Monitoring of optical properties of tumors during laser plasmon photothermal therapy.

We studied grafted tumors obtained by subcutaneous implantation of kidney cancer cells into male white rats. Gold nanorods with a plasmon resonance of about 800 nm were injected intratumorally for photothermal heating. Experimental irradiation of tumors was carried out percutaneously using a near-infrared diode laser. Changes in the optical properties of the studied tissues in the spectral range 350-2200 nm under plasmonic photothermal therapy (PPT) were studied. Analysis of the observed changes in the absorption bands of water and hemoglobin made it possible to estimate the depth of thermal damage to the tumor. A significant decrease in absorption peaks was observed in the spectrum of the upper peripheral part and especially the tumor capsule. The obtained changes in the optical properties of tissues under laser irradiation can be used to optimize laboratory and clinical PPT procedures.

Surface-enhanced Raman scattering from Au nanorods, nanotriangles, and nanostars with tuned plasmon resonances.

Electromagnetic theory predicts that the optimal value of the localized plasmon resonance (LPR) wavelength for the maximal SERS enhancement factor (EF) is half the sum of the laser and Raman wavelengths. For small Raman shifts, the theoretical EF scales as the fourth power of the local field. However, experimental data often disagree with these theoretical conclusions, leaving the question of choosing the optimal plasmon resonance for the maximal SERS signal unresolved. Here, we present experimental data for gold nanorods (AuNRs), gold nanotriangles (AuNTs), and gold nanostars (AuNSTs) simulating 1D, 2D, and 3D plasmonic nanostructures, respectively. The LPR wavelengths were tuned by chemical etching within 580-1020 nm at a constant concentration of the particles. The particles were functionalized with Cy7.5 and NBT, and the dependence of the intensity at 940 cm-1 (Cy7.5) and 1343 cm-1 (NBT) on the LPR wavelength was examined for laser wavelengths of 633 nm and 785 nm. The electromagnetic SERS EFs were calculated by averaging the product of the local field intensities at the laser and Raman wavelengths over the particle surface and their random orientations. The calculated SERS plasmonic profiles were redshifted compared to the laser wavelength. For 785 nm excitation, the calculated EFs were five to seven times higher than those for 633 nm excitation. With AuNR@Cy7.5 and AuNT@ Cy7.5, the experimental SERS was 35-fold stronger than it was with NBT-functionalized particles, but with AuNST@Cy7.5 and AuNST@NBT, the SERS responses were similar. With all nanoparticles tested, the SERS plasmonic profiles after 785 nm excitation were slightly blue-shifted, as compared with the laser wavelength, possibly owing to the inner filter effect. After 633 nm excitation, the SERS profiles were red-shifted, in agreement with EM theory. In all cases, the plasmonic EF profiles were much broadened compared to the calculated ones and did not follow the four-power law.

Enhanced Antibacterial Activity of Novel Fluorescent Glutathione-Capped Ag Nanoclusters.

Ag nanomaterials are promising candidates for the discovery of next-generation antibiotics with a high antibacterial effect against multi-drug resistant strains. This paper reports a simple synthesis of novel water-soluble glutathione-capped silver nanoclusters (GSH-Ag NCs) with an enhanced antibacterial activity. According to thin layer chromatography (TLC), the synthesized GSH-Ag NCs are an individual fraction of the same composition without any impurities. According to matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) and energy dispersive X-ray (EDX) analyses, the silver core of the GSH-Ag NCs contains approximately 35 silver atoms, and the molecular weight of these nanoclusters is about 11 kDa. The fabricated silver nanoclusters have a reddish fluorescence (λex/λem = 509/645 nm), with a large Stokes shift (>130 nm), and ultra-small size (less than 2 nm) according to transmission electron microscopy (TEM) data and dynamic light scattering (DLS) analysis. The antibacterial activity and minimal inhibitory concentrations of the silver nanoclusters towards Escherichia coli, Staphylococcus aureus, Bacillus cereus and Enterobacter cloacae were evaluated using the agar well-diffusion method and resazurin metabolism assay. The antibacterial activity of chelated silver in the nanoclusters was found to be significantly higher compared to the activity of free silver ions. To explain the possible mechanisms underlying the antibacterial actions of the GSH-Ag nanoclusters, molecular docking was performed, and prospective bacterial targets were identified using AutoDock.

