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Available online 24 October 2013 In this review, we report the most important contributions in the structure, synthesis, physicochemical
(surface adsorption, aggregation and phase behaviour) and biological properties (toxicity, antimicrobial
Keywords: activity and biodegradation) of Gemini natural amino acid-based surfactants, and some potential applications,
Gemini surfactants with an emphasis on the use of these surfactants as non-viral delivery system agents. Gemini surfactants derived
Natural amino acid-based surfactants from basic (Arg, Lys), neutral (Ser, Ala, Sar), acid (Asp) and sulphur containing amino acids (Cys) as polar head
Toxicity
groups, and Geminis with amino acids/peptides in the spacer chain are reviewed.
Aggregation
Delivery systems
© 2013 Elsevier B.V. All rights reserved.
Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134
2. Structure and synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135
2.1. Gemini surfactants with amino acids or peptides as headgroups . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135
2.1.1. Gemini surfactants from arginine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136
2.1.2. Gemini surfactants from lysine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138
2.1.3. Gemini surfactants from cystine and sulphur containing peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138
2.1.4. Gemini surfactants from neutral amino acids: β alanine, serine and sarcosine . . . . . . . . . . . . . . . . . . . . . . . 139
2.2. Gemini surfactants with amino acids/peptides in the spacer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140
3. Physicochemical properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140
3.1. Surfactant adsorption from solution and micellisation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140
3.2. Phase behaviour . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145
4. Biological properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146
4.1. Toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146
4.1.1. Hemolytic activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146
4.1.2. Lactate dehydrogenase release . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147
4.1.3. Alamar blue assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148
4.1.4. Keratinocytes or fibroblasts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148
4.1.5. MTT cell proliferation assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148
4.2. Antimicrobial activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149
4.3. Antifungal activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149
4.4. Aquatic toxicity and biodegradability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150
5. Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150
5.1. Gene transfection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150
5.2. Interactions with proteins and polymers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152
5.3. Biocompatible gels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153
6. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154
1. Introduction
⁎ Corresponding author. Tel.: +34 934006164; fax: +34 932045904. Numerous structural modifications to the surfactant structural
E-mail address: rosa.infante@iqac.csic.es (M.R. Infante). design have been carried out to increase hydrophobicity in an effort to
0001-8686/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.cis.2013.10.020
L. Pérez et al. / Advances in Colloid and Interface Science 205 (2014) 134–155 135
b
Fig. 3. Chemical structure of arginine. (*) Asymmetric carbon.
guanidyl type in place of the quaternary ammonium type. They are was very useful to prepare easily a large number of homologues.
termed Nα,Nω-bis(long chain Nα-acylarginine) α,ω-alkyldiamide salts However, this synthetic methodology did not meet the requirements
or bisArgs. They will be referred to as compounds 2 in the text or Cn necessary for a possible industrial development of these products.
(XA)2, where n is the number of methylene groups in the spacer 3 For this reason, a simple environmentally friendly synthetic alternative
(s= 1), 4 (s= 2), 6 (s= 4), 9 (s= 7), 10 (s=8), 12 (s= 10) and X refers that fulfilled the growing demand for cleaner technologies was
to fatty acyl chain length; Oleyl (8, n = 6), Caproyl (10, n = 8), and investigated. In this sense, we considered enzyme technology as a
Lauroyl (12, n = 10). putative alternative for the synthesis of amino acid-based surfactants
Compounds 2 were prepared to develop new effective surfactants [60,73–75].
with lower environmental impact, and for fundamental investigations Over the past few years, a better understanding of enzyme
in molecular self-assembly as a function of the spacer and hydrophobic functionality and catalytic behaviour, together with the progress of
alkyl chain length [71,72]. A large number of bisArgs homologues of molecular engineering has led to new applications for various types
8, 10 and 12 carbon atoms (Fig. 4, 2, n = 6, 8 and 10, respectively) of enzymes, such as proteases, acylases, oxidases, amylases, glycosidases,
using diaminoalkane spacer chains of varying length (Fig. 4, 2, s = cellulases or lipases. After a systematic study of the influence of
1–10) were prepared at multigram scale employing chemical several reaction variables (organic solvent, aqueous buffer concentration,
methodology [62]. support for enzyme deposition, and substrate concentration) on the
The method used for this first approach involved chemical protecting reaction performance and production yield, we reported a new and
groups, deprotection reactions, organic solvents, and chemical catalysts simple chemo-enzymatic strategy for the synthesis of bisArgs
obtaining the final compounds with yields of about 60%. This method (Scheme 2, 2) which consisted of two steps [63,76]. This method
was based on the fact that the endoprotease papain can accept bonds. The authors followed a multistep strategy which involves
long-chain Nα-acyl-arginine-alkyl ester derivatives as acyl-donor the tert-Butyloxycarbonyl group (Boc) protection and deprotection
ester substrates. Thus, firstly the quantitative acylation of one of different amino reactive groups using different synthetic pathways.
amino group of the α,ω diaminoalkane spacer by the carboxylic Basically, first condensation of the amino terminal groups of the
ethyl ester of the Nα-acyl-arginine (Scheme 2, 1′) took place spermidine with the fatty chain is conducted; subsequently, the
spontaneously, at the melting point of the α,ω diaminoalkane, in a protected amino acid is coupled to the secondary amino groups of
solvent-free system. The second step was the papain-catalyzed the spermidine. Ester bonds were also used to link the fatty chains
reaction between another Nα-acyl-arginine ethyl ester and the free to the spacer [82].
aliphatic amino group of the derivative formed in the first step Recently, and based on our bisArgs surfactants we have chemically
(Scheme 2, 1″). Reactions were carried out in solid-to-solid and synthesised Gemini cationic surfactants derived from Lys to study the
solution systems using low-toxicity potential solvents. The best effect of several structural parameters (hydrophobic chain length,
yields (70%) were achieved in solid-to-solid systems and in ethanol number and type of the cationic charge, spacer chain nature) on their
at a water content of 0.07. physicochemical properties and cellular toxicity [64].
