Fluorescein Isothiocyanate-labeled Bovine Serum Albumin Research Articles

Preparation of glycyrrhizic acid-modified BSA-nanoparticles and evaluation of their hepatic cellular distribution.

Orientation to specific cells is an important topic in active targeting strategy for nanoparticle-based drug delivery systems. While these administered nanoparticles will be sequestrated within the liver, their cellular distribution behaviors in the liver are not clear. The aim of this study was to fabricate glycyrrhizic acid (GL) modified BSA nanoparticles and evaluate their hepatic cellular distribution. GL-modified BSA (GL-BSA) was tailored according to the periodate oxidation method, then GL-BSA nanoparticles loaded with paclitaxel (PTX@GL-BSA NPs) were prepared through self-assembly approach. In vitro cellular uptake was assessed by FITC-labeled BSA nanoparticles and immunofluorescent analysis was performed to track their relative distribution in the liver. The fabricated PTX@GL-BSA NPs were spherical structure with the particle size of 179 nm and a negative potential (-17.3 mV). Flow cytometry (FCM) studies exhibited that the accumulation of GL-BSA nanoparticles was 5.3-fold compared with BSA nanoparticles in HepG2 cells. The Nanoparticles were preferentially accumulated in the sinusoidal endothelial cells rather than the Kupffer cells. This study provides useful information to understand the distribution of hepatic targeting nanoparticles when using GL-modified BSA nanoparticles, which helps to further use for effective treatment of liver disease.

A drug-incorporated-microparticle-eggshell-membrane-scaffold (DIMES) dressing: A novel biomaterial for localised wound regeneration

Chronic wounds affect millions of people annually and have emotional and financial implications in addition to health issues. The current treatment for chronic wounds involves the repeated use of bandages and drugs such as antibiotics over an extended period. A cost-effective and convenient solution for wound healing is the development of drug-incorporated bandages. This study aimed to develop a biocompatible bandage made of drug-incorporated poly (lactic-co-glycolic acid) (PLGA) microparticles (MPs) and eggshell membrane (ESM) for cornea wound healing. ESM has desirable properties for wound healing and can be isolated from eggshells using acetic acid or ethylenediaminetetraacetic acid (EDTA) protocols. Fluorescein isothiocyanate-labelled Bovine Serum Albumin (FITC-BSA) was used as a model drug, and the PLGA MPs were fabricated using a solvent extraction method. The MPs were successfully attached to the fibrous layer of the ESM using NaOH. The surface features of the ESM samples containing MPs were studied using a field emission scanning electron microscope (FESEM) and compared with blank ESM images. The findings indicated that the MPs were attached to the ESM fibres and had similar shapes and sizes as the control MPs. The fibre diameters of the MPs samples were assessed using Fiji-ImageJ software, and no significant changes were observed compared to the blank ESM. The surface roughness, Ra values, of the MPs incorporated ESM samples were evaluated and compared to the blank ESM, and no significant changes were found. Fourier transform infrared (FTIR) spectroscopy was used to analyse the chemical Composition of the bandage, and the spectra showed that the FBM were effectively incorporated into the ESM. The FTIR spectra identified the major peaks of the natural ESM and the PLGA polymer in the bandage. The bandage was transparent but had a reduced visibility in the waterproof test card method. The bandage achieved sustained drug release up to 10 days and was found to be biocompatible and non-toxic in a chorioallantoic membrane (CAM) assay. Overall, the drug-incorporated PLGA MPs-ESM bandage has great potential for treating chronic wounds.

Fabrication of Polysaccharide-Based Coaxial Fibers Using Wet Spinning Processes and Their Protein Loading Properties

