Investigating the Immunomodulatory Effects of Antigenic PLGA Nanoparticles and Nutritional Synergy in Caenorhabditis elegans

Abstract

This study explores the significance of antigenic nanoformulation in immunomodulation and in the interplay between immune response and nutrition. The work involves the development of a polylactic-co-glycolic acid (PLGA) biopolymer-based nanoparticle with immunogenic inclusions derived from Staphylococcus aureus cell wall and membrane (CWM) through a double emulsion method followed by their physio-chemical characterization and in vivo assessment in Caenorhabditis elegans (C. elegans). The prepared nanoparticles were monodispersed in nature and exhibited a diameter of ~ 25 nm with stable colloidal nature and a zeta potential of − 25 ± 2 mV. The inclusion release and carrier degradation profiling revealed controlled and steady kinetics supporting the sustained availability of the encapsulated payload. The immunomodulatory studies conducted in C. elegans revealed that the expression of the stress indicator gene viz., sodh-1 was significantly upregulated in the CWM-treated worms and was notably reduced in the worms treated with the nanoformulation indicative of the slow release of the antigen which does not trigger untoward stress responses. In contrast, the expression of host defense genes viz., clec-7, ilys-3, igg-1, and cyp-37B1 in response to the CWM treatment was found to be downregulated, while for the nanoformulation treatment, the extent of downregulation was relatively lesser. A notable observation emerged as these genes, previously downregulated, exhibited a significant upsurge when the nutritional supplementation was amplified. This highlighted the profound influence of nutrition in fine-tuning the immune responses. Our data offers insights that could pave the way for further research in designing nutritional strategies to augment immunomodulatory interventions, as well as advocate for nanoparticle-based immunomodulatory approaches to prevent immune stress.

1. Introduction

The oral delivery of immunomodulators represents a significant stride in medical science, offering a novel approach to regulate and modulate the responses of the immune system [1]. Immunomodulators are substances that can either enhance or suppress immune activity, and their successful oral delivery holds great potential for a variety of therapeutic applications in managing chronic inflammatory conditions. However, the oral delivery of immunoboosters encounters multifaceted challenges stemming from the harsh conditions of the gastrointestinal environment, including acidity and enzymatic degradation, which can compromise the stability and bioavailability of these agents [2]. Additionally, the risk of first-pass metabolism in the liver and limited absorption due to large molecular sizes further hampers their effectiveness. The potential for immunogenicity and tolerance issues within the gut, coupled with the need for precise formulation techniques, compounds the complexity of designing effective oral delivery systems.

Nanoparticles offer a promising avenue for overcoming the challenges associated with the oral delivery of immunoboosters [3, 4]. By encapsulating immunoboosters, nanoparticles provide protection against the harsh conditions of the gastrointestinal tract, safeguarding their integrity and bioactivity. Their small size and unique properties enhance bioavailability by facilitating absorption across the intestinal epithelium, increasing the likelihood of reaching the systemic circulation. Furthermore, nanoparticles enable controlled release, ensuring a sustained supply of immunoboosters and reducing the need for frequent dosing. Of the various BioNanoScience nanoparticulate systems available, polymer-based nanoparticles have revolutionized drug delivery, offering solutions to challenges that were once considered insurmountable [5, 6]. Poly lactic-co-glycolic acid (PLGA) is one such synthetic biopolymer that has gained significant attention in the fields of medicine, pharmaceuticals, and biotechnology [7].

In the present study, we have demonstrated the immunomodulatory and stress-mitigating properties of PLGA nanoparticles incorporated with Staphylococcus aureus cell wall and membrane extract (CWM) after oral delivery in Caenorhabditis elegans. These antigenic PLGA nanoparticles (aNPs) were prepared through a double emulsion method, and the physicochemical analyses were performed using FE-SEM, DLS-Zetasizer, and FTIR. The entrapment efficiency and release kinetics were assessed using ELISA specific for peptidoglycan, which is a major component of the immunogenic extract. The immunomodulation of the aNPs was evaluated by estimating the expression of stress indicator and host immunity genes in C. elegans viz., sodh-1, clec-7, ilys-3, igg-1, and cyp-37B1 in the presence of the natural diet of the nematode viz., E. coli OP50 strain in optimal and excess conditions. We observed a significant downregulation of the stress indicator gene sodh-1 in response to exposure to aNPs, as compared to the control nematodes treated with CWM alone. Furthermore, an increase in OP50 concentration exhibited a positive effect on the regulation of the sodh-1 gene, further enhancing the stress-downregulating potential of aNPs, indicating significant modulation of stress gene expression under nutritional abundance. The host defense genes viz., clec-7, ilys-3, igg-1, and cyp-37B1 were also significantly upregulated under nutritional abundance, indicating the potential influence of nutrition on immunomodulation at the organism level. Our data supports the idea that positive immunomodulation could be effectively achieved only if the nutrition factor is also optimally supplemented. This research would pave the way to in-depth explorations to understand the interplay between nutrition, gene expression, and immune function, offering insights that could revolutionize nutritional strategies to effectively augment oral delivery of immune boosters and vaccines.