BisArgs analogues of 8, 10 and 12 carbon atoms using 1, 3- Four Gemini surfactants (Fig. 6, 11–14) have been prepared in which
diaminopropane and 1, 3-diamino-2-hydroxy-propane as alkyldiamine the spacer chain and the number and type of cationic charges have been
spacers were prepared at the 6–7 g level employing the enzymatic regulated: (11) with two positive charges on the ε-amino groups of the
methodology developed. Products were purified using cation-exchange Lys and a methylene-based spacer chain; (12) with two positive charges
chromatography [77]. This strategy allows the preparation of amino on the α-amino groups of the Lys and a methylene-based spacer chain;
acid-based Gemini derivatives with two different polar heads or (13) with two positive charges on the two trimethylated α-amino
hydrophobic tails, rendering asymmetric compounds. groups of the quaternised Lys and a methylene-based spacer chain;
Vatively et al. [78] described a chemoenzymatic approach to prepare and finally (14) with a spermidine-based spacer and two positive
dimeric amino acid-based Gemini surfactants at analytical scale charges on the α-amino groups of the Lys but with a third positive
(not at multigram scale) with the amino acid head group bound to charge on the spacer chain. These compounds can be considered dimers
the spacer by ester linkages. Protected starting amino acids such as of the long-chain Nα- or Nε-lauroyl lysine derivatives [83]. They
N-Carbobenzyloxy(Cbz)-glycine, N-Cbz-phenylalanine, N-Cbz-L-tyrosine, were obtained at multigram scale with purity of 99% by chemical
N-Cbz-L-serine, N-Cbz-L-aspartic acid, N-Cbz-L-glutamic acid, Nα-Cbz-L- condensation of the single long-chain N-lauroyl-L-lysine, previously
lysine, Nα,Nε-di-Cbz-L-lysine, Nα,Nε-Di-Cbz-L-cystine and N,S-di-Cbz-L- protected, to the corresponding spacer in the presence of an activating
cysteine were condensed to α, ω polymethylenediol spacer chain by a agent. A final deprotection reaction was carried out to obtain the
lipase-catalyzed esterification reaction. desired cationic Gemini compound.
4
3
8
5
9
6
7
10
Fig. 5. Chemical structure of (3) Nα lauroyl lysine , (4) Nε lauroyl lysine, and different structures of lysine based Gemini surfactants (5) buthylendiamine NN′ dilauroyl lysine,
(6) Nα, Nω-bis(Nα-lauroyl-lysine) α,ω-hexylendiamide, (7) Nα, Nω-bis(Nα-lauroyl-lysine) α,ω-spermidindiamide, (8) Nα, Nω-bis(Nε-lauroyl-lysine) α,ω-hexylendiamide,
(9) Nα, Nω-bis(Nε-lauroyl-lysine) α,ω-spermidindiamide, (10) spermidine α,ω dioleoylamide NN lysinamide.
monomeric counterpart cysteine derivatives [91,92]. In fact, the anionic three lysines through their α or ε amino groups (α–α, ε–ε, α–ε)
ones have been the most investigated cystine Geminis. (Fig. 9, 24) [96].
Faustino et al. [93] reported anionic urea-based Gemini surfactants
(Fig. 8, 22) derived from L-cystine D-cystine and DL cystine and their 2.1.4. Gemini surfactants from neutral amino acids: β alanine, serine
monomeric counterparts cysteine, cysteic acid and metionine to and sarcosine
characterise their solution properties and to study Gemini surfactant- Neutral amino acids such as β-alanine, serine, and sarcosine have
protein and cyclodextrin interactions. also been used as raw materials to prepare Gemini type ionic surfactants.
A sophisticated family of cationic sulphur-containing peptide Kunieda et al. [65] investigated the phase behavior of a water-
Gemini surfactants with a spacer of thioether type instead of disulphide gemini surfactant, sodium 1,2-bis(N-dodecanoyl β-alanate)-N-ethane)
was chemically synthesised by Kirby et al. [94] and their aggregation (Fig. 10, 26) obtained by reaction of 1,2-bis(β-alanate)-N-ethane with
properties studied in order to obtain more stable compounds for lauroyl chloride. The properties were compared with the corresponding
drug delivery purposes with biocompatible properties [95]. The monomeric counterpart sodium N-dodecanoyl-N methyl β-alanate
compounds contain several amino acids in the molecule and a (Fig. 10, 25).
hydrophobic tail of lauryl, or oleyl groups. The synthesis of these Quaternary ammonium serine-based Gemini surfactants with
compounds consisted of a multistep methodology, attaching the alkyl chains of 12, 14, 16 and 18 and spacer chains (m) of 5, 10, 12
amino acids step by step using the N-hydroxysuccinimide active linked to the nitrogen atom of the serine (Fig. 10, 28) were obtained
ester method. The first step involves the reaction of L-cysteine by Silva et al. [97] by reductive amination of the corresponding α, ω
with dibromoethane to form the bisthioether and the subsequent dialdehyde with the monomeric serine counterpart (Fig. 10, 27).
incorporation of lysine and serine at the amino and carboxyl group Anionic Gemini surfactants from sarcosine with different alkyl chain
of each cysteine moiety. Finally, the fatty amine tails are attached length whose structures are shown in Fig. 10, 29 have also been
to the carboxyl group by amide covalent bonds (Fig. 9, 23). synthesised to compare their flotative efficiency with known flotative
Multicationic Gemini surfactants derived from the structure 23 were agents, N-dodecanoylsarcosine or in combination with alkylammonium
also prepared by a stepwise liquid-phase technique attaching two or salts as co-collector [98].
140 L. Pérez et al. / Advances in Colloid and Interface Science 205 (2014) 134–155
Fig. 6. Chemical structure of new cationic Gemini surfactants derived from Nα -lauroyl-lysine and Nε -lauroyl-lysine (Fig. 5, 3 and 4) respectively. Dihydrochloride salts of
(11) Nα, Nω-bis(Nα-lauroyl-lysine) α,ω-hexylendiamide; (12) Nε,Nω-bis(Nε-lauroyl-lysine) α,ω-hexylendiamide; (13) Nε,Nω-bis(Nε-lauroyl-Nα-trimethyl-lysine) α, ω-hexylendiamide;
(14) Nε,Nω-bis(Nε-lauroyl-lysine) α,ω-spermidindiamide.
2.2. Gemini surfactants with amino acids/peptides in the spacer their physicochemical properties studied in relation to their structure.
Compounds with the same structure were earlier described by
Peptide-based Gemini-like surfactants are compounds with two Infante et al. as mimetics of phospholipids but not as Gemini-like
hydrophobic groups in which the alkylene spacer and the head groups surfactants [102].
have been substituted by a peptide or amino acid, resulting, in most Asymmetrical cationic Gemini surfactant analogues from lysine, that
cases, in an asymmetrical molecule with cationic or anionic character. have two quaternary ammonium groups linked to the fluorinated or
They are usually synthesised for research purposes, although it is hydrocarbon alkyl chains through amide bonds and the lysine as spacer
possible to find them commercially as in Fig. 11, 30, where the anionic chain (Fig. 12, 34) were prepared to study their antimicrobial properties
tripeptide, long chain acyl Glu-Lys-Glu alkyl amide from Asahi Kasei by Xiao et al. [103].
Chemicals Co., Japan is illustrated [66,99].
