Aqueous Milieu Research Articles

In Vitro Investigation of Influences of Chitosan Nanoparticles on Fluorescein Permeation into Alveolar Macrophages.

Pulmonary infection namely tuberculosis is characterized by alveolar macrophages harboring a large microbe population. The chitosan nanoparticles exhibit fast extracellular drug release in aqueous biological milieu. This study investigated the matrix effects of chitosan nanoparticles on extracellular drug diffusion into macrophages. Oligo, low, medium and high molecular weight chitosan nanoparticles were prepared by nanospray drying technique. These nanoparticles were incubated with alveolar macrophages in vitro and had model drug sodium fluorescein added into the same cell culture. The diffusion characteristics of sodium fluorescein and nanoparticle behavior were investigated using fluorescence microscopy, scanning electron microscopy, differential scanning calorimetry and Fourier transform infrared spectroscopy techniques. The oligochitosan nanoparticles enabled macrophage membrane fluidization with the extent of sodium fluorescein entry into macrophages being directly governed by the nanoparticle loading. Using nanoparticles made of higher molecular weight chitosan, sodium fluorescein permeation into macrophages was delayed due to viscous chitosan diffusion barrier at membrane boundary. Macrophage-chitosan nanoparticle interaction at membrane interface dictates drug migration into cellular domains.

Oligomerization and Endocytosis of Hedgehog is Necessary for its Efficient Exovesicular Secretion

Hedgehog (Hh) is a secreted morphogen, involved in both short and long range signalling necessary for tissue patterning during development. It is unclear how this dually lipidated protein is transported over a long range in the aqueous milieu of interstitial spaces. We had previously shown that the long range signalling of Hh requires its oligomerization. Here we showthat Hh is secreted in the form of exovesicles. Fluorescence correlation spectroscopy (FCS) and electron microscopy based measurements confirmed Hh secretion on vesicles with a heterogeneous size distribution and a distinct biochemical composition. These are derived by the endocytic delivery of cell surface Hh to multi vesicular bodies (MVBs) via an endosomal sorting complex required for transport (ECSRT)-dependent process. Perturbations of ESCRT proteins have a selective effect on long-range Hh signalling in Drosophila wing imaginal discs. Importantly oligomerization-defective Hh is inefficiently incorporated into exovesicles and shows a narrower distribution of sizes. The defects in exovesicular secretion of this mutant directly correlate to its poor endocytic delivery to MVBs. These results provide evidence that nanoscale organization of Hh regulates the secretion of Hh on ESCRT-derived exovesicles, which in turn act as a vehicle for long range signalling.

Degradation of Endocrine Disrupting Chemical Nonylphenol in Aqueous Milieu using High Frequency Ultrasound

Degradation of Endocrine Disrupting Chemical Nonylphenol in Aqueous Milieu using High Frequency Ultrasound

Visualization of Endocannabinoids in the Cell.

A still unsolved, although critical, issue in endocannabinoid research is the mechanism by which the lipophilic compound anandamide (AEA) moves from its site of synthesis, crosses the aqueous milieu, and reaches the different intracellular compartments, where its metabolic and signaling pathways take place. The difficulty of studying intracellular AEA transport and distribution results from the lack of specific probes and techniques to track and visualize this bioactive lipid within the cell. Here, we describe the use of a biotinylated, non-hydrolyzable derivative of AEA (biotin-AEA, b-AEA) for visualizing the subcellular distribution of this endocannabinoid by means of confocal fluorescence microscopy.

PH-Regulated Reversible Transition Between Polyion Complexes (PIC) and Hydrogen-Bonding Complexes (HBC) with Tunable Aggregation-Induced Emission.

