Renzo Vanna | Fondazione Don C. Gnocchi (original) (raw)

Papers by Renzo Vanna

European Journal of Organic Chemistry, 2009

A highly efficient regioselective enzymatic preparation of penta-O-acetyl-1,5-anhydro-2-deoxy-3-h... more A highly efficient regioselective enzymatic preparation of penta-O-acetyl-1,5-anhydro-2-deoxy-3-hydroxy-4-O-β-galactopyranosyl-D-arabinohex-1-enitol has been successfully performed for the first time. This product was obtained in Ͼ99 % conversion (Ͼ95 % overall yield) by hydrolysis of per-O-acetylated lactal catalyzed by the lipase from Rhizomucor

Extracellular vesicles (EVs) from mesenchymal stromal cells (MSC) are emerging as valuable therap... more Extracellular vesicles (EVs) from mesenchymal stromal cells (MSC) are emerging as valuable therapeutic agents for tissue regeneration and immunomodulation, but their clinical applications have so far been limited by the technical restraints of current isolation and characterisation procedures. This study shows for the first time the successful application of Raman spectroscopy as label-free, sensitive and reproducible means of carrying out the routine bulk characterisation of MSC-derived vesicles before their use in vitro or in vivo, thus promoting the translation of EV research to clinical practice. The Raman spectra of the EVs of bone marrow and adipose tissue-derived MSCs were compared with human dermal fibroblast EVs in order to demonstrate the ability of the method to distinguish the vesicles of the three cytotypes automatically with an accuracy of 93.7%. Our data attribute a Raman fingerprint to EVs from undifferentiated and differentiated cells of diverse tissue origin, and provide insights into the biochemical characteristics of EVs from different sources and into the differential contribution of sphingomyelin, gangliosides and phosphatidilcholine to the Raman spectra themselves. Extracellular vesicles (EVs) are a heterogeneous group of membrane-bound vesicles that are constitutively released by cells of different tissue origins. Past controversies concerning nomenclature have now been resolved by the scientific community, which defines EVs as the group of particles made up of exosomes, microvesicles and apoptotic bodies 1. Exosomes (30–100 nm) and microvesicles (up to 1000 nm) differ in size and cellular origin, but both mediate intercellular communication within a tissue and among organs thanks to body fluid transportation 1. As is the case for most body cells, part of the secretome of mesenchymal stromal cells (MSCs) includes exosomes and microvesicles, which are currently being investigated because of their striking regenerative and immunomodulating potential. The bioactive molecules loaded onto/into EVs are involved in the paracrine effects of stem cells, and even the membrane constituents of vesicles seem to trigger intracellular protective/regener-ative pathways in recipient cells 2. It has been suggested that MSC-derived EVs may be sometimes even more therapeutically valuable than whole cells, because of their remarkable handling advantages, which can accelerate their clinical application in the so-called cell therapy without cells 3. The possibility of overcoming the cell therapy drawbacks of having to administer living, replicating and difficult to control cells is currently one of the main challenges facing regenerative medicine, and EVs can be an effective means of stimulating the restoration of organ function through tissue regeneration and repair in the context of an integrated strategy of regenerative rehabilitation 4. Over the last ten years, many studies have demonstrated the role that MSC-derived EVs can play in tissue repair and immunomodulation 5, 6 and, in 2014, EVs ability to influence the activity of recipient cells and regulate

Background: Identifying physiologically relevant binding partners of amyloid-(A) that modulate in... more Background: Identifying physiologically relevant binding partners of amyloid-(A) that modulate in vivo fibril formation may yield new insights into Alzheimer's disease (AD) etiology. Human cathelicidin peptide, LL-37, is an innate immune effector and modulator, ubiquitous in human tissues and expressed in myriad cell types. Objective: We present in vitro experimental evidence and discuss findings supporting a novel hypothesis that LL-37 binds to A 42 and can modulate A fibril formation. Methods: Specific interactions between LL-37 and A (with A in different aggregation states, assessed by capillary elec-trophoresis) were demonstrated by surface plasmon resonance imaging (SPRi). Morphological and structural changes were investigated by transmission electron microscopy (TEM) and circular dichroism (CD) spectroscopy. Neuroinflammatory and cytotoxic effects of LL-37 alone, A 42 alone, and LL-37/A complexes were evaluated in human microglia and neuroblastoma cell lines (SH-SY5Y). Results: SPRi shows binding specificity between LL-37 and A, while TEM shows that LL-37 inhibits A 42 fibril formation, particularly A's ability to form long, straight fibrils characteristic of AD. CD reveals that LL-37 prevents A 42 from adopting its typical-type secondary structure. Microglia-mediated toxicities of LL-37 and A 42 to neurons are greatly attenuated when the two peptides are co-incubated prior to addition. We discuss the complementary biophysical characteristics and AD-related biological activities of these two peptides. Conclusion: Based on this body of evidence, we propose that LL-37 and A 42 may be natural binding partners, which implies that balanced (or unbalanced) spatiotemporal expression of the two peptides could impact AD initiation and progression.