Recently, cationic Gemini tetrapeptide based compounds char- 3. Physicochemical properties
acterised to contain basic amino acids such as Arg and Lys have been
designed and synthesised as delivery carriers. Accordingly, Gemini 3.1. Surfactant adsorption from solution and micellisation
peptides such as Ser-Pro-Arg-Lys, Ser-Arg-Lys-Pro or Ser-Pro-Lys-
Arg, (this later represented in Fig. 11, 31) appended with oleoyl Several varying structures of amino acid-based Gemini surfactants
(condensed to the α-amino terminal) and oleyl amine (condensed have been characterised mainly by surface tension. In the following,
to the α-carboxyle terminal) tails have been described [100]. The we will use the μM scale for cmc values systematically (without
synthesis of the linear tetrapeptides was accomplished by Fmoc-based considering the negligible differences with the micromolal scale). The
solid phase methodologies. The tails are bound by amide bonds at values in the literature almost absolutely refer to 25 °C, we will only
their C and N termini with an alkyl amine and alkyl carboxylic acid, emphasise the temperature if it differs from this standard. We have
respectively. also rounded some of the literature values that we considered had too
The use of one amino acid as spacer to prepare Gemini surfactants many significant figures. In the following, we have grouped the results
has also been described. The architecture of these compounds is not following the amino acid from which they are derived. Most of the
exactly a dimeric structure but with a Gemini-like or pseudogemini surfactants have charges that may depend on the pH, protonated
configuration. These compounds have two alkyl chains and one amino amino groups for cationic species and carboxylate groups for anionic
acid as spacer (Fig. 12). Thus, several lysine-based amphiphiles with species, in some cases the positive charges have been fixed by
a Gemini-like configuration have been synthesised, with the amino quaternization. Also, we will consider aqueous solutions and brine,
acid side chain as the spacer group [101]. The molecules are either but not other solvents or additives like polymers [104].
esters (neutral, with C6–C12 even chains) (Fig. 12, 32) or sodium One of the first reports on Gemini surfactants derived from amino
carboxylates (anionic, with C6–C12 even chains) (Fig. 12, 33) and acids concerned cationic disulphur derivatives [86] (Fig. 7, 17 and 18).
L. Pérez et al. / Advances in Colloid and Interface Science 205 (2014) 134–155 141
Fig. 7. Cationic Gemini surfactants from cystine and cystamine. (17) di N lauryl N N dimethyl aminobetaine) N N cystinamide dimethyl ester salt; (18) di N lauryl N N dimethyl
aminobetaine) N N cystamide dimethyl ester salt, (19) cystine di dodecyl ester salt, (20) cystine dilauryl amide salt.
Those compounds showed very low cmc values, 21 and 41 μM order aggregates, like rods and vesicles, in line with the findings in
respectively and moderate to good adsorption efficacy with surface other amino acid and bisQuats Gemini surfactants.
tensions at cmc of 35 mNm−1 and 31 mNm−1 respectively. Also, the Fan et al. [91] studied the lauroyl and decanoyl homologues of
estimated areas per molecule were reasonable for dialkyl surfactants anionic Gemini N, N′ diacyl cystinate salt (Fig. 8, 21a). They found
with 1.0 and 1.2nm2 respectively [86,87]. Further studies with products the cmc to be 22 (at 40 °C) and 750 (at 30 °C) μM, respectively, by
with a somewhat lower purity (alleged presence of partial monoalkyl surface tension. They used the reversibility of the disulphur bond
derivatives) produced significantly bigger values of cmc, i.e. 65 and (by adding dithiothreitol or mercaptoethanol as dimer destroyer
120 μM, respectively, by using the Wilhelmy plate and 40 and 70 μM, and H2O2 as dimer promoter) to pulsate the surface tension of
respectively, by using a captive bubble technique [88]. In this last water with an amplitude around 20mNm−1 produced by the adsorption
study Pinazo et al. also reported the dynamics of adsorption using the of the dimeric surfactant (above its cmc) or the monomer (below the
pulsating bubble technique, concluding that the surfactant adsorption cmc) as dimerization is promoted or prevented. Accompanying these
is slower than the diffusion for these surfactants. changes in surface tension, formation and disappearance of vesicles
Aggregation properties of cystine anionic Gemini surfactants (Fig. 8, was demonstrated by electron microscopy.
21b) were studied by Yoshimura et al. [92]. They determined the cmc Enantiomerically pure and mixed didodecylcarbamic cystinate salts
in basic conditions (pH = 13) of homologues with 8, 10 and 12 carbon (Fig. 8, 22) were studied by Faustino et al. [93]. Their results show that
atoms per hydrophobic chain as 493, 139 and 1.4 μM, respectively, the cmc values estimated both from conductivity and surface tension
compared with the respective monomers as 6520, 424 and 180 μM, values do not reflect stereochemical effects, however, the adsorption
respectively. They also determined the efficacy in surface tension seemed influenced by stereochemistry. The area per molecule increased
reduction to be reduced as the surfactant chain length increased. from L to D to DL as 1.68, 1.88 and 2.14 nm2. The existence of
It was found that taking into account the standard free energies, stereochemical effects on the adsorbed layer should be related to
the adsorption at the air/water interface is promoted more than the the increased surface concentration as compared to the bulk 2D
micellization in the solution. From electron microscopy and SANS organisation of stereochemical molecules and may be favoured as
data they obtained the coexistence of spherical micelles with higher compared to the racemic mixtures.
142 L. Pérez et al. / Advances in Colloid and Interface Science 205 (2014) 134–155
Fig. 8. Anionic Gemini surfactants from cystine. (21a) N,N′ dilauroyl cystinate salt, (21b) N,N′-dilauryl cystinatesalt; (22) N,N′ didodecylcarbamic cystinate salt.
A significant effort has been devoted to the characterisation of chain surfactant LAM (structure 1 in Fig. 4). The most striking feature
cationic Arg Gemini surfactants. In Table 1, the cmc, surface tension at is the very low values of cmc, in the range of a few micromoles per
cmc (γcmc) and area per molecule (Amol) are shown for bisArgs (Fig. 4, litre, and also the strong difference (three orders of magnitude) as
2) with a hydrophobic chain length of C12 carbon atoms and spacer compared with the single chain compound. For comparison between
chain lengths between 2 and 10, and compared with the C12 single these values and those obtained for quaternary ammonium Gemini
surfactants, see Tables 1 and 3. As it can be observed, the dimerisation
of DTAB reduces the cmc value between one and two orders of
magnitude. The effect of 0.01 M NaCl on the adsorption behaviour is
shown in Table 2. Here we can observe the widely different behaviour
of the single chain surfactant (with a cmc decrease factor of 30) and
the dimeric surfactants with negligible effect. Moreover, it was observed
that bulk methods (conductivity, fluorescence and NMR) resulted
in different cmc values [108]. Those very different values of cmc
depending on whether the bulk or the surface tension was sensed
are difficult to reconcile. According to Pinazo et al. [106], “The low-
concentration cmc may be attributed to nonglobular aggregates
with small aggregation numbers, but perhaps larger than 2 or 3, to
account for the nearly constant monomer activity and the nearly
constant tension above the cmc1, and to small counterion binding
parameters. At concentrations higher than cmc2, strong and diverse
evidence shows that surfactants form micelle like globular aggregates,
that their degree of counterion binding is higher, that their molar
conductivity is smaller, and that the fluorophore solubilization behavior
is similar to those of conventional monomeric ionic surfactants.”
