Hybrid Assemblies of Fluorescent Nanocrystals and Membrane Proteins in Liposomes

Because of the growing potential of nanoparticles in biological and medical applications, tuning and directing their properties toward a high compatibility with the aqueous biological milieu is of remarkable relevance. Moreover, the capability to combine nanocrystals (NCs) with biomolecules, such as proteins, offers great opportunities to design hybrid systems for both nanobiotechnology and biomedical technology. Here we report on the application of the micelle-to-vesicle transition (MVT) method for incorporation of hydrophobic, red-emitting CdSe@ZnS NCs into the bilayer of liposomes. This method enabled the construction of a novel hybrid proteo–NC–liposome containing, as model membrane protein, the photosynthetic reaction center (RC) of <i>Rhodobacter sphaeroides</i>. Electron microscopy confirmed the insertion of NCs within the lipid bilayer without significantly altering the structure of the unilamellar vesicles. The resulting aqueous NC–liposome suspensions showed low turbidity and kept unaltered the wavelengths of absorbance and emission peaks of the native NCs. A relative NC fluorescence quantum yield up to 8% was preserved after their incorporation in liposomes. Interestingly, in proteo–NC–liposomes, RC is not denatured by Cd-based NCs, retaining its structural and functional integrity as shown by absorption spectra and flash-induced charge recombination kinetics. The outlined strategy can be extended in principle to any suitably sized hydrophobic NC with similar surface chemistry and to any integral protein complex. Furthermore, the proposed approach could be used in nanomedicine for the realization of theranostic systems and provides new, interesting perspectives for understanding the interactions between integral membrane proteins and nanoparticles, i.e., in nanotoxicology studies

Autophagy is altered during the transition from anagen to catagen.

<p>(A and B) Confocal fluorescence analysis revealed a marked reduction in the number of LC3B-positive fluorescent dots during the transition from anagen to catagen. (A) Representative confocal images of anagen and early/middle catagen organ-cultured HFs probed with specific anti-LC3B antibody/Alexa555 (red). Nuclei were stained with Hoechst 33342 (blue). (B) Quantification of the number of LC3B-flurescent dots per nuclei in the hair matrix region of anagen and early/middle catagen HFs, shown as the average of LC3B-fluorescent dots/nuclei ± SEM. **<i>P</i> < 0.01, catagen versus anagen. (C and D) In line with a reduction in autophagy-dependent protein degradation during HF cycle progression into catagen, the protein levels of SQSTM1 significantly increases in catagen. (C) Representative confocal images of anagen and early/middle catagen organ-cultured HFs probed with specific anti-SQSTM1 antibody/Alexa488 (green). Nuclei were stained with Hoechst 33342 (blue). (D) Quantification of SQSTM1-fluorescent signal in the hair matrix region of anagen and catagen HFs. Fluorescent intensity of anagen HFs was set to 1. Shown as relative integrated density ± SEM. *<i>P</i> < 0.05, catagen versus anagen. (E) Quantitative assessment of autophagic structures in TEM sections from anagen and catagen HFs showing the relative percentage of AVs per field. Shown as average ± SEM. **<i>P</i> < 0.01, catagen versus anagen. All quantifications were performed only on epithelial HF cells, specifically the hair matrix cells and precortical matrix, while the connective tissue sheath and dermal papilla were excluded from analysis. The underlying numerical data and statistical analysis for panels B, D, and E are provided in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002864#pbio.2002864.s005" target="_blank">S1 Data</a>. AV, autophagic vacuole; HF, hair follicle; LC3B, Light Chain 3B; MK, matrix keratinocyte; SQSTM1, sequestosome 1; TEM, transmission electron microscopy.</p

Sputtering-Enabled Intracellular X‑ray Photoelectron Spectroscopy: A Versatile Method To Analyze the Biological Fate of Metal Nanoparticles

The investigation of the toxicological profile and biomedical potential of nanoparticles (NPs) requires a deep understanding of their intracellular fate. Various techniques are usually employed to characterize NPs upon cellular internalization, including high-resolution optical and electron microscopies. Here, we show a versatile method, named sputtering-enabled intracellular X-ray photoelectron spectroscopy, proving that it is able to provide valuable information about the behavior of metallic NPs in culture media as well as within cells, directly measuring their internalization, stability/degradation, and oxidation state, without any preparative steps. The technique can also provide nanoscale vertical resolution along with semiquantitative information about the cellular internalization of the metallic species. The proposed approach is easy-to-use and can become a standard technique in nanotoxicology/nanomedicine and in the rational design of metallic NPs. Two model cases were investigated: silver nanoparticles (AgNPs) and platinum nanoparticles (PtNPs) with the same size and coating. We observed that, after 48 h incubation, intracellular AgNPs were almost completely dissolved, forming nanoclusters as well as AgO, AgS, and AgCl complexes. On the other hand, PtNPs were resistant to the harsh endolysosomal environment, and only some surface oxidation was detected after 48 h

Intrafollicular autophagic flux is active in MKs.