Plasmonics in Biology and Medicine XIII, 2016

Plasmonics in Biology and Medicine XIII, 2016

RSC Adv., 2015

Surface-enhanced Raman spectroscopy (SERS) widely improves the sensitivity of traditional Raman a... more Surface-enhanced Raman spectroscopy (SERS) widely improves the sensitivity of traditional Raman analysis, thus allowing this technique to be exploited for the development of new bio-analytical tests. In this work, 3D substrates made of zinc oxide tetrapods (ZnOTP) are decorated with branched gold nanoparticles by means of a new photochemical approach. The SERS enhancing properties of the obtained substrate are tested using different Raman dyes and apomorphine, a drug used for the management of Parkinson disease. The results prove that the enhancing properties depend on the shape of the gold nanoparticles grown on the branches of ZnO tetrapods. The optimized substrate here developed is characterized by an enhancing factor up to 7 × 10^6 and a detection limit for apomorphine of 1 μM. Finally, the new substrates are tested to study single cancer cells showing enhanced Raman signals related to the portion of the cell interacting with the 3D structure of the substrate.

A personalized drug administration method and a more reliable electrical interface for monitoring... more A personalized drug administration method and a more reliable electrical interface for monitoring biopotentials (electroencephalogram, electrocardiogram, electromyography) are pivotal matters in the clinical community. The aim of our research is to design, develop and test innovative transdermal devices, deriving from nano- and micro-technologies, for improving diagnosis and therapeutic treatments. In order to overcome, in a minimally invasive manner, the complex skin barrier, including stratum corneum, we explored a technological platform based on microneedles. In particular we worked on two projects: a ‘transdermal cartridge’ for drug delivery and a ‘dry electrodes’ device for the measurements of biosignals. We developed non-miniaturised prototypes with a hollow or solid microneedles matrix (max 1 mm tall). For testing purpose the former was connected to a syringe pump infusing a green fluorochrome labelled insulin (10 ll/h) and the latter to a biomechanical test station. We perfo...

Biomedical Vibrational Spectroscopy VI: Advances in Research and Industry, 2014

ABSTRACT Acute myeloid leukemia (AML) is a proliferative neoplasm, that if not properly treated c... more ABSTRACT Acute myeloid leukemia (AML) is a proliferative neoplasm, that if not properly treated can rapidly cause a fatal outcome. The diagnosis of AML is challenging and the first diagnostic step is the count of the percentage of blasts (immature cells) in bone marrow and blood sample, and their morphological characterization. This evaluation is still performed manually with a bright field light microscope. Here we report results of a study applying Raman spectroscopy for analysis of samples from two patients affected by two AML subtypes characterized by a different maturation stage in the neutrophilic lineage. Ten representative cells per sample were selected and analyzed with high-resolution confocal Raman microscopy by scanning 64x64 (4096) points in a confocal layer through the volume of the whole cell. The average spectrum of each cell was then used to obtain a highly reproducible mean fingerprint of the two different AML subtypes. We demonstrate that Raman spectroscopy efficiently distinguishes these different AML subtypes. The molecular interpretation of the substantial differences between the subtypes is related to granulocytic enzymes (e.g. myeloperoxidase and cytochrome b558), in agreement with different stages of maturation of the two considered AML subtypes . These results are promising for the development of a new, objective, automated and label-free Raman based methods for the diagnosis and first assessment of AML.

The Analyst, 2015

In clinical practice, the diagnosis and classification of acute myeloid leukaemia (AML) and myelo... more In clinical practice, the diagnosis and classification of acute myeloid leukaemia (AML) and myelodysplastic syndrome (MDS) start from the manual examination of stained smears of bone marrow (BM) and peripheral blood (PB) by using an optical microscope. This step is subjective and scarcely reproducible. Therefore, the development of subjective and potentially automatable methods for the recognition of typical AML/MDS cells is necessary. Here we have used Raman spectroscopy for distinguishing myeloblasts, promyelocytes, abnormal promyelocytes and erhytroblasts, which have to be counted for a correct diagnosis and morphological classification of AML and MDS. BM samples from patients affected by four different AML subtypes, mostly characterized by the presence of the four subpopulations selected for this study, were analyzed. First, each cell was scanned by acquiring 4096 spectra, thus obtaining Raman images which demonstrate an accurate description of morphological features characteristic of each subpopulation. Raman imaging coupled with hierarchical cluster analysis permitted the automatic discrimination and localization of the nucleus, the cytoplasm, myeloperoxidase containing granules and haemoglobin.

In this article, we describe how it is possible to tune the size and the aspect ratio of gold nan... more In this article, we describe how it is possible to tune the size and the aspect ratio of gold nanorods obtained using a highly efficient protocol based on the use of hydroquinone as a reducing agent by varying the amounts of CTAB and silver ions present in the “seed-growth” solution. Our approach not only allows us to prepare nanorods with a four times increased Au3+ reduction yield, when compared with the commonly used protocol based on ascorbic acid, but also allows a remarkable reduction of 50–60 % of the amount of CTAB needed. In fact, according to our findings, the concentration of CTAB present in the seed-growth solution do not linearly influence the final aspect ratio of the obtained nanorods, and an optimal concentration range between 30 and 50 mM has been identified as the one that is able to generate particles with more elongated shapes. On the optimized protocol, the effect of the concentration of Ag+ ions in the seed-growth solution and the stability of the obtained particles has also been investigated.

