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. 2025 May 14;25(9):70005. doi: 10.1002/mabi.202500165

Histidine Decapeptide‐Incorporating Lipid Nanoparticles with Low Ionizable Lipids Proportion for Efficient Small Interfering RNA Delivery

Yeonho Bae 1, Hyeondo Lee 1, Jun Hyuk Lee 1, Sangho Yeo 1, Hyejung Mok 1,✉
PMCID: PMC12434654  PMID: 40366254

Abstract

The diversification of lipid compositions in lipid nanoparticles (LNPs) is crucial for expanding their clinical applications and overcoming current limitations. In this study, LNPs with varying lipid compositions are fabricated using three different mixing processes (pipette, vortex, and microfluidic mixing) for small interfering RNA (siRNA) delivery. While both siRNA and hydrophobic fluorescent dye are successfully incorporated within LNPs using pipette‐ and vortex‐mixing, hydrophilic peptides cannot be encapsulated. Following optimization of ionizable lipid proportion via cost‐efficient vortex‐mixing method, LNPs with a lower ionizable lipid proportion (27.72%), termed LNP5, are selected and fabricated with histidine decapeptide (His10) during formulation via microfluidic mixing method to supplement the function of approximately half of the ionizable lipids by simple addition of His10. His10‐ incorporated LNP5 (LNP5H) exhibited a 1.6‐fold increase in gene silencing efficiency, compared to conventional LNPs (cLNPs; ionizable lipid proportion of 47.95%). Furthermore, LNP5H maintained siRNA potency for 4 weeks when stored in a 1% sucrose solution at −70 °C. Taken together, it fabricates potent LNP5H with low proportion of ionizable lipids via fast and easy processes, which can be applied to a variety of siRNA therapeutics for their efficient intracellular delivery.

Keywords: delivery; histidine decapeptide; lipid composition; lipid nanoparticle, small interfering RNA

Following optimization of ionizable lipid proportion via cost‐efficient vortex‐mixing method, lipid nanoparticles (LNPs) with a lower ionizable lipid proportion (27.72%), termed LNP5, are fabricated with histidine decapeptide (His10) during formulation via microfluidic mixing method. His10‐incorporated LNP5 (LNP5H) exhibited a 1.6‐fold increase in gene silencing efficiency, compared to conventional LNPs. Taken together, it fabricates potent LNP5H with low proportion of ionizable lipids via fast and easy processes, which can be applied to a variety of siRNA therapeutics for their efficient intracellular delivery.

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1. Introduction

Lipid nanoparticle (LNP) formulations composed of ionizable lipids, helper lipids, cholesterol, and PEGylated lipids have been successfully applied in COVID‐19 vaccines and small interfering RNA (siRNA) therapeutics.[ 1 ] Following this success, extensive investigations have sought to expand their applications in various diseases, including neurological disorders,[ 2 ] metabolic diseases,[ 3 ] and cardiovascular diseases.[ 4 ] However, limitations in conventional LNP (cLNP) compositions, particularly biosafety concerns following repeated administration and limited organ distribution, have hindered further development of diverse LNP‐based therapeutics. Unlike cholesterol and phospholipids, ionizable lipids, which account for half of the total lipids (≈50 molar %), could induce inflammatory responses and exhibit in vivo toxicity.[ 5 ] Although ionizable lipids were initially introduced to mitigate the toxicity of permanent cationic lipids, several studies have reported concerns regarding their safety. The binding of ionizable lipids to cellular toll‐like receptor 4 leads to increased nuclear factor kappa B expression and elevated levels of proinflammatory cytokines and chemokines, including interleukin‐6, monocyte chemoattractant protein‐1, and tumor necrosis factor‐alpha.[ 6 ] Furthermore, repeated administration of LNPs can result in their intracellular accumulation, triggering excessive reactive oxygen species (ROS) formation and activating inflammatory intracellular pathways, such as the nucleotide‐binding oligomerization domain, leucine‐rich repeat, and pyrin domain‐containing protein 3 inflammasome. This cascade subsequently increases inflammatory signaling, e.g., interleukin‐1 beta activation via a caspase‐dependent pathway.[ 6 , 7 ] These stimulated inflammatory responses could limit the therapeutic efficacy of the incorporated nucleic acids, causing unintended adverse effects, such as respiratory distress, tissue damage, and thrombosis.[ 8 , 9 ] In addition, patent‐related challenges regarding the use of ionizable lipids, LNP composition ratios, and manufacturing processes could also pose significant barriers.[ 1 , 10 ] Therefore, diversifying lipid compositions is essential for overcoming these limitations and broadening the clinical applicability of LNPs.

Various strategies have been reported to mitigate the toxicity of ionizable lipids in LNPs. Recent approaches include the synthesis of new components, such as lipid derivatives, and the development of new fabrication methods with optimized composition ratios. For instance, lipid‐like structures derived from known core molecules, such as vitamin B5 and Mildronate, have been developed to replace conventional ionizable lipids.[ 11 , 12 ] These newly synthesized ionizable lipids, which incorporate hydrogen bond donors and exhibit greater chemical flexibility, have demonstrated the potential to reduce the overall ionizable lipids content in LNPs while simultaneously improving transfection efficiency.[ 13 , 14 ] However, the synthesis and screening of such novel lipid structures is a time‐consuming and complex process. Therefore, alternative approaches involving simple and scalable methods are required to refine LNP formulations, minimize toxicity, and improve siRNAs delivery.

In this study, six new types of LNPs were fabricated for siRNA delivery to reduce the proportion of ionizable lipids in LNP formulations while simultaneously improving delivery efficiency. In addition, cost‐efficient mixing methods (pipette‐ and vortex‐mixing) were comparatively evaluated with microfluidic mixing for new LNP formulations. The newly formulated LNPs with different ionizable lipid proportions were characterized based on size, siRNA encapsulation efficiency (EE), and histidine loading efficiency (LE) using dynamic light scattering (DLS), RiboGreen assay, and fluorescamine assay, respectively. To complement the endosomal escape efficiency of LNPs with low ionizable lipid proportion, a histidine decapeptide (His10) was used for the formulation. To assess functional activity, the His10‐incorporated LNPs with low ionizable lipid proportion were incubated with green fluorescent protein (GFP)‐expressing prostate cancer 3 (PC3) cells (PC3‐GFP cells), and gene silencing efficiency of GFP siRNA was quantified via fluorescence intensity (FI) measurements using spectrofluorophotometer. To ensure long‐term stability, selected LNPs with His10 were stored in the presence of sucrose (up to 20% in phosphate‐buffered saline (PBS) solution) at ‐70 °C for four weeks, and their aqueous stability and siRNA bioactivity was evaluated using DLS and spectrofluorophotometry, respectively.

