Surface-Plasmon-Resonance Amplification of FMD Detection through Dendrimer Conjugation.

The amplification of the surface plasmon resonance (SPR) sensitivity for the foot-and-mouth disease (FMD) detection was studied using Poly(amidoamine) (PAMAM) succinamic-acid dendrimers. The dendrimers were conjugated with the complementary annealed with the aptamers capable of binding specifically to FMD peptides. The tethered layer of the dendrimer-conjugated double-stranded(ds)-aptamers was formed on the SPR sensor Au surface via a thiol bond between the aptamers and Au. After the tethered layer was formed, the surface was taken out of the SPR equipment. Then, the ds-aptamers on the surface were denatured to collect the dendrimer-conjugated single-stranded(ss)-complementary. The surface with only the remaining ss-aptamers was transferred again to the equipment. Two types of the injections, the FMD peptide only and the dendrimer-conjugated ss-complementary followed by the FMD peptides, were performed on the surface. The sensitivity was increased 20 times with the conjugation of the dendrimers, but the binding rate of the peptides became more than two times slower.

Binding Behavior between Transforming-Growth-Factor-Beta1 and Its Receptor Reconstituted in Biomimetic Membranes.

Transforming growth factor ß1 (TGF-ß1) is critical to cell differentiation, proliferation, and apoptosis. It is important to understand the binding affinity between TGF-ß1 and its receptors. In this study, their binding force was measured using an atomic force microscope. Significant adhesion was induced by the interaction between the TGF-ß1 immobilized on the tip and its receptor reconstituted in the bilayer. Rupture and adhesive failure occurred at a specific force around 0.4~0.5 nN. The relationship of the force to loading rate was used to estimate the displacement where the rupture occurred. The binding was also monitored in real time with surface plasmon resonance (SPR) and interpreted with kinetics to acquire the rate constant. Using the Langmuir adsorption, the SPR data were analyzed to estimate equilibrium and association constants to be approximately 107 M-1 and 106 M-1 s-1. These results indicated that the natural release of the binding seldom occurred. Furthermore, the degree of binding dissociation, confirmed by the rupture interpretation, supported that the reverse of the binding hardly happened.

Mechanical Properties of 3-Hydroxybutyric Acid-Induced Vesicles.

The vesicle mechanical behaviors were studied upon its exposure to 3-hydroxybutyric acid using an atomic force microscope (AFM). Dipalmitoylphosphatidylcholine (DPPC) and 3-hydroxybutyric acid were used to manufacture the vesicles at their desired ratio. The deflection of an AFM probe with respect to its displacement was measured after characterizing the vesicle adsorption. The movement was analyzed with the Hertzian model to understand the physical behavior of the vesicles. However, in the deflection just prior to the first penetration, the model was a good fit, and the vesicle mechanical moduli were calculated. The moduli became lower with the higher ratio of 3-hydroxybutyric acid to DPPC, but the moduli were saturated at 0.5 of the ratio. These results appear to be the basis for the function of the metabolism associated with 3-hydroxybutyric acid, i.e., anesthetization and glycemic control, on the physical properties of cell membranes.

Lyophilization of Curcumin-Albumin Nanoplex with Sucrose as Cryoprotectant: Aqueous Reconstitution, Dissolution, Kinetic Solubility, and Physicochemical Stability.

An amorphous curcumin (CUR) and bovine serum albumin (BSA) nanoparticle complex (nanoplex) was previously developed as a promising anticancer nanotherapy. The CUR-BSA nanoplex had been characterized in its aqueous suspension form. The present work developed a dry-powder form of the CUR-BSA nanoplex by lyophilization using sucrose as a cryoprotectant. The cryoprotective activity of sucrose was examined at sucrose mass fractions of 33.33, 50.00, and 66.66% by evaluating the lyophilized nanoplex's (1) aqueous reconstitution and (2) CUR dissolution and kinetic solubility. The physicochemical stabilizing effects of sucrose upon the nanoplex's 30-day exposures to 40 °C and 75% relative humidity were examined from (i) aqueous reconstitution, (ii) CUR dissolution, (iii) CUR and BSA payloads, (iv) amorphous form stability, and (v) BSA's structural integrity. The good cryoprotective activity of sucrose was evidenced by the preserved BSA's integrity and good aqueous reconstitution, resulting in a fast CUR dissolution rate and a high kinetic solubility (≈5-9× thermodynamic solubility), similar to the nanoplex suspension. While the aqueous reconstitution, CUR dissolution, and amorphous form were minimally affected by the elevated heat and humidity exposures, the treated nanoplex exhibited a lower BSA payload (≈7-26% loss) and increased protein aggregation postexposure. The adverse effects on the BSA payload and aggregation were minimized at higher sucrose mass fractions.

