Quality control and purification of ready-to-use conjugated gold nanoparticles to ensure effectiveness in biosensing

Introduction: 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

FFF-based high-throughput sequence shortlisting to support the development of aptamer-based analytical strategies

Aptamers are biomimetic receptors that are increasingly exploited for the development of optical and electrochemical aptasensors. They are selected in vitro by the SELEX (Systematic Evolution of Ligands by Exponential Enrichment) procedure, but although they are promising recognition elements, for their reliable applicability for analytical purposes, one cannot ignore sample components that cause matrix effects. This particularly applies when different SELEX-selected aptamers and related truncated sequences are available for a certain target, and the choice of the aptamer should be driven by the specific downstream application. In this context, the present work aimed at investigating the potentialities of asymmetrical flow field-flow fractionation (AF4) with UV detection for the development of a screening method of a large number of anti-lysozyme aptamers towards lysozyme, including randomized sequences and an interfering agent (serum albumin). The possibility to work in native conditions and selectively monitor the evolution of untagged aptamer signal as a result of aptamer-protein binding makes the devised method effective as a strategy for shortlisting the most promising aptamers both in terms of affinity and in terms of selectivity, to support subsequent development of aptamer-based analytical devices

Synthesis Monitoring, Characterization and Cleanup of Ag-Polydopamine Nanoparticles Used as Antibacterial Agents with Field-Flow Fractionation

Advances in nanotechnology have opened up new horizons in nanomedicine through the synthesis of new composite nanomaterials able to tackle the growing drug resistance in bacterial strains. Among these, nanosilver antimicrobials sow promise for use in the treatment of bacterial infections. The use of polydopamine (PDA) as a biocompatible carrier for nanosilver is appealing; however, the synthesis and functionalization steps used to obtain Ag-PDA nanoparticles (NPs) are complex and require time-consuming cleanup processes. Post-synthesis treatment can also hinder the stability and applicability of the material, and dry, offline characterization is time-consuming and unrepresentative of real conditions. The optimization of Ag-PDA preparation and purification together with well-defined characterization are fundamental goals for the safe development of these new nanomaterials. In this paper, we show the use of field-flow fractionation with multi-angle light scattering and spectrophotometric detection to improve the synthesis and quality control of the production of Ag-PDA NPs. An ad hoc method was able to monitor particle growth in a TLC-like fashion; characterize the species obtained; and provide purified, isolated Ag-PDA nanoparticles, which proved to be biologically active as antibacterial agents, while achieving a short analysis time and being based on the use of green, cost-effective carriers such as water

Celector®: An Innovative Technology for Quality Control of Living Cells

Among the in vitro and ex vivo models used to study human cancer biology, cancer cell lines are widely utilized. The standardization of a correct tumor model including the stage of in vitro testing would allow for the development of new high-efficiency drug systems. The poor correlation between preclinical in vitro and in vivo data and clinical trials is still an open issue, hence the need for new systems for the quality control (QC) of these cell products. In this work, we present a new technology, Celector®, capable of the label-free analysis and separation of cells based on their physical characteristics with full preservation of their native properties. Two types of cancer cell lines were used: HL60 as cells growing in suspension and SW620 as adherent cells. Cell lines in general show a growth variability depending on the passage and method of culture. Celector® highlights physical differences that can be correlated to cell viability. This work demonstrates the use of Celector® as an analytical platform for the QC of cells used for drug screening, with fundamental improvement of preclinical tests. Cells with a stable doubling time under analysis can be collected and used as standardized systems for high-quality drug monitoring

Quality Control Platform for the Standardization of a Regenerative Medicine Product

Adipose tissue is an attractive source of stem cells due to its wide availability. They contribute to the stromal vascular fraction (SVF), which is composed of pre-adipocytes, tissue-progenitors, and pericytes, among others. Because its direct use in medical applications is increasing worldwide, new quality control systems are required. We investigated the ability of the Non-Equilibrium Earth Gravity Assisted Dynamic Fractionation (NEEGA-DF) method to analyze and separate cells based solely on their physical characteristics, resulting in a fingerprint of the biological sample. Adipose tissue was enzymatically digested, and the SVF was analyzed by NEEGA-DF. Based on the fractogram (the UV signal of eluting cells versus time of analysis) the collection time was set to sort alive cells. The collected cells (F-SVF) were analyzed for their phenotype, immunomodulation ability, and differentiation potential. The SVF profile showed reproducibility, and the alive cells were collected. The F-SVF showed intact adhesion phenotype, proliferation, and differentiation potential. The methodology allowed enrichment of the mesenchymal component with a higher expression of mesenchymal markers and depletion of debris, RBCs, and an extracellular matrix still present in the digestive product. Moreover, cells eluting in the last minutes showed higher circularity and lower area, proving the principles of enrichment of a more homogenous cell population with better characteristics. We proved the NEEGA-DF method is a "gentle" cell sorter that purifies primary cells obtained by enzymatic digestion and does not alter any stem cell function

