Effect of Lyophilization on Stability of PEG-Protein Conjugate: A Case Study with Peginterferon alfa-2b

Abstract The purpose of the present study was to develop stable lyophilized formulation of peginterferon alfa-2b which is acquiescent to the short lyophilization process. The present study evaluates the effect of buffering components and cryoprotectant(s) on depegylation of the peginterferon alfa-2b in combination with lyophilization process. Finally, a short lyophilization process was identified which can produce a stable pharmaceutical form of peginterferon alfa-2b without any depegylation during long-term storage. Formulations were analyzed mainly for depegylation by HP-size exclusion chromatography and in-vitro antiviral activity. Residual moisture content in the lyophilized product was also used as a key indicating parameter to check its role with respect to depegylation upon storage under various temperature conditions. It was observed that the peginterferon alfa-2b when formulated in presence of cryoprotectant like sucrose requires longer lyophilization process of about 5 days, irrespective of the buffering components used, to reduce the level of residual moisture content and thereby to produce the stable formulation without depegylation. A stable formulation in presence of high concentration of lactose as a cryoprotectant was developed which can withstand stresses exerted to protein-polymer conjugate during lyophilization phases without any significant depegylation. A short lyophilization process of about 48 hours can be utilized for peginterferon alfa-2b when formulated in presence of lactose as a cryoprotectant through which a stable lyophilized formulation can be produced as against longer process required when sucrose is used a cryoprotectant, which is essential from commercial point of view as lyophilization is a costly process.

Cuantificación de compuestos con potencial bioactivo en Citrullus lanatus, Luffa cylindrica y Sicana odorifera

La sandía vanessa, la luffa y la cassabanana son cucurbitáceas que poseen compuestos con potencial bioactivo, esto es, presencia de compuestos que ejercen efectos benéficos para la salud. En Colombia, estas frutas son desaprovechadas, debido a su escasa popularidad; dar a conocer la información de sus compuestos nutricionales incentiva su aprovechamiento y consumo. El objetivo del presente estudio fue realizar la caracterización fisicoquímica y evaluar el efecto de la liofilización y la extracción asistida por ultrasonido sobre el contenido de vitamina C, polifenoles totales y capacidad antioxidante de las partes de cada fruto. Los desechos generados entre cortezas y semillas son de 28,3 % (sandía), 68,76 % (luffa) y 25,39 % (cassabanana); estos, a su vez, presentaron contenidos de polifenoles totales y capacidad antioxidante mayores que en la pulpa. El tratamiento de liofilización mejoró la extracción % en capacidad antioxidante, vitamina C y polifenoles totales, comparado con las muestras frescas. Por otro lado, tanto la corteza como la pulpa de luffa son una buena fuente de compuestos con capacidad antioxidante, mientras que la sandía y la cassabanana alcanzaron una buena aceptación sensorial, lo cual, se atribuye al contenido de sólidos solubles y el alto contenido de agua, que las hace frutas dulces y refrescantes.

Vanessa watermelon, luffa and cassabanana are cucurbits that have compounds with bioactive potential, that is, compounds that have beneficial effects on health. In Colombia, these fruits are underutilized due to their low popularity; making known the information on their nutritional compounds encourages their use and consumption. The objective of this study was to perform the physicochemical characterization and evaluate the effect of freeze-drying and ultrasound-assisted extraction on the vitamin C content, total polyphenols and antioxidant capacity of the parts of each fruit. The wastes generated between rinds and seeds are 28.3 % (watermelon), 68.76 % (luffa) and 25.39 % (cassabanana), these in turn presented higher total polyphenol contents and antioxidant capacity than in the pulp. The freeze-drying treatment improved the extraction % in antioxidant capacity, vitamin C and total polyphenols compared to fresh samples. On the other hand, both rind and pulp of luffa are a good source of compounds with antioxidant capacity, while watermelon and cassabanana reached a good sensory acceptance, which is attributed to the soluble solids content and the high-water content, which makes them sweet and refreshing fruits.

Optimization of a cationic liposomal gene delivery system and study of its endocytic pathway

Abstract A cationic liposomal gene delivery system comprising DOTAP, DOPE, and cholesterol was prepared and optimized. The results showed that the liposome/DNA (LP/DNA) system had spherical morphology, with a particle size of around 150 nm and zeta potential of approximately 30 mV. Cytotoxicity experiments showed that cells treated with all of the liposome carriers- with the exception of LP1-had more than 80% viability even at a weight ratio of 30. The in vitro transfection efficiency was measured using a Promega™ Luciferase Assay System. Of the tested lipoplexes, LP2/DNA showed the highest cell transfection efficiency (at a weight ratio of 10)-which was similar to or slightly lower than that of Lipofectamine® 2000 in HeLa, A549, and SPC-A1 cell lines. After freeze-drying, the cell transfection efficiency decreased slightly (P>0.05). The cell uptake mechanism study showed that LP/DNA lipoplexes mainly entered cells via clathrin-mediated and caveolin-mediated endocytic pathways. The results confirmed that LP2 has potential for use as an effective gene carrier, and provides experimental evidence to support its further development as a safe and effective gene delivery system.