According to their results the nonglobular aggregates formed at cmc1
have lower counterion binding and smaller aggregation numbers than
the globular aggregates (micelles) detected as cmc2. Also, and as a
difference to conventional micelles, the low concentration aggregates
tend to increase the molar conductivity compared to that of the pre-
cmc solution. This is different to the non-surface active premicellar
aggregates suggested by Tsubone et al. to explain their surface tension
results with alanine derivatives [109]. An alternative explanation of
the discrepancy relies on the acid–base behaviour of weak acid
Fig. 9. Structure of cationic sulphur-containing peptide Gemini surfactants. surfactants. At low concentrations, the basicity of water is enough
L. Pérez et al. / Advances in Colloid and Interface Science 205 (2014) 134–155 143
Fig. 10. Gemini surfactants from neutral amino acids (25) Sodium N-dodecanoyl-N methyl β-alanate; (26) sodium 1, 2-bis (N-dodecanoyl β -alanate)-N-ethane; (27) N-dodecyl serine
methyl ester ; (28) α, ω bis (N-dodecyl N-methyl serine methyl ester) quaternary ammonium salt; (29) Gemini surfactants from N-lauroyl sarcosinate salt.
to produce a small but significant amount of neutral species which (1 mM). The authors compared this value with that of the monomeric
adsorb to the surface well before any aggregation can be detected surfactant which by surface tension was found to be 7600 μM, this
by conductivity, NMR or fluorescent solubilisation. Also, the release corresponds to a 200 fold decrease by dimerisation, which is between
of protons justifies the increase of molar conductivities. This seems the decreased values observed for bisQuats and those of bisArgs. The
to be the case for diacyl glycerol amino acid derivatives as described increase of ionic strength implied a significant decrease of cmc as
by Pinazo et al. [61]. This behaviour, i.e. enhanced pKa shift coupled to observed from both techniques, different to what was observed for
non spherical aggregates at low concentrations seems to be common bisArgs. The authors also investigated the possible pKa shift for the
to other amino acid derived Gemini surfactants. Experiments on the Gemini surfactant. While the pKa obtained for the monomeric surfactant
dynamics of adsorption of bisArgs [110] resulted in slower adsorption was 6.8, the pKa of both carboxylic groups in the dimeric surfactant
as compared with the monomer but one thousand times more efficient showed significant pKa shifts with pKa1 = 8.5 and pKa2 = 4.1. Because
for the surface tension and around 20 times more efficient as foam of the concentrations used for pKa determinations, the monomeric
stabilisers, also in line with the observations of bisQuats [111]. surfactant is at a concentration below cmc, while the Gemini surfactant
Sodium 1,2-bis(N-dodecanoyl β-alanate)-N-ethane (Fig. 10, 26) is well above its cmc. This could make the comparison faulty. It is a bit
aggregation properties in the diluted regime were investigated by surprising that the pKa of the single chain surfactant differs so much
Tsubone and Ghosh [112]. They found a value of 37 μM by surface from that of short chain aliphatic carboxylic acids (~4.75) while
tension determinations (with a surface tension value of around measured below the cmc, where no major pKa shift is expected.
27 mN/m, at the cmc) which coincides with the determination of cmc Concerning the two pKa values, one of them is shifted positively and
(32μM) obtained from pyrene fluorescence results at low ionic strength the other one negatively, this implies that the one charge configuration
30 31
Fig. 11. Gemini surfactants with peptides in the spacer (30) N-dodecyl-Glu-Lys-Glu-O-dodecyl amide, (31) N-oleoyl-Ser-Pro-Lys-Arg-O-oleyl amide.
144 L. Pérez et al. / Advances in Colloid and Interface Science 205 (2014) 134–155
Fig. 12. Structure of lysine derived Gemini like surfactants, (32) non-ionic long chain diacyl lysine methyl ester and (33) anionic diacyl sodium lysinate, (34) quaternary
ammonium L-lysine Gemini surfactant.
for this surfactant is favoured at neutral pH. This, in part, contradicts surface tension in 100 mM NaCl as being 6 μM with a decrease of
another observation of the same authors concerning the practical two orders of magnitude from that of the monomeric surfactant
neutrality of the Gemini surfactant at low concentrations. which was 600 μM. The Gemini surfactant, with a surface tension of
Further studies on homologues of 26 (Fig. 10) varying both the 25 mN/m, is quite effective compared both with bisQuats and
hydrophobic length and the spacer length based on the alanine bisArgs. The interaction with sodium dodecyl sulphate (SDS) was
structure, showed that when the spacer contains two methylene studied and resulted in marginal synergism both for surface tension
groups, premicellar self-aggregation (both in water and in 10− 2 M and cmc value while the interaction of the Gemini surfactant with
NaCl) occurs when the hydrophobic group contains more than 12 the monomeric one had slight positive synergism for surface tension
carbon atoms. This was evidenced from surface tension measurements and slight negative effect for the cmc. By using pyrene fluorescence
of cmc. Those premicellar, non surface active agreggates, induce determinations, two transitions were observed by Tsubone and
deviations in the classical cmc-hydrophobic surfactant content behaviour, Ghosh for the same surfactant [114], the high concentration one
even showing an increase in the cmc value when the hydrophobic chain coinciding with that obtained from surface tension and a lower
increases. This can be observed in Table 4 where the values for the concentration one at around 1 μM. The authors attributed this to
derivatives with two and four methylene spacer groups are shown for the formation of premicellar aggregates.
different hydrophobic chain lengths. However, when the spacer contains Surface adsorption and cmc were determined from surface tension
four methylene groups, no premicellar aggregation was detected at any values for aspartate derivatives as a function of the hydrophobic chain
hydrophobic chain length. The deviations in the direction of larger cmc length with a homologue structure shown in Fig. 13. The main results
compared to that expected have been shown to be due to the formation are shown in Table 5 for water and 10 mM NaCl. It is also noteworthy
of small, nonsurface active premicellar soluble aggregates (dimers), to observe the small effect of the common salt on the cmc for most of
when the van der Waals attractive forces between the hydrophobic the derivatives, which coincides with observations for bisArgs [105].
groups become strong enough. In the same article, the discrepancy in However, there was a strong influence on the minimum area per
the measurement of cmc by surface tension and conductance was molecule as determined from the application of Gibbs adsorption
attributed to aggregation induced changes in surfactant protonation. isotherms. Note also the strong decrease obtained for the area per
Unfortunately, in the plots of pH-surfactant concentration on a linear molecule, areas that correspond to less than the minimum area
scale, the presence of a break in the cmc range cannot be observed. occupied by two alkyl chains which is roughly 0.4 nm. Therefore, these
Tsubone [113] also described the cmc of sodium 1,2-bis(N- values for areas per molecule could be due to either experimental
dodecanoyl β-aspartate)-N-ethane (Fig. 13, 35) as obtained from problems or they may not comply with the conditions applicable for
the Gibbs adsorption isotherm.
