Controlled Release of DNA from Surfaces

描述（由申请人提供）：该提案旨在开发新的材料和方法来设计超薄薄膜，允许对DNA从表面释放的空间和时间进行控制。该研究旨在验证由质粒DNA和可降解多胺交替层组成的超薄多层膜在体外和体内可用于促进对表面介导的细胞转染离子的广泛和可调控制的假设。我们的方法建立在初步观察的基础上，即当与细胞和组织接触时，由DNA和可降解阳离子聚合物交替层制成的超薄膜（例如100纳米厚）可促进细胞转染离子。这些重要的观察结果为在体外对DNA释放进行空间和时间控制的一般方法提供了基础，并为在体内从留置装置表面涂覆和局部递送DNA提供了便利的新方法。我们的初步研究验证了利用超薄多层膜进行DNA表面介导递送所需的原理。以下具体目标旨在系统地评估我们的假设，并评估该方法的全部潜力。它们是：1)表征聚合物结构对DNA释放速率的影响，以及由质粒DNA和可降解多胺交替层制成的超薄膜释放DNA的结构和形态；2)研究超薄膜包裹物与细胞接触时，聚合物结构和膜结构对细胞转染离子水平和体外活力的影响；3)证明在Aims 1和Aims 2下开发和评价的材料可用于血管内支架等留置装置的涂覆，薄膜涂层支架植入兔和猪的动脉后可在体内转染血管组织。我们设想，拟议的研究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转染提供新材料和工具；（ii）通过开发强大的新方法，从体内留置装置中定位递送DNA； （iii）为未来的研究和材料的选择提供基础(例如，特定释放谱)适合于治疗性候选基因的评估，针对广泛的不同治疗结果。

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.