Plasmonic nanobubbles (PNBs) are transient vapor nanobubbles generated in liquid around laser-overheated plasmonic nanoparticles. Unlike plasmonic nanoparticles, PNBs' properties are still largely unknown due to their highly nonstationary nature. Here we show the influence of the duration of the optical excitation on the energy efficacy and threshold of PNB generation. The combination of picosecond pulsed excitation with the nanoparticle clustering provides the highest energy efficacy and the lowest threshold fluence, around 5 mJ cm(-2), of PNB generation. In contrast, long excitation pulses reduce the energy efficacy of PNB generation by several orders of magnitude. Ultimately, the continuous excitation has the minimal energy efficacy, nine orders of magnitude lower than that for the picosecond excitation. Thus, the duration of the optical excitation of plasmonic nanoparticles can have a stronger effect on the PNB generation than the excitation wavelength, nanoparticle size, shape, or other "stationary" properties of plasmonic nanoparticles.
Chemoradiation-resistant cancers limit treatment efficacy and safety. We show here the cancer cell-specific, on-demand intracellular amplification of chemotherapy and chemoradiation therapy via gold nanoparticle- and laser pulse-induced mechanical intracellular impact. Cancer aggressiveness promotes the clustering of drug nanocarriers and gold nanoparticles in cancer cells. This cluster, upon exposure to a laser pulse, generates a plasmonic nanobubble, the mechanical explosion that destroys the host cancer cell or ejects the drug into its cytoplasm by disrupting the liposome and endosome. The same cluster locally amplifies external X-rays. Intracellular synergy of the mechanical impact of plasmonic nanobubble, ejected drug and amplified X-rays improves the efficacy of standard chemoradiation in resistant and aggressive head and neck cancer by 100-fold in vitro and 17-fold in vivo, reduces the effective entry doses of drugs and X-rays to 2-6% of their clinical doses and efficiently spares normal cells. The developed quadrapeutics technology combines four clinically validated components and transforms a standard macrotherapy into an intracellular on-demand theranostic microtreatment with radically amplified therapeutic efficacy and specificity.
We used complexes between a fourth generation polyamidoamine (PAMAM) dendrimer and one of two heterocyclic compounds - 1-(6-hydroxyhexyl)-3-(5-phenyl-isoxazole-3-yl)-urea or 5-phenyl-isoxazole-3-carboxylic acid - to reduce oxygen consumption in transverse slices of the hippocampus taken from 4-week old male rats. In vitro electrophysiological experiments revealed that the inhibitory effect of the hypoxic state on the evoked responses was enhanced in the presence of the complexes. The data were analyzed in terms of the potential antitumor effects of these complexes.
Malaria remains a widespread and deadly infectious human disease, with increasing diagnostic and therapeutic challenges due to the drug resistance and aggressiveness of malaria infection. Early detection and innovative approaches for parasite destruction are needed. The high optical absorbance and nano-size of hemozoin crystals have been exploited to detect and mechanically destroy the malaria parasite in a single theranostic procedure. Transient vapor nanobubbles are generated around hemozoin crystals in malaria parasites in infected erythrocytes in response to a single short laser pulse. Optical scattering signals of the nanobubble report the presence of the malaria parasite. The mechanical impact of the same nanobubble physically destroys the parasite in nanoseconds in a drug-free manner. Laser-induced nanobubble treatment of human blood in vitro results in destruction of up to 95% of parasites after a single procedure, and delivers an 8-fold better parasiticidal efficacy compared to standard chloroquine drug treatment. The mechanism of destruction is highly selective for malaria infected red cells and does not harm neighboring, uninfected erythrocytes. Thus, laser pulse-induced vapor nanobubble generation around hemozoin supports both rapid and highly specific detection and destruction of malaria parasites in one theranostic procedure.
