Albumin-fluorescein Isothiocyanate Research Articles

Redox-Responsive Hydrogels Loaded with an Antibacterial Peptide as Controlled Drug Delivery for Healing Infectious Wounds.

Infectious wounds occur when harmful microorganisms such as bacteria or viruses invade a wound site. Its problems associated include delayed healing, increased pain, swelling, and the potential for systemic infections. Therefore, developing new wound dressing materials with antibacterial effects is crucial for improving the healing process. Here a redox-degradable hydrogel loaded with an antibacterial peptide (vancomycin) in a straightforward gram-scale synthesis, is developed. The hydrogel structure consists of a disulfide bond-containing hyperbranched polyglycerol (SS-hPG) that is cross-linked by 4-arm polyethylene glycol-thiol (4-arm PEG-SH). The polymerization mechanism and full characterization of SS-hPG are described as this synthesis is reported for the first time. Rheology is used to ascertain the hydrogel's mechanical characteristics, such as stiffness, and self-healing, determining these properties for different ratios and concentrations of both gel components. The incorporation of disulfide bonds in the hydrogel is proved by conducting degradation experiments in reductive environments. Fluorescein isothiocyanate-albumin (FITC-BSA) and vancomycin both are loaded into the gel, and the guest release kinetics is assessed for both slow and on-demand releases. Finally, the in vitro and in vivo experiments prove that the vancomycin-loaded hydrogel acts as an antibacterial barrier for wound dressing and accelerates the healing of infectious wounds in a mouse model.

Functionalization of Carbon Electrode Surface Using Polyaniline and Gold Nanoparticles for Protein Immobilization

This study presents a screen-printed carbon electrode surface functionalization to immobilize proteins. The process involves the deposition of gold nanoparticles on the carbon electrode and subsequent surface modification to immobilize bovine serum albumin–fluorescein isothiocyanate conjugate (BSA-FITC). A combination of conducting polymer and gold nanoparticles was employed to enhance the electrode contact area and current transmission. The electrode surface morphology and properties were evaluated by scanning electron microscopy (SEM) and Raman spectroscopy, showing the porous polyaniline (PANI) film has been formed on the carbon electrode surface with the network of dendritic nanofibers. Gold nanoparticles were evenly distributed on the PANI film to form a nanocomposite material layer on the carbon electrode. The observed size of these gold nanoparticles was significantly smaller than those on the electrode without a PANI film. Additionally, the current transmission of this composite material layer has been verified by cyclic voltammetry (CV). The results show that the amplitude of the redox peak was doubled compared to the bare carbon electrode and gold nanoparticle-only deposited electrode. Moreover, the success of the process was verified by fluorescence and cyclic voltammetry. The fluorescent green color observed under the microscope indicated that BSA-FITC was on the electrode surface. The change of CV signal after each step verified the formation of a new layer on the electrode surface. These results are anticipated to play an important role in developing high-sensitivity immunosensors in the future.

AN EVALUATION OF A GOLD SURFACE FUNCTIONALIZATION PROCEDURE FOR ANTIBODY BINDING AND PROTEIN DETECTION USING 11-MERCAPTOUNDECANOIC ACID (11-MUA)

Immunosensors play an essential role in biological element recognition systems. Among the numerous types of electrodes utilized for immunosensor, gold electrodes with surface modification have attracted considerable attention and have experienced various improvements and developments due to their advantages, compromising high conductivity and chemical stability. Self-assembled monolayer (SAM) has been used as an effective approach for biomarker immobilization onto electrodes. In this study, two types of gold electrodes, namely, sputtered gold on a glass slide and screen-printed gold electrodes, were utilized to assess the performance of the proposed procedure utilizing 11-Mercaptoundecanoic acid (11-MUA). Bovine serum albumin–fluorescein isothiocyanate conjugate (BSA-FITC) and anti-albumin antibody (anti-BSA) were employed as the antigen–antibody pair. Microscope-based fluorescent, Raman spectroscopy and electrical measurements, including cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS), were conducted to validate the success of each step in the surface functionalization process. The results showed that BSA proteins were immobilized on the modified electrode, as indicated by the fluorescent green color observed under the microscope and the changes in surface impedance recorded in Raman spectroscopy, CV and EIS signal graphs. Additionally, the influence of the 11-MUA incubation time and the protein concentration on the performance of the gold electrode surface functionalization process were investigated. The results indicate that an incubation time exceeding 24 h and a protein concentration of 5 [Formula: see text]M was found to be the most optimal conditions. The screen-printed gold electrode exhibited a slightly superior performance in comparison to the sputtered gold electrode. This work has verified the proposed gold surface functionalization process and can serve as a solid foundation for future immunosensor research studies.

