Preparation and characterization of chitosan/ß-cyclodextrin nanoparticles containing plasmid DNA encoding interleukin-12.

Interleukin-12 (IL-12) as a cytokine has been proved to possess antitumor effects via stimulating the immune system. Non-viral gene delivery systems offer several advantages, including easiness in production, low cost, safety; low immunogenicity and can carry higher amounts of genetic material without limitation on their sizes.pUMVC3-hIL12 loaded Low Molecular Weight chitosan/ß-cyclodextrin (LMW CS/CD) nanoparticles were prepared using ionotropic gelation method and characterized in terms of size, zeta potential, polydispersity index, morphology, loading efficiency and cytotoxicity against the CT-26 colon carcinoma cell line.All prepared particles were spherical in shape and nano-sized (171.3±2.165 nm, PdI: 0.231±0.014) and exhibited a positive zeta potential (34.3±1.55). The nanoparticles demonstrated good DNA encapsulation efficiencies (83.315%±2.067). Prepared pUMVC3-hIL12 loaded LMW CS/CD nanoparticles showed no cell toxicity in murine CT-26 colon carcinoma cells. At the concentration of 0.1 µg/ml of nanoparticles, the transfection ability was obviously higher than that of the naked DNA.LMW CS/CD-plasmid DNA nanoparticles encoding IL-12 prepared using ionotropic gelation method with no toxic effect on the tested cells can be considered as a basis for further gene delivery studies both in vitro and in vivo to enhance the expression of IL-12.

Automatic segmentation in three-dimensional analysis of fibrovascular pigmentepithelial detachment using high-definition optical coherence tomography.

BACKGROUND/AIMS: A limited number of scans compromise conventional optical coherence tomography (OCT) to track chorioretinal disease in its full extension. Failures in edge-detection algorithms falsify the results of retinal mapping even further. High-definition-OCT (HD-OCT) is based on raster scanning and was used to visualise the localisation and volume of intra- and sub-pigment-epithelial (RPE) changes in fibrovascular pigment epithelial detachments (fPED). Two different scanning patterns were evaluated. METHODS: 22 eyes with fPED were imaged using a frequency-domain, high-speed prototype of the Cirrus HD-OCT. The axial resolution was 6 mum, and the scanning speed was 25 kA scans/s. Two different scanning patterns covering an area of 6 x 6 mm in the macular retina were compared. Three-dimensional topographic reconstructions and volume calculations were performed using MATLAB-based automatic segmentation software. RESULTS: Detailed information about layer-specific distribution of fluid accumulation and volumetric measurements can be obtained for retinal- and sub-RPE volumes. Both raster scans show a high correlation (p<0.01; R2>0.89) of measured values, that is PED volume/area, retinal volume and mean retinal thickness. Quality control of the automatic segmentation revealed reasonable results in over 90% of the examinations. CONCLUSION: Automatic segmentation allows for detailed quantitative and topographic analysis of the RPE and the overlying retina. In fPED, the 128 x 512 scanning-pattern shows mild advantages when compared with the 256 x 256 scan. Together with the ability for automatic segmentation, HD-OCT clearly improves the clinical monitoring of chorioretinal disease by adding relevant new parameters. HD-OCT is likely capable of enhancing the understanding of pathophysiology and benefits of treatment for current anti-CNV strategies in future.

Enhancement of dissolution rate and anti-inflammatory effects of piroxicam using solvent deposition technique.

Piroxicam solid depositions were prepared by means of the solvent deposition technique using different concentrations of microcrystalline cellulose as carrier material. The solvent deposition system (SDS) with drug to carrier ratio of 1:9 had a rapid dissolution rate in vitro. When this SDS was administered perorally to rats with a previous experimentally induced inflammation in their paws, it exhibited a pronounced anti-inflammatory action. X-ray diffraction and infrared (IR) spectroscopy showed no differences in the crystal state of piroxicam in SDS formulation and physical mixture of piroxicam and carrier. The increase in the dissolution rate and consequent enhancement of anti-inflammatory effect of piroxicam in SDS were attributed to the reduced particle size of drug deposited on the carrier and enhanced wettability of the particles brought about by the carrier.

Solubility prediction for furosemide in water-cosolvent mixtures using the minimum number of experiments.

