INTRODUCTION:

Vaccines have evolved into various categories, encompassing vaccines derived from entire microorganisms that have been either inactivated or attenuated, subunit vaccines, recombinant vaccines, and DNA vaccines¹. Subunit vaccines are composed of specific, isolated components derived from viruses or bacteria. These vaccines exhibit a high safety profile as their production does not necessitate the use of live pathogenic microorganisms. However, the antigens selected for subunit vaccines, despite being highly purified, frequently demonstrate suboptimal immunogenicity, thereby failing to elicit a robust immune response. The absence of the majority of microbial constituents, such as pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs), significantly contributes to this limitation. PAMPs and DAMPs are potent activators of the innate immune response, engaging with pattern recognition receptors (PRRs) on host cells that detect pathogenic microbes. The deficiency of PAMPs and DAMPs in subunit vaccines results in a diminished capacity to stimulate the innate immune system, which is crucial for the subsequent activation of the adaptive immune system and the establishment of immunity. To mitigate this limitation, subunit vaccines are frequently formulated with adjuvants that enhance their immunostimulatory properties².

Adjuvants, which are functional excipients, are categorized based on their function into delivery systems or immunostimulatory systems. Currently approved vaccine adjuvants often lack sufficient potency to elicit robust protective immune responses against various target pathogens, particularly in immunologically hyporesponsive populations such as the elderly and immunocompromised individuals. These populations tend to have a diminished antibody response due to impaired T cell function. Aluminium salt (alum)-based adjuvants, which are the most extensively used in human vaccines, have been associated with several adverse effects, including myalgia and pyrexia. Moreover, alum can induce granuloma formation at the injection site, with a heightened risk when vaccines are administered subcutaneously or intradermally. Consequently, the development of more effective adjuvants for next-generation vaccines is strongly recommended to mitigate these risks and enhance immunogenicity³. Adjuvants employed as antigen carriers and immunostimulatory agents in vaccines are frequently particulate in nature, including, but not limited to, liposomes, emulsion droplets, and immune-stimulating complexes⁴.

Liposomes represent a highly adaptable delivery platform for antigens, which can be meticulously engineered to achieve the desired immunological response through the integration of immunostimulatory elements and the fine-tuning of their composition, physicochemical properties, and antigen loading methodologies⁵. These nanocarriers are extensively utilized as vehicles to enhance the therapeutic efficacy of bioactive compounds, owing to their substantial loading capacity, targeted delivery capabilities, robust protection against degradative agents, excellent biocompatibility, versatility in structural modifications, and customizable attributes such as size, surface charge, membrane fluidity, and modes of agent incorporation⁶. Liposomes are under development as sophisticated multifunctional vaccine adjuvant delivery systems, characterized by their high capability to elicit the desired immune response⁷. This is attributed to their potential to specifically target immune cells, induce a delayed release via lysosomal pathways, and enhance the presentation of cross-linked antigens⁸. Consequently, these attributes collectively contribute to a marked improvement in the efficacy of vaccinations⁹. The integration of biopolymers into bilayer chains or liposomes is anticipated to enhance the structural integrity of the liposomal membrane, thereby rendering it more robust and stable¹⁰. This enhancement is further projected to augment the functional efficacy of liposomes when employed as vaccine adjuvants¹¹.

Alginate, a naturally occurring polymer, exhibits remarkable biocompatibility, biodegradability, non-irritant properties, and non-toxicity, rendering it an excellent candidate for complexation with liposomes ¹². Alginate has been utilized to engage with the immune cells of the body in biomedical applications. Alginates have been shown to stimulate lymphocyte proliferation and enhance the production of interleukin-1 (IL-1) and tumor necrosis factor-α (TNF-α)¹³. Furthermore, alginates augment both innate and adaptive immunity in a dose-dependent fashion¹⁴. Alginates incorporating pathogen-associated molecular patterns (PAMPs) are detected by the innate immune system's sensors, including Toll-like receptors (TLRs), nucleotide-binding oligomerization domain-like receptors (NOD-like receptors), and C-type lectins, which can initiate an immunological response¹⁵. Consequently, the incorporation of alginate enhances the efficacy of liposomal immunostimulants as vaccine adjuvants ¹⁶. Altogether, this study investigated the impact of incorporating alginate on the enhancement of the immunogenicity of liposomes, as assessed through in vitro nitric oxide (NO) release assays, which serve as an indicator of the immune response.

