Role of size, surface charge, and PEGylated lipids of lipid nanoparticles (LNPs) on intramuscular delivery of mRNA

doi:10.21203/rs.3.rs-4659748/v1

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Role of size, surface charge, and PEGylated lipids of lipid nanoparticles (LNPs) on intramuscular delivery of mRNA

https://doi.org/10.21203/rs.3.rs-4659748/v1

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Background

Lipid nanoparticles (LNPs) are currently the most commonly used non-viral gene delivery system. Their physiochemical attributes, encompassing size, charge and surface modifications, significantly affect their behaviors both in vivo and in vitro. Nevertheless, the effects of these properties on the transfection and distribution of LNPs after intramuscular injection remain elusive. In this study, LNPs with varying sizes, lipid-based charges and PEGylated lipids were formulated to study their transfection and in vivo distribution. Luciferase mRNA (mLuc) was loaded in LNPs as a model nucleic acid.

Results

In vivo and in vitro results indicated that smaller-sized LNPs and those with neutral potential presented superior transfection efficiency after intramuscular injection. Surprisingly, the sizes and charges did not exert a notable influence on the in vivo distribution of the LNPs. Furthermore, PEGylated lipids with shorter acyl chains contributed to enhanced transfection efficiency due to their superior cellular uptake and lysosomal escape capabilities. Notably, the mechanisms underlying cellular uptake differed among LNPs containing various types of PEGylated lipids, which was primarily attributed to the length of their acyl chain.

Conclusions

Together, these insights underscore the pivotal role of nanoparticle characteristics and PEGylated lipids in the intramuscular route. This study not only fills crucial knowledge gaps but also provides invaluable directions for the effective delivery of mRNA via LNPs.

lipid nanoparticles

size

surface charge

PEGylated lipids

transfection

distribution

Thanks to their numerous advantages, including precise targeting, low drug resistance, and high potency [1], a significant number of nucleic acid drugs have been successfully launched into markets [2]. For instance, Onpattro® and Inclisira® are small interfering RNA (siRNA) drugs targeting transthyretin and hyperlipidemia, respectively [3. 4]. Apart from metabolic diseases, messenger RNA (mRNA) vaccines have emerged as a pivotal tool in the fight against infectious diseases, particularly since the outbreak of COVID-19 [5]. mRNA vaccines are suitable for the treatment of multiple infectious diseases due to their rapid research and development [6]. Furthermore, mRNA undergoes transient translation in vivo and is subsequently degraded within a temporally precise yet regulatable duration [7], providing excellent safety for mutagenesis without integration into the host genome. Therefore, mRNA vaccines have become increasingly important in the research and development of vaccines for infectious diseases.

Nevertheless, the primary challenges associated with mRNA vaccines include their propensity for degradation and low transfection efficiency [8]. Delivery systems thus play pivotal roles in the efficacy of mRNA vaccines. Currently, lipid nanoparticles (LNPs) are recognized as the most effective and widely utilized non-viral vectors [1. 9]. The encapsulation of mRNA within LNPs serves as a protective shield, substantially mitigating mRNA degradation by RNase enzymes and facilitating the penetration of the mRNA into target tissues [10]. Additionally, LNPs significantly enhance both in vivo and in vitro transfection and protein expression [9]. It has been reported that the model mRNA encapsulated by LNPs is successfully delivered and translated into proteins, which persist in expression for a minimum duration of one week [11]. Moreover, the repeated intramuscular administration of antigen mRNA via LNPs leads to massive production of neutralizing antibodies [12] and antigen-specific T-cell immune responses [13].

LNPs are composed of four distinct lipid classes, including ionizable lipids, helper lipids, cholesterols, and PEGylated lipids [6. 8]. The particle size, charge, and lipid composition of nanoparticles have been shown to significantly influence their transfection capabilities and biodistribution following intravenous administration [11. 14. 15]. Physiochemical properties and PEGylated lipids are recognized as critical determinants in the functional performance of LNPs, influencing key processes such as transfection and systemic distribution [16]. LNPs with positive charges preferentially localize to the spleen following intravenous injection, whereas those with negative charges are more likely to accumulate in the lungs [17]. The primary mechanism underlying the distinct in vivo distribution patterns of charged LNPs is attributed to variations in the protein corona adsorbed onto the nanoparticles, which significantly affects cellular uptake [18]. Moreover, PEGylated lipids also play essential roles in LNPs for mRNA delivery [19. 20]. PEGylated lipids contribute to the evasion of macrophage capture, thereby enhancing nanoparticle stability and prolonging their efficacy in vivo [21. 22]. The presence of PEGylated lipids in LNPs extends their circulation time in vivo, which is particularly advantageous for intravenous injection [1. 23]. Upon entering the circulation, PEGylated lipids facilitate the interaction of LNPs with apolipoprotein E (ApoE) in serum, promoting subsequent binding to low-density lipoprotein receptor (LDLR), which is primarily located in the liver [20. 24]. However, studies on the impact of composition on the biofunctions of LNPs post-intramuscular administration are still lacking. Although numerous studies have been conducted on the impact of particle size, no consensus has been reached. For instance, some studies demonstrated that LNPs with a diameter of approximately 140 nm showed superior transfection both in vitro and in vivo [25], and smaller LNPs were prone to liver accumulation post-intramuscular injection [26. 27]. In contrast, a prior study indicated that mRNA-LNP vaccines with distinct sizes elicited comparably robust immune responses in non-human primates [28]. It has to admit that researches on these aspects are far from comprehensive and systematic.

