Effectiveness of Gold Nanorods of Different Sizes in Photothermal Therapy to Eliminate Melanoma and Glioblastoma Cells.

Gold nanorods are the most commonly used nanoparticles in photothermal therapy for cancer treatment due to their high efficiency in converting light into heat. This study aimed to investigate the efficacy of gold nanorods of different sizes (large and small) in eliminating two types of cancer cell: melanoma and glioblastoma cells. After establishing the optimal concentration of nanoparticles and determining the appropriate time and power of laser irradiation, photothermal therapy was applied to melanoma and glioblastoma cells, resulting in the highly efficient elimination of both cell types. The efficiency of the PTT was evaluated using several methods, including biochemical analysis, fluorescence microscopy, and flow cytometry. The dehydrogenase activity, as well as calcein-propidium iodide and Annexin V staining, were employed to determine the cell viability and the type of cell death triggered by the PTT. The melanoma cells exhibited greater resistance to photothermal therapy, but this resistance was overcome by irradiating cells at physiological temperatures. Our findings revealed that the predominant cell-death pathway activated by the photothermal therapy mediated by gold nanorods was apoptosis. This is advantageous as the presence of apoptotic cells can stimulate antitumoral immunity in vivo. Considering the high efficacy of these gold nanorods in photothermal therapy, large nanoparticles could be useful for biofunctionalization purposes. Large nanorods offer a greater surface area for attaching biomolecules, thereby promoting high sensitivity and specificity in recognizing target cancer cells. Additionally, large nanoparticles could also be beneficial for theranostic applications, involving both therapy and diagnosis, due to their superior detection sensitivity.

Microfluidic fabrication of vesicles with hybrid lipid/nanoparticle bilayer membranes.

Hybrid lipid/nanoparticle membranes are suitable model systems both to study the complex interactions between nanoparticles and biological membranes, and to demonstrate technological concepts in cellular sensing and drug delivery. Unfortunately, embedding nanoparticles into the bilayer membrane of lipid vesicles is challenging due to the poor control over the vesicle fabrication process of conventional methodologies and the fragility of the modified lipid bilayer assembly. Here, the utility of water-in-oil-in-water double emulsion drops with ultrathin oil shells as templates to form vesicles with hybrid lipid/nanoparticle membranes is reported. Moreover, upon bilayer formation, which occurs through dewetting of the oil solvent from the double emulsion drops, a phase separation is observed in the vesicle membrane, with solid-like nanoparticle-rich microdomains segregated into a continuous fluid-like nanoparticle-poor phase. This phase coexistence evidences the complex nature of the interactions between nanoparticles and lipid membranes. In this context, this microfluidic-assisted fabrication strategy may play a crucial role in thoroughly understanding such interactions given the uniform membrane properties of the resulting productions. Furthermore, the high encapsulation efficiency of both the vesicle membrane and core endows these vesicles with great potential for sensing applications and drug delivery.

pH-triggered endosomal escape of pore-forming Listeriolysin O toxin-coated gold nanoparticles.

BACKGROUND: A major bottleneck in drug delivery is the breakdown and degradation of the delivery system through the endosomal/lysosomal network of the host cell, hampering the correct delivery of the drug of interest. In nature, the bacterial pathogen Listeria monocytogenes has developed a strategy to secrete Listeriolysin O (LLO) toxin as a tool to escape the eukaryotic lysosomal system upon infection, allowing it to grow and proliferate unharmed inside the host cell. RESULTS: As a "proof of concept", we present here the use of purified His-LLO H311A mutant protein and its conjugation on the surface of gold nanoparticles to promote the lysosomal escape of 40 nm-sized nanoparticles in mouse embryonic fibroblasts. Surface immobilization of LLO was achieved after specific functionalization of the nanoparticles with nitrile acetic acid, enabling the specific binding of histidine-tagged proteins. CONCLUSIONS: Endosomal acidification leads to release of the LLO protein from the nanoparticle surface and its self-assembly into a 300 Å pore that perforates the endosomal/lysosomal membrane, enabling the escape of nanoparticles.

Mechanosensitive Gold Colloidal Membranes Mediated by Supramolecular Interfacial Self-Assembly.

