Affect of salt resolution on the physicochemical properties and in vitro/ in vivo expression of mRNA/LNP | Journal of Nanobiotechnology

March 20, 2025

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The impact of mRNA aqueous resolution on LNP physicochemical properties and transfection in vitro

Firstly, we investigated the affect of pH (pH = 3, 4, 5, 6), salt sort (citrate resolution (CS), acetate resolution (AS), CS plus NaCl, AS plus NaCl), and salt focus (10, 25, 50, 100 mM) on the physicochemical properties and mobile expression of LNPs (Fig. 1A and B). The precise sort of aqueous options was proven in Desk S3. We measured the hydrodynamic diameter, PDI, zeta potential, and EE of LNPs ready with completely different mRNA aqueous options (Desk S3). The hydrodynamic diameter of those LNPs ranged from 84 to 120 nm, with a PDI between 0.08 and 0.21, zeta potential from − 7.2 to 2.4 mV, and EE starting from 0 to 92%. The bottom EE was present in group A11 which the salt of mRNA aqueous resolution was 50 mM CS and 130 mM NaCl with pH equaling 6. Furthermore, the very best EE was achieved in group A13 with 91.8%, by which 100 mM CS with pH = 3 resolution was used to disperse mRNA. Furthermore, A13 additionally confirmed the very best measurement with 120.3 nm, whereas the smallest measurement was present in A1 (83.5 nm) which 10 mM CS (pH = 6) was used as mRNA aqueous resolution. Then, we tried to determine which issue impacted the physicochemical properties considerably and which issue or elements performed essentially the most essential position. Subsequently, we analyzed the correlation between the pH, salt focus, and the physicochemical properties (Fig. 1C). The outcomes confirmed a unfavourable correlation between the pH of mRNA aqueous resolution and the particle measurement, zeta potential and EE of the LNPs (Fig. 1C, D and E and Determine S1D).

Conversely, we solely noticed a constructive correlation between the salt focus of the mRNA aqueous resolution and the particle measurement of LNPs (Fig. 1F). Because the salt focus elevated, the particle measurement of LNPs barely elevated from 100 nm to 110 nm, doubtless because of the fusion between small vesicles below excessive ionic focus [42]. This indicated that the focus of salt had much less vital impact in contrast with pH. Moreover, we discovered that the kind of salt confirmed no impact on the physicochemical properties (Determine S1H). Furthermore, we discovered that EE and particle measurement elevated with rising zeta potential (Fig. 1G and Determine S1E). Moreover, Determine S1F and S1G demonstrated that EE and measurement had been negatively correlated with PDI. These outcomes instructed that the pH of mRNA aqueous resolution was essentially the most essential position of mRNA aqueous resolution affecting physicochemical properties which perhaps as a result of, because the pH elevated, the diploma of protonation LNPs decreased, resulting in the publicity of mRNA on the LNP floor and ensuing within the heterogeneous formation of mRNA/LNP complexes [35, 43, 44]. Consequently, EE and zeta potential decreased because the pH of the mRNA aqueous resolution elevated (Fig. 1C). Moreover, excessive pH values contributed to the formation of empty liposomes, resulting in smaller measurement and larger heterogeneity of LNPs [45] (Fig. 1C).

Subsequently, we examined the expression of those LNPs in AML12, DC2.4, and C2C12 cell traces (Fig. 1B, Determine S1A). The LNPs exhibited various mobile transfection effectivity throughout completely different cell traces. Particularly, LNPs from teams A1, A2, A6, A10, and A11 confirmed comparatively decrease luciferase expression, whereas group A8 demonstrated the very best mobile transfection efficacy. Subsequent, we investigated whether or not mobile expression correlated with the elements of the mRNA aqueous resolution. As proven in Fig. 1H and Determine S1B, the pH of the mRNA aqueous resolution considerably impacted LNP mobile expression. A lowering pattern in expression was noticed in DC2.4, AML12, and C2C12 cells because the pH elevated from 4 to six. In DC2.4 cells, mobile expression was 2.9 instances greater with a pH of 4 in comparison with pH = 6. In AML12 cells, the mobile expression of LNPs with mRNA aqueous options at pH = 5 and 6 decreased by 33% and 66% in comparison with pH = 4 (Fig. 1H). Equally, in C2C12 cells, luciferase expression with pH = 4 mRNA aqueous resolution was 3.9 instances greater than that of pH = 6 (Determine S1B). Curiously, LNPs with a pH of three didn’t comply with this pattern, presumably because of the instability of mRNA molecules at this low pH [46]. In distinction, the kind and focus of salts of the mRNA aqueous resolution didn’t present any vital affect on mobile expression (Fig. 1I, Determine S1C).

