heterocyclic cationic gemini surfactants: a comparative overview of their synthesis,...

Heterocyclic Cationic Gemini Surfactants:A Comparative Overview of Their

Synthesis, Self-assembling,Physicochemical, and Biological

Properties

Vishnu Dutt Sharma and Marc A. Ilies

Department of Pharmaceutical Sciences and Moulder Center for Drug Discovery Research, Temple UniversitySchool of Pharmacy, 3307 N Broad Street, Philadelphia, Pennsylvania 19140,

Published online in Wiley Online Library (wileyonlinelibrary.com).DOI 10.1002/med.21272

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Abstract: Gemini surfactants (GS) are presently receiving substantial attention due to their special self-assembling properties and unique interfacial activity. This comprehensive review is focused on positivelycharged heterocyclic GS, presenting their major synthetic access routes and examining the impact ofstructural elements on physicochemical and aggregation properties of this class of amphiphiles. Interactionof geminis surfactants with cells and their biological properties as novel transfection agents are emphasizedthrough a detailed structure–activity relationship analysis. Throughout the review we have also presentedthe properties of selected ammonium GS, simple surfactants and lipid congeners, in order to emphasizethe advantages conferred by using heterocyclic polar heads in GS design. C© 2012 Wiley Periodicals, Inc.

Med. Res. Rev., 00, No. 0, 1–44, 2012

Key words: gemini surfactant; cationic amphiphiles; self-assembling; transfection; gene delivery

1. INTRODUCTION: GENERAL STRUCTURE AND NOMENCLATURE OF GEMINISURFACTANTS

Twins have fascinated people throughout human history due to their unique, invisible bond,acting many times as two synergetic halves of a single mind. In surfactant chemistry, a similarsynergistic behavior was observed for dimeric surfactants, where an intrinsic link betweentwo surfactant molecules confers remarkable features and unique properties to this class of

Contract grant sponsor: Temple University—Dean’s Office; Contract grant sponsor: Temple University—Provost’sOffice.

Correspondence to: Dr. Marc A. Ilies, Department of Pharmaceutical Sciences, Temple University School ofPharmacy, 3307 N Broad Street, Philadelphia, PA 19140. E-mail: [email protected]

Medicinal Research Reviews, 00, No. 0, 1–44, 2012C© 2012 Wiley Periodicals, Inc.

2 � SHARMA AND ILIES

Figure 1. Cartoon depicting the general structure and main structural elements of gemini surfactants, in whichtwo (or more) individual surfactant molecules are chemically connected at the level of the polar head. Thepositioning of the linkage at (or very close) to the water/oil interface is essential: if the linkage between the twosurfactants is made far away from the polar head, the properties of the resulting compounds are completelydifferent and those compounds are not considered to belong to the class of gemini surfactants.

amphiphiles. This special behavior prompted Menger1 to coin the term “gemini surfactants”(GS) for amphiphiles consisting of two identical or different surfactants linked at the level ofthe polar head groups (Fig. 1), and sometimes at the level of the alkyl chains, yet very close tothe hydrophilic head.1–3

Historically, the research in the field of GS was rather sparse till early 90s. We have tomention the early work of Bunton et al.4 on “dicationic detergents,” of Devinsky et al.5 on“bisquaternary ammonium salts,” and of Okahara and co-workers6 on “sulfate group contain-ing amphiphatic compounds with two lipophilic chains.” The class of GS was later enlargedwith compounds that contain more than two hydrophilic heads and hydrophobic tails, forexample, trimeric amphiphiles.7

There are many ways to classify GS. Based on the polar head one may distinguish positivelycharged GS, such as ammonium, pyridinium; negatively charged GS, for example, phosphate,sulfate, and carboxylate polar heads; nonionic GS with sugar or peptidyl moieties at the aqueousinterface.8 The linker may be rigid (e.g., a stilbene moiety) or flexible (e.g., a hydrocarbon chain),with various sizes and polarities. The nomenclature m-s-m is generally used for these structures,where m is the number of alkyl carbon atoms in the hydrophobic tail and s is the number ofalkyl carbon atoms in the spacer.3 This review analyzes the synthesis, physicochemical andbiochemical properties of cationic GS, with emphasis on their heterocyclic members.

2. SYNTHETIC ROUTES TO ACCESS GEMINI SURFACTANTS

The structural diversity of GS is rather large, and consequently their synthesis and purifica-tion can vary significantly. For example, a simple ammonium GS 3 can be prepared easilyvia alkylation. Depending on the length of spacer and on the reactivity of the dibromide, wecan distinguish two main methods (Scheme 1). Thus, the first synthetic method (A) involvesreaction of a dibromide 1 with two fatty amines 2 to generate the cationic GS 3. It is particularlyuseful for reactive dibromides,2 which can be conveniently prepared from the correspondingdiols using PBr3.9 An alternative of this method involves alkylation of a tertiary amine with anepoxide; in this case the alkylation takes place at the less-substituted epoxide carbon atom.10

The second main synthetic route (method B) involves the reaction of fatty bromide 4 with alka-nediyl diamines 5 and is particularly advantageous for the formation of trimeric or oligomericamphiphiles from polyamines.7 This method is usually preferred for very short linkers (e.g., n =2, 3) since the first method will be less efficient due to high steric and electrostatic hindrance.7

Medicinal Research Reviews DOI 10.1002/med

HETEROCYCLIC CATIONIC GEMINI SURFACTANTS � 3

Scheme 1. Main synthetic strategies for accessing cationic ammonium GS.11,12 In method A the linker is intro-duced last, while in method B the hydrophobic chains are installed in the final steps. See text for details.

Comprehensive reviews on tetraalkylammonium GS synthesis are available,11, 12 providing fur-ther details for interested readers.

The choice of synthetic method depends also on the complexity of each structural element.Method A is predominantly used in conjunction with complex hydrophobic tails, which requiremultistep synthesis. Thus, Bhattacharya’s group synthesized novel cholesterol-based GS 8,with oxyethylene-type spacers, via alkylation of lipophilic tertiary amines 7 with various α,ω-dibromoalkoxyalkanes 6, in a screw-top pressure tube for up to 72 h (Scheme 2).13

Alternatively, method B becomes convenient when the linker is more complex than thehydrophobic tails, as exemplified by the recent GS synthesis from Badea’s group (Scheme 3).14

Scheme 2. Representative synthesis of cationic GS following synthetic method A.13

Scheme 3. Representative synthesis of cationic GS following synthetic method B.14



Heterocyclic cationic GS such as pyrolidinium,15 imidazolium,16–18, 32 or pyridinium19–24

are also accessible using these general alkylation strategies, but the reduced nucleophilicityof nitrogen-containing heterocycles requires longer reaction times and generate smaller finalyields as compared with ammonium GS. For example, the pyridinium GS with a tetramethylenespacer 15 has been synthesized from 1,4-dibromobutane 14 and α-alkyl pyridine 13 in ethanolicmedium at reflux (Scheme 4a). Repeated recrystallizations from acetone/ethanol yielded thepure compounds, which were characterized by standard methods and were compared in termsof physicochemical properties with their monomeric analogs (Scheme 4a).19

Engberts’ group synthesized a new series of pyridinium amphiphiles, including GS, using4-methylpyridine 16 as starting material.20, 21 Thus, 4-methylpyridine 16 was first deprotonatedwith two equivalents of lithium diisopropylamide, followed by the addition of two equivalentsof the appropriate alkyl iodide or alkyl bromide (Scheme 4b). Quaternization of the result-ing alkylated pyridine 18 with either methyl iodide or alkyl dibromides yielded the cationiclipids 19 and/or GS 20. Both amphiphiles were converted into the Cl− form by ion exchangechromatography (Scheme 4b).20, 21

Quagliotto et al. had prepared a novel series of pyridinium-based cationic GS by quaterniza-tion of the 2,2′-(α,ω-alkanediyl)bispyridines 24, 25, 27 or 4,4′-congeners 21 with N-alkylatingagents (alkylated halides, methanesulfonates, triflates), in order to correlate the geometry ofresulting GS with their physicochemical properties.22 The starting 2,2′- and 4,4′-bispyridineswere either commercially available or were made from 2-methylpyridine 23 or 2-vinylpyridine26 (Scheme 4c). The same group had prepared fluorinated versions of pyridinium GS fromthe same set of 2,2′-(α,ω-alkanediyl)bispyridines 24, 25, and 27 by refluxing them with fluori-nated alkyl methanesulfonates or trifluoromethanesulfonates, followed sometimes by counte-rion exchange.23 The fluorinated pyridinium GS 29 were thus obtained and their physiochemicalproperties were thoroughly investigated (Scheme 4c).

Another novel series of pyridinium GS (34) had been synthesized recently by quater-nization of pyridine using a pre-formed 1,2-bis(2-bromoalkylthio)ethane 33.24 The lipophilicdibromides were obtained by regioselective electrophilic cobromination of long-chain α-olefins31 using ethane-1,2-dithiol 30 and N-bromosuccinimide 32 (Scheme 4d). A similar syn-thetic strategy was reported recently by the same group for the generation of imidazoliumGS.18

Balaban, Ilies and co-workers proposed a new route to pyridinium cationic lipids and GSby condensing lipophilic pyrylium salts with primary diamines in the framework of the Bayer–Piccard reaction.25–28 This strategy allows the generation of the pyridinium polar head andlinker in a single, high yield, step. The structure of the lipophilic pyrylium salt 35 and diaminesused allow the control of the shape of the final surfactants. The reaction is versatile, allowingthe generation of GS 36, 38, 39, 41, 42, and 47 with different linker structures and polaritiesand also the access to trimeric 45 and oligomeric (mixed) pyridinium surfactants 43 and 44(Scheme 5).25–31

Following the general quaternization routes exemplified for pyridinium GS, Ao et al.32

synthesized new imidazolium GS 53 (Scheme 6) by alkylation of lipophilic imidazole derivatives52 with alkyl dibromides. The alkyl imidazoles were obtained through a reaction sequenceinvolving first the reaction of imidazole 48 with acrylonitrile 49 in methanol, subsequentalkylation of the imidazolium intermediate 50 with a long-chain alkyl bromide, followed bybasic cleavage of acrylonitrile moiety (Scheme 6).32 Other imidazolium-based GS were recentlysynthesized following similar procedures.16–18

In an elegant study, Menger’s group has synthesized the N-substituted diaza[12]annulenesGS 56 in moderate yields by using the one-pot reaction of N-(2,4-dinitrophenyl)pyridiniumchloride 54 with amines 55 discovered by Yamaguchi et al.,33 in which a 2,4-dinitrophenyl



Scheme 4. Representative syntheses of pyridinium GS based on quaternization of pyridines.19–24



Scheme 5. Synthesis of pyridinium GS from pyrylium salts28.



