1,721,159 research outputs found
Flow field-flow fractionation for the analysis of nanoparticles used in drug delivery
No full textStructured nanoparticles (NPs) with controlled size distribution and novel physicochemical features present fundamental advantages as drug delivery systems with respect to bulk drugs. NPs can transport and release drugs to target sites with high efficiency and limited side effects.Regulatory institutions such as the US Food and Drug Administration (FDA) and the European Commission have pointed out that major limitations to the real application of current nanotechnology lie in the lack of homogeneous, pure and well-characterized NPs, also because of the lack of well-assessed, robust routine methods for their quality control and characterization. Many properties of NPs are size-dependent, thus the particle size distribution (PSD) plays a fundamental role in determining the NP properties. At present, scanning and transmission electron microscopy (SEM, TEM) are among the most used techniques to size characterize NPs. Size-exclusion chromatography (SEC) is also applied to the size separation of complex NP samples. SEC selectivity is, however, quite limited for very large molar mass analytes such as NPs, and interactions with the stationary phase can alter NP morphology. Flow field-flow fractionation (F4) is increasingly used as a mature separation method to size sort and characterize NPs in native conditions. Moreover, the hyphenation with light scattering (LS) methods can enhance the accuracy of size analysis of complex samples. In this paper, the applications of F4-LS to NP analysis used as drug delivery systems for their size analysis, and the study of stability and drug release effects are reviewe
Field-Flow Fractionation: a physical method of separation in the macromolecular and supramolecular field
No full textMulti-environment and multi-parameter screening of stability and coating efficiency of gold nanoparticle bioconjugates in application media
Full text linkGold nanoparticles (AuNPs) and their biocompatible conjugates find wide use as transducers in (bio)sensors and as Nano-pharmaceutics. The study of the interaction between AuNPs and proteins in representative application media helps to better understand their intrinsic behaviors. A multi-environment, multi-parameter screening strategy is proposed based on asymmetric flow field flow fractionation (AF4)-multidetector. Citrate-coated AuNPs (AuCIT, 25.1 ± 0.2 nm) and PEG-coated AuNPs (AuPEG, 38.3 ± 0.8 nm) were employed with albumin as a model system. Attention was put in investigating the influence of Au/BSA mass ratios, that allowed to identify the yield-maximizing (1:1) and product-maximizing (2.5:1) conditions for the generation of AuNPs-protein conjugates. Furthermore, bioconjugate properties were thoroughly assessed across various saline media with different pH and ionic strengths. While AuNPs with PEG coating exhibit greater stability at high salinities, such as 30 mM, their conjugates are less stable over time. In contrast, although bare AuNPs are significantly affected by pH and salt concentration, once conjugates are formed, their stability surpasses that of the conjugates formed with AuPEG. The developed methodology can fill the vacancy of standard reference quality control (QC) procedures for bioconjugate synthesis and application in (bio)sensors and Nano-pharmaceutics, screening in a short time many combinations, easily scaling up to the semi-preparative scale or translating to different bioconjugates
Host-guest interactions in Fe(III)-trimesate MOF nanoparticles loaded with doxorubicin
No full textDoxorubicin (DOX) entrapment in porous Fe(III)-trimesate metal organic frameworks (MIL-100(Fe)) nanoparticles was investigated in neutral Tris buffer via UV-vis absorption, circular dichroism (CD), and fluorescence. The binding constants and the absolute spectra of the DOX-MIL-100(Fe) complexes were determined via absorption and fluorescence titrations. A binding model where DOX associates as monomer to the dehydrated Fe3O (OH)(H 2O)2 [(C6H3)(CO2) 3]2 structural unit in 1:1 stoichiometry, with apparent association constant of (1.1 to 1.8) × 104 M-1, was found to reasonably fit the experimental data. Spectroscopic data indicate that DOX binding occurs via the formation of highly stable coordination bonds between one or both deprotonated hydroxyl groups of the aglycone moiety and coordinatively unsaturated Fe(III) centers. Complete quenching of the DOX fluorescence and remarkable thermal and photochemical stability were observed for DOX incorporated in the MIL-100(Fe) framework
Quality control and purification of ready-to-use conjugated gold nanoparticles to ensure effectiveness in biosensing
Full text linkIntroduction: Gold nanoparticles (AuNPs) and their conjugates are used for
many applications in the field of sensors. Literature lacks procedures able to
separate, purify and characterize these species in native conditions without
altering them while assuring a high throughput. This technological gap can be
reduced by exploiting Asymmetrical Flow Field Flow Fractionation
multidetection platforms (AF4 multidetection).
