Intranasal delivery of bryostatin-1 using surface charge-engineered lipid nanoparticles to modulate mucosal defense for allergic rhinitis treatment

Abstract

Allergic rhinitis (AR), driven by immune imbalance and excessive IgE production, manifests with symptoms that significantly impair the patient’s quality of life. Current therapies mainly provide symptomatic relief without correcting the underlying immune dysregulation. Bryostatin-1 (bryo-1) is a promising candidate for the causal treatment of AR. It potently inhibits IgE-mediated allergic responses while enhancing nasal mucosal defense through the selective induction of IgA antibodies upon intranasal administration. However, the intranasal delivery of bryo-1 faces challenges, including high cost, chemical instability, and limited permeability across the nasal mucosal barrier. In this study, bryo-1 was incorporated with liposomes with varying surface charges. These LNPs exhibited stronger interactions with antigen-presenting cells and enhanced cellular uptake and delivery efficiency of bryo-1 in vitro. Notably, anionic LNPs achieved superior bryo-1 delivery to B cells, selectively promoting IgA class switching while suppressing IgE expression. In an AR mouse model, even the low-dose (0.5 ng) intranasal administration of bryo-1–loaded anionic LNPs elevated antigen-specific IgA levels in salivary secretions. These findings indicate that anionic LNPs enhance delivery efficiency, representing a promising platform for intranasal bryo-1 delivery to modulate mucosal immunity and treat AR.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-026-43174-8.

Introduction

Allergic rhinitis (AR) is a prevalent inflammatory disorder of the upper respiratory tract and is clinically characterized by sneezing, nasal congestion, and nasal itching, all of which can significantly impair a patient’s quality of life^1,2. According to the World Health Organization, allergic diseases are among the top three disorders requiring prioritized prevention and control in the twenty-first century. Despite this recognition, the global prevalence of AR continues to increase steadily.

Current treatments, including antihistamines and corticosteroids, primarily provide symptomatic relief without addressing the underlying immune dysregulation, and their long-term use is associated with side effects, such as tiredness and nasal dryness³. Sublingual immunotherapy is a therapeutic approach for allergic conditions; however, it is limited in scope, addressing only a select group of allergens, such as dust mites and pollen, and necessitates an extended duration of treatment^4,5. Moreover, surgical procedures such as inferior turbinate laser coagulotomy can mitigate nasal obstruction resulting from allergy-induced mucosal hypertrophy; however, their long-term efficacy remains limited⁶. Therefore, novel therapeutic strategies capable of modulating the mucosal immune environment and restoring the immune balance in patients with AR are needed urgently.

Our strategy is to suppress immunoglobulin E (IgE) related allergic diseases by inducing the selective class-switching of B cells to immunoglobulin A (IgA) instead of IgE. IgA antibodies play a crucial role in mucosal defense related to their ability to bind to antigens on mucosal surfaces, thereby preventing antigen penetration and subsequent allergic reactions⁷. Therefore, promoting selective class switch recombination (CSR) towards IgA can strengthen mucosal immunity, and the concurrent suppression of IgE production helps prevent IgE-mediated allergic symptoms.

Studies have reported that certain protein kinase C (PKC) activators regulate class switching^8,9. Although many classical PKC activators, notably phorbol esters, are carcinogenic^10,11, bryostatin-1 (bryo-1) is a non-carcinogenic PKC modulator that selectively enhances IgA class switching in B cells while downregulating IgE production (Fig. 1)⁸. Bryo-1 is a natural macrocyclic lactone isolated from the marine invertebrate Bugula neritina¹². As a potent PKC modulator, bryo-1 has attracted considerable interest related to its therapeutic potential and has demonstrated acceptable safety and tolerability in clinical studies¹³. Beyond its anticancer and antiviral effects, bryo-1 modulates immune cell functions through PKC-dependent pathways, regulating T- and B-cell activation and proliferation^14,15. Moreover, it has been reported to stimulate human monocytes and induce proinflammatory cytokines, such as interleukin-6 (IL-6)^16,17, which play an important role in IgA production^18,19. Most importantly, Morita et al.²⁰ demonstrated that bryo-improved the incorporation efficiency1 selectively promotes IgA class switching via the PKCδ-MEK/ERK-RUNX1 signaling pathway, while simultaneously inhibiting IgE class switching through the PKCδ-STAT5-ID2 axis. In the same study, the intranasal administration of 20 ng of bryo-1 significantly mitigated hay fever symptoms in mice, with the therapeutic effects enduring for a minimum of three months. Consequently, the established mechanism of bryo-1–mediated selective class switching and the therapeutic properties of bryo-1 highlight its potential for treating IgE-mediated allergic diseases of AR. Furthermore, GMP-grade bryo-1, when stored in a solid state, exhibits remarkable stability, underscoring its potential as a therapeutic agent.

Fig. 1.

Schematic illustration of bryostatin-1-mediated selective class switching to IgA, which can prevent allergic responses and enhance mucosal defense against antigens.

Despite these findings, the clinical application of bryo-1 faces significant challenges, including its high cost (ca. US $500/10 μg), and its tendency to adsorb onto glass and plastic surface in aqueous solution²¹. Additionally, the nasal mucosal barrier, rapid mucociliary clearance, and short residence time of the formulations within the nasal cavity complicate effective drug delivery to target mucosal immune cells²². Addressing these challenges requires an innovative delivery system; therefore, this study investigated the use of nanoparticle carriers to overcome the barriers to the clinical application of bryo-1. Nanoparticle carriers can enhance drug solubility, improve efficient delivery to cells, and reduce the frequency of drug administration²³. Previous studies have demonstrated that nanoparticle-based strategies, such as protein shell-lipophilic core nanoparticles²⁴, engineered extracellular vesicles²⁵, and targeted exosomes²⁶, can effectively encapsulate bryo-1, thereby enhancing its stability, delivery efficiency, and therapeutic efficacy. While various nanoparticle platforms, such as protein-based or exosome-derived systems, offer distinct advantages in drug delivery, their application to intranasal immunization presents specific challenges. For instance, the highly tunable surface properties of LNPs are particularly advantageous for intranasal delivery, where precise control of surface charge is essential for enhancing nasal retention and overcoming mucociliary clearance—factors that may be less flexible in fixed-architecture protein scaffolds²⁷. Furthermore, compared to complex biological systems like exosomes, LNP platforms offer structural simplicity and high synthetic reproducibility, which are critical for physicochemical standardization and scalable manufacturing²⁸. By leveraging these liposomal characteristics, we aimed to develop a robust and minimally invasive delivery system specifically optimized for the nasal mucosal environment, ensuring both consistent immune modulation and a favorable safety profile.

