The preservation of fungal cultures by lyophilization

A method for the preservation of fungal strains is presented. The cultures are grown on Sabouraud glucose agar in glass ampoules and lyophilized without further processing. By this method the macroscopical morphology of the cultures is preserved, so that these can be used immediately without recultivation as reference cultures. All tested mould and yeast strains remained viable over the six months duration of the experiment, whereas the dermatophyte strains could only partially be recultivated.     

Stabilization of Lyophilized Porcine Pancreatic Elastase

Porcine pancreatic elastase, a well-characterized serine protease, has been used as a model to assess the effects of excessive humidity on solid-state stability of the lyophilized protein. Elastase lyophilized without excipients retained full activity immediately after freeze-drying but became denatured upon continued storage at 40°C, 75% relative humidity. The extent of inactivation could be monitored through assays of amidolytic activity, as well as through changes in the circular dichroism (CD) and fluorescence spectra. Differential scanning calorimetry (DSC) was employed as a means of screening potential stabilizing additives; based on the results, sucrose and dextran 40 were selected for further evaluation. Both additives were effective in preventing denaturation. Possible mechanisms for the denaturation and stabilization of elastase are discussed.     

Cryopreserved and lyophilized cultured epidermal allografts in the treatment of leg ulcers: a pilot study

BACKGROUND: In the conservative therapy of venous leg ulcers modern types of dressings are used most frequently. In the past 20 years 'active wound dressings' - cultured epidermal keratinocytes as autografts and allografts - were also introduced in the management of leg ulcers. METHODS: The aim of our study was to compare the effect of cryopreserved and lyophilized cultured epidermal allografts in the treatment of venous leg ulcers. Evaluation of the therapy was documented as photodocumentation, planimetry, healing time and evaluation of pain relief over a 3-month period after application. Fifty patients with venous leg ulcers were selected. Twenty-five patients (group I) were treated with cryopreserved keratinocytes and 25 (group II) with lyophilized keratinocytes. RESULTS: The final evaluation 3 months after the application of allografts showed 84% of healed ulcers in group I and 80% in group II. The number of healed ulcers and the healing rate both showed no statistically significant differences. The size of the ulcer was reduced by half during the first week in both groups. The size differences during the first week are statistically significant in both groups and they are comparable (P < 0.001). The intensity of the pain was statistically significantly reduced during the first week after application in both groups (P < 0.001). CONCLUSIONS: The cryopreserved and lyophilized cultured allografts are comparable in healing rate, course of healing and relief of pain, and also in planimetric changes during the healing of venous leg ulcers. Lyophilized allografts are more convenient for routine use than cryopreserved allografts as they can be stored at room temperature. These results could give rise to the more frequent use of lyophilized allografts in slow-healing venous leg ulcers.     

Targeted Delivery of a Heme Oxygenase Inhibitor with a Lyophilized Liposomal Tin Mesoporphyrin Formulation

Tin mesoporphyrin (SnMP) is a competitive inhibitor of heme oxygenase being examined clinically for the treatment of hyperbilirubinemia. Since liposomes have been shown to target SnMP to the spleen and increase its efficacy (S. A. Landaw, G. S. Drummond, and A. Kappas, Pediatrics 84, 1091–1096, 1989), we began investigating the feasibility of the preparation and scaleup of a liposomal SnMP formulation for clinical use. SnMP liposomes were prepared by high-pressure homogenization of a suspension of SnMP and egg phosphatidylcholine (1:20, w/w) in lactose-phosphate buffer, resulting in SnMP liposomes that were less than 200 nm in diameter and had encapsulation efficiencies of up to 90% at pH 5. The SnMP liposomes could be sterile filtered and lyophilized in a 1-day cycle with retention of the encapsulation efficiency and particle size. Following injection into rats, the distribution of liposomal SnMP to spleen at 2 and 6 hr after dosing was 5–20 times higher than for aqueous SnMP. Lyophilized SnMP liposomes were also more effective than aqueous SnMP in decreasing bilirubin production in bile-cannulated rats. The results suggest the potential for producing a safe, sterile, and effective lyophilized formulation of SnMP liposomes for targeting of heme oxygenase inhibitors to the spleen.     

Bulk Dynamic Spray Freeze-Drying Part 1: Modeling of Droplet Cooling and Phase Change

In spray freeze-drying (SFD), the solution is typically dispersed into a gaseous cold environment producing frozen microparticles that are subsequently dried via sublimation. This technology can potentially manufacture bulk lyophilized drugs at higher rates compared with conventional freeze-drying in trays and vials because small frozen particles provide larger surface area available for sublimation. Although drying in SFD still has to meet the material collapse temperature requirements, the final characteristics of the respective products are mainly controlled by the spray-freezing dynamics. In this context, the main goal of this work is to present a single droplet spray-freezing model and validate it with previously published simulations and experimental data. For the investigated conditions, the droplet temperature evolutions predicted by the model agree with experiments within an error of ±10%. The proposed engineering-level modeling framework is intended to assist future development of efficient SFD processes and support scale up from laboratory to commercial scale equipment.     