Moreover, PEGylated lipids are implicated in the elicitation of side effects following intramuscular injection. Notably, serum anti-PEG-IgG levels have been reported to increase 13.1-fold post-vaccination with mRNA-1273, while anti-PEG-IgM levels have surged by 68.5-fold [29]. The induction of anti-PEG antibodies by PEGylated lipids may trigger hypersensitivity reactions by activating the complement system [20. 30. 31]. Consequently, it is also imperative to conduct studies on the impact of both the type and content of PEGylated lipids in LNPs on transfection following intramuscular injection.

In this study, a model mRNA, luciferase mRNA (mLuc), was loaded into LNPs to elucidate the impacts of sizes, charges, and PEGylated lipids on transfection efficiency and in vivo distribution. First, LNPs with varying sizes, charges, and various contents of PEGylated lipids were formulated. Subsequently, their transfection efficacy in vitro and in vivo was thoroughly evaluated. Meanwhile, fluorescence resonance energy transfer (FRET) technology, which labels intact nanoparticles, was utilized to monitor LNPs in vivo. Additionally, LNPs with different types of PEGylated lipids were assembled to assess their implications for transfection in vitro and in vivo. Finally, analyses of cellular uptake and lysosomal escape were conducted to elucidate the underlying mechanisms contributing to differences in transfection efficiency among various PEGylated lipid types in detail.

2.1. Materials

1-Octylnonyl 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoate (SM-102), 1,2-dioctadecanoyl-sn-glycero-3-phophocholine (DSPC), cholesterol, (2,3-dioxypropyl) trimethylammonium chloride (DOTAP), 1,2-distearoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DSPG), 1,2-dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol 2000 (DMG-PEG2k), diastearyl-rac-glycerol polyethylene glycol 2000 (DSG-PEG2k), and diethyl phosphatidylethanolamine polyethylene glycol 2000 (DSPE-PEG2k) were purchased from AVT Pharmaceuticals. Luciferase mRNA was purchased from APExbio. 1,1'-Dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine (DiD), 1,1'-Dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide (DiR) and LysoTracker Green were purchased from Meilunbio. The Quant-iT RiboGreen RNA Reagent and Kit were purchased from Thermo Fisher Scientific. Chlorpromazine (CPZ), filipin (FIL), cytochalasin D (CYTD), and wortmannin (WORT) were purchased from AbMol. Firefly luciferase reporter gene detection kit and D-Luciferin potassium salt were purchased from Beyotime Biotechnology. A polyacrylamide gel electrophoresis (PAGE) gel rapid preparation kit and Tris/Glycine/ Sodium dodecyl sulfate (SDS) electrophoretic buffer were purchased from Epizyme Biotech. GoldBand 3-color High Range Protein Marker and protein loading buffer were purchased from Yeasen Biotechnology. Fetal Bovine Serum (FBS) was purchased from Gibco.

2.2. Preparation of LNPs

LNPs with different sizes are formulated by microfluidic mixing [32–34]. Briefly, an ethanol phase containing SM102, DSPC, cholesterol, and DMG-PEG2k was mixed with an aqueous phase (citrate buffer, pH 4.0) containing mRNA at an N/P ratio of 10:1 in a microfluidic device (INano^™ E). The molar ratio of lipids (SM102:DSPC:cholesterol:DMG-PEG2k) was 50:10:48.5:1.5. The particle size of the LNPs was modulated by regulating the total flow rate and the flow ratio between the ethanol phase and aqueous phase (details are shown in Tab. S1). The obtained LNPs were further filtered using a 100 kDa ultrafiltration tube to exchange the buffer and stored at 4°C. DiD- or DiR-labeled LNPs were obtained by mixing DiD or DiR (1 mol% of total lipids) in the ethanol phase before microfluidic mixing.

LNPs with different charges are also formulated by microfluidic mixing. The details of the lipid molar ratios are shown in Tab. S2. The ethanol phase containing lipids was mixed with the aqueous phase containing mRNA at an N/P ratio of 10:1. A flow ratio of 3:1 and a total flow rate of 12 mL/min were applied to synthesize the LNPs.

LNPs containing varying contents of PEGylated lipids were formulated at molar ratios of PEGylated lipids ranging from 0.5 mol% to 3 mol%. LNPs with various types of PEG2k include DMG-PEG2k, DSG-PEG2k, and DSPE-PEG2k at a mole ratio of 1.5 mol%.

