Effect of pulsed laser parameters on photoacoustic flow cytometry efficiency in vitro and in vivo.

Photoacoustic flow cytometry is one of the most effective approaches to detect "alien" objects in the bloodstream, including circulating tumor cells, blood clots, parasites, and emboli. However, the possibility of detecting high-amplitude signals from these objects against the background of blood depends on the parameters of the laser pulse. So, the dependencies of photoacoustic signals amplitude and number on laser pulse energy (5-150 µJ), pulse length (1, 2, 5 ns), and pulse repetition rate (2, 5, 10 kHz) for the melanoma cells were investigated. First, the PA responses of a melanoma cell suspension in vitro were measured to directly assess the efficiency of converting laser light into an acoustic signal. After it, the same dependence with the developed murine model based on constant rate melanoma cell injection into the animal blood flow was tested. Both in vivo and in vitro experiments show that signal generation efficiency increases with laser pulse energy above 15 µJ. Shorter pulses, especially 1 ns, provide more efficient signal generation as well as higher pulse rates. A higher pulse rate also provides more efficient signal generation, but also leads to overheating of the skin. The results show the limits where the photoacoustic flow cytometry system can be effectively used for the detection of circulating tumor cells in undiluted blood both for in vitro experiments and for in vivo murine models.

Impaired cognitive control moderates the relation between the attribution of incentive salience and severity of consumption in patients with methamphetamine dependence.

BACKGROUND: Cognitive control and the attribution of incentive salience are two key neuropsychological processes proposed to explain substance use disorder (SUD). However, little is known about how they interact to influence the severity of drug use in people with SUD. OBJECTIVE: To determine if cognitive control exerts a moderating effect on the relation between the attribution of salience to drug/reward-related cues and the severity of drug use in SUD cases. METHOD: Sixty-nine SUD cases with methamphetamine as the main drug of consumption were selected and evaluated. Participants performed the Stroop, Go/No-Go, and Flanker tasks to identify a latent cognitive control factor, and the Effort-Expenditure for Reward task, as well as answering the Methamphetamine Incentive Salience Questionnaire to measure the attribution of incentive salience. Severity of drug use was determined by the KMSK scale and an exploratory clinical interview. RESULTS: As expected, higher incentive salience attribution predicted greater severity of methamphetamine use. Unexpectedly, however, we found a moderating effect of impaired cognitive control on the relations between higher incentive salience scores and higher monthly drug use, and between younger age at onset of systematic drug use and higher incentive salience scores. CONCLUSION: Results show the moderating role of cognitive control on the relation between incentive salience attribution and severity of drug use in SUD cases, and help explain the chronic, relapsing nature of addiction, knowledge necessary to develop more precise prevention and treatment strategies.

Targeted Therapy for Glomerulonephritis Using Arterial Delivery of Encapsulated Etanercept.

Complex immunosuppressive therapy is prescribed in medical practice to patients with glomerulonephritis to help them overcome symptoms and prevent chronic renal failure. Such an approach requires long-term systemic administration of strong medications, which causes severe side effects. This work shows the efficiency of polymer capsule accumulation (2.8 ± 0.4 µm) containing labeled etanercept (100 µg per dose) in the kidneys of mice. The comparison of injection into the renal artery and tail vein shows the significant superiority of the intra-arterial administration strategy. The etanercept retention rate of 18% and 8% ID in kidneys was found 1 min and 1 h after injection, respectively. The capsules were predominantly localized in the glomeruli after injection in mice using a model of acute glomerulonephritis. Histological analysis confirmed a significant therapeutic effect only in animals with intra-arterial administration of microcapsules with etanercept. The proposed strategy combines endovascular surgery and the use of polymer microcapsules containing a high molecular weight drug that can be successfully applied to treat a wide range of kidney diseases associated with glomerular pathology.

Effect of Surface Modification of Multifunctional Nanocomposite Drug Delivery Carriers with DARPin on Their Biodistribution In Vitro and In Vivo.

