HPMCAS-Coated Alginate Microparticles Loaded with Ctx(Ile21)-Ha as a Promising Antimicrobial Agent against Salmonella Enteritidis in a Chicken Infection Model.

Salmonella enterica subsp. enterica serovar Enteritidis (S. Enteritidis) in poultry is most often transmitted by the fecal-oral route, which can be attributed to high population density. Upon encountering the innate immune response in a host, the pathogen triggers a stress response and virulence factors to help it survive in the host. The aim of this study was to evaluate the effect of hypromellose acetate/succinate (HPMCAS)-coated alginate microparticles containing the Ctx(Ile21)-Ha antimicrobial peptide (AMP) on both intestinal colonization and systemic infection of laying hens challenged with S. Enteritidis. The applied AMP microsystem reduced the bacterial load of S. Enteritidis in the liver, with a statistical significance between groups A (control, no Ctx(Ile21)-Ha peptide) and B (2.5 mg of Ctx(Ile21)-Ha/kg) at 2 days postinfection (dpi), potentially indicating the effectiveness of Ctx(Ile21)-Ha in the first stage of infection by S. Enteritidis. In addition, the results showed a significant decrease in the S. Enteritidis counts in the spleen and cecal content at 5 dpi; remarkably, no S. Enteritidis counts were observed in livers at 5, 7, and 14 dpi, regardless of the Ctx(Ile21)-Ha dosage (p-value <0.0001). Using the Chi-square test, the effect of AMP microparticles on S. Enteritidis fecal excretion was also evaluated, and a significantly lower bacterial excretion was observed over 21 days in groups B and C, in comparison with the untreated control (p-value <0.05). In summary, the use of HPMCAS-Ctx(Ile21)-Ha peptide microcapsules in laying hens drastically reduced the systemic infection of S. Enteritidis, mainly in the liver, indicating a potential for application as a feed additive against this pathogen.

Quantification by Luminescence Tracking of Red Emissive Gold Nanoparticles in Cells.

Optical microscopy techniques are ideal for live cell imaging for real-time nanoparticle tracking of nanoparticle localization. However, the quantification of nanoparticle uptake is usually evaluated by analytical methods that require cell isolation. Luminescent labeling of gold nanoparticles with transition metal probes yields particles with attractive photophysical properties, enabling cellular tracking using confocal and time-resolved microscopies. In the current study, gold nanoparticles coated with a red-luminescent ruthenium transition metal complex are used to quantify and track particle uptake and localization. Analysis of the red-luminescence signal from particles is used as a metric of cellular uptake, which correlates to total cellular gold and ruthenium content, independently measured and correlated by inductively coupled plasma mass spectrometry. Tracking of the luminescence signal provides evidence of direct diffusion of the nanoparticles across the cytoplasmic membrane with particles observed in the cytoplasm and mitochondria as nonclustered "free" nanoparticles. Electron microscopy and inhibition studies identified macropinocytosis of clusters of particles into endosomes as the major mechanism of uptake. Nanoparticles were tracked inside GFP-tagged cells by following the red-luminescence signal of the ruthenium complex. Tracking of the particles demonstrates their initial location in early endosomes and, later, in lysosomes and autophagosomes. Colocalization was quantified by calculating the Pearson's correlation coefficient between red and green luminescence signals and confirmed by electron microscopy. Accumulation of particles in autophagosomes correlated with biochemical evidence of active autophagy, but there was no evidence of detachment of the luminescent label or breakup of the gold core. Instead, accumulation of particles in autophagosomes caused organelle swelling, breakdown of the surrounding membranes, and endosomal release of the nanoparticles into the cytoplasm. The phenomenon of endosomal release has important consequences for the toxicity, cellular targeting, and therapeutic future applications of gold nanoparticles.

Assisted delivery of anti-tumour platinum drugs using DNA-coiling gold nanoparticles bearing lumophores and intercalators: towards a new generation of multimodal nanocarriers with enhanced action.

New gold and lipoic based nanocarriers for the delivery of platinum(ii) and platinum(iv) drugs are developed, which allow enhanced loading of the drug on the surface of the nanocarriers and release in a pH-dependent fashion, with superior release at lower pHs which are associated with many tumours. The conjugate nanoparticles and their conjugates enter cells rapidly (within 3 hours). They tend to cluster in vesicles and are also observed by light and electron microscopies in the cytoplasm, endoplasmic reticulum and nucleus. We further incorporate aminoanthraquinone units that are both fluorophores and DNA intercalators. This results in nanocarriers that after drug release will remain surface decorated with DNA-binders challenging the conventional design of the nanocarrier as an inert component. The outcome is nanocarriers that themselves have distinctive, remarkable and unusual DNA binding properties being able to bind and wrap DNA (despite their anionic charge) and provide enhanced cytotoxic activity beyond that conferred by the platinum agents they release. DNA coiling is usually associated with polycations which can disrupt cell membranes; anionic nanoparticles that can cause novel and dramatic effects on DNA may have fascinating potential for new approaches to in-cell nucleic acid recognition. Our findings have implications for the understanding and interpretation of the biological activities of nanoparticles used to deliver other DNA-binding drugs including clinical drug doxorubicin and its formulations.

Iridium Nanoparticles for Multichannel Luminescence Lifetime Imaging, Mapping Localization in Live Cancer Cells.