2. Results and Discussion

2.1. Preparation of LNPs with Different Compositions

LNPs have been widely utilized for delivering functional nucleic acids, including siRNA, mRNA, and plasmid DNA into cells, by protecting them from enzymatic degradation and facilitating their intracellular uptake.[ 15 ] In this study, LNPs were prepared by three different mixing methods (pipette, vortex, and microfluidic mixing) to evaluate their suitability for siRNA delivery. The physicochemical properties of the fabricated LNPs were systematically evaluated, as shown in Figure  1a. Previous studies have successfully encapsulated polymeric nucleic acids, such as mRNA (molecular weight of 600–10,000 kDa), using vortex‐mixing and pipette‐mixing, with result comparable to those obtained via microfluidic mixing.[ 16 ] However, comparative studies on LNP formulations using three different mixing methods for the encapsulation of oligomeric nucleic acids, such as antisense oligodeoxynucleotides with a molecular weight of 4–10 kDa and siRNAs with a molecular weight of ≈14 kDa, have not yet been reported.[ 17 ] Since molecular characteristics of nucleic acids, including molecular weight and stiffness, could influence their encapsulation efficiency in LNPs, it is necessary to compare the amount of oligomeric nucleic acids incorporated into LNPs using three types of mixing methods. Therefore, this study systematically compares the feasibility of three different mixing methods for siRNA‐LNP formulation.

Figure 1.

Figure 1

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a) Schematic illustration of the preparation process for lipid nanoparticles (LNPs) using three types of mixing methods. b) Molar ratios of lipid components and histidine decapeptide (His10) in LNPs with new composition ratios.

LNP formulations consist of four types of lipid components each serving a distinct role in nucleic acids delivery: ionizable lipids (≈50%), helper lipids (10%), cholesterol (38.5–40%), and PEG‐lipids (1–2%).[ 18 ] Ionizable lipids (pKa of 6.0) can be positively charged in acidic buffers, allowing the incorporation of nucleic acids into LNPs during formulation and endosomal escape after cellular uptake. Helper lipids and cholesterol contribute to particle stability and reduce leakage, while PEG‐lipids prevent particle aggregation for proper colloidal stability.[ 19 ] As shown in Figure 1b, we fabricated six types of LNPs with different ionizable lipid proportions and total lipid concentrations. The molar ratios of ionizable lipids decreased to 37.95%, 27.95%, and 17.95% in LNP1, LNP2, and LNP3, respectively, with corresponding total lipid concentrations of 12.61, 10.86, and 9.54 mM, respectively. These were compared against conventional LNPs (cLNPs), which contain 47.95% ionizable lipid at a total lipid concentration of 15.04 mM. Similarly, the molar ratio of ionizable lipids in LNP4, LNP5, and LNP6 was decreased to 37.95%, 27.95%, and 17.95%, respectively, while maintaining a total lipid concentration of 15.04 mM, to determine the optimal ionizable lipid proportions and total lipid concentrations based on particle stability and gene inhibition activity of siRNA. To further enhance siRNA delivery efficiency, three selected LNPs were fabricated in the presence of additive His10 to prepare His10‐containing cLNP (cLNPH), His10‐containing LNP5 (LNP5H), and His10‐containing LNP6 (LNP6H) via vortex‐mixing and microfluidic mixing to comparatively examine the loading efficiency of siRNAs and His10.

2.2. Comparative Formulation of LNPs by Different Mixing Processes

Despite the successful and reproducible fabrication of nanosized and homogeneous LNPs, additional equipment is required for the microfluidic mixing of LNPs. For small‐scale and cost‐effective LNPs formulation, we comparatively examined the feasibility of using pipette and vortex‐mixing method for siRNA incorporation. Figure  2a shows the particle sizes of vacant cLNPs, DID‐incorporated cLNPs (DID‐cLNPs), and siRNA‐incorporated cLNPs (siRNA‐cLNPs) prepared by each mixing method. The particle size of vacant cLNPs after formulation by pipette, vortex, and microfluidic mixing was 111.82 ± 13.46, 111.4 ± 7.72, and 49.82 ± 2.23 nm, respectively. The particle size of siRNA‐cLNPs after formulation by pipette, vortex, and microfluidic mixing was 123.00 ± 5.23, 108.09 ± 17.27, and 67.52 ± 3.12 nm, respectively. These results indicate that the cLNPs and siRNA‐cLNPs obtained by pipette and vortex mixing were ≈2–2.5 folds bigger than those obtained by microfluidic mixing. Previous studies showed that mixing velocity during formulations influenced the physicochemical properties of LNPs, including the size, polydispersity, and morphology of the particles.[ 20 , 21 , 22 ] In particular, the size of the LNPs was reduced by approximately half when the flow rate of the solution was increased for fast mixing in a microfluidic device.[ 20 , 23 ] As a result, the small size of cLNPs and siRNA‐cLNPs produced by microfluidic mixing may be attributed to the fast mixing of solutions compared to pipette and vortex mixing. However, the polydispersity indices (PDI) of cLNPs and siRNA‐cLNPs produced by all three mixing methods were not significantly different. In addition, the sizes of the DiD‐cLNPs prepared by the three mixing methods were similar, probably because of the hydrophobic nature of DiD. The incorporated amounts of hydrophobic fluorescent dye (DiD) and hydrophilic siRNA within the cLNPs were quantitatively measured using spectrofluorophotometer and RiboGreen assays, respectively (Figure 2b). While the degree of siRNA incorporation was similar across the three mixing methods, DiD loading efficiency (LE) varied depending on the mixing process. As shown in Figure 2b, the DiD LE of DiD‐cLNPs after formulation by pipette, vortex, and microfluidic mixing was 46.87% ± 0.09%, 66.93% ± 2.33%, and 68.14% ± 1.64%, respectively. The siRNA encapsulation efficiency (EE) for siRNA‐cLNPs after formulation by pipette, vortex, and microfluidic mixing was 80.91% ± 5.45%, 82.16% ± 4.82%, and 83.70% ± 8.21%, respectively. These results may be due to the different loading mechanisms of DiD and siRNA in the LNPs. While siRNA is incorporated into LNPs through ionic interactions with ionizable lipids, DiD is dissolved in the ethanol phase and forms hydrophobic interactions with lipids for incorporation during formulation. Interestingly, siRNA was relatively less influenced by the mixing rate, probably because of the strong ionic interactions with ionizable lipids under acidic conditions.[ 16 , 24 ] In addition, cLNPs produced by vortex‐mixing demonstrated better aqueous stability during the first week than those produced by pipette‐mixing (Figure 2c). The size of cLNPs by pipette‐ and vortex‐mixing was 180.6 ± 6.66 and 133.25 ± 1.44 nm after incubation for 5 days at 4 °C. Considering the colloidal stability of the cLNPs, six new types of LNPs were formulated via vortex‐mixing in the following experiments.