2. Materials and Methods

2.1. Materials and Kits

The following materials and kits were used without further purification or modification: acid-terminated poly-lactide-co-glycolic acid (PLGA) copolymers with glycolic-to-lactic mole percentage ratio (50/50 ratio; Polysciences), ethanol (HiMedia), acetone (Sigma), sodium hydroxide (Sigma), phosphate-buffered saline pellets (Sigma), sodium chloride (Himedia), peptidoglycan monoclonal antibody (Invitrogen), goat anti-mouse IgG(γ) F(ab')2–HRP (Invitrogen), Tween 20 (Sigma), 2,2′-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (Sigma), and MilliQ water.

Bacterial culture

Staphylococcus aureus and E. coli OP50 (sourced from the Microbial Type Culture Collection, India) were grown in Nutrient broth and Luria Bertani broth, respectively, at a temperature of 37 °C, within a shaking incubator operating at 200 rpm to ensure proper aeration. All the materials and consumables required for microbial culture were procured from HiMedia, India.

Caenorhabditis elegans culture

Bristol N2 strain of C. elegans (sourced from CGC, University of Minnesota, USA) was maintained at 20 °C on solid Nematode Growth Medium (NGM) containing Escherichia coli OP50 as a food source. All the materials and consumables required for culture were procured from HiMedia, India.

2.2. Preparation of Bacterial Cell Wall/Membrane Extract

First, the bacterial culture, at the log phase (i.e., after ~ 16 h of incubation), was employed for deriving bacterial ghost cells as described by us earlier [8]. The ghost cells were then heat-treated at 95 °C for 10 min, followed by ultrasound sonication for 10 min at 40% amplitude for 7 s on a 3-s off cycle. The extract is then further diluted in 5-ml milliQ water and homogenized under sterile conditions, following which it was centrifuged at 10,000 rpm for 10 min at 4 °C, and the final pellet was lyophilized to estimate the dry weight.

2.3. Preparation and Optimization of Antigenic PLGA Nanoparticles

Antigenic PLGA nanoparticles (aNPs) were synthesized using the water/oil/water (W/O/W) double emulsion method. The cell wall and membrane (CWM) aqueous extract from a single-prep was added to PLGA (in acetone) at varying PLGA:CWM w/w ratios and vortexed thoroughly for 1 min to obtain the primary emulsion (water-in-oil). This was then dropped into 5-ml milliQ water under constant magnetic stirring to obtain a stable w/o/w emulsion. The formulation was kept under stirring for an additional 1 h for solvent evaporation and was then centrifuged at 10,000 rpm for 10 min at 4 °C. The resulting pellet was taken for further evaluation.

2.4. Indirect Peptidoglycan ELISA

For the estimation of entrapment efficiency, an indirect peptidoglycan ELISA was performed using 96-well ELISA plates, as reported previously [9]. The supernatant from the nanoformulation was collected and diluted in PBS. The wells were coated with 200 µL of the supernatant at 4 °C overnight, after which it was washed thrice with PBST (PBS BioNanoScience containing 0.1% Tween 20). Subsequently, 100 µL of the peptidoglycan monoclonal antibodies (mAbs), which were appropriately diluted in PBST, was introduced into each well of the plates. The plates were then incubated at 37 °C for a duration of 2 h. Following three PBST washes, 50 µL of a diluted solution (1 µg/mL) of goat anti-mouse IgG(γ) F(ab')2–HRP conjugate was introduced, and the plates were incubated for 1 h at 37 °C. After three washes with PBST, 100 µL of the substrate 2,2′-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) was dispensed into each well, and the absorbance at 405 nm was determined using a microplate reader, allowing for the quantification of the bound antibodies in each well.

2.5. Characterization of Antigenic PLGA Nanoparticles

2.5.1. Field Emission Scanning Electron Microscopy (FE-SEM) Analysis

Morphology and size characterization were performed using FE-SEM analysis. The nanoparticle suspension was appropriately diluted and dropped onto the carbon tape on a metal stub and air-dried overnight at room temperature, following which the sample was gold-sputtered. The imaging was done under the Field Emission Scanning Electron Microscope (Carl Zeiss Sigma, SMARTSEM).

2.5.2. Dynamic Light Scattering and Zeta Potential Analyses

Dynamic light scattering (DLS) is a rapid and non-intrusive technique that was used for assessing hydrodynamic size, size distribution, and stability of the suspension. We used the Horiba ZetaSizer (Horiba Nanopartica SZ-100) for the above measurements. The purified antigenic PLGA nanoformulation was appropriately diluted before analysis.

