abstract
Therapeutic approaches based on nucleic acids to modulate cell activity have recently gained attention. These molecules arise from complex biotechnological processes, requiring effective manufacturing strategies, high purity, and precise quality control to be used as biopharmaceuticals. One of the most critical and time-consuming steps for nucleic acids-based biotherapeutics manufacturing is their purification, mainly due to the complexity of the extracts. In this study, a simple, efficient, and reliable method to isolate and clarify plasmid DNA (pDNA) from complex samples is described. The method is based on the selective capture of RNA and other impurities, using pristine carbon nanotubes (CNTs). Multi-walled CNTs (MWCNTs) with different diameters were studied to determine their adsorption capacity and to address their ability to interact and distinguish between nucleic acids. The results revealed that MWCNTs preferentially interact with RNA and that smaller MWCNTs present a higher adsorption capacity, as expected by the higher specific surface area. Overall, this study showed that MWCNTs significantly reduce the levels of impurities, namely RNA, gDNA, and proteins, by approximately 83.6 % compared to their initial level, enabling the recovery of clarified pDNA in solution while maintaining its stability throughout the recovery process. This method facilitates the pre-purification of pDNA for therapeutic applications.
keywords
SOLID-PHASE EXTRACTION; GENE-THERAPY; IONIC LIQUID; VACCINES; PURIFICATION; ADSORPTION; FE3O4; RNA
subject category
Engineering
authors
Ferreira, P; Riscado, M; Bernardo, S; Freire, MG; Faria, JL; Tavares, A; Silva, C; Sousa, F
our authors
Ana Paula Mora Tavares
Assistant Researcher
Mara Guadalupe Freire Martins
Coordinating ResearcherProjects
CICECO - Aveiro Institute of Materials (UIDB/50011/2020)
CICECO - Aveiro Institute of Materials (UIDP/50011/2020)
Associated Laboratory CICECO-Aveiro Institute of Materials (LA/P/0006/2020)
acknowledgements
The authors thank Dr. Thomas Roberts for providing the pcDNA3-FLAG-p53 construct through Addgene, ref.: 10838. This work was supported by the project PTDC/BII-BBF/29496/2017, funded by FEDER, through COMPETE2020-POCI and by national funds, through FCT/MCTES. The authors also acknowledge the projects UIDB/00709/2020, UIDP/00709/2020, UIDB/50011/2020 and UIDP/50011/2020, UIDB/50020/2020 and UIDP/50020/2020, and LA/P/0045/2020 (ALiCE) and LA/P/0006/2020 (CICECO) financed by national funds through FCT/MCTES (PIDDAC) . Pedro Ferreira, Micaela Riscado, and A. P.M. Tavares acknowledge FCT for the PhD fellowships (2022.13803.BD), (2021.07658.BD) , and the research contract CEEC-IND/2020/01867, respectively.