MATERIALS AND METHODS:

Materials:

The materials utilized in this investigation encompassed sodium alginate [Sigma], calcium chloride [Merck], phosphatidylcholine (PC) [Sigma], cholesterol (CHO) [Sigma], lysozyme [Sigma], 1,2-Dipalmitoylphosphatidylcholine (DPPC) [Lipoid], parafilm, phosphate-buffered saline (PBS) [Gibco, Sigma], lipopolysaccharide (LPS), Griess reagent [Sigma-Aldrich], Bradford reagent [Sigma], sodium bicarbonate [Sigma-Aldrich], Dulbecco's Modified Eagle Medium High Glucose (DMEM-HG), MTT reagent (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide), Sodium Dodecyl Sulfate (SDS) [Merck], sodium nitrite [Sigma-Aldrich], RAW 264.7 cells, Fetal Bovine Serum (FBS) [Sigma], Penicillin-Streptomycin antibiotic, trypan blue, 0.22µm filter, ethanol [Merck], and Milli-Q water.

Preparation of lysozyme-loaded liposome:

A blend of PC/CHO/DPPC lipids (in a molar ratio of 4.5:4.5:1) at a lipid concentration of 10mg/mL, incorporating lysozyme as a model antigen, was utilized for the formulation of liposomes. The specific composition employed for the preparation of lysozyme-containing liposomes is detailed in Table 1.

Table 1. Formula of liposomes

Formula	Molecular weight (g/mol)	Molar ratio	Weight (mg)
Lipid phase (20 mg/2 mL)
Phosphatidylcholine (PC)*	314	4,5	7,2
Cholesterol*	386	4,5	8,9
1,2-Dipalmitoyl phosphatidylcholine (DPPC)*	734	1	3,78
Water phase
Lysozyme	14300		4
Phosphate buffer saline (PBS)	411		ad 2 mL

*in ethanol

A total of 291μL of a 25mg/mL PC stock solution, 358 μL of a 25mg/mL cholesterol stock solution, and 151μL of a 25mg/mL DPPC stock solution were combined in a microcentrifuge tube to constitute the lipid phase. Concurrently, lysozyme was dissolved in PBS within a beaker to form the aqueous phase. The lipid phase was subsequently introduced dropwise into the aqueous phase under magnetic stirring at 1000rpm, maintained at a temperature of 45℃. The resultant biphasic mixture was stirred continuously for 30minutes. Following the initial stirring period, the sample underwent sonication for an additional 30minutes to ensure thorough homogenization, culminating in the formation of a liposome sample.

Preparation of Sodium Alginate Solution:

The sodium alginate solution was prepared by accurately weighing 400mg of sodium alginate and subsequently dissolving it in 50mL of PBS within a beaker. The mixture was then subjected to continuous agitation using a magnetic stirrer set at 400rpm for a duration of 8 hours. Following this, the alginate solution was diluted to achieve varying concentrations of 0.2%, 0.4%, and 0.8% w/v.

Liposome Coating by Alginate:

A volume of 1mL of liposomes was transferred into a beaker. Subsequently, 0.8mL of alginate solution was incrementally added to the liposomes under continuous magnetic stirring at 600rpm for a duration of 15 minutes. Following this, 0.2mL of CaCl₂ solution was introduced into the alginate-liposome mixture and subjected to an additional 10 minutes of stirring. The resultant samples were then filtered through a 0.22μm filter membrane and stored at 4°C¹⁷. The formulation of the constituents incorporated into the liposomal-alginate adjuvant matrix is delineated in Table 2.

