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.

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.

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.

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.

Interactions of Cinnamycin-Immobilized Gold Nanorods with Biomimetic Membranes.

The behavior of the cinnamycin immobilized on the gold nanorod(AuNR) was investigated using surface plasmon resonance(SPR). For the comparison of the immobilized cinnamycin, the study for the free cinnamycin was also conducted. The bilayer was fabricated by tethering 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanols on a gold surface to form a monolayer and then using liposomes to adsorb an outer layer on the tethered-monolayer. The liposomes were prepared with a desired ratio of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine to 1,2-dioleoyl-sn-glycero-3-phosphocholine (0:100, 5:95, 10:90, 20:80, and 30:70). After the cinnamycin was injected on the bilayers, the specific binding between the cinnamycin and the bilayer was monitored with SPR. The inclusion of DOPE in the outer layer clearly led to the specific binding of the cinnamycin on the membranes. Specifically, the binding behavior of the immobilized was different from that of the free. For the free cinnamycin, the binding amount of cinnamycin at 10% was two times more than that at 5%. For the immobilized cinnamycin, the amounts were identical for both compositions. However, the rate was much faster for the immobilized cinnamycin at 10% than 5%, compared to that for the free at both compositions. This difference was attributed to the mean-molecular areas of the cinnamycin and DOPE, and the steric effect of the AuNR. For the effects of the heat and storage, the immobilized enzyme showed less decrease in the relative binding amount than the free one.

Trehalose-Induced Variation in Physical Properties of Fluidic Lipid Bilayer.

The effect of the trehalose on the physical properties of the fluidic lipid bilayer was studied using surface plasmon resonance (SPR) and cyclic voltammetry (CV). The bilayer was fabricated by tethering 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol on a gold surface to form a monolayer and then using liposomes to adsorb an upper layer on the tethered monolayer. The liposomes were prepared with a desired ratio (mol/mol) of trehalose to lipid, before the adsorption was performed. The formation of the adsorbed layer was monitored with SPR, and the SPR responses were converted to the surface density of the layer. In addition, the CV measurement was conducted to acquire the current-potential responses to evaluate the charge permeability of the layer. The surface density was gradually increased with the trehalose ratio up to 0.5, while the charge permeability was decreased. From these changes, the trehalose appears to be related to the curvature generation induced by the trehalose, which is consistent with the previous simulation results. In the identical measurements at glucose, little change in the properties was observed with even up to 2:1 ratio of glucose:lipid. These results seem attributed to the osmotic and volumetric effect on the headgroup packing disruption. The present study may provide a unique platform to control biological functions related to cellular processes.

Catabolism of (64)Cu and Cy5.5-labeled human serum albumin in a tumor xenograft model.

Human serum albumin (HSA), the most abundant protein in blood plasma, has been used as a drug carrier for the last few decades. Residualizingly radiolabeled serum albumin has been reported to be avidly taken up by tumors of sarcoma-bearing mice and to most likely undergo lysosomal degradation. In this study, we prepared (64)Cu-1,4,7,10-tetraazacyclododecane-N,N',N″,N'″-tetraacetic acid (DOTA) and Cy5.5-conjugated HSA (dual probe), and evaluated its tumor uptake and catabolism. Two dual probes were prepared using different DOTA conjugation sites of HSA (one via Lys residues and the other via the Cys residue). (64)Cu-DOTA-Lys-HSA-Cy5.5 (dual probe-Lys) exhibited higher uptake by RR1022 sarcoma cells in vitro than (64)Cu-DOTA-Cys-HSA-Cy5.5 (dual probe-Cys). In RR1022 tumor-bearing mice, the two dual probes showed a similar level of tumor uptake, but uptake of dual probe-Lys was reduced in the liver and spleen compared to dual probe-Cys, probably because of the presence of a higher number of DOTA molecules in the former. At 24 and 48 h after injection, dual probe-Lys was intact or partially degraded in blood, liver, kidney, and tumor samples, but (64)Cu-DOTA-Lys was observed in the urine using radioactivity detection. Similarly, Cy5.5-Lys was observed in the urine using fluorescence detection. These results indicate that dual probe-Lys may be useful for predicting the catabolic fate of drug-HSA conjugates.

