Key Factors Influencing the Stability of mRNA-Lipid Nanoparticle Drug Formulations

LNPs encapsulating Fluc mRNA (FLuc mRNA–LNPs) were manufactured using the NanoAssemblr^® Ignite^™ system through a microfluidic mixing approach (Precision NanoSystems Inc., Vancouver, BC, Canada). A freeze–thaw study was conducted to screen different concentrations of sucrose as a cryoprotectant in the LNP formulation. FLuc mRNA–LNPs composed of four lipids, namely DLin-MC3-DMA, Cholesterol, DSPC, and DMG-PEG2k (molar ratio 50/37.5/10/2.5) were prepared. Lipid solution in ethanol at a total lipid concentration of 50 mM, and FLuc mRNA at the target concentration in 10 mM sodium citrate buffer, pH 5.0, were used. LNPs were manufactured by mixing the lipid solution and FLuc mRNA solution at an optimized flow rate ratio of 1:3, while the total flow rate used was 12 mL/min. Obtained LNPs were diluted with sucrose (0–20% w/v) containing PBS buffer, followed by buffer exchange via ultracentrifugation (100 kDa molecular weight cut off, MWCO) (Millipore, Burlington, MA). LNP formulations were subjected to sterile filtration, filled in Schott 2R glass vials, and exposed to one overnight freeze–thaw cycle (−80°). Section 2.4 illustrates the analytical characterization performed on the formulated FLuc mRNA–LNPs.

After identifying an optimized sucrose content, various concentrations of Tris buffer were screened and compared with 1× PBS to narrow down the formulation buffers that could protect the drug product quality following two freeze–thaw cycles (1st cycle overnight, and 2nd cycle 2 h incubation at –80 °C before thawing the samples for 30 min at room temperature). NanoAssemblr^® Ignite^™ system (Precision NanoSystems Inc., Vancouver, BC, Canada) was used to formulate FLuc mRNA–LNPs containing various lipid combinations (Table 1). All five formulations (F1, F2, F3, F4, and F5) comprise an ionizable lipid/DSPC/cholesterol/PEG–lipid combination at a molar percent ratio of 50/10/37.5/2.5. These formulations were prepared using similar mixing conditions as described in Section 2.3.1. After microfluidic mixing, LNPs were diluted with either of the two buffers: 1× PBS or Tris (5, 10, 20, 30 mM) buffer, both containing 10% w/v sucrose, at pH 7.4. Diluted LNPs were subjected to ultracentrifugation for buffer exchange, and then sterile filtered to fill into 2R vials. FLuc mRNA–LNPs were characterized using different analytical techniques as mentioned in Section 2.4.

FLuc mRNA–LNPs were tested for long-term storage stability at different temperatures (−80 °C, −20 °C, 5 °C, and 25 °C) by incorporating the optimized formulation buffers and sucrose content from the aforementioned studies. FLuc mRNA–LNPs containing various lipid combinations were manufactured using microfluidic mixing as described in Section 2.3.1. Resultant LNPs were diluted with either of the two buffers: 20 or 30 mM Tris buffer containing 10% w/v sucrose, pH 7.4. Buffer exchange was performed, and LNPs were filtered by using a 0.2 µm syringe filter, followed by filling into Schott 2R glass vials (SCHOTT Pharma USA, Inc., Lebanon, PA, USA). Analytical characterization for all LNP formulations was performed as mentioned in Section 2.4.

Visual appearance of all the prepared formulations was assessed against a black background for their clarity, color, and visible particulates. pH value of the FLuc mRNA-LNP drug product was measured using a pH meter (Mettler Toledo, Columbus, OH, USA). Visual inspection and pH measurement of LNP formulations during long-term storage stability were performed on day 0 and the following 1, 3, 6, and 12 months when stored at different storage temperatures (−80 °C, −20 °C, 5 °C, and 25 °C).

Size and surface charge measurements of LNPs were measured using Malvern Zetasizer Ultra Red. Samples were diluted 10 times with sterile filtered 0.1× PBS pH 7.4 and transferred into a folded capillary cell (DTS1070). Measurements were performed in triplicate at 25 °C (refractive index of 1.45 and viscosity of 0.88 cP) using a 173° backscatter angle of detection. Particle size, polydispersity index, and zeta potential of LNP formulations on long-term storage stability were measured on day 0 and the following 1, 3, 6, and 12 months when stored at different storage temperatures (−80 °C, −20 °C, 5 °C, and 25 °C). For quality control of dynamic light scattering (DLS) size measurement by Zetasizer instrument, the 100 nm polystyrene nanoparticles, calibrated by NIST traceable standards, were used (Malvern Panalytical, Chipping Norton, NSW, Australia, LTX3100A). For Zeta potential quality control, the zeta potential transfer standard with −40 ± 5.8 mV was used prior to each measurement (Malvern Panalytical, ZTS1240).