Efficient permeation through the nasal mucosa and effective interactions with antigen-presenting cells (APCs) are essential for intranasal drug delivery systems²⁹. Particles within the size range of 100–200 nm exhibit higher nasal permeation, whereas the surface charge of nanoparticles influences their interaction with immune cells³⁰. Liposome-based lipid nanoparticles (LNPs) offer significant advantages as drug delivery vehicles^31,32. Their hydrophobic lipid bilayer provides a favorable environment for the efficient incorporation and stabilization of the moderately sized bryo-1 molecule, thereby improving its apparent solubility in physiological media³³. Furthermore, on the basis of their biocompatibility, biodegradability, and ability to prevent drug degradation, LNP-based delivery systems have gained clinical approval for applications such as cancer therapeutics and viral vaccines^34,35. Moreover, LNP formulations allow precise control over particle size and surface charge, further enhancing mucosal penetration and delivery efficiency³⁶.

Cationic LNPs enhance electrostatic interactions with the negatively charged nasal mucosa and cell membranes, thereby improving their retention and resistance to mucociliary clearance^29,37. In contrast, anionic LNPs facilitate cellular uptake via receptor-mediated pathways³⁸. These charge-dependent mechanisms contribute to the improved efficiency of intranasal drug delivery. On the basis of these considerations, we investigated the charge-dependent performance of LNPs for the intranasal delivery of bryo-1.

In this study, we prepared LNPs with different surface charges by mixing distinct phospholipids: neutral (1,2-dioleoyl-sn-glycero-3-phosphocholine; DOPC), cationic (1,2-dioleoyl-3-trimethylammonium-propane; DOTAP), and anionic (1,2-dioleoyl-sn-glycero-3-phospho-L-serine; DOPS). We evaluated the impact of the surface charge on the interaction between LNPs and APCs, their cytotoxicity, and CSR activity in vitro. Furthermore, the in vivo therapeutic efficacy of intranasally administered bryo-1@LNP formulations was assessed in a mouse model of AR. This study provides valuable insights into the rational design of LNP-based mucosal immunotherapies, highlights a promising therapeutic strategy for AR and other allergic diseases, and also provides a method for potentially increasing the therapeutic window of bryo-1.

Materials and methods

Animals and materials

The origins and manufacturers of materials not mentioned below can be found in the Supporting Information.

Cell culture and animal maintenance

Six-week-old female C57BL/6N mice weighting 14–19 g were purchased from Japan SLC Inc. (Shizuoka, Japan). The animals were housed 5–6 per cage. All animal experiments were performed according to the institutional guidance of Kyoto University on animal experimentation and were approved by the Animal Experiment Committee of the Faculty of Engineering, Kyoto University. This study is reported in accordance with ARRIVE guidelines. RAW264.7 cells (a mouse macrophage cell line) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin. Primary Splenic B cells were isolated from C57BL/6N mice. Mice were anesthetized with 2% isoflurane for 5 min, followed by euthanasia via cervical dislocation. The spleens were collected from mice and steeped in RPMI-1640 medium. Splenocytes were forced out of the spleen tissue using a glass slide and collected in a centrifuge tube after passing through a cell strainer. After resuspension in RPMI-1640 medium three times, B cells were purified using a B Cell Isolation Kit (Miltenyi Biotec., Bergisch Gladbach, Germany). The purity of the B cells was confirmed by pacific blue anti-mouse CD45R antibody staining. B cells were cultured in RPMI-1640 medium containing 10% FBS, 1% penicillin–streptomycin and 1 ng/mL anti-mouse CD40 antibody. Isolated B cells were preserved in a Cell Banker 1 (Takara Bio, Inc., Kusatsu, Japan) until use.

Preparation of blank LNPs and bryo-1@LNPs

Blank LNPs were prepared using the thin-film method³⁹. Briefly, lipids were mixed at a certain ratio, the solution was evaporated to form lipid films in a glass tube, and subjected to a vacuum overnight to remove the solvent. The lipid film was hydrated by the addition of HEPES buffer (0.1 M, pH 7.4) and incubated for 3 h at 37 °C. After vortexing, multilamellar vesicles were extruded through a 100-nm pore size to produce unilamellar vesicles. The LNPs were stored at 4 °C before use. For bryo-1@LNPs and rhodamine-PE (Rhod-PE) labeled LNPs, 0.005 mol% bryo-1 or 0.5 mol% Rhod-PE (relative to the total lipid content) dissolved in CHCl₃ was added to the lipid solution. After extrusion, additional centrifugation was performed using a 100 kDa membrane ultrafiltration at 14,000 × g for 10 min to remove any free bryo-1. LNPs with different surface charges were prepared using different molar ratios of neutral and ionic lipids. The neutral LNPs (N-LNPs) consisted of DOPC alone. The cationic LNPs (C-LNPs-5, -10, -20, and -40), corresponded to the additional molar percentage of the cationic lipid DOTAP to DOPC. The anionic LNPs (A-LNPs) followed the naming method for C-LNPs.

Characterization of blank LNPs and bryo-1@LNPs

The diameters and ζ-potentials of the LNPs were determined by dynamic and electrophoretic light scattering (DLS, ELS; Zetasizer Nano-ZS; Malvern Panalytical, Malvern, U.K.). The incorporation ratio of bryo-1 was determined by high-performance liquid chromatography (HPLC; Nexera; Shimadzu, Kyoto, Japan) and calculated using the following formula (Eq. 1), where n _{bryo-1 in LNPs} represents the amount (mol) of bryo-1 incorporated in the LNPs, and n _{total bryo-1} refers to the amount (mol) of bryo-1 added initially. For further evaluation, the amount of bryo-1 incorporated into the LNPs was determined as the incorporated amount of bryo-1 (n _{bryo-1 in LNPs}) divided by the total volume (L) of the prepared LNP solution.

1

Cell viability assays

Pre-cultured RAW264.7 cells (3 × 10⁴ cells/well, in a 96-well plate) were incubated with blank LNPs in Opti-MEM at concentrations of 25, 50, 100, 250, and 500 μM for 47 h. Cells without LNP treatment were used as blanks. The medium was removed, the cells were washed twice with PBS, and cultured with Cell Counting Reagent SF in DMEM (1:9) for an additional 2.5 h. Absorbance was measured at 450 nm using a plate reader (iMark; Bio-Rad, Hercules, CA, USA). Cell viability was calculated using the following formula based on the obtained OD value (Eq. 2): where the OD_test represents the OD value of cells treated with LNPs, OD_blank corresponds to the absorbance of the CCK-8 solution without cells, and OD_control represents the OD value of untreated cells.

