Review Article

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Review Article

Biocompatible surfactants from renewable hydrophiles

MâRosa Infante¹, Lourdes Pérez¹, MâCarmen Morán¹, Ramon Pons¹, Montserrat Mitjans², MâPilar Vinardell¹, MâTeresa Garcia¹and Aurora Pinazo¹

1Institut de Química Avanc¸ada de Catalunya, IQAC-CSIC, Barcelona, Spain

2Facultat de Farmacia, Departament Fisiologia, Universitat de Barcelona, Barcelona, Spain

Significant advances made by the authors in the field of cationic surfactants derived from arginine and lysine hydrophilic sources of different structure are reviewed. Linear, gemini and glycerolipid-like structures with polar groups consisting of Arg or Lys amino acids are described. Our multidisciplinary approach includes design, synthesis, adsorption at interfaces and self-assembly behavior, antimicrobial activity, and studies of biocompatibility including ecotoxicity, biodegradability and irritation effects.

Considering the green chemistry principles, the synthesis contemplates the preparation of more efficient and safer surfactants using renewable raw materials for the synthesis of surfactants: proteins, peptides, amino acids and triacylglycerols, using biocatalysis as condensating methodology, and designing for biodegradation. Also in order to increase the efficiency of these compounds, a complete characterization including self-assembly, ecotoxicity, biodegradability, and studies of mechanisms of toxicity has been carried out. The structure of the surfactant molecule affects micellization and not the nature of the polar head (Arg or Lys). The critical micelle concentrations of the gemini surfactants are three orders of magnitude lower than the linear ones for the same alkyl chain length. The presence of a cationic charge in the amino acid provides antimicrobial activity to these compounds.

Keywords: Arginine / Lipoaminoacid / Lysine surfactant / Renewable raw materials Received: May 8, 2009; accepted: September 24, 2009

DOI 10.1002/ejlt.200900110

1 Introduction

Society is increasingly concerned with safety issues while wishing for a sustainable future. In response to this concern, sustainable chemistry aspirates to raise the stake of less dan- gerous chemicals as well as production of environmentally friendy high-quality products from preferably renewable resources. There is a pressing need to adopt processes and technologies that follow Anastas’ 12 principles of green chemistry [1].

The use of renewable feedstocks is an important sustainable chemistry approach, and the production of useful compounds from biomass or waste is being accepted in many industrial processes, conciliating environmental regulations, innovation (new structures, properties, functionalities, etc.) and cost benefit requirements.

Manufacturers and consumers demand for novel environmentally friendly surfactants from renewable resources produced by clean and sustainable technologies (bio-based surfactants). The challenge is to find renewable surfactants with biocompatible and multifunctional capabilities. The use of hydrophilic renewable raw materials such as amino acids and vegetable oil derivatives to prepare biocompatible surfactants that grant mild reaction conditions, biodegradability, efficiency and multifunctional activity is an attractive research activity to prepare novel “natural” surfactants in which the sustainable issues are well matched with the industrial development.

Amino acid-based surfactants constitute an important class of natural surface-active bio-molecules of great interest to organic and physical chemists as well as to biologists, with an unpredictable number of basic and industrial applications [2]. Amino acids can be produced by protein hydrolysis, chemical synthesis, and microbiological synthesis. Lysine can be produced by fermentation of bacteria or their mutants [3, 4]. For nearly 50 years, constant efforts to increase production performance have been carried out both directed towards the Correspondence: M^aRosa Infante, IQAC-CSIC, c/ Jordi Girona 18–26,

08034 Barcelona, Spain.

E-mail: rimste@cid.csic.es Fax: 134 93 2045904

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microorganisms themselves and towards technical improve- ments of the respective processes [5]. Most L-arginine has been produced by the direct-fermentation method from natural carbon sources. The world leading manufacturer (Ajino- moto Co., Kawasaki, Japan) produces arginine by fermentation technology from corn starch. This technology produces very pure, high-quality amino acids of non-animal origin [6].

Structurally, lipoaminoacids are a very heterogeneous group of compounds, but with a common advantage: They are relatively easy to design and synthesize.

For more than 25 years, our group has been working on the fundamental and applied chemical investigation of novel environmentally friendly surfactants (products and processes) from natural renewable sources (amino acids and natural oil derivatives), as alternatives to conventional surfactants to be applied in cosmetic, textile, dermopharmaceu- tical, biomedicine and food industrial preparations. Con- sidering the green chemistry principles, this line has con- templated adopting its principles and progressing in the preparation of more efficient and safer surfactants, using renewable raw materials for the synthesis of surfactants (proteins, peptides, amino acids and triacylglycerols), employing safer solvents (water systems, solvent-free processes, concentrated emulsions) as reaction media, using biocatalysis as condensating methodology, and designing for biodegradation. Also, in order to increase the efficiency of these compounds, a complete characterization including self- assembly, ecotoxicity, biodegradability, and studies of mechanisms of toxicity has been carried out.