Drug delivery with melt-spun liquid-core fibers

Conventional local drug delivery systems often encounter issues such as burst-release, limited drug reservoirs, rigidity, and low mechanical performance. We present an innovative approach to local drug delivery utilizing melt-spun drug-loaded liquid-core filaments (LiCoFs). LiCoFs were produced, incorporating fluorescein sodium salt as the model drug, dissolved in various liquid core materials, and enveloped by poly(ε-caprolactone) as the sheath material. We assessed thermal, mechanical, and structural properties of LiCoFs and have conducted extensive drug diffusion trials. Some trials involved pre-exchange of the liquid core with ibuprofen, bovine serum albumin–fluorescein isothiocyanate and methylene blue solutions using a pumping device. It was observed that diffusion mechanisms and diffusion rates depend on temperature, core size/sheath thickness, sheath permeability, carrier liquid type, drug molecule properties, and the drug's affinity to the sheath polymer. These drug-loaded LiCoFs pave the way towards a new generation of medical textiles facilitating controlled, local drug delivery.

A novel technique to quantify the kinetics of blood clot contraction based on the expulsion of fluorescently labeled albumin into serum

BackgroundThe platelet-driven contraction or retraction of blood clots has been utilized to obtain blood serum for laboratory studies, but now, in vitro clot contraction assays are used in research laboratories and clinics to assess platelet functionality. The static final extent of clot contraction measured using a clot size or expelled serum volume can be supplemented substantially with a dynamic analysis. ObjectivesTo provide a step-by-step protocol for a relatively simple and affordable new automated methodology to follow the kinetics of blood clot contraction, which allows for simultaneous measurements of various samples at a time and requires only a fluorescence plate reader. MethodsThe kinetics of clot contraction in whole blood was assessed by continuously detecting the fluorescence intensity of fluorescein isothiocyanate-albumin added to a blood sample before clotting and expelled into the serum during clot shrinkage. ResultsThe clots are formed and fluorescence is measured in the wells of a black multiwell plate using a standard plate fluorescent reader. The specificity of this technique for clot contraction has been demonstrated by the strong inhibitory effects of blebbistatin, latrunculin A, and abciximab. To validate the new technique, increased fluorescence intensity in the contracting clots was measured in parallel with a visual decrease in clot size performed with the same blood samples. ConclusionThe resulting clot contraction dynamics based on the expulsion of fluorescein isothiocyanate-albumin can be quantified using a number of kinetic parameters as well as a phase kinetics analysis. The advantages and drawbacks of the new technique are discussed.

Exposure to ZnO nanoparticles induced blood-milk barrier dysfunction by disrupting tight junctions and cell injury

Zinc oxide nanoparticles (ZnO-NPs) are one of the most widely used nanomaterials with excellent chemical and biological properties. However, their widespread application has led to increased risk to the natural environment and public health. A growing number of studies have shown that ZnO-NPs deposited in target organs interact with internal barriers to trigger injurious responses. The underlying mechanism of ZnO-NPs on the blood-milk barrier dysfunction remains to be understood. Our results revealed that excessive accumulation of ZnO-NPs induced histopathological injuries in the mammary gland, leading to the distribution of ZnO-NPs in the milk of lactating mice. A prominent diffusion of blood-milk barrier permeability marker, albumin-fluorescein isothiocyanate conjugate (FITC-albumin) was observed at cell-cell junction after ZnO-NPs exposure. Meanwhile, ZnO-NPs weakened the blood-milk barrier function by altering the expression of tight junction proteins. The excessive accumulation of ZnO-NPs also induced inflammatory response by activating the NF-κB and MAPK signaling pathways, leading to the dysfunctional blood-milk barrier. Furthermore, we found that ZnO-NPs led to increased iron accumulation and lipid oxidation, thus increasing oxidative injury and ferroptosis in mammary glands. These results indicated that ZnO-NPs weaken the integrity of the blood-milk barrier by directly affecting tight junctions and cellular injury in different ways.

