
==== Front
J Nanobiotechnology
J Nanobiotechnology
Journal of Nanobiotechnology
1477-3155
BioMed Central London

2812
10.1186/s12951-024-02812-x
Research
Role of size, surface charge, and PEGylated lipids of lipid nanoparticles (LNPs) on intramuscular delivery of mRNA
Kong Weiwen 1
Wei Yuning 1
Dong Zirong 1
Liu Wenjuan 1
Zhao Jiaxin 1
Huang Yan 23
Yang Jinlong 1
Wu Wei 1
He Haisheng he_haisheng@fudan.edu.cn

1
Qi Jianping qijianping@fudan.edu.cn

1
1 grid.8547.e 0000 0001 0125 2443 School of Pharmacy, Key Laboratory of Smart Drug Delivery, Fudan University, Ministry of Education, Shanghai, 201203 China
2 https://ror.org/01zntxs11 grid.11841.3d 0000 0004 0619 8943 Department of Oncology, Shanghai Medical College of Fudan University, 270 Dong-an Road, Shanghai, 200032 China
3 https://ror.org/00my25942 grid.452404.3 0000 0004 1808 0942 Department of Gynecologic Oncology, Fudan University Shanghai Cancer Center, 270 Dong-an Road, Shanghai, 200032 China
11 9 2024
11 9 2024
2024
22 55329 6 2024
24 8 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
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 entraped in LNPs as a model nucleic acid molecule. 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. 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 significant directions for the effective delivery of mRNA via LNPs.

Graphical Abstract

Supplementary Information

The online version contains supplementary material available at 10.1186/s12951-024-02812-x.

Keywords

Lipid nanoparticles
Size
Surface charge
PEGylated lipids
Transfection
Distribution
http://dx.doi.org/10.13039/501100001809 National Natural Science Foundation of China 82073801 82073801 82073801 82073801 82073801 82073801 82073801 82073801 82073801 82073801 issue-copyright-statement© BioMed Central Ltd., part of Springer Nature 2024
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pmcIntroduction

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, 10]. 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 [11]. 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 [12]. Moreover, the repeated intramuscular administration of antigen mRNA via LNPs leads to massive production of neutralizing antibodies [13] and antigen-specific T-cell immune responses [14]. Furthermore, LNPs containing mRNA have been utilized into chimeric antigen receptor (CAR)-T cell therapy [15, 16]. Previous study has shown that LNPs containing mRNA encoding fibroblast activation protein (FAP) antibodies can achieve in vivo CAR-T therapy to treat cardiac injury [17], which further emphasized the importance of LNP-mRNA therapy.

LNPs are composed of four distinct lipid classes, including ionizable lipids, helper lipids, cholesterol, 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 [12, 18, 19]. 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 [20]. LNPs with positive charges preferentially localize to the lungs following intravenous injection, whereas those with negative charges are more likely to accumulate in the spleen [21]. 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 [22]. Moreover, PEGylated lipids also play essential roles in LNPs for mRNA delivery [23, 24]. PEGylated lipids contribute to the evasion of macrophage capture, thereby enhancing nanoparticle stability and prolonging their efficacy in vivo [25, 26]. The presence of PEGylated lipids in LNPs extends their circulation time in vivo, which is particularly advantageous for intravenous injection [1, 27]. 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 [24, 28]. 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 [29], and smaller LNPs were prone to liver accumulation post-intramuscular injection [30, 31]. In contrast, a prior study indicated that mRNA-LNP vaccines with distinct sizes elicited comparably robust immune responses in non-human primates [32]. 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 by 12.1 folds post-vaccination with mRNA-1273, while anti-PEG-IgM levels have surged by 67.5 folds [33]. The induction of anti-PEG antibodies by PEGylated lipids may trigger hypersensitivity reactions by activating the complement system [24, 34, 35]. 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 the differences in transfection efficiency among various PEGylated lipid types in detail.

Materials and methods

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-methoxypolyethylene glycol-2000 (DMG-PEG2k), 1,2-distearoyl-rac-glycerol-3-methoxypolyethylene glycol-2000 (DSG-PEG2k), and N-(Carbonyl-methoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycerol-3-phosphoethanolamine (DSPE-PEG2k) were purchased from AVT (Shanghai) Pharmaceuticals Tech Co., Ltd (Shanghai, China). Luciferase mRNA was purchased from APExbio (Houston, USA). 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 (Dalian, China). The Quant-iT RiboGreen RNA Reagent and Kit were purchased from Thermo Fisher Scientific (Waltham, USA). Chlorpromazine (CPZ), filipin (FIL), cytochalasin D (CYTD), and wortmannin (WORT) were purchased from AbMol (Shanghai, China). Firefly luciferase reporter gene detection kit and D-Luciferin potassium salt were purchased from Beyotime Biotechnology (Shanghai, China). A polyacrylamide gel electrophoresis (PAGE) gel rapid preparation kit and Tris/Glycine/Sodium dodecyl sulfate (SDS) electrophoretic buffer were purchased from Epizyme Biotech (Shanghai, China). GoldBand 3-color High Range Protein Marker and protein loading buffer were purchased from Yeasen Biotechnology (Shanghai, China). Dulbecco’s Modified Eagle Medium (DMEM), RPMI 1640 Medium, Fetal Bovine Serum (FBS) was purchased from Gibco (Waltham, USA). HEK293 cells and DC2.4 cells were purchased from National Collection of Authenticated Cell Cultures (Shanghai, China), cck-8 cell validation assay reagents were purchased from Biosharp Life Sciences (Anhui, China).

Preparation of LNPs

LNPs with different sizes are formulated by microfluidic mixing [36–38]. 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 Table 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 Table 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%.

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 [36].

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.

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 wavelength of 640 nm, respectively.

Cell culture

HEK293 cells were cultured in DMEM containing 10% FBS. DC2.4 cells were cultured in RPMI 1640 medium containing 10% FBS. Both HEK293 cells and DC2.4 cells were maintained at 37 °C, 5% CO2 in a humidified cell incubator.

In vitro transfection of mLuc

HEK293 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 mRNA 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).

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. All animals were housed in an SPF-grade animal facility which is temperature- and humidity- controlled environment with a 12-hour light/dark cycle. 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). Hearts, livers, spleens, lungs and kidneys of mice were harvested and imaged at 4 h after injection. 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).

Cellular uptake of LNPs

HEK293 cells and DC2.4 cells were seeded onto 24-well glass bottom plates for laser confocal imaging at a density of 1 × 105 cells per well and cultured at 37 °C overnight. Then, cells were treated with DiD-labeled mLuc LNPs at an mRNA concentration of 1 µg/mL. After incubation at 37 °C for 3 h, the cells were washed twice with 1× PBS and fixed with 4% polyformaldehyde (PFA) for 5 min. Then, the cells were stained with DAPI for 10 min in the dark and washed twice with 1× PBS. Images were captured by fluorescence microscopy (LSM710, Zeiss) at 20× magnification.