The nanoparticle tracking analysis (NTA) technique was employed to measure the LNP particle concentration using ZetaView^® (Particle Metrix Inc., Mebane, NC, USA). Prior to the measurements, samples were diluted with 0.1× PBS buffer and injected into the cell to avoid any air-bubbles. Sample dilutions were adjusted to maintain between 100 and 500 particles per frame (11 positions, 85 sensitivity) for accurate results. Particle concentration for LNP formulations on long-term storage stability was assessed on day 0 and the following one year at different storage conditions (−80 °C, −20 °C, 5 °C, and 25 °C).

Quant-IT^™ RiboGreen RNA Assay Kit (Invitrogen^™) was used to evaluate the encapsulation efficiency percentage (EE%) and total/free mRNA concentration present in FLuc mRNA–LNP formulations. Standard curves of FLuc mRNA (50–2000 ng/mL) were generated in 10 mM Tris/1 mM EDTA (TE) buffer and 2% w/v Triton x-100/TE (TR) buffers. To determine the free mRNA concentration, LNPs were diluted 10× in the TE buffer, and for total mRNA concentration evaluation, LNPs were diluted 25× in the TE buffer followed by a 1:1 dilution with the TR buffer. The plates were incubated at 37 °C for 10 min to allow LNP lysis for mRNA quantification. RiboGreen assay was then performed by adding the reagent to each standard/sample, and the fluorescence (Ex485/Em528) was measured using a plate reader (Synergy Neo2, Biotek, Winooski, VT, USA). The encapsulation efficiency in LNPs was calculated using the following formula:

Encapsulation efficiency and mRNA concentration analysis for FLuc mRNA–LNP formulations on long-term storage stability were determined on day 0 and the following 1, 3, 6, and 12 months when stored at different storage temperatures (−80 °C, −20 °C, 5 °C, and 25 °C).

The osmolality of LNPs was measured using the VAPRO^® Vapor Pressure Osmometer (ELITechGroup Inc., Logan, UT, USA) by following the manufacturer’s instructions. Briefly, a sample disk was placed in the sample holder, and 10 µL of the LNP solution was added onto the disk. The Open/Close key was pressed to have the sample placed in the measurement position. This allows automatic measurement of the sample osmolality. At least 3 measurements of each LNP sample were performed to consider an average value with an appropriate standard deviation. Osmolality of FLuc mRNA–LNP formulations subjected to long-term storage stability was measured on day 0 and the following one year at different storage conditions (−80 °C, −20 °C, 5 °C, and 25 °C).

FLuc mRNA and FLuc mRNA–LNP samples were diluted with 2% Triton x-100 to a final concentration of 5 µg/mL for further analysis. First, 80 µL of 1× Sample Buffer present in the RNA Pico Sensitivity Assay Kit (Perkin Elmer, Shelton, CT, USA) was added per well in a 96-well plate. Samples were denatured by heating in a 70 °C heating block followed by snap cooling on ice for a few min. RNA Pico ladder, gel-dye solution, and RNA LabChip^® were prepared by following the manufacturer’s protocol. The RNA LabChip was thoroughly cleaned, and the gel-dye solution and RNA Pico marker were then added to the appropriate wells. The assay was run using the instrument, LabChip^® GXII Touch^™ (Perkin Elmer, Shelton, CT, USA). Electropherograms for FLuc mRNA and FLuc mRNA–LNP samples were evaluated by calculating the main peak and fragmentation as a percentage of the total peak area. The purity and integrity of FLuc mRNA encapsulated within LNP formulations on long-term storage stability were determined on day 0 and the following 1, 3, 6, and 12 months when stored at different storage temperatures (−80 °C, −20 °C, 5 °C, 25 °C).

