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

Role of size, surface charge, and PEGylated lipids of lipid nanoparticles (LNPs) on intramuscular delivery of mRNA | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Role of size, surface charge, and PEGylated lipids of lipid nanoparticles (LNPs) on intramuscular delivery of mRNA Weiwen Kong, Yuning Wei, Zirong Dong, Wenjuan Liu, Jiaxin Zhao, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4659748/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 11 Sep, 2024 Read the published version in Journal of Nanobiotechnology → Version 1 posted 12 You are reading this latest preprint version Abstract 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 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Background 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. Materials and methods 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 1×10 5 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 4×10 5 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 4×10 5 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. Results and discussions 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. 1 A). 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. 1 A confirm that LNP-S, LNP-M, and LNP-L are spherical and monodispersed. Figure 1 B 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. 1 C, Fig. 1 D). The evaluation of these LNPs both in vitro and in vivo is illustrated in Fig. 1 E. The immortalized cell lines, HEK-293 and DC2.4, were transfected with LNP-S, LNP-M, or LNP-L. As shown in Fig. 1 F, 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. 1 G). 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. 1 H). Luminescence images of all the time and ex vivo images are presented in Fig. S1 A and Fig. S1 B. LNP-S exhibited the highest transfection efficiency in situ (Fig. 1 I, Fig. S1 C). 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. 1 J, Fig. S1 D). 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. 1 K). 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. 2 A. 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. 2 B). 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. 2 C). 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. 2 D). 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. 2 E). Comprehensive luminescence images and ex vivo assessments are provided in Fig. S2 A-B. Figure 2 F 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. 2 G-H, Fig. S2 C-D). Although variations in lipid composition were observed among the different nanoparticles, the in vivo distribution presented a homologous phenomenon (Fig. 2 F, 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. 2 I). 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. 3 A, 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. 3 B). The results revealed that the content of PEGylated lipids affected the stability of the encapsulation efficiency of the LNPs (Fig. 3 D-F). Specifically, the encapsulation efficiency of LNPs with 0.5 mol% DMG-PEG2k decreased significantly over a four-week period (Fig. 3 F). 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. 3 G). 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. 3 H). Additionally, the in vivo distribution of these nanoparticles, containing varying contents of DMG-PEG2k, did not significantly differ (Fig. 3 I). 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. 3 J-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. 3 K). 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. 4 A. LNPs with any type of PEGylated lipids showed comparable particle size, PDI, and zeta potential (Fig. 4 B-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 4 E 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. 4 E). 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. 4 F). Further investigations demonstrated that LNPs formulated with DMG-PEG2k exhibited enhanced transfection performance in vitro , as depicted in Fig. 4 F. This observation was corroborated by in vivo studies, where intramuscular injection of LNPs containing DMG-PEG2k showed superior transfection efficiency (Fig. 4 G-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. 4 E-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. 5 A). Specifically, LNPs containing DMG-PEG2k exhibited significantly greater cellular uptake than the other LNPs (Fig. 5 A). 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. 5 B). According to the results above, FBS affect cell transfection efficiency. Subsequent studies, shown in Fig. 5 C, 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. 5 D-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. 5 G-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. 5 J-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. 4. Conclusion 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. Abbreviations 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 Declarations 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 b Department of Oncology, Shanghai Medical College of Fudan University, 270 Dong-an Road, Shanghai, 200032, China c Department of Gynecologic Oncology, Fudan University Shanghai Cancer Center, 270 Dong-an Road, Shanghai, 200032, China References Samaridou, E, J Heyes, and P Lutwyche. 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Supplementary Files supplymentaryfigure.docx floatimage1.jpeg Graphical Abstract Cite Share Download PDF Status: Published Journal Publication published 11 Sep, 2024 Read the published version in Journal of Nanobiotechnology → Version 1 posted Editorial decision: Revision requested 24 Jul, 2024 Reviews received at journal 19 Jul, 2024 Reviews received at journal 18 Jul, 2024 Reviews received at journal 16 Jul, 2024 Reviewers agreed at journal 10 Jul, 2024 Reviewers agreed at journal 10 Jul, 2024 Reviewers agreed at journal 08 Jul, 2024 Reviewers agreed at journal 08 Jul, 2024 Reviewers invited by journal 08 Jul, 2024 Editor assigned by journal 07 Jul, 2024 Submission checks completed at journal 06 Jul, 2024 First submitted to journal 29 Jun, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4659748","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":331343739,"identity":"05a5ce3b-61fd-43dc-b2f7-8e3e7521ba6d","order_by":0,"name":"Weiwen Kong","email":"","orcid":"","institution":"Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Weiwen","middleName":"","lastName":"Kong","suffix":""},{"id":331343740,"identity":"8d37e859-7dfc-4cf1-943c-2c03f95c75b3","order_by":1,"name":"Yuning Wei","email":"","orcid":"","institution":"Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Yuning","middleName":"","lastName":"Wei","suffix":""},{"id":331343741,"identity":"3196d419-3761-4cdf-9c94-94fb0852d9d5","order_by":2,"name":"Zirong