In-vitro production and pre-validation of lyophilized canine platelet-rich plasma for therapeutic use

ABSTRACT: Platelet-rich plasma (PRP) has been considered a promising therapeutic alternative, since platelets are rich in growth factors that are used in the Regenerative Medicine field. However, fresh PRP cannot be stored for long periods. This study aimed to develop a protocol for obtaining lyophilized canine PRP capable of maintaining viability after its reconstitution. For that purpose, canine PRP extraction and lyophilization protocols were initially tested. Subsequently, assays were carried out to quantify the growth factors VEGF and TGF-, before and after the lyophilization process, gelation test and the three-dimensional gel structure analysis of the reconstituted lyophilized PRP by electron microscopy, as well as in vitro cell proliferation test in lyophilized PRP gel. Additionally, the immunogenicity test was performed, using allogeneic samples of lyophilized PRP. The results showed that the lyophilized PRP had adequate therapeutic concentrations of growth factors VEGF and TGF- (9.1pg/mL and 6161.6pg/mL, respectively). The reconstituted PRP gel after lyophilization showed an in vitro durability of 10 days. Its electron microscopy structure was similar to that of fresh PRP. In the cell proliferation test, an intense division process was verified in mesenchymal stem cells (MSCs) through the three-dimensional mesh structure of the lyophilized PRP gel. The immunogenicity test showed no evidence of an immune reaction. The findings were promising, suggesting the possibility of having a lyophilized canine PRP that can be marketed. New in vivo and in vitro studies must be carried out for therapeutic confirmation.

RESUMO: O plasma rico em plaquetas (PRP) é uma alternativa terapêutica promissora, pois as plaquetas são ricas em fatores de crescimento com ação na regeneração de tecidos. No entanto, o PRP fresco não pode ser armazenado por longos períodos. Esse trabalho teve como objetivo desenvolver um protocolo de obtenção de PRP liofilizado canino capaz de manter a viabilidade pós reconstituição. Portanto, foram testados diversos protocolos de extração e liofilização. Para validação do PRP canino liofilizado foi analisada a concentração dos fatores de crescimento VEGF e TGF- antes e após o processo de liofilização, a estrutura tridimensional do PRP liofilizado reconstituído em forma de gel por microscopia eletrônica e seu efeito in vitro na proliferação de células-tronco mesenquimais. Os resultados demonstraram que o PRP liofilizado apresentou concentrações terapêuticas adequadas dos fatores de crescimento VEGF e TGF- (9,1pg/ml e 6161,6pg/ml, respectivamente). O gel de PRP reconstituído após liofilização apresentou uma durabilidade in vitro de 10 dias, sua estrutura tridimensional mostrou-se semelhante ao PRP fresco e proporcionou intensa proliferação de células-tronco mesenquimais durante o cultivo. O teste de imunogenicidade não demonstrou evidências de reação imune. Os achados foram promissores, sugerindo a possibilidade de uso de PRP canino liofilizado para o mercado. Novos estudos in vivo e in vitro deverão ser conduzidos para comprovação terapêutica.

In-vitro production and pre-validation of lyophilized canine platelet-rich plasma for therapeutic use / Produção e validação de plasma rico em plaquetas canino liofilizado para fins de utilização terapêutica

Platelet-rich plasma (PRP) has been considered a promising therapeutic alternative, since platelets are rich in growth factors that are used in the Regenerative Medicine field. However, fresh PRP cannot be stored for long periods. This study aimed to develop a protocol for obtaining lyophilized canine PRP capable of maintaining viability after its reconstitution. For that purpose, canine PRP extraction and lyophilization protocols were initially tested. Subsequently, assays were carried out to quantify the growth factors VEGF and TGF-β, before and after the lyophilization process, gelation test and the three-dimensional gel structure analysis of the reconstituted lyophilized PRP by electron microscopy, as well as in vitro cell proliferation test in lyophilized PRP gel. Additionally, the immunogenicity test was performed, using allogeneic samples of lyophilized PRP. The results showed that the lyophilized PRP had adequate therapeutic concentrations of growth factors VEGF and TGF-β (9.1pg/mL and 6161.6pg/mL, respectively). The reconstituted PRP gel after lyophilization showed an in vitro durability of 10 days. Its electron microscopy structure was similar to that of fresh PRP. In the cell proliferation test, an intense division process was verified in mesenchymal stem cells (MSCs) through the three-dimensional mesh structure of the lyophilized PRP gel. The immunogenicity test showed no evidence of an immune reaction. The findings were promising, suggesting the possibility of having a lyophilized canine PRP that can be marketed. New in vivo and in vitro studies must be carried out for therapeutic confirmation.(AU)

