Proteins and nucleic acids derived from bioresources have become a novel medical modality.
However, the intrinsic features of proteins, such as their high molecular weight and polarity, impede their passage through the cell membrane.
In this study, to deliver proteins to cancer cells, we prepared conjugates consisting of bovine serum albumin (BSA) and hyaluronic acid (HA).
These conjugates contained multiple BSA and HA molecules and adopted a highly condensed structure by crosslinking through BSA.
When the interaction between the HA-BSA conjugates and recombinant CD44 (rCD44) was examined using a quartz crystal microbalance, the conjugates induced a larger decrease of frequency change than HA.
CT26 cells treated with FITC-labeled HA-BSA conjugates showed high fluorescence intensity. The uptake of the conjugates decreased upon adding excess HA.
Therefore, the conjugates and nanoparticles with densely packed HA structures could be a potent and effective platform for delivering proteins to cancer tissues.
Zebrafish (Danio rerio) larva as an in vivo vertebrate model to study renal function
There is an increasing interest in using the zebrafish (Danio rerio) larva as a vertebrate screening model to study drug disposition.
Since the pronephric kidney of zebrafish larvae shares high similarity with the anatomy of nephrons in higher vertebrates including humans, we explored in the present study whether 3 to 4 days old zebrafish larvae have a fully functional pronephron.
Intravenous injection of fluorescent polyethylene glycol and dextran derivatives of different molecular weight revealed a cut-off of 4.4 to 7.6 nm in hydrodynamic diameter for passive glomerular filtration, which is in agreement with corresponding values in rodents and humans.
Distal tubular reabsorption of a FITC-folate conjugate, covalently modified with PEG2000, via the folate receptor 1 was shown.
Transport experiments of fluorescent substrates were assessed in the presence and absence of specific inhibitors in the blood systems.
Thereby, functional expression in the proximal tubule of oat/slc22, mrp1/abcc1, mrp2/abcc2, mrp4/abcc4 and the zebrafish larvaes’ p-glycoprotein analog abcb4 was shown.
In addition, non-renal clearance of fluorescent substrates and plasmaprotein binding characteristics were assessed in vivo.
Results of transporter studies were confirmed by extrapolation to ex vivo experiments in killifish (Fundulus heteroclitus) proximal kidney tubules. We conclude that the zebrafish larva has a fully functional pronephron at 96 hours post fertilization and is therefore an attractive translational vertebrate screening model to bridge the gap between cell culture-based test systems and pharmacokinetic experiments in higher vertebrates.
Synthesis and Identification of Biologically Active Mono-Labelled FITC-Insulin Conjugate
Fluorescently labelling proteins such as insulin have wide ranging applications in a pharmaceutical research and drug delivery.
Human insulin (Actrapid®) was labelled with fluorescein isothiocyanate (FITC) and the synthesized conjugate identified using reverse phase high-performance liquid chromatography (RP-HPLC) on a C18 column and a gradient method with mobile phase A containing 0.1% trifluoroacetic acid (TFA) in Millipore water and mobile phase B containing 90% Acetonitrile, 10% Millipore water and 0.1% TFA.
Syntheses were carried out at varying reaction times between 4 and 20 h.
Mono-labelled FITC-insulin conjugate was successfully synthesised with labelling at the B1 position on the insulin chain using a molar ratio of 2:1 (FITC:insulin) at a reaction time of 18 h and confirmed by electrospray mass spectroscopy.
Reactions were studied across a pH range of 7-9.8 and the quantities switch from mono-labelled to di-labelled FITC-insulin conjugates at a reaction time of 2 h (2:1 molar ratio) at pH > 8. The conjugates isolated from the studies had biological activities in comparison to native insulin of 99.5% monoB1, 78% monoA1, 51% diA1B1 and 0.06% triA1B1B29 in HUVEC cells by examining AKT phosphorylation levels. MonoB1 FITC-insulin conjugate was also compared to native insulin by examining cell surface GLUT4 in C2C12 skeletal muscle cells.
No significant difference in the cellular response was observed for monoB1 produced in-house compared to native insulin.
Therefore mono-labelled FITC-insulin at the B1 position showed similar biological activity as native insulin and can potentially be used for future biomedical applications.
Establishment of a steroid binding assay for membrane progesterone receptor alpha (PAQR7) by using graphene quantum dots (GQDs)
Currently, semiconductor nanoparticles known as quantum dots (QDs) have attracted interest in various application fields such as those requiring sensing properties, binding assays, and cellular imaging and are the very important in the acceleration of drug discovery due to their unique photophysical properties.
Here, we applied graphene quantum dots (GQDs) for the binding assay of membrane progesterone receptor alpha (mPRα), one of the probable membrane receptors that have potential in drug discovery applications.