Cryo-TEM was used to determine the effect of freeze–thaw and storage temperature on the LNP’s morphology. Cryo-TEM analysis was performed for LNP formulations stored at different temperature conditions (−80 °C, −20 °C, 5 °C, and 25 °C), specifically at day 0 and following one month as well as one year of storage. Briefly, 3 µL of LNP solution was applied onto the mesh copper grids (Electron Microscopy Sciences, Hatfield, PA, USA), blotted, and vitrified by plunging into liquid ethane. Following vitrification, the grids were transferred to the autoloader of the microscope under liquid nitrogen for capturing images using a Glacios^™ Cryo-Transmission Electron Microscope (Thermo Fisher Scientific, Waltham, MA, USA). Images were captured at multiple scales for each grid by targeting the area of interest at lower magnifications (940x–8500x) before acquisition of movies at nominal magnifications of 28,000× (0.5 nm/pixel), 73,000× (0.2 nm/pixel), and 150,000× (0.1 nm/pixel), while highest magnification images (150,000x) were acquired with an electron dose between 12 and 25 e⁻/Ų.

The 5′-capping integrity of FLuc mRNA encapsulated within LNP formulations subjected to long-term storage stability was determined following one year at different storage conditions (−80 °C, −20 °C, 5 °C, 25 °C). For that, first the LNPs were treated with 2% triton-X100 to release the mRNA, then the released FLuc mRNA was analyzed using enzymatic treatment of mRNA–LNPs with a subsequent size-exclusion ultra performance chromatography (SE-UPLC) analysis. For enzymatic digestion of 5′-capping a sequential digestion using 5′ polyphosphatase (LGC Biosearch Technologies, Middlesex, UK, cat # RP8092H) and terminator 5′ phosphate-dependent exonuclease (LGC Biosearch Technologies, cat # TER51020↗) were used as previously described [7]. Following this enzymatic digestion, the mRNA with 5′ cap remained intact while uncapped transcripts were readily digested. Next, the intact transcripts and degraded mRNA fragments were resolved by an SE-UPLC method, and the percentage of capped mRNA was calculated using the peak areas by the following equation:

MFI measurements were performed with an MFI 5100 instrument featuring a flow cell depth of 400 μm (ProteinSimple). LNP samples were diluted 20-fold in a 0.2 μm-filtered 0.1× PBS. Data were acquired with MFI View System Software (MVSS) version 2-R4 and processed with MFI View Analysis Suite (MVAS) software version 1.4.0. A baseline measurement was performed with the sample diluent to ensure that less than 100 particles/mL were present. The optimizing illumination step was performed on the sample diluent, and then the samples were loaded into the sample port. The sample purge volume was set at 0.20 mL, and the sample volume analyzed was 0.571 mL. The reported particle size (in μm) is the equivalent circular diameter (ECD) detected by MFI following the guidance of United States Pharmacopeia (USP) Chapter <788>.

An ACQUITY UPLC I-Class system (Waters Corporation, Milford, MA, USA) with an Accucore^™ C30 column (2.6 µm, 150 Å, 2.1 mm × 150 mm, ThermoFisher Scientific, San Jose, CA, USA) was used for reversed-phase liquid chromatography (RPLC) to separate lipid components and degradants in each FLuc mRNA–LNP formulation under long-term storage stability conditions (−80 °C, −20 °C, 5 °C, 25 °C). Mobile Phase A consisted of 0.1% formic acid and 10 mM ammonium formate in a 40:60 water/acetonitrile mixture. Mobile Phase B consisted of 0.1% formic acid and 10 mM ammonium formate in a 10:90 acetonitrile/isopropanol mixture. The linear LC gradient was set up as follows: 30% B from 0 min to 1 min, 43% B at 3 min, 65% B at 13 min, 85% B at 19 min, 100% B from 21 min to 26 min, and 30% B from 26.1 min to 26 min. LC separation was conducted at a flow rate of 260 µL/min at 40 °C. Each LNP sample was diluted 200× and 1000× in 100% ethanol and analyzed in triplicate with a 4 µL injection volume.

Mass spectrometry analysis was performed using an Orbitrap Exploris^™ 480 mass spectrometer (ThermoFisher Scientific, Bremen, Germany) in positive electrospray ionization (ESI) mode. Instrument parameters included the following: capillary voltage of 3.5 kV, ion transfer tube temperature of 300 °C, sheath gas at 40, auxiliary gas at 10, sweep gas at 1. MS1 acquisitions were conducted at a resolution of 120,000, mass range 300–1200 m/z, normalized automatic gain control (AGC) target of 300%, and maximum injection time of 50 ms. MS2 acquisitions were performed at a resolution of 30,000, normalized AGC target of 100%, maximum injection time of 50 ms, isolation window of m/z = 1, and stepped higher-energy collisional dissociation (HCD) with collision energy of 15%, 30%, and 50%, using top10 data-dependent acquisition (DDA). Compound identification was performed using Compound Discoverer (version 3.3, ThermoFisher Scientific, San Jose, CA, USA) and relative quantification of LNP component and degradants was determined using TraceFinder (version 4.1, ThermoFisher Scientific, San Jose, CA, USA).