To study the mechanism of cellular uptake, HEK293 cells and DC2.4 cells were seeded onto 12-well plates at a density of 4 × 105 cells per well and further cultured overnight. The cells were pre-incubated with various endocytosis inhibitors, including 5 µM CPZ, 10 µM FIL, 3 µM CYTD, and 5 µM WORT, for 30 min according to methods in previous study [39]. 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).

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 °C. 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 °C 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).

Lysosomal escape

HEK293 cells and DC2.4 cells were seeded onto a 24-well glass-bottom plate at a density of 4 × 105 cells per well and further cultured for 24 h. Then, cells were treated with DiD-labeled LNPs for 4 h. Afterwards, cells were incubated with LysoTracker green at a dose of 100 nM LysoTracker Green for 2 h and stained with DAPI for 5 min. The colocalization of DiD and LysoTracker was imaged by fluorescence microscopy (SpinSR10, Olympus), and analyzed by ImageJ. The presence of LNPs within lysosomes was quantified by calculating the ratio of fluorescence colocalization between DiD and LysoTracker to the total fluorescence of DiD.

Cytotoxicity assay

HEK293 cells and DC2.4 cells were seeded onto 96-well plates at a density of 2 × 104 cells per well for DC2.4 cells, 2 × 104 cells per well for HEK293 cells and cultured for 24 h. For uptake inhibitors, cells were treated with cellular uptake inhibitors for 4 h. For positively charged LNPs, cells were treated for 24 h. The cck-8 cell viability assay reagents were then added to the cells and incubated for 1 h before detection using a plate reader at an absorbance of 450 nm.

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) or t-test. A p-value less than 0.05 was considered as statistically significant.

Results and discussions

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

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

Table 1 Particle size, PDI, zeta potential, and encapsulation efficiency (EE) of LNPs

	Particle size (nm)	PDI	Zeta potential (mV)	EE (%)	
LNP-S	94.61 ± 1.12	0.13 ± 0.02	-1.58 ± 0.37	90.4 ± 0.3	
LNP-M	121.93 ± 1.56**** (****p<0.0001 vs. LNP-S)	0.14 ± 0.01	-0.59 ± 0.02	95.4 ± 0.1	
LNP-L	167.37 ± 1.86**** (****p<0.0001 vs. LNP-S)	0.06 ± 0.01	1.26 ± 0.21	90.9 ± 0.1	

LNPs were labeled with the FRET pair (DiD-DiR), demonstrating a substantial FRET effect (Fig. 1C and D). The evaluation of these LNPs both in vitro and in vivo is illustrated in Fig. 1E. The immortalized cell lines, HEK293 and DC2.4, were transfected with LNP-S, LNP-M, or LNP-L. As shown in Fig. 1F, LNP-S exhibited the highest transfection efficiency in both cell lines. Specifically, in HEK293 cells, the transfection efficiencies of LNP-M and LNP-L were similar, whereas in DC2.4 cells, LNP-M outperformed LNP-L. Analogous results were observed following intramuscular injection (Fig. 1G). In addition, by labeling intact LNPs with a FRET pair (DiD-DiR), we found that the majority of the LNPs remained localized at the site of injection, insusceptible to the particle size (Fig. 1H). Luminescence images of all the time and ex vivo images are presented in Fig. S1A and Fig. S1B. LNP-S exhibited the highest transfection efficiency in situ (Fig. 1I, Fig. S1C). Notably, luciferase expression was detected in the liver across LNPs with all sizes, aligning with the previous finding [30]. Among these LNPs, no significant difference in luciferase expression was detected in the liver (Fig. 1J, Fig. S1D). Despite the observed significant differences in transfection efficiency in vivo, the in situ FRET ratio showed similar trends among LNP-S, LNP-M, and LNP-L (Fig. 1K).

As shown above, particle size significantly influences the transfection efficiency of LNPs in vitro. The observed differences in transfection efficiency between HEK293 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 [40]. 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 [29, 41]. 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.

Fig. 1 The effect of particle size on nanoparticle transfection and in vivo distribution. (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 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) In vitro and in vivo experimental flow chart of LNPs. A plate reader was exploited to detect luminescence in vitro, and IVIS was utilized to detect luminescence and fluorescence. (F) In vitro transfection of LNP-S, LNP-M, and LNP-L on HEK293 and DC2.4. The expression of firefly luciferase in cells was detected and analyzed. **p < 0.01, ***p < 0.001, ****p < 0.0001 (n = 3). (G) In vivo transfection of LNP-S, LNP-M, and LNP-L. Luminescence images obtained by IVIS, indicate the increased transfection with an order of LNP-S > LNP-M > LNP-L. (H) In vivo 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 < 0.05 vs. LNP-M and LNP-L. (J) A statistical graph of luminescence in the liver (n = 3). (K) A statistical graph of the FRET ratio on muscle (n = 3)

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

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

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

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

Fig. 2 The effect of surface charge on nanoparticle transfection and in vivo distribution. (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), ###p < 0.001 vs. neutral-LNP, ####p < 0.0001 vs. neutral-LNP, ***p < 0.001, ****p < 0.0001. (C) Particle size of LNPs with various charges (n = 3), ####p < 0.0001 vs. neutral-LNP. (D) In vitro transfection of LNPs with different charges on HEK293 and DC2.4. Expression of firefly luciferase in cells was detected and analyzed (n = 3), ####p < 0.0001 vs. neutral-LNP, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (E) In vivo transfection of LNPs with various charges. Luminescence was detected by IVIS. (F) In vivo 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 (n = 3), **p < 0.01 vs. 15% DSPG, 25% DSPG, 15% DOTAP, 25% DOTAP and 50% DOTAP, ****p < 0.0001 vs. 15% DSPG, 25% DSPG, 15% DOTAP, 25% DOTAP and 50% DOTAP. (H) Statistical analysis of the luminescence of the liver (n=3), *p < 0.05 vs. 15% DSPG, 25% DSPG, 15% DOTAP, 25% DOTAP and 50% DOTAP, **p < 0.01 vs. 15% DSPG, 25% DSPG, 15% DOTAP, 25% DOTAP and 50% DOTAP, ***p < 0.001 vs. 15% DSPG, 25% DSPG, 15% DOTAP, 25% DOTAP and 50% DOTAP, ****p < 0.0001 vs. 15% DSPG, 25% DSPG, 15% DOTAP, 25% DOTAP and 50% DOTAP. (I) The in vivo FRET ratio of LNPs with various charges was calculated and plotted (n = 3)

The effect of PEGylated lipid contents on the properties of LNPs

The impact of PEGylated lipids on the characteristics and functional attributes of LNPs was systematically evaluated by modulating the concentration of PEGylated lipids within the formulations. Specifically, LNPs were synthesized with 0.5 mol%, 1.5 mol%, and 3 mol% DMG-PEG2k to assess the effects on particle size, stability, and transfection efficiency, respectively. As shown in Fig. 3A, a notable decrease in particle size was observed with increasing DMG-PEG2k contents, potentially due to the expansion of the compression function attributed to the PEGylated lipids. The PDI of LNPs with varying contents of PEGylated lipids remained consistently below 0.3 (Fig. 3B). The results revealed that the content of PEGylated lipids affected the stability of the encapsulation efficiency of the LNPs (Fig. 3D-F). Specifically, the encapsulation efficiency of LNPs with 0.5 mol% DMG-PEG2k decreased significantly over a four-week period (Fig. 3F). These findings suggest that a reduction in PEGylated lipid contents may compromise the stability of LNPs, highlighting the critical role of these components in maintaining the structural integrity and functional efficacy of LNPs [42].

