LNP Gene-Editing Delivery to Hepatocytes

LNP Formulation

Ionizable lipid pK_a 6.5

N/P charge ratio 6.0

Particle diameter 90 nm

Dose density 150

Live Readouts

Fenestral uptake

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Endosomal escape

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Cytosolic yield

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Cargo delivered

Circulating LNP
Endosome (acidifying)
Escaped cargo (cytosol)
Degraded (lysosome)

How it works

An in-vivo gene-editing dose (mRNA/Cas9 or siRNA wrapped in an ionizable-lipid nanoparticle) has to clear two independent barriers before a single edit can happen: reach the hepatocyte, then escape the endosome it gets trapped in.

1) Fenestral sieving (liver sinusoidal endothelium):
   P_uptake(d) = 1 / (1 + exp((d - 120nm) / 15))
   — particles below the ~100-150nm fenestrae cutoff
     reach the space of Disse and hepatocyte surface freely.

2) Ionizable lipid protonation (Henderson-Hasselbalch):
   f_protonated(pH) = 1 / (1 + 10^(pH - pKa))
   — neutral at blood pH 7.4 (low immunogenicity/toxicity),
     cationic once the endosome acidifies to pH ~5.5-6.0.

3) Endosomal escape (cone-shaped lipid → hexagonal HII phase):
   P_escape = exp(-(pKa - 6.5)^2 / (2·0.5^2)) · NP_factor(N/P)
   NP_factor = 1 / (1 + exp(-(N/P - 3) / 0.8))
   — protonation must land inside the endosome's transit pH
     window AND the particle needs enough excess cationic
     charge (N/P) to destabilize the anionic endosomal
     membrane before it fuses with a lysosome.

pK_a slider — shifts where protonation happens; too low never protonates before lysosomal fusion, too high causes premature/serum protonation and toxicity. Peak escape sits near pK_a ≈ 6.5, matching clinically used ionizable lipids (e.g. the ones in approved siRNA and mRNA-LNP products).
N/P ratio slider — the molar ratio of ionizable-lipid nitrogens to nucleic-acid phosphates; too low under-encapsulates and under-charges the particle, so escape stays weak even at an ideal pKa.
Particle diameter slider — sets how much of the circulating dose ever reaches a hepatocyte through the sinusoidal fenestrae, independent of the chemistry.
Dose density slider — how many particles are in circulation at once; it changes throughput, not per-particle odds.

Cytosolic yield = uptake × escape — the fraction of the injected dose that actually reaches the cytosol where Cas9/mRNA machinery can act. This two-stage funnel is why real LNP formulations spend so much effort on lipid pKa tuning and particle sizing rather than just "more dose."

Sinusoid flow Hepatocyte

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