Cellular Uptake of Upconversion Nanoparticles Based on Surface Polymer Coatings and Protein Corona.

Upconversion nanoparticles (UCNPs) are materials that provide unique advantages for biomedical applications. There are constantly emerging customized UCNPs with varying compositions, coatings, and upconversion mechanisms. Cellular uptake is a key parameter for the biological application of UCNPs. Uptake experiments have yielded highly varying results, and correlating trends between cellular uptake with different types of UCNP coatings remains challenging. In this report, the impact of surface polymer coatings on the formation of protein coronas and subsequent cellular uptake of UCNPs by macrophages and cancer cells was investigated. Luminescence confocal microscopy and elemental analysis techniques were used to evaluate the different coatings for internalization within cells. Pathway inhibitors were used to unravel the specific internalization mechanisms of polymer-coated UCNPs. Coatings were chosen as the most promising for colloidal stability, conjugation chemistry, and biomedical applications. PIMA-PEG (poly(isobutylene-alt-maleic) anhydride with polyethylene glycol)-coated UCNPs were found to have low cytotoxicity, low uptake by macrophages (when compared with PEI, poly(ethylenimine)), and sufficient uptake by tumor cells for surface-loaded drug delivery applications. Inductively coupled plasma-optical emission spectroscopy (ICP-OES) studies revealed that PIMA-coated NPs were preferentially internalized by the clathrin- and caveolar-independent pathways, with a preference for clathrin-mediated uptake at longer time points. PMAO-PEG (poly(maleic anhydride-alt-1-octadecene) with polyethylene glycol)-coated UCNPs were internalized by energy-dependent pathways, while PAA- (poly(acrylic acid)) and PEI-coated NPs were internalized by multifactorial mechanisms of internalization. The results indicate that copolymers of PIMA-PEG coatings on UCNPs were well suited for the next-generation of biomedical applications.

Lanthanide-Doped Upconversion Nanoparticles: Exploring A Treasure Trove of NIR-Mediated Emerging Applications.

Lanthanide-doped upconversion nanoparticles (UCNPs) possess the remarkable ability to convert multiple near-infrared (NIR) photons into higher energy ultraviolet-visible (UV-vis) photons, making them a prime candidate for several advanced applications within the realm of nanotechnology. Compared to traditional organic fluorophores and quantum dots (QDs), UCNPs possess narrower emission bands (fwhm of 10-50 nm), large anti-Stokes shifts, low toxicity, high chemical stability, and resistance to photobleaching and blinking. In addition, unlike UV-vis excitation, NIR excitation is nondestructive at lower power intensities and has high tissue penetration depths (up to 2 mm) with low autofluorescence and scattering. Together, these properties make UCNPs exceedingly favored for advanced bioanalytical and theranostic applications, where these systems have been well-explored. UCNPs are also well-suited for bioimaging, optically modulating chemistries, forensic science, and other state-of-the-art research applications. In this review, an up-to-date account of emerging applications in UCNP research, beyond bioanalytical and theranostics, are presented including optogenetics, super-resolution imaging, encoded barcodes, fingerprinting, NIR vision, UCNP-assisted photochemical manipulations, optical tweezers, 3D printing, lasing, NIR-II imaging, UCNP-molecule nanohybrids, and UCNP-based persistent luminescent nanocrystals.

Unlocking Long-Term Stability of Upconversion Nanoparticles with Biocompatible Phosphonate-Based Polymer Coatings.

Achieving long-term (>3 months) colloidal stability of upconversion nanoparticles (UCNPs) in biologically relevant buffers has been a major challenge, which has severely limited practical implementation of UCNPs in bioimaging and nanomedicine applications. To address this challenge, nine unique copolymers formulations were prepared and evaluated as UCNP overcoatings. These polymers consisted of a poly(isobutylene-alt-maleic anhydride) (PIMA) backbone functionalized with different ratios and types of phosphonate anchoring groups and poly(ethylene glycol) (PEG) moieties. The syntheses were done as simple, one-pot nucleophilic addition reactions. These copolymers were subsequently coated onto NaYF4:Yb3+,Er3+ UCNPs, and colloidal stability was evaluated in 1 × PBS, 10 × PBS, and other buffers. UCNP colloidal stability improved (up to 4 months) when coated with copolymers containing greater proportions of anchoring groups and higher phosphonate valences. Furthermore, small molecules could be conjugated to these overcoated UCNPs by use of copper-free click chemistry, as was done to demonstrate suitability for sensor and bioprobe development.

