2.2. Antimicrobial Activity
The minimum inhibitory concentration (MIC; the concentration of gemini surfactant required to completely inhibit the growth of the microorganisms) against representative Gram-positive and Gram-negative bacteria was determined. The Gram-positive bacteria tested include some problematic microorganisms such us
L. monocytogenes; these bacteria cause listeriosis, a serious disease that has grown considerably in industrialized countries due to the increased ratio of immunosuppressed persons and the extensive use of refrigerated food. We have also tested the effectivity of these surfactants against methicillin-resistant
Staphylococcus aureus (MRSA), which is known as a major cause of hospital-acquired infections and, at this time, is a remarkable public health problem.
Figure 2 shows the MIC values for the gemini surfactants in which the alkyl chain and the spacer chain have been systematically varied. For comparison, this figure also includes the MIC values of a LAM (lauroyl arginine methyl ester) surfactant that can be considered the single-stranded homologue of these geminis and the BAC (benzalkonium ammonium chloride), a quaternary ammonium surfactant widely used as antiseptic.
These gemini surfactants show good antimicrobial activity against a wide spectrum of microorganisms. As expected, the activity depends on their molecular structure, number of carbon atoms in their hydrophobic moiety and in the spacer chain. Our results indicate that these amino acid-based surfactants exhibit a greater effectivity against Gram-positive bacteria. This behavior is common for antimicrobials exhibiting a detergent-like mechanism [
20]. The target of these compounds is the bacterial membrane. Given the excellent surface-active properties and the two positive charges present in their structure, it is expected that the mechanism of action of all these bis-Args involves the electrostatic interaction between the two positive charges of the protonated guanidine groups and the negative charges in the bacterial membranes, and then the hydrophobic interaction of the two alkyl chains in the intramembrane region. In fact, it was found that one of these geminis, the C
3(CA)
2, and the gemini-lysine surfactants exhibited this mode of action [
20]. The bacterial membrane of the Gram-negative bacteria has an external layer mainly composed of peptidoglycan, glicerophospholipids and lipopolisaccharides that hampers the interaction of surfactants with the cellular membranes [
21]. As has been mentioned, the mode of action of QACs, widely used biocides, involves the disruption of the bacterial membrane [
22], a fundamental target to bacterial survival. As such, in principle, the development of resistant microorganisms to these antimicrobials seems improbable. However, due to their wide use and their low biodegradability, their accumulation in the environment is inevitable. This means that bacterial populations are exposed to sub-lethal doses of these compounds for long periods of time. For this reason, in recent decades, the development of bacterial resistance to QASs and bis-QACs has increased alarmingly [
23]. The target of bis-Args is also the bacterial membrane, however, these compounds show good biodegradation levels [
24] and, therefore will not accumulate in the environment and, consequently, the development of bacteria resistance to these compounds will be very unlikely.
The bis-Args surfactants with C
10 and C
12 alkyl chains and a C
3 spacer showed better activity than their single counterpart N-lauroyl arginine methyl ester (LAM). Similar results have been already reported for numerous gemini surfactants of the bis-QACs type [
25] and amino acid-based gemini amphiphiles [
20]. The presence of two positive charges and two alkyl chains facilitates the electrostatic and hydrophobic interactions of these antimicrobials with the bacterial membranes. The pKa values of these compounds indicate that they have two positive charges at a physiological pH, which is a crucial characteristic for damaging bacterial membranes. In this regard, molecular dynamic models proved that arginine is the only natural amino acid residue that remains protonated inside the biological membranes [
26]. On the one hand, increasing the cationic charge density of the polar head is one common strategy to improve the antimicrobial efficiency. Haldar et al. [
27] prepared cationic surfactants containing one, two and three trimethylammonium head groups and found that the antimicrobial activity increased as the number of cationic groups increased. On the other hand, for the same cationic charge density, double-chain surfactants exhibit better antimicrobial activity than their corresponding single-chain homologues. For example, the 12-s-12 bis-QACs have the same cationic charge density than single-stranded homologue dodecyltrimethylammonium chloride (DTAB); however, the activity of these bis-QACs against
E. coli and
S. aureus is 2–5 times higher than that of DTAB [
25].