Table 2. Composition of liposome-alginate nanoparticles

Formula	Component (mg / 2 mL liposome)
Formula	Liposome	Alginate	CaCl₂
Formula 0	24
Formula 1	12	1.6	0.28
Formula 2		3.2	0.28
Formula 3		6.4	0.28

Characterization of liposome-alginate nanoparticles

The particle size and size distribution of liposome-alginate nanoparticles were quantified utilizing a Malvern Zetasizer Pro-Blue. A 50µL aliquot of the sample was diluted with Milli-Q water to a final volume of 1mL in a cuvette, subsequently analyzed using the Malvern Zetasizer Pro-Blue. The zeta potential determination was similarly conducted with the Malvern Zetasizer Pro-Blue. For this analysis, approximately 50 µL of the sample was diluted with 10mM KCl solution to a final volume of 1 mL in a cuvette before being subjected to measurement using the Malvern Zetasizer Pro-Blue¹⁸.

Encapsulation efficiency lysozyme in the liposome-alginate nanoparticles:

The encapsulation efficiency was quantified utilizing the Bradford reagent in conjunction with a Synergy HTX Multimode Microplate Reader. Liposome-alginate nanoparticle samples underwent centrifugation at 10,000 rpm for 15 minutes to separate the precipitate from the supernatant. Subsequently, 200μL of the supernatant was transferred to 96-well plates, followed by the addition of 50μL of Bradford reagent. The mixture was incubated for 1hour at ambient temperature in darkness. The absorbance of the samples was recorded at a wavelength of 595nm using the Synergy HTX Multimode Microplate Reader¹⁹.

Nitric oxide (NO) release in vitro assay:

The impact of incorporating alginate nanoparticles into liposomes on the interaction of lysozyme with macrophages was evaluated by monitoring the augmented release of nitric oxide (NO). The experimental samples were diluted into three concentration series (200, 400, and 800ppm) utilizing DMEM medium. RAW 264.7 cells (1.2 x 10⁵ cells/200 μL/well) were seeded into 96-well plates and cultured overnight. Subsequently, the medium (DMEM-HG) in each well was replaced with fresh DMEM-HG, followed by the addition of 20μL liposome-alginate nanoparticle samples, 20μL LPS (200, 400, 800ppm) as the positive control, and DMEM-HG alone as the negative control. The plates were incubated for 24hours. After the incubation period, 50μL of the culture supernatant was combined with 50μL of Griess reagent in new 96-well plates and incubated at room temperature for 20 minutes in the dark. The absorbance of the samples was determined using a Synergy HTX Multimode Microplate Reader at a wavelength of 540nm²⁰.

Cell viability of RAW 264.7 cell assay:

The assessment of the viability of liposome-alginate nanoparticle samples in RAW 264.7 cells was conducted utilizing the 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. RAW 264.7 murine macrophage cells (1 x 10⁴cells / 200μL/well) were cultured in 96-well plates. After a 24-hour incubation period, the cells were treated with liposome-alginate nanoparticle samples at varying concentrations of 20, 40, 60, and 160μg/mL and further incubated for 24 hours. Post-incubation, the medium containing the liposome-alginate nanoparticle samples was aspirated and replaced with MTT solution (phosphate-buffered saline containing 5mg/mL MTT) in DMEM medium, followed by an additional 4-hour incubation at 37˚C. Subsequently, 100μL of 10% SDS solution was added to each well, and the plates were incubated overnight in a shaker incubator. Finally, the absorbance was measured using the Synergy HTX Multimode Microplate Reader at a wavelength of 570nm²¹.

Data analysis:

The acquired data underwent a normality assessment utilizing the Shapiro-Wilk test. Upon confirmation of normal distribution, subsequent analysis was conducted via One-Way ANOVA. In the presence of a statistically significant difference, post hoc analysis was performed using Tukey's test to identify specific sample pairs exhibiting significant disparities. Statistical analyses were executed using GraphPad Prism version 9.5.0 software.

RESULT:

The findings pertaining to the particle size, size distribution, and zeta potential of the liposome-alginate nanoparticles are presented in Table 3. In general, each formulation exhibits a comparable particle size profile, with measurements ranging from 257 to 281.7nm. This observation is corroborated by the polydispersity index (PDI) values of each formulation, which denote a homogeneous distribution of the nanoparticles produced. Notably, formulation 2 demonstrates a significantly higher zeta potential value compared to the other formulations, approximately 2.4mV. (Table-1).

