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The pKa values and associated protonation states of ionizable lipids in lipid nanoparticle (LNP) formulations are strongly dependent on their chemical environment. This phenomenon leads to poorly understood structure-function relationships that influence payload delivery, tissue selective biodistribution, and manufacturing. For example, the charge- and biodistribution of an mRNA-loaded LNP can vary based on the type of ionizable lipid used, the molar ratio of its components, and its car-go. Yet, the spatial resolution of experimental protonation-state measurements is currently limited to the apparent charge of an ionizable lipid averaged over all environments/conformations of the LNP — best represented by the apparent pKa value. Such measurements are too coarse to capture the heterogenous charge distributions of ionizable lipids in LNPs, which influ-ence biocorona formation and interactions with the payload. Similar limitations are inherent to classical fixed-protonation-state in silico models that cannot capture the environment-dependent protonation states determining local pKa. To address this gap in experimental and computational tools available to accurately determine the local charge distributions in LNPs, this work now incorporates a scalable continuous constant pH molecular dynamics (CpHMD) model to simulate the self-assembly processes of five reported distinct LNP formulations. Parameters for ionizable lipids were generated from fitting fixed -state calculations performed with Hamiltonian replica exchange (HREX) to improve conformational sampling during parameterization. Simulated systems were composed of 100 ionizable lipids (50 mol%), cholesterol (40 mol%), dis-tearoylphosphatidylcholine (10 mol%), and mRNA (20 nucleotides) to model the interior of an LNP. Self-assembly was simulated for 100 ns at different pH values and integrated with bilayer models for neutral/basic pH conditions to validate the apparent pKa for each system. For our most accurate mixed self-assembly/bilayer model, the theoretically calculated apparent pKa values matched well with the experimental values (mean absolute error (MAE) = 0.32 pKa units (R2 = 0.52), and all systems exhibited pH-dependent structures. Overall, this work provides a new computational platform technology to (i) pre-dict the pKa values of ionizable lipids in different chemical environments and (ii) enable a structure-based way to model the heterogeneous, environment-dependent charge distributions of ionizable lipids in LNP systems typically encountered during LNP manufacturing and delivery.
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CpHMD Input Files and Supplementary Movies Showing Self-assembly and Bilayer CpHMD Trajectories of Different LNP Formulations at Varying Solution pH
Abstract: General note: a/b/c represent replicas 1, 2, and 3, respectively Movie S1a/b/c. Self-assembly of the DLin-KC2-DMA LNP formulation for 100 ns at pH = 3. Movie S2a/b/c. Self-assembly of the DLin-KC2-DMA LNP formulation for 100 ns at pH = 4. Movie S3a/b/c. Self-assembly of the DLin-KC2-DMA LNP formulation for 100 ns at pH = 5. Movie S4a/b/c. Self-assembly of the DLin-KC2-DMA LNP formulation for 100 ns at pH = 6. Movie S5a/b/c. Self-assembly of the DLin-KC2-DMA LNP formulation for 100 ns at pH = 7. Movie S6a/b/c. Self-assembly of the DLin-KC2-DMA LNP formulation for 100 ns at pH = 8. Movie S7a/b/c. Self-assembly of the DLin-KC2-DMA LNP formulation for 100 ns at pH = 9. Movie S8a/b/c. Self-assembly of the DLin-KC2-DMA LNP formulation for 100 ns at pH = 10. Movie S9a/b/c. Self-assembly of the DODAP LNP formulation for 100 ns at pH = 3. Movie S10a/b/c. Self-assembly of the DODAP LNP formulation for 100 ns at pH = 4. Movie S11a/b/c. Self-assembly of the DODAP LNP formulation for 100 ns at pH = 5. Movie S12a/b/c. Self-assembly of the DODAP LNP formulation for 100 ns at pH = 6. Movie S13a/b/c. Self-assembly of the DODAP LNP formulation for 100 ns at pH = 7. Movie S14a/b/c. Self-assembly of the DODAP LNP formulation for 100 ns at pH = 8. Movie S15a/b/c. Self-assembly of the DODAP LNP formulation for 100 ns at pH = 9. Movie S16a/b/c. Self-assembly of the DODAP LNP formulation for 100 ns at pH = 10. Movie S17a/b/c. Self-assembly of the DODMA LNP formulation for 100 ns at pH = 3. Movie S18a/b/c. Self-assembly of the DODMA LNP formulation for 100 ns at pH = 4. Movie S19a/b/c. Self-assembly of the DODMA LNP formulation for 100 ns at pH = 5. Movie S20a/b/c. Self-assembly of the DODMA LNP formulation for 100 ns at pH = 6. Movie S21a/b/c. Self-assembly of the DODMA LNP formulation