2

Uptake efficiency of LNPs

The uptake efficiency of LNPs was evaluated by measuring the fluorescence intensity using flow cytometry (LSR Fortessa Cell Analyzer, BD Biosciences, San Jose, CA, USA). Pre-cultured RAW264.7 cells (5 × 10⁴ cells/well in a 24-well plate) and B cells (2.5 × 10⁵ cells/well in a 24-well plate) were incubated with blank Rho-N-LNPs, -C-LNPs, and -A-LNPs in Opti-MEM. After a certain incubation period, the LNPs were removed and washed three times with PBS. The cells were dispersed in 0.5% BSA solution and passed through a cell strainer into a test tube. The test tubes were kept in the dark and on ice before measurements were performed.

Observation of uptake behavior using confocal laser scanning microscopy (CLSM)

RAW264.7 cells (3 × 10⁴ cells/well in u-slide 8-well high glass bottom plates) were incubated with 100 μM blank Rho-N-LNPs, -C-LNPs-5, and -A-LNPs-5 in Opti-MEM. After 3 h of incubation, LNPs were removed and washed with PBS. The nuclei were stained with Hoechst 33342 solution in PBS for 10 min. The distribution of LNP in cells was observed using CLSM (LSM780, Zeiss, Oberkochen, Germany).

Real-time qPCR assay

Splenic B cells (1 × 10⁶ cells/ well in a 6-well plate) were incubated with bryo-1 or bryo-1@LNPs (0.2 nM bryo-1) for 48 h. The concentration of bryo-1 in the LNPs was adjusted to equivalent bryo-1 levels as free bryo-1. All formulations had a final lipid concentration of approximately 8 μM. B cells were harvested, and total RNA was extracted using the RNeasy Plus Mini Kit (QIAGEN, Hilden, Germany). RT-qPCR was performed using the standard protocol with diluted RNA solution in triplicate for each sample using TB Green® Premix Ex Taq™ II FAST qPCR (Takara Bio, Inc., Kusatsu, Japan). Then, 10 ng RNA was used for each reaction. The primers used are listed in Table S1. Data were normalized to the housekeeping gene mouse β-actin.

Immunization and intranasal administration to mice

Mice were randomly divided into six groups (n = 3–8) as follows: PBS (negative control), ovalbumin (OVA, positive control), free bryo-1, bryo-1@N-LNPs, bryo-1@C-LNPs-10, and bryo-1@A-LNPs-40. n refers to number of animals. All procedures were performed under 2% isoflurane anesthesia. Primary immunization was performed on day 0. OVA (20 μg) was dissolved in 100 μL PBS and mixed with 100 μL aluminum hydroxide gel (aluminum hydroxide 19.8 mg/mL solution containing 0.05% sodium azide) as an adjuvant and injected intraperitoneally (200 μL) into mice. The PBS group was injected with aluminum hydroxide gel only. OVA was mixed with bryo-1 or bryo-1@LNPs and administered (total 20 μL, 10 μL/nostril) intranasally to mice. The doses of free bryo-1 and bryo-1@LNPs were adjusted to equivalent bryo-1 levels prior to use. The PBS group was intranasally administered 20 μL of PBS. The order of administration for each mouse within the same group was randomized each time. Administration started on day 7 and was performed weekly for 4 consecutive weeks.

Serum and saliva collection

Saliva samples were collected once a week from day 6 (i.e., 1 day before intranasal administration) for 5 weeks. All procedures were performed under 2% isoflurane anesthesia. Blood was collected from the submandibular vein of each mouse on day 41. After 30 min of blood collection, the samples were centrifuged at 3,500 rpm for 5 min. The serum was then collected and stored at − 20 °C before use. After anesthesia, the mice received an intraperitoneal injection (100 μL) of pilocarpine hydrochloride in PBS (0.25 mg/mL). After 6 min, approximately 40 μL of saliva was collected from each mouse and stored at − 20 °C before use. The order of blood collection, and saliva collection for each mouse within the same group was randomized each time.

Enzyme-linked immunosorbent assay (ELISA)

OVA-specific IgG and IgE titers in the serum and OVA-specific IgA in the saliva were determined by ELISA. OVA in PBS (5 mg/mL) was coated onto 96-well Maxisorp plates and incubated overnight at 4 °C. After each step, the plates were washed with 0.1% Tween 20 in PBS (PBST). The plate was blocked with 2% bovine serum albumin (BSA) in PBS for 2 h at 37 °C. Diluted serum and saliva samples were added to the plates and incubated overnight at 4 °C. The following day, biotin goat anti-mouse IgG, biotin anti-mouse IgE, and biotin anti-mouse IgA antibodies were added and incubated at 37 °C for 2 h. This was followed by incubation with HRP-conjugated streptavidin at room temperature for 2 h. Finally, tetramethylbenzidine (TMB) solution (Thermo Fisher Scientific, Waltham, MA, USA) was added at room temperature. The reactions for IgE and IgA measurements were stopped after 30 min by adding 2 M HCl, and those for IgG measurements were stopped after 5 min. The optical density was determined using a plate reader at an absorbance of 450 nm. For data analysis, animals with both IgE and IgG absorbance values below 0.36 and 1.32, respectively, were considered to have failed to establish the allergic model and were excluded from the analysis. These thresholds were defined as the lowest absorbance values in the positive control (OVA) group. This criterion was applied uniformly across all groups.

Mucus penetration study

The mucus penetration ability of Rhod-PE labeled LNPs (N-LNPs, C-LNPs-10, and A-LNPs-40) was investigated using a Transwell mucus diffusion model. Brifly, a 5% (w/v) mucin solution in PBS (pH 6.0) was added to the Transwell insert (Corning®, 24-well, cat#3422), and the receptor chamber was filled with 600 μL of PBS. The plate was incubated at 37 °C and shaken on the shaking board (PR-12, TAITEC, Osaka, Japan) for 30 min to remove air bubbles from the mucin layer. LNPs (0.4 mM, 100 μL) were loaded onto the mucin layer and incubated at 37 °C. At 1, 2, 3, 4, 5, and 6 h, PBS (100 µL) was withdrawn from the receptor chamber, and fluorescence intensity was measured using a spectrofluorometer (FP-6300, JASCO, Tokyo, Japan). After each sampling, fresh PBS (100 µL) was added to the receptor chamber to maintain a constant volume. Mucus penetration was expressed as the percentage of fluorescence intensity relative to the positive control (i.e., the same conditions without a mucin layer).

