Nanodelivery of Nisin by Homogeneous Protein-based Particles

Nanodelivery of Nisin by Homogeneous Protein-based Particles | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Nanodelivery of Nisin by Homogeneous Protein-based Particles Enrika Celitan, Ramunė Stanevičienė, Kamilė Vaišaitė, Algirdas Mikalkėnas, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8842944/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Nisin is a widely used antimicrobial peptide with strong inhibitory activity against Gram-positive bacteria and has attracted increasing interest for broader biomedical applications. However, its practical use is limited by susceptibility to environmental conditions, reduced stability, and rapid inactivation. Encapsulation has emerged as an effective strategy to overcome these limitations by protecting such bioactive peptides from environmental exposure, improving their stability, and enabling controlled release. Protein-based nanoparticles derived from viruses represent an attractive encapsulation platform due to their homogeneous size, biocompatibility, and ability to encapsulate and protect bioactive molecules without compromising structural integrity. In this study, yeast virus–like particles were explored as a nanodelivery system for nisin, and their antibacterial activity and cytotoxicity were evaluated. Results Nisin was successfully encapsulated into three types of yeast virus-like particles, achieving 77.1–89.3% encapsulation efficiency while preserving particle size and morphology. Nisin-loaded nanoparticles demonstrated antibacterial activity against Gram-positive bacteria, with the most pronounced inhibitory effect against Streptococcus pyogenes . The antibacterial activity against Gram-negative bacteria was nevertheless low. Compared with free nisin, encapsulated nisin exhibited moderately higher (1.2–10.1 fold) minimum inhibitory concentration values. Cytotoxicity studies involving the A549 human lung carcinoma epithelial cell line demonstrated that, while free nisin reduced cell viability, encapsulation in two differently prepared nanoparticles (Y-L-BC and B-L-BC) delayed nisin cytotoxicity. Conclusions Yeast virus–like nanoparticles represent a promising platform for nisin nanodelivery, preserving antibacterial activity while reducing immediate cytotoxicity through encapsulation. These findings highlight the potential for controlled nisin delivery in antimicrobial and biomedical applications. virus-like particles nisin antibacterial activity cytotoxicity nanodelivery yeast virus Figures Figure 1 Figure 2 Figure 3 Background Antimicrobial resistance has emerged as a significant global public health threat, primarily driven by the extensive and inappropriate use of antibiotics in food production, agriculture, and both human and veterinary medicine [ 1 ]. As antibiotic consumption continues to rise worldwide, it highlights the need for alternative antimicrobial strategies. Among the approaches being explored, antimicrobial peptides, such as bacteriocins, have attracted attention for their broad-spectrum antimicrobial activity [ 2 ]. However, the practical use of such peptides is often limited by susceptibility to environmental conditions and low in vivo stability [ 3 ]. Encapsulation has therefore emerged as a promising strategy to protect bacteriocins from degradation, increase their stability, and enable controlled release [ 4 ]. Nisin is the most extensively studied bacteriocin belonging to the Type A(I) lantibiotics, produced by Gram-positive bacteria such as Lactococcus lactis and some Streptococcus species [ 5 ]. It consists of 34 amino acids and has an isoelectric point above 8.5, making it cationic at neutral pH, an important characteristic for its application [ 6 ]. This antimicrobial peptide exhibits broad-spectrum inhibitory activity against Gram-positive bacteria by disrupting cell wall biosynthesis and forming pores in the cytoplasmic membrane [ 7 ]. Nisin is approved by the United States Food and Drug Administration, which granted it Generally Recognized as Safe (GRAS) status [ 8 ]. Due to its potency, natural origin, and established safe use as a food preservative, nisin has attracted considerable interest for applications in medicine and biotechnology. Studies show that it modulates oral and intestinal microbiomes, inhibits infection-associated bacteria, and exhibits anticancer activity [ 9 ]. Food-grade nisin has demonstrated antimicrobial activity against both Gram-positive and Gram-negative oral bacteria, reducing bacterial biofilm biomass and thickness without affecting human oral cell viability [ 10 ]. In combination with oxacillin, nisin effectively and synergistically inhibited the growth of methicillin-resistant Staphylococcus aureus (MRSA) - a pathogen responsible for skin and soft tissue infections - both in vitro and in vivo , and significantly reduced biofilm formation [ 11 ]. Several studies also showed that nisin is cytotoxic to various human cancer cells. For instance, it triggers apoptosis in colorectal adenocarcinoma cells (HT29) via Caspase 7 activation and p53 gene induction, leading to G0/G1 cell cycle arrest [ 12 ]. Nisin has also been shown to induce selective apoptosis and cell cycle arrest in human lung carcinoma cells (A549 and H1299), compared to healthy human embryonic kidney cells (HEK293), through mitochondrial dysfunction and the generation of reactive oxygen species [ 13 ]. Despite its potent antimicrobial and anticancer properties, the application of nisin remains challenging due to its strong pH dependence. While nisin is stable and soluble under acidic conditions, it undergoes rapid degradation and loss of activity at neutral or alkaline pH [ 14 ]. Encapsulation of nisin can therefore overcome these limitations by protecting it from both enzymatic degradation and unfavorable physicochemical conditions, thereby enhancing its stability and antimicrobial efficacy [ 15 ]. Various nisin encapsulation strategies have been explored, including microencapsulation by calcium alginate and guar gum [ 16 ]; liposomes [ 17 , 18 ]; lipid nanoparticles [ 19 ]; sulphated polysaccharides ulvan and fucoidan [ 20 , 21 ]; PLGA polymer [ 22 ]; egg white protein nanoparticles [ 23 ]; cyclodextrin-based nanosponges [ 24 ]. Despite extensive research on bacteriocin encapsulation, existing scaffolds - polymeric and lipid nanoparticles, liposomes, and polysaccharide-based carriers - face limitations such as low encapsulation efficiency, heterogeneous particle size, instability, and cytotoxicity [ 16 , 25 , 26 ]. Therefore, there is a profound need for stable and biocompatible nanocarriers capable of efficient encapsulation of bacteriocins while preserving their biological activity. Protein-based nanoparticles, such as virus-like particles, have gained increasing attention due to their homogenous size, biocompatibility, broad modification potential, and ability to encapsulate, deliver, and control the release of various biomolecules [ 27 ]. Our previous study demonstrated that nanoparticles derived from Saccharomyces cerevisiae L-BC virus can serve as an efficient encapsulation system for nisin due to electrostatic interaction between the positively charged nisin and the negatively charged particle interior [ 28 ]. This study has confirmed that L-BC virus-like particles (VLPs) are a promising delivery system due to the nanoparticle long-term stability under various conditions, resistance to harsh environments, and high encapsulation potential. In the present study, we expanded this approach by preparing three types of nisin-loaded yeast virus-like nanoparticles, evaluating their physicochemical properties and encapsulation efficiency using various analytical methods. To assess the potential applications, the antibacterial activity of the prepared nanoparticles was evaluated against six bacterial strains, and cytotoxicity was assessed in human lung carcinoma cells. We found that all three VLP-based systems efficiently encapsulate nisin, retain its antibacterial activity, and modulate its cytotoxicity, depending on the particle type, thus manifesting their potential as stable, biocompatible nanocarriers for controlled nisin delivery. Methods and materials Bacterial strains and culture conditions Gram-positive bacteria Bacillus subtilis ATCC 6633, Staphylococcus aureus ATCC 29213, Streptococcus pyogenes ATCC 19615 (Vilnius University, Lithuania), and Listeria innocua CECT 910T (University of Lisbon, Portugal), as well as Gram-negative bacteria Escherichia coli BL21 [F⁻ dcm ompT hsdS(rB⁻ mB⁻) gal λ(DE3)] (ThermoFisher Scientific, Vilnius, Lithuania) and Salmonella typhimurium LT2 (pyrF146) (Vilnius Gediminas Technical University, Lithuania), were used in this study. B. subtilis , S. aureus , E. coli , and S. typhimurium cells were propagated in Luria–Bertani (LB) medium (1% peptone, 0.5% yeast extract, 1% NaCl), whereas S. pyogenes and L. innocua were cultured in Tryptic Soy Agar medium (1.7% peptone from casein, 0.3% peptone from soymeal, 0.25% glucose, 0.5% NaCl, 0.25% K₂HPO₄, 2% agar; pH 7.3). All bacterial cultures were incubated for 16–18 h at 37°C with vigorous shaking. Preparation of nisin-loaded virus-like particles VLPs derived from Saccharomyces cerevisiae L-A and L-BC viruses were generated as previously described [ 28 ]. Briefly, for bacterial expression of the L-BC virus capsid protein Gag, the GAG gene was cloned into the inducible vector pET28a [ 29 ]. For yeast expression of L-A and L-BC Gag proteins, the inducible vector pFX7 [ 30 ] was used. VLPs were produced in Escherichia coli BL21-AI (Invitrogen) and Saccharomyces cerevisiae BY4741 [L-A⁻L-BC⁻] (generated in our laboratory, unpublished) strains and purified by ultracentrifugation through a sucrose cushion followed by cesium chloride gradient centrifugation. Buffer exchange of purified VLP samples was performed using 7K MWCO Zeba™ Spin Desalting Columns (Thermo Fisher Scientific, Lithuania) according to the manufacturer’s instructions. Encapsulation experiments were performed by using 220 µM, 22 µM, and 2.2 µM nisin Z (Handary, Belgium) aqueous solutions and 0.22 µM VLP solution in 2× citrate–phosphate buffer (pH 6) containing 300 mM NaCl and 20 mM EDTA. Equal volumes of VLP suspension and nisin solutions were combined to obtain VLP:nisin mixtures with molar ratios of 1:1000, 1:100, and 1:10. Prepared mixtures were incubated at 4°C for 16 h with gentle agitation to allow passive diffusion of nisin into the VLPs. Non-encapsulated nisin was removed by ultrafiltration using 100 kDa MWCO Pierce™ Protein Concentrators (Thermo Fisher Scientific, Lithuania), according to the manufacturer’s instructions. Retentates were washed once with 500 µl of 1× citrate–phosphate buffer (pH 6) containing 150 mM NaCl and 10 mM EDTA, and volumes were adjusted back to the initial with the same buffer. For negative control, equal volumes of 0.22 µM VLP solution in 2× citrate–phosphate buffer (pH 6) containing 300 mM NaCl and 20 mM EDTA and sterile water were combined. For positive controls, equal volumes of 220 µM, 22 µM, and 2.2 µM nisin Z aqueous solutions were combined with 2× citrate–phosphate buffer (pH 6) containing 300 mM NaCl and 20 mM EDTA. All final samples were prepared in 1× citrate–phosphate buffer (pH 6) containing 150 mM NaCl and 10 mM EDTA. Characterization of nisin-loaded virus-like particles Particle size and zeta potential were determined using the Zetasizer NanoZS instrument (Malvern Panalytical, UK) equipped with a 4 mW HeNe laser (633 nm). The intensity of the scattered light was measured at 22°C with a fixed angle of 173°. The data was analyzed by Malvern Zetasizer v8.02 software. The morphology of the particles was evaluated by transmission electron microscope (TEM) Talos™ L120C (Thermo Fisher Scientific, USA). Particles were applied on carbon-coated copper grids (Agar Scientific, UK) and stained with 2% uranyl acetate. Encapsulation efficiency After ultrafiltration, the amount of non-encapsulated nisin in filtrates was determined by Bradford protein assay. 