We present a targeted drug delivery system for therapy and diagnostics that is based on a combination of contrasting, cytotoxic, and cancer-cell-targeting properties of multifunctional carriers. The system uses multilayered polymer microcapsules loaded with magnetite and doxorubicin. Loading of magnetite nanoparticles into the polymer shell by freezing-induced loading (FIL) allowed the loading efficiency to be increased 5-fold, compared with the widely used layer-by-layer (LBL) assembly. FIL also improved the photoacoustic signal and particle mobility in a magnetic field gradient, a result unachievable by the LBL alone. For targeted delivery of the carriers to cancer cells, the carrier surface was modified with a designed ankyrin repeat protein (DARPin) directed toward the epithelial cell adhesion molecule (EpCAM). Flow cytometry measurements showed that the DARPin-coated capsules specifically interacted with the surface of EpCAM-overexpressing human cancer cells such as MCF7. In vivo and ex vivo biodistribution studies in FvB mice showed that the carrier surface modification with DARPin changed the biodistribution of the capsules toward epithelial cells. In particular, the capsules accumulated substantially in the lungs─a result that can be effectively used in targeted lung cancer therapy. The results of this work may aid in the further development of the "magic bullet" concept and may bring the quality of personalized medicine to another level.

The Positive Effect of the Rational Addiction Prevention Program (RAPP) on Adolescents with High Risk for Drug Consumption.

The aim of this longitudinal study was to determine the effect of an original prevention program (RAPP) on the behavioral and cognitive characteristics of adolescents with high risk for substance consumption. Seventy-six Mexican adolescents 12-15 years old (38 with high risk (HR) and 38 with low risk (LR)) were selected. RAPP was applied for 3 months. Resilience, social skills, attitudes towards substance use, ability to delay a reward, and inhibitory control were assessed in these adolescents, before and after the RAPP intervention. Both groups improved their scores; however, HR achieved greater changes than LR. Findings suggest that HR have behavioral characteristics that can be considered as risk factors for substance consumption (low levels of resilience, low social skills, little family support, positive attitudes towards substance use). RAPP proved to be an effective program for preventing these risk factors for substance use in adolescents.

Patterned Drug-Eluting Coatings for Tracheal Stents Based on PLA, PLGA, and PCL for the Granulation Formation Reduction: In Vivo Studies.

Expandable metallic stent placement is often the only way to treat airway obstructions. Such treatment with an uncoated stent causes granulation proliferation and subsequent restenosis, resulting in the procedure's adverse complications. Systemic administration of steroids drugs in high dosages slows down granulation tissue overgrowth but leads to long-term side effects. Drug-eluting coatings have been used widely in cardiology for many years to suppress local granulation and reduce the organism's systemic load. Still, so far, there are no available analogs for the trachea. Here, we demonstrate that PLA-, PCL- and PLGA-based films with arrays of microchambers to accommodate therapeutic substances can be used as a drug-eluting coating through securely fixing on the surface of an expandable nitinol stent. PCL and PLA were most resistant to mechanical damage associated with packing in delivery devices and making it possible to keep high-molecular-weight cargo. Low-molecular-weight methylprednisolone sodium succinate is poorly retained in PCL- and PLGA-based microchambers after immersion in deionized water (only 9.5% and 15.7% are left, respectively). In comparison, PLA-based microchambers retain 96.3% after the same procedure. In vivo studies on rabbits have shown that effective granulation tissue suppression is achieved when PLA and PLGA are used for coatings. PLGA-based microchamber coating almost completely degrades in 10 days in the trachea, while PLA-based microchamber films partially preserve their structure. The PCL-based film coating is most stable over time, which probably causes blocking the outflow of fluid from the tracheal mucosa and the aggravation of the inflammatory process against the background of low drug concentration. Combination and variability of polymers in the fabrication of films with microchambers to retain therapeutic compounds are suggested as a novel type of drug-eluting coating.

Indocyanine green dye based bimodal contrast agent tested by photoacoustic/fluorescence tomography setup.

