Instant Minimally Invasive Detection of Lung Cancer Through Fiber Optical Generation and Detection of Plasmonic Nanobubbles Around TiN Nanoparticles.

Chemotherapies are often impeded by drug resistance of cancer cells, high non-specific toxicity and low selectivity and efficacy of drug delivery. We developed a platform for the selective, fast, guided intracellular delivery of drugs into cancer cells with new cell-specific agents, plasmonic nanobubbles (PNBs). PNBs are not particles but transient events, vapor nanobubbles, induced by a short laser pulse around gold nanoparticles. Therapeutic effects of PNBs have a localized mechanical, non-thermal, nature and can be dynamically tuned in cancer cells to support non-invasive imaging, disruption of endosomes and drug carriers, injection of drug and mechanical cell ablation with single cell selectivity in a heterogeneous tissue. Simultaneous treatment of a bulk tissue activates those functions only in cancer cells while leaving adjacent normal cells intact. Optical and acoustical properties of PNBs provide a real time guidance of therapeutic effect. PNB mechanisms were evaluated for intracellular delivery of free and encapsulated doxorubicin into drug-resistant oral cavity squamous cell carcinoma (OCSCC) cells mixed in a co-culture with normal cells. Gold nanoshells were conjugated with Panitumumab antibody to target EGFR that is overexpressed by OCSCC. Receptor-endocytotic targeting resulted in cancer cell-specific clusters of nanoshells. These clusters selectively generated PNBs only in cancer cells while normal cells did not produce PNBs under simultaneous treatment with a single, short near-infrared laser pulse of low, biologically safe, energy. PNBs selectively delivered free extracellular drug only to cancer cells by creating transient nano-holes in cellular membranes and inbound nanojets that brought the extracellular drug into the cytoplasm. Thus PNBs worked as nano-injector of extracellular drug. Compared to a standard treatment with doxorubicin alone, PNB injection improved therapeutic efficacy by 12-fold and at the same time reduced drug dose by 10-fold and non-specific toxicity by 3-fold. Plasmonic nanobubble-enhanced endosomal escape (PNBEE) provided intracellular delivery of the liposome-encapsulated drug, Doxil, that was administered separately with gold nanoshells through a self-assembly of mixed nanoshell-Doxil nanoclusters by cancer cells. Small PNBs generated in these nanoclusters disrupted both liposomes and endosomes and ejected the released doxorubicin into cytoplasm. Cancer cell-specific generation of PNBs in a mixed co-culture of OCSCC and normal cells increased the therapeutic efficacy of Doxil by 31-fold and at the same time reduced drug dose by 20-fold, treatment time by 3-fold and non-specific toxicity by 10-fold. The described mechanisms of nano-injection and PNBEE are universal, can be applied to various cancers and provide a guided intracellular drug delivery in heterogeneous tissues through the application of plasmonic nanobubbles. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5693. doi:1538-7445.AM2012-5693

Titanium nitride (TiN) is a candidate material for several plasmonic applications, and pulsed laser ablation in liquids (PLAL) represents a rapid, scalable, and environmentally friendly approach for the large-scale production of nanomaterials with customized properties. In this work, the nanosecond PLAL process is developed, and we provide a concise understanding of the process parameters, such as the solvent and the laser fluence and pulse wavelength, to the size and structure of the produced TiN nanoparticles (NPs). TiN films of a 0.6 μm thickness developed by direct-current (DC) magnetron sputtering were used as the ablation targets. All laser process parameters lead to the fabrication of spherical NPs, while the laser pulse fluence was used to control the NPs' size. High laser pulse fluence values result in larger TiN NPs (diameter around 42 nm for 5 mJ and 25 nm for 1 mJ), as measured from scanning electron microscopy (SEM). On the other hand, the wavelength of the laser pulse does not affect the mean size of the TiN NPs (24, 26, and 25 nm for 355, 532, and 1064 nm wavelengths, respectively). However, the wavelength plays a vital role in the quality of the produced TiN NPs. Shorter wavelengths result in NPs with fewer defects, as indicated by Raman spectra and XPS analysis. The solvent type also significantly affects the size of the NPs. In aqueous solutions, strong oxidation of the NPs is evident, while organic solvents such as acetone, carbides, and oxides cover the TiN NPs.