2.3. Characterization of LNPs

The hydrodynamic size, polydispersity index (PDI), and zeta potential of the LNPs were measured using a Zeta-Sizer Nano (Malvern Instruments, Worcestershire, UK). The efficiency of mRNA encapsulation was determined using a modified Quant-iT RiboGreen RNA assay (Invitrogen). To measure the total amount of nucleic acid, including encapsulated and free mRNA, a solution of RNA quantification reagent with Triton X-100 was used to disrupt the LNPs. Moreover, a solution of the reagent without Triton X-100 was utilized to measure the amount of free mRNA. After loading with an equal volume of RiboGreen reagent, the fluorescence of the samples was measured using a microplate reader with excitation (Ex) and emission (Em) wavelengths set to 480 nm and 520 nm, respectively [32].

LNPs stained with phosphotungstic acid were dripped lightly dripped onto a copper siever with a supporting carbon film before observation. The morphology was observed via JEM-1230 transmission electron microscopy (TEM) (JEOL, Tokyo, Japan) at an acceleration voltage of 120 kV.

2.4. Spectroscopic investigation of DiD-DiR loaded LNPs

A total of 400 µL of LNPs labeled with DiD-DiR was taken in the fluorescence spectrophotometer sample cell. As a control, an equivalent concentration of DiD and DiR tetrahydrofuran mixed solution was utilized. The emission spectra were scanned and recorded using a slit width of 5, with an Ex wavelengths of 640 nm, respectively.

2.5. In vitro transfection of mLuc

HEK-293 cells and DC2.4 cells were seeded onto a 24-well plate at a density of 200,000 cells per well. After culture at 37°C overnight, mLuc-loaded LNPs were treated to cells at a concentration of 1 µg/mL prior to incubation for 24 h. Luciferase expression was evaluated by a firefly luciferase reporter gene detection kit (RG005, Beyotime), and total protein was measured by using a Bradford Protein Assay Kit (P0006, Beyotime).

2.6. In vivo expression and distribution of luciferase

All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) at the School of Pharmacy, Fudan University, China. BALB/c mice weighing 18 to 20 g were injected intramuscularly with LNPs containing mLuc at a dose of 3 µg mRNA per mouse. Mice were intraperitoneally injected with D-luciferin potassium salt (150 mg/kg) at different time intervals (0.5 h, 2 h, 4 h, 6 h, 8 h, 12 h, and 24 h) and imaged by an in vivo imaging system (IVIS) Lumina system (PerkinElmer). Bioluminescence values were quantified by measuring photon flux (photons/second) in the region of interest using Living Image Software (PerkinElmer). The FRET signals of the LNPs were recorded by an IVIS (Ex: 640 nm; Em of FRET: 780 nm; Em of donor: 680 nm).

2.7. Cellular uptake of LNPs

HEK-293 cells and DC2.4 cells were seeded onto 24-well glass bottom plates for laser confocal imaging at a density of 1×10⁵ cells per well and cultured at 37°C overnight. Then, cells were treated with DiD-labeled mLuc LNPs at an mRNA concentration of 1 µg/mL. After incubation at 37°C for 3 h, the cells were washed twice with 1× PBS and fixed with 4% polyformaldehyde (PFA) for 5 min. Then, the cells were stained with DAPI for 10 min in the dark and washed twice with 1× PBS. Images were captured by fluorescence microscopy (LSM710, Zeiss) at 20× magnification.

To study the mechanism of cellular uptake, HEK-293 cells and DC2.4 cells were seeded onto a 12-well plate at a density of 4×10⁵ cells per well and further cultured overnight. The cells were pre-incubated with various endocytosis inhibitors, including 5 µM CPZ, 10 µM FIL, 3 µM CYTD, and 5 µM WORT, for 30 minutes. Then, DiD-loaded mLuc LNPs were added to the cells at an mRNA concentration of 1 µg/mL and incubated for 4 h. Afterwards, the cells were digested with trypsin, washed twice with 1× PBS, and then resuspended in 1× PBS containing 1% FBS. Cellular uptake was quantitatively analyzed by a CytoFlex S flow cytometer (Beckman, Brea, CA, USA). The mean fluorescence intensity was measured by FlowJo 10.8.1 (FlowJo Software, Ashland, OR, USA).

2.8. Protein corona of LNPs with various PEGylated lipids

LNPs with various PEGylated lipids were prepared according to a previously described method and were concentrated to 1 g/L with 1× PBS. FBS was added to each LNP solution at a 1:3 volume ratio prior to incubation for 1 h at 37 ℃. The mixture of LNPs and plasma was loaded onto a 0.7 mol/L sucrose cushion of equal volume to the mixture and centrifuged at 15,300 × g and 4 ℃ for 1 h. The supernatant was removed, and the pellet was washed with 1× PBS. Then, the pellet was centrifuged at 15,300 × g and 4°C for 5 min, after which the supernatant was removed. The samples were washed a total of three times. Following the final wash, the pellet was resuspended in radioimmunoprecipitation assay (RIPA) buffer. The concentration of protein in each sample was quantified by the BCA Protein Assay Kit.

