Effect of sunflower oil nanoemulsions on the growth and lifespan extension of the Caenorhabditis elegans

Caenorhabditis elegans has been used as a central model system for broad - spectrum research studies across diverse modern biological domains. A study was conducted to evaluate the potential of sunflower oil - based nanoemulsions for lifespan enhancement in stressed C. elegans worms , that had been fed on nematode growth media (NGM) seeded with two strains of pathogenic bacteria ( Salmonella enterica serovar Typhimurium and Staphylococcus aureus ) . Nanoemulsions were fabricated using HI - CAP ® 100 modified food starch, and their stability was tested under different processing conditions (temperature, pH, ionic strength) over a period of 5 months. Lifespan assay results demonstrated that sunflower - oil - based nanoemulsions with droplet size 170.2±9.3 nm, significantly facilitated the


Introduction
Caenorhabditis elegans is a non-parasitic, small (1 mm in length), transparent, and free-living roundworm nematode with cosmopolitan distribution (Schulenburg and Félix 2017), a short life cycle of 3 days from egg stage to adulthood at 25 o C, and a total lifespan of between 2-3 weeks, permitting ease of genetic manipulation, and facilitation in terms of the study of its biological attributes (Alexander et al., 2014).C. elegans has been used as an in vivo, central model system across various biological disciplines (Zhang et al., 2017).C. elegans presents significant points of interest for research in comparison with other model organisms (Morelli et al., 2014), such as the convenience of use, large brood size, practicality, and ease of handling, as well as reduced costs from the perspective of research, rapid life cycle, and the ability to simulate the majority of human diseases (Zhang et al., 2020).Furthermore, genomic comparison studies of C. elegans and humans have revealed the presence of a large number of genes associated with major human diseases, and disease pathways (Kim et al., 2018).Also, the studies involving comparative proteomic analysis of close to 18,452 protein sequences from C. elegans have further revealed the existence of human gene analogs for approximately 83% of the worm's proteome (Kim et al., 2018), and as a consequence, humans and C. elegans share many conserved cellular pathways, and processes in the context of human biology and disease (Poupet et al., 2020).
Bacteria serve as the primary nutrition source for C. elegans in both natural as well as laboratory settings (Gottschling et al., 2019).Various species of bacteria serve as the dietary source for the worm, in natural settings, such as Pseudomonas medocina, Comomonas sp., Bacillus megaterium (soil), Acetobacter sp., Gluconobacter sp. and Enterobacter sp.(rotting fruits) (Montalvo-Katz et al., 2013).The dietary choices of C. elegans in the laboratory include more commonly Escherichia coli strain OP50, a uracil auxotroph, and to a lesser extent, the E. coli wild-type K12 strain, and its derivative strains, HB115 (D3), and HB101 (Zečić et al., 2019).The protein-carbohydrate-fat (energy %) relationship in this bacterial diet corresponds to about 80:10:10, and therefore the diet of C. elegans is essentially protein-rich, albeit lipid-, and carbohydratepoor (Gottschling et al., 2019).
Until recently, aging was not regarded as an actively regulated process (Uno and Nishida, 2016).However, gene-regulated signaling pathways have been attributed to playing a significant role in the lifespan extension of various organisms, including C. elegans (Dall and Faergeman, 2019).For instance, the mutations that reduce the activity of DAF-2, the insulin/insulin-like growth factor-1 (IGF-1) homolog of C. elegans, almost double the lifespan of the animal (Wrigley et al., 2017).Similarly, the mutations affecting the activity of AGE-1, the insulin/insulin-like growth factor signaling (IIS) downstream target, have also been associated with increasing the lifespan of C. elegans (Altintas et al., 2016).Likewise, the inhibition of the Target of Rapamycin (TOR) signaling pathway has also been shown to increase the longevity in C. elegans by regulating the process of autophagy, a significant factor in the lifespan extension of the worm (Bjedov and Rallis, 2020).