LNPs with 1.5 mol% DMG-PEG2k exhibited superior transfection efficiency in vitro (Fig. 3G). Luminescence images captured at all time points and ex vivo luminescence data are provided in Fig. S4A-B, providing a comprehensive view of the temporal and spatial dynamics of nanoparticle-mediated gene expression. In vivo assessments confirmed that LNPs with 1.5 mol% DMG-PEG2k demonstrated enhanced transfection capabilities (Fig. 3H). Additionally, the in vivo distribution of these nanoparticles, containing varying contents of DMG-PEG2k, did not significantly differ (Fig. 3I). Nanoparticles are mainly concentrated at the injection site (localized intramuscular region). It is obvious that LNPs with 1.5 mol% DMG-PEG2k facilitated notably excellent luciferase expression in situ and in the liver 4 h post-administration (Fig. 3J-K). Comprehensive luciferase expression in muscle and liver tissues is detailed in Fig. S4C-D. Despite similar particle sizes, LNPs with lower contents of PEGylated lipids showed better transfection efficiency. A notable observation was the delayed peak of luciferase expression in the liver when comparing LNPs with 1.5 mol% and 3 mol% DMG-PEG2k (Fig. 3K).

The impact of PEGylated lipid contents on the stability and transfection efficiency of LNPs represents a rarely explored area in nanoparticle research. Previous studies have suggested that the long circulation characteristics of PEGylated lipids are due to their resistance to cellular uptake [43]. As previously reported in the study, an elevated concentration of PEGylated lipids has been demonstrated to impair the efficacy of gene knockdown mediated by LNP-encapsulated siRNA [44]. These comply 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 [45]. 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.

Fig. 3 The effect of PEGylated lipid contents on the properties of LNPs. 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), ****p < 0.0001. 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), ***p < 0.001 vs. 1.5% PEG and 3% PEG, ****p < 0.0001 vs. 1.5% PEG and 3% PEG. (G) Statistical analysis of luciferase expression in HEK293 and DC2.4 after LNP transfection (n = 3). The graph on HEK293 is on the left, and that on DC2.4 is on the right. *p < 0.05, ****p < 0.0001. (H) In vivo 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) In vivo 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 < 0.05 vs. 0.5% PEG and 3% PEG, ***p < 0.001 vs. 0.5% PEG and 3% PEG. (K) A statistical chart of luminescence in the liver (n = 3), *p < 0.05 vs. 0.5% PEG and 3% PEG, **p < 0.005 vs. 0.5% PEG and 3% PEG. (L) A statistical chart of the FRET ratio on muscle (n = 3)

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

To investigate the roles of 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 [46]. DSG-PEG2k, a C18-PEG2k, was incorporated into LNPs to eliminate the interference of the acyl chain length [2]. The structural formulas of DMG-PEG2k, DSG-PEG2k, and DSPE-PEG2k are depicted in Fig. 4A. LNPs with any type of PEGylated lipids showed comparable particle size, PDI, and zeta potential (Fig. 4B-D). Additionally, LNPs composed of DMG-PEG2k, DSG-PEG2k, and DSPE-PEG2k exhibited good colloidal stability for a minimum duration of one week when stored at 4 °C (Fig. S5A-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. S5E-H).

Figure 4E illustrates the comparative analysis of transfection efficiency among LNPs incorporating DMG-PEG2k, DSG-PEG2k, and DSPE-PEG2k. Notably, luciferase activity significantly decreased within 72 h following transfection with LNPs containing DMG-PEG2k, in contrast to the other lipid types (Fig. 4E). Differences in the optimal timing of peak luciferase expression may be attributed to the variable shedding rates of PEGylated lipids. The accelerated dissociation of DMG-PEG2k potentially facilitated more rapid mRNA translation. In a buffer system containing 10% FBS, a decrease in luciferase activity was observed, indicating that serum components may adversely affect cellular transfection efficacy (Fig. 4F). Further investigations demonstrated that LNPs formulated with DMG-PEG2k exhibited enhanced transfection performance in vitro, as depicted in Fig. 4F. This observation was corroborated by in vivo studies, where intramuscular injection of LNPs containing DMG-PEG2k showed superior transfection efficiency (Fig. 4G-I, Fig. S6A-B). Overall, the total luciferase expression results suggest that LNPs equipped with DMG-PEG2k possess a notably greater capacity for mRNA delivery (Fig. S6C-D).

The impact of PEGylated lipid types on the in vitro and in vivo transfection efficiency of LNPs may be associated with variations in acyl chain length. This finding is supported by previous reports that LNPs with DMG-PEG2k have an enhanced ability to deliver siRNA than those with DSPE-PEG2k [43, 47]. The previous studies have indicated that acyl chain lengths of PEGylated lipids affect in vivo transfection efficiency during intravenous injection. This may be due to the rate at which acyl chains are shed from PEGylated lipids [22]. In accordance with the finding reported previously, the enhanced transfection efficiency observed in LNPs containing DMG-PEG2k can be attributed to its rapid dissociation from the nanoparticle complex, which subsequently facilitates greater protein delivery to the liver. Additionally, the functional groups of PEGylated lipids play crucial roles in cellular transfection efficiency in vitro, as evidenced by comparative analyses between LNPs formulated with DSG-PEG2k and those formulated with DSPE-PEG2k (shown in Fig. 4E-F). Such observations regarding the differential roles of functional groups in PEGylated lipids have rarely been reported in previous studies.