Surface-Enhanced Raman scattering of methylene blue on titanium nitride nanoparticles synthesized by laser ablation in organic solvents

Herein, we report the fabrication and characterization of novel polycaprolactone (PCL)-based nanofibers functionalized with bare (ligand-free) titanium nitride (TiN) nanoparticles (NPs) for tissue engineering applications. Nanofibers were prepared by a newly developed protocol based on the electrospinning of PCL solutions together with TiN NPs synthesized by femtosecond laser ablation in acetone. The generated hybrid nanofibers were characterised using spectroscopy, microscopy, and thermal analysis techniques. As shown by scanning electron microscopy measurements, the fabricated electrospun nanofibers had uniform morphology, while their diameter varied between 0.403 ± 0.230 µm and 1.1 ± 0.15 µm by optimising electrospinning solutions and parameters. Thermal analysis measurements demonstrated that the inclusion of TiN NPs in nanofibers led to slight variation in mass degradation initiation and phase change behaviour (Tm). In vitro viability tests using the incubation of 3T3 fibroblast cells in a nanofiber-based matrix did not reveal any adverse effects, confirming the biocompatibility of hybrid nanofiber structures. The generated hybrid nanofibers functionalized with plasmonic TiN NPs are promising for the development of smart scaffold for tissue engineering platforms and open up new avenues for theranostic applications.

Laser-synthesized TiN nanoparticles for biomedical applications: Evaluation of safety, biodistribution and pharmacokinetics

Owing to a strong photothermal response in the near-IR spectral range and very low toxicity, titanium nitride (TiN) nanoparticles (NPs) synthesized by pulsed laser ablation in liquids (PLAL) present a novel appealing object for photo-induced therapy of cancer, but the properties of these NPs still require detailed investigation. Here, we have elaborated methods of femtosecond laser ablation from the TiN target in a variety of liquid solutions, including acetonitrile, dimethylformamide, acetone, water, and H2O2, to synthesize TiN NPs and clarify the effect of liquid type on the composition and properties of the formed NPs. The ablation in all solvents led to the formation of spherical NPs with a mean size depending on the liquid type, while the composition of the NPs ranged from partly oxidized TiN to almost pure TiO2, which conditioned variations of plasmonic peak in the region of relative tissue transparency (670–700 nm). The degree of NP oxidation depended on the solvent, with much stronger oxidation for NPs prepared in aqueous solutions (especially in H2O2), while the ablation in organic solvents resulted in a partial formation of titanium carbides as by-products. The obtained results contribute to better understanding of the processes in reactive PLAL and can be used to design TiN NPs with desired properties for biomedical applications.

Titanium nitride (TiN) nanoparticles (NPs) prepared by methods of laser ablation in liquids present a novel object, which promises attractive biomedical applications. Here, we review our recent advances in the elaboration of femtosecond laser ablation technique from a TiN target in liquid medium (here, isopropanol) to maximize the efficiency of TiN NPs synthesis and optimize their characteristics. Our experiments showed that the synthesis productivity is dependent on lateral velocity of laser beam scanning during laser ablation with the achievement of productivity saturation at a certain relatively high velocity. The observed phenomenon was attributed to the interaction of laser pulses with cavitation bubbles generated during the ablation process. In addition, we assessed photo heating properties of the synthesized TiN NPs in the near-IR range. It was found that TiN NPs with sizes in 20-50 nm range have the highest heating rate and can be heated to maximal temperatures. As demonstrated by our recent tests in vitro and in vivo, this size range is optimal for biomedical applications, which promises successful applications of these nanoparticles in phototherapy and imaging modalities.