<p>(A) Representative confocal images of organ-cultured anagen HFs treated for 4 h with CQ (10 μM) or vehicle (water). An overlay of LC3B/Alexa555 (red) and Hoechst 33342 (blue) is shown (scale bar 20 μm). The boxed regions show high magnification images of LC3B-fluorescent dots in MKs (scale bar 5 μm). (B) CQ treatment significantly increased the number of LC3B-flurescent dots per nuclei in the hair matrix region compared with vehicle. Quantification was only performed on epithelial HF cells, specifically the hair matrix cells and precortical matrix, while the connective tissue sheath and dermal papilla were excluded from analysis. Shown as average of LC3B-fluorescent dots/nuclei ± SEM. **<i>P</i> < 0.01, CQ versus vehicle. (C) Representative TEM images from organ-cultured anagen HFs showing a continuous autophagic flux in MKs. (I–III) AVi’s with typical, well-visible bilayers separated by a narrower electron-lucent cleft (arrowheads). Predominantly, these AVi’s contained morphologically intact cytosol and organelles. Note the well-defined mitochondria in III (asterisk). (IV) AVd’s characterized by a partially or completely degraded internal membrane and electron dense cytoplasmic material and/or organelles at various stages of degradation (asterisks). IVa and IVb are higher magnifications of the boxed regions in IV. (V) Putative autophagolysosome characterized by lamellar internal membranes (arrowhead). Scale bars are 0.5 μm in C-I, C-III, C-IV, C-Iva, and C-IVb; 1 μm in C-II and C-V. (D) Quantitative assessment of autophagic structures in TEM sections showing the relative percentage of AVi and AVd in anagen HFs. Shown as average ± SEM. **<i>P</i> < 0.01, AVd versus AVi. The underlying numerical data and statistical analysis for panels B and D are provided in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002864#pbio.2002864.s005" target="_blank">S1 Data</a>. AVd, late/degradative autophagic vacuole; AVi, initial autophagic vacuole; CQ, chloroquine; cyt, cytoplasm; HF, hair follicle; LC3B, Light Chain 3B; MK, matrix keratinocyte; n, nucleus; TEM transmission electron microscopy.</p

The principal ingredients of an anti–hair loss product act as a potent inducer of intrafollicular autophagy.

