Formulation and characterization of a nanoemulsion-based vaccine adjuvant

Introduction

Nanoemulsions are colloidal dispersions consisting of two immiscible liquids stabilized by surfactants. Nanoemulsions exhibit unique physicochemical properties, including high surface area, optical transparency, enhanced dispersibility, and remarkable kinetic stability. Their long-term physical stability against flocculation, coalescence, and sedimentation makes them highly attractive for pharmaceutical and biomedical applications. This exceptional stability is largely attributed to steric stabilization imparted by non-ionic surfactants and polymers, resulting in systems that are often described as approaching thermodynamic stability (Tadros et al., 2004) [13] . Over the past two decades, nanoemulsions have emerged as versatile delivery platforms in pharmaceutical, cosmetic, food, and biotechnology industries. Compared with conventional emulsions and microemulsions, nanoemulsions possess several advantages, including enhanced physical stability, lower surfactant requirements, improved drug solubilization, and superior bioavailability of poorly water-soluble compounds. The extremely small droplet size minimizes gravitational separation and reduces the tendency for droplet aggregation and coalescence, thereby maintaining formulation homogeneity during storage. Furthermore, the large interfacial surface area facilitates rapid absorption and penetration of encapsulated compounds across biological barriers, making nanoemulsions particularly suitable for targeted and controlled delivery applications (Tharwat et al., 2003) [14] . Nanoemulsion-based formulations can be administered through multiple routes, including parenteral, oral, topical, nasal, pulmonary, and ocular pathways. Nanoemulsions have gained considerable attention as vaccine adjuvants owing to their ability to enhance antigen uptake, promote antigen presentation, and stimulate both humoral and cellular immune responses. Their nanoscale dimensions facilitate efficient interaction with antigen-presenting cells, resulting in improved immunogenicity and prolonged immune stimulation compared with conventional adjuvant systems.

Several studies have demonstrated the potential of nanoemulsion-based adjuvants in vaccine development. The oil-in-water nanoemulsion adjuvant W805EC has been shown to significantly enhance immune responses against influenza virus antigens, inducing both Th1-and Th17mediated immunity while maintaining a favorable safety profile (Hamouda et al., 2010) [5] . Similarly, nanoemulsion formulations combined with recombinant anthrax protective antigen and HIV gp120 elicited strong systemic and mucosal immune responses following intranasal administration in experimental animals (Bielinska et al., 2007; Bielinska et al., 2008) [2,1] . Furthermore, intranasal administration of W805EC formulated with hepatitis B surface antigen enhanced antigen-specific immune responses without inducing significant local or systemic adverse effects in mice, rats, guinea pigs, and dogs (Makidon et al., 2008) [9] . Studies in ferrets also demonstrated enhanced humoral immunity and antigensparing effects when nanoemulsion-adjuvanted influenza vaccines were administered intranasally (Hamouda et al. 2011) [6] . The increasing demand for safer and more effective vaccine adjuvants has intensified research into nanoemulsion-based delivery systems. In veterinary vaccinology, the development of stable, biocompatible, and immunologically effective adjuvants remains a critical requirement, particularly for recombinant and subunit vaccines that often exhibit limited intrinsic immunogenicity. Despite the promising potential of nanoemulsion adjuvants, their successful application depends on thorough physicochemical characterization and safety evaluation. Therefore, the present study was undertaken to develop a novel mineral oil-based nanoemulsion using a sonicationassisted emulsification technique and to characterize its physicochemical properties, including particle size distribution, zeta potential, surface morphology, and thermodynamic stability. In addition, the safety of the formulated nanoemulsion was evaluated in guinea pigs to assess its suitability as a potential vaccine adjuvant for veterinary applications.