Based mostly on these findings, the pH of the mRNA aqueous resolution emerged as essentially the most important issue influencing physicochemical properties and mobile expression of LNPs. Subsequently, mRNA aqueous resolution with pH = 4 was chosen for additional research.

The impact of Dilution on physicochemical properties and transfection of LNPs

Subsequently, we examined the impact of pH (pH = 4, 5, 6), salt sort (phosphate resolution (PS), CS, AS), and salt focus (10, 25, 50 mM) of the diluent on physicochemical properties and mobile expression of LNP (Fig. 2A). The dilutions had been listed in Desk S4. We examined the physicochemical properties of LNPs, luciferase expression on the mobile degree (Desk S4 and Fig. 2B), and explored the correlations amongst varied indicators (Fig. 2C). Firstly, we evaluated the affect of dilutions on physicochemical properties. The dimensions was roughly 84 nm and EE was round 91% (Desk S4) with little distinction between LNPs. Moreover, as proven in Fig. 2C and D, solely the salt focus of the diluent exhibited a unfavourable correlation with the zeta potential of LNPs. pH, alternatively, confirmed no vital correlation with the physicochemical properties, suggesting that the pH of the diluent was much less necessary than the pH of the mRNA aqueous resolution. Moreover, LNPs shaped at pH = 5 had been extra uniform, with a PDI of 0.07, in comparison with these shaped at pH = 4 and pH = 6 (pH = 4: PDI = 0.10; pH = 6: PDI = 0.14) (Fig. 2E). The salt sort didn’t considerably have an effect on the physicochemical properties of LNPs (Determine S2A). Based mostly on these outcomes, we concluded that the dilution had a smaller impact on the physicochemical properties of LNPs in comparison with the mRNA aqueous resolution.

By way of mobile expression, the pH, salt sort, and focus of the diluent had no notable results on LNP expression throughout completely different cell traces (Fig. 2C and Determine S2B-S2D), besides in C2C12 cells, the place the pH of the diluent influenced expression (Determine S2E). At pH = 4 (9.57*10⁶), LNP expression in C2C12 cells was 20% or 24% greater in comparison with pH = 5 (7.75*10⁶) or 6 (7.26*10⁶), a phenomenon not noticed within the different two cell traces. Furthermore, we discovered that the expression of LNPs in AML12 and DC2.4 cell traces was positively correlated with particle measurement (Fig. 2F and G).

Based mostly on these findings, there was no clear proof indicating that any specific issue of dilutions had a big affect on LNPs. Subsequently, we proceeded to check all elements of the dilutions together with different kinds of options in additional experiment.

The impact of change resolution on physicochemical properties and transfection of LNPs

We then studied the impact of various change options on the physicochemical properties and mobile expression of LNPs earlier than (indicated as course of A) and after (indicated as course of B) change with the ultimate storage resolution (PBS) (Fig. 3A). The composition of change options utilized in every group had been listed in Desk S5.