Fibers composed of polysaccharides are a promising candidate to be applied for biomaterials such as absorbable surgical sutures, textile fabrics, and hierarchical three-dimensional scaffolds. In this work, in order to fabricate biocompatible fibers with controlled-release abilities, the fabrication of coaxial fibers of calcium alginate (ALG-Ca) and polyion complexes (PICs) consisting of chitosan (CHI) and chondroitin sulfate C (CS), denoted as ALG-PIC fibers, by using a wet spinning process, and the evaluation of their molecular loading and release behavior were performed. The diameter and mechanical strength of the obtained ALG-PIC fibers increased with increasing concentrations of the CHI solution for PIC coatings. This indicated that higher concentrations of the CHI solution afforded a thicker PIC coating layer. Further, fluorescein isothiocyanate labeled-bovine serum albumin (FITC-BSA)-loaded ALG-PIC fibers were successfully prepared. The release behavior of FITC-BSA in the fibers exhibited a slower rate at the initial state than that in ALG-Ca, indicating that PIC coatings suppressed an initial burst release of the loading molecules. Accordingly, the fabricated coaxial fibers can be utilized as sustained-release drug carriers.

Intranasal administration of collagenase develops cerebral microbleeds and vascular dementia in mice: In vivo pilot study

ObjectiveTo establish a new animal model of lobar cerebral microbleeds (CMBs), we examined whether intranasal administration of collagenase induced CMBs in young mice which did not have hypertensive pathologic features. MethodsWe used thirty-two male CD1 mice (8–10 weeks old), which were received either phosphate-buffered saline (PBS) or collagenase by intranasal administration twice a day for 5 days. A dose response study was conducted, and motor function and number of CMBs was evaluated at one day after completing the intranasal administration. Temporal profile of cognitive impairment and number of CMBs through 7 days was evaluated after completing intranasal administration of 3.6U collagenase. ResultsThe number of CMBs showed an increasing trend in a dose-dependent manner and higher number of CMBs were observed with 3.6U collagenase. Cognitive impairment and CMBs was observed in 3.6U collagenase groups up to 7 days after administration. In addition, successful brain delivery via intranasal route was comfirmed by administering fluorescein isothiocyanate-labeled bovine serum albumin. ConclusionsIntranasal collagenase administration is a feasible and minimally invasive method to produce CMBs and cognitive impairment in mice. This model has potential for producing CMBs in animals with cerebral amyloid angiopathy such as transgenic mice models of Alzheimer’s disease.

Sessile Trichomes Play Major Roles in Prey Digestion and Absorption, While Stalked Trichomes Function in Prey Predation in Byblis guehoi

Carnivorous plants in the genus Byblis obtain nutrients by secreting viscous glue drops and enzymes that trap and digest small organisms. Here, we used B. guehoi to test the long-held theory that different trichomes play different roles in carnivorous plants. In the leaves of B. guehoi, we observed a 1:2.5:14 ratio of long-stalked, short-stalked, and sessile trichomes. We demonstrated that the stalked trichomes play major roles in the production of glue droplets, while the sessile trichomes secrete digestive enzymes, namely proteases and phosphatases. In addition to absorbing digested small molecules via channels/transporters, several carnivorous plants employ a more efficient system: endocytosis of large protein molecules. By feeding B. guehoi fluorescein isothiocyanate-labeled bovine serum albumin (FITC-BSA) to monitor protein transport, we found that sessile trichomes exhibited more endocytosis than long- and short-stalked trichomes. The uptaken FITC-BSA was delivered to the neighboring short epidermal cells in the same row as the sessile trichomes, then to the underlying mesophyll cells; however, no signals were detected in the parallel rows of long epidermis cells. The FITC control could be taken up by sessile trichomes but not transported out. Our study shows that B. guehoi has developed a well-organized system to maximize its food supply, consisting of stalked trichomes for prey predation and sessile trichomes for prey digestion. Moreover, the finding that sessile trichomes transfer large, endocytosed protein molecules to the underlying mesophyll, and putatively to the vascular tissues, but not laterally to the terminally differentiated epidermis, indicates that the nutrient transport system has evolved to maximize efficiency.

Marine-origin polysaccharides-based free-standing multilayered membranes as sustainable nanoreservoirs for controlled drug delivery.