Multimodal imaging systems are in high demand for preclinical research, experimental medicine, and clinical practice. Combinations of photoacoustic technology with other modalities including fluorescence, ultrasound, MRI, OCT have been already applied in feasibility studies. Nevertheless, only the combination of photoacoustics with ultrasound in a single setup is commercially available now. A combination of photoacoustics and fluorescence is another compelling approach because those two modalities naturally complement each other. Here, we presented a bimodal contrast agent based on the indocyanine green dye (ICG) as a single signalling compound embedded in the biocompatible and biodegradable polymer shell. We demonstrate its remarkable characteristics by imaging using a commercial photoacoustic/fluorescence tomography system (TriTom, PhotoSound Technologies). It was shown that photoacoustic signal of the particles depends on the amount of dye loaded into the shell, while fluorescence signal depends on the total amount of dye per particle. For the first time to our knowledge, a commercial bimodal photoacoustic/fluorescence setup was used for characterization of ICG doped polymer particles. Additionally, we conducted cell toxicity studies for these particles as well as studied biodistribution over time in vivo and ex vivo using fluorescent imaging. The obtained results suggest a potential for the application of biocompatible and biodegradable bimodal contrast agents as well as the integrated photoacoustic/fluorescence imaging system for preclinical and clinical studies.

Dynamic blood flow phantom for in vivo liquid biopsy standardization.

In vivo liquid biopsy, especially using the photoacoustic (PA) method, demonstrated high clinical potential for early diagnosis of deadly diseases such as cancer, infections, and cardiovascular disorders through the detection of rare circulating tumor cells (CTCs), bacteria, and clots in the blood background. However, little progress has been made in terms of standardization of these techniques, which is crucial to validate their high sensitivity, accuracy, and reproducibility. In the present study, we addressed this important demand by introducing a dynamic blood vessel phantom with flowing mimic normal and abnormal cells. The light transparent silica microspheres were used as white blood cells and platelets phantoms, while hollow polymeric capsules, filled with hemoglobin and melanin, reproduced red blood cells and melanoma CTCs, respectively. These phantoms were successfully used for calibration of the PA flow cytometry platform with high-speed signal processing. The results suggest that these dynamic cell flow phantoms with appropriate biochemical, optical, thermal, and acoustic properties can be promising for the establishment of standardization tool for calibration of PA, fluorescent, Raman, and other detection methods of in vivo flow cytometry and liquid biopsy.

"Smart" Polylactic Acid Films with Ceftriaxone Loaded Microchamber Arrays for Personalized Antibiotic Therapy.

Bacterial infections are a severe medical problem, especially in traumatology, orthopedics, and surgery. The local use of antibiotics-elution materials has made it possible to increase the effectiveness of acute infections treatment. However, the infection prevention problem remains unresolved. Here, we demonstrate the fabrication of polylactic acid (PLA) "smart" films with microchamber arrays. These microchambers contain ceftriaxone as a payload in concentrations ranging from 12 ± 1 µg/cm2 to 38 ± 8 µg/cm2, depending on the patterned film thickness formed by the different PLA concentrations in chloroform. In addition, the release profile of the antibiotic can be prolonged up to 72 h in saline. At the same time, on the surface of agar plates, the antibiotic release time increases up to 96 h, which has been confirmed by the growth suppression of the Staphylococcus aureus bacteria. The efficient loading and optimal release rate are obtained for patterned films formed by the 1.5 wt % PLA in chloroform. The films produced from 1.5 and 2 wt % PLA solutions (thickness-0.42 ± 0.12 and 0.68 ± 0.16 µm, respectively) show an accelerated ceftriaxone release upon the trigger of the therapeutic ultrasound, which impacted as an expansion of the bacterial growth inhibition zone around the samples. Combining prolonged drug elution with the on-demand release ability of large cargo amount opens up new approaches for personalized and custom-tunable antibacterial therapy.

In Vitro Bioeffects of Polyelectrolyte Multilayer Microcapsules Post-Loaded with Water-Soluble Cationic Photosensitizer.

