Sugar esters of aliphatic nonanoic acid along with a mixture of (R)-3-hydroxylated nonanoic acid and heptanoic acids (mPHN, derived from PHN) of glucose, galactose and lactose were obtained and characterized as described in our previous works. In search of effective anticancer agents—novel sugar esters based on polyhydroxyalkanoate Monomers (Snoch et al., 2021). The composition of new synthesized compounds is presented in Table 1, the yields and their purity are presented in Supplementary Table S1.

The surface tension has been measured for different batches of synthetized compounds by using pendant drop shape analysis method by two apparatus. First apparatus: a home-made experimental set-up described in detail in (Para et al., 2006), with experimental error equals to 2 mN/m, was used. The Young–Laplace capillary equation was fitted to the digitally recorded drop image. Measured surface tension value corresponds to the value of the best fit (note: it is as the only unknown parameter in this equation). The dynamic surface tension measurements were performed every 5 s. Measured equilibrium surface tension corresponded to the obtained steady-state time after adsorption, which was depended on surfactant concentration. The studied solutions of surfactant were mixtures of sugars mono and diesters, which can differ by number of hydrophobic hydrocarbon chains. As a consequence, the kinetic curves for different drops of the same solution did not often overlap. That is why the experimental values for one solution are the mean values from all the recorder dynamic curves. As second apparatus a commercial tensiometer (PAT–1M, Sinterface, Berlin, Germany), with experimental error of 0.2 mN/m, was used. (Kairaliyeva et al., 2017). The PAT-1M apparatus allows for an accurate control of the droplet area (or its volume) by a syringe pump, driven by a feedback loop software based on the drop imaging. The dynamic interfacial tension versus time on a freshly formed drop is measured during the ageing of the interface while keeping the drop volume constant (11 µL). The equilibrium interfacial tensions are obtained from these data at long period of time. The greatest experimental error is mainly connected with small differences in composition from batch to batch so we present all experimental points and determined the critical aggregation concentration (CAC). All surface tension measurements were performed at 295 K. For all the experiments, ultrapure water—produced from the Millipore Direct-Q ® 5UV purification system (18 MΩ cm⁻¹), was used. Its surface tension measurement provides a value of 72.5 ± 0.2 mN/m, stable for at least 2 hours at 20°C meaning negligible amount of surface-active impurities.

Hydrophilic-Lipophilic Balance was determined according Griffin method by using following equation (Griffin W.C., 1954) H L B = 20   x   h y d r o p h i l i c   g r o u p   m o l e c u l a r   w e i g h t t o t a l   s u r f a c t a n t   m o l e c u l a r   w e i g h t (1)

The ability of the SFAE to stabilize water–oil (W/O) systems was estimated by measuring time of phase separation in each performed emulsion. The experiment was conducted under following protocol: 5 g of each SFAE solutions were prepared as water phase and mixed with 0.5 g of oil phase: squalane or popular commercially available skincare oil- Bambino^® respectively. Concentrations of the SFAE in solutions were: 0.5 × CAC, 1.0 × CAC 1.5 × CAC respectively. The skincare oil consisted of: glycine soya oil, paraffinum liquidum, parfum, ethyl linolate, ethyl oleate, tocopherol, propylene glycol, propyl galate, citric acid, BHA, according to the manufacturer label in the unknown proportions. The mixtures were mixed in shaker at 25 C for 20 min and mixed vigorously with Vortex for another 1 min to ensure the phases were mixed sufficiently (An et al., 2019; Li and Xiang, 2019).

