Spray drying formulation of albendazole microspheres by experimental design. In vitro-in vivo studies.

Both an experimental design and optimization techniques were carried out for the development of chitosan-pectin-carboxymethylcellulose microspheres to improve the oral absorption of albendazole as a model drug. The effect of three different factors (chitosan, pectin and carboxy methyl cellulose concentrations) was studied on five responses: yield, morphology, dissolution rate at 30 and 60 min, and encapsulation efficiency of the microspheres. During the screening phase, the factors were evaluated in order to identify those which exert a significant effect. Simultaneous multiple response optimizations were then used to find out experimental conditions where the system shows the most adequate results. The optimal conditions were found to be: chitosan concentration, 1.00% w/v, pectin concentration 0.10% w/v and carboxymethylcellulose concentration 0.20% w/v. The bioavailability of the loaded drug in the optimized microspheres was evaluated in Wistar rats which showed an area under curve (AUC) almost 10 times higher than the pure drug.

Chitosan microparticles: influence of the gelation process on the release profile and oral bioavailability of albendazole, a class II compound.

Encapsulation of albendazole, a class II compound, into polymeric microparticles based on chitosan-sodium lauryl sulfate was investigated as a strategy to improve drug dissolution and oral bioavailability. The microparticles were prepared by spray drying technique and further characterized by means of X-ray powder diffractometry, infrared spectroscopy and scanning electron microscopy. The formation of a novel polymeric structure between chitosan and sodium lauryl sulfate, after the internal or external gelation process, was observed by infrared spectroscopy. The efficiency of encapsulation was found to be between 60 and 85% depending on the internal or external gelation process. Almost spherically spray dried microparticles were observed using scanning electron microscopy. In vitro dissolution results indicated that the microparticles prepared by internal gelation released 8% of the drug within 30 min, while the microparticles prepared by external gelation released 67% within 30 min. It was observed that the AUC and Cmax values of ABZ from microparticles were greatly improved, in comparison with the non-encapsulated drug. In conclusion, the release properties and oral bioavailability of albendazole were greatly improved by using spraydried chitosan-sodium lauryl sulphate microparticles.

Subzero nonfreezing storage of rat hepatocytes using UW solution and 1,4-butanediol. II- functional testing on rewarming and gene expression of urea cycle enzymes.

UNLABELLED: In the present study we have analyzed the viability and metabolic competence of isolated rat hepatocytes subjected first, to subzero nonfreezing storage (up to 120 h at -4 degrees C) in modified University of Wisconsin (UW) solution with 8% 1,4-butanediol, and then to a normothermic rewarming step (KHR media, 37 degrees C, up to 120 min, carbogen atmosphere). Results were compared with hepatocytes stored up to 120 h at 0 degrees C in modified UW solution and with freshly isolated hepatic cells. We have found that only cell suspensions stored in subzero nonfreezing conditions were able to finish the rewarming period with a viability comparable with the control group. Also, we have investigated the enzyme activities and the relative expression at messenger RNAs levels of two of the Urea cycle (UC) enzymes: Carbamyl phosphate synthetase I (CPSI) and ornithine transcarbamylase (OTC), during 60 min of rewarming. Results were compared with the ammonium removal efficiency of the three groups. IN CONCLUSION: These data indicated that hepatocytes preserved under cold or subzero conditions up to 120 h followed by 60 min of rewarming, maintain UC enzymes at levels similar to freshly isolated hepatocytes, allowing their use in bioartificial liver devices.

Morphological and biochemical analysis of human cardiac valve allografts after an increment of the cryostorage temperature.