Furthermore, the encapsulation efficiency of each formulation was meticulously evaluated utilizing the Bradford assay to determine the capacity of the nanoparticle formulation to sequester the encapsulated antigen from direct exposure. The assessment revealed that the inclusion of alginate in the formulation augmented the encapsulation efficiency of the nanoparticles by up to 30%, in comparison to the control formulation devoid of alginate (Figure 1). Notably, variations in alginate concentration did not significantly influence the percentage encapsulation efficiency.

Figure 1. The encapsulation efficiency percentage of lysozyme-loaded liposomal alginate nanoparticles.

In the subsequent phase, all formulations were evaluated for their biological activity utilizing the RAW 264.7 cell line. The employment of RAW 264.7, a murine macrophage cell line, constitutes a fundamental methodology, as this cell line is an established model for the primary immune response. According to the cell viability assay conducted with the MTT reagent, none of the liposome-alginate complex formulations exhibited cytotoxicity towards RAW 264.7 cells, with cell viability exceeding 50%. Notably, Formula 3 demonstrated superior cell viability across all lysozyme concentrations (refer to Figure 2). Furthermore, nitric oxide (NO) release by macrophages following treatment with the various complex formulations was quantified using the Griess reagent. Comparative analysis revealed that Formulas 1, 2, and 3, relative to the positive control and Formula 0 (lysozyme liposomes without alginate), significantly enhanced NO release stimulation (Figure 3). Formula 3 exhibited the highest efficacy in inducing macrophages to produce nitric oxide, particularly when encapsulating lysozyme at a concentration of 800 ppm. Formula 2 also yielded noteworthy results across all lysozyme concentrations, whereas Formula 1 elicited a significant effect only at the elevated lysozyme concentration of 800 ppm (Figure 3c).

DISCUSSION:

The results from the nanoparticle measurements indicate that the particle sizes span from 257 to 281.7 nm, falling within the typical dimensional spectrum of polymeric nanoparticles ²². The polydispersity index (PDI) of the liposome-alginate nanoparticles ranges from 0.248 to 0.389, indicating that the particle size distribution of the synthesized nanoparticles is relatively uniform ²³. The potential zeta values of formulations 1-3 range from -0.2 mV to -0.8 mV. These findings are consistent with the research conducted by Daemi and Barikani (2012), which demonstrated the presence of negative charges in alginate adjuvant samples. This is attributed to the carboxylic acid groups in the sodium alginate chains, which possess a negative (anionic) charge ²⁴. The incorporation of calcium chloride (CaCl₂), specifically the Ca²⁺ ions, into the liposome-alginate nanoparticle system induces a shift in the zeta potential towards a more positive charge ²⁵.

Free lysozyme was isolated from the liposomes to evaluate the encapsulation efficiency of lysozyme, serving as a model antigen within liposomal structures ²⁶. The separation process predominantly employed is centrifugation. The concentration of unencapsulated lysozyme present in the supernatant was quantified utilizing the Bradford assay ²⁷. The Bradford reagent binds to arginine and lysine residues in lysozyme, inducing a colorimetric shift from brown to blue ²⁸. Subsequently, absorbance measurements were conducted using a microplate reader at a wavelength of 595 nm. The encapsulation efficiency was then calculated with lysozyme as the reference standard ²⁹.

The findings regarding encapsulation efficiency revealed that an increase in alginate concentration correlates with heightened viscosity ³⁰. Consequently, this results in a thicker alginate film layer, which effectively decelerates the release of the encapsulated lysozyme ³¹. The incorporation of CaCl₂ significantly enhances the structural compactness of the alginate matrix. The interaction between alginate and Ca²⁺ ions, serving as a crosslinking agent, is considerably robust, thereby effectively inhibiting the premature release of encapsulated lysozyme ²⁵.

The viability of RAW 264.7 cells was assessed following a 24-hour incubation with liposome-alginate nanoparticles. Viable cells were quantified utilizing the MTT assay, which exploits the reduction of MTT reagent by mitochondrial enzymes in metabolically active cells, resulting in the formation of water-insoluble purple formazan crystals. Subsequently, the formazan was solubilized using SDS, and the absorbance was measured at 570 nm using a microplate reader ²¹. The results of the cell viability assay revealed that the liposome-alginate complex exhibited no cytotoxicity when introduced to RAW 264.7 cells, maintaining cell viability above 50%, even at the maximum concentration of 6.4 mg. This finding is consistent with the inherent non-toxic characteristics of the components utilized in the liposomal-alginate formulation ³².