These compounds can be classified as specialty surfactants with biodegradable, antimicrobial and low-toxicity pro- files, as well as characteristic self-aggregation properties [7].

In this paper, significant advances made by our group in the field of cationic surfactants derived from arginine (Arg) and lysine (Lys) hydrophilic sources of different structure are reviewed. Our multidisciplinary approach includes design, synthesis, adsorption at interfaces and self-assembly behavior, antimicrobial activity, and studies of biocompatibility including irritation effects, cytotoxicity, ecotoxicity and biodegradability.

2 Design and structures

Two interesting and novel strategies have been proposed to minimize the environmental effects of surfactants. One of these strategies involves the design of amphiphilic analogs that mimic the natural amphiphilic structures of glycolipids, phospholipids and lipoaminoacids [8] (Fig. 1).

The second strategy seeks to improve the efficiency and effectiveness of surfactants. The higher the level of performance, the lower is the amount of surfactant required and, consequently, the lower is the environmental impact produced. A number of surfactant structures with a variety of hydrophilic headgroups and hydrophobic tails of different

Figure 1. Chemical structures of naturally occurring lipids: (a) 1,2- dioleoyl-sn-glycerol, (b) 1,2-dipalmitoyl-sn-glycero-3-phospho- choline, (c) N-arachidonylglycine.

chain lengths have been reported in an effort to increase their surface-active properties [9, 10]. Surfactants made up of two identical amphiphilic moieties connected close to the headgroups by a spacer chain are referred to as bipolar gemini or dimeric surfactants (Fig. 2). These surfactants appear to be superior to the corresponding conventional monomeric surfactant, in most properties [11].

Lipoaminoacids of diverse structure and ionic nature, with monocatenary chains amino acids bearing at least one hydrophobic tail [12, 13], glycerolipids that consist of one polar head and one or two hydrophobic moieties linked together through a glycerol skeleton [14, 15], and gemini, dimeric amphipathic structures with two polar heads (i.e. two amino acids) and two hydrophobic tails per molecule [16], structures with multifunctional properties, have been synthesized and studied. These compounds were obtained from the con-

Figure 2. Schematic structures of gemini bis(Quats).

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densation of vegetable oil derivatives (fatty acids, amines and alcohols, mono- and diacylglycerols) with different amino acids for applications in the food and cosmetics sector. They have in common that they are chiral molecules in which the hydrophilic/hydrophobic moieties are linked by ester and amide linkages. These labile bonds were designed for easy degradation.

Since the middle of the last century, a lot of arginine and lysine surfactant molecules of different structure have been described. The presence of one carboxylate (anionic) and two basic, guanidinium or amino, moieties in arginine and lysine, respectively, makes it possible to design various types of surfactants of different ionic character (anionic, cationic, non- ionic and amphoteric derivatives) by introducing hydrophobic groups (fatty acid, fatty amine or fatty alcohol) into the molecule.

3 Synthetic aspects

3.1 Linear lipoaminoacids from Arg and Lys

Molecules with a cationic arginine residue as the hydrophilic moiety linked to a saturated alkyl chain of varying length through amide and ester bonds, which are susceptible to hydrolytic and biodegradable decomposition, were synthesized by chemical and enzymatic methodologies. Single-chain arginine-based cationic surfactants with chiral properties, such as hydrochloride salts of long-chain N-acyl arginine methyl ester (Fig. 3, series 1), N-alkyl arginine amide (Fig. 3, series 2) and arginine alkyl ester derivatives (Fig. 3, series 3) were prepared on a multigram scale by condensation of fatty

Figure 3. Chemical structures of linear arginine-based cationic surfactants. Hydrochloride salts of N-lauroyl-L-arginine methyl ester, LAM (series 1), N-lauryl-L-arginine amide, ALA (series 2), and

L-arginine lauryl ester, ALE (series 3) derivatives.

acid, fatty amine or fatty alcohol derivatives of different chain lengths, respectively, employing chemical and/or natural processes, using biocatalyst-based chemical transformations (enzymes) for efficiency and selectivity.