50 µl of each sample was mixed with 200 µl of Bradford reagent in 96-well plates, and absorbance was measured at 595 nm using an Infinite M Nano (Tecan, Switzerland) instrument. Nisin concentration was calculated from a standard calibration curve prepared with known nisin concentrations. The encapsulation efficiency (EE) was calculated according to the following formula: $$\:EE\left(\%\right)=\frac{(Total\:amount\:of\:nisin-Non\text{-}encapsulated\:nisin)}{Total\:amount\:of\:nisin}\times\:100$$ All measurements were performed in triplicate, and results are presented as the mean with standard deviation (± SD). Evaluation of antibacterial activity of free and encapsulated nisin The antibacterial activity of free and encapsulated nisin was evaluated using a direct survival assay. Overnight bacterial cultures were harvested by centrifugation at 6000×g for 5 min, washed twice with 1 mol/L sorbitol, and resuspended in 1 mol/L sorbitol at a final concentration of 1 OD 600 . For Gram-positive bacteria, 50 µl of the cell suspension was mixed with either an equal volume of buffer, nisin-loaded particles (Y-L-A, Y-L-BC, and B-L-BC), particles without nisin, or free nisin samples. For Gram-negative bacteria, the ratio of bacterial suspension and VLPs or nisin samples was adjusted to 1:3 (v/v). Samples were incubated at room temperature (22°C) for 24 h. Serial dilutions were performed in sterile 0.9% NaCl, and 50 µl of each solution was spread onto LB ( B. subtilis , S. aureus , E. coli , and S. typhimurium ) or Tryptic soy agar plates ( S. pyogenes and L. innocua ). Plates were incubated overnight at 37°C, and colonies were counted as colony-forming units (CFU). Results are presented as log 10 (CFU T /CFU C ), where CFU T is the CFU value of the cells treated with free nisin or nisin-loaded particles, and CFU C is the CFU value of the cells treated with buffer or empty VLPs, respectively. All experiments were performed with at least 3 biological replicates, and results are presented as the mean values with standard deviation (± SD). Determination of minimum inhibitory concentrations The minimum inhibitory concentration (MIC) of encapsulated nisin (in Y-L-A, Y-L-BC, and B-L-BC VLPs) and free nisin was determined using a broth microdilution assay. Fifty microliters of diluted overnight bacterial cultures (0.01 OD 600 ) were added to wells of a 96-well microplate. Nisin-loaded VLPs and free nisin samples were serially diluted with sterile water and mixed with Gram-positive bacteria in a ratio of 1:1 (v/v). For Gram-negative bacteria, 25 µl of bacterial suspension was combined with 75 µl of diluted sample. Wells containing only medium served as negative controls, and wells containing medium with bacteria were used as positive controls. Plates were covered and incubated at 37°C for 24 h. At the end of the incubation, turbidity was visually examined, and OD 600 values were measured using an Infinite M PLEX microplate reader (Tecan, Austria) operated with Tecan i-Control software (version 3.9.1.0). MIC was defined as the lowest nisin concentration that fully inhibited bacterial growth. Cytotoxicity The cytotoxicity of the samples was tested in A549 lung carcinoma epithelial cells using the MTT assay. A549 cells were cultured in DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin (Thermo Fisher Scientific, Lithuania). For the assay, 5×10 3 (plate 1), 2.3×10 3 (plate 2), and 1.5×10 3 (plate 3) cells per well were seeded in 96-well plates and incubated for 16–20 h at 37°C in a humidified CO 2 incubator (95% humidity, 5% CO 2 ). After incubation, the culture medium was replaced with fresh medium containing either buffer (control sample), free nisin, nisin-loaded VLPs, or empty VLPs, and the plates were incubated for 24, 48, and 72 h for plates 1, 2, and 3, respectively. Then, cells were washed twice with 150 µl of dPBS (0.9 mM CaCl 2 , 0.5 mM MgCl 2 , 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na 2 HPO 4 , 1.1 mM KH 2 PO 4 , pH 7.4). After washing, dPBS was removed, and 100 µl of culture medium containing 0.5 mg/ml MTT reagent (Merck, Germany) was added to each well. Plates were incubated for 4 h under the same conditions. After incubation, the medium was removed, 100 µl of isopropanol was added to each well, and the plates were incubated for 10 min at room temperature. The absorbance was measured at 565 nm on a Infinite M Nano microplate reader (Tecan, Switzerland). Background absorbance was subtracted from the sample and control values, and relative cell viability was calculated according to the following formula: $$\:Relative\:cell\:viability\left(\%\right)=\frac{Optical\:density\:at\:565\:nm\:of\:the\:sample}{Optical\:density\:at\:565\:nm\:of\:the\:control}\times\:100$$ A total of 4 biological replicates were performed, and results are presented as the mean with standard deviation (± SD). Statistical analysis Statistical differences among groups were obtained by one-way ANOVA followed by Tukey’s multiple pairwise comparison test, with statistical significance marked as follows: p ≤ 0.05(*), p ≤ 0.01(**), p ≤ 0.001(***), p ≤ 0.0001(****). Statistical analysis was performed using GraphPad Prism v.8.0.1 software. Results Characterization of nisin-loaded virus-like particles In previous work, we demonstrated that the antimicrobial peptide nisin Z can be successfully encapsulated into VLPs derived from Saccharomyces cerevisiae virus L-BC, using a passive-diffusion method [ 28 ]. In this study, we broadened the VLP repertoire by including nanoparticles derived from the S. cerevisiae L-A virus, which frequently coexists with the L-BC virus in yeast cells [ 31 ]. Altogether, three types of VLPs were examined: L-A VLPs purified from yeast (Y-L-A), L-BC VLPs purified from yeast (Y-L-BC), and from bacteria (B-L-BC). For encapsulation, three different molar ratios of VLP-to-nisin (1:1000, 1:100, and 1:10) were tested to ensure complete saturation of VLPs by nisin. First, we determined the average particle size and zeta potential of the empty and nisin-loaded VLPs (Table 1 ). Empty Y-L-A VLPs exhibited an average diameter of 42.2 ± 4.3 nm and a zeta potential of − 23.3 ± 0.2 mV. Following nisin encapsulation, no significant change in particle size was observed (p > 0.05), regardless of the VLP-to-nisin ratio. In contrast, the zeta potential of Y-L-A particles shifted toward more positive values after encapsulation, reflecting the presence of the cationic nisin peptide. The extent of this shift increased proportionally with the molar VLP-to-nisin ratio, reaching − 23.0 ± 0.6 mV for 1:10, − 21.8 ± 0.8 for 1:100, and − 15.2 ± 0.7 mV for 1:1000 ratio. The sample featuring a 1:1000 ratio displayed a statistically significant change in zeta potential compared to empty particles (p ≤ 0.001). A similar pattern was observed for Y-L-BC and B-L-BC nanoparticles, produced in yeast and bacteria, respectively. No statistically significant difference in particle size was detected between empty and nisin-loaded VLPs (p > 0.05). The size of empty and nisin-loaded Y-L-BC nanoparticles varied between 37.8 and 50.6 nm, while B-L-BC VLPs ranged from 35.0 to 41.6 nm. For Y-L-BC VLPs, after encapsulation, zeta potential increased slightly with higher peptide ratios: −23.2 ± 1.6 mV for 1:10, − 23.7 ± 2.5 mV for 1:100, and − 19.6 ± 2.4 mV for 1:1000. However, none of these values differed significantly (p > 0.05) from the empty particle control (− 21.2 ± 1.2 mV). B-L-BC VLPs observed a similar pattern, with zeta potential of − 26.7 ± 2.9 mV for 1:10, − 26.2 ± 3.0 mV for 1:100, and − 18.4 ± 1.7 mV for 1:1000, a significant shift observed only at the 1:1000 ratio (p ≤ 0.001). Together, these results indicate that a higher VLP-to-nisin ratio promotes incorporation of nisin, reflected through changes in zeta potential rather than particle size. As shown in Table 1 , for samples prepared at a 1:1000 VLP-to-nisin ratio, the encapsulation efficiency (EE further on) of nisin ranged from 76.8% to 83.1%, with slightly lower efficiency observed for Y-L-BC VLPs compared to the other two particle types. For the 1:100 ratio, EE reached 89.3% and 89.2% for Y-L-BC and B-L-BC VLPs, respectively, whereas Y-L-A VLPs exhibited a slightly lower EE of 77.1%. Encapsulation efficiency could not be determined for the samples prepared at a 1:10 VLP-to-nisin ratio, as encapsulated nisin concentrations were below the detection limit of the Bradford assay (see Table 1 , items marked N/A). Table 1 Particle size, zeta potential, and encapsulation efficiency of empty and nisin-loaded VLPs. Results are presented as mean (n = 3) with standard deviation (± SD). N/A – not available. Sample Molar conc. before filtration, µM Molar VLP-to-nisin ratio Hydrodynamic diameter, nm Zeta potential, mV EE, % VLPs Nisin Experiment with Y-L-A VLPs Y-L-A VLPs 0.11 0 - 42.2 ± 4.3 -23.3 ± 0.2 - Nisin-loaded Y-L-A VLPs 0.11 110 1:1000 43.7 ± 1.0 -15.2 ± 0.7 83.1 ± 3.9 11 1:100 44.7 ± 1.0 -21.8 ± 0.8 77.1 ± 6.9 1.1 1:10 42.0 ± 2.2 -23.0 ± 0.6 N/A Experiment with Y-L-BC VLPs Y-L-BC VLPs 0.11 0 - 50.6 ± 7.1 -21.2 ± 1.2 - Nisin-loaded Y-L-BC VLPs 0.11 110 1:1000 37.8 ± 2.0 -19.6 ± 2.4 76.8 ± 6.0 11 1:100 48.8 ± 6.2 -23.7 ± 2.5 89.3 ± 7.4 1.1 1:10 38.5 ± 3.1 -23.2 ± 1.6 N/A Experiment with B-L-BC VLPs B-L-BC VLPs 0.11 0 - 35.7 ± 5.4 -27.2 ± 2.2 - Nisin-loaded B-L-BC VLPs 0.11 110 1:1000 41.6 ± 6.0 -18.4 ± 1.7 81.9 ± 8.0 11 1:100 35.0 ± 7.0 -26.2 ± 3.0 89.2 ± 6.3 1.1 1:10 38.8 ± 4.0 -26.7 ± 2.9 N/A To visualize and evaluate potential morphological changes in VLPs, TEM was involved. Symmetrical spherical particles with an average size of 39 nm were observed in micrographs of Y-L-A VLPs (Fig. 1 A). Similarly, particles with average sizes of 40 nm and 43 nm were identified in micrographs of empty B-L-BC (Fig. 1 B) and Y-L-BC (Fig. 1 C) samples, respectively. These sizes were comparable to the average particle size of 42 nm observed for nisin-loaded Y-L-BC VLPs prepared at a 1:1000 VLP-to-nisin encapsulation ratio (Fig. 1 D). No visual difference in particle size and morphology was observed between the empty and nisin-loaded VLP samples. Antibacterial activity of nisin-loaded virus-like particles The antibacterial activity of free and encapsulated nisin was tested against Gram-positive ( S. pyogenes , S. aureus , B. subtilis , and L. innocua ) and Gram-negative ( E. coli and S. typhimurium ) bacteria. The results are summarized in Fig. 2 . Free nisin controls were prepared separately for each experiment corresponding to the three VLP types: Y-L-A, Y-L-BC, and B-L-BC. Significant differences between samples prepared at different encapsulation ratios are indicated in Fig. 2 . The results demonstrate that, for all tested bacteria, encapsulated nisin exhibited lower antibacterial activity than free nisin. As expected, the highest antibacterial activity of encapsulated nisin was achieved with the highest 1:1000 molar VLP-to-nisin ratio and was most comparable to that of free nisin. For Gram-positive bacteria treated with nisin-loaded VLPs prepared at a 1:1000 encapsulation ratio, the most pronounced antibacterial activity was observed against S. pyogenes (Fig. 2 A), resulting in a more than 5 log reduction in cell survival. S. aureus showed slightly lower sensitivity to encapsulated nisin, with viability reductions ranging from 4.7 to 5.2 log (Fig. 2 B). A further decrease in antibacterial efficacy was observed for B. subtilis , where encapsulated nisin induced a 3.2–3.9 log reduction of CFUs (Fig. 2 C). The lowest antibacterial effect of encapsulated nisin among the Gram-positive bacteria tested was detected against L. innocua. They were less susceptible to the nisin-loaded VLPs treatment, resulting in a 2 log reduction in cell survival (Fig. 2 D). The impact of encapsulated nisin on Gram-negative bacteria was limited. Within this group, treatment with encapsulated nisin at the 1:1000 ratio resulted in reductions of 0.37–0.46 log for E. coli (Fig. 2 E) and 0.85–1.10 log for S. typhimurium (Fig. 2 F). Among the three types of nisin-loaded VLPs prepared at a 1:1000 encapsulation ratio, Y-L-BC particles caused the greatest reduction in viability for three of the four tested Gram-positive bacteria ( S. pyogenes , S. aureus , and B. subtilis ), compared to Y-L-A and B-L-BC particles. In the case of L. innocua , the efficiency of different nisin-loaded VLP types was similar. At the lower VLP-to-nisin encapsulation ratios (1:100 or 1:10), the inhibitory activity of nisin was comparable among different types of VLPs. The antibacterial activity against Gram-negative bacteria varied by species: the greatest reduction in E. coli was achieved with nisin-loaded L-BC particles purified both from yeast and bacteria (B-L-BC and Y-L-BC VLPs), whereas the strongest inhibitory effect against S. typhimurium was observed with nisin-loaded Y-L-A particles. Notably, a statistically significant difference between particle types was observed only for S. aureus , between nisin-loaded Y-L-BC and B-L-BC particles (p ≤ 0.01) (not indicated in graphs). Minimal inhibitory concentration Since the samples prepared at a 1:1000 molar VLP-to-nisin ratio exhibited the highest antibacterial activity across the tested bacterial species, they were selected for MIC evaluation. The MIC values (µg/ml) of free and encapsulated nisin against Gram-positive and Gram-negative bacteria are presented in Table 2 . Table 2 Minimal inhibitory concentrations of free and encapsulated nisin. MIC of nisin, µg/ml Gram-positive bacteria Gram-negative bacteria S. pyogenes S. aureus B. subtilis L. innocua E. coli S. typhimurium Free nisin 0.60 3.85 1.93 7.71 30.83 41.11 Nisin-loaded Y-L-A VLPs 1.89 6.04 2.27 12.08 96.67 214.81 Nisin-loaded Y-L-BC VLPs 3.78 12.08 3.78 24.17 120.83 171.85 Nisin-loaded B-L-BC VLPs 6.04 24.17 3.02 12.08 96.67 161.11 Among the Gram-positive strains, the MIC of free nisin ranged from 0.60 to 7.71 µg/ml, with S. pyogenes being the most sensitive and L. innocua the least sensitive. For VLP-loaded nisin, the MIC values rose 1.2- to 10.1-fold compared to free nisin. The highest increase of MIC was observed for S. pyogenes , 3.2-, 6.3-, and 10.1-fold for Y-L-A, Y-L-BC, and B-L-BC VLPs, respectively. Despite higher MIC values, this bacterial species proved the most sensitive to encapsulated nisin among the bacteria studied. Overall, nisin-loaded Y-L-A VLPs consistently showed the lower MICs among the Gram-positive bacteria compared with the other two VLP types. B-L-BC VLPs encapsulated with nisin had the strongest effect on B. subtilis. The MIC value was determined to be 3.02 µg/ml. Meanwhile, Y-L-BC-loaded nisin more efficiently inhibits S. pyogenes and B. subtilis bacteria. The MIC values reached 3.78 µg/ml. As observed previously in the antibacterial activity assay (Fig. 2 ), MIC evaluation also confirmed the low sensitivity of Gram-negative bacteria to nisin (Table 2 ). The MICs of free nisin were 30.83 µg/ml for E. coli and 41.11 µg/ml for S. typhimurium . Encapsulation of nisin resulted in a 3.1- to 5.2-fold increase in MICs, with the smallest increase observed for B-L-BC VLPs. Based on MIC values, within the Gram-negative group, E. coli was more sensitive to both free and encapsulated nisin than S. typhimurium . Overall, our results demonstrate that encapsulation reduces nisin activity to varying degrees, depending on the VLP type and the bacterial strain. The impact of nisin-loaded VLPs on the relative viability of mammalian cells Our previous studies demonstrated that the nanoparticles employed in this investigation (Y-L-A, Y-L-BC, and B-L-BC VLPs) efficiently internalize into A549 lung carcinoma epithelial cells within 4 hours in the absence of transfection reagents (unpublished data). Based on these observations, the impact of both empty and nisin-loaded VLPs on A549 cell viability was assessed. Cells were incubated in mammalian culture medium supplemented with 100 pM of naïve or nisin-loaded VLPs for 24, 48, and 72 hours, and relative cell viability was evaluated by the MTT assay (Fig. 3 ). Cells incubated with buffer-supplemented medium served as controls. To evaluate the impact of nisin encapsulation, free nisin control (Nisin C ) was also