The development of long-lived luminescent nanoparticles for lifetime imaging is of wide interest as luminescence lifetime is environmentally sensitive detection independent of probe concentration. We report novel iridium-coated gold nanoparticles as probes for multiphoton lifetime imaging with characteristic long luminescent lifetimes based on iridium luminescence in the range of hundreds of nanoseconds and a short signal on the scale of picoseconds based on gold allowing multichannel detection. The tailor-made IrC6 complex forms stable, water-soluble gold nanoparticles (AuNPs) of 13, 25, and 100 nm, bearing 1400, 3200, and 22 000 IrC6 complexes per AuNP, respectively. The sensitivity of the iridium signal on the environment of the cell is evidenced with an observed variation of lifetimes. Clusters of iridium nanoparticles show lifetimes from 450 to 590 ns while lifetimes of 660 and 740 ns are an average of different points in the cytoplasm and nucleus. Independent luminescence lifetime studies of the nanoparticles in different media and under aggregation conditions postulate that the unusual long lifetimes observed can be attributed to interaction with proteins rather than nanoparticle aggregation. Total internal reflection fluorescence microscopy (TIRF), confocal microscopy studies and 3D luminescence lifetime stacks confirm the presence of bright, nonaggregated nanoparticles inside the cell. Inductively coupled plasma mass spectrometry (ICPMS) analysis further supports the presence of the nanoparticles in cells. The iridium-coated nanoparticles provide new nanoprobes for lifetime detection with dual channel monitoring. The combination of the sensitivity of the iridium signal to the cell environment together with the nanoscaffold to guide delivery offer opportunities for iridium nanoparticles for targeting and tracking in in vivo models.

Tailoring iridium luminescence and gold nanoparticle size for imaging of microvascular blood flow.

AIM: Imaging of blood flow in narrow channels and close to vessel walls is important in cardiovascular research for understanding pathogenesis. Our aim was to provide novel nanoprobes with visible emission and long lifetimes as trackers of flow. MATERIALS & METHODS: Gold nanoparticles coated with an iridium complex were prepared. Luminescence imaging was used to monitor their flows in different hematocrit blood and in murine tissues. RESULTS: The velocities are independent of hematocrit level and the nanoparticles entering blood circulation can be clearly detected in vessels in lungs, mesentery and the skeletal muscle. CONCLUSION: The work introduces for the first time iridium-based yellow-green luminescence with nanoparticle size of 100 nm for visualizing and monitoring flows with much higher resolution than conventional alternatives.

The deposition and imaging of silica sub-micron particles in dentine.

OBJECTIVES: Sub-micron particles may assist in the delivery of compounds into dentine tubules. The surface interactions of the particles with dentine may prevent them from entering the tubules. The aim of this study is to investigate whether silica particles, treated with surfactants improves dentine tubules occlusion using both artificial and human tooth models METHODS: Spherical silica particles (size 130-810nm) bearing an encapsulated ruthenium luminescent complex were coated with the following surfactants: Zonyl(®) FSA, Triton(®) X-100 and Tween20(®). The particles were prepared as 0.004% w/v and 0.04% w/v solutions with deionized water and were applied to the surface of; (1) in vitro model of PET ThinCert™ cell culture inserts; (2) 0.1mm thick sections of human molar teeth. RESULTS: Scanning electron and confocal fluorescence microscopy images show that particles without any coating and with TritonX-100 coating had the highest aggregation. Particles with Tween-20 are less aggregated on the surface and show inclusion in the tubules. Particles coated with fluorosurfactant Zonyl show a preference for aggregation at the tubule. With the ThinCert™ membranes high aggregation within the artificial tubules was increased by particle concentration. CONCLUSIONS: The use of silica sub-micron particles on hard dental tissues is dependent on the modification of the surface chemistry of both the particle and the dentine and the employment of the fluorοsurfactant may improve tubule occlusion. The use of ThinCerts™ membrane is useful in vitro model to mimic dentinal tubules and observe the ability of particles to occlude small channels. CLINICAL SIGNIFICANCE: The use of silica sub-micron particles on hard dentine tissues is dependent on the modification of the surface coating of the particles. This may influence how particles are incorporated in potential delivery vehicles applied to the dentine surface with the employment of a fluorosurfactant showing promise.

High coating of Ru(II) complexes on gold nanoparticles for single particle luminescence imaging in cells.

Gold nanoparticles are efficiently labelled with a luminescent ruthenium complex, producing 13 and 100 nm diameter, monodisperse red-emissive imaging probes with luminescence lifetimes prolonged over the molecular unit. Single, 100 nm particles are observed in whole cell luminescence imaging which reveals their biomolecular association with chromatin in the nucleus of cancer cells.

Characterisation of bone following ultrasonic cutting.

OBJECTIVES: Ultrasonic surgery is an increasingly popular technique for cutting bone, but little research has investigated how the ultrasonic tip oscillations may affect the cuts they produce in bone. The aim of this investigation was to evaluate the oscillation and cutting characteristics of an ultrasonic surgical device. MATERIALS AND METHODS: A Piezosurgery 3 (Mectron, Carasco, Italy) ultrasonic cutting system was utilised with an OP3 style tip. The system was operated with the tip in contact with porcine bone samples (loads of 50 to 200 g) mounted at 45° to the vertical insert tip and with a water flow of 57 ml/min. Tip oscillation amplitude was determined using scanning laser vibrometry. Bone surfaces defects were characterised using laser profilometry and scanning electron microscopy. RESULTS: A positive relationship was observed between the magnitude of tip oscillations and the dimensions of defects cut into the bone surface. Overloading the tip led to a reduction in oscillation and hence in the defect produced. A contact load of 150 g provided the greatest depth of cut. Defects produced in the bone came from two clear phases of cutting. CONCLUSIONS: The structure of the bone was found to be an important factor in the cut characteristics following piezosurgery. CLINICAL RELEVANCE: Cutting of bone with ultrasonics is influenced by the load applied and the setting used. Care must be used to prevent the tip from sliding over the bone at low loadings.