Sensitive Detection of Small Molecules by Competitive Immunomagnetic-Proximity Ligation Assay

A novel detection method of small molecules, competitive immunomagnetic-proximity ligation assay (CIPLA), was developed and described in this report. Through the proximity effect caused by special proximity probes we prepared, small molecules can be detected using only one monoclonal antibody. CIPLA overcomes the obstacle that the proximity ligation assay (PLA) cannot be used in small molecular detection, as two antibodies are unable to combine to one small molecule due to its small molecular structure. Two small molecular compounds, clenbuterol (CLE) and ractopamine (RAC), were selected as targets for CIPLA. The limit of detection (LOD) reached 0.01 ng mL^–1, which was 10–50-fold lower than ELISA. With 5 orders of magnitude of the dynamic range achieved, the excellent sensitivity and broad dynamic range of CIPLA are noted. It can be applied widely in the sensitive detection of many other small molecular materials such as pesticides, additives in food, drugs, and biological samples, which have great significance in both theoretical and practical aspects

<i>N</i>-Butyl-2-cyanoacrylate-based injectable and <i>in situ</i>-forming implants for efficient intratumoral chemotherapy

<p>The local delivery of chemotherapeutic drugs to tumor sites is an effective approach for achieving therapeutic drug concentrations in solid tumors. Injectable implants with the ability to form <i>in situ</i> represent one of the most promising technologies for intratumoral chemotherapy. However, many issues must be resolved before these implants can be applied in clinical practice. Herein, we report a novel injectable <i>in situ</i>-forming implant system composed of <i>n</i>-butyl-2-cyanoacrylate (NBCA) and ethyl oleate, and the sol–gel phase transition is activated by anions in body fluids or blood. This newly developed injectable NBCA ethyl oleate implant (INEI) is biodegradable, biocompatible, and non-toxic. INEI solidifies in several seconds after exposure to body fluids or blood, and the implant’s <i>in vivo</i> degradation time can be controlled. In addition, the pore sizes formed by the polymerization of NBCA can be decreased by increasing the NBCA concentration in the implants. Therefore, the drug retention/release time can be adjusted from a few weeks to several months by changing the concentration of NBCA in the implant formulation. Anti-tumor experiments in animal models showed that the average growth inhibition rate of xenografted human breast cancer cells by the paclitaxel-loaded INEI (40% NBCA) was 80%, and they also indicated that tumors in some of the mice were completely eliminated by just a single dosage injection. For the epirubicin-loaded INEI (50% NBCA), the average growth inhibition rate of xenografted human liver cancer cells was 58%. Thus, the chemotherapeutic drug-loaded INEIs exhibited excellent therapeutic efficacy for local chemotherapy.</p

The strategy of developing NAAA inhibitors.

<p>A–C, the chemical structures of classic NAAA inhibitors including PEA (A), CCP (B), and (S)-OOPP (C); (D) SAR study of 1-Pentadecanyl-carbonyl pyrrolidine; (E) Dose-dependent inhibition of 1-Pentadecanyl-carbonyl pyrrolidine on NAAA activity.</p

Characterization of compound 16 as a reversible and competitive NAAA inhibitor.

<p>(A) Effect of compound 16 (10 µM) on NAAA activity in HEK293 cells heterogeneously overexpressing NAAA. ***, P<0.001 vs. vehicle, n = 4. (B) Concentration-dependent inhibition of NAAA by compound 16 using NAAA recombinant protein derived from HEK293 cell heterogeneously expressing NAAA. (C) Rapid dilution NAAA assay in the presence of vehicle (1% DMSO, open circles) or compound 16 (closed circles). (D) Effect of NAAA activity in the presence of vehicle (open bars) or compound 16 (closed bars) before dialysis (0) and 8 hr after dialysis (8). ***, P<0.001 vs vehicle, n = 4; (E) Michaelis-Menten analysis of the NAAA reaction in the presence of vehicle (open circles) or compound 16 (closed circles). Insert is shown in a Lineweaver-Burk plot.</p

