Single Pulse Nanosecond Laser-Stimulated Targeted Delivery of Anti-Cancer Drugs from Hybrid Lipid Nanoparticles Containing 5 nm Gold Nanoparticles.

Encapsulating chemotherapeutic drugs like doxorubicin (DOX) inside lipid nanoparticles (LNPs) can overcome their acute, systematic toxicity. However, a precise drug release at the tumor microenvironment for improving the maximum tolerated dose and reducing side effects has yet to be well-established by implementing a safe stimuli-responsive strategy. This study proposes an integrated nanoscale perforation to trigger DOX release from hybrid plasmonic multilamellar LNPs composed of 5 nm gold (Au) NPs clustered at the internal lamellae interfaces. To promote site-specific DOX release, a single pulse irradiation strategy is developed by taking advantage of the resonant interaction between nanosecond pulsed laser radiation (527 nm) and the plasmon mode of the hybrid nanocarriers. This approach enlarges the amount of DOX in the target cells up to 11-fold compared to conventional DOX-loaded LNPs, leading to significant cancer cell death. The simulation of the pulsed laser interactions of the hybrid nanocarriers suggests a release mechanism mediated by either explosive vaporization of thin water layers adjacent to AuNP clusters or thermo-mechanical decomposition of overheated lipid layers. This simulation indicates an intact DOX integrity following irradiation since the temperature distribution is highly localized around AuNP clusters and highlights a controlled light-triggered drug delivery system.

Sensitive detection of electric field-induced second harmonic signals.

We demonstrate sensitive electric field measurements by coherent homodyne amplification of the electric field induced second harmonic generation (E-FISH) technique. In the process of E-FISH, an applied electric field breaks the centrosymmetry of an otherwise homogeneous medium, in turn promoting the generation of the second harmonic frequency of an incident field. Due to weak third-order hyperpolarizability and the requirement of an applied field to break the symmetry, the E-FISH technique has been mainly used to study high fields, also requiring a strong optical field and sensitive detection. Here we superimpose the E-FISH signal with an auxiliary beam, also termed a local oscillator (LO), at double the incident frequency. Coherent superposition of the LO and the E-FISH output (LOE-FISH) allows for a homodyne amplification of the otherwise weak nonlinear signal. We have demonstrated an increase of signal-to-noise by a factor of seven, which results in a measurement time reduction of a factor of 49. This technique, LOE-FISH, has a number of advantages: detection with intensified detectors is not required. Furthermore, instead of millijoule pulsed lasers, we can work with microjoule pulsed lasers, which allows measuring at repetition rates of megahertz and opens single shot and real-time capability. The LOE-FISH technique increases in sensitivity at lower electric field values. Our work is a demonstration of the principle. Already with our first results from the demonstration, one can see the high potential of LOE-FISH.

Coiled-Coil-Based Biofunctionalization of 100 nm Gold Nanoparticles with the Trastuzumab Antibody for the Detection of HER2-Positive Cancer Cells.

We compared different biofunctionalization strategies for immobilizing trastuzumab, an IgG targeting the HER2 biomarker, onto 100 nm spherical gold nanoparticles because of the E/K coiled-coil peptide heterodimer. First, Kcoil peptides were grafted onto the gold surface while their Ecoil partners were genetically encoded at the C-terminus of trastuzumab's Fc region, allowing for a strong and specific interaction between the antibodies and the nanoparticles. Gold nanoparticles with no Kcoil peptides on their surface were also produced to immobilize Ecoil-tagged trastuzumab antibodies via the specific adsorption of their negatively charged Ecoil tags on the positively charged gold surface. Finally, the nonspecific adsorption of wild-type trastuzumab on the gold surface was also assessed, with and without Kcoil peptides grafted on it beforehand. We developed a thorough workflow to systematically compare the immobilization strategies regarding the stability of nanoparticles, antibody coverage, and ability to specifically bind to HER2-positive breast cancer cells. All nanoparticles were highly monodisperse and retained their localized surface plasmon resonance properties after biofunctionalization. A significant increase in the amount of immobilized antibodies was observed with the two oriented coil-based strategies compared to nonspecific adsorption. Finally, all biofunctionalization strategies allowed for the detection of HER2-positive breast cancer cells, but among the investigated approaches, we recommend using the E/K coiled-coil-based strategy for gold nanoparticle biofunctionalization because it allows for the qualitative and quantitative detection of HER2-positive cells with a higher contrast compared to HER2-negative cells.