Rapamycin treatment induces tubular proteinuria: role of megalin-mediated protein reabsorption.

Introduction: Rapamycin is an immunosuppressor that acts by inhibiting the serine/threonine kinase mechanistic target of rapamycin complex 1. Therapeutic use of rapamycin is limited by its adverse effects. Proteinuria is an important marker of kidney damage and a risk factor for kidney diseases progression and has been reported in patients and animal models treated with rapamycin. However, the mechanism underlying proteinuria induced by rapamycin is still an open matter. In this work, we investigated the effects of rapamycin on parameters of renal function and structure and on protein handling by proximal tubule epithelial cells (PTECs). Methods: Healthy BALB/c mice were treated with 1.5mg/kg rapamycin by oral gavage for 1, 3, or 7days. At the end of each treatment, the animals were kept in metabolic cages and renal function and structural parameters were analyzed. LLC-PK1 cell line was used as a model of PTECs to test specific effect of rapamycin. Results: Rapamycin treatment did not change parameters of glomerular structure and function. Conversely, there was a transient increase in 24-h proteinuria, urinary protein to creatinine ratio (UPCr), and albuminuria in the groups treated with rapamycin. In accordance with these findings, rapamycin treatment decreased albumin-fluorescein isothiocyanate uptake in the renal cortex. This effect was associated with reduced brush border expression and impaired subcellular distribution of megalin in PTECs. The effect of rapamycin seems to be specific for albumin endocytosis machinery because it did not modify renal sodium handling or (Na++K+)ATPase activity in BALB/c mice and in the LLC-PK1 cell line. A positive Pearson correlation was found between megalin expression and albumin uptake while an inverse correlation was shown between albumin uptake and UPCr or 24-h proteinuria. Despite its effect on albumin handling in PTECs, rapamycin treatment did not induce tubular injury measured by interstitial space and collagen deposition. Conclusion: These findings suggest that proteinuria induced by rapamycin could have a tubular rather than a glomerular origin. This effect involves a specific change in protein endocytosis machinery. Our results open new perspectives on understanding the undesired effect of proteinuria generated by rapamycin.

Influence of PEGDA Molecular Weight and Concentration on the In Vitro Release of the Model Protein BSA-FITC from Photo Crosslinked Systems.

Novel 3D printing techniques enable the development of medical devices with drug delivery systems that are tailored to the patient in terms of scaffold shape and the desired pharmaceutically active substance release. Gentle curing methods such as photopolymerization are also relevant for the incorporation of potent and sensitive drugs including proteins. However, retaining the pharmaceutical functions of proteins remains challenging due to the possible crosslinking between the functional groups of proteins, and the used photopolymers such as acrylates. In this work, the in vitro release of the model protein drug, albumin-fluorescein isothiocyanate conjugate (BSA-FITC) from differently composed, photopolymerized poly(ethylene) glycol diacrylate (PEGDA), an often employed, nontoxic, easily curable resin, was investigated. Different PEGDA concentrations in water (20, 30, and 40 wt %) and their different molecular masses (4000, 10,000, and 20,000 g/mol) were used to prepare a protein carrier with photopolymerization and molding. The viscosity measurements of photomonomer solutions revealed exponentially increasing values with increasing PEGDA concentration and molecular mass. Polymerized samples showed increasing medium uptake with an increasing molecular mass and decreasing uptake with increasing PEGDA content. Therefore, the modification of the inner network resulted in the most swollen samples (20 wt %) also releasing the highest amount of incorporated BSA-FITC for all PEGDA molecular masses.