The ability to respond toward mechanical stimuli is a fundamental property of biological organisms at both the macroscopic and cellular levels, yet it has been considerably less observed in artificial supramolecular and colloidal homologues. An archetypal example in this regard is cellular mechanosensation, a process by which mechanical forces applied on the cell membrane are converted into biochemical or electrical signals through nanometer-scale changes in molecular conformations. In this article, we report an artificial gold nanoparticle (Au NP)-discrete π-conjugated molecule hybrid system that mimics the mechanical behavior of biological membranes and is able to self-assemble into colloidal gold nanoclusters or membranes in a controlled and reversible fashion by changing the concentration or the mechanical force (pressure) applied. This has been achieved by rational design of a small π-conjugated thiolated molecule that controls, to a great extent, the hierarchy levels involved in Au NP clustering by enabling reversible, cooperative non-covalent (π-π, solvophobic, and hydrogen bonding) interactions. In addition, the Au NP membranes have the ability to entrap and release aromatic guest molecules reversibly (Kb = 5.0 × 105 M-1) for several cycles when subjected to compression-expansion experiments, in close analogy to the behavior of cellular mechanosensitive channels. Not only does our hybrid system represent the first example of a reversible colloidal membrane, but it also can be controlled by a dynamic mechanical stimulus using a new supramolecular surface-pressure-controlled strategy. This approach holds great potential for the development of multiple colloidal assemblies within different research fields.

Reshaping, Fragmentation, and Assembly of Gold Nanoparticles Assisted by Pulse Lasers.

The vast majority of the outstanding applications of metal nanoparticles (NPs) developed during the last two decades have arisen from their unique optical properties. Within this context, rational synthesis and assembly of gold NPs have been the main research focus, aiming at the design of nanoplasmonic devices with tailored optical functionalities. The progress made in this field is thus to be ascribed to the understanding of the origin of the interaction between light and such gold nanostructures, the dynamics of which have been thoroughly investigated with significant contributions from short and ultrashort pulse laser technologies. We focus this Account on the potential of pulse lasers to provide new fundamental insights into the electron dynamics involved in the interaction of light with the free conduction electrons of Au NPs, that is, localized surface plasmon resonances (LSPRs). The excitation of LSPRs with a femtosecond pulse laser is followed by thermalization of the Au NP electrons and the subsequent relaxation of the nanocrystal lattice and the surrounding environment, which generally results in surface melting. By contrast, nanosecond irradiation usually induces AuNP fragmentation and uncontrolled melting due to overlapping excitation and relaxation phenomena. These concepts have been exploited toward the preparation of highly monodisperse gold nanospheres via pulse laser irradiation of polyhedral nanocrystal colloids, or in the fabrication of nanostructures with "written-in" optical properties. The applicability of pulsed coherent light has been extended toward the direct synthesis and manipulation of Au NPs. Through ablation of a gold target in a liquid with pulse lasers, spherical Au NPs can be synthesized with no need of stabilizing ligands, which is a great advantage in terms of reducing toxicity, rendering these NPs particularly suitable for medical applications. In addition, femtosecond laser irradiation has been proven a unique tool for the controlled welding of plasmonic gold nanostructures by electromagnetic field enhancement at the hot spots of assembled Au NPs. The combination of such nanostructures with pulse lasers promises significant chemical and biochemical advances, including the structural determination of organic reaction intermediates, the investigation of phase transitions in inorganic nanomaterials at mild reaction conditions, or the efficient photothermal destruction of cancer cells avoiding damage of surrounding tissue.

Remote Control by π-Conjugation of the Emissive Properties of Fischer Carbene-BODIPY Dyads.

The synthesis, structure, and complete characterization of mono- and bimetallic dyads joining Fischer carbene complexes and BODIPY chromophores are reported. In these organometallic species, the Fischer carbene complex is attached to the BODIPY moiety through a p-aminophenyl group linked at the C8 carbon atom of the BODIPY core. The photophysical properties, namely the corresponding UV/vis absorption and emission spectra of these new metal-carbene complexes, are analyzed and discussed. It is found that whereas the absorption of the considered dyads strongly resembles that of the parent 4-anilinyl-substituted BODIPY, the fluorescence emission is significantly reduced in these species, very likely as a result of a Förster-type energy transfer mechanism. At variance, the replacement of the pentacarbonyl-metal(0) fragment by a carbonyl group leads to high fluorescence emission intensity. In addition, the emissive properties of the BODIPY core in these organometallic dyads can be tuned by remote groups by means of π-conjugation, as supported by density functional theory calculations.

