2025 Volume 73 Issue 11 Pages 1018-1023
Lipid nanoparticles (LNPs) are crucial for delivering nucleic acid therapeutics, yet their complex and heterogeneous nature poses significant challenges for comprehensive quality assessment. Traditional characterization methods provide macroscopic data, failing to offer the molecular-level insights necessary for understanding LNP quality, stability, and in vivo performance. NMR spectroscopy emerges as a powerful, non-destructive tool to bridge this analytical gap. This review highlights the diverse applications of NMR in characterizing LNP formulations. NMR techniques, including solution-state and solid-state NMR, provide atomic-level information on chemical composition, molecular structure, dynamics, and interactions. Specifically, NMR can probe lipid dynamics and molecular organization, revealing how components like cholesterol and helper lipids influence the mobility and arrangement of ionizable lipids. It elucidates the impact of cargo encapsulation on LNP structure, showing how nucleic acids like small interfering RNA (siRNA) interact with and suppress the mobility of LNP constituents. Furthermore, NMR helps understand intermolecular interactions and spatial organization within LNPs, mapping the distribution of lipids and cargo. Finally, NMR can assess the influence of preparation methods on LNP heterogeneity and monitor kinetic processes such as shedding of polyethylene glycol-conjugated lipids. NMR spectroscopy is indispensable for rational LNP design, robust quality control, and ultimately, enhancing the therapeutic efficacy of LNP-based medicines.
Lipid nanoparticles (LNPs) have emerged as a promising platform in nucleic acid delivery, offering a practical solution to the longstanding challenges associated with the stability and cellular uptake of RNA-based therapeutics. LNPs have demonstrated particular efficacy in encapsulating and delivering nucleic acids such as mRNA and small interfering RNA (siRNA), thereby enabling advances in gene therapy, vaccinology, and RNA-based treatments.1–7) The success of mRNA-LNP vaccines during the coronavirus disease 2019 (COVID-19) pandemic has further underscored their potential, positioning LNPs as a critical component in the development of nucleic acid-based therapeutics.
Despite their considerable potential, the intrinsic complexity and heterogeneity of LNPs present significant obstacles to comprehensive quality assessment and a nuanced understanding of their behavior. LNPs typically consist of multiple lipid constituents, such as ionizable lipids, helper lipids, cholesterol, and PEGylated lipids (polyethylene glycol-conjugated lipids), along with an encapsulated cargo, resulting in a dynamic and often amorphous internal architecture. Although standard characterization techniques remain indispensable, they predominantly yield macroscopic or ensemble-averaged data. For example, dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA) are commonly employed to assess particle size distribution, while zeta potential measurements provide insights into surface charge. Cryogenic transmission electron microscopy (cryo-TEM) enables high-resolution visualization of LNP morphology and size in near-native hydrated states, offering detailed structural information.8,9) Techniques such as small-angle X-ray scattering (SAXS)10–14) and small-angle neutron scattering (SANS)15,16) can offer valuable information on internal organization and periodic structures. However, SAXS and SANS measurements sometimes require large-scale facilities like synchrotrons or neutron sources and remain a challenge regarding the sensitivity of lab-scale equipment. This requirement imposes considerable experimental constraints and limits their utility for routine quality control. As a result, critical molecular-level insights into LNP quality, stability, and in vivo performance remain largely inaccessible. This analytical gap, particularly regarding the molecular arrangement of lipids and the physical state and spatial distribution of the encapsulated cargo, hinders effective formulation development, process optimization, and the rigorous quality control essential for regulatory compliance.
Addressing this critical analytical gap necessitates a technique capable of delivering high-resolution, molecular-level insights into complex nanoscale systems. NMR spectroscopy emerges as a uniquely powerful and versatile method in this context. Its capacity to provide non-destructive, atomic-level information on chemical composition, molecular structure, dynamics, and interactions has been well demonstrated in the characterization and quality assessment of various complex pharmaceutical formulations dispersed in aqueous media, including liposomes17,18) and emulsions.19–21) Notably, NMR offers both solution-state techniques for mobile components and solid-state approaches for less mobile or solid-like regions, enabling a comprehensive molecular understanding of the internal structure of LNPs. These capabilities render NMR an indispensable tool for the robust quality evaluation of LNP formulations.