Porous Au-Ag Nanoparticles from Galvanic Replacement Applied as Single-Particle SERS Probe for Quantitative Monitoring.

Plasmonic nanostructures have raised the interest of biomedical applications of surface-enhanced Raman scattering (SERS). To improve the enhancement and produce sensitive SERS probes, porous Au-Ag alloy nanoparticles (NPs) are synthesized by dealloying Au-Ag alloy NP-precursors with Au or Ag core in aqueous colloidal environment through galvanic replacement reaction. The novel designed core-shell Au-Ag alloy NP-precursors facilitate controllable synthesis of porous nanostructure, and dealloying degree during the reaction has significant effect on structural and spectral properties of dealloyed porous NPs. Narrow-dispersed dealloyed NPs are obtained using NPs of Au/Ag ratio from 10/90 to 40/60 with Au and Ag core to produce solid core@porous shell and porous nanoshells, having rough surface, hollowness, and porosity around 30-60%. The clean nanostructure from colloidal synthesis exhibits a redshifted plasmon peak up to near-infrared region, and the large accessible surface induces highly localized surface plasmon resonance and generates robust SERS activity. Thus, the porous NPs produce intensely enhanced Raman signal up to 68-fold higher than 100 nm AuNP enhancement at single-particle level, and the estimated Raman enhancement around 7800, showing the potential for highly sensitive SERS probes. The single-particle SERS probes are effectively demonstrated in quantitative monitoring of anticancer drug Doxorubicin release.

Formation Pathways of Porous Alloy Nanoparticles through Selective Chemical and Electrochemical Etching.

Porous alloy nanomaterials are important for applications in catalysis, sensing, and actuation. Chemical and electrochemical etching are two methods to form porous nanostructures by dealloying bimetallic nanoparticles (NPs). However, it is not clear how the NPs evolve during these etching processes. Insight into the morphological and compositional transformations of the NPs during the etching is critical to understanding the nanoscale details of the dealloying process. Here, using in situ liquid phase transmission electron microscopy, the structural evolution of individual AuAg alloy NPs is tracked during both chemical and electrochemical etching of their Ag component. The observations show that the electrochemical etching produces NPs with more uniform pore sizes than the chemical etching and enables tuning the NPs porosity by modulating the electrochemical potential. The results show that at the initial stages of both etching methods, Au-rich passivation layer forms on the surface of the NPs, which is critical in preserving the NP's porous shell as pores form underneath this layer during the etching. These findings describing the selective etching and dealloying of AuAg NPs provide a critical insight needed to control the morphology and composition of porous multimetallic NPs, and paves the way for synthesizing nanomaterials with tailored chemical and physical properties for various applications.

Antibody-Functionalized Gold Nanostar-Mediated On-Resonance Picosecond Laser Optoporation for Targeted Delivery of RNA Therapeutics.

The rapid advances of genetic and genomic technology indicate promising therapeutic potential of genetic materials for regulating abnormal gene expressions causing diseases and disorders. However, targeted intracellular delivery of RNA therapeutics still remains a major challenge hindering the clinical translation. In this study, an elaborated plasmonic optoporation approach is proposed to efficiently and selectively transfect specific cells. The site-specific optoporation is obtained by tuning the spectral range of a supercontinuum pulsed picosecond laser in order for each individual cell binding gold nanostar with their unique resonance peak to magnify the local field strength in the near-infrared region and facilitate a selective delivery of small interfering RNA, messenger RNA, and Cas9-ribonucleoprotein into human retinal pigment epithelial cells. Numerical simulations indicate that optoporation is not due to a plasma-mediated process but rather due to a highly localized temperature rise both in time (few nanoseconds) and space (few nanometers). Taking advantage of the numerical simulation and fine-tuning of the optical strategy, the perforated lipid bilayer of targeted cells undergoes a membrane recovery process, important to retain their viability. The results signify the prospects of antibody functionalized nanostar-mediated optoporation as a simple and realistic gene delivery approach for future clinical practices.

Femtosecond nearly resonant self-focusing in gold nanorod colloids.

