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Use of X-ray crystallography for the characterization of single crystals grown in steroid containing transdermal drug delivery systems.

Target of the study was to characterize crystals which had grown in steroid-containing matrix patches during short-term storage and to thereby establish a rationale for the inhibition of crystal formation in those patches in general. Matrix type transdermal drug delivery systems (TDDS) containing either 2.2% gestodene or 3.3% estradiol were free of crystals directly after their production. However, crystals of up to 800 microns in length grew during 3 months of storage at ambient temperature. The application of several analytical methods did not help to identify the crystals. This was mainly due to the fact that the adhesive matrix surrounding the crystals could not be fully removed in the course of sample preparation with routine laboratory methods and thus impaired DSC, FTIR microscopy and hot stage polarized microscopy. However, within X-ray diffractometry, the residual amorphous patch matrix did not hamper the measurement of the crystals. Thus, they were identified as estradiol hemihydrate and gestodene form I, respectively. These results suggest that steroid-containing matrix TDDS should be stabilized against drug recrystallization e.g. by the addition of suitable crystallization inhibitors. Furthermore, systems containing estradiol may be stabilized by efficient removal water.

Administration, Cutaneous↗

A total sample preparation method for the measurement of airborne asbestos and other fibers by optical and electron microscopy.

A total sample preparation method utilizing one membrane filter for all or some of the measurements by phase contrast optical microscopy (PCM), optical microscopy (OM), dispersion staining polarized microscopy (DS/PLM), scanning electron microscopy (SEM), and transmission electron microscopy (TEM), was developed in order to evaluate airborne asbestos and other mineral fibers comprehensively, including fiber number, sizes and types, in various environments. This method consists of two alternate procedures: parallel and serial preparation. The former uses different portions of a filter for each measurement of PCM, OM, DS/PLM, SEM and TEM. The latter uses a single filter portion for all OM, SEM and TEM measurements by which the same area and same fibers on the filter can be observed. This allows fiber numbers to be directly compared using these measurements on the same filter area, and the fibers observed by OM can be also examined by SEM and/or TEM equipped with an energy dispersive X-ray spectrometer (EDX) to determine the types and precise sizes. Using this method, it was found that OM measurements developed in this study can visualize thinner fibers than PCM measurements and make it easier to count fibers. We also found that currently used PCM measurement can detect fibers thicker than about 0.4-0.5 microns in diameter. This total sample preparation method can be used not only for air samples, but also for liquid and biological tissue samples.

Air Pollutants, Occupational↗

Polarized light microscopy of the smooth muscle anococcygeus of the rat.

1. The birefringence of the rat anococcygeus muscle has been studied in the presence of solutions which dissolve myosin and actin filaments. About 80% of the muscle anisotropy disappear after this treatment. 2. The 'form' and 'intrinsic' components of the birefringence of muscles fixed either at rest or during maximal isometric contraction were studied by inhibition with media of different refractive indices. At rest, the 'form' birefringence accounts for about 67% of the total birefringence. In contraction, the increase of total birefringence is primarily (80%) due to an increase of the 'form' component, indicating that new filamentous material appears in this condition.

Actins↗

Identification of crystals in joint fluids.

The inflammatory process present in synovial arthritic specimens can be diagnosed by crystal-type identification performed in cytopathology laboratories by the use of simple methods. These methods involve wet coverslipped preparations of fresh fluid specimens or sediments and compensated polarizing microscopy, which can be performed with a routine microscope equipped with polarizing filters and a substitute compensator. Details of the methodology are described. The morphologic characteristics of the crystals are described in cases of gout, pseudogout and arthritis of other types in which secondary calcifications are present.

Adult↗

Niosomes as carriers for tretinoin. III. A study into the in vitro cutaneous delivery of vesicle-incorporated tretinoin.

