Institute for Soldier Nanotechnologies, Massachusetts Institute of Technology, Cambridge, MA 02139, United States; Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, United States; Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, United States.
Institute for Soldier Nanotechnologies, Massachusetts Institute of Technology, Cambridge, MA 02139, United States; Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, United States; Harvard-MIT Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA 02139, United States.
Acta Biomater. 2021 Nov;135:331-341. doi: 10.1016/j.actbio.2021.08.042. Epub 2021 Sep 1.
The ability to coat scaffolds and wound dressings with therapeutic short interfering RNA (siRNA) holds much potential for applications in wound healing, cancer treatment, and regenerative medicine. Layer-by-layer (LbL) technology is an effective method to formulate polyelectrolyte thin films for local delivery of siRNA; however, the formation and efficacy of LbL coatings as drug delivery systems are highly contingent on the assembly conditions. Here, we investigate the effects of LbL assembly parameters on film composition and consequent siRNA-mediated gene knockdown efficiency in vitro. Films comprising poly(β-amino ester) (PBAE) and siRNA were built on polyglactin 910 (Vicryl) sutures consisting of poly(10% L-lactide, 90% glycolide). A fractional factorial design was employed, varying the following LbL assembly conditions: pH, ionic strength, PBAE concentration, and siRNA concentration. Effects of these parameters on PBAE loading, siRNA loading, their respective weight ratios, and in vitro siRNA-mediated knockdown were elucidated. The parameter effects were leveraged to create a rationally designed set of solution conditions that was predicted to give effective siRNA-mediated knockdown, but not included in any of the original experimental conditions. This level of knockdown with our rationally designed loading conditions (47%) is comparable to previous formulations from our lab while being simpler in construction and requiring fewer film layers, which could save time and cost in manufacturing. This study highlights the importance of LbL solution conditions in the preparation of surface-mediated siRNA delivery systems and presents an adaptable methodology for extending these electrostatically-assembled coatings to the delivery of other therapeutic nucleic acids. STATEMENT OF SIGNIFICANCE: Short interfering RNA (siRNA) therapeutics are powerful tools to silence aberrant gene expression in the diseased state; however, the clinical utility of these therapies relies on effective controlled delivery approaches. Electrostatic self-assembly through the layer-by-layer (LbL) process enables direct siRNA release from surfaces, but this method is highly dependent upon the specific solution conditions used. Here, we use a fractional factorial design to illustrate how these assembly conditions impact composition of siRNA-eluting LbL thin films. We then elucidate how these properties mediate in vitro transfection efficacy. Ultimately, this work presents a significant step towards understanding how optimization of assembly conditions for surface-mediated LbL delivery can promote transfection efficacy while reducing the processing and material required.
用治疗性短干扰 RNA（siRNA）涂覆支架和伤口敷料的能力在伤口愈合、癌症治疗和再生医学中有很大的应用潜力。层层（LbL）技术是一种用于局部递送 siRNA 的有效方法，用于制定聚电解质薄膜；然而，LbL 涂层作为药物递送系统的形成和功效在很大程度上取决于组装条件。在这里，我们研究了 LbL 组装参数对体外薄膜组成和随后的 siRNA 介导的基因敲低效率的影响。由聚（β-氨基酯）（PBAE）和 siRNA 组成的薄膜构建在由聚（10% L-丙交酯，90%乙交酯）组成的聚甘醇酸 910（Vicryl）缝线。采用分数阶因子设计，改变以下 LbL 组装条件：pH 值、离子强度、PBAE 浓度和 siRNA 浓度。阐明了这些参数对 PBAE 负载、siRNA 负载、它们各自的重量比以及体外 siRNA 介导的敲低的影响。利用这些参数的影响创建了一组合理设计的溶液条件，这些条件被预测可以提供有效的 siRNA 介导的敲低，但不在任何原始实验条件中。用我们合理设计的加载条件（47%）获得的这种水平的敲低与我们实验室之前的制剂相当，而构建更简单，所需的薄膜层数更少，这可以节省制造过程中的时间和成本。本研究强调了 LbL 溶液条件在制备表面介导的 siRNA 递送系统中的重要性，并提出了一种可扩展的方法，将这些静电组装的涂层扩展到其他治疗性核酸的递送。意义声明：短干扰 RNA（siRNA）疗法是一种强大的工具，可以在疾病状态下沉默异常基因表达；然而，这些疗法的临床实用性依赖于有效的控制递药方法。通过层层（LbL）过程的静电自组装可以实现从表面直接释放 siRNA，但这种方法高度依赖于使用的特定溶液条件。在这里，我们使用分数阶因子设计来说明这些组装条件如何影响洗脱 siRNA 的 LbL 薄膜的组成。然后阐明这些特性如何介导体外转染效率。最终，这项工作朝着理解如何通过优化表面介导的 LbL 递药的组装条件来提高转染效率同时减少加工和材料要求迈出了重要一步。
