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High-Efficiency l‑PEI-Based Transfection of ARPE-19 Cells Using a Multiparametric Approach and Automated Polyplex

Daniel Keim1, Michaela Dehne2,3, Patricia Miller2

  • 1Process Biotechnology, University of Bayreuth, Universitätstraße 30, Bayreuth 95444, Germany.

Chem & Bio Engineering
|December 31, 2025
PubMed
Summary

Researchers optimized nonviral gene delivery for age-related macular degeneration (AMD) using linear poly-(ethylenimine) (l-PEI). This enhanced transfection efficiency in retinal cells, paving the way for scalable AMD therapies.

Keywords:
3D printinghuman retinal pigmented epithelia cellsl-PEImicrofluidicnonviral gene deliverypolycation

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Area of Science:

  • Biotechnology
  • Ophthalmology
  • Gene Therapy

Background:

  • Nonviral gene delivery is a promising strategy for age-related macular degeneration (AMD).
  • Genetic modification of retinal pigment epithelium (RPE) cells offers therapeutic potential for AMD.
  • Linear poly-(ethylenimine) (l-PEI) is explored as a nonviral vector for gene delivery.

Purpose of the Study:

  • To optimize nonviral gene delivery using linear poly-(ethylenimine) (l-PEI) for human ARPE-19 cells.
  • To enhance transfection efficiency (TE) and cell viability for potential AMD therapies.
  • To develop a semi-automated method for standardized polyplex formation using microfluidics.

Main Methods:

  • Multiparametric optimization of l-PEI/plasmid DNA (pDNA) polyplexes by adjusting N/P ratio, polymer density, volume, and contact time.
  • Development of a semi-automated polyplex formation method utilizing a 3D-printed microfluidic system.
  • Evaluation of transfection efficiency and cell viability under optimized and microfluidic conditions.

Main Results:

  • Optimized transfection protocol achieved 88% transfection efficiency with approximately 85% cell viability.
  • A semi-automated microfluidic system enabled standardized polyplex production.
  • The optimized protocol was successfully adapted to the microfluidic system without compromising TE or viability.

Conclusions:

  • Optimized nonviral gene delivery with l-PEI significantly enhances transfection efficiency and cell viability in RPE cells.
  • A 3D-printed microfluidic system provides a scalable and reproducible platform for l-PEI-based gene delivery.
  • This approach represents a significant advancement toward the clinical application of gene therapy for AMD.