included. The concentration of nisin in the control sample corresponded to the amount of nisin encapsulated in the respective VLPs, as determined by encapsulation efficiency measurements. Since the samples prepared at a 1:1000 molar VLP-to-nisin ratio exhibited the highest antibacterial activity, they were addressed in the viability assessment. Statistical significance was evaluated by comparing results obtained with cells treated with the sample buffer (control) and VLP- or free nisin-treated cells. Cytotoxicity assessment revealed clear differences between the three types of yeast virus-derived nanoparticles. Empty and nisin-loaded Y-L-BC particles did not significantly affect cell viability at any of the tested time points (24, 48, or 72 hours). Among B-L-BC particles, a modest but significant reduction in viability to 81.5 ± 7.14% (p ≤ 0.05) was observed only for empty B-L-BC particles after 48 hours, whereas nisin-loaded B-L-BC particles did not induce a significant decrease in viability at any time point (p > 0.05). In contrast, Y-L-A particles exhibited a pronounced cytotoxic effect. After 24 hours of incubation, both empty and nisin-loaded Y-L-A particles significantly reduced cell viability to 81.6 ± 4.53% (p ≤ 0.05) and 77.7 ± 8.35% (p ≤ 0.01), respectively. This effect persisted with prolonged incubation, with empty particles reducing viability to 62.0 ± 2.01% (p ≤ 0.0001) and 77.4 ± 4.68% (p ≤ 0.01) after 48 and 72 hours, respectively, while nisin-loaded Y-L-A particles reduced viability to 67.6 ± 11.0% (p ≤ 0.0001) and 72.4 ± 2.31% (p ≤ 0.0001). Compared to free nisin control, which significantly reduced viability to 80.9 ± 9.22% (p ≤ 0.05) after 72 hours of incubation, nisin encapsulated in Y-L-BC and B-L-BC particles did not significantly alter cell viability (p > 0.05). Overall, these results indicate that Y-L-A particles exert cytotoxicity on A549 cells, independent of nisin loading, whereas encapsulation of nisin in Y-L-BC and B-L-BC nanoparticles effectively reduced nisin cytotoxicity in mammalian cells. Discussion The aim of this study was to develop a VLP-based nanodelivery system for nisin and to evaluate its potential applications. For this purpose, yeast virus–like particles were selected, as our previous work demonstrated successful encapsulation of nisin into S. cerevisiae L-BC VLPs [ 28 ]. In the present study, VLPs derived from the S. cerevisiae viruses L-A and L-BC were investigated as carriers for nisin encapsulation. Formation of the L-A virus capsid requires N-acetylation of the capsid protein Gag [ 32 ]; therefore, L-A VLPs were produced using a S. cerevisiae expression system. Since N-acetylation of the L-BC Gag protein was found not essential [ 33 ], L-BC VLPs were produced in both S. cerevisiae and E. coli expression systems. The interiors of fungal dsRNA virus capsids are predominantly negatively charged [ 34 ], making them well-suited for encapsulating positively charged molecules such as nisin. Electrostatic interactions between the oppositely charged peptide and the VLP interior can enhance encapsulation efficiency [ 35 ]. Nisin encapsulation was achieved using a passive diffusion approach, facilitated by the small molecular size of nisin Z (~ 3 kDa) [ 36 ], which allows it to pass through pores located at the fivefold and threefold symmetry axes of the L-BC and L-A capsids [ 34 ]. Nisin Z was selected due to better solubility under neutral pH conditions (above pH 5) compared to nisin A [ 37 ], while L-BC VLPs were previously shown to maintain long term stability within the pH range of 6–8 [ 28 ]. As reported in previous encapsulation studies [ 38 – 41 ], encapsulation is commonly performed using a vast excess of cargo; therefore, several VLPs-to-nisin ratios were involved (1:1000, 1:100, and 1:10). After encapsulation, non-encapsulated nisin was filtered from the nisin-loaded VLPs by the ultrafiltration, as employed in other studies on nisin encapsulation [ 21 , 42 , 43 ]. For the characterization of naive and nisin-loaded VLPs, average particle size and zeta potential were addressed by a combination of electrophoretic mobility, dynamic light scattering (DLS), and transmission electron microscopy (TEM). DLS analysis demonstrated that the size of empty and nisin-loaded VLPs ranged from 35.0 to 50.6 nm, with no significant changes in particle size following the nisin encapsulation, regardless of VLP type (Y-L-A, Y-L-BC, or B-L-BC), or VLP-to-nisin encapsulation ratio (1:1000, 1:100, or 1:10) (p > 0.05). Consistently, TEM revealed neither morphological nor size difference between the empty and nisin-loaded VLPs. The absence of changes in particle size and morphology after loading suggests that cargo is incorporated within the interior of the VLPs rather than being absorbed onto their surface. At the same time, the initially negative zeta potential of VLPs shifted toward more positive values after encapsulation of the cationic peptide nisin, in a manner proportional to the VLP-to-nisin ratio, with the largest change observed for the 1:1000 samples. However, statistically significant changes in zeta potential were detected only for Y-L-A and B-L-BC VLPs. Similar shifts toward more positive zeta potential values have been reported in previous studies of nisin-loaded nanoparticles [ 21 , 44 , 45 ]. The value of zeta potential reflects the degree of electrostatic repulsion between adjacent, similarly charged particles in a dispersion and, therefore, is an indicator of colloidal stability. High zeta potential values (± 30 mV) are associated with increased stability, as particles tend to resist aggregation [ 46 ]. The zeta potential values of nisin-loaded nanoparticles prepared with the greatest nisin content (-15.2 to -19.6 mV) suggest notable electrostatic stabilization of the particles. Together with the preserved particle size and morphology, these results indicate good colloidal stability of the system, in line with previous observations of robust long-term stability of L-BC capsids [ 28 ]. Of note, the combination of a negative zeta potential and the small size of nisin-loaded VLPs (< 100 nm) is generally considered favorable for intravenously administered nanoparticles in drug delivery applications [ 47 ]. The encapsulation efficiency of nisin in yeast virus–like particles was 76.8–83.1% and 77.1–89.3% for samples prepared at 1:100 and 1:1000 ratios, respectively. These values are comparable to those reported for some of the most efficient nisin encapsulation systems described. For example, encapsulation efficiencies ranging from 80.5 ± 0.9% to 93.3 ± 0.2% were achieved for nisin-loaded bacterial cellulose nanocrystals via adsorption of the cationic peptide onto the anionic nanocrystal surface [ 48 ]. Similarly, an encapsulation efficiency of 78.8 ± 2.5% was reported for nisin-loaded monolaurin nanoparticles prepared using an emulsification-based method [ 49 ], while an even higher encapsulation efficiency of 94.12% was achieved for phosphatidylcholine nanovesicles produced using the thin-film hydration method [ 50 ]. Taken together, these results indicate that yeast virus-like particles, employed for passive diffusion, represent a promising and competitive platform for nisin encapsulation. Numerous studies have demonstrated that nisin displays broad antimicrobial activity against Gram-positive bacteria, with limited inhibitory activity against Gram-negative bacteria [ 20 , 21 , 51 ]. To evaluate the potential application of nisin-loaded virus-like particles, the antibacterial activity of the particles was tested against four Gram-positive ( S. pyogenes , S. aureus , B. subtilis , and L. innocua ) and two Gram-negative ( E. coli and S. typhimurium ) bacteria. S. pyogenes , also referred to as group A Streptococcus , is a human pathogen responsible for diseases such as bacteraemia, pharyngitis, cellulitis, necrotizing fasciitis, and toxic shock-like syndrome [ 52 ]. S. aureus is a clinically important pathogen associated with food contamination and capable of causing severe infections, including septicaemia, endocarditis, and pneumonia [ 53 ]. Highly resistant bacterial spores are a major contributor to food spoilage, contamination, and food poisoning. Therefore, B. subtilis was selected as a model organism for spore-forming bacteria [ 54 ]. Another foodborne pathogen, Listeria monocytogenes , causes listeriosis, a severe human disease with a high mortality - greater than 20% [ 55 ]. Due to biosafety considerations associated with the pathogenic nature of L. monocytogenes , L. innocua was used as a non-pathogenic model system. In Gram-negative bacteria, the presence of an outer membrane prevents nisin from binding to lipid II, thereby substantially reducing its antimicrobial activity. Thus, assessment of nisin inhibitory activity against Gram-negative bacteria requires additional outer membrane permeabilization [ 56 , 57 ]. As Gram-negative indicators, two bacterial species associated with foodborne gastrointestinal illnesses, E. coli and S. typhimurium , were selected [ 58 , 59 ]. The highest to lowest antibacterial activity of nisin-loaded yeast virus-like particles against Gram-positive bacteria was observed for S. pyogenes , S. aureus , B. subtilis , and L. innocua , respectively. Similar tendencies have been reported in previous studies. For example, MIC values of nisin, alone or in combination with natural organic compounds, were higher for L. innocua than for B. subtilis [ 21 , 60 ]. Wider inhibition zones were observed against S. aureus compared to B. subtilis when treated with citric acid-assisted nisin [ 61 ]. For nisin-loaded pectin particles, inhibition activity against S. pyogenes and S. aureus was reported to be similar [ 62 ]. Consistent with these observations, the lowest MIC of free nisin in the present study was detected for S. pyogenes , whereas the highest MIC was observed with L. innocua , indicating that S. pyogenes was the most sensitive and L. innocua the least sensitive to nisin. Compared to free nisin, encapsulation decreased antibacterial activity against all tested microbial strains across the three nisin-loaded nanoparticle types. However, the trend in antibacterial activity against the bacteria studied was similar for encapsulated and free nisin. Both cell survival results and MIC analysis confirmed that nisin-loaded VLPs have the greatest inhibitory impact on S. pyogenes . Encapsulated nisin antibacterial activity varies depending on VLP type. Based on the survival assay, the efficiency of nisin encapsulated in Y-L-BC was slightly higher against the majority of tested Gram-positive bacteria, but the MIC analysis demonstrated slightly greater activity of Y-L-A encapsulated nisin compared to other types of VLPs. These differences could be attributed to differences in testing methods: antibacterial activity was analyzed by direct survival assay, while MIC values were determined by a broth microdilution assay. As expected, nisin antibacterial activity against Gram-negative bacteria E. coli and S. typhimurium was much lower than against Gram-positive bacteria. This may be due to a barrier formed by the outer membrane, which prevents nisin from reaching receptors in the cytoplasmic membrane [ 63 ]. The lower antibacterial activity and increased MIC values observed for nisin-loaded nanoparticles may be associated with the encapsulation process. Encapsulation can protect the peptide from environmental exposure while slowing its release rate, thereby reducing its immediate availability and prolonging its action [ 64 , 65 ]. Nevertheless, all three types of nisin-loaded yeast virus–like particles retained antibacterial activity, indicating that they represent a suitable system for nisin nanodelivery. Recent studies indicate that, in addition to its antibacterial activity, nisin also bears potent anticancer properties, including induction of apoptosis, cell cycle arrest, and inhibition of cell proliferation [ 66 ]. It has been demonstrated that nisin cytotoxicity is confined by active concentration and the type of cancer cell line [ 45 ]. In the present study, the cytotoxic effects of free nisin and nisin-loaded yeast virus-like particles were evaluated using the A549 human lung carcinoma epithelial cell line, employed as a mammalian cancer cell model. Cytotoxicity analysis demonstrated that after 24 hours of incubation with free nisin, A549 cell viability decreased. Encapsulation of nisin resulted in up to 48-hour lasting alleviation of cytotoxicity for the nisin-loaded Y-L-BC and B-L-BC particles compared with free nisin, while a decrease in viability was recovered by 48 hours for nisin-loaded B-L-BC, and 72 hours for nisin-loaded Y-L-BC particles. In contrast, both empty and nisin-loaded Y-L-A VLPs exhibited a more profound cytotoxic effect on A549 cells during all time points. Similar results, with free nisin showing higher cytotoxicity against A549 cells than encapsulated nisin, were reported for nisin-loaded cyclodextrin-based nanosponges [ 24 ]. Along with reduced antibacterial activity, the delay in nisin cytotoxicity is expected to be driven by encapsulation, which deters the release and thereby reduces the immediate availability of nisin. Overall, these results suggest that yeast Y-L-BC and B-L-BC VLPs could serve as a safe and predictable platform for nisin encapsulation in mammalian cell applications, whereas Y-L-A should be considered for greater innate cytotoxicity. Importantly, the use of virus-like particles originating from yeast S. cerevisiae , generally recognised as safe (GRAS) by the Food and Drug Administration (FDA) in the USA and having qualified presumption of safety (QPS) status in Europe [ 67 ], further supports their potential safety and suitability for applications where biocompatibility and regulatory acceptance are essential. Conclusions A passive diffusion method enabled the encapsulation of the antimicrobial peptide nisin Z into three types of yeast virus–derived nanoparticles: Y-L-A, Y-L-BC, and B-L-BC. This approach achieved high encapsulation efficiency while preserving particle size and morphology, indicating the formation of a well-controlled and homogeneous nanocarrier system. Encapsulated nisin retained antibacterial activity against Gram-positive foodborne and clinically relevant bacteria, while the observed increase in MIC values is consistent with sustained release of the peptide from the nanoparticle interior. Encapsulation modulated nisin's release in a time-constrained manner while retaining its cytotoxicity, a critical consideration for potential biomedical applications of nisin-loaded nanoparticles. The previously demonstrated long-term stability of yeast virus–like particles under various conditions, along with their proteinaceous nature and origin, further supports their biocompatibility and potential for safe use in mammalian cell cultures. Overall, these findings highlight nisin-loaded yeast virus–like particles as a promising platform for the nanodelivery and controlled release of nisin in future biomedical applications. Declarations Acknowledgements Dr. Tatjana Kavleiskaja is acknowledged for introducing zeta potential measurements. Author contributions EC: Formal Analysis, Investigation, Methodology, Visualization, Writing–original draft. RS: Formal Analysis, Investigation, Visualization, Writing–review and editing. KV: Investigation. AM: Investigation. ES: Conceptualization, Data curation, Formal Analysis, Supervision, Validation, Visualization, Writing–review and editing. SS: Conceptualization, Data curation, Funding acquisition, Methodology, Project administration, Resources, Supervision, Validation, Writing–original draft, Writing–review and editing. Funding This work was supported by the Research Council of Lithuania (LMTLT), agreement [S-MIP-23-28]. Data availability Data is available upon request from the corresponding author. Ethics declarations No ethics declarations are required for this study. Consent for publication Not applicable. Competing interests The authors declare no competing interests. References Holmes AH, Moore LS, Sundsfjord A, Steinbakk M, Regmi S, Karkey A, Guerin PJ, Piddock LJV. 