Microencapsulation and targeted delivery of cytotoxic and antibacterial agents of photodynamic therapy (PDT) improve the treatment outcomes for infectious diseases and cancer. In many cases, the loss of activity, poor encapsulation efficiency, and inadequate drug dosing hamper the success of this strategy. Therefore, the development of novel and reliable microencapsulated drug formulations granting high efficacy is of paramount importance. Here we report the in vitro delivery of a water-soluble cationic PDT drug, zinc phthalocyanine choline derivative (Cholosens), by biodegradable microcapsules assembled from dextran sulfate (DS) and poly-l-arginine (PArg). A photosensitizer was loaded in pre-formed [DS/PArg]4 hollow microcapsules with or without exposure to heat. Loading efficacy and drug release were quantitatively studied depending on the capsule concentration to emphasize the interactions between the DS/PArg multilayer network and Cholosens. The loading data were used to determine the dosage for heated and intact capsules to measure their PDT activity in vitro. The capsules were tested using human cervical adenocarcinoma (HeLa) and normal human dermal fibroblast (NHDF) cell lines, and two bacterial strains, Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli. Our results provide compelling evidence that encapsulated forms of Cholosens are efficient as PDT drugs for both eukaryotic cells and bacteria at specified capsule-to-cell ratios.

Detection of Rare Objects by Flow Cytometry: Imaging, Cell Sorting, and Deep Learning Approaches.

Flow cytometry nowadays is among the main working instruments in modern biology paving the way for clinics to provide early, quick, and reliable diagnostics of many blood-related diseases. The major problem for clinical applications is the detection of rare pathogenic objects in patient blood. These objects can be circulating tumor cells, very rare during the early stages of cancer development, various microorganisms and parasites in the blood during acute blood infections. All of these rare diagnostic objects can be detected and identified very rapidly to save a patient's life. This review outlines the main techniques of visualization of rare objects in the blood flow, methods for extraction of such objects from the blood flow for further investigations and new approaches to identify the objects automatically with the modern deep learning methods.

Enabling magnetic resonance imaging of hollow-core microstructured optical fibers via nanocomposite coating.

Optical fibers are widely used in bioimaging systems as flexible endoscopes that are capable of low-invasive penetration inside hollow tissue cavities. Here, we report on the technique that allows magnetic resonance imaging (MRI) of hollow-core microstructured fibers (HC-MFs), which paves the way for combing MRI and optical bioimaging. Our approach is based on layer-by-layer assembly of oppositely charged polyelectrolytes and magnetite nanoparticles on the inner core surface of HC-MFs. Incorporation of magnetite nanoparticles into polyelectrolyte layers renders HC-MFs visible for MRI and induces the red-shift in their transmission spectra. Specifically, the transmission shifts up to 60 nm have been revealed for the several-layers composite coating, along with the high-quality contrast of HC-MFs in MRI scans. Our results shed light on marrying fiber-based endoscopy with MRI to open novel possibilities for minimally invasive clinical diagnostics and surgical procedures in vivo.

Influence of Heat Treatment on Loading of Polymeric Multilayer Microcapsules with Rhodamine B.

Layer-by-layer assembled polymeric multilayer capsules (PMC) of micrometer sizes are permeable for molecules below 1 KDa; therefore, the efficacy of such capsules in the delivery of low molecular weight water soluble bioactive compounds and drugs is frequently challenged. Thermally induced contraction of hollow PMC is explored here to enhance their loading efficacy with model compound, fluorescent rhodamine B (RhB). Four bilayered capsules obtained of poly(diallyldimethylammonium chloride)/polystyrene sulfonate ([PDADMAC/PSS]4 ) or poly-l-arginine/dextran sulfate ([PARG/DS]4 ) on sacrificial CaCO3 spherical microparticles are postloaded with RhB at ambient or elevated temperatures. The influence of heat on capsule loading is determined quantitatively by varying the amounts of capsules in the batch and keeping the concentration of RhB constant. The applied heat improves the loading efficacy of [PDADMAC/PSS]4 capsules at concentrations up to 2.25 × 109 capsules mL-1 , but has a reversed effect on [PARG/DS]4 capsules at all studied concentrations ((0-3.5) × 109 capsules mL-1 ).

