Poly(β-Amino Ester)-Nanoparticle Mediated Transfection of Retinal Pigment Epithelial Cells In Vitro and In Vivo.PDF

Purpose: Retinal pigment epithelial (RPE) cells are difficult to transfect, and existing nonviral reagents achieve only low efficiency in vitro. Improved nonviral gene delivery to RPE could benefit both laboratory studies and ocular gene therapy.

Hypothesis: A combinatorial library of end-modified poly(β-amino ester)s (PBAEs) can identify lead polymers for efficient, low-toxicity transfection of ARPE-19 and primary RPE cells. The lead polymer, B5-S5-E7, can mediate RPE transfection in vivo after subretinal injection, including when formulated as concentrated lyophilized nanoparticles.

Aims: Synthesize and screen an expanded PBAE library for transfection efficacy and toxicity in ARPE-19 cells. - Identify lead polymer structures and characterize molecular weight, degradation, nanoparticle size, and zeta potential. - Compare lead PBAE formulations with commercial reagents. - Validate in vivo efficacy by subretinal injection of lyophilized PBAE/DNA nanoparticles in mice.

Delivery system: Polymer class: Poly(β-amino ester)s (PBAEs), synthesized by two-step Michael addition. - Base monomers: diacrylates B3, B3b, B4, B5, B6; amino-alcohol side chains S3, S4, S5. - End-capping amines: E1–E12. - Lead polymer: B5-S5-E7 = 1-(3-aminopropyl)-4-methylpiperazine-end-modified poly(1,5-pentanediol diacrylate-co-5-amino-1-pentanol). - Nanoparticle type: PBAE/pDNA polyplex nanoparticles formed in 25 mM sodium acetate buffer, pH 5.0. - Payload: Plasmid DNA encoding eGFP under a CMV promoter. - Formulation: 30 or 60 w/w polymer:DNA; 600 ng DNA/well in vitro; 10 min self-assembly. - Targeting ligand: None. - Lyophilization: Sucrose added as cryoprotectant, flash-frozen, lyophilized, and resuspended to 1 mg/mL DNA for in vivo injection.

Approach: In vitro: ARPE-19 cells grown to confluence in 96-well plates, transfected in 10% serum with 600 ng pDNA/well, 4 h incubation. n = 4. Controls: Lipofectamine 2000, X-tremeGENE HP DNA, and 25 kDa branched PEI, tested at optimized ratios. - In vivo: 3-month-old C57Bl6 mice; subretinal injection of 1 µL lyophilized pDNA nanoparticles (60 w/w B5-S5-E7:DNA) at 1 mg/mL DNA. n = 5 nanoparticle-treated, n = 3 naked pDNA controls. Analysis 72 h post-injection. - No disease model; eGFP reporter only.

Key methods: Flow cytometry for % GFP+ cells. - MTS assay for metabolic activity/cytotoxicity. - NanoSight and DLS for hydrodynamic diameter; Zetasizer for zeta potential. - GPC for polymer molecular weight and degradation half-life. - NMR for polymer structure verification. - Subretinal injection followed by confocal flat-mount imaging and RT-PCR for GFP mRNA.

Key results: Lead in vitro efficacy: B5-S5-E7 at 60 w/w transfected 44 ± 5% of ARPE-19 cells with 23% cytotoxicity. - Commercial comparison: Lipofectamine 2000: 26 ± 7% transfection, 16% toxicity; X-tremeGENE HP: 22 ± 6%, 32% toxicity; branched PEI: 8 ± 1%, 25% toxicity. - Nanoparticle properties: Hydrodynamic diameter ~180 nm; zeta potential +26 mV; lyophilization retained size and zeta potential. - Polymer degradation: Free polymer half-life 4.6 h in PBS at 37 °C. - In vivo: Subretinal injection of lyophilized GFP-PBAE nanoparticles increased GFP mRNA by >1000-fold in both retina (p < 0.001) and RPE/choroid (p = 0.003) vs. naked pDNA. Fluorescence flat mounts showed increased GFP in 2 of 3 treated eyes, but aggregate difference was not statistically significant (p = 0.08).

Interpretation: PBAE nanoparticles, especially B5-S5-E7, enable efficient RPE transfection in vitro and in vivo, outperforming commercial reagents. Lyophilized nanoparticles can be concentrated for subretinal injection. This platform has potential for ocular gene therapy and laboratory studies of retinal disease.

Limitations: In vivo sample size was small (n = 3–5), and fluorescence quantification was not statistically significant. - Only an eGFP reporter was used; no therapeutic gene or disease model was tested. - Long-term expression, safety, and immune response were not evaluated. - Subretinal injection is invasive and not directly translatable to all clinical scenarios. - Polymer B5-S5-E7 was moderately polydisperse (PDI 3.2). - No active targeting ligand; delivery relies on local subretinal administration. - Human primary RPE in vivo was not tested.