Material and methods 2.1 Preparation of Nanoemulsion

A mineral oil-based nanoemulsion (NE) was prepared using soyabean oil and span20 surfactants obtained from the Quality Control and Quality Assurance (QC&QA) Unit. The oil phase was prepared by mixing the required volume of soyabean oil with a lipophilic surfactant and heating the mixture at 70 °C for 5 min. Simultaneously, the aqueous phase was prepared by dissolving the Tween-80 surfactant in distilled water. The aqueous phase was gradually added to the oil phase under continuous stirring to obtain a coarse emulsion. The resulting emulsion was subjected to ultrasonic homogenization using a probe sonicator (Q500, Qsonica, USA; 20 kHz). Sonication was performed for 6 cycles at 20 sec with 10 sec break with progressive increases in amplitude. To minimize heat-induced instability, the emulsion was maintained in an ice bath throughout the sonication process. The resulting nanoemulsion was stored at 4 °C until further characterization.

Stability under Different Storage Conditions

To evaluate storage stability, nanoemulsion samples were maintained at 4 °C for 6 months, -20 °C for 3 months, and -80 °C for 1 month. Samples were periodically examined for changes in appearance, phase separation, flocculation, creaming, or demulsification.

Thermodynamic Stability Studies

The thermodynamic stability of the nanoemulsion was assessed by centrifugation, autoclaving, and dilution tests. For centrifugation studies, samples were centrifuged at 3000 rpm for 30 min at 4 °C and examined for phase separation, flocculation, creaming, or demulsification. Autoclave stability was evaluated by exposing the nanoemulsion to 121 °C at 15 psi for 15 min. Following sterilization, samples were visually inspected for changes in physical appearance and phase stability. Dilution stability was determined by diluting the nanoemulsion with phosphate-buffered saline (PBS) at dilution ratios of 1:25, 1:50, 1:100, 1:200, and 1:1000. The diluted samples were examined for dispersibility, homogeneity, and phase separation.

Surface Morphology

The morphology of the nanoemulsion droplets was examined using Field Emission Scanning Electron Microscopy (FESEM) at the Jawaharlal Nehru Centre for Advanced Scientific Research (JNCASR), Bengaluru, India. A small volume of nanoemulsion was mounted on a specimen stub, air-dried, and examined under FESEM according to standard operating procedures.

Particle Size Analysis

The particle size distribution of both plain nanoemulsion and antigen-loaded nanoemulsion formulations was determined using a laser diffraction particle size analyzer (Mastersizer 2000, Malvern Instruments, UK). Samples were dispersed in Tris-NaCl buffer prior to analysis. Measurements were performed in triplicate, and the mean particle diameter was recorded.

Zeta Potential Analysis

The surface charge of the nanoemulsion droplets was determined using a Zetasizer Nano ZS instrument (Malvern Instruments, UK). Prior to measurement, samples were diluted in Tris-NaCl buffer. All measurements were performed in triplicate.

Results

Preparation of Nanoemulsion by Sonication method

The Nanoemulsion samples were prepared by sonication method as described in methods. The samples were prepared by using mineral oil & lipophilic, hydrophilic surfactant after standardizing the volume of surfactant, the stability of Nanoemulsion was tested in QC&QA lab.

Characterization of Nano-emulsion

Prepared samples were kept at various temperature and at various time interval like 4 °C for 6 months,-20 °C for 3 months,-80 °C for one month and that samples were observed frequently for phase separation, demulsification, and results of this study revealed no phase separation, (Fig. 1A). The pH of that sample was checked at each time of shifting the sample from freezing to thawing. The result showed that pH (7.4±0.2) of the sample was not changed by storing at various temperatures. Therefore the prepared formulations were tested for thermodynamic stability study by centrifugation at 3000 rpm for 30 mins at 4 °C and autoclaved at 121 °C for 15 mins at 15 lbs pressure. The stability of that Nano-emulsion was good and there was no phase separation or flocculation before and after centrifugation (Fig. 1B) as well as in autoclaving (Fig. 1B). The samples were diluted with PBS buffer at various dilutions like 1:25, 1:50, 1:100, 1:200 and 1:1000 were showing good dispersion (Fig. 1C). The emulsion were viewed under 40X were showing few micron size oil droplets, and its background were showing equal distribution of nano size oil droplets (Fig. 1D). The morphology of the nanoemulsion in Field Emmision-Scanning Electron Microscope (FE-SEM) was found to be spherical and size ranging from100-400nm (Fig. 1E). The mean droplet size of the Nano-emulsion preparation was found to be around 185.6 nm and the PDI was 0.245 in Zeta Sizer ® 3000-HS. The size distribution of the Nanoemulsion is shown in Table 1, Fig. 1F. The average zeta potential of the Nano-emulsion was 75.9 mV (Table .1).