This review aims to provide a comprehensive overview of the diverse applications of NMR-based molecular characterization techniques for the quality assessment of LNP formulations. We will explore how various NMR methodologies, ranging from standard solution-state experiments to advanced solid-state NMR, can unveil critical molecular details of LNP composition, internal structure, and cargo encapsulation. By highlighting the unique insights NMR offers, this paper seeks to demonstrate its indispensable role in bridging the analytical gap in LNP research and development, ultimately paving the way for more rational design, robust quality control, and enhanced therapeutic outcomes for next-generation LNP-based medicines.
Figure 1 summarizes the solution-state and solid-state NMR techniques employed to characterize the LNP formulations. The fundamental principle of NMR is based on the magnetic properties of certain atomic nuclei. When subjected to a strong magnetic field, these nuclei absorb and subsequently re-emit radiofrequency energy at specific resonance frequencies that are sensitive to their local chemical environments, thereby providing a molecular “fingerprint.” The technique involves the application of radiofrequency pulses and the detection of the re-emitted signals, which are processed to produce spectra with peaks corresponding to different atomic nuclei (e.g., 1H, 13C, 31P) in distinct chemical environments.
This table summarizes solution-state and solid-state NMR methods used in the physicochemical characterization of LNPs.
Solution-state NMR is typically employed for samples dissolved in a solvent, where rapid and isotropic molecular tumbling averages out anisotropic (direction-dependent) interactions, resulting in sharp, well-resolved spectral signals. In the context of LNPs, which are colloidal suspensions in aqueous media, solution NMR facilitates the analysis of more mobile components or regions exhibiting rapid motion, such as the flexible segments of lipid molecules within the LNP structure. Most ionizable lipids used in LNP formulations exist in a liquid-like state at room temperature due to their low melting points. Consequently, in empty LNPs, the ionizable lipid behaves akin to an “oil droplet-like” interior, enabling the direct detection of ionizable lipid peaks in solution-state 1H-NMR spectra.
For instance, Fig. 2 presents 1H-NMR spectra of LNP components dissolved in chloroform, alongside spectra of dioleoyl-dimethylammonium propane (DODMA)-based LNPs dispersed in an aqueous medium. In chloroform, each component is molecularly dissolved, producing sharp, well-resolved peaks. In contrast, in aqueous-dispersed LNPs, the lipid components are not individually dissolved but assembled into a nanoparticulate structure. Within this structure, certain lipid segments reside in relatively rigid environments, leading to restricted molecular motion and consequent NMR signal attenuation or invisibility. Therefore, only the relatively mobile regions, such as the disordered segments of ionizable lipids, the flexible PEG chains of PEGylated lipids, and the N-methyl protons of the headgroup in helper lipid, yield detectable signals in solution-state NMR. Notably, the relatively high molecular mobility of the ionizable lipid, the most critical component of LNPs for nucleic acid delivery, facilitates its detection, enabling direct characterization of its molecular state within the LNP.
(a) Spectra of individual LNP components, ionizable lipid, helper lipid, cholesterol, and PEGylated lipid, dissolved in chloroform. All components are molecularly dissolved, resulting in sharp and well-resolved peaks. (b) Spectrum of LNPs in an aqueous medium. Peaks corresponding to the ionizable lipid (DODMA) are clearly observed, whereas signals from the acyl chains of DSPC, PEGylated lipid, and cholesterol are absent, reflecting their restricted molecular mobility within the LNP structure. Resonances from the mobile PEG chains and the N-methyl group of the DSPC headgroup remain detectable.
Beyond compositional analysis, solution NMR provides critical insights into molecular dynamics and diffusion through measurements of relaxation rates and diffusion coefficients. Upon perturbation by radiofrequency pulses, nuclear spins return to equilibrium via relaxation processes, with relaxation rates being sensitive to molecular mobility and chemical exchange across various timescales.22,23) Diffusion NMR, particularly Pulsed Gradient Spin Echo (PGSE) methods, quantifies translational motion by applying magnetic field gradients that attenuate the NMR signal in proportion to molecular displacement over a defined time. Since diffusion coefficients are inversely related to molecular size, this technique can infer particle dimensions and reveal dynamic equilibria or heterogeneity within the sample.
