Control and enhancement of permselectivity of membrane‐based microcapsules for favorable biomolecular transport and immunoisolation

The success of membrane‐based, cell‐encapsulating microcapsules depends on the membrane permselectivity that provides efficient inward transport of nutrients, therapeutic protein egress, and complete exclusion of immunoglobulins. Microcapsules with a calcium crosslinked alginate core and a genipin‐crosslinked chitosan alginate (GCA) were prepared with good control over size, membrane thickness and density. Importantly, in this study, we report a novel approach of using three relevant biomolecules and investigating the effects of the membrane characteristics (thickness and density) and microcapsule size on biomolecular mass transport across the GCA microcapsules using mathematical models based on a balance of the chemical potential. Scaling analysis was used to interrelate the membrane thickness, chitosan–alginate reaction rate constant, and diffusion coefficient. The resistance to diffusion of the three biomolecules increased with membrane density and thickness. Interestingly, swelling in the large microcapsules resulted in an increase in permeability, allowing larger biomolecules (immunoglobulin and carbonic anhydrase) to diffuse more readily. In the case of the smaller biomolecule, vitamin B₁₂, a shorter diffusion path length in smaller microcapsules allowed better ingress. When compared with other microcapsules, the GCA microcapsules possess improved permselectivity for them to allow diffusion of small nutrient molecules and proteins, whereas completely excluding antibodies. Also, these results elucidate the importance of membrane properties and microcapsule size to realize favorable transport of biomolecules. © 2011 American Institute of Chemical Engineers AIChE J, 2011     

海藻酸钠-壳聚糖复合膜中溶氧扩散性能研究

氧气在微胶囊膜中的扩散行为将直接决定微囊内细胞的生长代谢行为.以海藻酸钠-壳聚糖聚电解质复合平板膜为研究模型,利用渗透池法,重点考察溶氧在膜中的扩散行为及其影响因素.结果显示:复合膜的扩散系数和孔隙率均低于海藻酸钙,复合膜中溶氧扩散系数为(7～13)×10-10m2·s-1,为水中的23.3％～43.3％,孔隙率为93％～97％;扩散系数随海藻酸钠特性黏度的增大而减小,随壳聚糖分子量的增大而减小.微胶囊膜是氧传质主要的阻力部位,孔隙率、三维结构和材料极性是影响扩散性能的重要因素,改变海藻酸钠特性黏度和壳聚糖分子量可以改变膜孔隙率、结构和材料极性,进而调节膜扩散性能.     

Oxygen diffusivity in alginate/chitosan microcapsules

BACKGROUND: Oxygen diffusion properties affect the proliferation and metabolism of cells cultured in microcapsules with a polyelectrolyte complex membrane. The effective diffusion coefficient (De) of oxygen in alginate/chitosan (AC) microcapsules under different preparation conditions was calculated, and a mathematic model was developed to investigate the effect of oxygen diffusion on cell loading in the microcapsules. RESULTS: Oxygen De in AC microcapsules was independent of alginate solution concentration, intrinsic viscosity of alginate and different polyelectrolyte complex membranes. De decreased from 2.1 ± 0.3 × 10^?9 to 0.17 ± 0.01 × 10^?9 m² s^?1 as microcapsule diameter decreased from 1800 to 45 0 µm. Microcapsule density was increased from 1.013 ± 0.000 to 1.034 ± 0.003 g mL^?1 as diameter decreased from 1775 to 430 µm. The mathematic model results showed that critical CHO cell loadings were 1.8 × 10⁸ or 1.1 × 10⁸ cells mL^?1 in microcapsules with 450 or 1800 µm diameter, respectively. CONCLUSIONS: No significant difference was found of oxygen De between calcium alginate beads and AC microcapsules. The decrease of De with diameter was attributed to the increasing density and compact degree on the surface. The model results indicated that risk on necrosis rose with the increasing diameter. Microcapsules with smaller diameters may have more advantages on cell culture. © 2012 Society of Chemical Industry     