Firstly, we in contrast the physicochemical properties of LNPs ready by the 2 processes (Desk S5). The info confirmed that LNPs ready by course of A had a notably smaller particle measurement (85 nm vs. 106 nm, p < 0.01) and EE (87% vs. 94%, p < 0.05) than these ready by course of B. Nevertheless, course of A LNPs exhibited the next zeta potential (0.87 mV vs. -0.58 mV, p < 0.01). No vital distinction in PDI was noticed between the 2 strategies (Fig. 3B). Subsequent, we sought to find out which elements of the change options contributed to those variations. We assessed the affect of pH (pH ranges of 4, 5, and 6), salt sort (PS, CS and AS), and salt focus (10, 25, and 50 mM) on particle measurement, zeta potential, and EE in each processes (Determine S3A). For particle measurement, LNPs ready by course of A with measurement beneath 100 nm, had been considerably smaller than these ready by course of B, which was bigger than 100 nm below all circumstances. This can be because of the pH rising from 4, 5, or 6 to 7 when including the ultimate storage solution-PBS. Concerning zeta potential, all LNPs in course of A excepting these exchanged with 50 mM salt focus or PS, confirmed considerably greater zeta potential than these in course of B. This can be attributed to the variations of floor cost arising from completely different pH circumstances. By way of EE, LNPs shaped by course of A confirmed considerably decrease EE than these shaped by course of B when the pH was 4 (79% vs. 94%, p < 0.0001), the salt focus was 50 mM (83% vs. 94%, p < 0.001), or the salt sort was PS (83% vs. 93%, p < 0.001). Below different circumstances, no vital variations had been noticed.

We then examined how the pH, salt sort, and salt focus affected the physicochemical properties of LNPs in every course of. The connection between pH, salt sort, focus, and physicochemical properties in course of A was analyzed (Fig. 3D and Determine S3B). We discovered that the pH of the change options was related to all of the examined physicochemical properties (Fig. 3D), aside from zeta potential (R² = 0.16, p = 0.27, Fig. 3H). As proven in Fig. 3E, particle measurement exhibited a powerful unfavourable correlation with pH (R² = 0.66, p = 0.008). When the pH was 4, the particle measurement was round 100 nm, which decreased to roughly 80 nm at pH = 5 and 6. These outcomes might be associated to the completely different particle constructions shaped below varied pH circumstances, as reported beforehand [35]. Moreover, EE decreased because the acidity of the change resolution elevated (Fig. 3F). At pH = 4, the EE was 14.8% decrease in comparison with pH = 6. As well as, the PDI decreased from 0.15 to 0.05 because the pH dropped from 5 or 6 to 4 (Fig. 3G). In distinction, the kind and focus of salt within the change options had no vital affect on the physicochemical properties of LNPs (Fig. 3D and Determine S3B).

On the mobile expression degree, pH was a important issue. A unfavourable correlation between pH and mobile expression was noticed in DC2.4, AML12 and C2C12 cells (Fig. 3D, Determine S3D and S3E), with greater mobile expression seen when the pH was 4 or 5 (Fig. 3I and J and Determine S3E). Notably, DC2.4 cells had been essentially the most delicate mannequin among the many three cell traces. Moreover pH, the luciferase sign in DC2.4 cells was additionally considerably influenced by particle measurement and EE (Fig. 3D and Ok), which the luciferase expression in DC2.4 was elevated with the rising of measurement (R² = 0.86, p = 0.0003) and lowering of EE (R² = 0.8, p = 0.0012). Evaluating with pH, different elements of change options similar to salt sort, focus confirmed restricted impact on the mobile expression of LNPs in each cells (Determine S3C).

In course of B, there was no vital correlation between the change options and the physicochemical properties or mobile expression of LNPs (Fig. 3L and M). Apart from C2C12, the luciferase sign was solely influenced by salt sort quite than pH and salt focus (Determine S3G). The best expression in C2C12 cells was noticed when PS was used because the salt which was 50% greater than CS because the salt (Determine S3F and S3G). Furthermore, the pH, salt sort, and focus confirmed restricted results on the expression of LNPs within the three cell traces (Determine S3F-I).

Based mostly on these findings, change resolution with 10 mM PS confirmed comparatively greater protein expression and was chosen as the popular change resolution for additional research.

The impact of storage options on physicochemical properties and transfection of LNPs

Lastly, we examined whether or not the physicochemical properties and mobile expression of LNPs had been influenced by the storage resolution (Fig. 4A). Particularly, we investigated the impact of salt sort (Tris, HEPES, PBS), salt focus (10, 20, 30 mM), and sucrose content material (0, 150, 300 mM) of the storage resolution (Desk S6) on LNPs. The precise compositions and the physicochemical properties of every group had been listed in Desk S6. The info revealed that the storage options had no vital impact on the physicochemical properties of LNPs (Fig. 4C). The dimensions of LNPs ranged from 79.5 to 99.6 nm, with near-neutral zeta potential, and EE ranged from 80.1 to 91.7% (Desk S6).

