Addressing food safety and detecting food fraud while fulfilling greenness requisites for analysis is a challenging but necessary task. The use of sustainable techniques, with limited pretreatment, non-toxic chemicals, high throughput results, is recommended. A combination of Field Flow Fractionation (FFF), working in saline carrier and with minimal preprocessing, and chemometrics was for the first time applied to bovine milk grouping. A set of 47 bovine milk samples was analyzed: a single analysis yielded a characteristic multidimensional colloidal dataset, that once processed with multivariate tools allowed simultaneously for different discriminations: fat content, thermal treatment, brand and manufacturing plant. The analytical methodology is fast, green, simple, and inexpensive and could offer great help in the field of quality control and frauds identification. This work represents also the first attempt to identify milk sub-typologies based on colloidal profiles, and the most complete study concerning multivariate analysis of FFF fingerprint.

Advanced therapy medicinal products (ATMP) are complex medicines based on gene therapy, somatic cell therapy, and tissue engineering. These products are rapidly arising as novel and promising therapies for a wide range of different clinical applications. The process for the development of well-established ATMPs is challenging. Many issues must be considered from raw material, manufacturing, safety, and pricing to assure the quality of ATMPs and their implementation as innovative therapeutic tools. Among ATMPs, cell-based ATMPs are drugs altogether. As for standard drugs, technologies for quality control, and non-invasive isolation and production of cell-based ATMPs are then needed to ensure their rapidly expanding applications and ameliorate safety and standardization of cell production. In this review, emerging approaches and technologies for quality control of innovative cell-based ATMPs are described. Among new techniques, microfluid-based systems show advantages related to their miniaturization, easy implementation in analytical process and automation which allow for the standardization of the final product.

A novel approach for the quantification of recycled polyethylene terephthalate (r-PET) in commercial bottles is presented. Fifty-eight bottle samples from several brands and producers containing different percentages of r-PET were purchased from the market. Samples were analyzed by two spectroscopic methods: near-infrared (NIR) spectroscopy and attenuated total reflection (ATR) spectroscopy in the mid-infrared (MIR) region. No chemical pre-treatment was applied before analyses. The spectra were analyzed by partial-least squares (PLS) regression, and two models for NIR and MIR data were computed. Then, a multi-block regression was applied to join the two datasets. All models were validated by cross-validation and by excluding and projecting onto the model the replicated spectra of one sample at a time. Results demonstrated the potential of this approach, especially considering the variability of commercial samples in terms of additives, shape, or thickness of the bottles: for samples close to the centroids of the models (i.e., from 10 to 50% r-PET), the predictions of multi-block method seldom departed from the expected values of ±10%. Only for samples with 0% declared r-PET, the models showed poor prediction abilities.

Aptamers are recognition elements increasingly used for the development of biosensing strategies, especially in the detection of proteins or small molecule targets. Lysozyme, which is recognized as an important biomarker for various diseases and a major allergenic protein found in egg whites, is one of the main analytical targets of aptamer-based biosensors. However, since aptamer-based strategies can be prone to artifacts and data misinterpretation, rigorous strategies for multifaceted characterization of the aptamer-target interaction are needed. In this work, a multitechnique approach has been devised to get further insights into the binding performance of the anti-lysozyme DNA aptamers commonly used in the literature. To study molecular interactions between lysozyme and different anti-lysozyme DNA aptamers, measurements based on a magneto-electrochemical apta-assay, circular dichroism spectroscopy, fluorescence spectroscopy, and asymmetrical flow field-flow fractionation were performed. The reliability and versatility of the approach were proved by investigating a SELEX-selected RNA aptamer reported in the literature, that acts as a positive control. The results confirmed that an interaction in the low micromolar range is present in the investigated binding buffers, and the binding is not associated with a conformational change of either the protein or the DNA aptamer. The similar behavior of the anti-lysozyme DNA aptamers compared to that of randomized sequences and polythymine, used as negative controls, showed nonsequence-specific interactions. This study demonstrates that severe testing of aptamers resulting from SELEX selection is the unique way to push these biorecognition elements toward reliable and reproducible results in the analytical field.

