Antibody-Loading of Biological Nanocarrier Vesicles Derived from Red-Blood-Cell Membranes.

Antibodies, disruptive potent therapeutic agents against pharmacological targets, face a barrier in crossing immune systems and cellular membranes. To overcome these, various strategies have been explored including shuttling via liposomes or biocamouflaged nanoparticles. Here, we demonstrate the feasibility of loading antibodies into exosome-mimetic nanovesicles derived from human red-blood-cell membranes, which can act as nanocarriers for intracellular delivery. Goat-antichicken antibodies are loaded into erythrocyte-derived nanovesicles, and their loading yields are characterized and compared with smaller dUTP-cargo molecules. Applying dual-color coincident fluorescence burst analyses, the loading yield of nanocarriers is rigorously profiled at the single-vesicle level, overcoming challenges due to size-heterogeneity and demonstrating a maximum antibody-loading yield of 38-41% at the optimal vesicle radius of 52 nm. The achieved average loading yields, amounting to 14% across the entire nanovesicle population, with more than two antibodies per loaded vesicle, are fully comparable to those obtained for the much smaller dUTP molecules loaded in the nanovesicles after additional exosome-spin-column purification. The results suggest a promising new avenue for therapeutic delivery of antibodies, potentially encompassing also intracellular targets and suitable for large-scale pharmacological applications, which relies on the exosome-mimetic properties, biocompatibility, and low-immunogenicity of bioengineered nanocarriers synthesized from human erythrocyte membranes.

Fluorescence Bar-Coding and Flowmetry Based on Dark State Transitions in Fluorescence Emitters.

Reversible dark state transitions in fluorophores represent a limiting factor in fluorescence-based ultrasensitive spectroscopy, are a necessary basis for fluorescence-based super-resolution imaging, but may also offer additional, largely orthogonal fluorescence-based readout parameters. In this work, we analyzed the blinking kinetics of Cyanine5 (Cy5) as a bar-coding feature distinguishing Cy5 from rhodamine fluorophores having largely overlapping emission spectra. First, fluorescence correlation spectroscopy (FCS) solution measurements on mixtures of free fluorophores and fluorophore-labeled small unilamellar vesicles (SUVs) showed that Cy5 could be readily distinguished from the rhodamines by its reversible, largely excitation-driven trans-cis isomerization. This was next confirmed by transient state (TRAST) spectroscopy measurements, determining the fluorophore dark state kinetics in a more robust manner, from how the time-averaged fluorescence intensity varies upon modulation of the applied excitation light. TRAST was then combined with wide-field imaging of live cells, whereby Cy5 and rhodamine fluorophores could be distinguished on a whole cell level as well as in spatially resolved, multiplexed images of the cells. Finally, we established a microfluidic TRAST concept and showed how different mixtures of free Cy5 and rhodamine fluorophores and corresponding fluorophore-labeled SUVs could be distinguished on-the-fly when passing through a microfluidic channel. In contrast to FCS, TRAST does not rely on single-molecule detection conditions or a high time resolution and is thus broadly applicable to different biological samples. Therefore, we expect that the bar-coding concept presented in this work can offer an additional useful strategy for fluorescence-based multiplexing that can be implemented on a broad range of both stationary and moving samples.

Local monitoring of photosensitizer transient states provides feedback for enhanced efficiency and targeting selectivity in photodynamic therapy.

Photodynamic therapy (PDT) fundamentally relies on local generation of PDT precursor states in added photosensitizers (PS), particularly triplet and photo-radical states. Monitoring these states in situ can provide important feedback but is difficult in practice. The states are strongly influenced by local oxygenation, pH and redox conditions, often varying significantly at PDT treatment sites. To overcome this problem, we followed local PDT precursor state populations of PS compounds, via their fluorescence intensity response to systematically varied excitation light modulation. Thereby, we could demonstrate local monitoring of PDT precursor states of methylene blue (MB) and IRdye700DX (IR700), and determined their transitions rates under different oxygenation, pH and redox conditions. By fiber-optics, using one fiber for both excitation and fluorescence detection, the triplet and photo-radical state kinetics of locally applied MB and IR700 could then be monitored in a tissue sample. Finally, potassium iodide and ascorbate were added as possible PDT adjuvants, enhancing intersystem crossing and photoreduction, respectively, and their effects on the PDT precursor states of MB and IR700 could be locally monitored. Taken together, the presented procedure overcomes current methodological limitations and can offer feedback, guiding both excitation and PDT adjuvant application, and thereby more efficient and targeted PDT treatments.

Photoisomerization of Heptamethine Cyanine Dyes Results in Red-Emissive Species: Implications for Near-IR, Single-Molecule, and Super-Resolution Fluorescence Spectroscopy and Imaging.