Evaluation of the Idexx ProCyte Dx® for analysis of canine cerebrospinal fluid.

OBJECTIVES: To assess the utility of the Idexx ProCyte Dx® haematology analyser for assessing total nucleated cell count and differential cell counts in canine cerebrospinal fluid. MATERIALS AND METHODS: Seventy-three client-owned dogs undergoing investigations for pyrexia and/or neurological signs were prospectively included. Cerebrospinal fluid samples were assessed using an Idexx ProCyte Dx® analyser and the results were compared to those obtained with the external laboratory reference standard. RESULTS: The Idexx ProCyte Dx® performed with good sensitivity (92.6%) and moderate specificity (67.4%) for total nucleated cell count when compared to the reference standard. Qualitative assessment of the Idexx ProCyte Dx® scatter plots, and quantitative assessment of differential cell counts where available, appeared to correlate well with the external laboratory manual differential cell counts, with a good-to-high agreement in 25 of 26 samples (96.2%). CLINICAL SIGNIFICANCE: The Idexx ProCyte Dx® analyser performed well in determining the total nucleated cell count and differential cell counts in canine cerebrospinal fluid when compared to a reference standard of external laboratory analysis, except for cell counts higher than ~1000/µL. As the Idexx ProCyte Dx® currently only provides a cell count in 10 cells/µL increments, software modification may improve agreement between the two methods. As in human medicine, automated methods may prove useful in the future for cerebrospinal fluid analysis in addition to manual assessment, particularly in an emergency setting.

In vitro initial stability of a stemless humeral implant.

BACKGROUND: Stemless humeral prostheses have been recently introduced. We measured for the first time their in vitro primary stability and analyzed the influence of three clinically important parameters (bone quality, implant size and post-operative loading) on micromotion. We also assessed if displacement sensors are appropriate to measure implant micromotion. METHODS: A stemless humeral implant (Sidus® Stem-Free Shoulder, Zimmer GmbH, Winterthur, Switzerland) was implanted in 18 cadaveric humeri. Three-dimensional motion of the implant was measured under dynamic loading at three load magnitudes with displacement sensors. Additionally, the relative motion at the bone-implant interface was measured with an optical system in four specimens. RESULTS: Micromotion values derived from the displacement sensors were significantly higher than those measured by the optical system (P<0.005). Analysis of variance (ANOVA) indicated that bone density (P<0.0005) and load (P<0.0001) had a significant effect on implant micromotion, however the effect of implant size was not statistically significant (P=0.123). INTERPRETATION: Micromotion of this stemless design was shown to be significantly dependent on cancellous bone density. Patients must therefore have adequate bone quality for this procedure. The influence of load magnitude on micromotion emphasizes the need for controlled post-operative rehabilitation. Measurements with displacement sensors overestimate true interface micromotion by up to 50% and correction by an optical system is strongly recommended.

Local and Systemic Therapies for Breast Cancer Patients: Reducing Short-term Symptoms with the Methods of Integrative Medicine.

With improved prognosis due to advances in the diagnosis and therapy of breast cancer, physicians and therapists now focus on aspects such as quality of life and the management of side effects from breast cancer treatment. Therapy- and disease-related side effects often reduce the patient's quality of life and can place a further burden on patients, with non-compliance or discontinuation of therapy a potential consequence. Study data have shown that therapy- and disease-related side effects can be reduced using the methods of integrative medicine. Reported benefits include improving patients' wellbeing and quality of life, reducing stress, and improving patients' mood, sleeping patterns and capacity to cope with disease. Examining the impact of integrative medicine on the side effects of cancer treatment would be beyond the scope of this review. This article therefore looks at short-term side effects of cancer treatment which are usually temporary and occur during or after local and systemic therapy. The focus is on mind-body medicine, acupuncture and classic naturopathic treatments developed by Sebastian Kneipp as complementary therapies. The latter includes hydrotherapy, phytotherapy, nutritional therapy, exercise therapy and a balanced lifestyle.