2.5.3. Fourier Transform Infrared Spectroscopy

The chemical integrity of the prepared antigenic PLGA nanoparticles was assessed using Fourier transform infrared (FTIR) spectroscopy, utilizing the Nicolet™ iS50 FTIR Spectrometer (Thermo Scientific, USA). The scanning was done for a wavenumber range spanning from 4000 to 500 cm⁻¹, and prior to scanning, the samples were uniformly mixed with KBr which served as a reference.

2.5.4. In Vitro Degradation of aNPs

To study the degradation profile of the aNPs under physiological conditions, an in vitro degradation assessment based on pH change of the degradation medium was performed in a time-dependent manner (0 h, 4 h, 24 h, 30 h, and 48 h). Freshly prepared aNPs were resuspended into 15-ml phosphate buffer solution (PBS) (pH = 7.2 ± 0.2) in a 50-ml conical flask and was shaken at 100 cycles/min in a shaking incubator kept at a constant temperature of 37.0 ± 0.2 °C. After specific incubation time points, the pH of the solution was monitored using a regular laboratory pH meter.

2.5.5. Antigen Release Study

To study the release profile of the antigenic inclusion (i.e., CWM), we performed a time-dependent release experiment (2 h, 4 h, 8 h, 12 h, 24 h, and 48 h). Freshly prepared antigenic PLGA nanoparticles were resuspended into 1-ml phosphate buffer solution (PBS) (pH = 7.2 ± 0.2) in a screw-capped tube and were shaken at 100 cycles/min in a shaking incubator kept at a constant temperature of 37.0 ± 0.2 °C. After specific incubation periods, 100 µl of the dissolution medium was withdrawn after being centrifuged at 10,000 rpm for 10 min at 4 °C. Following this, the indirect ELISA was performed as mentioned above to determine the peptidoglycan content in the dissolution medium for equating the amount of released antigen.

2.6. In Vivo Toxicity Evaluation

Caenorhabditis elegans was cultivated at 20 °C on solid Nematode Growth Medium (NGM) enriched with Escherichia coli OP50 at a regular/normal concentration of 1 × 10¹⁰ units/plates as a food source. We synchronized the age of the nematodes by the sodium hydroxide bleach method of synchronization previously reported [10]. The mature worms were collected by washing with M9 buffer and centrifuged at 2000 rpm for 2 min for pelletizing them. Following this, a cocktail of 0.5 ml of 5 M NaOH and 1 ml 4% bleach solution made up to 5 ml using double distilled water was poured onto the worms to eradicate them, leaving behind just the unhatched eggs. The suspension was then washed thoroughly to remove any remaining bleach and debris, leaving behind synchronized nematode eggs. These eggs are typically at the same developmental stage. The eggs were then transferred to a fresh NGM plate with E. coli OP50 lawn and allowed to hatch overnight. The newly hatched worms were continuously monitored until they reached the L4 larval stage in three days, after which they were collected using M9 buffer and washed twice with 5 mM PBS at pH 7.4 to eliminate OP50.

For in vivo toxicity testing, L4 stage C. elegans were grouped into equal numbers and subjected to treatment with CWM, aNP, and bare PLGA nanoparticles under regular OP50 concentrations as mentioned above and incubated at 20 °C. Worms subjected to only OP50 were considered BioNanoScience as the untreated control. The survival rates in each experimental group were determined by enumerating the live and active C. elegans on day 0 (start of treatment) and day 3 (72 h into treatment).

2.7. Quantitative Real-Time PCR (qRT-PCR)

The expression of the genes viz., sodh-1, clec-7, ilys-3, igg-1, and cyp-37B1 in response to the immunomodulation was assessed through qRT-PCR. The nematodes (approx. 2000 per plate) were plated onto NGM agar with a regular OP50 concentration of 1 × 10¹⁰ units/plates, simultaneously with CWM or aNP individually and incubated for 24 h, while worms subjected to only OP50 were treated as the control. In yet another experiment, the aNPs were plated along with double the concentration (i.e., 2 × 10¹⁰ units/plates) of regularly supplied OP50 and incubated for 24 h. This experiment was to check the influence of nutritional abundance on immunomodulation. After the incubation period, the C. elegans were washed with PBS and subsequently ground under liquid nitrogen. The resulting worm powder was then used for total RNA extraction using the RNeasy plus mini kit (Qiagen, GmbH). The RNA integrity and quality was verified using the NanoDrop spectrophotometer (NanoDropND-1000, Thermoscentific, USA) and agarose gel electrophoresis. The complementary DNA (cDNA) was synthesized from 1 µg of total RNA using SuperScript III reverse transcriptase kit and random hexamers (Invitrogen, Carlsbad, CA) in a total volume of 20 µl. SYBR Green RT-PCR Master Mix (Applied Biosystem, USA) was used for expression analysis of the gene. The quantitation of fold change was analyzed by the 2^−ΔΔCT method. All experiments were performed in two individual sets of triplicates. The primer sets used for the experiment are mentioned in Table 1.