Trehalose-Induced Variation in Mechanical Properties of Vesicles in Aqueous Solution.

The effect of the trehalose incorporation on the nanomechanical properties of dipalmitoylphosphatidylcholine vesicles was studied using atomic force microscope (AFM) on mica surface. The vesicles were prepared only with the variation in the trehalose concentration and adsorbed on the mica surface. After the morphology of the adsorbed vesicles was characterized, the behavior of an AFM tip into the vesicle was monitored using the plot of the tip displacement versus the tip deflection. It was observed that the breakthrough of the tip into the vesicles occurred two times. Each breakthrough represented each penetration of the tip into each layer. Force data prior to the first breakthrough fitted well with the Hertzian model to estimate Young's modulus and bending modulus of the vesicles. It was found that the Young's modulus and bending modulus decreased proportionally to the increase in the trehalose concentration up to 0.5 of trehalose to lipid. However, above 0.5, the moduli were a little varied with the increase. In the identical measurements at glucose, just a slight change in the moduli was observed with the increase in the glucose composition from 0 % glucose up to even 2:1 ratio of glucose:lipid. These results in the mechanical properties seem attributable to the osmotic and volumetric effects on the headgroup packing disruption.

Annexin V imaging detects diabetes-accelerated apoptosis and monitors the efficacy of benfotiamine treatment in ischemic limbs of mice.

The role of apoptosis imaging for monitoring treatment response in ischemic limbs has not been properly explored. In this study, we investigated the ability of annexin V (AnxV) imaging to assess the efficacy of antiapoptotic treatment in ischemic limbs of diabetic mice. Normal C57BL/6 mice and streptozotocin-induced diabetic mice were subject to hindlimb ischemia. AnxV-conjugated fluorescent streptavidin probes were intravenously injected, and optical imaging was performed. Tissue apoptosis was quantified by histochemistry and Western blotting. The AnxV probes showed specific targeting to apoptotic cells on confocal microscopy and flow cytometry. Intravenous AnxV probes displayed substantially greater accumulation in ischemic limbs of diabetic mice. Benfotiamine (BFT) treatment of diabetic mice led to better perfusion recovery on laser Doppler imaging and reduced AnxV binding on optical imaging. TUNEL staining and cleaved caspase-3 Western blots confirmed accelerated apoptosis by diabetes and its suppression by BFT treatment. Furthermore, AnxV-SAv-PEcy5.5 uptake in the ischemic limbs closely correlated to cleaved caspase-3 expression. Thus, AnxV imaging may be useful for monitoring the efficacy of therapeutic agents designed to suppress ischemia-induced apoptosis.

Dry Powder Inhaler Formulation of Lactobacillus rhamnosus GG Targeting Pseudomonas aeruginosa Infection in Bronchiectasis Maintenance Therapy.

The inhaled delivery of lactic acid bacteria (LAB) probiotics has been demonstrated to exert therapeutic benefits to the lungs due to LAB's immunomodulatory activities. The development of inhaled probiotics formulation, however, is in its nascent stage limited to nebulized LAB. We developed a dry powder inhaler (DPI) formulation of lactobacillus rhamnosus GG (LGG) intended for bronchiectasis maintenance therapy by spray freeze drying (SFD). The optimal DPI formulation (i.e., LGG: mannitol: lactose: leucine = 35: 45: 15: 5 wt.%) was determined based on the aerosolization efficiency (86% emitted dose and 26% respirable fraction) and LGG cell viability post-SFD (7 log CFU/mL per mg powder). The optimal DPI formulation was evaluated and compared to lyophilized naked LGG by its (1) adhesion capacity and cytotoxicity to human lung epithelium cells (i.e., A549 and 16HBE14o- cells) as well as its (2) effectiveness in inhibiting the growth and adhesion of Pseudomonas aeruginosa to lung cells. The optimal DPI of LGG exhibited similar non-cytotoxicity and adhesion capacity to lung cells to naked LGG. The DPI of LGG also inhibited the growth and adhesion of P. aeruginosa to the lung cells as effectively as the naked LGG. The present work established the feasibility of delivering the LAB probiotic by the DPI platform without adversely affecting LGG's anti-pseudomonal activities.