Changes in Mechanical Properties of Vesicles by Mucin in Aqueous Solution.

The mechanical properties of vesicles were investigated as they were prepared, according to the ratio of mucin to dipalmitoylphosphatidylcholine (DPPC), using an atomic force microscope (AFM). After the confirmation of the vesicle adsorption on a mica surface, an AFM-tip deflection, caused by the interaction between the tip and the vesicle, was measured. The deflection showed that the tip broke through into the vesicle twice. Each break meant a tip-penetration into the upper and lower portion of the vesicle. Only the first penetration allowed the Hertzian model available to estimate the vesicle mechanical moduli. Two moduli reduced as the ratio of mucin to DPPC increased to 0.5, but the moduli were little changed above the 0.5 ratio. These results seem to be a platform for the effect of the mucin on the plasma-membrane anchoring and cellular signaling.

Something's Fishy About It: How Opinion Congeniality and Explainability Affect Motivated Attribution to Artificial Intelligence Versus Human Comment Moderators.

An online experiment (N = 384) examined when and how the identity of the comment moderator (artificial intelligence [AI] vs. human) on a news website affects the extent to which individuals (a) suspect political motives for comment removal and (b) believe in the AI heuristic ("AI is objective, neutral, accurate, and fair"). Specifically, we investigated how the provision of an explanation for comment removal (none vs. real vs. placebic), and opinion congeniality between the remaining comments and the user's opinion (uncongenial vs. congenial) qualify social responses to AI. Results showed that news users were more suspicious of political motives for an AI (vs. human) moderator's comment removal (a) when the remaining comments were uncongenial, and (b) when no explanation was offered for deleted comments. Providing a real explanation (vs. none) attenuated participants' suspicion of political motives behind comment removal, but only for the AI moderator. When AI moderated the comments section, the exposure to congenial (vs. uncongenial) comments led participants to endorse the AI heuristic more strongly, but only in the absence of an explanation for comment removal. By contrast, the participants' belief in AI heuristic was stronger when a human moderator preserved uncongenial (vs. congenial) comments. Apparently, they considered AI as a viable alternative to a human moderator whose performance was unsatisfactory.

Benchmarking the Solubility Enhancement and Storage Stability of Amorphous Drug-Polyelectrolyte Nanoplex against Co-Amorphous Formulation of the Same Drug.

Amorphization, typically in the form of amorphous solid dispersion (ASD), represents a well-established solubility enhancement strategy for poorly soluble drugs. Recently, two amorphous drug formulations, i.e., the amorphous drug-polyelectrolyte nanoparticle complex (nanoplex) and co-amorphous system, have emerged as promising alternatives to circumvent the issues faced by ASD (i.e., large dosage requirement, high hygroscopicity). In the present work, the nanoplex was benchmarked against the co-amorphous system in terms of the preparation efficiency, drug payload, thermal stability, dissolution rate, supersaturation generation, and accelerated storage stability. Weakly acidic curcumin (CUR) and weakly basic ciprofloxacin (CIP) were used as the model poorly soluble drugs. The CUR and CIP nanoplexes were prepared using chitosan and sodium dextran sulfate as the polyelectrolytes, respectively. The co-amorphous CUR and CIP were prepared using tannic acid and tryptophan as the co-formers, respectively. The benchmarking results showed that the amorphous drug nanoplex performed as well as, if not better than, the co-amorphous system depending on the drug in question and the aspects being compared. The present work successfully established the nanoplex as an equally viable amorphous drug formulation as the more widely studied co-amorphous system to potentially serve as an alternative to ASD.