Table 1. List of primers used for the gene expression experiments on C. elegans.

Gene	Forward primer	Reverse primer
ilys-3	5’-AGATCAAACTCCCCTACTACTAC-3’	5’-TAGCGGTTGTAGGTAGTTCTC-3’
clec-7 [11]	5′-TTTATGGGACGATTCGACGG-3′	5′-GTCAATGCACCTTGTACGGA-3′
cyp-37B1[11]	5′-GAATGTATCCGTCAGTGCCA-3′	5′-TCGGACTCCTTTTGGGAAGA-3′
sodh-1[11]	5′-CTGGATGGCAACTTGGAGACAAAGC-3′	5′-GGTGGCAGAGTGGCTCGTGG-3′
igg-1[11]	5′-ACCATGACCACAATGGGACAACTC-3′	5′-ACACTTTCGTCACTGTAGGCGATG-3′
snb-1 (housekeeping)	5’-CGGAATCATGAAGGTGAAC-3’	5’-TGATGTTCTTCCACCAATAC-3’

2.8. Statistical Analysis

The data were analyzed using analysis of variance (ANOVA), and post hoc Tukey’s test was applied using GraphPad Prism, CA. The outcomes were presented as the mean ± SEM, and statistical significance was defined by differences with a p-value less than 0.05.

3. Results

3.1. aNP Preparation and Optimization

In the present study, we have formulated an immunomodulatory construct by incorporating S. aureus CWM extract into PLGA nanoparticles (referred to as antigenic nanoparticle, aNP). Firstly, we had prepared the CWM formula and found that the dry weight of the antigenic material extracted from one preparation was 2.42 ± 0.83 mg. Our synthesis employed the emulsion method (Fig. 1a), a well-established technique for the preparation of polymeric nanoparticles [12]. To optimize this nanoformulation, we meticulously explored various polymer-to-drug w/w ratios, specifically 2:1, 4:1, 6:1, and 8:1, as depicted in Fig. 1b. However, our investigation revealed that the 4:1 ratio yielded a stable colloid, evident from their sustained stability even after overnight standing at room temperature, and substantiated further by amplified antigen entrapment efficiency viz., 61.54 ± 9.1% (Fig. 1c).

Fig. 1. Nanoparticle preparation and optimization protocol.

a Schematic representation of antigenic nanoparticle (aNPs) preparation using PLGA as the matrix and S. aureus CWM as the antigenic payload, using the water-in-oil-in-water (W/O/W) emulsion method. b Photographs of the antigenic nanoformulation prepared using varying PLGA:CWM weight ratios. c Entrapment efficiency of the antigen in the various formulations quantified using indirect ELISA. Bars represent mean ± SEM. *p-value <0.05, **p-value <0.005, and ***p-value <0.001; ns: not significant

3.2. Chemical Characterization of aNPs

A comprehensive characterization of nanoparticles was conducted, encompassing assessments of their physico-chemical properties through various analytical techniques. We first performed the FE-SEM to evaluate the overall size of the antigenic nanoconstructs. Figure 2a, b shows varying magnifications of FE-SEM images depicting monodispersed and structurally well-defined spherical nanoparticles with a size of ~ 25 nm. Furthermore, we conducted DLS measurements to evaluate the hydrodynamic particle size (Fig. 2c), which revealed that the average hydrodynamic diameter of the aNP was ~ 60 nm and showed a narrow size distribution, which validated the SEM images. Beyond the considerations of size and morphology, the stability of nanoparticles is also a crucial factor for their effective functionality. For this, we performed the zeta analysis and found the zeta potential to be − 25 ± 2 mV, indicating a colloidally stable formulation with a negative surface charge (Fig. 2d).

Fig. 2. Characterization of optimized nanoparticles.

a, b Field emission scanning electron microscopy (FE-SEM) images of the antigenic nanoparticles (aNPs) captured at varying magnifications. c Dynamic light scattering analysis of aNPs showing a narrow size distribution and an average hydrodynamic particle size of ~ 60 nm. d The zeta potential of aNPs was found to be − 25 ± 2 mV, indicating a colloidally stable formulation

Furthermore, the chemical composition and functional groups of the aNPs were studied using FTIR spectroscopy. Three samples viz., CWM, PLGA NPs, and aNPs were compared based on their FTIR spectra. As seen in Fig. 3, spectrum A shows the vibrational bands of CWM at 3300–3250 cm⁻¹, 3100–3050 cm⁻¹, 2960–2880 cm⁻¹, and 2000–1000 cm⁻¹.

Fig. 3.

