Publications by authors named "Roberto Pini"

66 Publications

Photobiomodulation of Human Fibroblasts and Keratinocytes with Blue Light: Implications in Wound Healing.

Biomedicines 2021 Jan 5;9(1). Epub 2021 Jan 5.

Istituto di Fisica Applicata "Nello Carrara", Consiglio Nazionale delle Ricerche (CNR-IFAC), 50019 Florence, Italy.

In recent years, photobiomodulation (PBM) has been recognized as a physical therapy in wound management. Despite several published research papers, the mechanism underlying photobiomodulation is still not completely understood. The investigation about application of blue light to improve wound healing is a relatively new research area. Tests in selected patients evidenced a stimulation of the healing process in superficial and chronic wounds treated with a blue LED light emitting at 420 nm; a study in animal model pointed out a faster healing process in superficial wound, with an important role of fibroblasts and myofibroblasts. Here, we present a study aiming at evidencing the effects of blue light on the proliferation and metabolism in fibroblasts from healthy skin and keratinocytes. Different light doses (3.43, 6.87, 13.7, 20.6, 30.9 and 41.2 J/cm2) were used to treat the cells, evidencing inhibitory and stimulatory effects following a biphasic dose behavior. Electrophysiology was used to investigate the effects on membrane currents: healthy fibroblasts and keratinocytes showed no significant differences between treated and not treated cells. Raman spectroscopy revealed the mitochondrial Cytochrome C (Cyt C) oxidase dependence on blue light irradiation: a significant decrease in peak intensity of healthy fibroblast was evidenced, while it is less pronounced in keratinocytes. In conclusion, we observed that the blue LED light can be used to modulate metabolism and proliferation of human fibroblasts, and the effects in wound healing are particularly evident when studying the fibroblasts and keratinocytes co-cultures.
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http://dx.doi.org/10.3390/biomedicines9010041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7824830PMC
January 2021

Photostability of Contrast Agents for Photoacoustics: The Case of Gold Nanorods.

Nanomaterials (Basel) 2021 Jan 6;11(1). Epub 2021 Jan 6.

Istituto di Fisica Applicata Nello Carrara, IFAC-CNR, Via Madonna del Piano 10, 50019 Sesto Fiorentino, Italy.

Plasmonic particles as gold nanorods have emerged as powerful contrast agents for critical applications as the photoacoustic imaging and photothermal ablation of cancer. However, their unique efficiency of photothermal conversion may turn into a practical disadvantage, and expose them to the risk of overheating and irreversible photodamage. Here, we outline the main ideas behind the technology of photoacoustic imaging and the use of relevant contrast agents, with a main focus on gold nanorods. We delve into the processes of premelting and reshaping of gold nanorods under illumination with optical pulses of a typical duration in the order of few ns, and we present different approaches to mitigate this issue. We undertake a retrospective classification of such approaches according to their underlying, often implicit, principles as: constraining the initial shape; or speeding up their thermal coupling to the environment by lowering their interfacial thermal resistance; or redistributing the input energy among more particles. We discuss advantages, disadvantages and contexts of practical interest where one solution may be more appropriate than the other.
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http://dx.doi.org/10.3390/nano11010116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7825532PMC
January 2021

Experimental Study on Blue Light Interaction with Human Keloid-Derived Fibroblasts.

Biomedicines 2020 Dec 6;8(12). Epub 2020 Dec 6.

Istituto di Fisica Applicata "Nello Carrara", Consiglio Nazionale delle Ricerche (CNR-IFAC), 50019 Florence, Italy.

Keloids are an exuberant response to wound healing, characterized by an exaggerated synthesis of collagen, probably due to the increase of fibroblasts activity and to the reduction of their apoptosis rate: currently no standard treatments or pharmacological therapies are able to prevent keloid recurrence. To reach this goal, in recent years some physical treatments have been proposed, and among them the PhotoBioModulation therapy (PBM). This work analyses the effects of a blue LED light irradiation (410-430 nm, 0.69 W/cm power density) on human fibroblasts, isolated from both keloids and perilesional tissues. Different light doses (3.43-6.87-13.7-20.6-30.9 and 41.2 J/cm) were tested. Biochemical assays and specific staining were used to assess cell metabolism, proliferation and viability. Micro-Raman spectroscopy was used to explore direct effects of the blue LED light on the Cytochrome C (Cyt C) oxidase. We also investigated the effects of the irradiation on ionic membrane currents by patch-clamp recordings. Our results showed that the blue LED light can modulate cell metabolism and proliferation, with a dose-dependent behavior and that these effects persist at least till 48 h after treatment. Furthermore, we demonstrated that the highest fluence value can reduce cell viability 24 h after irradiation in keloid-derived fibroblasts, while the same effect is observed 48 h after treatment in perilesional fibroblasts. Electrophysiological recordings showed that the medium dose (20.6 J/cm) of blue LED light induces an enhancement of voltage-dependent outward currents elicited by a depolarizing ramp protocol. Overall, these data demonstrate the potentials that PBM shows as an innovative and minimally-invasive approach in the management of hypertrophic scars and keloids, in association with current treatments.
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http://dx.doi.org/10.3390/biomedicines8120573DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7762279PMC
December 2020

Ficoll as testing material for diffusion weighted imaging-quality assurance phantoms.

Magn Reson Imaging 2021 02 6;76:1-7. Epub 2020 Nov 6.

Université d'Orléans and Centre de Biophysique Moléculaire (CBM), CNRS UPR 4301, rue Charles Sadron, 45071 Orléans, France.