The influence of drug thermodynamic activity and niosome composition, size, lamellarity and charge on the (trans)dermal delivery of tretinoin (TRA) was studied. For this purpose, tretinoin was incorporated at saturated and unsaturated concentrations in both multilamellar (MLV) and unilamellar (UV) vesicular formulations using two different commercial mixtures of alkyl polyglucosides: octyl-decyl polyglucoside and decyl polyglucoside. Positively and negatively charged vesicular formulations were prepared using either stearylamine or dicetylphosphate as a charge inducer. Niosomes made with polyoxyethylene (4) lauryl ether and liposomes made with soy phosphatidylcholine were also prepared and studied. Vesicular formulations were characterised by transmission electron microscopy and optical and light polarized microscopy for vesicle formation and morphology, and by dynamic laser light scattering for size distribution. The effect of the vesicular incorporation of tretinoin on its (trans)dermal delivery through the newborn pig skin was also investigated in vitro using Franz cells, in comparison with a commercial formulation of the drug (RetinA). The amount of tretinoin delivered through and accumulated in the several skin layers was detected by HPLC. Overall, obtained results showed that tretinoin cutaneous delivery is strongly affected by vesicle composition and thermodynamic activity of the drug. In particular, small, negatively charged niosomal formulations, which are saturated with tretinoin, have shown to give higher cutaneous drug retention than both liposomes and commercial formulation. Moreover, interactions between skin and vesicles seem to depend on physico-chemical properties of the main component of the vesicular bilayer.

Administration, Cutaneous↗

Resolving complex atomic-scale spin structures by spin-polarized scanning tunneling microscopy.

The spin-polarized scanning tunneling microscope (SP-STM) operated in the constant current mode is proposed as a powerful tool to investigate complex atomic-scale magnetic structures of otherwise chemically equivalent atoms. The potential of this approach is demonstrated by successfully resolving the magnetic structure of Cr/Ag(111), which is predicted on the basis of ab initio vector spin-density calculations to be a coplanar noncollinear periodic 120 degrees Néel structure. Different operating modes of the SP-STM are discussed on the basis of the model of Tersoff and Hamann.

Journal Article↗

Diagnosis of trichothiodystrophy in 2 siblings.

Trichothiodystrophy (TTD) is a rare autosomal recessively inherited disorder which is characterized by sparse and brittle hair with low cystine content. It is often associated with physical and mental retardation. We report 2 cases of TTD in 2 sibs who were born to related parents. The children showed clinical features typical of TTD and in addition other symptoms such as epilepsy, ataxia, spasticity, strabismus, atopic dermatitis, dysarthria and hyperextensible fingerjoints. The sulfur content of hair was reduced to about 50% of normal values and scanning electron microscopy of hair showed trichorrhexis nodosa, trichoschisis, missing cuticle scales with weathering of hair shafts. Under polarizing microscopy an alternating dark and bright banding was found. The present cases show that the correct diagnosis of TTD in practice can be impeded for many years because of the heterogeneous clinical appearance and that the determination of the sulfur content in hair is a simple but indispensable method.

Ataxia↗

[Study of structural changes in muscle fiber contractile proteins using polarization ultraviolet fluorescent microscopy. IV. Several features of the conformational changes in F-actin during muscle fiber relaxation].

Using the polarized ultraviolet fluorescent microscopy in the presence of a fluorescence quencher (acrylamide, NO3-) it has been shown that actin tryptophanyls are oriented with their short axes perpendicular to the long axis of the thin filament. Fluorophores in hardly accessible sites of the macromolecule have a more pronounced anisotrophy of orientation in comparison with those in the protein sites easily available by a fluorescence quencher. During the muscle fiber relaxation, conformational changes of F-actin take place which embrace seemingly the sites of subunits, close to the surface of macromolecule, and make difficult the penetration of univalent ions and neutral molecules deep into the protein macromolecule. Some connection between conformational changes of the surface areas of F-actin subunits and the actin incapability of combining with HMM in the presence of ATP is assumed.

Acrylamides↗

Anaylsis of birefringence during wound healing and remodeling following alkali burns in rabbit cornea.