One-Shot Laser-Pulse Modification of Bare and Silica-Coated Gold Nanoparticles of Various Morphologies.

Gold nanoparticles are widely used in laser biomedical applications due to their favorable properties, mainly localized plasmon resonance. However, laser radiation can cause a change in the shape and size of plasmonic nanoparticles, thus resulting in an unwanted reduction of their photothermal and photodynamic efficiency due to a drastic alteration of optical properties. Most previously reported experiments were carried out with bulk colloids where different particles were irradiated by different numbers of laser pulses, thus making it difficult to accurately evaluate the laser power photomodification (PM) threshold. Here, we examine the one-shot nanosecond laser-pulse PM of bare and silica-coated gold nanoparticles moving in a capillary flow. Four types of gold nanoparticles, including nanostars, nanoantennas, nanorods, and SiO2@Au nanoshells, were fabricated for PM experiments. To evaluate the changes in the particle morphology under laser irradiation, we combine measurements of extinction spectra with electron microscopy. A quantitative spectral approach is developed to characterize the laser power PM threshold in terms of normalized extinction parameters. The experimentally determined PM threshold increases in series were as follows: nanorods, nanoantennas, nanoshells, and nanostars. An important observation is that even a thin silica shell significantly increases the photostability of gold nanorods. The developed methods and reported findings can be useful for the optimal design of plasmonic particles and laser irradiation parameters in various biomedical applications of functionalized hybrid nanostructures.

Immunization of Mice with Gold Nanoparticles Conjugated to Thermostable Cancer Antigens Prevents the Development of Xenografted Tumors.

Gold nanoparticles as part of vaccines greatly increase antigen stability, antigen accumulation in the lymph nodes, and antigen uptake by antigen-presenting cells. The use of such particles as part of anticancer vaccines based on heat shock proteins to increase vaccine effectiveness is timely. We prepared and characterized nanoconjugates based on 15-nm gold nanoparticles and thermostable tumor antigens isolated from MH22a murine hepatoma cells. The whole-cell lysate of MH22a cells contained the main heat shock proteins. BALB/c mice were injected with the conjugates and then received transplants of MH22a cells. The highest titer was produced in mice immunized with the complex of gold nanoparticles + antigen with complete Freund's adjuvant. The immunized mice showed no signs of tumor growth for 24 days. They also showed a decreased production of the INF-γ, IL-6, and IL-1 proinflammatory cytokines compared to the mice immunized through other schemes. This study is the first to show that it is possible in principle to use gold nanoparticles in combination with thermostable tumor antigens for antitumor vaccination. Antitumor vaccines based on thermostable tumor antigens can be largely improved by including gold nanoparticles as additional adjuvants.

Albumin-Based Nanocarriers for the Simultaneous Delivery of Antioxidant Gene and Phytochemical to Combat Oxidative Stress.

Human serum albumin (HSA) nanoparticles are promising biocompatible, nontoxic, and non-immunogenic platforms for biomedical applications such as bioimaging and drug and gene delivery. The development of nonviral gene delivery vectors is a great challenge for efficient and safe gene therapy. Sulforaphane (SF) can stimulate the expression of antioxidant genes via activation of a nuclear transcription factor, the erythroid-2 related factor 2 (Nrf-2). Here, we use polyethyleneimine (PEI)-stabilized HSA nanoparticles to stimulate endogenous antioxidant defense mechanisms in lung epithelial cells L-132 through the combinatorial effect of SF drug and antioxidant superoxide dismutase 1 gene (pSOD1 plasmid) delivered by HSA-PEI-SF-pSOD1 nanocomposites (NCs). The developed NCs demonstrated high biocompatibility (L-132 viability, >95%, MTT assay) and high antioxidant activity because of efficient entry of the SOD1 gene and SF-loaded NCs at a very low (3 µg) dose in L-132 cells. A high transfection efficiency of L-132 cells (∼66%, fluorescent microscopy) was obtained with the GFP-tagged transgene SOD1-GFP. We speculate that the antioxidant activity of HSA-PEI-SF-pSOD1 NCs in L-132 cells is due to the initial release of SF followed by subsequent SOD1 gene expression after three to four days of incubation. Hence, the developed HSA-based NCs can be efficient biocompatible nanocarriers for safe and effective drug and gene delivery applications to treat diseases with high oxidative stress due to combinatorial SF and SOD1 gene mechanisms.