Dong","email":"","orcid":"","institution":"Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Zirong","middleName":"","lastName":"Dong","suffix":""},{"id":331343742,"identity":"7d84efbe-c971-43a2-9c57-5203e64d3145","order_by":3,"name":"Wenjuan Liu","email":"","orcid":"","institution":"Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Wenjuan","middleName":"","lastName":"Liu","suffix":""},{"id":331343743,"identity":"b28bc549-f4b9-4098-a660-8dfc4f4ebe0f","order_by":4,"name":"Jiaxin Zhao","email":"","orcid":"","institution":"Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Jiaxin","middleName":"","lastName":"Zhao","suffix":""},{"id":331343744,"identity":"d5ad3de9-e0bd-41e8-89e1-4ca558c32191","order_by":5,"name":"Yan Huang","email":"","orcid":"","institution":"Shanghai Medical College of Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Yan","middleName":"","lastName":"Huang","suffix":""},{"id":331343745,"identity":"a77d96d9-dc00-4f33-b59e-3633d66d6ff8","order_by":6,"name":"Jinlong Yang","email":"","orcid":"","institution":"Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Jinlong","middleName":"","lastName":"Yang","suffix":""},{"id":331343746,"identity":"d829fffd-f8a6-495f-afa1-771d5bf9b1bf","order_by":7,"name":"Wei Wu","email":"","orcid":"","institution":"Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"Wu","suffix":""},{"id":331343747,"identity":"94d01c8f-a95d-48be-8735-cec6af3a620d","order_by":8,"name":"Haisheng He","email":"","orcid":"","institution":"Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Haisheng","middleName":"","lastName":"He","suffix":""},{"id":331343748,"identity":"8c23107e-4f52-481f-97bf-1301d329c140","order_by":9,"name":"Jianping Qi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/klEQVRIiWNgGAWjYDACZoaEA0CKB8g68IGBDSxmQKwWtsQZxGlBAB5D4rTwHWd4eODnjjsy5vxrPjbzlNnlMbA3b5NgqLmDU4vkYYaEg71nnvFYzni7sZnnXHIxA8+xMgmGY89wajEAajnA23aYx+DG2e2PeduYExskcswkGBsO49Vy8C9Yy5mHzbxt9YkN8m8IazkMtuV8DyNQy2GgLTz4tYD8cli27RnQFjbDxjnnjie28aQVWyQcw62F7/yZ5I9v2+7YG5w//LDhTVl1Yj/74Y03PtTg1sJwgCcBRDIwSCRABMBRk4BbA1Ax+wGIFv4D+JSNglEwCkbBSAYAmm1fQ4ugY/wAAAAASUVORK5CYII=","orcid":"","institution":"Fudan University","correspondingAuthor":true,"prefix":"","firstName":"Jianping","middleName":"","lastName":"Qi","suffix":""}],"badges":[],"createdAt":"2024-06-29 14:36:04","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4659748/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4659748/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12951-024-02812-x","type":"published","date":"2024-09-11T15:57:45+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":61528247,"identity":"2994cc00-09b7-43e1-851a-19604200cb35","added_by":"auto","created_at":"2024-07-31 21:20:39","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":943083,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe effect of particle size on nanoparticle transfection and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e distribution. \u003c/strong\u003e(A) A flow chart for the formulation of LNPs with different sizes, and TEM images (left) of LNP-S, LNP-M, and LNP-L. (B) A statistical chart for particle sizes of LNP-S, LNP-M, and LNP-L. (C) A fluorescence spectrum scanning chart of LNPs with DiD-DiR, Ex: 640 nm. (D) Fluorescence images of LNPs with DiD-DiR. FRET was detected by an IVIS (Ex: 640 nm, Em of FRET: 780 nm, Em of donor: 670 nm). (E) \u003cem\u003eIn vitro\u003c/em\u003eand \u003cem\u003ein vivo\u003c/em\u003e experimental flow chart of LNPs. A plate reader was exploited to detect luminescence \u003cem\u003ein vitro\u003c/em\u003e, and IVI\u003cu\u003eS\u003c/u\u003e was utilized to detect luminescence and fluorescence. (F) \u003cem\u003eIn vitro\u003c/em\u003e transfection of LNP-S, LNP-M, and LNP-L on HEK-293 and DC2.4. The expression of firefly luciferase in cells was detected and analyzed. ***: p\u0026lt;0.001, ****: p\u0026lt;0.0001, (n=3). (G) \u003cem\u003eIn vivo\u003c/em\u003e transfection of LNP-S, LNP-M, and LNP-L. Luminescence images harvested by IVIS, indicate the increased transfection with an order of LNP-S\u0026gt;LNP-M\u0026gt;LNP-L. (H) \u003cem\u003eIn vivo\u003c/em\u003e distribution of LNP-S, LNP-M, and LNP-L. LNPs were labeled with DiD-DiR, and IVIS was used to detect the distribution of fluorescence on the nanoparticles, Ex: 640 nm, Em of FRET: 780 nm, Em of donor: 670 nm. (I) A statistical graph of luminescence on muscle (n=3), **: P\u0026lt;0.01. (J) A statistical graph of luminescence in the liver (n=3). (K) A statistical graph of the FRET ratio on muscle (n=3).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4659748/v1/515e39bb85a6c07e162d9e35.png"},{"id":61528248,"identity":"d7379f79-7d4c-447a-8e28-99da40279b30","added_by":"auto","created_at":"2024-07-31 21:20:39","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":999462,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe effect of surface charge on nanoparticle transfection and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e distribution.\u003c/strong\u003e (A) A flow chart for the preparation and experiments of LNPs with various charges. (B) A statistical chart for the zeta potential of LNPs with different charges (n=3). (C) Particle size of LNPs with various charges (n=3). (D) \u003cem\u003eIn vitro\u003c/em\u003etransfection of LNPs with different charges on HEK-293 and DC2.4. Expression of firefly luciferase in cells was detected and analyzed (n=3). ***: p\u0026lt;0.001, ****: p\u0026lt;0.0001 (n=3). (E) \u003cem\u003eIn vivo\u003c/em\u003e transfection of LNPs with various charges. Luminescence was detected by IVIS. (F) \u003cem\u003eIn vitro\u003c/em\u003e distribution of LNPs with a positive charge. LNPs were labeled with DiD-DiR, and fluorescence images were acquired with an IVIS. Ex: 640 nm, Em of FRET: 780 nm, Em of donor: 670 nm. (G) Luminescence of muscle was analyzed and plotted, **: p\u0026lt;0,01, ****: p\u0026lt;0.0001. (H) Statistical analysis of the luminescence of the liver, *: P\u0026lt;0.05, **: p\u0026lt;0.01, ***: P\u0026lt;0.001, ****: P\u0026lt;0.0001. (I) The \u003cem\u003ein vivo\u003c/em\u003e FRET ratio of LNPs with various charges was calculated and plotted (n=3).\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4659748/v1/1bac0b082ce86c48cd89e89e.png"},{"id":61528446,"identity":"00abff32-db4e-4626-b015-3a710e5f211d","added_by":"auto","created_at":"2024-07-31 21:28:39","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":613594,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe effect of PEGylated lipid contents on the properties of LNPs.