FTIR spectra of components utilized in aNP formulation. Spectrum A depicts the chemical composition and functional groups of the cell wall membrane (CWM) extract, spectrum B represents the poly lactic-co-glycolic acid (PLGA) nanoparticles, and spectrum C illustrates the aNPs

Spectrum B (PLGA nanoparticles) shows vibration bands at 3000 cm⁻¹, 1770–1750 cm⁻¹, 1452 cm⁻¹, 1300–1150 cm⁻¹, 1250–1100 cm⁻¹, and 840 cm⁻¹. Spectrum C in Fig. 3 depicts the analysis of aNP and shows the characteristic peaks (1750 cm⁻¹, 1537 cm⁻¹, 1452 cm⁻¹, 1225 cm⁻¹, and 1087 cm⁻¹) of both the antigen (spectrum A) and those of PLGA (spectrum B).

3.3. Particle Degradation and Antigen Release

To explore the degradation kinetics of the PLGA nano-carrier, an in vitro assessment was conducted, monitoring the pH variation in the degradation medium. Figure 4a BioNanoScience depicts visual observations at various degradation time points revealing diminishing colloidal dispersion, and pH estimation of the degradation medium showcased a steady decrease (Fig. 4b). Commencing at pH 7.4, the pH declined to 6.75 ± 0.07 after 24 h and further decreased to 6.46 ± 0.05 after 48 h, indicating continual formation of acidic monomers. Subsequently, an evaluation of antigen release kinetics from the nanomatrix was performed. Figure 4c depicts the in vitro cumulative release of the encapsulated antigen at pH 7.4 using indirect ELISA. It showed that the antigen release progressively increased to approximately 41% within the first 24 h and remained sustained at 45% after 48 h.

Fig. 4. In vitro assessment of degradation and antigen release kinetics.

a Sequential images at significant time intervals displaying the diminishing colloid nature of aNPs in PBS solution, indicative of their degradation progression over time. b Graphical representation illustrating the pH changes at different incubation time points. c Time-dependent cumulative release profile of the antigen from aNPs recorded over 48 h of incubation. Error bars represent mean ± SEM

3.4. In Vivo Investigation on C. elegans

In our preliminary exploration of the toxicity profile of aNPs, we conducted a time-dependent survival analysis using C. elegans. The concentrations for testing were prepared by diluting the CWM (from a single preparation) four-fold in nematode medium, resulting in a final concentration of 605 μg/ml. Considering that CWM contains pathogen-associated molecular patterns, higher concentrations might induce toxicity. Therefore, we incorporated a dilution step before the experiments to ensure safer testing conditions. The concentration of aNP was adjusted to match the equivalent loading of CWM within them for comparative analysis. Visual representations in Fig. 5a, b illustrate various treatment conditions [CWM (antigen), PLGA nanoparticle (vehicle), and aNP (nano-antigen)] compared against a control group fed with E. coli OP50 alone on day 0 (initiation of treatment) and day 3 (72 h into treatment) respectively. We observed that the nematodes had progressed to adulthood and reproductive stages, as depicted in the microscopic images on day 3. Noticeable variations emerged in the nematode life cycle, meticulously assessed by enumerating distinct larval stages across each test plate and subsequently compared to the untreated control group. Figure 5c delineates a comparison of the nematode population between day 0 and day 3. Our results showed that the control, aNP, as well as PLGA-treated groups had a similar range of reproduction, as was evident from their population size at day 3. But notably, the population size for the CWM-treated group was only ~ 50%, which was significant when compared to untreated control, PLGA-treated, and aNP-treated groups.

Fig. 5. Microscopic images illustrating C. elegans under distinct treatment conditions: (i) control group, (ii) CWM-treated group, (iii) PLGA-treated group, and (iv) aNPs-treated group.

a Micrographs captured at day 0, magnified at 4 ×. b Micrographs captured at day 3, displayed at 1 × magnification. c Enumeration comparison of C. elegans populations at day 0 (D0) and day 3 (D3) across various treatment conditions. Bars represent mean ± SEM. *p-value < 0.05, **p-value < 0.005, and ***p-value < 0.001; ns: not significant

We conducted a gene expression analysis focusing on the stress-responsive marker sodh-1 across all treatment groups, comparing them with the untreated control. Notably, Fig. 6a illustrates a substantial upregulation of 20.14-fold in the sodh-1 gene expression in the bare CWM-treated group. Contrastingly, the aNP-treated group exhibited a significantly reduced expression of 13.42-fold, highlighting the pivotal role of nano-encapsulation in mitigating physiological stress responses. Following this, our investigation aimed to ascertain whether augmented nutritional supplementation could effectively mitigate stress levels and restore homeostasis by modulating the expression of the stress-responsive sodh-1 gene. Interestingly, our observations yielded a remarkable revelation: Upon a two-fold increase in OP50 concentration (double food, DF, i.e., 2 × 10¹⁰ cells/plate) in the CWM and aNP-exposed group, the expression of sodh-1 underwent a profound reduction, attaining a reduced level of 11.7-fold for CWM treatment and 6.5-fold for aNP group. These values were roughly half of the expression levels shown by the same treatments under regular OP50 supply (single food, SF, i.e., 1 × 10¹⁰ cells/plate).