The N-acylation of the amino-terminal arginine (Fig. 3, series 1) was performed by condensation of fatty acid chlorides to arginine methyl ester derivatives, using classical chemical methods in aqueous [2] or emulsion media [17]. The guanidine side chain of Arg is an extremely strong base, which in the unsubstituted state remains protonated under normal conditions for N-acylation. It is therefore possible to work with an unprotected arginine side chain by carefully controlling the pH. The application of biotechnological procedures was not efficient for these compounds [18]. N-Alkyl amide derivatives (Fig. 3, series 2) were at first prepared by chemical proce- dures [8]; however, papain from Carica papaya latex was found to be a suitable catalyst for the formation of amide (Fig. 3, series 2) and ester bonds (Fig. 3, series 3) between the N-benzyloxycarbonyl (Cbz)-protected arginine methyl ester salts, Cbz-Arg-OMe and various long-chain alkyl amines and fatty alcohols [19]. In all cases, papain deposited onto poly- amide was found to be the best biocatalyst configuration. The preparation of arginine alkyl esters (Series 3) was carried out in solvent-free systems using the same alcohol reagent. Both series were enzymatically synthesized at multigram scale with a purity higher than 99%. N-Alkyl amide and ester derivatives of N^a-protected amino acids have also been prepared by lipa- ses. In a study carried out with Candida antarctica and Rhizo- mucor miehei lipases, it was found that these enzymes could readily catalyze the condensation of a number of N^a-Cbz- amino acids witha,o-alkyldiamines or fatty alcohols [13].

In the case of Lys, different linear amphiphiles can be designed by hydrophobic modulation, although most of them are N^a-acyl lysine or N^e-acyl lysine ester derivatives [20]. In this case the necessary differentiation can be achieved given that the e-NH2-side chain amino group is more basic and nucleophilic than the a-NH2 one. The principal protecting group for the amino functions of Lys and homologues is Cbz, which can be easily removed by hydrogenolysis.

Recently, our group has synthesized linear cationic lysine amphiphiles as long-chain N^a-acyl lysine methyl esters (Fig. 4a) and N^e-acyl lysine methyl ester salts (Fig. 4b), with different alkyl chains to study the influence of the cationic charge nature (amino group ina or e position) on the biological properties [21].

The synthesis of these compounds was carried out fol- lowing the methodologies for the formation of amide functions in liquid phase, including peptide chemistry [22]. Ideal chemical conditions for the preparation of N-acyl amino acids would allow the acyl bond formation to be carried out rapidly and quantitatively under mild conditions, avoiding side-reac- tions, whilst maintaining all the adjacent chiral centers. How- ever, enzymatic synthesis has not yet been carried out. In practice, however, diverse methodologies have been devised to overcome the problems related to the reactivity and purity

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Figure 4. Chemical structures of linear lysine-based cationic sur- factants. (a) Hydrochloride salts of Nâ-lauroyl-L-lysine methyl ester, LLM. (b) Hydrochloride salts of Nê-lauroyl-L-lysine methyl ester, LKM; Nê-myristoyl lysine methyl ester, MKM; Nê-palmitoyl-L-lysine methyl ester, PKM.

processes. The procedure used for the synthesis of Nâ-acyl lysine methyl ester (Fig. 4a) and Nê-acyl lysine methyl ester salts (Fig. 4b) was easy and very efficient by reaction of Nê- Cbz-L-lysine methyl ester or Nâ-Cbz-lysine methyl ester, respectively, with the corresponding fatty acid chloride.

3.2 Glycerolipid-like lipoaminoacids

In the research for new multifunctional amino acid based- surfactants mimicking natural lipids, mono- and diacylglycerol arginine-based cationic surfactants [11–13] were synthesized by enzymatic and chemical procedures [23]. They can be considered analogs of mono- and diacylglycerols and phospholipids, exhibiting similar properties as well as a low- toxicity profile and antimicrobial activity. They consist of one or two aliphatic chains and one amino acid, as the polar head, linked together through ester bonds to the glycerol backbone.

Our group has synthesized mono- (Fig. 5; 1 and 2) and di- acylglycerol (Fig. 5; 3–7) derivatives from arginine and N^a- acetyl-arginine using chemical and chemo-enzymatic methodologies [9, 24]. The novel family of compounds proposed in this work would combine in one molecule the physicochemical properties of the glycerol derivatives and those of the polar arginine-based surfactants. It is also expected that they will be more hydrophilic than diacylglycerol derivatives and therefore will have higher water solubility. A dibasic amino acid such as

arginine was selected to introduce antimicrobial activity into the molecule.