\u003c/strong\u003e Particle size (A), PDI (B), and encapsulation efficiency (C) of LNPs with 0.5 mol%, 1.5 mol%, and 3 mol% DMG-PEG2k were analyzed statistically (n=3). Stability of LNPs with 0.5 mol%, 1.5 mol%, and 3 mol% DMG-PEG2k at 4 °C for four weeks. Particle size (D), PDI (E), and encapsulation efficiency (F) were analyzed and plotted (n=3). (G) Statistical analysis of luciferase expression in HEK-293 and DC2.4 after LNP transfection (n=3). The graph on HEK-293 is on the left, and that on DC2.4 is on the right. ****: p\u0026lt;0.0001. (H) \u003cem\u003eIn vivo\u003c/em\u003e transfection of LNPs with 0.5 mol%, 1.5 mol%, and 3 mol% DMG-PEG2k following intramuscular injection. Luminescence images were taken by IVIS; 0.5%: LNPs with 0.5 mol% DMG-PEG2k, 1.5%: LNPs with 1.5 mol% DMG-PEG2k, 3%: LNPs with 3 mol% DMG-PEG2k. (I) \u003cem\u003eIn vivo\u003c/em\u003e distribution of LNPs with 0.5 mol%, 1.5 mol% and 3 mol% DMG-PEG2k. LNPs were labeled with DiD-DiR, and fluorescence images were taken with an IVIS (Ex:640 nm, Em:780 nm, Em:680 nm). (J) Luminescence of LNPs with various contents of PEGylated lipids on muscle were measured and analyzed (n=3); *: p\u0026lt;0.05, ***: p\u0026lt;0.001, ****: p\u0026lt;0.0001. (K) A statistical chart of luminescence in the liver (n=3), *: p\u0026lt;0.05, **: p\u0026lt;0.005. (L) A statistical chart of the FRET ratio on muscle (n=3).\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-4659748/v1/98d511b036ae840fb5556655.png"},{"id":61528445,"identity":"1d0c5fe2-5be3-4c40-b4da-98fe6179f2a9","added_by":"auto","created_at":"2024-07-31 21:28:39","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":392595,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe impact of types of PEGylated lipids on the properties of LNPs.\u003c/strong\u003e (A) Structural formulas of DMG-PEG2k, DSG-PEG2k and DSPE-PEG2k. Statistical charts for particle size (B), PDI (C), and zeta potential (D) of LNPs with DMG-PEG2k, DSG-PEG2k and DSPE-PEG2k; DMG: LNPs with DMG-PEG2k; DSG: LNPs with DSG-PEG2k; DSPE: LNPs with DSPE-PEG2k. (E) \u003cem\u003eIn vitro\u003c/em\u003e transfection of LNPs with DMG-PEG2k, DSG-PEG2k, and DSPE-PEG2k on HEK-293 and DC2.4 at 12 h, 24 h, 48 h, and 72 h. The graph for HEK-293 is on the left, and that for DC2.4 is on the right (n=3); *: p\u0026lt;0.05, **: p\u0026lt;0.01, ***: p\u0026lt;0.001, ****: p\u0026lt;0.0001. (F) \u003cem\u003eIn vitro \u003c/em\u003etransfection of LNPs with DMG-PEG2k, DSG-PEG2k, and DSPE-PEG2k on HEK-293 and DC2.4 in 10% FBS or 0% FBS. Luciferase expression was analyzed and plotted (n=3). *: p\u0026lt;0.05, **: P\u0026lt;0.01, ***: P\u0026lt;0.001, ****: p\u0026lt;0.0001. (G) \u003cem\u003eIn vivo\u003c/em\u003etransfection of LNPs with DMG-PEG2k, DSG-PEG2k, and DSPE-PEG2k after intramuscular injection. Luminescence images were taken by IVIS. (H) Luminescence of LNPs with various types of PEGylated lipids on muscle was analyzed and plotted (n=3); *: p\u0026lt;0.05. (I) Luminescence of LNPs with various types of PEGylated lipids in the liver was analyzed and plotted (n=3); *: p\u0026lt;0.05, **: p\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-4659748/v1/bb396bba8718c69b67dc6511.png"},{"id":61528251,"identity":"8a8f3cc8-454e-42d6-97c5-d51fffb877e8","added_by":"auto","created_at":"2024-07-31 21:20:39","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1668748,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUnderlying cellular mechanisms affecting transfection by PEGylated lipids. \u003c/strong\u003e(A) Cellular uptake of LNPs with DMG-PEG2k, DSG-PEG2k, and DSPE-PEG2k in HEK-293 cells at 4 h, 8 h, and 12 h. LNPs were labeled with DiD (magenta), and the nucleus was stained with DAPI (blue). (B) Cellular uptake of LNPs with DSG-PEG2k or DSPE-PEG2k in HEK-293 and DC2.4 cells at 4 h. LNPs were labeled with DiD (magenta), and the nuclei were stained with DAPI (blue). (C) Protein adsorbed on LNPs with DMG-PEG2k, DSG-PEG2k, and DSPE-PEG2k in FBS, as determined by SDS‒PAGE. The protein bands had the same position. (D-I) Cellular uptake mechanism of LNPs with DMG-PEG2k, DSG-PEG2k, and DSPE-PEG2k in HEK-293 and DC2.4 cells. LNPs were labeled with DiD, and fluorescence was detected by FACs. The mean fluorescence intensity was calculated by FlowJo, and the MFI was analyzed and plotted (n=3). MOCK was used as the blank control, and DMSO was used as the control group without inhibitors, which was the comparison. *: P\u0026lt;0.05, **: P\u0026lt;0.01, ***: p\u0026lt;0.001, ****: p\u0026lt;0.0001. (J-K) Lysosomal escape in HEK-293 and DC2.4. LNPs were labeled with DiD (magenta), and lysosomes were stained with LysoTracker (green), and nuclei were stained with DAPI (blue). (L) The colocalization of LNPs and lysosomes was analyzed by ImageJ (n=3). The chart for HEK-293 is above, that for DC2.4 is below; ***: P\u0026lt;0.001, ****: p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-4659748/v1/c20c6f7ec03ded13ab5c55e9.png"},{"id":64619498,"identity":"f704683b-0d02-49f6-89c9-19b3e6b86630","added_by":"auto","created_at":"2024-09-16 16:15:15","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5691886,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4659748/v1/f19b1e79-5ff2-460f-ae72-6f8bd7f5ebb8.pdf"},{"id":61528254,"identity":"85c9010b-ca6b-4856-90ae-e58643bbf8b4","added_by":"auto","created_at":"2024-07-31 21:20:39","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2349173,"visible":true,"origin":"","legend":"","description":"","filename":"supplymentaryfigure.docx","url":"https://assets-eu.researchsquare.com/files/rs-4659748/v1/4e33722dc7b17f6cec34d497.docx"},{"id":61528447,"identity":"0387b0dc-ca4d-488d-ba45-cd63819b38d9","added_by":"auto","created_at":"2024-07-31 21:28:39","extension":"jpeg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1109888,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGraphical Abstract\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4659748/v1/9b8ea8fc342e9e70aee26137.jpeg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Role of size, surface charge, and PEGylated lipids of lipid nanoparticles (LNPs) on intramuscular delivery of mRNA","fulltext":[{"header":"1. Background","content":"\u003cp\u003eThanks 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\u0026reg; and Inclisira\u0026reg; 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 \u003cem\u003ein vivo\u003c/em\u003e 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.\u003c/p\u003e \u003cp\u003eNevertheless, 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 \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e 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 \u003cem\u003evia\u003c/em\u003e LNPs leads to massive production of neutralizing antibodies [12] and antigen-specific T-cell immune responses [13].