Catalpol relieved angiotensin II-induced blood–brain barrier destruction via inhibiting the TLR4 pathway in brain endothelial cells

Context Catalpol is a major bioactive constituent of Rehmannia glutinosa Libosch (Scrophulariaceae), a traditional Chinese medicine, which is widely used in multiple diseases, including hypertension. Objectives To explore whether catalpol protects against angiotensin II (Ang II)-triggered blood–brain barrier (BBB) leakage. Materials and methods The bEnd.3 cells and BBB models were pre-treated with or without catalpol (50, 200 and 500 μM) or TAK-242 (1 μM) for 2 h and then with Ang II (0.1 μM) or LPS (1 μg/mL) for 24 h. Cell viability was determined by the MTT assay. The levels of Toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), inducible nitric oxide synthase (iNOS), tumour necrosis factor-α (TNF-α), caveolin-1 (Cav-1) and p-eNOS/eNOS were tested by western blot. The BBB permeability was evaluated by the flux of bovine serum albumin-fluorescein isothiocyanate (BSA-FITC) across monolayers. nuclear factor kappa-B (NF-κB) p65 nuclear translocation was explored by immunofluorescence staining. Results Ang II (0.1 μM) decreased the cell viability to 86.52 ± 1.79%, elevated the levels of TLR4, MyD88, iNOS, TNF-α and Cav-1 respectively to 3.7-, 1.5-, 2.3-, 2.2- and 2.7-fold, reduced the level of p-eNOS/eNOS to 1.6-fold in bEnd.3 cells, and eventually increased BBB permeability. Catalpol dose-dependently reversed these changes at 50–500 μM. Meanwhile, catalpol (500 μM) inhibited the upregulated levels of TLR4 pathway-related proteins and NF-κB p65 nuclear translocation, decreased the enhanced transcytosis, and relieved the BBB disruption caused by both LPS (the TLR4 activator) and Ang II. The effects are same as TAK-242 (the TLR4 inhibitor). Conclusions Catalpol relieved the Ang II-induced BBB damage, which indicated catalpol has high potential for the treatment of hypertension-induced cerebral small vessel disease (cSVD).

Preparation and characterization of fast dissolving ulvan microneedles for transdermal drug delivery system

Dissolving microneedles made from natural polymers recently have gained much attention as an efficient transdermal drug delivery system (TDDS). For the first time, ulvan, a sulfated polysaccharide extracted from Ulva lactuca, was applied to fabricate dissolving microneedles through a two-step casting method. The ulvan microneedles (UMNs) made from 4% ulvan solution were in a pyramidal shape with an average height of 655 μm and an aspect ratio of 2.63. The in vitro skin insertion study showed the UMNs could totally penetrate into the porcine skin to the dermis layer and rapidly dissolved as the needle height was reduced by 90.3% after post-insertion of only 2 min. The rapid dissolution of UMNs in situ thus could release the loaded model drugs of rhodamine 6G (R6G) and bovine serum albumin–fluorescein isothiocyanate conjugate (FITC-BSA) in the skin tissue. The in vitro drug release profiles through porcine skin revealed the UMNs markedly enhanced the cumulative release of FITC-BSA. In addition, the UMNs had good biocompatibility towards normal cells of HaCaT and NIH3T3. Briefly, this study demonstrates the rapidly dissolving UMNs could effectively carry the drug into skin and thus can be developed as a potential TDDS in the pharmaceutical and cosmeceutical fields.

Feasibility of mucoadhesive chitosan maleimide-coated liposomes for improved buccal delivery of a protein drug

Chitosan-maleimide (CSMHA) coated liposomes were developed for the buccal delivery of protein. Albumin–fluorescein isothiocyanate conjugate (FITC-BSA) was used as a protein replica to be loaded in the liposomes. The liposomes were formed by a thin-film hydration technique before being decorated with CSMHA. The mucoadhesive capability of the CSMHA-coated liposomes on the porcine buccal mucosa, the loading efficiency and loading capacity, drug release, permeation through buccal mucosa, protein integrity, the cytotoxicity on oral fibroblast cells, and biocompatibility were investigated. The liposome formulations were obtained with sizes of 35–166 nm. CSMHA-coated FITC-BSA-loaded liposomes remained on the buccal tissue to the highest extent. Moreover, CSMHA-coated liposomes provided the highest buccal permeation of FITC-BSA and were safe for normal gingival fibroblast cells and buccal tissue. Therefore, the CSMHA-coated liposomes may have the capability to improve protein delivery via the buccal route.

Redox Regulation of Microvascular Permeability: IL-1β Potentiation of Bradykinin-Induced Permeability Is Prevented by Simvastatin.

