Bovine Serum Albumin Permeability Research Articles

Adsorption resin/polyethersulfone membrane used for plasma separation and middle molecular toxins adsorption

Accumulation of endogenous and exogenous toxins in patients with chronic kidney disease increases the kidney burden. The newly developed plasma separation adsorption membranes provide a new model for the removal of middle molecular toxins. In this study, the adsorption resin LX-1000H is physically pulverized and blended into polyethersulfone matrixes to prepare adsorption resin/polyethersulfone (AR/PES) membranes by nonsolvent-induced phase separation for plasma separation and middle molecular toxins absorption. The results show that the static adsorption capacity of AR/PES membrane for lysozyme (a typical mimic of middle molecular toxins) is up to 108.90 mg/g, which is 4 times that of AR. The maximum dynamic adsorption capacity reaches 102.80 mg/g, which is 93.4% of static adsorption. The adsorption process is more consistent with the Langmuir model and the quasi-first-order kinetic model. The permeability of bovine serum albumin in simulated blood surpasses 90% and the cattle blood separated by AR/PES membrane is colorless and transparent. The AR/PES membrane surface presents electronegative at pH 7.4 and the water wettability is enhanced with the doping amount. No hemolysis and negligible leakage indicated favorable hemocompatibility. This paper is devoted to providing a strategy for plasma separation and adsorption membranes.

Effect of Sericin Additive on Cellulose Acetate Membrane Morphology and Protein Rejection

Cellulose acetate (CA) membranes are synthesized for filtration purpose. The hydrophilic sericin macromolecule is blended with CA to analyze the enhancement of protein rejection. Increase in hydrophilicity, water permeation and protein rejection was studied by using contact angle, pure water flux (PWF) and Bovine serum albumin (BSA) permeation respectively. Surface modification was examined using SEM and AFM. The results show the homogenous blending of sericin in CA matrix and effective fabrication of membranes. In order to gauge the efficiency of casted membranes in protein filtration and separation, water flux and protein rejection were studied. The concentration of sericine protein was altered in CA polymeric solution that imparts variable properties to fabricated membrane samples. Increment in wt% of sericine up to 7.5 in polymer dope solution, enhances BSA rejection to the limit of 96%. The amino linkages between sericine and the BSA of feed resulted in the holding of protein by sericine, which leads to low permeation of BSA via CA-Sericine membrane M4.

Block Copolymer-Based Magnetic Mixed Matrix Membranes-Effect of Magnetic Field on Protein Permeation and Membrane Fouling.

In this study, we report the impact of the magnetic field on protein permeability through magnetic-responsive, block copolymer, nanocomposite membranes with hydrophilic and hydrophobic characters. The hydrophilic nanocomposite membranes were composed of spherical polymeric nanoparticles (NPs) synthesized through polymerization-induced self-assembly (PISA) with iron oxide NPs coated with quaternized poly(2-dimethylamino)ethyl methacrylate. The hydrophobic nanocomposite membranes were prepared via nonsolvent-induced phase separation (NIPS) containing poly (methacrylic acid) and meso-2,3-dimercaptosuccinic acid-coated superparamagnetic nanoparticles (SPNPs). The permeation experiments were carried out using bovine serum albumin (BSA) as the model solute, in the absence of the magnetic field and under permanent and cyclic magnetic field conditions OFF/ON (strategy 1) and ON/OFF (strategy 2). It was observed that the magnetic field led to a lower reduction in the permeate fluxes of magnetic-responsive membranes during BSA permeation, regardless of the magnetic field strategy used, than that obtained in the absence of the magnetic field. Nevertheless, a comparative analysis of the effect caused by the two cyclic magnetic field strategies showed that strategy 2 allowed for a lower reduction of the original permeate fluxes during BSA permeation and higher protein sieving coefficients. Overall, these novel magneto-responsive block copolymer nanocomposite membranes proved to be competent in mitigating biofouling phenomena in bioseparation processes.