In clinical practice, the diagnosis and classification of acute myeloid leukaemia (AML) and myelo... more In clinical practice, the diagnosis and classification of acute myeloid leukaemia (AML) and myelodysplastic syndrome (MDS) start from the manual examination of stained smears of bone marrow (BM) and peripheral blood (PB) by using an optical microscope. This step is subjective and scarcely reproducible. Therefore, the development of subjective and potentially automatable methods for the recognition of typical AML/MDS cells is necessary. Here we have used Raman spectroscopy for distinguishing myeloblasts, promyelocytes, abnormal promyelocytes and erhytroblasts, which have to be counted for a correct diagnosis and morphological classification of AML and MDS. BM samples from patients affected by four different AML subtypes, mostly characterized by the presence of the four subpopulations selected for this study, were analyzed. First, each cell was scanned by acquiring 4096 spectra, thus obtaining Raman images which demonstrate an accurate description of morphological features characteristic of each subpopulation. Raman imaging coupled with hierarchical cluster analysis permitted the automatic discrimination and localization of the nucleus, the cytoplasm, myeloperoxidase containing granules and haemoglobin.

ACS Nano, 2014

In our study, 2D nanopillar arrays with plasmonic crystal properties are optimized for surface-en... more In our study, 2D nanopillar arrays with plasmonic crystal properties are optimized for surface-enhanced Raman spectroscopy (SERS) application and tested in a biochemical assay for the simultaneous detection of multiple genetic leukemia biomarkers. The special fabrication process combining soft lithography and plasma deposition techniques allows tailoring of the structural and chemical parameters of the crystal surfaces. In this way, it has been possible to tune the plasmonic resonance spectral position close to the excitation wavelength of the monochromatic laser light source in order to maximize the enhancing properties of the substrate. Samples are characterized by scanning electron microscopy and reflectance measurements and tested for SERS activity using malachite green. Besides, as the developed substrate had been prepared on a simple glass slide, SERS detection from the support side is also demonstrated. The optimized substrate is functionalized with thiol-modified capture oligonucleotides, and concentration-dependent signal of the target nucleotide is detected in a sandwich assay with labeled gold nanoparticles. Gold nanoparticles functionalized with different DNA and various Raman reporters are applied in a microarray-based assay recognizing a disease biomarker (Wilms tumor gene) and housekeeping gene expressions in the same time on spatially separated microspots. The multiplexing performance of the SERS-based bioassay is illustrated by distinguishing Raman dyes based on their complex spectral fingerprints.

Colloidal Nanocrystals for Biomedical Applications VIII, 2013

ABSTRACT Surface Enhanced Raman Spectroscopy (SERS) is a popular method in bio-analytical chemist... more ABSTRACT Surface Enhanced Raman Spectroscopy (SERS) is a popular method in bio-analytical chemistry and a potentially powerful enabling technology for in vitro diagnostics. SERS combines the excellent chemical specificity of Raman spectroscopy with the good sensitivity provided by enhancement of the signal that is observed when a molecule is located on (or very close to) the surface of nanostructured metallic materials. Star-like gold nanoparticles (SGN) are a new class of multibranched nanoparticles that in the last few years have attracted the attention of SERS community for their plasmonic properties. In this work we present a new method to prepare star-like gold nanoparticles with a simple one step protocol at room temperature using hydroquinone as reducing agent. Besides we compare the enhancement of Raman signal of malachite green, a dye commonly employed as label in biological studies, by star-like gold nanoparticles having different size, directly in liquid. This study shows that SGN provide good enhancement of Raman signal and that the effect of their dimension is strongly dependent on the wavelength used. Moreover preliminary results suggest that SGN produced using this method are characterized by good physical-chemical properties and they can be functionalized using the standard thiol chemistry. Overall, these results suggest that star-like gold nanoparticles produced through this method could be used for the further development of highly specific and sensitive SERS-based bio-analytical tests.

Neuromelanins (NMs) are neuronal pigments of melanic-lipidic type which accumulate during aging. ... more Neuromelanins (NMs) are neuronal pigments of melanic-lipidic type which accumulate during aging. They are involved in protective and degenerative mechanisms depending on the cellular context, however their structures are still poorly understood. NMs from nine human brain areas were analyzed in detail. Elemental analysis led to identification of three types of NM, while infrared spectroscopy showed that NMs from neurons of substantia nigra and locus coeruleus, which selectively degenerate in Parkinson's disease, have similar structure but different from NMs from brain regions not targeted by the disease. Synthetic melanins containing Fe and bovine serum albumin were prepared to model the natural product and help clarifying the structure of NMs. Extensive nuclear magnetic resonance spectroscopy studies showed the presence of dolichols both in the soluble and insoluble parts of NM. Diffusion measurements demonstrated that the dimethyl sulfoxide soluble components consist of oligomeric precursors with MWs in the range 1.4-52 kDa, while the insoluble part contains polymers of larger size but with a similar composition. These data suggest that the selective vulnerability of neurons of substantia nigra and locus coeruleus in Parkinson's disease might depend on the structure of the pigment. Moreover, they allow to propose a pathway for NM biosynthesis in human brain. Citation: Engelen M, Vanna R, Bellei C, Zucca FA, Wakamatsu K, et al. (2012) Neuromelanins of Human Brain Have Soluble and Insoluble Components with Dolichols Attached to the Melanic Structure. PLoS ONE 7(11): e48490.