Table 1
Adsorption properties of bisArgs Cn(LA)2 (Fig. 4, 2) as a function of the spacer chain length
in water at 25 °C. Hydrophobic chain length of C12 carbon atoms, spacer chain length Table 2
between 2 and 10 (Fig. 4, 2; n = 10, s = 0–8) and compared with the C12 monomer Adsorption properties of bisArgs Cn(LA)2 (Fig. 4, 2) as a function of spacer length in 0.01 M
LAM (Fig. 4, 1 when n = 10) [105]. Values in parenthesis correspond to conductivity NaCl (brine) at 25 °C and compared with the monomer LAM (Fig. 4, 1 when n = 10).
data from Pinazo et al. [106]. From Pérez et al. [105].
Surfactant cmc (μM) γcmc (mNm−1) Amol (nm2) Surfactant cmc (μM) γcmc (mNm−1) Amol (nm2)
Table 3
Adsorption properties of dodecyl-α,ω-bis(dimethylalkylammonium bromide) bisQuats
surfactants as a function of spacer length (Fig. 1, s = 3–10) in water at 25 °C compared
with the monomer dodecyl trimethylammonium bromide (DTAB).
From Alami et al. [107].
Table 6 feature of these ternary phase diagrams was the swelling of the H1 to
Adsorption properties of serine derivatives (Fig. 10, 28) as a function of spacer and produce a discontinuous (Fm3m according to their peak findings)
hydrophobic chain lengths in water at 25 °C and compared with two monomers.
From Silva et al. [97].
cubic phase. Then, the formation of microemulsions was studied using
hexanol and butanol as cosurfactants, dodecane as oil, and sodium
Surfactant cmc (μM) γcmc (mNm−1) Amol (nm2) chloride solutions. In those cases, significant differences were obtained.
12-5-12 320 34.7 0.94 In the case of hexanol, only the single chain surfactant formed a wide
12-10-12 67 35.1 0.81 microemulsion region while the use of butanol promoted similarly
12-12-12 82 35.4 1.61
large microemulsion regions for both surfactants.
14-5-14 42 34.8 1.25
16-5-16 15 n/a n/a The aqueous binary phase behaviour of acyl N-dodecyl-Glu-Lys-
12 monomer 1900 29.2 0.46 Glu-dodecyl amide (Fig. 11, 30) and the homologues tetradecyl
14 monomer 1200 33.0 0.56 and hexadecyl was studied as a function of concentration and
temperature by Shrestha et al. [66]. The short chain derivatives
self-assemble into spherical micelles above the critical micelle
concentration (curiously enough, the value of cmc was not revealed
elongated micelles. This has been demonstrated also by electron by the authors) and transform to a micellar cubic phase with a space
microscopy [119] for the arginine derivatives. It was found that the group Pm3n at around 33 wt.% at 25 °C (according to the observed
single chain compound behaves as expected for a classic surfactant sequence of peaks 4½;5½;6½, with the notable absence of the peak
with a dodecyl hydrophobic chain (spherical micelles at moderate expected at 2½). The hexadecyl derivative presents similar behaviour
concentrations, b 8%, and elongated micelles in the concentrated but only at temperatures above 50 °C. At higher concentrations,
regime, N8%) while the dimeric surfactants with short spacer the sequence continues with hexagonal and lamellar phases. This
(Fig. 4, 2 when s = 1) formed spheroidal micelles at low sequence differs from that encountered for alanate derivatives [65]
concentrations (b4% but with the significant presence of some long in which the cubic phase is bicontinuous and is found between the
twisted ribbons) changing to predominant thread-like and disk-like hexagonal and lamellar phases.
aggregates at higher concentrations (N4%). Longer spacers decrease
the concentration at which predominant elongated structures are 4. Biological properties
observed, but still with some coexistence of small micelles and
elongated ones below 2%. In the case of the longest spacer (s = 10), Gemini surfactants derived from amino acids are a family of
no globular micelles were detected even at 0.1%. This behaviour is surfactants with a growing use in cosmetics, pharmaceutical and
similar to that found for the bisQuats [120]. biomedical applications. In general, surfactants derived from
The structure of the micelles produced by bisArgs was studied by amino acids are potentially non-toxic, eco-friendly with excellent
Pérez et al. [108] by means of SAXS, NMR and light scattering. The biodegradability. However, these properties must be checked
behaviour was compared to that of the monomer. The results agreed every time a new surfactant is developed.
with the observation of globular aggregates for the monomer as well
as for the short spacer derivative (s = 1) while a strong tendency to 4.1. Toxicity
form elongated micelles was determined for the longer spacer s = 4
and 7. For this last one, evolution of the samples with time was Most of the surfactant toxicity tests found in the literature describe
evidenced, forming more and more elongated structures. The authors in vitro alternatives with the goal of studying gene delivery [121]. This
made an effort to study the very low concentration regime by using kind of tests will be considered in the next section. Here, we focus on
light scattering. Their results indicated the presence of aggregates in tests whose goal is to find out the intrinsic toxicity of surfactants
the low concentration regime, that is, between the cmc determined without considering any specific applications. However, one should
by surface tension and that determined by conductivity, concluding keep in mind that it is unlikely that an in vitro system could ever be
that the highest concentration changes may correspond to micellar developed to mimic the complex cascade of reactions that occur in the
form changes. human organism. In vivo testing in human volunteers is, therefore, still
One of the compounds synthesised by Camilleri's group bearing crucial, at least to confirm in vitro results. We have identified five
two negative charges in the form of sulphonate groups [94] (analogues different categories of procedures used to measure surfactant toxicity,
of compound 23 in Fig. 9) had a cmc of 100 μM and formed globular say, Hemolytic activity, Lactate dehydrogenase release, Alamar blue
micelles (3–5 nm in diameter) coexisting with globular strings that assay, keratinocytes or fibroblasts, and MTT cell proliferation.
evolved in time to produce elongated micelles and fibrils showing
distinct characteristics of twisted ribbons, agreeing with electron 4.1.1. Hemolytic activity
microscopy of other amino acid-based Gemini surfactants. Hemolytic activity, also known as the red blood assay, is based on
Kunieda et al. [65] studied the phase behavior of a dimeric alanate spectroscopic measurement of the amount of hemoglobin after the
derivative (Fig. 10, 26). The authors compared the aqueous phase hemolysis of erythrocytes induced by surfactants. The percentage of
behavior to that of the monomer precursor (Fig. 10, 25) and found hemolysis is determined by comparing the hemoglobin amount present
parallel, classical behavior. That is, the sequence of aqueous micellar, in the samples treated with surfactant with that of the control totally
hexagonal (H1), bicontinuous cubic and lamellar phases was observed hemolysed with distilled water. Dose–response curves are determined
increasing the surfactant concentration. Characterization of these from hemolysis results and the concentration inducing 50% hemolysis
phases was performed by SAXS to obtain the area per surfactant (HC50) is calculated. Earlier toxicity tests were introduced by Draize
molecule only for the hexagonal and lamellar phases. In both phases, et al. in [122] where chemical compounds to be tested were applied
the resulting areas per molecule for the Gemini surfactant are about in vivo to rabbit eyes. Toxicity was measured as the extent of the damage
90% of what was anticipated from doubling that of the monomeric to the eye's cornea. More recently, Pape et al. [123] developed the in vitro
molecule. However, this difference did not induce notable differences hemolytic test that allowed to measure toxicity by assessing the
in the concentration of formation of the different phases. The difference hemolysis produced by chemical compounds on plasma membranes.
