In the field of mRNA vaccines and nucleic acid therapies, lipid nanoparticles (LNPs) serve as the core carriers for efficient nucleic acid delivery, with lipid materials being key determinants of LNP delivery efficiency and biosafety. However, the inherent instability of mRNA has long required LNP formulations to be stored at low temperatures. The ice crystals and osmotic stresses generated during freeze-thaw cycles can easily lead to LNP aggregation and mRNA leakage, severely limiting their practical application.

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A team from the Institute of Chemistry, Chinese Academy of Sciences, published research in Nature Communications, proposing a novel solution: leveraging the freeze-concentration phenomenon to actively incorporate betaine-based cryoprotectants (CPAs) into LNPs. This approach not only maintains formulation stability but also significantly enhances mRNA delivery efficiency. The study provides a new perspective for optimizing lipid-based formulations.

Research Background

mRNA is highly sensitive to hydrolysis, oxidation, and enzymatic degradation, necessitating sub-zero storage for stability. Clinically used mRNA vaccines, such as mRNA-1273 and BNT162b2, employ sucrose as a cryoprotectant. However, even with cryoprotectants, two core issues persist during freeze-thaw cycles:

• Physical Stability Disruption: Ice crystal formation and osmotic pressure changes can cause LNP fusion and aggregation, compromising their spherical structure and size uniformity.

• Reduced Delivery Efficiency: Structural damage to LNPs can lead to mRNA leakage and weakened endosomal escape, ultimately reducing mRNA expression efficiency in target cells.

While existing research primarily focuses on "passively stabilizing" LNPs, this study innovatively utilizes the freeze-thaw process itself to transform cryoprotectants from mere "stabilizers" into "functional enhancers," achieving dual improvements in stability and delivery efficiency.

Freeze-Concentration Drives BT-CPA Incorporation into LNPs

The research team developed a composite cryoprotectant (BT-CPA) composed of betaine and trehalose. Through a series of experiments, they demonstrated that freezing induces BT-CPA to actively incorporate into LNPs, achieving two key effects: (1) maintaining LNP structural integrity after freeze-thaw cycles, and (2) enhancing LNP endosomal escape capability, thereby boosting mRNA delivery efficiency.

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During freezing, water forms ice crystals, causing the concentrations of LNPs and cryoprotectants in the remaining liquid phase to sharply increase—a phenomenon termed "freeze-concentration." This process creates a steep concentration gradient across the LNP membrane, driving the passive diffusion of betaine and trehalose into LNP interiors through transient gaps in the lipid membrane (induced by mechanical stress from ice crystals and lipid phase transitions).

The researchers validated this mechanism using proton nuclear magnetic resonance (¹H NMR) and high-resolution mass spectrometry: In LNP+BT-CPA samples subjected to freeze-thaw cycles, characteristic signals of betaine (peaks at 3.28 ppm and 3.83 ppm) and trehalose (e.g., peak at 3.34 ppm) were detectable even after dialysis to remove external free BT-CPA. In contrast, LNP+BT-CPA samples not subjected to freeze-thaw cycles showed minimal BT-CPA encapsulation. This confirms that freezing is necessary for BT-CPA incorporation into LNPs, beyond simple surface adsorption.

Optimizing BT-CPA: Precise Control of Concentration and Ratio

To balance stability and delivery efficiency, the researchers systematically optimized BT-CPA concentration and ratio:

1. Composite Ratio Optimization: Using 25 mg/mL betaine alone maintained LNP stability, but mRNA encapsulation efficiency was slightly lower than with 87 mg/mL sucrose. Adding 25 mg/mL trehalose (i.e., BT-CPA: 25 mg/mL betaine + 25 mg/mL trehalose) resulted in minimal changes in LNP size after freeze-thaw, stable mRNA encapsulation efficiency, and a 1.4-fold increase in delivery efficiency compared to betaine alone, and a 2.4-fold increase over fresh LNPs.

2. Concentration Threshold Effect: Increasing betaine concentration from 10 mg/mL to 25 mg/mL significantly enhanced mRNA delivery efficiency, but further increases to 75 mg/mL yielded no additional gains, establishing 25 mg/mL as the optimal concentration.