\u003c/p\u003e \u003cp\u003eLNPs 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 \u003cem\u003ein vivo\u003c/em\u003e 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 \u003cem\u003ein vivo\u003c/em\u003e [21. 22]. The presence of PEGylated lipids in LNPs extends their circulation time \u003cem\u003ein vivo\u003c/em\u003e, 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 \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e [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.\u003c/p\u003e \u003cp\u003eMoreover, 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.\u003c/p\u003e \u003cp\u003eIn 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 \u003cem\u003ein vivo\u003c/em\u003e distribution. First, LNPs with varying sizes, charges, and various contents of PEGylated lipids were formulated. Subsequently, their transfection efficacy \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e was thoroughly evaluated. Meanwhile, fluorescence resonance energy transfer (FRET) technology, which labels intact nanoparticles, was utilized to monitor LNPs \u003cem\u003ein vivo\u003c/em\u003e. Additionally, LNPs with different types of PEGylated lipids were assembled to assess their implications for transfection \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. 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.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Materials\u003c/h2\u003e \u003cp\u003e1-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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Preparation of LNPs\u003c/h2\u003e \u003cp\u003eLNPs with different sizes are formulated by microfluidic mixing [32\u0026ndash;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\u003csup\u003e\u0026trade;\u003c/sup\u003e 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\u0026deg;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.\u003c/p\u003e \u003cp\u003eLNPs 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.\u003c/p\u003e \u003cp\u003eLNPs 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%.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Characterization of LNPs\u003c/h2\u003e \u003cp\u003eThe 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].\u003c/p\u003e \u003cp\u003eLNPs 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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Spectroscopic investigation of DiD-DiR loaded LNPs\u003c/h2\u003e \u003cp\u003eA total of 400 \u0026micro;L 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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. \u003cem\u003eIn vitro\u003c/em\u003e transfection of mLuc\u003c/h2\u003e \u003cp\u003eHEK-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\u0026deg;C overnight, mLuc-loaded LNPs were treated to cells at a concentration of 1 \u0026micro;g/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).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. \u003cem\u003eIn vivo\u003c/em\u003e expression and distribution of luciferase\u003c/h2\u003e \u003cp\u003e 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 \u0026micro;g 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 \u003cem\u003ein vivo\u003c/em\u003e 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).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Cellular uptake of LNPs\u003c/h2\u003e \u003cp\u003eHEK-293 cells and DC2.4 cells were seeded onto 24-well glass bottom plates for laser confocal imaging at a density of 1\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells per well and cultured at 37\u0026deg;C overnight. Then, cells were treated with DiD-labeled mLuc LNPs at an mRNA concentration of 1 \u0026micro;g/mL. After incubation at 37\u0026deg;C for 3 h, the cells were washed twice with 1\u0026times; 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\u0026times; PBS. Images were captured by fluorescence microscopy (LSM710, Zeiss) at 20\u0026times; magnification.\u003c/p\u003e \u003cp\u003eTo 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\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells per well and further cultured overnight. The cells were pre-incubated with various endocytosis inhibitors, including 5 \u0026micro;M CPZ, 10 \u0026micro;M FIL, 3 \u0026micro;M CYTD, and 5 \u0026micro;M WORT, for 30 minutes. Then, DiD-loaded mLuc LNPs were added to the cells at an mRNA concentration of 1 \u0026micro;g/mL and incubated for 4 h. Afterwards, the cells were digested with trypsin, washed twice with 1\u0026times; PBS, and then resuspended in 1\u0026times; 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).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Protein corona of LNPs with various PEGylated lipids\u003c/h2\u003e \u003cp\u003e LNPs with various PEGylated lipids were prepared according to a previously described method and were concentrated to 1 g/L with 1\u0026times; 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 \u0026times; g and 4 ℃ for 1 h. The supernatant was removed, and the pellet was washed with 1\u0026times; PBS. Then, the pellet was centrifuged at 15,300 \u0026times; g and 4\u0026deg;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.\u003c/p\u003e \u003cp\u003eThe protein adsorbed on the LNPs was mixed with protein sample loading buffer and boiled at 90\u0026deg;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).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9. Lysosomal escape\u003c/h2\u003e \u003cp\u003eHEK-293 cells and DC2.4 cells were seeded onto a 24-well glass-bottom plate at a density of 4\u0026times;10\u003csup\u003e5\u003c/sup\u003e 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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10. Statistical analysis\u003c/h2\u003e \u003cp\u003e \u003cem\u003eIn vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;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 \u003cem\u003ep\u003c/em\u003e-value less than 0.05 was considered as statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and discussions","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.1. The effects of particle size on nanoparticle transfection and \u003cem\u003ein vivo\u003c/em\u003e distribution\u003c/h2\u003e \u003cp\u003eTo highlight the influence of LNP size on transfection and \u003cem\u003ein vivo\u003c/em\u003e distribution, LNPs were engineered in three distinct sizes including small (S), medium (M), and large (L) particles (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). 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 \u0026minus;\u0026thinsp;5 to 5 mV range, demonstrating their neutral surface charge (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). 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.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA confirm that LNP-S, LNP-M, and LNP-L are spherical and monodispersed. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB illustrates the size distribution of LNP-S, LNP-M, and LNP-L.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eParticle size, PDI, zeta potential, and encapsulation efficiency (EE) of LNPs\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eParticle size (nm)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePDI\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eZeta potential (mV)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eEE (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLNP-S\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e94.61\u0026thinsp;\u0026plusmn;\u0026thinsp;1.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e-1.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e90.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLNP-M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e121.93\u0026thinsp;\u0026plusmn;\u0026thinsp;1.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e-0.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e95.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLNP-L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e167.37\u0026thinsp;\u0026plusmn;\u0026thinsp;1.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e1.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e90.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eLNPs were labeled with the FRET pair (DiD-DiR), demonstrating a substantial FRET effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). The evaluation of these LNPs both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e is illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE. The immortalized cell lines, HEK-293 and DC2.4, were transfected with LNP-S, LNP-M, or LNP-L. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF, 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.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). 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.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH). Luminescence images of all the time and \u003cem\u003eex vivo\u003c/em\u003e images are presented in Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA and Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB. LNP-S exhibited the highest transfection efficiency \u003cem\u003ein situ\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI, Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eC). 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.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eJ, Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eD). Despite the observed significant differences in transfection efficiency \u003cem\u003ein vivo\u003c/em\u003e, the \u003cem\u003ein situ\u003c/em\u003e FRET ratio showed similar trends among LNP-S, LNP-M, and LNP-L (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eK).\u003c/p\u003e \u003cp\u003eAs shown above, particle size significantly influences the transfection efficiency of LNPs \u003cem\u003ein vitro\u003c/em\u003e. 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.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.2. The effect of surface charge on nanoparticle transfection and \u003cem\u003ein vivo\u003c/em\u003e distribution\u003c/h2\u003e \u003cp\u003eTo 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 \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e experiments, is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA. 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.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). 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.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). LNPs with 15% DOTAP exhibited a charge of approximately\u0026thinsp;+\u0026thinsp;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 \u0026minus;\u0026thinsp;25.3 mV, a value consistent with that of LNPs prepared with 25% DSPG (Tab. S2).\u003c/p\u003e \u003cp\u003eNeutral LNPs demonstrated superior transfection efficiency compared to their counterparts mixed with DOTAP or DSPG \u003cem\u003ein vitro\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Interestingly, LNPs comprising 25% DOTAP exhibited superior \u003cem\u003ein vitro\u003c/em\u003e transfection efficiency relative to those with 50% DOTAP, despite having similar particle sizes. This trend was mirrored in transfection experiments \u003cem\u003ein vivo\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). Comprehensive luminescence images and \u003cem\u003eex vivo\u003c/em\u003e assessments are provided in Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA-B. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF illustrates the \u003cem\u003ein vivo\u003c/em\u003e distribution of LNPs labeled with FRET pairs. Neutral LNPs achieved the highest luciferase expression both \u003cem\u003ein situ\u003c/em\u003e and in the liver following intramuscular injection (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG-H, Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eC-D). Although variations in lipid composition were observed among the different nanoparticles, the \u003cem\u003ein vivo\u003c/em\u003e distribution presented a homologous phenomenon (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF, I). LNPs with a negative charge exhibited an enhanced FRET ratio \u003cem\u003ein situ\u003c/em\u003e, indicating the superior structural integrity of the nanoparticles. Meanwhile, positive-charged LNPs presented similar integrity with neutral-charged LNPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI).\u003c/p\u003e \u003cp\u003ePrevious 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 \u003cem\u003ein vivo\u003c/em\u003e 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.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.3 The effect of PEGylated lipid contents on the properties of LNPs\u003c/h2\u003e \u003cp\u003eThe 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.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, 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.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). The results revealed that the content of PEGylated lipids affected the stability of the encapsulation efficiency of the LNPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD-F). Specifically, the encapsulation efficiency of LNPs with 0.5 mol% DMG-PEG2k decreased significantly over a four-week period (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). 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].