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.

Lipid-matrix effects on tyrosinase immobilization in Langmuir and Langmuir-Blodgett films.

The immobilization of the enzyme tyrosinase (Tyr) in lipid matrices can be explored to produce biosensors for detecting polyphenols, which is relevant for the food industry. Herein, we shall demonstrate the importance of the lipid composition to immobilize the enzyme tyrosinase in Langmuir-Blodgett (LB) films. Tyr could be incorporated into Langmuir monolayers of arachidic acid (AA), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (sodium salt) (DPPG), having as the main effect an expansion in the monolayers. Results from polarization-modulated infrared reflection-absorption spectroscopy (PM-IRRAS) pointed to electrostatic interactions between the charged residues of Try and the lipid headgroups, in addition to changes in the order of lipid chains. The interaction between Tyr and DPPC in Langmuir monolayers can be correlated with the superior performance of DPPC/Tyr LB films used as biosensors to detect catechol by cyclic voltammetry. The molecular-level interactions assessed via PM-IRRAS are therefore believed to drive an immobilization process for Tyr in the lipid LB matrix and may serve as a general criterion to identify matrices that preserve enzyme activity.

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.

Correlating Artepillin C cytotoxic activity on HEp-2 cells with bioinspired systems of plasma membranes.

Artepillin C is the main compound present in propolis from Baccharis dracunculifolia, whose antitumor activity has been the focus of many studies. Herein, we shall investigate the Artepillin C mechanisms of action against cells derived from the oropharyngeal carcinoma (HEp-2). Cytotoxicity tests revealed that the concentrations of Artepillin C required to reduce cell viability by 50% (CC50) are dependent on the incubation time, decreasing from 40.7 × 10-5 mol/L to 15.7 × 10-5 mol/L and 9.05 × 10-5 mol/L considering 12, 24 and 48 h, respectively. Hydrophobic interactions on neutral species of Artepillin C induce aggregation over the HEp-2 plasma membrane, given the acid conditions of the cellular culture. Indeed, Langmuir monolayers mimicking cellular membranes of tumor cells revealed Artepillin C affinity to interact with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) containing 20 mol% of 1,2-dipalmitoyl-sn-glychero-3-phosphoserine (DPPS), leading aggregation on giant unilamellar vesicles (GUVs) at pH 3.2. Moreover, leakage experiments on GUVs have shown that the presence of DPPS enhances the efflux of the fluorescent probe signaling the membrane permeabilization, which is the origin of the necrotic pathway triggered in HEp-2 cells, as observed by flow cytometry assays.

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.

Breast cancer subtype specific biochemical responses to radiation.

External beam radiotherapy is a common form of treatment for breast cancer. Among patients and across different breast cancer subtypes, the response to radiation is heterogeneous. Radiation-induced biochemical changes were examined by Raman spectroscopy using cell lines that represent a spectrum of human breast cancer. Principal component analysis (PCA) and partial least squares discriminant analysis (PLSDA) revealed unique Raman spectral features in the HER2 and Ki67 subtype. The changes in Raman spectral profiles to different doses of radiation (0-50 Gy) included variations in the levels of proteins, lipids, nucleic acids and glycogen. Importantly, the differences in radiation-induced changes on the normal breast epithelial cell line MCF10A could be discriminated within and across the various breast tumor cell lines. These results demonstrate a novel approach to uncover differences between breast cancer cell subtypes and surrounding normal tissues by their biochemical variations in response to radiation.