Luigi Calzolai

• PhD
• Research Officer at European Commission

Aim: The microenvironment effect on the tumoral-derived Extracellular Vesicle release, which is of significant interest for biomedical applications, still represents a rather unexplored field. The aim of the present work is to investigate the interrelation between extracellular matrix (ECM) stiffness and the release of small EVs from cancer cells....

The French National Metrology Institute (LNE) initiated a series of events to identify priorities for test methods and their harmonisation that directly address regulatory needs in Nanomedicine. One of these workshops entitled “The International Standardisation Roadmap for Nanomedicine ” held in October 2023 (Paris, France) brought together key exp...

The approval of safe and effective LNP-mRNA vaccines during the SARS-CoV-2 pandemic is catalyzing the development of the next generation of mRNA therapeutics. Proper characterization methods are crucial for assessing the quality and efficacy of these complex formulations. Here, we show that analytical ultracentrifugation (AUC) can measure, simultan...

The emergence of Omicron lineages and descendent subvariants continues to present a severe threat to the effectiveness of vaccines and therapeutic antibodies. We have previously suggested that an insufficient mucosal immunoglobulin A (IgA) response induced by the mRNA vaccines is associated with a surge in breakthrough infections. Here, we further...

Analytical ultracentrifugation (AUC) analysis shows that the SARS-CoV-2 trimeric Spike (S) protein adopts different quaternary conformations in solution. The relative abundance of the “open” and “close” conformations is temperature-dependent, and samples with different storage temperature history have different open/close distributions. Neutralizin...

This study aims to highlight the impact of physicochemical properties on the behaviour of nanopharmaceuticals and how much carrier structure and physiochemical characteristics weigh on the effects of a formulation. For this purpose, two commercially available nanosimilar formulations of Doxil and their respective carriers were compared as a case st...

In the present study, we addressed the knowledge gaps regarding the agglomeration behavior and fate of food-grade titanium dioxide (E 171) in human gastrointestinal digestion (GID). After thorough multi-technique physicochemical characterization including TEM, single-particle ICP-MS (spICP-MS), CLS, VSSA determination and ELS, the GI fate of E 171...

Quercetin-loaded nano-liposomes were prepared by high-pressure homogenization (HPH) at different pressures (up to 150 MPa) and number of passes (up to 3) to define the best processing conditions allowing the lowest particle size and the highest encapsulation efficiency (EE). The process at 150 MPa for 1 pass was the best, producing quercetin-loaded...

The emergence of Omicron lineages and descendent subvariants continues to present a severe threat to the effectiveness of vaccines and therapeutic antibodies. We have previously suggested that an insufficient mucosal IgA response induced by the mRNA vaccines is associated with a surge in breakthrough infections. Here, we further show that the intra...

A deep and detailed understanding of drug-dendrimer conjugates key properties is needed to define the critical quality attributes that affect drug product performance. The characterization must be executed both in the formulation media and in biological matrices. This, nevertheless, is challenging on account of a very limited number of suitable, es...

Emergence of SARS-CoV-2 variants diminishes the efficacy of vaccines and antiviral monoclonal antibodies. Continued development of immunotherapies and vaccine immunogens resilient to viral evolution is therefore necessary. Using coldspot-guided antibody discovery, a screening approach that focuses on portions of the virus spike glycoprotein that ar...

Quality control of pharmaceutical and biopharmaceutical products, and verification of their safety and efficacy, depends on reliable measurements of critical quality attributes (CQAs). The task becomes particularly challenging for drug products and vaccines containing nanomaterials, where multiple complex CQAs must be identified and monitored. To r...

Unlabelled: Emergence of SARS-CoV-2 variants diminishes the efficacy of vaccines and antiviral monoclonal antibodies. Continued development of immunotherapies and vaccine immunogens resilient to viral evolution is therefore necessary. Using coldspot-guided antibody discovery, a screening approach that focuses on portions of the virus spike that ar...

PEGylated lipids are one of the four constituents of lipid nanoparticle mRNA COVID-19 vaccines. Therefore, various concerns have been raised on the generation of anti-PEG antibodies and their potential role in inducing hypersensitivity reactions following vaccination or in reducing vaccine efficacy due to anti-carrier immunity. Here, we assess the...

