The lipidated structure of INI-4001 (calculated logP of 8.5) makes it suitable for incorporation into various lipid-based formulations. In this work, various ionic liposomal formulations of the TLR7/8 agonist, INI-4001, of different composition and charge characteristics (Table 1) were prepared via a thin film hydration/sonication technique to examine the effect of these variables on the immunogenicity of INI-4001 adjuvanted H7 vaccines (Fig. 1, schematic representation of INI-4001 ionic liposomes). These liposomes were formulated at a relatively high INI-4001 concentration of 2 mg/ml to determine any limitation with adjuvant incorporation into these different liposome compositions. The developed liposomes were characterized for adjuvant loading, physicochemical properties and colloidal stability, and evaluated both in vitro and in vivo for safety and immunogenicity.

Characterization of liposomes

The physicochemical properties of all liposomal formulations were assessed by measuring particle size, polydispersity index (PDI), and zeta potential using DLS. The characterization results are summarized Table 2. Ionic liposomes with or without INI-4001 of various compositions were successfully prepared into uniform particles of less than 80 nm in diameter with narrow size distribution (PDI ranged from 0.118 to 0.274) as determined by DLS measurements (Table 2). Additionally, all ionic liposomes demonstrated the expected charge as depicted in Table 1 based on the corresponding composition where the loading of anionic INI-4001 resulted in a slight decrease in surface charge of the cationic liposomes (Table 2). The particle size, shape and lamellarity of these liposomes were characterized using cryo-TEM imaging (Fig. 2). The INI-4001-loaded formulations primarily formed unilamellar liposomes with a distinct bilayer except for DOTAP/Chol formulation which predominately formed multilamellar liposomes likely due to the distinct larger packing parameters of the DOTAP lipid [30] (Fig. 2f). No particulate aggregates were observed in any of the formulations which, along with the formation of liposomes with distinct bilayer, suggests that INI-4001 adjuvant is incorporated in the liposome bilayer. Similar to the DLS measurements, all formulations predominately formed particles smaller than 100 nm in diameter except for the DOTAP/Chol formulation (Fig. 2f) in which particles larger than 100 nm were predominant by the time of imaging (approximately four weeks after preparation and filtration through a 0.22 μm sterile filter). However, the inclusion of DDAB at a 1:9 molar ratio to DOTAP in this formulation resulted in the formation of smaller and more stable unilamelar liposomes (Fig. 2g). DOPC/Chol formulation (Fig. 2b) had a noticeable population of elongated vesicles. DOPC/DC-Chol (Fig. 2c) and DOTAP + DDAB/Chol (Fig. 2g) liposomes also showed the presence of similar elongated vesicles, albeit to a lesser extent. The appearance of elongated vesicles in these samples may be attributed to mechanical forces exerted during sterile filtration through a syringe filter, or it could be a result of colloidal instability that developed over the four weeks prior to when these images were taken.

Table 2 Summary of characterization results of the ionic liposomal formulations with or without INI-4001. Data were taken on day 1 after preparation and sterile filtration and presented as the mean ± sd (n = 3 independent replicates)Fig. 2

Cryo-TEM images of various INI-4001-loaded liposomal formulation with structural lipid: sterol lipid molar ratio of 2:1; A) DOPG/Chol, B) DOPC/Chol, C) DOPC/DC-Chol, D) DOPC/GL67, E) DOEPC/Chol, F) DOTAP/Chol, and G) DOTAP + DDAB/Chol. The data was obtained from samples analyzed approximately four weeks after preparation and filtration through a 0.22 μm sterile filter

Recovery of INI-4001 after sterile filtration

The palmitoyl lipidated nature of INI-4001 allows it to be stably incorporated within the liposomal bilayer. However, the charge and lipid composition of the liposome may also affect its encapsulation efficiency. To evaluate the effect of these parameters on adjuvant recovery, INI-4001 concentration in each formulation was measured via RP-HPLC post-filtration through a 0.22 μm pore size syringe filter (Table 2). The results demonstrated that liposome charge was not a determining factor in INI-4001 recovery where both cationic DOEPC/Chol and anionic DOPG/Chol liposomes showed the highest INI-4001 recovery of 85.6% and 81.3%, respectively. In contrast, formulations with larger particle sizes such as DOPC/Chol, DOTAP/Chol, and DOTAP + DDAB/Chol had the lowest adjuvant recovery of 46.6%, 45.2% and 35.3%, respectively. This could be due to the tendency of these formulations to aggregate or form larger particles during the preparation process, as well as after sterile filtration and storage, as confirmed by cryo-TEM imaging (Figs. 2b, g, f).

The ability of INI-4001 to incorporate into various liposomes with different charges and compositions highlights its versatility for use in lipid-based nanoparticles.

