Drug Delivery Team & Tissue Engineering Research Group, School of Pharmacy & Dept of Anatomy, RCSI, Dublin, Ireland; Trinity Centre for Bioengineering, Trinity College Dublin (TCD), Dublin, Ireland; Advanced Materials and Bioengineering Research (AMBER) Centre, RCSI & TCD, Ireland.
Centre for Research in Medical Devices (CURAM), RCSI, Dublin and National University of Ireland, Galway, Ireland; Department of Chemistry, RCSI, Dublin, Ireland.
J Control Release. 2019 Jun 28;304:191-203. doi: 10.1016/j.jconrel.2019.05.009. Epub 2019 May 8.
It is increasingly being recognised within the field of tissue engineering that the regenerative capacity of biomaterial scaffolds can be augmented via the incorporation of gene therapeutics. However, the field still lacks a biocompatible gene delivery vector which is capable of functionalizing scaffolds for tailored nucleic acid delivery. Herein, we describe a versatile, collagen based, gene-activated scaffold platform which can transfect autologous host cells in vivo via incorporation of star-shaped poly(˪-lysine) polypeptides (star-PLLs) and a plasmid DNA (pDNA) cargo. Two star-PLL vectors with varying number and length of poly(˪-lysine) arms were assessed. In vitro, the functionalization of a range of collagen based scaffolds containing either glycosaminoglycans (chondroitin sulfate or hyaluronic acid) or ceramics (hydroxyapatite or nano-hydroxyapatite) with star-PLL-pDNA nanomedicines facilitated prolonged, non-toxic transgene expression by mesenchymal stem cells (MSCs). We demonstrate that the star-PLL structure confers enhanced spatiotemporal control of nanomedicine release from functionalized scaffolds over a 28-day period compared to naked pDNA. Furthermore, we identify a star-PLL composition with 64 poly(˪-lysine) arms and 5 (˪-lysine) subunits per arm as a particularly effective vector, capable of facilitating a 2-fold increase in reporter transgene expression compared to the widely used vector polyethylenimine (PEI), a 44-fold increase compared to a 32 poly(˪-lysine) armed star-PLL and a 130-fold increase compared to its linear analogue, linear poly(˪-lysine) (L-PLL) from a collagen-chondroitin sulfate gene activated scaffold. In an in vivo subcutaneous implant model, star-PLL-pDNA gene activated scaffolds which were implanted cell-free exhibited extensive infiltration of autologous host cells, nanomedicine retention within the implanted construct and successful host cell transfection at the very early time point of just seven days. Overall, this article illustrates for the first time the significant ability of the star-PLL polymeric structure to transfect autologous host cells in vivo from an implanted biomaterial scaffold thereby forming a versatile platform with potential in numerous tissue engineering applications.
组织工程领域越来越认识到，通过基因治疗的方式可以增强生物材料支架的再生能力。然而，该领域仍然缺乏一种具有生物相容性的基因传递载体，使其能够对支架进行功能化，以实现定制化的核酸传递。在此，我们描述了一种通用的、基于胶原蛋白的基因激活支架平台，该平台可通过掺入星形聚（L-赖氨酸）多肽（星形 PLL）和质粒 DNA（pDNA）货物，在体内转染自体宿主细胞。评估了两种具有不同数量和长度聚（L-赖氨酸）臂的星形 PLL 载体。在体外，用星形 PLL-pDNA 纳米药物对包含糖胺聚糖（硫酸软骨素或透明质酸）或陶瓷（羟基磷灰石或纳米羟基磷灰石）的各种胶原蛋白支架进行功能化，促进间充质干细胞（MSCs）的长期、无毒转基因表达。我们证明，与裸 pDNA 相比，星形 PLL 结构可在 28 天内增强纳米药物从功能化支架中的时空控制释放。此外，我们确定了一种具有 64 个聚（L-赖氨酸）臂和每个臂 5 个（L-赖氨酸）亚基的星形 PLL 组成部分作为一种特别有效的载体，能够将报告基因的转基因表达提高 2 倍，与广泛使用的载体聚乙烯亚胺（PEI）相比，提高了 44 倍，与 32 个聚（L-赖氨酸）武装的星形 PLL 相比，提高了 130 倍，与线性类似物线性聚（L-赖氨酸）（L-PLL）相比，从胶原蛋白-硫酸软骨素基因激活支架中提高了 130 倍。在体内皮下植入模型中，无细胞植入的星形 PLL-pDNA 基因激活支架表现出自体宿主细胞的广泛浸润、植入结构内纳米药物的保留以及在仅 7 天的早期时间点成功转染宿主细胞。总的来说，本文首次说明了星形 PLL 聚合物结构在体内从植入生物材料支架转染自体宿主细胞的显著能力，从而形成了一种具有多种组织工程应用潜力的通用平台。