Several vaccines against COVID-19 use nanoparticles to protect the antigen cargo (either proteins or nucleic acids), increase the immunogenicity and ultimately the efficacy. The characterization of these nanomedicines is challenging due to their intrinsic complexity and requires the use of multidisciplinary techniques and competencies. The accurate...

The recent emergence of the Omicron variant has raised concerns on vaccine efficacy and the urgent need to study more efficient vaccination strategies. Here we observed that an mRNA vaccine booster in individuals vaccinated with two doses of inactivated vaccine significantly increased the plasma level of specific antibodies that bind to the recepto...

Information concerning the longevity of immunity to SARS-CoV-2 following natural infection may have considerable implications for durability of immunity induced by vaccines. Here, we monitored the SARS-CoV-2 specific immune response in COVID-19 patients followed up to 15 months after symptoms onset. Following a peak at day 15-28 post-infection, the...

Background There has been an unprecedented global effort to produce safe and effective vaccines against SARS-CoV-2. However, production challenges, supply shortages and unequal global reach, together with an increased number of breakthrough infections due to waning of immunity and the emergence of new variants of concern (VOC), have prolonged the p...

Prostate cancer (PCa), one of the leading causes of cancer‐related deaths, currently lacks effective treatment for advanced‐stage disease. Paclitaxel (PTX) is a highly active chemotherapeutic drug and the first‐line treatment for PCa; however, conventional PTX formulation causes severe hypersensitivity reactions and limits PTX use at high concentra...

Background Information concerning the longevity of immunity to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) following natural infection may have considerable implications for durability of immunity induced by vaccines. Here, we monitored the SARS-CoV-2 specific immune response in convalescent coronavirus disease-2019 (COVID-19) pati...

COVID-19 related morbidity and mortality have been often attributed to an exaggerated immune response. The role of cytokines and chemokines in COVID-19 and their contributions to illness severity are known, and thus their profiling from patient bronchoalveolar lavage (BAL) samples would help in understanding the disease progression. To date, limite...

Biocompatible coating based on bovine serum albumin (BSA) was applied on two different TiO2 nanoparticles (aeroxide P25 and food grade E171) to investigate properties and stability of resulting TiO2@BSA composites, under the final perspective to create a “Safe by Design” coating, able to uniform, level off and mitigate surface chemistry related phe...

Adjuvants have been used for decades to enhance the immune response to vaccines, in particular for the subunit-based adjuvants. Physicochemical properties of the adjuvant-protein antigen complexes, such as size, morphology, protein structure and binding, influence the overall efficacy and safety of the vaccine. Here we show how to perform an accura...

Neutralizing antibodies targeting the receptor binding domain (RBD) of the SARS-CoV-2 Spike (S) are among the most promising approaches against coronavirus disease 2019 (COVID-19)1,2. We developed a bispecific, IgG1-like molecule (CoV-X2) based on two antibodies derived from COVID-19 convalescent donors, C121 and C1353. CoV-X2 simultaneously binds...

Neutralizing antibodies targeting the receptor binding domain (RBD) of the SARS-CoV-2 Spike (S) are among the most promising approaches against coronavirus disease 2019 (COVID-19). We developed a bispecific, IgG1-like molecule (CoV-X2) based on two antibodies derived from COVID-19 convalescent donors, C121 and C135. CoV-X2 simultaneously binds two...

Neutralizing antibodies targeting the receptor binding domain (RBD) of the SARS-CoV-2 Spike (S) are among the most promising approaches against coronavirus disease 2019 (COVID-19). We developed a bispecific, IgG1-like molecule based on two antibodies derived from COVID-19 convalescent donors, C121 and C135. CoV-X2 simultaneously binds two independe...

The measurement of physicochemical properties of polydisperse complex biological samples, for example, extracellular vesicles, is critical to assess their quality, for example, resulting from their production and isolation methods. The community is gradually becoming aware of the need to combine multiple orthogonal techniques to perform a robust ch...

Hypothesis The implementation of the proposal from the European Chemical Agency (ECHA) to restrict the use of nanoplastics (NP) and microplastics (MP) in consumer products will require reliable methods to perform size and mass-based concentration measurements. Analytical challenges arise at the nanometre to micrometre interface, e.g., 800 nm–10 µm,...