Capsule permeability via polymer and protein ingress/egress

Static incubation tests, where microcapsules and beads are contacted with polymer and protein solutions, have been developed for the characterization of permselective materials applied for bioartificial organs and drug delivery. A combination of polymer ingress, detected by size‐exclusion chromatography, and protein ingress/egress, assessed by gel electrophoresis, provides information regarding the diffusion kinetics, molar mass cutoff (MMCO) and permeability. This represents an improvement over existing permeability measurements that are based on the diffusion of a single type of solute. Specifically, the permeability of capsules based on alginate, cellulose sulfate, polymethylene‐co‐guanidine were characterized as a function of membrane thickness. Solid alginate beads were also evaluated. The MMCO of these capsules was estimated to be between 80 and 90 kDa using polymers, and between 116–150 kDa with proteins. Apparently, the globular shape of the proteins (radius of gyration (R_g) of 4.2–4.6 nm) facilitates their passage through the membrane, comparatively to the polysaccharide coil conformation (R_g of 6.5–8.3 nm). An increase of the capsule membrane thickness reduced these values. The MMCO of the beads, which do not have a membrane limiting their permselective properties, was higher, between 110 and 200 kDa with dextrans, and between 150 and 220 kDa with proteins. Therefore, although the permeability estimated with biologically relevant molecules is generally higher due to their lower radius of gyration, both the MMCO of synthetic and natural water‐soluble polymers correlate well, and can be used as in vitro metrics for the immune protection ability of microcapsules and microbeads. This article shows, to the authors' knowledge, the first reported concordance between permeability measures based on model natural and biological macromolecules. © 2000 John Wiley & Sons, Inc. J Appl Polym Sci 75: 1165–1175, 2000     

重组人粒细胞-巨噬细胞集落刺激因子壳聚糖-海藻酸钠微囊的制备

目的以壳聚糖和海藻酸钠为原料,制备重组人粒细胞-巨噬细胞集落刺激因子(rhGM-CSF)微囊,探讨开发口服蛋白多肽类药物的可行性。方法以rhGM-CSF为药物模型,通过壳聚糖与海藻酸钠聚电解质的络合反应制备rhGM-CSF壳聚糖-海藻酸钠微囊,观察微囊的形态大小,测定其包封率,不同pH值下的膨胀度和体外释放率。结果制备的rhGM-CSF壳聚糖-海藻酸钠微囊呈均匀、完整的圆球形,平均直径1mm左右;包封率达80%以上;在模拟肠液(磷酸盐缓冲液,pH7.4)中浸泡3h,膨胀度可达600%,药物释放率达85%以上。结论壳聚糖-海藻酸钠微囊具有肠溶控释作用,有望成为rhGM-CSF等蛋白类口服药物的控释载体。     

Investigation of encapsulation of pancreatic beta cells and curcumin within alginate microcapsules

Cell encapsulation is an ideal approach for the replacement of pancreatic function in Type 1 diabetes. Poor biocompatibility of microcapsules generates an inflammatory response in the implantation site and induces fibrosis infiltration, which causes microencapsulated cell death and graft failure. To prevent inflammation after implantation, composite microcapsules that exhibit anti-inflammatory properties were designed. This study is about encapsulating beta cells and curcumin within 1.5% alginate by the jet-breaking regime of the syringe pump. The microcapsules’ size distribution and rate of the alginate solution were characterized to find uniform particles. Micro-size particles were attained at a rate of 25 mL/min. Uniform spherical microcapsules (200–300 μm) were created in large amounts in a short period. Microcapsule breakage was less than 3% during 7 days, which demonstrated the stability of the encapsulation method. Insulin secretion and cell viability assays were performed 1, 3, and 7 days after microencapsulation by glucose-stimulated insulin secretion (GSIS) and 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assays. No significant differences in the amount of insulin secretion and beta cell viability were observed among free cells, alginate microcapsules, and curcumin-alginate microcapsules during 7 days (p > 0.05). Therefore, the curcumin and alginate membrane did not show any harmful impacts on the function and survival of the beta cells.     

微流体数字化技术制备鱼卵微胶囊

基于微流体数字化技术,提出了一种结构简单、成本低、制备条件温和的细胞微胶囊制备方法.设计了以电磁铁为作动器的微流体脉冲驱动控制系统,通过控制电磁铁铁芯往复运动撞击固定有微喷嘴的振动体,为微喷嘴及其内部的海藻酸钠细胞悬浮液提供频率和大小可控的脉冲惯性力,实现了直径约为900μm的单个鱼卵细胞的稳定喷射和鱼卵细胞微胶囊的稳...     