Potential environmental impact of the investigated compounds was tested by treating Caenorhabditis elegans according to following protocol adapted from WormBook (Stiernagle, 2006) and (Djapovic et al., 2021). Briefly, synchronized worms (L4 stage) were suspended in a medium containing 95% M9 buffer (3.0 g of KH₂PO₄, 6.0 g of Na₂HPO₄, 5.0 g of NaCl, and 1 mL of 1 mol L^–1 MgSO₄ × 7 H₂O in 1 L of water), 5% LB broth (10 g L^–1 tryptone, 5 g L^–1 yeast extract, and 10 g L^–1NaCl), and 10 μg mL^–1 of cholesterol. The experiment was carried out in 96-well flat-bottomed microtiter plates (Sarstedt, Nümbrecht, Germany) in the final volume of 100 μL per well. Suspension of nematodes (25 μL containing 25–35 nematodes) was transferred to the wells of a 96-well microtiter plate, where 50 μL of the medium was previously added. Next, 25 μL of a solvent control (DMSO) or 25 μL of a concentrated solution was added to the test wells. The examined esters were dissolved in DMSO to obtain stock solutions than added to the wells with worms. The final concentrations of the compounds were 2.0, 1.5, 1.0, 0.5, 0.25 and 0.125 mg mL^–1. The final concentration of DMSO in each well was 1% (v/v) Subsequently, the plates were incubated at 25 C for 2 days. The fraction of dead worms was determined after 48 h by counting the number of dead worms and the total number of worms in each well, using a stereomicroscope (SMZ143-N2GG, Motic, Wetzlar, Germany). As a negative control experiment, nematodes were exposed to the medium containing 1% (v/v) DMSO.

Basing on a series of 5-min films and images taken up to 48 h of water/oil systems containing different concentrations of the tested SFAEs, it was not possible to measure the thickness of the emulsion layer and thus determine the stability index of the emulsion (Supplementary Figure S2). This was due to the short lifetime of the emulsion. However, depending on the type of ester, its concentration, and the composition of the oil phase, emulsion systems formed and maintained from several minutes to 1 hour (Table 3.). Emulsions containing squalene were less stable. W/O control systems containing no esters blurred after only a few minutes. On the other hand, systems containing SFAEs based on body care oil turned out to be more durable, as they had a diverse composition and contained co-surfactants such as alcohols. Furthermore, in most cases, differences in the durability and consistency of emulsions can be observed. They depend on the concentration of SFAEs in aqueous solutions. The higher the SFAE concentration, the more stable and homogeneous the system was. Systems containing C9-glu and C9-gal proved to be the most stable. The least stable system contained C9-lac. Emulsions based on mPHN-glu and mPHN-gal esters proved to be less stable than those originated from nonanoic acid. In contrast, the emulsions containing mPHN-lac exhibited a higher persistence than mPHN-glu, mPHN-gal and C9-lac. The maximum lifetime of the investigated homogeneous emulsion systems (Table 3 emulsion quality assessment 3, Supplementary Figure S2) was 60 min. These were systems containing a commercial baby care oil. However, some remaining emulsions were visible and even more stable: 180 min for mPHN-glu, mPHN-gal and mPHN-lac to 1440 min for C9-glu, C9-gal, C9-lac. In W/O systems that contained squalane, after 60 min, the quality rating were 2 or 1, respectively. These lifetimes are definitely too short to make them competitive against other surfactants used in the industry, such as Tween 80, Triton-X, Span 20, Span 60, polyethylene glycol (PEG). However, noteworthy is that commercially available products very often contain combinations of various surfactants e.g. Tween 80- polyoxoethylene sorbitan monooleate or Triton X-100-consisted of PEG and p-tert-octylphenol. Usually investigated W/O systems are based on combinations of two or more different ionic and/or nonionic surfactants and other co-surfactants i.e. alcohols and fatty acids, that make together the emulsions even more stable and extend their lifetime. (Watanabe et al., 2018). (Li and Friberg, 1982; McClements and Jafari, 2018). This approach should be investigated with our biocatalytically synthetized SFAE.