We evaluate the threshold power for self-focusing in gold nanorod colloids of varying concentration by a power limiting method in the femtosecond filamentation regime. The pulses are tuned near the longitudinal plasmon peak of the nanorods, leading to saturation of linear absorption and reshaping of the particles. We evaluated the last two effects by optical transmission measurements and spectroscopic analysis and estimated that considerable particle deformation does not occur before the collapse of the beam. We performed numerical simulations based on the experimental results, and evaluated only a subtle, monotonically increasing enhancement of the nonlinear refractive index of the host material (water) as the nanoparticles concentration increases. The role of higher-order contributions is discussed. Our work provides an alternative characterization approach of ultrafast nonlinearities in absorbing media. It further emphasizes that self-focusing of intense femtosecond pulses in gold nanocomposites is hampered by the ultrafast modulation of the susceptibility of the metal.

Single point single-cell nanoparticle mediated pulsed laser optoporation.

This article presents an optical platform for studying the dynamics of nanoparticle assisted pulsed laser optoporation of individual living cells. Here plasmonic nanoparticles (NPs) act as markers of the exact spatial position of living cell membranes and as an enhancer for localized pulsed laser perforation. High contrast NP imaging using reflected light microscopy (RLM) allows accurate and automatic laser targeting at individual NPs for spatially controlled laser optoporation of single cells at a single point. The NP imaging method is compatible with fluorescence microscopy and a cellular incubator that allows study of real-time perforation kinetics of live cells and the optomechanical interaction of NPs with membranes. These parameters are of great interest for the development and experimental implementation of the technology of pulsed laser optoporation and transfection applied to single living cells as well as to bulk-level assays.

Cost-effective side-illumination darkfield nanoplasmonic marker microscopy.

We present the development of an innovative technology for quantitative multiplexed cytology analysis based on the application of spectrally distinctive plasmonic nanoparticles (NPs) as optical probes and on cost-effective side-illumination multispectral darkfield microscopy (SIM) as the differential NP imaging method. SIM is based on lateral illumination by arrays of discrete color RGB light emitting diodes (LEDs) of spectrally adjusted plasmonic NPs and consecutive detection by the conventional CMOS color camera. We demonstrate the enhanced contrast and higher resolution of our method for individual NP detection in the liquid medium and of NP markers attached on the cell membrane in a cytology preparation by comparing it to the conventional darkfield microscopy (DFM). The proposed illumination and detection system is compatible with current clinical microscopy equipment used by pathologists and can greatly simplify the adaptation of plasmonic NPs as novel reliable and stable biological multiplexed chromatic markers for biodetection and diagnosis.

In Vivo Laser-Mediated Retinal Ganglion Cell Optoporation Using KV1.1 Conjugated Gold Nanoparticles.

Vision loss caused by retinal diseases affects hundreds of millions of individuals worldwide. The retina is a delicate central nervous system tissue stratified into layers of cells with distinct roles. Currently, there is a void in treatments that selectively target diseased retinal cells, and current therapeutic paradigms present complications associated with off-target effects. Herein, as a proof of concept, we introduce an in vivo method using a femtosecond laser to locally optoporate retinal ganglion cells (RGCs) targeted with functionalized gold nanoparticles (AuNPs). We provide evidence that AuNPs functionalized with an antibody toward the cell-surface voltage-gated K+ channel subunit KV1.1 can selectively deliver fluorescently tagged siRNAs or fluorescein isothiocyanate-dextran dye into retinal cells when irradiated with an 800 nm 100 fs laser. Importantly, neither AuNP administration nor irradiation resulted in RGC death. This system provides a novel, non-viral-based approach that has the potential to selectively target retinal cells in diseased regions while sparing healthy areas and may be harnessed in future cell-specific therapies for retinal degenerative diseases.

Rational Design of Plasmonic Nanoparticles for Enhanced Cavitation and Cell Perforation.

