Folate-mediated macromolecule delivery across the epithelium

Folate uses the natural endocytosis pathway via the folate receptor (FR) to enter the cells. Folate conjugation to small or macromolecular therapeutics has hence been exploited for intracellular delivery to, particularly, cancerous cells. This work reports on the expression and functionality of FR in polarised cell monolayer models of respiratory and gastrointestinal mucosa with the view to assess its potential for delivery of folate-modified macromolecular therapeutics either intracellularly or across the epithelium. Four cell lines representing bronchial and intestinal epithelium; cancer-derived intestinal Caco-2 and bronchial cell line Calu-3, and noncancerous intestinal and bronchial cell lines IEC-6 and HBEC were cultured on permeable membranes to produce polarised monolayers. Expression of FR was confirmed by RT-PCR and Western blot analysis for all the tested cell types and shown to be dependent on culturing time. The functionality of the receptor for endocytosis was demonstrated by a model macromolecular folate conjugate (fluorescent ovalbumin-folate (OVA-FA)), whereby significantly higher cellular uptake of the folate-conjugate, relative to non-folate control, was clearly demonstrated. Importantly the data showed that the expressed folate receptor was capable of mediating transport of the macromolecular folate conjugate across (transcytosis) the cells in the polarised monolayers. Preliminary studies led to investigation of the folate mediated uptake and transport of folate modified nanoparticles (NPs). It was shown that folate modified NPs traversed the Calu-3 layers and studies characterizing this transport indicated folate involvement in this process. Adsorption of OVA-FA on the surface of NPs was seen to promote their cellular uptake and transport across the cell layers. To examine the mechanism of cellular uptake and transport of folate modified nanoparticles, various endocytic inhibitors were employed. The study demonstrated an involvement of the caveolar pathway in internalization of folate modified nanoparticles; as judged from a significant reduction of internalization in filipin (inhibitor of caveolar pathway) treated cells. Moreover, the work also showed evidence of transport of folate-modified nanoparticles via the caveolar pathway, since translocation of nanoparticles across the cell monolayer was absent when this path was inhibited. Disruption of actin filament and microtubules caused no difference in cellular uptake of NPs but increased the transcytosis of folate modified NPs. Confocal microscopy, Transmission Electron Microscopy (TEM), Total Internal Reflection Microscopy (TIRM) and Total Internal Reflection Florescence microscopy (TIRFM) were used to confirm and visualize quantitative data. This study also investigated the effects of surface ligand distribution pattern (ligand clustering and density) on the internalization of nanoparticles by Calu-3 cells cultured as polarised layers. The density of the displayed ligand was manipulated by controlling the conjugation level of folate-ovalbumin, while ligand clustering was achieved by co-adsorption of varying mixtures of folate-ovalbumin conjugate (at different ligand density levels) and unconjugated ovalbumin. Increasing ligand density on the nanoparticle surface resulted in increased internalization of modified nanoparticles by the cells, up to a saturation level. Surface ligand density also affected the cellular uptake pathway; from predominantly clathrin to predominantly caveolae-mediated as the ligand density was increased. It was further demonstrated that surface clustering of the folate ligand enhanced cellular internalization of nanoparticles, relative to its dispersed surface distribution