As expected, the antimicrobial activity of these surfactants depends on the alkyl chain length. It is well known that the antimicrobial activity of cationic surfactants does not have a linear dependence with the length of their hydrophobic length and usually exhibits a cut-off effect [
25]. This behavior has been also observed for these gemini surfactants. The compound with the shortest alkyl chain, C
3(OA)
2, showed the lowest antimicrobial efficiency (highest MIC values). The alkyl chains of this surfactant are too short and give rise to weak hydrophobic interactions with the bacterial cell membranes. The incorporation of two methylene groups in the hydrophobic chain C
3(CA)
2 resulted in an important increase in the antimicrobial activity. A further increase in the alkyl chain C
3(LA)
2 produced a slight reduction in their efficacy. These results suggest that for these gemini homologues, the cut-off effect occurs for the C
10–C
12 derivatives; however, for the single chain arginine derivatives, the N-acyl arginine methyl esters, the most efficient surfactants for killing bacteria were the C
12–C
14 homologues [
20]. Usually, the cut-off effect for cationic gemini surfactants occurs at shorter alkyl chains than for their monocatenary homologues. The highest activity of the monocatenary QACs was observed for the C
14–C
16 homologues. However, the MIC of the QAC gemini surfactants reached the minimum value for the C
10–C
12 derivatives [
3]. The same tendency was observed for gemini histidine-based surfactants; in this case, the highest efficiency of the monocaternary homologues was observed for the C
12–C
14 homologues, while the C
10 derivative was the most effective gemini compound [
28]. This behavior could be ascribed to the higher hydrophobic character of the gemini surfactants and their extraordinarily low cmc values.
Figure 2 shows the influence of the spacer chain nature. It can be observed that the incorporation of one OH group in the spacer does not significantly affect the antimicrobial activity and the MIC values of C
3(LA)
2 and C
3(LA)
2OH are similar. However, the elongation of the spacer chain, the C
6 and C
9 derivatives, decreases the efficiency of these compounds. It has been observed that the activity of C
3(LA)
2 is slightly lower than that of C
3(CA)
2, suggesting that the hydrophobic character of this surfactant is in the cut-off limit for antimicrobial activity. Because of that, the C
6 and C
9 homologues with higher hydrophobic contents exhibited a lower efficiency than the C
3 derivative. The research regarding the influence of the spacer in the antimicrobial efficiency is still rather scarce. Zhang et al. found that for the same alkyl chain, the 12-s-12 compounds, the MIC values increased in long spacer chains [
29]. It has been also reported that the incorporation of an extra cationic charge in the spacer of bis-QACs improves the antimicrobial efficacy [
12].
The antimicrobial activity of BAC is similar to that obtained for C3(CA)2 and higher than that observed for all Cn(LA)2. BAC is not a pure cationic surfactant; it is a mixture of alkylbenzyldimethylammonium chlorides with alkyl chains of C8, C10, C12, C14, C16, C18; dodecyl and tetradecyl being the most abundant ones (about 40% C12, 50% C14). The good biocidal activity of this compound is associated with the mixture of C12 and C14 alkyl chain derivatives that gives the compound a very good water solubility. However, this compound is irritating to the skin and eyes and toxic to aquatic organisms. Moreover, BAC shows a low biodegradability; consequently, long-term exposure to this disinfectant can result in a change in microbial community and an increased antimicrobial resistance.
Our obtained results indicate that compound C3(CA)2 was the most active against all tested microorganisms. This surfactant showed very good activity against all Gram-positive and Gram-negative planktonic bacteria, including some problematic microorganisms such as MRSA, L. monocytogenes and P. aeruginosa bacteria that are commonly resistant to several antimicrobial agents.