Effect of a Controlled Release of Epinephrine Hydrochloride from PLGA Microchamber Array: In Vivo Studies.

This paper presents the synthesis of highly biocompatible and biodegradable poly(lactide- co-glycolide) (PLGA) microchamber arrays sensitive to low-intensity therapeutic ultrasound (1 MHz, 1-2 W, 1 min). A reliable method was elaborated that allowed the microchambers to be uniformly filled with epinephrine hydrochloride (EH), with the possibility of varying the cargo amount. The maximum load of EH was 4.5 µg per array of 5 mm × 5 mm (about 24 pg of EH per single microchamber). A gradual, spontaneous drug release was observed to start on the first day, which is especially important in the treatment of acute patients. Ultrasound triggered a sudden substantial release of EH from the films. In vivo real-time studies using a laser speckle contrast imaging system demonstrated changes in the hemodynamic parameters as a consequence of EH release under ultrasound exposure. We recorded a decrease in blood flow as a vascular response to EH release from a PLGA microchamber array implanted subcutaneously in a mouse. This response was immediate and delayed (1 and 2 days after the implantation of the array). The PLGA microchamber array is a new, promising drug depot implantable system that is sensitive to external stimuli.

Differential Improvement of Executive Functions During Abstinence in Cocaine-Dependent Patients: A Longitudinal Study.

BACKGROUND: In substance abusers, deficits in executive functioning (EF) are relevant to understanding the behavioral regulation of substance consumption and the failure to remain abstinent. OBJECTIVE: To determine the course of EF impairment, measured with traditional and ecological instruments, after 1 and 3 months of abstinence in patients with cocaine dependence. METHODS: 26 cocaine-only-dependent in-patients (cocaine-dependent group, CDG) and 24 nondependent controls (control group, CG) recruited from a typical population were assessed using several EF tests and questionnaires [Stroop, verbal and graphic fluency, Wisconsin Card-Sorting Test (WCST), Tower of London (TOL), and Behavior Rating Inventory of Executive Function-Adult Version (BRIEF-A)] after three periods of abstinence: 1 week, 1 month, and 3 months. RESULTS: At 1 week (baseline), CDG underperformed CG on most EF measures (WCST categories completed; TOL-total number of moves, execution time, and rule violations; and various BRIEF-A subscales). At 1 month, CDG moderately improved its performance on several measures, meanwhile the improvement in the 12 patients who would eventually complete the 3-month abstinence proved particularly significant when compared to their baseline values. In the a posteriori comparison of the baseline values of noncompleters versus completers, scores for the former were lower on the EF tasks, but higher on the BRIEF-A. Both groups, showed significantly worse scores than CG on both instruments. CONCLUSIONS/IMPORTANCE: Cocaine-dependent patients showed marked difficulties in the EF associated mainly with adaptive behavior to their environment. Cocaine-dependent patients with better EF at baseline improved significantly during abstinence and had better treatment adherence. Results suggest that executive improvement and treatment adherence in cocaine abusers in abstinence is related to intrinsic cognitive characteristics of patients.

The relationship between cognitive reserve and the clinical stage of HIV infection.

The objective of this study was to determine whether the effect of cognitive reserve (CR) on neuropsychological functioning differs according to the clinical stage of HIV infection. A sample of 34 HIV-positive individuals aged 23-49, with a minimum of 9 years of formal education, was assessed. Participants were grouped according to the Centers for Disease Control and Prevention's (CDC) clinical stages (A = 10, B = 16, C = 8). CR was calculated for each clinical stage group in accordance with estimates of premorbid IQ, years of education, and occupational attainment. The sum of these three variables was then transformed into z-scores. Individuals above the median were classified as having "High" CR (HCR), those below the median were classified as "Low" CR (LCR). Participants completed an evaluation of cognitive and executive functions based on selected, modified tasks from the HIV University of Miami Annotated Neuropsychological test in Spanish (HUMANS). Assessment included the following domains: attention, memory (visual, verbal, and working memory), executive functions (cognitive flexibility, switching), language (naming), and visual constructive skills (block design). HCR outperformed LCR in all cognitive domains. Comparison of HCR and LCR in each clinical stage revealed that the effect of CR was stronger in stage B than in stages A and C, suggesting that this effect does indeed vary among stages.