The mole fraction solubility of a poorly water soluble loop diuretic, furosemide, was determined in aqueous binary mixtures of ethanol, propylene glycol, and glycerol from 0% to 100% cosolvent concentrations at 25 degrees C. Solubility predictions based on the minimum number of experimental data points were performed using the commonly used accurate cosolvency models: the three-suffix excess free energy (3xEFE), the mixture response surface (MRS), the combined nearly ideal binary solvent/Redlich-Kister (CNIBS/R-K), and the general single model (GSM). This prediction method was tested using three sets of solubility data for furosemide generated in this study and 11 data sets collected from the literature. The average percentage deviations (APDs) were 8.4 +/- 3.8, 13.6 +/- 7.3, 7.4 +/- 2.8, and 7.6 +/- 2.9, respectively, for 3xEFE, MRS, CNIBS/R-K, and GSM models. Using 3xEFE, CNIBS/R-K, and GSM models, which are theoretically related, a mean predicted solubility (MPS) approach was also proposed. The APD for this method was 7.3 +/- 2.3. The mean differences between MRS and the others were statistically significant (p < .001). The results showed that one can employ solubility prediction based on a minimum of five experimental data points, and the expected APD is less than 10%.

Modeling of the structural features of integral-membrane proteins reverse-environment prediction of integral membrane protein structure (REPIMPS).

The Profiles-3D application, an inverse-folding methodology appropriate for water-soluble proteins, has been modified to allow the determination of structural properties of integral-membrane proteins (IMPs) and for testing the validity of solved and model structures of IMPs. The modification, known as reverse-environment prediction of integral membrane protein structure (REPIMPS), takes into account the fact that exposed areas of side chains for many residues in IMPs are in contact with lipid and not the aqueous phase. This (1) allows lipid-exposed residues to be classified into the correct physicochemical environment class, (2) significantly improves compatibility scores for IMPs whose structures have been solved, and (3) reduces the possibility of rejecting a three-dimensional structure for an IMP because the presence of lipid was not included. Validation tests of REPIMPS showed that it (1) can locate the transmembrane domain of IMPs with single transmembrane helices more frequently than a range of other methodologies, (2) can rotationally orient transmembrane helices with respect to the lipid environment and surrounding helices in IMPs with multiple transmembrane helices, and (3) has the potential to accurately locate transmembrane domains in IMPs with multiple transmembrane helices. We conclude that correcting for the presence of the lipid environment surrounding the transmembrane segments of IMPs is an essential step for reasonable modeling and verification of the three-dimensional structures of these proteins.

Relationship between potency and boiling point of general anesthetics: a thermodynamic consideration.

The most important group of nonspecific drugs is that of the general anesthetics. These nonspecific compounds vary greatly in structure, from noble gases such as Ar or Xe to complex steroids. Since the development of clinical anesthesia over a century ago, there has been a vast amount of research and speculation concerning the mechanism of action of general anesthetics. Despite these efforts, the exact mechanism remains unknown. Many theories of narcosis do not explain how unconsciousness is produced at a molecular level, but instead relate some physicochemical property of anesthetic agents to their anesthetic potencies. In this paper, we address some of those physicochemical properties, with more emphasis on correlating the anesthetic potency of volatile anesthetics to their boiling points based on thermodynamic principles.

Using multi-stimulus VEP source localization to obtain a retinotopic map of human primary visual cortex.

OBJECTIVE: The goal of this study was to acquire a detailed spatial and temporal map of primary visual cortex using a novel VEP stimulus and analysis technique. METHODS: A multi-stimulus array spanning the central 18 degrees of the visual field was used where each of 60 checkerboard stimulus 'patches' was simultaneously modulated with an independent binary m-sequence (Sutter, 1992). VEPs corresponding to each patch were recorded from 3 subjects using a dense posterior electrode array. For each stimulus patch, single dipole source localization was conducted to determine the location, magnitude, and time-function of the underlying neural activation. To reduce ambiguity in the solution, a common time-function was assumed for stimulus patches at the same visual eccentricity (defining an annulus). The analysis was conducted independently for each annulus composed of 4-12 patches. RESULTS: The loci of the dipole solutions followed a smooth retinotopic pattern across annuli consistent with the classical organization of primary visual cortex. Specifically, each dipole was found contralateral to the corresponding stimulus patch and field inversion was observed for all subjects. CONCLUSIONS: Using this technique, the most detailed spatial and temporal retinotopic map of primary visual cortex to date has been obtained.

Alpha 2-adrenergic receptor subtypes in rat dorsal root and superior cervical ganglion neurons.