Sirtuins are a family of nicotinamide adenine dinucleotide (NAD)-dependent protein deacetylase molecules with a direct association with NAD + -aided cellular nutrient signaling, and SIR-2.1 (the C. elegans homolog of Sir2), by virtue of its overexpression, has been outlined as a factor in lifespan extension in C. elegans (Zhao et al., 2020).Mediation of oxidative stress induced by reactive oxidative species (ROS) that are responsible for instigating damage in various biomolecules, for instance, DNA, lipids, and proteins, as well as promoting the deterioration of cellular structures, tissues, and, ultimately, the whole organism, is another avenue for regulation of aging-related phenomena, and consequently, lifespan extension in C. elegans (Hajam et al., 2022).In this regard, a recent study investigated the antioxidant effects of carotenoid pigments in C. elegans, whereby carotenoids imparted lifespan extension characteristics through inhibition of ROS by upregulating the expression of catalase and superoxide dismutase (SOD) (Lee et al., 2022).Similarly, another study demonstrated the anti-aging effects of saponin-rich extracts in C. elegans, whereby the extracts improved the lifespan of the worm, improved worm growth, enhanced stress resistance, and reduced biomarkers associated with aging (Kitisin et al., 2019).Future studies are aimed at identifying the genes that provide a link between various environmental factors, such as diet, environmental temperature, as well as oxidative status, and lifespan regulation in C. elegans worms (Uno and Nishida, 2016).
The hydrophobicity of lipid triglycerides (TGs), however, restricts their utilization as dietary components for C. elegans (Watts and Ristow, 2017).Potential strategies to overcome this impediment are a mechanical scattering of TGs, partial solvency of TGs in dimethyl sulfoxide (DMSO), TGs suspended in an E. coli OP50 stock, and scattering of TG nanoemulsions (NEs) in media.Mechanical scattering of TGs in hydrophilic media, without the inclusion of a surfactant, may bring about creaming/sedimentation of TGs (Rodrigues, Salgueiro, Bianchini et al., 2018).Furthermore, the hydrophobicity of TGs impairs their dissolvability in DMSO or other hydrophilic solvents, and the scattering of TGs in E. coli OP50 suspension may enzymatically alter them by way of the activity of E. coli layer lipases (Colmenares et al., 2016) before being consumed.
Consequently, NEs are a better alternative by way of incorporation of lipids into their compositional matrix, and, therefore, are naturally more suitable for delivering TGs into the C. elegans.NEs have increasingly been investigated as highly capable and efficient vehicles for the delivery of water-insoluble bioactives, such as lipidsoluble vitamins, omega-6 (ω-6) oils, and β-carotenoids (Dahlawi et al., 2020).Likewise, the concept of 'nanobait' has been explored, whereby C. elegans worms were fed nanocoated microbial cells, including E. coli, and the microalgae, Chlorella pyrenoidosa, via pharyngeal pumping, indicating an efficient delivery mechanism (Däwlätşina et al., 2013).
In this research, C. elegans were fed on two bacterial pathogens, S. enterica serovar Typhimurium, and Staphylococcus aureus.Caenorhabditis elegans consumes these pathogens by way of pharyngeal pumping, whereby the rate of pumping is regulated as per the availability of food sources (Ishita et al., 2020).Furthermore, C. elegans worms have special feeding behavior from the perspective of the water-loving nature of their preferred growth medium (Dimov and Maduro, 2019).Owing to their hydrophilic nature, bioactive components are rarely soluble in the growth media for C. elegans.To combat this limitation, a food-grade delivery system is vital.Food-grade NEs, therefore, could be a viable solution, allowing for the lipid component to be consumed and/or digested by the worms (Colmenares et al., 2016).This also provides an excellent opportunity in the context of potential application for developing antiaging mechanisms, by way of optimizing the delivery of bioactive substances capable of modulating molecular markers associated with the process of aging (Hernandez et al., 2020).

Materials and methods
Sunflower oil was purchased from the local market in Lahore, Pakistan.While Hi-Cap 100 (modified starch) was donated by Ingredion (Germany GmbH).For C. elegans Nematode Growth Medium (NGM) was prepared in the laboratory.HI-CAP 100 is an OSAmodified straight-chain starch and is highly suitable for encapsulation applications, in particular for the encapsulation of bioactive such as vitamins, flavor compounds, spices, clouds, as well as fatty esters, at high oil loading.NEs were prepared in two steps by using the highpressure homogenizer method described by Iqbal et al., 2020) with slight modifications.A coarse emulsion was prepared by combining the two phases (oil and aqueous).HI-CAP 100 5% (w/v) was dispersed with distilled water to generate the aqueous phase, while sunflower oil 10% (v/v) was utilized for the oil phase.Homogenization was achieved with the aid of a high throughput homogenizer (Troemner Talboys, Thorofare, NJ, USA) at 6000 rpm for 5 mins.The coarse emulsions were subjected to sonication (Ultra-Sonics 250W) at 25°C for 5 mins, followed by passage through a high-pressure disruption system (Constant Systems Ltd, Northamptonshire, UK) for 3 passes at 275 MPa and 25°C (Iqbal et al., 2020).

