Two-Step Preparation of Protein-Decorated Biohybrid Quantum Dot Nanoparticles for Cellular Uptake.

Decoration of nanoparticles with specific molecules such as antibodies, peptides, and proteins that preserve their biological properties is essential for the recognition and internalization of their specific target cells. Inefficient preparation of such decorated nanoparticles leads to nonspecific interactions diverting them from their desired target. We report a simple two-step procedure for the preparation of biohybrid nanoparticles containing a core of hydrophobic quantum dots coated with a multilayer of human serum albumin. These nanoparticles were prepared by ultra-sonication, crosslinked using glutaraldehyde, and decorated with proteins such as human serum albumin or human transferrin in their native conformations. These nanoparticles were homogeneous in size (20-30 nm), retained the fluorescent properties of quantum dots, and did not show a "corona effect" in the presence of serum. The uptake of transferrin-decorated quantum dot nanoparticles was observed in A549 lung cancer and SH-SY5Y neuroblastoma cells but not in non-cancerous 16HB14o- or retinoic acid dopaminergic neurons differentiated SH-SY5Y cells. Furthermore, digitoxin-loaded transferrin-decorated nanoparticles decreased the number of A549 cells without effect on 16HB14o-. Finally, we analyzed the in vivo uptake of these biohybrids by murine retinal cells, demonstrating their capacity to selectively target and deliver into specific cell types with excellent traceability.

IAEA Contribution to Nanosized Targeted Radiopharmaceuticals for Drug Delivery.

The rapidly growing interest in the application of nanoscience in the future design of radiopharmaceuticals and the development of nanosized radiopharmaceuticals in the late 2000's, resulted in the creation of a Coordinated Research Project (CRP) by the International Atomic Energy Agency (IAEA) in 2014. This CRP entitled 'Nanosized delivery systems for radiopharmaceuticals' involved a team of expert scientist from various member states. This team of scientists worked on a number of cutting-edge areas of nanoscience with a focus on developing well-defined, highly effective and site-specific delivery systems of radiopharmaceuticals. Specifically, focus areas of various teams of scientists comprised of the development of nanoparticles (NPs) based on metals, polymers, and gels, and their conjugation/encapsulation or decoration with various tumor avid ligands such as peptides, folates, and small molecule phytochemicals. The research and development efforts also comprised of developing optimum radiolabeling methods of various nano vectors using diagnostic and therapeutic radionuclides including Tc-99m, Ga-68, Lu-177 and Au-198. Concerted efforts of teams of scientists within this CRP has resulted in the development of various protocols and guidelines on delivery systems of nanoradiopharmaceuticals, training of numerous graduate students/post-doctoral fellows and publications in peer reviewed journals while establishing numerous productive scientific networks in various participating member states. Some of the innovative nanoconstructs were chosen for further preclinical applications-all aimed at ultimate clinical translation for treating human cancer patients. This review article summarizes outcomes of this major international scientific endeavor.

One Step Histological Detection and Staining of the PTEN Tumor Suppressor Protein by a Single Strand DNA.

Antibodies are the most used technological tool in histochemistry. However, even with monoclonal antibodies, their standardization is difficult due to variation of biological systems as well as to variability due to the affinity and amplification of the signal arising from secondary peroxidase detection systems. In this article we combined two synthetic molecules to facilitate the standardization of a detection protocol of protein markers in histological sections. The first molecule was an aptamer, a 50-base single-stranded DNA fragment, which recognizes a PTEN tumor suppressor. The second molecule used was also another single stranded 18-base aptamer DNA fragment, which forms a quadruplex structure guanine box. This G-quadruplex recognizes and attaches a molecule of hemin, increasing the catalytic capacity for the hydrogen peroxide. Our results show how the correct structural design of DNA combining an aptamer together with the peroxidase-like DNAzyme allows to detect proteins in histological sections. This tool offers the standardization of the detection of prognostic markers in cancer, in quality and quantity, due to its synthetic nature and its 1:1 antigen:enzyme ratio. This is the first time that reproducible results have been presented in histological sections staining a cancer marker using a single-stranded DNA molecule with dual function.

Effect of structure in ionised albumin based nanoparticle: Characterisation, Emodin interaction, and in vitro cytotoxicity.

