Differential Polymer Structure Tunes Mechanism of Cellular Uptake and Transfection Routes of Poly(β-amino ester) Polyplexes in Human Breast Cancer Cells

Purpose: Nonviral gene delivery must overcome cellular uptake, endosomal escape, and nuclear transport, but the cellular uptake mechanism of poly(β-amino ester) (PBAE) polyplexes was unknown. Triple-negative breast cancer (TNBC) also lacks effective targeted therapies, making improved nonviral gene delivery strategies desirable.

Hypothesis: PBAE polyplexes are internalized primarily through caveolae-mediated endocytosis, and small changes in polymer end group or molecular weight can tune the route of cellular uptake and the pathway that leads to successful transfection, even when nanoparticle size and zeta potential remain similar.

Aims: Identify the major endocytic pathway for cellular uptake of PBAE/DNA polyplexes in human TNBC cells. - Determine how polymer end group and molecular weight affect uptake route using three B4S4-based PBAE analogues. - Determine which endocytic routes lead to efficient transfection. - Compare PBAE behavior with PEI polyplexes and validate inhibitor specificity with pathway-specific positive controls.

Delivery system: Polymer: PBAEs based on the same base polymer poly(1,4-butanediol diacrylate-co-4-amino-1-butanol) (B4S4), synthesized at 1.2:1 acrylate:amine molar ratio. - 446L: B4S4 end-capped with E6 [2-(3-aminopropylamino)ethanol], ~13 kDa. - 447L: B4S4 end-capped with E7 [1-(3-aminopropyl)-4-methylpiperazine], ~13 kDa. - 447H: B4S4 end-capped with E7, ~34 kDa. - Nanoparticle: PBAE/DNA polyplexes formed by electrostatic self-assembly. - Payload: eGFP plasmid DNA; Cy3-labeled plasmid DNA for uptake tracking. - Formulation: PBAE at 60 w/w (≈70 N/P); PEI control at 2 w/w (≈15 N/P). - Targeting ligand: None. - Physicochemical properties: ~180–230 nm by NTA; zeta potential +10 to +17 mV.

Approach: In vitro only. Human triple-negative breast cancer MDA-MB-231 cells. - Cells synchronized by serum starvation for 24 h, then seeded at 15,000 cells/well in 96-well plates. - Polyplexes incubated with cells for 4 h in serum-containing medium; uptake analyzed immediately, transfection analyzed 48 h later. - Endocytosis inhibitors: chlorpromazine (42 µM; clathrin-mediated endocytosis, CME), genistein (300 µM; caveolae-mediated endocytosis, CvME), wortmannin (200 nM; macropinocytosis). Single and combination treatments; 1 h preincubation. - Positive controls: transferrin (CME), cholera toxin subunit B (CvME), dextran (macropinocytosis). - n ≥ 4 for flow cytometry and viability groups.

Key methods: DLS and nanoparticle tracking analysis (NTA) for particle size; zeta potential. - Flow cytometry for % Cy3+ cells (uptake), % GFP+ live cells (transfection), and geometric mean fluorescence intensity. - MTS assay for metabolic viability/cytotoxicity. - Endocytosis inhibition profiling with single and combined inhibitors. - GPC and ¹H NMR for polymer molecular weight and structure.

Key results: Uptake without inhibitors: 80–90% for all PBAE formulations. B4S4E6 and B4S4E7 gave 88% and 89% uptake and 46% and 52% transfection, respectively. - CvME was the major uptake pathway: genistein reduced uptake by 61% (446L), 83% (447L), and 91% (447H); PEI uptake decreased 71%. - CME played a minor uptake role: chlorpromazine reduced uptake by 13% (446L), 25% (447L), and 13% (447H); PEI decreased 52%. Wortmannin had no significant effect on uptake. - Transfection route depended on polymer structure: - 446L: CME inhibition reduced transfection 29%, but CvME inhibition reduced transfection only 8% despite a 61% uptake drop. - 447L: CME inhibition reduced transfection 49%, CvME 35%. - 447H: CvME inhibition reduced transfection 88%, CME 40%, macropinocytosis ~18%. - PEI: CvME inhibition reduced transfection 84%, CME 63%. - Combination inhibitors: nearly complete uptake inhibition occurred with all combinations except chlorpromazine + wortmannin, in which CvME remained functional. - Efficiency of pathways: CvME led to ~30% transfection; CME was most efficient for 447L (12%) and lowest for 447H (6%); macropinocytosis ~15% for 446L/447L and ~8% for 447H. - Overall: CvME was the main uptake route but often less efficient for transfection; CME was more efficient per internalized particle. E7 end group and higher molecular weight increased reliance on CvME for both uptake and transfection.

Interpretation: This is the first study to identify the cellular uptake mechanism of PBAE polyplexes. PBAEs can transfect TNBC MDA-MB-231 cells, and small changes to the same base polymer can alter uptake and transfection routes despite constant nanoparticle size and charge. CvME acts mainly as an uptake route that can be less productive for transfection, whereas CME is more efficient; the E7 end group and higher molecular weight favor CvME-mediated transfection. These findings support structure-based optimization of PBAEs for breast cancer gene delivery.

Limitations: Only one cell line (MDA-MB-231) and no in vivo validation. - Pharmacological endocytosis inhibitors may lack absolute specificity and can induce compensatory uptake pathways. - Transfection was tested only with an eGFP plasmid reporter. - Mechanistic conclusions rely on inhibitor studies rather than direct molecular perturbation. - No targeting ligand was used; delivery specificity was not addressed.