By coupling the amino groups of mPRα with GQDs, we prepared fluorogenic GQD-conjugated mPRα (GQD-mPRα). When mixed with a progesterone-BSA-fluorescein isothiocyanate conjugate (P4-BSA-FITC) to check the ligand receptor binding activity of GQD-mPRα, fluorescence at 520 nm appeared.
The fluorescence at 520 nm was reduced by the addition of free progesterone into the reaction mixture. GQD-coupled BSA (GQD-BSA) did not show a reduction in fluorescence at 520 nm. The results demonstrated the formation of a complex of GQD-mPRα and P4-BSA-FITC with ligand receptor binding.
We established a ligand binding assay for membrane steroid receptors that is applicable for high-throughput assays.
Silk-Mesoporous Silica-Based Hybrid Macroporous Scaffolds using Ice-Templating Method: Mechanical, Release, and Biological Studies
Development of biocompatible, biodegradable, and drug-eluting macroporous three-dimensional scaffolds that mimic the extracellular matrix of cells remains an important challenge in tissue engineering.
In this endeavor, we report the preparation of self-standing macroporous scaffold composed of the natural biopolymer silk fibroin and mesoporous silica particle using the ice-templating strategy.
Using methanol as a physical cross-linker, we were able to make self-standing scaffolds with very high mesoporous silica content (∼75% by weight) and with varying mechanical properties (38 ± 1.0 to 181 ± 5.9 kPa).
These macroporous scaffolds have ∼80% porosity with an average pore size of 60 μm. Scaffolds that encapsulated the small molecule doxorubicin (as a model drug) and macromolecule fluorescein isothiocyanate conjugate-bovine serum albumin (FITC-BSA) (as a model protein) were also prepared.
Prealbumin Protein Polyclonal Antibody, FITC Conjugated |
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| A57606 | EpiGentek |
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Prealbumin Protein Polyclonal Antibody, HRP Conjugated |
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| A57605 | EpiGentek |
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Prealbumin Protein Polyclonal Antibody, Biotin Conjugated |
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| A57607 | EpiGentek |
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Guinea pig anti-Human Prealbumin protein polyclonal Antibody, HRP conjugated |
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| MBS715990-005mg | MyBiosource | 0.05mg | 190 EUR |
Guinea pig anti-Human Prealbumin protein polyclonal Antibody, HRP conjugated |
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| MBS715990-01mg | MyBiosource | 0.1mg | 270 EUR |
Guinea pig anti-Human Prealbumin protein polyclonal Antibody, HRP conjugated |
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| MBS715990-5x01mg | MyBiosource | 5x0.1mg | 1205 EUR |
Guinea pig anti-Human Prealbumin protein polyclonal Antibody, Biotin conjugated |
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| MBS715602-005mg | MyBiosource | 0.05mg | 190 EUR |
Guinea pig anti-Human Prealbumin protein polyclonal Antibody, Biotin conjugated |
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| MBS715602-01mg | MyBiosource | 0.1mg | 270 EUR |
Guinea pig anti-Human Prealbumin protein polyclonal Antibody, Biotin conjugated |
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| MBS715602-5x01mg | MyBiosource | 5x0.1mg | 1205 EUR |
Prealbumin Conjugated Antibody |
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| C49356 | SAB | 100ul | 476.4 EUR |
Prealbumin Conjugated Antibody |
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| MBS9456984-01mLAF350 | MyBiosource | 0.1mL(AF350) | 480 EUR |
Prealbumin Conjugated Antibody |
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| MBS9456984-01mLAF405 | MyBiosource | 0.1mL(AF405) | 480 EUR |
Prealbumin Conjugated Antibody |
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| MBS9456984-01mLAF488 | MyBiosource | 0.1mL(AF488) | 480 EUR |
Prealbumin Conjugated Antibody |
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| MBS9456984-01mLAF555 | MyBiosource | 0.1mL(AF555) | 480 EUR |
Prealbumin Conjugated Antibody |
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| MBS9456984-01mLBiotin | MyBiosource | 0.1mL(Biotin) | 480 EUR |
Guinea pig anti-Human Prealbumin protein polyclonal Antibody, FITC |
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| MBS715956-005mg | MyBiosource | 0.05mg | 190 EUR |
We demonstrate that the release behavior of encapsulated molecules like doxorubicin (∼35% release) and FITC-BSA (∼47% release) is largely influenced by their interaction with the mesoporous silica particles and the silk fibroin.
The biodegradability property of silk hybrid scaffolds is also determined in the presence of protease enzyme, which demonstrates ∼90% degradation in 21 d.
Biological studies on ice-templated hybrid silk scaffolds demonstrate excellent biocompatibility, which indicates that hybrid scaffolds are promising candidate for therapeutically relevant repair and regeneration of soft tissues such as tendon and nascent bone.