Intracellular Drug Release from Curcumin-Loaded PLGA Nanoparticles Induces G2/M Block in Breast Cancer Cells

PLGA nanoparticles are among the most studied polymer nanoformulations for several drugs against different kinds of malignant diseases, thanks to their in vivo stability and tumor localization exploiting the well-documented “enhanced permeation and retention” (EPR) effect. In this paper, we have developed uniform curcumin-bearing PLGA nanoparticles by a single-emulsion process, which exhibited a curcumin release following a Fickian-law diffusion over 10 days in vitro. PLGA nanoparticles were about 120 nm in size, as determined by dynamic light scattering, with a surface negative charge of −30 mV. The loading ratio of encapsulated drug in our PLGA nanoformulation was 8 wt%. PLGA encapsulation provided efficient protection of curcumin from environment, as determined by fluorescence emission experiments. Next, we have investigated the possibility to study the intracellular degradation of nanoparticles associated with a specific G2/M blocking effect on MCF7 breast cancer cells caused by curcumin release in the cytoplasm, which provided direct evidence on the mechanism of action of our nanocomplex. This study was carried out using Annexin V-based cell death analysis, MTT assessment of proliferation, flow cytometry, and confocal laser scanning microscopy. PLGA nanoparticles proved to be completely safe, suggesting a potential utilization of this nanocomplex to improve the intrinsically poor bioavailability of curcumin for the treatment of severe malignant breast cancer

Structural Iridescent Tuned Colors from Self-Assembled Polymer Opal Surfaces

Structural colors are the object of a wide scientific interest, not only for the potential technical applications of their intriguing optical properties but also for the need of coloring agents to replace toxic and carcinogenic dyes. We present a simple methodology to obtain polymer opal surfaces of self-assembled core–shell nanoparticles with different degree of order for structural color applications. Polymer nanospheres prepared by surfactant-free emulsion radical copolymerization of an hydrophobic and an hydrophilic comonomer (styrene and methacrylic acid) spontaneously assemble into core–shell particles. Nanoparticles with identical composition and different diameters were prepared by modulating the degree of ionization of the weakly acidic comonomer. We report experimental results revealing how the synthesis parameters affect the properties of the core–shell particles and their influence on the optical properties of the final polymer opal surfaces, which depend on size, charge, and packing arrangement of the constituent nanoparticles

Additional file 2: of Association between admission anemia and long-term mortality in patients with acute myocardial infarction: results from the MONICA/KORA myocardial infarction registry

Table S1. Results of the sensitivity analysis (n = 2187). (DOCX 16 kb

Additional file 1: of Association between admission anemia and long-term mortality in patients with acute myocardial infarction: results from the MONICA/KORA myocardial infarction registry

Figure S1. Hazard ratios for long-term mortality in patients with AMI and anemia covering increasing observation periods. Reference: Non-anemia: Hemoglobin (Hb) concentration of ≥12 g/dL in women, Hb concentration of ≥13 g/dL in men. Mild anemia: Hb concentration of 11 g/dL to < 12 g/dL in women, Hb concentration of 11 g/dL to < 13 g/dL in men. Moderate to severe anemia: Moderate to severe anemia: Hb concentration of < 11 g/dL in women and men. 95% Confidence intervals (CI) are represented by vertical lines above and below the HR estimates; 95% CI for mild anemia: dashed line; 95% CI for moderate to severe anemia: continuous line. AMI, Acute myocardial infarction; CI, Confidence interval; Hb, Hemoglobin; HR, Hazard ratio. (PDF 49 kb

Peptide-Nanoparticle Ligation Mediated by <i>Cutinase</i> Fusion for the Development of Cancer Cell-Targeted Nanoconjugates

The relationship between the positioning of ligands on the surface of nanoparticles and the structural features of nanoconjugates has been underestimated for a long time, albeit of primary importance to promote specific biological recognition at the nanoscale. In particular, it has been formerly observed that a proper molecular orientation can play a crucial role, first optimizing ligand immobilization onto the nanoparticles and, second, improving the targeting efficiency of the nanoconjugates. In this work, we present a novel strategy to afford peptide-oriented ligation using genetically modified <i>cutinase</i> fusion proteins, which combines the presence of a site-directed “capture” module based on an enzymatic unit and a “targeting” moiety consisting of the ligand terminal end of a genetically encoded polypeptide chain. As an example, the oriented presentation of U11 peptide, a sequence specific for the recognition of urokinase plasminogen activator receptor (uPAR), was achieved by enzyme-mediated conjugation with an irreversible inhibitor of cutinase, an alkylphosphonate <i>p</i>-nitrophenol ester linker, covalently bound to the surface of iron oxide nanoparticles. The targeting efficiency of the resulting protein–nanoparticle conjugates was assessed using uPAR-positive breast cancer cells exploiting confocal laser scanning microscopy and quantitative fluorescence analysis of confocal images. Ultrastructural analysis of transmission electron micrographs provided evidence of a receptor-mediated pathway of endocytosis. Our results showed that, despite the small average number of targeting peptides presented on the nanoparticles, our ligand-oriented nanoconjugates proved to be very effective in selectively binding to uPAR and in promoting the uptake in uPAR-positive cancer cells

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

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

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

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