In vivo nasal residence studies and systemic safety assessment following intranasal administration

C57BL/6N mice (n = 4) were anesthetized intraperitoneally and administered 20 µL (10 µL per nostril) of PBS, free bryo-1 or Rhod-PE labeled bryo-1@LNPs (N-LNPs, C-LNPs-10, or A-LNPs-40) intranasally. The doses of free bryo-1 and bryo-1@LNPs were adjusted to equivalent bryo-1levels (0.5 ng) prior to use. Longitudinal imaging was performed in the same animal using an IVIS (Lumina LT, PerkinElmer, Waltham, MA, USA) with excitation at 550 nm and emission collected at 575–650 nm. Mice were imaged at 1, 2, 3, 6, 8, 24, and 48 h post-administration. Blood was collected 24 h post-administration, and serum was used to assess systemic safety. Systemic inflammatory cytokines (TNF-α, IFN-γ, and IL-10) were quantified using mouse TNF-α, IFN-γ, and IL-10 ELISA kits according to the manufacturers’ protocols, with serum samples diluted 40-fold. Potential organ toxicity was assessed by measuring AST and ALT using LabAssay™ kits.

Statistical analysis

Data are expressed as the mean ± SD of at least three independent experiments. Statistical analyses were performed using ANOVA followed by Tukey’s post-hoc test for multiple comparisons. Statistical significance was set at p < 0.05.

Results

Characterization of blank LNPs and bryo-1@LNPs (5 mol%)

Blank LNPs and bryo-1@LNPs were prepared using the thin-film hydration method³⁹. We characterized the particle size and surface zeta potential of empty LNPs and bryo-1 incorporated LNPs (bryo-1@LNPs) as well as the incorporation efficiency of bryo-1 (Table 1). These formulations were prepared using lipids of different compositions and surface charges.

Table 1.

Characterization of blank LNPs and bryo-1@LNPs

	Membrane composition	Rhod-PE labeled blank LNPs		Rhod-PE labeled bryo-1@LNPs
		Particle size (d. nm)	ζ potential (mV)	Particle size (d. nm)	ζ potential (mV)	Incorporation ratio (%)
N-LNPs	DOPC	146.0 ± 1.5	+ 0.7 ± 0.6	139.7 ± 0.9	-1.1 ± 0.6	94.5 ± 0.7
C-LNPs-5	5 mol% DOTAP + DOPC	130.6 ± 3.7	+ 15.1 ± 2.2	123.9 ± 8.4	+ 16.2 ± 1.3	93.5 ± 4.8
A-LNPs-5	5 mol% DOPS + DOPC	132.3 ± 2.2	-16.6 ± 0.5	121.1 ± 4.5	-16.8 ± 1.2	91.8 ± 7.1

N-LNPs were prepared by mixing the neutral DOPC lipid and rhodamine-PE (Rhod-PE) lipid, a fluorescence label. N-LNPs were characterized by a surface charge of + 0.7 and particle size of 146.0 nm. Based on the neutral N-LNPs, the addition of 5 mol% cationic lipid DOTAP or 5 mol% anionic lipid DOPS was used to form C-LNPs-5 or A-LNPs-5 (the suffix indicates the molar percentage of charged lipid added), with surface charges of 15.1 mV and − 16.6 mV, respectively. These results confirmed that the LNP surface charge can be modulated and controlled by the selection of ionic lipids. The addition of DOTAP or DOPS resulted in smaller particle sizes of 130.6 nm and 132.2 nm, likely because of the smaller hydrophilic head group in the DOTAP lipid⁴⁰ (Fig. 2A). After bryo-1 incorporation, a decrease in the particle size was observed across all formulations. This reduction may be attributable to bryo-1 partitioning into the lipid bilayer, as the insertion of hydrophobic molecules can modulate lipid packing²⁸ and membrane curvature⁴¹, thereby shifting vesicles toward smaller equilibrium diameters.

Fig. 2.

Characterization of LNPs. (A) Particle size distribution of blank LNPs. (B, C) Stability of the LNPs assessed by (B) particle size and (C) surface charge over different storage durations at 4 °C in HEPES buffer (pH 7.4, HEPES buffer, n = 3).

The incorporation ratio of bryo-1 into bryo-1@LNPs was evaluated using ultra-high-performance liquid chromatography (UHPLC). Free bryo-1 was separated from the formulations by centrifugation with a 100 kDa MWCO centrifugal filter. Incorporated bryo-1 was then released from N-LNPs, A-LNPs-5, and C-LNPs-5 by dissolving it in the UHPLC mobile phase. The incorporation ratios calculated based on the peak area of bryo-1 were 94.5%, 93.5%, and 91.8%, respectively. Notably, the addition of Rhod-PE improved the incorporation efficiency of bryo-1 from less than 30% to over 90% (Table S2).

Furthermore, to evaluate the stability of LNPs as drug delivery carriers, the LNPs were stored in PBS (pH 7.4) at 4 °C. Particle size distribution (Fig. 2B) and surface zeta potential (Fig. 2C) were measured on days 0, 1, 4, 8, and 18. The results demonstrated that the particle size and zeta potential remained stable over 18 days in storage. In addition, long-term stability studies demonstrated that N-LNPs and A-LNPs-5 remained stable for at least 105 days at 4 °C. In contrast, the cationic formulations (C-LNPs) tended to aggregate during prolonged storage (Figure S1). To minimize the impact of potential aggregation on the subsequent experiments, the particle size was confirmed prior to use. Rhod-PE labeled LNPs were used in the following in vitro and in vivo evaluations, and this designation will not be reiterated throughout the text.

Interaction of blank LNPs with RAW264.7 cells

The interaction between LNPs and APCs was investigated to confirm whether LNPs with a surface charge affect uptake efficiency. Neutral N-LNPs, cationic C-LNPs-5, and anionic A-LNPs-5 were incubated with RAW264.7 cells and fluorescence derived from Rhod-PE was observed by confocal laser scanning microscopy (Fig. 3A). As a result, red fluorescence from Rhod-PE was observed in the cell cytosol, indicating that the LNPs were taken into cells rather than attached to the surface of the cell membrane.

Fig. 3.