SERS and Indicator Paper Sensing of Hydrogen Peroxide Using Au@Ag Nanorods.

The detection of hydrogen peroxide and the control of its concentration are important tasks in the biological and chemical sciences. In this paper, we developed a simple and quantitative method for the non-enzymatic detection of H2O2 based on the selective etching of Au@Ag nanorods with embedded Raman active molecules. The transfer of electrons between silver atoms and hydrogen peroxide enhances the oxidation reaction, and the Ag shell around the Au nanorod gradually dissolves. This leads to a change in the color of the nanoparticle colloid, a shift in LSPR, and a decrease in the SERS response from molecules embedded between the Au core and Ag shell. In our study, we compared the sensitivity of these readouts for nanoparticles with different Ag shell morphology. We found that triangle core-shell nanoparticles exhibited the highest sensitivity, with a detection limit of 10-4 M, and the SERS detection range of 1 × 10-4 to 2 × 10-2 M. In addition, a colorimetric strategy was applied to fabricate a simple indicator paper sensor for fast detection of hydrogen peroxide in liquids. In this case, the concentration of hydrogen peroxide was qualitatively determined by the change in the color of the nanoparticles deposited on the nitrocellulose membrane.

Photothermal and Photodynamic Therapy of Tumors with Plasmonic Nanoparticles: Challenges and Prospects.

Cancer remains one of the leading causes of death in the world. For a number of neoplasms, the efficiency of conventional chemo- and radiation therapies is insufficient because of drug resistance and marked toxicity. Plasmonic photothermal therapy (PPT) using local hyperthermia induced by gold nanoparticles (AuNPs) has recently been extensively explored in tumor treatment. However, despite attractive promises, the current PPT status is limited by laboratory experiments, academic papers, and only a few preclinical studies. Unfortunately, most nanoformulations still share a similar fate: great laboratory promises and fair preclinical trials. This review discusses the current challenges and prospects of plasmonic nanomedicine based on PPT and photodynamic therapy (PDT). We start with consideration of the fundamental principles underlying plasmonic properties of AuNPs to tune their plasmon resonance for the desired NIR-I, NIR-2, and SWIR optical windows. The basic principles for simulation of optical cross-sections and plasmonic heating under CW and pulsed irradiation are discussed. Then, we consider the state-of-the-art methods for wet chemical synthesis of the most popular PPPT AuNPs such as silica/gold nanoshells, Au nanostars, nanorods, and nanocages. The photothermal efficiencies of these nanoparticles are compared, and their applications to current nanomedicine are shortly discussed. In a separate section, we discuss the fabrication of gold and other nanoparticles by the pulsed laser ablation in liquid method. The second part of the review is devoted to our recent experimental results on laser-activated interaction of AuNPs with tumor and healthy tissues and current achievements of other research groups in this application area. The unresolved issues of PPT are the significant accumulation of AuNPs in the organs of the mononuclear phagocyte system, causing potential toxic effects of nanoparticles, and the possibility of tumor recurrence due to the presence of survived tumor cells. The prospective ways of solving these problems are discussed, including developing combined antitumor therapy based on combined PPT and PDT. In the conclusion section, we summarize the most urgent needs of current PPT-based nanomedicine.

Multifunctional plasmonic gold nanostars for cancer diagnostic and therapeutic applications.