Fig. 4 The impact of types of PEGylated lipids on the properties of LNPs. (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) In vitro transfection of LNPs with DMG-PEG2k, DSG-PEG2k, and DSPE-PEG2k on HEK293 and DC2.4 at 12 h, 24 h, 48 h, and 72 h. The graph for HEK293 is on the left, and that for DC2.4 is on the right (n = 3), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (F) In vitro transfection of LNPs with DMG-PEG2k, DSG-PEG2k, and DSPE-PEG2k on HEK293 and DC2.4 in 10% FBS or 0% FBS. Luciferase expression was analyzed and plotted (n = 3), &&&&p < 0.0001 vs. DMG in 10% FBS, #p < 0.05 vs. DMG in 0% FBS, ***p < 0.001, ****p < 0.0001. (G) In vivo transfection 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 < 0.05 vs. DSG and DSPE. (I) Luminescence of LNPs with various types of PEGylated lipids in the liver was analyzed and plotted (n = 3), *p < 0.05 vs. DSG and DSPE, **p < 0.01 vs. DSG and DSPE

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 HEK293 cells increased with increasing incubation time (Fig. 5A). Specifically, LNPs containing DMG-PEG2k exhibited significantly greater cellular uptake than the other LNPs (Fig. 5A). This increased uptake may be influenced by the acyl chain length of the PEGylated lipids, suggesting that LNPs with longer acyl chains may exhibit reduced cellular uptake efficiency. The importance of acyl chain lengths of PEGylated lipids for the nucleic acid delivery of LNPs has been corroborated by a substantial body of research [2, 24]. The acyl chain of DMG-PEG2k is of a shorter length than that of the other two lipids, which contributes to a faster rate of shedding efficiency. Accordingly, the cellular uptake of LNPs formulated with DMG-PEG2k is more efficient, which is in accordance with the results presented in Fig. 5A. Functional groups of PEGylated lipids also affect cellular uptake in HEK293 and DC2.4 cells. Notably, LNPs formulated with DSG-PEG2k demonstrated markedly superior cellular uptake efficiency compared to those formulated with DSPE-PEG2k (Fig. 5B). According to the results above, FBS affect cell transfection efficiency. Subsequent studies, shown in Fig. 5C, investigated the protein corona adsorbed by LNPs containing DMG-PEG2k, DSG-PEG2k, and DSPE-PEG2k in the presence of FBS. The results revealed no significant differences in the type of protein corona formed among these LNPs. The observed variations in cellular uptake between different PEGylated lipids are primarily attributed to the length of the acyl chain and the specific functional groups, factors that appear to be independent of the protein corona composition. These findings highlight the complex interactions between the structural attributes of PEGylated lipids and their functional performance in cellular environments. To investigate the specific cellular uptake mechanisms of LNPs with various types of PEGylated lipids, a series of inhibitors, including CPZ, FIL, CYTD, and WORT, were utilized. In HEK293 cells, the uptake efficiency of LNPs with DMG-PEG2k was inhibited by CPZ and WORT, while the uptake of LNPs containing DSG-PEG2k and DSPE-PEG2k was inhibited by CPZ, FIL, and WORT (Fig. 5D-F). In DC2.4 cells, both CPZ and WORT impacted the cellular uptake of all tested PEGylated lipid types, and FIL also inhibited the cellular uptake of LNPs with DSG-PEG2k and DSPE-PEG2k (Fig. 5G-I). Cytotoxicity tests of HEK293 cells and DC2.4 cells treated with each inhibitor were exhibited in Fig. S6. It is evident that cell viability was all above 70% for each inhibitor, indicating that the differences in cellular uptake were not due to cell death.

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 HEK293 and DC2.4, LNPs incorporating DMG-PEG2k demonstrated superior lysosomal escape efficiencies (Fig. 5J-L).

Previous studies have reported that acyl chain length affects the adsorption rate of the protein corona [43]. However, it does not significantly affect the type of protein corona formed, which has not been previously explored. The results in HEK293 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. Acyl chain lengths influence cellular uptake efficiency by affecting endocytic pathways, which has not been reported previously. It is shown that the longer hydrophobic chains of PEGylated lipids in LNPs, the more likely LNPs are to enter cells via the caveolin system. This could serve as a hypothesis for future studies. 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 the 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 [43, 48], 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.

Fig. 5 Underlying cellular mechanisms affecting transfection by PEGylated lipids. (A) Cellular uptake of LNPs with DMG-PEG2k, DSG-PEG2k, and DSPE-PEG2k in HEK293 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 HEK293 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 HEK293 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 < 0.05 vs. DMSO, **p < 0.01 vs. DMSO, ***p < 0.001 vs. DMSO, ****p < 0.0001 vs. DMSO. (J-K) Lysosomal escape in HEK293 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 HEK293 cells is above, and that for DC2.4 cells is below; ***p < 0.001 vs. DMG, ****p < 0.0001 vs. DMG

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.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Material 1

Acknowledgements

Not applicable.

Author 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.

Funding

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

Data availability

No datasets were generated or analysed during the current study.

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.

Competing interests

The authors declare no competing interests.

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-methoxypolyethylene glycol-2000

DSG-PEG2k 1,2-distearoyl-rac-glycerol-3-methoxypolyethylene glycol-2000

DSPE-PEG2k N-(Carbonyl-methoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycerol-3-phosphoethanolamine

DMEM Dulbecco’s Modified Eagle Medium

DiD 1,1’-dioctadecyl-3,3,3’,3’-tetramethylindodicarbocyanine

DiR 1,1’-Dioctadecyl-3,3,3’,3’-Tetramethylindotricarbocyanine iodide

FIL Filipin

CPZ Chlorpromazine

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 Institutional Animal Care and Use Committee

PFA Polyformaldehyde

RIPA Radioimmunoprecipitation Assay

SEM Standard Error of Mean

ANOVA Analysis of Variance

EE Encapsulation Efficiencies

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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References