Asymmetric-flow field-flow fractionation (AF4) has been recognized as an invaluable tool for the characterisation of particle size, polydispersity, drug loading and stability of nanopharmaceuticals. However, the application of robust and high quality standard operating procedures (SOPs) is critical for accurate measurements, especially as these com...

Acute myeloid leukemia (AML), a blood/bone marrow cancer, is a severe and often fatal malignancy. AML cells are capable of impairing the anti-cancer activities of cytotoxic lymphoid cells. This includes the inactivation of natural killer (NK) cells and killing of T lymphocytes. Here we report for the first time that V-domain Ig-containing suppresso...

Specific targeting of fundamental biochemical/signaling pathways in human malignant cells with the purpose of recognition and delivery of cytotoxic drugs into them is a very promising but not well-developed strategic approach. It is a major focus of current molecular cancer research, nanomedicine, biotechnology, and synthetic biology. mTOR pathway...

The potential of analytical ultracentrifugation (AUC) in the analysis of the drug distribution of liposomal doxorubicin formulation (with nominal diameter of 85 nm) in the presence of human serum proteins is demonstrated using the absorbance detection function of the instrument. Based on the AUC measurement (and model fitting for molecular mass cal...

Liposomal formulations for the treatment of cancer and other diseases are the most common form of nanotechnology enabled pharmaceuticals (NEPs) submitted for market approval and in clinical application today. The accurate characterization of their physical-chemical properties is a key requirement; in particular, size, size distribution, shape, and...

This work is dealing with the use of polystyrene (PS) nanoparticles as substrates for bioanalytical specific interactions. Different techniques were used for the accurate characterization of the PS nanoparticles of 100 nm and 196 nm before coating them with a layer of antibodies against immunoglobulins of type E (aIgE), giving to the particle a spe...

An early dialogue between nanomedicine developers and regulatory authorities are of utmost importance to anticipate quality and safety requirements for these innovative health products. In order to stimulate interactions between the various communities involved in a translation of nanomedicines to clinical applications, the European Commission's Jo...

Calcineurin inhibitors potentially prevent pro-allergic mediator release from basophils and mast cells but are rarely used systemically due to ubiquitous expressions of target signaling proteins. However, specific targeting of allergic effector cells with these inhibitors could circumvent unwanted side effects. We recently demonstrated the biocompa...

The particle size distribution (PSD) and the stability of nanoparticles enabled medicinal products (NEP) in complex biological environments are key attributes to assess their quality, safety and efficacy. Despite its low resolution, dynamic light scattering (DLS) is the most common sizing technique since the onset of NEP in pharmaceutical technolog...

Introduction Highly specific targeting of human malignant cells with the purpose of recognition and delivery of specific drugs into them is a very promising but not well developed complex of diagnostic and therapeutic strategies. It is a major focus of current molecular cancer research, Nanomedicine, Biotechnology and Synthetic Biology. Material a...

Background Targeted radionuclide therapy is a highly efficient but still underused treatment modality for various types of cancers that uses so far mainly readily available β-emitting radionuclides. By using α-particle emitters several shortcomings due to hypoxia, cell proliferation and in the selected treatment of small volumes such as micrometast...

Multi-functionalized nanoparticles are of great interest in biotechnology and biomedicine, especially for diagnostic and therapeutic purposes. However, at the moment the characterization of complex, multi-functional nanoparticles is still challenging and this hampers the development of advanced nanomaterials for biological applications. In this wor...

In this study we used 5 nm gold nanoparticles as delivery platforms to target cancer cells expressing the immune receptor Tim-3 using single chain antibodies.

The use of nanotechnology in medical products has been demonstrated at laboratory scale, and many resulting nanomedicines are in the translational phase toward clinical applications, with global market trends indicating strong growth of the sector in the coming years. The translation of nanomedicines toward the clinic and subsequent commercializati...

Endocrine Disrupting Compounds (EDCs) are chemical substances shown to interfere with endogenous hormones affecting the endocrine, immune and nervous systems of mammals. EDCs are the causative agents of diseases including reproductive disorders and cancers. This highlights the urgency to develop fast and sensitive methods to detect EDCs, which are...