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Apoptotic and anti-inflammatory effect of nisin-loaded sodium alginate-gum arabic nanoparticles against colon cancer cells. Int J Biol Macromol. 2025;305(Pt 2):141747. Gronchi N, De Bernardini N, Cripwell RA, Treu L, Campanaro S, Basaglia M, Foulquié-Moreno MR, Thevelein JM, Van Zyl WH, Favaro L, Casella S. Natural Saccharomyces cerevisiae Strain Reveals Peculiar Genomic Traits for Starch-to-Bioethanol Production: the Design of an Amylolytic Consolidated Bioprocessing Yeast. Front Microbiol. 2022;12:768562. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8842944","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":591412325,"identity":"69a924e4-310c-4048-b222-81bcb6e71159","order_by":0,"name":"Enrika Celitan","email":"","orcid":"","institution":"Vilnius University","correspondingAuthor":false,"prefix":"","firstName":"Enrika","middleName":"","lastName":"Celitan","suffix":""},{"id":591412326,"identity":"22bcd15a-569d-4c07-b29e-7fb4ebcc538f","order_by":1,"name":"Ramunė Stanevičienė","email":"","orcid":"","institution":"Nature Research Centre","correspondingAuthor":false,"prefix":"","firstName":"Ramunė","middleName":"","lastName":"Stanevičienė","suffix":""},{"id":591412327,"identity":"6519cfab-708d-4555-8ef3-fd41076e3df3","order_by":2,"name":"Kamilė Vaišaitė","email":"","orcid":"","institution":"Vilnius University","correspondingAuthor":false,"prefix":"","firstName":"Kamilė","middleName":"","lastName":"Vaišaitė","suffix":""},{"id":591412328,"identity":"0a945988-251e-4da8-8988-adaa6c91f554","order_by":3,"name":"Algirdas Mikalkėnas","email":"","orcid":"","institution":"Vilnius University","correspondingAuthor":false,"prefix":"","firstName":"Algirdas","middleName":"","lastName":"Mikalkėnas","suffix":""},{"id":591412329,"identity":"b26b4cef-658c-4233-8d99-7fff11ac8af3","order_by":4,"name":"Elena Servienė","email":"","orcid":"","institution":"Nature Research Centre","correspondingAuthor":false,"prefix":"","firstName":"Elena","middleName":"","lastName":"Servienė","suffix":""},{"id":591412330,"identity":"85e6cba5-1a97-4b23-9911-8fbae35ad249","order_by":5,"name":"Saulius Serva","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6ElEQVRIiWNgGAWjYHACxgMJcHYFAz+YfkBAD5KWMwySDSA6IQG7UrgWhIVtRGiRbz9jcOBhWx2Q0ftM4ue8WgkGiRwDhsQfuLUYnMkxOJDYdhjIOG4m2bvtOEQLPlsMGMBaDjAYSKQxG/BuO1bHwHMGvxb5/jcgLUCHzX/GbPh3zjEJgloYboBtYQYy2Bgf8zbUSDCw9xBw2I1nBQcSzh3mMTiTxvhY5tgBCTb2NqBIGj6HJW98+KOsTk6+/RjDwTc1dRL8zMwbH3ywweMwKOCB0ocZ2BiQY4oIUEeK4lEwCkbBKBghAADAzk+XjmP5JAAAAABJRU5ErkJggg==","orcid":"","institution":"Vilnius University","correspondingAuthor":true,"prefix":"","firstName":"Saulius","middleName":"","lastName":"Serva","suffix":""}],"badges":[],"createdAt":"2026-02-10 15:56:46","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-8842944/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8842944/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":102938851,"identity":"5fab01cc-ecdd-4ae6-b519-c06a61fabdea","added_by":"auto","created_at":"2026-02-18 16:49:49","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":807220,"visible":true,"origin":"","legend":"\u003cp\u003eTEM micrographs of empty and nisin-loaded VLPs.\u003cstrong\u003e \u003c/strong\u003e(A) Empty Y-L-A VLPs, (B) empty B-L-BC VLPs, (C) empty Y-L-BC VLPs, and (D) nisin-loaded Y-L-BC VLPs. Scale-bar size: 200 nm.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8842944/v1/ca8b8574af43d077815925a7.png"},{"id":102964295,"identity":"ee7b0dfd-4cb7-4488-a17a-d1249bf62c44","added_by":"auto","created_at":"2026-02-19 04:22:01","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":212615,"visible":true,"origin":"","legend":"\u003cp\u003eViability of S. pyogenes (A), S. aureus (B), B. subtilis (C), L. innocua (D), E. coli (E), and S. typhimurium (F) bacteria after incubation with free nisin (Nisin\u003csub\u003eC\u003c/sub\u003e) or nisin-loaded VLPs (VLP:nisin).\u003cstrong\u003e \u003c/strong\u003eResults are expressed as log\u003csub\u003e10\u003c/sub\u003e(CFU\u003csub\u003eT\u003c/sub\u003e/CFU\u003csub\u003eC\u003c/sub\u003e), where CFU\u003csub\u003eT\u003c/sub\u003e is the value of the nisin-treated cells, and CFU\u003csub\u003eC\u003c/sub\u003e is the value of the negative control (cells treated with empty VLPs). Data are presented as mean (n=3) with standard deviation (±SD). Statistical significance between samples prepared at different encapsulation ratios marked above the columns as follows: p≤0.05(*), p≤0.01(**), p≤0.001(***), p≤0.0001(****).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8842944/v1/ce814bb17a865fb0b45024a4.png"},{"id":102938849,"identity":"a533c68e-1d60-455d-8e08-2cdc5614a2c9","added_by":"auto","created_at":"2026-02-18 16:49:49","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":37817,"visible":true,"origin":"","legend":"\u003cp\u003eRelative viability of lung carcinoma A549 line cells.\u003cstrong\u003e \u003c/strong\u003eCells were treated for 24, 48 and 72 hours with 100 pM of empty or nisin-loaded Y-L-A, Y-L-BC or B-L-BC VLPs prepared at 1:1000 ratio. Cells treated with sample buffer were used as the main controls. Results are presented as mean of four independent replicates with standard deviation (±SD). Statistical significance compared to buffer-treated control marked above the columns as follows: p≤0.05(*), p≤0.01(**), p≤0.001(***), p≤0.0001(****).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8842944/v1/378154e4304bc26309ba6b2a.png"},{"id":104399698,"identity":"df1b3a41-9301-471a-84d5-38b4aa526c77","added_by":"auto","created_at":"2026-03-11 12:07:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1993286,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8842944/v1/1c6adee8-e2fb-4113-9125-d6673b71eabf.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Nanodelivery of Nisin by Homogeneous Protein-based Particles","fulltext":[{"header":"Background","content":"\u003cp\u003eAntimicrobial resistance has emerged as a significant global public health threat, primarily driven by the extensive and inappropriate use of antibiotics in food production, agriculture, and both human and veterinary medicine [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. As antibiotic consumption continues to rise worldwide, it highlights the need for alternative antimicrobial strategies. Among the approaches being explored, antimicrobial peptides, such as bacteriocins, have attracted attention for their broad-spectrum antimicrobial activity [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. However, the practical use of such peptides is often limited by susceptibility to environmental conditions and low \u003cem\u003ein vivo\u003c/em\u003e stability [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Encapsulation has therefore emerged as a promising strategy to protect bacteriocins from degradation, increase their stability, and enable controlled release [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNisin is the most extensively studied bacteriocin belonging to the Type A(I) lantibiotics, produced by Gram-positive bacteria such as \u003cem\u003eLactococcus lactis\u003c/em\u003e and some \u003cem\u003eStreptococcus\u003c/em\u003e species [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. It consists of 34 amino acids and has an isoelectric point above 8.5, making it cationic at neutral pH, an important characteristic for its application [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. This antimicrobial peptide exhibits broad-spectrum inhibitory activity against Gram-positive bacteria by disrupting cell wall biosynthesis and forming pores in the cytoplasmic membrane [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Nisin is approved by the United States Food and Drug Administration, which granted it Generally Recognized as Safe (GRAS) status [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDue to its potency, natural origin, and established safe use as a food preservative, nisin has attracted considerable interest for applications in medicine and biotechnology. Studies show that it modulates oral and intestinal microbiomes, inhibits infection-associated bacteria, and exhibits anticancer activity [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Food-grade nisin has demonstrated antimicrobial activity against both Gram-positive and Gram-negative oral bacteria, reducing bacterial biofilm biomass and thickness without affecting human oral cell viability [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. In combination with oxacillin, nisin effectively and synergistically inhibited the growth of methicillin-resistant \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (MRSA) - a pathogen responsible for skin and soft tissue infections - both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e, and significantly reduced biofilm formation [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Several studies also showed that nisin is cytotoxic to various human cancer cells. For instance, it triggers apoptosis in colorectal adenocarcinoma cells (HT29) via Caspase 7 activation and p53 gene induction, leading to G0/G1 cell cycle arrest [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Nisin has also been shown to induce selective apoptosis and cell cycle arrest in human lung carcinoma cells (A549 and H1299), compared to healthy human embryonic kidney cells (HEK293), through mitochondrial dysfunction and the generation of reactive oxygen species [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Despite its potent antimicrobial and anticancer properties, the application of nisin remains challenging due to its strong pH dependence. While nisin is stable and soluble under acidic conditions, it undergoes rapid degradation and loss of activity at neutral or alkaline pH [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Encapsulation of nisin can therefore overcome these limitations by protecting it from both enzymatic degradation and unfavorable physicochemical conditions, thereby enhancing its stability and antimicrobial efficacy [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eVarious nisin encapsulation strategies have been explored, including microencapsulation by calcium alginate and guar gum [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]; liposomes [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]; lipid nanoparticles [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]; sulphated polysaccharides ulvan and fucoidan [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]; PLGA polymer [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]; egg white protein nanoparticles [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]; cyclodextrin-based nanosponges [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Despite extensive research on bacteriocin encapsulation, existing scaffolds - polymeric and lipid nanoparticles, liposomes, and polysaccharide-based carriers - face limitations such as low encapsulation efficiency, heterogeneous particle size, instability, and cytotoxicity [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Therefore, there is a profound need for stable and biocompatible nanocarriers capable of efficient encapsulation of bacteriocins while preserving their biological activity. Protein-based nanoparticles, such as virus-like particles, have gained increasing attention due to their homogenous size, biocompatibility, broad modification potential, and ability to encapsulate, deliver, and control the release of various biomolecules [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOur previous study demonstrated that nanoparticles derived from \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e L-BC virus can serve as an efficient encapsulation system for nisin due to electrostatic interaction between the positively charged nisin and the negatively charged particle interior [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. This study has confirmed that L-BC virus-like particles (VLPs) are a promising delivery system due to the nanoparticle long-term stability under various conditions, resistance to harsh environments, and high encapsulation potential. In the present study, we expanded this approach by preparing three types of nisin-loaded yeast virus-like nanoparticles, evaluating their physicochemical properties and encapsulation efficiency using various analytical methods. To assess the potential applications, the antibacterial activity of the prepared nanoparticles was evaluated against six bacterial strains, and cytotoxicity was assessed in human lung carcinoma cells. We found that all three VLP-based systems efficiently encapsulate nisin, retain its antibacterial activity, and modulate its cytotoxicity, depending on the particle type, thus manifesting their potential as stable, biocompatible nanocarriers for controlled nisin delivery.