I think the most overseen part, why this hypothesis is plausible.

The key lies in the physical forces of imbalance (a mutually reinforcing entropy): hydrophobic versus hydrophilic versus amphiphilic. Electrostatic forces drive neutral (but electron “smeared”) charged PEG versus negative charged DNA versus negative charged RNA. Van der Waals forces: repulsion (Pauli-Repulsion)* versus attraction + permanent dielectric proton exchange (see also ******)

The ionizable lipid is amphiphilic. It has a charged “head” (which attracts the DNA) and long hydrophobic “tails.” While the cholesterol and the lipid tails retreat from the aqueous phase into the core of the LNP (hydrophobic collapse), they pull the DNA along with them as if on a leash. The DNA is thereby physically squeezed into the interfaces between the aqueous pockets (where the RNA/DNA resides) and the massively dense, hydrophobic cholesterol/lipid domains.

Interestingly, Park et al.**demonstrated that the crystallization of cholesterol within phospholipid membranes does not proceed directly to the most thermodynamically stable state but instead adheres to Ostwald’s Rule of Stages***. This principle dictates that a system transitioning toward equilibrium will first occupy the metastable state closest in free energy to its initial state, evolving through a cascade of intermediate polymorphs before reaching a final crystalline form. Therefore, it seems reasonable to assume that cholesterol crystallization events occur, given the various phase transitions and the associated restructuring momentum of LNPs.

In short:

• Kinetically Trapped, not Thermodynamically Dead: The system possesses enough internal energy to undergo localized phase transitions.

• Predictable Evolution: These events follow a path of least resistance (Ostwald’s Rule), meaning they occur in stages rather than as a single, global collapse.

• Localized Impact: Because these transitions are spatially confined within the viscous, non-aqueous core of the LNP, they represent a internal reordering of the "frozen" state rather than a violation of its macroscopic stability.

Why this confirms the Liu/Ohmann observations:

Liu et al. and Ohmann et al. (****) forced cholesterol and DNA together via covalent bonds (tags) to see how the DNA disrupts the cholesterol lattice. In our LNP scenario, nanoprecipitation replaces this covalent bond with pure biophysical constraint: the DNA is irrevocably held in place by the amphiphilic ionizable lipid. (As the system reaches a kinetic minimum through hydrophobic-hydrophilic balancing, the DNA is forced into an 'optimal' kinetic position where it exerts the same structural disruption.) Given this as a plausible physicochemical assumption, it may follow that DNA within an LNP exerts the same disruptive pressure on cholesterol organization as observed in covalent models. The absence of a chemical bond seems to be irrelevant in this scenario; the biophysical constraint of the kinetically arrested state ensures that the DNA and lipid phases are inextricably linked, reinforcing the validity of the Liu/Ohmann observations within the real-world LNP scenario. Therefore, the argument regarding the steric void-filling effect–which is valid in bulk models–appears to be negligible and irrelevant in this context.

The hydrophobic collapse compresses the entire complex into a very small space (high packing density). To achieve the hydrophobic optimum, the lipid molecules and cholesterol must arrange themselves extremely densely around the nucleic acids. Nanoprecipitation is a dirty and fairly well-kept secret that is mostly found in exorbitantly expensive ACS articles.

Furthermore, the assumption of x molecules per LNP is not an experimental observation but a statistical average derived from bulk mass calculations. LNP populations are inherently heterogeneous, as demonstrated by the presence of at least three structurally distinct subpopulations with size differences of up to 5000 nm***** observed post-thaw in both BNT162b2 and mRNA-1273 preparations. This renders any Poisson-based loading assumption invalid: distinct subpopulations differ not only in particle number but in internal architecture, packing density, and likely lipid/RNA/DNA composition. The mean value therefore has little mechanistic relevance, given that nanoprecipitation is a stochastic event.

Interestingly, this could also be closely related to stochastic fluctuations in endosome escape rates–a phenomenon that warrants further investigation.