CD133 increases oxidative glucose metabolism of HT29 cancer cells by mitochondrial uncoupling and its inhibition enhances reactive oxygen species-inducing therapy.

OBJECTIVE: A better understanding of the metabolic phenotype of stem-like cancer cells could provide targets to help overcome chemoresistance. In this study, we hypothesized that colon cancer cells with the stem cell feature of CD133 expression have increased proton leakage that influences glucose metabolism and offers protection against reactive oxygen species (ROS)-inducing treatment. METHODS AND RESULTS: In HT29 colon cancer cells, 18 F-fluorodeoxyglucose (FDG) uptake was increased by CD133 selection and decreased by CD133 silencing. In CD133(+) cells, greater 18 F-FDG uptake was accompanied by increased oxygen consumption rate (OCR) and reduced mitochondrial membrane potential and mitochondrial ROS, indicating increased proton leakage. The uncoupling protein inhibitor genipin reversed the increased 18 F-FDG uptake and greater OCR of CD133(+) cells. The ROS-inducing drug, piperlongumine, suppressed CD133(-) cell survival by stimulating mitochondrial ROS generation but was unable to influence CD133(+) cells when used alone. However, cotreatment of CD133(+) cells with genipin and piperlongumine efficiently stimulated mitochondrial ROS for an enhanced antitumor effect with substantially reduced CD133 expression. CONCLUSION: These results demonstrate that mitochondrial uncoupling is a metabolic feature of CD133(+) colon cancer cells that provides protection against piperlongumine therapy by suppressing mitochondrial ROS generation. Hence, combining genipin with ROS-inducing treatment may be an effective strategy to reverse the metabolic feature and eliminate stem-like colon cancer cells.

Ectoine Effect on Mechanical Properties of Vesicles in Aqueous Solution.

The mechanical properties of the vesicles incorporated with ectoine were studied using atomic force microscope (AFM). The vesicles were prepared with dipalmitoylphosphatidylcholine (DPPC) by changing only the ratio of the ectoine to DPPC. After the vesicles were adsorbed on the mica substrate and their morphology were characterized, the plot of an AFM tip displacement versus the tip deflection was acquired by monitoring the behavior of the tip into the vesicle. The breakthrough of the tip into the vesicle was observed to occur twice. Each breakthrough represented a penetration of the tip into the top and bottom portions of the vesicle, respectively. The force data between the pre-contact and the first breakthrough were comparable with the Hertzian model to estimate Young's modulus and the bending modulus of the vesicles. Both moduli decreased proportionally with the increase in the ratio of ectoine to lipid up to 0.5. However, above 0.5, the moduli were slightly changed with the increase. These results of the mechanical properties appear to be due to the osmotic and volumetric effect on the headgroup packing disruption.

Amplification of urea detection based on pH-sensitive liposomes

BACKGROUND: This study aimed to develop an amplification method of urea detection based on pHsensitive liposomes. RESULTS: The urease covalently immobilized on the magnetic particles and the pH-sensitive liposomes encapsulating ferricyanide were added to the cyclic-voltammeter cell solution where urea was distributed. The conversion of urea into carbonic acid seemed to induce a pH decrease that caused a reduction in the electrostatic repulsion between the headgroups of weakly acidic 1,2-dipalmitoyl-sn-glycero3-succinate. The reduction induced the liposomes to release potassium ferricyanide that was encapsulated inside. The effects of urea concentration and pH value were investigated. A specific concentration (0.5 mg/mL) of the urea solution was set to observe the response. The activity of urease was reversible with respect to the pH change between 7 and 5. The sensitivity of this detection was almost identical to the comparable techniques such as an enzyme-linked immunosorbent assay and a field-effect transistor. CONCLUSIONS: In summary, the methodology developed in this study was feasible as a portable, rapid, and sensitive method.

Acetylcholine Detection Based on pH-Sensitive Liposomes.