Fig. 6. Gene expression profiling in C. elegans subjected to different treatment conditions.

a Comparison of stress indicator sodh-1 expression and its relative fold change across various treatments (UT – untreated, CWM – treated with SF, aNP + SF – antigenic nanoparticle with SF, CWM-treated with DF, aNP + DF – antigenic nanoparticles with DF). b Graphical representation illustrating the relative fold change in host defense gene expression in C. elegans treated with CWM (with SF or DF) and aNPs (with SF or DF). SF (single food, 1 × 10¹⁰ cells/plate); DF (double food, 2 × 10¹⁰ cells/plate). Bars represent mean ± SEM. *p-value < 0.05, **p-value < 0.005, and ***p-value < 0.001; ns: not significant

We next explored the effect of the aNPs on the host defense genes viz., clec-7, ilys-3, igg-1, and cyp-37B1 genes. To gauge the regulatory influence of the test samples on specific host defense genes, initial gene expression analyses were conducted on C. elegans exposed to CWM alone (bare antigen group), and comparing the results against a control group fed exclusively with a regular concentration of E. coli OP50 (i.e., single food, SF, 1 × 10¹⁰ cells/plate). In Fig. 6b, the displayed fold change in gene expression within the CWM-treated C. elegans group showcased a considerable downregulation in host defense genes in contrast to the untreated control group. Intriguingly, a downregulated status for the host defense genes was also noted in the aNP-treated group. Notably, our prior experiments had indicated significant upregulation of stress gene sodh-1 in both the CWM and aNP-treated groups, although the former was significantly higher than the latter (Fig. 6a).

Next, we conducted an experiment by providing a two-fold increased OP50 concentration (double food, DF, i.e., 2 × 10¹⁰ cells/plate) to the CWM and aNP-exposed group while maintaining the CWM and aNP concentration the same as above. As illustrated in Fig. 6b, we again observed a downregulated status for the CWM-treated group even upon doubling the food supply. But a notable upregulation in the expression of all the tested host defense genes was observed within the aNP-treated group under conditions of nutritional abundance (i.e., DF) in comparison to the regular OP50 cell concentration (SF). This nutritional augmentation significantly impacted gene expression in the case of aNP-treated groups, with the clec-7 gene exhibiting a 1.23 ± 0.06-fold upregulation, ilys-3 demonstrating a remarkable 2.79 ± 0.18-fold increase, igg-1 displaying an 3.03 ± 0.62-fold enhancement, and cyp-37B1 manifesting a 2.42 ± 0.37-fold amplification.

4. Discussion

An effective immune system is pivotal in defense against infectious and chronic ailments. Hence, the quest for novel immunomodulators, encompassing both synthetic and biological entities such as antibodies, enzymes, bacterial derivatives, and plant metabolites, remains at the forefront of scientific exploration [13]. Staphylococcus aureus (S. aureus) is a significant human pathogen, adept at causing a wide spectrum of clinical infections and displaying impressive adaptability even under harsh conditions characterized by elevated salinity, limited water resources, and osmotic stress [14, 15]. There were no effective vaccines yet developed against S. aureus owing to its complex biology, and therefore, the creation of a potent vaccine remains a crucial endeavor for the prevention of S. aureus infections [16, 17]. S. aureus-derived antigens were used by various researchers for investigating their immune-boosting potential [18, 19]. However, they demonstrated robust immune responses characterized by hyperactivated immune cells and excessive cytokine production, potentially leading to adverse effects on host cells [20]. In such a scenario, a slow and controlled release of antigen is very essential for prolonged immuno-boosting with lesser cytotoxicity, and therefore, its encapsulation into suitable biocompatible carriers is deemed fit.

In the present study, we have formulated an immunomodulatory construct by incorporating S. aureus CWM extract into PLGA nanoparticles (referred to as antigenic nanoparticle, aNP). PLGA is one of the most favorable biopolymers widely used for the encapsulation and controlled release of encapsulated payloads [21]. In our study, we decided to use PLGA as the carrier matrix as we intended to demonstrate a controlled and steady release of the extracted antigen. We have synthesized our antigenic nanoparticles (aNPs) through the double emulsion method, and their optimization was carried out using different polymer-to-drug ratios (Fig. 1). Notably, the entrapment efficiency did not exhibit a commensurate increase with higher polymer-to-drug ratios. Past studies have suggested that augmenting the polymer concentration in the solvent generally leads to greater encapsulation of the active compound [22]. Contrary to these findings, our observations did not manifest such a trend, intimating the plausible attainment of a saturation threshold by PLGA, limiting significant enhancements in encapsulation despite elevated polymer content. The size characterization performed for the aNPs showed monodispersed nanoparticles with appreciable BioNanoScience colloidal stability, which are essential attributes of a scalable therapeutic nanoformulation.

