Lipid nanoparticles for skin penetration enhancement-correlation to drug localization within the particle matrix as determined by fluorescence and parelectric spectroscopy.

With topical treatment of skin diseases, the requirement of a high and reproducible drug uptake often still is not met. Moreover, drug targeting to specific skin strata may improve the use of agents which are prone to cause local unwanted effects. Recent investigations have indicated that improved uptake and skin targeting may become feasible by means of nanoparticular systems such as solid lipid nanoparticles (SLN), nanostructured lipid carriers (NLC) and nanoemulsions (NE). Here we describe techniques to characterize drug loading to carrier systems and skin penetration profiles by using the lipophilic dye nile red as a model agent. Since the mode of drug association with the particle matrix may strongly influence the efficiency of skin targeting, parelectric spectroscopy (PS) was used to differentiate between matrix incorporation and attachment to the particle surface and fluorescence spectroscopy (FS) to solve dye distribution within NLC particles. Nile red was incorporated into the lipid matrix or the covering tensed shell, respectively, of SLN and NLC with all the lipids studied (Compritol, Precirol, oleic acid, Miglyol). In NLC, the dye was enriched in the liquid phase. Next, nile red concentrations were followed by image analysis of vertical sections of pigskin treated with dye-loaded nanoparticular dispersions and an oil-in-water cream for 4 and 8 h in vitro. Following the SLN dispersions, dye penetration increased about fourfold over the uptake obtained following the cream. NLC turned out less potent (相似文献   

Extracellular detection of K+ release during migration of transformed Madin-Darby canine kidney cells

 Madin Darby canine kidney cells transformed by alkaline stress (MDCK-F cells) constitutively migrate at a rate of about 1 μm·min^–1. Migration depends on the intermittent activity of a Ca²⁺-stimulated, 53-pS K⁺ channel (K_Ca channel) that is inhibitable by charybdotoxin. In the present study we examined whether this intermittent K_Ca channel activity results in a significant K⁺ loss across the plasma membrane. K⁺ efflux from MDCK-F cells should result in a transient increase of extracellular K⁺ ([K⁺]_e) in the close vicinity of a migrating cell. However, due to the rapid diffusion of K⁺ ions into the virtually infinite extracellular space, such a transient increase in [K⁺]_e was too small to be detected by conventional K⁺-selective electrodes. Therefore, we developed a ”shielded ion-sensitive microelectrode” (SIM) that limited diffusion to a small compartment, formed by a shielding pipette which surrounded the tip of the K⁺-sensitive microelectrode. The SIM improved the signal to noise ratio by a factor of at least three, thus transient increases of [K⁺]_e in the vicinity of MDCK-F cells became detectable. They occurred at a rate of 1.3 min^–1. The cell releases 40 fmol K⁺ during each burst of intermittent K_Ca channel activity, which corresponds to about 15% of the total cellular K⁺ content. Since transmembrane K⁺ loss must be accompanied by anion loss and therefore leads to a decrease of cell volume, these findings support the hypothesis that intermittent volume changes are a prerequisite for the migration of MDCK-F cells. Received: 15 April 1996 / Received after revision: 18 June 1996 / Accepted: 23 July 1996     

Biochemical engineering of the acyl side chain of sialic acids stimulates integrin-dependent adhesion of HL60 cells to fibronectin

Sialylation of glycoproteins and glycolipids plays an important role during development, regeneration and pathogenesis of several diseases. The physiological precursor of all sialic acids is N-acetyl- D-mannosamine. The N-acyl side chain of sialic acid can be modified by exposure of cells to synthetic N-acyl-modified D-mannosamines. In a new experimental approach cells were cultivated in the presence of N-propanoyl- D-mannosamine. This unnatural precursor of sialic acid is taken up by cells and efficiently metabolized to the respective N-acyl-modified neuraminic acid in vitro and in vivo. Here we report on the biological consequences of the incorporation of the unnatural N-propanoylneuraminic acid into glycoconjugates of HL60 cells. Biochemical engineering of the acyl side chain of neuraminic acids activates beta(1)-integrins (VLA4 or VLA5), resulting in an increased adhesion of HL60 cells to fibronectin.     

Volume dynamics in migrating epithelial cells measured with atomic force microscopy

Migration of transformed renal epithelial cells (transformed Madin-Darby canine kidney cells, MDCK-F cells) relies on the activity of a Ca(2+)-sensitive K+ channel (IK channel) that is more active at the rear end of these cells. We have postulated that intermittent IK channel activity induces local cell shrinkage at the rear end of migrating MDCK-F cells and thereby supports the cytoskeletal mechanisms of migration. However, due to the complex morphology of MDCK-F cells we have not yet been able to measure volume changes directly. The aim of the present study was to devise a new technique employing atomic force microscopy (AFM) to measure the volume of MDCK-F cells in their physiological environment and to demonstrate its dependence on IK channel activity. The spatial (x, y' and z) co-ordinates of each pixel of the three-dimensional image of MDCK-F cells allow calculation of the volume of the column "underneath" a given pixel. Thus, total cell volume is the sum of all pixel-defined columns. The mean volume of 17 MDCK-F cells was 2500+/-300 fl. Blockade of the IK channel with the specific inhibitor charybdotoxin (CTX) increased cell volume by 17+/-4%; activation of IK by elevating the intracellular [Ca2+] with the Ca2+ ionophore ionomycin decreased cell volume by 19+/-3%. Subtraction images (experimental minus control) reveal that swelling and shrinkage occur predominantly at the rear end of MDCK-F cells. In summary, our experiments show that AFM allows the measurement not only of total cell volume of living cells in their physiological environment but also the tracing of local effects induced by the polarized distribution of K+ channel activity.     