The pH-sensitive liposomes were employed to amplify the detection of acetylcholine (ACh). Acetylcholinesterase (AChE) covalently immobilized on the magnetic particles and the pH-sensitive liposomes encapsulating ferricyanide were added to a cyclic voltammetry cell solution where ACh was distributed. The conversion of ACh into acetic acid seemed to induce the pH decrease that caused the reduction in the electrostatic repulsion between the head groups of weakly acidic 1,2-dipalmitoyl-sn-glycero-3-succinate. The reduction generated liposome destabilization, which released potassium ferricyanide encapsulated inside the liposomes. The effects of the ACh concentration and pH were investigated. An addition of 10 µL of more than 0.5 mg/mL ACh concentration into 5 mL of a cyclic voltammetry cell solution was necessary to observe the response. The activity of AChE was reversible with respect to the pH change between 7 and 5. The sensitivity of this detection was almost identical to comparable techniques such as enzyme-linked immunosorbent assay, field-effect transistor, fluorescence, UV spectrometry, magnetic resonance imaging, and surface plasmon resonance. Therefore, the methodology developed in this study is feasible as a portable, rapid, and sensitive method.

89Zr anti-CD44 immuno-PET monitors CD44 expression on splenic myeloid cells and HT29 colon cancer cells.

CD44 is a cell-surface glycoprotein involved in cell-cell interaction, adhesion, and migration. CD44 is found on colon cancer cells and on immune cells. Previous studies of 89Zr PET imaging of CD44 have relied on an anti-human antibody (Ab), which can influence biodistribution in murine models. In this study, we used an Ab that cross-reacts with both human and mouse origin CD44 of all isoforms to unveil the type of leukocyte responsible for high splenic anti-CD44 uptake and investigate how its regulation can influence tumor immuno-PET. The Ab was site-specifically labeled with 89Zr-deferoxamine on cysteine residues. 89Zr-anti-CD44 demonstrated high-specific binding to HT29 human colon cancer cells and monocytic cells that showed CD44 expression. When 89Zr-anti-CD44 was administered to Balb/C nude mice, there was remarkably high splenic uptake but low SNU-C5 tumor uptake (1.2 ± 0.7%ID/g). Among cells isolated from Balb/C mouse spleen, there was greater CD44 expression on CD11b positive myeloid cells than lymphocytes. In cultured monocytic and macrophage cells, LPS stimulation upregulated CD44 expression and increased 89Zr-anti-CD44 binding. Similarly, normal Balb/C mice that underwent lipopolysaccharide (LPS) stimulation showed a significant upregulation of CD44 expression on splenic myeloid cells. Furthermore, LPS treatment stimulated a 2.44-fold increase of 89Zr-anti-CD44 accumulation in the spleen, which was attributable to splenic myeloid cells. Finally, in Balb/C nude mice bearing HT29 tumors, we injected 89Zr-anti-CD44 with greater Ab doses to reduce binding to splenic cells. The results showed lower spleen uptake and improved tumor uptake (2.9 ± 1.3%ID/g) with a total of 300 µg of Ab dose, and further reduction of spleen uptake and greater tumor uptake (5.7 ± 0.0%ID/g) with 700 µg Ab dose. Thus, using an 89Zr labeled Ab that cross-reacts with both human and mouse CD44, we demonstrate that CD44 immuno-PET has the capacity to monitor CD44 regulation on splenic myeloid cells and may also be useful for imaging colon tumors.

89Zr-Labeled Anti-PD-L1 Antibody PET Monitors Gemcitabine Therapy-Induced Modulation of Tumor PD-L1 Expression.