Femtosecond Laser-Controlled Tip-to-Tip Assembly and Welding of Gold Nanorods.

Directed assembly of gold nanorods through the use of dithiolated molecular linkers is one of the most efficient methodologies for the morphologically controlled tip-to-tip assembly of this type of anisotropic nanocrystals. However, in a direct analogy to molecular polymerization synthesis, this process is characterized by difficulties in chain-growth control over nanoparticle oligomers. In particular, it is nearly impossible to favor the formation of one type of oligomer, making the methodology hard to use for actual applications in nanoplasmonics. We propose here a light-controlled synthetic procedure that allows obtaining selected plasmonic oligomers in high yield and with reaction times in the scale of minutes by irradiation with low fluence near-infrared (NIR) femtosecond laser pulses. Selective inhibition of the formation of gold nanorod n-mers (trimers) with a longitudinal localized surface plasmon in resonance with a 800 nm Ti:sapphire laser, allowed efficient trapping of the (n - 1)-mers (dimers) by hot spot mediated photothermal decomposition of the interparticle molecular linkers. Laser irradiation at higher energies produced near-field enhancement at the interparticle gaps, which is large enough to melt gold nanorod tips, offering a new pathway toward tip-to-tip welding of gold nanorod oligomers with a plasmonic response at the NIR. Thorough optical and electron microscopy characterization indicates that plasmonic oligomers can be selectively trapped and welded, which has been analyzed in terms of a model that predicts with reasonable accuracy the relative concentrations of the main plasmonic species.

Nucleation of Amyloid Oligomers by RepA-WH1-Prionoid-Functionalized Gold Nanorods.

Understanding protein amyloidogenesis is an important topic in protein science, fueled by the role of amyloid aggregates, especially oligomers, in the etiology of a number of devastating human degenerative diseases. However, the mechanisms that determine the formation of amyloid oligomers remain elusive due to the high complexity of the amyloidogenesis process. For instance, gold nanoparticles promote or inhibit amyloid fibrillation. We have functionalized gold nanorods with a metal-chelating group to selectively immobilize soluble RepA-WH1, a model synthetic bacterial prionoid, using a hexa-histidine tag (H6). H6-RepA-WH1 undergoes stable amyloid oligomerization in the presence of catalytic concentrations of anisotropic nanoparticles. Then, in a physically separated event, such oligomers promote the growth of amyloid fibers of untagged RepA-WH1. SERS spectral changes of H6-RepA-WH1 on spherical citrate-AuNP substrates provide evidence for structural modifications in the protein, which are compatible with a gradual increase in ß-sheet structure, as expected in amyloid oligomerization.

Using inclusion complexes with cyclodextrins to explore the aggregation behavior of a ruthenium metallosurfactant.

The aggregation behavior of a chiral metallosurfactant, bis(2,2'-bipyridine)(4,4'-ditridecyl-2,2'-bipyridine)ruthenium(II) dichloride (Ru2(4)C13), synthesized as a racemic mixture was characterized by small-angle neutron scattering, light scattering, NMR, and electronic spectroscopies. The analysis of the SANS data indicates that micelles are prolate ellipsoids over the range of concentrations studied, with a relatively low aggregation number, and the micellization takes place gradually with increasing concentration. The presence of cyclodextrins (ß-CD and γ-CD) induces the breakup of the micelles and helps to establish that micellization occurs at a very slow exchange rate compared to the NMR time scale. The open structure of this metallosurfactant enables the formation of very stable complexes of 3:1 stoichiometry, in which one CD threads one of the hydrocarbon tails and two CDs the other, in close contact with the polar head. The complex formed with ß-CD, more stable than the one formed with the wider γ-CD, is capable of resolving the Δ and Λ enantiomers at high CD/surfactant molar ratios. The chiral recognition is possible due to the very specific interactions taking place when the ß-CD covers-via its secondary rim-part of the diimine moiety connected to the hydrophobic tails. A SANS model comprising a binary mixture of hard spheres (complex + micelles) was successfully used to study quantitatively the effect of the CDs on the aggregation of the surfactant.