1. Samaridou E Heyes J Lutwyche P Lipid nanoparticles for nucleic acid delivery: current perspectives Adv Drug Deliv Rev 2020 154–155 37 63 10.1016/j.addr.2020.06.002 32526452
Samaridou E, Heyes J, Lutwyche P. Lipid nanoparticles for nucleic acid delivery: current perspectives. Adv Drug Deliv Rev. 2020;154–155:37–63.32526452 10.1016/j.addr.2020.06.002
2. Suzuki T Suzuki Y Hihara T Kubara K Kondo K Hyodo K Yamazaki K Ishida T Ishihara H PEG shedding-rate-dependent blood clearance of PEGylated lipid nanoparticles in mice: faster PEG shedding attenuates anti-PEG IgM production Int J Pharm 2020 588 119792 10.1016/j.ijpharm.2020.119792 32827675
Suzuki T, Suzuki Y, Hihara T, Kubara K, Kondo K, Hyodo K, Yamazaki K, Ishida T, Ishihara H. PEG shedding-rate-dependent blood clearance of PEGylated lipid nanoparticles in mice: faster PEG shedding attenuates anti-PEG IgM production. Int J Pharm. 2020;588:119792.32827675 10.1016/j.ijpharm.2020.119792
3. Yang C, Lin ZI, Zhang X, Xu Z, Xu G, Wang YM, Tsai TH, Cheng PW, Law WC. K T Yong, and C K Chen. Recent advances in Engineering carriers for siRNA delivery. Macromol Biosci. 2023: e2300362.
4. Lamb YN Inclisiran First Approval Drugs 2021 81 3 389 95 33620677
Lamb YN, Inclisiran. First Approval Drugs. 2021;81(3):389–95.33620677
5. Aljabali AAA Bashatwah RM Obeid MA Mishra V Mishra Y Serrano-Aroca A Lundstrom K Tambuwala MM Current state of, prospects for, and obstacles to mRNA vaccine development Drug Discov Today 2023 28 2 103458 10.1016/j.drudis.2022.103458 36427779
Aljabali AAA, Bashatwah RM, Obeid MA, Mishra V, Mishra Y, Serrano-Aroca A, Lundstrom K, Tambuwala MM. Current state of, prospects for, and obstacles to mRNA vaccine development. Drug Discov Today. 2023;28(2):103458.36427779 10.1016/j.drudis.2022.103458
6. Pardi N Hogan MJ Porter FW Weissman D mRNA vaccines - a new era in vaccinology Nat Rev Drug Discov 2018 17 4 261 79 10.1038/nrd.2017.243 29326426
Pardi N, Hogan MJ, Porter FW, Weissman D. mRNA vaccines - a new era in vaccinology. Nat Rev Drug Discov. 2018;17(4):261–79.29326426 10.1038/nrd.2017.243
7. Pardi N Tuyishime S Muramatsu H Kariko K Mui BL Tam YK Madden TD Hope MJ Weissman D Expression kinetics of nucleoside-modified mRNA delivered in lipid nanoparticles to mice by various routes J Control Release 2015 217 345 51 10.1016/j.jconrel.2015.08.007 26264835
Pardi N, Tuyishime S, Muramatsu H, Kariko K, Mui BL, Tam YK, Madden TD, Hope MJ, Weissman D. Expression kinetics of nucleoside-modified mRNA delivered in lipid nanoparticles to mice by various routes. J Control Release. 2015;217:345–51.26264835 10.1016/j.jconrel.2015.08.007
8. Reichmuth AM, O M, Jaklenec A, Langer R, Blankschtein D. mRNA vaccine delivery using lipid nanoparticles. Ther Deliv. 2016;7(5):319–334.
9. Buschmann MD, Carrasco MJ, Alishetty S, Paige M, Alameh MG, Weissman D. Nanomaterial Delivery systems for mRNA vaccines. Vaccines (Basel). 2021;9(1):65.
10. Wang Q, Jiang Q, Li D, Yang Z, Gao L, Liu F, Li C, Feng Y, He Z, Luo C, Sun J. Elaborately engineering of lipid nanoparticle for targeting delivery of siRNA and suppressing acute liver injury. Chin Chem Lett. 2024;35(2):108683.
11. Schoenmaker L Witzigmann D Kulkarni JA Verbeke R Kersten G Jiskoot W Crommelin DJA mRNA-lipid nanoparticle COVID-19 vaccines: structure and stability Int J Pharm 2021 601 120586 10.1016/j.ijpharm.2021.120586 33839230
Schoenmaker L, Witzigmann D, Kulkarni JA, Verbeke R, Kersten G, Jiskoot W, Crommelin DJA. mRNA-lipid nanoparticle COVID-19 vaccines: structure and stability. Int J Pharm. 2021;601:120586.33839230 10.1016/j.ijpharm.2021.120586
12. Carrasco MJ Alishetty S Alameh MG Said H Wright L Paige M Soliman O Weissman D t Cleveland TE Grishaev A Buschmann MD Ionization and structural properties of mRNA lipid nanoparticles influence expression in intramuscular and intravascular administration Commun Biol 2021 4 1 956 10.1038/s42003-021-02441-2 34381159
Carrasco MJ, Alishetty S, Alameh MG, Said H, Wright L, Paige M, Soliman O, Weissman D, t Cleveland TE, Grishaev A, Buschmann MD. Ionization and structural properties of mRNA lipid nanoparticles influence expression in intramuscular and intravascular administration. Commun Biol. 2021;4(1):956.34381159 10.1038/s42003-021-02441-2
13. Hassett KJ Benenato KE Jacquinet E Lee A Woods A Yuzhakov O Himansu S Deterling J Geilich BM Ketova T Mihai C Lynn A McFadyen I Moore MJ Senn JJ Stanton MG Almarsson O Ciaramella G Brito LA Optimization of lipid nanoparticles for Intramuscular Administration of mRNA vaccines Mol Ther Nucleic Acids 2019 15 1 11 10.1016/j.omtn.2019.01.013 30785039
Hassett KJ, Benenato KE, Jacquinet E, Lee A, Woods A, Yuzhakov O, Himansu S, Deterling J, Geilich BM, Ketova T, Mihai C, Lynn A, McFadyen I, Moore MJ, Senn JJ, Stanton MG, Almarsson O, Ciaramella G, Brito LA. Optimization of lipid nanoparticles for Intramuscular Administration of mRNA vaccines. Mol Ther Nucleic Acids. 2019;15:1–11.30785039 10.1016/j.omtn.2019.01.013
14. Li C Lee A Grigoryan L Arunachalam PS Scott MKD Trisal M Wimmers F Sanyal M Weidenbacher PA Feng Y Adamska JZ Valore E Wang Y Verma R Reis N Dunham D O’Hara R Park H Luo W Gitlin AD Kim P Khatri P Nadeau KC Pulendran B Mechanisms of innate and adaptive immunity to the Pfizer-BioNTech BNT162b2 vaccine Nat Immunol 2022 23 4 543 55 10.1038/s41590-022-01163-9 35288714
Li C, Lee A, Grigoryan L, Arunachalam PS, Scott MKD, Trisal M, Wimmers F, Sanyal M, Weidenbacher PA, Feng Y, Adamska JZ, Valore E, Wang Y, Verma R, Reis N, Dunham D, O’Hara R, Park H, Luo W, Gitlin AD, Kim P, Khatri P, Nadeau KC, Pulendran B. Mechanisms of innate and adaptive immunity to the Pfizer-BioNTech BNT162b2 vaccine. Nat Immunol. 2022;23(4):543–55.35288714 10.1038/s41590-022-01163-9