O plasma rico em plaquetas (PRP) é uma alternativa terapêutica promissora, pois as plaquetas são ricas em fatores de crescimento com ação na regeneração de tecidos. No entanto, o PRP fresco não pode ser armazenado por longos períodos. Esse trabalho teve como objetivo desenvolver um protocolo de obtenção de PRP liofilizado canino capaz de manter a viabilidade pós reconstituição. Portanto, foram testados diversos protocolos de extração e liofilização. Para validação do PRP canino liofilizado foi analisada a concentração dos fatores de crescimento VEGF e TGF-β antes e após o processo de liofilização, a estrutura tridimensional do PRP liofilizado reconstituído em forma de gel por microscopia eletrônica e seu efeito in vitro na proliferação de células-tronco mesenquimais. Os resultados demonstraram que o PRP liofilizado apresentou concentrações terapêuticas adequadas dos fatores de crescimento VEGF e TGF- β (9,1pg/ml e 6161,6pg/ml, respectivamente). O gel de PRP reconstituído após liofilização apresentou uma durabilidade in vitro de 10 dias, sua estrutura tridimensional mostrou-se semelhante ao PRP fresco e proporcionou intensa proliferação de células-tronco mesenquimais durante o cultivo. O teste de imunogenicidade não demonstrou evidências de reação imune. Os achados foram promissores, sugerindo a possibilidade de uso de PRP canino liofilizado para o mercado. Novos estudos in vivo e in vitro deverão ser conduzidos para comprovação terapêutica.(AU)

Development of a new protocol for freeze-drying preservation of Pseudoalteromonas nigrifaciens and its protective effect on other marine bacteria

Background: Freeze-drying is known as one of the best methods to preserve bacterial strains. Protectant is the key factor affecting the survival rate of freeze-dried strains. In addition, salinity, bacterial suspension concentration, drying time, and other factors can also affect the survival rate of strains to varying degrees. At present, there are relatively few studies on freeze-drying preservation of marine bacteria. In the present study, we performed the freeze-drying protectant screening and optimized the preservation conditions for Pseudoalteromonas nigrifaciens, which is widely distributed in marine environment. The protective effects of the screened protectants were verified by 18 other marine bacterial strains. Results: The results indicated that the combination of 5.0% (w/v) lactose, 5.0% (w/v) mannitol, 5.0% (w/v) trehalose, 10.0% (w/v) skim milk powder, 0.5% (w/v) ascorbic acid and 0.5% (w/v) gelatin was the best choice for the preservation of P. nigrifaciens. The suggested salinity and concentration of initial cell suspension were 10 g/L NaCl and 1.0 × 109 CFU/mL, respectively. Furthermore, stationary-phase cells were the best choice for the freeze-drying process. The highest survival rate of P. nigrifaciens reached 52.8% when using 5­10% (w/v) skim milk as rehydration medium. Moreover, the other 18 marine strains belonging to Pseudoalteromonas, Vibrio, Photobacterium, Planomicrobium, Edwardsiella, Enterococcus, Bacillus, and Saccharomyces were freezedried under the abovementioned conditions. Their survival rates were 2.3­95.1%. Conclusion: Collectively, our results supported that the protectant mixture and parameters were beneficial for lyophilization of marine bacteria

Uso de disacáridos y carbón activado para preservar consorcios de bacterias ruminales celulolíticas liofilizadas / Use of disaccharides and activated carbon to preserve cellulolytic ruminal bacterial consortiums lyophilized

RESUMEN Objetivo. Determinar la fermentación in vitro de consorcios bacterianos ruminales celulolíticos (CBC) conservados por liofilización usando carbón activado, maltosa y lactosa como preservadores. Materiales y métodos. Un CBC se aisló de fluido ruminal de una búfala de agua en medios selectivos celulolíticos. Los CBC se liofilizaron con carbón activado (CA), lactosa (LA) o maltosa (MA) como preservadores y sin preservador (SP). El diseño experimental fue completamente al azar para medir biogás a diferentes intervalos de tiempo; así como, un diseño completamente al azar con arreglo factorial 4x3, los factores fueron preservadores (SP, CA, LA y MA) y tiempo de fermentación (24, 48 y 72 h) para pH, nitrógeno amoniacal (N-NH3), degradación de materia seca (DMS) y de fibra detergente neutro (DFDN), actividad enzimática celulasas y la población de bacterias totales. Resultados. LA produjo mayor biogás acumulado a las 72 h y parcial a partir de las 12 h (p≤0.05). SP no mostró diferencias (p>0.05) en celulasas, conteo de bacterias total, DMS y DFDN en los tiempos de fermentación evaluados con el resto de los preservadores. Conclusiones. La producción de biogás parcial y acumulada, el aumento en la tasa de degradación de 8.3 y 91.1 % en la DMS y DFDN de las 24 a 72 h (p≤0.05) con el preservador LA, muestran que la lactosa puede usarse como preservador de bacterias celulolíticas ruminales.