Figure 2.

Figure 2

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a) Particle size and polydispersity index (PDI) of three types of LNPs prepared by three different mixing processes. b) DID loading efficiency (LE) of DID‐LNPs and siRNA encapsulation efficiency (EE) of siRNA‐LNPs prepared by three different mixing processes. c) Particle sizes of cLNPs prepared by pipette‐ and vortex‐mixing methods during the incubation at 4 °C for 1 week. * p <0.05; *** p <0.001; ns: not significant.

2.3. Gene Silencing Activity by siRNA‐LNPs with Different Lipid Compositions

To examine whether different lipid compositions affect the physicochemical properties of LNPs, cLNPs and six new LNP formulations were prepared using vortex‐mixing. As shown in Figure  3a, the hydrodynamic sizes of cLNP, LNP1, LNP2, LNP3, LNP4, LNP5, and LNP6 were 109.06 ± 9.30, 108.59 ± 7.71, 120.95 ± 17.89, 116.95 ± 14.52, 111.09 ± 8.91, 128.63 ± 8.99 and 149.15 ± 8.04 nm, respectively. Interestingly, while the sizes of cLNP, LNP1, LNP2, LNP3, LNP4, LNP5, and LNP6 were similar when freshly prepared, a significant size increase was only observed in LNP2 and LNP3 after incubation for 24 h at 4 °C. The hydrodynamic sizes of cLNP, LNP1, LNP2, LNP3, and LNP4 were 186.78 ± 7.61, 180.10 ± 14.42, 265.58 ± 6.26, 283.80 ± 21.72, and 210.60 ± 11.49 nm, respectively. This size increase in LNP2 and LNP3 may be due to the reduced total lipid concentrations, potentially compromising the colloidal stability of these formulations.

Figure 3.

Figure 3

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a) Size of seven types of LNPs via vortex‐mixing methods. b) Agarose gel electrophoresis of seven types of LNPs before and after lysis. c) siRNA EE in different LNPs. d) Fluorescence microscopy images (scale bar: 100 µm) and e) Relative fluorescence intensity (FI) in PC3‐GFP cells treated with different GFP siRNA‐incorporating LNPs. f) Intracellular Cy5.5 FI in PC3 cells treated with different Cy5.5‐labeled siRNA‐incorporating LNPs. * p <0.05; ** p <0.01; *** p <0.001; ns: not significant.

Figure 3b shows the siRNAs incorporated into the agarose gel after lysis of each LNP. Following lysis, strong fluorescent signals were observed in agarose gel, indicating successful encapsulation of siRNAs within LNPs. To precisely determine the interior amount of siRNAs within the LNPs, incorporated siRNAs were quantitatively measured using a RiboGreen assay after lysis of the LNPs.[ 25 ] Notably, as shown in Figure 3c, the extent of the siRNA EE differed considerably depending on the ionizable lipid composition. The siRNA EE of cLNP, LNP1, LNP2, and LNP3 was 80.98 ± 8.40%, 72.90 ± 10.04%, 58.27 ± 19.96%, and 48.42 ± 24.16%, respectively. As the molar ratio of ionizable lipids decreased, the siRNA encapsulation efficiency also decreased, likely due to weaker interactions between siRNA and ionizable lipids at lower ionizable lipid ratios, particularly in LNP3 and LNP6 (ionizable lipid molar ratio of 17.95%). This reduction in ionizable lipids resulted in a lower siRNAs encapsulation efficiency (EE) compared to cLNPs (ionizable lipid molar ratio of 47.95%).

Considering the crucial role of ionizable lipids in endosomal escape after intracellular uptake, the biological activity of LNPs containing different amounts of ionizable lipids was examined. As shown in Figure 3d, different GFP siRNA incorporating‐LNPs were treated to PC3‐GFP cells for 5 h, and the degree of gene silencing by each LNP was observed by fluorescence microscopy. Cells treated with free siRNA showed strong fluorescence signals, whereas cells treated with siRNA‐LNPs showed substantially attenuated fluorescence signals due to RNA interference. To precisely quantify the extent of gene inhibition induced by siRNA‐LNPs, the FI in cell lysates was analyzed using a spectrofluorophotometer after treatment with LNPs for 5 h (Figure 3e). The relative FI of cLNP, LNP1, LNP2, LNP3, LNP4, LNP5, and LNP6 was 38.98 ± 8.87%, 49.01 ± 8.6%, 59.62 ± 8.58%, 59.51 ± 14.59%, 49.25 ± 12.21%, 47.92 ± 6.16%, and 46.52 ± 9.32% at a siRNA concentration of 187.8 nM, respectively. As expected, cLNP demonstrated the best gene inhibition among the LNPs, probably because of sufficient endosomal escape by high amounts of ionizable lipids.[ 26 , 27 ] In contrast, LNP2 and LNP3 exhibited poorer gene silencing activity than other LNPs. In a previous study, the extent of intracellular uptake of soft nanoparticles was lower than that of solid nanoparticles, which was attributed to the energetically unfavorable deformation of soft nanoparticles during cellular internalization.[ 28 ] It is conceivable that the soft and unstable particle formation of LNP2 and LNP3, due to low total lipid amounts, might result in reduced gene inhibition in Figure 3e. To verify this, intracellular uptake was quantitatively assessed using a fluorospectrophotometer following treatment of cells with different LNPs containing Cy5.5‐labeled siRNA. The intracellular Cy5.5 FI of cLNP, LNP1, LNP2 and LNP3 was 129.68 ± 5.10, 124.25 ± 5.47, 102.86 ± 10.58, and 91.51 ± 5.07%, as shown in Figure 3f. Significantly lower intracellular uptake was observed in cells treated with LNP3, compared to those treated with cLNPs. Consequently, LNP4, LNP5, and LNP6 were selected for further optimization to improve endosomal escape activity by incorporating an endosome escaper (His10) into the formulations.