Antioxidant effects of statins have been implicated in the reduction in microvascular permeability and edema formation in experimental and clinical studies. Bradykinin (Bk)-induced increases in microvascular permeability are potentiated by IL-1β; however, no studies have examined the protection afforded by statins against microvascular hyperpermeability. We investigated the effects of simvastatin pretreatment on albumin–fluorescein isothiocyanate conjugate (FITC-albumin) permeability in post-capillary venules in rat cremaster muscle. Inhibition of nitric oxide synthase with L-NAME (10µM) increased basal permeability to FITC-albumin, which was abrogated by superoxide dismutase and catalase. Histamine-induced (1 µM) permeability was blocked by L-NAME but unaffected by scavenging reactive oxygen species with superoxide dismutase (SOD) and catalase. In contrast, bradykinin-induced (1–100 nM) permeability increases were unaffected by L-NAME but abrogated by SOD and catalase. Acute superfusion of the cremaster muscle with IL-1β (30 pM, 10 min) resulted in a leftward shift of the bradykinin concentration–response curve. Potentiation by IL-1β of bradykinin-induced microvascular permeability was prevented by the nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase) inhibitor apocynin (1 µM). Pretreatment of rats with simvastatin (5 mg·kg−1, i.p.) 24 h before permeability measurements prevented the potentiation of bradykinin permeability responses by IL-1β, which was not reversed by inhibition of heme oxygenase-1 with tin protoporphyrin IX (SnPP). This study highlights a novel mechanism by which simvastatin prevents the potentiation of bradykinin-induced permeability by IL-1β, possibly by targeting the assembly of NADPH oxidase subunits. Our findings highlight the therapeutic potential of statins in the prevention and treatment of patients predisposed to inflammatory diseases.

Development of a 3D Printed Refillable Drug Delivery Device for Sustained Release to the Retina

Retinal degenerative diseases (RDD) such as age-related macular degeneration (AMD) and retinitis pigmentosa (RP) cause progressive loss of vision, affecting millions of people worldwide. Drug delivery system (DDS) such as vitreous injection and intraocular implant has been widely investigated to treat RDD. However, they are invasive and can risk infection of the eye. Thus, we have been working on the development of transscleral DDS, which are less invasive and can achieve more localized release of drug than the conventional methods [1]. We have previously reported capsule-type and sheet-type DDS devices that can be implanted on the transscleral (periocular) region of the eye [2] [3]. However, such devices need to be replaced once drug run out. Herein, we report a 3D printed refillable DDS device for long term delivery of drugs to the retina.The refillable device consisted of 3 sections: -polyvinyl alcohol gel chamber with openings to regulate drug release, the drug chamber as a reservoir for a test drug, and polydimethylsiloxane (PDMS) chamber with port holes to allow reinjection of the drug (Fig. 1a). The device was designed in SolidWorks 2018 (Dassault Systèmes, France) and 3D printed (Qidi Shadow 5.5S, QidiTech) using polyethylene glycol diacrylate (PEGDA) mixed with 1% photoinitiator (Irgacure819, w/w), and 1% photosensitizer (2-isopropylthioxanthone, w/w). PDMS and PVA hydrogel were manually introduced into the drug injection chamber and the drug release chamber, respectively. PDMS was cured by thermal curing and PVA was gelled by freeze-thawing. After introducing a model drug, Albumin-fluorescein isothiocyanate conjugate (FITC-Albumin), into the device, it was immersed in 5 mL of Dulbecco’s phosphate buffered saline(DPBS) and incubated at 37℃ inside a humidified incubator. The fluorescence intensities of DPBS solutions were measured by spectrofluorimetry to quantify diffusion of drugs from the capsule into the DPBS solution. The DPBS solutions were replaced at each measurement. In addition, drug reinjection and quantification of the released drug were performed. Quantification of the released drug was done by above-mentioned process.The refillable capsule-type DDS device was fabricated with a curved geometry such that it can fit the outer circumference of the eyeball (Fig. 1b). Specifically, the dimensions of the device were 20 mm inside diameter, 22 mm curvature diameter, 2 mm thickness and 4.4 mm width. By 3D printing, it was possible to fabricate a complex 3D shape that is not possible using conventional mold technique. Because the drug is impermeable to PEGDA, unidirectional release of the drug from the release ports was possible (Fig. 1c). The unidirectional release is important because it can reduce drug elimination by conjunctival clearance and increase efficiency of drug delivery. In vitro release tests of the device were performed with varying concentrations of PVA gel. The result showed that as the PVA concentration was increased, the drug release rate decreased. At 5 wt% PVA concentration, an extreme initial burst and complete release of FITC-Albumin in 7 days was observed whereas at 10 wt% PVA concentration, over 90% drug release in 30 days and complete release in 50 days were observed, and at 20 wt% PVA concentration, drug release over 100 days was observed. Similar drug release trends were observed when the drug was repeatedly injected into the same devices for 3 times. Also, no leakage of the drug from the PDMS port was confirmed during repeated injection.These in vitro experiment results show the potential of the device for future transscleral implants. As the next step, we are planning to implant the downsized device on rabbit’s eye and investigate biocompatibility and efficacy for treatment of RDD.