Tuning the photophysical properties of BODIPY molecules by π-conjugation with Fischer carbene complexes.

The synthesis, structure, and photophysical properties of novel BODIPY-Fischer alkoxy-, thio-, and aminocarbene dyads are reported. The BODIPY chromophore is directly attached to the carbene ligand by an ethylenic spacer, thus forming donor-bridge-acceptor π-extended systems. The extension of the π-conjugation is decisive in the equilibrium geometries of the dyads and is clearly reflected in the corresponding absorption and emission spectra. Whereas the BODIPY fragment is mainly isolated in aminocarbene complexes, it is fully conjugated in alkoxycarbene derivatives. The former thus exhibit the characteristic photophysical properties of BODIPY units, whereas complete suppression of the BODIPY fluorescence emission is observed in the latter, as a direct consequence of the strong electron-accepting character of the (CO)5M=C moiety. As the π-acceptor character of the metal-carbene group can be modified, the electronic properties of the conjugated BODIPY can be tuned. Density functional calculations have been carried out to gain insight into the photophysical properties.

Polyrotaxane-mediated self-assembly of gold nanospheres into fully reversible supercrystals.

The use of a thiol-functionalized nonionic surfactant to stabilize spherical gold nanoparticles in water induces the spontaneous formation of polyrotaxanes at the nanoparticle surface in the presence of the macrocycle α-cyclodextrin. Whereas using an excess of surfactant an amorphous gold nanocomposite is obtained, under controlled drying conditions the self-assembly between the surface supramolecules provides large and homogenous supercrystals with hexagonal close packing of nanoparticles. Once formed, the self-assembled supercrystals can be fully redispersed in water. The reversibility of the crystallization process may offer an excellent reusable material to prepare gold nanoparticle inks and optical sensors with the potential to be recovered after use.

From Multi- to Single-Hollow Trimetallic Nanocrystals by Ultrafast Heating.

Metal nanocrystals (NCs) display unique physicochemical features that are highly dependent on nanoparticle dimensions, anisotropy, structure, and composition. The development of synthesis methodologies that allow us to tune such parameters finely emerges as crucial for the application of metal NCs in catalysis, optical materials, or biomedicine. Here, we describe a synthetic methodology to fabricate hollow multimetallic heterostructures using a combination of seed-mediated growth routes and femtosecond-pulsed laser irradiation. The envisaged methodology relies on the coreduction of Ag and Pd ions on gold nanorods (Au NRs) to form Au@PdAg core-shell nanostructures containing small cavities at the Au-PdAg interface. The excitation of Au@PdAg NRs with low fluence femtosecond pulses was employed to induce the coalescence and growth of large cavities, forming multihollow anisotropic Au@PdAg nanostructures. Moreover, single-hollow alloy AuPdAg could be achieved in high yield by increasing the irradiation energy. Advanced electron microscopy techniques, energy-dispersive X-ray spectroscopy (EDX) tomography, X-ray absorption near-edge structure (XANES) spectroscopy, and finite differences in the time domain (FDTD) simulations allowed us to characterize the morphology, structure, and elemental distribution of the irradiated NCs in detail. The ability of the reported synthesis route to fabricate multimetallic NCs with unprecedented hollow nanostructures offers attractive prospects for the fabrication of tailored high-entropy alloy nanoparticles.

Surfactant (bi)layers on gold nanorods.

Gold nanorods in aqueous solution are generally surrounded by surfactants or capping agents. This is crucial for anisotropic growth during synthesis and for their final stability in solution. When CTAB is used, a bilayer has been evidenced from analytical methods even though no direct morphological characterization of the precise thickness and compactness has been reported. The type of surfactant layer is also relevant to understand the marked difference in further self-assembling properties of gold nanorods as experienced using 16-EO(1)-16 gemini surfactant instead of CTAB. To obtain a direct measure of the thickness of the surfactant layer on gold nanorods synthesized by the seeded growth method, we coupled TEM, SAXS, and SANS experiments for the two different cases, CTAB and gemini 16-EO(1)-16. Despite the strong residual signal from micelles in excess, it can be concluded that the thickness is imposed by the chain length of the surfactant and corresponds to a bilayer with partial interdigitation.

