Microbubble resonators for scattering-free absorption spectroscopy of nanoparticles.

We present a proof-of-concept experiment where the absorbance spectra of suspensions of plasmonic nanoparticles are accurately reconstructed through the photothermal conversion that they mediate in a microbubble resonator. This thermal detection produces spectra that are insensitive towards light scattering in the sample, as proved experimentally by comparing the spectra of acqueos gold nanorods suspensions in the presence or absence of milk powder. In addition, the microbubble system allows for the interrogation of small samples (below 40 nl) while using a low-intensity beam (around 20 µW) for their excitation. In perspective, this system could be implemented for the characterization of turbid biological fluids through their optical absorption, especially when considering that the microbubble resonator naturally interfaces to a microfluidic circuit and may easily fit within portable or on-chip devices.

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

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.

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

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.

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

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.

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

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.

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

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.

Photomobile Polymer-Piezoelectric Composite for Enhanced Actuation and Energy Generation.

In this study, we present an innovative approach to increase the quantum yield and wavelength sensitivity of photomobile polymer (PMP) films based on azobenzene by doping the polymer matrix with noble metal nanoparticles. These doped PMP films showed faster and more significant bending under both UV as well as visible and near-infrared light regardless of whether it was coherent, incoherent, polarized, or unpolarized irradiation, expanding the potential of PMP-based actuators. To illustrate their practical implications, we created a proof-of-concept model of power generation by coupling it to flexible piezoelectric materials under simulated sunlight. This model has been tested under real operating conditions, thus demonstrating the possibility of generating electricity with variable light exposure. Additionally, our synthetic protocol is solvent-free, which is another benefit of environmental relevance. Our research lays the groundwork for the development of sunlight-sensitive devices, such as photomechanical actuators and advanced photovoltaic modules, which may break ground in the thriving field of smart materials. We are confident that the presented findings will contribute to the ongoing discourse in the field and inspire additional advances in renewable energy applications.

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

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.

Cyanine-Doped Nanofiber Mats for Laser Tissue Bonding.

In spite of an extensive body of academic initiatives and innovative products, the toolkit of wound dressing has always revolved around a few common concepts such as adhesive patches and stitches and their variants. Our work aims at an alternative solution for an immediate restitutio ad integrum of the mechanical functionality in cutaneous repairs. We describe the fabrication and the application of electrospun mats of bioactive nanofibers all made of biocompatible components such as a natural polysaccharide and a cyanine dye for use as laser-activatable plasters, resembling the ultrastructure of human dermis. In particular, we investigate their morphological features and mechanical moduli under conditions of physiological relevance, and we test their use to bind a frequent benchmark of connective tissue as rabbit tendon and a significant case of clinical relevance as human dermis. Altogether, our results point to the feasibility of a new material for wound dressing combining translational potential, strength close to human dermis, extensibility exceeding 15% and state-of-art adhesive properties.

Paper-based genetic assays with bioconjugated gold nanorods and an automated readout pipeline.

Paper-based biosensors featuring immunoconjugated gold nanoparticles have gained extraordinary momentum in recent times as the platform of choice in key cases of field applications, including the so-called rapid antigen tests for SARS-CoV-2. Here, we propose a revision of this format, one that may leverage on the most recent advances in materials science and data processing. In particular, we target an amplifiable DNA rather than a protein analyte, and we replace gold nanospheres with anisotropic nanorods, which are intrinsically brighter by a factor of ~ 10, and multiplexable. By comparison with a gold-standard method for dot-blot readout with digoxigenin, we show that gold nanorods entail much faster and easier processing, at the cost of a higher limit of detection (from below 1 to 10 ppm in the case of plasmid DNA containing a target transgene, in our current setup). In addition, we test a complete workflow to acquire and process photographs of dot-blot membranes with custom-made hardware and regression tools, as a strategy to gain more analytical sensitivity and potential for quantification. A leave-one-out approach for training and validation with as few as 36 sample instances already improves the limit of detection reached by the naked eye by a factor around 2. Taken together, we conjecture that the synergistic combination of new materials and innovative tools for data processing may bring the analytical sensitivity of paper-based biosensors to approach the level of lab-grade molecular tests.

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

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.

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

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.

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

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 mm2/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 T1 and T2 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.

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

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.

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

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-1 for a magnetic gradient of 0.8 T m-1, the optical hyperthermia of cancer, with stability of photothermal conversion to temperatures exceeding 50∘C, 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.

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

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.

Fiber-cap biosensors for SERS analysis of liquid samples.

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-ß (Aß) neurotoxic biomarkers were realized by covalent grafting of Aß antibodies. As attested by fluorescence images and SERS measurements, the biosensors successfully exhibited enhanced Aß 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.

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

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.

Photothermally-induced disordered patterns of corneal collagen revealed by SHG imaging.

The loss of organization of the corneal collagen lattice induced by photothermal effects was analyzed by using second-harmonic generation (SHG) imaging. Porcine cornea samples were treated with low-power laser irradiation in order to get localized areas of tissue disorganization. The disorder induced within the irradiated area of corneal stroma was quantified by means of Discrete Fourier Transform, auto-correlation and entropy analyses of the SHG images. Polarization modulated SHG measurements allowed to probe the changes in the structural anisotropy of sub-micron hierarchical levels of the stromal collagen. Our results emphasize the great potential of the SHG imaging to detect subtle modifications in the collagen assembly. The proposed analytical methods may be used to track several genetic, pathologic, accidental or surgical-induced disorder states of biological tissues.

Photothermal effects in connective tissues mediated by laser-activated gold nanorods.

We report a study on the application of laser-activated nanoparticles in the direct welding of connective tissues, which may become a valuable technology in biomedicine. We use colloidal gold nanorods as new near-infrared chromophores to mediate functional photothermal effects in the eye lens capsules. Samples obtained ex vivo from porcine eyes are treated to simulate heterotransplants with 810-nm diode laser radiation in association with a stain of gold nanorods of aspect ratio approximately 4. This stain is applied at the interface between a patch of capsule from a donor eye and the capsule of a recipient eye. Then, by administration of laser pulses of 40 msec and approximately 100-140 J/cm(2), we achieved the local denaturation of the endogenous collagen filaments, which reveals that the treated area reached temperatures above 50 degrees C. The thermal damage is confined within 50-70 mum in a radial distance from the irradiated area.