2.4. Formulation of His10 Incorporating LNPs via Vortex‐Mixing Method

In previous studies, the addition of histidine oligomers and polymers to LNPs greatly elevated the gene inhibition activity owing to preferred endosomal escape.[ 29 , 30 ] To examine whether the reduced gene inhibition observed in LNP4, LNP5 and LNP6 could be restored by adding histidine peptide, His10 was added to each LNP to prepare cLNPH, LNP4H, LNP5H, and LNP6H via vortex‐mixing. After preparing these four types of His10‐incorporating LNPs, the hydrodynamic size was measured, as shown in Figure  4a. No notable differences in the particle size were observed for any of the LNPHs. However, lower siRNA encapsulation was observed in LNP6H, probably because of the poor ionic interactions between the siRNAs and ionizable lipids. The siRNA EE of cLNPH, LNP4H, LNP5H, and LNP6H was 74.76 ± 6.71, 67.54 ± 14.10, 60.98 ± 10.83, and 52.78 ± 12.12%, respectively (Figure 4b). Compared to our previous study, where cLNPH formulated using microfluidic mixing had an EE of ≈80%, the siRNA EE of cLNPH via vortex‐mixing was found to be comparable.[ 30 ] However, negligible amounts of His10 were incorporated into all LNPHs (Figure 4c). The His10 LE of cLNPH, LNP4H, LNP5H, and LNP6H was 4.46 ± 2.45, 2.89 ± 1.34, ‐0.43 ± 3.58, and 2.66 ± 1.73%, respectively. While the His10 LE of cLNPH was ≈30% after formulation by microfluidic mixing in our previous study, the His10 LE of cLNPH using the vortex‐mixing method was below 5%.[ 30 ] These results indicate that the vortex‐mixing method could not provide sufficiently vigorous and consistent mixing for the incorporation of hydrophilic peptide additives compared to the microfluidic mixing method.

Figure 4.

Figure 4

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a) Particle size, b) siRNA EE, and c) His10 LE of four types of His10‐incorporating LNP (LNPH)s prepared via vortex‐mixing methods. * p <0.05; ns: not significant.

2.5. Formulation of His10 Incorporating LNPs by Microfluidics‐Mixing

To examine whether His10 could be incorporated into the selected LNPs by microfluidic mixing, two types of LNPs (LNP5 and LNP6) with low ionizable lipid proportions were prepared by microfluidic mixing. Figure  5a shows that the particle size of LNP5, LNP5H, LNP6, and LNP6H was 88.80 ± 3.67, 113.00 ± 6.51, 114.8 ± 5.52, and 121.95 ± 8.41 nm, which were bigger than that of cLNP and cLNPHs. The siRNA EE of cLNP, cLNPH, LNP5, LNP5H, LNP6, and LNP6H was 79.04 ± 4.27%, 79.81 ± 1.79%, 68.20 ± 3.59%, 68.77 ± 3.36%, 49.54 ± 5.36%, and 58.09 ± 7.52%, respectively (Figure 5b). As expected, lower siRNA encapsulation was observed in the LNP5H and LNP6H. The incorporation of His10 was examined using a fluorescamine assay (Figure 5c). The histidine LE of cLNPH, LNP5H, and LNP6H was 31.19 ± 5.41%, 30.30 ± 2.26%, and 27.34 ± 5.85%, respectively, which indicates that additional molecules of His10 were successfully incorporated into LNP5 and LNP6 using microfluidic mixing. In addition, negligible differences in His10 LE were observed depending on lipid composition. This result demonstrates that the incorporation efficiency of additives into LNPs depends on the mixing method employed.

Figure 5.

Figure 5

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a) Particle size, b) siRNA EE, and c) His10 LE of LNPHs via microfluidic mixing methods. d) Relative FI in PC3‐GFP cells treated with different GFP siRNA incorporating LNPHs at different siRNA concentrations. e) The IC50 values of siRNA‐loaded LNPHs for GFP gene inhibition. * p <0.05; ** p <0.01; *** p <0.001; ns: not significant; n.d.: not determined.

