMA layers with a dry thickness of 5 ± 1 nm at 37 °C. After the cell medium cooled to below the lower critical solution temperature (LCST), PaTu 8988t cells showed a significantly decreased cell area. By contrast, there was no significant change in cell area for NIH 3T3 cells, while they still exhibited lamellipodia and filopodia. PaTu 8988t cells could be detached at 22 °C after flushing gently with the cell medium, while negligible cell detachment was observed for NIH 3T3 cells under identical conditions. This significant difference between PaTu 8988t and NIH 3T3 was due to the different cell adhesion on the culture-medium-derived protein covering the PDEGMA layer. Thus, cell separation from a coculture was achieved successfully by cooling the cell medium to 22 °C, resulting in the desorption of 95 ± 6% of PaTu 8988t cells, while the NIH 3T3 cells remained adherent. Our findings provide an effective, label-free, rapid, and simple approach, exploiting a thermoresponsive homopolymer layer to separate and enrich or isolate cells from mixed cell populations for basic biological research, medical diagnostics, tissue engineering, and cell therapy.