Our article dissects the problem of understanding the origin of size heterogeneity in polydispersed nanoparticle samples. A commercially available multimodal material representing a typical borderline case of the nano definition is characterised with various state of the art techniques. We focus on dimer (multimer) recognition capability of differe...

Peptide–lipid interactions support a variety of biological functions. Of particular interest are those that underpin fundamental mechanisms of innate immunity that are programmed in host defense or antimicrobial peptide sequences found virtually in all multicellular organisms. Here we synthetically modulate antimicrobial peptide–lipid interactions...

Nanomechanical monitoring of known mechanisms of membrane poration mediated by host defense peptides is reported. Quartz Crystal Microbalance with Dissipation monitoring used to probe the process of antimicrobial poration in model phospholipid bilayers offers a straightforward method for mechanically differential kinetic measurements of membrane-me...

Analytical ultracentrifugation (AUC) is a powerful tool for the study of particle size distributions and interactions with high accuracy and resolution. In this work, we show how the analysis of sedimentation velocity data from the AUC can be used to characterize nanocarrier drug delivery systems used in nanomedicine. Nanocarrier size distribution...

Ultraviolet (UV) radiation, temperature, and time can degrade proteins. Here, the authors show that gold nanoparticles significantly protect human serum albumin from denaturation when exposed to "stressing" conditions such as UV irradiation and sustained exposure in suboptimal conditions. In particular, the authors show that gold nanoparticles sign...

The detection and quantification of nanoparticles is a complex issue due to the need to combine "classical" identification and quantification of the constituent material, with the accurate determination of the size of submicrometer objects, usually well below the optical diffraction limit. In this work, the authors show that one of the most used an...

The spread of bacterial resistance to traditional antibiotics continues to stimulate the search for alternative antimicrobial strategies. All forms of life, from bacteria to humans, are postulated to rely on a fundamental host defense mechanism, which exploits the formation of open pores in microbial phospholipid bilayers. Here we predict that tran...

We demonstrate the use of inverse supercritical carbon dioxide (scCO2) extraction as a novel method of sample preparation for the analysis of complex nanoparticle-containing samples, in our case a model sunscreen agent with titanium dioxide nanoparticles. The sample was prepared for analysis in a simplified process using a lab scale supercritical f...

Asymmetric Flow Field-Flow Fractionation (AF4) combined with multidetector analysis form a promising technique in the field of nanoparticle characterization. This system is able to measure the dimensions and physicochemical properties of nanoparticles with unprecedented accuracy and precision. Here, for the first time, this technique is optimized t...

We propose a simple method to determine the structure and morphology of nanoparticle protein complexes. By combining a separation method with online size measurements, density measurements and circular dichroism, we could identify the number of proteins bound to each nanoparticle and their secondary structure changes in the complex. This method pro...

With the ever growing use of nanoparticles in a broad range of industrial and consumer applications there is increasing likelihood that such nanoparticles will enter the aquatic environment and be transported through freshwater systems, eventually reaching estuarine or marine waters. Due to silver's known antimicrobial properties and widespread use...

The estrogen receptor protein (ER) can bind a vast number of organic pollutants widely spread in the environment and collectively known as Endocrine Disrupting Chemicals, EDCs. Its broad selectivity makes it an ideal bio-recognition element for the detection of EDCs. Here we describe the strategy and rationale for the design of ER based biosensors...

In 2011 the European Commission published its recommendation for a definition for the term nanomaterial which requires the materials to be characterized in terms of the number size distribution of their constituent particles. More recently, the definition has begun to be applied to the labelling of food and cosmetic products where any components pr...

Different analytical techniques, Sedimentation Flow Field Fractionation (SdFFF), Asymmetrical Flow Field Flow Fractionation (AF4) and Centrifugal Liquid Sedimentation (CLS) and Dynamic Light Scattering (DLS) have been used to give complementary size information about suspensions of silver nanoparticles (AgNPs) in the size range of 20-100 nm by taki...

In this manuscript, we modulate the binding properties of estrogen receptor protein by rationally modifying the amino acid composition of its ligand binding domain. By combining sequence alignment and structural analysis of known estrogen receptor-ligand complexes with computational analysis, we were able to predict estrogen receptor mutants with a...

