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Development of Microparticulate DNA Vaccines for Pulmonary Delivery.

机译：用于肺部递送的微粒DNA疫苗的开发。

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摘要

DNA vaccines have emerged to be an alternative to conventional vaccines due to their increased safety compared with recombinant protein vaccines and live/attenuated vaccines. Pulmonary delivery of vaccines could offer additional advantages, because from an immunological point of view, there is a large presence of macrophages for antigen/DNA uptake and it also part of the mucosal immune system, which is important for effective immune protection. Encapsulating the DNA into a particulate delivery system would be advantageous for protection against degrading nucleases.;Microparticles were produced using poly(DL-lactide-co-glycolide) as the polymer due to its well-known biodegradable and biocompatibility characteristics, and polyvinyl alcohol (PVA) as the main stabiliser. Plasmid DNA encoding for luciferase protein was used as a model plasmid for studies. Water-in-oil-in-water (w1/o/w2) was used as the method for making particles.;Factorial experimental design was used to rapidly optimize a formulation that had the desired properties. Further studies examined the effect of adding various excipients (0.1M NaHCO3, 1% m/m Na2HPO 4) on the loading, diameters, morphology and charge on the microparticles.;Analytical (including TGA, NMR and DSC) and cell culture experiments - using a human alveolar cell line (A549) and mouse macrophage/monocyte cell line (J774A.1) - were carried out to analyse their physical and biological properties.;The aim of this project was to produce microparticles encapsulating plasmid DNA with a high loading efficiency, appropriate size suitable for pulmonary delivery and with the maintenance of plasmid DNA conformation.;The factorial experimental design aided in choosing a platform formulation, with particle diameters of 2-5 mum and loading of 66-72% m/m. Particles without added excipients had low plasmid DNA loading and the DNA had lost most of its original conformation. However, addition of buffers into the aqueous phases, particularly 1% m/v NaH2PO4, reduced the loss of conformation and enhanced loading efficiency.;Physicochemical studies gave mixed results on the formulations tested in terms of thermal stability, DCM and PVA levels and performance as a dry powder. Results demonstrated that microparticles made without buffer were more thermally stable, had lower residual DCM and PVA levels in contrast to those made with 1% m/v Na2HPO4 and 0.1M NaHCO3 . The microparticles did not aerosolise well as a dry powder - even with addition of surfactants such as lecithin and DPPC - with diameters between 69-95 mum being measured, which is in contrast to the diameters measured in a 'wet' state (2-5 mum).;Cell culture studies demonstrated that entrapped DNA (which was extracted out from the particles and delivered to cells using a commercial agent) was still biologically active, but the A549 cell line was much easier to transfect than the J774A.1 cells. However, the microparticles themselves were poor delivery vehicles of DNA in these models.;Overall, these experiments demonstrated the range of factors that need to be considered when trying to create suitable microparticulate carriers for pulmonary delivery of DNA vaccines.
机译：与重组蛋白疫苗和活/减毒疫苗相比，DNA疫苗具有更高的安全性，因此它们已成为传统疫苗的替代品。疫苗的肺部递送可能具有其他优势，因为从免疫学的角度来看，大量存在的巨噬细胞可用于抗原/ DNA的摄取，并且它也是粘膜免疫系统的一部分，这对于有效的免疫保护非常重要。将DNA封装到微粒输送系统中将有利于防止核酸酶降解。;由于聚（DL-丙交酯-乙交酯）作为聚合物，由于其众所周知的可生物降解和生物相容性特征以及聚乙烯醇（ PVA）作为主要的稳定剂。编码荧光素酶蛋白的质粒DNA被用作研究的模型质粒。水包油包水（w1 / o / w2）被用作制备颗粒的方法。;通过实验实验设计来快速优化具有所需性能的配方。进一步的研究检查了添加各种赋形剂（0.1M NaHCO3、1％m / m Na2HPO 4）对微粒的负载，直径，形态和电荷的影响。分析（包括TGA，NMR和DSC）和细胞培养实验-用人肺泡细胞系（A549）和小鼠巨噬细胞/单核细胞系（J774A.1）进行了分析，以分析它们的物理和生物学特性。该项目的目的是生产高负载质粒DNA的微粒高效，合适的大小适合肺部输送并维持质粒DNA构象。;析因实验设计有助于选择平台制剂，粒径为2-5 mum，负载为66-72％m / m。没有添加赋形剂的颗粒的质粒DNA载量低，DNA失去了大部分原始构象。但是，向水相中添加缓冲剂，尤其是1％m / v NaH2PO4，可以减少构象损失并提高装填效率。物理化学研究从热稳定性，DCM和PVA含量以及性能方面对测试的配方给出了混合结果作为干粉。结果表明，与使用1％m / v Na2HPO4和0.1M NaHCO3制成的微粒相比，无缓冲剂制成的微粒具有更高的热稳定性，较低的残留DCM和PVA含量。即使添加了表面活性剂（如卵磷脂和DPPC），微粒也无法像干粉一样很好地雾化，其直径在69-95微米之间，这与“湿”状态下的直径相反（2-5）细胞培养研究表明，被包裹的DNA（从颗粒中提取出来并使用商业试剂传递到细胞中）仍然具有生物活性，但是A549细胞系比J774A.1细胞更容易转染。但是，在这些模型中，微粒本身不是DNA的传递载体。总体而言，这些实验证明了尝试创建合适的微粒载体进行肺部传递DNA疫苗时需要考虑的因素范围。

著录项

  • 作者

    Tse, Man Tsuey.;

  • 作者单位

    University of London, University College London (United Kingdom).;

  • 授予单位 University of London, University College London (United Kingdom).;
  • 学科 Pharmaceutical sciences.
  • 学位 Ph.D.
  • 年度 2007
  • 页码 219 p.
  • 总页数 219
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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