Antibiofilm Activity
Nowadays, bacterial and fungal biofilms cause more than 65% of microbial infections and are one of the virulence factors promoting the development of resistant microorganisms [
6]. The development of a bacterial or fungal biofilm starts with the adhesion of microorganisms to surfaces. Once the biofilm is formed and matured, it is very hard to eliminate due the presence of the polymeric matrix [
5]. Therefore, it is crucial to design and synthesize effective antimicrobials capable of inhibiting biofilm formation and eradicating mature biofilms. In this work, we have investigated how the length of the alkyl chain and the nature of the spacer affect the ability of these gemini surfactants to inhibit and disrupt MRSA and
P. aeruginosa biofilms.
The ability of MRSA and
P. aeruginosa to form biofilms on propylene surfaces in the presence of bis-Args was determined. The two bacterial cells were kept in contact with the gemini surfactants at different concentrations from 2 to 128 μg/mL for 24 h. Then, the percentage of biofilm inhibition was determined.
Figure 3 shows the effect of the alkyl chain length on the capacity of these compounds to inhibit the growth of MRSA and
P. aeruginosa biofilms. The best activity was found for the compound with C
10 alkyl chains; this compound inhibits around 90% of the biofilm formation of these two bacteria at very low concentrations of around 4–8 μg/mL. By increasing the alkyl chain, the antibiofilm activity slightly decreased and the C
3(LA)
2 is able to inhibit the growth of both microorganisms at 32 μg/mL. A drastic decrease in the antibiofilm effectiveness was found for the gemini with the shortest alkyl chain, C
3(OA)
2; in this case, the biofilm inhibition is only reached at 128 μg/mL.
Figure 3 shows how the nature of the spacer chain affects the biofilm formation in presence of these surfactants. The results indicate that for a C
12 alkyl chain, the antibiofilm activity against MRSA decreases as the hydrophobicity of the molecule increases. The PAO1 biofilm inhibition follows the same behavior and the inhibition occurs at similar gemini concentrations.
All these results are consistent with the tendencies observed in the MIC values. The biofilm inhibition can be produced by two mechanisms: the compounds coat the polystyrene plate via hydrophobic interactions or/and the surfactants kill the planktonic bacteria in the solution. In this case, the inhibition occurs around the MIC values. These results suggest that the main mechanism involved in this inhibition is the interaction of these gemini with the planktonic cells. In this regard, it has been observed that bacteria die rapidly when kept in contact with these gemini surfactants at concentrations ≥MIC, so they cannot form biofilms.
The ability of these geminis to disperse and eradicate biofilms once formed on the propylene surfaces of the microtiter plates was evaluated using the same bacterial strains.
Figure 4 shows the percentage of biofilms eradicated by the tested compounds at concentrations ranging from 4 to 128 μg/mL. The susceptibility of both biofilms against the tested surfactants was similar. At a low concentration of 32 μg/mL, C
3(CA)
2 and C
3(LA)
2 were able to eradicate around 60–70% of preformed biofilm; however, at this concentration, the lowest hydrophobic compound, C
3(OA)
2, eradicated less than 30% of biofilm. At high concentrations, the eradication percentage is similar for all alkyl chains.
Figure 4 shows the spacer chain nature. In this case, at low concentrations, there is not a clear dependence between the antibiofilm properties and the spacer; however, at a high concentration, the best effectivity was obtained for the longer spacer chains.
The bis-Args concentration required to eradicate MRSA and
P. aeruginosa biofilms is higher than that required to inhibit their formation. It has already been observed that usually it is usually more difficult to eradicate biofilms than to inhibit their formation [
30]. At the MIC concentration, cationic surfactants usually inhibit biofilm formation because they kill bacteria. However, at this concentration, these compounds are not active against bacterial cells in mature biofilms [
31]. In this case, the mechanism of action of antimicrobials involves two steps: the disruption of the extracellular polymeric matrix by electrostatic interactions and then the elimination of the dispersed bacteria.
