Electrosprayable Levan-Coated Nanoclusters and Ultrasound-Responsive Drug Delivery for Cancer Therapy.

In this study, we synthesized levan shell hydrophobic silica nanoclusters encapsulating doxorubicin (L-HSi-Dox) and evaluated their potential as ultrasound-responsive drug delivery systems for cancer treatment. L-HSi-Dox nanoclusters were successfully fabricated by integrating a hydrophobic silica nanoparticle-doxorubicin complex as the core and an amphiphilic levan carbohydrate polymer as the shell by using an electrospray technique. Characterization analyses confirmed the stability, size, and composition of the nanoclusters. In particular, the nanoclusters exhibited a controlled release of Dox under aqueous conditions, demonstrating their potential as efficient drug carriers. The levanic groups of the nanoclusters enhanced the targeted delivery of Dox to specific cancer cells. Furthermore, the synergism between the nanoclusters and ultrasound effectively reduced cell viability and induced cell death, particularly in the GLUT5-overexpressing MDA-MB-231 cells. In a tumor xenograft mouse model, treatment with the nanoclusters and ultrasound significantly reduced the tumor volume and weight without affecting the body weight. Collectively, these results highlight the potential of the L-HSi-Dox nanoclusters and ultrasound as promising drug delivery systems with an enhanced therapeutic efficacy for biomedical applications.

Choroidal detachment and hypotony following selective laser trabeculoplasty: a case report.

BACKGROUND: Selective laser trabeculoplasty (SLT) is relatively safe and effective in lowering intraocular pressure (IOP). However, although rare, complications can occur after SLT. This report describes a patient with choroidal detachment due to hypotony following SLT without anterior chamber (AC) inflammation. CASE PRESENTATION: A 67-year-old man was referred for elevated IOP in his left eye with advanced glaucomatous visual field loss. He had previously been diagnosed with idiopathic uveitic glaucoma in the left eye, for which he underwent laser iridotomy, trabeculectomy, and cataract surgery. At the first visit, the IOP of his left eye measured by Goldmann tonometry was 28 mmHg despite maximally tolerated medical treatment. SLT was performed in his left eye, resulting in an IOP of 7 mmHg 7 days later. At 3 weeks post-procedure, the patient experienced ocular pain and decreased visual acuity in his left eye. Slit-lamp examination revealed deep anterior chamber depth and no inflammation reaction, but the IOP in his left eye was 4 mmHg, and both fundus and B-scan ultrasonography showed serous choroidal detachment. All anti-glaucoma agents were stopped, and the patient was started on treatment with oral prednisolone and cyclopentolate eye drops. Three weeks later, choroidal detachment had resolved and the IOP in his left eye had stabilized at 8 mmHg. Follow-up 3 months later showed that the IOP in his left eye remained stable. CONCLUSIONS: Choroidal detachment-related hypotony is a rare complication of SLT. This possible complication following SLT should be informed to the patients and considered when performing the procedure.

Protein, Amino Acid, Oil, Fatty Acid, Sugar, Anthocyanin, Isoflavone, Lutein, and Antioxidant Variations in Colored Seed-Coated Soybeans.

Different physiological and genetic studies show that the variations in the accumulation of pigment-stimulating metabolites result in color differences in soybean seed coats. The objective of this study was to analyze the nutrient contents and antioxidant potential in black, brown, and green seed-coated soybeans. Significant variations in protein (38.9-43.3%), oil (13.9-20.4%), total sugar (63.5-97.0 mg/g seed), total anthocyanin (3826.0-21,856.0 µg/g seed coat), total isoflavone (709.5-3394.3 µg/g seed), lutein (1.9-14.8 µg/g), total polyphenol (123.0-385.8 mg gallic acid/100 g seed), total flavonoid (22.1-208.5 mg catechin/100 g seed), 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid (ABTS; 275.0-818.8 mg Trolox/100 g seed), and 2,2-diphenyl-1-picrylhydrazyl (DPPH; 96.3-579.7 mg Trolox/100 g seed) were found among the soybean genotypes. Ilpumgeomjeong2 contained the lowest protein but the highest oil and total sugar. The lowest oil-containing Wonheug had the highest protein content. Socheong2 was rich in all four variables of antioxidants. Anthocyanins were detected only in black soybeans but not in brown and green soybeans. The variation in isoflavone content was up to 5-fold among the soybean genotypes. This study could be a valuable resource for the selection and improvement of soybean because an understanding of the nutrient content and antioxidant potentials is useful to develop effective strategies for improving the economic traits; for example, the major emphasis of soybean breeding for fatty acids is to enhance the oleic and linoleic acid contents and to decrease linolenic acid content.