The use of synthetic inhibitors of metalloproteinases (SIMP) or medroxyprogesterone (MP) can prevent or significantly delay the ulceration of alkali-injured corneas by influencing collagen degradation. We have examined the remodeling of rabbit corneal stroma following alkali injury and have assessed the effect of SIMP and MP treatment. Following a defined alkali injury to the rabbit cornea, animals were divided into three subgroups, one group treated with topical beta-mercaptomethyl tripeptide (SIMP), one treated by subconjunctival injection of MP and one treated with a control solution. The corneal tissue was taken at 3 days, 1, 2, 3, 4, 9 and 26 weeks after alkali injury and prepared for light microscopy and transmission electron microscopy (TEM). A quantitative measurement of birefringence, in terms of the optical path difference (OPD), was made using a modified polarized microscopy technique based on the analysis of interference colours. The results showed that SIMP effectively prevented deep corneal ulceration. MP could delay the ulceration and the corneas treated with MP appeared to have better transparency than the other groups. There was a significant difference of the OPD between the anterior (5.8 +/-0.3 nm) and posterior (7.8 +/-0.4 nm) stroma of the normal cornea (P<0.001). The OPD values from the central corneas from alkali-injured eyes were generally lower than normal during the first 4 weeks and then gradually recovered to the normal level or above, except for the posterior stroma of the MP-treated eyes. We found that the OPD changes were very dependent on the presence of corneal lesions. The stroma near corneal ulceration, scar tissue, calcified stroma and the retro-corneal collagen layer showed a significant reduction of birefringence (lower OPD values). These OPD values remained much lower than normal up to the end of the experiment. TEM showed disrupted corneal stroma in all three groups, with thinner scar tissue in the MP group. The fibril diameters did not change significantly 3 days and 1 week after the alkali burns (27.1+/-2.3 nm in the control group, 27.3+/-2.2 nm in the SIMP group and 27.7+/-2.1 nm in the MP group) and there were no differences compared with 29.7+/-1.7 nm of the normal cornea (P>0.05). After 2 weeks of tissue remodeling, the fibril diameters in alkali-injured corneas showed a large variation (the range was between 11.5 and 80 nm) with a bimodal distribution, especially in the control group. The technique presented here for birefringence evaluation can provide an alternative way to monitor wound healing and tissue remodeling, both visually and quantitatively.

Animals↗

Polarization microscopic investigation of collagen and acid glycosaminoglycans in the skin of progressive systemic sclerosis (PSS).

Collagen and acid glycosaminoglycans in the skin of progressive systemic sclerosis (PSS) were examined by polarization microscopy. Picrosirius Red and Toluidine Blue (pH 5.8) were used as stains. Digestion with chondroitinase ABC or streptomyces hyaluronidase were also employed. Under polarized light, the Picrosirius Red-stained collagen appeared green at any stage in PSS and orange in controls. Toluidine Blue-induced birefringence at stage I diminished in the presence of 0.2 M MgCl2 and in stage II in the presence of 0.3 M MgCl2. The collagen fibrils in PSS skin were significantly smaller in diameter than in controls. These results suggest that the change of polarization colours is due to the modulation of collagen thickness caused by an increased accumulation of acid glycosaminoglycans.

Adult↗

A NEW METHOD OF POLARIZATION MICROSCOPIC ANALYSIS. I. SCANNING WITH A BIREFRINGENCE DETECTION SYSTEM.

A new method of polarized light analysis is described in which a highly sensitive electronic detector specific for birefringence is used to identify the crystalline axes of an object and then measure its phase retardation due to birefringence. The microscopic system employed in the method consists of an electronic birefringence detection system (BDS), a microscope with strain-free lenses, and a driven stage for passing the specimen at appropriate velocities across the image of an aperture placed at the field stop and imaged in the specimen plane by the condenser. The detector registers retardations directly as voltage at a constant deflection sensitivity of ca. 1.1 v per angstrom unit over a range of 120 angstrom units. The basal rms noise level is 0.002 A for a spot 36 micro in diameter formed by a 95 x, N. A. 1.25 objective pair, and increases in proportion to the reciprocal of the diameter of the scanning spot. The increase in noise with high resolution scanning can be offset by increasing the instrumental time constant, which is adjustable in decades between 0.004 and 0.4 seconds. A number of difficult problems in high extinction polarization microscopy are avoided by the use of modulated light and a rapid electronic detector. For example: (a) The measured distribution of birefringence is unaffected by the usual diffraction anomaly; therefore polarization rectifiers are not required. (b) The detector is selective for birefringence, so that there is no problem in separating contrast due to different optical properties (e.g. dichroism, light scattering). (c) The speed and sensitivity are both increased by between one and two orders of magnitude over that attainable by visual or photographic methods, thereby rendering a vast number of weakly birefringent, light-scattering, and motile objects readily analyzable for the first time with polarized light.

Biophysical Phenomena↗

Scanning electron microscopy of collagen fibers in intestine.