It has been described that monocatenary cationic surfactants based on quaternary ammonium groups show remarkable antibiofilm activities [
30,
32,
33]. The antibiofilm activity of amino acid-based surfactants has been scarcely reported. Tac-seung et al. [
34] found that N-lauroyl arginine ethyl effectively eliminated biofilms from reverse osmosis membranes and Rodriguez Almeida et al. [
35] described small cationic peptide amphiphiles that reduced MRSA biofilm formation up to 69% at MIC (8 μg/mL) and up to 90% at 2ϗMIC, whereas Pinazo et al. demonstrated that single-chain arginine surfactants efficiently eradicate MRSA biofilms at higher values than that observed for these gemini arginine surfactants, at 32 μg/mL. The antibiofilm activity of cationic gemini surfactants is still poorly known. Jenning et al. [
12] found that the presence of two or three cationic charges on the polar head and two-alkyl-chain, bis(QACs) notably enhanced the biofilm-eradicating properties of quaternary ammonium surfactants against
S. aureus and
E. faecalis. The MBEC values obtained for the tested bis(QACs) ranged from 50 to 100 μM. Kozirog et al. found that a 12-6-12 gemini surfactant effectively prevented
A. lannensis biofilm growth in propylene surfaces and is also effective at eradicating biofilm in this bacteria [
31], and Oblak et al. reported that gemini quaternary ammonium salts inhibited the adhesion of
S. epidermis to a polystyrene surface and eradicated biofilm formed by PA01; they found that the best antibiofilm activity was obtained for C
12 derivatives. At 20 μM, these gemini destroyed about 50% of the biofilm [
36]. There are very few reports in the literature describing the antibiofilm activity of gemini surfactants based on amino acids. Alanine-based gemini quaternary ammonium salts with two carbon spacer groups and C
10–C
12 carbon alkyl chains (chlorides and bromides) dislodged the biofilms of
P. aeruginosa and
S. epidermidis and effectively reduced microbial adhesion by coating the polystyrene and silicone surfaces [
37].
2.3. Surface Pressure Molecular Area Isotherms and Elastic Modulus
Aimed at establishing models of surfactant–bacteria interactions, we have studied the interactions between three arginine-based surfactants and three different phospholipids. The studied surfactants included three gemini surfactants, namely C3(LA)2, C6(LA)2 and C9(LA)2. The study of the three geminal surfactants shall allow us to establish the influence of the spacer length on the interactions with phospholipids.
The phospholipids considered were DPPC (Dipalmitoylphosphatidylcholine), DPPE (Dipalmitoylphosphatidylethanolamine) and DPPG (dipalmitoylphosphatidylglycerol). DPPC and DPPG were selected because they are the main constituents of the bacterial membrane’s phospholipids, while DPPE is the main phospholipid in erythrocytes.
The surface pressure molecular area π-A isotherms (Langmuir isotherms) can be used as a tool to obtain information of the interaction between surfactants and phospholipids placed in an air–liquid interface. The shape of the compression isotherm shows the extent to which the surfactant is forced into the bulk subphase. If the surfactant is completely desorbed as the film is compressed, the resulting isotherm would match that of pure phospholipid. Thus, any deviation from this behavior can be attributed to an incomplete desorption of the surfactant [
38].
2.3.1. Langmuir Isotherms
The π-A isotherms for pure phospholipids and pure arginine gemini surfactants, as well as their mixtures, are shown in
Figure 5. Due to this redissolution, the registered areas per molecule of gemini surfactants are smaller than expected. The results were similar to those reported in previous work [
39].
First, we considered mixtures of gemini surfactants with DPPC. The profile of their isotherms corresponds to surfactants with partial solubility. Under compression, part of the molecules located at the interface redissolve either as aggregates or as monomers.
As mentioned, isotherms of pure phospholipid are used as references; hence, they are included within the phospholipid gemini surfactant π-A plots (
Figure 5). The isotherm of pure DPPC fully agrees with that reported in [
40]. It is noticed that mixtures of isotherms appear at areas lower than those of the pure DPPC. The plots shift toward smaller areas, according to the series
These shifts suggest two facts. First, both the length and flexibility of the spacer chain have an influence on the interactions with DPPC. Second, we can expect that in the mixed monolayer, the two components interact to form mixed aggregates which have a solubility higher than pure DPPC, favoring the phospholipid to move towards the bulk phase. C9(LA)2 is the surfactant with the largest spacer chain. The mixture C9(LA)2 -DPPC shows an isotherm almost parallel to that of the DPPC, which collapses at a molecular area slightly lower than that of the DPPC. Since C9(LA)2 has a large and flexible spacer chain, the interaction is favored, the compound remains in the monolayer and the formation and solubilization of mixed aggregates is not extensive.