The enzymatic synthesis of mono- and dilauroylated arginine acylglycerol-conjugated derivatives was achieved in the absence of solvents. The first step involves the enzymatic preparation of the arginine glyceryl ester derivative [rac-1-O- (N-acetyl-L-arginine) glycerol]. In the second step, the lipase- catalyzed acylation of the hydroxy groups of the arginine glyceryl ester by lauric acid is performed, obtaining the mono- (62%) or dilauroylated derivative (75%), depending on the excess of lauric acid used in each case (Fig. 6). Isolation of mono- or dilauroylated arginine acylglycerol was carried out by preparative HPLC. This methodology resulted in clean products, with a reasonable selectivity. For more details about the synthesis consult reference [14].

Recently, a novel class of cationic bisglycidol lysine-based surfactants of the type N^å,N^å-bis-2,3-dihydroxypropyl)-lysine methyl ester hydrochloride salts was prepared in our labora- tory by chemical methodologies [25]. They combine several hydroxyl functions and aliphatic chains of 12 or 14 carbon atoms. In Fig. 7, the structures of 3-monolauroyl-N^å,N^å-bis- 2,3-dihydroxypropyl)-lysine methyl ester and 3,3’-dilauroyl- N^å,N^å-bis-2,3-dihydroxypropyl)-lysine methyl ester hydrochloride are shown.

The main structural characteristics of these new cationic amphiphilic molecules are: a lysine methyl ester residue as polar group which gives the cationic character to the molecule, linked to a bisglycidol chain, and a residue of bis(2,3-dihy-

Figure 5. Chemical structures of glycerol-like arginine-based cat- ionic surfactants. (a) Monoacyl glycerol arginine conjugates: (1) 1- caprinoyl-3-(L-arginyl)-rac-glycerol; (2)L-lauroyl-3-(L-arginyl)-rac- glycerol; (b) diacyl glycerol arginine conjugates: (3) 1,2-dicapriloyl- 3-(L-arginyl)-rac-glycerol; (4) 1,2-dicaprinoyl-3-(L-arginyl)-rac-glycerol; (5) 1,2-dilauroyl-3-(L-arginyl)-rac-glycerol; (6) 1,2-dilauroyl-3- (N^a-acetyl-L-arginyl)-rac-glycerol; (7) 1,2-dimyristoyl-3-(N^a-acetyl-

L-arginyl)-rac-glycerol.

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Figure 6. Reaction pathway for the synthesis of mono- and dilauroylated glycerol arginine-based surfactants.

droxypropyl) which can carry one (Fig. 7a) or two (Fig. 7b) aliphatic chains as part of the hydrophobic moiety. The lysine is bonded to the polyol skeleton through an N-alkyl amine linkage (instead of an ester bond as in the case of arginine glycerolipids), which gives more stability to the molecule.

They are obtained by reaction of lysine with glycidol and subsequent chemical acylation of the hydroxyl functions with fatty acyl chlorides as acylating agents [25].

3.3 Geminal lipoaminoacids from Arg and Lys

Gemini surfactants, characterized by two hydrophobic chains with polar heads that are linked by a hydrophobic bridge, have very interesting properties compared to their monomeric equivalents. They are very effective in decreasing the surface tension of water. This efficiency is often quantified by the C₂₀ value (the concentration of surfactant needed to reduce the surface tension by 20 mN/m). Reduction of C₂₀depends on headgroup and spacer. For anionic surfactants, comparing

single-chain and gemini surfactants, a reduction of three orders of magnitude has been reported, while for cationic surfactants the observed reduction of C₂₀is only of two orders of magnitude [26]. Also, they are very effective in reducing the interfacial tension between oil- and water-based liquids (e.g.

values of 0.01 mN/m are reported for crude oil) [27] and have very low critical micelle concentrations (CMC) which are two or three orders of magnitude lower than those of their monomeric analogues [28, 29].

Gemini arginine-based surfactants were prepared by chemical and enzymatic technologies [30, 31], with the enzymatic technology being more sustainable due to the selectivity and the minimization of byproducts. They consist of two symmetrical long-chain N^a-acyl-L-arginine residues of 12 or 10 carbons atoms linked by amide covalent bonds to ana,o- alkenediamine spacer chain of various lengths. This particular alkenediamine spacer chain was chosen to control the distance between the charged sites of the cation that modify the inter- and intra-hydrophilic-hydrophobic interactions. They will be

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Figure 7. Chemical structure of bisglycidol lysine-based cationic surfactants. (a) (3-Monolauroyl-Nê,Nê-bis-2,3-dihydroxypropyl)- lysine methyl ester hydrochloride, (b) (3,3’-dilauroyl-Nê,Nê-bis-2,3- dihydroxypropyl)-lysine methyl ester hydrochloride.

termed N,N’-(alkane-diyl) bis(N^a-acyl arginine) amides or bis(Args) (Fig. 8). These amphiphiles are structural analogs of bis(Quats) compounds. Here, the headgoups are of the guanidyl type (from the natural amino acid arginine) in place of the quaternary ammonium type.