The layer-by-layer (LbL) assembly technology has been widely used to functionalise surfaces and precisely engineer robust multilayered bioarchitectures with tunable structures, compositions, properties, and functions at the nanoscale by resorting to a myriad of building blocks exhibiting complementary interactions. Among them, marine-origin polysaccharides are a sustainable renewable resource for the fabrication of nanostructured biomaterials for biomedical applications owing to their wide bioavailability, biocompatibility, biodegradability, non-cytotoxicity, and non-immunogenic properties. Chitosan (CHT) and alginate (ALG) have been widely employed as LbL ingredients to shape a wide repertoire of size- and shape-tunable electrostatic-driven multilayered assemblies by exploring their opposite charge nature. However, the insolubility of CHT in physiological conditions intrinsically limits the range of bioapplications of the as-developed CHT-based LbL structures. Herein, we report the preparation of free-standing (FS) multilayered membranes made of water-soluble quaternised CHT and ALG biopolymers for controlled release of model drug molecules. The influence of the film structure in the drug release rate is studied by assembling two distinct set-ups of FS membranes, having the model hydrophilic drug fluorescein isothiocyanate-labelled bovine serum albumin (FITC-BSA) either as an intrinsic building block or added as an outer layer after the LbL assembly process. Both FS membranes are characterised for their thickness, morphology, in vitro cytocompatibility, and release profile, with those having FITC-BSA as an intrinsic LbL ingredient denoting a more sustained release rate. This work opens up new avenues for the design and development of a wide array of CHT-based devices for biomedical applications, overcoming the limitations associated with the insolubility of native CHT under physiological conditions.

Enhanced immunoassay in a nanofluidic preconcentrator utilizing nano-interstices among self-assembled gold nanoparticles.

In this study, we developed a microfluidic device for a dual work of protein preconcentration and subsequent capture by an immunoassay system. The fabrication of nano-interstices (nanochannels) to generate ion concentration polarization effect (ICP) for the preconcentration was simply performed by exploiting the loose association of glass-on-modified AuNPs to the polydimethylsiloxane (PDMS) channel pads as well as spaces between the substrate and the PDMS pad caused by these AuNPs. As a result, 65-fold concentration enhancement was achieved when performed on a sample of fluorescein isothiocyanate-labeled bovine serum albumin (FITC-BSA). Furthermore, a modification procedure of antibodies responsible for capturing target proteins was performed on gold electrodes integrated into the proposed chip. After preconcentration, the immunoassay system was worked, and showed a good performance in capturing targets. Through this study, we demonstrated that the device can work efficiently for the dual purpose, has the potential to apply widely for the analysis and capture of various targets.

Targeting Plasma Kallikrein With a Novel Bicyclic Peptide Inhibitor (THR-149) Reduces Retinal Thickening in a Diabetic Rat Model.

PurposeTo investigate the effect of plasma kallikrein (PKal)-inhibition by THR-149 on preventing key pathologies associated with diabetic macular edema (DME) in a rat model.MethodsFollowing streptozotocin-induced diabetes, THR-149 or its vehicle was administered in the rat via either a single intravitreal injection or three consecutive intravitreal injections (with a 1-week interval; both, 12.5 µg/eye). At 4 weeks post-diabetes, the effect of all groups was compared by histological analysis of Iba1-positive retinal inflammatory cells, inflammatory cytokines, vimentin-positive Müller cells, inwardly rectifying potassium and water homeostasis-related channels (Kir4.1 and AQP4, respectively), vascular leakage (fluorescein isothiocyanate-labeled bovine serum albumin), and retinal thickness.ResultsSingle or repeated THR-149 injections resulted in reduced inflammation, as depicted by decreasing numbers and activation state of immune cells and IL-6 cytokine levels in the diabetic retina. The processes of reactive gliosis, vessel leakage, and retinal thickening were only significantly reduced after multiple THR-149 administrations. Individual retinal layer analysis showed that repeated THR-149 injections significantly decreased diabetes-induced thickening of the inner plexiform, inner nuclear, outer nuclear, and photoreceptor layers. At the glial–vascular interface, reduced Kir4.1-channel levels in the diabetic retina were restored to control non-diabetic levels in the presence of THR-149. In contrast, little or no effect of THR-149 was observed on the AQP4-channel levels.ConclusionsThese data demonstrate that repeated THR-149 administration reduces several DME-related key pathologies such as retinal thickening and neuropil disruption in the diabetic rat. These observations indicate that modulation of the PKal pathway using THR-149 has clinical potential to treat patients with DME.