in packing was attributed by the authors to the short spacer length used Vives et al. [124] applied for the first time the hemolysis test to measure
(−C2H4–). Furthermore, the authors studied ternary phase behaviour surfactant toxicity.
with dodecane and m-xylene where the differences between the In [71], Pérez et al. applied the hemolytic assay to determine the
Gemini and monomeric surfactant were also negligible. The main toxicity of bisArgs surfactants (Fig. 4, 2) in donated healthy human
L. Pérez et al. / Advances in Colloid and Interface Science 205 (2014) 134–155 147
efficacy represents a balance between the ability of the transfection 4.1.4. Keratinocytes or fibroblasts
agent and its associated cytotoxicity. The cytotoxicity assay based on keratinocytes or fibroblasts takes
advantage of the ability of viable cells to incorporate and bind neutral
4.1.3. Alamar blue assay red supravital dye. The neutral red is a weak cationic dye that readily
O'Brien et al. described for the first time the Alamar blue assay also diffuses through the plasma membrane and concentrates in lysosomes
known as resazurin reduction test, [129]. This assay measures the where it electrostatically binds to the anionic lysosomal matrix. Toxic
redox capacities of the cells due to the production of metabolites as a substances can alter the cell surface or the lysosomal membrane to
result of cell growth and allows the determination of viability over the cause lysosomal fragility and other adverse changes that gradually
period of culture without the detachment of adherent cells. Blue and become irreversible. Thus, cell death and/or inhibition of cell growth
nonfluorescent resazurin solutions are reduced to pink and highly decreases the amount of neutral red retained by the culture. Healthy
fluorescent resorufin, which is further reduced to hydroresorufin proliferating mammalian cells, when properly maintained in culture,
(uncoloured and nonfluorescent). This test has been used for about continuously divide and multiply over time. A toxic chemical, regardless
50 years to monitor bacterial and yeast contamination of milk, and of the site or mechanism of action, will interfere with this process
also for assessing semen quality. Recently, the dye has gained popularity and result in a reduction of the growth rate as reflected by cell
as a very simple and versatile way of measuring cell proliferation and number. Cytotoxicity is expressed as a concentration dependent
cytotoxicity. reduction of the uptake of NR after chemical exposure, thus providing
The Alamar blue assay has been applied by Calejo et al. in [130] a sensitive, integrated signal of both cell integrity and growth inhibition.
where authors study the suitability of arginine based surfactants in Martinez et al. in [133] applied the keratinocytes or fibroblasts assay to
the production of Ethyl(hyroxyethyl)cellulose-based thermoresponsive assess the integrity of membrane cells exposed to bisArgs surfactants
gel systems intended for human use. To this end the in vitro cytotoxicity (see structure 2 in Fig. 4) using the NCTC keratinocytes and 3 T6
of three arginine based surfactants, one single chain (LAM) and two fibroblast cells. The authors used HTAB commercial surfactant as
bisArgs (C6 (LA)2 and C9(LA)2 (Fig. 4, 1 and 2), was evaluated in a reference. Keratinocytes and fibroblasts cells were exposed to a
human cell line (HeLa) using the Alamar blue assay. The results are wide concentration range of bisArgs for 24 h. A clear dose–response
presented in terms of cell viability as a function of increasing surfactant relationship was established which allowed to calculate the con-
concentration and E50 values which is the surfactant concentration that centration of surfactant that caused 50% inhibition of growth for
induces a reduction of cell viability to 50%. The value of EC50 followed fibroblasts and keratinocytes. All bisArgs surfactants tested showed
the sequence LAM N C9(LA)2 N C6(LA)2. The lower toxicity observed an index of citotoxicity higher than the commercial surfactant. The
for the monomeric surfactant is probably related to its lower C3(OA)2 emerged as the least cytotoxic surfactant when compared
hydrophobicity, due to the sole presence of a single alkyl chain. In with the slightly irritant TB. Moreover, the cytotoxicity shown by
the case of Geminis the trend is not the same, C6(LA)2 has lower the bisArgs studied over cutaneous cells corroborate the observed
hydrophobic content but higher toxicity. The reasons for this can trend derived from the bisArgs hemolysis assay [126].
be related to the length of the spacer chain; it has been argued that
long hydrophobic spacer chains such as n = 9 become flexible
allowing penetration in the hydrophobic core avoiding contact 4.1.5. MTT cell proliferation assay
with the hydrophilic environment [105]. The reduction in the Measurement of cell viability and proliferation forms is the basis for
exposure of the hydrophobic moiety to the aqueous environment numerous in vitro assays of a cell population's response to external
might explain a lower cell membrane disturbance in the presence factors. The reduction of tetrazolium salts is now widely accepted as
of C9(LA)2 as compared to C6(LA)2. a reliable way to examine cell proliferation. The yellow tetrazolium
The cytotoxicities of a series of serine-based Gemini surfactants, MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide)
(Fig. 10, 28 and homologues), in human cell lines (HeLa cells) were is reduced by metabolically active cells, in part by the action of
evaluated by Silva et al. in [131]. Surfactants were studied in aqueous hydrogenase enzymes, to generate reducing equivalents such as
solutions at physiological pH by the colorimetric Alamar blue assay. NADH and NADPH. The resulting intracellular purple formazan
The toxicities of the surfactants at different concentrations were (products of the reduction of tetrazolium salts) can be solubilised
evaluated after 4, 8 or 24 h of incubation. Comparative in vitro studies and quantified by spectrophotometric means. The MTT cell proliferation
with HeLa cells show that the serine-based Gemini surfactants exhibit assay measures the cell proliferation rate and conversely, when
weaker cytotoxic effects than conventional bisQuats and monomeric metabolic events lead to apoptosis or necrosis, the reduction in cell
homologues, an important aspect in terms of potentional biological viability. The number of assay steps has been minimised as much as
applications. This is in contrast to what was found for Arg derivatives. possible to expedite sample processing. The MTT reagent yields low
Surfactant chain length and type of spacer linkage (amine, amide, and background absorbance values in the absence of cells. For each cell
ester) have marked influences on the cytotoxicity profiles exhibited type the linear relationship between cell number and signal produced
by the serine-based Gemini compounds. At all exposure times, the is established, thus allowing an accurate quantification of changes in
cytotoxicity displayed by the surfactants with the amide linkage is the rate of cell proliferation.
higher than those of its amine counterpart. The ester surfactant shows Recently Yang et al. [134] developed a series of Gemini surfactants in
intermediated toxicity values with respect to the other surfactants. An which the following amino acids or dipeptides were introduced in the
increase in chain length from 12 to 16 carbon atoms promotes a parallel spacer chain: glycine, lysine, glycyl–lysine and lysyl–lysyl (Fig. 15, 37).