The protein adsorbed on the LNPs was mixed with protein sample loading buffer and boiled at 90°C for 15 min. The protein samples were separated by electrophoresis via 12.5% polyacrylamide gel at a voltage of 120 V. Then, the gel was stained with a 0.25% Coomassie blue solution. After washing with Milli-Q water overnight, the gel was imaged with a gel imager (ChemiDoc, Bio-Rad).

2.9. Lysosomal escape

HEK-293 cells and DC2.4 cells were seeded onto a 24-well glass-bottom plate at a density of 4×10⁵ cells per well and further cultured for 24 h. Then, cells were treated with DiD-labeled LNPs for 4 h. Afterwards, cells were incubated with LysoTracker green at a dose of 100 nM LysoTracker Green for 2 h and stained with DAPI for 5 min. The colocalization of DiD and LysoTracker was imaged by fluorescence microscopy (SpinSR10, Olympus), and analyzed by ImageJ. The presence of LNPs within lysosomes was quantified by calculating the ratio of fluorescence colocalization between DiD and LysoTracker to the total fluorescence of DiD.

2.10. Statistical analysis

In vitro and in vivo data are presented as mean ± standard error of mean (SEM). The statistical analysis was conducted using GraphPad Prism 9.0 (GraphPad Software, La Jolla, CA, USA) with one-way analysis of variance (ANOVA). A p-value less than 0.05 was considered as statistically significant.

3.1. The effects of particle size on nanoparticle transfection and in vivo distribution

To highlight the influence of LNP size on transfection and in vivo distribution, LNPs were engineered in three distinct sizes including small (S), medium (M), and large (L) particles (Fig. 1A). The specifics of the particle synthesis are detailed in Tab. S1. Modifications in the flow ratio or rate during synthesis were employed to adjust the particle sizes, resulting in average diameters of 94.61 nm for small-sized LNPs (LNP-S), 121.93 nm for medium-sized LNPs (LNP-M), and 167.37 nm for large-sized LNPs (LNP-L). The PDI for all sizes was maintained below 0.2, indicating a uniform size distribution. The zeta potentials for each size group were in the − 5 to 5 mV range, demonstrating their neutral surface charge (Table 1). These LNPs with distinct particle sizes show good encapsulation of mRNA, as the encapsulation efficiencies (EEs) are all above 90%. TEM images shown in Fig. 1A confirm that LNP-S, LNP-M, and LNP-L are spherical and monodispersed. Figure 1B illustrates the size distribution of LNP-S, LNP-M, and LNP-L.

Table 1

Particle size, PDI, zeta potential, and encapsulation efficiency (EE) of LNPs
	Particle size (nm)	PDI	Zeta potential (mV)	EE (%)
LNP-S	94.61 ± 1.12	0.13 ± 0.02	-1.58 ± 0.37	90.4 ± 0.3
LNP-M	121.93 ± 1.56	0.14 ± 0.01	-0.59 ± 0.02	95.4 ± 0.1
LNP-L	167.37 ± 1.86	0.06 ± 0.01	1.26 ± 0.21	90.9 ± 0.1

LNPs were labeled with the FRET pair (DiD-DiR), demonstrating a substantial FRET effect (Fig. 1C, Fig. 1D). The evaluation of these LNPs both in vitro and in vivo is illustrated in Fig. 1E. The immortalized cell lines, HEK-293 and DC2.4, were transfected with LNP-S, LNP-M, or LNP-L. As shown in Fig. 1F, LNP-S exhibited the highest transfection efficiency in both cell lines. Specifically, in HEK-293 cells, the transfection efficiencies of LNP-M and LNP-L were similar, whereas in DC2.4 cells, LNP-M outperformed LNP-L. Analogous results were observed following intramuscular injection (Fig. 1G). In addition, by labeling intact LNPs with a FRET pair (DiD-DiR), we found that the majority of the LNPs remained localized at the site of injection, insusceptible to the particle size (Fig. 1H). Luminescence images of all the time and ex vivo images are presented in Fig. S1A and Fig. S1B. LNP-S exhibited the highest transfection efficiency in situ (Fig. 1I, Fig. S1C). Notably, luciferase expression was detected in the liver across LNPs with all sizes, aligning with the previous finding [26]. Among these LNPs, no significant difference in luciferase expression was detected in the liver (Fig. 1J, Fig. S1D). Despite the observed significant differences in transfection efficiency in vivo, the in situ FRET ratio showed similar trends among LNP-S, LNP-M, and LNP-L (Fig. 1K).

