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Article ; Online: Liposomes characterization for market approval as pharmaceutical products: Analytical methods, guidelines and standardized protocols.

Giordani, Stefano / Marassi, Valentina / Zattoni, Andrea / Roda, Barbara / Reschiglian, Pierluigi

Journal of pharmaceutical and biomedical analysis

2023 Volume 236, Page(s) 115751

Abstract: 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 ... ...

Abstract	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.
MeSH term(s)	Liposomes ; Drug Delivery Systems/methods ; Drug Liberation
Chemical Substances	Liposomes
Language	English
Publishing date	2023-09-27
Publishing country	England
Document type	Journal Article ; Review
ZDB-ID	604917-5
ISSN	1873-264X ; 0731-7085
ISSN (online)	1873-264X
ISSN	0731-7085
DOI	10.1016/j.jpba.2023.115751
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Article ; Online: Field-Flow Fractionation in Molecular Biology and Biotechnology.

Giordani, Stefano / Marassi, Valentina / Placci, Anna / Zattoni, Andrea / Roda, Barbara / Reschiglian, Pierluigi

Molecules (Basel, Switzerland)

2023 Volume 28, Issue 17

Abstract: 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 ... ...

Abstract	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.
MeSH term(s)	Biotechnology ; Fractionation, Field Flow ; Molecular Biology ; Food ; Industry
Language	English
Publishing date	2023-08-23
Publishing country	Switzerland
Document type	Journal Article ; Review
ZDB-ID	1413402-0
ISSN	1420-3049 ; 1431-5165 ; 1420-3049
ISSN (online)	1420-3049
ISSN	1431-5165 ; 1420-3049
DOI	10.3390/molecules28176201
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Article ; Online: Rapid and green discrimination of bovine milk according to fat content, thermal treatment, brand and manufacturer via colloidal fingerprinting.

Giordani, Stefano / Kassouf, Nicholas / Zappi, Alessandro / Zattoni, Andrea / Roda, Barbara / Melucci, Dora / Marassi, Valentina

Food chemistry

2023 Volume 440, Page(s) 138206

Abstract: 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 ... ...

Abstract	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.
MeSH term(s)	Animals ; Milk/chemistry ; Multivariate Analysis ; Fractionation, Field Flow ; Food Safety
Language	English
Publishing date	2023-12-13
Publishing country	England
Document type	Journal Article
ZDB-ID	243123-3
ISSN	1873-7072 ; 0308-8146
ISSN (online)	1873-7072
ISSN	0308-8146
DOI	10.1016/j.foodchem.2023.138206
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Article ; Online: Are Aptamers Really Promising as Receptors for Analytical Purposes? Insights into Anti-Lysozyme DNA Aptamers through a Multitechnique Study.

Toma, Lorenzo / Mattarozzi, Monica / Ronda, Luca / Marassi, Valentina / Zattoni, Andrea / Fortunati, Simone / Giannetto, Marco / Careri, Maria

Analytical chemistry

2024 Volume 96, Issue 6, Page(s) 2719–2726

Abstract: 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 ... ...

Abstract	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.
MeSH term(s)	Aptamers, Nucleotide/chemistry ; Muramidase ; Reproducibility of Results ; SELEX Aptamer Technique/methods ; Antibodies, Antinuclear
Chemical Substances	Aptamers, Nucleotide ; Muramidase (EC 3.2.1.17) ; Antibodies, Antinuclear
Language	English
Publishing date	2024-01-31
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	1508-8
ISSN	1520-6882 ; 0003-2700
ISSN (online)	1520-6882
ISSN	0003-2700
DOI	10.1021/acs.analchem.3c05883
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Article ; Online: Emerging Microfluidic Tools for Simultaneous Exosomes and Cargo Biosensing in Liquid Biopsy: New Integrated Miniaturized FFF-Assisted Approach for Colon Cancer Diagnosis.

Marassi, Valentina / Giordani, Stefano / Placci, Anna / Punzo, Angela / Caliceti, Cristiana / Zattoni, Andrea / Reschiglian, Pierluigi / Roda, Barbara / Roda, Aldo

Sensors (Basel, Switzerland)

2023 Volume 23, Issue 23

Abstract: 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 ... ...