Alginate core–shell microcapsule reduces the DMSO addition-induced osmotic damage to cells by inhibiting cellular blebs

In cryopreservation, the addition of cryoprotectant can change the intra- and extra-cellular osmotic pressure, affect the cell morphology, and induce blebs on the plasma membrane. In this study, the blebs of cells microencapsulated in the alginate microsphere induced by osmotic shock were studied, and the effects of microencapsulation on bleb size and cell viability were determined. Firstly, a coaxial co-flow focusing device was applied to generate cell-laden microcapsules using alginate hydrogel in this paper. Then, cellular blebs induced by DMSO with various concentrations under microencapsulation were com-pared with that when non-encapsulated, and the dynamic process of cellular bleb was investigated. Finally, the qualitative relationship between bleb size and cell viability in the presence of DMSO was built, and thus the effects of microencapsulation on bleb size and viability were evaluated. The results show that the bleb size is smaller and the cell viability is higher, and cell microencapsulation can signif-icantly inhibit the excessively large blebs generated on the cell membrane and reduce the osmotic dam-age to cells when loading cryoprotectant and then to improve cell viability during cryopreservation. This work can provide insights for optimizing cryoprotectant-loading protocols, offer a new avenue to study cell blebbing, and advance future research on cryopreservation of rare cells and biomaterials.     

Preparation of polyethersulfone–alginate microcapsules for controlled release

In this study, polyethersulfone (PES)–alginate microcapsules were prepared for drug‐controlled release, and vitamin B₁₂ (VB₁₂), rifampicin (RFP), and bovine serum albumin (BSA) were used as model drugs. Different microcapsules were prepared by the variation of the crosslinking degree of alginate and the variation of the chemical components of the microcapsule membrane, including the PES and polyethylene glycol (PEG) contents. Systematic experiments were carried out to study their influences on the release profile of the model drugs. The results showed that with the increase of the crosslinking degree of the alginate, the drug release rate increased; whereas with the increase of the PES concentration used to prepare the microcapsule membrane, the drug release rate decreased. The contents of the PEG in the microcapsule membrane also affected the drug release. This study enriched the methodology of the fabrication of the microcapsules, and the microcapsules may have a potential use for controlled release. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2009     

Confocal microscopy study of polymer microcapsules for enzyme immobilisation in paper substrates

The goal of this research is to develop the technology platform required for the production of bioactive paper based on enzymes as bioactive agents. The immobilization platform described here is based on microencapsulation, which consists in the entrapment of biomolecules in the core of hollow spheres made by a semipermeable membrane. The capsules containing the enzymes can be either deposited on paper or mixed with paper pulp to prepare a bioactive paper. The activity of encapsulated laccase was compared with that of free enzyme using its reaction with the o‐phenylenediamine (OPD) substrate. Confocal Laser Scanning Microscopy (CLSM) is used to study the location of protein in microcapsules and provides explanations for differences in activity of encapsulated laccase. The location of protein in microcapsules was determined using BSA modified with the fluorescent tag sulforhodamine. Polyethyleneimine microcapsules were modified with fluorescein isothiocyanate allowing the simultaneous identification of capsule walls and of encapsulated proteins. From CLSM analysis, proteins were found to favor the wall of the capsules because of strong ionic attraction with the charged polymer. BSA was found to some extent in the core of the capsules and encapsulation of higher loadings increased the proportion of core proteins. We will also present our results on the incorporation of microcapsules in a paper substrate. CLSM was used in this section to determine the distribution and density of tagged microcapsules in the paper substrate. The response of immobilized laccase to a common substrate will also be described. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2009     

Electrostatic Droplet Generator with 3-Coaxial-Nozzle Head for Microencapsulation of Living Cells in Hydrogel Covered by Synthetic Polymer Membranes

The current methods allow for encapsulation of cells inside spherical microcapsules made of a matrix covered by a permselective membrane using an electrostatic droplet generator with 1-nozzle or 2-nozzle heads. However, some potentially useful materials for the outer membranes cannot be put into direct contact with hydrophilic core filled by cells during the manufacturing process. Therefore, we designed a novel 3-coaxial-nozzle head that allows for the third fluid to separate the core material from the membrane material. The equipment was applied for manufacturing spherical microcapsules comprised of cell-friendly alginate core surrounded by semipermeable polyethersulfone membrane. The obtained microcapsules had a diameter between 0.84 mm and 1.79 mm, and the diameter correlated negatively with the applied electric voltage. The thickness of the membrane varied from 171 µm to 450 µm. The SEM images of the interior of microcapsules revealed highly porous membrane structure typical for synthetic membranes obtained by a wet phase inversion method. Bakery yeast cells encapsulated inside the alginate-polyethersulfone microcapsules retained their proliferation ability proving the effectiveness and safety of this encapsulation technique.     