Figure 5d shows gene inhibition by LNPs at different siRNA concentrations. The relative GFP FI in PC3‐GFP cells with cLNP, cLNPH, LNP5, LNP5H, LNP6, and LNP6H was 42.85 ± 5.23%, 39.80 ± 2.64%, 44.67 ± 3.53%, 33.00 ± 2.71%, 70.49 ± 2.50%, and 82.36 ± 7.52% at a siRNA concentration of 187.8 nM, respectively. Both cLNP and LNP5 showed similar gene inhibition activities, whereas significantly attenuated gene inhibition was observed with LNP6. In addition, cLNPH and LNP5H showed notably reduced GFP gene expression compared to cLNP and LNP5, respectively, at an siRNA concentration of 37.6 nM due to the addition of His10. Figure 5e shows IC50 of cLNP, cLNPH, LNP5, and LNP5H was 135.40 ± 8.00, 88.10 ± 6.90, 158.20 ± 19.50, and 84.40 ± 15.80 nM. Despite a lower ionizable lipid molar ratio of 27.72% in LNP5H compared to that in cLNPH (47.55%), the addition of His10 greatly improved gene inhibition activity, probably because of compensatory endosome escape. Notably, the IC50 of LNP5H was reduced by 1.6‐fold, compared to cLNP. However, IC50 values could not be determined for LNP6 and LNP6H, suggesting that a minimum ionizable lipid content is required for proper endosomal escape. To suppress highly overexpressed GFP reporter gene in cells, treatment with a higher concentration of siRNAs might be needed in this study, compared to suppression of endogenous genes in our previous study.[ 30 ] In this study, as a proof of concept, we optimized the ionizable lipid proportion in an easy, fast, and cost‐effective way using a vortex mixing method and fabricated new LNP5H formulation for the delivery of unmodified siRNAs. However, after sequence optimization, e.g., base pair mismatch in a sense strand and chemical modification, e.g., 2´‐O‐methyl‐ and 2´‐fluoro‐modifications of siRNAs, IC50 values of target gene inhibition by LNP5H might be further improved.[ 31 , 32 ] A previous study demonstrated that halving the ionizable lipids content markedly reduced non‐specific inflammation in vivo.[ 33 ] Therefore, reducing conventional ionizable lipids while incorporating peptides with endosomal escaping activity could be promising strategies for improving the safety profile of LNPs. In this study, to offset the potential reduction in endosomal escape efficiency caused by lower ionizable lipid content, His10 was incorporated to prepare LNP5H. Histidine, an amino acid with a pKa of ≈6.0, remains neutral at physiological pH 7.4 but becomes positively charged in the late endosome (pH 5.5–6.0).[ 29 , 30 ] This charge transition promotes membrane fusion and the proton‐sponge effect, enhancing endosomal escape.[ 30 , 34 ] It is also noted that histidine is a natural, essential, and biocompatible amino acid, offering additional safety benefits.[ 35 , 36 ] Accordingly, our results indicate that we successfully reduced the amount of ionizable lipids to 27.72% in total lipids for LNP5H using biocompatible peptides, which could induce gene inhibition by siRNA more efficiently than cLNPs.

2.6. Stability of LNPs at Different Storage Conditions

Particle stability of LNPs during long‐term storage is crucial for the clinical application of LNPs. Accordingly, storage conditions, such as temperature, freezing processes, and storage buffer have been reported to maintain the colloidal stability of LNPs during storage in previous studies.[ 37 , 38 , 39 ] To improve the long‐term stability of selected LNP5 and LNP5H for over four weeks, LNP solutions were frozen to reduce hydrolysis and oxidation of phospholipids.[ 40 , 41 ] In addition, sucrose was selected as a cryoprotectant during storage of the LNPs at −70 °C to maintain the stability of lipid membranes by forming hydrogen bonds with lipids and delaying ice crystal formation.[ 42 , 43 ] Figure  6a,b shows particle size of LNP5 and LNP5H during incubation for 28 days at ‐70 °C and thawing. The hydrodynamic size of LNP5 after incubation at ‐70 °C for 0, 1, 2, 3, and 4 weeks was 85.50 ± 0.21, 283.60 ± 19.31, 293.85 ± 1.20, 413.58 ± 43.59, and 372.20 ± 73.29 nm (Figure  6a). As expected, the LNP5 size increased more than threefold without a cryoprotectant after one‐time freezing and thawing. LNP5H also showed a significant size increase during incubation at −70 °C (Figure 6b). To improve the colloidal stability of LNP5H during storage at −70 °C until use, sucrose was added to the storage buffer as an additive at different concentrations (0–20% sucrose in PBS solution). In the presence of sucrose, negligible size changes were observed in LNP5 and LNP5H. The particle size of LNP5H in 0, 1, 3, 5, 10, and 20% sucrose buffer after 4 weeks was 330.15 ± 10.54, 136.33 ± 5.06, 119.54 ± 7.44, 95.11 ± 4.76, 87.11 ± 1.31, and 80.63 ± 5.02 nm, respectively. This result clearly demonstrated that sucrose concentration above 1% could effectively maintain the colloidal sizes of LNPs with low polydispersity index (PDI). Biocompatibility of LNP5H in sucrose solution was examined at different siRNA concentrations. It is well‐known that sucrose is a biocompatible and naturally degradable substance, which can be administered via intravenous injection up to 11.6 g kg−1.[ 44 ] Figure 6c shows that LNP5H in sucrose solution elicited negligible toxicity up to a siRNA concentration of 187.8 nM, which indicates that this storage condition is a promising and safe strategy. To examine siRNA activity during storage, the relative FI in cells treated with LNP5H at an siRNA concentration of 187.8 nM after 1 and 4 weeks of incubation at −70 °C was determined, as shown in Figure 6d. The FI of cells treated with LNP5H after storage in 0, 1, 3, 5, 10, and 20% sucrose buffer for 1 week were 27.73 ± 0.87%, 28.98 ± 1.37%, 39.69 ± 3.37%, 59.69 ± 10.51%, 62.22 ± 12.37%, and 61.60 ± 16.32%, which indicates that siRNA activity of LNP5H was not changed during storage. Interestingly, the extent of gene inhibition by LNP5H in 0% sucrose buffer was comparable to that by LNP5H in 1% sucrose buffer despite increased particle size. To investigate whether the efficient gene inhibition by LNP5H in 0% sucrose buffer was mediated by particle sedimentation or not, LNPs were incubated below PC3‐GFP cells using a transwell system (Figure 6e). The FI of cells treated with LNP5H at an siRNA concentration of 187.8 nM after storage in 4 °C control, 0, 1, and 3% sucrose buffer for 1 week were 62.26 ± 18.25%, 45.36 ± 7.37%, 39.60 ± 3.87%, and 54.32 ± 9.93%, respectively. This result evidently demonstrated that LNP5H in 1% sucrose buffer could be promising strategy for potent and efficient gene silencing compared with those in other buffers during storage.

Figure 6.