The mimicking of biological supramolecular interactions and their mutual transitions to fabricate intelligent artificial systems has been of increasing interest. Herein, we report the fabrication of supramolecular micellar nanoparticles consisting of quaternized poly(ethylene oxide)-b-poly(2-dimethylaminoethyl methacrylate) (PEO-b-PQDMA) and tetrakis(4-carboxylmethoxyphenyl)ethene (TPE-4COOH), which was capable of reversible transition between polyion complexes (PIC) and hydrogen bonding complexes (HBC) with tunable aggregation-induced emission (AIE) mediated by solution pH. At pH 8, TPE-4COOH chromophores can be directly dissolved in aqueous milieu without evident fluorescence emission. However, upon mixing with PEO-b-PQDMA, polyion complexes were formed by taking advantage of electrostatic interaction between carboxylate anions and quaternary ammonium cations and the most compact PIC micelles were achieved at the isoelectric point (i.e., [QDMA(+)]/[COO(-)] = 1), as confirmed by dynamic light scattering (DLS) measurement. Simultaneously, fluorescence spectroscopy revealed an evident emission turn-on and the maximum fluorescence intensity was observed near the isoelectric point due to the restriction of intramolecular rotation of TPE moieties within the PIC cores. The kinetic study supported a micelle fusion/fission mechanism on the formation of PIC micelles at varying charge ratios, exhibiting a quick time constant (τ1) relating to the formation of quasi-equilibrium micelles and a slow time constant (τ2) corresponding to the formation of final equilibrium micelles. Upon deceasing the pH of PIC micelles from 8 to 2 at the [QDMA(+)]/[COO(-)] molar ratio of 1, TPE-4COOH chromophores became gradually protonated and hydrophobic. The size of micellar nanoparticles underwent a remarkable decrease, whereas the fluorescence intensity exhibited a further increase by approximately 7.35-fold, presumably because of the formation of HBC micelles comprising cationic PQDMA coronas and PEO/TPE-4COOH hydrogen-bonded cores, an inverted micellar structures compared to initial PIC micelles. Moreover, the pH-mediated schizophrenic micellar transition from PIC to HBC with tunable AIE characteristic was reversible.

Oligomerization and endocytosis of Hedgehog is necessary for its efficient exovesicular secretion.

Hedgehog (Hh) is a secreted morphogen involved in both short- and long-range signaling necessary for tissue patterning during development. It is unclear how this dually lipidated protein is transported over a long range in the aqueous milieu of interstitial spaces. We previously showed that the long-range signaling of Hh requires its oligomerization. Here we show that Hh is secreted in the form of exovesicles. These are derived by the endocytic delivery of cell surface Hh to multivesicular bodies (MVBs) via an endosomal sorting complex required for transport (ECSRT)-dependent process. Perturbations of ESCRT proteins have a selective effect on long-range Hh signaling in Drosophila wing imaginal discs. Of importance, oligomerization-defective Hh is inefficiently incorporated into exovesicles due to its poor endocytic delivery to MVBs. These results provide evidence that nanoscale organization of Hh regulates the secretion of Hh on ESCRT-derived exovesicles, which in turn act as a vehicle for long-range signaling.

Abstract NG06: The Trojan Horse Strategy: Packaging chemotherapeutics can help alleviate toxicity

Abstract NG06: The Trojan Horse Strategy: Packaging chemotherapeutics can help alleviate toxicity

Analysis of the size dependence of macromolecular crowding shows that smaller is better

The aqueous milieu inside cells contains as much as 30-40% dissolved protein and RNA by volume. This large concentration of macromolecules is expected to cause significant deviations from solution ideality. In vivo biochemical reaction rates and equilibria might differ significantly from those measured in the majority of in vitro experiments that are performed at much lower macromolecule concentrations. Consequently crowding, a nonspecific phenomenon believed to arise from the large excluded volume of these macromolecules, has been studied extensively by experimental and theoretical methods. However, the relevant theory has not been applied consistently. When the steric effects of macromolecular crowders and small molecules like water and ions are treated on an equal footing, the effect of the macromolecules is opposite to that commonly believed. Large molecules are less effective at crowding than water and ions. There is also a surprisingly weak dependence on crowder size. Molecules of medium size, ∼5 Å radius, have the same effect as much larger macromolecules like proteins and RNA. These results require a reassessment of observed high-concentration effects and of strategies to mimic in vivo conditions with in vitro experiments.