Human amniotic mesenchymal stromal cells (hAMSCs) have unique immunomodulatory properties making them attractive candidates for regenerative applications in inflammatory diseases. Most of their beneficial properties are mediated through their secretome. The bioactive factors concurring to its therapeutic activity are still unknown. Evidence suggests synergy between the two main components of the secretome, soluble factors and vesicular fractions, pivotal in shifting inflammation and promoting self-healing. Biological variability and the absence of quality control (QC) protocols hinder secretome-based therapy translation to clinical applications. Moreover, vesicular secretome contains a multitude of particles with varying size, cargos and functions whose complexity hinders full characterization and comprehension.This study achieved a significant advancement in secretome characterization by utilizing native, FFF-based separation and characterizing extracellular vesicles derived from hAMSCs. This was accomplished by obtaining dimensionally homogeneous fractions then characterized based on their protein content, potentially enabling the identification of subpopulations with diverse functionalities.This method proved to be successful as an independent technique for secretome profiling, with the potential to contribute to the standardization of a qualitative method. Additionally, it served as a preparative separation tool, streamlining populations before ELISA and LC-MS characterization. This approach facilitated the categorization of distinctive and recurring proteins, along with the identification of clusters associated with vesicle activity and functions. However, the presence of proteins unique to each fraction obtained through the FFF separation tool presents a challenge for further analysis of the protein content within these cargoes.

Gold 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.

Red wine colloids, crucial in determining wine quality and stability, are understudied due to inadequate techniques for studying them effectively in the natural wine environment. Recently, Asymmetrical Flow Field-flow Fractionation (AF4) with online multidetection has emerged as a novel analytical tool for quantifying, fractionating, and characterizing red wine colloids in their native state. This study aimed to characterize the colloidal composition of 24 monovarietal Italian wines produced without filtration, oak contact, fining treatments, malolactic fermentation, macerating enzymes or ageing on yeast lees. AF4 analysis allowed quantification and characterization of wine colloids based on light scattering signal (MALS; gyration radius - Rg), size (hydrodynamic radius - Rh) and absorbance (A280 & A520 nm). The results showed that each wine contained up to five distinct colloids' populations, varying in size and gyration radii. Despite possessing very similar Rh, most colloids exhibited great differences in compactness, as indicated by their varying Rg values. Comparing the A280 signal of whole wines to those of wines containing only species larger than 5 kDa (considered colloids) allowed to calculate the percentage of molecules involved in colloidal particles assembly, ranging from 1 to 44 % of the total A280 absorbing compounds, reflecting the diversity among wines. The A520 signal indicated the presence of polymeric pigments in the colloidal fraction. Notably, colored colloids all had Rg > 20 nm, indicating their association with other colloidal-forming compounds. This observation led to the conclusion that, apart from free anthocyanins and polymeric pigments, the color of red wines is also due to colloidal particles formed by the latter bound to proteins, with their quantity being highly variable across wines of different origin. These findings, which highlight the fundamental role of proteins in shaping the colloidal status of red wines, were utilized to propose an updated hypothetical model for colloidal aggregation in red wine.

Rational design of analytical criteria to perform dissolution testing of nanomaterials: the relevance of juice composition for hazard identification and grouping.

Understanding the mechanism of toxicity of nanoparticles and their behavior in biological environments is crucial for designing materials with reduced side effects and improved performance. Among the factors influencing nanoparticle behavior in biological environments, the release and bioavailability of potentially toxic metal ions can alter equilibria and cause adverse effects. In this study, we applied two on-line Field-Flow Fractionation (FFF) strategies and compared the results with off-line benchmarking centrifugal ultrafiltration to assess a key descriptor, namely the solubility of zinc oxide (ZnO) nanoparticles. We found that, at the highest nanoparticle concentrations, the nanoparticle-ion ratio quickly reaches equilibrium, and the stability is not significantly affected by the separation technique. However, at lower concentrations, dynamic, non-equilibrium behavior occurs, and the results depend on the method used to separate the solid from the ionic fraction, where FFF yielded a more representative dissolution pattern. To support the (eco)toxicological profiling of the investigated nanoparticles, we generated experimental data on colloidal stability over typical (eco)toxicological assay durations. The Zeta Potential vs pH curves revealed two distinct scenarios typical of surfaces that have undergone significant modification, most likely due to pH-dependent dissolution and re-precipitation of surface groups. Finally, to enhance hazard assessment screening, we investigated ion-dependent toxicity and the effects of exposure to fresh water. Using an in vitro human skin model, we evaluated the cytotoxicity of fresh and aged ZnO nanoparticles (exposed for 72 h in M7), revealing time-dependent, dose-dependent, and nanoparticle-dependent cytotoxicity, with lower toxicity observed in the case of aged samples.