Atheroprotective Effect of Oleoylethanolamide (OEA) Targeting Oxidized LDL

<div><p>Dietary fat-derived lipid oleoylethanolamide (OEA) has shown to modulate lipid metabolism through a peroxisome proliferator-activated receptor-alpha (PPAR-α)-mediated mechanism. In our study, we further demonstrated that OEA, as an atheroprotective agent, modulated the atherosclerotic plaques development. <i>In vitro</i> studies showed that OEA antagonized oxidized LDL (ox-LDL)-induced vascular endothelial cell proliferation and vascular smooth muscle cell migration, and suppressed lipopolysaccharide (LPS)-induced LDL modification and inflammation. <i>In vivo</i> studies, atherosclerosis animals were established using balloon-aortic denudation (BAD) rats and ApoE^-/- mice fed with high-caloric diet (HCD) for 17 or 14 weeks respectively, and atherosclerotic plaques were evaluated by oil red staining. The administration of OEA (5 mg/kg/day, intraperitoneal injection, i.p.) prevented or attenuated the formation of atherosclerotic plaques in HCD-BAD rats or HCD-ApoE^−/− mice. Gene expression analysis of vessel tissues from these animals showed that OEA induced the mRNA expressions of PPAR-α and downregulated the expression of M-CFS, an atherosclerotic marker, and genes involved in oxidation and inflammation, including iNOS, COX-2, TNF-α and IL-6. Collectively, our results suggested that OEA exerted a pharmacological effect on modulating atherosclerotic plaque formation through the inhibition of LDL modification in vascular system and therefore be a potential candidate for anti-atherosclerosis drug.</p></div

Compound 16 interacted with NAAA protein.

<p>(A) Computational model illustrated docking of compound 16 at the active site of rat NAAA. (B) Effect of mutant Ala²⁰⁹-NAAA on NAAA activity. Mock, HEK293 cell heterogeneously overexpressing vector control; NAAA, HEK293 cell heterogeneously overexpressing NAAA; Ala209, HEK293 cell heterogeneously overexpressing mutant Ala²⁰⁹-NAAA. ***, P<0.001 vs. NAAA, n = 5.</p

Compound 16 reduced LPS-induced inflammation.

<p>(A) Effect of compound 16 (concentrations in µM) or Vehicle on PEA levels (A), mRNA expression levels of iNOS (B) and IL-6 (C) in RAW264.7 treated with vehicle (open bars) or LPS (closed bars). vehicle, 0.1% DMSO; LPS, 0.5 µg/mL. **, P<0.01; ***, P<0.001 vs. vehicle; ##, P<0.01; ###, P<0.001 vs. LPS control, n = 5.</p

OEA reduced atherosclerotic plaque formation in ApoE^−/− mice.

<p>Oil red O staining (A) and H & E staining (B–E) showed aortic atherosclerotic formation in wild-type C57mice fed with HCD (A₁, B), ApoE<b>⁻</b>^/<b>−</b> mice fed with normal diet (A₂, C), ApoE<b>⁻</b>^/<b>−</b> mice fed with HCD (A₃, D), and ApoE<b>⁻</b>^/<b>−</b>-HCD mice with OEA administration (5 mg/kg, i.p.) (A₄, E). Scale bar, 2 mm or 0.5 mm. (F–K), The effect of OEA on mRNA expression levels of PPAR-α (F), M-CSF (G), COX-2 (H), CRP (I), TNF-α (J) and IL-6 (K) in aorta tissues of wt-HCD mice, ApoE<b>⁻</b>^/<b>−</b>-ND mice and ApoE<b>⁻</b>^/<b>−</b>-HCD mice. Vehicle, 5% PEG/5% Tween-80 in saline; OEA, 5 mg/kg/day, i.p.; * p<0.05, ** p<0.01, *** p<0.001, one-way ANOVA, n = 6–8 mice/group.</p

OEA reduced ox-LDL-induced VSMC migration via PPAR-α signaling.

<p>(A–H), The effect of vehicle (A), LDL (B), ox-LDL (C) ,OEA (D) and MK886 (E–H) on VSMC migration, assessed by transwell assay; (I) Quantitation of chemotaxis of A–H. Vehicle, 0.1% DMSO; LDL, 50 μg/ml; ox-LDL, 50 μg/ml; OEA, 50 μM; MK886, 10 μM. *** p<0.001, one-way ANOVA, n = 6.</p

Inhibition of ox-LDL-induced inflammation by OEA was mediated by PPAR-α signaling.

<p>The effect of vehicle (0.1% DMSO), LDL (50 μg/ml), ox-LDL (50 μg/ml), OEA (50 μM) or MK886 (10 μM) on mRNA expression levels of PPAR-α (A, D), iNOS (B, E) and COX-2 (C, F) in mouse primary macrophages (A–C) or mouse macrophage RAW246.7 cells (D–F). N.S., not significant; * p<0.05, ** p<0.01, *** p<0.001, one-way ANOVA, n = 4–8.</p