Publications by authors named "Fulvio Ratto"

38 Publications

Enhanced Antitumoral Activity and Photoacoustic Imaging Properties of AuNP-Enriched Endothelial Colony Forming Cells on Melanoma.

Adv Sci (Weinh) 2021 Feb 21;8(4):2001175. Epub 2020 Dec 21.

Department of Experimental and Clinical Biomedical Sciences University of Florence Florence 50134 Italy.

Near infrared (NIR)-resonant gold nanoparticles (AuNPs) hold great promise in cancer diagnostics and treatment. However, translating the theranostic potential of AuNPs into clinical applications still remains a challenge due to the difficulty to improve the efficiency and specificity of tumor delivery in vivo as well as the clearance from liver and spleen to avoid off target toxicity. In this study, endothelial colony forming cells (ECFCs) are exploited as vehicles to deliver AuNPs to tumors. It is first demonstrated that ECFCs display a great capability to intake AuNPs without losing viability, and exert antitumor activity per se. Using a human melanoma xenograft mouse model, it is next demonstrated that AuNP-loaded ECFCs retain their capacity to migrate to tumor sites in vivo 1 day after injection and stay in the tumor mass for more than 1 week. In addition, it is demonstrated that ECFC-loaded AuNPs are efficiently cleared by the liver over time and do not elicit any sign of damage to healthy tissue.
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http://dx.doi.org/10.1002/advs.202001175DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7887578PMC
February 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

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

Microbubble Resonators for All-Optical Photoacoustics of Flowing Contrast Agents.

Sensors (Basel) 2020 Mar 18;20(6). Epub 2020 Mar 18.

CNR-IFAC, Istituto di Fisica Applicata "Nello Carrara", Consiglio Nazionale delle Ricerche, via Madonna del Piano 10, I50019 Sesto Fiorentino (FI), Italy.

In this paper, we implement a Whispering Gallery mode microbubble resonator (MBR) as an optical transducer to detect the photoacoustic (PA) signal generated by plasmonic nanoparticles. We simulate a flow cytometry experiment by letting the nanoparticles run through the MBR during measurements and we estimate PA intensity by a Fourier analysis of the read-out signal. This method exploits the peaks associated with the MBR mechanical eigenmodes, allowing the PA response of the nanoparticles to be decoupled from the noise associated with the particle flow whilst also increasing the signal-to-noise ratio. The photostability curve of a known contrast agent is correctly reconstructed, validating the proposed analysis and proving quantitative PA detection. The experiment was run to demonstrate the feasible implementation of the MBR system in a flow cytometry application (e.g., the detection of venous thrombi or circulating tumor cells), particularly regarding wearable appliances. Indeed, these devices could also benefit from other MBR features, such as the extreme compactness, the direct implementation in a microfluidic circuit, and the absence of impedance-matching material.
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http://dx.doi.org/10.3390/s20061696DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7175143PMC
March 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

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

Parametrical Optomechanical Oscillations in PhoXonic Whispering Gallery Mode Resonators.

Sci Rep 2019 May 9;9(1):7163. Epub 2019 May 9.

CNR-IFAC Institute of Applied Physics "N. Carrara", 50019, Sesto Fiorentino, Italy.

We report on the experimental and theoretical analysis of parametrical optomechanical oscillations in hollow spherical phoxonic whispering gallery mode resonators due to radiation pressure. The optically excited acoustic eigenmodes of the phoxonic cavity oscillate regeneratively leading to parametric oscillation instabilities.
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http://dx.doi.org/10.1038/s41598-019-43271-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6509163PMC
May 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

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

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

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

Organosilicon phantom for photoacoustic imaging.

J Biomed Opt 2015 Apr;20(4):46008

Istituto di Fisiologia Clinica, CNR, Via Giuseppe Moruzzi 1, Pisa 56124, ItalygFondazione CNR/Regione Toscana G. Monasterio, Via Giuseppe Moruzzi 1, Pisa 56124, Italy.

