Fe₃O₄ nanoparticles (MNP, a) synthesized in organic solvent and transferred to a water solution using PMA amphiphilic polymer (PMNP, b).

<p>MNP and PMNP were highly monodisperse in size as it is shown by TEM images (scale bars = 40 nm,). Part of the highly concentrated PMNP suspension (8 mg mL^–1) was incorporated in a w/o cream (0.8 wt % concentration) (c).</p

Cytofluorimetric analysis showing PMNP nanoparticles uptake by mouse skin and lymph node cells.

<p>PMNP suspension (a, upper panels). Skin CD45-positive and negative cells showing CFSE incorporation. Note that most of the skin cells uptake PMNP nanoparticles administered with the cream formulation. (a, lower panels) CFSE-positive cells in the lymph nodes of mice that received PMNP nanoparticles via cream formulation or via sc administration. Note that only with sc PMNP administration, nanoparticle-positive cells can be detected in the draining lymph nodes. (b) Lymph node macrophages and dendritic cells, identified as CD11b- and CD11c-positive cells respectively, showing CFSE incorporation. Note that only when PMNP are administered sc, CFSE positive macrophages and dendritic cells can be detected in the lymph nodes.</p

Histological microphotograph of normal human skin section.

<p>Haematoxylin and eosin staining (original magnification 40×) (a). <i>In vitro</i> diffusion studies of PMNP colloidal suspension or cream in human skin were carried out using Franz diffusion cells and diffused PMNP were quantified by ICP-OES analysis (b).</p

Fates of nanoparticles depending on the route of skin administration.

<p>Nanoparticle administered in a cream formulation are taken up by all the skin cell types and do no reach the draining lymph nodes. Nanoparticle administered with a sc injection in aqueous suspension are efficiently transported to the draining lymph nodes.</p

Assessing the <i>In Vivo</i> Targeting Efficiency of Multifunctional Nanoconstructs Bearing Antibody-Derived Ligands

A great challenge in nanodiagnostics is the identification of new strategies aimed to optimize the detection of primary breast cancer and metastases by the employment of target-specific nanodevices. At present, controversial proof has been provided on the actual importance of surface functionalization of nanoparticles to improve their <i>in vivo</i> localization at the tumor. In the present paper, we have designed and developed a set of multifunctional nanoprobes, modified with three different variants of a model antibody, that is, the humanized monocolonal antibody trastuzumab (TZ), able to selectively target the HER2 receptor in breast cancer cells. Assuming that nanoparticle accumulation in target cells is strictly related to their physicochemical properties, we performed a comparative study of internalization, trafficking, and metabolism in MCF7 cells of multifunctional nanoparticles (MNP) functionalized with TZ or with alternative lower molecular weight variants of the monoclonal antibody, such as the half-chain (HC) and scFv fragments (scFv). Hence, to estimate to what extent the structure of the surface bioligand affects the targeting efficiency of the nanoconjugate, three cognate nanoconstructs were designed, in which only the antibody form was differentiated while the nanoparticle core was maintained unvaried, consisting of an iron oxide spherical nanocrystal coated with an amphiphilic polymer shell. <i>In vitro</i>, <i>in vivo</i>, and <i>ex vivo</i> analyses of the targeting efficiency and of the intracellular fate of MNP-TZ, MNP-HC, and MNP-scFv suggested that the highly stable MNP-HC is the best candidate for application in breast cancer detection. Our results provided evidence that, in this case, active targeting plays an important role in determining the biological activity of the nanoconstruct

Antiproliferative Effect of ASC-J9 Delivered by PLGA Nanoparticles against Estrogen-Dependent Breast Cancer Cells

Among polymeric nanoparticles designed for cancer therapy, PLGA nanoparticles have become one of the most popular polymeric devices for chemotherapeutic-based nanoformulations against several kinds of malignant diseases. Promising properties, including long-circulation time, enhanced tumor localization, interference with “multidrug” resistance effects, and environmental biodegradability, often result in an improvement of the drug bioavailability and effectiveness. In the present work, we have synthesized 1,7-bis­(3,4-dimethoxyphenyl)-5-hydroxyhepta-1,4,6-trien-3-one (ASC-J9) and developed uniform ASC-J9-loaded PLGA nanoparticles of about 120 nm, which have been prepared by a single-emulsion process. Structural and morphological features of the nanoformulation were analyzed, followed by an accurate evaluation of the <i>in vitro</i> drug release kinetics, which exhibited Fickian law diffusion over 10 days. The intracellular degradation of ASC-J9-bearing nanoparticles within estrogen-dependent MCF-7 breast cancer cells was correlated to a time- and dose-dependent activity of the released drug. A cellular growth inhibition associated with a specific cell cycle G2/M blocking effect caused by ASC-J9 release inside the cytosol allowed us to put forward a hypothesis on the action mechanism of this nanosystem, which led to the final cell apoptosis. Our study was accomplished using Annexin V-based cell death analysis, MTT assessment of proliferation, radical scavenging activity, and intracellular ROS evaluation. Moreover, the intracellular localization of nanoformulated ASC-J9 was confirmed by a Raman optical imaging experiment designed <i>ad hoc</i>. PLGA nanoparticles and ASC-J9 proved also to be safe for a healthy embryo fibroblast cell line (3T3-L1), suggesting a possible clinical translation of this potential nanochemotherapeutic to expand the inherently poor bioavailability of hydrophobic ASC-J9 that could be proposed for the treatment of malignant breast cancer