Hepatotoxicosis in dogs consuming a diet of camel meat contaminated with indospicine.

BACKGROUND: Four dogs presented with clinical signs of severe hepatic disease after consuming a commercial camel meat diet. METHODS: Laboratory investigation revealed evidence of severe liver disease, including markedly increased serum alanine aminotransferase (ALT) activity and total bilirubin concentration, and prolonged clotting times. RESULTS: Two dogs deteriorated despite supportive therapy and were euthanased. Histologically, both livers appeared similar, with the main lesion being extensive periacinar necrosis and haemorrhage. Indospicine, a toxic amino acid of plant origin, was detected in the serum and/or plasma from all four dogs, as well as in tissues of a dog that was necropsied and in a sample of the camel meat fed to this animal. Serum biochemistry tests using blood samples collected from 15 additional dogs identified as having eaten the diet detected indospicine was in the serum of 14 and 3 had increased ALT activity. One of the latter dogs subsequently developed clinical signs of severe liver disease and was euthanased. CONCLUSION: To the authors' knowledge, this is the first published report of the detection of indospicine residues in camel meat and the occurrence of severe, sometimes fatal, liver disease in dogs that consumed this contaminated meat.

Presumptive Nocardia spp. infection in a dog treated with cyclosporin and ketoconazole.

CASE HISTORY: A dog that had received 8 months of cyclosporin and ketoconazole therapy for treatment of atopic dermatitis subsequently developed severe neurological disease, that failed to respond to treatment with trimethoprim-sulphadiazine and clindamycin. HISTOPATHOLOGICAL FINDINGS: Histopathological examination of the pulmonary parenchyma and spinal cord revealed loose aggregates of Gram-positive, partially acid-fast, fine, beaded, filamentous bacteria, most consistent with Nocardia spp. DIAGNOSIS: A presumptive diagnosis was made of disseminated nocardiosis of the spinal cord and lungs. CLINICAL RELEVANCE: Nocardia spp. is an opportunistic actinomycete that may cause disseminated disease, particularly in immunocompromised animals. Cyclosporin is used in veterinary medicine to control immune-mediated and allergic disorders, with few reported adverse side effects. This case gives further evidence that involvement of the spinal cord in nocardiosis of the central nervous system (CNS) carries a poor prognosis, and opportunistic infection by Nocardia spp. may be a potential complication of immunosuppressive cyclosporin therapy in the dog.

Computed tomography diagnosis of eight dogs with brain infarction.

OBJECTIVE: Medical records of eight dogs presenting with acute onset of neurological signs and a diagnosis of brain infarction as determined by computed tomography (CT) imaging were reviewed. DESIGN: Retrospective single-centre case review. RESULTS: Ischaemic infarction in the territory of the rostral cerebellar artery was identified in three spaniel-breed dogs. All cerebellar infarcts were non-haemorrhagic. Telencephalic infarcts were identified in five dogs, in the territories of the middle cerebral artery (2/5) and rostral cerebral artery (3/5). One of these dogs had an ischaemic infarction, but all other infarctions appeared haemorrhagic. All dogs were geriatric (≥ 8 years old), with concurrent medical conditions identified in six dogs. One dog was euthanased after diagnosis because of the severity of its neurological signs and one dog was euthanased as a result of associated renal disease 2 months after diagnosis. Six dogs were alive at least 1 year after diagnosis. CONCLUSIONS: CT is useful in the diagnosis of cerebrovascular accident in dogs, which can present as a spectrum of images with early changes in attenuation and subtle mass effects detected after infarction. CT is particularly sensitive for detecting haemorrhagic infarction, but under-represent ischaemic and lacunar infarctions when compared with MRI.

An extended cavity diode-pumped femtosecond Yb:KGW laser for applications in optical DNA sensor technology based on fluorescence lifetime measurements.

A reliable and power-scalable extended cavity diode-pumped passively mode-locked Yb:KGW laser generating ~200 fs long pulses at a repetition rate of 15 MHz was developed and characterized. The laser delivered up to 150 mW of average power at fundamental wavelength of 1040 nm, corresponding to a pulse energy of 10 nJ. The laser radiation was frequency-doubled in a single pass configuration within a nonlinear BIBO crystal to produce femtosecond green radiation at 520 nm with peak power of ~200 W. The generated second harmonic served as excitation source for optical DNA biosensor based on fluorescence lifetime measurements obtained using the time-correlated single photon counting technique.