Although the expected lifetime of a commercially acceptable emulsion should be counted in days up to 1 month, the prepared here W/O systems did not perform well more than 24 h. We can explain this either by poor stabilizing properties of the SFAEs or their too-low concentrations used in preparation W/O systems, as well as lack of other co-surfactants. The calculated concentrations of the investigated surfactants present in the emulsions were between 0.001% and 1.0% m/v respectively (from 0.5 to 1.5 x CAC) while usually used concentrations of surfactants from the literature are between 0.1% and 5.0% m/v (Feng et al., 2022) but rarely calculated on their CAC or CMC values. That makes our system difficult to compare. Moreover, literature mentions several different techniques used for emulsion preparation i.e. shaking, ultrasound mixing, microwave pulsing, (Taha et al., 2020; Hyde et al., 2021), magnetic mixing, high pressure homogenization (Li and Xiang, 2019), and syringe mixing (Koursari et al., 2020). In recent years, even use of solid stabilizing particles (Pickering emulsions) became more popular. Nanomaterials derived from natural sources are an interesting alternative or supplementary for this application. (Velásquez-Cock et al., 2021). In order to compare our surfactants for their feasibility in applications other than described below (drug delivery purposes), studies should be conducted in greater concentrations and with other additives.

The ideal W/O system used in the cosmetic or pharmaceutical industry should arise from natural sources, and also should be able to form stable and durable emulsions. Emulsions need to be stable enough to be stored at room temperature. Most importantly, they should be biodegradable and biocompatible. When it comes to drug delivery systems, their life time longer than 48–72 h is not advisable either. Another crucial factor is emulsion bioaccessibility. That means easy absorption by the epithelial surfaces, passing through cell membranes but not damaging them. (Production of green surfactants: Market prospects | Elsevier Enhanced Reader) The micelles protecting the structure of the drug from enzymes and/or pH changes should be able to release it gradually to the tissues. (Felzenszwalb et al., 2019). The micelle-building components ought to be easily degraded or removed from the body and easily decomposed in the environment. (Tovar-Sanchez et al., 2020) (Hunt, 2017) (Fagan and Portman, 2014). Undoubtedly, the studied SFAEs show some emulsion-stabilizing properties, but in order to give these W/O systems the desired longer lifespan, further optimization of SFAEs concentration, oil phase composition, mixing methods and addition of co-surfactants is required. (McCartney et al., 2019). The emulsion stabilizing properties of the investigated SFAE may not be spectacular, nevertheless sometimes desired if there is a need of administration of a less stable formulation to a patient prepared minutes prior injection/topical application. The used SFAE were already shown to exhibit anticancer properties, which increase their possibility to be applied in the medical industry (Tuvia et al., 2014).

Caenorhabditis elegans is a multicellular, non-parasitic model organism that is a valuable research object for testing the effects of various industrial chemicals such as anti-cancer drugs and antibiotics (Judy et al., 2019; Wittkowski et al., 2019). Every year, mankind supplies a huge amount of industrial wastewater and with it–surfactants (Production of green surfactants: Market prospects | Elsevier Enhanced Reader; Akbari et al., 2018; Felzenszwalb et al., 2019; Tovar-Sanchez et al., 2020). Therefore, there is a need for continuous monitoring of their impact on organisms living in soil and groundwaters (Ebele et al., 2017; Rathi et al., 2021). The usefulness of nematodes representatives is manifested in a fast life cycle, easy multiplication and obtaining a large number of individuals in subsequent generations, the possibility of long–term storage of larvae and eggs in laboratory conditions. They do not require continuous breeding, and the procedure for synchronizing the life cycles of individuals in a population is simple. In addition, Caenorhabditis elegans feed on an easily available source of food–bacteria E. coli. The most important from the human point of view, is the presence of simple organ systems: nervous (nerve ring), blood, protonfridial, gonads, and the ability to assess not only the size of the population under the microscope, but also the ability to actively move individuals in the population. Therefore, nematodes can be indirect bioindicators of the influence of the tested substances on the natural environment (Fagan and Portman, 2014; Hunt, 2017). Having the above in mind, toxicity of the tested sugar esters against Caenorhabditis elegans was assessed by observing the nematodes under a microscope after 48 h of exposure. Based on the mobility of Caenorhabditis elegans their viability was assessed. Actively moving organisms were considered living, non-moving organisms were considered dead, and barely moving organisms were considered alive as well (having in mind, that the compounds could have a negative effect on worms) (Figure 4).