Photoisomerization kinetics of the near-infrared (NIR) fluorophore Sulfo-Cyanine7 (SCy7) was studied by a combination of fluorescence correlation spectroscopy (FCS) and transient state (TRAST) excitation modulation spectroscopy. A photoisomerized state with redshifted emission was identified, with kinetics consistent with a three-state photoisomerization model. Combining TRAST excitation modulation with spectrofluorimetry (spectral-TRAST) further confirmed an excitation-induced redshift in the emission spectrum of SCy7. We show how this red-emissive photoisomerized state contributes to the blinking kinetics in different emission bands of NIR cyanine dyes, and how it can influence single-molecule, super-resolution, as well as Förster resonance energy transfer (FRET) and multicolor readouts. Since this state can also be populated at moderate excitation intensities, it can also more broadly influence fluorescence readouts, also readouts not relying on high excitation conditions. However, this additional red-emissive state and its photodynamics, as identified and characterized in this work, can also be used as a strategy to push the emission of NIR cyanine dyes further into the NIR and to enhance photosensitization of nanoparticles with absorption spectra further into the NIR. Finally, we show that the photoisomerization kinetics of SCy7 and the formation of its redshifted photoisomer depend strongly on local environmental conditions, such as viscosity, polarity, and steric constraints, which suggests the use of SCy7 and other NIR cyanine dyes as environmental sensors. Such environmental information can be monitored by TRAST, in the NIR, with low autofluorescence and scattering conditions and on a broad range of samples and experimental conditions.

Combined Fluorescence Fluctuation and Spectrofluorometric Measurements Reveal a Red-Shifted, Near-IR Emissive Photo-Isomerized Form of Cyanine 5.

Cyanine fluorophores are extensively used in fluorescence spectroscopy and imaging. Upon continuous excitation, especially at excitation conditions used in single-molecule and super-resolution experiments, photo-isomerized states of cyanines easily reach population probabilities of around 50%. Still, effects of photo-isomerization are largely ignored in such experiments. Here, we studied the photo-isomerization of the pentamethine cyanine 5 (Cy5) by two similar, yet complementary means to follow fluorophore blinking dynamics: fluorescence correlation spectroscopy (FCS) and transient-state (TRAST) excitation-modulation spectroscopy. Additionally, we combined TRAST and spectrofluorimetry (spectral-TRAST), whereby the emission spectra of Cy5 were recorded upon different rectangular pulse-train excitations. We also developed a framework for analyzing transitions between multiple emissive states in FCS and TRAST experiments, how the brightness of the different states is weighted, and what initial conditions that apply. Our FCS, TRAST, and spectral-TRAST experiments showed significant differences in dark-state relaxation amplitudes for different spectral detection ranges, which we attribute to an additional red-shifted, emissive photo-isomerized state of Cy5, not previously considered in FCS and single-molecule experiments. The photo-isomerization kinetics of this state indicate that it is formed under moderate excitation conditions, and its population and emission may thus deserve also more general consideration in fluorescence imaging and spectroscopy experiments.

Saliva Cortisol in Girls With Functional Abdominal Pain Disorders: A Randomized Controlled Dance and Yoga Intervention.

Introduction: Functional abdominal pain disorders (FAPDs) are common among girls and has been associated with stress. Cortisol is one of the major stress hormones. Dance and yoga have been shown to reduce abdominal pain among girls with FAPDs. Aim: To investigate the effect of an 8-month intervention with dance and yoga on cortisol levels in saliva among girls with FAPDs. Methods: A total of 121 girls aged 9-13 years with irritable bowel syndrome (IBS) or functional abdominal pain were included in the study. Participants were randomized into an intervention group and a control group. The intervention group attended a combined dance and yoga session twice a week for 8 months. Saliva samples were collected during 1 day, in the morning and evening, at baseline, and at 4 and 8 months. Subjective pain and stress were assessed as well. Results: No significant effects on saliva cortisol levels between groups were observed after completion of the intervention at 8 months. However, evening cortisol and evening/morning quotient were significantly reduced at 4 months in the intervention group compared to the control group (p = 0.01, p = 0.004). There was no association between cortisol quota and pain or stress. Conclusion: Improvements in cortisol levels were seen in the intervention group at 4 months but did not persist until the end of the study. This indicates that dance and yoga could have a stress-reducing effect during the ongoing intervention.

Coincident Fluorescence-Burst Analysis of the Loading Yields of Exosome-Mimetic Nanovesicles with Fluorescently-Labeled Cargo Molecules.

The possible targeting functionality and low immunogenicity of exosomes and exosome-like nanovesicles make them promising as drug-delivery carriers. To tap into this potential, accurate non-destructive methods to load them and characterize their contents are of utmost importance. However, the small size, polydispersity, and aggregation of nanovesicles in solution make quantitative characterizations of their loading particularly challenging. Here, an ad-hoc methodology is developed based on burst analysis of dual-color confocal fluorescence microscopy experiments, suited for quantitative characterizations of exosome-like nanovesicles and of their fluorescently-labeled loading. It is applied to study exosome-mimetic nanovesicles derived from animal extracellular-vesicles and human red blood cell detergent-resistant membranes, loaded with fluorescently-tagged dUTP cargo molecules. For both classes of nanovesicles, successful loading is proved and by dual-color coincident fluorescence burst analysis, size statistics and loading yields are retrieved and quantified. The procedure affords single-vesicle characterizations well-suited for the investigation of a variety of cargo molecules and biological nanovesicle combinations besides the proof-of-principle demonstrations of this study. The results highlight a powerful characterization tool essential for optimizing the loading process and for advanced engineering of biomimetic nanovesicles for therapeutic drug delivery.