ABSTRACT Objective. To determine in vitro fermentation of cellulolytic ruminal bacterial consortia (CBC) preserved by lyophilization using activated carbon, maltose and lactose as preservatives. Materials and methods. A CBC was isolated from the ruminal fluid of a female water buffalo in selective cellulolytic media. The CBC were lyophilized without preservative (SP), activated carbon (CA), lactose (LA) o maltose (MA) as preservatives. The experimental design was completely random to measure biogas at different time intervals; as well as completely random with 4x3 factorial arrangement, factors were preservative [SP, CA, LA and MA] and fermentation time (24, 48 and 72 h) for pH, ammoniacal nitrogen (NH3-N), dry matter degradation (DMD), neutral detergent fiber degradation (NDFD), enzymatic activity cellulases and total bacteria population. Results. LA produced higher accumulated biogas at 72 h and partial biogas after 12 h (p≤0.05). SP did not show differences (p>0.05) in cellulases, total bacteria population, DMD and NDFD in the fermentation times evaluated with the rest of the preservative. Conclusions. The production of partial and accumulated biogas, the increase in the degradation rate of 8.3 and 91.1% in the DMD and NDFD from 24 to 72 h (p≤0.05) in the LA preservative, show that lactose can be used as a preservative of ruminal cellulolytic bacteria.

Simultaneous Determination of Ten Banned Substances in Pig Urine by Liquid Chromatography-Tandem Mass Spectrometry / 分析化学

A simple, sensitive and reliable method was developed for simultaneous determination of ten banned drugs residues including zeranols(ZALs),chloroamphenicol,pentachlorophenol,etc. in swine urine using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The urine samples were pretreated using lyophilization and QuEChERS procedures, respectively. Acetonitrile and ammonium acetate (5 mmol/L) were chosen as mobile phases. Target compounds were separated well in ZorbaxSB-C18by following the optimized gradient elution program and determined by LC-MS/MS in negative electrospray ionization mode. The linearity of the matrix-matched standard curve of ten analytes in two methods was good in the range of the experimental concentration with correlation coefficients more than 0.99. The recoveries of ten drugs were in the range of 80.7%-107.7% and 73.5%-103.3% at the spiked levels of 5,10 and 20 μg/L by lyophilization and QuEChERS methods,respectively. The coefficients of variation were less than 15%. The limits of detection (LOD) and the limits of quantification (LOQ) from lyophilization and QuEChERS method were 0.1 to 2.0 μg/L and 0.2 to 5.0 μg/L,respectively.

Evaluación de diferentes métodos de extracción de ARN a partir del hongo nativo Xylaria sp / Evaluation of different RNA extraction methods from the native fungus Xylaria sp

RESUMEN La extracción de ARN de calidad constituye el primer paso para el análisis de la expresión génica. Sin embargo, su obtención no es sencilla debido a la susceptibilidad de esta molécula a la presencia de contaminantes como ARNasas, proteínas y polisacáridos. Adicionalmente, debido a la diversa composición de la pared celular de los hongos se requiere optimizar los procesos de extracción de ARN para organismos específicos. Este estudio evalúo el uso de diferentes metodologías de homogeneización de tejido (nitrógeno líquido y liofilización) y extracción de ARN (Trizol, CTAB y RNeasy mini kit) a partir del hongo nativo ascomiceto Xylaria sp. Se determinó la pureza, concentración e integridad del ARN obtenido por medio de espectrofotometría y electroforesis. Adicionalmente, se diseñaron cebadores de referencia para el gen β-Tubulina a partir del alineamiento de secuencias de este gen obtenidas de diferentes ascomicetes. Estos cebadores fueron utilizados para evaluar si el ARN extraído es amplificable mediante RT-PCR. Se determinó que la homogeneización de tejido por medio de liofilización generó mayores rendimientos de extracción independientemente del protocolo de extracción utilizado; sin embargo, éstos alteraron la integridad del ARN. Se obtuvo un ARN con mayor pureza con el protocolo CTAB y un mayor rendimiento con el RNeasy mini kit. Los resultados indican que el ARN extraído, independientemente de la metodología de homogeneización y extracción utilizada, es amplificable mediante RT-PCR. No obstante, se recomienda homogeneizar el tejido con nitrógeno líquido y extraer con RNeasy mini kit por la brevedad del protocolo de extracción y calidad obtenida.

