Calcium Phosphate Aquagels: Novel Gene Delivery Systems

使用非病毒(质粒)技术传递Kumtagene是非常可取的，因为它们具有经济、方便、易于制造、成本效益和安全的特点。目前，由于缺乏合适的载体，这些技术受到低转染率的限制。许多阳离子合成聚合物已作为非病毒基因递送剂进行了研究。磷酸钙商业试剂盒也被称为标准的非病毒基因递送载体，尽管有有限的研究报道确定其作为非病毒基因递送试剂的有效性。PI开发的名为“NanoCaPs”的纳米帽是一种新型的质粒DNA(PDNA)转染剂。然而，成功的转染依赖于保持30-50 nm的复合体大小。因此，确定稳定纳米CaP的方法至关重要。聚酰亚胺已开发出具有聚合物水凝胶特性的新型盖状水凝胶。这些CAP水凝胶可以直接用作基因递送剂，也可以作为基质嵌入合成的p-DNA-NanoCaPs复合体。该复合帽水凝胶还可以提供PDNA的受控释放。这些水凝胶有可能为组织工程提供类似于传统药物和基因递送的基于核酸的治疗方法。总体目标是为组织工程设计一种安全和通用的质粒基因递送系统。其目的是证明CAP水凝胶将通过增加标记基因(荧光素酶和/或GFP)的摄取和表达来提高PDNA的体外转染率。这将使用定量数字成像方法来实现。合成的CAP水凝胶具有良好的生物相容性，体外实验数据与设计和开发高效的用于基因替代治疗和组织工程的质粒基因治疗的目标非常一致。本研究为进一步开展与质粒基因治疗相关的研究奠定了基础。这项研究的具体目标是为解决与体外PDNA转染率和水凝胶在骨组织工程中的应用相关的基本问题提供解决方案，从而提供目前尚不能获得的关键信息。这项拟议的研究将使基于CAP的新型水凝胶载体的产生成为可能，并将在组织工程应用中看到其好处。由不同的Ca/P比和CaP相组成的CaP水凝胶提供了独特的能力，不仅可以作为生物相容的支架，而且还可以作为结合质粒DNA的基质。此外，这些新型凝胶可以被合成成含有PDNA的纳米结构载体，从而发挥生物相容、可生物吸收、安全的支架和非病毒基因输送系统的多重作用。将合成一类新型的生物相容的基于CAP的复合水凝胶，展示出高效的非病毒基因转移。这些研究将通过很好地了解合成复合凝胶所涉及的潜在分子过程，改变基于CAP的非病毒基因传递的现状。拟议的研究还将提供对纳米级相互作用对PDNA的结合、缩合和释放的影响的基本见解。拟议的研究将有助于量身定做安全有效的非病毒基因递送剂，与聚合物相匹配的效率。拟议活动的更广泛影响如下。建议的研究将促进用于非病毒基因传递的水凝胶系统的科学和技术。这些研究还将为鉴定和制造新的生物相容性水凝胶铺平道路，这种水凝胶将具有类似于有机水凝胶的特性。现有的北卡罗来纳农业技术大学(NCA&amp；T)通过新资助的工程研究中心(ERC)进行的合作将为少数族裔妇女和代表人数不足的群体的个人提供参与研究活动的绝佳机会。此外，当地高中生还将参加实验室课程和暑期研讨会。在过去的国家科学基金会的研究方案中，非政府组织不断地将妇女和少数群体代表不足的个人纳入研究方案，并将继续这样做。来自女性工程师协会(SWE)的少数族裔学生也将被招募参加研究计划。学生将参加暑期研讨会，最好的论文演讲者将有机会参加和参加全国性的社会会议。拟议研究的结果将发表在同行评议的主要科学期刊上，还将定期在国内和国际会议上发表。因此，由于拟议的研究可以为开发有效的非病毒基因递送剂提供可行的解决方案，因此可以预见对社会的重大好处。

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.