Tunable gold nanorod/NAO conjugates for selective drug delivery in mitochondria-targeted cancer therapy.

Nonyl acridine orange (NAO) is a lipophilic and positively charged molecule widely used as a mitochondrial fluorescent probe. NAO is cytotoxic at micromolar concentration and might be potentially used as a mitochondria-targeted drug for cancer therapy. However, the use of NAO under in vivo conditions would be compromised by the unspecific interactions with off-target cells and negatively charged proteins present in the bloodstream. To tackle this limitation, we have synthesized NAO analogues carrying an imidazole group for their specific binding to nitrilotriacetic (NTA) functionalized gold nanorods (AuNRs). We demonstrate that AuNRs provide 104 binding sites and a controlled delivery under acidic conditions. Upon incubation with mouse embryonic fibroblasts, the endosomal acidic environment releases the NAO analogues from AuNRs, as visualized through the staining of the mitochondrial network. The addition of the monoclonal antibody Cetuximab to the conjugates enhanced their uptake within lung cancer cells and the conjugates were cytotoxic at subnanomolar concentrations (c50 ≈ 0.06 nM). Moreover, the specific interactions of Cetuximab with the epidermal growth factor receptor (EGFR) provided a specific targeting of EGFR-expressing lung cancer cells. After intravenous administration in patient-derived xenografts (PDX) mouse models, the conjugates reduced the progression of EGFR-positive tumors. Overall, the NAO-AuNRs provide a promising strategy to realize membrane mitochondria-targeted conjugates for lung cancer therapy.

SERS chiral recognition and quantification of enantiomers through cyclodextrin supramolecular complexation.

We introduce here a simple approach in which a cyclodextrin, functionalized with thiols in the narrower rim, is assembled onto the silver surface of a SERS platform composed of polystyrene beads coated with silver nanoparticles. Trapping properties of the fabricated sensor are demonstrated through the retention of different enantiomers (R,R or/and S,S) of hydrobenzoin (HBZ), a molecule that has not been reported before in SERS because it has no affinity for coinage metal surfaces. Further, selective recognition of each enantiomer and semiquantification of its proportion in a racemic mixture are provided by the analysis of the SERS spectra of the HBZ-cyclodextrin complex, in full agreement with the surface selection rules.

Molecular organization and effective energy transfer in iridium metallosurfactant-porphyrin assemblies embedded in Langmuir-Schaefer films.

Mixed Langmuir monolayers and Langmuir-Schaefer (LS) films containing the cationic metallosurfactant bis(2-phenylpyridine)(4,4'-diheptadecyl-2,2'-bipyridine)-iridium(III) chloride (Ir-complex) and the anionic tetrakis(4-sulfonatophenyl)porphyrin (TSPP) in 4:1 molar ratio have been successfully prepared by the co-spreading method at the air-water interface. The presence of both luminescent species at the interface, as well as the organization of the TSPP underneath the Ir-complex matrix in Langmuir and LS films, is inferred by surface techniques such as π-A isotherms, reflection spectroscopy, Brewster angle microscopy (BAM) and UV-visible absorption spectroscopy. A red-shift in the absorption band of the porphyrin under the compression of the mixed monolayer suggests the J-aggregation of the TSPP under the Ir-complex matrix. To date, this is the first report of Langmuir and/or LS films containing these two types of species together. Furthermore, the intermolecular energy transfer between Ir-complex and TSPP molecules in solution and in transferred mixed films is investigated through steady-state fluorescence and lifetime measurements. These results indicate that effective intermolecular energy transfer occurs from the Ir-complex to the TSPP molecules in LS films. The influence of the spatial proximity of donor and acceptor molecules has been studied by the insertion of lipid interlayers among them.

Insights into colloidal nanoparticle-protein corona interactions for nanomedicine applications.