Cryopreserved human cardiac valve allografts could suffer lethal damages if the temperature is elevated during cryostorage. This work describes the functional and morphological alterations suffered by human cardiac valve allografts after a gradual increment of the cryostorage temperature from -147 degrees C to -47 degrees C due to a technical failure. Three experimental groups of human pulmonary and aortic allografts were compared: fresh, cryopreserved (-147 degrees C) and cryopreserved with temperature changes from -147 degrees C up to -47 degrees C and back to -147 degrees C. Fibroblast functionality was studied to asses the degree of valvular damages. Collagen network was also analyzed with bright light field and polarized microscopy; an immunohistochemistry for procollagen I was performed and the MTT colorimetric assay was used to evaluate fibroblast mitochondrial enzymatic activity. Porcine heart grafts valves were used to set the MTT colorimetric assay. With bright light field microscopy, disorganized collagen network was seen together with interstitial edema in cryopreserved groups. Polarized microscopy showed that fresh allografts had abundant collagen type I and III, cryopreserved group had less amount of collagen type I and in allografts that suffered cryopreservation temperature elevation collagen type I synthesis could not been demonstrated. Procollagen I was present in fibroblast cytoplasm of fresh group, but it was diminished in cryopreserved group and was absent in the group that suffered temperature elevation. Temperature changes during the cryopreservation period of human cardiac valve allografts induced fibroblast activity reduction. When the cryopreservation temperature is elevated during cryostorage, fibroblasts lost their functionality and the allografts may be not suitable for transplant.

Subzero nonfreezing storage of rat hepatocytes using modified University of Wisconsin solution (mUW) and 1,4-butanediol. I- effects on cellular metabolites during cold storage.

Various cryopreservation techniques have been investigated to extend the storage of isolated hepatocytes; however, most have a reduced viability after rewarming due to ice crystal formation. Subzero nonfreezing conditions could theoretically reduce organ metabolism without damage due to ice crystal formation. In the present work we evaluated the viability and metabolic parameters of isolated rat hepatocytes preserved in subzero nonfreezing condition. Cell suspensions were maintained in modified University of Wisconsin (mUW) solution using 8% - ,4-butanediol as cryoprotectant, up to 120 h at -4 masculineC. The time course evolution of hepatocytes viability were measured by LDH release and propidium iodide assay. The cellular concentrations of glutathione, ATP, glycogen and the lactate production during cold storage were also determined. Finally, results were compared with conventional hypothermic storage at 0 masculineC in mUW solution without cryoprotectant. After 5 days of subzero storage, we found an improvement in the ability of rat hepatocytes to maintain the metabolic resources in comparison with the cold preserved group.

Construction and performance of a minibioreactor suitable as experimental bioartificial liver.

This work deals with the construction and performance of a hollow fiber-based minibioreactor (MBR). Due to its simple design and the utilization of standard materials, it could serve as a suitable tool to evaluate the behavior and performance of cold preserved or cultured hepatocytes in bioartificial liver devices. The system consists of 140 fiber capillaries through which goat blood is pumped at a flow of 9 mL/min. The cell compartment contains 90 x 10(6) rat hepatocytes (volume 10 mL) and an internal oxygenator made of silicone tubing. To test the in vitro function of the system, 2-h perfusion experiments were performed, the evolution of hematocrit, plasma and extra-fiber fluid osmolality, and plasma urea and creatinine concentrations were evaluated. The detoxication efficiency of an ammonia overload was tested, showing that the system has enough capacity to remove ammonium. Also, the MBR oxygen transfer capacity to hepatocytes was tested, showing that the cells received an adequate oxygen supply.

The assessment of viability in isolated rat hepatocytes subjected to cold or subzero non-freezing preservation protocols using a propidium iodide modified test.

A rapid and simple assay (6 min, two steps) is described for determination of cell viability of hepatocytes subjected to cold preservation protocols. In this method, cells are incubated with the fluorescent marker propidium iodide (PI) and the fluorescence intensity is measured before (direct fluorescence--Fd) and after (total fluorescence--Ft) addition of digitonin, which allows the dye to enter the hepatocytes. The Fd originated from non-viable cells that have membrane damage and taken up PI. The Ft originated from all cells in the sample. The ratio between the two fluorescence values is used as an indicator of cell viability. The assay was challenged versus two classical viability tests: LDH retention and Trypan Blue exclusion. Our assay shows good correlation only with Trypan Blue test. In addition, a fluorescence confocal microscopy protocol was used to evaluate the possible toxicity of PI in hepatocyte suspensions.

Adenosine 5'triphosphate transport and accumulation during the cold preservation of rat hepatocytes in University of Wisconsin solution.

