Calcium Phosphate Aquagels: Novel Gene Delivery Systems

磷酸钙水凝胶：新型基因传递系统

• 批准号: 0933153
• 负责人: Prashant Kumta
• 金额: $ 30万
• 依托单位: University of Pittsburgh
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-01-01 至 2012-12-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0933153&HistoricalAwards=false
• 关键词: Calcium; Phosphate; Aquagels; Novel; Gene

0933153KumtaGene delivery using non-viral (plasmid) techniques are very desirable due to their economic, convenience, ease of manufacturing, cost-effectiveness, and safe characteristics. The techniques are currently limited by low transfection efficiencies due to lack of a suitable carrier. A number of cationic synthetic polymers have been studied as non-viral gene delivery agents. Calcium phosphate (CaP) commercial kits have also been known as standard non viral gene delivery vectors although there have been limited studies reported to ascertain their efficacy as non viral gene delivery agents. Nano-sized CaPs called "NanoCaPs" developed by the PI are novel delivery agents for plasmid DNA (pDNA) transfection. However, successful transfection is dependent on maintaining the complex size of 30-50 nm. It is hence critical to identify methods to stabilize the NanoCaPs. Novel CaP aquagels have been developed by the PI that display polymeric hydrogel characteristics. These CaP aquagels can be used directly as gene delivery agents or can serve as a matrix for embedding the synthesized p-DNA-NanoCaPs complex. The composite CaP aquagels can also provide controlled release of pDNA. These aquagels have the potential to provide nucleic-acid based therapeutics that closely resembles traditional pharmaceuticals and gene delivery for tissue engineering.The overall objective is to engineer a safe and versatile plasmid gene delivery system for tissue engineering. The goal is to demonstrate that CaP aquagels will enhance the in vitro transfection efficiency of pDNA by increasing the uptake and expression of marker genes (Luciferase and/or GFP). This will be achieved using quantitative digital imaging methods. The synthesized CaP aquagels are biocompatible and the in vitro data is in excellent agreement with the objective to design and develop an efficient plasmid gene therapy for gene replacement therapy and tissue engineering. The present study will provide the foundation for conducting further research related to plasmid gene therapy. The specific objectives of the research have been formulated to provide solutions to fundamental questions related to in vitro pDNA transfection efficiency and the application of the aquagels in bone tissue engineering thus providing key information currently not available. The proposed research will enable the generation of novel CaP based aquagel carriers for plasmid gene delivery, the benefits of which will be seen in tissue engineering applications. The CaP aquagels comprising various Ca/P ratios and CaP phases provide the unique ability to not only serve as biocompatible scaffolds but also matrices for binding plasmid DNA. In addition, these novel gels can be synthesized to contain nano-structured carriers of pDNA thus serving the multiple roles of biocompatible, bioresorbable, safe scaffolds and non-viral gene delivery systems.The intellectual merits of the proposed study are the following. A new class of biocompatible CaP based composite aquagels will be synthesized exhibiting efficient non-viral gene transfection. The studies will transform the current status of CaP based non-viral gene delivery by obtaining a good understanding of the underlying molecular processes involved in synthesizing the composite gels. The proposed studies will also provide fundamental insight into the influence of nanoscale interaction of the binding, condensation and release of pDNA. The proposed studies will help tailor safe and effective non viral gene delivery agents matching efficiencies of polymeric counterparts. The broader impacts of the proposed activity are the following. The proposed research will advance the science and technology of CaP aquagel systems for non viral gene delivery. The studies will also pave the way for the identification and fabrication of new biocompatible CaP based aquagels that will exhibit characteristics similar to organic hydrogels. The existing North Carolina Agriculture and Technical University (NCA&T) collaboration through the newly funded Engineering Research Center (ERC) will offer an excellent opportunity for minority women and individuals from underrepresented groups to participate in the research activity. Moreover, local high school students will engage in hands on laboratory courses and a summer symposia. The PI has continuously integrated women and individuals of underrepresented minority groups into past NSF research programs and will continue to do so. Minority students from the Society for Women Engineers (SWE) will also be activity recruited to participate in the research program. Students will participate in summer symposia and the best paper presenters will be given the opportunity to participate and attend national society meetings. Results of the proposed research study will be published in leading peer reviewed scientific journals and will also be periodically presented at national and international conferences. Thus significant benefits to society can be envisaged since the proposed research could offer a feasible solution to the development of efficient non-viral gene delivery agents.