Molecular Pharmaceutics, 2014

Among polymeric nanoparticles designed for cancer therapy, PLGA nanoparticles have become one of ... more Among polymeric nanoparticles designed for cancer therapy, PLGA nanoparticles have become one of the most popular polymeric devices for chemotherapeutic-based nanoformulations against several kinds of malignant diseases. Promising properties, including long-circulation time, enhanced tumor localization, interference with "multidrug" resistance effects, and environmental biodegradability, often result in an improvement of the drug bioavailability and effectiveness. In the present work, we have synthesized 1,7-bis(3,4-dimethoxyphenyl)-5-hydroxyhepta-1,4,6-trien-3-one (ASC-J9) and developed uniform ASC-J9-loaded PLGA nanoparticles of about 120 nm, which have been prepared by a single-emulsion process. Structural and morphological features of the nanoformulation were analyzed, followed by an accurate evaluation of the in vitro drug release kinetics, which exhibited Fickian law diffusion over 10 days. The intracellular degradation of ASC-J9-bearing nanoparticles within estrogen-dependent MCF-7 breast cancer cells was correlated to a time- and dose-dependent activity of the released drug. A cellular growth inhibition associated with a specific cell cycle G2/M blocking effect caused by ASC-J9 release inside the cytosol allowed us to put forward a hypothesis on the action mechanism of this nanosystem, which led to the final cell apoptosis. Our study was accomplished using Annexin V-based cell death analysis, MTT assessment of proliferation, radical scavenging activity, and intracellular ROS evaluation. Moreover, the intracellular localization of nanoformulated ASC-J9 was confirmed by a Raman optical imaging experiment designed ad hoc. PLGA nanoparticles and ASC-J9 proved also to be safe for a healthy embryo fibroblast cell line (3T3-L1), suggesting a possible clinical translation of this potential nanochemotherapeutic to expand the inherently poor bioavailability of hydrophobic ASC-J9 that could be proposed for the treatment of malignant breast cancer.

Nanoscale Imaging, Sensing, and Actuation for Biomedical Applications XI, 2014

ABSTRACT Surface Enhanced Raman Spectroscopy is a powerful analytical technique that combines the... more ABSTRACT Surface Enhanced Raman Spectroscopy is a powerful analytical technique that combines the excellent chemical specificity of Raman spectroscopy with the good sensitivity provided by the enhancement of the signal observed when a molecule is located on (or very close to) the surface of suitable nanostructured metallic materials. The availability of cheap, reliable and easy to use SERS substrates would pave the road to the development of bioanalytical tests that can be used in clinical practice. SERS, in fact, is expected to provide not only higher sensitivity and specificity, but also the simultaneous and markedly improved detection of several targets at the same time with higher speed compared to the conventional analytical methods. Here, we present the SERS activity of 2-D plasmonic crystals made by polymeric pillars embedded in a gold matrix obtained through the combination of soft-lithography and plasma deposition techniques on a transparent substrates. The use of a transparent support material allowed us to perform SERS detection from support side opening the possibility to use these substrates in combination with microfluidic devices. In order to demonstrate the potentialities for bioanalytical applications, we used our SERS active gold surface to detect the oxidation product of apomorphine, a well-known drug molecule used in Parkinson’s disease which has been demonstrated being difficult to study by traditional HPLC based approaches.

BioNanoScience, 2014

We present here the surface-enhanced Raman spectroscopic (SERS)-based detection of the Wilm's tum... more We present here the surface-enhanced Raman spectroscopic (SERS)-based detection of the Wilm's tumor gene (WT1) sequence using dye-labeled reporter oligonucleotide and magnetic core @ gold shell nanoparticles. Thiolated single-stranded DNA (ssDNA) complementers of the WT1 sequence were used to functionalize the gold shell with capture oligonucleotides in a facile and fast two-step method. The signal amplification performance of the core @ shell colloidal SERS substrate was tested using malachite green as label dye. The Raman signal enhancing efficacy of the magnetic core @ gold shell nanomaterial was compared with the efficacy of spherical gold particles produced using the conventional citrate reduction method. The core @ shell particles were found to be superior both regarding robustness in SERS and facile separation in a heterogeneous reaction system. The core @ shell particles functionalized with target specific oligonucleotide were able to capture the WT1 target and worked as Raman signal amplifiers in our assay system. The good physicochemical characteristics of these particles and the sensitivity observed in SERS experiments allow us to expect good performance in the further development steps of a novel, fast and reliable spectroscopic method for WT1 detection in minimal residual disease patients.

Vibrational Spectroscopy, 2013

Paper-based SERS active substrates were prepared adsorbing spherical and star-shaped gold nanopar... more Paper-based SERS active substrates were prepared adsorbing spherical and star-shaped gold nanoparticles on a standard filter paper support. Besides the deposition conditions, morphological parameters of the particles were found to strongly affect the enhancer properties of the substrates. The developed substrate was tested regarding surface homogeneity as well as in the quantitative analysis of malachite green, -a well documented Raman reporter dye -and proved to be capable also to detect the oxidation products of apomorphine, a well-known drug molecule used in Parkinson's disease. This material is simple to prepare, easy to handle and dispose and as such it could be a perfect target for further development of a new family of mass-produced, cheap solid SERS substrates.