\u003c/p\u003e"},{"header":"Methods and materials","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eBacterial strains and culture conditions\u003c/h2\u003e \u003cp\u003eGram-positive bacteria \u003cem\u003eBacillus subtilis\u003c/em\u003e ATCC 6633, \u003cem\u003eStaphylococcus aureus\u003c/em\u003e ATCC 29213, \u003cem\u003eStreptococcus pyogenes\u003c/em\u003e ATCC 19615 (Vilnius University, Lithuania), and \u003cem\u003eListeria innocua\u003c/em\u003e CECT 910T (University of Lisbon, Portugal), as well as Gram-negative bacteria \u003cem\u003eEscherichia coli\u003c/em\u003e BL21 [F⁻ dcm ompT hsdS(rB⁻ mB⁻) gal λ(DE3)] (ThermoFisher Scientific, Vilnius, Lithuania) and \u003cem\u003eSalmonella typhimurium\u003c/em\u003e LT2 (pyrF146) (Vilnius Gediminas Technical University, Lithuania), were used in this study.\u003c/p\u003e \u003cp\u003e \u003cem\u003eB. subtilis\u003c/em\u003e, \u003cem\u003eS. aureus\u003c/em\u003e, \u003cem\u003eE. coli\u003c/em\u003e, and \u003cem\u003eS. typhimurium\u003c/em\u003e cells were propagated in Luria\u0026ndash;Bertani (LB) medium (1% peptone, 0.5% yeast extract, 1% NaCl), whereas \u003cem\u003eS. pyogenes\u003c/em\u003e and \u003cem\u003eL. innocua\u003c/em\u003e were cultured in Tryptic Soy Agar medium (1.7% peptone from casein, 0.3% peptone from soymeal, 0.25% glucose, 0.5% NaCl, 0.25% K₂HPO₄, 2% agar; pH 7.3). All bacterial cultures were incubated for 16\u0026ndash;18 h at 37\u0026deg;C with vigorous shaking.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePreparation of nisin-loaded virus-like particles\u003c/h3\u003e\n\u003cp\u003eVLPs derived from \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e L-A and L-BC viruses were generated as previously described [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Briefly, for bacterial expression of the L-BC virus capsid protein Gag, the \u003cem\u003eGAG\u003c/em\u003e gene was cloned into the inducible vector pET28a [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. For yeast expression of L-A and L-BC Gag proteins, the inducible vector pFX7 [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] was used. VLPs were produced in \u003cem\u003eEscherichia coli\u003c/em\u003e BL21-AI (Invitrogen) and \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e BY4741 [L-A⁻L-BC⁻] (generated in our laboratory, unpublished) strains and purified by ultracentrifugation through a sucrose cushion followed by cesium chloride gradient centrifugation. Buffer exchange of purified VLP samples was performed using 7K MWCO Zeba\u0026trade; Spin Desalting Columns (Thermo Fisher Scientific, Lithuania) according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003cp\u003eEncapsulation experiments were performed by using 220 \u0026micro;M, 22 \u0026micro;M, and 2.2 \u0026micro;M nisin Z (Handary, Belgium) aqueous solutions and 0.22 \u0026micro;M VLP solution in 2\u0026times; citrate\u0026ndash;phosphate buffer (pH 6) containing 300 mM NaCl and 20 mM EDTA. Equal volumes of VLP suspension and nisin solutions were combined to obtain VLP:nisin mixtures with molar ratios of 1:1000, 1:100, and 1:10. Prepared mixtures were incubated at 4\u0026deg;C for 16 h with gentle agitation to allow passive diffusion of nisin into the VLPs. Non-encapsulated nisin was removed by ultrafiltration using 100 kDa MWCO Pierce\u0026trade; Protein Concentrators (Thermo Fisher Scientific, Lithuania), according to the manufacturer\u0026rsquo;s instructions. Retentates were washed once with 500 \u0026micro;l of 1\u0026times; citrate\u0026ndash;phosphate buffer (pH 6) containing 150 mM NaCl and 10 mM EDTA, and volumes were adjusted back to the initial with the same buffer. For negative control, equal volumes of 0.22 \u0026micro;M VLP solution in 2\u0026times; citrate\u0026ndash;phosphate buffer (pH 6) containing 300 mM NaCl and 20 mM EDTA and sterile water were combined. For positive controls, equal volumes of 220 \u0026micro;M, 22 \u0026micro;M, and 2.2 \u0026micro;M nisin Z aqueous solutions were combined with 2\u0026times; citrate\u0026ndash;phosphate buffer (pH 6) containing 300 mM NaCl and 20 mM EDTA. All final samples were prepared in 1\u0026times; citrate\u0026ndash;phosphate buffer (pH 6) containing 150 mM NaCl and 10 mM EDTA.\u003c/p\u003e\n\u003ch3\u003eCharacterization of nisin-loaded virus-like particles\u003c/h3\u003e\n\u003cp\u003eParticle size and zeta potential were determined using the Zetasizer NanoZS instrument (Malvern Panalytical, UK) equipped with a 4 mW HeNe laser (633 nm). The intensity of the scattered light was measured at 22\u0026deg;C with a fixed angle of 173\u0026deg;. The data was analyzed by Malvern Zetasizer v8.02 software. The morphology of the particles was evaluated by transmission electron microscope (TEM) Talos\u0026trade; L120C (Thermo Fisher Scientific, USA). Particles were applied on carbon-coated copper grids (Agar Scientific, UK) and stained with 2% uranyl acetate.\u003c/p\u003e\n\u003ch3\u003eEncapsulation efficiency\u003c/h3\u003e\n\u003cp\u003eAfter ultrafiltration, the amount of non-encapsulated nisin in filtrates was determined by Bradford protein assay. 50 \u0026micro;l of each sample was mixed with 200 \u0026micro;l of Bradford reagent in 96-well plates, and absorbance was measured at 595 nm using an Infinite M Nano (Tecan, Switzerland) instrument. Nisin concentration was calculated from a standard calibration curve prepared with known nisin concentrations. The encapsulation efficiency (EE) was calculated according to the following formula:\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:EE\\left(\\%\\right)=\\frac{(Total\\:amount\\:of\\:nisin-Non\\text{-}encapsulated\\:nisin)}{Total\\:amount\\:of\\:nisin}\\times\\:100$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eAll measurements were performed in triplicate, and results are presented as the mean with standard deviation (\u0026plusmn;\u0026thinsp;SD).\u003c/p\u003e\n\u003ch3\u003eEvaluation of antibacterial activity of free and encapsulated nisin\u003c/h3\u003e\n\u003cp\u003eThe antibacterial activity of free and encapsulated nisin was evaluated using a direct survival assay. Overnight bacterial cultures were harvested by centrifugation at 6000\u0026times;g for 5 min, washed twice with 1 mol/L sorbitol, and resuspended in 1 mol/L sorbitol at a final concentration of 1 OD\u003csub\u003e600\u003c/sub\u003e. For Gram-positive bacteria, 50 \u0026micro;l of the cell suspension was mixed with either an equal volume of buffer, nisin-loaded particles (Y-L-A, Y-L-BC, and B-L-BC), particles without nisin, or free nisin samples. For Gram-negative bacteria, the ratio of bacterial suspension and VLPs or nisin samples was adjusted to 1:3 (v/v). Samples were incubated at room temperature (22\u0026deg;C) for 24 h. Serial dilutions were performed in sterile 0.9% NaCl, and 50 \u0026micro;l of each solution was spread onto LB (\u003cem\u003eB. subtilis\u003c/em\u003e, \u003cem\u003eS. aureus\u003c/em\u003e, \u003cem\u003eE. coli\u003c/em\u003e, and \u003cem\u003eS. typhimurium\u003c/em\u003e) or Tryptic soy agar plates (\u003cem\u003eS. pyogenes\u003c/em\u003e and \u003cem\u003eL. innocua\u003c/em\u003e). Plates were incubated overnight at 37\u0026deg;C, and colonies were counted as colony-forming units (CFU). Results are presented as log\u003csub\u003e10\u003c/sub\u003e(CFU\u003csub\u003eT\u003c/sub\u003e/CFU\u003csub\u003eC\u003c/sub\u003e), where CFU\u003csub\u003eT\u003c/sub\u003e is the CFU value of the cells treated with free nisin or nisin-loaded particles, and CFU\u003csub\u003eC\u003c/sub\u003e is the CFU value of the cells treated with buffer or empty VLPs, respectively. All experiments were performed with at least 3 biological replicates, and results are presented as the mean values with standard deviation (\u0026plusmn;\u0026thinsp;SD).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of minimum inhibitory concentrations\u003c/h2\u003e \u003cp\u003eThe minimum inhibitory concentration (MIC) of encapsulated nisin (in Y-L-A, Y-L-BC, and B-L-BC VLPs) and free nisin was determined using a broth microdilution assay. Fifty microliters of diluted overnight bacterial cultures (0.01 OD\u003csub\u003e600\u003c/sub\u003e) were added to wells of a 96-well microplate. Nisin-loaded VLPs and free nisin samples were serially diluted with sterile water and mixed with Gram-positive bacteria in a ratio of 1:1 (v/v). For Gram-negative bacteria, 25 \u0026micro;l of bacterial suspension was combined with 75 \u0026micro;l of diluted sample. Wells containing only medium served as negative controls, and wells containing medium with bacteria were used as positive controls. Plates were covered and incubated at 37\u0026deg;C for 24 h. At the end of the incubation, turbidity was visually examined, and OD\u003csub\u003e600\u003c/sub\u003e values were measured using an Infinite M PLEX microplate reader (Tecan, Austria) operated with Tecan i-Control software (version 3.9.1.0). MIC was defined as the lowest nisin concentration that fully inhibited bacterial growth.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCytotoxicity\u003c/h3\u003e\n\u003cp\u003eThe cytotoxicity of the samples was tested in A549 lung carcinoma epithelial cells using the MTT assay. A549 cells were cultured in DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin (Thermo Fisher Scientific, Lithuania). For the assay, 5\u0026times;10\u003csup\u003e3\u003c/sup\u003e (plate 1), 2.3\u0026times;10\u003csup\u003e3\u003c/sup\u003e (plate 2), and 1.5\u0026times;10\u003csup\u003e3\u003c/sup\u003e (plate 3) cells per well were seeded in 96-well plates and incubated for 16\u0026ndash;20 h at 37\u0026deg;C in a humidified CO\u003csub\u003e2\u003c/sub\u003e incubator (95% humidity, 5% CO\u003csub\u003e2\u003c/sub\u003e). After incubation, the culture medium was replaced with fresh medium containing either buffer (control sample), free nisin, nisin-loaded VLPs, or empty VLPs, and the plates were incubated for 24, 48, and 72 h for plates 1, 2, and 3, respectively. Then, cells were washed twice with 150 \u0026micro;l of dPBS (0.9 mM CaCl\u003csub\u003e2\u003c/sub\u003e, 0.5 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e, 1.1 mM KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, pH 7.4). After washing, dPBS was removed, and 100 \u0026micro;l of culture medium containing 0.5 mg/ml MTT reagent (Merck, Germany) was added to each well. Plates were incubated for 4 h under the same conditions. After incubation, the medium was removed, 100 \u0026micro;l of isopropanol was added to each well, and the plates were incubated for 10 min at room temperature. The absorbance was measured at 565 nm on a Infinite M Nano microplate reader (Tecan, Switzerland). Background absorbance was subtracted from the sample and control values, and relative cell viability was calculated according to the following formula:\u003cdiv id=\"Equb\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equb\" name=\"EquationSource\"\u003e\n$$\\:Relative\\:cell\\:viability\\left(\\%\\right)=\\frac{Optical\\:density\\:at\\:565\\:nm\\:of\\:the\\:sample}{Optical\\:density\\:at\\:565\\:nm\\:of\\:the\\:control}\\times\\:100$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eA total of 4 biological replicates were performed, and results are presented as the mean with standard deviation (\u0026plusmn;\u0026thinsp;SD).\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical differences among groups were obtained by one-way ANOVA followed by Tukey\u0026rsquo;s multiple pairwise comparison test, with statistical significance marked as follows: p\u0026thinsp;\u0026le;\u0026thinsp;0.05(*), p\u0026thinsp;\u0026le;\u0026thinsp;0.01(**), p\u0026thinsp;\u0026le;\u0026thinsp;0.001(***), p\u0026thinsp;\u0026le;\u0026thinsp;0.0001(****). Statistical analysis was performed using GraphPad Prism v.8.0.1 software.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eCharacterization of nisin-loaded virus-like particles\u003c/h2\u003e \u003cp\u003eIn previous work, we demonstrated that the antimicrobial peptide nisin Z can be successfully encapsulated into VLPs derived from \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e virus L-BC, using a passive-diffusion method [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. In this study, we broadened the VLP repertoire by including nanoparticles derived from the \u003cem\u003eS. cerevisiae\u003c/em\u003e L-A virus, which frequently coexists with the L-BC virus in yeast cells [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Altogether, three types of VLPs were examined: L-A VLPs purified from yeast (Y-L-A), L-BC VLPs purified from yeast (Y-L-BC), and from bacteria (B-L-BC). For encapsulation, three different molar ratios of VLP-to-nisin (1:1000, 1:100, and 1:10) were tested to ensure complete saturation of VLPs by nisin.\u003c/p\u003e \u003cp\u003eFirst, we determined the average particle size and zeta potential of the empty and nisin-loaded VLPs (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Empty Y-L-A VLPs exhibited an average diameter of 42.2\u0026thinsp;\u0026plusmn;\u0026thinsp;4.3 nm and a zeta potential of \u0026minus;\u0026thinsp;23.