toxicity decrease. C18 shows lower cytotoxicity than C16. The authors In [135] these compounds were tested in rabbit epithelial cells using
give a rationale based on the cut-off point which is defined as the a plasmid model and a helper lipid. The MTT assay was used to evaluate
point at which biological activity reaches a maximum value and then cell toxicity. An overall low toxicity is observed for all with no significant
starts to decline with further increases in molecular size. differences among substituted and unsubstituted compounds. The dye
Calejo et al. in [132] devotes a rather short section to study the exclusion assay suggests a more balanced protection of the DNA
cytotoxicity of three lysine-derived surfactants with a Gemini like by the glycine and glycyl–lysine substituted compounds. The higher
structure (Fig. 12, 33). Toxicity, evaluated on Hela cells, increased with transfection efficiency is attributed to the greater biocompatibility and
the chain length from C6 to C10. The C6 chain length surfactant was flexibility of the amino acid/peptide-substituted Gemini surfactant and
shown to be less cytotoxic than sodium dodecyl sulphate and the demonstrates the feasibility of using amino acid-substituted Gemini
lysine-derived surfactants studied were less toxic than the cationic surfactants as gene carriers. These results agree with those reported
hexadecyl trimethylammonium bromide (HTAB). by Castro et al. [12].
L. Pérez et al. / Advances in Colloid and Interface Science 205 (2014) 134–155 149
Fig. 17. Structure–activity dependence on hydrocarbon tail length and peptide head section. (a) Transfection activity as a function of increasing Gemini concentration; highest activity was
generally observed at 4 μM, and data for other compounds (panels b and c) represent measurements in the presence of 4 μM surfactant. (b) Effect of varying chain length for the series with
ε, ε-linked trilysine GS2, GS5, GS7, GS11. (c) Effect of varying trilysine linkage GS12, GS13, GS9, GS11; data are shown for compounds with the oleyl (C18) side-chain and (inset) for the
corresponding C12 derivatives.
From reference [96]. Reprinted adapted with permission from reference [96]. Copyright 2013 American Chemical Society.
avoid the steric effects than can limit the formation of ion pairs with Cationic Gemini surfactants consisting of two N-acyl-lysine linked
anionic molecules [143]. through a diamine spacer (Fig. 5, 6 or Fig. 6, 11) show also transfection
Physico-chemical studies of these cysteine Gemini surfactants activity, but in this case the activity was similar to changing spacer
indicated that the formation of micelles was not necessary for length. This behavior could be due to the distance between the cationic
complexation to DNA given that the cmc values were well above charges and the spacer. In these compounds, even for the shortest
the concentration used in DNA-binding and transfection experiments. spacer there is a substantial distance between the two positive cationic
A correlation between aggregate morphology and transfection efficiency charges. Recently, a study has been reported [146] on lipoplexes formed
was observed. Gemini surfactants that formed arrays or fibrils were by Gemini surfactants of the type 11, shown in Fig. 6 with C12 chains
ineffective vectors, while those lacking these structures were more and a spacer of 6 carbon atoms, C6(LA)2, the neutral helper lipid 1,2-
effective [144]. dioleyl-sn-glycero-3-phosphatidylethanolamine (DOPE), and either of
Amino acid Gemini surfactants based on spermine have been the two DNA types, a plasmid DNA or a commercial linear DNA has
proposed for gene delivery. Castro [12] described the synthesis of a been reported [147]. The electrochemical studies revealed that the
series of spermidine-derived cationic Gemini surfactants based on plasmid DNA is compacted with a larger number of Na+ counterions
different diamino acid lysine homologues (Fig. 5, 10). The presence than the linear DNA and, consequently, has a much lower effective
of two symmetrical oleyl chains is essential for obtaining high negative charge. This implies that lower quantity of cationic lipid
transfection levels. Two different groups of spermine Gemini is necessary and accordingly the potential toxicity is reduced. The
surfactants have also been prepared by Ronsin et al. [145] one with composition of the mixed liposomes plays an important role in the
the peptide head groups (AAm) linked to the terminal amino groups
of the spermine and the alkyl chains to the middle amino groups
(Fig. 18, 39a) and the other with the peptide group linked to the
middle amino groups of the spermine and the alkyl chains to the
terminal ones (Fig. 18, 39b). Transfection efficiency was tested
against four different cell lines. The oleyl chain generally gave the
most efficient transfection. The efficiency of compounds of the 39b
is considerably higher than those of 39a. In general, the most active
surfactants of 39b are those containing only one lysine linked to
every NH middle group of the spermine, however, in the case of a
mouse muscle cell line, the most active is the compound with a
peptide (three Lys and one Ser) linked to these amino groups of the
spermine. This means that it is also necessary to adapt the type of
cationic vectors to the cell type targeted.
The compaction of DNA with cationic lipids seems to involve an
initial interaction between a small group of surfactant molecules
and DNA. After that, compaction is enhanced through electrostatic
and hydrophobic interactions. This process could be favored by low
critical aggregation concentrations. In the case of the bisQuats of
the type 12-s-12, the minimum in compaction efficiency was found
at s = 6 and, a maximum in the critical aggregation concentration Fig. 18. Cationic Gemini surfactants from spermine with different positions for the peptide,
was found at s = 5 [146]. AAm, and alkyl chain [145].
152 L. Pérez et al. / Advances in Colloid and Interface Science 205 (2014) 134–155
final morphology of the lipoplex and, due to the chirality of these pathway of the aggregates but improved buffering capacity and
surfactants, different aggregates, from ribbon-type to cluster-type, conferred a pH-dependent increase of particle size. This is probably
have been observed (Fig. 19). the cause of a more efficient endosomial escape of the aggregates
Cationic Gemini surfactants based on tartaric acid have also been with the subsequent improvement of gene expression [149].
prepared to transport DNA into the cells. They consist of three Geminis An important physical property of amino acid-based surfactants
with palmitoyl chains in which the head group size has been enlarged: is their pH sensitivity. Xu and al. [150] prepared two cationic lipids
a) the smallest has one ethylendiamine linked to the tartaric acid, b) the that consisted of two N-oleyl cysteines linked through a peptide
other one has a lysine moiety, c) and the biggest contains one lysine and (histidine–lysine–histidine or lysine–triptophane–histidine). The
one ethylendiamine. While surfactant c) forms only stable micelles in nanoparticles prepared with these lipids and DNA or RNA exhibited
aqueous dispersion, a) and b) form non-stable vesicles that precipitated pH-sensitive hemolysis when the pH decreased from 7.5 to 5.5 which
after 15 min or one day. Compound a) did not show transfection is the endosomal–lysosomal pH. The amphiphilic carriers were more
activity whereas b) and c) displayed moderate transfection efficiency. effective for RNA delivery although both resulted in high cellular uptake
Moreover, these compounds showed high toxicity against the cells of the two nucleic acids. The results obtained suggested that the pH
transfected. The toxicity was attributed to the high hydrolysis of one sensitivity at the endosomal–lysosomal pH is an important factor for
of the ester bonds of the molecules which gives rise to intermediates efficient transfection and that amino acid Gemini surfactants are
with higher surfactant properties and greater toxicity [148]. promising carriers for this application. The structure–activity studies
BisQuats surfactants in which different amino acids or peptides until now indicate that it is not easy to establish structure–activity
(AAm) were introduced in the spacer chain a) glycine, b) lysine, relationships given that minor changes in structure can have major
c) glycyl-lysine or d) lysyl-lysine, (Fig. 15, 37), were tested as effects on biological activity and structure activity correlations are the
transfecting agents using different epithelial cells in order to know exception even when closely similar structures are compared.