Purpose: The aim of this work is to test the use of aqueous solutions of Ficoll®**, a highly branched polymer displaying crowding properties, to build a phantom suitable for Diffusion Weighted Imaging (DWI) in Magnetic Resonance Imaging (MRI).

Methods: We developed a test object made of a cylindrical plastic container with a precise geometrical arrangement suitable for measuring several samples at the same time. The container was designed to host single vials with variable geometry and number, and to fit inside common commercial head coils for MRI scanners. In our experiments, vials were filled with 8 aqueous solutions of Ficoll 70 and Ficoll 400 spanning a range of polymer concentration from 5 to 30% by weight. Vials containing ultra-pure water were also used as reference. Experiments were performed on both 1.5 and 3 T clinical scanners (GE, Philips and Siemens), under the conditions of a standard clinical examination.

Results: The geometry of the phantom provided reduced imaging artifacts, especially image distortions at magnetic interfaces. We found that the Apparent Diffusion Coefficient (ADC) varied in the range of 0.00125-0.00223 mm/s and decreased with Ficoll concentration. ADC vs Ficoll concentration exhibited a linear trend. Results were consistent over time and among different MRI clinical scanners, showing an average variability of 3% at 1.5 T and of 7.5% at 3 T. Moreover, no substantial difference was found between Ficoll 70 and 400. By varying Ficoll concentration, ADC can be modulated to approach tissue-mimicking values. Preliminary results for relaxation measurements proved that both T and T decreased with Ficoll concentration in the ranges 1.3-2.4 s and 150-800 ms respectively.

Conclusions: In this work, we propose a 3D phantom design based on the widespread crowding agent Ficoll, which is suitable for DWI quality assurance purposes in MRI acquisitions. Aqueous Ficoll solutions provide good performance in terms of stability, ease of preparation, and safety.
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http://dx.doi.org/10.1016/j.mri.2020.11.001DOI Listing
February 2021

A bionic shuttle carrying multi-modular particles and holding tumor-tropic features.

Mater Sci Eng C Mater Biol Appl 2020 Dec 7;117:111338. Epub 2020 Aug 7.

Istituto di Fisica Applicata "Nello Carrara", Consiglio Nazionale delle Ricerche, Via Madonna del Piano 10, 50019 Sesto Fiorentino, FI, Italy.

The systemic delivery of composite nanoparticles remains an outstanding challenge in cancer nanomedicine, and the principal reason is a complex interplay of biological barriers. In this regard, adaptive cell transfer may represent an alternative solution to circumvent these barriers down to the tumor microenvironment. Here, tumor-tropic macrophages are proposed as a tool to draw and vehiculate modular nanoparticles integrating magnetic and plasmonic components. The end result is a bionic shuttle that exhibits a plasmonic band within the so-called therapeutic window arising from as much as 40 pg Au per cell, magnetization in the order of 150 pemu per cell, and more than 90% of the pristine viability and chemotactic activity of its biological component, until at least two days of preparation. Its synergistic combination of plasmonic, magnetic and tumor-tropic functions is assessed in vitro for applications as magnetic guidance or sorting, with a propulsion around 4 μm s for a magnetic gradient of 0.8 T m, the optical hyperthermia of cancer, with stability of photothermal conversion to temperatures exceeding 50C, and the photoacoustic imaging of cancer under realistic conditions. These results collectively suggest that a bionic design may be a promising roadmap to reconcile the efforts for multifunctionality and targeted delivery, which are both key goals in nanomedicine.
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http://dx.doi.org/10.1016/j.msec.2020.111338DOI Listing
December 2020

Optically activated and interrogated plasmonic hydrogels for applications in wound healing.

J Biophotonics 2020 09 25;13(9):e202000135. Epub 2020 Jun 25.

Istituto di Fisica Applicata "Nello Carrara", Consiglio Nazionale delle Ricerche, Sesto Fiorentino, Florence, Italy.

We disclose the use of hybrid materials featuring Au/Ag core/shell nanorods in porous chitosan/polyvinyl alcohol scaffolds for applications in tissue engineering and wound healing. The combination of Au and Ag in a single construct provides synergistic opportunities for optical activation of functions as near infrared laser tissue bonding, and remote interrogation to return parameters of prognostic relevance in wound healing monitoring. In particular, the bimetallic component ensures optical tunability, enhanced shelf life and photothermal stability, serves as a reservoir of germicidal silver cations, and changes in near-infrared and visible color according to the environmental level of oxidative stress. At the same time, the polymeric blend is ideal to bind connective tissue upon photothermal activation, and to support fabrication processes that provide high porosity, such as electrospinning, thus putting all the premises for cellular repopulation and antimicrobial protection.
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http://dx.doi.org/10.1002/jbio.202000135DOI Listing
September 2020

Impact of Kapitza resistance on the stability and efficiency of photoacoustic conversion from gold nanorods.

J Colloid Interface Sci 2020 Oct 6;578:358-365. Epub 2020 Jun 6.

Istituto di Fisica Applicata Nello Carrara, IFAC-CNR, Via Madonna del Piano 10, Sesto Fiorentino 50019, Italy.

