Raman spectroscopy uncovers biochemical tissue-related features of extracellular vesicles from mesenchymal stromal cells.

Extracellular vesicles (EVs) from mesenchymal stromal cells (MSC) are emerging as valuable therapeutic agents for tissue regeneration and immunomodulation, but their clinical applications have so far been limited by the technical restraints of current isolation and characterisation procedures. This study shows for the first time the successful application of Raman spectroscopy as label-free, sensitive and reproducible means of carrying out the routine bulk characterisation of MSC-derived vesicles before their use in vitro or in vivo, thus promoting the translation of EV research to clinical practice. The Raman spectra of the EVs of bone marrow and adipose tissue-derived MSCs were compared with human dermal fibroblast EVs in order to demonstrate the ability of the method to distinguish the vesicles of the three cytotypes automatically with an accuracy of 93.7%. Our data attribute a Raman fingerprint to EVs from undifferentiated and differentiated cells of diverse tissue origin, and provide insights into the biochemical characteristics of EVs from different sources and into the differential contribution of sphingomyelin, gangliosides and phosphatidilcholine to the Raman spectra themselves.

Multiple epitope presentation and surface density control enabled by chemoselective immobilization lead to enhanced performance in IgE-binding fingerprinting on peptide microarrays.

Multiple ligand presentation is a powerful strategy to enhance the affinity of a probe for its corresponding target. A promising application of this concept lies in the analytical field, where surface immobilized probes interact with their corresponding targets in the context of complex biological samples. Here we investigate the effect of multiple epitope presentation (MEP) in the challenging context of IgE-detection in serum samples using peptide microarrays, and evaluate the influence of probes surface density on the assay results. Using the milk allergen alpha-lactalbumin as a model, we have synthesized three immunoreactive epitope sequences in a linear, branched and tandem form and exploited a chemoselective click strategy (CuAAC) for their immobilization on the surface of two biosensors, a microarray and an SPR chip both modified with the same clickable polymeric coating. We first demonstrated that a fine tuning of the surface peptide density plays a crucial role to fully exploit the potential of oriented and multiple peptide display. We then compared the three multiple epitope presentations in a microarray assay using sera samples from milk allergic patients, confirming that a multiple presentation, in particular that of the tandem construct, allows for a more efficient characterization of IgE-binding fingerprints at a statistically significant level. To gain insights on the binding parameters that characterize antibody/epitopes affinity, we selected the most reactive epitope of the series (LAC1) and performed a Surface Plasmon Resonance Imaging (SPRi) analysis comparing different epitope architectures (linear versus branched versus tandem). We demonstrated that the tandem peptide provides an approximately twofold increased binding capacity with respect to the linear and branched peptides, that could be attributed to a lower rate of dissociation (Kd).

Cream formulation impact on topical administration of engineered colloidal nanoparticles.

In order to minimize the impact of systemic toxicity of drugs in the treatment of local acute and chronic inflammatory reactions, the achievement of reliable and efficient delivery of therapeutics in/through the skin is highly recommended. While the use of nanoparticles is now an established practice for drug intravenous targeted delivery, their transdermal penetration is still poorly understood and this important administration route remains almost unexplored. In the present study, we have synthesized magnetic (iron oxide) nanoparticles (MNP) coated with an amphiphilic polymer, developed a water-in-oil emulsion formulation for their topical administration and compared the skin penetration routes with the same nanoparticles deposited as a colloidal suspension. Transmission and scanning electron microscopies provided ultrastructural evidence that the amphiphilic nanoparticles (PMNP) cream formulation allowed the efficient penetration through all the skin layers with a controllable kinetics compared to suspension formulation. In addition to the preferential follicular pathway, also the intracellular and intercellular routes were involved. PMNP that crossed all skin layers were quantified by inductively coupled plasma mass spectrometry. The obtained data suggests that combining PMNP amphiphilic character with cream formulation improves the intradermal penetration of nanoparticles. While PMNP administration in living mice via aqueous suspension resulted in preferential nanoparticle capture by phagocytes and migration to draining lymph nodes, cream formulation favored uptake by all the analyzed dermis cell types, including hematopoietic and non-hematopoietic. Unlike aqueous suspension, cream formulation also favored the maintenance of nanoparticles in the dermal architecture avoiding their dispersion and migration to draining lymph nodes via afferent lymphatics.

Antibody-engineered nanoparticles selectively inhibit mesenchymal cells isolated from patients with chronic lung allograft dysfunction.

AIMS: Chronic lung allograft dysfunction represents the main cause of death after lung transplantation, and so far there is no effective therapy. Mesenchymal cells (MCs) are primarily responsible for fibrous obliteration of small airways typical of chronic lung allograft dysfunction. Here, we engineered gold nanoparticles containing a drug in the hydrophobic section to inhibit MCs, and exposing on the outer hydrophilic surface a monoclonal antibody targeting a MC-specific marker (half-chain gold nanoparticles with everolimus). MATERIALS & METHODS: Half-chain gold nanoparticles with everolimus have been synthesized and incubated with MCs to evaluate the effect on proliferation and apoptosis. RESULTS & DISCUSSION: Drug-loaded gold nanoparticles coated with the specific antibody were able to inhibit proliferation and induce apoptosis without stimulating an inflammatory response, as assessed by in vitro experiments. CONCLUSION: These findings demonstrate the effectiveness of our nanoparticles in inhibiting MCs and open new perspectives for a local treatment of chronic lung allograft dysfunction.

Polymer nanopillar-gold arrays as surface-enhanced Raman spectroscopy substrate for the simultaneous detection of multiple genes.