Assessment of colloidal stability of the lead INI-4001 liposomes

The effect of storage time and temperature on the colloidal stability of the liposomes was determined via monitoring the changes in particle size and zeta potential over three months of storage at 4, 25 and 40 °C. Except for DOTAP-based liposomes, all formulations demonstrated great stability for at least three months when stored at 4 and 25 °C with little or no change in particle size and zeta potential (Fig. 3). The addition of DDAB lipid to DOTAP/Chol liposomes considerably improved its stability. The average particle size and PDI of formulations containing cationic sterol lipids including DC-Chol and GL-67 remained stable at 40 °C despite a decrease in zeta potential. In contrast, liposomes containing cationic structural lipid (DOEPC, DOTAP, DDAB) lost their structural integrity and demonstrated larger particle sizes after two weeks of storage at 40 °C. This elevated temperature was used as a stress condition to assess the robustness of the formulations beyond standard storage conditions. The reduced stability of liposomes containing cationic structural lipids such as DOEPC, DOTAP, and DDAB is likely due to the labile nature and higher packing parameters of these lipids, which may promote vesicle fusion, formation of non-lamellar structures, or aggregation under thermal stress [31,32,33]. Despite demonstrating excellent particle size and PDI stability, INI-4001-loaded anionic DOPG/Chol liposomes exhibited a consistent decrease in zeta potential over time, even when stored at 4 °C, dropping from approximately − 46 mV on day one to around − 21 mV after four weeks of storage (Fig. 3). This may be due to the dynamic rearrangement of lipid/adjuvant components, changing the exposure of the ionic groups and their density at the lipid bilayer surface. Although we did not evaluate the chemical stability of INI-4001 when formulated into liposomes in this study, the observed colloidal stability over three months at 4 °C and 25 °C suggests the structural integrity of the liposomal formulations was maintained. Future studies are warranted to assess long-term chemical stability of INI-4001 under accelerated and real-time conditions.

Based on these results, the DOTAP/Chol formulation was eliminated from subsequent experiments due to its poor colloidal stability, even when stored at 4 °C (Fig. 3).

Fig. 3

Monitoring of z-average particle size, PDI, and zeta potential of INI-4001-loaded ionic liposomes over 12 weeks of storage at 4, 25 and 40 °C. Stability data are presented as the mean ± SD (n = 3 independent replicates)

In vitro cytotoxicity and innate cytokine production from hPBMCs

Particle charge and lipid structure can influence the uptake of liposomes by immune cells, thereby affecting trafficking, activity and cytotoxicity [34]. Cellular uptake of cationic liposomes occurs through electrostatic interaction between the anionic surface of the cell membrane and the cationic lipid, whereas anionic liposomes are taken up by macrophages through scavenger receptors [35]. Furthermore, Takano et al. [36] showed there was a significant correlation between cationic liposomal association with cells and degree of apoptosis. Studies have also indicated that reactive oxygen species (ROS) production and intracellular thiol levels are key factors in induction and regulation of apoptosis by cationic liposomes via a mitochondrial pathway [37, 38]. The cytotoxicity and adjuvant activity of the INI-4001 liposomal formulations were determined in vitro via treatment of hPBMCs with serial dilutions of INI-4001-loaded liposomes for 1 day followed by evaluation of IFN-α (a primary readout for TLR7 activation) and TNF-α (a primary readout for TLR8 activation) by ELISA and viability was determined by Cell Titer Glo assay (Fig. 4).

The neutral (DOPC/Chol) and anionic (DOPG/Chol) liposomes demonstrated the highest cell viability relative to the cationic liposomes (Fig. 4a). The viability of the cells treated with up to 100 µM of these INI-4001 liposomal formulations was comparable to the untreated control and vehicle treated cells. Among the cationic liposomes, DOPC/DC-Chol with moderate cationic charge (< 20 mV) induced no toxicity up to 11 µM adjuvant concentration. The other cationic liposomes showed no toxicity up to 3.5 µM of adjuvant but their toxicity increased considerably at higher concentrations. These findings suggest that the composition of the cationic liposomes may play a key role in determining the liposome’s effect on cell viability.