PH responsive switchable protein adsorption properties of poly(2-methyl-2-oxazoline)/ poly(4-vinyl pyridine) based coatings: Influence of poly(4-vinyl pyridine) chain length and ionic strength

Protein adsorption at interfaces is a key for many applications especially in the biomedical field, biotechnologies, biocatalysis, food industry, etc. The development of surface layers that allow to control and manipulate protein adsorption is thus highly desired. To achieve this desired goal, we designed a smart thin stimuli responsive coating based on poly[(2-methyl-2-oxazoline)-random-glycidyl methacrylate)] (PMOXA-r-GMA) comb copolymer and poly(4-vinyl pyridine)-block-poly(glycidyl methacrylate) (P4VP-b-PGMA) block copolymer and scrutinized its ability to control protein adsorption properties by unraveling the effect of ionic strength (I) along with the pH of the surrounding medium and variation of chain length of P4VP. Firstly, PMOXA/P4VP based coatings were prepared by spin coating the mixture of PMOXA-r-GMA and P4VP-b-PGMA copolymer solutions onto silicon substrates followed by annealing at 110 °C. The surface composition and morphology of the PMOXA/P4VP based coatings were evaluated through X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). The switchable ability of the coating with the variation of chain length of P4VP upon pH and ionic strength trigger was characterized by water contact angle (WCA) and variable angle spectroscopic ellipsometry (VASE). The experimental observations illustrated that the change in the water contact angle (ΔWCA) and thickness (Δh) were found to be maximum when the chain length of P4VP was almost three times that of PMOXA while turning the environmental condition from pH 3, with low ionic strength (I = 10−3 M) to pH 9 with high ionic strength (I = 10-1 M). Finally, the adsorption and desorption behavior of mixed polymer coating against bovine serum albumin (BSA) protein was analyzed qualitatively and quantitatively using the fluorescein isothiocyanate-labelled BSA (FITC-BSA) assay and quartz crystal microbalance with dissipation monitoring (QCM-D), respectively. The low ionic strength (I = 10-3 M) at pH 3, caused to increase the adsorption of BSA and this increment was enhanced by increasing the chain length of P4VP in PMOX/P4VP based coatings. Subsequently, the adsorbed protein was then efficiently desorbed by changing the conditions to pH 9, I = 10-1 M, as well as the maximum desorption percentage (> 92 %) could be obtained at these optimized conditions in PMOXA/P4VP based polymer coatings.

A receptor-binding radiopharmaceutical for imaging of traumatic brain injury in a rodent model: [99mTc]Tc-tilmanocept

IntroductionBlood-brain barrier (BBB) disruption and subsequent neuro-inflammation occur following traumatic brain injury (TBI), resulting in a spectrum of human nervous system disorders. [99mTc]Tc-tilmanocept is a receptor-binding radiopharmaceutical FDA-approved for sentinel lymph node mapping. We hypothesize that after an intravenous (i.v.) injection, [99mTc]Tc-tilmanocept, will traverse a disrupted BBB and bind to CD206-bearing microglial cells. MethodsAge-matched mice were divided into three groups: 5-days post TBI (n = 4), and 5-days post sham (n = 4), and naïve controls (n = 4). IRDye800CW-labeled [99mTc]Tc-tilmanocept (0.15 nmol per gram body weight) and FITC-labeled bovine serum albumin (FITC-BSA) were injected (i.v.) into each mouse. Mice were imaged with a high-resolution gamma camera for 45 min. Immediately after imaging, the brains were perfused with fixative, excised, imaged with a fluorescence scanner, assayed for radioactivity, and prepared for histology. ResultsIn vivo nuclear imaging, ex vivo fluorescence imaging, ex vivo gamma well counting, and histo-microscopy demonstrated enhanced tilmanocept uptake in the TBI region. The normalized [99mTc]Tc-tilmanocept uptake value from nuclear imaging and the maximum pixel intensity from fluorescence imaging of the TBI group (1.12 ± 0.12 and 2288 ± 278 a.u., respectively) were significantly (P < 0.04) higher than the sham group (0.64 ± 0.28 and 1708 ± 101 a.u., respectively) and the naive group (0.76 ± 0.24 and 1643 ± 391 a.u., respectively). The mean [99mTc]Tc-tilmanocept scaled uptake in the TBI brains (0.058 ± 0.013%/g) was significantly (P < 0.010) higher than the scaled brain uptake of the sham group (0.031 ± 0.011%/g) and higher (P = 0.04) than the uptake of the naïve group (0.020 ± 0.002%/g). Fluorescence microscopy demonstrated increased uptake of the IRDye800CW-tilmanocept and FITC-BSA in the TBI brain regions. Conclusion[99mTc]Tc-tilmanocept traverses disrupted blood-brain barrier and localizes within the injured region. Advances in knowledge and implications for patient care[99mTc]Tc-tilmanocept could serve as an imaging biomarker for TBI-associated neuroinflammation and any disease process that involves a disruption of the blood-brain barrier.