Subsequent, we evaluated the affect of the storage options on mobile expression. In DC2.4 cells, a constructive correlation was noticed between sucrose content material and expression, with the very best LNP expression at 300 mM sucrose focus, as proven in Fig. 4C and D. When the sucrose content material elevated from 0 to 300 mM, the luciferase sign elevated by 31%. In distinction, the kind and focus of salt had no vital impact on expression (Fig. 4E). In AML12 cells, nevertheless, solely the kind of salt influenced expression, with decrease mobile expression noticed when HEPES was used in comparison with PBS and Tris (Determine S4A). Moreover, it was intriguing to notice that EE was negatively correlated with mobile expression in AML12 cells (Fig. 4F). This might be on account of comparatively low EE selling the discharge of mRNA from LNPs, thereby facilitating expression. Furthermore, the kind of storage options had no impact on the expression of LNPs in C2C12 cell line (Determine S4B).

mRNA-based medication are unstable below vigorous shaking and long-term storage in resolution type [47, 48], and consequently, mRNA medication are usually preserved and transported after freezing or lyophilization. Moreover, below frozen situation, mRNA degradation happens extra slowly. Nevertheless, the freeze-thaw course of could disrupt the steadiness of mRNA/LNP formulations, resulting in vital modifications of their physicochemical properties and decreased expression each in vitro and in vivo [34]. Printed research had indicated that the storage options considerably impacted the freeze-thaw stability of mRNA/LNP medication [40]. Subsequently, we investigated the impact of various storage options on the steadiness of mRNA/LNP throughout the freeze-thaw course of, offering a basis for the storage situation of mRNA-based medication (Fig. 4G-J).

We discovered that sucrose focus considerably influenced each the modifications in physicochemical properties and mobile expression of LNPs after the freeze-thaw course of (Fig. 4Ok and N). Particularly, LNPs with out sucrose safety tended to mixture, forming bigger particles and leaking mRNA, which decreased the EE after freeze-thaw (Fig. 4Ok and L). After freezing, LNPs within the 0% sucrose group skilled a 300% enhance in measurement and a 20% lower in EE. In distinction, when the sucrose focus was elevated to 150 mM or 300 mM, no vital modifications in particle measurement or EE had been noticed (Fig. 4Ok and L).

Moreover, sucrose focus affected the expression of LNPs in DC2.4 cells after freeze-thaw, with the change in expression inversely proportional to the sucrose focus (Fig. 4C and M). This phenomenon was not noticed in AML12 cells, which might be associated to cell-specific elements (Fig. 4N). Moreover, the kind and focus of salt within the storage options had no vital affect on particle measurement, EE, or mobile expression of LNPs earlier than or after freeze-thaw (Determine S4C-F).

On this examine, just like earlier experiences [34], sucrose was recognized as a important issue influencing the steadiness of mRNA/LNPs. Though mRNA/LNPs with out sucrose exhibited vital modifications in measurement and EE, cell expression enchancment was noticed in DC2.4 cell traces. This might be attributed to the desire of DCs for phagocytosing the aggregates [49]. Based mostly on these findings, a sucrose focus of 300 mM was chosen for higher freeze-thaw stability. Tris was chosen as salt species, as a result of it may present extra safety for ionizable lipids [33] and confirmed no vital distinction in comparison with different salts. The salt focus was set to twenty mM to align with industrial formulations.