As shown above, particle size significantly influences the transfection efficiency of LNPs in vitro. The observed differences in transfection efficiency between HEK-293 and DC2.4 can be attributed to cell types. In addition, particle sizes play a crucial role in transfection rather than the distribution of LNPs following intramuscular injection. Smaller nanoparticles may penetrate cells more readily and enter the blood circulation more rapidly [35]. Protein translated from mRNA is also found in the liver, a phenomenon that occurs independently of nanoparticle-mediated delivery following intramuscular injection. It is possible that the protein expressed at the injection site is transported to the liver by other cells, such as dendritic cells or macrophages. The observed consistency in FRET ratios across different particle sizes may result from a desynchronization between the disintegration of nanoparticles and the expression of the encoded protein. This phenomenon could be explained by previous findings, which have indicated that the structural properties of larger LNPs differ significantly from those of smaller ones [25. 36]. This structural variation could lead to a hypothesis that a similar number of nanoparticles release different quantities of mRNA. Investigations should be conducted in the future to prove such a hypothesis.

3.2. The effect of surface charge on nanoparticle transfection and in vivo distribution

To systematically investigate the impact of the LNP charge, formulations were prepared by incorporating varying concentrations of either DOTAP or DSPG. A schematic representation of the methodological approach, including both in vitro and in vivo experiments, is shown in Fig. 2A. The detailed compositions of these LNPs are tabulated in Tab. S2. The incorporation of DOTAP into the LNP formulation results in a net positive charge, whereas the inclusion of DSPG results in a net negative charge (Fig. 2B). The sizes of positive-charged LNPs slightly exceeded those of the neutrally charged LNPs, while negative-charged LNPs were comparable in size to neutral-charged counterparts (Fig. 2C). LNPs with 15% DOTAP exhibited a charge of approximately + 12.83 mV. Increasing the DOTAP content to 25% and 50% results in a correspondingly greater charge. Conversely, LNPs containing 15% DSPG showed a charge of approximately − 25.3 mV, a value consistent with that of LNPs prepared with 25% DSPG (Tab. S2).

Neutral LNPs demonstrated superior transfection efficiency compared to their counterparts mixed with DOTAP or DSPG in vitro (Fig. 2D). Interestingly, LNPs comprising 25% DOTAP exhibited superior in vitro transfection efficiency relative to those with 50% DOTAP, despite having similar particle sizes. This trend was mirrored in transfection experiments in vivo (Fig. 2E). Comprehensive luminescence images and ex vivo assessments are provided in Fig. S2A-B. Figure 2F illustrates the in vivo distribution of LNPs labeled with FRET pairs. Neutral LNPs achieved the highest luciferase expression both in situ and in the liver following intramuscular injection (Fig. 2G-H, Fig. S2C-D). Although variations in lipid composition were observed among the different nanoparticles, the in vivo distribution presented a homologous phenomenon (Fig. 2F, I). LNPs with a negative charge exhibited an enhanced FRET ratio in situ, indicating the superior structural integrity of the nanoparticles. Meanwhile, positive-charged LNPs presented similar integrity with neutral-charged LNPs (Fig. 2I).

Previous findings have underscored the critical role of particle size in the functional properties of LNPs. In assessing LNPs with neutral, positive, and negative charges, the influence of particle size cannot be excluded due to the larger size of positive-charged LNPs. Notably, an increase in the positive charge, achieved by incorporating 15%, 25%, and 50% DOTAP, correlates with a decrease in transfection efficiency. The accumulation of almost all intact nanoparticles at the injection site suggests that the in vivo distribution of LNPs during intramuscular injection is largely independent of charge. The enhanced structural integrity of negative-charged LNPs appears to hinder effective mRNA release, thereby reducing transfection efficiency. The observation of a slower decrease in the FRET ratio in LNPs containing 50% DOTAP, which corresponds with reduced lysis, corroborates the previously discussed results. This new phenomenon underscores the unique interactions within these specific nanoparticle formulations. Future investigations are warranted to elucidate the underlying mechanisms responsible for these novel findings, thereby enhancing our understanding of nanoparticle behaviors in biological systems.

3.3 The effect of PEGylated lipid contents on the properties of LNPs

The impact of PEGylated lipids on the characteristics and functional attributes of LNPs was systematically evaluated by modulating the concentration of PEGylated lipids within the formulations. Specifically, LNPs were synthesized with 0.5 mol%, 1.5 mol%, and 3 mol% DMG-PEG2k to assess the effects on particle size, stability, and transfection efficiency, respectively. As shown in Fig. 3A, a notable decrease in particle size was observed with increasing DMG-PEG2k contents, potentially due to the expansion of the compression function attributed to the PEGylated lipids. The PDI of LNPs with varying contents of PEGylated lipids remained consistently below 0.3 (Fig. 3B). The results revealed that the content of PEGylated lipids affected the stability of the encapsulation efficiency of the LNPs (Fig. 3D-F). Specifically, the encapsulation efficiency of LNPs with 0.5 mol% DMG-PEG2k decreased significantly over a four-week period (Fig. 3F). These findings suggest that a reduction in PEGylated lipid contents may compromise the stability of LNPs, highlighting the critical role of these components in maintaining the structural integrity and functional efficacy of LNPs [37].