Fluid inclusions are a window into deep geological fluids, providing unique access to their nature and composition. The isotopic composition of CO2 and CH4 hosted in fluid inclusions is a powerful proxy to assess the origin and transformation of deep geological fluids, giving insights into carbon sources, fluxes, and degassing in a wide variety of geodynamic settings. Over the last 5 decades, techniques have been developed to extract fluid inclusions from their host minerals and measure their bulk composition. These techniques are often challenged by analytical artifacts including high blank levels of CO2 and CH4, fluid re-speciation, gas adsorption, and diffusion. Since these processes may alter the pristine composition of gases liberated from fluid inclusions, rigorous protocols are needed in order to evaluate the isotopic integrity of the extracted volatile species. In this study, we introduce new protocols for bulk off-line fluid inclusion extraction for the analysis of δ13C-CH4 and δ13C-CO2 using a Cavity Ring-Down Spectroscopy (CRDS) analyser (Picarro G2201-i). Two mechanical fluid extraction techniques are compared: ball milling in ZrO2 jars and sample crushing in a stainless steel sealed tube under a hydraulic press. Blanks and isotopically labelled tests with the ball milling technique suggest that rotation speed, grinding stock filling degree and filling type alter the CH4 and CO2 concentrations and isotopic compositions measured by the CRDS analyser. In contrast, the crushing technique does not generate measurable quantities of blank CH4 and CO2. The protocols presented in this study allow to extract, detect, and analyse δ13C of CH4 and CO2 for concentrations above 10 and 1,000 ppm respectively. Interlaboratory experiments allowed to replicate previously measured δ13C-CH4 values in natural fluid inclusions within 1‰ with both extraction techniques. This study highlights the potential of combining simple bulk off-line fluid inclusion extraction techniques with a CRDS analyser for δ13C analysis of CO2 and CH4 without gas separation being required.

Instrument automation, technological advancements and improved computational power made separation science an extremely data-rich approach, requiring the use of statistical and data analysis tools that are able to optimize processes and combine multiple outputs. The use of chemometrics is growing, greatly improving the ability to extract meaningful information. Separation–multidetection generates multidimensional data, whose elaboration should not be left to the discretion of the operator. However, some applications or techniques still suffer from the lack of method optimization through DoE and downstream multivariate analysis, limiting their potential. This review aims at summarizing how chemometrics can assist analytical chemists in terms of data elaboration and method design, focusing on what can be achieved by applying chemometric approaches to separation science. Recent applications of chemometrics in separation analyses, in particular in gas, liquid and size-exclusion chromatography, together with field flow fractionation, will be detailed to visualize the state of the art of separation chemometrics, encompassing volatile, soluble and solid (colloidal) analytes. The samples considered will range from food chemistry and environmental chemistry to bio/pharmaceutical science.

Melanin-inspired nanomaterials offer unique photophysical, electronic and radical scavenging properties that are widely explored for health and environmental preservation, or energy conversion and storage. The incorporation of functional melanin building blocks in more complex nanostructures or surfaces is typically achieved via a bottom-up approach starting from a molecular precursor, in most cases dopamine. Here we demonstrate that indeed, the oxidative polymerization of dopamine, for the synthesis of melanin-like polydopamine (PDA), leads to the simultaneous formation of more than one nanosized species with different compositions, morphologies and properties. In particular, a low-density polymeric structure and dense nanoparticles (NP) are simultaneously formed. The two populations could be separated and analyzed in real time using a chromatographic technique free of any stationary phase (flow field fractionation, FFF). The results following the synthesis of melanin-like PDA showed that the NP are formed only during the first 6 hours as a result of a supramolecular self-assembly-driven polymerization, while the formation of the polymer continues for about 36 hours. The two populations were also separated and characterized using TEM, UV-vis absorption spectroscopy, fluorescence and light scattering spectroscopy, DLS, FTIR, ζ-potential measurements, gel electrophoresis and pH titrations. Interestingly, very different properties between the two populations were observed: in particular the polymer contains a higher number of catechol units (8 mmol g-1 -OH) with respect to the NP (1 mmol g-1 -OH) and presents a much higher antioxidant activity. The attenuation of light by NP is more efficient than that by the polymer especially in the Vis-NIR region. Moreover, while the NP scatter light with an efficiency up to 27% they are not fluorescent, and the polymer does not scatter light but shows an excitation wavelength-dependent fluorescence typical of multi-fluorophoric uncoupled systems.