Solid-state NMR is applied to systems in which molecular motion is restricted, such as crystalline solids and gels. In these samples, the lack of rapid, isotropic tumbling means that anisotropic interactions are not averaged out, resulting in broad and often poorly resolved spectral lines. To enhance resolution and extract detailed structural information, specialized techniques such as Magic Angle Spinning (MAS) are commonly employed. MAS involves spinning the sample rapidly at a precise angle (54.74°) relative to the external magnetic field, which averages out anisotropic interactions and significantly sharpens the resulting NMR signals.24) This enables the investigation of rigid or immobilized components inaccessible through solution-state NMR. A standard approach in solid-state NMR is the use of cross-polarization (CP) experiments, which enhance sensitivity for low-abundance nuclei such as 13C or 31P by transferring polarization from abundant 1H nuclei through heteronuclear dipolar couplings. CP is particularly effective for probing structurally rigid regions. In contrast, Insensitive Nuclei Enhanced by Polarization Transfer (INEPT) experiments exploit scalar couplings and are suited for detecting more mobile regions. The complementary use of CP and INEPT techniques allows for a nuanced analysis of both rigid and dynamic domains within complex systems.
Chemical Shift Anisotropy (CSA) is a fundamental NMR parameter that arises from the orientation-dependent shielding of a nucleus by its surrounding electron cloud. In solution-state NMR, rapid isotropic molecular tumbling averages the CSA to yield a single, sharp isotropic chemical shift. In contrast, under solid-state conditions, particularly in systems with restricted molecular mobility, CSA is preserved and manifests as a broad, characteristic “powder pattern” line shape. The geometry of this pattern provides valuable insights into molecular orientation and the extent of anisotropic motion experienced by the nucleus. In the context of LNPs, the CSA of 31P nuclei in phospholipids such as distearoylphosphatidylcholine (DSPC) serves as a direct probe of membrane dynamics. Specifically, the 31P CSA reflects membrane fluidity and the degree of molecular packing, making it a sensitive reporter of the physical state and structural organization within lipid bilayers.
Dynamic Nuclear Polarization (DNP) is an advanced technique employed to significantly enhance the sensitivity of NMR experiments, particularly in the solid state. It achieves this by transferring the high polarization of unpaired electron spins to nearby nuclear spins, typically via microwave irradiation. This process can result in signal enhancements of several orders of magnitude, enabling the acquisition of high-quality spectra from dilute, low-sensitivity, or otherwise challenging samples in substantially reduced experimental times. This is particularly advantageous in studying LNPs, where the formulations often consist of multiple components dispersed in aqueous media at relatively low concentrations, making conventional NMR analysis challenging without sensitivity enhancement.
This chapter will delve into the diverse applications of NMR in elucidating key physicochemical properties of LNPs, providing specific examples across several critical areas. We will explore how NMR can probe lipid dynamics and molecular organization within these complex nanostructures, shed light on the impact of cargo encapsulation on LNP structure, unravel intermolecular interactions and spatial organization, and investigate the influence of preparation methods through kinetic studies. These NMR-driven insights are crucial for rational LNP design, quality control, and the development of highly effective nanomedicines.