The affect of built-in Preparation options on physicochemical properties and transfection of LNPs

After systematically evaluating the person results of varied preparation options on the physicochemical properties and transfection effectivity of mRNA/LNPs, we noticed a number of intriguing phenomena. Nevertheless, these findings had been derived below circumstances the place different preparation options remained fixed, elevating the query of whether or not these outcomes would persist when a number of preparation parameters had been concurrently diverse. Moreover, sure outcomes from earlier experiments exhibited limitations in parameter ranges, which may compromise the robustness of the conclusions. To deal with these uncertainties, we built-in the optimized preparation circumstances primarily based on the efficiency of physicochemical properties and in vitro transfection effectivity, and subsequently redesigned the preparation course of (Fig. 5A, Desk S7). Given the demonstrated advantages of pH 4 mRNA options, we chosen a variety of pH 4 mRNA options, various in salt concentrations (10, 25, 50, 100 mM) and salt sorts (CS, CS + NaCl, AS, AS + NaCl), as candidates for additional analysis. Though no vital variations had been noticed within the dilution resolution, we expanded the screening scope by incorporating various salt species (PS, CS, AS, Tris), salt concentrations (10, 25, 50 mM), and pH ranges (4, 5, 6, 7) to make sure complete protection.

Moreover, primarily based on preliminary findings indicating that the pH of the change resolution considerably influenced mRNA/LNP formation, we narrowed the screening parameters to verify this statement by designing change options with pH 4, 5, 6, and seven PS. Lastly, whereas 300 mM sucrose was recognized as a important part for stabilizing mRNA/LNPs throughout freeze-thaw cycle, the potential interplay between sucrose and different excipients within the storage resolution remained unexplored. To deal with this, we mixed 300 mM sucrose with Tris options of various pH ranges to additional assess the affect of storage resolution composition on mRNA/LNP stability and efficiency. As a management, we employed generally used preparation circumstances, the place 10 mM CS (pH 4) served because the mRNA aqueous resolution, and PBS was used for dilution, change, and storage. We investigated the physicochemical properties, in vitro transfection effectivity (Fig. 5B and Determine S5A), and in vivo expression (Fig. 5C, D and G) of LNPs shaped by completely different options and analyzed their correlation (Fig. 6A).

By way of physicochemical properties, the hydrodynamic diameter of those LNPs ranged from 93.0 to 122.6 nm, with a PDI between 0.05 and 0.19, zeta potential starting from 0.4 to 2.7 mV, and EE starting from 93.5 to 97.4%, which had been greater than these of the management group (EE = 88.2%, Desk S7). Then we analyzed the correlation between physicochemical properties of LNPs and elements of options. Determine 6A and B confirmed that the pH of the change resolution was essentially the most important issue, exhibiting a constructive relationship with PDI and EE (p = 0.04). Nevertheless, the kind and focus of salt within the mRNA aqueous resolution, in addition to the addition of sodium chloride, had no impact on the particle measurement, PDI, or EE of LNPs (Determine S5B), besides when the salt sort was CS, by which case the EE decreased by 6% in comparison with when AS was used. The salt focus within the diluent, change resolution and storage resolution had no affect on the physicochemical properties of LNPs (knowledge not confirmed).

Concerning mobile expression, the management group demonstrated comparatively higher efficiency in all cell traces, significantly in AML12 and C2C12, by which the management group exhibited the very best luciferase expression (Fig. 5B and Determine S5A). In DC2.4 cells, solely E1, E5, E6, and E16 confirmed comparable luciferase expression to the management group (Fig. 5B). Nevertheless, we didn’t discover any clearly correlation between elements of options and mobile expression as confirmed in Fig. 6A.

Subsequently, we investigated the expression of various LNPs in mice after i.v. or i.m. injection. We discovered that, with the identical lipid formulation, altering the options used throughout the LNP preparation course of may considerably affect the expression depth in mice (Fig. 5C and D-G). After i.v. injection, E4, E7, E8, and E10 exhibited comparable expression to the management group, whereas E1, E6, E9, and E16 teams demonstrated considerably decreased luciferase alerts (Fig. 5D).

Concerning i.m. administration, the outcomes of luciferase expression differed significantly from these noticed with i.v. administration. For luciferase sign on the injection web site, teams E1, E2, E5, and E6 exhibited considerably decrease expression in comparison with the management group (Fig. 5E). By way of luciferase expression at liver after i.m. injection, all teams besides E10 confirmed considerably decrease luciferase expression than the management group (Fig. 5F). Consequently, most experimental teams, besides E15, had considerably decrease biodistribution outdoors the injection web site after i.m. administration (Fig. 5G). These findings indicated that the options used to type LNPs not solely affected their physicochemical properties and in vitro transfection effectivity but in addition influenced protein translation in vivo.