Cold isopropanol precipitation was used to isolate mRNA from the FLuc mRNA–LNP formulation [8]. Specifically, 900 µL of 60 mM ammonium acetate isopropanol solution was added to a 100 µL aliquot of the sample. After a brief vortex, the mixture was centrifuged at 14,000 g for 15 min at 4 °C, and the supernatant was discarded. The pellet was then washed twice with 1000 µL of cold isopropanol, vacuum dried, and rehydrated in 100 µL of ultrapure water for quantification by UV Nanodrop 2000 spectrophotometer (260 nm, ThermoFisher Scientific, San Jose, CA, USA). Subsequently, the isolated mRNA (approximately 2 µg) was enzymatically digested to nucleosides for the LC-MS analysis. In brief, the mRNA samples were incubated with 10 units of nuclease P1 in 25 mM Tris-HCl (pH 7.2), 50 mM NaCl, 1 mM ZnCl₂ (New England Biolabs, Ipswich, MA, USA), 2 units of phosphodiesterase I (Sigma-Aldrich, Inc., St. Louis, MO, USA), and 2 units of quick calf intestinal alkaline phosphatase in 25 mM Tris-HCl (pH 7.5), 1 mM MgCl₂ and 0.1 mM ZnCl₂ (New England Biolabs, Ipswich, MA, USA). The digestion reaction was carried out at 37 °C for 1 h.

Afterward, nucleoside analysis was conducted using the UPLC I-Class system (Waters Corporation, Milford, MA, USA) with an Atlantis Premier BEH Z-HILIC Column (1.7 µm, 2.1 mm × 150 mm, Waters Corporation, Milford, MA, USA) at a column temperature of 50 °C. This setup was used to quantify the four nucleosides in each mRNA–LNP formulation under long-term storage stability conditions (−80 °C, −20 °C, 5 °C, and 25 °C). The mobile phases were 200 mM ammonium acetate aqueous solution (Mobile Phase A) and acetonitrile (Mobile Phase B). Separation was achieved at a flow rate of 0.5 mL/min with a step gradient: 100% B from 0 to 1 min, 50% B at 11 min, 50% B from 11 to 12 min, and 100% B at 15 min. Each mRNA sample was analyzed in triplicate with a 5 µL injection volume. Mass spectrometry scans were acquired using an Orbitrap Exploris 480 mass spectrometer (ThermoFisher Scientific, Bremen, Germany) in both positive and negative ESI modes. The instrument parameters were as follows: capillary voltage of 3.2 kV in positive mode and 2.5 kV in negative mode. The ion transfer tube temperature was set to 260 °C. The sheath gas was set at 8, the auxiliary gas at 5, and the sweep gas at 1. Both MS1 and MS2 acquisitions were conducted at a resolution of 60,000, with a mass range of 60–760 m/z. The AGC target was 100%, and the maximum injection time was 100 ms. MS2 acquisitions were performed with an isolation window of 1.5 m/z. Stepped HCD collision energies of 20%, 35%, and 50% were used with top10 DDA. Compound identification and quantification were performed using FreeStyle (Version 1.3, ThermoFisher Scientific, San Jose, CA, USA).

Throughout 12 months of storage, the pH of all four formulations (F1–F4) stayed between 7.0 and 7.5 with no meaningful drift, confirming that 20 mM and 30 mM Tris + 10% sucrose effectively buffer the system irrespective of temperature or lipid composition (Figure S3). Formulations remained clear and no visible particulates were detected at −80 °C, −20 °C, 5 °C, or 25 °C (a representative image of the LNPs’ visual appearance is shown as Figure S4). Also, osmolality was consistently 340–360 mmol kg⁻¹ across conditions over 12 months (Figure S5).