LNPs with 1.5 mol% DMG-PEG2k exhibited superior transfection efficiency in vitro (Fig. 3G). Luminescence images captured at all time points and ex vivo luminescence data are provided in Fig. S3A-B, providing a comprehensive view of the temporal and spatial dynamics of nanoparticle-mediated gene expression. In vivo assessments confirmed that LNPs with 1.5 mol% DMG-PEG2k demonstrated enhanced transfection capabilities (Fig. 3H). Additionally, the in vivo distribution of these nanoparticles, containing varying contents of DMG-PEG2k, did not significantly differ (Fig. 3I). Nanoparticles are mainly concentrated at the injection site (localized intramuscular region). It is obvious that LNPs with 1.5 mol% DMG-PEG2k facilitated notably excellent luciferase expression in situ and in the liver 4 hours post-administration (Fig. 3J-K). Comprehensive luciferase expression in muscle and liver tissues is detailed in Fig. S3C-D. Despite similar particle sizes, LNPs with lower contents of PEGylated lipids showed better transfection efficiency. A notable observation was the delayed peak of luciferase expression in the liver when comparing LNPs with 1.5 mol% and 3 mol% DMG-PEG2k (Fig. 3K).

The impact of PEGylated lipid contents on the stability and transfection efficiency of LNPs represents a rarely explored area in nanoparticle research. Previous studies have suggested that the long circulation characteristics of PEGylated lipids are due to their resistance to cellular uptake [38]. This complies with our findings that LNPs with 3 mol% DMG-PEG2k show diminished transfection efficiency compared to those with 1.5 mol% DMG-PEG2k. Additionally, LNPs formulated with a lower concentration of PEGylated lipids (0.5 mol%) exhibited larger particle sizes, which further contributed to decreased transfection efficiency both in vitro and in vivo. The observed delay in the peak of luciferase expression in the liver might be attributed to the slower rate at which PEGylated lipids are shed from the nanoparticle structure. The slight differences observed in the FRET ratio among LNPs containing various contents of PEGylated lipids suggest the desynchronization between mRNA expression and nanoparticle degradation [39]. This suggests that while PEGylated lipids extend nanoparticle circulation, they may also impact the timing and efficiency of the intended gene expression within target tissues. Further investigations are necessary to fully elucidate the mechanisms by which PEGylation influences the biodistribution and functional efficacy of LNPs.

3.4. The impact of types of PEGylated lipids on the functions of LNPs

To investigate the roles of lipid nanoparticles (LNPs) formulated with different PEGylated lipids, DSPE-PEG2k was utilized in place of DMG-PEG2k, while maintaining identical molar ratios. Previous studies have indicated that C14-PEG2k lipids are shed more easily from LNPs, a critical process for effective mRNA delivery in vivo [40]. DSG-PEG2k, a C18-PEG2k, was incorporated into LNPs to eliminate the interference of the acyl chain length [2]. The structural formulas of DMG-PEG2k, DSG-PEG2k, and DSPE-PEG2k are depicted in Fig. 4A. LNPs with any type of PEGylated lipids showed comparable particle size, PDI, and zeta potential (Fig. 4B-D). Additionally, LNPs composed of DMG-PEG2k, DSG-PEG2k, and DSPE-PEG2k exhibited good colloidal stability for a minimum duration of one week when stored at 4°C (Fig. S4A-D). Concurrently, in PBS containing 10% FBS, LNPs with three distinct types of PEGylated lipids showed similar trends in particle size, PDI, zeta potential, and mRNA encapsulation efficiency (Fig. S4E-H).

Figure 4E illustrates the comparative analysis of transfection efficiency among LNPs incorporating DMG-PEG2k, DSG-PEG2k, and DSPE-PEG2k. Notably, luciferase activity significantly decreased within 72 hours following transfection with LNPs containing DMG-PEG2k, in contrast to the other lipid types (Fig. 4E). Differences in the optimal timing of peak luciferase expression may be attributed to the variable shedding rates of PEGylated lipids. The accelerated dissociation of DMG-PEG2k potentially facilitated more rapid mRNA translation. In a buffer system containing 10% FBS, a decrease in luciferase activity was observed, indicating that serum components may adversely affect cellular transfection efficacy (Fig. 4F). Further investigations demonstrated that LNPs formulated with DMG-PEG2k exhibited enhanced transfection performance in vitro, as depicted in Fig. 4F. This observation was corroborated by in vivo studies, where intramuscular injection of LNPs containing DMG-PEG2k showed superior transfection efficiency (Fig. 4G-I, Fig. S5A-B). Overall, the total luciferase expression results suggest that LNPs equipped with DMG-PEG2k possess a notably greater capacity for mRNA delivery (Fig. S5C-D).