ABSTRACT Obtaining high quality RNA is the first step for gene expression analysis. However, the low stability of this molecule and high presence of contaminants such as RNases, proteins and polysaccharides may trouble extractions. Fungi cell wall composition is highly diverse; therefore optimizing RNA extraction procedures is necessary when studying specific organisms. In this study, different methods of tissue homogenization (liquid nitrogen and lyophilization) and RNA extraction (Trizol, CTAB and RNeasy mini kit) were assessed with a native ascomycete, Xylaria sp. RNA purity, concentration and integrity were determined by spectrophotometry and electrophoresis. In addition, a set of housekeeping gene primers was designed targeting the β-tubulin gene. The primers were used to determine if the RNA extracted allowed RT-PCR amplification. It was demonstrated that homogenization of tissue by lyophilization allowed higher yields of RNA regardless of the extraction protocol used, however, the RNA integrity was affected. The higher RNA purity was obtained using CTAB and the higher yields using the RNeasy mini kit. The extracted RNA is amplifiable by RT-PCR regardless of the homogenization and extraction methodology used. However, it is recommended to homogenize the tissue with liquid nitrogen and to extract RNA with the RNeasy mini kit due the shortness and efficiency of these protocols.

Development and characterization of oriented scaffolds derived from cartilage extracellular matrix / 华西口腔医学杂志

<p><b>OBJECTIVE</b>This study aimed to prepare oriented scaffolds derived from a cartilage extracellular matrix (CECM) and to investigate their physicochemical property and compatibility with adipose-derived stem cells (ADSCs).</p><p><b>METHODS</b>A fresh porcine articular cartilage was cut into pieces. Cartilage nanofibers with diameters of 50-500 nm were collected through homogenization and centrifugation. These nanofibers were then decellularized by using Triton X-100 to produce 6% CECM. The oriented scaffolds derived from the nanoscale CECM were fabricated through unidirectional solidification and lyophilization. Afterward, these scaffolds were crosslinked. The physical and chemical performances and cell compatibility of CECM-oriented scaffolds were evaluated.</p><p><b>RESULTS</b>The cross-sections of the scaffolds contained homogeneous reticular porous structures with nanofibers on the walls of the pores, and the longitudinal sections revealed vertical tubular structures. Hematoxylin-eosin staining revealed that the scaffolds were red without blue. Toluidine blue, safranin O, and Sirius red staining showed positive results. The porosity, water absorption rate, and vertical compressive elastic modulus of the scaffolds were 95.455%±0.910%, 95.889%±1.071%, and (40.208±5.097) kPa, respectively.</p><p><b>CONCLUSIONS</b>The components of the oriented scaffolds derived from CECM are similar to those of native cartilage with favorable biocompatibility. The porous structures and sizes of the scaffolds are suitable for the adhesion, proliferation, and infiltration of ADSCs. The oriented scaffolds derived from CECM are relatively optimal for cartilage tissue engineering. 
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Analysis of ADR/ADE cases caused by Sanqi Panax notoginseng for injection / 中国基层医药

Objective To analyze the related factors of ADR/ADE cases caused by Sanqi Panax notoginseng for injection(lyophilization),in order to provide reference for clinical rational use of Sanqi Panax notoginseng for injection.Methods Adopted the method of retrospective study,this paper statistically analyzed ADR/ADE cases caused by Sanqi Panax notoginseng for injection(lyophilization)in the database of Adverse Drug Reaction Monitoring Center in Qingdao from January 2015 to December 2015.Results There were altogether 123 ADR/ADE cases caused by Sanqi Panax notoginseng for injection(lyophilization)in 2015,with 90 cases in general,12 new cases, 21 severe cases;more female patients than male patients;more middle -aged and old patients;irrational use of the drug accounting for 45.53% of the total number of the report;and there was a higher proportion of irrational drug use in grass -roots medical institutions.Conclusion Irrational drug use is an important factor leading to ADR/ADE cases caused by Sanqi Panax notoginseng for injection(lyophilization),so the injection of Sanqi Panax notoginseng (lyophilization)should be regulated in order to reduce the occurrence of ADR/ADE cases.