Cryo-TEM revealed that all formulations retained spherical morphology after one year at −80 °C (Figure 2 and Figure S6). At −20 °C, only F4 preserved its morphology, while the rest of the formulations showed early aggregation, large internal blebs, and size growth, detectable within one month and pronounced by 12 months (Figure 2 and Figure S6). Storage at 5 °C maintained morphology for the first month, but at 12 months, all samples displayed reduced particle counts (based on the visual observation of the samples in the scanned regions under cryo-TEM) and distorted shapes. Here, F1 showed moderate aggregation, while F4 formed oversized (>200 nm) vesicles lacking internal structure, indicative of advanced instability; F2 and F3 showed few distinct ultrastructural alterations (Figure 2 and Figure S6). At 25 °C, all formulations showed early signs of particle growth under cryo-TEM at one month of storage, though the other physical characteristics, such as pH and osmolality, did not indicate any significant changes during this time.

Figure 3, Figures S7 and S8 summarize the physicochemical characterization of four tested mRNA–LNP formulations. Particle size distribution (measured by DLS) showed no meaningful difference over 12 months (Figure 3). MC3-based formulations (F1 and F3) remained ~20 nm larger than their ALC-0315 counterparts (F2 and F4) across all temperatures, and both Tris concentrations with no variation were detected over this time period. Polydispersity indices remained low overall across the study. DMG-PEG2k formulations (F1 and F2) consistently showed the narrowest distributions (PDI < 0.10), whereas ALC-0159 formulations (F3 and F4) exhibited modest fluctuations with occasional values higher than 0.18, consistent with Figure S7.

Zeta potential values varied from −10 mV to +10 mV and were largely unaffected by temperature or buffer concentration (). ALC-0315 systems (F2 and F4) trended slightly more negative than MC3 analogs (F1 and F3), but differences were <5 mV and did not change with storage. Figure S8

Encapsulation efficiency (EE) was maintained above 95% for MC3 LNPs and 80–90% for ALC-0315 LNPs throughout the study, independent of temperature or buffer, except for F3 (MC3 with ALC-0159) at −80 °C, which declined ~40% by month 12 (Figure 3). Notably, this EE% drop occurred without parallel changes in size or PDI, indicating possible selective mRNA leakage rather than gross particle destabilization. More evaluation is needed to determine the observed discrepancy between EE% and the size and PDI data observations for the F3 formulation.

One of the key observations of this study was the changes observed in subvisible particle counts measured by MFI. These results showed that after 12 months, all samples contained less than 600 particles mL⁻¹ of ≥10 µm except F3 at −20 °C (≈1200 particles mL⁻¹, Figure 4). The highest incidence of LNP aggregates followed the order −20 °C > 5 °C > 25 °C > −80 °C.

Across formulations, 20 mM Tris generated fewer subvisible particles than 30 mM Tris. Together, the data highlights F3 as the most aggregation-prone formulation and reaffirm −80 °C with 20 mM Tris as the optimal storage condition for maintaining both nanoparticle integrity and minimal subvisible particulate burden.

Lipid content and integrity of LNPs were evaluated by LC-MS post-one year storage at different temperatures. This LC-MS profiling at month 12 showed that the lipid composition of every formulation was essentially unchanged at −80 °C and −20 °C (≤10% deviation from baseline;) when 20 mM or 30 mM tris/sucrose buffer was used. At 5 °C and 20 mM tris/sucrose, total lipid loss reached 10–25%, and at 25 °C as much as 40%, possibly driven by degradation of DSPC and PEG-lipids. Notably, DSPC content fell to ~70% of baseline in F1 and 60% in F3, while DMG-PEG2k in F1 decreased by ~20%. When the tris concentration increased to 30 mM, a significantly higher amount of lipid loss was observed for F1 and F3 (MC3-based formulations), even at −20 °C, while F2 and F4 (ALC-0315-based formulations) were surprisingly unaffected. Additionally, we observed that the lipid content of F2 and F4 was not affected at 20 mM tris buffer over the storage time by 12 months. Figure S11

At the same time, oxidative markers showed an increase in parallel to the lipid loss. Using 20 mM tris, the potentially mRNA reactive DSPC-C₁₈H₃₄O increased 2–6 fold at 5 °C and >10-fold at 25 °C (Figure 5). Increasing the tris concentration to 30 mM caused a severe increase in this reactive DSPC derivative at 5 °C and 25 °C by approximately 10–20-fold higher compared to the frozen samples at −80 °C and −20 °C. MC3 N-oxide, a predominant MC3 reactive impurity in MC3-containing F1 and F3, climbed up sixfold at 25 °C in 20 mM tris and up to threefold when 30 mM tris was used. On the contrary, the oxidized ALC-0315 in F2 and F4 showed either no significant changes at any temperature and tris concentration or rose only by <0.5-fold, indicating the greater stability of the ALC-0315 head group against oxidation. Collectively, these results confirm that frozen storage preserves the lipid integrity, while DSPC hydrolyzation can be the earliest indicator of chemical degradation at elevated temperatures. Lastly, as indicated by DSPC and MC3/ALC-0315 lipid data, the buffer concentration exhibits a mixed effect on each of the oxidized species of lipids and overall, we have observed less susceptibility of ALC-0315-based formulation lipid loss and oxidation compared to MC3-based LNPs.