Plasmonic particles have been proposed for a broad variety of optical and hybrid applications, including the photothermal ablation and photoacoustic imaging of cancer, or their integration in photonic sensors. Here, we address the effect of thermal resistance at the gold-water interface, or Kapitza resistance, on the performance of photoacoustic conversion of gold nanorods. Our findings point to possible strategies for the optimization of plasmonic particles as contrast agents for imaging, or even as transducers for biosensing. We perform numerical simulations that project a simultaneous increase of efficiency and stability of photoacoustic conversion with a decrease of Kapitza resistance. We suggest an effective approach to modulate Kapitza resistance by including underresolved features as roughness or the presence of adsorbates. Inspired by this idea, we synthesize a rough variant of gold nanorods by the deposition and galvanic replacement of a silver shell, where roughness provides higher photoacoustic signals by about 70% and damage thresholds by 120%. In addition, we coat our particles with a protein corona and find a decrease of photoacoustic signals with shell thickness, which may inspire new solutions for biosensors based on a mechanism of photoacoustic transduction. Both our findings are consistent with an effective modulation of Kapitza resistance, which decreases upon roughening, due to an underlying increase of specific surface area, and increases upon coating with a protein shell that may act as a thermal insulation. We discuss possible directions to gain more advantage of our concept for topical applications at the crossroads of plasmonics, biomedical optics and biosensing.
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http://dx.doi.org/10.1016/j.jcis.2020.05.108DOI Listing
October 2020

Fiber-cap biosensors for SERS analysis of liquid samples.

J Mater Chem B 2020 02;8(8):1629-1639

European Laboratory for Non-Linear Spectroscopy (LENS), Via Nello Carrara 1, 50019, Sesto Fiorentino, Italy. and National Institute of Optics, National Research Council (INO-CNR), Via Nello Carrara 1, 50019 Sesto Fiorentino, Italy and Department of Physics, University of Florence, Via G. Sansone 1, 50019 Sesto Fiorentino, Italy.

Optical detection techniques based on surface enhanced Raman spectroscopy (SERS) are a powerful tool for biosensing applications. Meanwhile, due to technological advances, different approaches have been investigated to integrate SERS substrates on the tip of optical fibres for molecular probing in liquids. To further demonstrate the perspectives offered by SERS-on-fiber technology for diagnostic purposes, in this study, novel cap-shaped SERS sensors for reversible coupling with customized multimodal probes were prototyped via low-cost polymer casting of polydimethylsiloxane (PDMS) and further assembly of gold nanoparticles (Au NPs) of varied sizes and shapes. To demonstrate the feasibility of liquid sensing with cap sensors using backside illumination and detection, the spectra of rhodamine were acquired by coupling the caps with the fiber. As expected by UV-vis, the highest SERS efficiency was observed for NP-decorated substrates with plasmonic properties in resonance with the irradiation wavelength. Then, SERS biosensors for the specific detection of amyloid-β (Aβ) neurotoxic biomarkers were realized by covalent grafting of Aβ antibodies. As attested by fluorescence images and SERS measurements, the biosensors successfully exhibited enhanced Aβ affinity compared to the bare sensors without ligands. Finally, these versatile (bio)sensors are a powerful tool to transform any milli-sized fibers into functional (bio)sensing platforms with plasmonic and biochemical properties tailored for specific applications.
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http://dx.doi.org/10.1039/c9tb01866bDOI Listing
February 2020

TiO nanoparticles in a biosolid-amended soil and their implication in soil nutrients, microorganisms and Pisum sativum nutrition.

Ecotoxicol Environ Saf 2020 Mar 20;190:110095. Epub 2019 Dec 20.

Research Institute on Terrestrial Ecosystems (IRET-CNR), Via G. Moruzzi, 1, 56124, Pisa, Italy. Electronic address:

The wide use of nanoparticles (NPs), gives concern about their possible negative implications in the environment and living organisms. In particular, titanium dioxide (TiO) NPs are accumulated in biosolids (Bs) coming from wastewater treatment plants, which in turn are used as farm soil amendments and are becoming an important way of NPs entrance in the terrestrial ecosystems. In this study, to simulate a low and cumulative load of TiO NPs, 80 and 800 mg TiOper Kg of soil were spiked in the Bs prior to its addition to soil. The effects of different crystal phases of TiO NPs (pure anatase and pure rutile or their mixture) and their non-coated bulk counterparts (larger particles) on the availability of mineral nutrients and on the status of the bacterial communities together with the nutritional status of Pisum sativum L. plants were evaluated. Results showed the reduction, to different extents, on the availability of important soil mineral nutrients (e.g. Mn 65%, Fe 20%, P 27%, averagely), in some cases size- (e.g. P) and dose-dependent. Bacterial biodiversity was also affected by the presence of high TiO dose in soil. The mineral nutrition of pea plants was also altered, showing the main reduction in Mn (80% in the roots and 50% in the shoots), K, Zn, P (respectively, 80, 40, and 35% in the roots), and an increase of N in the shoots, with possible consequences on the quality of the crop. The present study gives new integrated data on the effects of TiO NPs in the soil-plant system, on the soil health and on the nutritional quality of crops, rising new implications for future policies and human health.
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http://dx.doi.org/10.1016/j.ecoenv.2019.110095DOI Listing
March 2020

Blue LED light modulates inflammatory infiltrate and improves the healing of superficial wounds.

Photodermatol Photoimmunol Photomed 2020 03 10;36(2):166-168. Epub 2019 Dec 10.

Institute of Applied Physics, National Research Council (IFAC-CNR), Sesto Fiorentino, Italy.

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http://dx.doi.org/10.1111/phpp.12527DOI Listing
March 2020

Hybrid organosilicon/polyol phantom for photoacoustic imaging.

Biomed Opt Express 2019 Aug 2;10(8):3719-3730. Epub 2019 Jul 2.