Lyophilized allografts without pre-treatment with glutaraldehyde are more suitable than cryopreserved allografts for pulmonary artery reconstruction

Various methods are available for preservation of vascular grafts for pulmonary artery (PA) replacement. Lyophilization and cryopreservation reduce antigenicity and prevent thrombosis and calcification in vascular grafts, so both methods can be used to obtain vascular bioprostheses. We evaluated the hemodynamic, gasometric, imaging, and macroscopic and microscopic findings produced by PA reconstruction with lyophilized (LyoPA) grafts and cryopreserved (CryoPA) grafts in dogs. Eighteen healthy crossbred adult dogs of both sexes weighing between 18 and 20 kg were used and divided into three groups of six: group I, PA section and reanastomosis; group II, PA resection and reconstruction with LyoPA allograft; group III, PA resection and reconstruction with CryoPA allograft. Dogs were evaluated 4 weeks after surgery, and the status of the graft and vascular anastomosis were examined macroscopically and microscopically. No clinical, radiologic, or blood-gas abnormalities were observed during the study. The mean pulmonary artery pressure (MPAP) in group III increased significantly at the end of the study compared with baseline (P=0.02) and final [P=0.007, two-way repeat-measures analysis of variance (RM ANOVA)] values. Pulmonary vascular resistance of groups II and III increased immediately after reperfusion and also at the end of the study compared to baseline. The increase shown by group III vs group I was significant only if compared with after surgery and study end (P=0.016 and P=0.005, respectively, two-way RM ANOVA). Microscopically, permeability was reduced by ≤75% in group III. In conclusion, substitution of PAs with LyoPA grafts is technically feasible and clinically promising.

Ascorbic acid from lyophilized camu-camu fruit: stability and quality control of hard capsules

Camu-camu, a fruit found in the Amazon Basin river banks and lake shores, is known for its high ascorbic acid content together with, other antioxidants. This feature shows high potential for being exploited in agribusiness industry and pharmaceutical processes. However, its high acidity, as well as peel bitterness associated with the phenolic substances content, has discouraged its consumption while still fresh. The development of alternative forms for consuming this fruit, while still preserving its ascorbic acid and polyphenol content, in addition to its great potential for maintaining human health, has become a major economic activity in coastal communities. The present study evaluated the ascorbic acid stability found in camu-camu capsules. Lyophilization was performed with the fruit pulp and peel. Both freeze-dried fruit and powder-flled capsules were stored at 5° C. Ascorbic acid stability was monitored for 90 days using HPLC assay technique. The encapsulation process of freezedried pulp was considered satisfactory in the ascorbic acid conservation, since there was only a loss of 10% of its initial concentration throughout the study period for 60 days.(AU)

Optimization of a freeze-drying cycle of a viral vaccine based on changes in temperature, time and geometry of the vials.

The freeze-drying process is used in pharmaceutical industry, however in terms of process management, the process must be avoided at all cost because it has several disadvantages such as high equipment investment, high energy demand, a process that requires long times and products easily to moisturize and fragile and needs to be carefully packed and stored. This work aims at reducing the freeze-drying cycle time of a viral product and consider loading this product at subzero temperatures to increase productivity of this product. Experiments were carried out with freeze-drying cycles with less 15 and 20h in comparison to the original cycle. The experiments were performed with the same formulation of the commercial batch and using a pilot freeze dryer. Modifications were made in the physical properties of the current freeze-drying cycle (temperature, pressure and time) and the loading temperature of the product, without changing the formulation of the vaccine or primary loading. Samples of the lyophilized product trial were analyzed for their appearance, performance, residual moisture, strength and accelerated thermostability in the amount currently used by the company quality control. All the results were within the specifications of the company were close to or better when compared to commercial batches.

Evaluación del cultivo liofilizado de Candida albicans utilizado en esquemas de certificaciones de calidad / Evaluation of the lyophilized culture of Candida albicans used in quality certification schemes

Introducción: el laboratorio de control microbiológico de la UEB Laboratorios Liorad dispone de una colección de cultivos microbianos para la conservación de microorganismos, donde se encuentra depositada la levadura Candida albicans que se emplea en esquemas de certificaciones de calidad establecidos para la evaluación de ensayos como: promoción de crecimiento de los medios de cultivos, validación de técnicas microbiológicas, entre otros. Objetivo: evaluar los resultados de la conservación de esta cepa por el método de liofilización durante un periodo de ocho años. Métodos: para el crecimiento de la cepa se utilizó el medio de cultivo Caldo Saboraud y variantes de sustancias lioprotectoras puras como: (leche descremada al 20 por ciento, glicerol 20 por ciento, sacarosa al 10 por ciento y peptona 5 por ciento) así como la mezcla de lioprotectores (leche 10 por ciento, sacarosa 5 por ciento, glicerol 5 por ciento). Se evaluó viabilidad, pureza y estabilidad genética de esta cepa durante el tiempo objeto de estudio. Resultados: las características propias de la especie estudiada se mantuvieron inalterables con un elevado grado de pureza en todas las variantes estudiadas. En cuanto a la supervivencia, cuando se emplearon las sustancias lioprotectoras puras se evidenció una marcada disminución de la viabilidad. No así al emplear la mezcla de lioprotectores que mantuvo niveles de viabilidad por encima del límite establecido durante todo el tiempo objeto de estudio. Conclusiones: los valores obtenidos en cuanto a la supervivencia de este microorganismo permiten inferir que para la conservación por largos periodos de tiempo la variante donde se empleó mezclas de lioprotectores resultó una buena opción para la conservación de C. albicans(AU)

