Fluorescent Nanoparticle Adhesion Assay: a Novel Method for Surface p<i>K</i>_a Determination of Self-Assembled Monolayers on Silicon Surfaces

Since the computer industry enables us to generate smaller and smaller structures, silicon surface chemistry is becoming increasingly important for (bio‑)­analytical and biological applications. For controlling the binding of charged biomacromolecules such as DNA and proteins on modified silicon surfaces, the surface p<i>K</i>_a is an important factor. Here we present a fluorescent nanoparticle adhesion assay as a novel method to determine the surface p<i>K</i>_a of silicon surfaces modified with weak acids or bases. This method is based upon electrostatic interactions between the modified silicon surface and fluorescent nanoparticles with an opposite charge. Silicon slides were modified with 3-aminopropyltriethoxysilane (APTES) and were further derivatized with succinic anhydride. Layer thickness of these surfaces was determined by ellipsometry. After incubating the surfaces with an amine-reactive fluorescent dye, fluorescence microscopy revealed that the silicon surfaces were successfully modified with amine- and carboxyl-groups. Two surface p<i>K</i>_a values were found for APTES surfaces by the fluorescent nanoparticle adhesion assay. The first surface p<i>K</i>_a (6.55 ± 0.73) was comparable with the surface p<i>K</i>_a obtained by contact angle titration (7.3 ± 0.8), and the second surface p<i>K</i>_a (9.94 ± 0.19) was only found by using the fluorescent nanoparticle adhesion assay. The surface p<i>K</i>_a of the carboxyl-modified surface by the fluorescent nanoparticle adhesion assay (4.37 ± 0.59) did not significantly differ from that found by contact angle titration (5.7 ± 1.4). In conclusion, we have developed a novel method to determine the surface p<i>K</i>_a of modified silicon surfaces: the fluorescent nanoparticle adhesion assay. This method may provide a useful tool for designing pH-dependent electrostatic protein and particle binding/release and to design surfaces with a pH-dependent surface charge for (bio‑)­analytical lab-on-a-chip devices or drug delivery purposes

Nasal application of low-dose mB29a-PLGA containing particles reduces severity of arthritis.

<p><b>A and B.</b> Effect of mB29a-nanoparticles on nasally induced suppression of PG-induced arthritis in BALB/c mice. Mice received 30 µg of mB29a peptide i.n. dissolved in PBS or encapsulated in PLGA or PLGA-TMC nanoparticles prior to arthritis induction. Arthritis scores of mB29a-PBS (black squares), PLGA (black circles) or PLGA-TMC (black triangles) treated mice as assessed by swelling and redness of the paws. Data are shown as the mean arthritis scores ± SEM. of n = 3 mice per group. Statistically significant: **, <i>P</i><0.01.</p

Nanoparticle mediated enhanced antigen presentation capacity of BMDCs <i>in vitro</i>.

<p>BMDC were incubated in the presence of sOVA-FITC or OVA-FITC encapsulated into PLGA, PLGA-TMC or TMC-TPP nanoparticles at different concentrations. External FITC signaling was silenced by trypan blue. <b>A.</b> The ΔMFI of OVA-FITC was assessed by subtraction of FITC signaling at 4°C from 37°C. <b>B.</b> OVA-FITC uptake by BMDC shown as the net percentage of OVA-FITC positive cells. <b>C.</b> CFSE-labeled CD4⁺ T-cells were incubated with BMDC stimulated with sOVA or OVA encapsulated in nanoparticles. Gray filled histograms; unstimulated CD4⁺ T-cells, Black overlays; CD4⁺ T-cell division patterns at different OVA concentrations after 72 hours. <b>D–F.</b> Cytokine concentrations of IL-2, IFN-γ and IL-10 (ng/ml) were determined in culture supernatants, after 72 h of culture. Data are representative for 3 independent experiments; mean ± SEM. Statistically significant: *, <i>P</i><0.05; **, <i>P</i><0.01.</p

Onset of disease and maximum arthritis scores.

<p>Hsp70-mB29a peptide loaded PLGA, PLGA-TMC nanoparticles or PBS control (10 µg) were given i.n. on day −7, −5 and −3 and arthritis was induced by PG/DDA immunization on day 0 and 21. Arthritis symptoms were scored as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026684#s4" target="_blank">materials and methods</a>. Day of onset and maximum arthritis scores were depicted as mean ± SEM. of n = 3 mice per group of one experiment.</p

Enhanced OVA-specific CD4⁺ T-cell proliferation, after nanoparticle administration.

<p><b>A and D.</b> OVA-specific CFSE labeled CD4⁺ T-cells were transferred to BALB/c recipient mice one day prior to vaccination. Mice received a single i.n. application of 30 µg of sOVA or OVA encapsulated into PLGA, PLGA-TMC or TMC-TPP nanoparticles. For induction of a non-mucosal response, mice received a single i.m. immunization in the hind limbs. At 72 h post OVA administration, <i>in vivo</i> T-cell division was addressed in spleen, nose-draining NALT and CLN as well as the thigh-draining ILN. Data are representative for at least 3 i.n. and 2 i.m. independent transfer studies. <b>B, C and E.</b> Total mRNA was purified from single cell suspensions from NALT, CLN, and ILN. Relative mRNA expression to HPRT of Foxp3 was determined 72 h post OVA application. Cells isolated from NALT were pooled per group. LN data are representative for at least 3 to 5 mice per group; mean ± SEM. Statistically significant: *, <i>P</i><0.05.</p

Complement activation by IVIg fractions.