Hollow-fiber flow field-flow fractionation and multi-angle light scattering investigation of the size, shape and metal-release of silver nanoparticles in aqueous medium for nano-risk assessment

open11siAvailable online 22 November 2014 The research leading to these results has received funding from the European Community's Seventh Framework Programme (FP7/2007–2013) through the project SANOWORK under Grant Agreement no. 280716. The HRTEM has been made available under the INSPIRE programme, funded by Irish Government's Programme for Research in Third Level Institutions, Cycle 4, National Development Plan 2007–2013, which is supported by European Union Structural Fund. Drs. Abbasi Gandhi and Vishnu Mogili of the University of Limerick are acknowledged for generating HRTEM data.Due to the increased use of silver nanoparticles in industrial scale manufacturing, consumer products and nanomedicine reliable measurements of properties such as the size, shape and distribution of these nano particles in aqueous medium is critical. These properties indeed affect both functional properties and biological impacts especially in quantifying associated risks and identifying suitable risk-mediation strategies. The feasibility of on-line coupling of a fractionation technique such as hollow-fiber flow field flow fractionation (HF5) with a light scattering technique such as MALS (multi-angle light scattering) is investigated here for this purpose. Data obtained from such a fractionation technique and its combination thereof with MALS have been compared with those from more conventional but often complementary techniques e.g. transmission electron microscopy, dynamic light scattering, atomic absorption spectroscopy, and X-ray fluorescence. The combination of fractionation and multi angle light scattering techniques have been found to offer an ideal, hyphenated methodology for a simultaneous size-separation and characterization of silver nanoparticles. The hydrodynamic radii determined by fractionation techniques can be conveniently correlated to the mean average diameters determined by multi angle light scattering and reliable information on particle morphology in aqueous dispersion has been obtained. The ability to separate silver (Ag+) ions from silver nanoparticles (AgNPs) via membrane filtration during size analysis is an added advantage in obtaining quantitative insights to its risk potential. Most importantly, the methodology developed in this article can potentially be extended to similar characterization of metal-based nanoparticles when studying their functional effectiveness and hazard potential.partially_openembargoed_20151122Marassi, Valentina; Casolari, Sonia; Roda, Barbara; Zattoni, Andrea; Reschiglian, Pierluigi; Panzavolta, Silvia; Tofail, Syed A.M.; Ortelli, Simona; Delpivo, Camilla; Blosi, Magda; Costa, Anna LuisaMarassi, Valentina; Casolari, Sonia; Roda, Barbara; Zattoni, Andrea; Reschiglian, Pierluigi; Panzavolta, Silvia; Tofail, Syed A.M.; Ortelli, Simona; Delpivo, Camilla; Blosi, Magda; Costa, Anna Luis

The Challenges of O2 Detection in Biological Fluids: Classical Methods and Translation to Clinical Applications

Dissolved oxygen (DO) is deeply involved in preserving the life of cellular tissues and human beings due to its key role in cellular metabolism: its alterations may reflect important pathophysiological conditions. DO levels are measured to identify pathological conditions, explain pathophysiological mechanisms, and monitor the efficacy of therapeutic approaches. This is particularly relevant when the measurements are performed in vivo but also in contexts where a variety of biological and synthetic media are used, such as ex vivo organ perfusion. A reliable measurement of medium oxygenation ensures a high-quality process. It is crucial to provide a high-accuracy, real-time method for DO quantification, which could be robust towards different medium compositions and temperatures. In fact, biological fluids and synthetic clinical fluids represent a challenging environment where DO interacts with various compounds and can change continuously and dynamically, and further precaution is needed to obtain reliable results. This study aims to present and discuss the main oxygen detection and quantification methods, focusing on the technical needs for their translation to clinical practice. Firstly, we resumed all the main methodologies and advancements concerning dissolved oxygen determination. After identifying the main groups of all the available techniques for DO sensing based on their mechanisms and applicability, we focused on transferring the most promising approaches to a clinical in vivo/ex vivo settin