Istituto di Fisica Applicata 'Nello Carrara' IFAC-CNR, Via Madonna del Piano, 10, 50019 Sesto Fiorentino (FI), Italy.

The rapid development of hardware and software for photoacoustic technologies is urging the establishment of dedicated tools for standardization and performance assessment. In particular, the fabrication of anatomical phantoms for photoacoustic imaging remains an open question, as current solutions have not yet gained unanimous support. Here, we propose that a hybrid material made of a water-in-oil emulsion of glycerol and polydimethylsiloxane may represent a versatile platform to host a broad taxonomy of hydrophobic and hydrophilic dyes and recapitulate the optical and acoustic features of bio tissue. For a full optical parameterization, we refer to Wróbel, [ Biomed. Opt. Express7, 2088 (2016)], where this material was first presented for optical imaging. Instead, here, we complete the picture and find that its speed of sound and acoustic attenuation resemble those of pure polydimethylsiloxane, i.e. respectively 1150 ± 30 m/s and 3.5 ± 0.4 dB/(MHz·cm). We demonstrate its use under a commercial B-mode scanner and a home-made A-mode stage for photoacoustic analysis to retrieve the ground-truth encoded in a multilayer architecture containing indocyanine green, plasmonic particles and red blood cells. Finally, we verify the stability of its acoustic, optical and geometric features over a time span of three months.
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http://dx.doi.org/10.1364/BOE.10.003719DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6701555PMC
August 2019

1064-nm-resonant gold nanorods for photoacoustic theranostics within permissible exposure limits.

J Biophotonics 2019 10 2;12(10):e201900082. Epub 2019 Jun 2.

Istituto di Fisica Applicata 'Nello Carrara', Consiglio Nazionale delle Ricerche IFAC-CNR, Sesto Fiorentino, Italy.

Therapeutic and diagnostic methods based on photomechanical effects are attracting much current attention in contexts as oncology, cardiology and vascular surgery, for such applications as photoacoustic imaging or microsurgery. Their underlying mechanism is the generation of ultrasound or cavitation from the interaction of short optical pulses with endogenous dyes or targeted contrast agents. Among the latter, gold nanorods are outstanding candidates, but their use has mainly been reported for photoacoustic imaging and photothermal treatments. Conversely, much less is still known about their value as a precision tool for photomechanical manipulations, such as to impart local damage with high spatial resolution through the expansion and collapse of microbubbles. Here, we address the feasibility of gold nanorods exhibiting a distribution of surface plasmon resonances between about 900 to above 1100 nm as a contrast agent for photoacoustic theranostics. After testing their cytotoxicity and cellular uptake, we discuss their photostability and use to mediate cavitation and the photomechanical destruction of targeted cells. We find that the choice of a plasmonic band peaking around 1064 nm is key to enhance the translational potential of this approach. With respect to the standard alternative of 800 nm, at 1064 nm, relevant regulations on optical exposure are less restrictive and the photonic technology is more mature.
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http://dx.doi.org/10.1002/jbio.201900082DOI Listing
October 2019

In situ diode laser fenestration: An ex-vivo evaluation of irradiation effects on human aortic tissue.

J Biophotonics 2019 09 30;12(9):e201900032. Epub 2019 Jun 30.

Institute of Applied Physics Nello Carrara, National Research Council, Florence, Italy.

The in situ laser fenestration is an interesting option for the endovascular treatment of short-necked aneurysms with an intraoperative modification of a standard endograft. According to literature evidence, diode laser emitting in the near-infrared wavelength (810 nm) can be successfully used to fenestrate the endograft fabric. This paper describes a three-dimensional navigation system for the accurate targeting of the fenestration site, then reports results of an ex vivo study to assess whether the laser operative conditions, which ensure the fabric fenestration, are harmless for the biological tissue surrounding the endoprosthesis. Two hundred twenty-five samples of human aorta, including healthy specimens and abdominal aortic aneurysm samples, were irradiated ex vivo using a 810 nm diode laser. Energy and pulse duration were varied. Irradiated tissues were fixed in formaldehyde, sectioned and subjected to histological examination. Only 7.5% of the irradiated samples exhibited a thermal damage, which was always confined to the contact point between the laser fiber tip and the aortic wall. These experiments suggest that the diode laser can be safely used for the proposed surgical application.
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http://dx.doi.org/10.1002/jbio.201900032DOI Listing
September 2019

Morpho-mechanics of human collagen superstructures revealed by all-optical correlative micro-spectroscopies.

Commun Biol 2019 26;2:117. Epub 2019 Mar 26.

1National Institute of Optics, National Research Council (CNR-INO), Via Nello Carrara 1, I-50019 Sesto Fiorentino, Italy.

In every biological tissue, morphological and topological properties strongly affect its mechanical features and behaviour, so that ultrastructure, composition and mechanical parameters are intimately connected. Overall, it is their correct interplay that guarantees the tissue functionality. The development of experimental methods able to correlate these properties would open new opportunities both in the biological and the biomedical fields. Here, we report a correlative study intended to map supramolecular morphology, biochemical composition and viscoelastic parameters of collagen by all-optical microscopies. In particular, using human corneal tissue as a benchmark, we correlate Second-Harmonic Generation maps with mechanical and biochemical imaging obtained by Brillouin and Raman micro-spectroscopy. The study highlights how subtle variations in supramolecular organization originate the peculiar mechanical behavior of different subtypes of corneal lamellae. The presented methodology paves the way to the non-invasive assessment of tissue morpho-mechanics in biological as well as synthetic materials.
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http://dx.doi.org/10.1038/s42003-019-0357-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6435656PMC
May 2020

Anvil-profiled penetrating keratoplasty: load resistance evaluation.