To determine which alpha 2-adrenergic receptor subtypes are present in primary afferent and sympathetic postganglionic neurons we have performed in situ hybridization and immunohistochemistry experiments on rat dorsal root and superior cervical ganglia. Reverse transcriptase polymerase chain reaction was used as a preliminary screen for the presence of mRNA encoding alpha 2-adrenergic subtypes in dorsal root and superior cervical ganglia; polymerase chain reaction primers amplified distinct regions of the rat alpha 2A-(RG20), alpha 2B-(RNG) and alpha 2C-(RG10) adrenergic receptor subtypes in mRNA extracted from lumbar dorsal root and superior cervical ganglia. To localize receptors to cell types in the ganglia, in situ hybridization was performed on cryosections of dorsal root and superior cervical ganglia with oligonucleotide probes designed to distinguish between mRNA encoding for alpha 2-adrenergic receptor subtypes. Immunohistochemistry was performed with a polyclonal antibody against the alpha 2A-adrenergic receptor subtype. Our results with reverse transcriptase polymerase chain reaction indicate that all three alpha 2-adrenergic receptor subtypes are expressed in dorsal root and superior cervical ganglia. Data from the in situ hybridization experiments indicated that the mRNA detected with the reverse transcriptase polymerase chain reaction was present in neuronal cell bodies, except for the mRNA encoding the alpha 2A-adrenergic receptor which was not detectable in dorsal root ganglia. The distribution of mRNA encoding alpha 2B- and alpha 2C-adrenergic receptor subtypes among dorsal root ganglion neurons and alpha 2A-, alpha 2B- and alpha 2C-adrenergic receptor subtypes among superior cervical ganglion neurons suggests that multiple adrenergic receptor subtypes are present in a single neuron. Neuronal cell bodies in both the dorsal root and superior cervical ganglion consistently demonstrated alpha 2A-adrenergic receptor-like immunoreactivity. The apparent co-expression of multiple alpha 2-adrenergic receptor subtypes in dorsal root and superior cervical ganglion neurons enables a single transmitter to produce a number of effects in the same neuron; which receptors are functionally active may vary with the presence of nerve injury, inflammation or other physiological and pathophysiological conditions.

Selective attenuation of mu-opioid receptor-mediated effects in rat sensory neurons by intrathecal administration of antisense oligodeoxynucleotides.

To test the hypothesis that the expression of specific proteins on peripheral terminals of primary afferents can be attenuated by intrathecal administration of antisense oligodeoxynucleotides (ODNs), we administered ODNs antisense to the mu-opioid receptor to male Sprague-Dawley rats via chronically implanted intrathecal cannulae. Antisense but not mismatch ODN treatment significantly decreased peripheral (D-Ala2, N-Me-Phe4, Gly5-ol)-enkephalin (DAMGO) inhibition of prostaglandin E2 (PGE2) hyperalgesia. Antisense treatment affected neither the magnitude of PGE2 hyperalgesia nor the antinociception produced by a peripherally administered adenosine A1-agonist. The antinociceptive effects of DAMGO was fully recovered 8 days after cessation of ODN treatment. DAMGO-induced inhibition of voltage-gated Ca2+ currents (VGCC), in cultured dorsal root ganglion (DRG) neurons from rats treated with ODNs, was also significantly reduced by antisense but not mismatch ODNs. Taken together, these observations suggest that intrathecal administration of antisense ODNs can be used to study the function of proteins present in the peripheral terminals of primary afferent neurons.

Co-expression of nociceptor properties in dorsal root ganglion neurons from the adult rat in vitro.

The cell body of sensory neurons in vitro has been used as a model to study the electrophysiological properties of afferent terminals. A limitation of this approach has been the ability to identify the function of the neuron studied. In the present study, we have tested the hypothesis that a putative nociceptor can be identified in vitro based on the expression of properties associated with nociceptors in vivo. A combination of patch-clamp electrophysiological and immunohistochemical techniques were used to describe the expression of nociceptor properties in acutely cultured dorsal root ganglion neurons from the adult rat. These properties include: a small cell body diameter; the presence of the neuropeptides substance P and calcitonin-gene related peptide; a shoulder (inflection) on the falling phase of the somal action potential, a response to the algogenic agent capsaicin, and sensitization in response to prostaglandin E2. Our results indicate that the frequency of expression of each of these properties varies in a manner consistent with that predicted from observations made in vivo, and that when one property is present in any given neuron, the other properties are also likely to be present. These data support the suggestion that the cell body of adult rat dorsal root ganglion neurons in vitro can be used to study the electrophysiological properties of nociceptors.