Long-alkyl chain cationic gemini surfactants from lysine with different structures have been proposed for gene trans- fection [32]. Recently, our group synthesized chemically a new class of gemini cationic surfactants derived from lysine to study the effect of several structural parameters (hydrophobic chain length, number and type of the cationic charge, spacer chain nature) on their physicochemical properties and cellular toxicity. These compounds can be considered dimers of the long-chain Nâ- or Nê-acyllysine derivatives. They consist of two symmetrical long-chain Nâ- or Nê-acyl lysine residues linked by amide bonds through an 1,6-hexyl diamine spacer chain of different lengths and chemical nature. In Fig. 9, the chemical structure of the N,N’-(hexane-1,6-diyl) bis(Nâ-

Figure 8. N,N’-(Alkane-diyl) bis(N^a-acyl arginine) amides.

lauroyl-lysine) amide, C₆(LM)₂, and N,N’-(hexane-1,6-diyl) bis(N^e-lauroyl-lysine) amide, C₆(LK)₂, are shown. They were obtained with a purity of 99% by chemical condensation of the single long-chain N-lauroyl-L-lysine previously protected to the corresponding spacer in the presence of an activating agent. A final deprotection reaction was carried out to obtain the cationic gemini compounds [33].

4 Physicochemical properties: Self-assembly

A systematic study to characterize the influence of chemical structure on the physicochemical properties was carried out for all three families of linear, gemini and glycerolipid-like compounds. The equilibrium surface tension was studied and the CMC was calculated from the surface tension versus con- centration plots. Table 1 shows the CMC in mM concentration of lipoaminoacids with 12 carbon atoms of linear, geminal and glycerolipid structure from Arg and Lys.

The CMC value obtained for LKM (lysine derivative with a fatty chain condensed to thee-amino group) is similar to that obtained for LAM (arginine derivative with the same number of carbon atoms in the hydrophobic part). The results indicate that the cationic charge type in these ionic amino acid surfactants does not affect the micellization process; this process is mainly controlled by the hydrophobic part [15].

The structure of the surfactant molecule affects micellization and not the nature of the polar head (Arg or Lys). Com- pared to LAM or LKM, the gemini C₃(LA)₂or C₆(LK)₂in water show very small CMC values. Micellization takes places at concentrations in the range of 10^–6M, about three orders of

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Figure 9. Chemical structure of (a) N,N’-(hexane-1,6- diyl) bis(N^a-lauroyl-lysine) amide, C₆(LM)₂, and (b) N,N’- (hexane-1,6-diyl) bis(N^e-lauroyl-lysine) amide, C₆(LK)₂.

Table 1. CMC in mM concentration at 25 7C of lipoaminoacids with 12 carbon atoms of linear, geminal and glycerolipid structure from Arg and Lys, by surface tension method.

Linear Gemini Glycerolipid

Arg Lys Arg Lys Arg Lys

LAM LKM C3(LA)2 C6(LK)2 1212R (5)^§ LGGdi12

6 5.5 0.005 0.51 0.3 0.5

§See Fig. 5.

magnitude lower than those of LAM [34]. This extraordinary ability to aggregate is a common feature of the gemini-type surfactants. The low CMC values of gemini surfactants are due to the greater total number of carbon atoms in the two alkyl chains of the molecules and not to the hydrophobic/

lipophilic balance (HLB) per molecule. Notice that, con- sidering the chemical structure, LAM and, e.g., C₃(LA)₂have comparable HLB values. The solubility of surfactant molecules decreases as the hydrocarbon number of the hydrophobic chain increases; at the same time the Kraft temperatures increase, and the combination limits the surface activity by the increasing hydrophobic chain length. However, due to the close proximity and interaction, the solubility of gemini surfactants is bigger than would correspond to a single hydrophobic chain with the same total carbon number. When comparing gemini surfactants with single-chain surfactants with the same chain length, the former have very low CMC values. Comparison of gemini surfactants with single-chain surfactants with the same total number of carbon atoms in the hydrophobic moiety is not generally possible because of the high Kraft temperatures as the carbon number increases [20].

The use of other methods to determine the CMC, i.e. pri- marily ion activity (of Cl^–), conductivity, fluorescence using pyrene as fluorophore, and NMR, reveals the existence of a second CMC for C₃(LA)₂ at 0.05 mM. The aggregates formed at surfactant concentrations above the surface tension CMC, or first CMC (CMC₁), are not traditional micelles [35].