A γ-irradiated bovine albumin serum-based nanoparticle was characterised structurally, and functionally. The nanoparticle was characterised by A.F.M., D.L.S, zeta potential, T.E.M., gel-electrophoresis, and spectroscopy. We studied the stability of the nanoparticle at different pH values and against time, by fluorescence spectroscopy following the changes in the tryptophan environment in the nanoparticle. The nanoparticle was also functionalized with Folic Acid, its function as a nanovehicle was evaluated through its interaction with the hydrophobic drug Emodin. The binding and kinetic properties of the obtained complex were evaluated by biophysical methods as well as its toxicity in tumor cells. According to its biophysics, the nanoparticle is a spherical nanosized vehicle with a hydrodynamic diameter of 70 nm. Data obtained describe the nanoparticle as nontoxic for cancer cell lines. When combined with Emodin, the nanoparticle proved to be more active on MCF-7 cancer cell lines than the nanoparticle without Emodin. Significantly, the albumin aggregate preserves the main activity-function of albumin and improved characteristics as an excellent carrier of molecules. More than carrier properties, the nanoparticle alone induced an immune response in macrophages which may be advantageous in vaccine and cancer therapy formulation.

Albumin Nanocarriers, γ- Irradiated Crosslinked, Combined with Therapeutic Drugs for Cancer Therapy>.

Albumin polymeric Nanoparticles (NPs) have opened a great expectancy as for controlled drug delivery due to their therapeutic potency. Concomitantly biodegradable NPs technologies with target linked structures to pave the way of personalised medicine are becoming increasingly important in sight of a therapeutically effective research technology. This is particularly attractive for nanoparticle-based cancer delivery systems, based on the known limitations and efforts to overcome. This new group of gamma irradiated-NPs inherited both the protein delivery properties and robustness of polymer forming structures, and gamma irradiation techniques that leave clean, innocuous and biodegradable NPs. These protein NPs made of serum albumin are referred to SA NPs that possesses several characteristics making them especially attractive to be considered as a drug delivery system. This review focused on methodologies actually being used in the synthesis and characterisation of albumin NPs and different author's opinions on strategic ways to treat cancerous cell-lines with NPs. Utterly, challenges being overthrown by researchers are brought up to anneal an effective, all in one targeted albumin NPs to passed through in vitro and preclinical trials.

Immobilized palladium(II) ion affinity chromatography for recovery of recombinant proteins with peptide tags containing histidine and cysteine.

Fusion of peptide-based tags to recombinant proteins is currently one of the most used tools for protein production. Also, immobilized metal ion affinity chromatography (IMAC) has a huge application in protein purification, especially in research labs. The combination of expression systems of recombinant tagged proteins with this robust chromatographic system has become an efficient and rapid tool to produce milligram-range amounts of proteins. IMAC-Ni(II) columns have become the natural partners of 6xHis-tagged proteins. The Ni(II) ion is considered as the best compromise of selectivity and affinity for purification of a recombinant His-tagged protein. The palladium(II) ion is also able to bind to side chains of amino acids and form ternary complexes with iminodiacetic acid and free amino acids and other sulfur-containing molecules. In this work, we evaluated two different cysteine- and histidine-containing six amino acid tags linked to the N-terminal group of green fluorescent protein (GFP) and studied the adsorption and elution conditions using novel eluents. Both cysteine-containing tagged GFPs were able to bind to IMAC-Pd(II) matrices and eluted successfully using a low concentration of thiourea solution. The IMAC-Ni(II) system reaches less than 20% recovery of the cysteine-containing tagged GFP from a crude homogenate of recombinant Escherichia coli, meanwhile the IMAC-Pd(II) yields a recovery of 45% with a purification factor of 13.

An arsenic fluorescent compound as a novel probe to study arsenic-binding proteins.