The Importance of Understanding the Freezing Step and Its Impact on Freeze-Drying Process Performance

The freeze-drying process is a combination of 2 equally important processes, freezing, and drying. In the past, the effort was mainly focused on optimizing the drying process without considering the possible effects of the freezing step. During freezing, a solution undergoes several physical changes, including a supercooling state. The degree of supercooling of a solution dictates the ice habit (size, number, and morphology) during freezing, which impacts the subsequent drying process, such as the resistance to water vapor flow. Therefore, heterogeneous degree of supercooling leads to heterogeneous ice habits and, in turn, to heterogeneous drying behavior. This poses significant challenges during freeze-drying process development, optimization, and scale up. Hence, controlling the degree of supercooling significantly improves freeze-drying process design. The aim of the current review is to gather existing information on the physicochemical phenomena involved in the freezing process and how these phenomena impact the subsequent drying step of the freeze-drying process. In addition, modification of the freezing process and different techniques used to actively control the degree of supercooling during freezing will be reviewed and discussed. Their impact on freeze-drying process performance will be also addressed.     

冻干重组人三突变型HIF-1α腺病毒生物学效应考察

目的研究冻干对重组人三突变型HIF-1α腺病毒(Ad-HIF-1α-564/402/803)生物学效应的影响。方法将前期构建的Ad-HIF-1α-564/402/803在HEK293A细胞中进行扩增,用氯化铯浓度梯度离心法进行腺病毒纯化,X-Gal染色法测定重组腺病毒转染效率,在适宜的条件下制成冻干剂。采用MTS试剂盒观察冻干前后Ad-HIF-1α-564/402/803对hMVECs增殖的影响。分别在冻干前、冻干后1 d、6月、12月四个时间点提取病毒DNA,进行PCR及PCR产物测序鉴定重组人三突变型HIF-1α腺病毒基因;并在4个时间点以Ad-HIF-1α-564/402/803转染hMVECs,提取蛋白进行western blot检测HIF-1α蛋白的表达。结果 X-gal染色显示MOI为100pfu/cell时,转染效率趋于稳定。冻干前后重组人三突变型HIF-1α腺病毒对hMVECs增殖影响无显著差异(P>0.05)。经PCR及基因测序鉴定,在四个时间点,腺病毒所携带的目的基因信息无丢失或突变,HIF-1α蛋白表达无差异(P>0.05)。结论冻干能够长期保持重组人三突变型HIF-1α腺病毒的高生物活性。     

载EPO腺病毒的PLGA纳米纤维支架在体内促进骨缺损修复作用

目的: 将促红细胞生成素(EPO)腺病毒局限在应用部位,观察其在体内的促进骨形成作用。方法: 采用冷冻干燥法,将增强型绿色荧光蛋白(EGFP)腺病毒与聚乳酸羟基乙酸共聚物(PLGA)纳米纤维支架复合作为实验组(PLGA/Ad-EGFP组),以等剂量的EGFP腺病毒为对照组,分别感染骨髓基质细胞(BMSCs),以BMSCs感染EGFP的荧光细胞面积比例检测病毒活力。采用Picogreen dsDNA定量检测试剂盒检测病毒释放动力学。将EPO腺病毒与PLGA纳米纤维支架冻干复合作为实验组(PLGA/Ad-EPO组),以单纯骨缺损为对照组,植入大鼠颅骨缺损处,在第4和8周采用Micro CT及组织学HE染色检测EPO腺病毒的新生骨面积百分比。结果: PLGA/Ad-EGFP组荧光细胞面积比例明显高于对照组(P<0.05);腺病毒在PLGA上呈突释曲线,第1小时存留53.0%±5.6%,第16小时存留20.0%±3.3%。与对照组比较,PLGA/Ad-EPO组在第4周促进红细胞生成,并在第4及8周大鼠颅骨缺损修复,新生骨面积百分比达到25.0%±5.7%及35.0%±6.3%(P<0.05)。结论: 应用冻干法将腺病毒与PLGA复合能够有效保存病毒活性, PLGA /Ad-EPO纳米纤维支架能够在一定程度上促进骨缺损修复。     

海藻糖联合葡萄糖负载红细胞后溶血效果

目的：探讨海藻糖和葡萄糖联合负载红细胞的效果,为冷冻干燥红细胞提供新的保存剂。方法：分别采用0、0.125、0.25、0.5和1 mol/L的海藻糖、葡萄糖以及海藻糖联合葡萄糖37℃负载红细胞6 h。然后采用邻甲苯胺法检测上清液中游离血红蛋白含量。结果：负载液浓度为1 mol/L时,3组的细胞溶血程度较重,即上清中游离血红蛋白浓度较高。在浓度低于1 mol/L时,海藻糖联合葡萄糖组与单独海藻糖负载组溶血程度没有明显差别,但是2者低于葡萄糖组。结论：在浓度小于1 mol/L时,海藻糖联合葡萄糖负载红细胞后对细胞的损伤较小,能够满足冻存的负载要求。     

Stabilization of Chimeric BR96-Doxorubicin Immunoconjugate

Chimeric BR96-doxorubicin conjugate (BR96-DOX) is an immunoconjugate designed to specifically target and kill certain tumor cells. The linker between the chimeric BR96 antibody and DOX is an acid-labile hydrazone group which was designed to undergo lysosomal hydrolysis to release DOX in vivo. Stability studies indicated that acid-catalyzed hydrazone hydrolysis was the major degradation route in vitro. Even under optimal conditions of pH and temperature, the stability of BR96-DOX in solution was not acceptable for long-term storage. Lyophilization of BR96-DOX in the presence of added sugars, such as lactose or sucrose, and subsequent storage of the lyophile under refrigeration significantly improved the stability. Therefore lyophilization appears to be a viable approach for achieving long-term stabilization of BR96-DOX.