Fucoidan- and carrageenan-based biosynthetic poly(vinyl alcohol) hydrogels for controlled permeation

Since the permeation of the inflammatory cytokines into hydrogel scaffolds has been shown to cause dysfunction of encapsulated cells, appropriate design strategies to circumvent this are essential. In the present work, it was hypothesized that highly crosslinked PVA-fucoidan and PVA-carrageenan hydrogels can control permeation of the trefoil-shaped inflammatory cytokine IL-1β while allowing the permeation of the globular protein albumin. PVA, fucoidan, and carrageenans were functionalized with methacrylate groups and the functionalized polymers were co-crosslinked by UV photopolymerization. The resultant hydrogels were characterized physicochemically and the release of fucoidan and carrageenans was quantified by developing a colorimetric assay, which was validated by XPS analysis. The permeability characteristics of the hydrogels were evaluated using bovine serum albumin (BSA), IgG, and IL-1β. The results demonstrated an increase in hydrogel swelling through the incorporation of the polysaccharides with minimal overall mass loss. The release studies showed hydrogel stability, where the formulations exhibited ~43% retention of fucoidan and ~60–80% retention of carrageenans consistently up to 7 days. The permeation data revealed very low permeation of IgG and IL-1β through the hydrogels, with <1% permeation after 24 h, while allowing >6% permeation of BSA. These data indicate that such hydrogels can be used as the basis for cytokine-protective implantable devices for clinical applications.

Preparation of Filtration Membrane by Grafting of Poly(N-vinylpyrrolidone) onto Polyethersulfone and Its Influence on Pollution Resistance of Membrane

In this study, a new modified polymer by grafting poly(N-vinylpyrrolidone) onto polyethersulfone was prepared. The polymerization was carried out in a homogenous phase by using N-methyl pyrrolidone as solvent. Crucial parameters affecting the graft copolymerization, such as monomer concentration and absorbed dose were studied. The existence of grafted chains was confirmed by FTIR spectroscopy. Pristine polyethersulfone and graft copolymer with different grafting degrees were then cast into membranes through phase inversion methodology. The contact angle of the membranes was measured to study transformation of the hydrophilic property on the surface. The cross-section morphology, thicknesses, water permeability, bovine serum albumin permeability and rejection of the membranes were studied. Finally, the membranes made of grafted copolymer (PES-g-PVP) showed an improvement of the antifouling property after BSA solution flux attenuation test.

Bovine serum albumin selective integral asymmetric isoporous membrane

Separation of bovine serum albumin (BSA) and hemoglobin (Hb), two proteins with almost identical molecular weight, has been a big challenge for more than two decades. Using traditional ultrafiltraion membranes separation of these proteins is possible only at the isoelectric point of one protein via electrostatic repulsion between the membrane and the other protein. Here we introduce an integral asymmetric isoporous membrane from polystyrene-block-poly(2-hydroxyethyl methacrylate-ran-2-(succinyloxy)ethyl methacrylate) (PS-b-P(HEMA-r-SEMA)) having random –OH and –COOH groups along the pore walls prepared by a method which combines solvent induced self-assembly of the block copolymer and nonsolvent induced phase separation (SNIPS). The membrane consists of soft isoporous channels due to swelling of the P(HEMA-r-SEMA) blocks in a hydrated state. The effective pore size of the membrane in a hydrated state is significantly lower compared to that in a dry state. These soft channels allow the permeation of BSA while retaining Hb at constant pH 7.4 where both proteins are negatively charged. In comparison to 22 commercial and in-house prepared membranes the PS-b-P(HEMA-r-SEMA) membrane presents unprecedented ideal selectivity of BSA over Hb (ψBSA/Hb=16). Unlike the conventional technique in this case the electroneutrality of one protein is not mandatory which will provide significantly easier handling.

Assessment of increased glomerular permeability associated with recurrent focal segmental glomerulosclerosis using an in vitro model of the glomerular filtration barrier.