The early-stage diagnosis of cancer is a crucial clinical need. The inadequacies of surgery tissue biopsy have prompted a transition to a less invasive profiling of molecular biomarkers from biofluids, known as liquid biopsy. Exosomes are phospholipid bilayer vesicles present in many biofluids with a biologically active cargo, being responsible for cell-to-cell communication in biological systems. An increase in their excretion and changes in their cargo are potential diagnostic biomarkers for an array of diseases, including cancer, and they constitute a promising analyte for liquid biopsy. The number of exosomes released, the morphological properties, the membrane composition, and their content are highly related to the physiological and pathological states. The main analytical challenge to establishing liquid biopsy in clinical practice is the development of biosensors able to detect intact exosomes concentration and simultaneously analyze specific membrane biomarkers and those contained in their cargo. Before analysis, exosomes also need to be isolated from biological fluids. Microfluidic systems can address several issues present in conventional methods (i.e., ultracentrifugation, size-exclusion chromatography, ultrafiltration, and immunoaffinity capture), which are time-consuming and require a relatively high amount of sample; in addition, they can be easily integrated with biosensing systems. A critical review of emerging microfluidic-based devices for integrated biosensing approaches and following the major analytical need for accurate diagnostics is presented here. The design of a new miniaturized biosensing system is also reported. A device based on hollow-fiber flow field-flow fractionation followed by luminescence-based immunoassay is applied to isolate intact exosomes and characterize their cargo as a proof of concept for colon cancer diagnosis.

The future of biomaterial production will leverage biotechnology based on the domestication of cells as biological factories. Plants, algae, and bacteria can produce low-environmental impact biopolymers. Here, two strategies were developed to produce a biopolymer derived from a bioengineered vacuolar storage protein of the common bean (phaseolin; PHSL). The cys-added PHSL* forms linear-structured biopolymers when expressed in the thylakoids of transplastomic tobacco leaves by exploiting the formation of inter-chain disulfide bridges. The same protein without signal peptide (ΔPHSL*) accumulates in Escherichia coli inclusion bodies as high-molar-mass species polymers that can subsequently be oxidized to form disulfide crosslinking bridges in order to increase the stiffness of the biomaterial, a valid alternative to the use of chemical crosslinkers. The E. coli cells produced 300 times more engineered PHSL, measured as percentage of total soluble proteins, than transplastomic tobacco plants. Moreover, the thiol groups of cysteine allow the site-specific PEGylation of ΔPHSL*, which is a desirable functionality in the design of a protein-based drug carrier. In conclusion, ΔPHSL* expressed in E. coli has the potential to become an innovative biopolymer.

Field-flow fractionation (FFF) is a family of single-phase separative techniques exploited to gently separate and characterize nano- and microsystems in suspension. These techniques cover an extremely wide dynamic range and are able to separate analytes in an interval between a few nm to 100 µm size-wise (over 15 orders of magnitude mass-wise). They are flexible in terms of mobile phase and can separate the analytes in native conditions, preserving their original structures/properties as much as possible. Molecular biology is the branch of biology that studies the molecular basis of biological activity, while biotechnology deals with the technological applications of biology. The areas where biotechnologies are required include industrial, agri-food, environmental, and pharmaceutical. Many species of biological interest belong to the operational range of FFF techniques, and their application to the analysis of such samples has steadily grown in the last 30 years. This work aims to summarize the main features, milestones, and results provided by the application of FFF in the field of molecular biology and biotechnology, with a focus on the years from 2000 to 2022. After a theoretical background overview of FFF and its methodologies, the results are reported based on the nature of the samples analyzed.