To shortcut a summary: All the basic assumptions of a stoichiometric interaction framework fail because, in this case, the framework is not defined by thermodynamic equilibrium but by a frozen kinetic minimum. In other terms position, geometry, and interfacial forces govern structural outcome, not molecular mass ratios.

(Incidentally, this hypothesis should be understood for what it is: a complementary perspective, not an attempt to rule out the bioactivity of DNA contaminants in general.)

*Marzuoli I, Margreitter C, Fraternali F. Lipid Head Group Parameterization for GROMOS 54A8: A Consistent Approach with Protein Force Field Description. J Chem Theory Comput. 2019;15(10):5175-5193. doi:10.1021/acs.jctc.9b00509

*Müller P, Bonthuis DJ, Miller R, Schneck E. Ionic Surfactants at Air/Water and Oil/Water Interfaces: A Comparison Based on Molecular Dynamics Simulations. J Phys Chem B. 2021;125(1):406-415. doi:10.1021/acs.jpcb.0c08615

**Park S, Sut TN, Ma GJ, Parikh AN, Cho NJ. Crystallization of Cholesterol in Phospholipid Membranes Follows Ostwald's Rule of Stages. J Am Chem Soc. 2020;142(52):21872-21882. doi:10.1021/jacs.0c10674

***Donnelly ME , Teeratchanan P , Bull CL , Hermann A , Loveday JS . Ostwald's rule of stages and metastable transitions in the hydrogen-water system at high pressure. Phys Chem Chem Phys. 2018;20(42):26853-26858. doi:10.1039/c8cp04464c

***Nag K, Sarker MEH, Kumar S, et al. DoE-derived continuous and robust process for manufacturing of pharmaceutical-grade wide-range LNPs for RNA-vaccine/drug delivery. Sci Rep. 2022;12(1):9394. Published 2022 Jun 7. doi:10.1038/s41598-022-12100-z

****Liu J, Chen L, Zhai T, Li W, Liu Y, Gu H. Programmable Assembly of Amphiphilic DNA through Controlled Cholesterol Stacking. J Am Chem Soc. 2022;144(36):16598-16603. doi:10.1021/jacs.2c06610

****Ohmann A, Göpfrich K, Joshi H, Thompson RF, Sobota D, Ranson NA, Aksimentiev A, Keyser UF. Controlling aggregation of cholesterol-modified DNA nanostructures. Nucleic Acids Res. 2019 Dec 2;47(21):11441-11451. doi: 10.1093/nar/gkz914. PMID: 31642494; PMCID: PMC6868430.

*****Hermosilla J, Alonso-García A, Salmerón-García A, Cabeza-Barrera J, Medina-Castillo AL, Pérez-Robles R, Navas N. Analysing the In-Use Stability of mRNA-LNP COVID-19 Vaccines Comirnaty™ (Pfizer) and Spikevax™ (Moderna): A Comparative Study of the Particulate. Vaccines (Basel). 2023 Oct 25;11(11):1635. doi: 10.3390/vaccines11111635. PMID: 38005967; PMCID: PMC10675537.

******I wonder if Grotthuss proton shuttling will play also a role in vivo?:

Song B, Yuan H, Pham SV, Jameson CJ, Murad S. Nanoparticle permeation induces water penetration, ion transport, and lipid flip-flop. Langmuir. 2012;28(49):16989-17000. doi:10.1021/la302879r

Wolf MG, Grubmüller H, Groenhof G. Anomalous surface diffusion of protons on lipid membranes. Biophys J. 2014 Jul 1;107(1):76-87. doi: 10.1016/j.bpj.2014.04.062. PMID: 24988343; PMCID: PMC4119267.

Would be really interesting what DANIEL SANTIAGO RPh, PharmD thinks.

I hope Anandamide (Kevin McKernan) will find the time and inspiration to discuss this a bit more in detail with The Offsc℞ipt Pharmacist, Daniel Santiago and me. It’s about softmatter physics.