Figure 6

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Stability of LNP5 and LNP5H after storage at −70 °C. Particle size (left panel) and polydispersity index (PDI; right panel) of a) LNP5 and b) LNP5H at different sucrose concentrations after incubation at −70 °C. c) Cell viability of LNP5H after incubation in different sucrose solutions for 3 days. d) Relative FI in PC3‐GFP cells on 24‐well plate treated with LNP5H after incubation in different sucrose solutions for 1 and 4 weeks. e) Transwell‐based gene inhibition assay. Relative FI in PC3‐GFP cells on transwell treated with LNP5H after incubation in different sucrose solutions for 4 weeks. * p < 0.05; ** p < 0.01; *** p < 0.001; n.s.: not significant.

In this study, we aimed to develop new lipid composition ratios for LNPs by adjusting the total lipid amounts and the proportion of ionizable lipids, which could be time‐consuming and costly. However, the vortex‐mixing method was employed during the selection processes in this study, which allowed for fast and small‐scale screening of LNPs. In addition, we successfully reduced the proportion of ionizable lipids without synthesizing new lipid structures. The decrease in ionizable lipids in the LNPs was compensated by proportionally increasing the amount of helper lipids and His10. It is notable that a potent LNP5H was developed by adjusting the composition ratios of existing lipids without the synthesis of entirely new lipid structures, which could save time and cost. In addition, while conventional lipids have substantial data regarding their toxicity, pharmacokinetics, and pharmacodynamics, newly synthesized lipids may pose unforeseen safety issues. Accordingly, commercially available and approved lipids and peptides were employed and optimized in this study, which may provide a reasonable and effective strategy for designing new LNPs.

3. Conclusion

In this study, both siRNA and hydrophobic fluorescent dye were successfully incorporated within LNPs by pipette and vortex mixing processes. After optimizing the ionizable lipids proportion in the total lipid composition via screening using vortex‐mixing methods, the selected LNP5 demonstrated comparable stability, siRNA EE, and transfection efficiency to cLNP despite having a reduced proportion of ionizable lipids. In addition, LNP5H, which incorporated His10 as an endosomal escape enhancer, exhibited a significant 1.6‐fold improvement in gene silencing activity compared to cLNP and LNP5. In addition, LNP5H in a 1% sucrose solution was successfully stored for 4 weeks at −70 °C without loss of potency. Thus, this potent new formulation, LNP5H, could be harnessed for the effective delivery of diverse siRNA therapeutics, and these developmental processes could provide valuable insights into the development of versatile LNP‐based therapeutics.

4. Experimental Section

Materials

Phosphate‐buffered saline (PBS) powder, diethyl pyrocarbonate (DEPC), ethanol, cholesterol (Chol), acetic acid, sodium acetate, agarose, ethidium bromide, chloroform, formaldehyde solution, fluorescamine, Triton X‐100 (Tx‐100), and mounting medium Fluoroshield were obtained from Sigma–Aldrich (St. Louis, MO, USA). His10 (> 90% purity) was obtained from Peptron (Daejeon, South Korea). The 1,2‐distearoyl‐sn‐glycero‐3‐phosphocholine (DSPC) was purchased from Avanti Polar Lipids Inc. (Alabaster, AL, USA). The 1,2‐dimyristoyl‐rac‐glycero‐3‐methoxypolyethylene glycol‐2000 (DMG‐PEG) and 2‐hexyl‐decanoic acid, 1,1′‐[[(4‐hydroxybutyl)imino]di‐6,1‐hexanediyl] ester (ALC‐0315) were purchased from Cayman Chemical Company (Ann Arbor, MI, USA). Fetal bovine serum (FBS) and penicillin/streptomycin (P/S) were purchased from Gibco BRL (Grand Island, NY). Roswell Park Memorial Institute (RPMI) 1640 medium was purchased from Welgene (Gyeongsan, South Korea). Cell counting kit‐8 (CCK‐8) was obtained from Dojindo Laboratories (Kumamoto, Japan). The Quant‐iT RiboGreen Assay Kit, Lipofectamine 2000 Transfection Reagent, and 1,1′‐dioctadecyl‐3,3,3′,3′‐tetramethylindodicarbocyanine perchlorate (DiD) were sourced from Invitrogen (Waltham, MA, USA). The GFP siRNA and cyanine 5.5 (Cy5.5)‐labeled GFP siRNA were purchased from Bioneer (Daejeon, South Korea). GFP siRNA sequences used in the experiment were as follows (underlined letters represent deoxyribonucleotides): 5′‐GCAAGCUGACCCUGAAGUUTT‐3′ (sense), 5′‐AACUUCAGGGUCAGCUUGCTT‐3′ (antisense).

Preparation of cLNPs Using Different Formulation Processes

Three types of cLNPs, vacant cLNPs, DID‐cLNPs, and siRNA‐cLNPs, were fabricated using different mixing processes (pipette, vortex, and microfluidics mixing).[ 16 ] Briefly, 0.29 mg of lipids (ionizable lipid: helper lipid: Chol: PEG‐lipid molar ratio = 47.95:10.50:40.01:1.54) in ethanol and sodium acetate buffer (pH 4.2) were mixed by pipette or vortex mixing for 30 s (volume ratio of aqueous phase: ethanol phase = 3:1) to prepare vacant cLNPs. For the preparation of DID‐cLNPs and siRNA‐cLNPs, DID (4 µg) or siRNA (14.4 µg) was dissolved in ethanol or sodium acetate buffer at an ionizable lipids/siRNA weight ratio of 12.8, respectively. In addition, the ethanol phase containing 7.5 mg of lipids with the same molar ratio as previously described and the aqueous phase containing 0.35 mg of siRNA were infused into the NanoAssembler Ignite microfluidic mixing device (Precision Nanosystems, Vancouver, BC, Canada) at a total flow rate of 12 mL min−1. The formulation was dialyzed using a dialysis membrane (molecular weight cut‐off 25 kDa) against a PBS solution (pH 7.4) at 4 °C with stirring overnight. After dialysis, the hydrodynamic particle size of the LNPs was analyzed via DLS using a Malvern Nano‐S device (Malvern Instruments, Malvern, UK).