The presence of circulating permeability factors (cPFs) has been hypothesized to be associated with recurrence of focal segmental glomerulosclerosis (rFSGS) in renal allografts. The available methods to detect cPFs are complex, not easily repeatable and inappropriate to represent the anatomical characteristics of the three-layer glomerular filtration barrier (GFB). Here we describe a novel method which measures the permeability to bovine serum albumin (BSA) through a three-layer device (3LD). The 3 layers comprise: (1) conditionally immortalized human podocytes (HCiPodo), (2) collagen type IV coated porous membrane and (3) human glomerular endothelial cells (HCiGEnC). Using this method, we found that sera from all rFSGS patients increased albumin permeability, while sera from non recurrent (nrFSGS) and genetic (gFSGS) forms of FSGS did not. The mechanisms underlying the increase of albumin permeability are probably due to endothelial cell damage as an initial event, which was demonstrated by the decrease of Platelet endothelial cell adhesion molecule (PECAM-1 or CD31), while the podocytes' expressions of synaptopodin and podocin were normal. Furthermore, we also found that the plasmapheretic treatment (PPT) eliminated the effect of increasing BSA permeability in sera from rFSGS patients. These preliminary data suggest that our in vitro GFB model could not only be useful in predicting the recurrence of FSGS after renal transplantation (RTx), but also be a valuable in vitro model to study podocyte and endothelial cell biology.

Development of pH-responsive organic-inorganic hybrid nanocomposites as an effective oral delivery system of protein drugs

This research aimed to develop a pH-responsive organic-inorganic hybrid nanocomposite as an effective oral delivery system for protein drugs. Three different nanocomposites were prepared by using bovine serum albumin (BSA) as a model protein. A nanocomplex of BSA with 3-aminopropyl functionalized magnesium phyllosilicate (AC-BSA) was obtained via the spontaneous co-assembly and then sequentially coated with glycol-chitosan (GAC-BSA) and the pH sensitive polymer, Eudragit®L100–55 (EGAC-BSA). These organic-inorganic hybrid nanocomposites exhibited high entrapment efficiency (86–99%) and their structural characteristics were confirmed by using energy dispersive X-ray spectroscopy, Fourier transform infrared spectroscopy, and circular dichroism analysis, indicating that the secondary structure of BSA was well retained in the nanocomposites. At pH 1.2, AC-BSA achieved rapid drug release of about 80% within 2 h, while GAC-BSA and EGAC-BSA exhibited slow drug release of 30% and 15%, respectively, indicating that the surface-coated nanocomposites were more stable in the gastric condition. Furthermore, the conformational stability of BSA entrapped in EGAC-BSA was well retained in the presence of proteolytic enzymes, suggesting that EGAC-BSA should be effective in protecting the protein against gastrointestinal harsh environment. Compared to free BSA, all of tested nanocomposites demonstrated 2.1–3.8-fold higher cellular uptake in Caco-2 cells. Furthermore, energy-dependent endocytosis and paracellular pathway contributed to the cellular transport of nanoparticles. After oral administration in rats, EGAC-BSA significantly enhanced the intestinal permeation of BSA compared to free BSA. In conclusion, EGAC-BSA appears to be promising as an effective oral delivery system for proteins with enhanced intestinal absorption.

Evaluation of a Methylcellulose and Hyaluronic Acid Hydrogel as a Vehicle for Rectal Delivery of Biologics.

Biologics have changed the management of Inflammatory Bowel Disease (IBD), but there are concerns regarding unexpected systemic toxicity and loss of therapeutic response following administration by injection. Local delivery of biologics directly to the inflamed mucosa via rectal enema administration addresses the problems associated with systemic administration. Hydrogels are potentially useful delivery vehicles enabling rectal administration of biologics. Here, we prepared a hydrogel system based on methylcellulose (MC) and hyaluronic acid (HA), which possesses mucosal healing properties, incorporating a model macromolecular drug, namely (fluorescently-labeled) bovine serum albumin (BSA). The BSA-loaded MCHA hydrogel showed temperature-dependent gelation (liquid-like at 20 °C and gel-like at 37 °C) and shear thinning behavior, with these being important and desirable characteristics for rectal application (enabling easy application and retention). BSA release from the MCHA system at 37 °C was linear, with 50% of the loaded drug released within 2 h. The system demonstrated acceptable toxicity towards intestinal (colon) Caco-2 epithelial cells, even at high concentrations. Importantly, application of the BSA-loaded MCHA hydrogel to polarized Caco-2 monolayers, with or without an exemplar absorption enhancer, resulted in transintestinal permeability of BSA. The study therefore indicates that the MCHA hydrogel shows potential for topical (rectal) delivery of biologics in IBD.