Magnetic nanoparticle decorated anodic alumina nanotubes for fluorescent detection of cathepsin B

In this work, we present the process to provide anodic alumina nanotubes with magnetic responsivity based on magnetic nanoparticles. We demonstrate the possibility to cause the motion of these composite nanotubes under magnetic field, providing them with guided mobility. The obtained magnetic anodic alumina nanotubes are completely characterized and their potential to undergo selective and effective functionalization, and stimuli-responsive load release is demonstrated. For this purpose, protease-triggered release of fluorescent molecules loaded inside the magnetic anodic alumina nanotubes (MAANTs) by selective functionalization is performed. The inner walls of the MAANTs were selectively covered with protein padding of albumin-fluorescein isothiocyanate conjugate (FITC-BSA) through means of silanization. Protein functionalization was designed to undergo proteolytic hydrolysis in presence of cathepsin B- protease highly expressed during growth and initial stages of tumor metastasis – in order to cleave peptide bond of albumin and release fluorescent fragments of the protein. Proteolytic reaction with the enzyme is performed under acidic conditions. Presented arrangement is an exemplary combination of functionalities – which are vast – and of value for applications like drug delivery and biosensing applications.

The Beneficial Effect of HES on Vascular Permeability and Its Relationship With Endothelial Glycocalyx and Intercellular Junction After Hemorrhagic Shock.

BackgroundVascular leakage is a common complication of hemorrhagic shock. Endothelial glycocalyx plays a crucial role in the protection of vascular endothelial barrier function. Hydroxyethyl starch (HES) is a commonly used resuscitation fluid for hemorrhagic shock. However, whether the protective effect of HES on vascular permeability after hemorrhagic shock is associated with the endothelial glycocalyx is unclear.MethodsUsing hemorrhagic shock rat model and hypoxia treated vascular endothelial cells (VECs), effects of HES (130/0.4) on pulmonary vascular permeability and the relationship to endothelial glycocalyx were observed.ResultsPulmonary vascular permeability was significantly increased after hemorrhagic shock, as evidenced by the increased permeability of pulmonary vessels to albumin-fluorescein isothiocyanate conjugate (FITC-BSA) and Evans blue, the decreased transendothelial electrical resistance of VECs and the increased transmittance of FITC-BSA. The structure of the endothelial glycocalyx was destroyed, showing a decrease in thickness. The expression of heparan sulfate, hyaluronic acid, and chondroitin sulfate, the components of the endothelial glycocalyx, was significantly decreased. HES (130/0.4) significantly improved the vascular barrier function, recovered the thickness and the expression of components of the endothelial glycocalyx by down-regulating the expression of heparinase, hyaluronidase, and neuraminidase, and meanwhile increased the expression of intercellular junction proteins ZO-1, occludin, and VE-cadherin. Degradation of endothelial glycocalyx with degrading enzyme (heparinase, hyaluronidase, and neuraminidase) abolished the beneficial effect of HES on vascular permeability, but had no significant effect on the recovery of the expression of endothelial intercellular junction proteins induced by HES (130/0.4). HES (130/0.4) decreased the expression of cleaved-caspase-3 induced by hemorrhagic shock.ConclusionsHES (130/0.4) has protective effect on vascular barrier function after hemorrgic shock.The mechanism is mainly related to the protective effect of HES on endothelial glycocalyx and intercellular junction proteins. The protective effect of HES on endothelial glycocalyx was associated with the down-regulated expression of heparinase, hyaluronidase, and neuraminidase. HES (130/0.4) had an anti-apoptotic effect in hemorrhagic shock.