The surfactant C3(LA)2 has a short spacer chain; thus, when mixed with DPPC, forms mixed aggregates that, under compression, move to the bulk phase and results in a plot shift larger than that of C9(LA)2. The isotherm plots of C3LA-DPPC and C9(LA)2-DPPC are very close to each other. This small shift suggests that both surfactants interact with DPPC through one arginine group. The six carbon atoms included in the spacer chain of C6(LA)2 conform to rigid spatial structures that can interact through its two arginine groups, with the DPPC forming mixed aggregates that move to the bulk phase, resulting in the largest plot shift.
Mixtures of gemini surfactants with DPPE are also shown in
Figure 5. The pure DPPE isotherm agrees with as is reported in the literature [
40]. The isotherms of three mixtures considered were shifted toward lower areas, according to the series
A rationale for this behavior is that interactions between one of the guanidine groups of the gemini surfactants and DPPE could lead to the formation of cationic aggregates which are soluble in the subphase.
Clearly, isotherms of mixtures with DPPC are different from those with DPPE. Although both phospholipids are zwitterionic, DPPC carries in its polar head a quaternary ammonium group, while DPPE carries an ammonium group. Differences in the effects that phospholipids induce in the mixtures should be mainly attributed to the fact that the DPPC has three methyl groups in its polar head, thus showing a large hydrophobic character.
The interactions of gemini surfactants with an anionic phospholipid, DPPG, have been also studied. Plots of the DPPG/arginine mixture isotherms are shown in
Figure 5. Since DPPG has an anionic charge in its polar head, interactions with arginine cationic surfactants are strong and result in conformational changes in the molecules at the interface, but this does not imply their dissolution in the bulk. The presence of a significant number of molecules at the interface gives rise to high values in collapse pressure.
Isotherms for three mixtures shifted to lower areas according the series
2.3.2. Mechanical Properties of the Mixed Monolayers
The rigidity of a monolayer under pressure depends on the interfacial cohesive forces and can be measured using the elastic modulus: a mechanical concept defined as
where A is the area and π is the pressure exerted on A. The higher the elastic modulus, the greater the material rigidity. Therefore, once the π–A isotherms are known, the elastic modulus E associated with the monolayer can be calculated. Clearly, as the E value associated with the monolayer grows, the monolayer elasticity decreases due to increasing interfacial cohesive forces. Values of E in the range 100–200 m/mN are typical of condensed phases [
41].
To avoid secondary effects, measurements of E have been carried out at π = 10 mN/m and π = 20 mN/m when the monolayers studied are in an expanded phase and structural differences of the mixture components are clearly identified.
Figure 6 shows E values measured for arginine surfactants and their mixtures with DPPC, DPPE and DPPG. Under compression, monolayers of pure gemini arginine surfactants form expanded gas and liquid phases. At π = 10 mN/m, E values correspond to typical values of expanded monolayers for the four surfactants studied. At π = 20 mN/m, the maximum observed value is 80 mN/m, which corresponds to the compound C
6(LA)
2. This value is close to the elasticity values presented by the condensed monolayers [
39]. The E values of the mixtures of these surfactants with DPPC are all around 40 m/mN, which is slightly lower than those of the surfactants alone. Therefore, the mixed monolayer is less compacted, more disordered and more fluid than the pure surfactant monolayer. When compared to the E values of pure DPPC, a slight increase in the mixtures was observed. These values indicate that arginine and DPPC interact to form a mixed monolayer. The values of the modulus of elasticity obtained for the DPPC agree with those previously reported [
42].
Mixtures of C3(LA)2 with DPPC showed E values similar to mixtures with DPPE, while for C6(LA)2 and C9(LA)2, the E values were slightly higher. The E values for DPPE alone are close to 100 m/mN, thus reflecting the formation of a condensed phase monolayer.
These results show that the interactions of arginine-derived surfactants with amphoteric phospholipids depend, on the one hand, on the hydrophobic component of the surfactant and, on the other hand, on the nature of the cationic charge on the polar head of the phospholipid.
