Formulation of Synthetic Vectors as Lyophilized Products

人工合成多核苷酸递送系统的发展受到极不稳定的 这些胶体悬浮液作为液体配方。这种不稳定性也会通过迫使 科学家将进行易于分批显著的载体制备实验 可变性。因此，人们对开发允许合成载体被 配制成稳定的脱水制剂，可大批量制备，并发运和 在常温下储存。这里提出的工作集中在开发脱水方法上。 合成载体，使其物理特性和生物活性在这两个过程中保持 急性冻干应激和长时间贮藏。以前对这些问题的研究已经表明， 在急性冻干应激期间，可以通过使用高剂量的糖来实现稳定。 不幸的是，稳定所需的糖分含量与肌肉中的渗透压不相容。 或皮下注射；是许多应用的首选给药方法，例如DNA疫苗。 我们建议进行机制研究来确定载体聚集的因果效应(主要 急性冻干胁迫期间的损伤机制)，并寻求在 等渗渗透压。这些初步研究的结果将被应用于研究两个 增强存储稳定性的方法。在第一种方法中，配方将被直接评估其 在储存过程中减少活性氧积累的能力。这些实验利用了 新的荧光技术，第一次允许在干燥的 需要监测的准备工作。第二种方法与最近的报告和对 固态稳定性领域的传统教条是“越干越好”。归根结底，这些结果 这些方法将结合在对完全优化的配方进行的为期两年的储存稳定性研究中。认识 不同类型的合成载体正在针对基因/多核苷酸传递进行优化，这些 实验使用了不同的模型向量(例如，脂复合体和多维复合体)，以开发合理的 一般适用于大分子络合物稳定的配方指南(例如， 疫苗、病毒、纳米颗粒)在冷冻干燥和储存期间。

The development of synthetic polynucleotide delivery systems is hampered by the extreme instability of these colloidal suspensions as liquid formulations. This instability also affects basic research by forcing scientists to conduct experiments with vector preparationsthat are prone to significant batch-to-batch variability. As a result, there is interest in developing methods that would allow synthetic vectors to be formulated as stable, dehydrated preparations that could be prepared in large batches, and shipped and stored at ambient temperatures. The work proposed here focuses on developing methods of dehydrating synthetic vectors such that their physical characteristics and biological activity are maintained during both acute lyophilization stress and prolonged storage. Previous work on these problems has demonstratedthat stabilization can be achieved during acute lyophilization stress by employing high amounts of sugars. Unfortunately, the amount of sugar needed for stabilization is not osmotically compatible with intramuscular or subcutaneous injection; the preferred method of administration for many applications, e.g., DNAvaccines. We propose mechanistic studies to determine the causative effects of vector aggregation (the major mechanism of damage) during acute lyophilization stress, and pursue strategies that achieve stability at isotonic osmolalities. The findings from these initial studies will be applied to experiments investigating two approaches to enhance storage stability. In the first approach, formulations will be directly assessed for their ability to reduce the accumulation of reactive oxygen species during storage. These experiments utilize a novel fluorescence technique that, for the first time, allows the generation of oxygen radicals in dried preparations to be monitored. The second approach is consistent with recent reports and questions the conventional dogma in the field of solid-state stability that "drier is better". Ultimately, the results from these approaches will be combined in a 2-year storage stability study on fully-optimized formulations. Recognizing that different types of synthetic vectors are being optimized for gene/polynucleotide delivery, these experiments employ different model vectors (e.g., lipoplexes and polyplexes) in order to develop rational formulation guidelines that are generally applicable to the stabilization of macromolecular complexes (e.g., vaccines, viruses, nanoparticles) during freeze-drying and storage.