Abstract	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.
MeSH term(s)	Humans ; Exosomes/chemistry ; Microfluidics ; Liquid Biopsy/methods ; Biomarkers/analysis ; Colonic Neoplasms/diagnosis ; Cell Communication
Chemical Substances	Biomarkers
Language	English
Publishing date	2023-11-27
Publishing country	Switzerland
Document type	Journal Article ; Review
ZDB-ID	2052857-7
ISSN	1424-8220 ; 1424-8220
ISSN (online)	1424-8220
ISSN	1424-8220
DOI	10.3390/s23239432
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Article: FFF-based high-throughput sequence shortlisting to support the development of aptamer-based analytical strategies

Marassi, Valentina / Mattarozzi, Monica / Toma, Lorenzo / Giordani, Stefano / Ronda, Luca / Roda, Barbara / Zattoni, Andrea / Reschiglian, Pierluigi / Careri, Maria

Analytical and bioanalytical chemistry. 2022 July, v. 414, no. 18

2022

Abstract: 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 ... ...

Abstract	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.
Keywords	analytical chemistry ; aptasensors ; biomimetics ; electrochemistry ; fractionation ; lysozyme ; oligonucleotides ; serum albumin ; systematic evolution of ligands by exponential enrichment
Language	English
Dates of publication	2022-07
Size	p. 5519-5527.
Publishing place	Springer Berlin Heidelberg
Document type	Article
ISSN	1618-2642
DOI	10.1007/s00216-022-03971-2
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Article: Characterization of red wine native colloids by asymmetrical flow field-flow fractionation with online multidetection

Marassi, Valentina / Marangon, Matteo / Zattoni, Andrea / Vincenzi, Simone / Versari, Andrea / Reschiglian, Pierluigi / Roda, Barbara / Curioni, Andrea

Food hydrocolloids. 2021 Jan., v. 110

2021

Abstract: Colloids are an important component of wines, but their study is challenging due to their instability. Asymmetrical Flow-Field Flow Fractionation (AF4)-multidetection is here proposed as a suitable approach to isolate and characterize red wine colloids ... ...

Abstract	Colloids are an important component of wines, but their study is challenging due to their instability. Asymmetrical Flow-Field Flow Fractionation (AF4)-multidetection is here proposed as a suitable approach to isolate and characterize red wine colloids in native state. AF4 provided size-separation and enabled quantification of the colloidal content of two wines. The gyration radius of colloids was determined by multi-angle light scattering, and ranged between 25 and 50 nm. Analysis of the collected AF4-fractions showed that proteins, polysaccharides and phenolics were present in different proportions among fractions. The composition of AF4-fractions differed between wines. SDS-PAGE analysis of AF4-fractions indicated the presence of protein-phenolics sub-aggregates only in the fractions containing colloids with small radius. The results allowed proposing a model for red wine colloids structure, which comprises two coexisting entities, one made of covalently linked proteins-phenolics sub-aggregates interacting by non-covalent forces with polysaccharides, and a second in which only polysaccharides and phenolics are present. The proposed model is consistent with the reported relative stability of red wine proteins, a fact that can be due to the stabilizing activity of polysaccharides. Given that enological practices affect the extraction of proteins, polysaccharides and phenolics, AF4-technique represents a very promising tool to investigate the effects of these practices on wine colloidal aggregation and behavior.
Keywords	chemical bonding ; fractionation ; hydrocolloids ; models ; phenolic compounds ; polyacrylamide gel electrophoresis ; polysaccharides ; red wines
Language	English
Dates of publication	2021-01
Publishing place	Elsevier Ltd
Document type	Article
Note	NAL-AP-2-clean
ZDB-ID	742742-6
ISSN	1873-7137 ; 0268-005X
ISSN (online)	1873-7137
ISSN	0268-005X
DOI	10.1016/j.foodhyd.2020.106204
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Article ; Online: Application of Af4-Multidetection to Liraglutide in Its Formulation: Preserving and Representing Native Aggregation.

Marassi, Valentina / Macis, Marco / Giordani, Stefano / Ferrazzano, Lucia / Tolomelli, Alessandra / Roda, Barbara / Zattoni, Andrea / Ricci, Antonio / Reschiglian, Pierluigi / Cabri, Walter

Molecules (Basel, Switzerland)

2022 Volume 27, Issue 17

Abstract: Aggregation is among the most critical parameters affecting the pharmacological and safety profile of peptide Active Pharmaceutical Ingredients (APIs). For this reason, it is of utmost importance to define the exact aggregation state of peptide drugs, ... ...