Hydrophilic microenvironment required for the channel-independent insertase function of YidC protein

The recently solved crystal structure of YidC protein suggests that it mediates membrane protein insertion by means of an intramembrane cavity rather than a transmembrane (TM) pore. This concept of protein translocation prompted us to characterize the native, membrane-integrated state of YidC with respect to the hydropathic nature of its TM region. Here, we show that the cavity-forming region of the stage III sporulation protein J (SpoIIIJ), a YidC homolog, is indeed open to the aqueous milieu of the Bacillus subtilis cells and that the overall hydrophilicity of the cavity, along with the presence of an Arg residue on several alternative sites of the cavity surface, is functionally important. We propose that YidC functions as a proteinaceous amphiphile that interacts with newly synthesized membrane proteins and reduces energetic costs of their membrane traversal.

Sonic hedgehog multimerization: a self-organizing event driven by post-translational modifications?

Sonic hedgehog (Shh) is a morphogen active during vertebrate development and tissue homeostasis in adulthood. Dysregulation of the Shh signalling pathway is known to incite carcinogenesis. Due to the highly lipophilic nature of this protein imparted by two post-translational modifications, Shh's method of transit through the aqueous extracellular milieu has been a long-standing conundrum, prompting the proposition of numerous hypotheses to explain the manner of its displacement from the surface of the producing cell. Detection of high molecular-weight complexes of Shh in the intercellular environment has indicated that the protein achieves this by accumulating into multimeric structures prior to release from producing cells. The mechanism of assembly of the multimers, however, has hitherto remained mysterious and contentious. Here, with the aid of high-resolution optical imaging and post-translational modification mutants of Shh, we show that the C-terminal cholesterol and the N-terminal palmitate adducts contribute to the assembly of large multimers and regulate their shape. Moreover, we show that small Shh multimers are produced in the absence of any lipid modifications. Based on an assessment of the distribution of various dimensional characteristics of individual Shh clusters, in parallel with deductions about the kinetics of release of the protein from the producing cells, we conclude that multimerization is driven by self-assembly underpinned by the law of mass action. We speculate that the lipid modifications augment the size of the multimolecular complexes through prolonging their association with the exoplasmic membrane.

Further Closing the Resolution Gap: Integrating Cryo-Soft X-Ray and Light Microscopies

Further Closing the Resolution Gap: Integrating Cryo-Soft X-Ray and Light Microscopies

Annular floral nectary with oil-producing trichomes in Salvia farinacea (Lamiaceae): Anatomy, histochemistry, ultrastructure, and significance.

Many angiosperms produce nectar that entices pollinator visits. Each floral nectary tends to embody a singular form, such as the receptacular ring arising beneath the ovary in mint flowers (Lamiaceae). Exceptionally, the annular floral nectary in Salvia farinacea possesses modified stomata plus secretory trichomes. This first study of nectary ultrastructure within the largest genus of Lamiaceae examined this unusual condition. Nectary anatomy, histochemistry, and ultrastructure were investigated from fresh and fixed material using light microscopy and scanning electron and transmission electron microscopy. The annular nectary encircled the ovary plus extended ventrally as a projection. Modified stomata occurred only in the projection's abaxial epidermis. Conversely, peltate trichomes with a basal cell, a stalk cell, and 4-7 head cells were interspersed among the ovary lobes and covered the projection's adaxial surface. Phloem and xylem supplied the nectary interior, where parenchyma cells had numerous mitochondria and plastids with little starch, but few dictyosomes and little endoplasmic reticulum. Nectar accumulated as a drop opposite the projection's abaxial surface, escaping through stomatal pores and probably the cuticle. However, the annular nectary's glistening trichomes secreted a Sudan-positive product largely retained below the distended cuticle, but not nectar. This first ultrastructural study of co-occurring secretory trichomes and modified stomata on a mint nectary suggests multiple interactive functions for this atypical structure. These trichomes-possibly generating a substance informative to pollinators or as an ovarian defense against phytophagy-produced oil in an aqueous milieu, rather than contributing fluid to nectar.