In this study we demonstrate for the first time a new approach for highly specific targeting of viable human leukocytes by functionalised gold nanoconjugates made on the basis of citrate-stabilised gold nanoparticles. This immobilisation involves highly specific recognition of the cells of interest by plasma membrane-associated receptor ligands or...

The Recommendation (2011/696/EU) of the European Commission (EC) on the definition of the term 'nanomaterial' includes an article on its review in the light of experience and of scientific and technological developments by December 2014. This report collects information on scientific-technical issues that should be considered when reviewing the cur...

This report provides the JRC assessment of feedback on the experiences of stakeholders with the EC nanomaterial definition, published in 2011 (EC Recommendation 2011/696/EU). The report is a follow-up report of the previous JRC report (EUR 26567 EN, 2014), which compiled feedback collected by JRC in 2013 and early 2014, partly through a dedicated s...

http://www.ttl.fi/en/publications/Electronic_publications/Nanosafety_in_europe_2015-2025/Documents/nanosafety_2015-2025.pdf

Gold nanoparticles (AuNPs) can interfere with some of the biochemical processes of macrophage cells but the mechanisms of action of these potentially medically-relevant effects are still unclear. Here we study the fate of AuNPs interacting with cells derived from the innate immune system. To visualize AuNPs with nanometer resolution without losing...

Interleukin 1 beta (IL-1β)-dependent inflammatory disorders, such as rheumatoid arthritis and psoriasis, pose a serious medical burden worldwide, where patients face a lifetime of illness and treatment. Organogold compounds have been used since the 1930s to treat rheumatic and other IL-1β-dependent diseases and, though their mechanisms of action ar...

Antibodies play an increasing pivotal role in both basic research and the biopharmaceutical sector, therefore technology for characterizing and improving their properties through rational engineering is desirable. This is a difficult task thought to require high-resolution x-ray structures, which are not always available. We, instead, use a combina...

Binding assay (ELISA) for all the antibody mutants designed to alter its properties in a predictable manner and mentioned in the main text. The antibody concentration is on the x axis; increased y values correspond to increased binding. DIII from each Dengue serotype was immobilized on a surface in the presence of different amounts of antibodies as...

(TIF)

15N HSQC spectra of DIII of the four Dengue serotypes free (blue) and in complex with antibody DV32.6 (red). Residue V324 is affected by complex formation and shows chemical shift changes in DenV1, DenV3 and DenV4 but not DenV2. (TIF)

NMR validation and docking results for the DV32.6/DIII complexes chosen as representative for each different antibody homology model used for docking (Model1 to Model10 and PIGS). Models in agreement with the NMR epitope mapping data and chosen as final result for the structural analysis (see main text) are highlighted in green. If an antigen resid...

DV32.6 has a stronger binding affinity for its epitope on DIII of DenV4 than for the other serotypes (panel a) but it is less efficient at neutralizing it (panel c). a) Binding assay (ELISA) for wt DV32.6 on DIII. The antibody concentration is on the x axis and increased y values correspond to increased binding. DIII from each Dengue serotype was i...

15N HSQC spectra of DIII of the four Dengue serotypes free (blue) and in complex with antibody DV32.6 (red). Residue K310 shows chemical shift changes upon complex formation in DenV4 and DenV1 (smaller changes). The peak corresponding to the bound state disappears in DenV2 and DenV3, revealing that the residue is affected by antibody binding. (TIF)

Ultraviolet radiation can cause the unfolding and destabilization of proteins. By using high energy photons from a synchrotron radiation source, we show that the UV-induced destabilization of human serum albumin (HSA) can be detected and monitored by measuring the circular dichroism spectrum of the protein. The high flux radiation source damages th...

The behavior and toxicological properties of nanoparticles (NP) in biological medium depends heavily on their interactions with proteins. In return, the structure, stability and biological properties of the proteins that interact with the nanoparticles are strongly affected by this interaction. Unfortunately, the mechanisms of interaction and their...

Protein-nanoparticle associations have important applications in nanoscience and nanotechnology such as targeted drug delivery and theranostics. However, the mechanisms by which proteins recognize nanoparticles and the determinants of specificity are still poorly understood at the microscopic level. Gold is a promising material in nanoparticles for...