The E values of the mixtures with DPPG are lower than those obtained with DPPC and DPPE. The negative charge on the polar head of DPPG forms an ion pair with the positive charge of the guanidino group present in arginine-derived surfactants. The values obtained for C6(LA)2 suggest that it is only one guanidino group of a molecule that interacts with DPPG. C3(LA)2 and C6(LA)2 show smaller values of E, thus reflecting a greater disorder in the monolayer, probably because the guanidino groups that interact belong to adjacent molecules.
In general, interactions of cationic surfactants derived from the amino acid arginine with simple membrane models strongly depend on the structure of the surfactants. Modifying the structure of the surfactant, either in the polar head or in the hydrophobic part, entails changes in the interactions. The gemini structure surfactants studied here have two cationic charges so they can form aggregates via ion pairs, which are then solubilized in the sub phase under compression. As a result, isotherms are shifted towards smaller areas. However, there are other factors that influence the surfactant membrane model’s interactions. Mixed monolayers of gemini surfactants derived from the amino acid Lysine [
43] as well as the amino acid Histidine [
28] carry two cationic charges on the polar head; however, isotherms of mixtures of each surfactant with phospholipids are displaced towards larger areas with respect to those of the phospholipid alone. This difference can be attributed to the fact that the polar heads of both Lysine and Histidine are more hydrophobic than the polar head of Arginine, resulting in an increase in the overall hydrophobic character, consequently having a significant influence on the interactions with phospholipids.
The number of fatty chains and their length are also key structural factors in surfactant–phospholipids interactions. Synergy is observed in interactions of surfactants with two fatty chains mimetic to a phospholipid [
44]; moreover, under compression, stable monolayers are formed. However, the stability of monolayers formed by surfactants derived from arginine which have just one fatty chain [
45] is clearly smaller, according to the corresponding values of the elastic module measured.
2.4. Anti-Enzymatic Inhibitory Activities
Several proteolytic enzymes are involved in the skin- and connective tissue-repairing mechanisms. For instance, collagenase and elastase are responsible for the degradation of collagen and elastin fibers in the extracellular matrix [
46,
47]. Controlling their activity plays a key role in skin aging, while the modulation between collagen production and degradation is also important to ensure adequate and shorter wound healing [
48,
49]. Hyaluronidase is involved in the inflammatory activity and more importantly in the degradation of hyaluronic acid, which is an important dermic filler [
50]. In view of these characteristics, the effect of the surfactants in the inhibition of these enzymes was evaluated to explore the potential applications of these compounds.
The inhibitory activities of the surfactants over key enzymes enrolled in the skin repairing processes were evaluated. C
6(LA)
2, immediately followed by C
9(LA)
2 and C
3(LA)
2, presented moderate anti-collagenase activity (
Figure 7a), ranging from approximately 40 to 50%, similar to that found for the positive control EGCG (54.2%). Therefore, the surfactants were found to moderately inhibit the enzyme collagenase. These molecules can be important during the second phase of wound healing to regulate the deposition of proteins during the fibroblast proliferation phase, promoting its modelling. This is also another important aspect to be explored in alternative skin care applications.
The inhibition of the enzyme elastase was far more discrete. C
3(LA)
2, C
6(LA)
2 and C
9(LA)
2 presented anti-elastase activity limited to 17, 10.5 and 15.9%, respectively. In contrast, EGCG presented 67.3% of inhibition (
Figure 7b). Therefore, the surfactants did not affect the activity of this enzyme.
Hyaluronidase reverse depolymerizes hyaluronic acid found in the cementum around the cells of the connective tissue. Unlike the previous enzymes, the activity of hyaluronidase was boosted by the presence of these surfactants, demonstrated by the negative values observed. C
3(LA)
2, C
6(LA)
2 and C
9(LA)
2 increased the enzymatic activity by 48.3, 47.2 and 29.9%, respectively (
Figure 7c). EGCG presented an inhibitory activity of 13%, while the enzyme control resulted in 32% inhibition. Therefore, the surfactants would contribute synergically with the enzyme hyaluronidase for the removal of hyaluronic acid in specific cases, such as when aesthetic procedures need to be corrected and in special clinical conditions when tissues are formed inadequately and need to be molded. This enzyme also increases the absorption and reduces the pain caused by subcutaneous or intramuscular administration of liquids, enhances the absorption of extravasated liquids and blood on the tissues and improves the efficacy of local anesthesia. In this case, the surfactants could also be useful as an adjuvant to the parenteral administration of drugs, also considering its solubilization and wettability properties.