Folate-mediated macromolecule delivery across the epithelium

Folate uses the natural endocytosis pathway via the folate receptor (FR) to enter the cells. Folate conjugation to small or macromolecular therapeutics has hence been exploited for intracellular delivery to, particularly, cancerous cells. This work reports on the expression and functionality of FR in polarised cell monolayer models of respiratory and gastrointestinal mucosa with the view to assess its potential for delivery of folate-modified macromolecular therapeutics either intracellularly or across the epithelium. Four cell lines representing bronchial and intestinal epithelium; cancer-derived intestinal Caco-2 and bronchial cell line Calu-3, and noncancerous intestinal and bronchial cell lines IEC-6 and HBEC were cultured on permeable membranes to produce polarised monolayers. Expression of FR was confirmed by RT-PCR and Western blot analysis for all the tested cell types and shown to be dependent on culturing time. The functionality of the receptor for endocytosis was demonstrated by a model macromolecular folate conjugate (fluorescent ovalbumin-folate (OVA-FA)), whereby significantly higher cellular uptake of the folate-conjugate, relative to non-folate control, was clearly demonstrated. Importantly the data showed that the expressed folate receptor was capable of mediating transport of the macromolecular folate conjugate across (transcytosis) the cells in the polarised monolayers. Preliminary studies led to investigation of the folate mediated uptake and transport of folate modified nanoparticles (NPs). It was shown that folate modified NPs traversed the Calu-3 layers and studies characterizing this transport indicated folate involvement in this process. Adsorption of OVA-FA on the surface of NPs was seen to promote their cellular uptake and transport across the cell layers. To examine the mechanism of cellular uptake and transport of folate modified nanoparticles, various endocytic inhibitors were employed. The study demonstrated an involvement of the caveolar pathway in internalization of folate modified nanoparticles; as judged from a significant reduction of internalization in filipin (inhibitor of caveolar pathway) treated cells. Moreover, the work also showed evidence of transport of folate-modified nanoparticles via the caveolar pathway, since translocation of nanoparticles across the cell monolayer was absent when this path was inhibited. Disruption of actin filament and microtubules caused no difference in cellular uptake of NPs but increased the transcytosis of folate modified NPs. Confocal microscopy, Transmission Electron Microscopy (TEM), Total Internal Reflection Microscopy (TIRM) and Total Internal Reflection Florescence microscopy (TIRFM) were used to confirm and visualize quantitative data. This study also investigated the effects of surface ligand distribution pattern (ligand clustering and density) on the internalization of nanoparticles by Calu-3 cells cultured as polarised layers. The density of the displayed ligand was manipulated by controlling the conjugation level of folate-ovalbumin, while ligand clustering was achieved by co-adsorption of varying mixtures of folate-ovalbumin conjugate (at different ligand density levels) and unconjugated ovalbumin. Increasing ligand density on the nanoparticle surface resulted in increased internalization of modified nanoparticles by the cells, up to a saturation level. Surface ligand density also affected the cellular uptake pathway; from predominantly clathrin to predominantly caveolae-mediated as the ligand density was increased. It was further demonstrated that surface clustering of the folate ligand enhanced cellular internalization of nanoparticles, relative to its dispersed surface distribution

Thin-film transducers for the detection and imaging of Brillouin oscillations in transmission on cultured cells

Mechanical imaging and characterisation of biological cells has been a subject of interest for the last twenty years. Ultrasonic imaging based on the scanning acoustic microscope (SAM) and mechanical probing have been extensively reported. Large acoustic attenuation at high frequencies and the use of conventional piezo-electric transducers limit the operational frequency of a SAM. This limitation results in lower resolution compared to an optical microscope. Direct mechanical probing in the form of applied stress by contacting probes causes stress to cells and exhibits poor depth resolution. More recently, laser ultrasound has been reported to detect ultrasound in the GHz range via Brillouin oscillations on biological cells. This technique offers a promising new high resolution acoustic cell imaging technique. In this work, we propose, design and apply a thin-film based opto-acoustic transducer for the detection in transmission of Brillouin oscillations on cells. The transducer is used to generate acoustic waves, protect the cells from laser radiation and enhance signal-to-noise ratio (SNR). Experimental traces are presented in water films as well as images of the Brillouin frequency of phantom and fixed 3T3 fibroblast cells

Sub-100 nm resolution microscopy based on proximity projection grating scheme

Structured illumination microscopy (SIM) has been widely used in life science imaging applications. The maximum resolution improvement of SIM, compared to conventional bright field system is a factor of 2. Here we present an approach to structured illumination microscopy using the proximity projection grating scheme (PPGS), which has the ability to further enhance the SIM resolution without invoking any nonlinearity response from the sample. With the PPGS-based SIM, sub-100 nm resolution has been obtained experimentally, and results corresponding to 2.4 times resolution improvement are presented. Furthermore, it will be shown that an improvement of greater than 3 times can be achieved