Spatially controlled coating of continuous liquid interface production microneedles for transdermal protein delivery

Microneedle patches, arrays of micron-scale projections that penetrate skin in a minimally invasive manner, are a promising tool for transdermally delivering therapeutic proteins. However, current microneedle fabrication techniques are limited in their ability to fabricate microneedles rapidly and with a high degree of control over microneedle design parameters. We have previously demonstrated the ability to fabricate microneedle patches with a range of compositions and geometries using the novel additive manufacturing technique Continuous Liquid Interface Production (CLIP). Here, we establish a method for dip coating CLIP microneedles with protein cargo in a spatially controlled manner. Microneedle coating mask devices were fabricated with CLIP and utilized to coat polyethylene glycol-based CLIP microneedles with model proteins bovine serum albumin, ovalbumin, and lysozyme. The design of the coating mask device was used to control spatial deposition and loading of coated protein cargo on the microneedles. CLIP microneedles rapidly released coated protein cargo both in solution and upon insertion into porcine skin. The model enzyme lysozyme was shown to retain its activity throughout the CLIP microneedle coating process, and permeation of bovine serum albumin across full thickness porcine skin was observed after application with coated CLIP microneedles. Protein-coated CLIP microneedles were applied to live mice and showed sustained retention of protein cargo in the skin over 72 h. These results demonstrate the utility of a versatile coating platform for preparation of precisely coated microneedles for transdermal therapeutic delivery.

ATRP-grown protein-polymer conjugates containing phenylpiperazine selectively enhance transepithelial protein transport

Despite its patient-friendliness, the oral route is not yet a viable strategy for the delivery of biomacromolecular therapeutics. This is, in part, due to the large size of proteins, which greatly limits their absorption across the intestinal epithelium. Although chemical permeation enhancers can improve macromolecular transport, their positive impact is often accompanied by toxicity. One element potentially contributing to this toxicity is the lack of co-localization of the enhancer with the protein drug, which can result in non-specific permeation of the intestine as well as enhancer overdosing in some areas due to non-uniform distribution. To circumvent these issues, this study describes a new way of increasing protein permeability via a polymer conjugation process that co-localizes permeation enhancer with the protein. Based on previous reports demonstrating the utility of 1-phenylpiperazine as an intestinal permeation enhancer, we synthesized protein-polymer conjugates with a phenylpiperazine-containing polymer using polymer-based protein engineering. A novel phenylpiperazine acrylamide monomer was synthesized and chain extended using atom transfer radical polymerization from the model protein bovine serum albumin (BSA). At non-cytotoxic doses, the protein-polymer conjugates induced a dose dependent reduction in the trans-epithelial electrical resistance of Caco-2 monolayers and an impressive ~30-fold increase in BSA permeability. Furthermore, this permeability increase was selective, as the permeability of the small molecule calcein co-incubated with the protein-polymer conjugate increased only 5-fold. Together, these data represent an important first step in the development of protein polymer conjugates that facilitate selective protein transport across membranes that are typically impermeable to macromolecules.

NMDA receptors participate in the progression of diabetic kidney disease by decreasing Cdc42-GTP activation in podocytes.