Nuclear pore function viewed with atomic force microscopy

In this review we focus on studies using atomic force microscopy (AFM) to describe the function of nuclear pore complexes (NPC). After a short introduction of AFM we follow the route of cargo molecules from the cytosol into the nucleus. AFM visualizes cargo before translocation into the nucleoplasm, cargo docking at the cytoplasmic NPC surface, cargo passing through the NPC and changes in NPC conformation in response to ATP, Calcium and pH. We discuss AFM experiments on nuclear envelopes on the basis of previous data obtained with more conventional techniques such as electron microscopy, confocal microscopy and other imaging techniques. Finally we draw attention to the recently developed nuclear hourglass technique that serves as a new electrophysiological approach to studying the structure-function relationship of NPC in combination with AFM at a molecular level.     

Validation of the Johnson Anti-Shear Accessory as an Accurate and Effective Clinical lsokinetic Instrument

This article reports two separate investigations into the validity of the Johnson Anti- Shear Accessory (JASA) as an isokinetic evaluation and exercise instrument. Experiment I attempted to verify the shear prevention aspect of the device on 10 anterior cruciate ligament injury patients and experiment I1 sought to establish the testing accuracy of the tool in comparison to the standard Cybex(R) knee input shaft (CKIS) on a qualified sample of eight subjects. A Cybex II-CDRC system protocol of five test knee extension/flexion repetitions at velocity spectrum speeds of 60, 120, 180,240, and 300 degrees /sec, preceded by a warm-up of three gradient submaximal and one maximal repetitions, was used for both studies. The null hypothesis was rejected in experiment 1 as an ANOVA test revealed significant factoral differences and Pearson correlation tests showed noncomparison between the JASA (r = 0.79 quadriceps, r = 0.89 hamstrings) and the CKIS (r = 0.23 quadriceps, r = 0.91 hamstrings) relative to the design control. The null hypothesis was accepted in experiment I1 as ANOVA testing failed to show a significant factoral difference while the JASA and CKIS demonstrated strong correlations (r = 0.97 quadriceps, r = 0.99 hamstrings). It was concluded that the JASA effectively controls anterior tibial shear during isokinetic activity and that the JASA is an accurate and valid isokinetic evaluation and exercise device. J Orthop Sports Phys Ther 1986;7(6):298-303.     

Deciphering the molecular effects of non-ablative Er:YAG laser treatment in an in vitro model of the non-keratinized mucous membrane

Lasers in Medical Science -     

Urinary calprotectin,NGAL, and KIM-1 in the differentiation of primarily inflammatory vs. non-inflammatory stable chronic kidney diseases

Introduction It has been demonstrated that urinary neutrophil gelatinase-associated lipocalin (NGAL) and calprotectin are helpful biomarkers in the differentiation of intrinsic and prerenal acute kidney injury.Objective The present cross-sectional study investigates, whether urinary biomarkers are able to differentiate primarily inflammatory from non-inflammatory entities in chronic kidney disease (CKD).Methods Urinary calprotectin, NGAL, and kidney injury molecule-1 (KIM-1) concentrations were assessed in a study population of 143 patients with stable CKD and 29 healthy controls. Stable renal function was defined as an eGFR fluctuation ≤5 ml/min/1.73 m² in the past 12 months. Pyuria, metastatic carcinoma, and renal transplantation were regarded as exclusion criteria. Diabetic nephropathy, hypertensive nephropathy, and polycystic kidney disease were categorized as ‘primarily non-inflammatory renal diseases’ (NIRD), whereas glomerulonephritis and vasculitis were regarded as ‘primarily inflammatory renal diseases’ (IRD).Results Urinary calprotectin and NGAL concentrations significantly differed between CKD and healthy controls (p < 0.05 each), whereas KIM-1 concentrations did not (p = 0.84). The three biomarkers did neither show significant differences in-between the individual entities, nor the two categories of IRD vs. NIRD (calprotectin 155.7 vs. 96.99 ng/ml; NGAL 14 896 vs. 11 977 pg/ml; KIM-1 1388 vs. 1009 pg/ml; p > 0.05 each). Albumin exceeds the diagnostic power of the investigated biomarkers by far.Conclusions The urinary biomarkers calprotectin, NGAL, and KIM-1 have no diagnostic value in the differentiation of primarily inflammatory vs. non-inflammatory etiologies of CKD.