Non destructive evaluation of biological cells

© 2019 Author(s). Regenerative medicine promises to be the next revolution in health care. This technology, which will be the first systematic manufacturing of biological parts for human consumption, requires non destructive evaluatioin (NDE) thechniques for the inspection and quality assurance of products such as tissue and organs. Ultrasound is the technology of choice due to its low invasiveness however state of the art is limited in resolution and not able to inspect single cells. In this paper we present a novel NDE technique applicable for single cells that uses sound, offers contrast provided by mechanical properties, does not require toxic chemical labels and can achieve optical or higher resolution. This will be required for tissue manufacturing as the current technologies used for research on the life-sciences heavily depends on toxic chemicals and radiation. This will stablish ultrasound as a single platform for the inspection of biological products at all scales

Cell imaging by phonon microscopy: sub-optical wavelength ultrasound for non-invasive imaging

The mechanical properties of cells play an important role in cell function and behavior. This paper presents recent developments that have enabled the use of laser-generated phonons (ultrasound) with sub-optical wavelengths to look inside living cells. The phonons reveal contrast from changes in the elasticity of the cell and can provide high resolution three dimensional images

Thin-film transducers for the detection and imaging of Brillouin oscillations in transmission on cultured cells

Mechanical imaging and characterisation of biological cells has been a subject of interest for the last twenty years. Ultrasonic imaging based on the scanning acoustic microscope (SAM) and mechanical probing have been extensively reported. Large acoustic attenuation at high frequencies and the use of conventional piezo-electric transducers limit the operational frequency of a SAM. This limitation results in lower resolution compared to an optical microscope. Direct mechanical probing in the form of applied stress by contacting probes causes stress to cells and exhibits poor depth resolution. More recently, laser ultrasound has been reported to detect ultrasound in the GHz range via Brillouin oscillations on biological cells. This technique offers a promising new high resolution acoustic cell imaging technique. In this work, we propose, design and apply a thin-film based opto-acoustic transducer for the detection in transmission of Brillouin oscillations on cells. The transducer is used to generate acoustic waves, protect the cells from laser radiation and enhance signal-to-noise ratio (SNR). Experimental traces are presented in water films as well as images of the Brillouin frequency of phantom and fixed 3T3 fibroblast cells

Prevalence, Related Factors, and Levels of Burnout Syndrome Among Nurses Working in Gynecology and Obstetrics Services: A Systematic Review and Meta-Analysis

Background: Although burnout levels and the corresponding risk factors have been studied in many nursing services, to date no meta-analytical studies have been undertaken of obstetrics and gynecology units to examine the heterogeneity of burnout in this environment and the variables associated with it. In the present paper, we aim to determine the prevalence, levels, and related factors of burnout syndrome among nurses working in gynecology and obstetrics services. Methods: A systematic review and meta-analysis of the literature were carried out using the following sources: CINAHL (Cumulative Index of Nursing and Allied Health Literature), LILACS (Latin American and Caribbean Health Sciences Literature), Medline, ProQuest (Proquest Health and Medical Complete), SciELO (Scientific Electronic Library Online), and Scopus. Results: Fourteen relevant studies were identified, including, for this meta-analysis, n = 464 nurses. The following prevalence values were obtained: emotional exhaustion 29% (95% CI: 11–52%), depersonalization 19% (95% CI: 6–38%), and low personal accomplishment 44% (95% CI: 18–71%). The burnout variables considered were sociodemographic (age, marital status, number of children, gender), work-related (duration of the workday, nurse-patient ratio, experience or number of miscarriages/abortions), and psychological (anxiety, stress, and verbal violence). Conclusion: Nurses working in obstetrics and gynecology units present high levels of burnout syndrome. In over 33% of the study sample, at least two of the burnout dimensions considered are apparent.This research was funded by the Excellence Research Project (P11HUM-7771) provided by the Andalusian Government (Spain)

Picosecond ultrasonics for elasticity-based imaging and characterization of biological cells