For decades, the search for terrestrial abiotic CH4 has been a central quest in geology and astrobiology. Most of this research has focused on crustal fluid samples collected at surface seeps, hydrothermal vents, and wells. Nonetheless, in such open systems processes like mixing with shallow biotic CH4, oxidation, and diffusion can lead to large uncertainties in the initial composition of deeply originated CH41. Extracting natural CH4 from fluid inclusions entrapped in minerals could overcome the effects of shallow contamination occurring in natural open systems and shed light on the origin of deep CH4 in a wide variety of geodynamic settings2,3. In this abstract, we present a novel approach for bulk off-line fluid inclusion extraction for the analysis of δ13C-CH4 using a Cavity Ring-Down Spectroscopy (CRDS) analyzer (Picarro G2201-i). Two fluid extraction techniques were compared: ball milling in ZrO2 jars and sample crushing in a stainless-steel sealed tube under a piston. The accuracy and precision of the different protocols was evaluated with blanks, CH4 isotopic labelling and interlaboratory comparisons. Blanks and isotopically labelled tests with the ball milling technique suggested that milling speed, and duration, and sample mass and type may strongly affect the CH4 concentrations and isotopic compositions measured by the CRDS analyser. These effects are mainly ruled by the blank production of CH4 –demonstrated by gas chromatography analyses– and potentially other molecules induced by frictional heating. The blank gases may cause interference effects on the absorption bands detected by the CRDS analyser. This effect was marked by a large offset in the δ13C-CH4 measured in the high-range analytical modes of the Picarro G2201-i. The magnitude of the interference was inversely correlated to the CH4 concentration.Other processes such as CH4 diffusion and adsorption may play a role in the observed changes in CH4 concentrations and isotopic compositions. Experimental conditions involving high CH4 concentrations and sample mass could well reproduce CH4 isotopic composition. Crushing in a sealed stainless-steel tube produces lower blank levels compared to ball milling, nevertheless the crushing efficiency and CH4 release is lower due to smaller rock sample size. The presented ball milling protocol provides a simple and fast way to extract and accurately analyse CH4 hosted in fluid inclusions even if great care should be considered for samples with low CH4 concentrations where contamination and interferences can potentially alter the CH4 isotopic signature of natural samples. References 1 - Young et al. (2017). 10.1016/j.gca.2016.12.041 2 - Klein et al. (2019). 10.1073/pnas.1907871116 3 - Grozeva et al. (2020). 10.1098/rsta.2018.0431 This work is part of project that has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (Grant agreement No. 864045).

Liposomes are nano-sized lipid-based vesicles widely studied for their drug delivery capabilities. Compared to standard carries they exhibit better properties such as improved site-targeting and drug release, protection of drugs from degradation and clearance, and lower toxic side effects. At present, scientific literature is rich of studies regarding liposomes-based systems, while 14 types of liposomal products have been authorized to the market by EMA and FDA and many others have been approved by national agencies. Although the interest in nanodevices and nanomedicine has steadily increased in the last two decades the development of documentation regulating and standardizing all the phases of their development and quality control still suffers from major inadequacy due to the intrinsic complexity of nano-systems characterization. Many generic documents (Type 1) discussing guidelines for the study of nano-systems (lipidic and not) have been proposed while there is a lack of robust and standardized methods (Type 2 documents). As a result, a widespread of different techniques, approaches and methodologies are being used, generating results of variable quality and hard to compare with each other. Additionally, such documents are often subject to updates and rewriting further complicating the topic. Within this context the aim of this work is focused on bridging the gap in liposome characterization: the most recent standardized methodologies suitable for liposomes characterization are here reported (with the corresponding Type 2 documents) and revised in a short and pragmatical way focused on providing the reader with a practical background of the state of the art. In particular, this paper will put the accent on the methodologies developed to evaluate the main critical quality attributes (CQAs) necessary for liposomes market approval.

The possibility of incorporating H2S slow-release donors inside biomimetic scaffolds can pave the way to new approaches in the field of tissue regeneration and anti-inflammatory treatment. In the present work, GYY4137, an easy-to-handle commercially available Lawesson's reagent derivative, has been successfully incorporated inside biomimetic silk fibroin-based electrospun scaffolds. Due to the instability of GYY4137 in the solvent needed to prepare silk fibroin solutions (formic acid), the electrospinning of the donor together with the silk fibroin turned out to be impossible. Therefore, a multilayer structure was realized, consisting of a PLGA mat containing GYY4137 sandwiched between two silk fibroin nanofibrous layers. Before their use in the multilayer scaffold, the silk fibroin mats were treated in ethanol to induce crystalline phase formation, which conferred water resistance and biomimetic properties. The morphological, thermal, and chemical properties of the obtained scaffolds were thoroughly characterized by SEM, TGA, DSC, FTIR, and WAXD. Multilayer devices showing two different concentrations of the H2S donor, i.e., 2 and 5% w/w with respect to the weight of PLGA, were analyzed to study their H2S release and biological properties, and the results were compared with those of the sample not containing GYY4137. The H2S release analysis was carried out according to an "ad-hoc" designed procedure based on a validated high-performance liquid chromatography method. The proposed analytical approach demonstrated the slow-release kinetics of H2S from the multilayer scaffolds and its tunability by acting on the donor's concentration inside the PLGA nanofibers. Finally, the devices were tested in biological assays using bone marrow derived mesenchymal stromal cells showing the capacity to support cell spreading throughout the scaffold and prevent cytotoxicity effects in serum starvation conditions. The resulting devices can be exploited for applications in the tissue engineering field since they combine the advantages of controlled H2S release kinetics and the biomimetic properties of silk fibroin nanofibers.

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.

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 long-term 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.