Introduction: the microbiological control laboratory at the Basic Enterprise Unit Liorad Laboratories stores a collection of microbial cultures for the preservation of microorganisms, including the Candida albicans yeast used in the quality certification schemes established for the evaluation of assays such as fostering of culture medium growth and validation of microbiological techniques, among others. Objective: evaluate the results obtained in the preservation of this strain by the lyophilization method during a period of eight years. Methods: for strain growth, use was made of Saboraud broth culture medium as well as variants of pure lyoprotective substances such as 20 percent skimmed milk, 20 percent glycerol, 10 percent saccharose and 5 percent peptone, and the mixture of lyoprotectors (10 percent milk, 5 percent saccharose, 5 percent glycerol). An evaluation was conducted of the viability, purity and genetic stability of the strain during the study period. Results: characteristics typical of the study species remained unchanged with a high degree of purity in all the variants examined. As to survival, a marked reduction in viability was observed when pure lyoprotective substances were used, but not with the mixture of lyoprotectors, in which case viability levels exceeded the established limit during the entire study period. Conclusions: the survival values obtained for this microorganism indicate that preservation for long periods with mixtures of lyoprotectors was a good option for the preservation of C. albicans(AU)

Investigation of two kinds of solidification methods for felodipine nanosuspension: flocculation versus lyophilization / 中国药学杂志

OBJECTIVE: To solve the stability problem of felodipine nanosuspension which is a physically unstable colloidal dispersion by solidification method.

Effects of fermentation substrates and conservation methods on the viability and antimicrobial activity of weissella confusa and its metabolites

Lactic acid bacteria produce metabolites with antagonistic activity against other bacteria. However, growth conditions and conservation methods may reduce the viability and antimicrobial activity of lactic acid bacteria. This study evaluated the effects of fermentation substrate, lyophilization (freeze-drying) and refrigeration on the viability and antimicrobial activity of Weissella confusa strain and its metabolites against pathogens responsible for bovine mastitis. W. confusa strain was grown in MRS broth and milk supplemented with yeast extract and glucose (MYEG). The collected fractions were preserved by lyophilization or under refrigeration at 4ºC. Every seven days, the viability of W. confusa strain and the stability of its metabolites were evaluated against Staphylococcus aureus and Streptococcus agalactiae by disc diffusion assays. In both fermentation substrates, the combination of lyophilized strain and metabolites retained antimicrobial activity against the two pathogens for 42 days. Also, W. confusa strain retained adequate viability and antimicrobial activity when grown in MYEG and stored under refrigeration conditions. It was concluded that MYEG and refrigeration are acceptable low cost options to preserve the viability of W. confusa for its potential commercial use in the prevention and treatment of bovine mastitis.

An improved method for genomic DNA extraction from strawberry leaves / Otimização de um método para extração de DNA genômico a partir de folhas de morangueiro

Several extraction methods of genomic DNA for identification and characterization of genetic diversity in different plant species are routinely applied during molecular analysis. However, the presence of undesirable compounds such as polyphenols and polysaccharides is one of the biggest problems faced during the isolation and purification of high quality DNA in plants. Therefore, achievement of fast and accurate methods for DNA extraction is crucial in order to produce pure samples. Leaves of strawberry genotypes (Fragaria ananassa) have high contents of polysaccharides and polyphenols which increase the sample viscosity and decrease the DNA quality, interfering with the PCR performance. Thereby, in this study we evaluated the quality and amount of genomic DNA extracted from young leaves of strawberry after tissue lyophilization and maceration in presence of polivinilpirrolidone (PVP). The CTAB method was used as reference procedure and it was modified to improve the DNA extraction. The modifications consisted of tissue lyophilization overnight until it was completely freeze-dried and addition of PVP during the tissue maceration in liquid nitrogen. The results showed the efficiency and reliability of the modified method compared to the unmodified method, indicating that combination of lyophilization and PVP improve the quality and amount of the DNA extracted from strawberry leaves.

