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DNA encapsulation within membrane-coated alginate beads.

机译：DNA包裹在膜包裹的藻酸盐珠中。

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Alginate beads produced by both external and internal gelation techniques were Studied so as to determine the optimal bead matrix structure within which DNA can be immobilized for in vim application. Alginates were characterized for guluronic/marmuronic acid (G/M) content and average molecular weight. Non-homogeneous calcium, alginate and DNA distributions were found within gels made by the external gelation method because of the external calcium source used. In contrast, the internal gelation method produces more homogeneous gels. The encapsulation yield of DNA was over 97 and 80% for beads formed using external and internal gelation methods, respectively. DNA molecular weight cut-off was determined to be 394 kDa for both external and internal gelled beads.; DNA alginate beads produced using both external and internal calcium sources were coated with chitosan, poly-L-lysine or co-guanidine membranes. For chitosan and poly-L-lysine, membrane thickness increased with decreasing molecular weight and increasing degree of deacetylation (chitosan). Co-guanidine membranes were shown to form intact ionically complexed membranes on alginate beads, serving as an alternative to the commonly used polymers; poly-L-lysine and chitosan. The co-guanidine membranes thickness increased with increasing concentration and coating time. The beads were assayed with DNAse nuclease to determine optimal membrane matrix combinations offering the highest level of DNA protection from nucleic acid hydrolysis, simulating gastrointestinal exposure. An extractive electrophoresis method was also developed to extract and assay intracapsular DNA by measuring its molecular size range.; Almost total hydrolysis of DS-DNA was observed in alginate beads and chitosan coated beads following nuclease exposure. The casting of membranes reduced the permeability of alginate beads, shown by the enhanced retention of DNA residuals (i.e. double- and single-stranded DNA, polynucleatides, bases) after DNAse exposure. The highest level of DNA protection was obtained with high molecular weight (197.1 kDa) poly-L-lysine and high concentration (5 mg/ml) co-guanidine membranes coated on beads formulated using an external calcium source, where over 80 and 90% of the DS-DNA remained after 40 min of DNAse exposure. Guanidine membranes were capable of fully excluding DNAse nuclease with a molecular weight of 31 kDa. Lyophilization and rehydration of DNA beads also reduced permeability to nucleases, resulted in DNA recoveries following nuclease exposure of 60% for chitosan coated, 90% for poly-L-lysine coated, and 95% for alginate beads.; Soluble chitosan and poly-L-lysine are readily hydrolyzed using lysozyme or chitmanase for chitosan, and trypsin, chymotrypsin or proteinase K for poly-L-lysine. In contrast, chitosan and poly-L-lysine membranes were almost inert to the respective hydrolytic enzymes, with less than 2% of the membrane weight being hydrolyzed. It appeared that either membrane material would be stable for in vivo application, and in particular in the protection of DNA during gastrointestinal transit; Intracapsular DNA was accessible to the carcinogen ethidium bromide, which showed a 4 fold increase in uptake in uncoated beads and 2 fold uptake in co-guanidine coated beads compared to beads lacking DNA. Low molecular weight cut-off co-guanidine membranes appear to provide free diffusional access to low molecular weight carcinogens or mutagens.
机译：研究了通过外部和内部凝胶化技术生产的藻酸盐珠子，以确定可以固定DNA用于vim应用的最佳珠子基质结构。表征藻酸盐的古洛糖醛/ marmuronic酸（G / M）含量和平均分子量。由于使用外部钙源，在通过外部凝胶化方法制备的凝胶中发现钙，藻酸盐和DNA分布不均匀。相反，内部凝胶化方法产生更均匀的凝胶。对于使用外部和内部凝胶化方法形成的珠子，DNA的包封率分别超过97％和80％。对于外部和内部凝胶化的珠子，测定的DNA分子量截留值均为394 kDa。使用外部和内部钙源生产的DNA藻酸盐珠子均用壳聚糖，聚L-赖氨酸或共胍膜包被。对于壳聚糖和聚-L-赖氨酸，膜厚度随着分子量的降低和脱乙酰基（壳聚糖）的增加而增加。已显示共胍膜在藻酸盐珠上形成完整的离子络合膜，可作为常用聚合物的替代品。聚-L-赖氨酸和壳聚糖。共胍膜的厚度随着浓度和涂覆时间的增加而增加。用DNAse核酸酶分析珠子，以确定最佳的膜基质组合，从而提供最高水平的DNA保护以免受核酸水解，从而模拟胃肠道暴露。还开发了一种提取电泳方法，通过测量其分子大小范围来提取和测定囊内DNA。核酸酶暴露后，在藻酸盐珠和壳聚糖包被的珠中观察到DS-DNA几乎完全水解。 DNAse暴露后，DNA残渣（即双链和单链DNA，多核苷酸，碱基）的保留增强，表明膜的浇铸降低了藻酸盐珠的渗透性。用高分子量（197.1 kDa）聚L-赖氨酸和高浓度（5 mg / ml）共胍膜包被在使用外部钙源配制的磁珠上可获得最高水平的DNA保护，其中80％和90％以上DNAse暴露40分钟后，剩下的DS-DNA仍然保留下来。胍膜能够完全排除分子量为31 kDa的DNAse核酸酶。 DNA珠的冻干和再水化也降低了对核酸酶的渗透性，在核酸酶暴露下，脱乙酰壳多糖包被的60％，聚-L-赖氨酸包被的90％和藻酸盐小珠的95％导致DNA回收。可溶性壳聚糖和聚L赖氨酸很容易被溶菌酶或壳聚糖酶水解为壳聚糖，而胰蛋白酶，胰凝乳蛋白酶或蛋白酶K则被水解为聚L赖氨酸。相反，壳聚糖膜和聚-L-赖氨酸膜对各自的水解酶几乎是惰性的，其中不到2％的膜重量被水解。似乎任何一种膜材料对于体内的[斜体]应用都是稳定的，尤其是在胃肠道运输过程中对DNA的保护方面。致癌物溴化乙锭可接近囊内DNA，与没有DNA的珠子相比，未包被的珠子的摄取量增加了4倍，而共胍盐包被的珠子的摄取量增加了2倍。低分子量截止共胍膜似乎提供了向低分子量致癌物或诱变剂的自由扩散途径。

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