Biomech Model Mechanobiol 2019 Apr 31;18(2):319-325. Epub 2018 Oct 31.

Istituto Nazionale di Ottica, Consiglio Nazionale delle Ricerche, Via G. Moruzzi 1, 56124, Pisa, Italy.

The aim of this study was to qualitatively evaluate the biomechanical load resistance of different surgical wound configurations (mushroom, zig-zag, anvil and conventional trephination) in penetrating keratoplasty (PK) by designing a 2D and a 3D finite-element biomechanical model of the cornea. A mathematical model of the human cornea was developed, and different geometric configurations for PK were designed. The internal pressure was raised until the wound misaligned; wound prolapse then occurred. Better wound resistance was found in all the laser trephined profiles tested in comparison with the conventional straight one. The anvil profile was more resistant to the increasing internal pressure than was the mushroom or the zig-zag pattern. Thanks to its greater mechanical load resistance, the anvil profile made possible the apposition of a restricted number of sutures and early suture removal. These advantages can contribute to a faster visual recovery in patients undergoing penetrating keratoplasty.
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http://dx.doi.org/10.1007/s10237-018-1083-yDOI Listing
April 2019

Nanoscale Discrimination between Toxic and Nontoxic Protein Misfolded Oligomers with Tip-Enhanced Raman Spectroscopy.

Small 2018 09 9;14(36):e1800890. Epub 2018 Aug 9.

IFAC-CNR, Institute of Applied Physics "Nello Carrara,", National Research Council, Via Madonna del Piano 10, I-50019, Sesto Fiorentino, Italy.

Highly toxic protein misfolded oligomers associated with neurological disorders such as Alzheimer's and Parkinson's diseases are nowadays considered primarily responsible for promoting synaptic failure and neuronal death. Unraveling the relationship between structure and neurotoxicity of protein oligomers appears pivotal in understanding the causes of the pathological process, as well as in designing novel diagnostic and therapeutic strategies tuned toward the earliest and presymptomatic stages of the disease. Here, it is benefited from tip-enhanced Raman spectroscopy (TERS) as a surface-sensitive tool with spatial resolution on the nanoscale, to inspect the spatial organization and surface character of individual protein oligomers from two samples formed by the same polypeptide sequence and different toxicity levels. TERS provides direct assignment of specific amino acid residues that are exposed to a large extent on the surface of toxic species and buried in nontoxic oligomers. These residues, thanks to their outward disposition, might represent structural factors driving the pathogenic behavior exhibited by protein misfolded oligomers, including affecting cell membrane integrity and specific signaling pathways in neurodegenerative conditions.
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http://dx.doi.org/10.1002/smll.201800890DOI Listing
September 2018

Culture and horticulture: Protecting soil quality in urban gardening.

Sci Total Environ 2018 Dec 3;644:45-51. Epub 2018 Jul 3.

CNR, Institute of Ecosystem Studies, Via Moruzzi 1, 56124 Pisa, Italy. Electronic address:

Urban cultivation for food production is of growing importance. The quality of urban soil can be improved by tillage and the incorporation of organic matter, or can be degraded by chemical treatments. Urban gardeners have a role in this process, through the selection of various cultivation techniques. Our study focuses on an allotment area in the town of Pisa (Italy), which since 1995 has been run as a municipal vegetable garden by the residents. We analysed the soil and compared the data with those collected five years previously, to verify the possible changes in soil properties and fertility. We also interviewed the gardeners regarding their backgrounds, motivations and cultivation practices. We looked for possible changes in the soil quality attributable to the cultivation techniques. We found that the allotment holders influenced the soil quality through the cultivation techniques. Organic carbon, electrical conductivity and the content of copper increased unevenly in relation to the gardeners' cultivation practices. At the same time the study highlights that the urban gardeners were not completely aware of how to protect and enhance the fertility and the quality of urban soil. We believe that town councils should be responsible for providing correct information to the allotment holders and thus prevent the possible misuse of urban soil to grow food, as this can affect everyone's health.
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http://dx.doi.org/10.1016/j.scitotenv.2018.06.289DOI Listing
December 2018

Polylysine as a functional biopolymer to couple gold nanorods to tumor-tropic cells.

J Nanobiotechnology 2018 May 31;16(1):50. Epub 2018 May 31.

Institute of Applied Physics 'N. Carrara', National Research Council of Italy, Via Madonna del Piano, 10, 50019, Sesto Fiorentino, Italy.

Background: The delivery of plasmonic particles, such as gold nanorods, to the tumor microenvironment has attracted much interest in biomedical optics for topical applications as the photoacoustic imaging and photothermal ablation of cancer. However, the systemic injection of free particles still crashes into a complexity of biological barriers, such as the reticuloendothelial system, that prevent their efficient biodistribution. In this context, the notion to exploit the inherent features of tumor-tropic cells for the creation of a Trojan horse is emerging as a plausible alternative.

Results: We report on a convenient approach to load cationic gold nanorods into murine macrophages that exhibit chemotactic sensitivity to track gradients of inflammatory stimuli. In particular, we compare a new model of poly-L-lysine-coated particles against two alternatives of cationic moieties that we have presented elsewhere, i.e. a small quaternary ammonium compound and an arginine-rich cell-penetrating peptide. Murine macrophages that are exposed to poly-L-lysine-coated gold nanorods at a dosage of 400 µM Au for 24 h undertake efficient uptake, i.e. around 3 pg Au per cell, retain the majority of their cargo until 24 h post-treatment and maintain around 90% of their pristine viability, chemotactic and pro-inflammatory functions.