Hepatocellular carcinoma dually-targeted nanoparticles for reduction triggered intracellular delivery of doxorubicin

Hepatocellular carcinoma (HCC) dual targeted stimuli responsive nanoparticles (NPs) for intracellular delivery of doxorubicin (DOX) were developed based on a reduction cleavable hyaluronic acid–glycyrrhetinic acid conjugate (HA-Cyst-GA). HA-Cyst-GA conjugate readily formed NPs in aqueous milieu and exhibited a high drug loading capacity (33.9%). The NPs redox responsiveness evaluation showed a tendency to lose their structural integrity in response to a reductive stimulus while remaining stable at physiological conditions, and that drug release was dramatically accelerated in presence of an intracellular level of glutathione. Moreover, cellular uptake studies highlighted the affinity of hepatoma cells (HepG2) toward the NPs as compared to breast cancer cells (MDA-MB-231). HA-Cyst-GA DOX-NPs displayed an increased cytotoxic potency over their non-responsive counterparts and free DOX with IC50 of 5.75, 9.33 and 10.23μg/mL, respectively. CLSM observations showed that HA-Cyst-GA DOX-NPs mediated a faster intracellular release and nuclear delivery of DOX as compared to the insensitive control. In vivo imaging study performed on H22 tumor bearing mice revealed a selective accumulation of DiR labeled NPs in the tumor and liver upon systemic administration. The antitumor efficacy was evaluated in HepG2 tumor xenograft model. Overall HA-Cyst-GA NPs appear as a potential HCC targeted intracellular delivery platform for DOX.

Hybrid Assemblies of Fluorescent Nanocrystals and Membrane Proteins in Liposomes

Because of the growing potential of nanoparticles in biological and medical applications, tuning and directing their properties toward a high compatibility with the aqueous biological milieu is of remarkable relevance. Moreover, the capability to combine nanocrystals (NCs) with biomolecules, such as proteins, offers great opportunities to design hybrid systems for both nanobiotechnology and biomedical technology. Here we report on the application of the micelle-to-vesicle transition (MVT) method for incorporation of hydrophobic, red-emitting CdSe@ZnS NCs into the bilayer of liposomes. This method enabled the construction of a novel hybrid proteo-NC-liposome containing, as model membrane protein, the photosynthetic reaction center (RC) of Rhodobacter sphaeroides. Electron microscopy confirmed the insertion of NCs within the lipid bilayer without significantly altering the structure of the unilamellar vesicles. The resulting aqueous NC-liposome suspensions showed low turbidity and kept unaltered the wavelengths of absorbance and emission peaks of the native NCs. A relative NC fluorescence quantum yield up to 8% was preserved after their incorporation in liposomes. Interestingly, in proteo-NC-liposomes, RC is not denatured by Cd-based NCs, retaining its structural and functional integrity as shown by absorption spectra and flash-induced charge recombination kinetics. The outlined strategy can be extended in principle to any suitably sized hydrophobic NC with similar surface chemistry and to any integral protein complex. Furthermore, the proposed approach could be used in nanomedicine for the realization of theranostic systems and provides new, interesting perspectives for understanding the interactions between integral membrane proteins and nanoparticles, i.e., in nanotoxicology studies.

Bioavailability of Dietary Carotenoids: Intestinal Absorption and Metabolism

Various carotenoids with diverse structures are present in foods and have been reported to have benefi cial effects on human health. Owing to their hydrophobicity, however, poor solubilization in the aqueous milieu of the digestive tract restricts their intestinal absorption. Fats and oils were found to increase the solubilization of carotenoids into mixed-micelles, which would therefore enhance their bioavailability. The uptake of carotenoids solubilized in the micelles by intestinal cells are thought to be mediated by simple diffusion and/or facilitated diffusion through scavenger receptors. Lipids that constituted the mixed-micelles affected the uptake of carotenoids. In particular, lysophosphatidylcholine significantly enhanced uptake. Highly polar carotenoids, meanwhile accumulated in mice but not in humans, suggesting discriminate absorption and metabolism in the latter. The metabolism of provitamin A carotenoids is well known to be mediated by the central cleavage enzyme. Recently, another cleavage enzyme was found to cleave various carotenoids asymmetrically into apocarotenoids. Xanthophylls were found to be oxidized to unstable keto-carotenoids in mice. These metabolic conversions are thought to affect the levels of bioavailable carotenoids in tissues. In this article, the solubilization into mixed-micelles, intestinal absorption and oxidative metabolism of carotenoids are reviewed to understand the factors that determine the bioavailability of carotenoids ingested from fruit and vegetables.

