Kumta Prashant N, Sfeir Charles, Lee Dong-Hyun, Olton Dana, Choi Daiwon
Department of Materials Science and Engineering, Biomedical Engineering, Carnegie Mellon University, Pittsburgh, PA 15213, USA.
Acta Biomater. 2005 Jan;1(1):65-83. doi: 10.1016/j.actbio.2004.09.008.
Materials play a key role in several biomedical applications, and it is imperative that both the materials and biological aspects are clearly understood for attaining a successful biological outcome. This paper illustrates our approach to implement calcium phosphates as gene delivery agents. Calcium phosphates (CaP) belong to the family of biocompatible apatites and there are several CaP phases, the most ubiquitous being hydroxyapatite (HAp, Ca(10)(PO(4))(6)(OH)(2). Other CaP structures include brushite (B, CaHPO(4).2H(2)O) and tricalcium phosphate (TCP, Ca(3)(PO(4))(2)). Several low and high temperature approaches have been reported for synthesizing HAp and brushite, while TCP is primarily synthesized using high temperature methods. Novel low temperature chemical methods have been developed by us to synthesize nanostructured HAp, brushite and TCP phases. The new low temperature approach results in the formation of stoichiometric and nanosized HAp under physiological conditions. Moreover, the synthesis methods were designed to be biocompatible with biological systems such as cells, DNA and proteins so that the CaP structures can be studied for gene delivery. The use of HAp type CaP phases for gene delivery is well known but to our knowledge, other forms of CaP have not been studied for gene delivery due to the lack of a biocompatible synthesis method. In addition to the biocompatible synthesis of CaP structures, we have also performed ion substitution that would provide us the appropriate tools to study the DNA-to-particle interactions and assess how these ionic substitutions would affect the level of DNA uptake by the cell and then its release to the cell nucleus. Substitution of calcium by 14% magnesium results in the formation of crystalline ( approximately 20 mum) brushite platelets that remains stable at pH 7.5. Further substitution results in unique nanostructured spherical morphologies of brushite from which rosette shaped high specific surface area ( approximately 200 m(2)/g) nanocrystals ( approximately 80 nm) of beta-TCMP phase can be grown. The novelty lies in the formation of stable phases of HAp, brushite and beta-TCMP under physiological conditions making them potential candidates for use as carriers for non-viral gene delivery or more generally in biological systems. The resultant nanocrystalline phosphates have been characterized for their structure, morphology, thermal stability, and composition. Results of the in vitro transfection are also described.
材料在多种生物医学应用中起着关键作用，为了获得成功的生物学结果，必须清楚地了解材料和生物学方面的知识。本文阐述了我们将磷酸钙用作基因传递载体的方法。磷酸钙（CaP）属于生物相容性磷灰石家族，有多种CaP相，最常见的是羟基磷灰石（HAp，Ca(10)(PO(4))(6)(OH)(2)）。其他CaP结构包括透钙磷石（B，CaHPO(4).2H(2)O）和磷酸三钙（TCP，Ca(3)(PO(4))(2)）。已经报道了几种低温和高温方法来合成HAp和透钙磷石，而TCP主要通过高温方法合成。我们开发了新型低温化学方法来合成纳米结构的HAp、透钙磷石和TCP相。这种新的低温方法在生理条件下可形成化学计量比的纳米级HAp。此外，合成方法设计为与细胞、DNA和蛋白质等生物系统具有生物相容性，以便研究CaP结构用于基因传递。使用HAp型CaP相进行基因传递是众所周知的，但据我们所知，由于缺乏生物相容性合成方法，其他形式的CaP尚未用于基因传递研究。除了CaP结构的生物相容性合成外，我们还进行了离子取代，这将为我们提供合适的工具来研究DNA与颗粒的相互作用，并评估这些离子取代如何影响细胞对DNA的摄取水平以及随后向细胞核的释放。用14%的镁取代钙会形成结晶（约20μm）的透钙磷石薄片，在pH 7.5时保持稳定。进一步取代会导致透钙磷石形成独特的纳米结构球形形态，从中可以生长出β-TCMP相的玫瑰花形高比表面积（约200 m(2)/g）纳米晶体（约80 nm）。其新颖之处在于在生理条件下形成了HAp、透钙磷石和β-TCMP的稳定相，使其成为非病毒基因传递载体或更广泛地在生物系统中使用的潜在候选物。所得的纳米晶磷酸盐已对其结构、形态、热稳定性和组成进行了表征。还描述了体外转染的结果。