The micelle formation of mono- and diacylglycerol surfactants from arginine (see Fig. 5) was evaluated by conductivity,

surface tension and fluorescence measurements [36, 37]. From the conductivity/concentration curves, the CMC of the diacyl arginine acylglycerol surfactants of 12 carbon atoms is 0.3 mM for 5 and 0.12 mM for the acetylated homolog 6. The CMC of the diacyl arginine acylglycerol surfactants are one order of magnitude higher than those published for the short-chain phospholipids [38] with similar alkyl chain lengths. The second alkyl chain increases the hydrophobic content of the molecule and, consequently, the CMC of these compounds is lower than those corresponding to the monoacyl arginine acylglycerols with the same alkyl chain: 1.3 mM for compound 2 in Fig. 5. As expected, the CMC decreases when the alkyl chain increases, as a consequence of the higher hydrophobic content of the molecule. Similarly to gemini surfactants, unconventional aggregation behavior and two CMC were inferred from surface tension, conductivity and fluorescence technique.

As in conventional surfactants, lipoaminoacids aggregate in solution to form micelles because of the hydrophobic effect.

At high concentration, the micelles become ordered, forming lyotropic liquid crystals. Self-assembly of linear Arg and Lys surfactants in water/surfactant systems shows the presence of spherical micelles and hexagonal, cubic and lamellar liquid crystals [39]. Interestingly, systems formed with ALA (Fig. 3, series 2, a linear cationic arginine surfactant) with a linear anionic surfactant are capable to form liposome, hexasome and cubosome structures [40].

The aggregation behavior of gemini cationic surfactants and their corresponding single-chain homologs has been investigated using small-angle X-ray scattering (SAXS), dy- namic light scattering (LS), and pulsed-gradient spin-echo

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NMR (PGSE-NMR) spectroscopy [41]. From SAXS and NMR experiments, it was found that the gemini compounds form cylinder micelles whereas the monomeric surfactant LAM forms spherical aggregates. The results obtained by SAXS and NMR spectroscopy are in good qualitative agree- ment with the results obtained by cryo-transmission electron microscopy (TEM) observations. The area per molecule of the gemini surfactants is smaller than double that of the single- chain counterparts; this produces a displacement of the pre- ferred packing properties of the gemini surfactants towards cylindrical and lamellar structures [35]. From cryo-TEM, it was concluded that the structure of surfactants also influences the micellar or microstructures formed in aqueous solutions [42]. LAM, at a concentration of 6% forms spheroidal micelles of 3–5 nm; C₃(LA)₂at.1.3% forms thread-like micelles. Also the spacer chain of geminis influences the shape of aggregates:

C₆(LA)₂at 1.3–1.5% produces coexisting single twisted ribbons with spheroidal micelles, C₉(LA)₂at 0.1% flat and twisted ribbons, and C₁₂(LA)₂at 0.05% twisted ribbons (Fig. 10).

The phases of the water binary systems of compounds 6 and 7 (see Fig. 5) were determined by visual observation of the samples through crossed polarized microscopy. Qualita- tive phase behavior studies applying the flooding method revealed the formation of anisotropic phases in all the binary surfactant systems studied. Compound 6 forms lamellar liquid crystals at room temperature (25 7C), and this structure is stable as long as the temperatures are moderate [43]. As in the case of diacylphosphatidyl choline, compound 6 is a dou- ble-chained surfactant with a large headgroup area. These structural characteristics imply the formation of vesicles the size of which depends on the preparation method. Low-shear methods produce large multilamellar vesicles several microns in diameter [37], while sonication leads to unilamellar and oligolamellar vesicles 25–100 nm in diameter [44]. For TEM images consult the indicated references.

5 Biological properties

The antimicrobial activities were determined “in vitro” on the basis of the minimum inhibitory concentration (MIC) values, defined as the lowest concentration of antimicrobial agent that inhibits the development of visible growth after 24 h of incu-

bation at 37 7C. The dilution antimicrobial susceptibility test was carried out and the MIC values were determined. In Table 2, the MIC values of the most active compound of each family are shown. As the CMC, the antimicrobial activity depends on the structure and the alkyl chain length of the surfactant. For linear lipoaminoacids, the maximum corre- sponds to the compound with 12 carbon atoms in the alkyl chain, for geminis to the homolog with 10 carbon atoms, and for the arginine-based diacylglycerolipid cationic surfactants to the compound with 8 carbon atoms in the alkyl chains.