We developed an 89Zr-labeled anti-programmed death ligand 1 (anti-PD-L1) immune PET that can monitor chemotherapy-mediated modulation of tumor PD-L1 expression in living subjects. Methods: Anti-PD-L1 underwent sulfohydryl moiety-specific conjugation with maleimide-deferoxamine followed by 89Zr radiolabeling. CT26 colon cancer cells and PD-L1-overexpressing CT26/PD-L1 cells underwent binding assays, flow cytometry, and Western blotting. In vivo pharmacokinetics, biodistribution, and PET imaging were evaluated in mice. Results:89Zr-anti-PD-L1 synthesis was straightforward and efficient. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that reduction produced half-antibody fragments, and matrix-assisted laser desorption ionization time-of-flight analysis estimated 2.18 conjugations per antibody, indicating specific conjugation at the hinge-region disulfide bonds. CT26/PD-L1 cells showed 102.2 ± 6.7-fold greater 89Zr-anti-PD-L1 binding than that of weakly expressing CT26 cells. Excellent target specificity was confirmed by a drastic reduction in binding by excess cold antibody. Intravenous 89Zr-anti-PD-L1 followed biexponential blood clearance. PET/CT image analysis demonstrated decreases in major organ activity over 7 d, whereas high CT26/PD-L1 tumor activity was maintained. Again, this was suppressed by excess cold antibody. Treatment of CT26 cells with gemcitabine for 24 h augmented PD-L1 protein to 592.4% ± 114.2% of the control level and increased 89Zr-anti-PD-L1 binding, accompanied by increased AKT (protein kinase B) activation and reduced phosphatase and tensin homolog (PTEN). In CT26 tumor-bearing mice, gemcitabine treatment substantially increased tumor uptake from 1.56% ± 0.48% to 6.24% ± 0.37% injected dose per gram (tumor-to-blood ratio, 34.7). Immunoblots revealed significant increases in tumor PD-L1 and activated AKT and a decrease in PTEN. Conclusion:89Zr-anti-PD-L1 showed specific targeting with favorable imaging properties. Gemcitabine treatment upregulated cancer cell and tumor PD-L1 expression and increased 89Zr-anti-PD-L1 uptake. 89Zr-anti-PD-L1 PET may thus be useful for monitoring chemotherapy-mediated tumor PD-L1 modulation in living subjects.

Genipin enhances the antitumor effect of elesclomol in A549 lung cancer cells by blocking uncoupling protein-2 and stimulating reactive oxygen species production.

The uncoupling protein-2 (UCP2) serves a role in tumor aggressiveness and anticancer resistance, which is considered to be associated with its ability to attenuate reactive oxygen species (ROS) production. We hypothesized that UCP2 may protect cancer cells from elesclomol-induced cytotoxicity, and that this may be overcome by blocking UCP2 function with genipin. In A549 lung cancer cells that exhibited high UCP2 expression, treatment with elesclomol alone induced limited changes in glucose uptake, ROS production and cell survival. By contrast, both UCP2 knockdown and genipin treatment mildly reduced glucose uptake, increased ROS production and decreased cell survival. Combining genipin and elesclomol further reduced glucose uptake and increased cellular and mitochondrial ROS production. Moreover, co-treatment with genipin and elesclomol reduced the colony forming capacity to 50.6±7.4% and the cell survival to 42.0±3.4% of that in the control cells (both P<0.001). Suppression of cell survival by treatment with elesclomol and genipin was enhanced in the presence of an exogenous ROS inducer and attenuated by a ROS scavenger. The cytotoxic effects of combining genipin and elesclomol were accompanied by reduced mitochondrial membrane potential and occurred through apoptosis as demonstrated by Annexin V assay and increased protein cleavage of PARP and caspase-3. Finally, in an A549 ×enograft mouse model, tumor growth was only modestly retarded by treatment with elesclomol or genipin alone, but was markedly suppressed by combining the two drugs compared with that in the control group (P=0.008). Therefore, high UCP2 expression may limit the antitumor effect of elesclomol by attenuating ROS responses, and this may be overcome by co-treatment with genipin; combining elesclomol and genipin may be an effective strategy for treating cancers with high UCP2.

Targeting poor proteasomal function with radioiodine eliminates CT26 colon cancer stem cells resistant to bortezomib therapy.

We tested the hypothesis that tumor response to conventional bortezomib (BTZ) treatment is enhanced by targeted radiotherapy of resistant cancer stem cells (CSCs) that have characteristically poor proteasome function. This was accomplished by augmenting 131I uptake through expression of a sodium-iodide symporter (NIS) fusion protein that accumulates in cells with low proteasome activity. The NIS gene fused with the C-terminal of ornithine decarboxylase degron (NIS-cODC) was cloned. Stably expressing CT26/NIS-cODC cells and tumorsphere-derived CSCs were evaluated for NIS expression and radioiodine uptake. CT26/NIS-cODC cells implanted into mice underwent PET imaging, and tumor-bearing mice were treated with BTZ alone or with BTZ plus 131I. CT26/NIS-cODC cells accumulated NIS protein, which led to high radioiodine uptake when proteasome activity was inhibited or after enrichment for stemness. The cell population that survived BTZ treatment was enriched with CSCs that were susceptible to 131I treatment, which suppressed stemness features. Positron emission tomography and uptake measurements confirmed high 124I and 131I uptake of CT26/NIS-cODC CSCs implanted in living mice. In CT26/NIS-cODC tumor-bearing mice, whereas BTZ treatment modestly retarded tumor growth and increased stemness markers, combining 131I therapy suppressed stemness features and achieved greater antitumor effects. The NIS-cODC system offer radioiodine-targeted elimination of CSCs that are tolerant to proteasome inhibition therapy.