Salvianolic acid A increases the accumulation of doxorubicin in brain tumors through Caveolae endocytosis

Brain glioma is one of the most common brain tumors in the central nervous system (CNS). The blood-brain tumor barrier (BTB) restricts the delivery of anti-tumor drugs into tumor tissue in the brain. Therefore, improving the transportation of antineoplastic drugs across the BTB is essential to ameliorate treatment of brain tumors. The present study was performed to explore the effect and mechanism of salvianolic acid A (Sal A) on transportation of doxorubicin (Dox) across the BTB in vivo and in vitro. By creating a brain C6 glioma model in rats, we demonstrated that Sal A significantly increased the level of Dox in brain tumor tissue as shown by liquid chromatograph mass spectrometry. Interestingly, we found that Sal A increased transendothelial electrical resistance (TEER) values of the BTB and decreased the permeability of FITC-Dextran (4kD) across the BTB in vitro. Furthermore, the expression of tight junction proteins (TJs) in glioma endothelial cells (GECs) and brain tumor microvessels were also increased, suggesting that Sal A enhanced delivery of Dox across the BTB independent of the paracellular pathway. Next, we detected that Sal A had an effect on transcellular transport of compounds across the BTB. The accumulation of FITC-labeled bovine serum albumin (FITC-BSA) was significantly increased in GECs after treatment with Sal A (10 μM) for 6h, which was inhibited after pre-treatment with methyl-β-cyclodextrin (MβCD) for 30 min. The increased delivery of Dox across the BTB was also reduced after treatment with MβCD. In addition, phosphorylation levels of protein kinase B (PKB) and tyrosine protein kinase–Src family (Src) were increased in the Sal A treatment group. Sal A up-regulated the expression level of the phosphorylation of Caveolin-1 (pCaveolin-1), and this effect was reversed by a PKB or Src inhibitor. Taken together, our study showed for the first time that Sal A facilitated the delivery of antitumor drugs into brain tumor tissues by targeting the PKB/Src/Caveolin-1 signaling pathway.

Online sample clean-up and enrichment of proteins from salty media with dynamic double gradients on a paper fluidic channel

Paper-based analytical device (PAD) has received more and more attention in the field of point-of-care test (POCT) due to its low cost, portability and simple operation. Sensitivity and selectivity are two important aspects in clinical diagnostic analysis. However, low sensitivity of a PAD limits its wider application for POCT. Here we introduced a PAD that can clean and enrich the protein content from salty media with both electric field (E) and pH gradient (double E/pH gradients), with which 100-fold enrichment effect could be achieved within 70 s as demonstrated by fluorescein isothiocyanate labeled bovine serum albumin (FITC-BSA) from artificial urine media. With post staining of the protein stacking band with bromophenol blue (BPB), selective colorimetric detection of human serum albumin (HSA) was achieved simply with smartphone camera in the clinically significant range of 10–300 mg‧L−1 (R2 = 0.99) with a limit of detection (LOD) of 4.9 mg‧L−1. Detection of microalbuminuria (MAU) of diabetic patients was demonstrated with this method without difference (ɑ = 0.01) to that by the clinical method (immunoturbidimetry). This work demonstrated the potential of this PAD method in online sample pretreatment and detection of target component from complex physiological samples.