Interaction of LNPs with RAW264.7 cells. (A) Confocal laser scanning microscopic (CLSM) images of RAW264.7 cells treated with Rhod-PE labeled LNPs. The scale bar represents 10 μm. (B) Concentration-dependent uptake and (C) Time-dependent uptake of LNPs by RAW264.7 cells (n = 3). Data are presented as the mean ± SD. Data were analyzed by two-way ANOVA with Tukey’s post-hoc test (***p < 0.001).

The fluorescence intensity observed for Rhod-PE reflects the cellular uptake efficiency of the LNPs. Among the prepared LNPs, A-LNPs-5 exhibited a stronger fluorescence intensity than the neutral and cationic LNPs, indicating their enhanced uptake efficiency.

We then investigated whether the LNP concentration or incubation time affected uptake behavior. LNPs at varying concentrations (0.1 mM, 0.25 mM, and 0.5 mM) were incubated with RAW264.7 cells for 3 h. Following incubation, the Rhod-PE-derived fluorescence intensity of cells was measured by flow cytometry and reported as the median fluorescence intensity (Fig. 3B). The fluorescence intensity increased in a concentration-dependent manner, with no significant difference observed between N-LNPs and C-LNPs-5. In contrast, A-LNPs-5 had significantly enhanced cellular uptake, consistent with the CLSM results. However, with increasing incubation time (2, 4, and 24 h), the fluorescence intensity of cells treated with N-LNPs and cationic C-LNPs-5 improved uptake in a time-dependent manner, whereas the uptake of anionic A-LNPs-5 remained relatively constant (Fig. 3C).

Optimization of the surface charge of LNPs

Considering that a higher surface charge may enhance cellular uptake on APCs, cationic and anionic LNPs containing 10, 20, or 40 mol% DOTAP or DOPS ionic lipids were prepared (Table S3) and designated as C-LNPs-10, C-LNPs-20, and C-LNPs-40, respectively, according to their molar ratios. The surface charge and particle size were characterized, and the results are summarized in Fig. 4A. The surface charge increased with the increasing molar ratios of cationic DOTAP or anionic DOPS lipids. The particle size of cationic C-LNPs decreased with increasing DOTAP content, likely because of its smaller hydrophilic head group, whereas the particle size of DOPS-containing LNPs remained relatively constant. The incorporation ratio of bryo-1 in all formulations was maintained above 80%, and the concentration of bryo-1 was determined to be 1.5–2.0 μg/mL. The dosages of both free bryo-1 and bryo-1@LNPs were standardized to ensure that equivalent quantities of bryo-1 were utilized across all experimental conditions.

Fig. 4.

Cell viability assays. (A) Zeta potential (represented as bar graphs) and particle size (represented as circles) of the prepared LNPs (n = 3). RAW264.7 cells were treated with (B) C-LNPs and (C) A-LNPs lipid concentrations of 0.025, 0.05, 0.1, 0.25, and 0.5 mM. After 48 h of incubation, a CCK-8 assay was performed to test cell viability (n = 6). Data are presented as the mean ± SD.

Subsequently, we evaluated the cytotoxicity of the prepared C-LNPs and A-LNPs on RAW264.7 cells according to the cell viability after 48 h of co-culture (Fig. 4B, C). The results indicated that neutral (Figure S2) and anionic LNPs were nontoxic, even at high concentrations. In contrast, C-LNPs bearing a cationic surface charge induced dose-dependent cytotoxicity, which correlated with the surface charge and administered lipid concentration.All the prepared LNPs exhibited no detectable cytotoxicity at a lipid concentration of 0.025 mM, which was subsequently used for further evaluation.

Cellular uptake of LNPs and induction of class switch recombination in antigen-presenting cells

We then evaluated the cellular uptake efficiency of the prepared LNPs. After a 3-h co-incubation with LNPs, fluorescence derived from Rhod-PE in B cells were measured by flow cytometry, and the relative fluorescence intensity was quantified relative to the untreated control group (Fig. 5A). The uptake efficiency of C-LNPs-10 with a surface charge of approximately + 30 mV exhibited the highest uptake efficiency, achieving a 2.5-fold increase compared with C-LNPs-5 and neutral N-LNPs, and an approximately twofold increase relative to all anionic A-LNPs. However, the fluorescence intensity of C-LNPs-20 and C-LNPs-40 was not detected because of their toxicity to B cells.

Fig. 5.

Effect of LNP surface charge on cellular uptake and bryo-1–induced class switching in APCs. Cellular uptake determined by FACS of (A) B cells and (B) RAW264.7 cells. Relative gene expression of germline transcripts (C) GLTα and (D) GLTε in splenic B cells after 48 h of co-culture with free bryo-1 or bryo-1@ LNPs, each containing an equivalent bryo-1 concentration of 0.2 nM with varying surface charges. The expression levels were quantified by RT-qPCR. The positive controls included TGF-β1 (for GLTα) and IL-4 (for GLTε). Cells without bryo-1 or bryo-1@LNPs treatments were used as negative controls. All groups were cultured under stimulation with anti-mouse CD40 antibody. Data are presented as the mean ± SD (n = 3). Statistical analysis was performed by one-way ANOVA with Tukey’s multiple comparisons test (*p < 0.05, **p < 0.01, ***p < 0.001).

In contrast, RAW264.7 cells showed greater tolerance to the cationic surface charge. Specifically, C-LNPs-20, with a surface charge of approximately + 50 mV, achieved a sevenfold increase in uptake efficiency compared with C-LNPs-10, indicating that differences in cationic surface charge can strongly and differentially affect cellular uptake among distinct types of APCs (Fig. 5B).

For the anionic A-LNPs, increasing the negative surface charge had a minimal impact on the uptake efficiency, likely because of the saturation of scavenger receptors on cells. Of note, with the exception of C-LNPs-20 and C-LNPs-40, cationic C-LNPs-10 exhibited higher uptake in B cells than all anionic A-LNP formulations. In contrast, C-LNPs-10 showed comparable or lower uptake efficiency than other A-LNPs in RAW264.7 cells.

We evaluated bryo-1 delivery to mouse splenic B cells using the same set of formulations as in the uptake study (bryo-1@N-LNPs, bryo-1@C-LNPs, and bryo-1@A-LNPs) to enable a direct comparison of the formulation-dependent delivery efficiency. Delivery efficiency was evaluated by quantifying the expression levels of germline α transcripts (GLTα) and germline ε transcripts (GLTε), which serve as indicators of class switching to IgA and IgE, respectively.