15. Billingsley MM Hamilton AG Mai D Patel SK Swingle KL Sheppard NC June CH Mitchell MJ Orthogonal Design of experiments for optimization of lipid nanoparticles for mRNA Engineering of CAR T cells Nano Lett 2022 22 1 533 42 10.1021/acs.nanolett.1c02503 34669421
Billingsley MM, Hamilton AG, Mai D, Patel SK, Swingle KL, Sheppard NC, June CH, Mitchell MJ. Orthogonal Design of experiments for optimization of lipid nanoparticles for mRNA Engineering of CAR T cells. Nano Lett. 2022;22(1):533–42.34669421 10.1021/acs.nanolett.1c02503
16. Billingsley MM Singh N Ravikumar P Zhang R June CH Mitchell MJ Ionizable lipid nanoparticle-mediated mRNA delivery for Human CAR T Cell Engineering Nano Lett 2020 20 3 1578 89 10.1021/acs.nanolett.9b04246 31951421
Billingsley MM, Singh N, Ravikumar P, Zhang R, June CH, Mitchell MJ. Ionizable lipid nanoparticle-mediated mRNA delivery for Human CAR T Cell Engineering. Nano Lett. 2020;20(3):1578–89.31951421 10.1021/acs.nanolett.9b04246
17. Rurik JG Tombacz I Yadegari A Mendez Fernandez PO Shewale SV Li L Kimura T Soliman OY Papp TE Tam YK Mui BL Albelda SM Pure E June CH Aghajanian H Weissman D Parhiz H Epstein JA CAR T cells produced in vivo to treat cardiac injury Science 2022 375 6576 91 6 10.1126/science.abm0594 34990237
Rurik JG, Tombacz I, Yadegari A, Mendez Fernandez PO, Shewale SV, Li L, Kimura T, Soliman OY, Papp TE, Tam YK, Mui BL, Albelda SM, Pure E, June CH, Aghajanian H, Weissman D, Parhiz H, Epstein JA. CAR T cells produced in vivo to treat cardiac injury. Science. 2022;375(6576):91–6.34990237 10.1126/science.abm0594
18. Okuda K Sato Y Iwakawa K Sasaki K Okabe N Maeki M Tokeshi M Harashima H On the size-regulation of RNA-loaded lipid nanoparticles synthesized by microfluidic device J Control Release 2022 348 648 59 10.1016/j.jconrel.2022.06.017 35716883
Okuda K, Sato Y, Iwakawa K, Sasaki K, Okabe N, Maeki M, Tokeshi M, Harashima H. On the size-regulation of RNA-loaded lipid nanoparticles synthesized by microfluidic device. J Control Release. 2022;348:648–59.35716883 10.1016/j.jconrel.2022.06.017
19. Chatzikleanthous D D T O’Hagan Lipid-based nanoparticles for delivery of Vaccine adjuvants and antigens: toward Multicomponent vaccines Mol Pharm 2021 18 8 2867 88 10.1021/acs.molpharmaceut.1c00447 34264684
Chatzikleanthous D, D T O’Hagan. Lipid-based nanoparticles for delivery of Vaccine adjuvants and antigens: toward Multicomponent vaccines. Mol Pharm. 2021;18(8):2867–88.34264684 10.1021/acs.molpharmaceut.1c00447
20. Jackman MJ Li W Smith A Workman D Treacher KE Corrigan A Abdulrazzaq F Sonzini S Nazir Z Lawrence MJ Mahmoudi N Cant D Counsell J Cairns J Ferguson D Lenz E Baquain S Madla CM van Pelt S Moss J Peter A Puri S Ashford M Mazza M Impact of the physical-chemical properties of poly(lactic acid)-poly(ethylene glycol) polymeric nanoparticles on biodistribution J Control Release 2024 365 491 506 10.1016/j.jconrel.2023.11.043 38030083
Jackman MJ, Li W, Smith A, Workman D, Treacher KE, Corrigan A, Abdulrazzaq F, Sonzini S, Nazir Z, Lawrence MJ, Mahmoudi N, Cant D, Counsell J, Cairns J, Ferguson D, Lenz E, Baquain S, Madla CM, van Pelt S, Moss J, Peter A, Puri S, Ashford M, Mazza M. Impact of the physical-chemical properties of poly(lactic acid)-poly(ethylene glycol) polymeric nanoparticles on biodistribution. J Control Release. 2024;365:491–506.38030083 10.1016/j.jconrel.2023.11.043
21. Cheng Q Wei T Farbiak L Johnson LT Dilliard SA Siegwart DJ Selective organ targeting (SORT) nanoparticles for tissue-specific mRNA delivery and CRISPR-Cas gene editing Nat Nanotechnol 2020 15 4 313 20 10.1038/s41565-020-0669-6 32251383
Cheng Q, Wei T, Farbiak L, Johnson LT, Dilliard SA, Siegwart DJ. Selective organ targeting (SORT) nanoparticles for tissue-specific mRNA delivery and CRISPR-Cas gene editing. Nat Nanotechnol. 2020;15(4):313–20.32251383 10.1038/s41565-020-0669-6
22. Dilliard SA, Cheng Q, Siegwart DJ. On the mechanism of tissue-specific mRNA delivery by selective organ targeting nanoparticles. Proc Natl Acad Sci U S A. 2021;118(52):e2109256118.
23. Ryals RC Patel S Acosta C McKinney M Pennesi ME Sahay G The effects of PEGylation on LNP based mRNA delivery to the eye PLoS ONE 2020 15 10 e0241006 10.1371/journal.pone.0241006 33119640
Ryals RC, Patel S, Acosta C, McKinney M, Pennesi ME, Sahay G. The effects of PEGylation on LNP based mRNA delivery to the eye. PLoS ONE. 2020;15(10):e0241006.33119640 10.1371/journal.pone.0241006
24. Shi D Beasock D Fessler A Szebeni J Ljubimova JY Afonin KA Dobrovolskaia MA To PEGylate or not to PEGylate: immunological properties of nanomedicine’s most popular component, polyethylene glycol and its alternatives Adv Drug Deliv Rev 2022 180 114079 10.1016/j.addr.2021.114079 34902516
Shi D, Beasock D, Fessler A, Szebeni J, Ljubimova JY, Afonin KA, Dobrovolskaia MA. To PEGylate or not to PEGylate: immunological properties of nanomedicine’s most popular component, polyethylene glycol and its alternatives. Adv Drug Deliv Rev. 2022;180:114079.34902516 10.1016/j.addr.2021.114079
25. Hashiba K Sato Y Harashima H pH-labile PEGylation of siRNA-loaded lipid nanoparticle improves active targeting and gene silencing activity in hepatocytes J Control Release 2017 262 239 46 10.1016/j.jconrel.2017.07.046 28774839
Hashiba K, Sato Y, Harashima H. pH-labile PEGylation of siRNA-loaded lipid nanoparticle improves active targeting and gene silencing activity in hepatocytes. J Control Release. 2017;262:239–46.28774839 10.1016/j.jconrel.2017.07.046
26. Grenier P Chenard V Bertrand N The mechanisms of anti-PEG immune response are different in the spleen and the lymph nodes J Control Release 2023 353 611 20 10.1016/j.jconrel.2022.12.005 36493950