Observations of the nematodes allowed us to conclude that the obtained SFAEs do not pose a major threat to Caenorhabditis elegans at the given time of exposure. Only the highest concentrations (2.0 mg ml^–1) of the mixtures reduced nematodes populations up to 15% but 1.5 mg ml^–1 was already not effective. The concentrations of the esters per the well ranged from 0.0625 to 2.0 mg ml^–1 which correspond to their molar concentrations of: C9-glu 0.186.14–5.69 mmol L^–1; C9-gal 0.162–5.312 mmol L^–1; C9-lac 0.125–4.0 mmol L^–1; mPHN-glu 0.142–4.544 mmol L^–1; mPHN-gal 0.148–4.744 mmol L^–1; mPHN-lac 0.119–3.816 mmol L^–1, respectively. We have previously reported that SFAE esters based on nonanoic acid and PHN monomers show anticancer potential. The reported consternations (IC₅₀) of these compounds that were found to be effective against certain cancer lines (In Search of Effective Anticancer Agents—Novel Sugar Esters Based on Polyhydroxyalkanoate Monomers-W. Snoch et al., 2021. pdf) are at the levels of being non-toxic to Caenorhabditis elegans. For example, the effective IC₅₀ for Du145 and HTB140 cell lines after 24 h exposures to all tested SFAE were below 0.25 mg ml^–1 (i.e. IC₅₀ range respectively 0.09–1.5 mmol L^–1for cancer cells and 0.5–2.5 mmol L^–1, respectively for reference healthy cells).

Sugar esters belong to the group of non-ionic surfactants. The presence of a hydrophobic fatty acid tail and a hydrophilic sugar head give them an amphiphilic character. The chemical structure itself does not, in general, pose a direct threat to the entire organism of nematode. However, this amphiphilicity and the ability of compounds to lower the surface tension of solutions may permeabilize cell membranes (Tuvia et al., 2014; McCartney et al., 2019). On the other hand, the presence of long carbon chains (such as c18 steric acid) increases the solubility of individual compounds in water (e.g. in industrial wastewaters) (Vinarov et al., 2018). All this together causes the entry of undesirable substances from the environment into the cells, an increase in the concentration of H⁺ ions, free oxygen radicals, herbicides, pesticides or salts (which can cause an osmotic shock), which in turn may direct the cells to the apoptotic pathway (Shao et al., 2018; Cavanagh et al., 2019; Appah et al., 2020; Liu et al., 2021; Martins-Gomes et al., 2022). In addition, the hydrophobic components of the SFAE tested do not exceed 9 carbon atoms, which may be another advantage compared to other commonly used esters based on acids with a chain length of c12, c16 or c18 (e.g. Span 20, Span 80, PEG 20– Sorbitan monolaurate (SPEG), Tween 20, Tween 80). Since increasing the chain length of the hydrophobic component also increases the toxicity of SFAE (Bunchongprasert and Shao, 2020). For comparison, the toxicity of other structurally similar allose-based esters to Caenorhabditis elegans with various carbon chain lengths n = 2, 4, 6, 8 was in the between 0.2 and 1.0 mmol L^–1 (Sakoguchi et al., 2019).

In addition, every cosmetic or drug carrier potentially used in the industry must be thoroughly tested in terms of toxicology, before it goes to preclinical research (the smallest, largest harmful dose, and chronic administration of the compound), at every possible stage, not only cellular, but also more complex living organisms (Dent et al., 2018; Bopp et al., 2019). The highest tested levels of the SFAE (2.0 mg ml^–1 around 6.0 mmol L^–1) that reduced Caenorhabditis elegans populations ∼15% were near to CMC values. At the same time, the range of surfactants concentrations used in the water phase, to design model emulsion systems, were 0.5–10-fold CAC. Information obtained about minor negative effects of the esters on tested nematode in this concentration range opens the possibility for further application trials. However, it should be remembered that the SFAE molecules building micelles have a different concentration and organization than those dispersed in the buffers in which the Caenorhabditis elegans were used. They may behave differently and show a different level of toxicity, therefore more detailed investigation is foreseen for the future (Khamkar, 2011; Kaur et al., 2016; Rusanov, 2018; Bunchongprasert and Shao, 2020).