Vários métodos de extração de DNA genômico para a identificação e caracterização da diversidade genética em diferentes espécies de plantas são rotineiramente aplicados durante a análise molecular. Entretanto, a presença de compostos indesejáveis, tais como polifenóis e polissacarídeos, é um dos maiores problemas que ocorrem durante o isolamento e purificação de DNA de alta qualidade em plantas. Dessa forma, o sucesso no desenvolvimento de métodos de extração de DNA rápidos e acurados é crucial para produzir amostras puras. Folhas de genótipos de morangueiro (Fragaria ananassa) têm elevado conteúdo de polissacarídeos e polifenóis que aumentam a viscosidade da amostra e reduzem a qualidade do DNA, interferindo no desempenho da PCR. Neste estudo, avaliamos a qualidade e a quantidade de DNA genômico extraído de folhas jovens de morangueiro após a liofilização do tecido e a maceração na presença de polivinilpirrolidona (PVP). O método CTAB foi utilizado como procedimento de referência e foi modificado para melhorar a extração do DNA. As modificações consistiram na liofilização do tecido a baixa temperatura até que ele tivesse sido desidratado completamente, associada à adição de PVP durante a maceração do tecido no nitrogênio líquido. Os resultados demonstraram a eficiência e a confiabilidade do método modificado comparado ao método não modificado, indicando que a combinação da liofilização com PVP melhora a qualidade e quantidade do DNA extraído de folhas de morangueiro.

Determination of moisture content in lyophilized mannitol through intact glass vials using NIR micro-spectrometers / Determinação do teor de umidade em manitol liofilizado através de frascos de vidro intactas usando micro-espectrômetros NIR

Determination of moisture content in lyophilized solids is fundamental to predict quality and stability of freeze-dried products, but conventional methods are time-consuming, invasive and destructive. The aim of this study was to develop and optimize a fast, inexpensive, noninvasive and nondestructive method for determination of moisture content in lyophilized mannitol, based on an NIR micro-spectrometer instead of a conventional NIR spectrometer. Measurements of lyophilized mannitol were performed through the bottom of rotating glass vials by means of a reflectance probe. The root mean standard error of prediction (RMSEP) and the correlation coefficient (R²pred), yielded by the pre-treatments and calibration method proposed, was 0.233 percent (w/w) and 0.994, respectively.

A determinação do conteúdo de umidade em sólidos liofilizados é fundamental para se prever a qualidade e a estabilidade de produtos liofilizados, mas os métodos convencionais consomem muito tempo, são invasivos e destrutivos. O objetivo desse estudo foi desenvolver e otimizar um método rápido, econômico, não invasivo e não destrutivo para a determinação do conteúdo de umidade em manitol liofilizado, com base em microespectrômetro de infravermelho próximo ao invés de um espectrômetro de infravermelho próximo convencional. As medidas de manitol liofilizado foram realizadas através do fundo de recipiente de vidro em rotação por meio de sonda de reflectância. A raíz do erro médio padrão de predição (RMSEP) e o coeficiente de correlação (R²pred) obtidos pelo prétratamento e pelo método de calibração proposto foram, respectivamente, 0,233 por cento (p/p) e 0,994.

PRODUCCIÓN DE MATRICES DE QUITOSANO EXTRAÍDO DE CRUSTÁCEOS / PRODUCTION OF SCAFFOLDS USING CHITOSAN EXTRACTED FROM CRUSTACEANS

El quitosano está presente en el caparazón de los crustáceos, y desde hace algún tiempo ha sido utilizado en el campo de la medicina y la ingeniería de tejidos para la fabricación de matrices de crecimiento celular. En este estudio se extrajo quitosano de caparazón de crustáceos y se propuso un método sencillo para fabricar matrices con microestructura controlada. Las matrices fueron preparadas por congelación y liofilización de soluciones de quitosano y luego fueron caracterizadas por microscopía electrónica de barrido. La difracción de rayos X del quitosano extraído mostró un espectro acorde con una fuente comercial del material, evidenciando la efectividad del protocolo de extracción. La microscopía mostró poros ovalados y circulares distribuidos en todo el volumen de las muestras, con diámetros de poros entre 100 µm y 150 µm. Lo anterior demuestra que el método de producción propuesto proporciona un punto de partida para la fabricación de matrices de crecimiento celular.

Chitosan is present in crustacean shells and it has been used in the fields of medicine and tissue engineering for the construction of scaffolds that support cell growth. In this study, chitosan was extracted from crustacean shells and processed into scaffolds with controlled microstructure using a simple processing method presented herein. The scaffolds were prepared by freezing and lyophilization of chitosan solutions and were characterized by scanning electron microscopy. The results showed a chitosan with an X-ray diffraction spectrum similar to that of a commercial chitosan, thus demonstrating the effectiveness of the extraction protocol. Microscopy showed oval and circular pores distributed on the bulk sample, with pore diameters between 100 µm and 150 µm. This shows that the proposed fabrication method provides a starting point for the construction of porous scaffolds that may support cell growth.