Composite SERS-based satellites navigated by optical tweezers for single cell analysis.

Herein, we have designed composite SERS-active micro-satellites, which exhibit a dual role: (i) effective probes for determining cellular composition and (ii) optically movable and easily detectable markers. The satellites were synthesized by the layer-by-layer assisted decoration of silica microparticles with metal (gold or silver) nanoparticles and astralen in order to ensure satellite SERS-based microenvironment probing and satellite recognition, respectively. A combination of optical tweezers and Raman spectroscopy can be used to navigate the satellites to a certain cellular compartment and probe the intracellular composition following cellular uptake. In the future, this developed approach may serve as a tool for single cell analysis with nanometer precision due to the multilayer surface design, focusing on both extracellular and intracellular studies.

Optical heating and temperature determination of core-shell gold nanoparticles and single-walled carbon nanotube microparticles.

The real-time temperature measurement of nanostructured materials is particularly attractive in view of increasing needs of local temperature probing with high sensitivity and resolution in nanoelectronics, integrated photonics, and biomedicine. Light-induced heating and Raman scattering of single-walled carbon nanotubes with adsorbed gold nanoparticles decorating silica microparticles are reported, by both green and near IR lasers. The plasmonic shell is used as nanoheater, while the single-walled carbon nanotubes are Raman active and serve as a thermometer. Stokes and Anti-Stokes Raman spectra of single-walled carbon nanotubes serve to estimate the effective light-induced temperature rise on the metal nanoparticles. The temperature rise is constant with time, indicating stability of the adsorption density. The effective temperatures derived from Stokes and Anti-Stokes intensities are correlated with those measured in a heating stage. The resolution of the thermal experiments in our study was found to be 5-40 K.

Cognitive and behavioral phenotype of a young man with a chromosome 13 deletion del(13)(q21.32q31.1).

Cognitive, emotional, and behavioral characterizations have been reported for patients with a few chromosomal imbalances, but not for patients with a 13q deletion. We report the neuropsychological profile and specific linguistic, visual, spatial, constructional, and behavioral disabilities of a young man with a de novo chromosome 13 deletion (13)(q21.32)(q31.1). Karyotyping at 550 G-band resolution showed that the patient's parents did not share the deletion. According to array-comparative genomic hybridization, the deletion spanned about 14 Mb and included 27 genes. A fluorescence in situ hybridization assay revealed an intact 13q telomere on the partially deleted chromosome. The patient had multiple morphologic and ophthalmologic anomalies. A brain magnetic resonance imaging study did not show gross brain defects. Neuropsychological testing showed an acceptable use of everyday language, but mild mental retardation, executive dysfunction, and very poor performance on visual, visuospatial, and constructional tasks. Establishing a neuropsychological profile for a patient with a specific genetic defect can help clinicians, parents, and teachers work to meet the patient's medical, academic, and behavioral needs.

Gender differences in cognitive development.

The potential effect of gender on intellectual abilities remains controversial. The purpose of this research was to analyze gender differences in cognitive test performance among children from continuous age groups. For this purpose, the normative data from 7 domains of the newly developed neuropsychological test battery, the Evaluación Neuropsicológica Infantil [Child Neuropsychological Assessment] (Matute, Rosselli, Ardila, & Ostrosky-Solis, 2007), were analyzed. The sample included 788 monolingual children (350 boys, 438 girls) ages 5 to 16 years from Mexico and Colombia. Gender differences were observed in oral language (language expression and language comprehension), spatial abilities (recognition of pictures seen from different angles), and visual (Object Integration Test) and tactile perceptual tasks, with boys outperforming girls in most cases, except for the tactile tasks. Gender accounted for only a very small percentage of the variance (1%-3%). Gender x Age interactions were observed for the tactile tasks only. It was concluded that gender differences during cognitive development are minimal, appear in only a small number of tests, and account for only a low percentage of the score variance.