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.

PCR-tips for rapid diagnosis of bacterial pathogens.

Micropipette tips are currently among the most used disposable devices in bioresearch and development laboratories. Their main application is the fractionation of solutions. New functionalities have recently been added to this device, widening their applications. This paper analyzed disposable micropipette tips as reagent holders of PCR reagents. PCR has become a prevalent and often indispensable technique in biological laboratories for various applications, such as the detection of coronavirus and other infectious diseases. A functional micropipette tip was implemented to simplify PCR analysis and reduce the contamination chances of deoxynucleotides and specific primers. This disposable device is prepared by tip coating processes of reagents, using polyvinyl alcohol polymer and additives. The coated layer is optimized to load and release PCR reagents efficiently. As a proof of concept, we show that the detection of Bordetella pertussis, the etiological agent of whooping cough whose diagnostic relies on PCR, can be quickly done using practical-functional tips. This device is an excellent example of testing the functionality and contribution of molecular diagnostic PCR tips. KEY POINTS: • Functional micropipette tips are prepared by coating with dNTPs and primers. • Functional tips are used to replace dNTPs and primers in the PCR master mix. • PCR diagnostic of Bordetella pertussis is performed using functional tips.

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.

Media interaction and stability of a gamma irradiated albumin nanoparticle.

Protein nanoparticle designing at the nanoscale is challenging because of protein vulnerability to chemical and physical degradation during processing and biodistribution. We present a crosslinked gamma irradiated albumin-based nanoparticle as a potential drug delivery system. The focus was set on the study of the nanoparticle under adverse experimental conditions: different pH values, SDS, tween 80 and urea. The albumin-based nanoparticle was also tested against time stabilityand ionic strength solutions Regarding its stability against pH, the nanoparticle showed similarity to the behaviour of albumin, whilst the stability of the nanoparticle improved in urea and Tween 80. The nanoparticle was stable for 15 days, and presented no protein degradation in solutions up to 2 M salt concentration. Moreover, it presented a better and controlled drug release at slightly acidic pH values than at physiological pH. Results highlight the potentiality of the nanoparticle due to its biophysical properties as a drug delivery system. The hydrophobic character of the albumin molecules changes when they are in aggregating conditions, and treated with gamma irradiation. Our results reveal that stability of the nanoparticle can result from a competition between short-range attraction and long-range repulsion. They provide a framework for understanding the stability and functioning of nanoparticles. Most interesting, the results here serve as a platform for improving the design of the nanoparticle for future in vivo testing.

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.

Albumin-based nanoparticle trehalose lyophilisation stress-down to preserve structure/function and enhanced binding.

The aim of this study was to preserve albumin nanoparticle structure/function during the lyophilisation process. Bovine serum albumin nanoparticles were obtained by γ-irradiation. Nanoparticles were lyophilised in buffer, miliQ water or in trehalose/miliQ solution. The size and charge of the nanoparticles were tested after lyophilisation by light scattering and Z potential. The most relevant results in size of BSA nanoparticle were those lyophilised in PBS between 20 and 350nm, assembled in different aggregates, and negative Z potential obtained was 37±8mV in all, and those nanoparticles lyophilised with trehalose had a size range of 70±2nm and a negative Z potential of 20±5mV. Structure determination of surface aminoacids SH groups in the BSA NP lyophilised in PBS showed an increase in the free SH groups. Different aggregates had different amount of SH groups exposure from 55 to 938 (from smaller to bigger aggregates), whereas BSA NP lyophilised with trehalose showed no significant difference if compared with BSA NP. The binding properties of the BSA nanoparticle with a theragnostic probe (merocyanine 540) were studied after lyophilisation. Results showed more affinity between the BSA NP lyophilised with trehalose than that observed with non lyophilised BSA NP. As a result, the lyophilisation condition in trehalose 100µM solution is the best one to preserve the BSA NP structure/function and the one with the enhance binding affinity of the BSA NP.

High-level fed-batch fermentative expression of an engineered Staphylococcal protein A based ligand in E. coli: purification and characterization.