\u003c/p\u003e \u003cp\u003eLNPs with 1.5 mol% DMG-PEG2k exhibited superior transfection efficiency \u003cem\u003ein vitro\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). Luminescence images captured at all time points and \u003cem\u003eex vivo\u003c/em\u003e luminescence data are provided in Fig. S3A-B, providing a comprehensive view of the temporal and spatial dynamics of nanoparticle-mediated gene expression. \u003cem\u003eIn vivo\u003c/em\u003e assessments confirmed that LNPs with 1.5 mol% DMG-PEG2k demonstrated enhanced transfection capabilities (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH). Additionally, the \u003cem\u003ein vivo\u003c/em\u003e distribution of these nanoparticles, containing varying contents of DMG-PEG2k, did not significantly differ (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI). 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 \u003cem\u003ein situ\u003c/em\u003e and in the liver 4 hours post-administration (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eJ-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.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eK).\u003c/p\u003e \u003cp\u003eThe 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 \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. 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.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.4. The impact of types of PEGylated lipids on the functions of LNPs\u003c/h2\u003e \u003cp\u003eTo 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 \u003cem\u003ein vivo\u003c/em\u003e [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.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA. LNPs with any type of PEGylated lipids showed comparable particle size, PDI, and zeta potential (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB-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\u0026deg;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).\u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE 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.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). 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.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). Further investigations demonstrated that LNPs formulated with DMG-PEG2k exhibited enhanced transfection performance \u003cem\u003ein vitro\u003c/em\u003e, as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF. This observation was corroborated by \u003cem\u003ein vivo\u003c/em\u003e studies, where intramuscular injection of LNPs containing DMG-PEG2k showed superior transfection efficiency (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG-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).\u003c/p\u003e \u003cp\u003eThe impact of PEG2k lipid types on the \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e 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 \u003cem\u003ein vitro\u003c/em\u003e, as evidenced by comparative analyses between LNPs formulated with DSG-PEG2k and those formulated with DSPE-PEG2k (shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE-F). Such observations regarding the differential roles of functional groups in PEGylated lipids have rarely been reported in previous studies.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Underlying cellular mechanisms for transfection affected by PEGylated lipids\u003c/h2\u003e \u003cp\u003eTo 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.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Specifically, LNPs containing DMG-PEG2k exhibited significantly greater cellular uptake than the other LNPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). 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.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). According to the results above, FBS affect cell transfection efficiency. Subsequent studies, shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, 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.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD-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.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG-I).\u003c/p\u003e \u003cp\u003eThe 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.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eJ-L).\u003c/p\u003e \u003cp\u003ePrevious 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.\u003c/p\u003e \u003cp\u003eBased 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.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eIn 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 \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. 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.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eLNPs \u0026nbsp; \u0026nbsp; Lipid nanoparticles\u003c/p\u003e\n\u003cp\u003emLuc \u0026nbsp; \u0026nbsp; Luciferase mRNA \u0026nbsp;\u003c/p\u003e\n\u003cp\u003esiRNA \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Small interfering RNA\u003c/p\u003e\n\u003cp\u003emRNA \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Messenger RNA\u003c/p\u003e\n\u003cp\u003eApoE \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Apolipoprotein E\u003c/p\u003e\n\u003cp\u003eLDLR \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Low-density lipoprotein receptor\u003c/p\u003e\n\u003cp\u003eFRET \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Fluorescence resonance energy transfer\u003c/p\u003e\n\u003cp\u003eSM-102 \u0026nbsp; \u0026nbsp; 1-octylnonyl 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoate\u003c/p\u003e\n\u003cp\u003eDSPC \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; 1,2-dioctadecanoyl-sn-glycero-3-phophocholine\u003c/p\u003e\n\u003cp\u003eDOTAP \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;(2,3-dioxypropyl) trimethylammonium chloride\u003c/p\u003e\n\u003cp\u003eDSPG \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;1,2-distearoyl-sn-glycero-3-phospho-(1\u0026apos;-rac-glycerol)\u003c/p\u003e\n\u003cp\u003eDMG-PEG2k. \u0026nbsp; \u0026nbsp;1,2-dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol 2000\u003c/p\u003e\n\u003cp\u003eDSG-PEG2k. \u0026nbsp; \u0026nbsp; Diastearyl-rac-glycerol polyethylene glycol 2000\u003c/p\u003e\n\u003cp\u003eDSPE-PEG2k. \u0026nbsp; \u0026nbsp; Diethyl phosphatidylethanolamine polyethylene glycol 2000\u003c/p\u003e\n\u003cp\u003eDiD. \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;1,1\u0026apos;-dioctadecyl-3,3,3\u0026apos;,3\u0026apos;-tetramethylindodicarbocyanine\u003c/p\u003e\n\u003cp\u003eDiR. \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;1,1\u0026apos;-Dioctadecyl-3,3,3\u0026apos;,3\u0026apos;-Tetramethylindotricarbocyanine