Crocin alleviates lipopolysaccharide-induced acute respiratory distress syndrome by protecting against glycocalyx damage and suppressing inflammatory signaling pathways.

ObjectiveTo explore the mechanisms of crocin against glycocalyx damage and inflammatory injury in lipopolysaccharide (LPS)-induced acute respiratory distress syndrome (ARDS) mice and LPS-stimulated human umbilical vein endothelial cells (HUVECs).MethodsMice were randomly divided into control, LPS, and crocin + LPS (15, 30, and 60 mg/kg) groups. HUVECs were separated into eight groups: control, crocin, matrix metalloproteinase 9 inhibitor (MMP-9 inhib), cathepsin L inhibitor (CTL inhib), LPS, MMP-9 inhib + LPS, CTL inhib + LPS, and crocin + LPS. The potential cytotoxic effect of crocin on HUVECs was mainly evaluated through methylthiazolyldiphenyl-tetrazolium bromide assay. Histological changes were assessed via hemotoxylin and eosin staining. Lung capillary permeability was detected on the basis of wet–dry ratio and through fluorescein isothiocyanate-albumin assay. Then, protein levels were detected through Western blot analysis, immunohistochemical staining, and immunofluorescence.ResultsThis study showed that crocin can improve the pulmonary vascular permeability in mice with LPS-induced ARDS and inhibit the inflammatory signaling pathways of high mobility group box, nuclear factor κB, and mitogen-activated protein kinase in vivo and in vitro. Crocin also protected against the degradation of endothelial glycocalyx heparan sulfate and syndecan-4 by inhibiting the expressions of CTL, heparanase, and MMP-9 in vivo and in vitro. Overall, this study revealed the protective effects of crocin on LPS-induced ARDS and elaborated their underlying mechanism.ConclusionCrocin alleviated LPS-induced ARDS by protecting against glycocalyx damage and suppressing inflammatory signaling pathways.

Controlling the Release Profile Through Phase Control of Calcium Phosphate-Alginate Core-shell Nanoparticles in Gene Delivery

In this study, we aimed to control the drug release rate from calcium phosphate-alginate (CaP-Alg) core-shell nanoparticle (NPs) using CaP phases (brushite (DCPD) [Bru] and hydroxyapatite [HA]) generated under different pH conditions. Core-shell NPs consisted of an inorganic CaP core and an organic Alg shell and were synthesized by the water-in-oil emulsification and precipitation method. CaP-Alg NPs were synthesized under different pH conditions, resulting in the mineralization of CaP with Bru and HA in the core region of core-shell NPs. Albumin-fluorescein isothiocyanate conjugate (FITC-BSA) was used as a model drug for in-vitro drug release studies. CaP-Alg (Bru-Alg and HA-Alg) NPs exhibited a higher loading capacity and encapsulation efficiency than Ca-Alg NPs. The release behavior of synthesized core-shell NPs showed different patterns due to the pH-sensitivity of Alg and CaP. At physiological pH, Ca-Alg NPs exhibited an initial burst release behavior, while CaP-Alg showed controlled release behavior. Our results demonstrate that HA-Alg NPs are more suitable for controlled intracellular delivery while Bru-Alg NPs are more suitable for extracellular compartment delivery. Therefore, CaP-Alg NPs could be potential candidates for controlled gene and biomolecule delivery into cells for therapeutic purposes.