Trends in the development of nucleic acid biosensors for medical diagnostics.

Some of the recent advances in the field of biosensors for nucleic acid analysis in medical diagnostic applications are highlighted. Particular attention is paid in this review to the progress made in two key areas of development: (i) enhancements achieved in device selectivity, and (ii) enhancements achieved in device sensitivity.

Rapid detection of single nucleotide polymorphisms associated with spinal muscular atrophy by use of a reusable fibre-optic biosensor.

Rapid (<2 min) and quantitative genotyping for single nucleotide polymorphisms (SNPs) associated with spinal muscular atrophy was done using a reusable (approximately 80 cycles of application) fibre-optic biosensor over a clinically relevant range (0-4 gene copies). Sensors were functionalized with oligonucleotide probes immobilized at high density (approximately 7 pmol/cm2) to impart enhanced selectivity for SNP discrimination and used in a total internal reflection fluorescence detection motif to detect 202 bp PCR amplicons from patient samples. Real-time detection may be done over a range of ionic strength conditions (0.1-1.0 M) without stringency rinsing to remove non-selectively bound materials and without loss of selectivity, permitting a means for facile sample preparation. By using the time-derivative of fluorescence intensity as the analytical parameter, linearity of response may be maintained while allowing for significant reductions in analysis time (10-100-fold), permitting for the completion of measurements in under 1 min.

A reusable optical nucleic acid biosensor applied to the rapid detection of single nucleotide polymorphisms associated with Spinal Muscular Atrophy.

Extract: Current methodologies for single nucleotide polymorphism (SNP) screening often require amplification, followed by time-consuming enzymatic manipulation (e.g., digestion or extension) and separation of the resultant products. Advancements in real-time PCR methods permit simultaneous amplification and quantification. Unfortunately, the ability to provide quantitative determinations can be made difficult by factors such as non-specific amplification and alterations in amplification efficiency owing to secondary-structure and sample matrix effects. Newer sensor and microarray technologies attempt to facilitate SNP analysis but usually require several hours for hybridization and data analysis. Furthermore, microarrays often cannot be reused as a result of limitations in the chemistries more routinely employed for nucleic acid immobilization. Difficulties associated with control of homogeneity of probe distribution, probe density and fidelity of the probe sequences (particularly when assembled by in-situ synthesis) can result in variations in the binding energetics of interfacial hybridization from spot to spot on an array, or even within individual array elements. Regions on the substrate may therefore exhibit variations in the selectivity, kinetics and dynamic range of response. The biosensor technology developed by our group presents an alternative to such an approach by use of controlled immobilization methodologies in a sensor format that provides for a reusable and quantitative technology where selectivity and kinetics may be more finely tuned.

The effects of steroids on vestibular compensation and vestibular nucleus neuronal activity in the guinea pig.

Recent studies have suggested that steroids such as dexamethasone and methylprednisolone might be useful in the treatment of vestibular disorders, irrespective of whether inflammatory processes are involved. The aim of this study was to investigate the effects of systemic administration of dexamethasone on vestibular compensation of spontaneous nystagmus (SN) in guinea pig, and the effects of dexamethasone and methylprednisolone on extracellularly recorded spontaneous activity of medial vestibular nucleus (MVN) neurons in brainstem slices in vitro. In the behavioral study, none of the 3 doses of dexamethasone (5, 10, or 40 mg/kg i.p., delivered at 0, 12, 24, and 36 h following a unilateral surgical labyrinthectomy (UL)) resulted in a significant change in the frequency or compensation of SN, relative to the vehicle control group. In the in vitro study, only a minority of MVN neurons showed any response to 1 microM dexamethasone (1 out of 9 neurons), or 10 nM (3 out of 13), or 0.1 microM methylprednisolone (3 out of 7). These results suggest, contrary to previous evidence, that dexamethasone may not accelerate compensation of SN following surgical UL and that dexamethasone and methylprednisolone may have a direct action only on a minority of MVN neurons.