Gold nanostar (AuNSt) has gained great attention in bioimaging and cancer therapy due to their tunable surface plasmon resonance across the visible-near infrared range. Photothermal treatment and imaging capabilities including fluorescence lifetime imaging at two-photon excitation (TP-FLIM) and dark-field microscopic imaging are considered in this work. Two types of AuNSts having plasmon absorption peaks centred at 600 and 750 nm wavelength were synthesized and studied. Both NSts exhibited low cytotoxicity on A549 human lung carcinoma cells. A strong emission at two-photon excitation was observed for both NSts, well-distinguishable from lifetimes of bio-object autofluorescence. High efficiency in raising the temperature in the NSts environment with the irradiation of near infrared, AuNSts triggered photothermal effect. The decreased cell viability of A549 observed via MTT test and the cell membrane damaging was demonstrated with trypan blue staining. These results suggest AuNSts can be agents with tunable plasmonic properties for imaging and photothermal therapy.

Lateral Flow Immunoassay of SARS-CoV-2 Antigen with SERS-Based Registration: Development and Comparison with Traditional Immunoassays.

The current COVID-19 pandemic has increased the demand for pathogen detection methods that combine low detection limits with rapid results. Despite the significant progress in methods and devices for nucleic acid amplification, immunochemical methods are still preferred for mass testing without specialized laboratories and highly qualified personnel. The most widely used immunoassays are microplate enzyme-linked immunosorbent assay (ELISA) with photometric detection and lateral flow immunoassay (LFIA) with visual results assessment. However, the disadvantage of ELISA is its considerable duration, and that of LFIA is its low sensitivity. In this study, the modified LFIA of a specific antigen of the causative agent of COVID-19, spike receptor-binding domain, was developed and characterized. This modified LFIA includes the use of gold nanoparticles with immobilized antibodies and 4-mercaptobenzoic acid as surface-enhanced Raman scattering (SERS) nanotag and registration of the nanotag binding by SERS spectrometry. To enhance the sensitivity of LFIA-SERS analysis, we determined the optimal compositions of SERS nanotags and membranes used in LFIA. For benchmark comparison, ELISA and conventional colorimetric LFIA were used with the same immune reagents. The proposed method combines a low detection limit of 0.1 ng/mL (at 0.4 ng/mL for ELISA and 1 ng/mL for qualitative LFIA) with a short assay time equal to 20 min (at 3.5 h for ELISA and 15 min for LFIA). The results obtained demonstrate the promise of using the SERS effects in membrane immuno-analytical systems.

Metal-Specific Response of High-Resolution ICP-MS for Proteins Binding to Gold Nanoparticles in Human Serum.

Metalloproteins have many different functions such as storage and transport of proteins, enzymes, signal transduction proteins, etc. Herein, for a selection of gold nanoparticles differing in shape, size, charge, and surface modification, the binding behavior in human serum was assessed with respect to metal-containing proteins. Our results based on sector-field ICP-MS measurements and a simple calculation algorithm indicate the possible involvement of proteins, incorporating Cu and Fe, in the formation of the biomolecular layer around the particle surface. Given that such binding encompasses a substantial amount of copper and iron within the serum proteome (>50%) at a calculated nanoparticle dose, it may result in depleting their biological functions and should be taken into account when selecting lead candidates with an improved biocompatibility.

Extinction, absorption, and scattering of light by plasmonic spheres embedded in an absorbing host medium.

Although the general Lorenz-Mie formalism for spheres in an absorbing host has been developed, no correct analytical expressions in the small-particle limit have been published so far. Here, we derive two sets of analytical expressions for the extinction, absorption, and far- and near-field scattering cross sections of small particles embedded in an absorbing host. One set is a modification of the electrostatic approximation (EA) for an absorbing host, whereas the other represents an improved electrostatic approximation (IEA) based on the generalized Lorenz-Mie theory and a new form of Mie coefficients for the internal field expansion. To illustrate the accuracy of the derived approximations, we consider Au and Ag nanospheres embedded in model hosts (real part of the refractive index, 1.33; imaginary part, 0-0.3), in a lossless poly(methyl methacrylate) (PMMA), and a lossy poly(3-hexylthiophene) (P3HT) matrix. In general, the IEA cross sections agree with those calculated using Lorenz-Mie theory if the particle diameter is not greater than 50 nm. Two small-particle limits are found for the near-field scattering cross sections. When host absorption is negligible, the scattering efficiency scales as the fourth power of the size parameter. In contrast, for nonzero absorption, the scattering efficiency scales as the first power of the size parameter. For a spectrally independent host, an increase in host absorption broadens and suppresses plasmonic peaks. We found an exception to this general tendency for near-field scattering by small (10-50 nm) particles; for these, an increase in host absorption increases the scattering peak. This surprising behavior is explained analytically. For 10-30 nm Au particles in the PMMA and P3HT matrixes, the EA and IEA data perfectly agree with the exact Lorenz-Mie simulations, in contrast to the previously reported conclusions. In particular, replacing PMMA with P3HT shifts the plasmonic peaks of the 10 nm particles from 540 nm to 650 nm and strongly enhances near- and far-field scattering. However, far-field scattering does not contribute to the extinction derived from the generalized optical theorem.