Correlation between composition of the outer layer and phase asymmetry for vesicles ruptured by phospholipase D.

Spherical phospholipid bilayers, vesicles, were prepared by using the layer-by-layer double emulsion technique, which allows individual layers to be formed asymmetrically. Phases of the layers were adjusted by selecting the lipid tail group. The head group composition of the vesicle outer layer varied 0-100 % of phosphatidylcholine (PC) by 10 % under the condition that the diameter of the vesicle was kept constant. On the outer layer of the vesicle, the phospholipase D (PLD) reacted to convert PC to phosphatidic acid. The reaction induced a curvature change of the vesicles, which eventually led them to rupture. Response time from the PLD injection to the rupture was measured against the different compositions of the outer layer at each phase (solid and liquid) using the fluorescence intensity change of pH-sensitive dye encapsulated in the vesicles. From this measurement, the rupture caused by the PLD reaction was analyzed with respect to the phase asymmetry of the layers and the composition of the outer layer. These results were interpreted with the lipid density and stability of the layers. It was observed that the solid phase of the outer layer had a variance in response time according to the phase of the inner layer, whereas the liquid phase did not. Additionally, the response of the solid phase of the outer layer at the liquid phase of the inner layer was faster than at the solid phase of the inner layer as a result of its stability.

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.

Effect of mixed-phospholipid layer on phospholipase D reaction-induced vesicle rupture.

Spherical phospholipid bilayers, or vesicles, were prepared layer by layer using a double-emulsion technique, which allows the outer layer of the vesicles to be formed with two phospholipids that have different head groups: phosphatidylcholine (PC) and phosphatidylethanolamine. At the outer layer of the vesicles, the phospholipase D (PLD) catalyzed for the conversion of PC to phosphatidic acid. The reaction caused by PLD induced the curvature change of the vesicles, which eventually led to the rupture of the vesicles. Before the investigation, the ratio of dioleoylphosphatidylethanolamine to oleoylhydroxyphosphatidylethanolamine was found as a condition such that the vesicles made with the mixed lipids were as stable as those made with pure dioleoylphosphatidylcholine. Response time from the PLD injection to vesicle rupture was monitored by the composition of the outer layer by the fluorescence intensity change of pH-sensitive dye encapsulated in the vesicles. The response time began to be slowed at approximately 30 % PC. The response times for the compositions were associated with the surface density of PC at the outer layer. These results also seem to be determined by the size of PLD, specifically the PLD active site.

Hydrazinonicotinamide prolongs quantum dot circulation and reduces reticuloendothelial system clearance by suppressing opsonization and phagocyte engulfment.

In this study, we investigated the effects of hydrazinonicotinamide (HYNIC)-a bifunctional crosslinker widely used to (99m)Tc radiolabel protein and nanoparticles for imaging studies-on quantum dot opsonization, macrophage engulfment and in vivo kinetics. In streptavidin-coated quantum dots (SA-QDots), conjugation with HYNIC increased the net negative charge without affecting the zeta potential. Confocal microscopy and fluorescence-activated cell sorting showed HYNIC attachment to suppress SA-QDot engulfment by macrophages. Furthermore, HYNIC conjugation suppressed surface opsonization by serum protein including IgG. When intravenously injected into mice, HYNIC conjugation significantly prolonged the circulation of SA-QDots and reduced their hepatosplenic uptake. Diminished reticuloendothelial system clearance of SA-QDots and aminoPEG-QDots by HYNIC conjugation was also demonstrated by in vivo and ex vivo optical imaging. The effects of HYNIC on the opsonization, phagocytosis and in vivo kinetics of quantum dots were reversed by removal of the hydrazine component from HYNIC. Thus, surface functionalization with HYNIC can improve the in vivo kinetics of quantum dots by reducing phagocytosis via suppression of surface opsonization.

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.

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.

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.