Arsenic-binding proteins are under continuous research. Their identification and the elucidation of arsenic/protein interaction mechanisms are important because the biological effects of these complexes may be related not only to arsenic but also to the arsenic/protein structure. Although many proteins bearing a CXXC motif have been found to bind arsenic in vivo, new tools are necessary to identify new arsenic targets and allow research on protein/arsenic complexes. In this work, we analyzed the performance of the fluorescent compound APAO-FITC (synthesized from p-aminophenylarsenoxide, APAO, and fluorescein isothiocyanate, FITC) in arsenic/protein binding assays using thioredoxin 1 (Trx) as an arsenic-binding protein model. The Trx-APAO-FITC complex was studied through different spectroscopic techniques involving UV-Vis, fluorescence, atomic absorption, infrared and circular dichroism. Our results show that APAO-FITC binds efficiently and specifically to the Trx binding site, labeling the protein fluorescently, without altering its structure and activity. In summary, we were able to study a protein/arsenic complex model, using APAO-FITC as a labeling probe. The use of APAO-FITC in the identification of different protein and cell targets, as well as in in vivo biodistribution studies, conformational studies of arsenic-binding proteins, and studies for the design of drug delivery systems for arsenic anti-cancer therapies, is highly promising.

Synthesis of a fluorescently labeled compound for the detection of arsenic-induced apoptotic HL60 cells.

Arsenic compounds have shown medical usefulness since they proved to be effective in causing complete remission of acute promyelocytic leukemia. In this work we obtained a fluorescently labeled arsenic compound that can be used with current fluorescence techniques for basic and applied research, focused on arsenic-induced apoptosis studies. This compound is an arsanilic acid bearing a covalently linked FITC that was chemically synthesized and characterized by fluorescence, UV-Vis, mass and FTIR spectrometry. In addition, we assessed its apoptotic activity as well as its fluorescent labeling properties in HL60 cell line as a leukemia cell model through flow cytometry. We obtained a compound with a 1:1 FITC:arsenic ratio and a 595 m/z, confirming its structure by FTIR. This compound proved to be useful at inducing apoptosis in the leukemia cell model and labeling this apoptotic cell population, in such a way that the highest FITC fluorescence correlated with the highest arsenic amount.

Iron protoporphyrin-modified monolithic support for on-line preconcentration of angiotensin prior to CE analysis.

An on-line preconcentration method using a polymeric monolithic support is proposed for the retention of the decapeptide angiotensin I and its subsequent analysis by CZE. Monolithic capillary columns were prepared in fused-silica (FS) capillaries of 150 microm id by ionizing radiation-initiated in situ polymerization and cross-linking of diethylene glycol dimethacrylate and glycidyl methacrylate, and chemically modified with iron protoporphyrin IX (Fe-ProP). Monolithic microcolumns (8 mm long) were coupled on-line to the inlet of the separation capillary (FS capillary, 75 microm id x10 cm from the inlet to the microcolumn and 27 cm from the microcolumn to the detector). Angiotensin I was released from the sorbent by a 50 mM sodium phosphate, pH 2.5/ACN, 75:25 v/v solution and then analyzed by CZE with UV absorption detection at 214 nm. The concentration LOQ (CLOQ) was 0.5 ng/mL. The Fe-ProP-derivatized monolithic microcolumn coupled to the separation capillary exhibited a high retention capacity for peptide angiotensin I, and showed as much as 10,000-fold improvement in concentration sensitivity.

On-line affinity selection of histidine-containing peptides using a polymeric monolithic support for capillary electrophoresis.

An on-line affinity selection method using a polymeric monolithic support is proposed for the retention of histidine-containing peptides and their subsequent separation by capillary zone electrophoresis (CZE). Monolithic capillary columns were prepared in fused-silica capillaries of 150 mum inner diameter (ID) by ionizing radiation-initiated in situ polymerization and cross-linking of diethylene glycol dimethacrylate and glycidyl methacrylate, and chemically modified with iminodiacetic acid (IDA) and copper ion. Monolithic microextractors were coupled on-line near the inlet of the separation capillary (fused-silica capillary, 75 mum ID x 28 cm from the microextractor to the detector). Model peptide mixtures of histidine-containing and histidine-noncontaining peptides were assessed. Peptides were released from the sorbent by a 5 mM imidazole solution and then separated by CZE with ultraviolet detection. Relative standard deviation values for migration times and corrected peak areas were found to be lower than 5.8 and 10.5%, respectively. IDA-Cu(II) ion modified monolithic microextractors showed a chromatographic behavior and could be reused at least 25 times. The use of monolithic supports proved to be an advantageous alternative to packed particles for the preparation of microextractors.