Metallic nanoparticles are routinely used as nanoscale antenna capable of absorbing and converting photon energy with subwavelength resolution. Many applications, notably in nanomedicine and nanobiotechnology, benefit from the enhanced optical properties of these materials, which can be exploited to image, damage, or destroy targeted cells and subcellular structures with unprecedented precision. Modern inorganic chemistry enables the synthesis of a large library of nanoparticles with an increasing variety of shapes, composition, and optical characteristic. However, identifying and tailoring nanoparticles morphology to specific applications remains challenging and limits the development of efficient nanoplasmonic technologies. In this work, we report a strategy for the rational design of gold plasmonic nanoshells (AuNS) for the efficient ultrafast laser-based nanoscale bubble generation and cell membrane perforation, which constitute one of the most crucial challenges toward the development of effective gene therapy treatments. We design an in silico rational design framework that we use to tune AuNS morphology to simultaneously optimize for the reduction of the cavitation threshold while preserving the particle structural integrity. Our optimization procedure yields optimal AuNS that are slightly detuned compared to their plasmonic resonance conditions with an optical breakdown threshold 30% lower than randomly selected AuNS and 13% lower compared to similarly optimized gold nanoparticles (AuNP). This design strategy is validated using time-resolved bubble spectroscopy, shadowgraphy imaging and electron microscopy that confirm the particle structural integrity and a reduction of 51% of the cavitation threshold relative to optimal AuNP. Rationally designed AuNS are finally used to perforate cancer cells with an efficiency of 61%, using 33% less energy compared to AuNP, which demonstrate that our rational design framework is readily transferable to a cell environment. The methodology developed here thus provides a general strategy for the systematic design of nanoparticles for nanomedical applications and should be broadly applicable to bioimaging and cell nanosurgery.

Plasma-mediated photothermal effects in ultrafast laser irradiation of gold nanoparticle dimers in water.

The intention of this paper is to study the physical mechanism underlying the response of gold nanoparticle (AuNP) dimers to a near-infrared off-resonance femtosecond pulse laser in aqueous medium. We show that the strongly localized field enhancement in the gap distance and around nanoparticles significantly reduces the laser fluence threshold to achieve an optical breakdown in comparison with an AuNP monomer. This optical breakdown results from highly localized plasma in surrounding media where the nanoparticles stay intact. Also the impact of the gap distance, field polarization, laser fluence and pulse duration on the energy deposition in plasma is presented. These results can be used to make nanoscale plasmonic devices for variety of absorption-based applications.

Hyperspectral reflected light microscopy of plasmonic Au/Ag alloy nanoparticles incubated as multiplex chromatic biomarkers with cancer cells.

A hyperspectral microscopy system based on a reflected light method for plasmonic nanoparticle (NP) imaging was designed and compared with a conventional darkfield method for spatial localization and spectroscopic identification of single Au, Ag and Au/Ag alloy NPs incubated with fixed human cancer cell preparations. A new synthesis protocol based on co-reduction of Au and Ag salts combined with the seeded growth technique was used for the fabrication of monodispersed alloy NPs with sizes ranging from 30 to 100 nm in diameter. We validated theoretically and experimentally the performance of 60 nm Au, Ag and Au/Ag (50 : 50) NPs as multiplexed biological chromatic markers for biomedical diagnostics and optical biosensing. The advantages of the proposed reflected light microscopy method are presented for NP imaging in a complex and highly diffusing medium such as a cellular environment. The obtained information is essential for the development of a high throughput, selective and efficient strategy for cancer detection and treatment.

Mechanisms of plasmon-enhanced femtosecond laser nanoablation of silicon.

We perform plasmon-enhanced femtosecond laser ablation of silicon using gold nanorods to produce sub-diffraction limit features. While the observed hole shape seems inconsistent with calculated field distribution, we show that using a carrier diffusion-based model, both shape and depth of the nanoholes can be reliably explained. The laser energy is first deposited into electron-hole pairs that are created in the nanostructure's enhanced near-field. Those carriers then diffuse and transfer their energy to the silicon lattice, producing ablation. Increased importance of the carrier diffusion process is shown to arise from the extreme localization of the deposited energy around the nanostructure, due to the plasmonic effect. The characteristic shape of holes is revealed as a striking signature of the screened charge carriers-phonon coupling that is shown to channel the heat transfer to the lattice and control ablation.

Coherently-enabled environmental control of optics and energy transfer pathways of hybrid quantum dot-metallic nanoparticle systems.