Mouse splenic B cells were stimulated with bryo-1 or bryo-1@LNPs in the presence of a CD40 ligand and TGF-β1⁴² or IL-4⁴³ to induce GLTα or GLTε expression, respectively. Each formulation contained an equivalent bryo-1 concentration of 0.2 nM. Total RNA was isolated, and the expression levels of GLTα and GLTε were quantified by quantitative reverse transcription polymerase chain reaction (RT-qPCR), represented as the relative expression versus the housekeeping gene β-actin⁴⁴.

The administration of 0.2 nM free bryo-1 to B cells induced IgA class switching to a level comparable to that of the TGF-β1-treated positive control, as indicated by the similar GLTα expression (Fig. 5C). GLTα expression was further upregulated following treatment with bryo-1@LNPs. The extent of GLTα enhancement in the cationic and anionic LNP groups closely mirrored their cellular uptake efficiency. In addition, GLTα expression increased in a concentration-dependent manner (Figure S3 A,B). Notably, cationic C-LNPs-10 did not induce stronger GLTα upregulation than anionic LNPs did. Higher-charge cationic formulations, such as C-LNPs-20 and C-LNPs-40, exhibited reduced GLTα expression compared to lower-charge formulations. Regarding GLTε expression, treatment with bryo-1 inhibited IL-4–mediated GLTε transcription, and this inhibitory effect was further enhanced when bryo-1 was delivered via LNPs (Fig. 5D). To exclude any effect of Rhod-PE on CSR activity, we compared the bryo-1@N-LNPs formulated with and without Rhod-PE (Figure S3 C,D). No differences were observed in GLTα induction or GLTε suppression between the two formulations, indicating that Rhod-PE served as a fluorescent tracer without affecting CSR activity.

AR therapeutic effect of bryo-1@LNPs on IgE-mediated allergies

Finally, we investigated the in vivo therapeutic effects of the intranasal administration of free bryo-1 and bryo-1@LNPs in an OVA-induced mouse model of AR. Based on the results of cellular uptake, CSR activity, and cytotoxicity evaluations, we selected one representative formulation from each surface-charge group (neutral, cationic, and anionic) for subsequent studies. N-LNPs served as neutral control. Among the cationic formulations, C-LNPs-10 was selected because it achieved high cellular uptake without detectable cytotoxicity, whereas C-LNPs-20 and C-LNPs-40 were toxic at higher concentrations. For the anionic group, as all formulations were non-toxic, A-LNPs-40 was chosen for its strongest uptake efficiency and enhanced class switching modulation. Therefore, bryo-1@N-LNPs, bryo-1@C-LNPs-10, and bryo-1@A-LNPs-40 were used for all further evaluations.

AR model mice received the intranasal administration of 0.5 ng or 2 ng free bryo-1 four times over 4 weeks, with saliva and blood samples collected according to the schedule shown in Figure S4. OVA-specific IgA and IgE levels were quantified by ELISA. By day 60, 2 ng bryo-1-treated mice exhibited a significant increase in saliva OVA-specific IgA levels and decrease serum OVA-specific IgE levels. In contrast, mice that received a low dose of 0.5 ng of bryo-1 exhibited minimal OVA-specific IgA induction and no significant suppression of OVA-specific IgE.

Subsequently, we assessed whether LNP carriers enhanced the therapeutic efficacy of a subtherapeutic 0.5 ng dose of bryo-1. Mice received the intranasal administration of PBS, OVA alone, or OVA combined with 0.5 ng of free bryo-1 or bryo-1@LNPs (all normalized to 0.5 ng bryo-1) following the schedule shown in Fig. 6. Saliva was collected every 7 days, and OVA-specific IgA levels were quantified (Fig. 6A). Mice treated with bryo-1@LNPs exhibited higher OVA-specific IgA titers than those treated with free bryo-1 on day 41 after primary sensitization. Among the tested LNPs, anionic A-LNPs-40 exhibited the strongest IgA response and significantly increased IgA titers at day 41 compared with other LNPs.

Fig. 6.

(A) OVA-specific IgA titers in saliva 6, 13, 20, 27, 33, and 41 days after primary sensitization, (B) OVA-specific IgE titers and (C) OVA-specific IgG titers in serum 41 days after primary sensitization measured by OVA-specific ELISA for PBS, OVA, free bryo-1, bryo-1@N-LNPs, bryo-1@C-LNPs-10, and bryo-1@A-LNPs-40. (D) Evaluation of LNP safety in mice, based on changes in body weight, which was recorded weekly prior to saliva sampling. Mice were initially sensitized intraperitoneally with Al(OH)₃ and 40 μg OVA, followed by four intranasal immunizations at 7-day intervals with 20 μg OVA and free bryo-1 or bryo-1@LNPs (total 20 μL, 10 μL/nostril) per mouse. The doses of free bryo-1 and bryo-1@LNPs were adjusted to equivalent bryo-1 levels (0.5 ng) prior to use. Individual data in panels A–C are shown as open circles (○). Data are presented as the mean ± SD. Sample sizes were as follows: PBS group (n = 6), OVA group (n = 3), free bryo-1 group (n = 7), bryo-1@N-LNPs group (n = 7), bryo-1@C-LNPs-10 (n = 8), bryo-1@A-LNPs-40 (n = 6). Statistical analysis was performed by one-way ANOVA with Tukey’s multiple comparisons test (*p < 0.05, **p < 0.01, ***p < 0.001).

We also investigated the serum OVA-specific IgE (Fig. 6B) and IgG (Fig. 6C) levels on day 41. Free bryo-1 did not reduce IgE titers, whereas bryo-1@LNPs produced a modest decrease; however, the difference between each LNP group was not statistically significant. In addition, the elevated OVA-specific IgG titers relative to those in the PBS control group confirmed that the immune system was successfully activated in all bryo-1-treated groups. Furthermore, we confirmed the safety of the LNPs, as evidenced by the stable increase in body weight (Fig. 6D).

Discussion

In this study, we evaluated the treatment of allergic rhinitis (AR) by intranasal administration of bryostatin-1 (bryo-1) using liposome-based lipid nanoparticles (LNPs) with a tunable surface charge. To design the carrier, we first prepared blank LNPs with different surface charges using either DOPC alone or DOPC in combination with 5 mol% of the cationic lipid DOTAP or anionic lipid DOPS. The addition of these ionic lipids enabled precise control of the particle surface charge and influenced the particle size owing to differences in the ionic hydrophilic head groups (Table 1). All formulations exhibited excellent incorporation efficiency. Notably, the addition of Rhod-PE improved the incorporation efficiency of bryo-1. We hypothesized that this effect may be attributed to the enhanced CH–π interactions between bryo-1 and the aromatic moiety of Rhod-PE, which enabled bryo-1 incorporation within the lipid bilayer and at the particle surface.