the potential application of these compounds in scleroderma, atopic
dermatitis and wound healing. The spacer substituents are hydrophilic 5.2. Interactions with proteins and polymers
and make the spacer more hydrated than the non-substituted bisQuats,
therefore, the amino acid-based surfactants possess higher critical Interactions between proteins and Gemini surfactants from amino
micellar concentrations than non-substituted bisQuats. BisQuats acids have been also studied because they are important in industrial,
surfactants substituted with amino acids in the spacer displayed biological, pharmaceutical and cosmetic systems. This type of studies
higher transfection efficiency than the unsubstituted ones. These can help to understand the action of surfactants as denaturants and
studies indicated that the introduction of the amino acids improves solubilizing agents for proteins. It has been observed that Gemini
the delivery and release of DNA into the cytoplasm due to different surfactants from glutamic acid exhibit different interactions with
interactions with the nucleotide. BisQuats interact with DNA via hemoglobin than their corresponding single chain homologue. The
strong electrostatic interactions while Gemini surfactants could Gemini surfactants showed lower denaturing ability to hemoglobin,
interact via softened van der Waals and hydrogen bonding forces probably due to their bigger size, and the denaturation degree
increasing the flexibility of the aggregates. Moreover, the increase decreased when the spacer length increased. It was also observed
in hydrophilicity could lead to greater biocompatibility and lower that when the Gemini surfactants' content is low, the secondary
cytotoxicity [134,135]. Subsequent studies showed that the amino structure of hemoglobin can be stabilised. [151]. Different behavior
acid substitution in the spacer did not alter the cellular uptake has been observed for bisQuats surfactants of the C12CsC12 type and
Fig. 19. Crio TEM micrographs of C6(LA)2/DOPE/p-DNA lipoplexes showing the presence of ribbon type lipoplexes (a–e) in coexistence with cluster type lipoplexes (f).
From reference [147].]. Reproduced by permission of the Royal Society of Chemistry.
L. Pérez et al. / Advances in Colloid and Interface Science 205 (2014) 134–155 153
their single chain surfactant counterpart (dodecyl trimethyl ammonium When the monomeric surfactant is used, very high concentrations
bromide DTAB). In this case, the Gemini surfactants have much stronger of surfactant are needed to induce a sol–gel transition. The Gemini
binding ability with bovine serum albumin (BSA) to induce the surfactant showed superior gel forming efficiency. They can form
denaturation of BSA at a very low C12CsC12/BSA ratio. The results were thermoresponsive gels at concentrations one thousand times lower
attributed to the enhanced hydrophobic and electrostatic interactions than the monomeric ones. BisArgs were able to induce gelation at
associated to the chemical structure of the dimeric surfactants [152]. concentrations lower than the EC50; hence, these systems can be
Interactions between BSA and Gemini surfactants derived from particularly interesting for applications requiring temperature-
cysteine, 22 in Fig. 8, have also been reported. The interactions induced thickening. [130]. Aqueous dispersions of pure Gemini
with this protein were influenced by temperature and pH. The surfactants from arginine or mixtures of bisArgs with phospholipids
stereochemistry of the Gemini surfactants also influenced their also originate stable cationic colloidal systems with potential
interaction with BSA: the association of enantiomerically pure applications as drug delivery systems. Single chain surfactants and
surfactants is favored when compared to the racemic mixture, and Gemini with short spacer chains gave rise to solutions with micellar
pure L-stereochemistry is favored over D-stereochemistry [153]. aggregates while Gemini with long spacers formed big aggregates as
Takeda et al. reported the protective effect of Gemini surfactants on twisted ribbons that give rise to viscous solutions or gels (Fig. 20).
thermal denaturation of BSA. The Gemini surfactant studied by these Antimicrobial and hemolytic activity was found to be strongly affected
authors consists of two glutamic acids as polar heads and a lysine as by the size of the aggregates; small aggregates penetrate more deeply
spacer. At 25 °C, Gemini surfactants produced a higher decrease in the into the cell surface and have lower MIC and HC50 values. Gemini with
helicity of the protein than the anionic sodium dodecyl sulphate long spacer chains give rise to big aggregates and have more difficulties
(SDS). For the Gemini surfactant, the protection of the recovery of the to interact with biological membranes and show lower cytotoxicity. The
helicity of BSA appeared at lower concentration due to the higher HC50 of LAM, C6(LA)2 and C9(LA)2 are 80 μM, b 20 μM and 210 μM,
hydrophobicity of these compounds. In fact, the secondary structure of respectively. The concentration of the stock solution used to carry out
the protein mostly recovered to the original state in the presence of the biological tests also influences the biological properties of these
very small quantities of Gemini surfactant upon cooling to 25 °C from systems [128].
temperatures below 75 °C [154].
The interaction of surfactants with polymers is of increasing interest
6. Conclusions
because of their capacity as viscosity modulators and their solvent
capacity towards fats and oil. La Mesa et al. [104] studied systematically
Gemini natural amino acid-based surfactants present adsorption
the interaction of two different polymers (a homopolymer and
properties similar to that of general Gemini surfactants, that is, cmc
a hydrophobically modified graft copolymer, with three Gemini
values well below that of the monomeric constituents. The molecules
surfactants that differ in polar head groups: sulphonate, quaternary
whose charge is due to pH dependent groups, may present extremely
ammonium and arginine groups). These Gemini surfactants did
low cmc values, down to three orders of magnitude lower than that of
not interact with the homopolymer but for the hydrophobically
the monomeric constituent molecules as determined from surface
modified copolymer, the interaction was significant for all Gemini.
tension measurements. Bulk measurements result in more moderate
It was found that the arginine derivative was the least effective in
decreases, one to two orders of magnitude, parallel to those found
binding to the polymer, probably due to the smaller spacer chain
with simpler headgroups. The origin of this discrepancy (different
length of this surfactant.
from the two cmc phenomena found for quaternary ammonium
Gemini surfactants) has been attributed to strong pKa shifts induced
5.3. Biocompatible gels by non spherical micelle formation. However, the comprehension of
these phenomena is still lacking a general interpretation. The scarce
Recently, it has been reported that mixtures of the ethyl(hydro- literature on surfactant phase behaviour on amino acid derived
xyethyl)cellulose polysaccharide and both, monomeric and Gemini Gemini surfactants shows that they present phase behaviour similar
surfactants bisArgs 2 originate biocompatible thermoresponsive gels. to that of general Gemini surfactants, that is, formation of elongated
LAM C6(LA)2
C9(LA)2
Fig. 20. Size distribution profiles of LAM, C6(LA)2 and C9(LA)2 formulations at 4 mM [128].
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