Conclusions: With respect to previous models of cationic coatings, poly-L-lysine is a competitive solution for the preparation of biological vehicles of gold nanorods, especially for applications that may require longer life span of the Trojan horse, say in the order of 24 h. This biopolymer combines the cost-effectiveness of small molecules and biocompatibility and efficiency of natural peptides and thus holds potential for translational developments.
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http://dx.doi.org/10.1186/s12951-018-0377-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5984317PMC
May 2018

Triggering molecular assembly at the mesoscale for advanced Raman detection of proteins in liquid.

Sci Rep 2018 01 18;8(1):1033. Epub 2018 Jan 18.

Institute of Applied Physics 'Nello Carrara', National Research Council (IFAC-CNR), via Madonna del Piano 10, Sesto Fiorentino, Italy.

An advanced optofluidic system for protein detection based on Raman signal amplification via dewetting and molecular gathering within temporary mesoscale assemblies is presented. The evaporation of a microliter volume of protein solution deposited in a circular microwell precisely follows an outward-receding geometry. Herein the combination of liquid withdrawal with intermolecular interactions induces the formation of self-assembled molecular domains at the solid-liquid interface. Through proper control of the evaporation rate, amplitude of the assemblies and time for spectral collection at the liquid edge are extensively raised, resulting in a local enhancement and refinement of the Raman response, respectively. Further signal amplification is obtained by taking advantage of the intense local electromagnetic fields generated upon adding a plasmonic coating to the microwell. Major advantages of this optofluidic method lie in the obtainment of high-quality, high-sensitivity Raman spectra with detection limit down to sub-micromolar values. Peculiarly, the assembled proteins in the liquid edge region maintain their native-like state without displaying spectral changes usually occurring when dried drop deposits are considered.
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http://dx.doi.org/10.1038/s41598-018-19558-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5773671PMC
January 2018

A multifunctional organosilica cross-linker for the bio-conjugation of gold nanorods.

Colloids Surf B Biointerfaces 2017 Sep 31;157:174-181. Epub 2017 May 31.

Institute of Applied Physics, National Research Council of Italy, Sesto Fiorentino, Italy.

We report on the use of organosilica shells to couple gold nanorods to functional peptides and modulate their physiochemical and biological profiles. In particular, we focus on the case of cell penetrating peptides, which are used to load tumor-tropic macrophages and implement an innovative drug delivery system for photothermal and photoacoustic applications. The presence of organosilica exerts subtle effects on multiple parameters of the particles, including their size, shape, electrokinetic potential, photostability, kinetics of endocytic uptake and cytotoxicity, which are investigated by the interplay of colorimetric methods and digital holographic microscopy. As a rule of thumb, as the thickness of organosilica increases from none to ∼30nm, we find an improvement of the photophysical performances at the expense of a deterioration of the biological parameters. Therefore, detailed engineering of the particles for a certain application will require a careful trade-off between photophysical and biological specifications.
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http://dx.doi.org/10.1016/j.colsurfb.2017.05.068DOI Listing
September 2017

Site-Selective Surface-Enhanced Raman Detection of Proteins.

ACS Nano 2017 01 20;11(1):918-926. Epub 2016 Dec 20.

Institute of Applied Physics "Nello Carrara", National Research Council , via Madonna del Piano 10, 50019 Sesto Fiorentino, Italy.

Strategies for protein detection via surface-enhanced Raman spectroscopy (SERS) currently exploit the formation of randomly generated hot spots at the interfaces of metal colloidal nanoparticles, which are clustered together by intrusive chemical or physical processes in the presence of the target biomolecule. We propose a different approach based on selective and quantitative gathering of protein molecules at regular hot spots generated on the corners of individual silver nanocubes in aqueous medium at physiological pH. Here, the protein, while keeping its native configuration, experiences an intense local E-field, which boosts SERS efficiency and detection sensitivity. Uncontrolled signal fluctuations caused by variable molecular adsorption to different particle areas or inside clustered nanoparticles are circumvented. Advanced electron microscopy analyses and computational simulations outline a strategy relying on a site-selective mechanism with superior Raman signal enhancement, which offers the perspective of highly controlled and reproducible routine SERS detection of proteins.
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http://dx.doi.org/10.1021/acsnano.6b07523DOI Listing
January 2017

Three-dimensional mapping of the orientation of collagen corneal lamellae in healthy and keratoconic human corneas using SHG microscopy.

J Biophotonics 2017 01 29;10(1):75-83. Epub 2016 Jul 29.

National Institute of Optics, National Research Council (INO-CNR), Via Nello Carrara 1, 50019, Sesto Fiorentino, Italy.