AR induces excessive mucus secretion, wherein the mucus barrier, composed of a mucin network with an average pore size of 20–200 nm, can significantly hinder the diffusion of nanoparticles exceeding this size⁴⁵. Therefore, the particle size of the prepared LNPs (< 150 nm) was expected to enable their effective penetration through the mucus layer and access to the underlying nasal epithelium and immune cells. Furthermore, these LNPs remained stable for at least 18 days in PBS (pH 7.4) at 4 °C, indicating that their size and surface charge were compatible with practical storage conditions (Fig. 2B,C).

CLSM and FACS analysis revealed the internalization and uptake of LNPs, with ionic LNPs exhibiting higher uptake efficiency than neutral LNPs (Fig. 3). Interestingly, contrary to the typical expectation of strong interactions from cationic NPs due to electrostatic attraction to negatively charged cell membrane, anionic A-LNPs-5 demonstrated the strongest fluorescence intensity and higher uptake efficiency than cationic C-LNPs-5. This unexpected enhancement is likely attributed to the anionic surface charge and the presence of phosphatidylserine (PS) of A-LNPs. The anionic charge of A-LNPs can be specifically recognized by class A scavenger receptors (SR-A) expressed on RAW264.7 cells because of their polyanionic nature⁴⁶. In addition, PS functions as an “eat-me” signal that is recognized by phagocytic receptors, thereby promoting rapid internalization⁴⁷. These features are considered to contribute to the higher uptake efficiency of A-LNPs. FACS analysis revealed a concentration-dependent increase across all formulations, with higher LNP concentrations resulting in greater internalization (Fig. 3B,C). Consistent with the CLSM findings, A-LNPs-5 exhibited the highest overall cellular uptake. Regarding time-dependent uptake, N-LNPs and C-LNPs-5 showed increased internalization with longer incubation times. In contrast, the fluorescence intensity of A-LNPs-5 remained relatively constant, which may reflect saturation due to the expression levels of scavenger receptors on the surface of RAW264.7 cells.

To examine whether an increased surface charge enhances cellular uptake by APCs, we prepared LNPs with higher surface charges by increasing the molar ratio of ionic to neutral lipids (Fig. 4, Table S3). As expected, the zeta potential increased with increasing molar fractions of the ionic lipids, whereas the bryo-1 encapsulation efficiency remained high for all formulations, indicating that the modulation of the surface charge did not compromise drug loading.

Cytotoxicity assays demonstrated that the neutral and anionic LNPs were non-toxic within the tested concentration range, whereas cationic LNPs containing more than 20 mol% ionic lipids induced dose-dependent cytotoxicity. The cytotoxicity of cationic C-LNPs contributes to strong electrostatic interactions between the positively charged LNPs and the anionic cell membrane, which may disrupt the cell membrane via enhanced fusion events⁴⁸.

Using the nontoxic lipid concentration for all formulations (0.025 mM), the cellular uptake of the prepared LNPs in B cells and RAW264.7 cells was investigated (Fig. 5A,B). C-LNPs-10 exhibited the highest uptake efficiency in B cells. This result indicates a strong enhancement of cellular interactions driven by the electrostatic attraction between the negatively charged cell membrane and the increased cationic surface charge of the nanoparticles. Further increases in the surface charge of C-LNPs-20 and C-LNPs-40 led to significant B cell death, which may be related to their higher surface-area-to-volume ratio; smaller cells are more likely to interact with and internalize NPs, thereby amplifying nanoparticle-induced cytotoxicity⁴⁹. In contrast to B cells, RAW264.7 cells showed higher tolerance to the cationic surface charge. In RAW264.7 cells, C-LNPs-20 (≈ + 45 mV) yielded nearly a sevenfold increase in uptake compared to C-LNPs-10, suggesting that the optimal cationic surface charge for maximizing uptake differs substantially among APC subsets. For anionic A-LNPs, increasing the negative surface charge had only a minimal impact on uptake efficiency, likely because the scavenger receptors on the cell surface were saturated. Among the anionic formulations, A-LNPs-40, which exhibited the highest uptake efficiency, showed a 1.6-fold higher uptake than C-LNPs-10 in RAW264.7 cells, but only 0.6-fold that of C-LNPs-10 in B cells. This difference may be attributable to the lower expression of scavenger receptors on B cells than on RAW264.7 cells.

We assessed this by evaluating immunoglobulin (Ig) class-switch recombination activity to gain deeper insights into the delivery efficiency of LNP carriers (Fig. 5C,D). Ig class switching occurs when IgM-expressing B cells are activated via signals such as the CD40 ligand secreted by activated T cells. Subsequently, cytokines such as transforming growth factor-β1 (TGF-β1) and interleukin-4 (IL-4) induce the expression of germline α transcripts (GLTα) and germline ε transcripts (GLTε), respectively, leading to the production of IgA and IgE⁵⁰. Thus, the expression levels of GLTα and GLTε can be used as indicators of class switching of IgA and IgE.

The markedly upregulated GLTα and downregulated GLTε expression between bryo-1 and bryo-1@LNPs demonstrated that bryo-1 was effectively delivered to B cells by LNPs, promoting IgA class switching and suppressing IgE class switching (Fig. 5C,D). The close correspondence between GLTα upregulation and the uptake profiles of cationic and anionic LNPs suggests that the surface charge influences bryo-1 delivery to B cells, thereby modulating class switching. The absence of greater GLTα induction by cationic C-LNPs-10 compared with anionic LNPs, and the reduced GLTα expression observed for C-LNPs-20 relative to lower-charge formulations, may be attributable to undesired electrostatic interactions between cationic particles and serum components or other proteins in the culture medium, which likely reduce the fraction of LNPs available for effective intracellular delivery of bryo-1. In the uptake experiments, reduced-serum medium was used because of the shorter incubation time and lower nutritional requirements, which mitigated these interactions. The diminished efficacy of C-LNPs-40 is likely attributable to protein-mediated aggregation, as supported by the observed particle aggregation after 2 days of incubation at 37 °C in PBS containing 10% FBS (Table S4). Regarding GLTε expression, treatment with bryo-1 inhibited IL-4–mediated GLTε transcription, which was further enhanced when bryo-1 was delivered via LNPs. This suppression is expected to contribute to a decrease in IgE-mediated allergic responses. These results suggest that LNPs efficiently delivered bryo-1 to B cells and selectively activated class switching in B cells.