Method: This work describes a complete set of strategies based on the AF4
system, from nanoparticle synthesis to separative method optimization to
conjugates screening and characterization, achieving quantitative control
and purification of ready-to-use conjugated Gold nanoparticles and
ensuring effectiveness in biosensing.
Results and Discussion: AF4-multidetection was used to study AuNPs with
different types of surface coating [Poly ethylene glycol, (PEG) and Citrate], their
binding behaviour with protein (Bovine serum albumin, BSA) and their stability
after conjugation to BSA. A robust but flexible method was developed, able to
be applied to different AuNPs and conjugating molecules. The morphology and
conjugation mechanism of AuNPs-BSA conjugates were evaluated by
combining online Multiangle light scattering (MALS) and offline Dynamic
Light Scattering (DLS) measures, which provided an important feature for the
quality control required to optimize bio-probe synthesis and subsequent
bioassay
A new analytical platform based on field-flow fractionation and olfactory sensor to improve the detection of viable and non-viable bacteria in food
No full textAn integrated sensing system is presented for the first time, where a metal oxide semiconductor sensor-based electronic olfactory system (MOS array), employed for pathogen bacteria identification based on their volatile organic compound (VOC) characterisation, is assisted by a preliminary separative technique based on gravitational field-flow fractionation (GrFFF). In the integrated system, a preliminary step using GrFFF fractionation of a complex sample provided bacteria-enriched fractions readily available for subsequent MOS array analysis. The MOS array signals were then analysed employing a chemometric approach using principal components analysis (PCA) for a first-data exploration, followed by linear discriminant analysis (LDA) as a classification tool, using the PCA scores as input variables. The ability of the GrFFF-MOS system to distinguish between viable and non-viable cells of the same strain was demonstrated for the first time, yielding 100 % ability of correct prediction. The integrated system was also applied as a proof of concept for multianalyte purposes, for the detection of two bacterial strains (Escherichia coli O157:H7 and Yersinia enterocolitica) simultaneously present in artificially contaminated milk samples, obtaining a 100 % ability of correct prediction. Acquired results show that GrFFF band slicing before MOS array analysis can significantly increase reliability and reproducibility of pathogen bacteria identification based on their VOC production, simplifying the analytical procedure and largely eliminating sample matrix effects. The developed GrFFF-MOS integrated system can be considered a simple straightforward approach for pathogen bacteria identification directly from their food matrix. [Figure not available: see fulltext.