Successful diagnosis, screening, and elimination of malaria critically depend on rapid and sensitive detection of this dangerous infection, preferably transdermally and without sophisticated reagents or blood drawing. Such diagnostic methods are not currently available. Here we show that the high optical absorbance and nanosize of endogenous heme nanoparticles called "hemozoin," a unique component of all blood-stage malaria parasites, generates a transient vapor nanobubble around hemozoin in response to a short and safe near-infrared picosecond laser pulse. The acoustic signals of these malaria-specific nanobubbles provided transdermal noninvasive and rapid detection of a malaria infection as low as 0.00034% in animals without using any reagents or drawing blood. These on-demand transient events have no analogs among current malaria markers and probes, can detect and screen malaria in seconds, and can be realized as a compact, easy-to-use, inexpensive, and safe field technology.
Cancer chemotherapies suffer from multi drug resistance, high non-specific toxicity and heterogeneity of tumors. We report a method of plasmonic nanobubble-enhanced endosomal escape (PNBEE) for the selective, fast and guided intracellular delivery of drugs through a self-assembly by cancer cells of separately targeted gold nanoparticles and encapsulated drug (Doxil). The co-localized with Doxil plasmonic nanobubbles optically generated in cancer cells released the drug into the cytoplasm thus increasing the therapeutic efficacy against these drug-resistant cells by 31-fold, reducing drug dose by 20-fold, the treatment time by 3-fold and the non-specific toxicity by 10-fold compared to standard treatment. Thus the PNBEE mechanism provided selective, safe and efficient intracellular drug delivery in heterogeneous environment opening new opportunities for drug therapies.
The synergistic physical and biological effects of selective targeting and activation of plasmonic nanoparticles were studied for a transient vapor nanobubble mode. Simultaneous optical activation of two plasmon resonances in multi-nanoparticle clusters significantly improved the selectivity and efficacy of the nanobubble generation through and was termed "rainbow plasmonic nanobubbles." The rainbow nanobubble mechanism has been studied in water and in living cells in vitro. This mechanism provided maximal selectivity of the nanobubble generation in both models and therefore, can the therapeutic selectivity and optical contrast of gold nanoparticles in a heterogeneous physiological microenvironment at cell level.
This review is focused on a novel cellular probe, the plasmonic nanobubble (PNB), which has the dynamically tunable and multiple functions of imaging, diagnosis, delivery, therapy and, ultimately, theranostics. The concept of theranostics was recently introduced in order to unite the clinically important stages of treatment, namely diagnosis, therapy and therapy guidance, into one single, rapid and highly accurate procedure. Cell level theranostics will have far-reaching implications for the treatment of cancer and other diseases at their earliest stages. PNBs were developed to support cell level theranostics as a new generation of on-demand tunable cellular probes. A PNB is a transient vapor nanobubble that is generated within nanoseconds around an overheated plasmonic nanoparticle with a short laser pulse. In the short term, we expect that PNB technology will be rapidly adaptable to clinical medicine, where the single cell resolution it provides will be critical for diagnosing incipient or residual disease and eliminating cancer cells, while leaving healthy cells intact. This review discusses mechanisms of plasmonic nanobubbles and their biomedical applications with the focus on cancer cell theranostics.
Theranostic applications require coupling of diagnosis and therapy, a high degree of specificity and adaptability to delivery methods compatible with clinical practice. The tunable physical and biological effects of selective targeting and activation of plasmonic nanobubbles (PNB) were studied in a heterogeneous biological microenvironment of prostate cancer and stromal cells. All cells were targeted with conjugates of gold nanoparticles (NPs) through an antibody-receptor-endocytosis-nanocluster mechanism that produced NP clusters. The simultaneous pulsed optical activation of intracellular NP clusters at several wavelengths resulted in higher optical contrast and therapeutic selectivity of PNBs compared with those of gold NPs alone. The developed mechanism was termed "rainbow plasmonic nanobubbles." The cellular effect of rainbow PNBs was tuned in situ in target cells, thus supporting a theranostic algorithm of prostate cancer cell detection and follow-up guided destruction without damage to collateral cells. The specificity and tunability of PNBs is promising for theranostic applications and we discuss a fiber optic platform that will capitalize on these features to bring theranostic tools to the clinic.
