Quinine copolymer reporters promote efficient intracellular DNA delivery and illuminate a protein-induced unpackaging mechanism

Abstract

Polymeric vehicles that efficiently package and controllably release nucleic acids enable the development of safer and more efficacious strategies in genetic and polynucleotide therapies. Developing delivery platforms that endogenously monitor the molecular interactions, which facilitate binding and release of nucleic acids in cells, would aid in the rational design of more effective vectors for clinical applications. Here, we report the facile synthesis of a copolymer containing quinine and 2-hydroxyethyl acrylate that effectively compacts plasmid DNA (pDNA) through electrostatic binding and intercalation. This polymer system poly(quinine-co-HEA) packages pDNA and shows exceptional cellular internalization, transgene expression, and low cytotoxicity compared to commercial controls for several human cell lines, including HeLa, HEK 293T, K562, and keratinocytes (N/TERTs). Using quinine as an endogenous reporter for pDNA intercalation, Raman imaging revealed that proteins inside cells facilitate the unpackaging of polymer–DNA complexes (polyplexes) and the release of their cargo. Our work showcases the ability of this quinine copolymer reporter to not only facilitate effective gene delivery but also enable diagnostic monitoring of polymer–pDNA binding interactions on the molecular scale via Raman imaging. The use of Raman chemical imaging in the field of gene delivery yields unprecedented insight into the unpackaging behavior of polyplexes in cells and provides a methodology to assess and design more efficient delivery vehicles for gene-based therapies.

Polymeric Delivery of Therapeutic Nucleic Acids

“…(4) Although biodegradable polymers are considered most favorably in the light of regulatory approval, the long-term safety profile of these vehicles must be evaluated, and the immune responses to degradation products must be examined in detail. (5) Synthetic chemists must develop monomers that possess theranostic capabilities, by coupling delivery functionalities with imaging capabilities (such as Raman imaging, 1504 magnetic resonance imaging (MRI), or aggregation-induced emission (AIE)). Theranostic polyplexes will combine delivery efficiency with a detailed mechanistic view of intracellular events that are often challenging to monitor via traditional microscopy.…”

Polycation Architecture Affects Complexation and Delivery of Short Antisense Oligonucleotides: Micelleplexes Outperform Polyplexes

“…The physical properties of micelleplexes complexed with pDNA, − short interfering RNA (siRNA), − messenger RNA (mRNA), − and even CRISPR/Cas9 ,, have been studied in detail, and the complex relationships between physical properties and gene delivery have been elucidated. Yet, it is not well understood how short (<30 base pairs) single-stranded nucleic acids assemble with different polymer architectures and how the physical behavior and colloidal stability are affected by polymer composition, architecture, and formulation parameters .…”

Materiomically Designed Polymeric Vehicles for Nucleic Acids: Quo Vadis?

“…Surface-enhanced Raman scattering is ideal to interrogate cellular responses to polyplexes, particularly when applied to probe the extracellular environment and to analyze metabolite chemical gradients . Applied in tandem with theranostic tags and data processing approaches, such as principal component analysis, it is possible to identify promising polycationic vehicles and simultaneously map intracellular unpackaging mechanisms. , Raman chemical imaging combines molecular spectroscopy with high-resolution spatial information to quantitatively represent the intracellular distribution of Raman reporters . With the advent of stimulated Raman scattering (SRS), both the acquisition speed and the spatial resolution of chemical maps of intracellular polymer and nucleic acid distribution will improve greatly .…”