Assessment of physical and chemical stability of nanoemulsions 2.2.1 Droplet size analysis
The measurement of the droplet size of the oil-inwater (O/W) NEs was achieved by way of dynamic light scattering (DLS) utilizing a Malvern Zetasizer Nano ZS90 (Malvern Instruments Inc., Worcestershire, UK).The refractive index obtained for NEs was 1.35.The NEs were subjected to 50 dilutions prior to the measurements.The readings were recorded in triplicates.

Measurement of zeta (ζ)-potential
A Malvern Zetasizer Nano ZS90 (Malvern Instruments Inc., Worcestershire, UK) was used for the measurement of electric charge on the surface of the NEs (25°C, 3.9V).Samples were subjected to 50 dilutions using double-distilled water (Rodrigues, Diniz, Sousa et al., 2018).The readings were recorded in triplicates.

Effect of pH
The human digestive system experiences varying pH values (Beasley et al., 2015).Accordingly, the NEs were stability tested at different pH levels (2, 5, and 7) using 1N hydrochloric acid (HCl), followed by storage at ambient temperature for 1h, before analysis was initiated (Iqbal et al., 2020).

Effect of thermal treatments
NE samples were collected in an Eppendorf tube (1.5mL).Samples were subjected to two thermal treatments by placement in a water bath (30 mins, 63°C, and 10 mins, 95°C), followed by cooling at ambient temperature for 60 mins, and subsequent measurement for mean particle size (Iqbal et al., 2020, Khalid et al., 2017).

Effect of NaCl
The stability of NEs at varying salt concentrations was tested by collecting NEs in a beaker, NaCl concentration adjustments were made between 0.1M and 1M through the addition of NaCl solution.Samples were subjected to gentle mixing, with subsequent storage at ambient temperature for 60 mins prior to analysis (Khalid et al., 2017).

Storage stability of nanoemulsions
The storage stability of NEs was measured by way of centrifuging NEs at 1300×g for 30 mins at 5°C.The NEs were later stored at ambient temperature for 5 months at pH 7. The measurement of the mean particle size of stored NEs was undertaken on a monthly basis (Iqbal et al., 2020).

Application of nanoemulsions on Caenorhabditis elegans 2.3.1 Culturing of Caenorhabditis elegans with Staphylococcus aureus
C. elegans were procured from the collection maintained by the departmental laboratory.According to the method devised by Stiernagle, ( 2006), the NGM plates were prepared and seeded with S. aureus (100 μL) instead of E. coli OP-50 (as a food source for worms).Worms were then transferred to NGM plates for initiation of the growth of worms.

Culturing of Caenorhabditis elegans with
Salmonella enterica serovar Typhimurium C. elegans were procured from the collection maintained by the departmental laboratory.Per the method devised by Stiernagle (2006), the NGM plates were prepared and seeded with S. enterica ser.Typhimurium (100 μL) instead of E. coli OP-50 (as a food source for worms).The worms were once again transferred to NGM plates for propagation.

Lifespan assay of Caenorhabditis elegans with nanoemulsions
The survival lifespan of C. elegans worms was assayed using the protocol outlined by Sutphin and Kaeberlein (2009) with slight modifications.A total of twenty-five worms of identical size were selected and transferred to new culture plates, followed by inoculation of the NEs (100 µL).The preparation of the control plate involved the addition of worms without inoculation with NEs.For the observations related to survival rates, after every 2 days, the worms were transferred to new culture plates with identical conditions outlined above.), and the results for survival analysis were recorded.