<p>(A) Representative confocal images of anagen organ-cultured HFs treated with the principal ingredients (core mix) of the topic formulation of an anti–hair loss product on the market, which includes N¹-methyspermidine, a metabolically stable analog of the well-recognized autophagy-promoting agent, spermidine [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002864#pbio.2002864.ref036" target="_blank">36</a>–<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002864#pbio.2002864.ref039" target="_blank">39</a>]. An overlay of LC3B/Alexa555 (red) and Hoechst 33342 (blue) is shown. Scale bars are 20 μm. Insets show high magnification of LC3B-fluorescent dots in MKs (scale bar 5 μm). (B) Quantification of the number of LC3B-fluorescent dots per nuclei in the hair matrix region of core mix–and vehicle-treated HFs (connective tissue sheath and dermal papilla were excluded from analysis), shown as average of LC3B-fluorescent dots/nuclei ± SEM. ***<i>P</i> < 0.001, core mix versus vehicle. (C) Representative confocal images of core mix–and vehicle-treated HFs probed with specific anti-SQSTM1 antibody/Alexa488 (green). Nuclei were stained with Hoechst 33342 (blue). Scale bars are 20 μm. (D) Quantification of SQSTM1-fluorescent signal in the hair matrix region of HFs treated with core mix or vehicle. Fluorescent intensity of vehicle HFs was set to 1. Shown as relative integrated density ± SEM. **<i>P</i> < 0.001, core mix versus vehicle. (E) Quantitative assessment of autophagic structures in TEM sections from HFs treated with core mix or vehicle. Shown as average number of AVs per field ± SEM. *<i>P</i> < 0.05, core mix versus vehicle. (F and G). Pro-autophagic activity of N¹-methyspermidine was tested in an in vitro cellular assay previously adopted to demonstrate the ability of spermidine in inducing autophagy [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002864#pbio.2002864.ref036" target="_blank">36</a>]. Cultured human U2OS cells were treated 6 h with vehicle or equimolar doses of spermidine and N¹-methyspermidine (100 μM). (F) The levels of lipidated LC3B (LC3-II) and SQSTM1 were then assessed by immunoblotting analysis with specific antibody. Actin signals were adopted as a loading control. (G) Densitometry analysis of protein signals is reported as relative protein levels normalized by ACTIN. Vehicle sample value was set to 1. Shown as mean ± SEM, <i>n</i> = 3; **<i>P</i> < 0.01, compounds versus vehicle. The underlying numerical data and statistical analysis for panels B, D, E, and G are provided in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002864#pbio.2002864.s005" target="_blank">S1 Data</a>. ACTIN, actin beta; AV, autophagic vacuole; HF, hair follicle; LC3, Light Chain 3; LC3B, Light Chain 3B; MK, matrix keratinocyte; spe, spermidine; SQSTM1, sequestosome 1; TEM, transmission electron microscopy; U2OS, human bone osteosarcoma epithelial U2OS cell line; veh, vehicle.</p

Mesoscale Assemblies of Iron Oxide Nanocubes as Heat Mediators and Image Contrast Agents

Iron oxide nanocubes (IONCs) represent one of the most promising iron-based nanoparticles for both magnetic resonance image (MRI) and magnetically mediated hyperthermia (MMH). Here, we have set a protocol to control the aggregation of magnetically interacting IONCs within a polymeric matrix in a so-called magnetic nanobead (MNB) having mesoscale size (200 nm). By the comparison with individual coated nanocubes, we elucidate the effect of the aggregation on the specific adsorption rates (SAR) and on the <i>T</i>₁ and <i>T</i>₂ relaxation times. We found that while SAR values decrease as IONCs are aggregated into MNBs but still keeping significant SAR values (200 W/g at 300 kHz), relaxation times show very interesting properties with outstanding values of <i>r</i>₂/<i>r</i>₁ ratio for the MNBs with respect to single IONCs

Silencing of the essential autophagy gene, <i>ATG5</i>, functionally reduces intrafollicular autophagy.

<p>(A) Organ-cultured HFs were transfected with pooled siRNA sequences against the autophagy gene, <i>ATG5</i> (siATG5), or a nontargeting pool (Control). Forty-eight hours after transfection, HFs were processed for indirect IF analysis with a specific anti-ATG5 antibody/Alexa555 (red). Hoechst 33342 was used to stain nuclei (blue). Demonstrating the efficient knock-down of ATG5 in HFs, ATG5-fluorescence signals were undetectable in siATG5 HFs. (B–E) ATG5 silencing actually impaired autophagy in organ-cultured HFs. (B) Representative confocal images of siATG5 and control HFs probed with specific anti-LC3B antibody/Alexa555 (red). Nuclei were stained with Hoechst 33342 (blue). Insets show high magnification of LC3B-fluorescent dots in MKs. Scale bars are 10 μm. (C) Quantification of the number of LC3B-fluorescent dots per nuclei in the hair matrix region of siATG5 and control HFs. Shown as average of LC3B-fluorescent dots/nuclei ± SEM. *<i>P</i> < 0.05, siATG5 versus control HFs. (D) Representative confocal images of siATG5 and control organ-cultured HFs probed with specific anti-SQSTM1 antibody/Alexa488 (green). Nuclei were stained with Hoechst 33342 (blue). Scale bars are 20 μm. (E) Quantification of SQSTM1-fluorescent signal in hair matrix region of siATG5 and control HFs. Fluorescent intensity of control HFs was set to 1. Shown as relative integrated density ± SEM. *<i>P</i> < 0.05, siATG5 versus control HFs. All quantifications were performed only on epithelial HF cells, specifically the hair matrix cells and precortical matrix, while the connective tissue sheath and dermal papilla were excluded from analysis. The underlying numerical data and statistical analysis for panels C and E are provided in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002864#pbio.2002864.s005" target="_blank">S1 Data</a>. <i>ATG5</i>, autophagy-related gene 5; HF, hair follicle; IF, immunofluorescence; MK, matrix keratinocyte; siRNA, small interfering RNA; SQSTM1, sequestosome 1.</p

Core mix–related autophagic stimulation is instrumental in its anagen promoting activity.

