Competing two enzymatic reactions realizing one‐step preparation of cell‐enclosing duplex microcapsules

The usefulness of cell‐enclosing microcapsules in biomedical and biopharmaceutical fields is widely recognized. In this study, we developed a method enabling the preparation of microcapsules with a liquid core in one step using two enzymatic reactions, both of which consume H₂O₂ competitively. The microcapsule membrane prepared in this study is composed of the hydrogel obtained from an alginate derivative possessing phenolic hydroxyl moieties (Alg‐Ph). The cell‐enclosing microcapsules with a hollow core were obtained by extruding an aqueous solution of Alg‐Ph containing horseradish peroxidase (HRP), catalase, and cells into a co‐flowing stream of liquid paraffin containing H₂O₂. Formation of the microcapsule membrane progressed from the surface of the droplets through HRP‐catalyzed cross‐linking of Ph moieties by consuming H₂O₂ supplied from the ambient liquid paraffin. A hollow core structure was induced by catalase‐catalyzed decomposition of H₂O₂ resulting in the center region being at an insufficient level of H₂O₂. The viability of HeLa cells was 93.1% immediately after encapsulation in the microcapsules with about 250 µm diameter obtained from an aqueous solution of 2.5% (w/v) Alg‐Ph, 100 units mL^?1 HRP, 9.1 × 10⁴ units mL^?1 catalase. The enclosed cells grew much faster than those in the microparticles with a solid core. In addition, the thickness of microcapsule membrane could be controlled by changing the concentrations of HRP and catalase in the range of 13–48 µm. The proposed method could be versatile for preparing the microcapsules from the other polymer derivatives of carboxymetylcellulose and gelatin. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1528–1534, 2013     

海藻酸钠漂浮微囊的制备以及载药应用研究

目的:本文研究了一种海藻酸钠漂浮微囊的制备方法用以实现胃部持续给药。方法:采用微胶囊发生器制备海藻酸钠漂浮微囊,壁材为海藻酸钠,芯材为食用油的漂浮微囊,衡量不同的制备参数对微囊的理化特性影响;采用克拉霉素作为模型脂溶性药物,测量漂浮药物递送系统的控制释放性质、以及微囊载药特性和小鼠体内漂浮验证。结果:成功制备出了具有漂浮特性的海藻酸钠微囊,其中泵送速度对微囊性质的影响最大。制备出的微囊具有低细胞毒性,可以实现90%的药物包埋率。此外,微囊可以在小鼠的胃中保存超过6小时,具有良好的漂浮特性。结论:海藻酸钠漂浮微囊是一种有效的胃部药物递送系统,可明显延长药物在胃部的滞留时间。     

Cancer stem cell marker‐expressing cell‐rich spheroid fabrication from PANC‐1 cells using alginate microcapsules with spherical cavities templated by gelatin microparticles

Cancer stem‐like cells (CSCs) are rare subpopulations of cancer cells. The development of three‐dimensional tissues abundant in CSCs is important to both the understanding and establishment of novel therapeutics targeting them. Here, we describe the fabrication of multicellular tumor spheroids (MTSs) abundant in CSCs by employing alginate microcapsules with spherical cavities templated by cell‐enclosing gelatin microparticles. Encapsulated human pancreatic cancer cell line PANC‐1 cells grew for 14 days until they filled the cavities. The percentage of cells expressing reported CSC markers CD24, CD44, and epithelial‐specific antigen (ESA), increased during this growth period. The percentage at 24 days of incubation, 22%, was 1.6 times higher than that of MTSs formed on a nonadherent surface in the same period of incubation. The MTSs in microcapsules could be cryopreserved in liquid nitrogen using a conventional method. No significant difference in the content of CSC marker‐expressing cells was detected at 3 days of incubation when thawed after cryopreservation for 2 weeks, compared with cells incubated without prior cryopreservation. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1071–1076, 2015     

膜材与制备过程对血红蛋白微胶囊粒径和包埋率的影响

以单甲氧基聚乙二醇聚乳酸共聚物（PELA）为膜材用复乳溶剂扩散法制备了包含牛血红蛋白（BHb）的微胶囊,微胶囊中BHb的P₅₀和Hill系数分别为3 466 Pa和2.4左右,接近于天然BHb的生物活性。研究发现膜材种类对BHb微胶囊包埋率和粒径的影响最大,使用MPEG2000为亲水性嵌段的PELA共聚物时,包埋率最高,达到90％以上,粒径为3～5 μm左右;随着膜材浓度的增大,微胶囊包埋率和粒径均增加;随着外水相NaCl浓度的增大,微胶囊包埋率升高、粒径减小;随着外水相稳定剂PVA浓度的增大,微胶囊粒径减小,包埋率先升高后降低,在较低浓度下（10 g/L、20 g/L）包埋率较高;初乳化搅拌速率的增大,有利于包埋率的提高,但对粒径影响不大;复乳化搅拌速率的影响较复杂,当复乳液体积较大时,复乳化搅拌速率对微胶囊制备的影响规律性不明显。当固定膜材和初乳化搅拌速率时,包埋率和粒径之间存在着类似抛物线的关系,包埋率随着粒径的减小而降低。     

A new method for targeted drug delivery using polymeric microcapsules

Recent research and clinical evidence suggest that thalidomide could potentially be used to treat inflammation associated with Crohn's disease. However, systemic side effects associated with large doses of this drug have limited its widespread use. Treatment, with thalidomide would prove more efficacious if the drug could be delivered directly to target areas in the gut, thereby reducing systemic circulation. Microcapsule encapsulation could enable direct delivery of the drug. To assess the latter, we designed and tested drug-targeting release characteristics of alginate-poly-l-lysine-alginate (APA) microcapsules in simulated gastrointestinal environments. The results show that APA capsules enabled delivery of thalidomide in the middle and distal portions of the small intestine. We also compared the APA membrane formulation with an earlier designed alginate chitosan (AC) membrane thalidomide formulation. The results show that both APA and AC capsules allow for successful delivery of thalidomide in the gut and could prove beneficial in the treatment of Crohn's disease. However, further research is required.     