IFN-α is a cytokine capable of enhancing antiviral immunity and induction of Th1 polarized immune responses. TLR7 mediated induction of an IFN-α response can upregulate the expression of interferon regulatory factors transcription factors which mediate innate and adaptive immunity [39]. IFN-α production appears to be influenced by liposome surface charge and lipid composition. INI-4001 neutral (DOPC/Chol) and cationic sterol lipid-based (DOPC/GL-67 and DOPC/DC-Chol) liposomes demonstrated lower potency in IFN-α production compared to the anionic (DOPG/Chol) and cationic structural lipid-based (DOEPC/Chol and DOTAP + DDAB/Chol) liposomes (Fig. 4b). TNF-α is involved in controlling both innate and adaptive immune responses to viral infections. As an early cytokine, TNF-α is generated in response to viral infections through pathogen recognition receptors like TLR8. hPBMCs treated with the cationic sterol lipid-based (DOPC/GL-67 and DOPC/DC-Chol) liposomes showed the highest level of TNF-α production among all the formulations tested (Fig. 4c). The other cationic liposomes (DOEPC/Chol and DOTAP + DDAB/Chol) with higher surface charge exhibited the lowest levels of TNF-α release. Interestingly, at concentrations exceeding 10 µM, DOPG/Chol liposomes with a negative surface charge induced robust TNF-α production, though not to the same extent as the sterol lipid-based cationic liposomes (Fig. 4c). This observation suggests that cellular uptake of the formulations and TNF-α production was more influenced by the composition of ionic lipid rather than its charge. Although it is expected that cationic liposomes have a better cellular uptake due electrostatic interaction between the anionic surface of the cell and the cationic surface charge of liposome, anionic liposomes have also shown a strong uptake by macrophages through scavenger receptors as mentioned earlier [40].

Fig. 4

INI-4001 ionic liposomes impact cell viability (A) as well as IFN-α (B) and TNF-α (C) induction in hPBMCs. Cell viability was assessed using Cell Titer Glo. Freshly isolated hPBMCs were stimulated for 24 hours with vehicle (liposome hydration buffer (LHB), pH 7.4) or ionic liposomes containing INI-4001. IFN-α and TNF-α were quantified by ELISA from hPBMC supernatants. Data displayed is representative of three biological replicates as the mean ± SD (n = 3).

An inverse correlation was observed between IFN-α and TNF-α induction among the tested cationic liposomes. DOEPC/Chol and DOTAP + DDAB/Chol liposomes were strong inducers of IFN-α but elicited minimal TNF-α. In contrast, DOPC/DC-Chol and DOPC/GL-67 liposomes induced higher levels of TNF-α than IFN-α. These differences between IFN-α and TNF-α activation may arise from the liposomes being taken up by various types of APCs found within the hPBMC populations, which can result in unequal stimulation of TLR7 versus TLR8. TLR7 is predominantly expressed in plasmacytoid DCs (pDCs) and regulate the expression of IFN-α whereas TLR8 is primarily expressed in macrophages, monocytes, mast cells, mDCs, and neutrophils in humans [41]. Our observation confirmed that the capability of INI-4001 as a TLR7/8 agonist in inducing immunogenicity, via IFN-α and TNF-α production, can be influenced by the physicochemical properties of the formulation especially the lipid structure and composition. These properties can determine the fate of liposome in terms of targeting specific APCs as well as dictating the rate and mechanism of cellular uptake (membrane fusion or endocytosis).

In vivo immunogenicity evaluation

It was hypothesized that the incorporation of INI-4001 in liposomes of various charge and lipid composition would impact the immunogenicity and safety of INI-4001 adjuvanted H7 vaccines and could further improve the antigen-specific humoral and cellular immune responses. Previous studies have shown that cationic liposomes, in particular, can enhance the potency of TLR7/8 agonists [42,43,44]. To investigate this hypothesis, a series of four cationic (DOPC/DC-Chol, DOPC/GL67, DOEPC/Chol, DOTAP + DDAB/Chol), one anionic (DOPG/Chol), and one neutral (DOPC/Chol) INI-4001 liposomes and their non-adjuvanted counterparts were combined with the H7 influenza antigen and administered intramuscularly to mice in a series of three injections 14 days apart. Vaccination was conducted using a 1 µg dose of H7 and 10 µg dose of INI-4001. Blood was collected 14 days post-primary (14dp1) and 14 days post-secondary (14dp2) injections to determine H7-specific antibody responses. Five days post-tertiary injection, lymph nodes were harvested to characterize H7-specific T cell responses induced by each formulation.