壳聚糖分子量对酵母细胞固定化培养的影响

以海藻酸钠-壳聚糖微胶囊（AC微胶囊）为研究对象,以酵母细胞S.cerevisiae BY4741为细胞模型,分别考察了相同成膜反应时间和相同成膜厚度两种情况下,壳聚糖分子量对微囊膜膨胀行为、微囊内细胞增殖情况及微囊化细胞培养过程中细胞泄漏的影响规律。研究结果显示,在成膜反应时间相同的情况下,壳聚糖分子量越小,向海藻酸钙凝胶网络扩散越容易,交联成膜越厚,微胶囊膜越能耐受渗透压膨胀。但在控制相同膜厚的情况下,大分子量壳聚糖由于分子链长,与凝胶微球表面海藻酸钠分子的作用位点多,易纠结相邻海藻酸钠分子链形成更致密的络合交联,呈现更高的耐受渗透压膨胀的特性。AC微囊化细胞培养48 h后,小分子量壳聚糖充分成膜反应制备的AC微胶囊,虽然膨胀不显著,但由于膜结构占据了更多微胶囊体积,既缩小了酵母细胞的增殖空间,又极大增加了传质阻力,反而限制了细胞增殖。培养过程中,各组微胶囊均未发现明显的细胞泄漏现象。     

干扰素壳聚糖/海藻酸钠微囊控释制剂载体的初步研究

目的研究蛋白质类药物口服控释给药的可行性。方法壳聚糖与海藻酸钠通过聚电解质络合反应制备成壳聚糖/海藻酸钠微囊,以干扰素为模型药物,研究不同pH条件下,药物的控制释放情况。结果微囊的粒径为1 mm左右,其干扰素的包封率达90.0%以上,微囊在模拟胃液(pH1.0)中,3h药物释放不到5%;在模拟肠液(pH7.4)中,3h药物释放近100%。结论壳聚糖/海藻酸钠微囊有可能成为蛋白质类药物口服控释制剂的载体。     

二甲酸钾微胶囊的构建与缓释抑菌性

以自制沸石分子筛为载体,通过水热法将二甲酸钾负载形成芯材,海藻酸钠、壳聚糖为壳材,以复凝聚法制备出缓释微胶囊。通过FTIR、XPS对微胶囊进行了结构表征,通过缓释及抑菌率测试对其性能进行表征。结果表明:沸石分子筛与二甲酸钾之间可形成复配效应,构成微胶囊的芯材;壳聚糖和海藻酸钠发生静电作用,形成聚电解质膜,构成微胶囊的壳材;制备的微胶囊在pH=2.0的模拟胃环境中稳定,在pH=7.2的模拟肠道环境中二甲酸钾得到了释放,并在3 h内可释放完全;微胶囊包封率为68.33%,二甲酸钾的释放量为137.50 mg/g。微胶囊对大肠杆菌的生长具有一定的抑制作用,并随着微胶囊浓度的增大对大肠杆菌的抑制率呈现增长的趋势,最高抑菌率可以达到85%。     

海藻酸钠微囊的制备及应用进展

微囊化技术作为一项发展迅速的新技术,具有精确给药、芯材控释等特点,在生物医药、食品、化工等领域均得到成功应用。海藻酸钠是从海洋藻类提取的功能独特的植物多糖,具有良好的溶解性、成膜性和凝胶性等优势,已被广泛用作微囊的包膜材料。但海藻酸钠微囊易受到基质材料、交联剂和生产工艺参数的影响,性质难以调控,所以微囊的生产仍存在配方不完善、制备工艺不稳定等问题。为解决上述问题,本综述对海藻酸钠的离子交换性、pH敏感性、凝胶特性等性质和微囊制备过程的影响因素进行了总结。论述了海藻酸钠微囊在包封细胞、药物以及精油方面的应用,指出今后的研究方向应集中于改进微囊的制备工艺,探明海藻酸钠成膜机理与机械性能的关系,提高海藻酸钠微囊强度与韧性,继续推进海藻酸钠与其他高分子材料的复配研究,以期扩大海藻酸钠微囊的应用范围,加快海藻酸钠微囊的工业化进程。     

MEMBRANE EMULSIFICATION SYSTEMS

Microcapsules (or microspheres) with small size and narrow size distribution have potential applications as carriers of proteins and polypeptides. However the conventional preparation methods severely limit their real applications. Membrane emulsification technology may become a new preparation method of microcapsules with monodisperse droplets, mild conditions, good stability, low energy consumption and easy to realize mass production. In this paper, studies on membrane emulsification systems and the possible existing problems are summarized, and primary attempts on preparing alginate/chitosan microcapsules are conducted.     