Podocytes play important roles in the progression of diabetic kidney disease (DKD) and these roles are closely associated with cytoskeletal actin dynamics. N-Methyl-d-aspartate receptors (NMDARs), which consist of two functional NR1 subunits and two regulatory NR2 subunits, are widely expressed in the brain but are also found in podocytes. Here, we found increased NR1 expression in two diabetic mouse models and in podocytes incubated in high glucose (HG). In diabetic mice, knockdown of NR1 using lentivirus carrying NR1-shRNA ameliorated the pathological features associated with DKD, and reversed the decreased expression of synaptopodin and Wilms' tumour-1. In podocytes incubated with HG, NR1 was secreted from the endoplasmic reticulum and this was blocked by bisindolylmaleimide I. NR1 knockdown decreased the cell shape remodelling, cell collapse, bovine serum albumin permeability, and migration induced by HG. After HG incubation, levels of cell division control protein 42 (Cdc42) and its active form increased, and a significantly higher Cdc42-GTP level, increased Cdc42 translocation onto the leading edges, and lower migration ability were found in podocytes with NR1 knockdown. Increases in the number and length of filopodia were found in podocytes with NR1 knockdown but these were abolished by Cdc42-GTP blockade with ML141. In conclusion, the activation of NMDARs plays an important role in DKD by reducing Cdc42-GTP activation. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

PREPARATION, CHARACTERIZATION AND PERFORMANCE STUDIES OF ACTIVE PVDF ULTRAFILTRATION-SURFACTANTS MEMBRANES CONTAINING PVP AS ADDITIVE

The role of surfactants in the formation of active Poly (vinylidene fluoride) (PVDF) ultrafiltration (AUF) membranes was studied. The effect combination of surfactants that are Sodium dodecyl sulfate (SDS)/Tween 80 and Tween 80/Triton X-100 formulations on performance and morphological structures were investigated for the first time. The influence of surfactants blends on the membrane pores was also examined. Experimental data showed that combination of Tween 80/Triton X-100 give the highest BSA permeation flux which value 285.51 Lm-2h-1. With combination of SDS/Tween 80, the AUF membrane showed the highest protein rejection up to 93% and 79% for Bovine Serum Albumin (BSA) and Egg Albumin (EA), respectively. Moreover, membranes characterization demonstrated that the addition of SDS/Tween 80 and Tween 80/Triton X-100 were found to affect the performance, surface morphologies and membrane pores of AUF PVDF membranes.

Preparation, Characterization and Performance Studies of Active PVDF Ultrafiltration-Surfactants Membranes Containing PVP as Additive

The role of surfactants in the formation of active Poly (vinylidene fluoride) (PVDF) ultrafiltration (AUF) membranes was studied. The effect combination of surfactants that are Sodium dodecyl sulfate (SDS)/Tween 80 and Tween 80/Triton X-100 formulations on performance and morphological structures were investigated for the first time. The influence of surfactants blends on the membrane pores was also examined. Experimental data showed that combination of Tween 80/Triton X-100 give the highest BSA permeation flux which value 285.51 Lm-2h-1. With combination of SDS/Tween 80, the AUF membrane showed the highest protein rejection up to 93% and 79% for Bovine Serum Albumin (BSA) and Egg Albumin (EA), respectively. Moreover, membranes characterization demonstrated that the addition of SDS/Tween 80 and Tween 80/Triton X-100 were found to affect the performance, surface morphologies and membrane pores of AUF PVDF membranes.

Potential of D-Octaarginine-Linked Polymers as an in Vitro Transfection Tool for Biomolecules.