Photoacoustic imaging is an emerging technique. Although commercially available photoacoustic imaging systems currently exist, the technology is still in its infancy. Therefore, the design of stable phantoms is essential to achieve semiquantitative evaluation of the performance of a photoacoustic system and can help optimize the properties of contrast agents. We designed and developed a polydimethylsiloxane (PDMS) phantom with exceptionally fine geometry; the phantom was tested using photoacoustic experiments loaded with the standard indocyanine green dye and compared to an agar phantom pattern through polyethylene glycol-gold nanorods. The linearity of the photoacoustic signal with the nanoparticle number was assessed. The signal-tonoiseratio and contrast were employed as image quality parameters, and enhancements of up to 50 and up to 300%, respectively, were measured with the PDMS phantom with respect to the agar one. A tissue-mimicking (TM)-PDMS was prepared by adding TiO2 and India ink; photoacoustic tests were performed in order to compare the signal generated by the TM-PDMS and the biological tissue. The PDMS phantom can become a particularly promising tool in the field of photoacoustics for the evaluation of the performance of a PA system and as a model of the structure of vascularized soft tissues.
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http://dx.doi.org/10.1117/1.JBO.20.4.046008DOI Listing
April 2015

In vitro assessment of antibody-conjugated gold nanorods for systemic injections.

J Nanobiotechnology 2014 Dec 5;12:55. Epub 2014 Dec 5.

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

Background: The interest for gold nanorods in biomedical optics is driven by their intense absorbance of near infrared light, their biocompatibility and their potential to reach tumors after systemic administration. Examples of applications include the photoacoustic imaging and the photothermal ablation of cancer. In spite of great current efforts, the selective delivery of gold nanorods to tumors through the bloodstream remains a formidable challenge. Their bio-conjugation with targeting units, and in particular with antibodies, is perceived as a hopeful solution, but the complexity of living organisms complicates the identification of possible obstacles along the way to tumors.

Results: Here, we present a new model of gold nanorods conjugated with anti-cancer antigen 125 (CA125) antibodies, which exhibit high specificity for ovarian cancer cells. We implement a battery of tests in vitro, in order to simulate major nuisances and predict the feasibility of these particles for intravenous injections. We show that parameters like the competition of free CA125 in the bloodstream, which could saturate the probe before arriving at the tumors, the matrix effect and the interference with erythrocytes and phagocytes are uncritical.

Conclusions: Although some deterioration is detectable, anti-CA125-conjugated gold nanorods retain their functional features after interaction with blood tissue and so represent a powerful candidate to hit ovarian cancer cells.
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http://dx.doi.org/10.1186/s12951-014-0055-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4266900PMC
December 2014

Size dependent biological profiles of PEGylated gold nanorods.

J Mater Chem B 2014 Sep 7;2(36):6072-6080. Epub 2014 Aug 7.

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

The perspective of introducing plasmonic particles for applications in biomedical optics is receiving much interest. However, their translation into clinical practices is delayed by various factors, which include a poor definition of their biological interactions. Here, we describe the preparation and the biological profiles of gold nanorods belonging to five different size classes with average effective radii between ∼5 and 20 nm and coated with polyethylene glycol (PEG). All these particles exhibit decent stability in the presence of representative proteins, low cytotoxicity and satisfactory compatibility with intravenous administration, in terms of their interference with blood tissue. However, the suspension begins to become unstable after a few days of exposure to blood proteins. Moreover, the cytotoxicity is a little worse for smaller particles, probably because their purification is more critical, while undesirable interactions with the mononuclear phagocyte system are minimal in the intermediate size range. Overall, these findings hold implications of practical relevance and suggest that PEGylated gold nanorods may be a versatile platform for a variety of biomedical applications.
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http://dx.doi.org/10.1039/c4tb00991fDOI Listing
September 2014

A simple method to disentangle nanoparticle optical properties by darkfield microspectroscopy.

Microsc Res Tech 2014 Nov 28;77(11):886-95. Epub 2014 Jul 28.

