Sub-micrometer-sized biodegradable particles of poly(L-lactic acid) via the gas antisolvent spray precipitation process.

Sub-micrometer-sized particles of poly(L-lactic acid) may be formed by using near-critical or supercritical carbon dioxide as an antisolvent to precipitate poly(L-lactic acid) from droplets of methylene chloride solution sprayed into a carbon dioxide continuous phase. Particle sizes may be controlled by varying the density of the carbon dioxide; at constant temperature in the supercritical region, higher carbon dioxide densities yield larger particles. Two methods (one batch and one continuous) for introducing the poly(L-lactic acid) solutions into carbon dioxide are demonstrated. Although the two methods use very different mechanisms for forming the droplets, similar particle sizes are observed as a function of carbon dioxide density. We suggest that mass transport, rather than jet breakup and hydrodynamics, controls particle sizes in the near-critical and supercritical regions.

Antagonism of selected ethanol-enhanced brain stimulation properties by Ro 15-4513.

Low doses of ethanol increase responding for brain stimulation. Recently, other intoxicating effects of ethanol have been reversed by the imidazobenzodiazepine, Ro 15-4513. Possibly, Ro 15-4513 blockade also acts on reward-enhancing properties of ethanol. Rats trained to alternately shuttle between nose poke and lever operanda for rewarding stimulation to the medial forebrain bundle, were tested following intragastric intubations of ethanol (18%, 1.35 g/kg), Ro 15-4513 (3 mg/kg in 18% ethanol), or vehicle. Ro 15-4513 reversed ethanol-enhanced effects on reinforced responses. Because Ro 15-4513 did not completely block instrumental responding for brain stimulation, we conclude its effects on ethanol were not acting on the same reward substrate as the current and consummatory response.

Effects of high molecular weight urinary macromolecules on crystallization of calcium oxalate dihydrate.

Timed urinary collections from 8 normal (Nl) persons of 11 stone forming (SF) patients were passed through ultrafiltration apparatus to remove macromolecules in the ranges 1000-30,000 d, 30,000-50,000 d, and over 50,000 d. No macromolecules could be recovered from either group in the 30,000-50,000 d range, and no low molecular weight macromolecules (LMWMM) (less than 30,000 d) were recovered from stone forming urines. Significant amounts of LMWMM (mean 105.8 +/- 17.63 mg./l.) were recovered from normal urine, but these extracts had no effects on calcium oxalate dihydrate (COD) nucleation (Bo) or linear growth (G) rates in a continuous crystallization (MSMPR) system. Urines from SF contained nearly twice the concentration of high molecular weight macromolecules (HMWMM) when compared to Nl urines. SF HMWMM differed from Nl in immunoelectrophoresis separation by absence of a dense band that was present in Nl extracts. This band reappeared in SF extract after boiling. Comparison of effects of addition of SF or Nl HMWMM to the COD-MSMPR crystallization system revealed no major quantitative differences in Bo or G, but SF HMWMM had a remarkable stabilizing effect on total mass (MT) of COD crystals produced. This effect was confirmed by analysis of oxalate residual supersaturation after crystallization. We conclude that SF excrete higher concentrations of HMWMM and almost no LMWMM when compared to normals. This higher concentration of HMWMM must contribute to increased Bo and decreased G noted in SF urine additive experiments previously reported. The mechanism of rapid removal of oxalate (i,e, stabilization) noted in experiments with SF HMWMM is not obvious at this time.

Observations upon calcium oxalate crystallization kinetics in simulated urine.

Two major etiologic theories of urinary stones are excessive saturation of urine with crystallizable substances or defects in inhibitors that allow relative supersaturation to occur. To date, it has been difficult to confirm the supersaturation theory in experiments using diffusion-limited crystallization systems because direct measurements of the nucleation process of crystallization could not be performed. We used well developed, continuous crystallizer techniques and adapted them from industrial use to the study of stone disease. Data derived from the experiments allow the absolute measurement of crystal growth rate and determination of nucleation rate. These methods were applied to study the calcium oxalate dihydrate (weddellite) system in artificial urine that lacked only proteinaceous components. Based on these experiments it was not possible to grow crystals large enough within 5 to 20 minutes to obstruct the collecting ducts of the kidney. Therefore, it appears that other processes, such as aggregation or stasis within tissues, may well be related to initiation of stone disease. Under the experimental conditions of this study nucleation rate exceeded growth rate. Therefore, multiple small particles are created at the expense of allowing larger particles to grow. Inhibitors can be tested rapidly in this system by adding them in concentrations compatible with those found in urine.

Effects of human urine on aggregation of calcium oxalate crystals.

The importance of aggregation in calcium oxalate urolithiasis, although not fully understood, has long been postulated. Previous investigators of calcium oxalate crystal aggregation have applied static crystallization rather than continuous flow techniques to their studies. We describe the use of a Couette agglomerator in series with our previously reported continuous flow mixed suspension-mixed product removal crystallization system. We compared synthetic urine controls with 5 per cent volume-in-volume human urine additions from normal persons or patients with calcium oxalate stones. There was no significant difference in nucleation, linear crystal growth rate or total crystal mass between normal persons and those with stones. Control nucleation rate was significantly higher than in either human urine addition group. Comparison of aggregator particle size distributions revealed significant differences in aggregation among the control, normal and stone groups. We concluded that urine inhibitors to aggregation are somewhat deficient in patients with stones, resulting in the generation of larger particle masses or eventually stones.

Determination of GOT activity on nucleation and crystal growth of calcium oxalate.

Crystal Size Distribution (CSD) and the yield of Calcium Oxalate Crystals in solutions with an admixture of 5 normal and 3 stone forming urines, were determined. A positive correlation was found between the median size, the number of particles and the overall inhibitory potentials of the urines toward calcium oxalate precipitation in vitro as reflected by Discriminating Index (DI) measurements. Incubation of two samples of stone formers' (SF) urines with glutamic-oxalacetic-transaminase (GOT) caused a reduction of aspartic acid concentration, an increase in glutamic acid concentration and a parallel decrease in the DI values. After 90 min of SF urine incubation with GOT the DI in three samples was improved and both the median size and number of particles reduced, by 28% and 45% respectively. These results could indicate that GOT activity changes the inhibitory power of the SF urine by transforming aspartic acid into glutamic acid, having thus most probably a part in the inhibition of CaOx stone formation.

The paradox of inhibition and enhancement of the formation of urinary stones.

Nucleation (Bo) and linear crystal growth (G) rates, average particle size (L1,0) and total mass (MT) of calcium oxalate dihydrate crystals were measured in artificial urine with and without polylysine, polyglutamic acid or heparin. The purpose of the study was to see if any of these polymers had effects on crystallisation similar to those created by addition of 5% natural urine to artificial urine, wherein Bo had increased but G, L1,0 and MT decreased. Polylysine addition had insignificant effects. Heparin increased Bo and decreased G, L1,0 and MT significantly, and polyglutamate had similar but more marked effects than did heparin. It is concluded that properly structured organic polymers can significantly inhibit calcium oxalate dihydrate crystallisation by paradoxical enhancement of nucleation. It is possible that such polymers may act as nucleation substrates.