A novel cytomedical vehicle capable of protecting cells against complement

We have developed "Cytomedicine," which consists of functional cells entrapped in semipermeable polymer, and previously reported that APA microcapsules could protect the entrapped cells from injury by cellular immune system. However, microencapsulated cells were not protected from humoral immune system. Here, we developed a novel APA microcapsule, in which APA microbeads (APA(Ba) microbeads) were modified to contain a barium alginate hydrogel within their centers in an attempt to make it more difficult for antibody and complement to permeate the microcapsules. The permeability of APA(Ba) microbeads was clearly less than that of APA microcapsules, presumably due to the presence of barium alginate hydrogel. Cells encapsulated within APA(Ba) microbeads were protected against treatment with xenogeneic anti-serum. Furthermore, murine pancreatic beta-cells encapsulated in APA(Ba) microbeads remained viable and continued to secrete insulin in response to glucose. Therefore, APA(Ba) microbeads may be a useful carrier for developing anti-complement device for cytomedical therapy.     

纤维素硫酸钠-聚二甲基二烯丙基氯化铵微胶囊固定化Candida krusei产甘油条件优化

研究了利用一种新型的纤维素硫酸钠／聚二甲基二烯丙基氯化铵（Nacs／PDMDAAc）中空微胶囊包埋Can—dida krusei ZJU5205制备甘油的过程。通过摇瓶培养过程中对初始包菌量、胶囊和胶珠、初始甘油浓度、胶囊大小、胶囊体积／发酵液体积等关键固定化参数和培养条件的优化,确立了NaCS／PDMDAAC微胶囊固定化Candida krusei的最佳工艺参数为包菌量为0．6g／L,发酵初加入20g／L甘油,胶囊体积／发酵液体积为0．4。     

克雷伯氏杆菌的固定化及其在微胶囊中生长状况的研究

采用注滴法制备载克雷伯氏杆菌的海藻酸盐微胶囊,对微胶囊制备条件进行优化,并考察了各种因素对克雷伯氏杆菌微囊化的影响。实验结果表明：控制海藻酸钠溶液的质量分数为2%、氯化钙溶液的质量分数为4.5%、凝胶反应4 h以及菌体与海藻酸钠溶液体积比为1：30,所制备的微胶囊及微囊内菌体的生长效果最好。还初步探讨了壳聚糖对海藻酸盐微胶囊的影响,发现微胶囊表面复合一层壳聚糖半透膜,可以提高微胶囊的强度,但弹性有所降低。另外进行了微胶囊中克雷伯氏菌代谢途径的探索。     

Advances in polymer-based cell encapsulation and its applications in tissue repair

Cell microencapsulation is a more widely accepted area of biological encapsulation. In most cases, it involves fixing cells in polymer scaffolds or semi-permeable hydrogel capsules, providing the environment for protecting cells, allowing the exchange of nutrients and oxygen, and protecting cells against the attack of the host immune system by preventing the entry of antibodies and cytotoxic immune cells. Hydrogel encapsulation provides a three-dimensional (3D) environment similar to that experienced in vivo, so it can maintain normal cellular functions to produce tissues similar to those in vivo. Embedded cells can be genetically modified to release specific therapeutic products directly at the target site, thereby eliminating the side effects of systemic treatments. Cellular microcarriers need to meet many extremely high standards regarding their biocompatibility, cytocompatibility, immunoseparation capacity, transport, mechanical, and chemical properties. In this article, we discuss the biopolymer gels used in tissue engineering applications and the brief introduction of cell encapsulation for therapeutic protein production. Also, we review polymer biomaterials and methods for preparing cell microcarriers for biomedical applications. At the same time, in order to improve the application performance of cell microcarriers in vivo, we also summarize the main limitations and improvement strategies of cell encapsulation. Finally, the main applications of polymer cell microcarriers in regenerative medicine are summarized.     

Two-step modification of silica for efficient adsorption of phosphatidylcholine in water and application in phospholipase D-catalyzed transphosphatidylation

An efficient and green aqueous–solid system was introduced for phospholipase D-mediated transphosphatidylation. γ-(2,3-epoxypropoxy) propytrimethoxysilane was covalently bound to silica and esterified by acetic acid, which acted as an anchor molecule to facilitate the adsorption of phosphatidylcholine (PC) in aqueous solutions. Obtained silica-adsorbed PC was successfully used for transphosphatidylation to produce phosphatidylserine (PS). The PC loading and PS yield reached 98.8 and 98.3%, respectively. A new model was proposed to illustrate the adsorption and enzymatic processes. Moreover, this aqueous–solid system provides a promising way for the continuous production. Four kinds of phospholipids were biosynthesized in the pack-bed reactor. The stability of the aqueous–solid system was excellent, as demonstrated by its use 30 times without any loss of the productivity. The product was eluted by coconut oil and manufactured into microcapsules. Toxic agents were completely avoided in the whole production process.