使用非病毒（质粒）技术的KumtaGene递送由于其经济、方便、易于制造、成本效益和安全特性而非常理想。由于缺乏合适的载体，这些技术目前受到低转染效率的限制。 已经研究了许多阳离子合成聚合物作为非病毒基因递送剂。磷酸钙（CaP）商业试剂盒也被称为标准的非病毒基因递送载体，尽管已经有有限的研究报道来确定它们作为非病毒基因递送剂的功效。由PI开发的称为“NanoCaPs”的纳米尺寸的CaPs是用于质粒DNA（pDNA）转染的新型递送剂。然而，成功的转染依赖于维持30-50 nm的复合物尺寸。因此，确定稳定NanoCaP的方法至关重要。PI已经开发了新型CaP水凝胶，其显示聚合物水凝胶特性。这些CaP水凝胶可以直接用作基因递送剂，或者可以用作嵌入合成的p-DNA-NanoCaPs复合物的基质。复合CaP水凝胶还可以提供pDNA的受控释放。这些水凝胶具有提供与传统药物和组织工程基因递送非常相似的基于核酸的治疗剂的潜力。总体目标是设计一种安全且通用的组织工程质粒基因递送系统。目的是证明CaP水凝胶将通过增加标记基因（荧光素酶和/或GFP）的摄取和表达来增强pDNA的体外转染效率。这将使用定量数字成像方法来实现。所合成的CaP水凝胶具有生物相容性，并且体外数据与设计和开发用于基因替代疗法和组织工程的有效质粒基因疗法的目标非常一致。本研究为进一步开展质粒基因治疗的相关研究奠定了基础。 该研究的具体目标已经制定，以提供解决方案的基本问题有关的体外pDNA转染效率和水凝胶在骨组织工程中的应用，从而提供关键的信息，目前还没有。拟议的研究将能够产生新的CaP为基础的水凝胶载体质粒基因传递，其好处将在组织工程应用中看到。包含各种Ca/P比和CaP相的CaP水凝胶提供了不仅用作生物相容性支架而且用作结合质粒DNA的基质的独特能力。此外，这些新的凝胶可以合成含有纳米结构的pDNA载体，从而服务于生物相容性，生物可吸收性，安全的支架和非病毒基因传递系统的多重角色。将合成一类新的生物相容性的基于CaP的复合水凝胶，其表现出有效的非病毒基因转染。这些研究将通过对合成复合凝胶所涉及的潜在分子过程的良好理解，改变基于CaP的非病毒基因递送的现状。所提出的研究还将提供对pDNA的结合、缩合和释放的纳米级相互作用的影响的基本见解。拟议的研究将有助于定制安全和有效的非病毒基因传递剂匹配聚合物对应物的效率。拟议活动的广泛影响如下。这项研究将推动CaP水凝胶系统用于非病毒基因递送的科学和技术。这些研究还将为鉴定和制造新的生物相容性CaP基水凝胶铺平道路，这些水凝胶将表现出与有机水凝胶相似的特性。现有的北卡罗来纳州农业和技术大学（NCA T）通过新资助的工程研究中心（ERC）的合作将提供一个很好的机会，少数民族妇女和个人从代表性不足的群体参与研究活动。此外，当地高中生将参与实验室课程和夏季研讨会。PI不断将妇女和代表性不足的少数群体的个人纳入过去的NSF研究计划，并将继续这样做。来自女工程师协会（SWE）的少数民族学生也将被招募参加研究计划。学生将参加夏季研讨会，最好的论文演讲者将有机会参加和出席国家学会会议。拟议研究的结果将发表在领先的同行评审科学期刊上，并将定期在国家和国际会议上发表。因此，可以设想对社会的重大利益，因为拟议的研究可以提供一个可行的解决方案，以开发有效的非病毒基因递送剂。