The therapeutic effects of intranasal administration of different doses of free bryo-1, bryo-1@N-LNPs, bryo-1@C-LNPs-10, and bryo-1@A-LNPs-40 were evaluated in an OVA-induced mouse model (Fig. 6). A 2 ng dose of free bryo-1 significantly enhanced mucosal OVA-specific IgA while maintaining low serum OVA-specific IgE levels, indicating improved mucosal defense and suppression of IgE-mediated allergic responses. In contrast, 0.5 ng bryo-1 did not induce sufficient IgA or reduce IgE levels, suggesting that higher doses of free bryo-1 are necessary to achieve a therapeutic effect.

Incorporating bryo-1 into LNPs enhanced the therapeutic effect of a subtherapeutic dose of bryo-1, promoting IgA production even at 0.5 ng dose (Fig. 6A). The higher IgA titers in the A-LNPs-40 group suggests that LNPs efficiently deliver bryo-1 to target cells and confer therapeutic benefits, even at low doses, highlighting their potential as a carrier system. In contrast, IgA induction by the cationic C-LNPs-10 was lower than that achieved with the anionic A-LNPs-40 s, which may be attributable to electrostatic interactions with mucins that hinder mucus penetration and limit delivery of bryo-1 to target cells⁵¹. To confirm this interpretation, we performed in vitro mucus penetration and in vivo nasal residence studies. The mucus penetration assay showed that N-LNPs and A-LNPs-10 exhibited higher muco-penetration, whereas C-LNPs-10 displayed the lowest muco-penetration, consistent with partial retention of cationic LNPs within the mucin layer (Figure S5). In vivo nasal residence time was assessed by longitudinal IVIS imaging, with mice imaged at 1, 2, 3, 6, 8, 24, and 48 h post-administration (Figure S6). N-LNPs showed a trend toward clearance into oral cavity via nose-throat path (strong oral cavity fluorescence) and exhibited lower overall fluorescence intensity than the other two formulations, indicating more rapid transit through the nasal cavity. In contrast, both C-LNPs-10 and A-LNPs-40 remained localized at the initial administration site for at least 8 h, suggesting that surface charge contributes to enhanced nasal retention. Combining mucus penetration and nasal residence result, the prolonged retention of cationic LNPs is consistent with mucin entrapment, whereas anionic LNPs combine sustained nasal residence with higher mucus penetration, supporting reduced mucin trapping and improved bryo-1 delivery. Together, these findings reveal A-LNPs-40 as an optimized formulation that exhibits the highest delivery efficiency to promote IgA production. Our results support the utility of this anionic nanocarrier to potentiate the effects of low-dose bryo-1 for mucosal immune modulation.

OVA-specific IgE demonstrated that the bryo-1@LNPs exhibited a modest decrease in IgE, indicating the suppression of IgE-related allergic reactions (Fig. 6B). The absence of significant differences in IgE reduction among the various LNP formulations implies that, under the present dosing regimen, the surface charge had a limited impact on systemic IgE modulation, despite its pronounced effects on cellular uptake and GLTε expression in vitro. The increase in OVA-specific IgG titers across all bryo-1-treated groups indicated that the overall antigen-specific immune response was preserved rather than being globally suppressed (Fig. 6C). In addition, the steady increase in body weight throughout the study period (Fig. 6D), no increase in serum level of inflammatory cytokines (TNF-α, IFN-γ, and IL-10) was observed (Figure S7), and liver injury markers (AST and ALT) within the normal range⁵² at 24 h post-administration (Figure S8) support the systemic safety of bryo-1@LNPs under the tested conditions.

While our findings provide a proof-of concept for mucosal delivery platform for bryo-1, several limitations should be considered. As inter-animal variability was substantial in the IgA response and the sample size was small, the consistency of the delivery efficacy needs further validation in a larger sample size. In addition, although we observed a clear trend in IgA induction increase after the administration of bryo-1@A-LNPs-40, the lack of statistically significant differences in IgE suppression among the LNP formulations indicates that the current platform may primarily function as a potent mucosal immune modulator rather than an immediate suppressor of established allergic cascades. While we have provided evidence of mucosal penetration via in vitro models and confirmed systemic safety, further nasal tissue analyses, such as nasal tissue histopathology and pharmacokinetic/biodistribution analyses, are required to better characterize mucosal distribution and to assess the local safety profile of these formulations. Therefore, future studies utilizing standardized disease endpoints and longitudinal safety assessments are essential to fully bridge the gap between these foundational results and their clinical applications in AR.

In summary, the incorporation of bryo-1 into anionic A-LNPs-40 enhanced mucosal absorption from the nasal cavity compared with cationic LNPs, resulting in elevated OVA-specific IgA levels in saliva and a modest reduction in serum OVA-specific IgE. Of note, when using the LNP carrier, as little as 0.5 ng of bryo-1 was sufficient to induce IgA production, significantly reducing the required dose and overall cost. These findings support the potential of anionic LNPs to enhance bryo-1 delivery to mucosal immune cells.

Conclusion

In this study, we systematically evaluated LNPs with varying surface charges for intranasal delivery of bryo-1 and examined the charge-dependent modulation of mucosal IgA induction. By precisely controlling the ratio of ionic lipids, we engineered formulations with high bryo-1 encapsulation efficiencies. Notably, anionic LNPs demonstrated the superior delivery of bryo-1 to B cells, selectively promoting IgA class switching, while suppressing IgE expression. In a mouse model of AR, the intranasal administration of bryo-1@LNPs significantly increased antigen-specific IgA levels in the saliva, even at low doses, supporting an immunomodulatory effect on mucosal IgA responses. These findings suggest that anionic DOPS-based LNPs can evade mucin entrapment and enhance delivery efficiency to APCs, thus representing a promising platform for intranasal delivery.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

J.L. carried out the experiments, analyzed the data and wrote the manuscript. J.L., N.M. and R.M. designed the experiments. Y.K., R.S. and T.K. supervised and discussed the study and contributed to the experimental design. All authors contributed to the writing and revision of the paper; all authors contributed to manuscript editing.

Funding

This work was supported by JSPS KAKENHI Grant-in-Aid for JSPS Fellows (Grant Number JP24KJ1430).

Data availability

All data generated or analyzed during this study are included in this published article and its supplementary information files.

Declarations

Competing interests

The authors declare no competing interests.