Sintered electrospun polycaprolactone for controlled model drug delivery

Electrospinning has been used widely for drug delivery applications due to its versatility and ease of modification of spun fiber properties. Net drug loading and release is typically limited by the inherent surface-area of the sample. In a relatively novel approach, sintering of electrospun fiber was used to create a capsule favoring long-term delivery. We showed that electrospun polycaprolactone (PCL) retained its initial morphology out to 1042 days of in vitro exposure, illustrating its potential for extended performance. Sintering decreased the electrospun pore size by 10- and 28-fold following 56 and 57 °C exposures, respectively. At 58 and 59 °C, the PCL capsules lost all apparent surface porosity, but entrapped pores were observed in the 58 °C cross-section. The use of Rhodamine B (RhB, 479.02 g mol−1), Rose Bengal (RB, 1017.64 g mol−1) and albumin-fluorescein isothiocyanate conjugate from bovine serum (BSA-FITC, ~66,000 g mol−1) as model compounds demonstrated that release (RhB > RB ≫ BSA-FITC) is controlled both by molecular weight and available porosity. Interestingly, the ranking of release following sintering was 57 > 56 > 59 > 58 °C; COMSOL simulations explored the effects of capsule wall thickness and porosity on release rate. It was hypothesized that model drug adsorption on the available fiber surface-area (57 versus 56 °C) and entrapped porosity (59 versus 58 °C) could have also attributed to the observed ranking of release rates. While the 56 and 57 °C exposures allowed the bulk of the release to occur in <1 day, the capsules sintered at 58 and 59 °C exhibited release that continued after 12 days of exposure.

Spatiotemporal Expression of GRP78 in the Blood Vessels of Rats Treated With 3-Nitropropionic Acid Correlates With Blood-Brain Barrier Disruption.

Glucose-regulated protein (GRP78) or BiP, a 78-kDa chaperone protein located in the endoplasmic reticulum (ER), has recently been reported to be involved in the neuroglial response to ischemia-induced ER stress. The present study was designed to study the expression patterns of this protein and the cell types involved in the induction of GRP78 expression in rats treated with the mitochondrial toxin 3-nitropropionic acid (3-NP). GRP78 immunoreactivity was almost exclusively localized to striatal neurons in saline-treated controls, but GRP78 expression was induced in activated glial cells, including reactive astrocytes and activated microglia/macrophages, in the striata of rats treated with 3-NP. In the lesion core, increased GRP78 immunoreactivity was observed in the vasculature; this was evident in the lesion periphery of the core at 3 days after lesion induction, and was evenly distributed throughout the lesion core by 7 days after lesion induction. Vascular GRP78 expression was correlated, both temporally and spatially, with infiltration of activated microglia into the lesion core. In addition, this was coincident with the time and pattern of blood–brain barrier (BBB) leakage, detected by the extravasation of fluorescein isothiocyanate-albumin, an established BBB permeability marker. Vascular GRP78-positive cells in the lesion core were identified as endothelial cells, smooth muscle cells, and adventitial fibroblast-like cells, in which GRP78 protein was specifically localized to the cisternae of the rough ER and perinuclear cisternae, but not to other organelles such as mitochondria or nuclei. Thus, our data provide novel insights into the phenotypic and functional heterogeneity of GRP78-positive cells within the lesion core, suggesting the involvement of GRP78 in the activation/recruitment of activated microglia/macrophages and its potential role in BBB impairment in response to a 3-NP-mediated neurotoxic insult.

A microfluidic chip with a staircase pH gradient generator, a packed column and a fraction collector for chromatofocusing of proteins.

A microfluidic device for pH gradient chromatofocusing is presented, which performs creation of a micro‐column, pH gradient generation, and fraction collection in a single device. Using a sieve micro‐valve, anion exchange particles were packed into a microchannel in order to realize a solid‐phase absorption column. To fractionate proteins according to their isoelectric points, elution buffer solutions with a stepwise pH gradient were prepared in 16 parallel mixing reactors and flowed through the micro‐column, wherein a protein mixture was previously loaded. The volume of the column is only 20 nL, hence it allows extremely low sample consumption and fast analysis compared with a conventional system. We demonstrated separation of two proteins, albumin–fluorescein isothiocyanate conjugate (FITC‐BSA) and R‐Phycoerythrin (R‐PE), by using a microcolumn of commercial charged polymeric particles (Source 15Q). The microfluidic device can be used as a rapid diagnostic tool to analyse crude mixtures of proteins or nucleic acids and determine adsorption/desorption characteristics of various biochemical products, which can be helpful for scientific fundamental understanding as well as instrumental in various industrial applications, especially in early stage screening and process development.