Abu-Awwad Hosam Al-Deen M, Thiagarajan Lalitha, Dixon James E
Wolfson Centre for Stem Cells, Tissue Engineering, and Modelling (STEM), Centre of Biomolecular Sciences, School of Pharmacy, University of Nottingham, Nottingham NG7 2RD, UK.
Wolfson Centre for Stem Cells, Tissue Engineering, and Modelling (STEM), Centre of Biomolecular Sciences, School of Pharmacy, University of Nottingham, Nottingham NG7 2RD, UK.
Acta Biomater. 2017 Jul 15;57:225-237. doi: 10.1016/j.actbio.2017.04.028. Epub 2017 Apr 27.
Controlled release systems for therapeutic molecules are vital to allow the sustained local delivery of their activities which direct cell behaviour and enable novel regenerative strategies. Direct programming of cells using exogenously delivered transcription factors can by-pass growth factor signalling but there is still a requirement to deliver such activity spatio-temporally. We previously developed a technology termed GAG-binding enhanced transduction (GET) to efficiently deliver a variety of cargoes intracellularly, using GAG-binding domains which promote cell targeting, and cell penetrating peptides (CPPs) which allow cell entry. Herein we demonstrate that GET system can be used in controlled release systems to mediate sustained intracellular transduction over one week. We assessed the stability and activity of GET peptides in poly(dl-lactic acid-co-glycolic acid) (PLGA) microparticles (MPs) prepared using a S/O/W double emulsion method. Efficient encapsulation (∼65%) and tailored protein release profiles could be achieved, however intracellular transduction was significantly inhibited post-release. To retain GET peptide activity we optimized a strategy of co-encapsulation of l-Histidine, which may form a complex with the PLGA degradation products under acidic conditions. Simulations of the polymer microclimate showed that hydrolytic acidic PLGA degradation products directly inhibited GET peptide transduction activity, and use of l-Histidine significantly enhanced released protein delivery. The ability to control the intracellular transduction of functional proteins into cells will facilitate new localized delivery methods and allow approaches to direct cellular behaviour for many regenerative medicine applications.
The goal for regenerative medicine is to restore functional biological tissue by controlling and augmenting cellular behaviour. Either Transcription (TFs) or growth factors (GFs) can be presented to cells in spatio-temporal gradients for programming cell fate and gene expression. Here, we have created a sustained and controlled release system for GET (Glycosaminoglycan-enhanced transducing)-tagged proteins using S/O/W PLGA microparticle fabrication. We demonstrated that PLGA and its acidic degradants inhibit GET-mediated transduction, which can be overcome by using pH-activated l-Histidine. l-Histidine inhibits the electrostatic interaction of GET/PLGA and allows enhanced intracellular transduction. GET could provide a powerful tool to program cell behaviour either in gradients or with sustained delivery. We believe that our controlled release systems will allow application of GET for tissue regeneration directly by TF cellular programming.
治疗性分子的控释系统对于实现其活性的持续局部递送至关重要，这些活性可指导细胞行为并促成新的再生策略。使用外源性递送的转录因子对细胞进行直接编程可以绕过生长因子信号传导，但仍然需要在时空上递送这种活性。我们之前开发了一种称为糖胺聚糖结合增强转导（GET）的技术，利用促进细胞靶向的糖胺聚糖结合结构域和允许细胞进入的细胞穿透肽（CPP），有效地将多种货物递送至细胞内。在此，我们证明GET系统可用于控释系统，以介导长达一周的持续细胞内转导。我们评估了使用S/O/W双乳液法制备的聚（dl-乳酸-共-乙醇酸）（PLGA）微粒（MPs）中GET肽的稳定性和活性。可以实现高效包封（约65%）和定制的蛋白质释放曲线，然而释放后细胞内转导受到显著抑制。为了保留GET肽的活性，我们优化了一种共包封l-组氨酸的策略，l-组氨酸在酸性条件下可能与PLGA降解产物形成复合物。聚合物微环境的模拟表明，水解产生的酸性PLGA降解产物直接抑制GET肽的转导活性，而使用l-组氨酸可显著增强释放蛋白的递送。控制功能性蛋白质向细胞内转导的能力将促进新的局部递送方法，并为许多再生医学应用提供指导细胞行为的方法。
再生医学的目标是通过控制和增强细胞行为来恢复功能性生物组织。转录因子（TFs）或生长因子（GFs）都可以以时空梯度呈现给细胞，以编程细胞命运和基因表达。在此，我们使用S/O/W PLGA微粒制造技术创建了一种用于GET（糖胺聚糖增强转导）标记蛋白的持续控释系统。我们证明PLGA及其酸性降解产物会抑制GET介导的转导，这可以通过使用pH激活的l-组氨酸来克服。l-组氨酸抑制GET/PLGA的静电相互作用并增强细胞内转导。GET可以提供一个强大的工具，以梯度或持续递送的方式对细胞行为进行编程。我们相信我们的控释系统将允许通过TF细胞编程直接将GET应用于组织再生。