Abstract P5-17-02: Efficiency of methylene blue nanoparticles (nanoMB) as local injectable agent for photodynamic therapy in breast cancer

Abstract Photodynamic therapy (PDT), a non-invasive and alternative method for the treatment of cancer, is a light-activated treatment modality for breast cancer. Destruction of cancerous cells by PDT is achieved by a combination of photosensitizer and light of an appropriate wavelength for the photosensitizer. Methylene blue (MB) is a blue dye clinically being used and is known to show efficient photosensitizing activity with a very high yield of singlet oxygen generation (65%), where singlet oxygen is the actual therapeutic agent for PDT. However MB shows low cell uptake efficiency by itself and thus a low PDT efficacy. In this regard, we developed a nanoformulation of MB (nanoMB) to improve cancer cell uptake efficiency while keeping the high efficiency of singlet oxygen generation. NanoMB is composed of ternary components that are physically assembled in an aqueous milieu. In this study, we investigated the cellular uptake of nanoMB and cancer cell apoptosis induced by nanoMB PDT in MDA-MB-231 human breast cancer cells in vitro and in vivo. In vitro, nanoMB was indeed avidly taken up by MDA-MB231 cells, unlike free MB showing negligible cellular uptake. NanoMB formulation preserved the photosensitization activity of MB under laser irradiation at 655 nm. Taken together, it was revealed from the in vitro microscopic observation that nanoMB can efficiently destroy live MDA-MB231 cells even under red lamp illumination. In the control group, cells were treated with free MB and did not show phototoxic influence under the same light. In vivo phototoxicity evaluation, the locally injected nanoMB was internalized into cancer cells. Upon annexin V treatment after laser irradiation at 655 nm, apoptosis of cancer cells was clearly observed from the spot where nanoMB and laser were applied together. The PDT-induced cell apoptosis was visualized in a simple mouse model by using fluorescently labeled annexinV that has high affinity toward apoptotic cells. Briefly, cancer cells were inoculated in muscle on opposite sides and nanoMB was applied to both the inoculation sites. After some time, only one side was laser-treated and fluorescent annexinV was injected to both. Only the dual-treated side (nanoMB + laser) showed retention of annexinV after 1 hour, indicating the occurrence of apoptosis by the PDT treatment. Free MB and nanoMB were applied by subcutaneous injection around an early tumor tissue (not intratumoral injection). After 1 hour, free MB signals disappeared whereas nanoMB was retained at the tumor, implying that nanoMB penetrated into the tumor through the basement membrane. Laser irradiation was done one hour after sample injection for both free MB and nanoMB. This treatment (sample + laser) was repeated seven times. According to the results, only nanoMB showed the tumor growth suppression effect, demonstrating the potential of nanoMB as a local injectable PDT agent. In this study, nanoMB (MBOF) presented avid internalization into live cancer cells while keeping the high photosensitizing efficiency of MB. Consequently, highly efficient PDT of cancer cells was demonstrated in vitro and in vivo. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-17-02.

Dynamic Turn Conformation of a Short Tryptophan-Rich Cationic Antimicrobial Peptide and Its Interaction with Phospholipid Membranes

Cationic antimicrobial peptides are promising sources for novel therapeutic agents against multi-drug-resistant bacteria. HHC-36 (KRWWKWWRR) is a simple but effective antimicrobial peptide with similar or superior activity compared with several conventional antibiotics. In this biophysical study, unique conformational properties of this peptide and some of its analogs as well as its interaction with lipid membranes are investigated in detail. Circular dichroism (CD) and molecular dynamics modeling studies of HHC-36 in different environments reveal a dynamic amphipathic structure composed of competing turn conformations with free energies lower than that of the unfolded state, implying a strong influence of tryptophan interactions in formation of the turns. CD spectra and gel electrophoresis also show strong evidence of self-association of this peptide in aqueous milieu and interaction with both neutrally and negatively charged lipid membrane systems. Isothermal titration calorimetry and acrylamide fluorescence quenching experiments emphasize the preference of HHC-36 for negatively charged vesicles. In addition, dye leakage experiments suggest that this peptide functions through a surface-associated mechanism with weak lytic activity against bacterial model membranes.