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 mV. Following nisin encapsulation, no significant change in particle size was observed (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05), regardless of the VLP-to-nisin ratio. In contrast, the zeta potential of Y-L-A particles shifted toward more positive values after encapsulation, reflecting the presence of the cationic nisin peptide. The extent of this shift increased proportionally with the molar VLP-to-nisin ratio, reaching\u0026thinsp;\u0026minus;\u0026thinsp;23.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6 mV for 1:10, \u0026minus;\u0026thinsp;21.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8 for 1:100, and \u0026minus;\u0026thinsp;15.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7 mV for 1:1000 ratio. The sample featuring a 1:1000 ratio displayed a statistically significant change in zeta potential compared to empty particles (p\u0026thinsp;\u0026le;\u0026thinsp;0.001).\u003c/p\u003e \u003cp\u003eA similar pattern was observed for Y-L-BC and B-L-BC nanoparticles, produced in yeast and bacteria, respectively. No statistically significant difference in particle size was detected between empty and nisin-loaded VLPs (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). The size of empty and nisin-loaded Y-L-BC nanoparticles varied between 37.8 and 50.6 nm, while B-L-BC VLPs ranged from 35.0 to 41.6 nm. For Y-L-BC VLPs, after encapsulation, zeta potential increased slightly with higher peptide ratios: \u0026minus;23.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6 mV for 1:10, \u0026minus;\u0026thinsp;23.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5 mV for 1:100, and \u0026minus;\u0026thinsp;19.6\u0026thinsp;\u0026plusmn;\u0026thinsp;2.4 mV for 1:1000. However, none of these values differed significantly (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) from the empty particle control (\u0026minus;\u0026thinsp;21.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2 mV). B-L-BC VLPs observed a similar pattern, with zeta potential of \u0026minus;\u0026thinsp;26.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2.9 mV for 1:10, \u0026minus;\u0026thinsp;26.2\u0026thinsp;\u0026plusmn;\u0026thinsp;3.0 mV for 1:100, and \u0026minus;\u0026thinsp;18.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7 mV for 1:1000, a significant shift observed only at the 1:1000 ratio (p\u0026thinsp;\u0026le;\u0026thinsp;0.001). Together, these results indicate that a higher VLP-to-nisin ratio promotes incorporation of nisin, reflected through changes in zeta potential rather than particle size.\u003c/p\u003e \u003cp\u003eAs shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, for samples prepared at a 1:1000 VLP-to-nisin ratio, the encapsulation efficiency (EE further on) of nisin ranged from 76.8% to 83.1%, with slightly lower efficiency observed for Y-L-BC VLPs compared to the other two particle types. For the 1:100 ratio, EE reached 89.3% and 89.2% for Y-L-BC and B-L-BC VLPs, respectively, whereas Y-L-A VLPs exhibited a slightly lower EE of 77.1%. Encapsulation efficiency could not be determined for the samples prepared at a 1:10 VLP-to-nisin ratio, as encapsulated nisin concentrations were below the detection limit of the Bradford assay (see Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, items marked N/A).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eParticle size, zeta potential, and encapsulation efficiency of empty and nisin-loaded VLPs. Results are presented as mean (n\u0026thinsp;=\u0026thinsp;3) with standard deviation (\u0026plusmn;\u0026thinsp;SD). N/A \u0026ndash; not available.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eMolar conc. before filtration, \u0026micro;M\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eMolar \u003c/p\u003e \u003cp\u003eVLP-to-nisin ratio\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eHydrodynamic diameter, nm\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eZeta potential, mV\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eEE, %\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVLPs\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNisin\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"7\" nameend=\"c7\" namest=\"c1\"\u003e \u003cp\u003eExperiment with Y-L-A VLPs\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eY-L-A VLPs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e42.2\u0026thinsp;\u0026plusmn;\u0026thinsp;4.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-23.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eNisin-loaded Y-L-A VLPs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e0.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e110\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1:1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e43.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-15.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e83.1\u0026thinsp;\u0026plusmn;\u0026thinsp;3.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1:100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e44.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-21.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e77.1\u0026thinsp;\u0026plusmn;\u0026thinsp;6.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1:10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e42.0\u0026thinsp;\u0026plusmn;\u0026thinsp;2.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-23.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"7\" nameend=\"c7\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eExperiment with Y-L-BC VLPs\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eY-L-BC VLPs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e50.6\u0026thinsp;\u0026plusmn;\u0026thinsp;7.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-21.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eNisin-loaded Y-L-BC VLPs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e0.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e110\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1:1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e37.8\u0026thinsp;\u0026plusmn;\u0026thinsp;2.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-19.6\u0026thinsp;\u0026plusmn;\u0026thinsp;2.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e76.8\u0026thinsp;\u0026plusmn;\u0026thinsp;6.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1:100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e48.8\u0026thinsp;\u0026plusmn;\u0026thinsp;6.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-23.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e89.3\u0026thinsp;\u0026plusmn;\u0026thinsp;7.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1:10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e38.5\u0026thinsp;\u0026plusmn;\u0026thinsp;3.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-23.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"7\" nameend=\"c7\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eExperiment with B-L-BC VLPs\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eB-L-BC VLPs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e35.7\u0026thinsp;\u0026plusmn;\u0026thinsp;5.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-27.2\u0026thinsp;\u0026plusmn;\u0026thinsp;2.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eNisin-loaded B-L-BC VLPs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e0.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e110\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1:1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e41.6\u0026thinsp;\u0026plusmn;\u0026thinsp;6.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-18.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e81.9\u0026thinsp;\u0026plusmn;\u0026thinsp;8.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1:100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e35.0\u0026thinsp;\u0026plusmn;\u0026thinsp;7.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-26.2\u0026thinsp;\u0026plusmn;\u0026thinsp;3.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e89.2\u0026thinsp;\u0026plusmn;\u0026thinsp;6.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1:10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e38.8\u0026thinsp;\u0026plusmn;\u0026thinsp;4.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-26.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eTo visualize and evaluate potential morphological changes in VLPs, TEM was involved. Symmetrical spherical particles with an average size of 39 nm were observed in micrographs of Y-L-A VLPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Similarly, particles with average sizes of 40 nm and 43 nm were identified in micrographs of empty B-L-BC (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) and Y-L-BC (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC) samples, respectively. These sizes were comparable to the average particle size of 42 nm observed for nisin-loaded Y-L-BC VLPs prepared at a 1:1000 VLP-to-nisin encapsulation ratio (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). No visual difference in particle size and morphology was observed between the empty and nisin-loaded VLP samples.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eAntibacterial activity of nisin-loaded virus-like particles\u003c/h2\u003e \u003cp\u003eThe antibacterial activity of free and encapsulated nisin was tested against Gram-positive (\u003cem\u003eS. pyogenes\u003c/em\u003e, \u003cem\u003eS. aureus\u003c/em\u003e, \u003cem\u003eB. subtilis\u003c/em\u003e, and \u003cem\u003eL. innocua\u003c/em\u003e) and Gram-negative (\u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eS. typhimurium\u003c/em\u003e) bacteria. The results are summarized in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Free nisin controls were prepared separately for each experiment corresponding to the three VLP types: Y-L-A, Y-L-BC, and B-L-BC. Significant differences between samples prepared at different encapsulation ratios are indicated in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The results demonstrate that, for all tested bacteria, encapsulated nisin exhibited lower antibacterial activity than free nisin. As expected, the highest antibacterial activity of encapsulated nisin was achieved with the highest 1:1000 molar VLP-to-nisin ratio and was most comparable to that of free nisin.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFor Gram-positive bacteria treated with nisin-loaded VLPs prepared at a 1:1000 encapsulation ratio, the most pronounced antibacterial activity was observed against \u003cem\u003eS. pyogenes\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), resulting in a more than 5 log reduction in cell survival. \u003cem\u003eS. aureus\u003c/em\u003e showed slightly lower sensitivity to encapsulated nisin, with viability reductions ranging from 4.7 to 5.2 log (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). A further decrease in antibacterial efficacy was observed for \u003cem\u003eB. subtilis\u003c/em\u003e, where encapsulated nisin induced a 3.2\u0026ndash;3.9 log reduction of CFUs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). The lowest antibacterial effect of encapsulated nisin among the Gram-positive bacteria tested was detected against \u003cem\u003eL. innocua.\u003c/em\u003e They were less susceptible to the nisin-loaded VLPs treatment, resulting in a 2 log reduction in cell survival (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). The impact of encapsulated nisin on Gram-negative bacteria was limited. Within this group, treatment with encapsulated nisin at the 1:1000 ratio resulted in reductions of 0.37\u0026ndash;0.46 log for \u003cem\u003eE. coli\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE) and 0.85\u0026ndash;1.10 log for \u003cem\u003eS. typhimurium\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003eAmong the three types of nisin-loaded VLPs prepared at a 1:1000 encapsulation ratio, Y-L-BC particles caused the greatest reduction in viability for three of the four tested Gram-positive bacteria (\u003cem\u003eS. pyogenes\u003c/em\u003e, \u003cem\u003eS. aureus\u003c/em\u003e, and \u003cem\u003eB. subtilis\u003c/em\u003e), compared to Y-L-A and B-L-BC particles. In the case of \u003cem\u003eL. innocua\u003c/em\u003e, the efficiency of different nisin-loaded VLP types was similar. At the lower VLP-to-nisin encapsulation ratios (1:100 or 1:10), the inhibitory activity of nisin was comparable among different types of VLPs. The antibacterial activity against Gram-negative bacteria varied by species: the greatest reduction in \u003cem\u003eE. coli\u003c/em\u003e was achieved with nisin-loaded L-BC particles purified both from yeast and bacteria (B-L-BC and Y-L-BC VLPs), whereas the strongest inhibitory effect against \u003cem\u003eS. typhimurium\u003c/em\u003e was observed with nisin-loaded Y-L-A particles. Notably, a statistically significant difference between particle types was observed only for \u003cem\u003eS. aureus\u003c/em\u003e, between nisin-loaded Y-L-BC and B-L-BC particles (p\u0026thinsp;\u0026le;\u0026thinsp;0.01) (not indicated in graphs).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eMinimal inhibitory concentration\u003c/h2\u003e \u003cp\u003eSince the samples prepared at a 1:1000 molar VLP-to-nisin ratio exhibited the highest antibacterial activity across the tested bacterial species, they were selected for MIC evaluation. The MIC values (\u0026micro;g/ml) of free and encapsulated nisin against Gram-positive and Gram-negative bacteria are presented in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMinimal inhibitory concentrations of free and encapsulated nisin.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"6\" nameend=\"c7\" namest=\"c2\"\u003e \u003cp\u003eMIC of nisin, \u0026micro;g/ml\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e \u003cp\u003eGram-positive bacteria\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003eGram-negative bacteria\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eS. pyogenes\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eS. aureus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eB. subtilis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eL. innocua\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cem\u003eS. typhimurium\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFree nisin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e30.