Hence, the anti-enzymatic activities were identified to support the use of the surfactants in the treatment of specific skin disorders. While collagenase and elastase inhibition may be helpful in maintaining and stimulating the resistance and elasticity of the skin and also the skin repairing, the increased hyaluronidase activity found may be useful in the connective tissues or skin remodeling, for instance, treating hypertrophic and keloid lesions, based on their anti-inflammatory effect.
2.5. Molecular Docking Results
Molecular docking is a computational tool capable of identifying several connection sequences and predicting the binding affinity of new compounds at the target receptor binding sites. Molecular docking simulations were carried out to understand the observed enzymatic activities of the studied surfactants and to shed light on the binding modes between docked ligands and enzymatic targets.
The likeliest docked positions of surfactants and EGCG ligands with the best binding affinity for ligand complexes in the active site of the targeted receptors are shown in
Figure 8 and
Figure 9. All surfactants showed a good affinity to the receptors pocket (
Table 2,
Table 3 and
Table 4), with free energy binding values between −10 and −12.8 kcal/mol for surfactants and between −9.2 and −10.9 kcal/mol for EGCG. Three types of interaction were found in the modes of action for all ligands: hydrogen, electrostatic and hydrophobic interactions, with the exception of a Pi-sulfide type interaction, which was observed in the interaction mode of EGCG against the enzyme elastase.
Regarding collagenase and elastase, the experimental part showed that the EGCG presented a higher inhibition rate (54%) than the surfactants (from 40% to 50%). This aspect corroborates the high affinity of EGCG with these two enzymes in molecular docking. Based on their modes of interaction (
Figure 8 and
Figure 9), EGCG shows more hydrogen-type interactions compared to surfactants, due to the existence of more hydroxyl groups on its molecular structure, contributing to the proton labile of the OH group to interact with the residues of the enzyme collagenase and elastase. The same aspect was also observed for hyaluronidase in molecular docking, i.e., the two types of molecules, EGCG and surfactants, show in their interaction modes different bonding-types against many residues in the hyaluronidase structure. Unlike the other two enzymes, the addition of surfactants increased the activity of hyaluronidase, as evidenced by the negative values found. Hyaluronidase is a naturally occurring enzyme capable of the local degradation of hyaluronic acid [
51]. Hyaluronidase hydrolyzes hyaluronic acid by splitting the bond between the C
1 of an N-acetyl-glucosamine moiety and the C
4 of a glucuronic acid moiety [
52]. The surfactants C
3(LA)
2, C
6(LA)
2 and C
9(LA)
2 enhanced the enzymatic activity by 48.3, 47.2 and 29.9%, respectively. EGCG presented a 13% inhibitory activity, while the enzyme control had a 32% inhibition. The experimental data indicate that the surfactants enhanced the degradation of hyaluronic acid, enhancing the enzymatic activity. Two hypotheses are possible: either the surfactants hyaluronidase interactions made the enzyme more active or the presence of surfactants made the hyaluronic acid more reactive by solubilizing or exposing the enzymatic target found in the N-acetyl-glucosamine moiety. Unlike the surfactants, EGCG shows hydrogen-type interactions over hyaluronidase (
Figure 9). The presence of OH groups on its structure promotes many hydrogen-type interactions. Therefore, this results in a greater affinity, competitively, over the enzyme compared to hyaluronic acid, which makes it less likely to be degraded in the presence of EGCG molecules. In contrast, the surfactants also show a limited hydrogen-type interaction. The molecular structure of hyaluronic acid has a lot of carbonyls and some hydroxyl groups, which can favor the affinity of hyaluronidase to its substrate over the surfactants. This difference may be one of the reasons why, in the presence of surfactants, hyaluronidase becomes more active, resulting in a more extended degradation of hyaluronic acid.