The amount of incorporated DID was quantified by measuring the fluorescence intensity (FI) after destabilization of the LNPs with Tx‐100‐containing PBS solution (final concentration: 1% v/v) using a spectrofluorophotometer (Gemini EM microplate reader, Molecular Devices, CA, USA) at excitation and emission wavelengths of 640 and 705 nm, respectively. The FI of DID in Tx‐100‐containing PBS solution was used as the standard. The LE of DID was calculated using the following equation: loaded amount of DID / feed amount of DID × 100. The amount of siRNA encapsulated in the LNPs was determined using the Quant‐iT RiboGreen Assay Kit, according to the manufacturer's protocol. After incubating the samples in PBS solution and Tx‐100‐containing PBS solution (final Tx‐100 concentration: 1% v/v), the fluorescence intensity of the solutions was measured using a spectrofluorophotometer at excitation and emission wavelengths of 480 and 520 nm, respectively. The EE of the siRNA was calculated using the following equation: (total amount of siRNA‐unloaded amount of siRNA) / total amount of siRNA × 100.

Preparation and Characterization of LNPs with Different Compositions

Seven types of LNPs with different compositions, namely cLNP, LNP1, LNP2, LNP3, LNP4, LNP5, and LNP6, were formulated using the vortex‐mixing method. The molar ratios of the lipids (molar ratios of ionizable lipid: helper lipid: Chol: PEG‐lipid) are shown in Figure 1b. In brief, lipids (0.29 mg) in ethanol and siRNA (14.4 µg) in sodium acetate buffer (pH 4.2) were mixed vigorously using vortexing for 30 s. The formulation was dialyzed using a dialysis membrane (molecular weight cut‐off 25 kDa) against a PBS solution (pH 7.4) at 4 °C with stirring overnight. After dialysis, the hydrodynamic particle size of the LNPs was analyzed via DLS using a Malvern Nano‐S device (Malvern Instruments, Malvern, UK). The siRNAs encapsulated in LNPs were also examined using a gel retardation assay. The seven types of LNPs, each containing 1 µg of siRNA, were incubated in PBS solution and lysis solution (1% (v/v) Tx‐100 in PBS solution) for 10 min, respectively. The resulting solution was loaded onto 2% agarose gel. Free siRNA (1 µg) was used as a control. After running the gel for 15 min, the siRNA bands were visualized using a UV transilluminator at 312 nm after ethidium bromide staining. The encapsulation efficiency of the siRNA was determined using the Quant‐iT RiboGreen Assay Kit, as described above.

Gene Inhibition Assay

PC3‐GFP cells were plated in 6‐well plates at a density of 5 × 105 cells per well for 24 h. Cells were then treated with seven types of LNPs at a siRNA concentration of 187.8 nM for 5 h at 37 °C. The culture medium was replaced with fresh medium containing 10% FBS, and the cells were incubated for an additional 43 h. After incubation, the cells were washed twice with PBS solution and fixed in 3.7% formaldehyde in PBS solution. After covering with a mounting medium, the cells were analyzed by fluorescence microscopy (Olympus, Shinjuku, Tokyo, Japan). To quantify the relative fluorescence intensity (FI), PC3‐GFP cells were plated onto 12‐well plates at a density of 2 × 105 cells per well for 24 h. Cells were treated with seven types of LNPs at different siRNA concentrations (0, 37.6, and 187.8 nM) for 5 h at 37 °C. The culture medium was replaced with fresh medium containing 10% FBS, and the cells were incubated for an additional 43 h. After incubation, the cells were washed twice with PBS and incubated with cell lysis solution (1% (v/v) Tx‐100 in PBS solution) for 20 min at room temperature. The cell lysis solution was centrifuged at 13,000 rpm, 4 °C for 10 min to remove cell debris. FI in the supernatant was quantitatively measured using a spectrofluorophotometer at excitation and emission wavelengths of 489 and 519 nm, respectively.

Intracellular Uptake of LNPs

To prepare seven types of LNPs with Cy5.5‐labeled siRNAs, lipids (0.29 mg) in ethanol and Cy5.5‐labeled siRNA (14.4 µg) in sodium acetate buffer (pH 4.2) were mixed vigorously using vortexing for 30 s. After dialysis of the formulation in a PBS solution, the amount of incorporated Cy5.5‐labeled siRNA in LNPs was quantified by measuring the Cy5.5 FI in the presence of Tx‐100 (final concentration: 1% v/v) using a spectrofluorophotometer at excitation and emission wavelengths of 640 and 705 nm, respectively. PC3 cells were plated in 24‐well plates at a density of 1.0 × 105 cells per well for 24 h. Cells were then treated with seven types of Cy5.5‐containing LNPs at a Cy5.5 concentration of 0.3 µg mL−1 for 6 h at 37 °C. After incubation, the cells were washed three times with 5% FBS‐containing PBS solution and incubated with cell lysis solution (1% (v/v) Tx‐100 in PBS solution) for 20 min at room temperature. The cell lysis solution was centrifuged at 13 000 rpm for 10 min at 4 °C to remove cell debris. Cy5.5 FI in the supernatant was quantitatively measured using a spectrofluorophotometer at excitation and emission wavelengths of 640 and 705 nm, respectively.

Preparation and Characterization of His10‐Containing LNPs with Different Compositions

Four types of His10‐containing LNPs (cLNPH, LNP4H, LNP5H, and LNP6H) were prepared using a vortex‐mixing method. The molar ratios of ionizable lipids, helper lipids, Chol, and PEG‐lipids in ethanol used for the formulation of the LNPs are shown in Figure 1b. His10 (5.76 µg; His/siRNA weight ratio of 0.4) was dissolved with siRNA (14.4 µg) in sodium acetate buffer. The samples in sodium acetate buffer were mixed with lipids (0.29 mg) in ethanol by vortexing for 30 s. The formulation was dialyzed using a dialysis membrane (molecular weight cut‐off 25 kDa) against a PBS solution (pH 7.4) at 4 °C with stirring overnight. After dialysis, the hydrodynamic particle size of the LNPs was analyzed via DLS using a Malvern Nano‐S device (Malvern Instruments, Malvern, UK). The EE of siRNA in the LNPs was determined using the Quant‐iT RiboGreen Assay Kit, as described above.