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e41.11\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNisin-loaded \u003c/p\u003e \u003cp\u003eY-L-A VLPs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e12.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e96.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e214.81\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNisin-loaded \u003c/p\u003e \u003cp\u003eY-L-BC VLPs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e24.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e120.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e171.85\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNisin-loaded B-L-BC VLPs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e24.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e12.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e96.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e161.11\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eAmong the Gram-positive strains, the MIC of free nisin ranged from 0.60 to 7.71 \u0026micro;g/ml, with \u003cem\u003eS. pyogenes\u003c/em\u003e being the most sensitive and \u003cem\u003eL. innocua\u003c/em\u003e the least sensitive. For VLP-loaded nisin, the MIC values rose 1.2- to 10.1-fold compared to free nisin. The highest increase of MIC was observed for \u003cem\u003eS. pyogenes\u003c/em\u003e, 3.2-, 6.3-, and 10.1-fold for Y-L-A, Y-L-BC, and B-L-BC VLPs, respectively. Despite higher MIC values, this bacterial species proved the most sensitive to encapsulated nisin among the bacteria studied. Overall, nisin-loaded Y-L-A VLPs consistently showed the lower MICs among the Gram-positive bacteria compared with the other two VLP types. B-L-BC VLPs encapsulated with nisin had the strongest effect on B. subtilis. The MIC value was determined to be 3.02 \u0026micro;g/ml. Meanwhile, Y-L-BC-loaded nisin more efficiently inhibits \u003cem\u003eS. pyogenes\u003c/em\u003e and \u003cem\u003eB. subtilis\u003c/em\u003e bacteria. The MIC values reached 3.78 \u0026micro;g/ml.\u003c/p\u003e \u003cp\u003eAs observed previously in the antibacterial activity assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), MIC evaluation also confirmed the low sensitivity of Gram-negative bacteria to nisin (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The MICs of free nisin were 30.83 \u0026micro;g/ml for \u003cem\u003eE. coli\u003c/em\u003e and 41.11 \u0026micro;g/ml for \u003cem\u003eS. typhimurium\u003c/em\u003e. Encapsulation of nisin resulted in a 3.1- to 5.2-fold increase in MICs, with the smallest increase observed for B-L-BC VLPs. Based on MIC values, within the Gram-negative group, E. coli was more sensitive to both free and encapsulated nisin than \u003cem\u003eS. typhimurium\u003c/em\u003e. Overall, our results demonstrate that encapsulation reduces nisin activity to varying degrees, depending on the VLP type and the bacterial strain.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eThe impact of nisin-loaded VLPs on the relative viability of mammalian cells\u003c/h2\u003e \u003cp\u003eOur previous studies demonstrated that the nanoparticles employed in this investigation (Y-L-A, Y-L-BC, and B-L-BC VLPs) efficiently internalize into A549 lung carcinoma epithelial cells within 4 hours in the absence of transfection reagents (unpublished data). Based on these observations, the impact of both empty and nisin-loaded VLPs on A549 cell viability was assessed. Cells were incubated in mammalian culture medium supplemented with 100 pM of na\u0026iuml;ve or nisin-loaded VLPs for 24, 48, and 72 hours, and relative cell viability was evaluated by the MTT assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Cells incubated with buffer-supplemented medium served as controls. To evaluate the impact of nisin encapsulation, free nisin control (Nisin\u003csub\u003eC\u003c/sub\u003e) was also included. The concentration of nisin in the control sample corresponded to the amount of nisin encapsulated in the respective VLPs, as determined by encapsulation efficiency measurements. Since the samples prepared at a 1:1000 molar VLP-to-nisin ratio exhibited the highest antibacterial activity, they were addressed in the viability assessment. Statistical significance was evaluated by comparing results obtained with cells treated with the sample buffer (control) and VLP- or free nisin-treated cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCytotoxicity assessment revealed clear differences between the three types of yeast virus-derived nanoparticles. Empty and nisin-loaded Y-L-BC particles did not significantly affect cell viability at any of the tested time points (24, 48, or 72 hours). Among B-L-BC particles, a modest but significant reduction in viability to 81.5\u0026thinsp;\u0026plusmn;\u0026thinsp;7.14% (p\u0026thinsp;\u0026le;\u0026thinsp;0.05) was observed only for empty B-L-BC particles after 48 hours, whereas nisin-loaded B-L-BC particles did not induce a significant decrease in viability at any time point (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). In contrast, Y-L-A particles exhibited a pronounced cytotoxic effect. After 24 hours of incubation, both empty and nisin-loaded Y-L-A particles significantly reduced cell viability to 81.6\u0026thinsp;\u0026plusmn;\u0026thinsp;4.53% (p\u0026thinsp;\u0026le;\u0026thinsp;0.05) and 77.7\u0026thinsp;\u0026plusmn;\u0026thinsp;8.35% (p\u0026thinsp;\u0026le;\u0026thinsp;0.01), respectively. This effect persisted with prolonged incubation, with empty particles reducing viability to 62.0\u0026thinsp;\u0026plusmn;\u0026thinsp;2.01% (p\u0026thinsp;\u0026le;\u0026thinsp;0.0001) and 77.4\u0026thinsp;\u0026plusmn;\u0026thinsp;4.68% (p\u0026thinsp;\u0026le;\u0026thinsp;0.01) after 48 and 72 hours, respectively, while nisin-loaded Y-L-A particles reduced viability to 67.6\u0026thinsp;\u0026plusmn;\u0026thinsp;11.0% (p\u0026thinsp;\u0026le;\u0026thinsp;0.0001) and 72.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2.31% (p\u0026thinsp;\u0026le;\u0026thinsp;0.0001). Compared to free nisin control, which significantly reduced viability to 80.9\u0026thinsp;\u0026plusmn;\u0026thinsp;9.22% (p\u0026thinsp;\u0026le;\u0026thinsp;0.05) after 72 hours of incubation, nisin encapsulated in Y-L-BC and B-L-BC particles did not significantly alter cell viability (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Overall, these results indicate that Y-L-A particles exert cytotoxicity on A549 cells, independent of nisin loading, whereas encapsulation of nisin in Y-L-BC and B-L-BC nanoparticles effectively reduced nisin cytotoxicity in mammalian cells.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe aim of this study was to develop a VLP-based nanodelivery system for nisin and to evaluate its potential applications. For this purpose, yeast virus\u0026ndash;like particles were selected, as our previous work demonstrated successful encapsulation of nisin into \u003cem\u003eS. cerevisiae\u003c/em\u003e L-BC VLPs [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. In the present study, VLPs derived from the \u003cem\u003eS. cerevisiae\u003c/em\u003e viruses L-A and L-BC were investigated as carriers for nisin encapsulation. Formation of the L-A virus capsid requires N-acetylation of the capsid protein Gag [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]; therefore, L-A VLPs were produced using a \u003cem\u003eS. cerevisiae\u003c/em\u003e expression system. Since N-acetylation of the L-BC Gag protein was found not essential [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], L-BC VLPs were produced in both \u003cem\u003eS. cerevisiae and E. coli\u003c/em\u003e expression systems.\u003c/p\u003e \u003cp\u003eThe interiors of fungal dsRNA virus capsids are predominantly negatively charged [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], making them well-suited for encapsulating positively charged molecules such as nisin. Electrostatic interactions between the oppositely charged peptide and the VLP interior can enhance encapsulation efficiency [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Nisin encapsulation was achieved using a passive diffusion approach, facilitated by the small molecular size of nisin Z (~\u0026thinsp;3 kDa) [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], which allows it to pass through pores located at the fivefold and threefold symmetry axes of the L-BC and L-A capsids [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Nisin Z was selected due to better solubility under neutral pH conditions (above pH 5) compared to nisin A [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], while L-BC VLPs were previously shown to maintain long term stability within the pH range of 6\u0026ndash;8 [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. As reported in previous encapsulation studies [\u003cspan additionalcitationids=\"CR39 CR40\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], encapsulation is commonly performed using a vast excess of cargo; therefore, several VLPs-to-nisin ratios were involved (1:1000, 1:100, and 1:10). After encapsulation, non-encapsulated nisin was filtered from the nisin-loaded VLPs by the ultrafiltration, as employed in other studies on nisin encapsulation [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFor the characterization of naive and nisin-loaded VLPs, average particle size and zeta potential were addressed by a combination of electrophoretic mobility, dynamic light scattering (DLS), and transmission electron microscopy (TEM). DLS analysis demonstrated that the size of empty and nisin-loaded VLPs ranged from 35.0 to 50.6 nm, with no significant changes in particle size following the nisin encapsulation, regardless of VLP type (Y-L-A, Y-L-BC, or B-L-BC), or VLP-to-nisin encapsulation ratio (1:1000, 1:100, or 1:10) (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Consistently, TEM revealed neither morphological nor size difference between the empty and nisin-loaded VLPs. The absence of changes in particle size and morphology after loading suggests that cargo is incorporated within the interior of the VLPs rather than being absorbed onto their surface. At the same time, the initially negative zeta potential of VLPs shifted toward more positive values after encapsulation of the cationic peptide nisin, in a manner proportional to the VLP-to-nisin ratio, with the largest change observed for the 1:1000 samples. However, statistically significant changes in zeta potential were detected only for Y-L-A and B-L-BC VLPs. Similar shifts toward more positive zeta potential values have been reported in previous studies of nisin-loaded nanoparticles [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. The value of zeta potential reflects the degree of electrostatic repulsion between adjacent, similarly charged particles in a dispersion and, therefore, is an indicator of colloidal stability. High zeta potential values (\u0026plusmn;\u0026thinsp;30 mV) are associated with increased stability, as particles tend to resist aggregation [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. The zeta potential values of nisin-loaded nanoparticles prepared with the greatest nisin content (-15.2 to -19.6 mV) suggest notable electrostatic stabilization of the particles. Together with the preserved particle size and morphology, these results indicate good colloidal stability of the system, in line with previous observations of robust long-term stability of L-BC capsids [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Of note, the combination of a negative zeta potential and the small size of nisin-loaded VLPs (\u0026lt;\u0026thinsp;100 nm) is generally considered favorable for intravenously administered nanoparticles in drug delivery applications [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe encapsulation efficiency of nisin in yeast virus\u0026ndash;like particles was 76.8\u0026ndash;83.1% and 77.1\u0026ndash;89.3% for samples prepared at 1:100 and 1:1000 ratios, respectively. These values are comparable to those reported for some of the most efficient nisin encapsulation systems described. For example, encapsulation efficiencies ranging from 80.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9% to 93.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2% were achieved for nisin-loaded bacterial cellulose nanocrystals via adsorption of the cationic peptide onto the anionic nanocrystal surface [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Similarly, an encapsulation efficiency of 78.8\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5% was reported for nisin-loaded monolaurin nanoparticles prepared using an emulsification-based method [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e], while an even higher encapsulation efficiency of 94.12% was achieved for phosphatidylcholine nanovesicles produced using the thin-film hydration method [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Taken together, these results indicate that yeast virus-like particles, employed for passive diffusion, represent a promising and competitive platform for nisin encapsulation.