<p>Detection of fluid-phase complement activation by measuring C4b/c in serum incubated at 37°C together with IVIg, dimeric, monomeric fraction and heated IgG (aggregates), or no stimulus, incubated at 37°C or on ice. One-way ANOVA; *** p < 0.001.</p

Neutrophil activation by IVIg and fractions thereof.

<p>Neutrophil activation, measured as elastase release of resting neutrophils, induced by filtered IVIg fractions at concentrations between 0.3 and 5 mg/mL in the presence of poloxamer (a), or using a previously established protocol without filtration and poloxamer (b). As a positive control, neutrophils were also stimulated by coated IgG, which leads to more than 80% degranulation at concentrations as low as 0.5 mg/mL (c). Activation of primed neutrophils using cytochalacin B (d) or TNF (e) by filtered IVIg fractions in the presence of poloxamer. Similarly, oxidation burst was evaluated in the presence of filtered IVIg fractions in the presence of poloxamer (f). One-way ANOVA (a, d, e, f) or Student’s t test (b, c); * p < 0.05, ** p < 0.01, *** p < 0.001.</p

Binding to FcγR by IVIg fractions.

<p>Sensograms of the binding of dimeric fraction (a,d) and monomeric fraction (b,e) to FcγRIIIb (a-b) or FcγRIIa (d-e) coupled to a biosensor chip through its C-terminal His-tag. Steady state apparent affinities calculated from the sensogram data were obtained from fitting a Langmuir binding model as shown in the inset panels, to calculate the apparent Kd values to FcγRIIIb (c) or FcγRIIa (f). One-way ANOVA; ** p < 0.01.</p

A New Strategy To Stabilize Oxytocin in Aqueous Solutions: II. Suppression of Cysteine-Mediated Intermolecular Reactions by a Combination of Divalent Metal Ions and Citrate

A series of studies have been conducted to develop a heat-stable liquid oxytocin formulation. Oxytocin degradation products have been identified including citrate adducts formed in a formulation with citrate buffer. In a more recent study we have found that divalent metal salts in combination with citrate buffer strongly stabilize oxytocin in aqueous solutions (Avanti, C.; et al. <i>AAPS J.</i> <b>2011</b>, <i>13</i>, 284–290). The aim of the present investigation was to identify various degradation products of oxytocin in citrate-buffered solution after thermal stress at a temperature of 70 °C for 5 days and the changes in degradation pattern in the presence of divalent metal ions. Degradation products of oxytocin in the citrate buffer formulation with and without divalent metal ions were analyzed using liquid chromatography–mass spectrometry/mass spectrometry (LC–MS/MS). In the presence of divalent metal ions, almost all degradation products, in particular citrate adduct, tri- and tetrasulfides, and dimers, were greatly reduced in intensity. No significant difference in the stabilizing effect was found among the divalent metal ions Ca²⁺, Mg²⁺, and Zn²⁺. The suppressed degradation products all involve the cysteine residues. We therefore postulate that cysteine-mediated intermolecular reactions are suppressed by complex formation of the divalent metal ion and citrate with oxytocin, thereby inhibiting the formation of citrate adducts and reactions of the cysteine thiol group in oxytocin

Cathodic Corrosion of a Bulk Wire to Nonaggregated Functional Nanocrystals and Nanoalloys

A key enabling step in leveraging the properties of nanoparticles (NPs) is to explore new, simple, controllable, and scalable nanotechnologies for their syntheses. Among “wet” methods, cathodic corrosion has been used to synthesize catalytic aggregates with some control over their size and preferential faceting. Here, we report on a modification of the cathodic corrosion method for producing a range of nonaggregated nanocrystals (Pt, Pd, Au, Ag, Cu, Rh, Ir, and Ni) and nanoalloys (Pt₅₀Au₅₀, Pd₅₀Au₅₀, and Ag_<i>x</i>Au_{100–<i>x</i>}) with potential for scaling up the production rate. The method employs poly­(vinylpyrrolidone) (PVP) as a stabilizer in an electrolyte solution containing nonreducible cations (Na⁺, Ca²⁺), and cathodic corrosion of the corresponding wires takes place in the electrolyte under ultrasonication. The ultrasonication not only promotes particle–PVP interactions (enhancing NP dispersion and diluting locally high NP concentration) but also increases the production rate by a factor of ca. 5. Further increase in the production rate can be achieved through parallelization of electrodes to construct comb electrodes. With respect to applications, carbon-supported Pt NPs prepared by the new method exhibit catalytic activity and durability for methanol oxidation comparable or better than the commercial benchmark catalyst. A variety of Ag_<i>x</i>Au_{100–<i>x</i>} nanoalloys are characterized by ultraviolet–visible absorption spectroscopy and high-resolution transmission electron microscopy. The protocol for NP synthesis by cathodic corrosion should be a step toward its further use in academic research as well as in its practical upscaling