Characterization of nanoemulsions
NEs were formulated by using 5% (w/w) HI-CAP 100 as a natural emulsifier containing 10% (w/w) sunflower oil. Figure 1 shows that O/W NEs stabilized with HI-CAP 100, exhibited slight monomodal droplet size distribution, and a mean droplet average diameter (d av ) of 170.2±9.3 nm.Furthermore, the NEs recorded a higher value of zeta potential -28.3±0.2 with a lower value of the polydispersity index (PDI), i.e. 0.325.It has been reported that the relatively lower value of the PDI is attributed to the higher stability of NEs during prolonged storage (Sari et al., 2015).

Effect of pH
The NEs demonstrated relative stability following changes in pH from 2 to 7, albeit with a slight increase in the d av , and without any marked separation for NEs as compared to the control (Figure 2a).Also, the pH modifications had a minimal effect on the droplet size of NEs.However, in the case of ζ-potential, NEs exhibited significant changes, in particular at pH 2 (Figure 2b).As the decrease in pH consequently increases the concentration of hydrogen ions (H + ), this, in turn, induces a net positive charge on NE droplets, ultimately attributable to the negatively charged carboxylic groups of the modified starch molecules (Abbas et al., 2014).In the context of food systems and the human digestive system in general, the pH value does not go below 2, as is evident from the findings associated with the stability of NEs over a pH range of 2-7 in current research.

Effect of temperature
In order to simulate real-world scenarios, the temperature ranges commonly employed during processing operations in the food and beverage industry were used in current research for appraising the instability of NEs and any physicochemical changes brought on by temperature modifications.Fairly significant changes in both d av and PDI of the formulated food-grade emulsions were observed when these NEs were exposed to two different temperatures, i.e., pasteurization (63 o C for 30 mins), and near-boiling point (95 o C for 10 mins) (Figure 3a).Regarding d av , at 63 o C, a small increase was recorded, with the increase much more marked at 95 o C, which can be attributed to possible thinning of the emulsifier interface surrounding the droplet, and droplet aggregation as a consequence of weakening of the emulsifier interface respectively (Banasaz et al., 2020).NEs formulated with synthetic emulsifiers have shown greater stability at elevated temperatures as compared to protein emulsifiers or surfactants, which tend to undergo coagulation when subjected to higher temperatures (Sari et al., 2015) proteins get denatured, and consequently unfold upon exposure to elevated temperatures (Lapidus, 2017), thereby also exposing the sulfhydryl and hydrophobic groups, which, in turn, induces aggregation of proteins (Andlinger et al., 2021) and ultimately results in destabilizing the NEs.In this regard, the NEs stabilized with modified starches have exhibited improved stability at elevated temperatures (Zhao et al., 2019).
Additionally, the ζ-potential of the formulated NEs decreased significantly at 95 o C (Figure 3b), indicating a possible weakening of the emulsion interface, and subsequent release of ions from the interface.

Effect of ionic strength
Salt (sodium chloride/NaCl) and their associated ingredients are essential components of the food and beverage formulations, and therefore, have the tendency to influence both the functional performance as well as the stability of the delivery systems, pre, or postingestion (Aswathanarayan and Vittal 2019).Keeping this in view, the effect of various NaCl concentrations on the stability of NEs was investigated.As can be evident from Figure 4a, the effect of increasing NaCl concentrations had a nominal effect on the d av of the NEs, whereby a slight increase in the d av values was observed as the concentration of NaCl was increased.The increase in NaCl concentration, however, induced a decrease in the magnitude of the negative charges for the formulated NEs (Figure 4b).This decrease might be attributable to the weakening of the electrostatic interactions existing in the NEs, arguably owing to the electrostatic screening effects (McClements, 2018).Interestingly, however, despite the decrease in the ζpotential, and consequently, the magnitude of negative charges, the NEs remained stable.This can be alluded that the magnitude of negative charges on the emulsion droplets contributed to the maintenance of the electrostatic interactions in the presence of NaCl, bringing about an overall charge stabilization effect, and, therefore, induced stability of NEs by suppressing Ostwald ripening.Another factor that could have significantly contributed toward the stability of NEs in this study is the steric hindrance induced by the modified starch (HI-Cap 100).

Long-term stability
One of the most common problems associated with NE formulations is their instability against various factors (de Oca-Ávalos et al., 2017), particularly, concerning their application and storage for extended periods (Barkat et al., 2020).Some of the major mechanisms responsible for inducing instability in NEs include gravitational separation (sedimentation or creaming), coalescence, flocculation, Ostwald ripening, and phase separation (Liu et al., 2019;Sheth et al., 2020).The formulated NEs in this research were found to be stable for a duration of 5 months (Figure 5) and were repeatedly characterized for centrifugation and particle-size distribution (PSD) throughout the duration of storage.