Viscous Dynamics of Lyme Disease and Syphilis Spirochetes Reveal Flagellar Torque and Drag

The spirochetes that cause Lyme disease (Borrelia burgdorferi) and syphilis (Treponema pallidum) swim through viscous fluids, such as blood and interstitial fluid, by undulating their bodies as traveling, planar waves. These undulations are driven by rotation of the flagella within the periplasmic space, the narrow (∼20–40 nm in width) compartment between the inner and outer membranes. We show here that the swimming speeds of B. burgdorferi and T. pallidum decrease with increases in viscosity of the external aqueous milieu, even though the flagella are entirely intracellular. We then use mathematical modeling to show that the measured changes in speed are consistent with the exertion of constant torque by the spirochetal flagellar motors. Comparison of simulations, experiments, and a simple model for power dissipation allows us to estimate the torque and resistive drag that act on the flagella of these major spirochetal pathogens.

Intraosseous monitoring and guiding by ultrasound: A feasibility study

An efficient method for navigation within bone tissue boundaries is essential for performance of surgical procedures, e.g. without damaging of adjacent vulnerable organs. The application of sonographic measuring methods for this purpose promises to be effective in the ability to distinguish soft trabecular bone from dense cortical bone, owing to an order of magnitude difference in acoustic density between these structures. For this purpose, a specific method was developed that utilizes propagation of a 5MHz ultrasonic wave through an aqueous milieu. Using this method a 0.2mm resolution in measurements is achieved. This resolution is in an order of magnitude lower than is required for a clinical use. A three-stage experimental approach was adopted: measurements in a cubic “phantom” made of a transparent plastic material, in samples of fresh porcine femora, and in a clinical setting of drilling in the upper and lower jaw during dental implantation surgery in nine patients. Two patterns of the detected ultrasound wave reflections were found: low amplitude reflections from the aqueous surrounding and trabecular bone and highly reflected ultrasound waves from the cortical bone. We show that trabecular and cortical bones are distinguishable by real-time ultrasonic measurement. The distances of the drilled tracts, in the range of 58.0–122.0mm for the “phantom” experiment, 22.6–35.5mm for the ex vivo experiment and 10.0–11.5mm in the clinical experiment, and residual distances to the opposite edge of the tested samples and organs, in the range of 21.0–82.0mm for the “phantom” experiment, 3.8–11.36mm for the ex vivo experiment and 2.1–6.9mm in the clinical experiment, were measured by the presented sonographic method and compared statistically, using linear correlation and Bland Altman plot, to the mechanical and/or radiographic measurements in all three stages of the experiment. A correlation coefficient above 0.95 was considered an indication of high correlation, while a value of 0.75–0.94 was considered intermediate, and a value below 0.75 was considered poor.A very high correlation (p<0.001) and agreement between the sonographic and the “gold standard” measurements techniques, either mechanical or radiographic depending on the experimental setting, were found. Therefore the presented method of intraosseous sonographic measurements may provide an improved method for the monitoring of intraosseous drilling in respect of the currently used mechanical and/or radiographic clinical methods.

Development of Nile red-functionalized magnetic silica nanoparticles for cobalt ion sensing and entrapping

A new type of hybrid nanoparticles (NPs) with combined magnetic and fluorescent properties in single particle was developed by incorporating magnetic silica NPs with highly fluorescent Nile red dyes. These NPs clearly exhibit Co2+ ion entrapping ability in aqueous milieu and Co2+-induced fluorescence enhancement features with high selectivity owing to the Co2+-triggered inhibition on the photoinduced electron transfer progress in the efficient fluorophore (Nile red derivative). Moreover, these dual-functional NPs display superparamagnetic features and the motion of these fluorescent NPs can be induced by the application of an external magnetic field, enabling a facile separation/removal of toxic Co2+ ion from the aqueous milieu and real-time monitoring via fluorescence measurements.