Gene delivery and transfection of eukaryotic cells are widely used for research and for developing gene cell therapy. However, the existing methods lack selectivity, efficacy and safety when heterogeneous cell systems must be treated. We report a new method that employs plasmonic nanobubbles (PNBs) for delivery and transfection. A PNB is a novel, tunable cellular agent with a dual mechanical and optical action due to the formation of the vapor nanobubble around a transiently heated gold nanoparticle upon its exposure to a laser pulse. PNBs enabled the mechanical injection of the extracellular cDNA plasmid into the cytoplasm of individual target living cells, cultured leukemia cells and human CD34+ CD117+ stem cells and expression of a green fluorescent protein (GFP) in those cells. PNB generation and lifetime correlated with the expression of green fluorescent protein in PNB-treated cells. Optical scattering by PNBs additionally provided the detection of the target cells and the guidance of cDNA injection at single cell level. In both cell models PNBs demonstrated a gene transfection effect in a single pulse treatment with high selectivity, efficacy and safety. Thus, PNBs provided targeted gene delivery at the single cell level in a single pulse procedure that can be used for safe and effective gene therapy.
The photothermal (PT) outputs of individual gold nanoparticles (NP) were compared at room (cold) and high transient (hot) temperatures. High temperatures were induced in NPs by a single 0.5 ns laser pulse. All NPs with near-infrared plasmon resonances (rods, shells and bi-pyramids) exhibited a significant decrease in their photothermal output at the resonant wavelengths under high temperature, while non-resonant excitation of the same NPs provided several times higher PT efficacy of the hot NPs. This "inversion" of the PT efficacy of hot plasmonic NPs near their plasmon resonances might have been caused by damping of their resonances due to heating and surface melting. Therefore, photothermal output of plasmonic nanoparticles significantly depends upon their thermal state including the shift in excitation wavelength in hot nanoparticles. In particular, NPs with near-infrared resonances perform several times more efficiently at non-resonant excitation wavelengths rather than at the resonant ones.
Cell theranostics is a new approach that unites diagnosis, therapy and confirmation (guidance) of the results of therapy in one single process at cell level, thus principally improving both the rapidity and precision of treatment. The ideal theranostic agent will support all three of the above functions in vivo with cellular resolution, allowing individual assessment of disease state and the elimination of diseased cells while leaving healthy cells intact. We have developed and evaluated plasmonic nanobubbles (PNBs) as an in vivo tunable theranostic cellular agent in zebrafish hosting prostate cancer xenografts. PNBs were selectively generated around gold nanoparticles in cancer cells in the zebrafish with short single laser pulses. By varying the energy of the laser pulse, we dynamically tuned the PNB size in a theranostic sequence of two PNBs: an initial small PNB detected a cancer cell through optical scattering, followed by a second bigger PNB, which mechanically ablated this cell without damage to surrounding tissue, while its optical scattering confirmed the destruction of the cell. Thus PNBs supported the diagnosis and guided ablation of individual human cancer cells in a living organism without damage to the host.
We have used short laser pulses to generate transient vapor nanobubbles around plasmonic nanoparticles. The photothermal, mechanical, and optical properties of such bubbles were found to be different from those of plasmonic nanoparticle and vapor bubbles, as well. This phenomenon was considered as a new complex nanosystem-plasmonic nanobubble (PNB). Mechanical and optical scattering properties of PNB depended upon the nanoparticle surface and heat capacity, clusterization state, and the optical pulse length. The generation of the PNB required much higher laser pulse fluence thresholds than the explosive boiling level and was characterized by the relatively high lower threshold of the minimal size (lifetime) of PNB. Optical scattering by PNB and its diameter (measured as the lifetime) has been varied with the fluence of laser pulse, and this has demonstrated the tunable nature of PNB.
A new method of optically guided controlled release was experimentally evaluated with liposomes containing a molecular load and gold nanoparticles (NPs). NPs were exposed to short laser pulses to induce transient vapor bubbles around the NPs, plasmonic nanobubbles, in order to disrupt the liposome and eject its molecular contents. The release efficacy was tuned by varying the lifetime and size of the nanobubble with the fluence of the laser pulse. Optical scattering by nanobubbles correlated to the molecular release and was used to guide the release. The release of two fluorescent proteins from individual liposomes has been directly monitored by fluorescence microscopy, while the generation of the plasmonic nanobubbles was imaged and measured with optical scattering techniques. Plasmonic nanobubble-induced release was found to be a mechanical, nonthermal process that requires a single laser pulse and ejects the liposome contents within a millisecond timescale without damage to the molecular cargo and that can be controlled through the fluence of laser pulse.