iodide\u003c/p\u003e\n\u003cp\u003eCPZ. \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Chlorpromazine\u003c/p\u003e\n\u003cp\u003eFIL. \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Filipin\u003c/p\u003e\n\u003cp\u003eCYTD. \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;CytochalasinD\u003c/p\u003e\n\u003cp\u003eWORT. \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Wortmannin\u003c/p\u003e\n\u003cp\u003ePAGE. \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Polyacrylamide gel electrophoresis\u003c/p\u003e\n\u003cp\u003eSDS. \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Sodium dodecyl sulfate\u003c/p\u003e\n\u003cp\u003eFBS. \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Fetal Bovine Serum\u003c/p\u003e\n\u003cp\u003ePDI. \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Polydispersion index\u003c/p\u003e\n\u003cp\u003eTEM. \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Transmission electron microscopy\u003c/p\u003e\n\u003cp\u003eIACUC. \u0026nbsp; \u0026nbsp; \u0026nbsp;Institutional Animal Care and Use Committee\u003c/p\u003e\n\u003cp\u003eIVIS. \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003cem\u003eIn vivo\u003c/em\u003e imaging system\u003c/p\u003e\n\u003cp\u003ePFA. \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Polyformaldehyde\u003c/p\u003e\n\u003cp\u003eRIPA. \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Radioimmunoprecipitation assay\u003c/p\u003e\n\u003cp\u003eSEM. \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Standard error of mean \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eANOVA. \u0026nbsp; \u0026nbsp; \u0026nbsp;Analysis of variance\u003c/p\u003e\n\u003cp\u003eEE. \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Encapsulation efficiencies\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal procedures are conducted in compliance with the Guide for the\u003c/p\u003e\n\u003cp\u003eCare and Use of Laboratory Animals published by the National Institutes of\u003c/p\u003e\n\u003cp\u003eHealth (NIH Publication No. 85\u0026minus;23, 1996, revised 2011) and approved by the Institutional Animal Care and Use Committee (IACUC) at the School of Pharmacy, Fudan University, China.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData will be made available on request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe work was supported by the National Natural Science Foundation of China (No. 82073801).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eW.K.:\u0026nbsp;\u003c/strong\u003eData curation, Formal analysis, Investigation, Writing-original draft. \u003cstrong\u003eY.W.:\u003c/strong\u003e Data curation, Investigation. \u003cstrong\u003eZ.D.:\u003c/strong\u003e Data curation. \u003cstrong\u003eW. L.\u003c/strong\u003e: Data curation.\u003cstrong\u003e\u0026nbsp;J. Z.:\u003c/strong\u003e Data curation. \u003cstrong\u003eY.H.:\u003c/strong\u003e Validation, Visualization. \u003cstrong\u003eJ.Y.:\u003c/strong\u003e Validation, Visualization.\u003cstrong\u003e\u0026nbsp;W.W.:\u003c/strong\u003e Validation, Visualization. \u003cstrong\u003eH.H.:\u0026nbsp;\u003c/strong\u003eValidation, Writing \u0026ndash; review \u0026amp; editing. \u003cstrong\u003eJ.Q.:\u0026nbsp;\u003c/strong\u003eFormal analysis, Methodology, Project administration, Supervision, Writing \u0026ndash; review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003ea\u003c/sup\u003e Key Laboratory of Smart Drug Delivery of MOE, School of Pharmacy, Fudan University, Shanghai 201203, China\u003c/p\u003e\n\u003cp\u003e\u003csup\u003eb\u0026nbsp;\u003c/sup\u003eDepartment of Oncology, Shanghai Medical College of Fudan University, 270 Dong-an Road, Shanghai, 200032, China\u003c/p\u003e\n\u003cp\u003e\u003csup\u003ec\u0026nbsp;\u003c/sup\u003eDepartment of Gynecologic Oncology, Fudan University Shanghai Cancer Center, 270 Dong-an Road, Shanghai, 200032, China\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSamaridou, E, J Heyes, and P Lutwyche. 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Structure, activity and uptake mechanism of siRNA-lipid nanoparticles with an asymmetric ionizable lipid\u003cem\u003e.\u003c/em\u003e Int J Pharm. 2016; 510(1): 350-8.\u003c/li\u003e\n\u003cli\u003eSahay, G, W Querbes, C Alabi, A Eltoukhy, S Sarkar, C Zurenko, E Karagiannis, K Love, D Chen, R Zoncu, Y Buganim, A Schroeder, R Langer, and D G Anderson. Efficiency of siRNA delivery by lipid nanoparticles is limited by endocytic recycling\u003cem\u003e.\u003c/em\u003e Nat Biotechnol. 2013; 31(7): 653-8.\u003c/li\u003e\n\u003cli\u003eCui, L, M R Hunter, S Sonzini, S Pereira, S M Romanelli, K Liu, W Li, L Liang, B Yang, N Mahmoudi, and A S Desai. Mechanistic Studies of an Automated Lipid Nanoparticle Reveal Critical Pharmaceutical Properties Associated with Enhanced mRNA Functional Delivery In Vitro and In Vivo\u003cem\u003e.\u003c/em\u003e Small. 2022; 18(9): e2105832.\u003c/li\u003e\n\u003cli\u003eDi, J, Z Du, K Wu, S Jin, X Wang, T Li, and Y Xu. Biodistribution and Non-linear Gene Expression of mRNA LNPs Affected by Delivery Route and Particle Size\u003cem\u003e.\u003c/em\u003e Pharm Res. 2022; 39(1): 105-14.\u003c/li\u003e\n\u003cli\u003eManolova, V, A Flace, M Bauer, K Schwarz, P Saudan, and M F Bachmann. Nanoparticles target distinct dendritic cell populations according to their size\u003cem\u003e.\u003c/em\u003e Eur J Immunol. 2008; 38(5): 1404-13.\u003c/li\u003e\n\u003cli\u003eHassett, K J, J Higgins, A Woods, B Levy, Y Xia, C J Hsiao, E Acosta, O Almarsson, M J Moore, and L A Brito. Impact of lipid nanoparticle size on mRNA vaccine immunogenicity\u003cem\u003e.\u003c/em\u003e J Control Release. 2021; 335: 237-46.\u003c/li\u003e\n\u003cli\u003eJu, Y, W S Lee, E H Pilkington, H G Kelly, S Li, K J Selva, K M Wragg, K Subbarao, T H O Nguyen, L C Rowntree, L F Allen, K Bond, D A Williamson, N P Truong, M Plebanski, K Kedzierska, S Mahanty, A W Chung, F Caruso, A K Wheatley, J A Juno, and S J Kent. Anti-PEG Antibodies Boosted in Humans by SARS-CoV-2 Lipid Nanoparticle mRNA Vaccine\u003cem\u003e.\u003c/em\u003e ACS Nano. 2022; 16(8): 11769-80.\u003c/li\u003e\n\u003cli\u003eWalsh, E E, R W Frenck, Jr., A R Falsey, N Kitchin, J Absalon, A Gurtman, S Lockhart, K Neuzil, M J Mulligan, R Bailey, K A Swanson, P Li, K Koury, W Kalina, D Cooper, C Fontes-Garfias, P Y Shi, O Tureci, K R Tompkins, K E Lyke, V Raabe, P R Dormitzer, K U Jansen, U Sahin, and W C Gruber. Safety and Immunogenicity of Two RNA-Based Covid-19 Vaccine Candidates\u003cem\u003e.\u003c/em\u003e N Engl J Med. 2020; 383(25): 2439-50.\u003c/li\u003e\n\u003cli\u003eEstape Senti, M, C A de Jongh, K Dijkxhoorn, J J F Verhoef, J Szebeni, G Storm, C E Hack, R M Schiffelers, M H Fens, and P Boross. Anti-PEG antibodies compromise the integrity of PEGylated lipid-based nanoparticles via complement\u003cem\u003e.\u003c/em\u003e J Control Release. 2022; 341: 475-86.