Purpose: Endosomal escape is a key but poorly quantified step for intracellular nucleic acid delivery. Existing methods are low-throughput, indirect, or fail to distinguish endosomal disruption from actual cytosolic cargo delivery. A reliable, sensitive method is needed to rationally optimize polymeric mRNA delivery vehicles.
Hypothesis: If a deactivated Renilla luciferase–derived probe (ddRLuc-Fc) is co-encapsulated with mRNA in the same polymeric vehicle, then its cytosolic activation by NGLY1 can quantitatively measure endosomal escape. Endosomal escape and mRNA encapsulation, rather than cellular uptake, will be the primary determinants of mRNA transfection efficiency across a library of poly(amine-co-ester) (PACE) polymers with different end groups.
Aims: Develop and validate ddRLuc-Fc as a molecular probe for quantifying endosomal escape. - Synthesize a library of 31 PACE polymers with identical backbones but different end groups. - Quantify mRNA encapsulation, cellular uptake, endosomal escape, and transfection efficiency for each polymer. - Correlate each step with mRNA transfection efficiency. - Evaluate in vivo mRNA expression and tissue distribution for top-performing polymers.
Delivery system:
Component: Polymer class; Details: Poly(amine-co-ester) (PACE) terpolymers; biodegradable, cationic
Component: Backbone; Details: Synthesized enzymatically from cationic diamine, lactone (10% ω-pentadecalactone), and diacid monomers
Component: Molecular weight; Details: ~5 kDa
Component: End-group library; Details: 31 different amine-containing small molecules conjugated via CDI chemistry
Component: Payloads; Details: mRNA encoding firefly luciferase (FLuc); ddRLuc-Fc protein probe; Cy5-labeled mRNA for uptake
Component: Nanoparticle type; Details: Polyplex nanoparticles; hydrodynamic diameters 130–330 nm
Component: Targeting ligand; Details: None
Component: Key feature; Details: ddRLuc-Fc co-encapsulated with mRNA to measure endosomal escape in same particle/cell population
Approach: In vitro: Exp293F cells; transfection for 7 h; dual-luciferase assay for FLuc (transfection) and RLuc (endosomal escape). - Encapsulation: Ribogreen assay. - Uptake: FACS with Cy5-labeled mRNA after 1 h. - In vivo: Mice injected intravenously or intraperitoneally with top 10 polymers; luminescence imaging. - Controls: TransIT commercial reagent normalized to 1; untreated controls. - No disease model, no tumor efficacy, no toxicity study. - Replicates: n = 3–4 for most assays.
Key methods: Ribogreen assay: mRNA encapsulation efficiency. - FACS: Cellular uptake of Cy5-mRNA. - Dual-luciferase assay: FLuc activity for transfection; RLuc activity for endosomal escape via ddRLuc-Fc activation. - DLS and TEM: Nanoparticle size and morphology. - Agarose gel electrophoresis: Co-encapsulation confirmation. - In vitro translation (IVT): Assess mRNA accessibility/dissociation. - In vivo luminescence imaging: Tissue distribution after IV and IP administration. - Linear regression: Correlate encapsulation, uptake, adjusted uptake, endosomal escape, adjusted escape with transfection efficiency.
Key results: Endosomal escape vs. transfection: Strong correlation, R² = 0.9334. - Encapsulation vs. transfection: Moderate correlation, R² = 0.5976; most high-transfection polymers encapsulated mRNA at >80%. - Uptake vs. transfection: Moderate correlation, R² = 0.6887; adjusted uptake correlation weak, R² = 0.1534. - Adjusted endosomal escape vs. transfection: Still strong, R² = 0.7603. - In vivo: Top 10 polymers mediated mRNA expression primarily in the spleen after IV injection, independent of end group. After IP injection, expression concentrated in intestinal lymph node–like structures. - Probe validation: ddRLuc-Fc was co-encapsulated with mRNA and activated only after cytosolic NGLY1-mediated deglycosylation, enabling quantitative endosomal escape measurement.
Interpretation: The authors claim that ddRLuc-Fc is an effective tool to quantitatively measure endosomal escape and that endosomal escape and mRNA encapsulation are primary determinants of mRNA transfection efficiency. Cellular uptake alone is not predictive. The PACE polymers transfect mRNA in vitro and in vivo, with spleen and lymph node–like distribution suggesting potential for mRNA vaccine delivery.
Limitations: In vitro model: Exp293F cells only; no primary cells or disease-relevant cell types. - No therapeutic efficacy: Reporter genes only (FLuc, RLuc); no vaccine or therapeutic outcome. - No toxicity or safety data. - One polymer family: PACE library only; generalizability to lipid nanoparticles or other carriers not established. - Probe dependence: ddRLuc-Fc activation requires NGLY1 and Fc-mediated uptake; may not apply universally. - In vivo mechanism: Spleen/lymph node distribution observed but not mechanistically resolved. - No long-term expression or repeated dosing. - No human translation. - End-group effects on in vivo distribution were minimal; material-specific backbone effects dominate.