Keratoconus is an eye disorder that causes the cornea to take an abnormal conical shape, thus impairing its refractive functions and causing blindness. The late diagnosis of keratoconus is among the principal reasons for corneal surgical transplantation. This pathology is characterized by a reduced corneal stiffness in the region immediately below Bowman's membrane, probably due to a different lamellar organization, as suggested by previous studies. Here, the lamellar organization in this corneal region is characterized in three dimensions by means of second-harmonic generation (SHG) microscopy. In particular, a method based on a three-dimensional correlation analysis allows to probe the orientation of sutural lamellae close to the Bowman's membrane, finding statistical differences between healthy and keratoconic samples. This method is demonstrated also in combination with an epi-detection scheme, paving the way for a potential clinical ophthalmic application of SHG microscopy for the early diagnosis of keratoconus. SHG image acquired with sagittal optical sectioning (A) of a healthy cornea and (B) of a keratoconic cornea. Scale bars: 30 μm.
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http://dx.doi.org/10.1002/jbio.201600122DOI Listing
January 2017

Statistical detection of nanoparticles in cells by darkfield microscopy.

Phys Med 2016 Jul 2;32(7):938-43. Epub 2016 Jul 2.

Department of Experimental and Clinical Biomedical Sciences "Mario Serio", University of Florence, viale Pieraccini 6, I-50139 Florence, Italy.

In the fields of nanomedicine, biophotonics and radiation therapy, nanoparticle (NP) detection in cell models often represents a fundamental step for many in vivo studies. One common question is whether NPs have or have not interacted with cells. In this context, we propose an imaging based technique to detect the presence of NPs in eukaryotic cells. Darkfield images of cell cultures at low magnification (10×) are acquired in different spectral ranges and recombined so as to enhance the contrast due to the presence of NPs. Image analysis is applied to extract cell-based parameters (i.e. mean intensity), which are further analyzed by statistical tests (Student's t-test, permutation test) in order to obtain a robust detection method. By means of a statistical sample size analysis, the sensitivity of the whole methodology is quantified in terms of the minimum cell number that is needed to identify the presence of NPs. The method is presented in the case of HeLa cells incubated with gold nanorods labeled with anti-CA125 antibodies, which exploits the overexpression of CA125 in ovarian cancers. Control cases are considered as well, including PEG-coated NPs and HeLa cells without NPs.
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http://dx.doi.org/10.1016/j.ejmp.2016.06.007DOI Listing
July 2016

Preparation and Photoacoustic Analysis of Cellular Vehicles Containing Gold Nanorods.

J Vis Exp 2016 05 2(111). Epub 2016 May 2.

Institute of Applied Physics, Italian National Research Council.

Gold nanorods are attractive for a range of biomedical applications, such as the photothermal ablation and the photoacoustic imaging of cancer, thanks to their intense optical absorbance in the near-infrared window, low cytotoxicity and potential to home into tumors. However, their delivery to tumors still remains an issue. An innovative approach consists of the exploitation of the tropism of tumor-associated macrophages that may be loaded with gold nanorods in vitro. Here, we describe the preparation and the photoacoustic inspection of cellular vehicles containing gold nanorods. PEGylated gold nanorods are modified with quaternary ammonium compounds, in order to achieve a cationic profile. On contact with murine macrophages in ordinary Petri dishes, these particles are found to undergo massive uptake into endocytic vesicles. Then these cells are embedded in biopolymeric hydrogels, which are used to verify that the stability of photoacoustic conversion of the particles is retained in their inclusion into cellular vehicles. We are confident that these results may provide new inspiration for the development of novel strategies to deliver plasmonic particles to tumors.
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http://dx.doi.org/10.3791/53328DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4942024PMC
May 2016

Controlled graphene oxide assembly on silver nanocube monolayers for SERS detection: dependence on nanocube packing procedure.

Beilstein J Nanotechnol 2016 6;7:9-21. Epub 2016 Jan 6.

Department of Chemistry and CSGI, University of Florence, Via della Lastruccia 3-13, I-50019 Sesto Fiorentino, Italy.

Hybrid graphene oxide/silver nanocubes (GO/AgNCs) arrays for surface-enhanced Raman spectroscopy (SERS) applications were prepared by means of two procedures differing for the method used in the assembly of the silver nanocubes onto the surface: Langmuir-Blodgett (LB) transfer and direct sequential physisorption of silver nanocubes (AgNCs). Adsorption of graphene oxide (GO) flakes on the AgNC assemblies obtained with both procedures was monitored by quartz crystal microbalance (QCM) technique as a function of GO bulk concentration. The experiment provided values of the adsorbed GO mass on the AgNC array and the GO saturation limit as well as the thickness and the viscoelastic properties of the GO film. Atomic force microscopy (AFM) measurements of the resulting samples revealed that a similar surface coverage was achieved with both procedures but with a different distribution of silver nanoparticles. In the GO covered LB film, the AgNC distribution is characterized by densely packed regions alternating with empty surface areas. On the other hand, AgNCs are more homogeneously dispersed over the entire sensor surface when the nanocubes spontaneously adsorb from solution. In this case, the assembly results in less-packed silver nanostructures with higher inter-cube distance. For the two assembled substrates, AFM of silver nanocubes layers fully covered with GO revealed the presence of a homogeneous, flexible and smooth GO sheet folding over the silver nanocubes and extending onto the bare surface. Preliminary SERS experiments on adenine showed a higher SERS enhancement factor for GO on Langmuir-Blodgett films of AgNCs with respect to bare AgNC systems. Conversely, poor SERS enhancement for adenine resulted for GO-covered AgNCs obtained by spontaneous adsorption. This indicated that the assembly and packing of AgNCs obtained in this way, although more homogeneous over the substrate surface, is not as effective for SERS analysis.
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http://dx.doi.org/10.3762/bjnano.7.2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4734306PMC
February 2016

One-pot synthesis of magnesium nanoparticles embedded in a chitosan microparticle matrix: a highly biocompatible tool for in vivo cancer treatment.