The nitric oxide (NO) release assay was executed to evaluate the impact of varying alginate concentrations on the efficacy of liposome adjuvant in inducing NO release ³³. The findings of this investigation corroborate the prior research conducted by Ueno et al. (2015), which demonstrated that the incorporation of alginate polymer enhances the stimulation of NO release from RAW 264.7 macrophage cells ³⁴. Alginate comprises flagellin and peptidoglycan, constituents of pathogen-associated molecular patterns (PAMPs) that are identifiable by the host's immune system, thereby eliciting an innate immune response ¹⁵. The release of nitric oxide (NO) serves as a biochemical marker indicative of immunological activity within the organism ³⁵. As the concentration of alginate was augmented, an observed enhancement in the stimulation of nitric oxide (NO) release was noted. Remarkably, this enhancement persisted even at a lysozyme concentration of 800 ppm within formulation 3, achieving a higher level compared to the control group of cells solely induced by lipopolysaccharide (LPS). These findings are consistent with the research conducted by Zhang et al. (2014), which posited that the integration of dual adjuvant modalities, specifically polymeric nanoparticles and liposomes, can potentiate the immunogenicity of the adjuvant ³⁶.

In this study, all formulations were evaluated in comparison to lipopolysaccharide (LPS), which is recognized as an inducer or activator of macrophages during bacterial infection. The observed similarity between the effects of all formulations and LPS on nitric oxide (NO) release suggests that liposome–alginate complex nanoparticles may operate via a mechanism analogous to that of LPS ³⁷. Lipopolysaccharide (LPS) is capable of inducing macrophage activation via the Toll-like receptor 4 (TLR-4), a pivotal receptor in the innate immune system ³⁸. The activation of Toll-Like Receptor 4 (TLR-4) signaling facilitates the translocation of Nuclear Factor Kappa B (NF-κB) from the cytoplasm to the nucleus, where it subsequently binds to DNA sequences to enhance the transcription of pro-inflammatory genes ³⁹. A study conducted by Yang and Jones (2008) proposed that alginate autonomously induces the innate immune response by activating the NF-κB signaling pathway ⁴⁰. The specific receptor targeted by alginate and its associated signaling pathway remain inadequately characterized. Furthermore, the integration with liposomes could potentially yield a distinct and specialized trajectory.

CONCLUSION:

Based on the research findings, it can be inferred that the incorporation of alginate nanoparticles into liposomes as a vaccine adjuvant significantly modulates the characteristics of liposomes by enhancing encapsulation efficiency, reducing particle size, and augmenting the zeta potential. The integration of alginate nanoparticles into liposomal formulations as an adjuvant has been demonstrated to elevate the efficacy of liposomal adjuvants in stimulating the release of nitric oxide (NO) compounds and shows a tendency to improve the viability of RAW 264.7 cells in vitro.

CONFLICT OF INTEREST:

The authors declared no conflict of interest.

ACKNOWLEDGMENTS:

Thanks to the Research Centre for Vaccine and Drug, National Research and Innovation Agency (BRIN)- Indonesia under the scheme of “Rumah Program” for the research funding of Fiscal Year 2024.

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Received on 04.11.2024 Revised on 25.03.2025

Accepted on 29.05.2025 Published on 01.12.2025

Available online from December 06, 2025

Research J. Pharmacy and Technology. 2025;18(12):5633-5639.

DOI: 10.52711/0974-360X.2025.00813

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

S. No.	Formula	Content (mg)			Z-average (nm)	PDI	Zeta Potential (mV)
S. No.	Formula	Liposome	Alginate	CaCl2	Z-average (nm)	PDI	Zeta Potential (mV)
1.	Formula 1	12	1.6	0.28	281.7	0.389	-0.876
2.	Formula 2	12	3.2	0.28	257	0.248	2.404
3.	Formula 3	12	6.4	0.28	274.1	0.365	-0.256