The impact of PEG2k lipid types on the in vitro and in vivo transfection efficiency of LNPs may be associated with variations in acyl chain length. The enhanced transfection efficiency observed in LNPs containing DMG-PEG2k can be attributed to its rapid dissociation from the nanoparticle complex, which subsequently facilitates greater protein delivery to the liver. Additionally, the functional groups of PEGylated lipids play crucial roles in cellular transfection efficiency in vitro, as evidenced by comparative analyses between LNPs formulated with DSG-PEG2k and those formulated with DSPE-PEG2k (shown in Fig. 4E-F). Such observations regarding the differential roles of functional groups in PEGylated lipids have rarely been reported in previous studies.

3.5. Underlying cellular mechanisms for transfection affected by PEGylated lipids

To elucidate the mechanisms underlying the differences in transfection efficiency observed among LNPs formulated with various types of PEGylated lipids, the nanoparticles were labeled with DiD to study cellular uptake. Confocal microscopy revealed that the cellular uptake of LNPs by HEK-293 cells increased with increasing incubation time (Fig. 5A). Specifically, LNPs containing DMG-PEG2k exhibited significantly greater cellular uptake than the other LNPs (Fig. 5A). This increased uptake may be influenced by the acyl chain length of the PEGylated lipids, suggesting that LNPs with longer acyl chains may exhibit reduced cellular uptake efficiency. Functional groups of PEGylated lipids also affect cellular uptake in HEK-293 and DC2.4 cells. Notably, LNPs formulated with DSG-PEG2k demonstrated markedly superior cellular uptake efficiency compared to those formulated with DSPE-PEG2k (Fig. 5B). According to the results above, FBS affect cell transfection efficiency. Subsequent studies, shown in Fig. 5C, investigated the protein corona adsorbed by LNPs containing DMG-PEG2k, DSG-PEG2k, and DSPE-PEG2k in the presence of FBS. The results revealed no significant differences in the type of protein corona formed among these LNPs. The observed variations in cellular uptake between different PEGylated lipids are primarily attributed to the length of the acyl chain and the specific functional groups, factors that appear to be independent of the protein corona composition. These findings highlight the complex interactions between the structural attributes of PEGylated lipids and their functional performance in cellular environments. To investigate the specific cellular uptake mechanisms of LNPs with various types of PEGylated lipids, a series of inhibitors, including CPZ, FIL, CYTD, and WORT, were utilized. In HEK-293 cells, the uptake efficiency of LNPs with DMG-PEG2k was inhibited by CPZ and WORT, while the uptake of LNPs containing DSG-PEG2k and DSPE-PEG2k was inhibited by CPZ, FIL, and WORT (Fig. 5D-F). In DC2.4 cells, both CPZ and WORT impacted the cellular uptake of all tested PEGylated lipid types, and FIL also inhibited the cellular uptake of LNPs with DSG-PEG2k and DSPE-PEG2k (Fig. 5G-I).

The critical function of lysosomal escape in mRNA delivery is increasingly recognized. This study aimed to elucidate the differences in lysosomal escape among LNPs formulated with various types of PEGylated lipids. LysoTracker dye was used to specifically label lysosomes within cells, while DiD was used to mark the LNPs. The colocalization of LysoTracker and DiD signals was used to identify LNPs residing within lysosomes. To mitigate the influence of differential cellular uptake on the results, LNPs in the lysosome were analyzed by fluorescence colocalization/cellular uptake. In the cell lines HEK-293 and DC2.4, LNPs incorporating DMG-PEG2k demonstrated superior lysosomal escape efficiencies (Fig. 5J-L).

Previous studies have reported that acyl chain length affects the adsorption rate of the protein corona [38]. However, it does not significantly affect the type of protein corona formed, which has not been previously explored. The results in HEK-293 cells suggest that LNPs with DMG-PEG2k are primarily internalized via clathrin-mediated endocytosis and macropinocytosis. In contrast, macropinocytosis, along with phagocytosis and caveolin-mediated pathways, are implicated in the uptake of LNPs containing DSG-PEG2k and DSPE-PEG2k. These findings underscore the role of acyl chain length in modulating the cellular uptake mechanisms of LNPs. Moreover, the similar uptake mechanisms observed for LNPs with DSG-PEG2k and DSPE-PEG2k suggest that the specific functional groups of the PEGylated lipids do not significantly influence these processes. Nonetheless, the specific receptors and cellular pathways involved in these mechanisms warrant further investigation to fully understand the interactions at the cellular level.

Based on the findings presented above, the types of PEGylated lipids affect the lysosomal escape of LNPs, primarily depending on the acyl chain length rather than the functional groups of the PEGylated lipids. Specifically, a decrease in lysosomal escape efficiency was observed with increasing acyl chain length, a trend parallel to that of cellular uptake. Lysosomal escape is as crucial as cellular uptake for transfection among LNPs with various types of PEGylated lipids. Therefore, LNPs with DMG-PEG2k showed superior transfection efficiency over their counterparts. These findings have not been reported previously. Although some studies have suggested that acyl chains of PEGylated lipids may be shed upon cellular entry [38. 41], it remains unresolved whether this shedding occurs for all acyl chains of PEGylated lipids. Further research is required to clarify this mechanism and its implications for LNP design and functional performance in gene delivery applications.

