Bishop Corey J, Majewski Rebecca L, Guiriba Toni-Rose M, Wilson David R, Bhise Nupura S, Quiñones-Hinojosa Alfredo, Green Jordan J
Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Translational Tissue Engineering Center, Baltimore, MD 21231, USA.
Department of Materials Science and Engineering, Johns Hopkins University, Baltimore, MD 21231, USA.
Acta Biomater. 2016 Jun;37:120-30. doi: 10.1016/j.actbio.2016.03.036. Epub 2016 Mar 24.
Non-viral, biomaterial-mediated gene delivery has the potential to treat many diseases, but is limited by low efficacy. Elucidating the bottlenecks of plasmid mass transfer can enable an improved understanding of biomaterial structure-function relationships, leading to next-generation rationally designed non-viral gene delivery vectors. As proof of principle, we transfected human primary glioblastoma cells using a poly(beta-amino ester) complexed with eGFP plasmid DNA. The polyplexes transfected 70.6±0.6% of the cells with 101±3% viability. The amount of DNA within the cytoplasm, nuclear envelope, and nuclei was assessed at multiple time points using fluorescent dye conjugated plasmid up to 24h post-transfection using a quantitative multi-well plate-based flow cytometry assay. Conversion to plasmid counts and degradation kinetics were accounted for via quantitative PCR (plasmid degradation rate constants were determined to be 0.62h(-1) and 0.084h(-1) for fast and slow phases respectively). Quantitative cellular uptake, nuclear association, and nuclear uptake rate constants were determined by using a four-compartment first order mass-action model. The rate limiting step for these poly(beta-amino ester)/DNA polyplex nanoparticles was determined to be cellular uptake (7.5×10(-4)h(-1)) and only 0.1% of the added dose was taken up by the human brain cancer cells, whereas 12% of internalized DNA successfully entered the nucleus (the rate of nuclear internalization of nuclear associated plasmid was 1.1h(-1)). We describe an efficient new method for assessing cellular and nuclear uptake rates of non-viral gene delivery nanoparticles using flow cytometry to improve understanding and design of polymeric gene delivery nanoparticles.
In this work, a quantitative high throughput flow cytometry-based assay and computational modeling approach was developed for assessing cellular and nuclear uptake rates of non-viral gene delivery nanoparticles. This method is significant as it can be used to elucidate structure-function relationships of gene delivery nanoparticles and improve their efficiency. This method was applied to a particular type of biodegradable polymer, a poly(beta-amino ester), that transfected human brain cancer cells with high efficacy and without cytotoxicity. A four-compartment first order mass-action kinetics model was found to model the experimental transport data well without requiring external fitting parameters. Quantitative rate constants were identified for the intracellular transport, including DNA degradation rate from polyplexes, cellular uptake rate, and nuclear uptake rate, with cellular uptake identified as the rate-limiting step.
非病毒、生物材料介导的基因传递有治疗多种疾病的潜力，但受限于低效率。阐明质粒传质的瓶颈能够增进对生物材料结构-功能关系的理解，从而设计出下一代合理设计的非病毒基因传递载体。作为原理验证，我们使用与增强绿色荧光蛋白（eGFP）质粒DNA复合的聚（β-氨基酯）转染人原发性胶质母细胞瘤细胞。该多聚体转染了70.6±0.6%的细胞，细胞活力为101±3%。在转染后长达24小时的多个时间点，使用基于定量多孔板的流式细胞术检测法，通过荧光染料偶联质粒评估细胞质、核膜和细胞核内的DNA量。通过定量聚合酶链反应（PCR）计算质粒数量和降解动力学（快速和慢速阶段的质粒降解速率常数分别确定为0.62h⁻¹和0.084h⁻¹）。使用四房室一级质量作用模型确定定量细胞摄取、核结合和核摄取速率常数。这些聚（β-氨基酯）/DNA多聚体纳米颗粒的限速步骤确定为细胞摄取（7.5×10⁻⁴h⁻¹），人脑癌细胞仅摄取了0.1%的添加剂量，而内化DNA的12%成功进入细胞核（核相关质粒的核内化速率为1.1h⁻¹）。我们描述了一种高效的新方法，使用流式细胞术评估非病毒基因传递纳米颗粒的细胞摄取和核摄取速率，以增进对聚合物基因传递纳米颗粒的理解和设计。
在这项工作中，开发了一种基于定量高通量流式细胞术的检测方法和计算建模方法，用于评估非病毒基因传递纳米颗粒的细胞摄取和核摄取速率。该方法具有重要意义，因为它可用于阐明基因传递纳米颗粒的结构-功能关系并提高其效率。该方法应用于一种特定类型的可生物降解聚合物——聚（β-氨基酯），它能高效转染人脑癌细胞且无细胞毒性。发现一个四房室一级质量作用动力学模型能很好地模拟实验转运数据，无需外部拟合参数。确定了细胞内转运的定量速率常数，包括多聚体中DNA的降解速率、细胞摄取速率和核摄取速率，细胞摄取被确定为限速步骤。
