Controlled Release of DNA from Surfaces

描述(由申请人提供)：这项提案寻求开发新的材料和方法来设计允许空间和时间控制DNA从表面释放的超薄膜。这项拟议的研究试图验证这样一种假设，即由交替的质粒DNA和可降解的多胺组成的超薄多层膜可以在体外和体内促进对表面介导的细胞转染离子的广泛和可调的控制。我们的方法建立在初步观察的基础上，即由交替的DNA层和可降解的阳离子聚合物制备的超薄膜(例如，100 nm厚)在与细胞和组织接触时促进细胞转染离子。这些关键的观察结果表明了允许在体外对DNA释放进行空间和时间控制的一般方法的基础，以及从体内留置装置表面涂覆和局部传递DNA的简便新方法。我们的初步研究验证了利用超薄多层膜进行表面介导的DNA输送所需的原理。以下具体目标旨在系统地评估我们的假设，并评估这种方法的全部潜力。它们是：1)表征聚合物结构对DNA释放速率和DNA释放的结构和形态的影响；2)研究聚合物结构和膜结构对细胞转染率和体外细胞存活率的影响；3)证明根据AIMS 1和2开发和评价的材料可用于血管内支架等导管置入器的涂层，以及薄膜涂层支架可用于体内血管组织的转染。我们预计，拟议研究的结果将通过以下方式产生重大的基础和应用影响：(I)为基础生物医学研究和体外表面介导的转染法提供新的材料和工具，(Ii)开发强有力的新方法，用于体内留置装置的DNA局部输送，以及(Iii)为未来的研究和材料的选择提供基础，这些材料具有适合评估针对广泛不同治疗结果的治疗基因候选的特性(例如，特定释放谱)。 公共卫生相关性：控制DNA从表面释放的方法的潜在影响范围从基础生物医学研究的应用到最终在临床上实现基于基因的疗法。这项提案中描述的研究将通过提供体外表面介导的DNA传递的新工具和通过体内留置装置局部传递DNA的新方法来影响公众健康，从而提高基于基因的治疗的有效性并减少副作用。

DESCRIPTION (provided by applicant): This proposal seeks to develop new materials and approaches to the design of ultrathin films that permit spatial and temporal control over the release of DNA from surfaces. The proposed research seeks to test the hypothesis that ultrathin multilayered films composed of alternating layers of plasmid DNA and degradable polyamines can be used to promote broad and tunable control over surface-mediated cell transfect ion in vitro and in vivo. Our approach is founded on preliminary observations that ultrathin films (e.g., 100 nm thick) fabricated from alternating layers of DNA and degradable cationic polymers promote cell transfect ion when placed in contact with cells and tissues. These critical observations suggest the basis of general methods that permit spatial and temporal control over the release of DNA in vitro and facile new methods for the coating and localized delivery of DNA from the surfaces of indwelling devices in vivo. Our preliminary investigations validate the principles required to exploit ultrathin multilayered films for the surface-mediated delivery of DNA. The following Specific Aims are designed to systematically evaluate our hypothesis and evaluate the full potential of this approach. They are: 1) To characterize the influence of polymer structure on rates of release of DNA and the structure and morphology of DNA released from ultrathin films fabricated from alternating layers of plasmid DNA and degradable polyamines; 2) To investigate the influence of polymer structure and film architecture on levels of cell transfect ion and viability in vitro when objects coated with ultrathin films are placed in contact with cells; and 3) To demonstrate that materials developed and evaluated under Aims 1 and 2 can be used to coat indwelling devices such as intravascular stents, and that film-coated stents can be used transfect vascular tissue in vivo when implanted in the arteries of rabbits and pigs. We envisage the outcomes of the proposed research as having substantial fundamental and applied impacts by (i) providing new materials and tools for basic biomedical research and surface-mediated transfection in vitro, (ii) by developing robust new methods for the localized delivery of DNA from indwelling devices in vivo, and (iii) by providing a foundation for future research and the selection of materials with properties (e.g., specific release profiles) suited for the evaluation of therapeutic gene candidates directed toward a broad range of different therapeutic outcomes. Public Health Relevance: The potential impact of methods that provide control over the release of DNA from surfaces ranges from applications in basic biomedical research to, ultimately, the realization of gene-based therapies in the clinic. The research described in this proposal will impact public health by providing new tools for surface-mediated DNA delivery in vitro and new methods for localized delivery of DNA from indwelling devices in vivo that could improve the efficacies and reduce the side effects of gene-based therapies.