Poly(2-methyl-2-oxazoline) and poly(4-vinyl pyridine) based mixed brushes with switchable ability toward protein adsorption

In this work, poly[(2-methyl-2-oxazoline)-random-glycidyl methacrylate] (PMOXA-r-GMA) and poly(4-vinyl pyridine)-block-poly(glycidyl methacrylate) (P4VP-b-PGMA) were synthesized by cationic ring opening polymerization (CROP) of 2-methyl-2-oxazoline (MOXA) and reversible addition-fragmentation chain transfer (RAFT) polymerization of 4-vinyl pyridine (4VP) followed by their random and block copolymerization with glycidyl methacrylate (GMA), respectively. Then poly(2-methyl-2-oxazoline)/poly(4-vinyl pyridine) (PMOXA/P4VP) based binary polymer brushes on silicon/glass substrate were fabricated using spin coating of the mixture of PMOXA-r-GMA and P4VP-b-PGMA copolymer solution followed by annealing process at 110 °C. Afterwards, these polymer brushes were characterized via water contact angle (WCA), X-ray photoelectron spectroscopy (XPS), variable angle spectroscopic ellipsometry (VASE), and atomic force microscopy (AFM) techniques. The results showed that the surface composition on the substrate surface could be adjusted by tuning the composition of the mixture of polymer solution used for the preparation of mixed brushes. Moreover, it also showed the swelling and de-swelling of mixed polymer brushes in a systematic way between two specific points during varying the pH of the surrounding medium from 2 to 10. Finally, the tunable behavior of PMOXA/P4VP based mixed brushes against bovine serum albumin (BSA) adsorption/desorption was investigated by fluorescein isothiocyanate-labelled BSA (FITC-BSA) assay and ellipsometry measurements. The results indicated that by changing the pH value from 2 to 10, the PMOXA/P4VP based mixed brushes could adsorb and desorb protein from very low to high amount depending upon the composition of PMOXA-r-GMA and P4VP-b-PGMA in preparing solution used for the PMOXA/P4VP based coatings, and ~89% of the adsorbed protein could be desorbed under the selected conditions.

Use of a Mouse Model and Human Umbilical Vein Endothelial Cells to Investigate the Effect of Arsenic Exposure on Vascular Endothelial Function and the Associated Role of Calpains.

Background:Arsenic (As) is a well-known environmental contaminant. Chronic exposure to As is known to increase the risk of cardiovascular diseases, including atherosclerosis, hypertension, diabetes, and stroke. However, the detailed mechanisms by which As causes vascular dysfunction involving endothelial integrity and permeability is unclear.Objectives:Our goal was to investigate how exposure to As leads to endothelial dysfunction.Methods:Arsenic trioxide (ATO) was used to investigate the effects and mechanisms by which exposure to As leads to endothelial dysfunction using a mouse model and cultured endothelial cell monolayers.Results:Compared with the controls, mice exposed chronically to As (10 ppb in drinking water supplied by ATO) exhibited greater vascular permeability to Evans blue dye and fluorescein isothiocyanate–labeled bovine serum albumin (FITC-BSA). In addition, endothelial monolayers treated with ATO ( As) exhibited greater intracellular gaps and permeability to low-density lipoprotein or transmigrating THP-1 cells. Furthermore, activity and protein levels of calpain-1 (CAPN-1) were significantly higher in aortas and human umbilical vein endothelial cells (HUVECs) treated with ATO. These results were consistent with effects of ATO treatment and included a rapid increase of intracellular calcium () and higher levels of CAPN-1 in the plasma membrane. Endothelial cell dysfunction and the proteolytic disorganization of vascular endothelial cadherin (VE-cadherin) in HUVECs in response to ATO were partially mitigated by treatment with a CAPN-1 inhibitor (ALLM) but not a CAPN-2 inhibitor (Z-LLY-FMK).Conclusions:This study found that in mice and HUVEC models, exposure to ATO led to CAPN-1 activation by increasing and CAPN-1 translocation to the plasma membrane. The study also suggested that inhibitor treatment may have a role in preventing the vascular endothelial dysfunction associated with As exposure. The findings presented herein suggest that As-induced endothelial dysfunction involves the hyperactivation of the CAPN proteolytic system. https://doi.org/10.1289/EHP4538