Correction: A novel concept of two-component dielectric function for gold nanostars: theoretical modelling and experimental verification.

Correction for 'A novel concept of two-component dielectric function for gold nanostars: theoretical modelling and experimental verification' by Nikolai G. Khlebtsov et al., Nanoscale, 2020, 12, 19963-19981, DOI: 10.1039/D0NR02531C.

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.

A novel concept of two-component dielectric function for gold nanostars: theoretical modelling and experimental verification.

Rational design of AuNST morphology requires adequate computational models. The bulk dielectric function is not applicable to sharp nanostar spikes. We suggest a two-component dielectric function in which the nanostar core is treated as a bulk material, whereas the size-corrected dielectric function of the spikes is treated by a modified Coronado-Schatz model. In addition to the strong broadening of plasmonic peaks, the simulated absorption and scattering spectra show unusual properties, which are not observed with bulk dielectric functions. The effect of NIR water absorption on nanostar spectra is small, and the absorption peak demonstrates the expected small decrease in the absorbing media. Surprisingly, however, water absorption increases the scattering peak by 30%. For the common surfactant-free Vo-Dinh AuNSTs, we report, for the first time, very intense SWIR plasmonic peaks around 1900 nm, in addition to the common strong peak in the UV-vis-NIR band (here, at 1100 nm). For bilayers of AuNSTs in air, we recorded two similarly intense peaks near 800 and 1500 nm. To simulate the experimental extinction spectra of colloids and bilayers on glass in air, we develop a statistical model that includes the major fraction of typical Vo-Dinh AuNSTs and two minor fractions of sea urchins and particles with protrusions. In contrast to the general belief, we show that the common UV-vis-NIR plasmonic peak of surfactant-free AuNSTs is related to short spikes on a spherical core, whereas long spikes produce an intense SWIR plasmonic mode. Such a structural assignment of vis-NIR and SWIR peaks does not seem to have been reported previously for surfactant-free nanostars. With our model, we demonstrate good agreement between simulated and measured spectra of colloids and bilayers on glass in air.

Advantages of Highly Spherical Gold Nanoparticles as Labels for Lateral Flow Immunoassay.

The use of lateral flow immunoassays (LFIAs) for rapid on-site testing is restricted by their relatively high limit of detection (LoD). One possible way to decrease the LoD is to optimize nanoparticle properties that are used as labels. We compare two types of Au nanoparticles: usual quasispherical gold nanoparticles (C-GNPs), obtained by the Turkevich-Frens method, and superspherical gold nanoparticles (S-GNPs), obtained by a progressive overgrowth technique. Average diameters were 18.6-47.5 nm for C-GNPs and 20.2-90.4 nm for S-GNPs. Cardiomarker troponin I was considered as the target analyte. Adsorption and covalent conjugation with antibodies were tested for both GNP types. For C-GNPs, the minimal LoD was obtained with 33.7 nm nanoparticles, reaching 12.7 ng/mL for covalent immobilization and 9.9 ng/mL for adsorption. The average diameter of S-GNPs varied from 20.2 to 64.5 nm, which resulted in a decrease in LoD for an LFIA of troponin I from 3.4 to 1.2 ng/mL for covalent immobilization and from 2.9 to 2.0 ng/mL for adsorption. Thus, we obtained an 8-fold decrease in LoD (9.9 to 1.2 ng/mL) by using S-GNPs. This effect can be related to more effective antibody immobilization and improved S-GNP optical properties. The obtained results can improve LFIAs for various practically significant analytes.

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.

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.