Gomes Carla P, Varela-Moreira Aida, Leiro Victoria, Lopes Cátia D F, Moreno Pedro M D, Gomez-Lazaro Maria, Pêgo Ana P
INEB - Instituto de Engenharia Biomédica, Universidade do Porto, Rua Alfredo Allen 208, 4200-135 Porto, Portugal; i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal; Faculdade de Engenharia da Universidade do Porto, R. Dr. Roberto Frias, 4200-465 Porto, Portugal.
INEB - Instituto de Engenharia Biomédica, Universidade do Porto, Rua Alfredo Allen 208, 4200-135 Porto, Portugal; i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal; Faculdade de Medicina da Universidade do Porto, Alameda Prof. Hernâni Monteiro, 4200-319 Porto, Portugal.
Acta Biomater. 2016 Dec;46:129-140. doi: 10.1016/j.actbio.2016.09.037. Epub 2016 Sep 26.
By using imaging flow cytometry as a powerful statistical high-throughput technique we investigated the impact of degradation on the biological performance of trimethyl chitosan (TMC)-based nanoparticles (NPs). In order to achieve high transfection efficiencies, a precise balance between NP stability and degradation must occur. We altered the biodegradation rate of the TMC NPs by varying the degree of acetylation (DA) of the polymer (DA ranged from 4 to 21%), giving rise to NPs with different enzymatic degradation profiles. While this parameter did not affect NP size, charge or ability to protect plasmid DNA, NPs based on TMC with an intermediate DA (16%) showed the highest transfection efficiency. Subsequently, by means of a single quantitative technique, we were able to follow, for each tested formulation, major steps of the NP-mediated gene delivery process - NP cell membrane association, internalization and intracellular trafficking, including plasmid DNA transport towards the nucleus. NP cytotoxicity was also possible to determine by quantification of cell apoptosis. Overall, the obtained data revealed that the biodegradation rate of these NPs affects their intracellular trafficking and, consequently, their efficiency to transfect cells. Thus, one can use the polymer DA to modulate the NPs towards attaining different degradation rates and tune their bioactivity according to the desired application. Furthermore, this novel technical approach revealed to be a valuable tool for the initial steps of nucleic acid vector design.
By changing the biodegradation rate of trimethyl chitosan-based nanoparticles (NPs) one was able to alter the NP ability to protect or efficiently release DNA and consequently, to modulate their intracellular dynamics. To address the influence of NP degradation rate in their transfection efficiency we took advantage of imaging flow cytometry, a high-throughput bioimaging technique, to unravel some critical aspects about NP formulation such as the distinction between internalized versus cell-associated/adsorbed NP, and even explore NP intracellular localization. Overall, our work provides novel information about the importance of vector degradation rate for gene delivery into cells, as a way to tune gene expression as a function of the desired application, and advances novel approaches to optimize nanoparticle formulation.
通过使用成像流式细胞术这一强大的统计高通量技术，我们研究了降解对基于三甲基壳聚糖（TMC）的纳米颗粒（NP）生物学性能的影响。为了实现高转染效率，NP稳定性和降解之间必须达到精确平衡。我们通过改变聚合物的乙酰化程度（DA）来改变TMC NPs的生物降解速率（DA范围为4%至21%），从而产生具有不同酶降解特性的NP。虽然该参数不影响NP的大小、电荷或保护质粒DNA的能力，但基于DA为中间值（16%）的TMC的NP显示出最高的转染效率。随后，通过单一的定量技术，我们能够跟踪每种测试制剂中NP介导的基因传递过程的主要步骤——NP与细胞膜的结合、内化和细胞内运输，包括质粒DNA向细胞核的转运。通过定量细胞凋亡也能够确定NP的细胞毒性。总体而言，所获得的数据表明这些NP的生物降解速率影响其细胞内运输，进而影响其转染细胞的效率。因此，人们可以利用聚合物DA来调节NP以获得不同的降解速率，并根据所需应用调整其生物活性。此外，这种新颖的技术方法被证明是核酸载体设计初始步骤的宝贵工具。
通过改变基于三甲基壳聚糖的纳米颗粒（NP）的生物降解速率，能够改变NP保护或有效释放DNA的能力，从而调节其细胞内动力学。为了研究NP降解速率对其转染效率的影响，我们利用成像流式细胞术这一高通量生物成像技术，揭示了有关NP制剂的一些关键方面，如内化的NP与细胞相关/吸附的NP之间的区别，甚至探索NP的细胞内定位。总体而言，我们的工作提供了关于载体降解速率对基因传递到细胞中的重要性的新信息，作为根据所需应用调节基因表达的一种方式，并推进了优化纳米颗粒制剂的新方法。