Theranostic Nanocages for Imaging and Photothermal Therapy of Prostate Cancer Cells by Active Targeting of Neuropeptide‑Y Receptor

Gold nanocages (AuNCs) have been shown to be a useful tool for harnessing imaging and hyperthermia therapy of cancer, thanks to their unique optical properties, low toxicity, and facile surface functionalization. Herein, we use AuNCs for selective targeting of prostate cancer cells (PC3) via specific interaction between neuropeptide Y (NPY) receptor and three different NPY analogs conjugated to AuNCs. Localized surface plasmon resonance band of the nanoconjugates was set around 800 nm, which is appropriate for in vivo applications. Long-term stability of nanoconjugates in different media was confirmed by UV–vis and DLS studies. Active NPY receptor targeting was observed by confocal microscopy showing time-dependent AuNCs cellular uptake. Activation of ERK1/2 pathway was evaluated by Western blot to confirm the receptor-mediated specific interaction with PC3. Cellular uptake kinetics were compared as a function of peptide structure. Cytotoxicity of nanoconjugates was evaluated by MTS and Annexin V assays, confirming their safety within the concentration range explored. Hyperthermia studies were carried out irradiating the cells, previously incubated with AuNCs, with a pulsed laser at 800 nm wavelength, showing a heating enhancement ranging from 6 to 35 °C above the culture temperature dependent on the irradiation power (between 1.6 and 12.7 W/cm²). Only cells treated with AuNCs underwent morphological alterations in the cytoskeleton structure upon laser irradiation, leading to membrane blebbing and loss of microvilli associated with cell migration. This effect is promising in view of possible inhibition of proliferation and invasion of cancer cells. In summary, our Au-peptide NCs proved to be an efficient theranostic nanosystem for targeted detection and activatable killing of prostate cancer cells

Biochemical behaviour of AR in ADSCs and ADSCKs.

<p>(A) High resolution fluorescence microscopy analysis (63X magnification) performed on ADSCs and ADSCKs in absence (–T) or in presence (+T) of 10 nM of testosterone for 48 hours in basal condition or after treatment with 10 µM of MG132 for 24 hours. Nuclei were stained with DAPI. Scale bar 10 µm. (B) High resolution fluorescence microscopy analysis (63X magnification) performed on ADSCKs in the presence (+T) of 10 nM of testosterone for 48 hours after treatment with 10 µM of MG132 for 24 hours. Fluorescence microscopy localization of AR (green) and Hsp70 (red). Nuclei were stained with DAPI. (C) High resolution fluorescence microscopy analysis (63X magnification) performed on ADSCKs in presence (+T) of 10 nM of testosterone for 48 hours after treatment with 10 µM of MG132 for 24 hours. Fluorescence microscopy localization of AR (green) and ubiquitin (red). Nuclei were stained with DAPI. Panel B and C represent only the nuclear area of the cells where the aggregates were observed. The arrows indicate the ARpolyQ inclusions co-stained with Hsp70 and ubiquitin, respectively.</p

Characterization of ADSC and ADSCK cell cultures.

<p>(A) Growth curve of two representative ADSC and ADSCK primary cultures. Values were expressed as cumulative population doublings calculated with the formula reported by Avanzini et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112746#pone.0112746-Avanzini1" target="_blank">[26]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112746#pone.0112746-Kotaja1" target="_blank">[44]</a>. (B) Cell viability after freezing and thawing evaluated by trypan blue dye exclusion assay comparing the number of viable cells after thawing to the number of cells previously frozen. Data are expressed as mean±SEM (n = 9 per group; three independent experiments for each cell culture; *P<0.05 vs ADSCs). (C) FACs analysis performed at passage 3 on ADSC (black bar) and on ADSCK (grey bar) to evaluate immunophenotypically mesenchymal markers. Data are expressed as mean±SD (n = 3 per group).</p

Characterization of AR expression in ADSCs and ADSCKs.

<p>(A) AR mRNA levels in ADSCs and ADSCKs were determined by real time quantitative PCR. SHSY-5Y cells were used as negative control. (n = 3 per group; *P<0.05, **P<0.01 <i>vs</i> negative control; °P<0.05 <i>vs</i> ADSCs). (B) Western Blot on ADSCs and ADSCKs in absence (–T) or in presence (+T) of 10 nM of testosterone for 48 hours. Alpha-tubulin was used to normalize for protein loading.</p