Purpose: DNA- and mRNA-based therapies are transforming biomedicine, but intracellular delivery—especially to hard-to-transfect, clinically relevant cells such as primary human T lymphocytes—remains a major barrier. Existing non-viral methods often suffer from low transfection efficiency, high toxicity, or complex manufacturing requirements. A safe, efficient, serum-compatible non-viral platform is needed.
Hypothesis: If polyelectrolyte core–shell nanoparticles are assembled using nanosized vaterite calcium carbonate templates and layer-by-layer deposition of poly-L-arginine (PARG) and dextran sulfate (DEXS), then they will efficiently encapsulate and deliver mRNA and plasmid DNA to hard-to-transfect primary human T lymphocytes, achieving higher transfection efficiency than electroporation with minimal cytotoxicity and without specialized equipment or serum-free conditions.
Aims: Develop a scalable synthesis of 50–150 nm vaterite CaCO₃ nanoparticles as sacrificial templates. - Assemble biocompatible polyelectrolyte core–shell nanoparticles via layer-by-layer (LbL) deposition for mRNA and pDNA loading. - Characterize particle size, morphology, crystal phase, zeta potential, and cargo encapsulation. - Evaluate cellular uptake, viability, and transfection efficiency in primary human T lymphocytes compared with electroporation. - Demonstrate transfection in serum-containing medium without special conditions.
Delivery system:
Component: Template; Details: Vaterite calcium carbonate (CaCO₃) nanoparticles, 50–150 nm
Component: Core–shell assembly; Details: Layer-by-layer (LbL) polyelectrolyte deposition
Component: Polyelectrolytes; Details: Poly-L-arginine hydrochloride (PARG, cationic); Dextran sulfate (DEXS, anionic)
Component: Zero layer; Details: BSA-FITC loaded by freezing-induced loading for visualization
Component: Payload; Details: eGFP-encoding mRNA and eGFP plasmid DNA; first layer is mRNA/PARG or pDNA/PARG complex
Component: Particle size; Details: ~50–100 nm core–shell nanoparticles
Component: Targeting ligand; Details: None
Component: Cell model; Details: Primary human T lymphocytes (hard-to-transfect, clinically relevant)
Component: Key feature; Details: Serum-compatible, no electroporation, high mRNA/pDNA transfection
Approach: In vitro only. No in vivo animal studies. - Cell type: Primary human T lymphocytes isolated by magnetic separation. - Cargo: eGFP mRNA or pDNA. - Transfection: Incubated with core–shell nanoparticles in serum-containing basic culture medium; no special supplements or equipment. - Doses: 0.15–25 µg/mL nanoparticles. - Controls: Electroporation; untreated cells. - Assays: Uptake, viability, transfection efficiency, confocal imaging, Z-stack.
Key methods: Particle synthesis/characterization: SEM, TEM, DLS, XRD, Raman spectroscopy, zeta potential. - Cargo loading: Spectrophotometry; encapsulation efficiency. - Cellular uptake and localization: Confocal laser scanning microscopy (CLSM) with Z-stack; flow cytometry. - Viability: AlamarBlue staining with flow cytometry. - Transfection: Flow cytometry for GFP expression; confocal microscopy. - Comparison: Electroporation as standard method.
Key results: Particle size: Core–shell nanoparticles ~50–100 nm; vaterite templates 50–150 nm. - Encapsulation efficiency: ~98% for mRNA; >95% for pDNA. - Uptake: Up to ~99% of primary human T lymphocytes internalized nanoparticles; dose-dependent increase up to ~100% at 10 µg. - Viability: >90% viability; mRNA- and pDNA-loaded nanoparticles exerted minimal cytotoxicity (~94% viability). - Transfection efficiency: mRNA: 90% vs 51% for electroporation; pDNA: 62% vs 39% for electroporation. - Serum compatibility: Transfections performed in serum-containing medium without special conditions or equipment.
Interpretation: The authors claim that biocompatible polyelectrolyte core–shell nanoparticles represent a promising universal non-viral platform for efficient and safe gene delivery to hard-to-transfect, clinically relevant cells. The system provides high mRNA and pDNA transfection, low toxicity, serum stability, and ease of use, with potential for clinical translation in gene and cell therapy, including T-cell-based immunotherapies.
Limitations: In vitro only: No in vivo validation, biodistribution, or therapeutic efficacy. - No targeting ligand: Delivery relies on nonspecific internalization. - Primary T lymphocytes only: Generalizability to other hard-to-transfect cells not fully established. - Transient expression: mRNA and pDNA expression is transient; no stable integration or long-term expression data. - No direct comparison with viral vectors. - No long-term toxicity or safety data. - Accepted Manuscript: Final peer-reviewed version may differ in minor details. - No animal disease model or tumor efficacy data.