Next, we tested the mRNA integrity at one year of storage at different temperatures. MCE for mRNA purity showed that frozen storage preserved mRNA integrity, where all formulations retained ~80% purity after 12 months at −80 °C or −20 °C (Figure 6). At 5 °C, purity began to decline after month three and fell to 30–40% by month 12, regardless of buffer strength. At 25 °C, purity dropped by 40–60% within three months and continued to degrade, confirming rapid hydrolytic/oxidative decay at ambient temperature.

LC-MS quantification of enzymatically digested nucleosides partially corroborated these findings. Adenosine, cytidine, guanosine, and uridine levels were unchanged at −80 °C, −20 °C, and 5 °C but fell slightly at 25 °C (), indicating mRNA backbone (ribose-phosphodiester structure) fragmentation rather than selective base loss for the liquid formulations. Figure S12

Ion-pair RP-HPLC revealed near-quantitative 5′-capping retention (>95%) in 20 mM Tris after one year at both frozen temperatures. The 30 mM Tris buffer reduced 5′-cap levels by 5–15% at both frozen temperatures, suggesting mild cap hydrolysis at higher ionic strength (Table 3, Figures S13 and S14). Additionally, capping analysis was not possible for 5 °C and 25 °C samples because bulk mRNA was already degraded below the assay’s threshold (Figures S13 and S14). Together, these data reinforce the need for sub-zero storage, preferably −80 °C, in a low-ionic-strength buffer to protect both the chemical and structural integrity of encapsulated mRNA.

The transfection efficiency (TE) results (Figure 6) indicated that, in general, the baseline TE% ranked F1 ≈ F2 ≈ F3 ≫ F4. The three higher performing formulations produced ~10⁷ mean luminescence intensity (MLI) in HEK-293 cells, whereas F4 reached only ~10⁶ MLI, consistent with its slightly lower encapsulation efficiency (80–90%, as indicated in Figure 3).

TE was largely preserved for all formulations stored at −80 °C and −20 °C, showing ≤0.2-log variation over 12 months. At 25 °C, TE% declined in parallel with mRNA purity, dropping one log by month three and becoming negligible by month 12. The 5 °C condition showed a biphasic pattern, where transfection remained comparable to the frozen controls through six months, despite measurable mRNA purity loss, but fell sharply between 6 and 12 months. This lag suggests that a critical threshold of intact mRNA must be crossed before in vitro functional activity is compromised. Overall, −80 °C and −20 °C storage fully preserves in vitro TE, while even mild refrigeration (5 °C) affords short-term (up to six months) protection for luciferase mRNA–LNPs.

We next evaluated the in vivo transfection trend of LNPs over one year of storage at different temperatures at 30 mM tris composition. Long-term storage temperature exerted a clear, formulation composition-independent hierarchy on in vivo potency (Figure 7). Intravenous dosing of the four LNPs into CD1 mice showed that −80 °C preserved whole-body, liver, and spleen luminescence similar to the day one levels for every formulation. At −20 °C, formulations F2–F4 largely maintained in vivo activity over 12 months, while F1 showed a modest ~15–20% reduction in liver signal relative to its corresponding −80 °C LNPs but not statistically significant (Figure 7c, One-way ANOVA, p-value = 0.73). Also, the spleen expression was unaffected for F1, suggesting slight instability specific to this lipid blend. LNPs stored at 5 °C stayed stable up to six months (Figure S16) but showed a uniform ~10-fold drop in liver and spleen signals by month 12 (Figure 7), mirroring the late decline in the in vitro TE%. Finally, 25 °C storage led to near-complete loss of detectable luminescence by month six for all formulations, confirming functional failure at this temperature. These results align with mRNA integrity data and reinforce the obvious requirement for frozen storage, preferably −80 °C, to ensure the retention of systemic transfection efficiency.