Effective Label-Free Sorting of Multipotent Mesenchymal Stem Cells from Clinical Bone Marrow Samples

Mesenchymal stem cells (MSC) make up less than 1% of the bone marrow (BM). Several methods are used for their isolation such as gradient separation or centrifugation, but these methodologies are not direct and, thus, plastic adherence outgrowth or magnetic/fluorescent-activated sorting is required. To overcome this limitation, we investigated the use of a new separative technology to isolate MSCs from BM; it label-free separates cells based solely on their physical characteristics, preserving their native physical properties, and allows real-time visualization of cells. BM obtained from patients operated for osteochondral defects was directly concentrated in the operatory room and then analyzed using the new technology. Based on cell live-imaging and the sample profile, it was possible to highlight three fractions (F1, F2, F3), and the collected cells were evaluated in terms of their morphology, phenotype, CFU-F, and differentiation potential. Multipotent MSCs were found in F1: higher CFU-F activity and differentiation potential towards mesenchymal lineages compared to the other fractions. In addition, the technology depletes dead cells, removing unwanted red blood cells and non-progenitor stromal cells from the biological sample. This new technology provides an effective method to separate MSCs from fresh BM, maintaining their native characteristics and avoiding cell manipulation. This allows selective cell identification with a potential impact on regenerative medicine approaches in the orthopedic field and clinical applications

Native Study of the Behaviour of Magnetite Nanoparticles for Hyperthermia Treatment during the Initial Moments of Intravenous Administration

Magnetic nanoparticles (MNPs) present outstanding properties making them suitable as therapeutic agents for hyperthermia treatments. Since the main safety concerns of MNPs are represented by their inherent instability in a biological medium, strategies to both achieve longterm stability and monitor hazardous MNP degradation are needed. We combined a dynamic approach relying on flow field flow fractionation (FFF)-multidetection with conventional techniques to explore frame-by-frame changes of MNPs injected in simulated biological medium, hypothesize the interaction mechanism they are subject to when surrounded by a saline, protein-rich environment, and understand their behaviour at the most critical point of intravenous administration. In the first moments of MNPs administration in the patient, MNPs change their surrounding from a favorable to an unfavorable medium, i.e., a complex biological fluid such as blood; the particles evolve from a synthetic identity to a biological identity, a transition that needs to be carefully monitored. The dynamic approach presented herein represents an optimal alternative to conventional batch techniques that can monitor only size, shape, surface charge, and aggregation phenomena as an averaged information, given that they cannot resolve different populations present in the sample and cannot give accurate information about the evolution or temporary instability of MNPs. The designed FFF method equipped with a multidetection system enabled the separation of the particle populations providing selective information on their morphological evolution and on nanoparticle– proteins interaction in the very first steps of infusion. Results showed that in a dynamic biological setting and following interaction with serum albumin, PP-MNPs retain their colloidal properties, supporting their safety profile for intravenous administration

Unravelling Heterogeneity of Amplified Human Amniotic Fluid Stem Cells Sub-Populations

Human amniotic fluid stem cells (hAFSCs) are broadly multipotent immature progenitor cells with high self-renewal and no tumorigenic properties. These cells, even amplified, present very variable morphology, density, intracellular composition and stemness potential, and this heterogeneity can hinder their characterization and potential use in regenerative medicine. Celector\uae (Stem Sel ltd.) is a new technology that exploits the Non-Equilibrium Earth Gravity Assisted Field Flow Fractionation principles to characterize and label-free sort stem cells based on their solely physical characteristics without any manipulation. Viable cells are collected and used for further studies or direct applications. In order to understand the intrapopulation heterogeneity, various fractions of hAFSCs were isolated using the Celector\uae profile and live imaging feature. The gene expression profile of each fraction was analysed using whole-transcriptome sequencing (RNAseq). Gene Set Enrichment Analysis identified significant differential expression in pathways related to Stemness, DNA repair, E2F targets, G2M checkpoint, hypoxia, EM transition, mTORC1 signalling, Unfold Protein Response and p53 signalling. These differences were validated by RT-PCR, immunofluorescence and differentiation assays. Interestingly, the different fractions showed distinct and unique stemness properties. These results suggest the existence of deep intra-population differences that can influence the stemness profile of hAFSCs. This study represents a proof-of-concept of the importance of selecting certain cellular fractions with the highest potential to use in regenerative medicine