The major platform for high level recombinant protein production is based on genetically modified microorganisms like Escherichia coli (E. coli) due to its short dividing time, ability to use inexpensive substrates and additionally, its genetics is comparatively simple, well characterized and can be manipulated easily. Here, we investigated the possibilities of finding the best media for high cell density fermentation, by analyzing different media samples, focusing on improving fermentation techniques and recombinant protein production. Initial fermentation of E. coli BL21 DE3:pAV01 in baffled flasks showed that high cell density was achieved when using complex media, Luria-Bertani (LB) and Terrific medium broth (TB) (10 and 14 g/L wet weight, respectively), as compared to mineral media M9, modified minimal medium (MMM) and Riesenberg mineral medium (RM) (7, 8 and 7 g/L, respectively). However, in fed-batch fermentation processes when using MMM after 25 h cultivation, it was possible to yield an optical density (OD600) of 139 corresponding to 172 g/L of wet biomass was produced in a 30 L TV Techfors-S Infors HT fermenter, with a computer controlled nutrient supply (glucose as a carbon source) delivery system, indicating nearly 1.5 times that obtained from TB. Upon purification, a total of 1.65 mg/g of protein per gram cell biomass was obtained and the purified AviPure showed affinity for immunoglobulin. High cell density fed batch fermentation was achieved by selecting the best media and growth conditions, by utilizing a number of fermentation parameters like media, fermentation conditions, chemical concentrations, pO2 level, stirrer speed, pH level and feed media addition. It is possible to reach cell densities higher than shake flasks and stirred tank reactors with the improved oxygen transfer rate and feed.

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.

High capacity cryogel-type adsorbents for protein purification.

Cryogel bodies were modified to obtain epoxy groups by graft-copolymerization using both chemical and gamma irradiation initiation techniques. The free epoxy adsorbents were reacted further to introduce diethylaminoethanol (DEAE) functionalities. The resulting weak anion-exchange cryogel adsorbents showed dynamic binding capacities of ca. 27±3mg/mL, which was significantly higher than previously reported for this type of adsorbent material. Gamma irradiated grafting initiation showed a 4-fold higher capacity for proteins than chemical grafting initiation procedures. The phosphate capacity for these DEAE cryogels was 119mmol/L and also showed similar column efficiency as compared to commercial adsorbents. The large pores in the cryogel structure ensure convective transport of the molecules to active binding sites located on the polymer-grafted surface of cryogels. However, as cryogels have relatively large pores (10-100µm), the BET area available for surface activation is low, and consequently, the capacity of the cryogels is relatively low for biomolecules, especially when compared to commercial beaded adsorbents. Nevertheless, we have shown that gamma ray mediated surface grafting of cryogel matrices greatly enhance their functional and adsorptive properties.

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.

Preparation, characterization, and process performance of composite fibrous adsorbents as cation exchangers for high throughput and high capacity bioseparations.

Fibrous materials are proposed as novel chromatographic supports depicting high throughput and high product capacity. In this work, a composite fiber harboring strong cation-exchange moieties has been investigated. Such materials were characterized by a plethora of physical methods including degree of swelling (DS), scanning electron microscope (SEM), confocal laser scanning microscopy (CLSM), and Fourier transform infrared spectroscopy-attenuated total reflection (FTIR-ATR). The composite showed a high degree of grafting (∼30%) and exhibited a high swelling ratio (∼300%). Moreover, homogenous grafting and the development of an internal (functional) hydrogel were observed. The fibrous adsorbent was packed utilizing a designed "double roll" supported-structure and subsequently tested for packing efficiency and chromatography performance. The mentioned system showed similar packing efficiency of height equivalent to a theoretical plate (HETP) value and higher permeability coefficient (0.92 × 10⁻7 cm²) than commercial resins. Experimentally determined Peclet number (Pe) values were within the range 60-90, suggesting a close-to-plug-flow condition. Total ionic capacity of the fibrous adsorbent was determined by the transition pH method. A capacity of 6.5 mequiv./g was obtained. Moreover, a high dynamic binding capacity for lysozyme was found to be 283 mg/g. On the other hand, a bed of randomly packed fiber also demonstrated high-resolution ability when a mixture of model protein was utilized to that end. Resolution was maintained at high flow rates (up to 900 cm/h) and utilizing shorter gradient development routines. Direct sequestration of a model protein (lysozyme) was also possible from an artificial mixture containing 1.5% yeast homogenate. Summarizing, the composite fibrous adsorbents exhibited superior performance during early protein capture and intermediate-resolution applications.