Abstract	Aggregation is among the most critical parameters affecting the pharmacological and safety profile of peptide Active Pharmaceutical Ingredients (APIs). For this reason, it is of utmost importance to define the exact aggregation state of peptide drugs, particularly when the API is marketed as a ready-to-use solution. Consequently, appropriate non-destructive techniques able to replicate the peptide environment must be employed. In our work, we exploited Asymmetrical Flow Field-Flow Fractionation (AF4), connected to UV, dRI, fluorescence, and MALS detectors, to fully characterize the aggregation state of Liraglutide, a peptide API used for the treatment of diabetes type 2 and chronic obesity. In previous studies, Liraglutide was hypothesized to assemble into hexa-octamers in phosphate buffer, but no information on its behavior in the formulation medium was provided up to now. The method used allowed researchers to work using formulation as the mobile phase with excellent recoveries and LoQ/LoD, discerning between stable and degraded samples, and detecting, when present, aggregates up to 10
MeSH term(s)	Fractionation, Field Flow/methods ; Liraglutide
Chemical Substances	Liraglutide (839I73S42A)
Language	English
Publishing date	2022-08-26
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	1413402-0
ISSN	1420-3049 ; 1431-5165 ; 1420-3049
ISSN (online)	1420-3049
ISSN	1431-5165 ; 1420-3049
DOI	10.3390/molecules27175485
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Article: Synthesis Monitoring, Characterization and Cleanup of Ag-Polydopamine Nanoparticles Used as Antibacterial Agents with Field-Flow Fractionation.

Marassi, Valentina / Casolari, Sonia / Panzavolta, Silvia / Bonvicini, Francesca / Gentilomi, Giovanna Angela / Giordani, Stefano / Zattoni, Andrea / Reschiglian, Pierluigi / Roda, Barbara

Antibiotics (Basel, Switzerland)

2022 Volume 11, Issue 3

Abstract: 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 ...

Abstract	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.
Language	English
Publishing date	2022-03-08
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2681345-2
ISSN	2079-6382
ISSN	2079-6382
DOI	10.3390/antibiotics11030358
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Article ; Online: A Green Analytical Method Combined with Chemometrics for Traceability of Tomato Sauce Based on Colloidal and Volatile Fingerprinting.

Zappi, Alessandro / Marassi, Valentina / Kassouf, Nicholas / Giordani, Stefano / Pasqualucci, Gaia / Garbini, Davide / Roda, Barbara / Zattoni, Andrea / Reschiglian, Pierluigi / Melucci, Dora

Molecules (Basel, Switzerland)

2022 Volume 27, Issue 17

Abstract: Tomato sauce is a world famous food product. Despite standards regulating the production of tomato derivatives, the market suffers frpm fraud such as product adulteration, origin mislabelling and counterfeiting. Methods suitable to discriminate the ... ...

Abstract	Tomato sauce is a world famous food product. Despite standards regulating the production of tomato derivatives, the market suffers frpm fraud such as product adulteration, origin mislabelling and counterfeiting. Methods suitable to discriminate the geographical origin of food samples and identify counterfeits are required. Chemometric approaches offer valuable information: data on tomato sauce is usually obtained through chromatography (HPLC and GC) coupled to mass spectrometry, which requires chemical pretreatment and the use of organic solvents. In this paper, a faster, cheaper, and greener analytical procedure has been developed for the analysis of volatile organic compounds (VOCs) and the colloidal fraction via multivariate statistical analysis. Tomato sauce VOCs were analysed by GC coupled to flame ionisation (GC-FID) and to ion mobility spectrometry (GC-IMS). Instead of using HPLC, the colloidal fraction was analysed by asymmetric flow field-fractionation (AF4), which was applied to this kind of sample for the first time. The GC and AF4 data showed promising perspectives in food-quality control: the AF4 method yielded comparable or better results than GC-IMS and offered complementary information. The ability to work in saline conditions with easy pretreatment and no chemical waste is a significant advantage compared to environmentally heavy techniques. The method presented here should therefore be taken into consideration when designing chemometric approaches which encompass a large number of samples.
MeSH term(s)	Chemometrics ; Gas Chromatography-Mass Spectrometry/methods ; Lycopersicon esculentum ; Principal Component Analysis ; Volatile Organic Compounds/analysis
Chemical Substances	Volatile Organic Compounds
Language	English
Publishing date	2022-08-27
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	1413402-0
ISSN	1420-3049 ; 1431-5165 ; 1420-3049
ISSN (online)	1420-3049
ISSN	1431-5165 ; 1420-3049
DOI	10.3390/molecules27175507
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