Engineering of episomal plasmid structure to enhance non-viral Poly(beta-amino ester) nanoparticle gene delivery to liver and brain cancer cells
Joanna Yang, Jack Kollings, Ethan Idnani, David R. Wilson, Isabella G. Cozzone, Shanelle Mendes, Mahita Varanasi, Stephany Y. Tzeng , Jordan J. Green
Abstract
The objective of this study was to create and evaluate episomal plasmids for use in non-viral polymeric gene delivery to cancer cells. A comparative analysis was conducted utilizing poly(beta-amino ester)s (PBAEs) as the nanocarrier vector and various hepatocellular carcinoma (HCC) and brain cancer cell lines. A total of fourteen reporter plasmids that varied in promoter (CMV, CAG, EF1α), backbone (Z1, pUNO1, Nanoplasmid [NanoP]), length (~2000 to ~7000 base-pairs), and antibiotic selection marker (Kanamycin, Zeocin, Blasticidin, or none) were constructed, characterized, and evaluated for gene delivery performance.
Introduction
Non-viral gene delivery is a rapidly evolving, modular platform for cellular engineering and translatable therapeutics [1–4]. However, non-viral systems face intracellular barriers, including serum instability, endosomal escape, cytosolic transport, and nuclear entry, which together limit transgene levels and reproducibility across cell lines [5–9].
Materials and method
Plasmid construct cloning
The pUNO1 constructs were generated using the pUNO1 backbone (Invivogen, catalog no. pUNO1-mcs), and clonal genes for GFP, luciferase, and mCherry were purchased from Twist Biosciences (S1 Table). The pUNO1 backbone and clonal genes were double digested with NheI-HF (NEB [New England Biolabs], catalog no. R3131) and BamHI-HF (NEB, catalog no. R3136). Ligation was performed with T4 DNA Ligase (NEB, catalog no. M0202S), and plasmid sequences were verified via Sanger sequencing.
Results
Human hepatocellular carcinoma (Hep3b) cells were transfected with 5-3-6 PBAE nanoparticles [30], while human meningioma cells (IOMM-Lee) were transfected with 4-5-39 PBAE nanoparticles (Fig 1A). Percent transfection by formulation w/w is shown in Fig 1B. GFP geometric mean fluorescence intensity (gMFI) and viability are also reported (Fig 1C-1D). As w/w increases, transfection efficacy and GFP gMFI increase while viability decreases.
Discussion
In this study, we systematically compared a panel of plasmid backbones encoding a fluorescent protein or luciferase. Plasmid designs differed in promoter type, antibiotic resistance genes, and total plasmid size. They were evaluated for transfection efficacy across six HCC cell lines derived from human, mouse, and pig, as well as three brain cancer lines derived from human and mouse. By holding the PBAE delivery platform constant within each cancer model, this study was designed to evaluate how plasmid architecture and cellular context influence reporter gene expression.
Acknowledgments
Fig 2A, 4A, and 5A was created with BioRender.com using drag-and-drop tools and without using AI tools. No AI or similar tools were used for any part of this manuscript.
Citation: Yang J, Kollings J, Idnani E, Wilson DR, Cozzone IG, Mendes S, et al. (2026) Engineering of episomal plasmid structure to enhance non-viral Poly(beta-amino ester) nanoparticle gene delivery to liver and brain cancer cells. PLoS One 21(7): e0352468. https://doi.org/10.1371/journal.pone.0352468
Editor: Chen Ling, Fudan University, CHINA
Received: February 20, 2026; Accepted: June 10, 2026; Published: July 23, 2026
Copyright: © 2026 Yang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript and its Supporting Information files.
Funding: The authors thank the Goldhirsh-Yellin Foundation (JG) and NIH (R01CA228133 (JG), R37CA246699 (ST), P41EB024495 (JG), R01EY031097 (JG), P41EB028239 (JG)) for support of this work. The sponsors did not play any role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: Johns Hopkins filed patents related to the polymer technology discussed in the manuscript with J.J.G and S.Y.T. as co-inventors. J.J.G. is also a board member, CSO, and co-founder of Cove Therapeutics, a manager, CTO, and co-founder of Dome Therapeutics, and a manager and co-founder of OncoSwitch Therapeutics. S.Y.T. is a manager and co-founder of OncoSwitch Therapeutics. Any potential conflicts of interest are managed by the Johns Hopkins University Committee on Outside Interests. There are no competing interests from the other authors.