Effect of titanium nitride nanoparticles on grain size stabilization and consolidation of cryomilled titanium

We have studied the effect of chromium disilicide and titanium nitride nanoparticles on the expression level of gene FABP4 (fatty acid binding protein 4), which reflects the cell stress condition, including the endoplasmic reticulum stress. It was shown that treatment of mice by titanium nitride and chromium disilicide nanoparticles (20 nm; 20 mg with food every working day for 2 months) upregulated the expression of FABP4 gene in mouse liver but effect of chromium disilicide was much stronger. It was also found reduced level of free radicals production in the liver of mice, which indicating depletion of the antioxidant system. In particular, it was shown the decrease of catalase activity in 2 times upon treatment by both chromium disilicide and titanium nitride nanoparticles. Present study demonstrates that chromium disilicide and titanium nitride nanoparticles affects the expression of FABP4 gene in mouse liver, which possibly reflects genotoxic activities of both types of nanoparticles, but molecular mechanisms of their action on the genome warrant further investigation.

Combining photonic excitation and acoustic detection, photoacoustic imaging (PAI) presents one of the most promising noninvasive biomedical diagnostic modalities, but this technique still lacks efficient nano-sized contrast agents absorbing light in the region of relative tissue transparency (630–900 nm). Here, we explore the use of titanium nitride (TiN) nanoparticles (NPs) fabricated by methods of pulsed laser ablation in liquids as a contrast agent in PAI. When prepared in acetone, the NPs are spherical, have an average size of 25 nm, and exhibit a broad plasmonic absorption peak around 700 nm. We show that solutions of these NPs render possible a strong nonlinear photoacoustic response and the generation of photoacoustic images with 67 μm resolution within the biological transparency window. The observed effect is explained by a plasmonically enhanced two-photon absorption process in TiN NPs. Combined with earlier demonstrated capability of generating photothermal therapeutic effect, relative chemical purity, and excellent biocompatibility, laser-synthesized TiN NPs promise attractive applications in biomedical theranostics involving imaging modalities based on photoacoustics microscopy or tomography.

The in vivo performance of plasmonic nanobubbles as cell theranostic agents in zebrafish hosting prostate cancer xenografts

In this work, a promising strategy to increase the broadband solar light absorption was developed by synthesizing a composite of metal-free carbon nitride-carbon dots (C3N4-C dots) and plasmonic titanium nitride (TiN) nanoparticles (NPs) to improve the photoelectrochemical water-splitting performance under simulated solar radiation. Hot-electron injection from plasmonic TiN NPs to C3N4 played a role in photocatalysis, whereas C dots acted as catalysts for the decomposition of H2O2 to O2. The use of C dots also eliminated the need for a sacrificial reagent and prevented catalytic poisoning. By incorporating the TiN NPs and C dots, a sixfold improvement in the catalytic performance of C3N4 was observed. The proposed approach of combining TiN NPs and C dots with C3N4 proved effective in overcoming low optical absorption and charge recombination losses and also widens the spectral window, leading to improved photocatalytic activity.

Optically guided controlled release from liposomes with tunable plasmonic nanobubbles

Titanium nitride (TiN)‐modified hydrophobic‐treated carbon paper (CP) was prepared by impregnating CP with polytetrafluoroethylene (PTFE) emulsion containing TiN nanoparticles (NPs). Electrochemical measurements showed that CP witnessed an increase in corrosion resistance and electrical resistivity after treated with 5 wt% PTFE emulsion. Since the sacrifice in electrical conductivity is not beneficial to the application in proton exchange membrane fuel cells (PEMFCs), conductive ceramic NPs – TiN NPs were introduced into the CP to further improve its electrical conductivity. It was found that after adding TiN NPs, the electrical resistivity of CP (immersed in 5 wt% PTFE emulsion containing 8 wt% TiN NPs) decreased to a level (3.8 mΩ cm) similar to that of untreated CP (3.5 mΩ cm), and meanwhile there is a further improvement in corrosion resistance, where the corrosion current density (at 1.4 V) of TiN‐modified CP stabilises at the lowest value of 5.50 μA/cm 2 , as compared with 32.8 and 12.7 μA/cm 2 for raw CP and PTFE‐treated CP. According to microstructure characterisation, these improvements could be attributable to the formation of conductive networks and improved PTFE distribution due to the addition of TiN NPs.