European Journal of Organic Chemistry, 2009

A highly efficient regioselective enzymatic preparation of penta-O-acetyl-1,5-anhydro-2-deoxy-3-h... more A highly efficient regioselective enzymatic preparation of penta-O-acetyl-1,5-anhydro-2-deoxy-3-hydroxy-4-O-β-galactopyranosyl-D-arabinohex-1-enitol has been successfully performed for the first time. This product was obtained in Ͼ99 % conversion (Ͼ95 % overall yield) by hydrolysis of per-O-acetylated lactal catalyzed by the lipase from Rhizomucor

Extracellular vesicles (EVs) from mesenchymal stromal cells (MSC) are emerging as valuable therap... more Extracellular vesicles (EVs) from mesenchymal stromal cells (MSC) are emerging as valuable therapeutic agents for tissue regeneration and immunomodulation, but their clinical applications have so far been limited by the technical restraints of current isolation and characterisation procedures. This study shows for the first time the successful application of Raman spectroscopy as label-free, sensitive and reproducible means of carrying out the routine bulk characterisation of MSC-derived vesicles before their use in vitro or in vivo, thus promoting the translation of EV research to clinical practice. The Raman spectra of the EVs of bone marrow and adipose tissue-derived MSCs were compared with human dermal fibroblast EVs in order to demonstrate the ability of the method to distinguish the vesicles of the three cytotypes automatically with an accuracy of 93.7%. Our data attribute a Raman fingerprint to EVs from undifferentiated and differentiated cells of diverse tissue origin, and provide insights into the biochemical characteristics of EVs from different sources and into the differential contribution of sphingomyelin, gangliosides and phosphatidilcholine to the Raman spectra themselves. Extracellular vesicles (EVs) are a heterogeneous group of membrane-bound vesicles that are constitutively released by cells of different tissue origins. Past controversies concerning nomenclature have now been resolved by the scientific community, which defines EVs as the group of particles made up of exosomes, microvesicles and apoptotic bodies 1. Exosomes (30–100 nm) and microvesicles (up to 1000 nm) differ in size and cellular origin, but both mediate intercellular communication within a tissue and among organs thanks to body fluid transportation 1. As is the case for most body cells, part of the secretome of mesenchymal stromal cells (MSCs) includes exosomes and microvesicles, which are currently being investigated because of their striking regenerative and immunomodulating potential. The bioactive molecules loaded onto/into EVs are involved in the paracrine effects of stem cells, and even the membrane constituents of vesicles seem to trigger intracellular protective/regener-ative pathways in recipient cells 2. It has been suggested that MSC-derived EVs may be sometimes even more therapeutically valuable than whole cells, because of their remarkable handling advantages, which can accelerate their clinical application in the so-called cell therapy without cells 3. The possibility of overcoming the cell therapy drawbacks of having to administer living, replicating and difficult to control cells is currently one of the main challenges facing regenerative medicine, and EVs can be an effective means of stimulating the restoration of organ function through tissue regeneration and repair in the context of an integrated strategy of regenerative rehabilitation 4. Over the last ten years, many studies have demonstrated the role that MSC-derived EVs can play in tissue repair and immunomodulation 5, 6 and, in 2014, EVs ability to influence the activity of recipient cells and regulate

Background: Identifying physiologically relevant binding partners of amyloid-(A) that modulate in... more Background: Identifying physiologically relevant binding partners of amyloid-(A) that modulate in vivo fibril formation may yield new insights into Alzheimer's disease (AD) etiology. Human cathelicidin peptide, LL-37, is an innate immune effector and modulator, ubiquitous in human tissues and expressed in myriad cell types. Objective: We present in vitro experimental evidence and discuss findings supporting a novel hypothesis that LL-37 binds to A 42 and can modulate A fibril formation. Methods: Specific interactions between LL-37 and A (with A in different aggregation states, assessed by capillary elec-trophoresis) were demonstrated by surface plasmon resonance imaging (SPRi). Morphological and structural changes were investigated by transmission electron microscopy (TEM) and circular dichroism (CD) spectroscopy. Neuroinflammatory and cytotoxic effects of LL-37 alone, A 42 alone, and LL-37/A complexes were evaluated in human microglia and neuroblastoma cell lines (SH-SY5Y). Results: SPRi shows binding specificity between LL-37 and A, while TEM shows that LL-37 inhibits A 42 fibril formation, particularly A's ability to form long, straight fibrils characteristic of AD. CD reveals that LL-37 prevents A 42 from adopting its typical-type secondary structure. Microglia-mediated toxicities of LL-37 and A 42 to neurons are greatly attenuated when the two peptides are co-incubated prior to addition. We discuss the complementary biophysical characteristics and AD-related biological activities of these two peptides. Conclusion: Based on this body of evidence, we propose that LL-37 and A 42 may be natural binding partners, which implies that balanced (or unbalanced) spatiotemporal expression of the two peptides could impact AD initiation and progression.