© 2020 Author(s). Characterization of the elasticity of biological cells is growing as a new way to gain insight into cell biology. Cell mechanics are related to most aspects of cellular behavior, and applications in research and medicine are broad. Current methods are often limited since they require physical contact or lack resolution. From the methods available for the characterization of elasticity, those relying on high frequency ultrasound (phonons) are the most promising because they offer label-free, high (even super-optical) resolution and compatibility with conventional optical microscopes. In this Perspective contribution, we review the state of the art of picosecond ultrasonics for cell imaging and characterization, particularly for Brillouin scattering-based methods, offering an opinion for the challenges faced by the technology. The challenges are separated into biocompatibility, acquisition speed, resolution, and data interpretation and are discussed in detail along with new results

Folate-mediated macromolecule delivery across the epithelium

Folate uses the natural endocytosis pathway via the folate receptor (FR) to enter the cells. Folate conjugation to small or macromolecular therapeutics has hence been exploited for intracellular delivery to, particularly, cancerous cells. This work reports on the expression and functionality of FR in polarised cell monolayer models of respiratory and gastrointestinal mucosa with the view to assess its potential for delivery of folate-modified macromolecular therapeutics either intracellularly or across the epithelium. Four cell lines representing bronchial and intestinal epithelium; cancer-derived intestinal Caco-2 and bronchial cell line Calu-3, and non- cancerous intestinal and bronchial cell lines IEC-6 and HBEC were cultured on permeable membranes to produce polarised monolayers. Expression of FR was confirmed by RT-PCR and Western blot analysis for all the tested cell types and shown to be dependent on culturing time. The functionality of the receptor for endocytosis was demonstrated by a model macromolecular folate conjugate (fluorescent ovalbumin-folate (OVA-FA)), whereby significantly higher cellular uptake of the folate-conjugate, relative to non-folate control, was clearly demonstrated. Importantly the data showed that the expressed folate receptor was capable of mediating transport of the macromolecular folate conjugate across (transcytosis) the cells in the polarised monolayers. Preliminary studies led to investigation of the folate mediated uptake and transport of folate modified nanoparticles (NPs). It was shown that folate modified NPs traversed the Calu-3 layers and studies characterizing this transport indicated folate involvement in this process. Adsorption of OVA-FA on the surface of NPs was seen to promote their cellular uptake and transport across the cell layers. To examine the mechanism of cellular uptake and transport of folate modified nanoparticles, various endocytic inhibitors were employed. The study demonstrated an involvement of the caveolar pathway in internalization of folate modified nanoparticles; as judged from a significant reduction of internalization in filipin (inhibitor of caveolar pathway) treated cells. Moreover, the work also showed evidence of transport of folate-modified nanoparticles via the caveolar pathway, since translocation of nanoparticles across the cell monolayer was absent when this path was inhibited. Disruption of actin filament and microtubules caused no difference in cellular uptake of NPs but increased the transcytosis of folate modified NPs. Confocal microscopy, Transmission Electron Microscopy (TEM), Total Internal Reflection Microscopy (TIRM) and Total Internal Reflection Florescence microscopy (TIRFM) were used to confirm and visualize quantitative data. This study also investigated the effects of surface ligand distribution pattern (ligand clustering and density) on the internalization of nanoparticles by Calu-3 cells cultured as polarised layers. The density of the displayed ligand was manipulated by controlling the conjugation level of folate-ovalbumin, while ligand clustering was achieved by eo-adsorption of varying mixtures of folate-ovalbumin conjugate (at different ligand density levels) and unconjugated ovalbumin. Increasing ligand density on the nanoparticle surface resulted in increased internalization of modified nanoparticles by the cells, up to a saturation level. Surface ligand density also affected the cellular uptake pathway; from predominantly clathrin to predominantly caveolae-mediated as the ligand density was increased. It was further demonstrated that surface clustering of the folate ligand enhanced cellular internalization of nanoparticles, relative to its dispersed surface distribution.EThOS - Electronic Theses Online ServiceGBUnited Kingdo