Subsequent, we analyzed which elements of the preparation options may have an effect on the expression of LNPs in vivo. The info confirmed that rising the pH of the change resolution enhanced mRNA expression at liver, no matter whether or not the administration by means of i.v. (p = 0.008) or i.m. (p = 0.0015) (Fig. 6C). Nevertheless, the mRNA aqueous resolution, dilution, change resolution and storage resolution had no vital impact on in vivo expression (Determine S5C-E). Furthermore, there was a powerful correlation between protein expression on the injection web site and that at liver, with a p-value of 0.017 after i.m. injection (Fig. 6D).

Moreover, we discovered that the expression of LNPs in AML12 cells in vitro was extremely according to that noticed at liver after i.v. administration in vivo (p = 0.048, Fig. 6E), suggesting that AML12 cells might be used to foretell LNP expression in vivo.

The steadiness of LNPs shaped with completely different options after freeze-thaw

Subsequently, the steadiness of LNPs ready utilizing completely different preparation options was evaluated below freeze-thaw stress. We discovered that the scale of the management group, which lacked sucrose safety, elevated greater than 10 instances after freeze-thaw (Fig. 7A). In distinction, the scale of some LNPs dispersed in a Tris/sucrose resolution modified by lower than 25% in comparison with the preliminary measurement, aside from E4, E7, and E13 (Fig. 7A), which might be associated to the pH of the change resolution (Fig. 7E). Moreover, a lot of the modifications of EE had been beneath 10%, aside from E5 (12.0%), E6 (14.3%), E8 (11.2%), and the management group (17.6%) (Fig. 7B).

Subsequent, expression modifications in vitro had been evaluated to evaluate the resistance of LNPs to the freeze-thaw course of. To our shock, though the physicochemical modifications of most LNPs met the suitable standards, greater than 25% of transfection discount was noticed in each DC2.4 and AML12 cells, aside from E11, E13, and E15 (Fig. 7C and D). Moreover, we discovered that the change ratio of expression in AML12 and DC2.4 cells was positively correlated with the change ratio of EE (Fig. 7F and G). Among the many completely different LNP formulations, LNP E15 exhibited the most effective stability.

We then sought to find out why LNPs with the identical lipid composition however completely different preparation options exhibited various physicochemical properties and expression profiles in vitro and in vivo. We hypothesized that completely different preparation options could affect the construction of LNPs, subsequently influencing their efficiency. To check this speculation, samples from E1, E7, E9, E15, and the management group — chosen for his or her distinct efficiency — had been additional characterised utilizing cryo-TEM. As proven in Fig. 7H, the construction of LNPs from completely different preparation circumstances diverse considerably. For instance, particles from the management group exhibited almost 10% of bleb constructions (5.7% of particles with blebs occupying greater than one-third of the LNP quantity, and 4.4% of particles with blebs occupying lower than one-third of the LNP quantity). Along with the bleb construction, a notable variety of empty LNPs smaller than 30 nm had been noticed within the E1 group. Moreover, the next proportion (> 50%) of LNPs carrying blebs was noticed within the E7 group in comparison with different teams. Though particles from the E9 and E15 teams exhibited a extra homogeneous construction than these from E1, E7, and the management group, the form and inner options of the LNPs from E1 and E7 require additional investigation to know the variations. The structural variations between the LNPs could partially clarify the various efficiency of LNPs ready below completely different circumstances, however extra experiments are wanted to confirm this speculation.

Affect of salt resolution on the physicochemical properties and in vitro/ in vivo expression of mRNA/LNP | Journal of Nanobiotechnology

The impact of mRNA aqueous resolution on LNP physicochemical properties and transfection in vitro

The impact of Dilution on physicochemical properties and transfection of LNPs

The impact of change resolution on physicochemical properties and transfection of LNPs

The impact of storage options on physicochemical properties and transfection of LNPs

The affect of built-in Preparation options on physicochemical properties and transfection of LNPs

The steadiness of LNPs shaped with completely different options after freeze-thaw

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