A new approach for the separation, characterization and testing of potential prionoid protein aggregates through hollow-fiber flow field-flow fractionation and multi-angle light scattering
No full textProtein misfolding and aggregation are the common mechanisms in a variety of aggregation-dependent diseases. The compromised proteins often assemble into toxic, accumulating amyloid-like structures of various lengths and their toxicity can also be transferred both in vivo and in vitro a prion-like behavior. The characterization of protein interactions, degradation and conformational dynamics in biological systems still represents an analytical challenge in the prion-like protein comprehension. In our work, we investigated the nature of a transferable cytotoxic agent, presumably a misfolded protein, through the coupling of a multi-detector, non-destructive separation platform based on hollow-fiber flow field-flow fractionation with imaging and downstream in vitro tests. After purification with ion exchange chromatography, the transferable cytotoxic agentwas analyzed with Atomic Force Microscopy and statistical analysis, showing that the concentration of protein dimers and low n-oligomer forms was higher in the cytotoxic sample than in the control preparation. To assess whether the presence of these species was the actual toxic and/or self-propagating factor, we employed HF5 fractionation, with UV and Multi-Angle Light Scattering detection, to define proteins molar mass distribution and abundance, and fractionate the sample into size-homogeneous fractions. These fractions were then tested individually in vitro to investigate the direct correlation with cytotoxicity. Only the later-eluted fraction, which contains high-molar mass aggregates, proved to be toxic onto cell cultures. Moreover, it was observed that the selective transfer of toxicity also occurs for one lower-mass fraction, suggesting that two different mechanisms, acute and later induced toxicity, are in place. These results strongly encourage the efficacy of this platform to enable the identification of protein toxicants.Protein misfolding and aggregation are the common mechanisms in a variety of aggregation-dependent diseases. The compromised proteins often assemble into toxic, accumulating amyloid-like structures of various lengths and their toxicity can also be transferred both in vivo and in vitro a prion-like behavior. The characterization of protein interactions, degradation and conformational dynamics in biological systems still represents an analytical challenge in the prion-like protein comprehension. In our work, we investigated the nature of a transferable cytotoxic agent, presumably a misfolded protein, through the coupling of a multi-detector, non-destructive separation platform based on hollow-fiber flow field-flow fractionation with imaging and downstream in vitro tests. After purification with ion exchange chromatography, the transferable cytotoxic agentwas analyzed with Atomic Force Microscopy and statistical analysis, showing that the concentration of protein dimers and low n-oligomer forms was higher in the cytotoxic sample than in the control preparation. To assess whether the presence of these species was the actual toxic and/or self-propagating factor, we employed HF5 fractionation, with UV and Multi-Angle Light Scattering detection, to define proteins molar mass distribution and abundance, and fractionate the sample into size-homogeneous fractions. These fractions were then tested individually in vitro to investigate the direct correlation with cytotoxicity. Only the later-eluted fraction, which contains high-molar mass aggregates, proved to be toxic onto cell cultures. Moreover, it was observed that the selective transfer of toxicity also occurs for one lower-mass fraction, suggesting that two different mechanisms, acute and later induced toxicity, are in place. These results strongly encourage the efficacy of this platform to enable the identification of protein toxicants
Hollow-fiber flow field-flow fractionation and multi-angle light scattering as a new analytical solution for quality control in pharmaceutical nanotechnology
No full textThe rapid development of nanoproducts in pharmaceutical field highlights the need for robust analytical methods to ensure their quality and stability. Nanoparticles of different nature and composition (NPs) are employed for many purposes, such as the improvement of drug solubility/bioavailability and the controlled delivery of drugs. Among NPs features, particle size distribution (PSD) plays a fundamental role in determining NPs properties. Nevertheless the high development of different NPs, authorities such as the FDA and the European Union highlight the lack of robust characterization methods and quality control for nanomaterials.
Among the techniques for the size-characterization of particles, Field-Flow Fractionation (FFF) represents a competitive choice: due to the absence of a stationary phase, the separation mechanism is gentle with total maintenance of the native properties of the analytes. In this paper the microcolumn variant of FFF, the Hollow-Fiber Flow FFF (HF5), is coupled on-line with Multi-Angle Light Scattering (MALS) for the development of methods for the characterization of NPs as quality control in new pharmaceutical field. The HF5-MALS was applied to the size characterization in different preservation conditions of silver polyvinylpyrrolidone-stabilized NPs (AgPVP) used for their antimicrobial activity. The ratio of gyration and hydrodynamic radii of AgPVP was evaluated for fresh and aged NPs, suggesting in this case aggregation rearrangement. The influence of different PVP coating and dilution factor was also studied. Finally the metal ion release was determined in relationship to these shape modifications. The HF5-MALS method is robust and reproducible and it can be considered as an important tool for the development of analytical platform for quality control of NPs
Sviluppo ed applicazione di metodologie di spettrometria di massa nello studio di macromolecole di interesse biologico
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