We have been investigating the potential use of cell-penetrating peptide-linked polymers as a novel penetration enhancer. Since previous in vivo studies demonstrated that poly(N-vinylacetamide-co-acrylic acid) bearing D-octaarginine, a typical cell-penetrating peptide, enhanced membrane permeation of biomolecules, its potential as an in vitro transfection tool was evaluated in this study. A plasmid DNA encoding green fluorescent protein (pGFP-C1), β-galactosidase, and bovine serum albumin (BSA) were used as model biomolecules. Anionic pGFP-C1 interacted electrostatically with cationic d-octaarginine-linked polymers. When the ratio of mass concentration of polymers to that of pGFP-C1 reached 2.5, complexes whose size and zeta potential were approximately 200 nm and 15 mV, respectively, were obtained. GFP expression was observed in cells incubated with complexes prepared under conditions in which the polymer/pDNA concentration ratio exceeded 2.5. The expression level elevated with an increase in the concentration ratio, but physicochemical properties of the complexes remained unchanged. Results suggested that free polymers contributed to pGFP-C1 internalization. Another cell study demonstrated that β-galactosidase premixed with polymers was taken up into cells in its active tetrameric form. Similar electrostatic interaction-driven complex formation was observed for BSA charged negatively in neutral solution. However, it appeared that the internalization processes of BSA differed from those of pGFP-C1. A mass concentration-dependent increase in internalized BSA was observed, irrespective of the polymer/protein concentration ratio. Due to frail interactions, polymers that were released from the complexes and subsequently immobilized on cell membranes might also contribute to membrane permeation of BSA.

Delivery of large molecular protein using flat and short microneedles prepared using focused ion beam (FIB) as a skin ablation tool.

Many studies have been reported in the literature on the effects of various geometries and lengths of microneedles (MNs) on transdermal drug delivery using a variety of drug molecules. In particular, sharp-tipped MNs have been used to disrupt the top layer of the skin, namely, stratum corneum (SC). It has also been shown that short- and flat-tipped MNs can pierce the SC and they have the potential to increase drug permeability. However, there is little work that explores MNs as a skin ablative tool with a view to increasing skin permeability. To address this point, well-defined small patterns (size of individual pattern 10–20 μm) on the tip of flat MN (tip radius of individual MN ∼250 μm) were created and their effects evaluated on the permeability of bovine serum albumin (BSA), which is chosen as a model drug of high molecular weight. The patterns on the tip of flat MN act as rough surfaces (e.g. like sand paper) which when applied on the surface of the skin ablate the SC layer. Focused ion beam (FIB) has been used as the fabrication technique for the MNs. The permeability data are then compared with the other data for flat- and sharp-tipped MN. The permeability data from passive diffusion experiments are used as the reference case. The exact number of MNs or patterns in the flat and patterned MN patches is not considered as important as they have not been designed to pierce the skin. However, this is an important consideration in the case of sharp MNs as they pierce and create cavities in the skin. It is found that the delivery of BSA with the fabricated flat and patterned MNs gave similar but somewhat lower drug permeation profile in comparison to the sharp MNs. Passive diffusion showed no permeation, as would be expected due to the large size of the chosen molecule.

Permeation of biological compounds through porous poly(l-lactic acid) (PLLA) microtube array membranes (MTAMs)

In this study, the permeation of two biological compounds through a novel porous poly(l-lactic acid) (PLLA) microtube array membrane (MTAM) were investigated. Glucose and bovine serum albumin (BSA) were permeated through PLLA MTAMs to simulate the transportation of biological compounds during nerve regeneration. PLLA MTAMs with various porous wall structures were prepared by the addition of porogen. Electrospinning process was conducted to produce solid MTAMs. Porous MTAMs were obtained by washing off the porogen. Permeation of BSA and glucose were investigated by utilizing a home-made permeation unit at 37°C for both inflow and outflow. The permeation rate increased with the porosity of the MTAM; with the inflow rate faster than the outflow rate for both compounds suggesting a diffusive permeation. The rate of BSA permeation was faster than that of glucose. Fourier transfer infra-red (FTIR) spectra of the two compounds permeated through PLLA membranes were obtained to elucidate this phenomena.