To determine the amount of incorporated His10, the primary amine groups on the histidine molecules were quantified using a fluorescamine reagent according to the manufacturer's protocol. LNPs in PBS solution and Tx‐100‐containing PBS solution (final concentration: 1% v/v), respectively, were mixed with fluorescamine in acetone (2 mg mL−1). The number of histidine molecules encapsulated in each particle (cLNPH, LNP4H, LNP5H, and LNP6H) was quantified using His10 as a standard. The FIs of the solutions were measured using a spectrofluorophotometer at excitation and emission wavelengths of 390 and 475 nm, respectively.

Preparation and Characterization of Selected LNPs Using Microfluidic Device

Three types of siRNA‐containing LNPs were formulated according to our previous study, with slight modifications, using a microfluidic device.[ 30 ] In brief, 7.5 mg of lipids with molar ratios in ethanol and siRNA (0.35 mg) in sodium acetate buffer (pH 4.2) at an ionizable lipids/siRNA weight ratio of 12.8 were injected into the NanoAssembler Ignite microfluidic mixing device (Precision Nanosystems, Vancouver, BC, Canada) at a total flow rate of 12 mL min−1 (volume ratio of aqueous phase: organic phase = 3:1). To formulate three types of His10‐incorporating LNPs, His10 (0.14 mg; His/siRNA weight ratio of 0.4) were dissolved with siRNA (0.35 mg) in sodium acetate buffer and injected with lipids (7.5 mg) in ethanol into the microfluidic mixing device. Each formulation was dialyzed using a dialysis membrane (molecular weight cut‐off 25 kDa) against a PBS solution (pH 7.4) at 4 °C with stirring overnight. After dialysis, the hydrodynamic particle size of the LNPs was analyzed via DLS using a Malvern Nano‐S device (Malvern Instruments, Malvern, UK). The amounts of siRNA and His10 incorporated were quantified as described above.

For gene inhibition assay, PC3‐GFP cells were plated onto 24‐well plates at a density of 1 × 105 cells per well for 24 h. Cells were treated with six types of LNPs at different siRNA concentrations (0, 3.76, 18.8, 37.6, and 187.8 nM) for 5 h at 37 °C. The culture medium was replaced with fresh medium containing 10% FBS, and the cells were incubated for an additional 43 h. After incubation, the cells were washed twice with PBS and incubated with cell lysis solution (1% (v/v) Tx‐100 in PBS solution) for 20 min at room temperature. The cell lysis solution was centrifuged at 13 000 rpm, 4 °C for 10 min to remove cell debris. FI in the supernatant was quantitatively measured using a spectrofluorophotometer at excitation and emission wavelengths of 489 and 519 nm, respectively.

Long Term Stability of LNP5 and LNP5H

For long‐term stability, LNP5 and LNP5H were diluted with 40% sucrose (w/v) in PBS solution to obtain five sucrose concentrations (1, 3, 5, 10, and 20% (w/v) sucrose).[ 39 ] After vortexing the mixture for 30 s, the prepared LNP solution was rapidly frozen in liquid nitrogen and, stored in a deep freezer (−70 °C), thawed at different periods (0, 1, 2, 3, and 4 weeks). After the predetermined time intervals, the LNP solution was thawed at room temperature and diluted with PBS. After diluting each LNP solution with PBS, the hydrodynamic size and PDI were measured by DLS at different viscosity values. To assess the cytotoxicity of the LNP5H in sucrose solutions, a CCK‐8 assay was performed. PC3 cells were plated onto 96‐well plates at a density of 2 × 104 cells per well for 24 h. Cells were treated with LNP5H in sucrose solutions at six different concentrations (0, 1, 3, 5, 10, and 20% (w/v) sucrose) and six different siRNA concentrations (0, 3.76, 18.8, 37.6, 93.9, and 187.8 nM) for 5 h at 37 °C. The culture medium was replaced with fresh medium containing 10% FBS, and the cells were incubated for an additional 19 h. After incubation, the cells were treated with the CCK‐8 reagent and cell cytotoxicity was determined according to the manufacturer's protocol. To examine the biological activity of melted LNP5H in sucrose solutions, LNP5H frozen for 1 and 4 weeks were melted and treated to PC3‐GFP cells in 24‐well plates at a siRNA concentration of 187.8 nM for 5 h at 37 °C. The culture medium was replaced with fresh medium containing 10% FBS, and the cells were incubated for an additional 43 h. After incubation, the cells were washed twice with PBS and incubated with cell lysis solution (1% (v/v) Tx‐100 in PBS solution) for 20 min at room temperature. The cell lysis solution was centrifuged at 13 000 rpm, 4 °C for 10 min to remove cell debris. FI in the supernatant was quantitatively measured using a spectrofluorophotometer at excitation and emission wavelengths of 489 and 519 nm, respectively. For the transwell‐based gene inhibition assay, PC3‐GFP cells were plated on the membrane of SPLInsert(SPL, Pocheon, South Korea) at a density of 2 × 104 cells per well and incubated for 24 h. After incubation, LNP5H frozen for 4 weeks were melted and treated to the PC3‐GFP cells in the upper chambers at a siRNA concentration of 187.8 nM for 5 h at 37 °C. The outer chambers were also adjusted to the same siRNA concentration. Subsequent procedures were performed as previously described.

Statistical Analysis

The data represent the mean values of independent measurements, with error bars indicating the standard deviation of each experiment. Statistical analysis was conducted using one‐way ANOVA, and statistical significance was assigned for P < 0.05 (95% confidence level).

Conflict of Interest

The authors declare no conflict of interest.

Acknowledgements

Y.B. and H.L. contributed equally to this work. This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean Government (NRF‐2023R1A2C1005538).

Bae Y., Lee H., Lee J. H., Yeo S., and Mok H., “Histidine Decapeptide‐Incorporating Lipid Nanoparticles with Low Ionizable Lipids Proportion for Efficient Small Interfering RNA Delivery.” Macromol. Biosci. 25, no. 9 (2025): 25, 70005. 10.1002/mabi.202500165

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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