Plasmonics in Biology and Medicine XIII, 2016

Plasmonics in Biology and Medicine XIII, 2016

RSC Adv., 2015

Surface-enhanced Raman spectroscopy (SERS) widely improves the sensitivity of traditional Raman a... more Surface-enhanced Raman spectroscopy (SERS) widely improves the sensitivity of traditional Raman analysis, thus allowing this technique to be exploited for the development of new bio-analytical tests. In this work, 3D substrates made of zinc oxide tetrapods (ZnOTP) are decorated with branched gold nanoparticles by means of a new photochemical approach. The SERS enhancing properties of the obtained substrate are tested using different Raman dyes and apomorphine, a drug used for the management of Parkinson disease. The results prove that the enhancing properties depend on the shape of the gold nanoparticles grown on the branches of ZnO tetrapods. The optimized substrate here developed is characterized by an enhancing factor up to 7 × 10^6 and a detection limit for apomorphine of 1 μM. Finally, the new substrates are tested to study single cancer cells showing enhanced Raman signals related to the portion of the cell interacting with the 3D structure of the substrate.

A personalized drug administration method and a more reliable electrical interface for monitoring... more A personalized drug administration method and a more reliable electrical interface for monitoring biopotentials (electroencephalogram, electrocardiogram, electromyography) are pivotal matters in the clinical community. The aim of our research is to design, develop and test innovative transdermal devices, deriving from nano- and micro-technologies, for improving diagnosis and therapeutic treatments. In order to overcome, in a minimally invasive manner, the complex skin barrier, including stratum corneum, we explored a technological platform based on microneedles. In particular we worked on two projects: a ‘transdermal cartridge’ for drug delivery and a ‘dry electrodes’ device for the measurements of biosignals. We developed non-miniaturised prototypes with a hollow or solid microneedles matrix (max 1 mm tall). For testing purpose the former was connected to a syringe pump infusing a green fluorochrome labelled insulin (10 ll/h) and the latter to a biomechanical test station. We perfo...

Biomedical Vibrational Spectroscopy VI: Advances in Research and Industry, 2014

ABSTRACT Acute myeloid leukemia (AML) is a proliferative neoplasm, that if not properly treated c... more ABSTRACT Acute myeloid leukemia (AML) is a proliferative neoplasm, that if not properly treated can rapidly cause a fatal outcome. The diagnosis of AML is challenging and the first diagnostic step is the count of the percentage of blasts (immature cells) in bone marrow and blood sample, and their morphological characterization. This evaluation is still performed manually with a bright field light microscope. Here we report results of a study applying Raman spectroscopy for analysis of samples from two patients affected by two AML subtypes characterized by a different maturation stage in the neutrophilic lineage. Ten representative cells per sample were selected and analyzed with high-resolution confocal Raman microscopy by scanning 64x64 (4096) points in a confocal layer through the volume of the whole cell. The average spectrum of each cell was then used to obtain a highly reproducible mean fingerprint of the two different AML subtypes. We demonstrate that Raman spectroscopy efficiently distinguishes these different AML subtypes. The molecular interpretation of the substantial differences between the subtypes is related to granulocytic enzymes (e.g. myeloperoxidase and cytochrome b558), in agreement with different stages of maturation of the two considered AML subtypes . These results are promising for the development of a new, objective, automated and label-free Raman based methods for the diagnosis and first assessment of AML.

The Analyst, 2015

In clinical practice, the diagnosis and classification of acute myeloid leukaemia (AML) and myelo... more In clinical practice, the diagnosis and classification of acute myeloid leukaemia (AML) and myelodysplastic syndrome (MDS) start from the manual examination of stained smears of bone marrow (BM) and peripheral blood (PB) by using an optical microscope. This step is subjective and scarcely reproducible. Therefore, the development of subjective and potentially automatable methods for the recognition of typical AML/MDS cells is necessary. Here we have used Raman spectroscopy for distinguishing myeloblasts, promyelocytes, abnormal promyelocytes and erhytroblasts, which have to be counted for a correct diagnosis and morphological classification of AML and MDS. BM samples from patients affected by four different AML subtypes, mostly characterized by the presence of the four subpopulations selected for this study, were analyzed. First, each cell was scanned by acquiring 4096 spectra, thus obtaining Raman images which demonstrate an accurate description of morphological features characteristic of each subpopulation. Raman imaging coupled with hierarchical cluster analysis permitted the automatic discrimination and localization of the nucleus, the cytoplasm, myeloperoxidase containing granules and haemoglobin.

In this article, we describe how it is possible to tune the size and the aspect ratio of gold nan... more In this article, we describe how it is possible to tune the size and the aspect ratio of gold nanorods obtained using a highly efficient protocol based on the use of hydroquinone as a reducing agent by varying the amounts of CTAB and silver ions present in the “seed-growth” solution. Our approach not only allows us to prepare nanorods with a four times increased Au3+ reduction yield, when compared with the commonly used protocol based on ascorbic acid, but also allows a remarkable reduction of 50–60 % of the amount of CTAB needed. In fact, according to our findings, the concentration of CTAB present in the seed-growth solution do not linearly influence the final aspect ratio of the obtained nanorods, and an optimal concentration range between 30 and 50 mM has been identified as the one that is able to generate particles with more elongated shapes. On the optimized protocol, the effect of the concentration of Ag+ ions in the seed-growth solution and the stability of the obtained particles has also been investigated.