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

We present a darkfield optical microspectroscopy technique devoted to the disentangled measurement of the absorption and scattering cross sections of nanoparticle (NP) samples with variable concentration. The robustness of the method, including the needed instrumental calibrations, is examined in detail by analyzing and quantifying the major sources of statistic and systematic errors. As an exemplary case, results are presented on a gold NP colloid. The technique takes advantage of a simple inverted microscope, coupled with a spectrograph and equipped with a darkfield condenser and a variable numerical aperture objective to obtain spectra either in darkfield or brightfield optical configurations. By adopting the Lambert-Beer (LB) equation modeling, we were able to disentangle and measure with a single setup the absorption, scattering, and extinction coefficients of the same sample by combining three spectra, obtained by opportunely varying the objective numerical aperture. Typical plasmonic resonances were recognized at approximately 520 and 750 nm. Optical coefficients were measured as a function of particle number density (0.04-3.94 µm(-3), corresponding to 40 µM-4 mM nominal Au concentration) and good linearity was verified up to ∼1.5 µm(-3) (∼1 mM Au). Moreover, extinction and scattering cross sections were quantified and the validity of the LB approximation was reviewed. Besides its applications to plasmonic NPs, this method may be appropriate for any colloid, provided there exists a characteristic spectral feature in the ultraviolet-visible-near infrared range. This technique may be exploited to localize NPs in biological samples.
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http://dx.doi.org/10.1002/jemt.22411DOI Listing
November 2014

Quantitative readout of optically encoded gold nanorods using an ordinary dark-field microscope.

Nanoscale 2013 Oct;5(20):9645-50

INO-CNR, National Institute of Optics-CNR, Largo E. Fermi 6, Florence, Italy.

In this paper we report on a new use for dark-field microscopy in order to retrieve two-dimensional maps of optical parameters of a thin sample such as a cryptograph, a histological section, or a cell monolayer. In particular, we discuss the construction of quantitative charts of light absorbance and scattering coefficients of a polyvinyl alcohol film that was embedded with gold nanorods and then etched using a focused mode-locked Ti:Sapphire oscillator. Individual pulses from this laser excite plasmonic oscillations of the gold nanorods, thus triggering plastic deformations of the particles and their environment, which are confined within a few hundred nm of the light focus. In turn, these deformations modify the light absorbance and scattering landscape, which can be measured with optical resolution in a dark-field microscope equipped with an objective of tuneable numerical aperture. This technique may prove to be valuable for various applications, such as the fast readout of optically encoded data or to model functional interactions between light and biological tissue at the level of cellular organelles, including the photothermolysis of cancer.
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http://dx.doi.org/10.1039/c3nr00726jDOI Listing
October 2013

In vivo laser assisted microvascular repair and end-to-end anastomosis by means of indocyanine green-infused chitosan patches: a pilot study.

Lasers Surg Med 2013 Jul 5;45(5):318-25. Epub 2013 Jun 5.

Institute of Neurosurgery, Catholic University School of Medicine, Rome, 00100, Italy.

Background And Objectives: Laser-based repairing techniques offer several advantages respect to standard suturing in microsurgery. In this work we evaluate the applicability and feasibility of two innovative laser-based approaches for microvascular repair and anastomoses: (1) laser-assisted vascular repair (LAVR); (2) laser-assisted end-to-end vascular anastomosis (LAVA). All these procedures have been executed by the use of diode laser irradiation and chitosan-patches infused with Indocyanine Green (ICG).

Study Design/materials And Methods: Experiments were performed on 30 rabbits. Twenty animals underwent LAVR and 10 end-to-end LAVA procedures. In the LAVR group, a 5-mm longitudinal cut was performed on the common carotid artery (CCA), then an ICG-infused chitosan patch was topically applied and laser-soldered over the arterial lesion. In the LAVA group the end-to-end anastomosis was executed on CCA by means of application of the three interrupted sutures and subsequent laser soldering of the ICG-infused patch. Animals underwent different follow-up periods (2, 7, 30, and 90 days). At the end of every follow-up, the animals were re-anesthetized and a microdoppler analysis was performed in order to check patency of the treated vessels. Then soldered segments were excised and subjected to histological and ultrastructural evaluations.