In summary, this study conducted a systematic investigation into the influence of various nanoparticle characteristics on transfection efficacy. Our findings reveal that nanoparticle sizes, charges, and PEGylated lipids play crucial roles in transfection efficiency both in vitro and in vivo. In detail, smaller-LNPs, neutral-potential LNPs, and LNPs with 1.5 mol% PEGylated lipids demonstrated superior transfection performance. Moreover, the length of the acyl chain and functional groups are also significant factors influencing both the cellular uptake and lysosomal escape of LNPs, reaffirming the previously proposed mechanism that the length of the acyl chain could influence cellular uptake by modulating their shedding rate. The insights gained from this study are anticipated to guide future optimizations of LNPs and advance the understanding of PEGylated lipids in gene delivery systems.

LNPs Lipid nanoparticles

mLuc Luciferase mRNA

siRNA Small interfering RNA

mRNA Messenger RNA

ApoE Apolipoprotein E

LDLR Low-density lipoprotein receptor

FRET Fluorescence resonance energy transfer

SM-102 1-octylnonyl 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoate

DSPC 1,2-dioctadecanoyl-sn-glycero-3-phophocholine

DOTAP (2,3-dioxypropyl) trimethylammonium chloride

DSPG 1,2-distearoyl-sn-glycero-3-phospho-(1'-rac-glycerol)

DMG-PEG2k. 1,2-dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol 2000

DSG-PEG2k. Diastearyl-rac-glycerol polyethylene glycol 2000

DSPE-PEG2k. Diethyl phosphatidylethanolamine polyethylene glycol 2000

DiD. 1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine

DiR. 1,1'-Dioctadecyl-3,3,3',3'-Tetramethylindotricarbocyanine iodide

CPZ. Chlorpromazine

FIL. Filipin

CYTD. CytochalasinD

WORT. Wortmannin

PAGE. Polyacrylamide gel electrophoresis

SDS. Sodium dodecyl sulfate

FBS. Fetal Bovine Serum

PDI. Polydispersion index

TEM. Transmission electron microscopy

IACUC. Institutional Animal Care and Use Committee

IVIS. In vivo imaging system

PFA. Polyformaldehyde

RIPA. Radioimmunoprecipitation assay

SEM. Standard error of mean

ANOVA. Analysis of variance

EE. Encapsulation efficiencies

Ethics approval and consent to participate

All animal procedures are conducted in compliance with the Guide for the

Care and Use of Laboratory Animals published by the National Institutes of

Health (NIH Publication No. 85−23, 1996, revised 2011) and approved by the Institutional Animal Care and Use Committee (IACUC) at the School of Pharmacy, Fudan University, China.

Consent for publication

Not applicable

Availability of data and materials

Data will be made available on request.

Competing interests

The authors declare no competing interests

Funding

The work was supported by the National Natural Science Foundation of China (No. 82073801).

Authors’ contributions

W.K.: Data curation, Formal analysis, Investigation, Writing-original draft. Y.W.: Data curation, Investigation. Z.D.: Data curation. W. L.: Data curation. J. Z.: Data curation. Y.H.: Validation, Visualization. J.Y.: Validation, Visualization. W.W.: Validation, Visualization. H.H.: Validation, Writing – review & editing. J.Q.: Formal analysis, Methodology, Project administration, Supervision, Writing – review & editing.

The authors declare no conflict of interests.

Acknowledgments

Not applicable

Author details

^a Key Laboratory of Smart Drug Delivery of MOE, School of Pharmacy, Fudan University, Shanghai 201203, China

^bDepartment of Oncology, Shanghai Medical College of Fudan University, 270 Dong-an Road, Shanghai, 200032, China

^cDepartment of Gynecologic Oncology, Fudan University Shanghai Cancer Center, 270 Dong-an Road, Shanghai, 200032, China

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Role of size, surface charge, and PEGylated lipids of lipid nanoparticles (LNPs) on intramuscular delivery of mRNA

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Abstract

Figures

1. Background

2. Materials and methods

2.1. Materials

2.2. Preparation of LNPs

2.3. Characterization of LNPs

2.4. Spectroscopic investigation of DiD-DiR loaded LNPs

2.5. In vitro transfection of mLuc

2.6. In vivo expression and distribution of luciferase

2.7. Cellular uptake of LNPs

2.8. Protein corona of LNPs with various PEGylated lipids

2.9. Lysosomal escape

2.10. Statistical analysis

3. Results and discussions

3.1. The effects of particle size on nanoparticle transfection and in vivo distribution

3.2. The effect of surface charge on nanoparticle transfection and in vivo distribution

3.3 The effect of PEGylated lipid contents on the properties of LNPs

3.4. The impact of types of PEGylated lipids on the functions of LNPs

3.5. Underlying cellular mechanisms for transfection affected by PEGylated lipids

4. Conclusion

Abbreviations

Declarations

References

Additional Declarations

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