As shown in Fig. 5a, the anionic INI-4001 DOPG/Chol liposomes induced the highest H7-specific IgG serum titers 14dp1, which was significantly improved compared to antigen alone and DOPG/Chol liposomes. This suggests that anionic liposomes can lead to a more rapid increase in humoral immunity following a single vaccination, which may be essential to immunization in an influenza pandemic setting. This response could also be particularly valuable for vaccines targeting other RNA viruses that require rapid protection, including emerging or re-emerging pathogens such as SARS-CoV-2, RSV, or Zika virus. Anionic liposomes are preferentially taken up by macrophages through scavenger receptors [35], and some studies have shown that the uptake of anionic liposomes by macrophages was higher than that of cationic liposomes [45]. However, the robust induction of both IFN-α and TNF-α from hPBMCs (Fig. 5) suggests that DOPG/Chol liposomes were also efficiently taken up by other immune cells, including pDCs, mDCs, and monocytes. The strong induction of H7-specific IgG serum titers by anionic INI-4001 DOPG/Chol liposomes may be attributed to this enhanced uptake, leading to a more robust immune response. As expected, IgG titers increased 14dp2 immunization (Fig. 5b) compared to 14dp1 immunization. Except for DOTAP + DDAB/Chol, loading of INI-4001 into the tested ionic liposomes resulted in higher IgG titers 14dp2 relative to the corresponding control liposomes. Among all the evaluated formulations, the cationic INI-4001 DOPC/DC-Chol and to a lower extent DOPC/DC-Chol (without INI-4001) and INI-4001 DOEPC/Chol liposomes demonstrated significantly higher induction of H7-specific IgG serum titers relative to the H7 alone group. These results indicate that the extent of TLR7/8 agonist-induced humoral immune response can be altered by changing the liposome composition.

Fig. 5

Immunogenicity evaluation of ionic liposomes with INI-4001 (solid shapes) or without INI-4001 (open shapes) 14-day post vaccination. Serum IgG antibody titers determined by ELISA (n = 7–8). (A) 14-day post-primary titers, (B) 14-day post-secondary titers. One-way ANOVA with post hoc Dunnett test; comparisons shown are between H7 and the different formulations. * p < 0.05; *** p < 0.001; **** p < 0.0001

ELISA-based binding antibody titers provide valuable insights into the immunogenicity of the evaluated liposomal vaccine formulations, especially when used to compare relative responses across groups. However, binding does not correlate with antibody effector function. In future studies, we plan to perform Hemagglutination inhibition assays (HAI) and other functional assays to fully characterize the protective potential of the immune responses.

In addition to humoral immunity, the cellular immune response to these liposomal formulations was also investigated by measuring the production of IFN-γ, TNF-α, IL-5 and IL-17 A in response to antigen-restimulation of cells from the LNs of vaccinated mice. Considering the different impacts of formulations on the antigen-specific IgG, post-harvest antigen re-stimulation of LNs was performed to further investigate the Th1/Th2/Th17 cytokines induction by these various liposomes. Figure 6 represents the antigen-specific T cell responses in LNs harvested 5 days post-tertiary vaccination and re-stimulated ex vivo with H7 influenza antigen. Secreted cytokines were detected by MesoScale Discovery (MSD) multiplex cytokine array. The H7 antigen alone vaccinated mice were used as a control to determine the effect of adjuvant on the immune response more accurately.

With the exception of the INI-4001 DOTAP + DDAB/Chol liposomes, all the other INI-4001 liposomal formulations exhibited significantly higher H7-specific IFN-γ induction compared to the H7 alone or the non-adjuvanted liposome vaccinated mice (Fig. 6), suggestive of a Th1-biased response. TNF-α is produced by many different CD4 T-helper cell types, often described as multifunctional antigen specific T-cells. It has a role as an early inducer of inflammation to direct anti-viral responses through multiple pathways [46]. During adaptive immune responses, T cells produce TNF-α to aid in clearing the virus and restoring homeostasis in infected tissues [47, 48]. TNF-α production was also significantly increased in INI-4001 DOPC/DC-Chol liposomes vaccinated mice. Additionally, all the INI-4001-loaded liposomes resulted in less Th2-associated IL-5 production compared to the H7 alone or the non-adjuvanted liposomes. Among the evaluated liposomes, INI-4001-loaded DOPC/DC-Chol with a moderate cationic charge induced the highest production of both IFN-γ and TNF-α that was significant over the antigen alone group, indicative of a Th1-biased immunity without inducing Th-17-associated IL-17A production (Fig. 6).

Fig. 6

Five-day post-tertiary vaccine T-cell antigen stimulated cytokine analysis of INI-4001 ionic liposomes vaccinated mice. Cytokines were quantified from the dLNs of vaccinated mice using MSD, n = 7–8 mice per group. One-way ANOVA with post hoc Dunnett test; comparisons shown are between H7 and the different formulations. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001

Taken together, the early innate cytokine profiles, such as IFN-α and TNF-α induced in hPBMCs, seem to correlate with the magnitude and quality of the adaptive immune responses observed in vivo, including antibody titers and T cell cytokine production. Additionally, these results indicate that surface charge and lipid composition of liposomes can play a determining role in the immunomodulatory effect of TLR7/8 agonists. The DOPC/DC-Chol liposomes with moderate cationic surface charge increased both humoral and cellular immunity without significant cytotoxicity in contrast to the other liposomal formulations with higher cationic surface charges.