3. Freeze-Thaw Cycle Optimization: 1–2 cycles enhanced delivery efficiency without damaging LNP structure, while 6 cycles caused LNP aggregation, mRNA leakage, and reduced efficiency, identifying 2 cycles as optimal.

BT-CPA Enhances mRNA Delivery by Boosting Endosomal Escape

Through cellular experiments and mechanistic validation, the researchers clarified that BT-CPA improves delivery efficiency not by affecting cellular uptake but by enhancing endosomal escape to boost mRNA expression.

1. Unchanged Cellular Uptake, Enhanced Endosomal Escape: Flow cytometry showed that cellular uptake efficiency for SM-102-LNPs treated with BT-CPA and freeze-thaw (BT-CPA-LNPs) was nearly identical to fresh SM-102-LNPs. However, confocal microscopy revealed significantly reduced overlap between Cy5-labeled mRNA (red) and lysosomal markers (green) in BT-CPA-LNPs, with a significantly lower Pearson correlation coefficient (PCC) than fresh LNPs, indicating enhanced endosomal escape.

2. Molecular Mechanism: Betaine Protonation Mediates Membrane Fusion: Betaine, a zwitterionic molecule, undergoes protonation in the acidic endosomal environment, becoming positively charged and promoting electrostatic interactions with negatively charged endosomal membranes to facilitate fusion. Membrane fusion experiments using NBD-PE and Rhod-PE-labeled model endosomes showed stronger NBD fluorescence dequenching in BT-CPA-LNPs, confirming enhanced fusion capability.

3. Universality: Compatibility with Multiple Lipids: The enhancing effect of BT-CPA is not limited to SM-102 but applies to various clinically used ionizable lipids (e.g., ALC-0315, MC3). LNPs prepared with these lipids showed significantly improved mRNA delivery efficiency after BT-CPA treatment and freeze-thaw, with particle sizes below 200 nm and PDI < 0.2, demonstrating broad applicability.

In Vivo Validation: Stronger Immune Response and Dose-Sparing Advantage

In C57BL/6 mice, BT-CPA-LNPs exhibited significant advantages:

• Higher mRNA Expression: At 4 h and 24 h post-intramuscular injection, luciferase expression in BT-CPA-LNPs was 2.3-fold and 1.7-fold higher than in fresh LNPs, respectively.

• Stronger Immune Response: BT-CPA-LNPs encapsulating ovalbumin mRNA (mOVA) induced significantly higher OVA-specific IgG antibody titers at doses of 0.1 μg and 1 μg, and ELISpot assays showed increased IFN-γ-positive CD4⁺ and CD8⁺ T cells.

• Excellent Long-Term Stability: After 6 months at -80°C, BT-CPA-LNPs maintained high mRNA expression efficiency with low cytotoxicity (DC2.4 cell viability > 90%).

Research Significance
This study not only proposes a novel LNP cryoprotection strategy but also redefines cryoprotectants as "active components" that can incorporate into LNPs during freezing to regulate function. Key contributions include:

1. Optimizing Storage Protocols: Addressing stability challenges in low-temperature LNP storage and transport, supporting global distribution of mRNA vaccines and nucleic acid drugs.

2. Expanding Lipid Applications: Demonstrating that lipids like SM-102, ALC-0315, and MC3 can achieve enhanced delivery via BT-CPA, aiding applications in cancer immunotherapy and gene therapy.

3. Innovating Formulation Design: Pioneering the concept of "leveraging freezing for functional enhancement," offering a new paradigm for LNP development.

Conclusion

By strategically utilizing the freezing process, this research transforms LNP storage challenges into functional advantages, providing a valuable technical solution for optimizing formulations of core lipids like SM-102, ALC-0315, and MC3, with promising implications for disease treatment.

SINOPEG supplies lipids used in the study (SM-102, ALC-0315, MC3), offering one-stop liposome excipients and a range of proprietary lipid excipients. We also provide high-quality polysaccharides and polyzwitterionic products to meet diverse R&D and production needs.

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Reference
Cheng X, Zheng X, Tao K, et al. Freezing induced incorporation of betaine in lipid nanoparticles enhances mRNA delivery. Nature Communications. 2025;16(1):4700.

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