This article is focused on the optical generation and detection of photothermal vapor bubbles around plasmonic nanoparticles. We report physical properties of such plasmonic nanobubbles and their biomedical applications as cellular probes. Our experimental studies of gold nanoparticle-generated photothermal bubbles demonstrated the selectivity of photothermal bubble generation, amplification of optical scattering and thermal insulation effect, all realized at the nanoscale. The generation and imaging of photothermal bubbles in living cells (leukemia and carcinoma culture and primary cancerous cells), and tissues (atherosclerotic plaque and solid tumor in animal) demonstrated a noninvasive highly sensitive imaging of target cells by small photothermal bubbles and a selective mechanical, nonthermal damage to the individual target cells by bigger photothermal bubbles due to a rapid disruption of cellular membranes. The analysis of the plasmonic nanobubbles suggests them as theranostic probes, which can be tuned and optically guided at cell level from diagnosis to delivery and therapy during one fast process.
Transient photothermal phenomena in the environment of light-absorbing plasmonic nanoparticles, heating and evaporation, were shown to influence the optical scattering efficacy of such nanoparticles, when they absorb and scatter the light. The heating of the environment suppresses the optical scattering, while the evaporation enhances the scattering by the nanoparticles. These opposite effects have transient, local, and thermal nature and significantly (more than 10 times) influence the optical contrast of the nanoparticles as shown for gold spheres in water.
Laser-induced generation of the vapor bubbles in water around plasmonic nanoparticles was experimentally studied with optical scattering methods. Nanoparticle-generated bubbles temporally and spatially localize laser-induced thermal field and also amplify optical scattering relatively to that of gold nanoparticles. Bubble lifetimes and threshold fluencies were determined as functions of the laser (pulse duration, fluence, inter-pulse interval), nanoparticle (size, shape, aggregation state) and sample chamber parameters so to optimize bubble generation around plasmonic nanoparticles. Nanoparticle-generated bubbles are suggested as nano-scaled optical sensors and sources of localized thermal and mechanical impact.
Current methods of cell processing for gene and cell therapies use several separate procedures for gene transfer and cell separation or elimination, because no current technology can offer simultaneous multifunctional processing of specific cell subsets in highly heterogeneous cell systems. Using the cell-specific generation of plasmonic nanobubbles of different sizes around cell-targeted gold nanoshells and nanospheres, we achieved simultaneous multifunctional cell-specific processing in a rapid single 70 ps laser pulse bulk treatment of heterogeneous cell suspension. This method supported the detection of cells, delivery of external molecular cargo to one type of cells and the concomitant destruction of another type of cells without damaging other cells in suspension, and real-time guidance of the above two cellular effects.
The transient 100-fold enhancement and spectral narrowing to 2 nm of the photothermal conversion by solid gold nanospheres under near-infrared excitation with a short laser pulse is reported. This non-stationary effect was observed for a wide range of optical fluences starting from 10 mJ cm(-2) for single nanospheres, their ensembles and aggregated clusters in water, in vitro and in vivo.
The resistance of residual cancer cells after oncological resection to adjuvant chemoradiotherapies results in both high recurrence rates and high non-specific tissue toxicity, thus preventing the successful treatment of such cancers as head and neck squamous cell carcinoma (HNSCC). The patients survival rate and quality of life therefore depend upon the efficacy, selectivity and low non-specific toxicity of the adjuvant treatment. We report a novel, theranostic in vivo technology that unites both the acoustic diagnostics and guided intracellular delivery of anti-tumor drug (liposome-encapsulated doxorubicin, Doxil) in one rapid process, namely a pulsed laser-activated plasmonic nanobubble (PNB). HNSCC-bearing mice were treated with gold nanoparticle conjugates, Doxil, and single near-infrared laser pulses of low energy. Tumor-specific clusters of gold nanoparticles (solid gold spheres) converted the optical pulses into localized PNBs. The acoustic signals of the PNB detected the tumor with high specificity and sensitivity. The mechanical impact of the PNB, co-localized with Doxil liposomes, selectively ejected the drug into the cytoplasm of cancer cells. Cancer cell-specific generation of PNBs and their intracellular co-localization with Doxil improved the in vivo therapeutic efficacy from 5-7% for administration of only Doxil or PNBs alone to 90% thus demonstrating the synergistic therapeutic effect of the PNB-based intracellular drug release. This mechanism also reduced the non-specific toxicity of Doxil below a detectable level and the treatment time to less than one minute. Thus PNBs combine highly sensitive diagnosis, overcome drug resistance and minimize non-specific toxicity in a single rapid theranostic procedure for intra-operative treatment.