for 100 ns at pH = 7. Movie S22a/b/c. Self-assembly of the DODMA LNP formulation for 100 ns at pH = 8. Movie S23a/b/c. Self-assembly of the DODMA LNP formulation for 100 ns at pH = 9. Movie S24a/b/c. Self-assembly of the DODDMA LNP formulation for 100 ns at pH = 10. Movie S25a/b/c. Self-assembly of the DLin-DMA LNP formulation for 100 ns at pH = 3. Movie S26a/b/c. Self-assembly of the DLin-DMA LNP formulation for 100 ns at pH = 4. Movie S27a/b/c. Self-assembly of the DLin-DMA LNP formulation for 100 ns at pH = 5. Movie S28a/b/c. Self-assembly of the DLin-DMA LNP formulation for 100 ns at pH = 6. Movie S29a/b/c. Self-assembly of the DLin-DMA LNP formulation for 100 ns at pH = 7. Movie S30a/b/c. Self-assembly of the DLin-DMA LNP formulation for 100 ns at pH = 8. Movie S31a/b/c. Self-assembly of the DLin-DMA LNP formulation for 100 ns at pH = 9. Movie S32a/b/c. Self-assembly of the DLin-DMA LNP formulation for 100 ns at pH = 10. Movie S33a/b/c. Self-assembly of the DLin-MC3-DMA LNP formulation for 100 ns at pH = 3. Movie S34a/b/c. Self-assembly of the DLin-MC3-DMA LNP formulation for 100 ns at pH = 4. Movie S35a/b/c. Self-assembly of the DLin-MC3-DMA LNP formulation for 100 ns at pH = 5. Movie S36a/b/c. Self-assembly of the DLin-MC3-DMA LNP formulation for 100 ns at pH = 6. Movie S37a/b/c. Self-assembly of the DLin-MC3-DMA LNP formulation for 100 ns at pH = 7. Movie S38a/b/c. Self-assembly of the DLin-MC3-DMA LNP formulation for 100 ns at pH = 8. Movie S39a/b/c. Self-assembly of the DLin-MC3-DMA LNP formulation for 100 ns at pH = 9. Movie S40a/b/c. Self-assembly of the DLin-MC3-DMA LNP formulation for 100 ns at pH = 10. Movie S41a/b/c. Simulated pH change of the acidic DODAP structure to pH = 7.4. Movie S42a/b/c. Mixed self-assembly/bilayer simulations of DLin-KC2-DMA for 50 ns at pH = 7, started from the corresponding pH 10 structures, which were equilibrated for 100 ns. Movie S43a/b/c. Mixed self-assembly/bilayer simulation of DLin-KC2-DMA for 50 ns at pH = 8, started from the corresponding pH 10 structure, which was equilibrated for 100 ns. Movie S44a/b/c. Mixed self-assembly/bilayer simulation of DLin-KC2-DMA for 50 ns at pH = 9, started from the corresponding pH 10 structure, which was equilibrated for 100 ns. Movie S45a/b/c. Mixed self-assembly/bilayer simulations of DLin-MC3-DMA for 50 ns at pH = 7, started from the corresponding pH 10 structures, which were equilibrated for 100 ns. Movie S46a/b/c. Mixed self-assembly/bilayer simulation of DLin-MC3-DMA for 50 ns at pH = 8, started from the corresponding pH 10 structure, which was equilibrated for 100 ns. Movie S47a/b/c. Mixed self-assembly/bilayer simulation of DLin-MC3-DMA for 50 ns at pH = 9, started from the corresponding pH 10 structure, which was equilibrated for 100 ns. Movie S48a/b/c. Mixed self-assembly/bilayer simulations of DODMA for 50 ns at pH = 7, started from the corresponding pH 10 structures, which were equilibrated for 100 ns. Movie S49a/b/c. Mixed self-assembly/bilayer simulation of DODMA for 50 ns at pH = 8, started from the corresponding pH 10 structure, which was equilibrated for 100 ns. Movie S50a/b/c. Mixed self-assembly/bilayer simulation of DODMA for 50 ns at pH = 9, started from the corresponding pH 10 structure, which was equilibrated for 100 ns. Movie S51a/b/c. Mixed self-assembly/bilayer simulations of DLin-DMA for 50 ns at pH = 7, started from the corresponding pH 10 structures, which were equilibrated for 100 ns. Movie S52a/b/c. Mixed self-assembly/bilayer simulation of DLin-DMA for 50 ns at pH = 8, started from the corresponding pH 10 structure, which was equilibrated for 100 ns. Movie S53a/b/c. Mixed self-assembly/bilayer simulation of DLin-DMA for 50 ns at pH = 9, started from the corresponding pH 10 structure, which was equilibrated for 100 ns. Movie S54a/b/c. Mixed self-assembly/bilayer simulation of DODAP for 50 ns at pH = 7, started from the corresponding pH 10 structure, which was equilibrated for 100 ns. Movie S55a/b/c. Mixed self-assembly/bilayer simulation of DODAP for 50 ns at pH = 8, started from the corresponding pH 10 structure, which was equilibrated for 100 ns. Movie S56a/b/c. Mixed self-assembly/bilayer simulations of DODAP for 50 ns at pH = 9, started from the corresponding pH 10 structures, which were equilibrated for 100 ns.
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- Award ID(s):
- 2317652
- PAR ID:
- 10700619
- Publisher / Repository:
- Zenodo
- Date Published:
- Format(s):
- Medium: X
- Right(s):
- Creative Commons Attribution 4.0 International
- Sponsoring Org:
- National Science Foundation
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