Dance and yoga reduced functional abdominal pain in young girls: A randomized controlled trial.

BACKGROUND: Functional abdominal pain disorders (FAPDs) affect children, especially girls, all over the world. The evidence for existing treatments is mixed, and effective accessible treatments are needed. Dance, a rhythmic cardio-respiratory activity, combined with yoga, which enhances relaxation and focus, may provide physiological and psychological benefits that could help to ease pain. OBJECTIVES: The aim of this study was to evaluate the effect of a dance and yoga intervention on maximum abdominal pain in 9- to 13-year- old girls with FAPDs. METHODS: This study was a prospective randomized controlled trial with 121 participants recruited from outpatient clinics as well as the general public. The intervention group participated in dance and yoga twice weekly for 8 months; controls received standard care. Abdominal pain, as scored on the Faces Pain Scale-Revised, was recorded in a pain diary. A linear mixed model was used to estimate the outcomes and effect sizes. RESULTS: Dance and yoga were superior to standard health care alone, with a medium to high between-group effect size and significantly greater pain reduction (b = -1.29, p = 0.002) at the end of the intervention. CONCLUSIONS: An intervention using dance and yoga is likely a feasible and beneficial complementary treatment to standard health care for 9- to 13-year-old girls with FAPDs. SIGNIFICANCE: FAPDs affect children, especially girls, all over the world. The negative consequences such as absence from school, high consumption of medical care and depression pose a considerable burden on children and their families and effective treatments are needed. This is the first study examining a combined dance/yoga intervention for young girls with FAPDs and the result showed a reduction of abdominal pain. These findings contribute with new evidence in the field of managing FAPDs in a vulnerable target group.

Dance intervention for adolescent girls: Effects on daytime tiredness, alertness and school satisfaction. A randomized controlled trial.

INTRODUCTION: Daytime tiredness is a risk factor for poor health and well-being in the short and long term and is often associated with sleep problems, stress-related mental health complaints and decreased school functioning. This study investigates the effect of an 8 month dance intervention study on daytime tiredness, alertness, sleep duration, sleep quality and school satisfaction. METHODS: Randomized controlled intervention study that included a total of 112 girls aged 13-18 years old with stress-related somatic and mental health problems. Dance intervention with focus on enjoyment were performed semiweekly for 8 months. Questionnaire-based measurements regarding self-reported daytime tiredness, alertness, school satisfaction, sleep duration and quality were evaluated at baseline and at 8-, 12- and 20- month follow-ups. RESULTS: Daytime tiredness decreased significantly in the dance group compared to control group at all follow-ups. Alertness increased significant within the dance group, but there were no significant difference compared to the controls. Significant improvements were found in all sleep quality items within the dance group, between groups only one significant result was found. No conclusive changes in sleep duration were observed. School satisfaction increased significantly in the dance intervention group; however, its significance compared to that of the control group faded after adjustment for differences between groups at baseline. CONCLUSIONS: This study suggests that a dance intervention focusing on enjoyment can be effective in decreasing daytime tiredness. This study also adds to the growing body of evidence for the benefits of using nonpharmacological interventions to decrease stress-related problems among adolescents.

Local redox conditions in cells imaged via non-fluorescent transient states of NAD(P)H.

The autofluorescent coenzyme nicotinamide adenine dinucleotide (NADH) and its phosphorylated form (NADPH) are major determinants of cellular redox balance. Both their fluorescence intensities and lifetimes are extensively used as label-free readouts in cellular metabolic imaging studies. Here, we introduce fluorescence blinking of NAD(P)H, as an additional, orthogonal readout in such studies. Blinking of fluorophores and their underlying dark state transitions are specifically sensitive to redox conditions and oxygenation, parameters of particular relevance in cellular metabolic studies. We show that such dark state transitions in NAD(P)H can be quantified via the average fluorescence intensity recorded upon modulated one-photon excitation, so-called transient state (TRAST) monitoring. Thereby, transitions in NAD(P)H, previously only accessible from elaborate spectroscopic cuvette measurements, can be imaged at subcellular resolution in live cells. We then demonstrate that these transitions can be imaged with a standard laser-scanning confocal microscope and two-photon excitation, in parallel with regular fluorescence lifetime imaging (FLIM). TRAST imaging of NAD(P)H was found to provide additional, orthogonal information to FLIM and allows altered oxidative environments in cells treated with a mitochondrial un-coupler or cyanide to be clearly distinguished. We propose TRAST imaging as a straightforward and widely applicable modality, extending the range of information obtainable from cellular metabolic imaging of NAD(P)H fluorescence.