The difference between LAM, LKM, and LLM lies in the polar group (see Fig. 3, series 1 and Fig. 4a, b for the structures). The main difference between these compounds is the pK_aassociated with the protonated amino group on the polar heads (the guanidine function of LAM has a pK_aof 12.4, the e-amino group of LKM a pKaof 10.5, and thea-amino group of LLM a pK_aof 8.9) [15]. These results allow us to state that the antimicrobial properties of pH-sensitive amino acid surfactants correlate with the pH of the medium. As for the effect of the compound net charge on its bactericidal activity, numerous studies show that the electrostatic interactions play a key role in the action of cationic systems, and that a decrease in the charge density of the cationic compound results in a reduction in adsorption and bactericidal effects [45]. Accord- ingly, the antibacterial activity varies with the pH in systems in which the net charge depends on the pH.

In contrast to short-chain lecithins, the glycerolipids exhibited antimicrobial activity [18, 30, 37, 46]. The more active compound is the homolog 3 in Fig. 5 with eight carbon atoms. As expected, the gram-negative bacteria were more resistant than the gram-positive bacteria. This antimicrobial activity can make them suitable for the subsequent biodegradation of these surfactants. It is well known that some gram-negative bacteria are relatively insensitive because their outer membranes are impermeable to some hydrophobic/

hydrophilic compounds.

Comparing all three families, the antimicrobial activity of the gemini C3(CA)2with ten carbon atoms in the alkyl chains is superior to the rest of the lipoaminoacids in the table.

Acute toxicity tests on freshwater crustaceans (Daphnia magna) were carried out to assess the aquatic toxicity of the new cationic surfactants [18, 30, 37, 40]. Concentration values that cause immobilization in 50% of the Daphnia after

Figure 10. Twisted ribbons of C₁₂(LA)₂by cryo-TEM.

(A, B) 0.05% concentration.

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Table 2. MIC values (mm/mL) for linear LAM, LKM and LLM, gemini C₃(CA)₂and arginyl acylglycerol 88R (3)^§.

Microorganism LAM LKM LLM C3(CA)2 88R (3)^§

Gram positive

Bacillus cereus var. mycoides 16 64 – 16 64

Micrococcus luteus 16 128 4 4 16

Staphilococcus aureus 16 128 32 2 4

Bacillus subtilis 64 128 16 16 64

Staphilococcus epidermis 8 64 16 32 8

Candida albicans 64 64 – 2 16

Gram negative

Klebsiella pneumonae 32 128 32 8 8

Escherichia coli 64 64 16 8 8

Salmonella typhimurium 64 R 32 16 16

Pseudomonas aeuruginosa 64 R 64 32 64

Bordetella bronchiseptica 64 R 16 4 0.25

Enterobacter aerogenes 64 R – 16 32

§See Fig. 5.

24 h of exposure (IC₅₀) were determined. Values of IC₅₀for the linear LAM and LKM, and gemini C₃(CA)₂, together with those of the diacylglycerol of arginine 1010R with ten carbon atoms in the chains, are summarized in Table 3. Values reported for hexadecyltrimethylammonium bromide (HTAB) are also indicated. The higher the IC₅₀, the lower is the toxicity.

Against Daphnia magna, all three families of cationic lipoaminoacids are much less toxic than the conventional HTAB (differences in IC₅₀values about two orders of magnitude). The introduction of a second alkyl chain in the surfac- tant molecule (from 1 to 4) (see Fig. 5) increases the acute toxicity.

The biodegradability of the arginine-based glycerolipidic cationic surfactants prepared in this study was evaluated by applying two ready biodegradation tests, the modified screening test and the closed bottle test [47]. In these tests, ultimate biodegradation or mineralization of the surfactants, i.e. the microbial transformation of the parent chemical into inorganic final products of the degradation process, such as carbon dioxide, water, and assimilated biomass, was determined. The terms “ready” and “inherent biodegradability”, as defined by the OECD, were applied to classify the surfactants studied as a function of the results obtained in the biodegradation tests. In the biodegradation test used in this work, a result of more than 20% biodegradation can be regarded as evidence for inherent biodegradability, and a result of more than 70% biodegradation is evidence of ready biodegradability. The results in Table 4 indicate that all compounds are easily biodegradable.

Toxicity of lipoaminoacids to human cells was evaluated measuring the hemolytic activity, HC₅₀, which is the concentration of surfactant that causes 50% hemolysis of red blood cells from healthy human donors, and the HC₅₀/D ratio,

Table 3. Aquatic toxicity values of Daphnia magna for the linear LAM, and LKM, the gemini C₃(CA)₂, the didecyl arginyl acylglycerol 1010R, the monodecyl arginylacylglycerol, and the commercial HTAB.