Glycan chip based on structure-switchable DNA linker for on-chip biosynthesis of cancer-associated complex glycans.

On-chip glycan biosynthesis is an effective strategy for preparing useful complex glycan sources and for preparing glycan-involved applications simultaneously. However, current methods have some limitations when analyzing biosynthesized glycans and optimizing enzymatic reactions, which could result in undefined glycan structures on a surface, leading to unequal and unreliable results. In this work, a glycan chip is developed by introducing a pH-responsive i-motif DNA linker to control the immobilization and isolation of glycans on chip surfaces in a pH-dependent manner. On-chip enzymatic glycosylations are optimized for uniform biosynthesis of cancer-associated Globo H hexasaccharide and its related complex glycans through stepwise quantitative analyses of isolated products from the surface. Successful interaction analyses of the anti-Globo H antibody and MCF-7 breast cancer cells with on-chip biosynthesized Globo H-related glycans demonstrate the feasibility of the structure-switchable DNA linker-based glycan chip platform for on-chip complex glycan biosynthesis and glycan-involved applications.

Surface Design of Eu-Doped Iron Oxide Nanoparticles for Tuning the Magnetic Relaxivity.

Relaxivity tuning of nanomaterials with the intrinsic T1- T2 dual-contrast ability has great potential for MRI applications. Until now, the relaxivity tuning of T1 and T2 dual-modal MRI nanoprobes has been accomplished through the dopant, size, and morphology of the nanoprobes, leaving room for bioapplications. However, a surface engineering method for the relaxivity tuning was seldom reported. Here, we report the novel relaxivity tuning method based on the surface engineering of dual-mode T1- T2 MRI nanoprobes (DMNPs), along with protein interaction monitoring with the DMNPs as a potential biosensor application. Core nanoparticles (NPs) of europium-doped iron oxide (EuIO) are prepared by a thermal decomposition method. As surface materials, citrate (Cit), alendronate (Ale), and poly(maleic anhydride- alt-1-octadecene)/poly(ethylene glycol) (PP) are employed for the relaxivity tuning of the NPs based on surface engineering, resulting in EuIO-Cit, EuIO-Ale, and EuIO-PP, respectively. The key achievement of the current study is that the surface materials of the DMNP have significant impacts on the r1 and r2 relaxivities. The correlation between the hydrophobicity of the surface material and longitudinal relaxivity ( r1) of EuIO NPs presents an exponential decay feature. The r1 relaxivity of EuIO-Cit is 13.2-fold higher than that of EuIO-PP. EuIO can act as T1- T2 dual-modal (EuIO-Cit) or T2-dominated MRI contrast agents (EuIO-PP) depending on the surface engineering. The feasibility of using the resulting nanosystem as a sensor for environmental changes, such as albumin interaction, was also explored. The albumin interaction on the DMNP shows both T1 and T2 relaxation time changes as mutually confirmative information. The relaxivity tuning approach based on the surface engineering may provide an insightful strategy for bioapplications of DMNPs and give a fresh impetus for the development of novel stimuli-responsive MRI nanoplatforms with T1 and T2 dual-modality for various biomedical applications.

Selection of affinity peptides for interference-free detection of cholera toxin.

Cholera toxin is a major virulent agent of Vibrio cholerae, and it can rapidly lead to severe dehydration, shock, causing death within hours without appropriate clinical treatments. In this study, we present a method wherein unique and short peptides that bind to cholera toxin subunit B (CTX-B) were selected through M13 phage display. Biopanning over recombinant CTX-B led to rapid screening of a unique peptide with an amino acid sequence of VQCRLGPPWCAK, and the phage-displayed peptides analyzed using ELISA, were found to show specific affinities towards CTX-B. To address the use of affinity peptides in development of the biosensor, sequences of newly selected peptides were modified and chemically synthesized to create a series of affinity peptides. Performance of the biosensor was studied using plasmonic-based optical techniques: localized surface plasmon resonance (LSPR) and surface-enhanced Raman scattering (SERS). The limit of detection (LOD) obtained by LSPR with 3σ-rule was 1.89ng/mL, while SERS had a LOD of 3.51pg/mL. In both cases, the sensitivity was much higher than the previously reported values, and our sensor system was specific towards actual CTX-B secreted from V. cholera, but not for CTX-AB5.

Versatile signal peptide of Flavobacterium-originated organophosphorus hydrolase for efficient periplasmic translocation of heterologous proteins in Escherichia coli.