Collagen fibers of rat intestine were observed with and without mechanical stress in the scanning electron microscope. Observations were correlated with the previous results obtained by optical polarized microscopy to provide further insight into the organization and morphology of intestinal collagen. Larger fibers, approximately 4 micrometers in diameter, are densely packed in parallel undulating arrays. The initial response to stress is straightening of the original fibers. The extended fibers are biaxially oriented at +30 degrees and -30 degrees to the longitudinal direction. These large fibers appear as assemblies of subfibers. At higher magnifications, the larger fibers appear to be enmeshed in a network of small randomly oriented fibers approximately 0.2 micrometer in diameter. Study of the effect of age on fiber morphology showed that the length of the large fiber undulations increases during maturation but remains constant during aging. The diameters of large and small fibers appear not to change with age, but more of the 4 micrometers fibers are loosely associated into larger fibers which can be observed at both the optical microscope and scanning electron microscope levels. A hierarchical organization of intestinal collagen is proposed.

Aging↗

Utility of biliary microscopy for the prediction of the chemical composition of gallstones and the outcome of dissolution therapy with ursodeoxycholic acid.

To learn whether microcrystalline solids in bile could predict gallstone composition, the findings determined by polarizing microscopy of gallbladder bile were related to stone type at cholecystectomy in 53 patients. Cholesterol crystals were present in 36 of 39 cholesterol stone bile and absent in 12 of 14 bile from non-cholesterol stones. Fifteen cholesterol stones (eight radiopaque) contained calcium carbonate, and characteristic vaterite microspheroliths were observed in 53% of their bile. In another study, crystals in basal duodenal bile were related to the outcome of gallstone dissolution treatment with ursodeoxycholic acid, 10 mg/kg X day. In 39 patients treated for 1 yr, efficacy (complete gallstone dissolution) was 41% overall and 52% in patients with stones less than or equal to 10 mm in diameter. In connection with the findings of biliary microscopy, efficacy was 93% in 14 patients with cholesterol crystals in bile, and 27% in 11 patients with microspheroliths in bile. Cholecystectomies in 9 patients with dissolution failure revealed 4 cases of non-cholesterol stones and 5 cases (including 3 with on-therapy calcification) of calcium carbonate-rich cholesterol stones with a surface/interior mineral ratio greater than 3. The results confirm that cholesterol crystals in bile are a sensitive measure of cholesterol gallstones. They also show that vaterite microspheroliths in bile indicate the presence of calcium carbonate in gallstones. Both findings suggest that biliary crystals reflect gallstone composition, and it is demonstrated that this information is useful in predicting the success or failure of cholelitholysis with ursodeoxycholic acid. Finally, the data show that radiologically undetectable stone calcification reduces the probability of dissolution, and that the calcified structures appearing in some stones during treatment are composed of calcium carbonate.

Bile↗

Liposomes as carriers for dermal delivery of tretinoin: in vitro evaluation of drug permeation and vesicle-skin interaction.

The influence of liposome composition, size, lamellarity and charge on the (trans)dermal delivery of tretinoin (TRA) was studied. For this purpose we studied both multilamellar (MLV) or unilamellar (UV) liposomes. Positively or negatively charged liposomes were obtained using either hydrogenated (Phospholipon90H) or non-hydrogenated soy phosphatidylcholine (Phospholipon90) and cholesterol, in combination with stearylamine or dicetylphosphate. Liposomal formulations were characterized by transmission electron microscopy (TEM) and optical and light polarized microscopy for vesicle formation and morphology, and by dynamic laser light scattering for size distribution. In order to obtain more information about the stability and the thermodynamic activity of the liposomal tretinoin, TRA diffusion through a lipophilic membrane was investigated. The effect of the vesicular incorporation of tretinoin on its accumulation into the newborn pig skin was also studied. The experiments were performed in vitro using Franz cells in occlusive conditions and were compared to three different controls. The tretinoin amount delivered through and accumulated in the several skin layers was detected by HPLC. Furthermore, TEM in combination with osmium tetroxide was used to visualize the skin structure after the liposomal administration. Overall obtained results showed that liposomes may be an interesting carrier for tretinoin in skin disease treatment, when appropriate formulations are used. In particular, negatively charged liposomes strongly improved newborn pig skin hydration and TRA retention, though no evidence of intact vesicle penetration was found.

Animals↗