Discussion

Nanoemulsion-based adjuvants have emerged as promising vaccine delivery systems because of their ability to enhance antigen uptake, improve antigen presentation, and stimulate both humoral and cell-mediated immune responses. In the present study, a mineral oil-based nanoemulsion was successfully formulated using a combination of hydrophilic and lipophilic surfactants through ultrasonic emulsification. The formulated nanoemulsion exhibited desirable physicochemical characteristics, including long-term stability, nanoscale particle size distribution, and spherical morphology, supporting its suitability as a potential vaccine adjuvant.

The stability of nanoemulsions is a critical factor influencing their practical application in vaccine formulations. In the present study, the nanoemulsion remained stable under different storage conditions and exhibited no evidence of phase separation, flocculation, creaming, or demulsification following centrifugation and autoclaving. Similar findings have been reported that the ultrasonication-generated nanoemulsions had efficient surfactant coverage at the oil-water interface contributed to enhanced kinetic stability and prolonged shelf life (Ganesan et al., 2023) [4] . The observed stability indicates that the surfactant system employed in the present formulation effectively prevented droplet aggregation during storage and processing. Particle size and size distribution play crucial roles in determining the stability and biological performance of nanoemulsion-based vaccine adjuvants. The formulated nanoemulsion exhibited a mean droplet size of approximately 185.6 nm with a polydispersity index (PDI) of 0.245, indicating a relatively homogeneous distribution. Nanoemulsions with PDI values below 0.3 are generally considered to possess acceptable uniformity and physical stability. Similar observations were reported by Ganesan et al., (2023) [4] , who developed ultrasonication-based nanoemulsions with narrow particle size distributions and enhanced storage stability. The relatively low PDI obtained in the present study suggests efficient droplet formation during the sonication process and reduced susceptibility to coalescence.

Zeta potential is commonly used to evaluate colloidal stability. The zeta potential values of the present nanoemulsion ranged from -2.08 to + 3.28 mV. Although these values were relatively low, the formulation remained physically stable throughout the study period. This finding suggests that steric stabilization rather than electrostatic repulsion was the primary mechanism responsible for maintaining stability. Similar observations have been reported for sterically stabilized nanoemulsions, where low absolute zeta potential values did not adversely affect longterm stability because of effective surfactant-mediated interfacial protection (Khan et al., 2024; Herneisey et al., 2023) [8,7] . Morphological analysis by FESEM revealed predominantly spherical droplets ranging from 100 to 700 nm in diameter. Spherical morphology is a characteristic feature of stable nanoemulsion systems and is considered advantageous for enhanced dispersion and cellular interaction. Comparable morphological characteristics have been reported in nanoemulsion-based delivery systems and vaccine nanoadjuvants, where spherical nanoparticles exhibited improved physicochemical stability and biological performance (Sun et al., 2024; Filipić et al., 2023) [12,3] . The growing interest in nanoemulsion-based adjuvants is primarily attributed to their ability to improve antigen delivery and immune stimulation. Recent studies have demonstrated that nanoemulsion formulations facilitate efficient uptake by antigen-presenting cells, promote lymphatic transport, and enhance both humoral and cellular immune responses (Nooraei et al., 2023; Zhao et al., 2023) [10, 15] . These characteristics are particularly important for recombinant and subunit vaccines, which often require potent adjuvants to overcome their limited intrinsic immunogenicity. Therefore, the favorable physicochemical characteristics observed in the present study suggest that the formulated nanoemulsion could serve as an effective immunomodulatory platform for vaccine delivery.

Conclusion

Overall, the present findings demonstrate that sonicationbased emulsification successfully generated a stable mineral oil-based nanoemulsion with desirable physicochemical properties. The formulation maintained its stability under various stress conditions and exhibited particle size characteristics suitable for vaccine applications. These findings justify its further evaluation as a vaccine adjuvant, including studies on antigen-loading efficiency, cytokine responses, cellular immunity, and protective efficacy in target animal species.