Crosslinked Hydrogel Capsules for Cell Encapsulation Formed Using Amino/Betaine Dual-Functional Semibatch Copolymers

Robust hydrogel capsules consisting of calcium alginate beads with shells reinforced via crosslinked amino/betaine copolymers of N-(3-aminopropyl) methacrylamide hydrochloride (APM) with 2-methacryloyloxyethyl phosphorylcholine (MPC) are described. These capsules are designed to support future use in immunoisolation of therapeutic cells for transplant purposes. The poly[APM-co-MPC] copolymers were prepared by semibatch free radical copolymerization to reduce the significant compositional drift and the resulting diffusional spread within alginate beads, observed for analogous batch copolymers. Fluorescent labeling was used to track copolymer distribution within the alginate hydrogels. Semibatch copolymers led to better shell confinement and lower cationic charge density on the capsule surface. Three reagents were used to covalently cross-link the resulting poly(amino/betaine) shells: cationic tetrakis(hydroxymethyl)phosphonium chloride (THPC), charge-neutral genipin and anionic partially hydrolyzed poly(methyl vinyl ether-alt-maleic anhydride) (PMM50). Genipin-crosslinked capsules were found to be remarkably robust in pipet aspiration tests. Selected capsules were exposed to FITC-labeled bovine serum albumin to assess the relative degree of protein adhesion, as a proxy for immune recognition; genipin crosslinked capsules were found to have very low levels of BSA binding and good mechanical robustness.

Silk fibroin nanoparticles for enhanced bio-macromolecule delivery to the retina

The aim of this study was to investigate intravitreal injection of silk fibroin nanoparticles (SFNs) encapsulating bio-macromolecules, achieving enhanced drug bioavailability, and extended retention in retina. SFNs were prepared with regenerated silk fibroin using desolvation method with fluorescein isothiocyanate labeled bovine serum albumin (FITC-BSA) as bio-macromolecular model drug encapsulated. In vitro physicochemical properties and in vitro drug release of FITC-BSA loaded SFNs (FITC-BSA-SFNs) were evaluated. Cytotoxicity, cellular uptake, and retention of FITC-BSA-SFNs were determined in human retinal pigment epithelial cell line (ARPE-19). In addition, in vivo distribution and safety of intravitreally administered FITC-BSA-SFNs were investigated in New Zealand white rabbits. The particle size of FITC-BSA-SFNs was 179.1 ± 3.7 nm with polydispersity index of 0.102 ± 0.033 and the zeta potential was greater than −25 mV. FITC-BSA-SFNs exhibited excellent biocompatibility with no cytotoxicity observed within 24 and 48 h in AREP-19 cells. Compared to FITC-BSA solution, FITC-BSA-SFNs showed enhanced cellular uptake and prolonged retention. Furthermore, FITC-BSA-SFNs achieved accumulated distribution and extended retention in retina in vivo following intravitreal injection compared to a single administration of free drug solution. Therefore, this bio-macromolecule delivery platform based on SFNs could have great potential in the treatment of posterior segment disorders.

Nanocrystalline Hydroxyapatite-Based Scaffold Adsorbs and Gives Sustained Release of Osteoinductive Growth Factor and Facilitates Bone Regeneration in Mice Ectopic Model

Nanocrystalline hydroxyapatite (NHA) is a biocompatible, biodegradable, and osteoconductive bone graft material; however, it lacks osteoinductivity. The present study is aimed at investigating the feasibility of nanocrystalline hydroxyapatite (NHA) as an osteoinductive growth factor carrier. Bone morphogenic protein 2 (BMP2), an osteoinductive growth factor, was incorporated into NHA (BMP2-NHA) using a simple adsorption method. The growth factor loading and release kinetics were profiled using fluorescein-isothiocyanate-labeled bovine serum albumin (FITC-BSA) as a mimic of the osteoinductive growth factor BMP2. The effect of BMP2-NHA on the osteogenic differentiation of C2C12 cells and ectopic bone formation in mice were tested. Confocal laser-scanning microscopy showed that FITC-BSA was diffused throughout the porous structure of NHA. FITC-BSA was efficiently loaded in NHA and sustained release was observed up to 35 days in vitro. BMP2-NHA enhanced the expression of osteogenic markers Runx2, Osterix, Alp, and Col1α1 and ALP activity in C2C12 cells compared to NHA. Similarly, μ-CT and histological examinations showed that BMP2-NHA robustly induced ectopic bone formation in mice. This study suggests that NHA could be used as an effective carrier of osteoinductive growth factors, which ensures osteoinductivity of NHA via sustained release of the growth factor.