2.6. Cytotoxicity
Although these surfactants were found to be very potent in the biological activities tested, some limitations can be found when they are incorporated together with other adjuvants. Moreover, they are commonly associated with the most irritative category of surfactants. Recently, we have demonstrated that nanoencapsulation was a suitable strategy to reduce the hemolytic activity of these compounds, leaving the antimicrobial activities unaltered [
39]. In view of these characteristics, in this work, the cytotoxicity of both free surfactants and surfactants loaded to zein nanoparticles was comparatively evaluated. The arginine-based nanoparticles were obtained and characterized according to our previously published paper [
39].
The cytotoxicity was assessed by the colorimetric methods (MTT and NRU assays) over immortal human keratinocyte (HaCaT) (
Figure 10a,b) and squamous cell carcinoma (A431) (
Figure 10c,d), comparing the activity of the bulk surfactants and the corresponding nanoparticles with the negative control cells in the absence of any treatment.
The cytotoxic response differed between methods as a consequence of their different interaction with cells. Thus, while the neutral red uptake (NRU) assay relies on the ability of living cells to incorporate and bind neutral red dye though the membranes in lysosomes, the colorimetric assay is based on the reduction in yellow tetrazolium salt (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to purple formazan crystals by metabolically active cells. The viable cells contain NADPH-dependent oxidoreductase enzymes, which reduce the MTT to formazan. Bearing in mind that the surfactants inhibited some enzymes and also adhere to the cellular membranes, the MTT findings may be limited as the enzymes were not totally available due the inhibition that may have been caused by the molecules assayed. For this reason, the MTT method promoted cell viability values always lower than those obtained in the case of the NRU method.
According to the NRU method, when the cellular response of the surfactants in solution was evaluated, the results demonstrated the same cytotoxicity pattern: C
9(LA)
2 > C
3(LA)
2 > C
6(LA)
2 in both cellular lines (
Figure 10a,b). However, while the nanoencapsulation did not markedly alter the cytotoxicity of the surfactants over HaCaT cells (
Figure 10c), this procedure seems to enhance the cytotoxicity over A431 squamous carcinoma cells (
Figure 10d). Therefore, they were more selective over the cells than the surfactants in the solution. The HaCaT cells remained stable after contact with C
6(LA)
2 with a cellular viability of 99.3%, while the viability was reduced to 66.2 and 60.7% after treatment with C
3(LA)
2 and C
9(LA)
2, respectively (
Figure 10a). The nanoparticles of C
6(LA)
2 presented a viability of 79.0% over HaCaT cells, immediately followed by C
3(LA)
2 (75.2%) and C
9(LA)
2 (55.0%), although without statistical significance (
p > 0.05). Over the A431 cell line, C
9(LA)
2 maintained its cytotoxicity with a viability of 60.7%, in contrast to C
3(LA)
2 and C
6(LA)
2, which did not cause any apparent cytotoxicity (
Figure 10c). In contrast, the nanoparticles hindered a more cytotoxic effect over the A431 cells with the highest cytotoxicity caused by C
6(LA)
2 (ca. 40%), followed by C
9(LA)
2 (24%) and C
3(LA)
2 (12%), without statistical significance (
p > 0.05) between the nanoparticles (
Figure 10d). In addition, C
6(LA)
2 nanoparticles were more cytotoxic than the bulk surfactant solution (
p < 0.05) over A431 cells (
Figure 10b,d).
The biomedical applications of these gemini surfactants depend on their ability to selectively kill bacteria without toxic effects on mammalian cells. This selectivity is determined by the IC50/MIC ratio, named the therapeutic index (TI). The TI of these surfactants depends on the bacteria strain and on the end point method used to evaluate the cytotoxicity. Considering the NRU method and the HaCaT cell line, it can be assumed that the IC50 of all surfactants is higher than 35.6 µg/mL. Then, the TI for the C3(LA)2 against the Gram-positive bacteria and E. coli and PA01 is higher than one.
From the obtained results, it can be assessed that the C3(LA)2 surfactant is the safest C12 derivative to be used in applications that require contact with HaCaT cells. Interestingly, C3(LA)2 has antimicrobial activity at concentrations below that, producing toxicity against HaCaT cells (IC50 is always higher than the highest tested concentration, i.e., 35.6 µg/mL).