\u003c/p\u003e \u003cp\u003eNumerous studies have demonstrated that nisin displays broad antimicrobial activity against Gram-positive bacteria, with limited inhibitory activity against Gram-negative bacteria [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. To evaluate the potential application of nisin-loaded virus-like particles, the antibacterial activity of the particles was tested against four Gram-positive (\u003cem\u003eS. pyogenes\u003c/em\u003e, \u003cem\u003eS. aureus\u003c/em\u003e, \u003cem\u003eB. subtilis\u003c/em\u003e, and \u003cem\u003eL. innocua\u003c/em\u003e) and two Gram-negative (\u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eS. typhimurium\u003c/em\u003e) bacteria. \u003cem\u003eS. pyogenes\u003c/em\u003e, also referred to as group A \u003cem\u003eStreptococcus\u003c/em\u003e, is a human pathogen responsible for diseases such as bacteraemia, pharyngitis, cellulitis, necrotizing fasciitis, and toxic shock-like syndrome [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. \u003cem\u003eS. aureus\u003c/em\u003e is a clinically important pathogen associated with food contamination and capable of causing severe infections, including septicaemia, endocarditis, and pneumonia [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Highly resistant bacterial spores are a major contributor to food spoilage, contamination, and food poisoning. Therefore, \u003cem\u003eB. subtilis\u003c/em\u003e was selected as a model organism for spore-forming bacteria [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Another foodborne pathogen, \u003cem\u003eListeria monocytogenes\u003c/em\u003e, causes listeriosis, a severe human disease with a high mortality - greater than 20% [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Due to biosafety considerations associated with the pathogenic nature of \u003cem\u003eL. monocytogenes\u003c/em\u003e, \u003cem\u003eL. innocua\u003c/em\u003e was used as a non-pathogenic model system. In Gram-negative bacteria, the presence of an outer membrane prevents nisin from binding to lipid II, thereby substantially reducing its antimicrobial activity. Thus, assessment of nisin inhibitory activity against Gram-negative bacteria requires additional outer membrane permeabilization [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. As Gram-negative indicators, two bacterial species associated with foodborne gastrointestinal illnesses, \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eS. typhimurium\u003c/em\u003e, were selected [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe highest to lowest antibacterial activity of nisin-loaded yeast virus-like particles against Gram-positive bacteria was observed for \u003cem\u003eS. pyogenes\u003c/em\u003e, \u003cem\u003eS. aureus\u003c/em\u003e, \u003cem\u003eB. subtilis\u003c/em\u003e, and \u003cem\u003eL. innocua\u003c/em\u003e, respectively. Similar tendencies have been reported in previous studies. For example, MIC values of nisin, alone or in combination with natural organic compounds, were higher for \u003cem\u003eL. innocua\u003c/em\u003e than for \u003cem\u003eB. subtilis\u003c/em\u003e [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. Wider inhibition zones were observed against \u003cem\u003eS. aureus\u003c/em\u003e compared to \u003cem\u003eB. subtilis\u003c/em\u003e when treated with citric acid-assisted nisin [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. For nisin-loaded pectin particles, inhibition activity against \u003cem\u003eS. pyogenes\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e was reported to be similar [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. Consistent with these observations, the lowest MIC of free nisin in the present study was detected for \u003cem\u003eS. pyogenes\u003c/em\u003e, whereas the highest MIC was observed with \u003cem\u003eL. innocua\u003c/em\u003e, indicating that \u003cem\u003eS. pyogenes\u003c/em\u003e was the most sensitive and \u003cem\u003eL. innocua\u003c/em\u003e the least sensitive to nisin. Compared to free nisin, encapsulation decreased antibacterial activity against all tested microbial strains across the three nisin-loaded nanoparticle types. However, the trend in antibacterial activity against the bacteria studied was similar for encapsulated and free nisin. Both cell survival results and MIC analysis confirmed that nisin-loaded VLPs have the greatest inhibitory impact on \u003cem\u003eS. pyogenes\u003c/em\u003e. Encapsulated nisin antibacterial activity varies depending on VLP type. Based on the survival assay, the efficiency of nisin encapsulated in Y-L-BC was slightly higher against the majority of tested Gram-positive bacteria, but the MIC analysis demonstrated slightly greater activity of Y-L-A encapsulated nisin compared to other types of VLPs. These differences could be attributed to differences in testing methods: antibacterial activity was analyzed by direct survival assay, while MIC values were determined by a broth microdilution assay. As expected, nisin antibacterial activity against Gram-negative bacteria \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eS. typhimurium\u003c/em\u003e was much lower than against Gram-positive bacteria. This may be due to a barrier formed by the outer membrane, which prevents nisin from reaching receptors in the cytoplasmic membrane [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. The lower antibacterial activity and increased MIC values observed for nisin-loaded nanoparticles may be associated with the encapsulation process. Encapsulation can protect the peptide from environmental exposure while slowing its release rate, thereby reducing its immediate availability and prolonging its action [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e, \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. Nevertheless, all three types of nisin-loaded yeast virus\u0026ndash;like particles retained antibacterial activity, indicating that they represent a suitable system for nisin nanodelivery.\u003c/p\u003e \u003cp\u003eRecent studies indicate that, in addition to its antibacterial activity, nisin also bears potent anticancer properties, including induction of apoptosis, cell cycle arrest, and inhibition of cell proliferation [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. It has been demonstrated that nisin cytotoxicity is confined by active concentration and the type of cancer cell line [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. In the present study, the cytotoxic effects of free nisin and nisin-loaded yeast virus-like particles were evaluated using the A549 human lung carcinoma epithelial cell line, employed as a mammalian cancer cell model. Cytotoxicity analysis demonstrated that after 24 hours of incubation with free nisin, A549 cell viability decreased. Encapsulation of nisin resulted in up to 48-hour lasting alleviation of cytotoxicity for the nisin-loaded Y-L-BC and B-L-BC particles compared with free nisin, while a decrease in viability was recovered by 48 hours for nisin-loaded B-L-BC, and 72 hours for nisin-loaded Y-L-BC particles. In contrast, both empty and nisin-loaded Y-L-A VLPs exhibited a more profound cytotoxic effect on A549 cells during all time points. Similar results, with free nisin showing higher cytotoxicity against A549 cells than encapsulated nisin, were reported for nisin-loaded cyclodextrin-based nanosponges [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Along with reduced antibacterial activity, the delay in nisin cytotoxicity is expected to be driven by encapsulation, which deters the release and thereby reduces the immediate availability of nisin. Overall, these results suggest that yeast Y-L-BC and B-L-BC VLPs could serve as a safe and predictable platform for nisin encapsulation in mammalian cell applications, whereas Y-L-A should be considered for greater innate cytotoxicity. Importantly, the use of virus-like particles originating from yeast \u003cem\u003eS. cerevisiae\u003c/em\u003e, generally recognised as safe (GRAS) by the Food and Drug Administration (FDA) in the USA and having qualified presumption of safety (QPS) status in Europe [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e], further supports their potential safety and suitability for applications where biocompatibility and regulatory acceptance are essential.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eA passive diffusion method enabled the encapsulation of the antimicrobial peptide nisin Z into three types of yeast virus\u0026ndash;derived nanoparticles: Y-L-A, Y-L-BC, and B-L-BC. This approach achieved high encapsulation efficiency while preserving particle size and morphology, indicating the formation of a well-controlled and homogeneous nanocarrier system. Encapsulated nisin retained antibacterial activity against Gram-positive foodborne and clinically relevant bacteria, while the observed increase in MIC values is consistent with sustained release of the peptide from the nanoparticle interior. Encapsulation modulated nisin's release in a time-constrained manner while retaining its cytotoxicity, a critical consideration for potential biomedical applications of nisin-loaded nanoparticles. The previously demonstrated long-term stability of yeast virus\u0026ndash;like particles under various conditions, along with their proteinaceous nature and origin, further supports their biocompatibility and potential for safe use in mammalian cell cultures. Overall, these findings highlight nisin-loaded yeast virus\u0026ndash;like particles as a promising platform for the nanodelivery and controlled release of nisin in future biomedical applications.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDr. Tatjana Kavleiskaja is acknowledged for introducing zeta potential measurements.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEC: Formal Analysis, Investigation, Methodology, Visualization, Writing–original draft. RS: Formal Analysis, Investigation, Visualization, Writing–review and editing. KV: Investigation. AM: Investigation. ES: Conceptualization, Data curation, Formal Analysis, Supervision, Validation, Visualization, Writing–review and editing. SS: Conceptualization, Data curation, Funding acquisition, Methodology, Project administration, Resources, Supervision, Validation, Writing–original draft, Writing–review and editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Research Council of Lithuania (LMTLT), agreement [S-MIP-23-28].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData is available upon request from the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo ethics declarations are required for this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eHolmes AH, Moore LS, Sundsfjord A, Steinbakk M, Regmi S, Karkey A, Guerin PJ, Piddock LJV. 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J Dairy Sci. 2020;103(3):2041-2052.\u003c/li\u003e\n\u003cli\u003eSoans SH, Chonche MJ, Sharan K, Srinivasan A, Archer AC. Apoptotic and anti-inflammatory effect of nisin-loaded sodium alginate-gum arabic nanoparticles against colon cancer cells. Int J Biol Macromol. 2025;305(Pt 2):141747.\u003c/li\u003e\n\u003cli\u003eGronchi N, De Bernardini N, Cripwell RA, Treu L, Campanaro S, Basaglia M, Foulqui\u0026eacute;-Moreno MR, Thevelein JM, Van Zyl WH, Favaro L, Casella S. Natural \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e Strain Reveals Peculiar Genomic Traits for Starch-to-Bioethanol Production: the Design of an Amylolytic Consolidated Bioprocessing Yeast. Front Microbiol. 2022;12:768562.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"virus-like particles, nisin, antibacterial activity, cytotoxicity, nanodelivery, yeast virus","lastPublishedDoi":"10.21203/rs.3.rs-8842944/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8842944/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eNisin is a widely used antimicrobial peptide with strong inhibitory activity against Gram-positive bacteria and has attracted increasing interest for broader biomedical applications. However, its practical use is limited by susceptibility to environmental conditions, reduced stability, and rapid inactivation. Encapsulation has emerged as an effective strategy to overcome these limitations by protecting such bioactive peptides from environmental exposure, improving their stability, and enabling controlled release. Protein-based nanoparticles derived from viruses represent an attractive encapsulation platform due to their homogeneous size, biocompatibility, and ability to encapsulate and protect bioactive molecules without compromising structural integrity. In this study, yeast virus\u0026ndash;like particles were explored as a nanodelivery system for nisin, and their antibacterial activity and cytotoxicity were evaluated.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eNisin was successfully encapsulated into three types of yeast virus-like particles, achieving 77.1\u0026ndash;89.3% encapsulation efficiency while preserving particle size and morphology. Nisin-loaded nanoparticles demonstrated antibacterial activity against Gram-positive bacteria, with the most pronounced inhibitory effect against \u003cem\u003eStreptococcus pyogenes\u003c/em\u003e. The antibacterial activity against Gram-negative bacteria was nevertheless low. Compared with free nisin, encapsulated nisin exhibited moderately higher (1.2\u0026ndash;10.1 fold) minimum inhibitory concentration values. Cytotoxicity studies involving the A549 human lung carcinoma epithelial cell line demonstrated that, while free nisin reduced cell viability, encapsulation in two differently prepared nanoparticles (Y-L-BC and B-L-BC) delayed nisin cytotoxicity.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eYeast virus\u0026ndash;like nanoparticles represent a promising platform for nisin nanodelivery, preserving antibacterial activity while reducing immediate cytotoxicity through encapsulation. These findings highlight the potential for controlled nisin delivery in antimicrobial and biomedical applications.\u003c/p\u003e","manuscriptTitle":"Nanodelivery of Nisin by Homogeneous Protein-based Particles","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-18 16:49:42","doi":"10.21203/rs.3.rs-8842944/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c226c270-b457-4a32-9612-62f581fdef67","owner":[],"postedDate":"February 18th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-03-25T17:09:12+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-18 16:49:42","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8842944","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8842944","identity":"rs-8842944","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}