Multifunctional self-assembled monolayers via microcontact printing and degas-driven flow guided patterning

Soft lithography-based patterning techniques have been developed to investigate biological and chemical phenomena. Until now, micropatterning with various materials required multiple procedural steps such as repeating layer-by-layer patterning, aligning of stamps, and incubating printed inks. Herein, we describe a facile micropatterning method for producing chemically well-defined surface architectures by combining microcontact (µCP) and microfluidic vacuum-assisted degas-driven flow guided patterning (DFGP) with a poly(dimethylsiloxane) (PDMS) stamp. To demonstrate our concept, we fabricated a bi-composite micropatterned surface with different functional molecular inks such as fluorescein isothiocyanate labelled bovine serum albumin (FITC-BSA) and polyethylene glycol (PEG)-silane for a biomolecule array, and 3-aminopropyltriethoxysilane (APTES) and PEG-silane pattern for a self-assembled colloid gold nanoparticle monolayer. With a certain composition of molecular inks for the patterning, bi-composite surface patterns could be produced by this µCP-DFGP approach without any supplementary process. This patterning approach can be used in microfabrication and highly applicable to biomolecules and nanoparticles that spread as a monolayer.

Delivery of proteins encapsulated in chitosan-tripolyphosphate nanoparticles to human skin melanoma cells.

We have successfully encapsulated two proteins, bovine serum albumin (BSA) and p53, in chitosan-tripolyphosphate (TPP) nanoparticles at various pH values from 5.5 to 6.5 and delivered the particles to human melanoma cells. The particles have diameters ranging from 180 nm to 280 nm and a zeta potential of +15 to + 40 mV. Cellular uptake of the particles by human skin melanoma cells was evaluated by: (i) fluorescence microscopy and (ii) gel electrophoresis showing that FITC-labeled BSA and p53 could be recovered in the soluble cell fraction after lysis of the cells. Our data also show that the highest cellular uptake takes place at the lowest pH as the particles have the highest positive charge under these conditions. The method we describe appears to be a general method for delivery of proteins to cells using chitosan-TPP nanoparticles as a drug delivery system, since structurally unrelated proteins such as BSA and p53 with different isoelectrical points can be encapsulated in the chitosan-TPP nanoparticles and be effectively internalized by the cells.

Charge-Controlled Surface Properties of Native and Fluorophore-Labeled Bovine Serum Albumin at the Air-Water Interface.

Proteins at interfaces are important for protein formulations and in soft materials such as foam. Here, interfacial stability and physicochemical properties are key elements, which drive macroscopic foam properties through structure–property relations. Native and fluorescein isothiocyanate-labeled bovine serum albumin (BSA) were used to modify air–water interfaces as a function of pH. Characterizations were performed with tensiometry and sum-frequency generation (SFG). SFG spectra of O–H stretching vibrations reveal a phase reversal and a pronounced minimum in O–H intensity at pH values of 5.3 and 4.7 for native and labeled BSA, respectively. This minimum is attributed to the interfacial isoelectric point (IEP) and is accompanied by a minimum in surface tension and negligible ζ-potentials in the bulk. Interfacial proteins at pH values close to the IEP can promote macroscopic foam stability and are predominately located in the lamellae between individual gas bubbles as evidenced by confocal fluorescence microscopy. Different from the classical stabilization mechanisms, for example, via the electrostatic disjoining pressure, we propose that the presence of more close-packed BSA, because of negligible net charges, inside the foam lamellae is more effective in reducing foam drainage as compared to a situation with strong repulsive electrostatic interactions.