Curvature Effect of a Phosphatidylethanolamine-Included Membrane on the Behavior of Cinnamycin on the Membrane.

The behavior of cinnamycin on a biomimetic membrane was studied with respect to the curvature of a phosphatidylethanolamine (PE)-included membrane with the adhesion measured using an atomic force microscope (AFM). The membrane was formed through vesicle fusion on the hydrophobic surface of silica spheres, which was used to define the curvature of the membrane. The hydrophobicity was generated by the reaction of alkyl-silane and analyzed with an X-ray photoelectron spectrometer. The cinnamycin, immobilized covalently to the AFM tip coated with 1-mercapto-1-undecanol that was observed to be inert to any adhesion to the membrane, showed that the adhesion became stronger with the increase in the curvature. The correlation between the adhesion and the curvature was linearly proportional. Since it was found that the cinnamycin was bound to a PE headgroup and the binding was enhanced by the interaction of the hydrophobic area located at one side of the cinnamycin, the linear proportionality seems to suggest that the interaction is related to the one dimensional orientation of the binding.

Kinetic and thermodynamic studies of cinnamycin specific-adsorption on PE-Included-Membranes using surface plasmon resonance.

The binding of the cinnamycin on the biomimetic membrane was studied with respect to time using the surface plasmon resonance(SPR). The membrane was composed of the inner layer tethered on the gold surface and the outer layer formed on the inner layer, which was at the desired ratio of dioleoylphosphatidylethanolamine(DOPE) to dioleoylphosphatidyl- choline(DOPC). On the bilayer, the cinnamycin solution was injected and showed different behavior of the binding with respect to time up on its concentration. For kinetic analysis, the behavior was converted to the coverage fraction with respect to time, which was ratio to the saturated response of 5 µM cinnamycin solution. The fraction change with respect to time was function of the available-site, which was eventually the subtraction of the fraction from one. With the fitting of the first order of the available site, the rate constant was acquired into 6∼7 × 10-3 s-1. Furthermore, the reciprocals of the fraction and the concentration were fitted with the Langmuir adsorption isotherm. From the fitting, the equilibrium constant was between 1 × 107 and 5 × 107 M-1.

Facile Synthesis of SiO2/CMC/Ag Hybrids Derived from Waste Biomass (Sugarcane Bagasse) Having Special Medical Application.

This research is focused on the use of sol gel technique to synthesize amorphous SiO2 hybrids derived from lab made CMC (made from sugarcane bagasse) and tetraethoxysilane (TEOS) comprising silver nanoparticles (Ag-NPs). The dominant absorption peak in the order of 425 nm confirms the presence of Ag-NPs hybrid group owing to the surface Plasmon resonance (SPR). Ag-NPs hybrid characterization of was performed by Ultra violet-visible spectra (UV-Vis), Scanning electronic microscopy (SEM), Transmission electron microscopy (TEM), Particle size analyser (PSA), Energy Dispersive X-ray spectroscopy (EDX), X-ray diffractometer (XRD) and Fourier transform infrared spectroscopy (FTIR). The antibacterial action of Ag-NPs in contrast to Gram-negative bacteria (Escherichia coli) (ATCC 433) and Gram-positive bacteria (Bacillus subtilis) (ATCC 1688) was analyzed by using the method of agar disk diffusion technique. Ag-NPs hybrids extracted from lab-made CMC confirm higher adverse bacterial action through Gram-positive bacteria as well as Gram-negative bacteria related to synthetic CMC acquired from the market.