Grenier P, Chenard V, Bertrand N. The mechanisms of anti-PEG immune response are different in the spleen and the lymph nodes. J Control Release. 2023;353:611–20.36493950 10.1016/j.jconrel.2022.12.005
27. Suzuki Y Ishihara H Structure, activity and uptake mechanism of siRNA-lipid nanoparticles with an asymmetric ionizable lipid Int J Pharm 2016 510 1 350 8 10.1016/j.ijpharm.2016.06.124 27374199
Suzuki Y, Ishihara H. Structure, activity and uptake mechanism of siRNA-lipid nanoparticles with an asymmetric ionizable lipid. Int J Pharm. 2016;510(1):350–8.27374199 10.1016/j.ijpharm.2016.06.124
28. Sahay G Querbes W Alabi C Eltoukhy A Sarkar S Zurenko C Karagiannis E Love K Chen D Zoncu R Buganim Y Schroeder A Langer R Anderson DG Efficiency of siRNA delivery by lipid nanoparticles is limited by endocytic recycling Nat Biotechnol 2013 31 7 653 8 10.1038/nbt.2614 23792629
Sahay G, Querbes W, Alabi C, Eltoukhy A, Sarkar S, Zurenko C, Karagiannis E, Love K, Chen D, Zoncu R, Buganim Y, Schroeder A, Langer R, Anderson DG. Efficiency of siRNA delivery by lipid nanoparticles is limited by endocytic recycling. Nat Biotechnol. 2013;31(7):653–8.23792629 10.1038/nbt.2614
29. Cui L Hunter MR Sonzini S Pereira S Romanelli SM Liu K Li W Liang L Yang B Mahmoudi N Desai AS Mechanistic studies of an automated lipid nanoparticle reveal critical Pharmaceutical Properties Associated with enhanced mRNA functional delivery in Vitro and in vivo Small 2022 18 9 e2105832 10.1002/smll.202105832 34914866
Cui L, Hunter MR, Sonzini S, Pereira S, Romanelli SM, Liu K, Li W, Liang L, Yang B, Mahmoudi N, Desai AS. Mechanistic studies of an automated lipid nanoparticle reveal critical Pharmaceutical Properties Associated with enhanced mRNA functional delivery in Vitro and in vivo. Small. 2022;18(9):e2105832.34914866 10.1002/smll.202105832
30. Di J Du Z Wu K Jin S Wang X Li T Xu Y Biodistribution and non-linear gene expression of mRNA LNPs affected by Delivery Route and particle size Pharm Res 2022 39 1 105 14 10.1007/s11095-022-03166-5 35080707
Di J, Du Z, Wu K, Jin S, Wang X, Li T, Xu Y. Biodistribution and non-linear gene expression of mRNA LNPs affected by Delivery Route and particle size. Pharm Res. 2022;39(1):105–14.35080707 10.1007/s11095-022-03166-5
31. Manolova V Flace A Bauer M Schwarz K Saudan P Bachmann MF Nanoparticles target distinct dendritic cell populations according to their size Eur J Immunol 2008 38 5 1404 13 10.1002/eji.200737984 18389478
Manolova V, Flace A, Bauer M, Schwarz K, Saudan P, Bachmann MF. Nanoparticles target distinct dendritic cell populations according to their size. Eur J Immunol. 2008;38(5):1404–13.18389478 10.1002/eji.200737984
32. Hassett KJ Higgins J Woods A Levy B Xia Y Hsiao CJ Acosta E Almarsson O Moore MJ Brito LA Impact of lipid nanoparticle size on mRNA vaccine immunogenicity J Control Release 2021 335 237 46 10.1016/j.jconrel.2021.05.021 34019945
Hassett KJ, Higgins J, Woods A, Levy B, Xia Y, Hsiao CJ, Acosta E, Almarsson O, Moore MJ, Brito LA. Impact of lipid nanoparticle size on mRNA vaccine immunogenicity. J Control Release. 2021;335:237–46.34019945 10.1016/j.jconrel.2021.05.021
33. Ju Y Lee WS Pilkington EH Kelly HG Li S Selva KJ Wragg KM Subbarao K Nguyen THO Rowntree LC Allen LF Bond K Williamson DA Truong NP Plebanski M Kedzierska K Mahanty S Chung AW Caruso F Wheatley AK Juno JA Kent SJ Anti-PEG antibodies boosted in humans by SARS-CoV-2 lipid nanoparticle mRNA vaccine ACS Nano 2022 16 8 11769 80 10.1021/acsnano.2c04543 35758934
Ju Y, Lee WS, Pilkington EH, Kelly HG, Li S, Selva KJ, Wragg KM, Subbarao K, Nguyen THO, Rowntree LC, Allen LF, Bond K, Williamson DA, Truong NP, Plebanski M, Kedzierska K, Mahanty S, Chung AW, Caruso F, Wheatley AK, Juno JA, Kent SJ. Anti-PEG antibodies boosted in humans by SARS-CoV-2 lipid nanoparticle mRNA vaccine. ACS Nano. 2022;16(8):11769–80.35758934 10.1021/acsnano.2c04543
34. Walsh EE Frenck RW Jr Falsey AR Kitchin N Absalon J Gurtman A Lockhart S Neuzil K Mulligan MJ Bailey R Swanson KA Li P Koury K Kalina W Cooper D Fontes-Garfias C Shi PY Tureci O Tompkins KR Lyke KE Raabe V Dormitzer PR Jansen KU Sahin, and W C Gruber. Safety and Immunogenicity of two RNA-Based Covid-19 vaccine candidates N Engl J Med 2020 383 25 2439 50 10.1056/NEJMoa2027906 33053279
Walsh EE, Frenck RW Jr., Falsey AR, Kitchin N, Absalon J, Gurtman A, Lockhart S, Neuzil K, Mulligan MJ, Bailey R, Swanson KA, Li P, Koury K, Kalina W, Cooper D, Fontes-Garfias C, Shi PY, Tureci O, Tompkins KR, Lyke KE, Raabe V, Dormitzer PR, Jansen KU. Sahin, and W C Gruber. Safety and Immunogenicity of two RNA-Based Covid-19 vaccine candidates. N Engl J Med. 2020;383(25):2439–50.33053279 10.1056/NEJMoa2027906
35. Estape Senti M C A de Jongh K Dijkxhoorn JJF Verhoef J Szebeni G Storm CE Hack RM Schiffelers MH Fens Boross P Anti-PEG antibodies compromise the integrity of PEGylated lipid-based nanoparticles via complement J Control Release 2022 341 475 86 10.1016/j.jconrel.2021.11.042 34890719
Estape Senti M, C A de Jongh K, Dijkxhoorn JJF, Verhoef J, Szebeni G, Storm CE, Hack RM, Schiffelers MH, Fens, Boross P. Anti-PEG antibodies compromise the integrity of PEGylated lipid-based nanoparticles via complement. J Control Release. 2022;341:475–86.34890719 10.1016/j.jconrel.2021.11.042
36. Bailey-Hytholt CM, Ghosh P, Dugas J, Zarraga IE, Bandekar A. Formulating and characterizing lipid nanoparticles for gene delivery using a microfluidic mixing platform. J Vis Exp. 2021;(168):e62226.
37. Chen D Love KT Chen Y Eltoukhy AA Kastrup C Sahay G Jeon A Dong Y Whitehead KA Anderson DG Rapid discovery of potent siRNA-containing lipid nanoparticles enabled by controlled microfluidic formulation J Am Chem Soc 2012 134 16 6948 51 10.1021/ja301621z 22475086