In clinical practice, the diagnosis and classification of acute myeloid leukaemia (AML) and myelo... more In clinical practice, the diagnosis and classification of acute myeloid leukaemia (AML) and myelodysplastic syndrome (MDS) start from the manual examination of stained smears of bone marrow (BM) and peripheral blood (PB) by using an optical microscope. This step is subjective and scarcely reproducible. Therefore, the development of subjective and potentially automatable methods for the recognition of typical AML/MDS cells is necessary. Here we have used Raman spectroscopy for distinguishing myeloblasts, promyelocytes, abnormal promyelocytes and erhytroblasts, which have to be counted for a correct diagnosis and morphological classification of AML and MDS. BM samples from patients affected by four different AML subtypes, mostly characterized by the presence of the four subpopulations selected for this study, were analyzed. First, each cell was scanned by acquiring 4096 spectra, thus obtaining Raman images which demonstrate an accurate description of morphological features characteristic of each subpopulation. Raman imaging coupled with hierarchical cluster analysis permitted the automatic discrimination and localization of the nucleus, the cytoplasm, myeloperoxidase containing granules and haemoglobin.

ACS Nano, 2014

In our study, 2D nanopillar arrays with plasmonic crystal properties are optimized for surface-en... more In our study, 2D nanopillar arrays with plasmonic crystal properties are optimized for surface-enhanced Raman spectroscopy (SERS) application and tested in a biochemical assay for the simultaneous detection of multiple genetic leukemia biomarkers. The special fabrication process combining soft lithography and plasma deposition techniques allows tailoring of the structural and chemical parameters of the crystal surfaces. In this way, it has been possible to tune the plasmonic resonance spectral position close to the excitation wavelength of the monochromatic laser light source in order to maximize the enhancing properties of the substrate. Samples are characterized by scanning electron microscopy and reflectance measurements and tested for SERS activity using malachite green. Besides, as the developed substrate had been prepared on a simple glass slide, SERS detection from the support side is also demonstrated. The optimized substrate is functionalized with thiol-modified capture oligonucleotides, and concentration-dependent signal of the target nucleotide is detected in a sandwich assay with labeled gold nanoparticles. Gold nanoparticles functionalized with different DNA and various Raman reporters are applied in a microarray-based assay recognizing a disease biomarker (Wilms tumor gene) and housekeeping gene expressions in the same time on spatially separated microspots. The multiplexing performance of the SERS-based bioassay is illustrated by distinguishing Raman dyes based on their complex spectral fingerprints.

Colloidal Nanocrystals for Biomedical Applications VIII, 2013

ABSTRACT Surface Enhanced Raman Spectroscopy (SERS) is a popular method in bio-analytical chemist... more ABSTRACT Surface Enhanced Raman Spectroscopy (SERS) is a popular method in bio-analytical chemistry and a potentially powerful enabling technology for in vitro diagnostics. SERS combines the excellent chemical specificity of Raman spectroscopy with the good sensitivity provided by enhancement of the signal that is observed when a molecule is located on (or very close to) the surface of nanostructured metallic materials. Star-like gold nanoparticles (SGN) are a new class of multibranched nanoparticles that in the last few years have attracted the attention of SERS community for their plasmonic properties. In this work we present a new method to prepare star-like gold nanoparticles with a simple one step protocol at room temperature using hydroquinone as reducing agent. Besides we compare the enhancement of Raman signal of malachite green, a dye commonly employed as label in biological studies, by star-like gold nanoparticles having different size, directly in liquid. This study shows that SGN provide good enhancement of Raman signal and that the effect of their dimension is strongly dependent on the wavelength used. Moreover preliminary results suggest that SGN produced using this method are characterized by good physical-chemical properties and they can be functionalized using the standard thiol chemistry. Overall, these results suggest that star-like gold nanoparticles produced through this method could be used for the further development of highly specific and sensitive SERS-based bio-analytical tests.

Neuromelanins (NMs) are neuronal pigments of melanic-lipidic type which accumulate during aging. ... more Neuromelanins (NMs) are neuronal pigments of melanic-lipidic type which accumulate during aging. They are involved in protective and degenerative mechanisms depending on the cellular context, however their structures are still poorly understood. NMs from nine human brain areas were analyzed in detail. Elemental analysis led to identification of three types of NM, while infrared spectroscopy showed that NMs from neurons of substantia nigra and locus coeruleus, which selectively degenerate in Parkinson's disease, have similar structure but different from NMs from brain regions not targeted by the disease. Synthetic melanins containing Fe and bovine serum albumin were prepared to model the natural product and help clarifying the structure of NMs. Extensive nuclear magnetic resonance spectroscopy studies showed the presence of dolichols both in the soluble and insoluble parts of NM. Diffusion measurements demonstrated that the dimethyl sulfoxide soluble components consist of oligomeric precursors with MWs in the range 1.4-52 kDa, while the insoluble part contains polymers of larger size but with a similar composition. These data suggest that the selective vulnerability of neurons of substantia nigra and locus coeruleus in Parkinson's disease might depend on the structure of the pigment. Moreover, they allow to propose a pathway for NM biosynthesis in human brain. Citation: Engelen M, Vanna R, Bellei C, Zucca FA, Wakamatsu K, et al. (2012) Neuromelanins of Human Brain Have Soluble and Insoluble Components with Dolichols Attached to the Melanic Structure. PLoS ONE 7(11): e48490.