Results: At the end of surgery no bleeding from the treated segment was observed; all the treated vessels were patent. At the end of follow-up periods, no signs of perivascular haemorrhage were found. An intraoperative microdoppler evaluation assessed the patency of all the treated vessels. Histology showed a good reorganization of the vascular wall structures and an early endothelial regeneration was observed by SEM.

Conclusions: Our study demonstrated the efficacy of laser tissue soldering by means of ICG-infused chitosan patches for the in vivo repairing of microvascular lesions and end-to-end anastomoses. This approach offers several advantages over conventional suturing methods and is technically easy to perform, minimizing the surgical trauma to vessels.
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http://dx.doi.org/10.1002/lsm.22145DOI Listing
July 2013

Photothermally activated hybrid films for quantitative confined release of chemical species.

Angew Chem Int Ed Engl 2013 Jun 9;52(23):5956-60. Epub 2013 May 9.

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

Illuminating films of a porous chitosan matrix containing gold nanorods and thermosensitive micelles loaded with a chemical stimulates local photothermal conversion of the gold nanorods. The heat produced activates the ejection of the chemical from the micelles (see scheme), and causes the transient permeabilization of adjacent cell membranes, resulting in a selective cellular uptake of the released chemical with control over spatiotemporal parameters and dosage.
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http://dx.doi.org/10.1002/anie.201207986DOI Listing
June 2013

Light-responsive nanocomposite sponges for on demand chemical release with high spatial and dosage control.

J Mater Chem B 2013 Feb 7;1(8):1096-1100. Epub 2013 Jan 7.

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

We present a biocompatible device for on demand chemical release in the form of a light-activated sponge-like nanocomposite scaffold, which ensures excellent control over the principal parameters of chemical release and dosage in order to sustain effective therapeutic action. The sponge consists of a porous biopolymer scaffold containing a dispersion of gold nanorods, which acts as an absorber of the incoming laser light, and of thermosensitive micelles, which serve as a reservoir for the drug molecules to be released. The photothermal response of the nanoparticles contained inside the sponge triggers a contraction in proximal micelles, thus promoting the expulsion of the drug that in turn is released from the sponge to the external environment. The peculiar physiochemical and structural properties of the nanocomposite sponges impart a number of interesting features to the proposed drug release system, including the possibility of spatially confining the therapeutic treatment as well as precise control of the amount of released drug as a function of duration and power of the excitation light.
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http://dx.doi.org/10.1039/c2tb00310dDOI Listing
February 2013

Thermal transitions of fibrillar collagen unveiled by second-harmonic generation microscopy of corneal stroma.

Biophys J 2012 Sep;103(6):1179-87

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

The thermal transitions of fibrillar collagen are investigated with second-harmonic generation polarization anisotropy microscopy. Second-harmonic generation images and polarization anisotropy profiles of corneal stroma heated in the 35-80°C range are analyzed by means of a theoretical model that is suitable to probe principal intramolecular and interfibrillar parameters of immediate physiological interest. Our results depict the tissue modification with temperature as the interplay of three destructuration stages at different hierarchical levels of collagen assembly including its tertiary structure and interfibrillar alignment, thus supporting and extending previous findings. This method holds the promise of a quantitative inspection of fundamental biophysical and biochemical processes and may find future applications in real-time and postsurgical functional imaging of collagen-rich tissues subjected to thermal treatments.
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http://dx.doi.org/10.1016/j.bpj.2012.07.055DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3446693PMC
September 2012

Hybrid nanocomposite films for laser-activated tissue bonding.

J Biophotonics 2012 Nov 17;5(11-12):868-77. Epub 2012 Aug 17.