In our study, 2D nanopillar arrays with plasmonic crystal properties are optimized for surface-enhanced Raman spectroscopy (SERS) application and tested in a biochemical assay for the simultaneous detection of multiple genetic leukemia biomarkers. The special fabrication process combining soft lithography and plasma deposition techniques allows tailoring of the structural and chemical parameters of the crystal surfaces. In this way, it has been possible to tune the plasmonic resonance spectral position close to the excitation wavelength of the monochromatic laser light source in order to maximize the enhancing properties of the substrate. Samples are characterized by scanning electron microscopy and reflectance measurements and tested for SERS activity using malachite green. Besides, as the developed substrate had been prepared on a simple glass slide, SERS detection from the support side is also demonstrated. The optimized substrate is functionalized with thiol-modified capture oligonucleotides, and concentration-dependent signal of the target nucleotide is detected in a sandwich assay with labeled gold nanoparticles. Gold nanoparticles functionalized with different DNA and various Raman reporters are applied in a microarray-based assay recognizing a disease biomarker (Wilms tumor gene) and housekeeping gene expressions in the same time on spatially separated microspots. The multiplexing performance of the SERS-based bioassay is illustrated by distinguishing Raman dyes based on their complex spectral fingerprints.

Antiproliferative effect of ASC-J9 delivered by PLGA nanoparticles against estrogen-dependent breast cancer cells.

Among polymeric nanoparticles designed for cancer therapy, PLGA nanoparticles have become one of the most popular polymeric devices for chemotherapeutic-based nanoformulations against several kinds of malignant diseases. Promising properties, including long-circulation time, enhanced tumor localization, interference with "multidrug" resistance effects, and environmental biodegradability, often result in an improvement of the drug bioavailability and effectiveness. In the present work, we have synthesized 1,7-bis(3,4-dimethoxyphenyl)-5-hydroxyhepta-1,4,6-trien-3-one (ASC-J9) and developed uniform ASC-J9-loaded PLGA nanoparticles of about 120 nm, which have been prepared by a single-emulsion process. Structural and morphological features of the nanoformulation were analyzed, followed by an accurate evaluation of the in vitro drug release kinetics, which exhibited Fickian law diffusion over 10 days. The intracellular degradation of ASC-J9-bearing nanoparticles within estrogen-dependent MCF-7 breast cancer cells was correlated to a time- and dose-dependent activity of the released drug. A cellular growth inhibition associated with a specific cell cycle G2/M blocking effect caused by ASC-J9 release inside the cytosol allowed us to put forward a hypothesis on the action mechanism of this nanosystem, which led to the final cell apoptosis. Our study was accomplished using Annexin V-based cell death analysis, MTT assessment of proliferation, radical scavenging activity, and intracellular ROS evaluation. Moreover, the intracellular localization of nanoformulated ASC-J9 was confirmed by a Raman optical imaging experiment designed ad hoc. PLGA nanoparticles and ASC-J9 proved also to be safe for a healthy embryo fibroblast cell line (3T3-L1), suggesting a possible clinical translation of this potential nanochemotherapeutic to expand the inherently poor bioavailability of hydrophobic ASC-J9 that could be proposed for the treatment of malignant breast cancer.

How many people are able to control a P300-based brain-computer interface (BCI)?

An EEG-based brain-computer system can be used to control external devices such as computers, wheelchairs or Virtual Environments. One of the most important applications is a spelling device to aid severely disabled individuals with communication, for example people disabled by amyotrophic lateral sclerosis (ALS). P300-based BCI systems are optimal for spelling characters with high speed and accuracy, as compared to other BCI paradigms such as motor imagery. In this study, 100 subjects tested a P300-based BCI system to spell a 5-character word with only 5 min of training. EEG data were acquired while the subject looked at a 36-character matrix to spell the word WATER. Two different versions of the P300 speller were used: (i) the row/column speller (RC) that flashes an entire column or row of characters and (ii) a single character speller (SC) that flashes each character individually. The subjects were free to decide which version to test. Nineteen subjects opted to test both versions. The BCI system classifier was trained on the data collected for the word WATER. During the real-time phase of the experiment, the subject spelled the word LUCAS, and was provided with the classifier selection accuracy after each of the five letters. Additionally, subjects filled out a questionnaire about age, sex, education, sleep duration, working duration, cigarette consumption, coffee consumption, and level of disturbance that the flashing characters produced. 72.8% (N=81) of the subjects were able to spell with 100% accuracy in the RC paradigm and 55.3% (N=38) of the subjects spelled with 100% accuracy in the SC paradigm. Less than 3% of the subjects did not spell any character correctly. People who slept less than 8h performed significantly better than other subjects. Sex, education, working duration, and cigarette and coffee consumption were not statistically related to differences in accuracy. The disturbance of the flashing characters was rated with a median score of 1 on a scale from 1 to 5 (1, not disturbing; 5, highly disturbing). This study shows that high spelling accuracy can be achieved with the P300 BCI system using approximately 5 min of training data for a large number of non-disabled subjects, and that the RC paradigm is superior to the SC paradigm. 89% of the 81 RC subjects were able to spell with accuracy 80-100%. A similar study using a motor imagery BCI with 99 subjects showed that only 19% of the subjects were able to achieve accuracy of 80-100%. These large differences in accuracy suggest that with limited amounts of training data the P300-based BCI is superior to the motor imagery BCI. Overall, these results are very encouraging and a similar study should be conducted with subjects who have ALS to determine if their accuracy levels are similar.