Declaration Conflict of Interest

On behalf of all author declare that no conflict of interest with the publication

Ethics Statement

The animals used in this study were in accordance with the ethics of the IVRI, Izatnagar institutional animal ethics committee (IAEC).

Acknowledgement

I gratefully acknowledge and thankful to the IVRI, Deemed University for funding and support during entire study period.

References

1. Bielinska AU, Chepurnov AA, Landers JJ, Janczak KW, Chepurnova TS, Luker GD, et al. A novel killedvirus nasal vaccinia virus vaccine. Clinical and Vaccine Immunology. 2008;15(2):348-358.
2. Bielinska AU, Janczak KW, Landers JJ, Makidon P, Sower LE, Peterson JW. Mucosal immunization with a novel nanoemulsion-based recombinant anthrax protective antigen vaccine protects against Bacillus anthracis spore challenge. Infection and Immunity. 2007;75(8):4020-4029.
3. Filipić B, Pantelić I, Nikolić I. Nanoparticle-based adjuvants and delivery systems for modern vaccines. Vaccines. 2023;11(7):1172.
4. Ganesan NG, Singh RD, Dwivedi D, Rangarajan V. Synergy evaluation between diverse biosurfactants toward the formulation of green oil-in-water nanoemulsions by ultrasonication method. Journal of Cleaner Production. 2023;400:136735.
5. Hamouda T, Chepurnov A, Mank N, Knowlton J, Chepurnova T, Myc A. Efficacy, immunogenicity and stability of a novel intranasal nanoemulsion adjuvanted influenza vaccine in a murine model. Human Vaccines. 2010;6(7):585-594.
6. Hamouda T, Sutcliffe JA, Ciotti S, Baker JR. Intranasal immunization of ferrets with commercial trivalent influenza vaccines formulated in a nanoemulsion-based adjuvant. Clinical and Vaccine Immunology. 2011;18(7):1167-1175.
7. Herneisey M, Janjic JM. Multiple linear regression predictive modeling of colloidal and fluorescence stability of theranostic perfluorocarbon nanoemulsions. Pharmaceutics. 2023;15(4):1103.
8. Khan BA, Ahmad N, Alqahtani A, Baloch R, Rehman AU, Khan MK. Formulation development of pharmaceutical nanoemulgel for transdermal delivery of febuxostat: Physical characterization and in vivo International Journal of Advanced Biochemistry Research https://www.biochemjournal.com evaluation. European Journal of Pharmaceutical Sciences. 2024;195:106665.
9. Makidon PE, Bielinska AU, Nigavekar SS, Janczak KW, Knowlton J, Scott AJ, et al. Pre-clinical evaluation of a novel nanoemulsion-based hepatitis B mucosal vaccine. PLOS ONE. 2008;3(8):e2954.
10. Nooraei S, Lotfabadi AS, Akbarzadehmoallemkolaei M, Rezaei N. Immunogenicity of different types of adjuvants and nano-adjuvants in veterinary vaccines: A comprehensive review. Vaccines. 2023;11(2):453.
11. Rachman ES, Soeratri W, Erawati TM. Characteristics and physical stability of nanoemulsion as a vehicle for anti-aging cosmetics: A systematic review. Journal Farmasi dan Ilmu Kefarmasian Indonesia. 2023;10(1):62-85.
12. Sun M, Pratama AC, Qiu H, Liu Z, He F. Toward innovative veterinary nanoparticle vaccines. Animal Diseases. 2024;4:119.
13. Tadros T, Izquierdo P, Esquena J, Solans C. Formation and stability of nanoemulsions. Advances in Colloid and Interface Science. 2004;108-109:303-318.
14. Tharwat F. Colloids in cosmetics and personal care. 2003;4:36-39.
15. Zhao T, Cai Y, Jiang Y, He X, Wei Y, Yu Y, et al. Vaccine adjuvants: Mechanisms and platforms. Signal Transduction and Targeted Therapy. 2023;8(1):283.