Organophosphorus hydrolase (OPH) from Flavobacterium species is a membrane-associated homodimeric metalloenzyme and has its own signal peptide in its N-terminus. We found that OPH was translocated into the periplasmic space when the original signal peptide-containing OPH was expressed in recombinant Escherichia coli even though its translocation efficiency was relatively low. To investigate the usability of this OPH signal peptide for periplasmic expression of heterologous proteins in an E. coli system, we employed green fluorescent protein (GFP) as a cytoplasmic folding reporter and alkaline phosphatase (ALP) as a periplasmic folding reporter. We found that the OPH signal peptide was able to use both twin-arginine translocation (Tat) and general secretory (Sec) machineries by switching translocation pathways according to the nature of target proteins in E. coli. These results might be due to the lack of Sec-avoidance sequence in the c-region and a moderate hydrophobicity of the OPH signal peptide. Interestingly, the OPH signal peptide considerably enhanced the translocation efficiencies for both GFP and ALP compared with commonly used TorA and PelB signal peptides that have Tat and Sec pathway dependences, respectively. Therefore, this OPH signal peptide could be successfully used in recombinant E. coli system for efficient periplasmic production of target protein regardless of the subcellular localization where functional folding of the protein occurs. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:848-854, 2016.

Surface-independent antibacterial coating using silver nanoparticle-generating engineered mussel glue.

During implant surgeries, antibacterial agents are needed to prevent bacterial infections, which can cause the formation of biofilms between implanted materials and tissue. Mussel adhesive proteins (MAPs) derived from marine mussels are bioadhesives that show strong adhesion and coating ability on various surfaces even in wet environment. Here, we proposed a novel surface-independent antibacterial coating strategy based on the fusion of MAP to a silver-binding peptide, which can synthesize silver nanoparticles having broad antibacterial activity. This sticky recombinant fusion protein enabled the efficient coating on target surface and the easy generation of silver nanoparticles on the coated-surface under mild condition. The biosynthesized silver nanoparticles showed excellent antibacterial efficacy against both Gram-positive and Gram-negative bacteria and also revealed good cytocompatibility with mammalian cells. In this coating strategy, MAP-silver binding peptide fusion proteins provide hybrid environment incorporating inorganic silver nanoparticle and simultaneously mediate the interaction of silver nanoparticle with surroundings. Moreover, the silver nanoparticles were fully synthesized on various surfaces including metal, plastic, and glass by a simple, surface-independent coating manner, and they were also successfully synthesized on a nanofiber surface fabricated by electrospinning of the fusion protein. Thus, this facile surface-independent silver nanoparticle-generating antibacterial coating has great potential to be used for the prevention of bacterial infection in diverse biomedical fields.

Highly purified mussel adhesive protein to secure biosafety for in vivo applications.

BACKGROUND: Unique adhesive and biocompatibility properties of mussel adhesive proteins (MAPs) are known for their great potential in many tissue engineering and biomedical applications. Previously, it was successfully demonstrated that redesigned hybrid type MAP, fp-151, mass-produced in Gram-negative bacterium Escherichia coli, could be utilized as a promising adhesive biomaterial. However, purification of recombinant fp-151 has been unsatisfactory due to its adhesive nature and polarity which make separation of contaminants (especially, lipopolysaccharide, a toxic Gram-negative cell membrane component) very difficult. RESULTS: In the present work, we devised a high resolution purification approach to secure safety standards of recombinant fp-151 for the successful use in in vivo applications. Undesirable impurities were remarkably eliminated as going through sequential steps including treatment with multivalent ion and chelating agent for cell membrane washing, mechanical cell disruption, non-ionic surfactant treatment for isolated inclusion body washing, acid extraction of washed inclusion body, and ion exchange chromatography purification of acid extracted sample. Through various analyses, such as high performance liquid chromatographic purity assay, limulus amoebocyte lysate endotoxin assay, and in vitro mouse macrophage cell tests on inflammation, viability, cytotoxicity, and apoptosis, we confirmed the biological safety of bacterial-derived purified recombinant fp-151. CONCLUSIONS: Through this purification design, recombinant fp-151 achieved 99.90% protein purity and 99.91% endotoxin reduction that nearly no inflammation response was observed in in vitro experiments. Thus, the highly purified recombinant MAP would be successfully used as a safety-secured in vivo bioadhesive for tissue engineering and biomedical applications.

Photolabile micropatterned surfaces for cell capture and release.

A method for capture and release of cells was developed using a photolabile linker and antibody-attached glass surface with a poly(ethylene glycol) (PEG)-pattern.

The use of glass substrates with bi-functional silanes for designing micropatterned cell-secreted cytokine immunoassays.