Lifespan assessment
From Figure 6a, it was evident that the lifespan of C. elegans in the presence of S. enterica ser.Typhimurium Typhimurium and other S. enterica serovars are pathogenic and ultimately, lethal for C. elegans, as reported in various research studies (Knodler, 2015).Furthermore, it can be noted that there was a gradual decline in the growth of C. elegans from a 100% survival rate on day 1 with only 20% surviving on day 10.In contrast to these results, the addition of an NE delivery system significantly increased the lifespan of C. elegans, with the worms' survival rate under stress showing marked improvement and increasing from the previously established 11 days (initial findings of the study) to 26 days (post-NE feeding) in current research.The presence of NEs, therefore, aided C. elegans to survive for an additional 15 days under stress conditions, thereby hinting at the anti-aging attributes imparted by NE formulations.This anti-aging can be attributed to the phenomenon of autophagy, a major lifespan determinant in various longevity models.Aging can be regarded as the progressive failure of cellular repair mechanisms over time, contributing to an increase in cellular and molecular damage, and accumulation of aberrant macromolecules, ultimately culminating in the loss of function (Gelino and Hansen, 2013).In contrast, autophagy has been shown to mitigate various stressinduced scenarios (nutrient deprivation, hypoxia and hypothermia) by way of recycling the damaged cellular components (the autophagic cargo) into re-usable construction materials for the synthesis of proteins, and cellular repair (Chun and Kim, 2018).The TOR signaling pathway is another major factor that affects aging, and its nutrient-dependent activation induces a pronounced metabolic shift towards cell proliferation (Bjedov and Rallis, 2020).In the case of C. elegans, the nutrient sensor TOR homolog, let-363, negatively regulates autophagy (Blackwell et al., 2019).This regulation is induced at the transcriptional level as well, through inhibition of the nuclear translocation of transcription factors such as PHA-4/FOXA, DAF-16/ FOXO, and HLH-30/TFEB, responsible for increasing the expression of genes associated with autophagy and lysosomes, factors vital for extension of lifespan in C. elegans (Denzel et al., 2019).Transcription factor HLH-30/TFEB also plays its role in the regulation of genes responsible for lipid metabolism, implying that under nutrient-stressed conditions, the survival of an organism is reliant on the synergistic coordination between the pathways regulating autophagy and lipid metabolism (Zhang et al., 2020).This relationship could be a crucial factor in contributing toward lifespan extension in C. elegans, given that the food-grade delivery systems evaluated in this research study are sunflower oil-based NEs, aimed at the provision of lipids to the worm.
The NEs in this research study showed excellent results for C. elegans in terms of enhanced survival rates when fed on S. aureus.The lifespan of C. elegans, in this case, increased from 15 days to 28 days under stress conditions (Figure 6b).The probable reason for improved survival can be attributed to the increased solubility of lipids manifested by NE formulations administered to the worms (Banasaz et al., 2020).Previous research has indicated that C. elegans can ingest lipid nanoparticles and researchers have confirmed that due to the small particle size of NEs, worms were able to easily ingest the NE formulations (Shen et al., 2019).Lipid-based NEs can easily be metabolized by the lipase enzymes due to their large surface area (Mehanna and Mneimneh 2021) resulting in the formation of omega (ω)-6 fatty acids.These lipase-generated ω-6 fatty acids have been shown to promote autophagy, thereby contributing to an increased lifespan in C. elegans (Seah et al., 2016).This study augments the previous research studies (Mokoena et al., 2020;O'Rourke et al., 2013) in this regard, and proposes that supplementation of these ω -6 fatty acids to C. elegans or humans can increase the lifespan of organisms.

Conclusion
Genomic comparison studies indicate that a large number of genes from the human genome have homologs in C. elegans.Similarly, the presence in C. elegans, of the genes associated with human diseases, and their respective pathways, proves that the worm can be utilized as a key model system for studies related to

Figure 2 .
Figure 2. Effect of pH on the stability of sunflower oil NEs (a) droplet size (b) ζ -potential.