Efficiency of Cytosolic Delivery with Poly(beta-amino ester) Nanoparticles is Dependent on the Effective pKa of the Polymer

Purpose: The mechanism by which cationic polymers with titratable amines mediate endosomal escape and cytosolic delivery of nucleic acids remains poorly understood. Buffering capacity alone has often failed to predict transfection efficacy, so the study examines whether the effective pKa of poly(beta-amino ester)s (PBAEs) governs cytosolic delivery and transfection.

Hypothesis: PBAE-mediated transfection and cytosolic delivery are highly dependent on the polymer’s effective pKa. Acidic environments that fully protonate PBAEs will inhibit transfection downstream of uptake, whereas neutral-to-basic environments that preserve unprotonated amines and buffering capacity will enhance transfection. PEI, with broad buffering and no clear effective pKa, will be less pH-sensitive.

Aims: Characterize buffering capacity and effective pKa of selected PBAEs (446, 447, 746) versus linear and branched PEI. - Measure pH-dependent DNA binding, nanoparticle size/zeta potential, and vesicle membrane leakage. - Assess transfection and cellular uptake across extracellular pH 5–8 in multiple cell lines. - Determine whether pH sensitivity occurs at uptake or at a downstream intracellular delivery step.

Delivery system: Polymers: PBAEs: - 446: B4-S4-E6 - 447: B4-S4-E7 - 746: B7-S4-E6 - Controls: Linear PEI 4 kDa (LPEI) and branched PEI 25 kDa (BPEI). - Nanoparticle type: Polyplex nanoparticles formed by electrostatic self-assembly with plasmid DNA. - Payload: CMV-eGFP-N1 plasmid for transfection; 20% Cy5-labeled plasmid DNA for uptake tracking. - Formulation: PBAE 446/447 at 60 w/w (N/P 21.0 and 25.6); PBAE 746 at 40 w/w (N/P 10.4); PEI at 2 w/w (6.9 N/P). DNA dose 5 µg/mL in 120 µL/well. - Targeting ligand: None. - Buffers: PBAEs in 25 mM sodium acetate pH 5.0; PEI in 150 mM NaCl. pH-specific DMEM with 20 mM phosphate instead of carbonate for pH modulation.

Approach: In vitro only. Cell lines: HEK293T (human embryonic kidney), B16-F10 (murine melanoma), and GB319 (human glioblastoma). - Cells seeded at 15,000 cells/well in 96-well plates one day prior. - Nanoparticles incubated with cells for 2 h; transfection assessed at 24–48 h by flow cytometry; viability assessed. - pH-dependent experiments: extracellular media adjusted to pH 5, 6, 7, or 8 only during the 2 h nanoparticle incubation; cells then returned to normal carbonate-buffered DMEM. - Controls: untreated cells, LPEI, BPEI. Experiments repeated at least twice; representative results shown; typically four wells per condition.

Key methods: ¹H NMR and GPC for polymer identity and molecular weight. - Acid–base titration for buffering capacity and effective pKa. - Yo-Pro-1 iodide competition assay for pH-dependent DNA binding. - DLS and zeta potential for nanoparticle characterization at different pH values. - POPC vesicle ANTS/DPX leakage assay for membrane disruption potential. - Flow cytometry for eGFP expression and Cy5-DNA uptake. - One-way ANOVA with linear trend testing for pH dependence.

Key results: Transfection efficacy: PBAEs achieved up to 99% in HEK293T, 65% in B16-F10, and 90% in GB319 cells, with high viability. - Effective pKa: PBAE 746 = 6.16, PBAE 446 = 6.95, PBAE 447 = 7.15. PEI showed broad buffering with no clear effective pKa peak. - DNA binding: PBAE 447 binding changed by ~100-fold between pH 4 and pH 8; PEI binding was minimally pH-dependent. - Vesicle leakage: PBAE 447 induced only minor leakage at relevant concentrations; BPEI showed no meaningful leakage. Neither polymer efficiently formed small-molecule-permeable pores in POPC vesicles. - pH dependence of transfection: PEI transfection slightly decreased with increasing pH. PBAE transfection showed the opposite trend: increased significantly at pH 7–8 and was inhibited at pH 5–6. - Uptake: Cellular uptake was minimally affected by extracellular pH for PBAEs (slope near zero) and slightly decreased with pH for PEI. Thus, pH sensitivity occurred after uptake, at an intracellular delivery step. - pKa correlation: PBAE 746 became ineffective below its effective pKa (pH 6 vs pH 7), supporting pKa-dependent transfection.

Interpretation: PBAE-mediated cytosolic delivery and transfection depend strongly on the polymer’s effective pKa and buffering behavior. A neutral-to-basic environment that preserves unprotonated amines is necessary for efficient transfection, whereas acidic conditions below the pKa inhibit delivery after uptake. PEI is less pH-sensitive due to broad buffering. The authors conclude that tuning polymer pKa is critical for rational design of polymeric gene delivery vectors and for understanding endosomal escape.

Limitations: Entirely in vitro; no in vivo validation. - POPC vesicle leakage assay is not a cellular endosomal membrane and may underestimate or misrepresent membrane interactions. - Extracellular pH modulation is a proxy; it does not directly measure endosomal pH or proton sponge effects. - Nanoparticle size and zeta potential changed with pH, which could confound interpretation; authors note future work should uncouple these parameters. - Only selected PBAEs and PEI controls were tested; findings may not generalize to all cationic polymers. - No direct measurement of endosomal escape efficiency or cytosolic DNA bioavailability.