It is often desirable to sequester cells in specific locations on the surface and to integrate sensing elements next to the cells. In the present study, surfaces were fabricated so as to position cytokine sensing domains inside non-fouling poly(ethylene glycol) (PEG) hydrogel microwells. Our aim was to increase sensitivity of micropatterned cytokine immunoassays through covalent attachment of biorecognition molecules. To achieve this, glass substrates were functionalized with a binary mixture of acrylate- and thiol-terminated methoxysilanes. During subsequent hydrogel photopatterning steps, acrylate moieties served to anchor hydrogel microwells to glass substrates. Importantly, glass attachment sites within the microwells contained thiol groups that could be activated with a hetero-bifunctional cross-linker for covalent immobilization of proteins. After incubation with fluorescently-labeled avidin, microwells fabricated on a mixed acryl/thiol silane layer emitted ∼ 6 times more fluorescence compared to microwells fabricated on an acryl silane alone. This result highlighted the advantages of covalent attachment of avidin inside the microwells. To create cytokine immunoassays, micropatterned surfaces were incubated with biotinylated IFN-γ or TNF-α antibodies (Abs). Micropatterned immunoassays prepared in this manner were sensitive down to 1 ng/ml or 60 pM IFN-γ. To further prove utility of this biointerface design, macrophages were seeded into 30 µm diameter microwells fabricated on either bi-functional (acryl/thiol) or mono-functional silane layers. Both types of microwells were coated with avidin and biotin-anti-TNF-α prior to cell seeding. Short mitogenic activation followed by immunostaining for TNF-α revealed that microwells created on bi-functional silane layer had 3 times higher signal due to macrophage-secreted TNF-α compared to microwells fabricated on mono-functional silane. The rational design of cytokine-sensing surfaces described here, will be leveraged in the future for rapid detection of multiple cytokines secreted by individual immune cells.

High and compact formation of baculoviral polyhedrin-induced inclusion body by co-expression of baculoviral FP25 in Escherichia coli.

Previously, we found that baculoviral polyhedrin (Polh) can successfully be used in Escherichia coli as a fusion partner for the expression of special foreign proteins as inclusion bodies, and the resulting, easily isolatable Polh-induced fusion inclusion bodies had almost the same characteristics as the native Polh. Here, we investigated the effects of co-expression of baculoviral FP25 protein on Polh-induced inclusion-body production in an E. coli expression system, as FP25 is known to be involved specifically in polyhedra formation. Using several analytical tools, including SDS-PAGE, pronase proteolysis, solubilization under alkaline conditions, and electron microscopy, we found that co-expressed FP25 was associated with Polh-induced inclusion bodies and that its co-expression led to formation of compact inclusion bodies as well as high production levels. We confirmed that FP25 co-expression induced higher production levels of other heterologous protein, antimicrobial peptide Hal18, fused with aggregation-prone Polh. Therefore, co-expression of baculoviral FP25 can be promisingly used to increase the levels of baculoviral Polh-fused foreign proteins, especially harmful proteins, expressed as inclusion bodies in an E. coli expression system.

Facilitation of expression and purification of an antimicrobial peptide by fusion with baculoviral polyhedrin in Escherichia coli.

Several fusion strategies have been developed for the expression and purification of small antimicrobial peptides (AMPs) in recombinant bacterial expression systems. However, some of these efforts have been limited by product toxicity to host cells, product proteolysis, low expression levels, poor recovery yields, and sometimes an absence of posttranslational modifications required for biological activity. For the present work, we investigated the use of the baculoviral polyhedrin (Polh) protein as a novel fusion partner for the production of a model AMP (halocidin 18-amino-acid subunit; Hal18) in Escherichia coli. The useful solubility properties of Polh as a fusion partner facilitated the expression of the Polh-Hal18 fusion protein ( approximately 33.6 kDa) by forming insoluble inclusion bodies in E. coli which could easily be purified by inclusion body isolation and affinity purification using the fused hexahistidine tag. The recombinant Hal18 AMP ( approximately 2 kDa) could then be cleaved with hydroxylamine from the fusion protein and easily recovered by simple dialysis and centrifugation. This was facilitated by the fact that Polh was soluble during the alkaline cleavage reaction but became insoluble during dialysis at a neutral pH. Reverse-phase high-performance liquid chromatography was used to further purify the separated recombinant Hal18, giving a final yield of 30% with >90% purity. Importantly, recombinant and synthetic Hal18 peptides showed nearly identical antimicrobial activities against E. coli and Staphylococcus aureus, which were used as representative gram-negative and gram-positive bacteria, respectively. These results demonstrate that baculoviral Polh can provide an efficient and facile platform for the production or functional study of target AMPs.