<p>(A) Pro-anagen activity of the principal ingredients of an anti–hair loss product on the market (core mix). The hair cycle stage of anagen VI HFs from diverse donors treated with core mix or vehicle was assessed by morphological analysis [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002864#pbio.2002864.ref029" target="_blank">29</a>]. Compared with vehicle, core mix treatment increased the percentage of anagen HFs (green) from all donors. Notably, the anagen-promoting effects of the core mix was observed even in catagen-primed HFs (donor 3), as indicated by the fact that all vehicle-treated HFs had transitioned into catagen at the end of organ culture. (B) Pro-anagen activity of the core mix is impaired in HFs silenced for the essential autophagy gene, <i>ATG5</i>. Anagen VI HFs from three donors were transfected with pool siRNA sequences against <i>ATG5</i> gene (<i>siATG5 #1 and ATG5 #2</i>) or with nontargeting scrambled oligonucleotides (<i>siControl</i>) and then treated with core mix or vehicle. Consistent with the analysis shown in (A), subsequent evaluation of the hair cycle stage revealed that siControl HFs treated with the core mix had an increased percentage of anagen HFs compared with vehicle-treated HFs. In marked contrast, core mix treatment failed to promote anagen in ATG5-silenced HFs from two independent donors. The underlying numerical data and statistical analysis are provided in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002864#pbio.2002864.s005" target="_blank">S1 Data</a>. <i>ATG5</i>, autophagy-related gene 5; HF, hair follicle; siRNA, small interfering RNA.</p

Functionalization of Strongly Interacting Magnetic Nanocubes with (Thermo)Responsive Coating and Their Application in Hyperthermia and Heat-Triggered Drug Delivery

Herein, we prepare nanohybrids by incorporating iron oxide nanocubes (cubic-IONPs) within a thermoresponsive polymer shell that can act as drug carriers for doxorubicin­(doxo). The cubic-shaped nanoparticles employed are at the interface between superparamagnetic and ferromagnetic behavior and have an exceptionally high specific absorption rate (SAR), but their functionalization is extremely challenging compared to bare superparamagnetic iron oxide nanoparticles as they strongly interact with each other. By conducting the polymer grafting reaction using reversible addition–fragmentation chain transfer (RAFT) polymerization in a viscous solvent medium, we have here developed a facile approach to decorate the nanocubes with stimuli-responsive polymers. When the thermoresponsive shell is composed of poly­(<i>N</i>-<i>iso</i>propylacrylamide-<i>co</i>-polyethylene glycolmethyl ether acrylate), nanohybrids have a phase transition temperature, the lower critical solution temperature (LCST), above 37 °C in physiological conditions. Doxo loaded nanohybrids exhibited a negligible drug release below 37 °C but showed a consistent release of their cargo on demand by exploiting the capability of the nanocubes to generate heat under an alternating magnetic field (AMF). Moreover, the drug free nanocarrier does not exhibit cytotoxicity even when administered at high concentration of nanocubes (1g/L of iron) and internalized at high extent (260 pg of iron per cell). We have also implemented the synthesis protocol to decorate the surface of nanocubes with poly­(vinylpyridine) polymer and thus prepare pH-responsive shell coated nanocubes

Additional file 1: Figure S1. of Metformin promotes tau aggregation and exacerbates abnormal behavior in a mouse model of tauopathy

Expression levels of key molecules in the insulin pathway in WT and P301S mouse cortex. Figure S2. Expression levels of key molecules in the insulin pathway in the cortex of P301S mouse treated with or without metformin. Figure S3. Expression levels of AMPK, S6, pS6, and tau in primary cortical neurons treated with or without metformin and/or specific blockers. Figure S4. Body weight, glycemia, water and food consumption in P301S mice treated with or without metformin. Table S1. Concentration of metformin in the plasma and brain of WT and P301S mice after treatment for 7 days. Table S2. Neuronal viability after pharmacological treatment for 6 h. Table S3. Primers used for qRT-PCR. (PDF 5110 kb