Improved separation of bovine serum albumin and lactoferrin mixtures using charged ultrafiltration membranes

The aim of this work is to assess the isoelectric separation of a binary protein mixture containing bovine serum albumin (BSA) and lactoferrin (LF) using charged ultrafiltration membranes. First, a thorough characterization of the protein properties that could affect their separation, i.e., the zeta potential under different working conditions, the molecular size, and the tendency to form aggregates, was carried out. The influence of the following operational variables on the separation selectivity was then analyzed: (i) BSA/LF initial concentration ratio, (ii) protein isoelectric point (Ip), and (iii) membrane charge using three composite regenerated cellulose (CRC) membranes: negatively charged, positively charged, and unmodified. The results were used to identify conditions that yielded the optimum separation of proteins. Under those conditions, LF was completely retained in the feed mixture, BSA was characterized by an Ip of pH 5.0, and the use of an unmodified membrane yielded a maximum BSA permeation flux of 30.31gm−2h−1. By contrast, BSA was completely retained by the negatively charged membrane at an LF Ip of pH 9.0, and LF yielded a maximum permeation flux of 1.07gm−2h−1. This work provides theoretical and experimental insights into the phenomena that influence the membrane separation of mixtures of similarly sized milk proteins. This work also offers a phenomenological explanation of the main features of the separation process.

Activation of the Nrf2-ARE Pathway Attenuates Hyperglycemia-Mediated Injuries in Mouse Podocytes

Background: Damage to podocytes caused by excessive reactive oxygen species (ROS) contributes to onset and progression of diabetic kidney disease (DKD). Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a redox-sensing transcription factor that can induce the expression of antioxidant enzymes. We explored whether activation of Nrf2 pathway attenuated hyperglycemia-induced injuries in mouse podocytes. Methods: Tert-Butylhydroquinone (tBHQ) and small interfering RNAs (siRNAs) were used to regulate Nrf2 expression. Apoptosis and intracellular superoxide anion production were measured by flow cytometry. The activity of the Nrf2 antioxidant pathway was measured by an antioxidant response element (ARE)-driven luciferase reporter gene assay, and Nrf2 expression was assessed by real-time PCR and western blot analyses. Results: Podocytes incubated with high-glucose (HG) medium had higher intracellular superoxide anion and hydrogen peroxide production, higher apoptosis rate, higher bovine serum albumin (BSA) permeability and lower synaptopodin expression compared with podocytes exposed normal glucose (NG) (p<0.05). tBHQ increased the activity of the Nrf2 antioxidant pathway and enhanced nuclear Nrf2 expression, reduced intracellular superoxide anion and hydrogen peroxide production, apoptosis rate and BSA permeability, and restored synaptopodin expression in podocytes exposed to HG (p<O.05). Podocytes with Nrf2 siRNAs showed higher intracellular superoxide anion and hydrogen peroxide production, apoptosis and BSA permeability as well as lower synaptopodin expression compared with podocytes exposed to HG (p<0.05). Conclusions: Our findings suggest that protection against activation of the Nrf2-ARE pathway in podocytes exposed to hyperglycemia. Thus, regulation of the Nrf2-ARE pathway could be a therapeutic option to combat oxidative stress and inhibit the development of DKD.

Permeability Enhancement for Transdermal Delivery of Large Molecule Using Low-Frequency Sonophoresis Combined with Microneedles

Transdermal drug delivery is limited by the high resistance of skin towards diffusion of high-molecular-weight drugs. This is mainly because of the fact that the outer layer of the skin, that is the stratum corneum, can prevent diffusion of molecules whose molecular weight is greater than 500 Da. Sonophoresis can be used to enhance the permeability of the skin. However, in the delivery of large molecules, ultrasound alone cannot provide sufficient permeability enhancement. In addressing this issue, we propose optimised ultrasound combined with microneedles to further increase the permeation rates. In this paper, we use porcine ear skin to simulate human skin and treat the skin samples with both ultrasound and microneedles. Further, bovine serum albumin (BSA) is used as a model of larger molecular weight molecule. Our results show that the permeability of BSA is increased to 1 μm/s with the combination of 1.5 mm microneedles patch and 15-W ultrasound output which is about 10 times higher than the permeability obtained in passive diffusion. Diffusion with only microneedles or ultrasound pre-treatment is also tested. The maximum permeability from microneedles and ultrasound treatment reached 0.43 and 0.4 μm/s, respectively.