3.1. Probing Lipid Dynamics and Molecular OrganizationNMR relaxometry is a robust technique for quantitatively assessing the molecular mobility of LNP components. 1H spin–spin relaxation rate (R2) analysis has demonstrated that incorporating neutral lipids such as DSPC and cholesterol into LNPs reduces the overall molecular mobility of ionizable lipids.25) Specifically, DSPC reduces the overall mobility of ionizable lipids, whereas cholesterol selectively restricts the motion of their hydrophobic tails, likely by occupying inter-tail gaps. This decrease in molecular mobility and the resulting alteration in lipid orientation are thought to stabilize the stacked bilayer arrangement of siRNA and ionizable lipids, thereby improving siRNA encapsulation efficiency.25)
Further investigations of lipid dynamics using solid-state NMR, specifically through 13C INEPT and CP signals analyses, reveal that ionizable lipids exhibit substantially greater mobility on the nanosecond timescale compared to other lipid components in both empty and siRNA-loaded LNPs.26) In contrast, DSPC and cholesterol demonstrate markedly slower dynamics, with cholesterol being the most rigid. Above its liquid-crystalline phase transition temperature, DSPC undergoes uniaxial rotational motion, indicative of behavior consistent with a fluid bilayer structure.26)
The 31P CSA parameters were shown to be a sensitive indicator of the molecular assembly of DSPC lipids within LNPs, providing structural and dynamic information about their phospholipid envelope.27) The δCSA parameter directly reports on membrane fluidity; lower values indicate a more fluid membrane, while higher values suggest a more rigid one. The η (asymmetry) parameter reflects the uniaxial motion of lipid systems, where a value of 0 indicates a perfect bilayer in the liquid-crystalline phase (above phase transition temperature), and η > 0 suggests a gel phase. Increasing the PEGylated lipid in the lipid membranes content decreases 31P δCSA and causes the η value to approach zero, indicating that PEGylated lipid impacts LNP bilayer fluidity, allowing lipids to move more freely and influencing bilayer rigidity depending on its concentration.27)
3.2. The Impact of Cargo Encapsulation on LNP StructureThe encapsulation of siRNA markedly affects the molecular state and dynamics of LNP components. 1H-NMR analysis of ionizable lipids reveals peak broadening and downfield shifts, attributed to their interaction with siRNA and the reduced mobility associated with the formation of a stacked bilayer structure28) (Fig. 3). Additionally, 31P solution NMR serves as a useful tool for confirming siRNA encapsulation, evidenced by the broadening of 31P signals from siRNA upon encapsulation.29) This signal broadening indicates diminished siRNA mobility, suggesting its localization within the lipid phase rather than in freely mobile aqueous compartments.
The N/P charge ratios (ratios of the positive charge on the protonated form of the ionizable lipid to the negative charge on the siRNA oligonucleotide) are indicated in the figure. Ionizable lipid protons are labeled to designate NMR peak assignments. Upon siRNA encapsulation, the Hα and Hβ protons of the ionizable lipid exhibit downfield shifts and peak broadening, suggesting interactions between the tertiary amine groups of the ionizable lipid and the siRNA.
Furthermore, NMR relaxometry has quantitatively demonstrated that siRNA encapsulation significantly suppresses the molecular mobility of ionizable lipids, particularly at the tertiary amine head group, the primary site of interaction with siRNA.25) This suppression indicates the formation of a stacked bilayer structure comprising siRNA and ionizable lipids. A strong correlation between the 1H-R2 of the tertiary amine protons and the amount of encapsulated siRNA has been observed, highlighting this parameter as a valuable indicator of siRNA loading.25) Complementary evidence from 13C CP spectra indicates reduced mobility of ionizable lipids upon siRNA encapsulation, manifested as enhanced 13C CP signal intensity in siRNA-loaded LNPs across various temperatures compared to empty LNPs.26) These findings support a dense core model for siRNA-loaded LNPs, wherein ionizable lipids are tightly associated with the nucleic acid cargo. Additionally, solution 1H-NMR measurements reveal alterations in the N-methyl proton peak of DSPC upon siRNA encapsulation, suggesting that a subset of DSPC molecules may directly participate in complex formation with siRNA in the LNP core, while others remain localized at the LNP interface.25)