It is well-known that optical properties of semiconductor quantum dots can be controlled using optical cavities or near fields of localized surface plasmon resonances (LSPRs) of metallic nanoparticles. In this paper we study the optics, energy transfer pathways, and exciton states of quantum dots when they are influenced by the near fields associated with plasmonic meta-resonances. Such resonances are formed via coherent coupling of excitons and LSPRs when the quantum dots are close to metallic nanorods and driven by a laser beam. Our results suggest an unprecedented sensitivity to the refractive index of the environment, causing significant spectral changes in the Förster resonance energy transfer from the quantum dots to the nanorods and in exciton transition energies. We demonstrate that when a quantum dot-metallic nanorod system is close to its plasmonic meta-resonance, we can adjust the refractive index to: (i) control the frequency range where the energy transfer from the quantum dot to the metallic nanorod is inhibited, (ii) manipulate the exciton transition energy shift of the quantum dot, and (iii) disengage the quantum dot from the metallic nanoparticle and laser field. Our results show that near meta-resonances the spectral forms of energy transfer and exciton energy shifts are strongly correlated to each other.

An improved refractive index sensor based on genetic optimization of plasmon waveguide resonance.

Plasmon waveguide resonance (PWR) sensors are particularly useful for biosensing due to their unique ability to perform sensing with two different polarizations. In this paper we report a comprehensive performance comparison between the surface plasmon resonance (SPR) sensor and the PWR sensor in terms of the sensitivity and the refractive index resolution. Both sensors were optimized using a genetic algorithm to acquire their best performance for bulk sensing applications. The experimental results show that the PWR sensor has a refractive index resolution of 5 × 10(-7) RIU which is 6 times smaller than that of the optimized SPR sensor. The TE polarization in the PWR sensor has a resolution of 1.4 × 10(-6) RIU which is smaller than the SPR sensor. The polarization diversity in the PWR sensor is another advantage which can be used to improve the measurement reliability.

Quantum dot-metallic nanorod sensors via exciton-plasmon interaction.

We investigate quantum nanosensors based on hybrid systems consisting of semiconductor quantum dots and metallic nanorods in the near-infrared regime. These sensors can detect biological and chemical substances based on their impact on the coherent exciton-plasmon coupling and molecular resonances supported by such systems when they interact with a laser field. We demonstrate that the ultrahigh sensitivity of such molecular resonances on environmental conditions allows dramatic and nearly instantaneous changes in the total field experienced by the semiconductor quantum dot via minuscule variations of the local refractive indices of the quantum dot or nanorod. The proposed nanosensors can utilize quantum effects to control the sense (or direction) of the changes in the quantum dot emission, allowing us to have bistable switching from dark to bright states or vice versa via adsorption (or detachment) of biomolecules. These sensors can also offer detection of ultra-small variations in the local dielectric constant of the quantum dots or metallic nanorods via coherent induction of time delays in the effective field experienced by the quantum dots when the hybrid systems interact with time-dependent laser fields. This leads to unprecedented bulk refractive index sensitivities. Our results show that one can utilize quantum phase to control the coherent exciton-plasmon dynamics in these sensors such that introduction of a biomolecule can increase or decrease the time delay. These results offer novel ways to detect single biomolecules via application of quantum coherence to convert their impact into spectacular optical events.

Coherent confinement of plasmonic field in quantum dot-metallic nanoparticle molecules.

Interaction of a hybrid system consisting of a semiconductor quantum dot and a metallic nanoparticle (MNP) with a laser beam can replace the intrinsic plasmonic field of the MNP with a coherently normalized field (coherent-plasmonic or CP field). In this paper we show how quantum coherence effects in such a hybrid system can form a coherent barrier (quantum cage) that spatially confines the CP field. This allows us to coherently control the modal volume of this field, making it significantly smaller or larger than that of the intrinsic plasmonic field of the MNP. We investigate the spatial profiles of the CP field and discuss how the field barrier depends on the collective states of the hybrid system.

Plasma mediated off-resonance plasmonic enhanced ultrafast laser-induced nanocavitation.

The generation of nanobubbles around plasmonic nanostructures is an efficient approach for imaging and therapy, especially in the field of cancer research. We show a novel method using infrared femtosecond laser that generates ≈800 nm bubbles around off-resonance gold nanospheres using 200 mJ/cm(2) 45 fs pulses. We present experimental and theoretical work that demonstrate that the nanobubble formation results from the generation of a nanoscale plasma around the particle due to the enhanced near-field rather than from the heating of the particle. Energy absorbed in the nanoplasma is indeed more than 11 times the energy absorbed in the particle. When compared to the usual approach that uses nanosecond laser to induce the extreme heating of in-resonance nanoparticles to initiate bubble formation, our off-resonance femtosecond technique is shown to bring many advantages, including avoiding the particles fragmentation, working in the optical window of biological material and using the deposited energy more efficiently.