Commercial Linear Gemini Glycerolipid

HTAB LAM LKM C3(CA)2 1010R (4)^§

100R (1)^§

0.36 37 12 16 30

170

§See Fig. 5.

Table 4. Ultimate biodegradation (% total organic carbons removal) of linear LAM, gemini C₃(CA)₂, and glycerolipid 1010R.

Linear Gemini Glycerolipid

LAM C3(CA)2 1010R (4)^§

90 87 79

§See Fig. 5.

where D is the hemoglobin denaturing index (DI). The HC₅₀/ D or L/D is used for predicting the potential ocular irritation relative to sodium dodecyl sulfate (SDS) (L/DSDS: 0.44; irritant) [48]. For comparison’s sake, two soft commercial surfactants, betaine and APG, were tested. According to the results of the L/D ratio, the amino acid-based surfactants in Table 5 have no or only slight irritant effects on the eyes [22, 28, 49].

Interestingly, the linear and gemini long-chain N^e-acyl lysine methyl ester series LKM, MKM and PKM (see Fig. 4b) and C₆(LK)₂showed significantly lower hemolytic

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Table 5. Classification of ocular irritation induced by the Lys and Arg derivatives and the two commercial compounds betaine and APG, according to their L/D or HC₅₀/D ratios calculated from HC₅₀ and the denaturation index (DI).

Compound HC50 DI L/D Classification

LLM 75.5

LKM 143.3 5.9 26.5 slight irritant

MKM 501.8 8.7 77.1 slight irritant

PKM 629.7 2.5 274.3 non-irritant

C6(LK)2 450.9

LAM 59 22.5 2.6 moderate

C3(CA)2 48 12 4 moderate

C3(LA)2 12 8.2 1.5 moderate

Betaine^§ 34 14.4 2.4 moderate

APG^$ 251.9 1.2 17.8 slight

§ Decyl Glucoside – Plantacare^®2000UP from Cognis GmbH.

$ Cocoamidopropylbetaine (Tego-betaine T-50; TB), Goldschmidt AG (Germany).

activity than the N^a-acyl lysine (LLM) and N^a-acyl arginine (LAM) compounds. The positive charge nature could affect the capability of these surfactants to disrupt the fragile cellular surface of the erythrocytes. Surfactant intercalation into the membrane leads to changes in the membrane molecular organization and an increase of the membrane permeability which concludes with cell lysis. However, hemolysis depends on the adsorption of the surfactant to components of the membrane surface, which is influenced by electrostatic attraction between the surfactant molecules and membrane components, among other factors. In this sense, Shalel et al.

[50] demonstrated that the ionic strength of the solution diminishes the effects of the erythrocyte negative surface charge at physiological pH and thus decreases the rate and amount of surfactant incorporated into the cell membrane, and consequently reduces the membrane permeability. The decrease of the hemolytic activity could be associated with the decrease of the antimicrobial activity of these surfactants. The change of the positive charge from thee-amino group to the a- amino group in the amino acid belongs to compounds with moderate antimicrobial activity and lower cellular toxicity.

Curiously, the hemolytic activity of these lysine surfactants decreases with increasing length of the hydrophobic tail. The decrease of the hemolytic activity with increasing alkyl chain length has been reported for partially fluorinated pyridinium bromides [51]. On the other hand, the hemolytic activity shown for these lysine derivatives is clearly lower than those found for classical cationic quaternary ammonium surfactants as alkyltrimethylammonium salts, hexadecyltrimethylammonium bromide (CTAB) and dioctadecyldimethyl ammonium bromide (DODAB) and for new gemini quaternary ammonium compounds (QAC) [52]. These results show that the erythrocyte-disrupting ability of these surfactants is correlated to the cationic charge type and to the alkyl chain length.

6 Conclusions

To design safer and healthier surfactants from amino acids and vegetable oil derivatives using molecular design and the principles of toxicity and environmental mechanism of action to minimize the intrinsic toxicity/ecotoxicity of the product while maintaining its efficacy and function has resulted in sustainable approaches to prepare environmentally friendly surfactants. Their multifunctional performance (rich phase behavior, capability to form vesicles/lamellar structures, efficiency, antimicrobial activity) and their low toxicity in con- junction with their remarkable biodegradability properties make amino acid-based surfactants high-added-value compounds from renewable raw materials for multipurpose applications.

Acknowledgments

Financial support from the Spanish CYCYT, ref. CTQ2006- 01582, CICYT ref. CQT2007-60749/PPQ and CIRIT 2005GR00143 is gratefully acknowledged.

Conflict of interest statement

The authors have declared no conflict of interest.

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