含微胶囊相变材料的海藻酸钙大胶囊的制备及其性能

采用原位聚合法合成了微胶囊相变材料,并通过锐孔法制备了海藻酸钙包覆多个微胶囊相变材料的大胶囊。用FTIR分析了大胶囊的化学结构,采用游标卡尺测量在不同质量分数的海藻酸钠水溶液和氯化钙水溶液中制备的大胶囊的平均粒径;用SEM和DSC对微胶囊相变材料的微观形貌和热性能进行了分析,对大胶囊的热性能进行了考察,讨论了在不同海藻酸钠质量分数下制备的大胶囊经甲苯萃取30 m in后的热性能。结果表明,微胶囊呈粒径均一,表面光滑,密封较好的球体;其相变温度为34.1℃,相变潜热为143.8 J/g。随着微胶囊相变材料加入量的增加,大胶囊的相变潜热逐渐增加;当w(CaC l2)=2%时,随着海藻酸钠质量分数的增加,大胶囊的粒径由1.36 mm逐渐增加至1.96 mm并趋于平缓,且海藻酸钠水溶液质量分数不宜超过3%;随着氯化钙质量分数的增加,大胶囊平均粒径逐渐增长,但幅度较其随海藻酸钠质量分数变化的幅度小;甲苯对大胶囊壳材的渗透能力随海藻酸钠质量分数的增加而减小。     

Poly(L‐histidine)‐chitosan/alginate complex microcapsule as a novel drug delivery agent

Novel poly(L ‐histidine)‐chitosan/alginate complex microcapsules were prepared from biodegradable polymers poly(L ‐histidine) (PLHis) in the presence of chitosan at acetate buffer solution pH 4.6. Microcapsules obtained are spherical and well‐dispersed with a smooth surface and a narrow size distribution. The microcapsules can encapsulate the protein model drug hemoglobin (Hb) efficiently. The results show that the complex microcapsules with low, medium, or high molecular weight of chitosan (0.05%, w/v), the highest encapsulation efficiencies obtained are 91.3%, 85.9%, and 94.2% with loading efficiencies of 47.8%, 44.3%, and 39.7%, respectively. The release profiles indicate that Hb‐loaded microcapsules conform to first‐order release kinetic in whole procedure, and 84.8%, 71.4%, and 87.3% of Hb were released during 72‐h incubation in PBS pH6.8 for microcapsules with low, medium, and high molecular weight chitosan (0.05%, w/v), respectively. The results also indicate that particle size and drug loading efficiency have a significant influence on the release profile and encapsulation efficiency. Our results reveal that the PLHis‐chitosan/alginate complex microcapsules are able to encapsulate and release Hb and are potential carriers for protein drugs. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2012     

Combined method of complex coacervation and electrospray for encapsulate preparation

Encapsulate beads composed of alginate and chitosan as shell and bovine serum albumin (BSA) as core were prepared by combined method of complex coacervation and electrospray. The main objective of this work was to produce mono‐sized and spherical capsule of chitosan‐alginate with controlled sizes of capsules and shell. However, the effects of applied voltage, flow rate, and molecular weight of chitosan were investigated on the size, size distribution, membrane thickness of the prepared capsules, as well as the release rate of BSA. The results revealed that by the method developed in this study, it was possible to produce spherical capsules with controlled size and narrow size distribution. Increasing the voltage and decreasing the flow rate reduced the radius of capsule and its shell thickness from 2.09 mm to 750 μm and from 1.31 mm to 490 μm, respectively. Furthermore, the molecular weight of chitosan had no significant effect on the capsules' size and the release rate of BSA, whereas the rate of BSA release was increased with increase of the voltage. The later effect would be due to the increase of shell porosity at the higher voltages. © 2010 Wiley Periodicals, Inc. J Appl Polym Sci, 2010     

滴丁·乙-脲醛微囊悬浮剂的制备与表征

以原位聚合脲醛树脂的方法制备了滴丁·乙-微囊悬浮剂。利用UV,激光粒度仪,光学显微镜,SEM,FT-IR等对制剂进行表征,结果表明:所制备的微囊悬浮剂中,2,4-D丁酯和乙草胺的包封率分别为85%和89.9%,微囊平均粒径为3.79μm,粒度分布较窄,表面光滑致密,红外光谱分析表明微囊由脲醛树脂壁材与滴丁·乙芯材所组成。