MUC1 is a large, heavily glycosylated transmembrane glycoprotein that is proposed to create a protective microenvironment in many adenocarcinomas. Here we compare MUC1 and the well studied cell surface receptor target, EGFR, as gold nanoparticle (AuNP) targets and their subsequent vapor nanobubble generation efficacy in the human epithelial cell line, HES. Although EGFR and MUC1 were both highly expressed in these cells, TEM and confocal images revealed MUC1 as a superior target for nanoparticle intracellular accumulation and clustering. The MUC1-targeted AuNP intracellular clusters also generated significantly larger vapor nanobubbles. Our results demonstrate the promising opportunities MUC1 offers to improve the efficacy of targeted nanoparticle based approaches.
Optimal cell therapies require efficient, selective and rapid delivery of molecular cargo into target cells without compromising their viability. Achieving these goals ex vivo in bulk heterogeneous multi-cell systems such as human grafts is impeded by low selectivity and speed of cargo delivery and by significant damage to target and non-target cells. We have developed a cell level approach for selective and guided transmembrane injection of extracellular cargo into specific target cells using transient plasmonic nanobubbles (PNB) as cell-specific nano-injectors. As a technical platform for this method we developed a laser flow cell processing system. The PNB injection method and flow system were tested in heterogeneous cell suspensions of target and non-target cells for delivery of Dextran-FITC dye into squamous cell carcinoma HN31 cells and transfection of human T-cells with a green fluorescent protein-encoding plasmid. In both models the method demonstrated single cell type selectivity, high efficacy of delivery (96% both for HN31 cells T-cells), speed of delivery (nanoseconds) and viability of treated target cells (96% for HN31 cells and 75% for T-cells). The PNB injection method may therefore be beneficial for real time processing of human grafts without removal of physiologically important cells.
The limited specificity of nanoparticle (NP) uptake by target cells associated with a disease is one of the principal challenges of nanomedicine. Using the threshold mechanism of plasmonic nanobubble (PNB) generation and enhanced accumulation and clustering of gold nanoparticles in target cells, we increased the specificity of PNB generation and detection in target versus non-target cells by more than one order of magnitude compared to the specificity of NP uptake by the same cells. This improved cellular specificity of PNBs was demonstrated in six different cell models representing diverse molecular targets such as epidermal growth factor receptor, CD3 receptor, prostate specific membrane antigen and mucin molecule MUC1. Thus PNBs may be a universal method and nano-agent that overcome the problem of non-specific uptake of NPs by non-target cells and improve the specificity of NP-based diagnostics, therapeutics and theranostics at the cell level.
The photothermal efficacy of near-infrared gold nanoparticles (NP), nanoshells, and nanorods was studied under pulsed high-energy optical excitation in plasmonic nanobubble (PNB) mode as a function of the wavelength and duration of the excitation laser pulse. PNBs, transient vapor nanobubbles, were generated around individual and clustered overheated NPs in water and living cells. Transient PNBs showed two photothermal features not previously observed for NPs: the narrowing of the spectral peaks to 1 nm and the strong dependence of the photothermal efficacy upon the duration of the laser pulse. Narrow red-shifted (relative to those of NPs) near-infrared spectral peaks were observed for 70 ps excitation laser pulses, while longer sub- and nanosecond pulses completely suppressed near-infrared peaks and blue shifted the PNB generation to the visual range. Thus, PNBs can provide superior spectral selectivity over gold NPs under specific optical excitation conditions.
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