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.

Synthesis and performance of 3D-megaporous structures for enzyme immobilization and protein capture.

The preparation of megaporous bodies, with potential applications in biotechnology, was attempted by following several strategies. As a first step, naive and robust scaffolds were produced by polymerization of selected monomers in the presence of a highly soluble cross-linker agent. Ion-exchange function was incorporated by particle embedding, direct chemical synthesis, or radiation-induced grafting. The total ionic capacity of such systems was 1.5 mmol H(+)/g, 1.4 mmol H(+)/g, and 17 mmol H(+)/g, respectively. These values were in agreement with the ability to bind model proteins: observed dynamic binding capacity at 50% breakthrough was ≅7.2 mg bovine serum albumin/g, ≅7.4 hen egg-white lysozyme (HEWL) mg/g, and ≅108 HEWL mg/g. In the later case, total (static) binding capacity reached 220 mg/g. It was observed that the structure and size of the megapores remained unaffected by the grafting procedure which, however, allowed for the highest protein binding capacity. Lysozyme supported on grafted body showed extensive clarification activity against a Micrococcus lysodekticus suspension in the flow-through mode, i.e., 90% destruction of suspended microbial cells was obtained with a residence time ≈ 18 min. Both protein capture and biocatalysis applications are conceivable with the 3D-megaporous materials described in this work.

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.

Improved hollow-fibre membranes for dye-affinity chromatography.

Hollow-fibre membranes with different degrees of surface hydrophilicity were obtained by grafting mixtures of glycidyl methacrylate (GMA) and dimethyl acrylamide (DMAA) in various proportions, and Cibacron Blue F3G-A was attached to them through ammonia or glucamine spacers. Membrane hydrophilicity increased with the amount of dimethyl acrylamide in the grafted polymer. As the hydrophilicity increased the permeability decreased from 352 mL/cm2 min MPa for membranes grafted with GMA with ammonia spacer to 12.7 mL/cm2 min MPa for membranes grafted with GMA/DMAA 1/3 with glucamine spacer. Membranes grafted with GMA/DMAA 1/3 with ammonia spacer showed the best performance for BSA and lysozyme adsorption: maximum capacity was 15.3 +/- 2.2 mg BSA/mL membrane and 58.3 +/- 6.6 mg lysozyme/mL membrane while dissociation constants were 0.27 +/- 0.16 and 0.13 +/- 0.12 mg/mL, respectively. Over 80% of adsorbed proteins could be eluted with 2 M NaCl + 20% isopropanol in 20 mM sodium phosphate buffer, pH 7.0.

On the molecular interaction between lactoferrin and the dye Red HE-3b. A novel approach for docking a charged and highly flexible molecule to protein surfaces.

Lactoferrin (Lf) is a non-heme, iron binding protein present in many physiological fluids of vertebrates where its main role is the microbicidal activity. It has been isolated by different methods, including dye-affinity chromatography. Red HE-3B is one of the most common triazinic dyes applied in protein purification, but scant knowledge is available on structural details and on the energetics of its interaction with proteins. In this work we present a computational approach useful for identifying possible binding sites for Red HE-3B in apo and holo forms of Lfs from human and bovine source. A new geometrical description of Red HE-3B is introduced which greatly simplifies the conformational analysis. This approach proved to be of particular advantage for addressing conformational ensembles of highly flexible molecules. Predictions from this analysis were correlated with experimentally observed dye-binding sites, as mapped by protection from proteolysis in Red HE-3B/Lf complexes. This method could bear relevance for the screening of possible dye-binding sites in proteins whose structure is known and as a potential tool for the design of engineered protein variants which could be purified by dye-affinity chromatography.