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

We report new advancements in the biomedical exploitation of plasmonic nanoparticles as an effective platform for the photothermal repair of biological tissue. Chitosan films are loaded with gold nanorods with intense optical absorption in the "therapeutic window" of deepest light penetration through the body, and then activated by near infrared laser excitation to give adhesion with adjacent connective tissues. The adhesion consists of 0.07 mm(2) welds of ~20 kPa tensile strength at the film/tissue interface, which are obtained by administration of pulses with duration in the hundreds of millisecond timescale from a diode laser at ~130 J cm(-2). We investigate the adhesive effect as a function of pulse power and duration and identify an optimal operative window to achieve effective and reproducible welds with minimal detrimental superheating. These results may prove valuable to standardize laser bonding techniques and meet current needs for new knowledge which is urged by the penetration of nanotechnology into biomedical optics.
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http://dx.doi.org/10.1002/jbio.201200115DOI Listing
November 2012

Emerging concepts of laser-activated nanoparticles for tissue bonding.

J Biomed Opt 2012 Jan;17(1):010701

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

We report recent achievements and future perspectives of minimally invasive bonding of biological tissues triggered by laser light. In particular, we review new advancements in the biomedical exploitation of near-infrared absorbing gold nanoparticles as an original solution for the photothermal closure of surgical incisions. Advanced concepts of laser tissue bonding involving the application of hybrid nanocomposites obtained by inclusion of nanochromophores into biopolymer scaffolds are also introduced. The perspectives of tissue bonding are discussed in the following aspects: (1) tissue bonding with highly-stabilized nanochromophores, (2) enhanced tissue bonding with patterned nanocomposites, (3) real-time monitoring of temperature distributions, (4) tracking of tissue regeneration based on the optical resonances of gold nanoparticles.
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http://dx.doi.org/10.1117/1.JBO.17.1.010701DOI Listing
January 2012

Application of ultrafast gold luminescence to measuring the instrument response function for multispectral multiphoton fluorescence lifetime imaging.

Opt Express 2011 Jul;19(15):13848-61

Department of Physics, Imperial College London, UK.

When performing multiphoton fluorescence lifetime imaging in multiple spectral emission channels, an instrument response function must be acquired in each channel if accurate measurements of complex fluorescence decays are to be performed. Although this can be achieved using the reference reconvolution technique, it is difficult to identify suitable fluorophores with a mono-exponential fluorescence decay across a broad emission spectrum. We present a solution to this problem by measuring the IRF using the ultrafast luminescence from gold nanorods. We show that ultrafast gold nanorod luminescence allows the IRF to be directly obtained in multiple spectral channels simultaneously across a wide spectral range. We validate this approach by presenting an analysis of multispectral autofluorescence FLIM data obtained from human skin ex vivo.
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http://dx.doi.org/10.1364/OE.19.013848DOI Listing
July 2011

In vivo carotid artery closure by laser activation of hyaluronan-embedded gold nanorods.

J Biomed Opt 2010 Jul-Aug;15(4):041508

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

We prove the first application of near-infrared-absorbing gold nanorods (GNRs) for in vivo laser closure of a rabbit carotid artery. GNRs are first functionalized with a biopolymeric shell and then embedded in hyaluronan, which gives a stabilized and handy laser-activable formulation. Four rabbits undergo closure of a 3-mm longitudinal incision performed on the carotid artery by means of a 810-nm diode laser in conjunction with the topical application of the GNRs composite. An effective surgery is obtained by using a 40-W/cm(2) laser power density. The histological and electron microscopy evaluation after a 30-day follow-up demonstrates complete healing of the treated arteries with full re-endothelization at the site of GNRs application. The absence of microgranuloma formation and/or dystrophic calcification is evidence that no host reaction to nanoparticles interspersed through the vascular tissue occurred. The observation of a reshaping and associated blue shift of the NIR absorption band of GNRs after laser treatment supports the occurrence of a self-terminating process, and thus of additional safety of the minimally invasive laser procedure. This study underlines the feasibility of using GNRs for in vivo laser soldering applications, which represents a step forward toward the introduction of nanotechnology-based therapies in minimally invasive clinical practices.
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http://dx.doi.org/10.1117/1.3449574DOI Listing
January 2011