J Mater Chem B 2016 Jan 15;4(2):207-211. Epub 2015 Dec 15.

Division of Surgical Oncology, Department of Surgery, University of Louisville, Louisville, KY 40202, USA.

A novel highly biocompatible nanosystem made up of a chitosan matrix and filled with magnesium nanoparticles was synthesized using a simple and one-pot strategy, and tested as a promising, well-tolerated tool for photothermal therapy. Moreover, in vivo a proof of concept on hepatocarcinoma-bearing mice is presented.
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http://dx.doi.org/10.1039/c5tb02499dDOI Listing
January 2016

Observation of an improved healing process in superficial skin wounds after irradiation with a blue-LED haemostatic device.

J Biophotonics 2016 06 12;9(6):645-55. Epub 2016 Jan 12.

National Institute of Optics, National Research Council (INO-CNR), Florence, 50125, Italy.

The healing process of superficial skin wounds treated with a blue-LED haemostatic device is studied. Four mechanical abrasions are produced on the back of 10 Sprague Dawley rats: two are treated with the blue-LED device, while the other two are left to naturally recover. Visual observations, non-linear microscopic imaging, as well as histology and immunofluorescence analyses are performed 8 days after the treatment, demonstrating no adverse reactions neither thermal damages in both abraded areas and surrounding tissue. A faster healing process and a better-recovered skin morphology are observed: the treated wounds show a reduced inflammatory response and a higher collagen content. Blue LED induced photothermal effect on superficial abrasions.
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http://dx.doi.org/10.1002/jbio.201500191DOI Listing
June 2016

Controlled Veiling of Silver Nanocubes with Graphene Oxide for Improved Surface-Enhanced Raman Scattering Detection.

ACS Appl Mater Interfaces 2016 Feb 21;8(4):2628-34. Epub 2016 Jan 21.

Institute of Applied Physics , National Research Council, via Madonna del Piano 10, I-50019 Sesto Fiorentino, Italy.

Hybrid graphene oxide (GO)/metal nanocomposites have been recently proposed as novel surface-enhanced Raman scattering (SERS) substrates. Despite an increasing interest in these systems, standardization in their fabrication process is still lacking but urgently required to support their use for real-life applications. In this work we investigate how the assembly of GO should be conducted to control adsorption geometry and optical properties at the interface with plasmonic nanostructures as monolayer assemblies of silver nanocubes, by tuning main experimental parameters including GO concentration and self-assembly time. We finally identified the experimental conditions for building up a close-fitting soft dressing of the plasmonic surface, which shows optimal characteristics for flexible and reliable SERS detection.
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http://dx.doi.org/10.1021/acsami.5b10438DOI Listing
February 2016

ESPRESSO: A novel device for laser-assisted surgery of the anterior eye segment.

Minim Invasive Ther Allied Technol 2016 1;25(2):70-8. Epub 2015 Oct 1.

a The BioRobotics Institute, Scuola Superiore Sant'Anna , Pisa , Italy.

Introduction: Merging robotics with laser eye surgery could enhance precision, repeatability and automation. During some eye laser procedures the patient is awake, thus eye stabilization is desired to avoid movements that could affect the treatment.

Material And Methods: The ESPRESSO platform has a two-stage actuation system to position a stabilization tool on the eye, a proximity sensing unit to monitor the stabilization tool position, and a sensing unit to monitor the pressure exerted on the eye. The platform is tested in-vitro and ex-vivo with clinicians. A maximum pressure to be exerted on the eye is defined with expert ophthalmic surgeons to be 22 mmHg: physiological intraocular pressure (IOP) range is 10-21 mmHg. This pressure corresponds to a force of 0.3 N.

Results: The necessary contact force to have eye fixation (according to the clinicians' feedback) is evaluated: maximum values resulted always below 0.3 N. A maximum IOP increase of 4.67 mmHg is observed, that is a slight variation with respect to the performance of other platforms (IOP elevations up to 328 mmHg).

Conclusion: Design and initial assessment of the platform is presented. Eye stabilization is performed without exceeding the critical contact force value and causing large/sudden IOP increases.
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http://dx.doi.org/10.3109/13645706.2015.1092450DOI Listing
January 2017

An "All-laser" Endothelial Transplant.

J Vis Exp 2015 Jul 6(101):e52939. Epub 2015 Jul 6.

U.O. Oculistica, Nuovo Ospedale Santo Stefano.

The "all laser" assisted endothelial keratoplasty is a procedure that is performed with a femtosecond laser used to cut the donor tissue at an intended depth, and a near infrared diode laser to weld the corneal tissue. The proposed technique enables to reach the three main goals in endothelial keratoplasty: a precise control in the thickness of the donor tissue; its easy insertion in the recipient bed and a reduced risk of donor lenticule dislocation. The donor cornea thickness is measured in the surgery room with optical coherence tomography (OCT), in order to correctly design the donor tissue dimensions. A femtosecond laser is used to cut the donor cornea. The recipient eye is prepared by manual stripping of the descemetic membrane. The donor endothelium is inserted into a Busin-injector, the peripheral inner side is stained with a proper chromophore (a water solution of Indocyanine Green) and then it is pulled in the anterior chamber. The transplanted tissue is placed in the final and correct location and then diode laser welding is induced from outside the eyeball. The procedure has been performed on more than 15 patients evidencing an improvement in surgery performances, with a good recovery of visual acuity and a reduced donor lenticule dislocation event.
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http://dx.doi.org/10.3791/52939DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4545165PMC
July 2015