Colloidal nanoparticles (NPs) have attracted significant attention due to their unique physicochemical properties suitable for diagnosing and treating different human diseases. Nevertheless, the successful implementation of NPs in medicine demands a proper understanding of their interactions with the different proteins found in biological fluids. Once introduced into the body, NPs are covered by a protein corona (PC) that determines the biological behavior of the NPs. The formation of the PC can eventually favor the rapid clearance of the NPs from the body before fulfilling the desired objective or lead to increased cytotoxicity. The PC nature varies as a function of the different repulsive and attractive forces that govern the NP-protein interaction and their colloidal stability. This review focuses on the phenomenon of PC formation on NPs from a physicochemical perspective, aiming to provide a general overview of this critical process. Main issues related to NP toxicity and clearance from the body as a result of protein adsorption are covered, including the most promising strategies to control PC formation and, thereby, ensure the successful application of NPs in nanomedicine.

Controlled pDNA Release in Gemini Cationic Lipoplexes by Femtosecond Laser Irradiation of Gold Nanostars.

The design of nanovectors able to overcome biological barriers is one of the main challenges in biomedicine. Gemini cationic lipids are considered potential candidates for gene therapy due to their high biocompatibility and capacity to condense nucleic acids safely in the form of lipoplexes. However, this approach presents difficulties regarding genetic unpacking and, therefore, control over this process becomes crucial to ensure successful transfection. In this work, gemini cationic lipoplexes were prepared in the presence of plasmonic gold nanostars (AuNSs) to afford a nanovector that efficiently releases plasmid DNA (pDNA) upon irradiation with near-infrared femtosecond laser pulses. A critical AuNSs concentration of 50 pM and optimized laser power density of 400 mW led to successful pDNA release, whose efficiency could be further improved by increasing the irradiation time. Agarose gel electrophoresis was used to confirm pDNA release. UV-Vis-NIR spectroscopy and transmission electron microscopy studies were performed to monitor changes in the morphology of the AuNSs and lipoplexes after irradiation. From a physicochemical point of view, this study demonstrates that the use of AuNSs combined with gemini cationic lipoplexes allows control over pDNA release under ultrafast laser irradiation.

Gemini Cationic Lipid-Type Nanovectors Suitable for the Transfection of Therapeutic Plasmid DNA Encoding for Pro-Inflammatory Cytokine Interleukin-12.

Ample evidence exists on the role of interleukin-12 (IL-12) in the response against many pathogens, as well as on its remarkable antitumor properties. However, the unexpected toxicity and disappointing results in some clinical trials are prompting the design of new strategies and/or vectors for IL-12 delivery. This study was conceived to further endorse the use of gemini cationic lipids (GCLs) in combination with zwitterionic helper lipid DOPE (1,2-dioleoyl-sn-glycero-3-phosphatidyl ethanol amine) as nanovectors for the insertion of plasmid DNA encoding for IL-12 (pCMV-IL12) into cells. Optimal GCL formulations previously reported by us were selected for IL-12-based biophysical experiments. In vitro studies demonstrated efficient pCMV-IL12 transfection by GCLs with comparable or superior cytokine levels than those obtained with commercial control Lipofectamine2000*. Furthermore, the nanovectors did not present significant toxicity, showing high cell viability values. The proteins adsorbed on the nanovector surface were found to be mostly lipoproteins and serum albumin, which are both beneficial to increase the blood circulation time. These outstanding results are accompanied by an initial physicochemical characterization to confirm DNA compaction and protection by the lipid mixture. Although further studies would be necessary, the present GCLs exhibit promising characteristics as candidates for pCMV-IL12 transfection in future in vivo applications.

Efficient photoinduced energy transfer mediated by aromatic homoconjugated bridges.

A new donor-bridge-acceptor (D-B-A) dyad consisting of ruthenium(II) and iridium(III) species separated by an homoconjugated bridge derived from 7,7-diphenylnorbornane [Ir-Nor-Ru](3+) has been synthesised. The photophysical and electrochemical properties of the heterodinuclear complex have been compared with those of the analogous homodinuclear complexes [Ru-Nor-Ru](4+) and [Ir-Nor- Ir](2+) . Transient absorption spectra on the nanosecond and sub-picosecond timescales show, for the first time, that an homoconjugated bridge can mediate efficiently in the photoinduced energy transfer from the iridium(III) to the ruthenium(II) centres according to a Dexter-type mechanism.