\u003c/li\u003e\n\u003cli\u003eBailey-Hytholt, C M, P Ghosh, J Dugas, I E Zarraga, and A Bandekar. Formulating and Characterizing Lipid Nanoparticles for Gene Delivery using a Microfluidic Mixing Platform\u003cem\u003e.\u003c/em\u003e J Vis Exp. 2021; (168).\u003c/li\u003e\n\u003cli\u003eChen, D, K T Love, Y Chen, A A Eltoukhy, C Kastrup, G Sahay, A Jeon, Y Dong, K A Whitehead, and D G Anderson. Rapid discovery of potent siRNA-containing lipid nanoparticles enabled by controlled microfluidic formulation\u003cem\u003e.\u003c/em\u003e J Am Chem Soc. 2012; 134(16): 6948-51.\u003c/li\u003e\n\u003cli\u003eMaeki, M, S Uno, A Niwa, Y Okada, and M Tokeshi. Microfluidic technologies and devices for lipid nanoparticle-based RNA delivery\u003cem\u003e.\u003c/em\u003e J Control Release. 2022; 344: 80-96.\u003c/li\u003e\n\u003cli\u003eWisse, E, F Jacobs, B Topal, P Frederik, and B De Geest. The size of endothelial fenestrae in human liver sinusoids: implications for hepatocyte-directed gene transfer\u003cem\u003e.\u003c/em\u003e Gene Ther. 2008; 15(17): 1193-9.\u003c/li\u003e\n\u003cli\u003eHunter, M R, L Cui, B T Porebski, S Pereira, S Sonzini, U Odunze, P Iyer, O Engkvist, R L Lloyd, S Peel, A Sabirsh, D Ross-Thriepland, A T Jones, and A S Desai. Understanding Intracellular Biology to Improve mRNA Delivery by Lipid Nanoparticles\u003cem\u003e.\u003c/em\u003e Small Methods. 2023; 7(9): e2201695.\u003c/li\u003e\n\u003cli\u003eKim, J, A Jozic, Y Lin, Y Eygeris, E Bloom, X Tan, C Acosta, K D MacDonald, K D Welsher, and G Sahay. Engineering Lipid Nanoparticles for Enhanced Intracellular Delivery of mRNA through Inhalation\u003cem\u003e.\u003c/em\u003e ACS Nano. 2022; 16(9): 14792-806.\u003c/li\u003e\n\u003cli\u003eBerger, M, M Degey, J Leblond Chain, E Maquoi, B Evrard, A Lechanteur, and G Piel. Effect of PEG Anchor and Serum on Lipid Nanoparticles: Development of a Nanoparticles Tracking Method\u003cem\u003e.\u003c/em\u003e Pharmaceutics. 2023; 15(2).\u003c/li\u003e\n\u003cli\u003eMuller, J A, N Schaffler, T Kellerer, G Schwake, T S Ligon, and J O Radler. Kinetics of RNA-LNP delivery and protein expression\u003cem\u003e.\u003c/em\u003e Eur J Pharm Biopharm. 2024; 197: 114222.\u003c/li\u003e\n\u003cli\u003eWilson, S C, J L Baryza, A J Reynolds, K Bowman, M E Keegan, S M Standley, N P Gardner, P Parmar, V O Agir, S Yadav, A Zunic, C Vargeese, C C Lee, and S Rajan. Real time measurement of PEG shedding from lipid nanoparticles in serum via NMR spectroscopy\u003cem\u003e.\u003c/em\u003e Mol Pharm. 2015; 12(2): 386-92.\u003c/li\u003e\n\u003cli\u003eSarode, A, Y Fan, A E Byrnes, M Hammel, G L Hura, Y Fu, P Kou, C Hu, F I Hinz, J Roberts, S G Koenig, K Nagapudi, C C Hoogenraad, T Chen, D Leung, and C W Yen. Predictive high-throughput screening of PEGylated lipids in oligonucleotide-loaded lipid nanoparticles for neuronal gene silencing\u003cem\u003e.\u003c/em\u003e Nanoscale Adv. 2022; 4(9): 2107-23.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-nanobiotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jnan","sideBox":"Learn more about [Journal of Nanobiotechnology](http://jnanobiotechnology.biomedcentral.com)","snPcode":"12951","submissionUrl":"https://submission.nature.com/new-submission/12951/3","title":"Journal of Nanobiotechnology","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"lipid nanoparticles, size, surface charge, PEGylated lipids, transfection, distribution","lastPublishedDoi":"10.21203/rs.3.rs-4659748/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4659748/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLipid 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 \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e. 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 \u003cem\u003ein vivo\u003c/em\u003e distribution. Luciferase mRNA (mLuc) was loaded in LNPs as a model nucleic acid.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eIn vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e 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 \u003cem\u003ein vivo\u003c/em\u003e 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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTogether, 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 \u003cem\u003evia\u003c/em\u003e LNPs.\u003c/p\u003e","manuscriptTitle":"Role of size, surface charge, and PEGylated lipids of lipid nanoparticles (LNPs) on intramuscular delivery of mRNA","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-31 21:20:34","doi":"10.21203/rs.3.rs-4659748/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-07-24T16:25:44+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-19T14:34:03+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-18T16:04:36+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-16T15:59:14+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"107992412584090193092752685984423373147","date":"2024-07-11T00:18:48+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"17251110744122970304894988706833332407","date":"2024-07-10T22:16:02+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"116230307013191044084571812394037228642","date":"2024-07-08T11:43:18+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"170932577959949394352271016827306187448","date":"2024-07-08T06:21:02+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-07-08T05:00:01+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-07-08T00:12:32+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-07-06T18:40:24+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Nanobiotechnology","date":"2024-06-29T14:34:23+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-nanobiotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jnan","sideBox":"Learn more about [Journal of Nanobiotechnology](http://jnanobiotechnology.biomedcentral.com)","snPcode":"12951","submissionUrl":"https://submission.nature.com/new-submission/12951/3","title":"Journal of Nanobiotechnology","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f61ee6ba-7ff5-4dd2-80e2-0947db56a0e9","owner":[],"postedDate":"July 31st, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-09-16T16:08:56+00:00","versionOfRecord":{"articleIdentity":"rs-4659748","link":"https://doi.org/10.1186/s12951-024-02812-x","journal":{"identity":"journal-of-nanobiotechnology","isVorOnly":false,"title":"Journal of Nanobiotechnology"},"publishedOn":"2024-09-11 15:57:45","publishedOnDateReadable":"September 11th, 2024"},"versionCreatedAt":"2024-07-31 21:20:34","video":"","vorDoi":"10.1186/s12951-024-02812-x","vorDoiUrl":"https://doi.org/10.1186/s12951-024-02812-x","workflowStages":[]},"version":"v1","identity":"rs-4659748","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4659748","identity":"rs-4659748","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}