AIM: We used isolated hepatocytes to investigate how different concentrations of ATP in the University of Wisconsin (UW) solution affected both cellular ATP content and cell viability during the cold storage and the rewarming step. The mechanism involved in ATP transport and accumulation in hypothermia was also determined. METHODS: The cells were preserved up to 72 h in different conditions: UW solution without ATP (a-group), UW+5 mmol/L ATP (b-group), and UW+10 mmol/L ATP (c-group). The ATP content and the cell viability (LDH release) were determined during the cold storage and the rewarming step. In the groups a and c, the respiratory function of the cells at rewarming was studied. In addition, the cell volume of hepatocytes and the mechanism involved in ATP transport and accumulation were assessed. The extracellular degradation of exogenous nucleotides during transport experiments was investigated by a HPLC technique. RESULTS: After three days of cold storage a loss of cellular ATP content was observed in hepatocytes preserved either without nucleotides (a-group) or with 5 mmol/L ATP (b-group). In contrast, 10 mmol/L ATP (c-group) was able to maintain a normal ATP cellular content, with only a 6% diminution after 72 h of cold storage. The respiratory function was significantly different in hepatocytes preserved with 10 mmol/L ATP than without ATP. No significant change was detected for the three groups in cellular volume during the cold storage. We also report that the time course accumulation of (3H)-ATP by cold stored hepatocytes is a rapid process that is completed after 180 s with linear dependence on the extracellular ATP concentration (linear fitting results in a slope of 0.5624+/-0.1179 mmol/L ATP intracell/mmol/L ATP extracell). CONCLUSION: Our results show that, during hypothermic storage in UW solution, hepatocytes are permeable to ATP by a diffusive mechanism. Also, we found that it is ATP the main extracellular nucleotide available for transport and it is not the breakdown products.

Preservacion hipotermica del higado: evaluacion de la solucion Eurocollins utilizando cortes de higado de rata / Liver hypothermic preservation: evaluation of Eurocollins solution using rats liver slices

Se analizan los efectos producidos en el hígado por la isquemia fría en solución eurocollins (EC). Para ello se evalúa la función de cortes provenientes de hígados preservados en EC. Se utilizaron hígados de ratas Wistar, perfundidos con Krebs-Henseleit (KH) luego con EC y conservados a 4º en la misma durante 7, 24 y 48 hs. Finalizados los períodos de preservación, se hicieron cortes, los que fueron incubados 1 hora a 37ºC en KH y se determinaron los parámetros: distribución del agua y electrolitos tisulares, liberación de LDH al medio de incubación, producción de sustancias Ac. Tiobarbitúrico reactivas (SATBR) y Ureagénesis. Los cortes provenientes de hígados preservados desde 7hs, mostraron un incremento del agua tisular, expresado en la expansión del espacio extracelular. También se observaron, una disminución progresiva de la conc. tisular de K+, aumentos de SATBR de la liberación de LDH con el incremento del tiempo de presevación. No se observaron cambios en la ureogénesis. Estos resultados indican que la isquemia fría en EC, provoca cambios en la permeabilidad de membranas, fenómeno que afecta severamente el mecanismos de regulación del volumen tisular y compromete la viabilidad funcional del órgano a transplantar

Preservacion hipotermica del higado: evaluacion de la solucion Eurocollins utilizando cortes de higado de rata / Liver hypothermic preservation: evaluation of Eurocollins solution using rats liver slices

Se analizan los efectos producidos en el hígado por la isquemia fría en solución eurocollins (EC). Para ello se evalúa la función de cortes provenientes de hígados preservados en EC. Se utilizaron hígados de ratas Wistar, perfundidos con Krebs-Henseleit (KH) luego con EC y conservados a 4º en la misma durante 7, 24 y 48 hs. Finalizados los períodos de preservación, se hicieron cortes, los que fueron incubados 1 hora a 37ºC en KH y se determinaron los parámetros: distribución del agua y electrolitos tisulares, liberación de LDH al medio de incubación, producción de sustancias Ac. Tiobarbitúrico reactivas (SATBR) y Ureagénesis. Los cortes provenientes de hígados preservados desde 7hs, mostraron un incremento del agua tisular, expresado en la expansión del espacio extracelular. También se observaron, una disminución progresiva de la conc. tisular de K+, aumentos de SATBR de la liberación de LDH con el incremento del tiempo de presevación. No se observaron cambios en la ureogénesis. Estos resultados indican que la isquemia fría en EC, provoca cambios en la permeabilidad de membranas, fenómeno que afecta severamente el mecanismos de regulación del volumen tisular y compromete la viabilidad funcional del órgano a transplantar (AU)