Event Abstract Back to Event Phage-templated gold nano-aggregates for cancer treatment Esen Sokullu1, Aziz Berchtikou1, Amy Blum2, Tsuneyuki Ozaki1 and Marc A. Gauthier1 1 Institut national de la recherche scientifique (INRS), Énergie, Matériaux et Télécommunications (EMT), Canada 2 McGill University, Chemistry Department, Canada Theranostic is an approach combining diagnosis and therapy to increase the precision and efficacy of cancer treatment. Different nanoparticle (NP) platforms have been engineered to obtain both functions by uniting them in one agent; however none of these can overcome the limitation in specificity of NP targeting, which is still a major challenge in cancer treatment. Plasmonic nanobubbles (PNB) are a new class of nano-agent that was developed to overcome this limitation. A PNB is a vapor nanobubble transiently generated around superheated gold NPs by a short laser pulse. The rapidly expanding bubble mechanically disrupts the surrounding tissue and achieves micrometer precision in tissue ablation. In general, PNB therapy relies on receptor-mediated endocytosis of gold NPs and formation of gold clusters within the cells [1],[2]. However, variability in the cellular uptake and subsequent aggregation of gold NPs may be detrimental to reproducibility of PNB. In this study, M13 and T4 phage display platforms are used to synthesize well-defined gold nano-aggregates and their efficiency in formation of gold nano-aggregates are evaluated. Two different T4 phage templates were designed by displaying cysteine and gold binding A3 peptide (AYSSGAPPMPPF) (A: Alanine, Y: Tyrosine, S: Serine, G: Glycine, P: Proline, M: Methionine, F: Phenylalanine, L: Leucine, K: Lysine, H: Histidine, R: Arginine, V: Valine, D: Aspartic acid) on small outer capsid (SOC) protein of phage through the homologous recombination between T4-Z1 mutant phage and pRH recombination plasmid. Ph.D. Peptide Display Cloning System (Biolabs Inc.) was used to design M13 phage templates. Self assembly of gold NP was directed by displaying gold binding peptide sequence (VSGSSPDS) on p8 major coat protein of phage. M13 phage templates were also engineered to display RGD peptides (ACDCRGDCFC, ACRGDGWC, GRGDSP) (C; Cysteine, W; Tryptophan) on p3 minor coat protein to target cancer cells through integrin protein. Recombinant phages were analyzed by PCR and also verified via DNA sequencing. The affinities of RGD displaying M13 phages were tested against human integrin alpha 5 protein fragment by phage-ELISA and M13 phages displaying cyclic RGD peptides (with two and four cysteines) showed significant binding affinity to integrin. Gold NPs were synthesized according to the protocol of Zahr and Blum and formation of gold nano-aggregates was initially studied by using cysteine bearing T4 phage template [3]. Conjugation of gold NPs to phage capsid was monitored by using UV-Vis spectrometer and the growth of plasmon at 600 nm was recorded. After 3 days of reaction, the samples were visualized by transmission electron microscopy (TEM) and formation of phage-templated gold nano-aggregates was confirmed. Herein, it was shown that cysteine-displaying T4 phage was successfully used as a template to develop well-defined gold nano-aggregates. The number of functional moieties on phage surface can be controlled at gene level and make us to avoid the use of irreproducible functionalization steps. The future work will focus on optimization of gold nano-aggregate formation for each phage design and their efficiency in PNB generation will be investigated. We would like to acknowledge that T4Z1 mutant phage and pRH recombination plasmid were kindly supplied by Prof. Lindsay W. Black (University of Maryland, US).