3.3. Intermolecular Interactions and Spatial Organization within LNPsBeyond characterizing individual component dynamics, NMR techniques also provide insights into intermolecular contacts and the overall spatial organization of LNP constituents. Two-dimensional 1H–1H Nuclear Overhauser Effect Spectroscopy (NOESY) NMR has revealed specific intermolecular interactions among LNP lipid components.30) Notably, interactions were detected between DSPC and cholesterol with the methylene protons of PEGylated lipids, suggesting their localization at the lipid–water interface. Contacts were also observed between ionizable lipids and the methylene protons of PEGylated lipids in empty LNPs, indicating that a fraction of the ionizable lipid may reside in the outer layer rather than being exclusively sequestered within the core.30)
NMR findings collectively suggest that LNPs comprise an outer layer enriched with all lipid components, including DSPC, cholesterol, PEGylated lipids, and even a fraction of the ionizable lipid, encasing a core region where DSPC primarily adopts a bilayer configuration.30) This structural model is further supported by dynamic DNP NMR spectroscopy, including relayed DNP experiments and solid-state NMR analyses of 1H, 13C, and 31P nuclei.31) These advanced techniques indicate that DSPC and PEGylated lipid predominantly localize to a surface-rich layer, whereas cholesterol and the ionizable lipid are mainly embedded within the core. Notably, the experimental data do not support a homogeneous core model with uniform distribution of all components.31)
The incorporation of neutral lipids such as DSPC and cholesterol has also been shown, via NMR relaxometry, to enhance the flexibility of PEG chains at the LNP interface.25) Remarkably, even low levels of DSPC significantly increase PEG chain mobility, likely by intercalating between the hydrophobic tails of PEGylated lipids and promoting an upright orientation of PEG chains toward the aqueous phase.25) Increasing the proportion of PEGylated lipids generally results in a decreased 13C CP signal of other LNP components, indicating enhanced bilayer dynamics, particularly for cholesterol, likely due to increased membrane fluidity and possibly elevated membrane curvature.26)
3.4. Influence of Preparation Methods and Kinetic StudiesNMR is also instrumental in elucidating the influence of LNP preparation methods and dynamic behaviors. 1H-NMR has revealed molecular-level heterogeneity in the LNP core depending on the preparation strategy.28) In siRNA-pre-mixed LNPs, siRNA is uniformly distributed within the core, with ionizable lipids interacting homogeneously with the nucleic acid, and no ionizable lipid populations resembling those in empty LNPs. In contrast, siRNA-post-mixed LNPs exhibit regions devoid of siRNA as well as localized enrichment of siRNA within the core.28)
Additionally, PGSE NMR has been employed to directly monitor PEGylated lipid shedding from siRNA-loaded LNPs in real time within rat serum.32) This label-free, molecularly specific method differentiates between PEGylated lipids associated with LNPs, exhibiting slower diffusion, and free PEGylated lipids, exhibiting faster diffusion. PGSE NMR studies have shown that LNPs stabilized by PEGylated lipids with shorter hydrophobic tails shed more readily than those with longer tails, a result attributed to weaker hydrophobic interactions that facilitate PEGylated lipid dissociation from the LNP membrane.32)
LNPs are transforming the landscape of medicine, particularly in the delivery of genetic therapeutics. However, their structural and compositional complexity necessitates the use of advanced analytical methodologies. While conventional techniques provide valuable macroscopic insights, they often fall short in capturing critical molecular-level details. In this context, NMR spectroscopy emerges as an indispensable tool. As demonstrated in this review, the breadth of NMR techniques offers atomistic resolution into the dynamics, organization, and interactions of lipid components and the structural consequences of cargo encapsulation and preparation methods. As a molecular microscope, NMR enables detailed mapping of LNP architecture and behavior, thereby informing rational formulation design, improving quality control processes, and ultimately advancing the therapeutic efficacy of LNP-based drug delivery systems.
Finally, I would like to express my deepest gratitude to Professor Kunikazu Moribe and Associate Professor Kenjirou Higashi of the Graduate School of Pharmaceutical Sciences, Chiba University, for their continuous and invaluable guidance and support throughout the research presented in this manuscript. This work was made possible through the collaboration of many researchers and the dedicated efforts of students in our laboratory, to whom I extend my sincere appreciation. This research was supported by JSPS KAKENHI Grants-in-Aid for Scientific Research (Young Scientists (B): 16K18859, Young Scientists: 19K16334, Scientific Research (C): 22K06545), for which I am also deeply grateful.
The author declares no conflict of interest.
This review of the author’s work was written by the author upon receiving the 2025 Pharmaceutical Society of Japan Award for Young Scientists.