Chen D, Love KT, Chen Y, Eltoukhy AA, Kastrup C, Sahay G, Jeon A, Dong Y, Whitehead KA, Anderson DG. Rapid discovery of potent siRNA-containing lipid nanoparticles enabled by controlled microfluidic formulation. J Am Chem Soc. 2012;134(16):6948–51.22475086 10.1021/ja301621z
38. Maeki M Uno S Niwa A Okada Y Tokeshi M Microfluidic technologies and devices for lipid nanoparticle-based RNA delivery J Control Release 2022 344 80 96 10.1016/j.jconrel.2022.02.017 35183654
Maeki M, Uno S, Niwa A, Okada Y, Tokeshi M. Microfluidic technologies and devices for lipid nanoparticle-based RNA delivery. J Control Release. 2022;344:80–96.35183654 10.1016/j.jconrel.2022.02.017
39. Huang JL Jiang G Song QX Gu X Hu M Wang XL Song HH Chen LP Lin YY Jiang D Chen J Feng JF Qiu YM Jiang JY Jiang XG Chen HZ Gao XL Lipoprotein-biomimetic nanostructure enables efficient targeting delivery of siRNA to ras-activated glioblastoma cells via macropinocytosis Nat Commun 2017 8 15144 10.1038/ncomms15144 28489075
Huang JL, Jiang G, Song QX, Gu X, Hu M, Wang XL, Song HH, Chen LP, Lin YY, Jiang D, Chen J, Feng JF, Qiu YM, Jiang JY, Jiang XG, Chen HZ, Gao XL. Lipoprotein-biomimetic nanostructure enables efficient targeting delivery of siRNA to ras-activated glioblastoma cells via macropinocytosis. Nat Commun. 2017;8:15144.28489075 10.1038/ncomms15144
40. Wisse E Jacobs F Topal B Frederik P De Geest B The size of endothelial fenestrae in human liver sinusoids: implications for hepatocyte-directed gene transfer Gene Ther 2008 15 17 1193 9 10.1038/gt.2008.60 18401434
Wisse E, Jacobs F, Topal B, Frederik P, De Geest B. The size of endothelial fenestrae in human liver sinusoids: implications for hepatocyte-directed gene transfer. Gene Ther. 2008;15(17):1193–9.18401434 10.1038/gt.2008.60
41. Hunter MR Cui L Porebski BT Pereira S Sonzini S Odunze U Iyer P Engkvist O Lloyd RL Peel S Sabirsh A Ross-Thriepland D Jones AT Desai. Understanding Intracellular Biology to improve mRNA delivery by lipid nanoparticles Small Methods 2023 7 9 e2201695 10.1002/smtd.202201695 37317010
Hunter MR, Cui L, Porebski BT, Pereira S, Sonzini S, Odunze U, Iyer P, Engkvist O, Lloyd RL, Peel S, Sabirsh A, Ross-Thriepland D, Jones AT. Desai. Understanding Intracellular Biology to improve mRNA delivery by lipid nanoparticles. Small Methods. 2023;7(9):e2201695.37317010 10.1002/smtd.202201695
42. Kim J Jozic A Lin Y Eygeris Y Bloom E Tan X Acosta C MacDonald KD Welsher KD Sahay G Engineering lipid nanoparticles for enhanced intracellular delivery of mRNA through Inhalation ACS Nano 2022 16 9 14792 806 10.1021/acsnano.2c05647 36038136
Kim J, Jozic A, Lin Y, Eygeris Y, Bloom E, Tan X, Acosta C, MacDonald KD, Welsher KD, Sahay G. Engineering lipid nanoparticles for enhanced intracellular delivery of mRNA through Inhalation. ACS Nano. 2022;16(9):14792–806.36038136 10.1021/acsnano.2c05647
43. Berger M, Degey M, Leblond Chain J, Maquoi E, Evrard B, Lechanteur A, Piel G. Effect of PEG anchor and serum on lipid nanoparticles: development of a nanoparticles tracking method. Pharmaceutics. 2023;15(2):597.
44. Mui BL Tam YK Jayaraman M Ansell SM Du X Tam YY Lin PJ Chen S Narayanannair JK Rajeev KG Manoharan M Akinc A Maier MA Cullis P Madden TD Hope MJ Influence of polyethylene glycol lipid desorption rates on pharmacokinetics and pharmacodynamics of siRNA lipid nanoparticles Mol Ther Nucleic Acids 2013 2 12 e139 10.1038/mtna.2013.66 24345865
Mui BL, Tam YK, Jayaraman M, Ansell SM, Du X, Tam YY, Lin PJ, Chen S, Narayanannair JK, Rajeev KG, Manoharan M, Akinc A, Maier MA, Cullis P, Madden TD, Hope MJ. Influence of polyethylene glycol lipid desorption rates on pharmacokinetics and pharmacodynamics of siRNA lipid nanoparticles. Mol Ther Nucleic Acids. 2013;2(12):e139.24345865 10.1038/mtna.2013.66
45. Muller JA Schaffler N Kellerer T Schwake G Ligon TS Radler JO Kinetics of RNA-LNP delivery and protein expression Eur J Pharm Biopharm 2024 197 114222 10.1016/j.ejpb.2024.114222 38387850
Muller JA, Schaffler N, Kellerer T, Schwake G, Ligon TS, Radler JO. Kinetics of RNA-LNP delivery and protein expression. Eur J Pharm Biopharm. 2024;197:114222.38387850 10.1016/j.ejpb.2024.114222
46. Wilson SC Baryza JL Reynolds AJ Bowman K Keegan ME Standley SM Gardner NP Parmar P Agir VO Yadav S Zunic A Vargeese C Lee CC Rajan S Real time measurement of PEG shedding from lipid nanoparticles in serum via NMR spectroscopy Mol Pharm 2015 12 2 386 92 10.1021/mp500400k 25581130
Wilson SC, Baryza JL, Reynolds AJ, Bowman K, Keegan ME, Standley SM, Gardner NP, Parmar P, Agir VO, Yadav S, Zunic A, Vargeese C, Lee CC, Rajan S. Real time measurement of PEG shedding from lipid nanoparticles in serum via NMR spectroscopy. Mol Pharm. 2015;12(2):386–92.25581130 10.1021/mp500400k
47. Hattori Y Tamaki K Sakasai S Ozaki KI Onishi H Effects of PEG anchors in PEGylated siRNA lipoplexes on in vitro gene–silencing effects and siRNA biodistribution in mice Mol Med Rep 2020 22 5 4183 96 33000194
Hattori Y, Tamaki K, Sakasai S, Ozaki KI, Onishi H. Effects of PEG anchors in PEGylated siRNA lipoplexes on in vitro gene–silencing effects and siRNA biodistribution in mice. Mol Med Rep. 2020;22(5):4183–96.33000194
48. Sarode A Fan Y Byrnes AE Hammel M Hura GL Fu Y Kou P Hu C Hinz FI Roberts J Koenig SG Nagapudi K Hoogenraad CC Chen T Leung D Yen CW Predictive high-throughput screening of PEGylated lipids in oligonucleotide-loaded lipid nanoparticles for neuronal gene silencing Nanoscale Adv 2022 4 9 2107 23 10.1039/D1NA00712B 36133441
Sarode A, Fan Y, Byrnes AE, Hammel M, Hura GL, Fu Y, Kou P, Hu C, Hinz FI, Roberts J, Koenig SG, Nagapudi K, Hoogenraad CC, Chen T, Leung D, Yen CW. Predictive high-throughput screening of PEGylated lipids in oligonucleotide-loaded lipid nanoparticles for neuronal gene silencing. Nanoscale Adv. 2022;4(9):2107–23.36133441 10.1039/D1NA00712B