Molecular Pharmaceutics, 2014

Among polymeric nanoparticles designed for cancer therapy, PLGA nanoparticles have become one of ... more Among polymeric nanoparticles designed for cancer therapy, PLGA nanoparticles have become one of the most popular polymeric devices for chemotherapeutic-based nanoformulations against several kinds of malignant diseases. Promising properties, including long-circulation time, enhanced tumor localization, interference with "multidrug" resistance effects, and environmental biodegradability, often result in an improvement of the drug bioavailability and effectiveness. In the present work, we have synthesized 1,7-bis(3,4-dimethoxyphenyl)-5-hydroxyhepta-1,4,6-trien-3-one (ASC-J9) and developed uniform ASC-J9-loaded PLGA nanoparticles of about 120 nm, which have been prepared by a single-emulsion process. Structural and morphological features of the nanoformulation were analyzed, followed by an accurate evaluation of the in vitro drug release kinetics, which exhibited Fickian law diffusion over 10 days. The intracellular degradation of ASC-J9-bearing nanoparticles within estrogen-dependent MCF-7 breast cancer cells was correlated to a time- and dose-dependent activity of the released drug. A cellular growth inhibition associated with a specific cell cycle G2/M blocking effect caused by ASC-J9 release inside the cytosol allowed us to put forward a hypothesis on the action mechanism of this nanosystem, which led to the final cell apoptosis. Our study was accomplished using Annexin V-based cell death analysis, MTT assessment of proliferation, radical scavenging activity, and intracellular ROS evaluation. Moreover, the intracellular localization of nanoformulated ASC-J9 was confirmed by a Raman optical imaging experiment designed ad hoc. PLGA nanoparticles and ASC-J9 proved also to be safe for a healthy embryo fibroblast cell line (3T3-L1), suggesting a possible clinical translation of this potential nanochemotherapeutic to expand the inherently poor bioavailability of hydrophobic ASC-J9 that could be proposed for the treatment of malignant breast cancer.

Nanoscale Imaging, Sensing, and Actuation for Biomedical Applications XI, 2014

ABSTRACT Surface Enhanced Raman Spectroscopy is a powerful analytical technique that combines the... more ABSTRACT Surface Enhanced Raman Spectroscopy is a powerful analytical technique that combines the excellent chemical specificity of Raman spectroscopy with the good sensitivity provided by the enhancement of the signal observed when a molecule is located on (or very close to) the surface of suitable nanostructured metallic materials. The availability of cheap, reliable and easy to use SERS substrates would pave the road to the development of bioanalytical tests that can be used in clinical practice. SERS, in fact, is expected to provide not only higher sensitivity and specificity, but also the simultaneous and markedly improved detection of several targets at the same time with higher speed compared to the conventional analytical methods. Here, we present the SERS activity of 2-D plasmonic crystals made by polymeric pillars embedded in a gold matrix obtained through the combination of soft-lithography and plasma deposition techniques on a transparent substrates. The use of a transparent support material allowed us to perform SERS detection from support side opening the possibility to use these substrates in combination with microfluidic devices. In order to demonstrate the potentialities for bioanalytical applications, we used our SERS active gold surface to detect the oxidation product of apomorphine, a well-known drug molecule used in Parkinson’s disease which has been demonstrated being difficult to study by traditional HPLC based approaches.

BioNanoScience, 2014

We present here the surface-enhanced Raman spectroscopic (SERS)-based detection of the Wilm's tum... more We present here the surface-enhanced Raman spectroscopic (SERS)-based detection of the Wilm's tumor gene (WT1) sequence using dye-labeled reporter oligonucleotide and magnetic core @ gold shell nanoparticles. Thiolated single-stranded DNA (ssDNA) complementers of the WT1 sequence were used to functionalize the gold shell with capture oligonucleotides in a facile and fast two-step method. The signal amplification performance of the core @ shell colloidal SERS substrate was tested using malachite green as label dye. The Raman signal enhancing efficacy of the magnetic core @ gold shell nanomaterial was compared with the efficacy of spherical gold particles produced using the conventional citrate reduction method. The core @ shell particles were found to be superior both regarding robustness in SERS and facile separation in a heterogeneous reaction system. The core @ shell particles functionalized with target specific oligonucleotide were able to capture the WT1 target and worked as Raman signal amplifiers in our assay system. The good physicochemical characteristics of these particles and the sensitivity observed in SERS experiments allow us to expect good performance in the further development steps of a novel, fast and reliable spectroscopic method for WT1 detection in minimal residual disease patients.

Vibrational Spectroscopy, 2013

Paper-based SERS active substrates were prepared adsorbing spherical and star-shaped gold nanopar... more Paper-based SERS active substrates were prepared adsorbing spherical and star-shaped gold nanoparticles on a standard filter paper support. Besides the deposition conditions, morphological parameters of the particles were found to strongly affect the enhancer properties of the substrates. The developed substrate was tested regarding surface homogeneity as well as in the quantitative analysis of malachite green, -a well documented Raman reporter dye -and proved to be capable also to detect the oxidation products of apomorphine, a well-known drug molecule used in Parkinson's disease. This material is simple to prepare, easy to handle and dispose and as such it could be a perfect target for further development of a new family of mass-produced, cheap solid SERS substrates.