Scheme 6. The synthetic route used for preparation of imidazolium GS 5332

Scheme 7. Synthesis of the first annulene GS, by Menger’s group34—a rather extreme case in which the polarhead and the linker of the GS cannot be clearly distinguished.

moiety is used as a leaving group, and, interestingly, a pyridinium moiety functions as a C5synthetic equivalent (Scheme 7).34

3. SELF-ASSEMBLING AND PHYSIOCHEMICAL PROPERTIES OF GEMINISURFACTANTS

A. Critical Micelle Concentration

For simple surfactants the critical micelle concentration (CMC) is defined as the concentra-tion above which monomeric amphiphiles molecules placed in water spontaneously assembleinto supramolecular aggregates, quantitatively describing the propensity of the surfactant toassemble in this solvent. The lower the CMC, the stronger is the tendency of a surfactantto assemble.11, 35 For these amphiphilic molecules, the CMC decreases with the increase ofthe chain length (Table I). Other important structural parameters are the size and chargeof the polar head and of the counterions.36 CMC can be determined by various meth-ods such as calorimetry,37 conductometry,22 ESR spectroscopy, gonimetry, microscopy, lightscattering,2 neutron scattering,9 NMR spectroscopy,2 rheometry, surface tension method,38

and spectrophotometry.39

Focusing on GS, one may observe that they can display lower CMC values as compared withcorresponding single chain surfactants of same chain length, polar head, and counterion (e.g.,58 and 59, Chart 1 , Table I). The linker joining the two (or more) amphiphilic structural unitsrestricts their individual mobilities and lowers the CMC of the GS irrespective of the structure



Table I. CMC Values of Representative Cationic GS

Entry Surfactant Counterion CMC (mM) Reference

1 C12H25N+(CH3)3 Br− 16 40

2 C12H25N+(CH3)3 Cl− 22 41

3 C14H29N+(CH3)3 Br− 3.7 42

4 C16H33N+(CH3)3 Br− 1 43

5 1-C12H25-Py+ Cl− 17.1 22

6 1-C12H25-Py+(4)CH3 Cl− 15.1 44

7 1-C12H25-Im+(3)CH3 Br− 10.9 32

8 C8H17N+(CH3)2-(CH2)3-N+(CH3)2C8H17 2Br− 55 45

9 C10H21N+(CH3)2-(CH2)4-N+(CH3)2C10H21 2Br− 8.78 11

10 C12H25N+(CH3)2-(CH2)3-N+(CH3)2C12H25 2Cl− 1.36 35

11 C12H25N+(CH3)2-(CH2)3-N+(CH3)2C12H25 2Cl− 0.98 10

12 C12H25N+(CH3)2-(CH2)3-N+(CH3)2C12H25 2Br− 1.1 5

13 C12H25N+(CH3)2-(CH2)4-N+(CH3)2C12H25 2Br− 1 46

14 C12H25N+(CH3)2-(CH2)5-N+(CH3)2C12H25 2Br− 1.09 3

15 C12H25N+(CH3)2-(CH2)6-N+(CH3)2C12H25 2Br− 1.03 3

16 C12H25N+(CH3)2-(CH2)8-N+(CH3)2C12H25 2Br− 0.80 47

17 C12H25N+(CH3)2-(CH2)16-N+(CH3)2C12H25 2Br− 0.12 3

18 C14H29N+(CH3)2-(CH2)2-N+(CH3)2C14H29 2Br− 0.137 48

19 C14H29N+(CH3)2-(CH2)8-N+(CH3)2C14H29 2Br− 0.018 47

20 C16H33N+(CH3)2-(CH2)2-N+(CH3)2C16H33 2Br− 0.003 49

21 C12H25N+(CH3)2-(CH2)2-O-(CH2)2-N+(CH3)2C12H25

2Cl− 0.5 10

22 C12H25N+(CH3)2-CH2–CH(OH)-CH2-N+(CH3)2C12H25

2Br− 0.8 10

23 C12H25N+(CH3)2-CH2–C6H4-CH2-N+(CH3)2C12H25

Cl− 0.03 38

24 C12H25N+(CH3)2-CH2–CH(OH)-CH(OH)-CH2-N+(CH3)2C12H25

2Br− 0.7 50

25 C12H25N+(CH3)2-CH2–CH(OH)-CH2-N+(CH3)-CH2-CH(OH)-CH2-N+(CH3)2C12H25

3Cl− 0.5 10

26 C16H33N+(CH3)2-(CH2)5-N+(CH3)2C16H33 2Br− 0.009 51

27 C16H33N+(CH3)2-(CH2)2–O(CH2)2-N+(CH3)2C16H33

2Br− 0.004 51

28 C16H33N+(CH3)2-CH2-(CH2–O-CH2)3-CH2-N+(CH3)2C16H33

2Br− 0.02 9

29 C10H21N+(CH3)2-CH2-C≡C-CH2-N+(CH3)2C10H21

2Br− 1.39 52

30 2-C10H21-Py+(1)-(CH2)4-(1)Py+-2-C10H21 2Br− 2.69 19

31 2-C10H21-Py+(1)-(CH2)6-(1)Py+-2-C10H21 2Br− 2.00 19

32 2-C12H25-Py+(1)-(CH2)4-(1)Py+-2-C12H25 2Br− 0.56 19

33 2-C12H25-Py+(1)-(CH2)6-(1)Py+-2-C12H25 2Br− 0.54 19

34 1-C12H25-Py+(2)-(CH2)2-(2)Py+-1-C12H25 2CH3SO3− 2.07 22

35 1-C12H25-Py+(2)-(CH2)3-(2)Py+-1-C12H25 2Cl− 1.51 22

36 1-C12H25-Py+(2)-(CH2)4-(2)Py+-1-C12H25 2Cl− 1.28 22

37 1-C12H25-Py+(2)-(CH2)8-(2)Py+-1-C12H25 2Cl− 1.11 22

38 1-C12H25-Py+(2)-(CH2)12-(2)Py+-1-C12H25 2Cl− 0.22 22

39 1-C6F13-C2H4-Py+(2)-(CH2)3-(2)Py+-1-C2H4-C6F13

2Cl− 1.19 23



Table I. Continued

Entry Surfactant Counterion CMC (mM) Reference

40 1-C6F13-C2H4-Py+(2)-(CH2)4-(2)Py+-1-C2H4-C6F13

2Cl− 1.86 23

41 1-C6F13-C2H4-Py+(2)-(CH2)8-(2)Py+-1-C2H4-C6F13

2Cl− 1.29 23

42 1-C6F13-C2H4-Py+(2)-(CH2)12-(2)Py+-1-C2H4-C6F13

2Cl− 1.10 23

43 Py+-(1)-CH(C10H21)-CH2S(CH2)2SCH2-CH(C10H21)-(1)Py+

2Br− 0.43 24


2Br− 0.10 24


2Br− 0.03 24

46 1-C10H21-Im+(3)-(CH2)4-(3)Im+-1-C10H21 2Br− 4.50 32

47 1-C12H25-Im+(3)-(CH2)2-(3)Im+-1-C12H25 2Br− 0.55 32

48 1-C12H25-Im+(3)-(CH2)4-(3)Im+-1-C12H25 2Br− 0.72 32

49 1-C12H25-Im+(3)-(CH2)6-(3)Im+-1-C12H25 2Br− 0.78 32

50 1-C14H29-Im+(3)-(CH2)4-(3)Im+-1-C14H29 2Br− 0.10 32

51 C10H21Pyrr+-(CH2)4-Pyrr+C10H21 2Br− 5.76 15




55 C12H25[N+[12]annuleneN+]C12H25 2Cl− 7.7 34



Note. CMC Values for Selected Simple Surfactants are Given as Reference.

Chart 1.

of the polar head. For example, the CMC of pyridinium GS, [1-C12H25-Py+(2)-(CH2)3-(2)Py+-1-C12H25] 2Cl− was found to be 1.51 mM, which is ten times lower than the CMC value of15.1 mM corresponding to the monomeric surfactant [1-C12H25-Py+(4)CH3] Cl−.22, 23 Thesame trend was found valid for the imidazolium GS (Table I, compare entries 7 and 47–49).32

All the structural elements of GS will influence their CMC, although their impact is differentand rather complex. The conformation of the individual GS into the micelle has an importantimpact on the aggregation number (AN) and ultimately on the CMC. This conformation is theresultant of several attractive and repulsive forces that are acting simultaneously on differentparts of the molecule. Thus, the two alkyl chains tend to pack together as tightly as possibleand close the distance between polar heads. However, the coulombic repulsion in between thecharged heads opposes this approach. The linker can mediate in between these opposite forces.Rigid linkers will position the polar head at fixed distances and therefore will have a greaterimpact on CMC than the flexible ones. The later ones allow more mobility for the polar headsand in this case their length and polarity becomes important.



Chart 2.

Charge density has also a big impact on the self-assembling properties of the surfactants.For example, the pyridinium polar head in GS 15 was found to generate lower CMC values thanammonium polar head in GS 60 having the same structural elements (Chart 2, Table I). GS withimidazolium polar head (53b) exhibited an intermediate CMC value in between pyridinium- andammonium-based congeners, showing that CMC decreases monotonously with the decreasein charge density (Table I, see entries 13, 32, and 48). Interestingly, pyrrolidinium GS recentlyintroduced by Dong and co-workers displayed CMCs in between quaternary ammonium andimidazolium congeners (Table I, compare entries 9, 51, 46; 13, 52, 48).15

As mentioned above, the GS self-assembling was found to be more responsive to tail lengththan aggregation of conventional surfactants. Variation of CMC with the length of alkyl chainm can be written for simple surfactants as53

log CMC = A− Bm,

where A is a constant for the particular temperature and for the homologous series considered,and B is an empirical constant.

The same correlation formula can be applied to GS.54 For the m-s-m type GS, the variationof log CMC with m was found to be linear up to m = 16 for the series of m-2-m (2Br−),48 m-3-m(2 Cl−),10 m-4-m (2Br−),11 m-5-m (2Br−),5 and m-6-m (2Br−).3, 5, 45 Further increase in spacerlength induces a departure from the linearity, as observed by Zana for the series m-8-m, startingat m = 14.47 It is believed that longer, flexible, hydrophobic spacers penetrate the interior of themicelles, enhancing the self-assembling of these amphiphiles. Heterocyclic GS are following thesame trend, as exemplified by imidazolium series 53 (Chart 3). On the other hand, branchingof alkyl chains translates into an increase of the CMC values as compared with conventionalsurfactants due to bigger steric demands.55 Fluorination of alkyl chains was shown to decreaseCMC due to the fluorophobic effect. Quagliotto et al. revealed that the fluorinated pyridiniumGS displayed lower CMC values as compared to the congeners with regular alkyl chains(Table I, see entries 34–42).23

The effect of the spacer (linker) on the CMC of GS can be analyzed as presented aboveby taking into consideration the spacer length, rigidity, and polarity. For the same linker type,the impact of the linker’s length on the value of CMC is not as prominent as the impact ofthe alkyl chain length when the linkers are relatively short (up to six carbon atoms; Table I,see entries 12–15). However, further elongation of the linker was shown to decrease the CMCsignificantly. For example, a long hydrocarbon spacer of 16 methylene groups (61) reduces the

Chart 3.



Chart 4.

CMC almost tenfold relative to short spacer GS 59 and 60 (Chart 4), due to its contributionto hydrophobicity of the GS via penetration in the interior of the micelle. The same trendswere confirmed recently for a series of pyridinium GS,22, 56 where the representatives with twoto four carbon atoms in the linker displayed about the same CMC. Further elongation of thelinker to 8 and 12 carbon atoms resulted in a steep decrease of CMC (Table I, entries 34–38). Asimilar trend can be achieved by enhancing the lipophilicity of the linker through insertion oflipophilic atoms; for example, the pyridinium GS having an ethane-1,2-dithiol spacer displayedlower CMC values as compared to other pyridinium GS.24

Hydrophilic, flexible, spacers as in GS 62 provide a water-compatible interface and werefound to allow a more closely packed micelle structure than the hydrophobic ones, thus reducingthe CMC of the surfactants with similar linker lengths (Chart 5, Table I, compare entries 12with 22; 13 with 24; or 26 with 27).57 However, the CMC decrease is rather small since thehydrophobic effect at the level of the linker is limited.

Rigid linkers were observed to have lower CMC than their congeners with hydrophobicflexible linkers. For ammonium 10-s-10 GS, Menger et al.52 have shown that GS with acetylenelinker (64) has significantly lower CMC than its congener 63 (Chart 6). Low CMC valueswere observed for GS having triazole rigid linkers.58 The CMC of annulene GS 56 was foundto be significantly higher than conventional ammonium congeners, a fact that was attributedto packing difficulties encountered by annulene molecules.34 This unusual GS constitutes theultimate blending of rigid spacers with charge delocalization, bordering traditional ammoniumGS, and their heterocyclic congeners.

Chart 5.



Chart 6.

B. Counterion Effect

Simple, permanently charged surfactants (either positively charged or negatively charged),placed in aqueous media, face at least two opposite forces during the process of spontaneous self-assembling: (i) associative ones, due to the hydrophobic effect and (ii) dissociative, electrostaticones, due to their similar charge of the polar head. Charged surfactants possess counterions thatcomplement the ionic polar heads and maintain the overall electric neutrality of the system.These counterions are efficiently buffering the electrostatic repulsions between the chargedpolar heads, favoring self-assembling (Fig. 2).

A counterion is associated with the surfactant polar head with a certain strength, which de-pends on the structure and electronic properties of both the counterion and the surfactant polarhead. The immediate layer of counterions, firmly attached to the polar heads, forms the Sternlayer. Remaining counterions that will tend to associate with the polar heads will encounterrepulsion from Stern layer, and consequently will localize farther away from it, forming thediffuse layer (Fig. 2). The degree of counterion binding β is the estimation of the counterionspresent in the Stern layer. These counterions will effectively reduce the coulombic repulsiveforces encountered when two similarly charged amphiphiles are getting in close proximity dur-ing self-assembling. Parameter β can be calculated indirectly, using the conductivity method.In this method, the degree of counterion dissociation α is first calculated from the variationof conductivity with surfactant concentration. Then, the degree of counterion binding β can

Figure 2. Cartoon depicting the self-assembling of GS, showing the counterion impact on supramolecularstructures. The Stern layer of counterions is depicted with a dotted line in the micelle cross-section.



be determined knowing that α + β = 1.59 Another method for determining α is via quenchingof 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ) probe fluorescence by halide anions, whichare frequently used counterions of cationic GS. Proposed by Kuwamoto et al.,60 this is an“absolute method,” better performing than the conductivity method. Thus, SPQ has greatwater solubility and a low octanol/water partition coefficient. It tends to be partitioned in theaqueous bulk phase and not inside the micelles, which makes its fluorescence insensitive tosurfactant monomers and also to surfactant micelles. The degree of counterion dissociation α

is the ratio of the Stern–Volmer constant KSV above and below the CMC when plotting thevariation of the fluorescence intensity of the probe with the concentration of halide anion [X−](from the Stern–Volmer relation of I0/I = 1 + KSV[X−], where I0 and I are the fluorescenceintensities in the absence and in the presence of quencher, respectively).60 The third reliablemethod to determine β is from the slope of Corrin–Harkins plot, representing the logarithmicCMC at different counterion concentration against logarithmic concentration of counterion,determined at a specific temperature.61

The degree of counterion binding β is dependent on the nature of polar head, spacerlength, and hydrophobic tail of GS, and on the temperature. For the same counterion andhydrophobic chain length, two parameters have to be considered when comparing the β valuesfor the monomeric surfactants and for GS: (i) the nature of the polar head and (ii) the linkerlength. The charge density of the polar head determines the relative equilibrium position inbetween two individual surfactant molecules in a micelle. Dilution of the charge density allowsa closer micellar packing and reduces the average distance in between polar heads. This tighterpacking will increase the effective charge density of the micelle and counterions will be retainedstronger. Therefore, the value of β will increase while CMC will decrease. For example, the β

value of 58 (C12H25N+(CH3)3 Br−) is 0.7562 while β of 65 (1-C12H25-Im+(3)CH3 Br−) is 0.7632

(Chart 7).Individual surfactant molecules self-assemble in water forming supramolecular assemblies

(e.g., micelles) in which the individual molecules are separated by thermodynamic equilibriumdistance d. When two individual surfactant molecules are joined with a linker forming a GS,the distance in between the two polar heads will be determined by this linker. When thelinker is shorter than d, then the effective charge density will increase, attracting stronger thecounterions and increasing the β parameter. Oppositely, when the linker is longer than d, theeffective charge density will decrease, counterions will be less bounded and β will decrease. Forexample, the C12H25N+(CH3)3 Br− has a β = 0.75;62 the β value of C12H25N+(CH3)2-(CH2)3-N+(CH3)2C12H25 2Br− is 0.78, while for the GS with longer linkers, it decreases monotonouslyfrom 0.74 for C12H25N+(CH3)2-(CH2)4-N+(CH3)2C12H25 2Br− to 0.46 for C12H25N+(CH3)2-(CH2)8-N+(CH3)2C12H25 2Br−.3 The same observations were made for pyridinium surfactants.Thus, the β value of monomeric 1-dodecylpyridinium chloride is 0.60, while for the pyridiniumGS 28b and 28c was found to be 0.69 and 0.67 respectively. Elongation of the linker to 8 carbonatoms (28d) decreases the β value to 0.44 (Chart 8).22 As a consequence of these facts, GShaving very short linkers often assemble in nonspherical micelles because of high counterion

Chart 7.



Chart 8.

binding, whereas GS with long linkers generally form spherical micelles due to smaller β andhigher conformational freedom.

The degree of counterion binding can be changed by chemically modifying the hydrophobicmoiety of surfactant; for example, the introduction of fluorine in the alkyl chain of pyridiniumGS thus yielding fluorinated pyridinium amphiphiles results in higher degree of counterionbinding β for chloride counterions as compared to methanesulfonate counterions, opposite tothe trend found in the n-alkyl pyridinium GS congeners.23

Temperature has a substantial effect on β: increase in temperature results in a decreasein counterion binding due to increase in the entropy of molecules. The effect can be clearlyillustrated in imidazolium based GS 1-C12H25-Im+(3)-(CH2)4-(3)Im+-1-C12H25 2Br− with n =2, 4, and 6, where the β value decreases with the increase of temperature (Chart 9).63 As micellesize is directly related to β value, higher temperature promote lower counterion binding in[C12-s-C12Im]2+ 2Br− micelle resulting in smaller micelle sizes. The opposite is found valid forthe GS at low temperature.

Hydration of ionic species present in solution represents another important parameter tobe taken into consideration when assessing the counterion impact on micellization process.Polar surfactant heads and counterions, both hydrated, conjoin reversibly to form neutral ionpairs that have reduced hydration. Several water molecules are released in the process, with sub-stantial entropic gain. The amount of water of hydration liberated in the process and the overallfree energy effect is greatly influenced by the structure of the two ionic partners. Shape, volume,and charge density will directly impact the polarizability of ions and will determine their hydra-tion properties. Thus, Hofmeister series classifies ions into chaotropes and kosmotropes.64, 65

Chaotropes are represented by large ions with low charge density (SCN−, ClO4−, I−, Br−,

NO3−, Cs+, K+) that disrupt the intermolecular forces between water molecules. These

poorly hydrated ions can dehydrate rather easily and are bound more strongly to the mi-cellar ionic surface when used as counterions, promoting aggregation and micellar growth viathe above-described mechanisms. Kosmotropes are small ions with high charge density (F−,SO4

2−, HPO42−, Li+, Mg2+) that enhance the intermolecular forces between water molecules,

Chart 9.



Figure 3. Various counterions tested in combination with ammonium GS and their key physicochemical char-acteristics that impact GS micellization process. Adapted from Manet et al.68

stabilizing the water structure. They are well hydrated and the energy penalty for dehydrationis very big. In consequence they are only loosely associated with micellar ionic surface and donot efficiently shield the electrostatic repulsions of the charged polar heads, thus reducing theaggregation propensity and raising the CMC.9, 54

The impact of counterions on the micellization of conventional monomeric surfactants66

and on cationic GS was recently studied in great detail.67, 68 After a very comprehensive study,Oda and co-workers68 concluded that the effect of the counterions on micellization behavior ofGS C14H29N+(CH3)2-(CH2)2-N+(CH3)2C14H29 is a complex interplay between hydrophobicityof anions, counterion hydration, ionic morphology, and the interfacial packing of ions.

Four categories of counterions were selected (Fig. 3). The first group comprised smallcounterions selected from the Hofmeister series of chaotropes and kosmotropes. In the secondgroup were included aliphatic carboxylate counterions in which the hydrophobicity of the anionwas tailored by increasing the aliphatic chain length. The third group consists of aromatic car-boxylate counterions with hydroxyl substituents in various positions. The last group containedstructurally related counterions of physiologic and/or pharmaceutical relevance. The authorshave shown for the first series of counterions that the CMC is increasing with the decrease inlyotropic number N69 from chaotropic ions (N = 13.25 for SCN−) to kosmotropic ions (N =4.8 for F−).65, 70 In the case of halide ions, the trend is related to their ionic size, which is directlyproportional to hydrophobicity and leads to a decrease in CMC while passing from fluoride to



iodide counterions (vide supra). However, the hydration number, ionization degree, and pKa

were also shown to be important for polyatomic counterions, such as acetate, nitrate, and sul-fate, and can cause the values of CMC to deviate from expected trend.68 The same conclusionswere drawn in a related study, where the CMC of C12H25N+(CH3)2-(CH2)6-N+(CH3)2C12H25

with simple counterions such as F−, Cl−, Ac−, Br−, NO3−, SO4

2− were found to be 1.84,1.33, 1.10, 0.98, 0.89, and 0.68 respectively, showing a decrease in CMC with increase in ionicsize of counterions.67 Elongating chain length of aliphatic carboxylate counterions, from ac-etate to octanoate was shown to decrease CMC spontaneously due to the increase of thehydrophobicity of counterion. For long-chain counterions (more than three carbon atoms)the hydrophobic effect dominates over kosmotropy and for alkyl carboxylates longer than sixcarbon atoms a substantial part of the counterions is embedded in the apolar domain of themicelle.48, 68

The third group comprises aromatic carboxylate counterions and is influenced by hy-drophobicity and interfacial packing of counterions. In this group, the number and the positionof substituent(s) impact directly the hydrophobicity of the counterions. Thus, Manet et al.68 hasshown that the order of CMC for C14H29N+(CH3)2-(CH2)2-N+(CH3)2C14H29 with aromaticcarboxylate counterion at 60◦C was as follows: salicylate (SAL) < benzoate (BENZ) < metahy-droxybenzoate (MHB) < phenylacetate (PA) < mandelate (MAND) < parahydroxybenzoate(PHB) < dihydroxybenzoate (DHB). The lower CMC of MAND than PHB and DHB, despiteits higher hydrophobicity, can be explained by the interfacial packing of counterions.68 Theposition of hydroxyl group with respect to the carboxylate moiety is essential for micelle sta-bility (Fig. 4).66 If the hydroxyl group is located inside the apolar core of the micelle (PHB),the micelle will be destabilized (MHB counterion can tilt at the CMC as compared with PHB[Fig. 4]).66, 68 For the last group of counterions it was shown that GS C14H29N+(CH3)2-(CH2)2-N+(CH3)2C14H29 trifluoroacetate displayed a CMC of 0.14 mM, which is much lower thanthe CMC induced by acetate counterion (0.61 mM). This effect has been attributed to higherhydrophilicity of TFA as compared with acetate. For the same GS, lactate counterion induceda lower CMC (0.19 mM) than propionate (CMC = 0.42 mM), despite being more hydrophilicthan the later. In this case, the decrease in CMC was attributed to the presence of intramolecularhydrogen bonding in the core of lactate anion. Switching from lactate to tartrate diminishessubstantially the CMC due to stronger electrostatic interactions of the double charged tartratewith cationic micelle surface as compared with single-charged lactate.68

The counterion binding has also an impact on the DNA association process. DNA bindingto micelle is favored by the large entropy gain due to release of the small counterions from themicelles–DNA interaction.67 In the case of pyridinium GS having ethane-1,2-dithiol spacer,

Figure 4. Cartoon depicting the orientation of hydroxyl moiety with respect to carboxylate group in severalaromatic counterions at the aqueous/organic interface. Placing one or more (polar) hydroxy groups inside thehydrophobic part of the micelle destabilizes the micelle and increases the CMC of parent GS. Adapted fromreferences66 and68



the increase in the chain length of surfactant results in a decrease in β, an increase in bindingaffinity toward DNA, and a lower cytotoxic effect.24

C. Aggregation Numbers

A supramolecular assembly is a well defined complex of molecules held together by noncovalentbonds. The AN is defined as the number of monomers present in the stable supramolecularassembly once the CMC has been reached. It can be measured by various methods includinglight or neutron scattering, fluorescence quenching method, etc. The light scattering methodrequires extrapolation to low concentrations, close to CMC, due to intermicellar interactions.However, decrease in the surfactant concentration changes the micelle size, and may givefalse results.71 Small angle neutron scattering (SANS) method provides information on theAN and shape of the supramolecular assembly of GS.72 Unfortunately, this method is quiteexpensive and requires complex facilities.9 The fluorescence probing method73–78 is a simple andconvenient method for determining AN. It involves a fluorescent probe, generally pyrene, and aquencher of the pyrene fluorescence, solubilized in the micelles. For the steady-state fluorescencequenching, the fluorescence decay curves are analyzed to yield the molar concentration ratioR = [quencher]/[micelle]. The AN can then easily be determined using the equation below.

AN = R ([Surfactant] − CMC)/[quencher]. (1)

The shape and intensity of the fluorescence emission of pyrene is sensitive to its microen-vironment at the site of solubilization of fluorophore. Importantly, for reliable AN data, boththe fluorescent probe and quencher must be able to move relatively freely into the micellarcore. This is rather difficult within long filomicelles and in these cases the method of time-resolved fluorescence quenching can provide more accurate values for AN since it can accountfor the rapid exchange of the probe and/or the quencher among micelles and water, or amongmicelles.72–74

The AN of a given GS is affected by the concentration of GS, polarity of head group, natureof spacer, spacer length, and by the temperature. Thus, similar to the case of conventional ionicsurfactants,79 an increase in concentration of surfactant supports the micelle growth, elevatingthe AN of micelle. The trend is more pronounced for the GS having hydrophilic flexible spacersas compared with the ones possessing hydrophobic ones and reveals their propensity towardmicelle growth.57 On the other hand, an increase in the polarity of the GS cationic head grouptranslates into better hydration, and into a higher AN. Thus, Borse et al.80 increased the polarityof head group by introducing hydroxyl groups in ammonium polar head of 12–4-12 GS 60,yielding GS 68 and 69. The authors observed a rise in AN from 60 to 75 with the increase inhead group polarity from amphiphile 60 to 69 (Chart 10).80

As mentioned above, spacer plays a critical role in micellization process. In hydrophilicflexible linkers, one may observe a decrease in the coulombic repulsion between polar heads

Chart 10.



Chart 11.

Chart 12.

due to hydration around hydrophilic spacer. The GS with hydrophilic flexible spacers will alwaysassemble more strongly than congeners with hydrophobic rigid ones, leading to higher micelleANs (Chart 11).57

On the other hand, variation in spacer length of GS changes the conformation of spacerat the water–micelle interface. Short spacers maintain the extended conformation in the mi-celle, whereas long spacers form a loop extended toward the hydrophobic core or toward thehydrophilic environment (depending upon the nature of the linker). For example, an increasein spacer length for the 12–4-12 68 from four to six carbon atoms (GS 73) decreases the ANmarkedly (from 108 to 56), whereas little change was observed while elongating the linker fromsix to eight carbon atoms (GS 74, Chart 12).80 The same effect was observed for pyridinium19

and imidazolium GS.63 As expected, an increase in the temperature also changes the aggregationbehavior due to breaking of inter/intra molecular H bonds at higher temperature.80

D. Shape of Supramolecular Assembly

The shape of the supramolecular assemblies formed by various surfactant molecules affects theirphysiochemical and rheological properties. It can be determined via light scattering methods,81

cryo-transmission electron microscopy (cryo-TEM),82 and by SANS method.83 Nowadays, it isknown that aqueous solutions of surfactants, including GS, can contain spherical or spheroidalmicelles, elongated (prolate) micelles or disklike (oblate) micelles, filomicelle (threadlike mi-celles, worm micelles), and bilayers (vesicles).12, 84

A straightforward structure–shape correlation is difficult to elaborate due to many fac-tors that contribute to the shape of supramolecular assemblies. Concentration of the GS hasa significant impact on micellar shape. A very good example is the study done by Talmonand co-workers,85 who analyzed the variation in shape of GS 12–2-12 (C12H25N+(CH3)2-(CH2)3-N+(CH3)2C12H25 2Br−) while increasing amphiphiles concentration. At low concen-trations, the surfactant prefers to self-assemble into spherical micelles, which are converted into



Table II. Effect of Concentration on the Shape of Supramolecular Assemblies of GS 12–2-12 2Br−

Entry Concentration of 12–2-12 2Br− Shape of the supramolecular assemblies

1 0.26 wt% Spherical micelles2 0.5 wt% Spheroidal and threadlike micelles3 0.5–1.0 wt% Threadlike (many), spherical micelles4 1.0 wt% Branched and closed-ring threadlike micelles5 1.5 wt% Network of threadlike micelles, closed ring micelles

Note. Compiled from Bernheim-Groswasser et al.85

Figure 5. Cryo-TEM images of 12–2-12 2Br− solutions at 25◦C. (A) At 0.26 wt% only spheroid micelles (darkdots) and some cylindrical micelles were observed; (B) at 0.5 wt%; (C) at 0.62 wt%; and (D) at 0.74 wt% boththe number of cylindrical micelles and their length is increasing; the number of spherical micelles is decreasing;inset in (D) is showing that the endcap diameter is larger than the cylindrical-body diameter; bar = 25 nm); (E) at1 wt%, there are very few spheroid micelles and endcaps but many extremely long micelles; notice the existenceof branching points (arrows) and rings (arrowheads); (F) at 1.5 wt%, a saturated network of branched (arrows)cylindrical micelles were observed; (g) at the same surfactant concentration many close rings (arrowheads) inaddition to “normal” branching points were detected. Adapted with permission from Bernheim-Grosswater etal.85 Copyright (2000) American Chemical Society.

threadlike micelles (linear, branched, closed-ring) with the increase in concentration (Table II,Fig. 5).12, 85, 86

Shape of supramolecular assembly of GS is also influenced by the structure of polar head,hydrophobic tail and by the nature and length of the linker. The impact of these structuralparameters is quite complex. A detailed analysis is possible using the packing parameter “P”



Figure 6. (A) Structural parameters defining the packing parameter of a surfactant87,94; (B) supramolecularassemblies formed by amphiphiles with different packing parameters.94–96

introduced by Israelachvili87 (Fig. 6A).

P = v/aml, (2)

where v is the volume of the surfactant moiety, made of one or two alkyl chains, l is the chainlength in the fully extended conformation, and am is the surface area occupied by the surfactanthead group at the water/micelle hydrophobic core interface.87, 88 The dimensionless parametergives information about the possible shape of micelle (Fig. 6B).87, 89

Thus, surfactants having large polar heads (large “a," small “P”) form spherical shapemicelles at moderate concentration. Many heterocyclic GS fall into this category due to thelarge size of their heterocyclic polar head. Interestingly, the charge of polar head of GS hasno noticeable influence on the micelle shape as long as overall size of the polar head remainsunchanged. For the 16–3-16 GS, it has been reported that both cationic and anionic GS have asimilar disklike shape.90

The variation of linker length and structure can change the packing parameter dramaticallyand can induce a large variety of shapes for GS. It is known that short linkers reside at micellesurface irrespective of their nature (hydrophilic/hydrophobic).46 Quaternary ammonium GSwith short linkers (two to three carbon atoms) generally form threadlike micelles.86 If the lengthof linker is increased, value of “a” increases rapidly, with “l” and “v” remaining unchanged.This induces a decrease of “P” value and results in the transition from an elongated shapeto a spherical shape. Further increase in linker length results in formation of a loop, whichpenetrates into the micelle. This process increases “v” and decreases “a,” thus increasing thepacking parameter and causing the transition from spherical micelle into vesicles (bilayers).The above-mentioned trend is illustrated for the GS 10-s-10, 12-s-12, and 16-s-16 in Table III.

The spacer polarity also affects the shape of supramolecular assembly. Maiti andChowdhury94 has demonstrated that hydrophilic spacers form spherical micelles and causea decrease in CMC with the increase in spacer rigidity as presented in the previous section. Onthe other hand, hydrophobic linkers form threadlike or rodlike micelle depending on the linkerlength and increase the CMC with the increase in spacer rigidity.94

By elongating the hydrophobic tails in a homologous GS series, one can observe the forma-tion of less-curved supramolecular assemblies, similarly to the case of conventional surfactants.For the quaternary ammonium GS m-3-m, the shape of micelle changes from spherical to



Table III. Effect of Spacer Length on the Shape of Supramolecular Assemblies of Representative GS

EntryGemini

surfactantLength ofspacer “s”

Shape of supramolecular assemblies(concentration) Reference

1 10-s-10 s = 2, 3 Prolate spheroidal micelles 91,92

s = 4, 6, 8, 10 Spheroidal micelles2 12-s-12 s = 2 Threadlike micelles (20 mM) 84

s = 3 Threadlike micelles (110 mM),s = 4 Spheroidal micelles (30 mM)s = 8 Spheroidal micelles (78 mM)s = 10 Spheroidal micelles (71 mM)s = 12 Spheroidal micelles (138 mM)s = 16 Spheroidal micelles (66, 133 mM)s = 20 Vesicles

Vesicles3 16-s-16 s = 3 Threadlike micelles + Vesicles (12 mM)

s = 4 Threadlike + Spheroidal micelles (45 mM) 84, 93

s = 6 Elongated and spheroidal micelles (51 mM)s = 8 Spheroidal (61 mM)s = 10 Spheroidal (50 mM)s = 12 Spheroidal (50 mM)

Note. Concentration of GS was mentioned in each case.

Table IV. Effect of Alkyl Chain Length on Micelle Shape for GS of identical linker length

Entry Gemini surfactant Shape of micelle Reference

1 10–3-10 Spheroidal 92

2 12–3-12 Threadlike (110 mM) 84

Spheroidal (30 mM)3 16–3-16 Threadlike (0.5 mM) 9

Disklike (2.5 mM)

filomicelle, and then to disklike micelle, with the increase in alkyl chain length from 10 to 12carbon atoms (Table IV).

Temperature and the presence of additives can also change the shape of GS assemblies.For 16–3-16 GS, Aswal et al.9 demonstrated the transition of disklike micelle into threadlikemicelle with the increase in temperature from 30◦C to 45◦C. Addition of n-hexanol to GS12–2-12 was also shown to change the shape of micelle from threadlike micelle to vesicles.95 Forvesicle-shaped GS 12–20-12, the addition of spherical micelle-forming surfactant (12–10-12)changes the shape of the assembly to a spherical micelle,96 whereas the addition of threadlikemicelle-forming surfactant (12–2-12) converts it into threadlike micelles.85

E. Surface Activity

It is primarily defined as the ability of a surfactant to reduce the surface tension of water.The surface activity of various surfactants is measured in terms of C20 value, representingthe surfactant concentration that reduces the surface tension by an arbitrary 20 m Nm−1.97

GS in general and cationic GS in particular have higher surface activity than correspondingmonomeric surfactants.10, 50 For example, the C20 value of pyridinium-based GS Py-12–4-12 is0.23 mM, which is about one order of magnitude lower than the value for the corresponding



Table V. C20 values of Representative GS

Entry Surfactant Counterion C20 value (mM) Reference

1 C8H17N+(CH3)2-(CH2)4-N+(CH3)2C8H17 2Br− 19.8 52

2 C10H21N+(CH3)2-(CH2)4-N+(CH3)2C10H21 2Br− 2.49 52

3 C12H25N+(CH3)2-(CH2)4-N+(CH3)2C12H25 2Br− 0.26 52

4 C8H17N+(CH3)2-CH2-C≡C-CH2-N+(CH3)2C8H17 2Br− 10.8 52

5 C10H21N+(CH3)2-CH2-C≡C-CH2-N+(CH3)2C10H21 2Br− 0.60 52

6 1-C12H25-Im+(3)-(CH2)2-(3)Im+-1-C12H25 2Br− 0.02 63

7 1-C12H25-Im+(3)-(CH2)4-(3)Im+-1-C12H25 2Br− 0.11 63

8 1-C12H25-Im+(3)-(CH2)6-(3)Im+-1-C12H25 2Br− 0.18 63

9 2-C10H21-Py+(1)-(CH2)4-(1)Py+-2-C10H21 2Br− 0.76 19

10 2-C10H21-Py+(1)-(CH2)6-(1)Py+-2-C10H21 2Br− 0.74 19

11 2-C12H25-Py+(1)-(CH2)4-(1)Py+-2-C12H25 2Br− 0.23 19

12 2-C12H25-Py+(1)-(CH2)6-(1)Py+-2-C12H25 2Br− 0.21 19

monomer py-12–2 (2.90 mM).19 This behavior is believed to be due to a more efficient packingand a more coherent interfacial film formed at the oil/water interface by GS as compared withtheir monomeric congeners.11, 46 The packing density can be estimated by the surface areasAmin occupied by the various surfactants.98 The Gibbs absorption equation can be used forcalculating saturation concentration Гmax at the air/aqueous interface.19, 38, 40, 46

�CMC = 1020

NA Amin= − 1

2.303n RT

(dγ

d(log C)

)T

,

where n represents the number of species at the interface whose concentration varies with sur-factant concentration, R is the gas constant (8.314 J·mol−1·K−1), T is the absolute temperature(K), C is the surfactant concentration, and NA is Avogadro’s number.

Considering the polar head, it is natural for heterocyclic GS to have a higher surface activitycompared to ammonium ones (Table V, compare entries 2 and 9; 3 with 11 or 7). An increase intail length will promote self-assembling, will decrease the CMC (as mentioned in the previoussection) and will increase the surface activity of an amphiphile (compare entries 1–3; 9 and11; 10 and 12 in Table V). The linker impact on surface activity was extensively studied.19, 63

For pyridinium GS, Zhou et al.19 has shown a decrease in C20 values and an increase in thesurface activity with elongation of linker (compare entries 9 and 10; 11 and 12), as expected.Surprisingly, Ao et al.63 have shown a decrease in surface activity with the increase in the linkerlength (Table V, compare entries 6–8). Rigidifying the linker improves surface activity as shownby Menger et al.52 (compare entries 1 and 4; 2 and 5 in Table V).

4. BIOLOGICAL ACTIVITY

Interaction of self-assembled systems containing GS with living cells is of significant interestfor pharmaceutical and biotechnological applications. Due to their amphiphilic nature andexcellent aggregation properties, GS can perturb significantly cellular components. A majorimpact can be envisaged at the level of external and internal membranes, involving membranedisruption, temporary membrane poration, and denaturation of physiologic macromolecules.These processes can induce metabolic inhibition, altered homeostasis, and ultimately cell death.The mass transfer between the host bilayer and the foreign amphiphile-based supramolecu-lar assembly can occur immediately or it can be delayed via processes such as endocytosis.



Regardless of the event time frame, the physicochemical parameters of the GS and host mem-branes (which define cell type and morphology), the concentration of the amphiphile, tempera-ture, nature of formulation, and pH, are several important factors defining this interaction andits outcome.99, 100

The packing parameter of the amphiphile has a very strong impact on the membrane dis-ruption process. Amphiphiles with a P < 1/3 (detergent and other micelle-forming compoundsincluding GS) can change dramatically the local membrane curvature as the concentration ofthe amphiphile increases, triggering membrane poration.101–103 Due to their membrane destabi-lization properties, cationic surfactants, including GS, are used as antimicrobials and biocides.Related with human administration, it must be emphasized that route of administration im-pacts significantly the observed cytotoxic effect, besides their structural and physicochemicalparameters. For example, when administered orally, cationic surfactants are poorly absorbedin the intestinal tract thus exhibiting low cytotoxicity. Their toxicity increases 10–100 timeswhen administered i.v. due to interaction with figurative elements of blood such as red bloodcells, white blood cells, albumin, etc. Topical administration of positively charged surfactantsalters the water binding capacity of the skin, the skin permeability, and can cause proteindenaturation of stratum corneum. This phenomenon can be used to increase the absorptionof a co-administered drug. GS were proposed as skin permeation enhancers,100 and structure–activity relationship studies were conducted in order to quantify their benefic and unwantedeffects such as irritation and long-term damage of the skin.100, 104

The unique properties of cationic amphiphiles in general and cationic GS in particular,recommend them in biotechnological applications as efficient transfection agents.105 In a typicaltransfection process (Fig. 7), cationic amphiphile supramolecular assemblies (micelle, liposomesetc.) first combine with DNA and form a amphiphile–DNA complex (lipoplex).106 Under thiscomplexed form, DNA is protected from the action of exogenous nucleases. The lipoplexusually contains an excess positive charge that will allow its association with the negatively

Figure 7. Schematic representation of cationic amphiphile-mediated gene delivery, emphasizing the majorsteps of transfection process.



charged sialic acid residues on the surface of the target cells. This binding is usually followed byendocytosis, with the lipoplexes ending into endosomes.107 The lipoplex outer shell will makecontact with endosomal membrane and the cationic amphiphile from the lipoplex will startmixing with anionic lipid in endosomes. The positive charge of cationic amphiphile will beneutralized and as a consequence, DNA will be released in the cytoplasm.108

Since the nucleic acid is no longer complexed with lipids, it is susceptible to degradationby cytoplasm DNAs.29, 30, 109 It must reach the nucleus as soon as possible before degradationoccurs. Nuclear entry remains one of the biggest barriers for gene delivery and various strategiesinvolving a codelivery of a nuclear localization signal peptide were performed in order toovercome this hurdle.29, 30, 110

Essentially, the cationic GS is actively involved in the packing of DNA, its protection duringpassage from the point of administration to the target cells, association with target cells, and inendosomal escape. It is hard for a single amphiphile to ensure an optimum balance for all thesesteps. In practice, colipids such as cholesterol and dioleoylphosphatidylethanolamine (DOPE)and other additives are added to achieve a better balance over these delivery stages. Theselipid formulations display superior biological activity as compared with the components takenalone, in a typical case of synergism. All the structural elements of the cationic component andcolipid have an impact on the biological activity of the formulation. The important moleculardescriptors are the size and shape of the amphiphiles (related to packing parameter and Tc) andtheir ability to interact with other physiological relevant lipids from external and internal cellmembranes. In what will follow, we will examine the effect of main structural elements (polarhead, hydrophobic tail, and linker) to the transfection efficiency of GS, with an emphasis onheterocyclic GS.

A. Effect of Polar Head

Due to presence of positively charged polar groups, GS efficiently compact the negativelycharged molecules of nucleic acids, allowing the formation of particles small enough to beendocytosed by the cell.105, 111 The electrostatic attraction of the positively charged GS andnegatively charged DNA is affected by the ratio of cationic surfactant and DNA that areassociating into the lipoplex. Cell membrane is usually negatively charged, so the lipoplexhas to carry a surplus of positive charge for effective binding and internalization. However,excess positive charge also leads to toxicity (cationic lipids are not natural components of thecell) and to premature inactivation of lipoplexes due to interaction with negatively chargedproteins in blood when administered systemically. Therefore, positive to negative (+/−) chargeratio is critical for efficient gene transfection. For example, Cardoso et al.112 showed that theammonium based GS 75, when formulated with cholesterol in 1:1 molar ratio has the maximalbiological activity at 4/1 +/− charge ratio (Chart 13). The most transfection efficient chargeratio for the same amphiphile coformulated with both cholesterol and DOPE at 1:1:1 molar

Chart 13.



Figure 8. (A) Effect of lipoplex composition and +/− charge ratio on luciferase gene expression (A) and viabilityin TSA cells (B, through Alamar Blue assay). GS 75 was formulated alone (empty bars), with cholesterol (1:1,black bars) or with cholesterol and DOPE (1:1:1, gray bars), and mixed with DNA at charge ratio indicated;adapted with permission from Cardoso et al. (reference112). Copyright (2011) Elsevier.

ratio was found to be 8/1. The two formulations have similar toxicities at 4/1 charge ratio, whileat 8/1 +/− ratio the ternary one was slightly more toxic (but also more efficient) than the binaryformulation (Fig. 8).112 Interestingly, for the pyridinium GS 20b (SAINT 3) coformulated withDOPE at 1:1 molar ratio, the optimal lipid to DNA charge ratio for efficient gene deliverywas found to be 2.5/1.20, 21 Moreover, Ilies et al.28 have shown that the transfection activity of36a:chol (1:1) was optimal at 2/1 charge ratio in NCI-H23 cells, whereas for related 75:chol(1:1), the optimal activity was found to be at 4/1 +/− charge ratio.112 This charge ratio inducedan activity about ten times higher than the same ammonium GS based formulation at the 2/1charge ratio on TSA cells.112

Obviously, the charge density of the head group affects the overall charge density of theformulation and its gene transfection efficiency. The monoionized phosphate group that linksthe individual nucleotides in nucleic acids is essentially a softer anion than carboxylate113 and,according to Collins theory,114 will interact strongly with soft charged systems such as theheterocyclic ones, including pyridinium. The interaction occurs at the aqueous/organic inter-face and involves large scale amphiphile rearrangement, driven by the release of counterionsand weakly bound hydration water.29, 115–117 In pyridinium-based GS, this strong amphiphile–DNA interaction ensures genetic material compaction and structural sturdiness. Ammonium-based GS display higher charge densities and have more tightly bound hydration water thanpyridinium or imidazolium, therefore efficient compaction of DNA occurs at higher chargeratios.

B. Effect of Tail length and Characteristics

As mentioned before, the CMC and aggregation properties of GS decrease with the increaseof tail length. In other words, for a given number of molecules of surfactant, the propensity toassemble and the stability of supramolecular assemblies generated increase with the elongationof tail length. Thus, it is not surprising that tail elongation also translates into increased trans-fection efficiency as the stability of the lipoplexes will increase. This behavior was observed forboth the 16–3-16 and 18:1–3-18:1 irrespective of presence or absence of DOPE.118, 119 However,an increase in tail length will translate into an increase in size that might affect endocytosisrate. Tail elongation (for the same spacer length) will change (decrease) the packing parameterP and trigger unwanted morphological changes of the self-assembly. These changes can be



Chart 14.

leveled/buffered to some extent by using colipids, in which case the size mismatch becomesan important parameter to be considered. As the stability of self-assembly increases with thetail elongation, so does the critical temperature; the assembly becomes stiffer at physiologicaltemperature and critical steps such as lipid mixing in the endosomes and endosomal rupturewith DNA release are slowed down. Usually, a common strategy to harvest just the benefitsis to use long but unsaturated tails that induce low CMCs and low Tc. Introduction of unsat-urations in alkyl chains decreases Tc below physiological temperature and therefore increasesthe susceptibility of supramolecular aggregate to morphological changes at 37◦C. This strategycan translate into an increased biological efficiency.105 The same result was found to be validon sugar based pro-cationic GS.120 The orientation, cis and trans, and bond multiplicity ofunsaturated group also have a marked impact on biological activity, as they modulate Tc andP. For pyridinium cationic lipids Py-18:1 19 it was observed that the trans-orientation hashigher transfection efficiency as compared with the cis-analog.21 Also, the decrease in Tc dueto enhanced molecular asymmetry (with respect to alkyl chain length of the GS) has markedimpact on the binding with DNA. For example, for ammonium based m-6-n 70, 76–78 withm + n = 24 and m = 12, 14, 16, 18, the increase in m/n ratio (asymmetric degree) resultsin a decrease of critical aggregation concentration (CAC) and saturation concentration (C2;Chart 14). This is due to an enhancement in intermolecular hydrophobic interactions betweensurfactant molecules, making the aggregation process more spontaneous while m/n increases.However, the Gibbs free energy for interaction between GS and DNA was shown to become lessnegative with increased m/n, thus revealing a weaker interaction between GS and the nucleicacid.121

Branching of tails is another strategy to reduce CMC and Tc. Introduction of phytanyl andother similar groups has a strong impact on the biological properties of GS. Thus, replacementof C16 chain from 16–3-16 (79) with a phytanyl moiety (80) translates in lower CMC, higherdegree of micellization, and a transfection activity that doubled as compared with parent am-monium GS.122 It should be mentioned that the GS 79 was used successfully in vivo for topicaltransfection of IFNγ plasmid.123, 124 Bhattacharya and co-workers have used successfully thecholesteryl hydrophobic moiety (81a–e) in the design of new GS for gene delivery with im-proved biocompatibility. The authors have shown that when coformulated with DOPE at 1:4or 1:5 molar ratios these surfactants were superior to their monomeric lipid counterparts, alsosurpassing the efficiency of commercially available formulations (Chart 15).125 The efficiencyof this strategy was confirmed by Seu and co-workers.126 Another strategy to improve biocom-patibility was proposed by Fisicaro et al.127 who inserted biodegradable ester groups in thealkyl chain of standard ammonium based m-2-m, yielding the bis-betaine derivative 82 andits inferior homologs derived from dodecanol and decanol. Maximum transfection efficiencywas obtained when m = 12, in the presence of DOPE (2:1 molar ratio to GS), and this for-mulation was found to be more efficient than GenePORTER commercial transfection system.



Chart 15.

Interestingly, the representative 82 was able to transfect DNA plasmids alone, without anycolipid (Fig. 9).127

C. Effect of Spacers

In the previous section it was shown that the shape, size, and CMC of GS can be dramaticallyaffected by the length and nature of their spacers. The spacer determines the effective distancebetween the polar heads of GS and can modulate their self-assembling (vide supra).

When the positive charged GS interacts with the negative charged DNA, the repulsiveelectrostatic interactions within the GS and in between the GS molecules are counterbalancedby the attractive interactions between cationic GS and the anionic nucleic acid backbone.The attractive electrostatic interaction is maximal if the charge density on the surface of GSassemblies matches perfectly the DNA local charge density.

On the other hand, it is also known that charged molecules cause electrostatic contraction(electrostriction) of water molecules in their vicinity, also reducing their mobility.128, 129 Asmentioned before, when GS associate with DNA, this partially ordered water surroundingthe two charged species will be released with entropic gain.129, 130 Since the hydration of theDNA is relatively constant, the dynamics of dehydration/hydration of GS polar head becomesdeterminant for the DNA compaction/unwrapping process. If one increases the linker lengthof a GS, the distance between cationic heads increases, reducing their electrostatic repulsion.However, the linker is located at the water–oil interface and its hydration starts to play animportant role. In the case of hydrophobic linkers, hydration of linkers occurs with entropicpenalty; elongation of these spacers over a certain threshold causes their bending and insertioninto the oil phase, decreasing the equilibrium distance between the positively charged heads andthus reducing the amount of ordered water at the oil/water interface (vide supra). Hydrophilicspacers are expected to display a more homogeneous behavior, as they can efficiently stabilizethe water–oil interface.

MacDonald and co-workers111 have synthesized two series of GS with saturated (octadecyl)or unsaturated (oleyl) tails, 83 and 84 (Chart 16). The hydrophobic linkers separating thetetraalkylammonium polar heads comprised two, three, or six methylene units. The authors



Figure 9. Transfection of RD-4 cells with lipoplexes generated from GS 82 and its inferior homologs derived fromdodecanol and decanol, without or with colipid DOPE at different molar ratios, and only DOPE: (A) fluorescencemicroscopy of transfected cells (as shown by green cells expressing GFP); (B) efficiency of transfection (fiverandom fields were examined from each well, and each experiment was done in triplicate. The biological effectof standard commercial transfection reagent GenePORTERTM is also shown. Statistical differences betweentreatments were calculated with Student’s test and multifactorial ANOVA. Adapted with permission from Fisicaroet al. (reference127). Copyright (2011) Elsevier.

Chart 16.



found that the linker had a significant influence on the self-assembling of unsaturated GS 84.The HexEce 84c was micellar in both water and saline, PropEce 84b was micellar in water butlamellar in saline, while TmedEce 84a was lamellar in both water and saline. The saturatedanalogs 83 were found to retain a lamellar-type crystalline array structure upon hydration witheither water or saline. The biological data have shown that the saturated GS formulated alonewere more efficient than the formulations with DOPE in transfecting DNA to BHK cells in vitro.The second most efficient formulation had a 1:2 molar ratio GS to DOPE. The linker had alsoa strong impact on the biological properties of GS 83, with the HexAce 83c derivative being themost efficient, followed by TmedAce 83a and PropAce 83b. These differences in transfectionvanished in the case of unsaturated GS 84, where all three compounds had about the sametransfection efficiency at a 3:1 GS/DNA weight ratio. The unsaturation of the hydrophilictails, which decreases the Tc and increases the fluidity of their DNA complexes, becomes thepredominant factor in this case, with linker length having a negligible impact.

Interestingly, the initial GS assemblies (micelles, liposomes) seem to be also less relevant forunsaturated compounds 84. For saturated GS 83, the linker length becomes the dominant factorfor transfection, showing the combined impact of all structural elements of GS on the biologicalproperties. Mention should be made that the efficiency of both saturated and unsaturated GS83 and 84 was lost in the presence of serum, probably due to significant interaction withamphiphilic serum proteins.

The same trend of increased transfection with elongation of linker was observed for thepyridinium GS of type 36 coformulated with cholesterol at 1:1 molar ratio,28 except that themaximum of transfection was obtained with an eight carbon atoms linker. This is probablydue to bigger steric demands of pyridinium polar head. Importantly, the biological effect ofthis octamethylene representative was surpassed by the congener having a very short 2C atomslinker, which has a behavior closer to traditional (monomeric) cationic lipids.

The combined effect of linker and hydrophobic tail is also evident in the studies done byBhattacharya and co-workers.125, 131 Thus, Bajaj et al.131 synthesized a set of 15 tetraalkylam-monium GS of type 88–90 having 3,4-dialkoxybenzyl hydrophobic chains (C12, C14, C16)with linkers of 3, 4, 5, 6, and 12 methylene units. Their monomeric congeners 85–87 were alsosynthesized and tested in the same conditions for direct comparison (Chart 17). The C12 GS88 formulated at 1:2 GS/DOPE molar ratio and at 1:1 cationic amphiphile/DNA charge ratio,were able to efficiently compact green fluorescent protein (GFP)-reporter plasmid DNA andtransfect it into HeLa and HT1080 cell lines in vitro. The hydrophobic, flexible, linker did notleave a significant impact on transfection, expressed as percentage of GFP (60–70%) and meanfluorescent intensity MFI (20–25%). Elongating the chain to C14 homolog 89 translated into aslight decrease in percentage of GFP but a substantial increase in the MFI. The percentage ofGFP decreases slightly with the elongation of the linker from n = 5 to 6, then more dramaticallyto n = 12. The MFI is significantly higher for C14 derivatives 89 than to C12 derivatives 88

Chart 17.



Figure 10. The combined effect of tail and spacer lengths on DNA transfection ability of GS 88–90 (4–6 in thefigure), at N/P ratio of 0.75, in HT1080 cell line, in the absence of serum (-FBS-FBS). Transfection efficiencyof structurally related cationic lipids 85–87 (1–3 in the figure) is also provided for comparison. Adapted withpermission from Bajaj et al. (reference131). Copyright (2007) American Chemical Society.

(∼60 vs. ∼20) and is relatively insensitive to elongation of the linker. Further elongation ofthe alkoxy chain length to C16 generated GS 90 that required more DOPE in the formulation(probably due to a higher Tc of the GS). The transfection efficiencies with these formulationswere inferior to the ones obtained with C14 representatives 89 in both percentage of GFPand MFI. The most efficient linkers in this case were the pentamethylene, hexamethylene, anddodecamethylene ones. These linkers were consistently efficient throughout the series, probablydue to a good match with the local charge density on the DNA backbone (vide supra).

The most efficient GS was the C14 representative 89c with a pentamethylene linker, whichalso transfected more cells than the corresponding monomeric cationic lipid 85–87, whiledisplaying about the same MFI. Importantly, the monomeric cationic lipids were most efficientthan GS with the shorter and longer linkers than n = 5, emphasizing again the combinedeffect of these two structural elements for an optimum P and transfection efficiency. It must bestressed out that for HT1080 cell line, the GS with n = 5, 6 in combination with a C12 chain(88c and 88d) were the most efficient, showing thus a preference of this cell line for shorter alkylchains (Fig. 10). The C12, C14 GS representatives having penta- and hexamethylene linkers(88c, 88d and 89c, 89d) were also the most efficient in the presence of elevated levels of serum.This linker length was found to be optimal in the GS series of type 81, synthesized by the samegroup.125

Similar to Bhattacharya’s GS of type 88–90 are the pyridinium GS 20a (SAINT-12),20b (SAINT 3), and 20c (SAINT 13) synthesized by Engberts’ group (Chart 18).20, 21 Theirstructure comprises four hexadecyl chain (two per monomeric unit), with linkers from threeto five methylene units. The DOTMA/DOPE was used as a reference for comparing thetransfection efficiency and toxicity of these GS and their corresponding monomer 19 in COS

Chart 18.



7 cells. It was observed that the monomeric analog SAINT 1 displayed similar transfectionactivity as compared to the reference, when coformulated with DOPE at 1:1 molar ratio. TheGS 20 have shown increased transfection efficiency as compared with 19, transfection thatincreased with elongation of linker length from three methylene unit to four methylene unit.Further increase in the linker length to five methylene unit did not increase the transfectionefficiency. Toxicity of the GS increased monotonously with the elongation of linker, being lowerthan that of DOTMA/DOPE. The toxicity of 19/DOPE formulation was also found to besmaller than that of the reference. The best formulation was found to be 20b/DOPE (1:1) withtransfection efficiency 2.5 times higher than the reference while displaying half the toxicity ofDOTMA/DOPE.

For understanding the effect of the nature of the linker on gene transfection, Bhattacharyaand co-workers132 have synthesized three thiocholesterol-derived GS having hydrophobic-flexible (91), hydrophobic-rigid (92), and hydrophilic flexible (93) linkers (Chart 19, Fig. 11).The group had compared the transfection efficiency in the form of percentage of GFP and MFIin four different cell lines, that is, HeLa, HT1080, PC3AR, and HaCat cell lines. The GS werecoformulated with DOPE and optimized for best transfection efficiency. For the GS 91 and 93,the optimized GS/DOPE molar ratio was found to be 1:5, whereas for GS 92, the optimizedratio was found to be 1:4. In term of charge ratio, the new GS based formulations were found tobe able to fully condense plasmid DNA starting with 1/1 positive to negative ratio (N/P ratio).Interestingly, this finding was recently confirmed by Bhadani and Singh18 for DNA complex-ation by novel imidazolium GS bearing thioether linkers. Furthermore, when tested in HeLacells, GS 91–93 had a rather similar behavior, with GS 92 exhibiting maximum percentage ofGFP expression, followed by GS 93 and 91 at the N/P ratio of 1:1. The transfection efficiencydecreases with increase in N/P ratio. For the HT1080 cell line, the hydrophilic linker was foundto be slightly better with almost 60% GFP and ∼50 MFI. The transfection efficiency was in theorder of 93 > 91 > 92 at 1:1 N/P ratio. In PC3AR cell line, GS 92 with hydrophobic aromaticrigid linker possess maximum transfection efficiency with 61% GFP and ∼418 MFI, followedby GS 91 with hydrophobic flexible linker. The GS 93 containing hydrophilic linker had shown

Chart 19.



Figure 11. Transfection efficiencies of optimized DOPE-based lipoplexes of GS 91–93 ((A and D) 91, (B andE) 92, (C and F) 93) in HeLa (A)–(C) and HT1080 cells (D)–(F) at various N/P ratios in the absence and inthe presence of serum. Adapted with permission from Bajaj et al. (reference132). Copyright (2008) AmericanChemical Society.

least transfection activity. For HaCat cell lines, GS 91 and 92 displayed ∼20% GFP and thehydrophilic linker showed 5% GFP with ∼18 MFI, a rather good result for a hard to transfectcell line.

The good results obtained with the GS of type 93 having a polar linker, prompted Bhat-tacharya and co-workers to expand the cholesteryl GS series with congeners of type 94,having oligoxyethylene spacers (Chart 20).13 The 3-oxapentane-1,5-diyl 94a was again verytransfection-efficient when coformulated with DOPE at 1:4 molar ratio. The biological effectwas retained even in the presence of elevated levels of serum. Higher homologs were alsoefficient, but further elongation of the polar linker had not translated into more active formu-lations.

Chart 20.



Interestingly, when cholesteryl hydrophobic anchor was replaced with 3,4-dialkoxylbenzylmoieties in GS 95 and 96 (Chart 20),133 the trend was reversed. The most efficient formulationcomprised GS of coformulated with DOPE at 1:6 molar ratio. Its efficiency was maintained inthe presence of elevated levels of serum, similar to their oligooxyethylene congeners presentedbefore. The efficiency of oligooxyethylene linkers was also recently confirmed for pyridiniumGS of type 97 and congeners.134

Incorporation of pro-cationic linkers derived from polyamines in the structure of pyri-dinium GS of type 39 and 42 did not have a benefic effect on transfection efficiency, as shownby Balaban, Ilies and co-workers (Chart 21).28 GS of type 39a (which have a lower pKa of theNH group) were more efficient than GS 39b and 39c having more basic linkers (thus positivelycharged at physiological pH). GS 39a had about the same efficiency with its congener 39 with

Chart 21.



Figure 12. Transfection data for GS 36, 39, 42 (A) and their Boc-protected precursors 38 and 41 (B) comparedwith cationic lipid DOTAP as reference, on NCI-H23 cell lines. All GS were formulated with cholesterol as colipid,at 1:1 molar ratio. An electrostatic charge ratio cationic amphiphile/DNA of 2:1 was used in all cases. Figure andlegend adapted with permission from Ilies et al. (reference28). Copyright (2006) American Chemical Society.

a pentamethylene linker, while 39b and 39c displayed just a fraction of the efficiency of theircongeners 36 with hydrophobic linkers. The spermine derived GS 42 was the most efficientfrom the pro-cationic set, but its transfection capacity was less than half of the biological effi-ciency of its octamethylene congener 36g (Fig. 12A). All GS were formulated with cholesterolas helper lipid at 1:1 molar ratio, and were assessed at 2:1 +/− charge ratio. This behaviorwas suspected to be due to a very strong compaction of the DNA plasmid, which cannot bereleased efficiency after being intercalated inside the transfected cells. To test this hypothesis,the authors had reverted to the Boc-protected synthetic precursors of GS 39 and 42, namelyGS 38 and 41. Experimental results confirmed their hypothesis, with 38 and 41 being about fiveand ten times more efficient than 39 and 42 (Fig. 12B).28 Since in the case of 41 the lipophilicBoc groups can dive into the oil phase enhancing self-assembling, the authors have synthesizedthe multi-chain representatives 43, 44, and 45. Transfection data for these heterocyclic com-pounds, related to GS, dendrons, and lipid oligomers, revealed that they are more efficient whencoformulated with DOPE than with cholesterol, and that the optimum ratio GS/DOPE was1:1. Coformulated compounds were more efficient than the cationic amphiphiles acting alone,with the tapered-shaped 44 surpassing the efficiency of Lipofectamine R© when coformulatedwith DOPE at 1/1 molar ratio. It must be noted that trimeric derivative 45 was able to transfectNCI-H23 cell line alone, displaying about half of Lipofectamine R© transfection power. Otherattempts to integrate reduction-sensitive disulfide groups in the structure of the linker (GS47) were not successful, although similar strategies proved efficient for tetraalkylammoniumcongeners.105, 135

The efficiency of pro-cationic polar linkers can also be evaluated considering the studyof Wettig et al.136 that followed the one from Ilies et al.28 Foldvari and co-workers synthe-sized a series of tetraalkylammonium GS 98–101 with C12 alkyl chains and having methy-lamino or unsubstituted amino polar groups into their linkers (Chart 22).137 The most ef-ficient GS was representative 101, having the least basic and the most hydrophobic linker(Fig. 13).136 The authors proved that the amino group is not charged significantly at physi-ologic pH. On the other hand, methylation of the amino group yielded the more basic con-gener 100, which displayed about one-third of the transfection efficiency of 101.136 The othermethylated GS of type 98 and type 99 were even less efficient, confirming the conclusionsof Ilies et al.28 that a hydrophilic charged linker is not benefic to transfection efficiency dueto a very strong interaction with the DNA that hampers its release inside the cells to betransfected.



Chart 22.

Figure 13. (A) In vitro transfection of COS7 cells with lipoplexes generated from gemini surfactants 98–101 inthe absence (black bars) or in the presence of colipid DOPE (white bars), at +/− charge ratio = 10 (n = 6,error bars = standard deviation (SD)). Data are also presented for cationic lipid DC-Chol formulated with (whilebars) or without DOPE (black bars), and for Lipofectamine (shaded bar), as controls. (B) Mean cell viability (n =4, error bar = SD) of COS7 cells transfected with DOPE-containing lipoplexes of GS 98–101 (white bars), withLipofectamine (gray bar). Data are also presented for untransfected cells and cells transfected with plasmidonly (black bars, n = 4, bars = SD). Columns connected by a solid line show no significant difference; thoseconnected with a dashed line show a significant difference (p < 0.01). Adapted with permission from Wettig etal. (reference136). Copyright (2007) John Wiley and Sons.

5. CONCLUSIONS AND PERSPECTIVES

As discussed above, GS have great technological potential due to their low CMC, high surfaceactivity and special biological properties. In our review, we have summarized the work doneon cationic heterocyclic GS in a comparative way, emphasizing the differences between themand their tetraalkylammonium congeners. These compounds are now gaining momentum inworldwide research due to their superior properties generated by their softer charge, increased



hydrophobicity, and special hydration properties. New synthetic routes to heterocyclic GSbecame available in recent years, offering convenient access to new members and ensuring awide chemical diversity within this class of amphiphiles. Biotechnological applications towardefficient biocides and transfection agents can justify their higher cost and have fueled theresearch in the field. Based on these reasons, we envisage a promising future for this classof compounds, and we hope that our work will help the design of new GS with improvedproperties.

6. ABBREVIATIONS

AN = aggregation numberBENZ = benzoate

Bn = benzylC2 = saturation concentration

CAC = critical aggregation concentrationChol = cholesterol

CMC = critical micelle concentrationcryo-TEM = cryo-transmission electron microscopy

DHB = dihydroxybenzoateDOPE = dioleoylphosphatidylethanolamine

ESR = electron spin resonanceGFP = green fluorescent protein

GS = gemini surfactant(s)Im = imidazolium

MAND = mandelateMHB = metahydroxybenzoateMFI = mean fluorescence intensity

NMR = nuclear magnetic resonancePA = phenylacetate

PHB = parahydroxybenzoatePTSA = p-toluenesulfonic acid

Py = pyridiniumPyrr = pyrrolidiniumSAL = salicylate

SANS = small angle neutron scatteringTc = critical temperatureTr = triphenylmethyl

TFA = trifluoroacetate

ACKNOWLEDGMENTS

VDS also acknowledges Temple Graduate School and TUSP Graduate Office for a UniversityScholarship. The authors are grateful to the reviewers of this material for valuable suggestionsand corrections.



REFERENCES

1. Menger FM, Littau CA. Gemini surfactants—Synthesis and properties. J Am Chem Soc1991;113(4):1451–1452.

2. Menger FM, Littau CA. Gemini surfactants—A new class of self-assembling molecules. J Am ChemSoc 1993;115(22):10083–10090.

3. Zana R, Benrraou M, Rueff R. Alkanediyl-alpha,omega-bis(dimethylalkylammonium bromide)surfactants. 1. Effect of the spacer chain-length on the critical micelle concentration and micelleionization degree. Langmuir 1991;7(6):1072–1075.

4. Bunton CA, Robinson L, Schaak J, Stam MF. Catalysis of nucleophilic substitutions by micelles ofdicationic detergents. J Org Chem 1971;36(16):2346–2350.

5. Devinsky F, Masarova L, Lacko I. Surface-activity and micelle formation of some new bisquaternaryammonium-salts. J Coll Interface Sci 1985;105(1):235–239.

6. Zhu Y, Masuyama A, Okahara M. Preparation and surface-active properties of amphipathic com-pounds with two sulfate groups and two lipophilic alkyl chains. J Am Oil Chem Soc 1990;67(7):459–463.

7. Esumi K, Goino M, Koide Y. Adsorption and adsolubilization by monomeric, dimeric, or trimericquaternary ammonium surfactant at silica/water interface. J Coll Interface Sci 1996;183(2):539–545.

8. Castro MJL, Kovensky J, Cirelli AF. Gemini surfactants from alkyl glucosides. Tetrahedron Lett1997;38(23):3995–3998.

9. Aswal VK, De S, Goyal PS, Bhattacharya S, Heenan RK. Transition from disc to rod-like shapeof 16–3-16 dimeric micelles in aqueous solutions. J Chem Soc-Faraday Trans 1998;94(19):2965–2967.

10. Kim TS, Kida T, Nakatsuji Y, Hirao T, Ikeda I. Surface-active properties of novel cationic surfac-tants with two alkyl chains and two ammonio groups. J Am Oil Chem Soc 1996;73(7):907–911.

11. Menger FM, Keiper JS. Gemini surfactants. Angew Chem Int Ed Engl 2000;39(11):1907–1920.

12. Ikeda I. Synthesis of gemini (dimeric) and related surfactants. In Zana R, Xia J editors. ZanaGemini Surfactants: Synthesis, Interfacial and Solution-Phase Behavior, and Applications. NewYork: Marcel Dekker, Inc; 2004. p 9–35.

13. Bajaj A, Kondaiah P, Bhattacharya S. Synthesis and gene transfer activities of novel serumcompatible cholesterol-based gemini lipids possessing oxyethylene-type spacers. Bioconj Chem2007;18(5):1537–1546.

14. Yang P, Singh J, Wettig S, Foldvari M, Verrall RE, Badea I. Enhanced gene expression in ep-ithelial cells transfected with amino acid-substituted gemini nanoparticles. Eur J Pharm Biopharm2010;75(3):311–320.

15. Cai B, Li X, Yang Y, Dong J. Surface properties of gemini surfactants with pyrrolidinium headgroups. J Coll Interface Sci 2012; 370:111–116.

16. Casal-Dujat L, Rodrigues M, Yague A, Calpena AC, Amabilino DB, Gonzales-Linares J, Borras M,Perez-Garcia L. Gemini imidazolium amphiphiles for the synthesis, stabilization, and drug deliveryfrom gold nanoparticles. Langmuir 2012, 28:2368–2381.

17. Sunitha S, Reddy PS, Prasad RBN, Kanjilal S. Synthesis and evaluation of new imidazolium-basedaromatic ether functionalized cationic mono and gemini surfactants. Eur J Lipid Sci Technol 2011;113:756–762.

18. Bhadani A, Singh S. Synthesis and properties of thioether spacer containing gemini imidazoliumsurfactants. Langmuir 2011; 27:14033–14044.

19. Zhou LM, Jiang XH, Li YT, Chen Z, Hu XQ. Synthesis and properties of a novel class of geminipyridinium surfactants. Langmuir 2007;23(23):11404–11408.

20. Meekel AAP, Wagenaar A, Smisterova J, Kroeze JE, Haadsma P, Bosgraaf B, Stuart MCA, BrissonA, Ruiters MHJ, Hoekstra D, Engberts JBFN. Synthesis of pyridinium amphiphiles used for



transfection and some characteristics of amphiphile/DNA complex formation. Eur J Org Chem2000;(4):665–673.

21. VanDerWoude I, Wagenaar A, Meekel AAP, TerBeest MBA, Ruiters MHJ, Engberts JBFN, Hoek-stra D. Novel pyridinium surfactants for efficient, nontoxic in vitro gene delivery. Proc Nat AcadSci USA 1997;94(4):1160–1165.

22. Quagliotto P, Viscardi G, Barolo C, Barni E, Bellinvia S, Fisicaro E, Compari C. Gemini pyridiniumsurfactants: Synthesis and conductometric study of a novel class of amphiphiles. J Org Chem2003;68(20):7651–7660.

23. Quagliotto P, Barolo C, Barbero N, Barni E, Compari C, Fisicaro E, Viscardi G. Synthesis and char-acterization of highly fluorinated gemini pyridinium surfactants. Eur J Org Chem 2009;(19):3167–3177.

24. Bhadani A, Singh S. Novel gemini pyridinium surfactants: Synthesis and study of their surfaceactivity, DNA binding, and cytotoxicity. Langmuir 2009;25(19):11703–11712.

25. Ilies MA, Seitz WA, Caproiu MT, Wentz M, Garfield RE, Balaban AT. Pyridinium-based cationiclipids as gene-transfer agents. Eur J Org Chem 2003:2645–2655.

26. Ilies MA, Seitz WA, Ghiviriga I, Johnson BH, Miller A, Thompson EB, Balaban AT. Pyri-dinium cationic lipids in gene delivery: A structure-activity correlation study. J Med Chem2004;47(15):3744–3754.

27. Ilies MA, Johnson BH, Makori F, Miller A, Seitz WA, Thompson EB, Balaban AT. Pyridiniumcationic lipids in gene delivery: An in vitro and in vivo comparison of transfection efficiency versusa tetraalkylammonium congener. Arch Biochem Biophys 2005;435(1):217–226.

28. Ilies MA, Seitz WA, Johnson BH, Ezell EL, Miller AL, Thompson EB, Balaban AT. Lipophilicpyrylium salts in the synthesis of efficient pyridinium-based cationic lipids, gemini surfactants, andlipophilic oligomers for gene delivery. J Med Chem 2006;49(13):3872–3887.

29. Ilies MA, Balaban AT. Recent developments in cationic lipid-mediated gene delivery and genetherapy. Expert Opin Ther Patents 2001;11(11):1729–1752.

30. Ilies MA, Seitz WA, Balaban AT. Cationic lipids in gene delivery: Principles, vector design andtherapeutical applications. Curr Pharm Design 2002;8(27):2441–2473.

31. Balaban AT, Ilies MA, Eichhofer A, Balaban TS. Molecular and crystal structure of a self-assembling pyridinium cationic lipid. J Mol Struct 2010;984:228–231.

32. Ao MQ, Xu GY, Zhu YY, Bai Y. Synthesis and properties of ionic liquid-type Gemini imidazoliumsurfactants. J Coll Interface Sci 2008;326(2):490–495.

33. Yamaguchi I, Gobara Y, Sato M. One-pot synthesis of N-substituted diaza[12] annulenes. Org Lett2006;8(19):4279–4281.

34. Shi L, Lundberg D, Musaev DG, Menger FM. [12]Annulene gemini surfactants: Structure andself-assembly. Angew Chem Int Ed Engl 2007;46(31):5889–5891.

35. Menger FM, Keiper JS, Mbadugha BNA, Caran KL, Romsted LS. Interfacial compositionof gemini surfactant micelles determined by chemical trapping. Langmuir 2000;16(23):9095–9098.

36. Rosen MJ. Surfactants and Interfacial Phenomena. Hoboken, NJ: Wiley-Interscience; 2004.

37. Bai GY, Wang JB, Yan HK, Li ZX, Thomas RK. Thermodynamics of molecular self-assemblyof cationic gemini and related double chain surfactants in aqueous solution. J Phys Chem B2001;105(15):3105–3108.

38. Song LD, Rosen MJ. Surface properties, micellization, and premicellar aggregation of gemini sur-factants with rigid and flexible spacers. Langmuir 1996;12(5):1149–1153.

39. Panda L, Behera GB. Thermodynamics of micellization. Part 1. Micelle Formation of CTAB,NaLS and Triton-X-100 in DMF-H2O, DMSO-H2O, Dioxane-H2O and MeOH-H2O systems bydye incorporation method. J Indian Chem Soc 1985;62(1):44–49.

40. Perez L, Pinazo A, Rosen MJ, Infante MR. Surface activity properties at equilibrium of novel geminicationic amphiphilic compounds from arginine, Bis(Args). Langmuir 1998;14(9):2307–2315.



41. Benrraou M, Zana R, Varoqui R, Pefferkorn E. Study of the interaction between dodecyltrimethy-lammonium bromide and poly(maleic acid-co-alkyl vinyl ether) in aqueous-solution by potentiom-etry and fluorescence probing. J Phys Chem 1992;96(3):1468–1475.

42. Evans DF, Allen M, Ninham BW, Fouda A. Critical micelle concentrations for alkyltrimethylam-monium bromides in water from 25 to 160◦C. J Sol Chem 1984;13(2):87–101.

43. Dam T, Engberts JBFN, Karthauser J, Karaborni S, vanOs NM. Synthesis, surface properties andoil solubilisation capacity of cationic gemini surfactants. Coll Surf A 1996;118(1-2):41–49.

44. Quagliotto P, Barbero N, Barolo C, Artuso E, Compari C, Fisicaro E, Viscardi G. Synthesis andproperties of cationic surfactants with tuned hydrophylicity. J Coll Interface Sci 2009;340(2):269–275.

45. Frindi M, Michels B, Levy H, Zana R. Alkanediyl-alpha,omega-bis(dimethylalkylammonium bro-mide) surfactants. 4. Ultrasonic-absorption studies of amphiphile exchange between micelles andbulk phase in aqueous micellar solutions. Langmuir 1994;10(4):1140–1145.

46. Alami E, Beinert G, Marie P, Zana R. Alkanediyl-alpha,omega-bis(dimethylalkylammonium bro-mide) surfactants. 3. Behavior at the air-water-interface. Langmuir 1993;9(6):1465–1467.

47. Zana R. Alkanediyl-alpha,omega-bis(dimethylalkylammonium bromide) surfactants—10. Behav-ior in aqueous solution at concentrations below the critical micellization concentration: An electricalconductivity study. J Coll Interface Sci 2002;246(1):182–190.

48. Zana R, Levy H. Alkanediyl-alpha,omega-bis(dimethylalkylammonium bromide) surfactants(dimeric surfactants). 6. CMC of the ethanediyl-1,2-bis(dimethylalkylammonium bromide) series.Coll Surf A 1997;127(1-3):229–232.

49. Zhao J, Christian SD, Fung BM. Mixtures of monomeric and dimeric cationic surfactants. J PhysChem B 1998;102(39):7613–7618.

50. Rosen MJ, Liu LT. Surface activity and premicellar aggregation of some novel diquaternary geminisurfactants. J Am Oil Chem Soc 1996;73(7):885–890.

51. Devinsky F, Lacko I, Bittererova F, Tomeckova L. Relationship between structure, surface-activity,and micelle formation of some new bisquaternary isosteres of 1,5-pentanediammonium dibromides.J Coll Interface Sci 1986;114(2):314–322.

52. Menger FM, Keiper JS, Azov V. Gemini surfactants with acetylenic spacers. Langmuir2000;16(5):2062–2067.

53. Klevens HB. Structure and Aggregation in dilute solutions of surface active agents. J Am Oil ChemSoc 1953;30(2):74–80.

54. Zana R. Gemini (dimeric) surfactants in water: Solubility, cmc, thermodynamics of micellization,and interaction with water-soluble polymers. In: Zana R, Xia J editors. Gemini Surfactants: Syn-thesis, Interfacial and Solution-Phase Behavior and Applications. New York: Marcel Dekker; 2004.p 108–138.

55. Devinsky F, Lacko I, Mlynarcik D, Svajdlenka E, Borovska V. Quaternary ammonium-salts. 33.QSAR of antimicrobially active niketamide derivatives. Chem Papers 1990;44(2):159–170.

56. Fisicaro E, Compari C, Biemmi M, Duce E, Peroni M, Barbero N, Viscardi G, QuagliottoP. Unusual behavior of the aqueous solutions of gemini bispyridinium surfactants: Apparentand partial molar enthalpies of the dimethanesulfonates. J Phys Chem B 2008;112(39):12312–12317.

57. Wang XY, Wang JB, Wang YL, Yan HK, Li PX, Thomas RK. Effect of the nature of the spacer onthe aggregation properties of gemini surfactants in an aqueous solution. Langmuir 2004;20(1):53–56.

58. Qiu L-G, Xie A-J, Shen Y-H. Micellar effects of a triazole-based cationic gemini surfactant on therate of a nucleophilic aromatic substitution reaction. Colloid Polym Sci 2005; 283:1343–1348.

59. Evans HC. Alkyl sulphates. Part 1. Critical micelle concentrations of the sodium salts. J Chem Soc1956:579–586.

60. Kuwamoto K, Asakawa T, Ohta A, Miyagishi S. Degree of micelle ionization and micellar growth forgemini surfactants detected by 6-methoxy-N-(3-sulfopropyl)quinolinium fluorescence quenching.Langmuir 2005; 21:7691–7695.



61. Sugihara G, Arakawa Y, Tanaka K, Lee S, Moro Y. Micelle formation and counterion bindingof dodecylammonium alkanesulfonates in water at different temperatures. J Coll Interface Sci1995;170(2):399–406.

62. Barry BW, Wilson R. CMC counterion binding and thermodynamics of ethoxylated anionic andcationic surfactants. Coll Polymer Sci 1978;256(3):251–260.

63. Ao MQ, Huang PP, Xu GY, Yang XD, Wang YJ. Aggregation and thermodynamic propertiesof ionic liquid-type gemini imidazolium surfactants with different spacer length. Coll Polym Sci2009;287(4):395–402.

64. Collins KD, Washabaugh MW. The Hofmeister effect and the behaviour of water at interfaces. QRev Biophys 1985;18(4):323–422.

65. Nostro PL, Nostro AL, Ninham BW, Pesavento G, Fratoni L, Baglioni P. Hofmeister specific ioneffects in two biological systems. Curr Opin Colloid Interface Sci 2004;9(1–2):97–101.

66. Bijma K, Engberts JBFN. Effect of counterions on properties of micelles formed by alkylpyri-dinium surfactants.1. Conductometry and 1H-NMR chemical shifts. Langmuir 1997;13(18):4843–4849.

67. Jiang N, Li PX, Wang YL, Wang JB, Yan HK, Thomas RK. Micellization of cationic geminisurfactants with various counterions and their interaction with DNA in aqueous solution. J PhysChem B 2004;108(39):15385–15391.

68. Manet S, Karpichev Y, Bassani D, Kiagus-Ahmad R, Oda R. Counteranion effect on micel-lization of cationic gemini surfactants 14–2-14: Hofmeister and other counterions. Langmuir2010;26(13):10645–10656.

69. Voet A. Quantative lyotropy. Chem Rev 1937;20(2):169–179.

70. Schott H. Lyotropic numbers of anions from cloud point changes of nonionic surfactants. ColloidsSurf 1984;11(1-2):51–54.

71. Underwood AL, Anacker EW. Organic counterions and micellar parameters—Methyl-substituted,chloro-substituted and phenyl-substituted acetates. J Coll Interface Sci 1984;100(1):128–135.

72. Zana R, Ed. Surfactant Solutions: New Methods of Investigation, Vol. 22. New York: MarcelDekker, Inc.; 1987. Chapter 2, p 57.

73. Alargova RG, Kochijashky II, Zana R. Fluorescence study of the aggregation behavior of dif-ferent surfactants in aqueous solutions in the presence and in the absence of gas. Langmuir1998;14(7):1575–1579.

74. Infelta PP. Fluorescence quenching in micellar solutions and its application to the determination ofaggregation numbers. Chem Phys Lett 1979;61(1):88–91.

75. Barzykin AV, Almgren M. On the distribution of surfactants among mixed micelles. Langmuir1996;12(20):4672–4680.

76. Vethamuthu MS, Almgren M, Mukhtar E, Bahadur P. Fluorescence quenching studies of the aggre-gation behavior of the mixed micelles of bile-salts and cetyltrimethylammonium halides. Langmuir1992;8(10):2396–2404.

77. Reekmans S, Bernik D, Gehlen M, Vanstam J, Vanderauweraer M, Deschryver FC. Change in themicellar aggregation number or in the size distribution—A dynamic fluorescence quenching studyof aqueous cetyltrimethylammonium chloride. Langmuir 1993;9(9):2289–2296.

78. Rosen MJ, Mathias JH, Davenport L. Aberrant aggregation behavior in cationic gemini surfac-tants investigated by surface tension, interfacial tension, and fluorescence methods. Langmuir1999;15(21):7340–7346.

79. Missel PJ, Mazer NA, Benedek GB, Young CY, Carey MC. Thermodynamic analysis of the growthof sodium dodecyl-sulfate micelles. J Phys Chem 1980;84(9):1044–1057.

80. Borse M, Sharma V, Aswal VK, Goyal PS, Devi S. Effect of head group polarity and spacerchain length on the aggregation properties of gemini surfactants in an aquatic environment. J CollInterface Sci 2005;284(1):282–288.

81. Candau SJ, Ed. Surfactant Solutions: New Methods of Investigation, Vol. 22. New York: MarcelDekker; 1987. Chapter 3.



82. Danino D, Kaplun A, Talmon Y, Zana R. Cryo-Transmission Electron Microscopy Investigationsof Unusual Amphiphilic Systems in Relation to Their Rheological Properties. Structure and Flow inSurfactant Solutions, Vol. 578. ACS Symposium Series. Washington: American Chemical Society;1994. p 105–119.

83. Cabane B, Ed. Surfactant Solutions: New Method of Investigation, Vol. 22. New York: MarcelDekker; 1987. Chapter 2.

84. Danino D, Talmon Y, Zana R. Alkanediyl-alpha,omega-bis(dimethylalkylammonium bromide)surfactants (dimeric surfactants). 5. Aggregation and microstructure in aqueous-solutions. Lang-muir 1995;11(5):1448–1456.

85. Bernheim-Groswasser A, Zana R, Talmon Y. Sphere-to-cylinder transition in aqueousmicellar solution of a dimeric (gemini) surfactant. J Phys Chem B 2000;104(17):4005–4009.

86. Zana R, Talmon Y. Dependence of aggregate morphology on structure of dimeric surfactants.Nature 1993;362(6417):228–230.

87. Israelachvili JN, Mitchell DJ, Ninham BW. Theory of self-assembly of hydrocarbon amphiphilesinto micelles and bilayers. J Chem Soc 1976;72:1525–1568.

88. Tanford C. The Hydrophobic Effect: Formation of Micelles and Biological Membranes. Somerset:Wiley; 1980.

89. Balazs DA, Godbey W. Liposomes for use in gene delivery. J Drug Del 2011(326497):1–12.

90. De S, Aswal VK, Goyal PS, Bhattacharya S. Characterization of new gemini surfactant micelleswith phosphate headgroups by SANS and fluorescence spectroscopy. Chem Phys Lett 1999;303(3–4):295–303.

91. Hattori N, Hirata H, Okabayashi H, Furusaka M, O’Connor CJ, Zana R. Small-angle neutron-scattering study of bis(quaternaryammonium bromide) surfactant micelles in water. Effect of thelong spacer chain on micellar structure. Coll Polym Sci 1999;277(1):95–100.

92. Hirata H, Hattori N, Ishida M, Okabayashi H, Furusaka M, Zana R. Small-angle neutron-scatteringstudy of bis(quaternaryammonium bromide) surfactant micelles in water. Effect of the spacer chainlength on micellar structure. J Phys Chem 1995; 99: 17778–17784.

93. De S, Aswal VK, Goyal PS, Bhattacharya S. Role of spacer chain length in dimeric micellar organi-zation. Small angle neutron scattering and fluorescence studies. J Phys Chem 1996;100(28):11664–11671.

94. Maiti PK, Chowdhury D. Micellar aggregates of gemini surfactants: Monte Carlo simulation of amicroscopic model. Europhys Lett 1998;41(2):183–188.

95. Oda R, Bourdieu L, Schmutz M. Micelle to vesicle transition induced by cosurfactant: Rheologicalstudy and direct observations. J Phys Chem B 1997;101(31):5913–5916.

96. Danino D, Talmon Y, Zana R. Vesicle-to-micelle transformation in systems containing dimericsurfactants. J Coll Interface Sci 1997;185(1):84–93.

97. Rosen MJ, Ed. Surfactants and interfacial phenomena. 2nd Ed. New York: John Wiley & Sons Inc;1989.

98. Laschewsky A, Wattebled L, Arotcarena M, