Photochemical outcomes triggered by gold shell-isolated nanorods on bioinspired nanoarchitectonics for bacterial membranes.

Boosted by the indiscriminate use of antibiotics, multidrug-resistance (MDR) demands new strategies to combat bacterial infections, such as photothermal therapy (PTT) based on plasmonic nanostructures. PTT efficiency relies on photoinduced damage caused to the bacterial machinery, for which nanostructure incorporation into the cell envelope is key. Herein, we shall unveil the binding and photochemical mechanisms of gold shell-isolated nanorods (AuSHINRs) on bioinspired bacterial membranes assembled as Langmuir and Langmuir-Schaefer (LS) monolayers of DOPE, Lysyl-PG, DOPG and CL. AuSHINRs incorporation expanded the isotherms, with stronger effect on the anionic DOPG and CL. Indeed, FTIR of LS films revealed more modifications for DOPG and CL owing to stronger attractive electrostatic interactions between anionic phosphates and the positively charged AuSHINRs, while electrostatic repulsions with the cationic ethanolamine (DOPE) and lysyl (Lysyl-PG) polar groups might have weakened their interactions with AuSHINRs. No statistical difference was observed in the surface area of irradiated DOPE and Lysyl-PG monolayers on AuSHINRs, which is evidence of the restricted nanostructures insertion. In contrast, irradiated DOPG monolayer on AuSHINRs decreased 4.0 % in surface area, while irradiated CL monolayer increased 3.7 %. Such results agree with oxidative reactions prompted by ROS generated by AuSHINRs photoactivation. The deepest AuSHINRs insertion into DOPG may have favored chain cleavage while hydroperoxidation is the mostly like outcome in CL, where AuSHINRs are surrounding the polar groups. Furthermore, preliminary experiments on Escherichia coli culture demonstrated that the electrostatic interactions with AuSHINRs do not inhibit bacterial growth, but the photoinduced effects are highly toxic, resulting in microbial inactivation.

Immunoassay platform with surface-enhanced resonance Raman scattering for detecting trace levels of SARS-CoV-2 spike protein.

The early diagnosis of Coronavirus disease (COVID-19) requires either an accurate detection of genetic material or a sensitive detection of viral proteins. In this work, we designed an immunoassay platform for detecting trace levels of SARS-CoV-2 spike (S) protein. It is based on surface-enhanced resonance Raman scattering (SERRS) of methylene blue (MB) adsorbed onto spherical gold nanoparticles (AuNPs) and coated with a 6 nm silica shell. The latter shell in the SERRS nanoprobe prevented aggregation and permitted functionalization with SARS-CoV-2 antibodies. Specificity of the immunoassay was achieved by combining this functionalization with antibody immobilization on the cover slides that served as the platform support. Different concentrations of SARS-CoV-2 antigen could be distinguished and the lack of influence of interferents was confirmed by treating SERRS data with the multidimensional projection technique Sammon's mapping. With SERRS using a laser line at 633 nm, the lowest concentration of spike protein detected was 10 pg/mL, achieving a limit of detection (LOD) of 0.046 ng/mL (0.60 pM). This value is comparable to the lowest concentrations in the plasma of COVID-19 patients at the onset of symptoms, thus indicating that the SERRS immunoassay platform may be employed for early diagnosis.

Mechanisms of hypericin incorporation to explain the photooxidation outcomes in phospholipid biomembrane models.

Cell membranes are the first barriers for drug binding and key for the action of photosensitizers (PS). Herein, we report on the incorporation of the PS hypericin into Langmuir monolayers of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS) to represent eukaryotic cell membranes, and 1,2-dioleoyl-sn-glycero-3-phospho(1'-rac-glycerol) (DOPG) to mimic bacterial membranes. Surface pressure (π) vs mean molecular area (Å) isotherms showed a high degree of interaction (binding, penetration and relative solubilization) of hypericin into DPPC and DOPC monolayers. On the other hand, electrostatic repulsions govern the interactions with DOPG and DOPS, favoring hypericin self-aggregation, as visualized by Brewster angle microscopy (BAM). Indeed, the larger domains in BAM were consistent with the greater expansion of DOPG monolayers with incorporated hypericin, owing to stronger electrostatic repulsions. In contrast to DPPC, light-irradiation of DOPC monolayers containing hypericin induced loss of material due to hydrocarbon chain cleavage triggered by contact-dependent reactions between triplet excited state of hypericin and chain unsaturations. The mild effects noted for both irradiated DOPS and DOPG monolayers are attributed to hypericin self-aggregation, which may have decreased the singlet oxygen quantum yield (Φ1O2) via self-quenching, despite the increased instability induced in the monolayers.

The efficiency of photothermal action of gold shell-isolated nanoparticles against tumor cells depends on membrane interactions.

Photoinduced hyperthermia with nanomaterials has been proven effective in photothermal therapy (PTT) of tumor tissues, but a precise control in PTT requires determination of the molecular-level mechanisms. In this paper, we determined the mechanisms responsible for the action of photoexcited gold shell-isolated nanoparticles (AuSHINs) in reducing the viability of MCF7 (glandular breast cancer) and especially A549 (lung adenocarcinoma) cells in vitro experiments, while the photoinduced damage to healthy cells was much smaller. The photoinduced effects were more significant than using other nanomaterials, and could be explained by the different effects from incorporating AuSHINs on Langmuir monolayers from lipid extracts of tumoral (MCF7 and A549) and healthy cells. The incorporation of AuSHINs caused similar expansion of the Langmuir monolayers, but Fourier-transform infrared spectroscopy (FTIR) data of Langmuir-Schaefer films (LS) indicated distinct levels of penetration into the monolayers. AuSHINs penetrated deeper into the A549 extract monolayers, affecting the vibrational modes of polar groups and carbon chains, while in MCF7 monolayers penetration was limited to the surroundings of the polar groups. Even smaller insertion was observed for monolayers of the healthy cell extract. The photochemical reactions were modulated by AuSHINs penetration, since upon irradiation the surface area of A549 monolayer decreased owing to lipid chain cleavage by oxidative reactions. For MCF7 monolayers, hydroperoxidation under illumination led to a ca. 5% increase in surface area. The monolayers of healthy cell lipid extract were barely affected by irradiation, consistent with the lowest degree of AuSHINs insertion. In summary, efficient photothermal therapy may be devised by producing AuSHINs capable of penetrating the chain region of tumor cell membranes.

Plasma membrane permeabilization to explain erythrosine B phototoxicity on in vitro breast cancer cell models.

Lipid oxidation is ubiquitous in cell life under oxygen and essential for photodynamic therapy (PDT) of carcinomas. However, the mechanisms underlying lipid oxidation in rather complex systems such as plasma membranes remain elusive. Herein, Langmuir monolayers were assembled with the lipid extract of glandular breast cancer (MCF7) cells and used to probe the molecular interactions allowing adsorption of the photosensitizer (PS) erythrosine B and subsequent photooxidation outcomes. Surface pressure (π) versus area (cm2/mL) isotherms of MCF7 lipid extract shifted to larger areas upon erythrosine incorporation, driven by secondary interactions that affected the orientation of the carbonyl groups and lipid chain organization. Light-irradiation increased the surface area of the MCF7 lipid extract monolayer containing erythrosine owing to the lipid hydroperoxidation, which may further undergo decomposition, resulting in the chain cleavage of phospholipids and membrane permeabilization. Incorporation of erythrosine by MCF7 cells induced slight toxic effects on in vitro assays, differently of the severe phototoxicity caused by light-irradiation, which significantly decreased cell viability by more than 75% at 2.5 × 10-6 mol/L of erythrosine incubated for 3 and 24 h, reaching nearly 90% at 48 h of incubation. The origin of the phototoxic effects is in the rupture of the plasma membrane shown by the frontal (FSC) and side (SSC) light scattering of flow cytometry. Consistent with hydroperoxide decomposition, membrane permeabilization was also confirmed by cleaved lipids detected in mass spectrometry and subsidizes the necrotic pathway of cell death.

Modulating photochemical reactions in Langmuir monolayers of Escherichia coli lipid extract with the binding mechanisms of eosin decyl ester and toluidine blue-O photosensitizers.

Photodynamic damage to the cell envelope can inactivate microorganisms and may be applied to combat super-resistance phenomenon, empowered by the indiscriminate use of antibiotics. Efficiency in microbial inactivation is dependent on the incorporation of photosensitizers (PS) into the bacterial membranes to trigger oxidation reactions under illumination. Herein, Langmuir monolayers of Escherichia coli lipid extract were built to determine the binding mechanisms and oxidation outcomes induced by eosin decyl ester (EosDEC) and toluidine blue-O (TBO) PSs. Surface-pressure isotherms of the E. coli monolayers were expanded upon EosDEC and TBO, suggesting incorporation of both PSs. Fourier-transform infrared spectroscopy (FTIR) of Langmuir-Schaefer (LS) films reveled that the EosDEC and TBO binding mechanisms are dominated by electrostatic interactions with the anionic polar groups, with limited penetration into the chains. Light-irradiation reduced the relative area of E. coli monolayer on TBO, indicating an increased loss of material to the subphase owing to the chain cleavage, generated by contact-dependent reactions with excited states of TBO. In contrast, the increased relative area of E. coli monolayers containing EosDEC suggests lipid hydroperoxidation, which is PS contact-independent. Even considering a small chain penetration, the saturated EosDEC may have partitioned towards saturated reach domains, avoiding direct contact with membrane unsaturations.

Chain Cleavage of Bioinspired Bacterial Membranes Photoinduced by Eosin Decyl Ester.

Photodynamic therapy (PDT) is promising for bacterial inactivation since cellular internalization of photosensitizers (PS) is not crucial for the treatment effectiveness. Photoinduced damage in the lipid envelope may already induce microbial inactivation, which requires PS capable of easily penetrating into the membrane. Herein, we report on the insertion of the PS eosin decyl ester (EosDec) into Langmuir films of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG), and cardiolipin (CLP) used as mimetic systems of bacterial membranes. Surface pressure isotherms and polarization-modulated infrared reflection absorption spectroscopy (PM-IRRAS) indicated that the hydrophobic nature of EosDec favored deeper penetration in all the phospholipid monolayers. The incorporation of EosDec led to monolayer expansion, especially in the anionic DOPG and CLP owing to repulsive electrostatic interactions, and induced disorder in the lipid chains. Light irradiation of DOPE, DOPG, and CLP monolayers containing EosDec increased the rate of material loss to the subphase, which is attributed to cleavage of lipid chains triggered by contact-dependent reactions between excited states of EosDec and lipid unsaturations. The latter is key for membrane permeabilization and efficiency in microbial inactivation.

Molecular-level effects on cell membrane models to explain the phototoxicity of gold shell-isolated nanoparticles to cancer cells.

Metallic nanoparticles are promising agents for photothermal cancer therapy (PTT) owing to their photostability and efficient light-to-heat conversion, but their possible aggregation remains an issue. In this paper, we report on the photoinduced heating of gold shell-isolated nanoparticles (AuSHINs) in in vitro experiments to kill human oropharyngeal (HEp-2) and breast (BT-474 and MCF-7) carcinoma cells, with cell viability reducing below 50 % with 2.2 × 1012 AuSHINs/mL and 6 h of incubation. This toxicity to cancer cells is significantly higher than in previous works with gold nanoparticles. Considering the AuSHINs dimensions we hypothesize that cell uptake is not straightforward, and the mechanism of action involves accumulation on phospholipid membranes as the PTT target for photoinduced heating and subsequent generation of reactive oxygen species (ROS). Using Langmuir monolayers as simplified membrane models, we confirmed that AuSHINs have a larger effect on 1,2-dioleoyl-sn-glycero-3-phospho-l-serine (DOPS), believed to represent cancer cell membranes, than on 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) taken as representative of healthy eukaryotic cells. In particular, data from polarization-modulated infrared reflection absorption spectroscopy (PM-IRRAS) revealed an increased conformational order of DOPS tails due to the stronger adsorption of AuSHINs. Furthermore, light irradiation reduced the stability of AuSHINs containing DOPC and DOPS monolayers owing to oxidative reactions triggered by ROS upon photoinduced heating. Compared to DOPC, DOPS lost nearly twice as much material to the subphase, which is consistent with a higher rate of ROS formation in the vicinity of the DOPS monolayer.

Photo-Induced Necrosis on Oropharyngeal Carcinoma (HEp-2) Cells Mediated by the Xanthene Erythrosine.

The photodynamic therapy (PDT) has been outstanding as a promising alternative for treating different carcinomas. However, the lack of detailed knowledge on the mechanisms of action prevents exploitation of the therapy full potential. Herein we shall evaluate not only the photodynamic efficiency but the mechanism of cell death triggered by the photoactivated erythrosine in oropharyngeal cancer cells (HEp-2). Cytotoxic assays were performed by MTT at distinct concentrations (10-3 to 10-6 mol/L) and incubation time (3, 24 and 48 h) of erythrosine in HEp-2 in vitro culture. In addition to the cytotoxic effect, the mechanisms of cell death were evaluated by flow cytometry following the annexin V/propidium iodide double staining protocol. Erythrosine was incorporated by HEp-2 cells in a dose- and time-dependent pathway. The incubation of erythrosine in dark has not shown any significant effect over the culture until 24 h and 1.25×10-6 mol/L concentration, from which a small portion (<25% and statistically significant) of the cell population have undergone apoptosis. On the other hand, 50% of cell viability is reduced mainly by necrosis when 10, 3.75 and 1.9×10-6 mol/L of erythrosine concentrations at 3, 24 and 48 h of incubation are photoactivated, respectively. Bioinspired models of tumor membrane based on Langmuir monolayers of 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS) mixture reveled that electrostatic interactions with the lipid head groups are the main driving forces allowing the erythrosine adsorption. Furthermore, light-induced hydroperoxidation significantly increased the surface area of the monolayers, which might be the origin of the necrotic pathway triggered in HEp-2 cells.

Surface Plasmon Resonances in Silver Nanostars.

The recent development of silver nanostars (Ag-NSs) is promising for improved surface-enhanced sensing and spectroscopy, which may be further exploited if the mechanisms behind the excitation of localized surface plasmon resonances (LSPRs) are identified. Here, we show that LSPRs in Ag-NSs can be obtained with finite-difference time-domain (FDTD) calculations by considering the nanostars as combination of crossed nanorods (Ag-NRs). In particular, we demonstrate that an apparent tail at large wavelengths ( λ ≳ 700 nm) observed in the extinction spectra of Ag-NSs is due to a strong dipolar plasmon resonance, with no need to invoke heterogeneity (different number of arms) effects as is normally done in the literature. Our description also indicates a way to tune the strongest LSPR at desired wavelengths, which is useful for sensing applications.

Evidence of photoinduced lipid hydroperoxidation in Langmuir monolayers containing Eosin Y.

Photodynamic therapy (PDT) efficiency depends on many factors including the incorporation of the photosensitizer (PS) in cell membranes and possible lipid hydroperoxidation. In this study, we show that hydroperoxidation may be photoinduced when eosin Y is incorporated into Langmuir monolayers that serve as cell membrane models. This occurs for Langmuir monolayers of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), which have unsaturation in their hydrophobic chains. In contrast, light irradiation had no effect on monolayers of saturated 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). Evidence of hydroperoxidation was obtained from the area increase in eosin-containing DOPC and POPC monolayers upon irradiation, which was accompanied by a decrease in monolayer thickness according to grazing incidence X-ray off-specular scattering (GIXOS) data. Furthermore, the changes in polarization-modulated infrared reflection absorption spectroscopy (PM-IRRAS) induced by irradiation were consistent with hydroperoxide migration toward the lipid hydrophilic heads.. In summary, this combination of experimental methods allowed us to determine the effects of eosin Y interaction with cell membrane models under irradiation, which may be associated with the underlying mechanisms of eosin Y as photosensitizer in PDT.

New trends in plasmonic (bio)sensing.

The strong enhancement and localization of electromagnetic field in plasmonic systems have found applications in many areas, which include sensing and biosensing. In this paper, an overview will be provided of the use of plasmonic phenomena in sensors and biosensors with emphasis on two main topics. The first is related to possible ways to enhance the performance of sensors and biosensors based on surface plasmon resonance (SPR), where examples are given of functionalized magnetic nanoparticles, magnetoplasmonic effects and use of metamaterials for SPR sensing. The other topic is focused on surface-enhanced Raman scattering (SERS) for sensing, for which uniform, flexible, and reproducible SERS substrates have been produced. With such recent developments, there is the prospect of improving sensitivity and lowering the limit of detection in order to overcome the limitations inherent in ultrasensitive detection of chemical and biological analytes, especially at single molecule levels.

Zika Immunoassay Based on Surface-Enhanced Raman Scattering Nanoprobes.

Zika virus (ZIKV) was considered a public health emergency of international concern after the 2015 outbreak. Serological tests based on immunoassay platforms is one of the methods applied on the diagnosis of ZIKV and Dengue virus (DENV). However, the high limits of detection (LOD) and the cross-reactivity between ZIKV and DENV are still limitations in immunological tests. In order to tackle these issues, we have designed an immune-specific assay based on surface-enhanced Raman scattering (SERS) nanoprobes. Gold shell-isolated nanoparticles (Au-SHINs) were synthesized with 100 nm Au nanoparticles and 4 nm silica shell thickness coated with Nile Blue (Raman reporter). Then, the SERS nanoprobes were wrapped in a final silica shell and functionalized with monoclonal anti-ZIKV NS1 antibodies. Concentrations of ZIKV NS1 down to 10 ng/mL were probed free of cross-reactivity with DENV NS1 antigens.

New trends in plasmonic (bio)sensing

Abstract The strong enhancement and localization of electromagnetic field in plasmonic systems have found applications in many areas, which include sensing and biosensing. In this paper, an overview will be provided of the use of plasmonic phenomena in sensors and biosensors with emphasis on two main topics. The first is related to possible ways to enhance the performance of sensors and biosensors based on surface plasmon resonance (SPR), where examples are given of functionalized magnetic nanoparticles, magnetoplasmonic effects and use of metamaterials for SPR sensing. The other topic is focused on surface-enhanced Raman scattering (SERS) for sensing, for which uniform, flexible, and reproducible SERS substrates have been produced. With such recent developments, there is the prospect of improving sensitivity and lowering the limit of detection in order to overcome the limitations inherent in ultrasensitive detection of chemical and biological analytes, especially at single molecule levels.

Co-Deposition of Gold Nanoparticles and Metalloporphyrin Using the Langmuir-Blodgett (LB) Technique for Surface-Enhanced Raman Scattering (SERS).

The synergistic effect produced by metallic nanoparticles when incorporated into different systems empowers a research field that is growing rapidly. In addition, organometallic materials are at the center of intensive research with diverse applications such as light-emitting devices, transistors, solar cells, and sensors. The Langmuir-Blodgett (LB) technique has proven to be suitable to address challenges inherent to organic devices, since the film properties can be tuned at the molecular level. Here we report a strategy to incorporate gold nanoparticles (AuNPs) into the LB film by co-deposition in order to achieve surface-enhanced Raman scattering (SERS) of the zinc(II)-protoporphyrin (IX) dimethyl ester (ZnPPIX-DME). Prior to the LB co-deposition, the properties of the Langmuir monolayer of ZnPPIX-DME at the air-water interface, containing AuNPs in the subphase, are studied through the surface-pressure versus mean molecular area (π-A) isotherms. The ZnPPIX-DME+AuNPs π-A isotherm presented a significant shift to higher molecular area, suggesting an interaction between both ZnPPIX-DME molecules and AuNPs. Those interactions are a key factor allowing the co-deposition of both AuNPs and ZnPPIX-DME molecules onto a solid substrate, thus forming the LB film. SERS of ZnPPIX-DME was successfully attained, ensuring the spatial distribution of the AuNPs. Higher enhancement factors were found at AuNP aggregates, as a result of the intense local electromagnetic field found in the metal nanoparticle aggregates. The main vibrational bands observed in the SERS spectra suggest a physical adsorption of the ZnPPIX-DME onto the surface of AuNPs. The latter is not only in agreement with the interactions pointed out by the π-A isotherms but also suggests that this interaction is kept upon LB film co-deposition.