The FTIR analysis helped to qualitatively decipher the interactions between the functional groups of the payload and the carrier in terms of the vibrational bands [23]. The spectrum of CWM indicates peaks at 3300–3250 cm⁻¹ corresponding to -OH groups from absorbed water, while shorter peaks between 3100 and 3050 cm⁻¹ were associated with = C-H groups found in unsaturated fatty acid chains, as is commonly found in the phospholipid bilayer of bacterial cell membranes. Additionally, peaks around 2960 cm⁻¹ and 2880 cm⁻¹ were attributed to the vibrations of -CH3 groups, representing alkane groups [24]. Alkane groups are commonly associated with the hydrophobic tails of phospholipids in the bacterial cell membrane, where they contribute to its fluidity and stability, and any shift in the position of these alkane peaks is believed to reflect changes in the cell wall/membrane structure [25]. We also observed four notable peaks within the 2000–1000 cm⁻¹ range that indicated amides, carboxyl, and glycosidic linkages as found in bacteria [26]: (1) vibrational stretching band centered at 1635 cm⁻¹ corresponding to C = 0 vibration within amide group indicating the presence of peptidoglycan and the interlinking peptides which are prominent cell wall material in S.aureus; (2) vibrations of amino acid side chains at 1520 cm⁻¹; (3) peaks between 1500 and 1300 cm⁻¹ attributed to the vibrations of -CH2 and -CH3 groups found in lipids and proteins; and (4) vibrational band at 1225 cm⁻¹ corresponding to glycosidic linkage which could be found plentifully in peptidoglycan. Spectrum B shows the PLGA peaks showing the stretching vibrations of the carbonyl groups present in the two monomers of PLGA, which is depicted by the intense bands between 1770 and 1750 cm⁻¹ [27]. Additionally, medium-intensity signals between 1300 and 1150 cm⁻¹ corresponding to asymmetric and symmetric C-C(= O)-O stretches, respectively, in esters were also observed. The minor twin peaks near 3000 cm⁻¹ corresponded to -CH, -CH2, and -CH3 groups in PLGA. Also, the identifying signal at 1182 cm⁻¹ can be linked to the presence of the ether group. The characteristic bands at 1130 cm⁻¹ and 1452 cm⁻¹ are indicative of the C-O-C group and the C-H bond within the methyl group, respectively. The harmonic bands of the C-C-O group appeared around 840 cm⁻¹, while the absorption bands at 1100–1250 cm⁻¹ and 1750–1760 cm⁻¹ represent the esters and carbonyl groups specific for PLGA [28, 29]. Spectrum C of aNPs shows the characteristic peaks of both antigen and PLGA. Though the common peaks were observed, their intensities and positions were slightly varied, indicating inter- and intra-molecular interactions [29]. The characteristic peaks of PLGA at 1750 cm⁻¹ and 1087 cm⁻¹, indicating carbonyl group and C-O stretch, respectively, were clearly evident for aNPs with the same intensity and mild positional shift, potentially due to the interaction of PLGA with the CWM components. The characteristic peaks of the antigen CWM at 1537 cm⁻¹, 1452 cm⁻¹, and 1225 cm⁻¹ corresponding to amino acids, C-H bonds, and glycosidic bonds, respectively, were evident in the spectra obtained for aNPs [30]. Interestingly, these spectral bands exhibited reduced absorption intensity, which serves as an indicative marker of the presence of PLGA coating enveloping the antigen, rendering them vibrationally less accessible for analysis.

Achieving a sustained and controlled drug release hinges upon the orchestrated degradation of the carrier matrix. PLGA stands as a widely employed biodegradable polymer in diverse particle formulations [12, 21, 31–33]. Its hydrolytic degradation yields lactic acid and glycolic acid monomers [34, 35]. This degradation, initiated under physiological pH conditions, induces a gradual decline in pH over time due to accumulating acidic fragments [36]. In vitro degradation study reveals that within a stipulated time period, complete degradation of particles occurs in physiological conditions. PLGA is known to undergo hydrolytic degradation into acidic monomers which could contribute to the acidification of the release medium. The burst release from PLGA formulations is considered a draw-back which could be deleterious to the host [37]. But, in our study, we observed a more gradual and controlled antigen release kinetics. This observed steady and controlled antigen release aligns with the gradual matrix degradation, indicating congruence between release kinetics and carrier degradation. Our deliberate pursuit of precisely controlled antigen release, exemplified by the selected PLGA carrier, highlights our primary objective to potentially leverage this platform for finely modulated immunomodulatory applications.

In our subsequent experiments, we embarked upon an exploration into the effects of post-oral administration of aNPs on host defense and stress genes in C. elegans, a remarkably simplistic yet resilient nematode. C. elegans has garnered widespread acclaim as an exceptionally versatile animal model in the realm of scientific investigation, spanning diverse research domains encompassing aging, neurodegenerative disorders, and, most notably, the intricate nuances of the innate immune system [38]. This model organism lacks certain facets of the adaptive immune system commonly found in vertebrates, rendering it a particularly invaluable platform for the meticulous dissection of pivotal facets pertaining to innate immunity and the intricate interplay between host and microbe [39–41]. Apart from its scientific value, employing C. elegans as an experimental model presents various practical benefits such as cost-effectiveness, a shortened generation time, easy cultivation protocols, and the ability to bypass extensive animal experimentation during initial screening stages [42].

In our preliminary exploration of the toxicity profile of aNPs, we conducted a time-dependent survival analysis BioNanoScience using C. elegans. Our data indicated that the bare antigen treatment could lead to lesser reproductive acumen in the test group with a decrease in larval instars – a sign suggestive of induced stress response directly from the antigen exposure, decelerating life cycle progression [43]. Particularly noteworthy was the heightened compatibility observed with aNPs compared to the antigen group. This enhanced biocompatibility might stem from the gradual and sustained antigen release from aNPs, a stark contrast to the abrupt exposure experienced with bare CWM. These outcomes highlighted the protective effects of encapsulation technology, facilitating controlled antigen release and mitigating the immediate immune response associated with the naked antigen [44].

In light of the observed adverse effects on the nematode life cycle due to exposure to bare CWM in our previous experiment, our inquiry aimed to assess the status of the stress indicator gene sodh-1. Interestingly, the CWM-treated group had shown drastic upregulation of sodh-1, validating our observation on the link between stress and reproductive acumen. Also, the decreased levels of sodh-1 in the aNP-treated nematodes corroborated with the overall assessment in this aspect. However, an interesting factor was the observation that, upon doubling the food supply, the levels of stress were further downregulated. This phenomenon may be attributed to the fact that a surplus of nutrients offers the necessary metabolic substrates and energy, enabling the immune system to mount a more robust and effective response [45]. In situations of nutritional abundance, the nano-immunobooster can capitalize on these favorable conditions to boost the immune response, ultimately yielding improved outcomes.

We next explored the effect of the aNPs on the host defense genes viz., clec-7, ilys-3, igg-1, and cyp-37B1 genes. Our observations suggested that the bare antigen (i.e., CWM) as well as aNPs under regular food (single food) supply were not competent enough to upregulate the host defense mechanisms in the organism. Similarly, the CWM-treated group also did not show any enhancement in the host defense, even upon doubling the food supply for the organism. Consequently, we hypothesize that the heightened expression state of sodh-1 might have contributed to the observed reduction in host defense gene expression levels. This interrelation suggests a potential link between stress responses and the modulation of immune defense genes, warranting further investigation into their intricate regulatory dynamics. Upon examining the gene expression patterns alongside the stress indicator gene sodh-1, an inverse relationship between physiological stress and the immune defense capability of the host was observed in the CWM group. However, interestingly, we found that the aNP-treated group under double food supply showed significant upregulation of the host defense genes, providing substantial evidence supporting the role of nutritional abundance in the modulation of immune function [46]. While our data strongly advocates for the inclusion of nanocarrier-mediated delivery of immunoboosters for avoiding stress induction due to bare antigens, they also call for improvisation in the nutritional strategies that should be followed while undergoing immunomodulatory therapies.

5. Conclusion

Our study delves into the intricate relationship between stress, host defense response, and nutrition driven by the oral delivery of an antigenic PLGA nanoparticle developed by encapsulating bacterial antigen extract from the cell wall and membrane. The investigations have been on the model organism, Caenorhabditis elegans, where we understood the stress-management benefits that the nanoformulation could render over the bare antigen counterpart. More interestingly, our observations on how nutritional sufficiency had a role to play in immunomodulation were insightful, potentially opening up new avenues for personalized nutritional interventions that could augment immunomodulatory strategies. Notably, the significance of our observations lies in its implications for immunomodulatory treatments, such as vaccination or immunotherapy, where the provision of nutritional sufficiency could emerge as a pivotal factor in augmenting immunity and ameliorating stress responses within the organism while in the intervention. Moreover, the innovative approach of oral delivery of nano-immune boosters provides a promising avenue for the development of more effective and targeted immunotherapies. Nevertheless, our aspirations extend toward the pursuit of comprehensive studies within higher organisms, to further validate synergistic relationships between nutrition and success in immunomodulatory strategies.

Data Availability

No datasets were generated or analyzed during the current study.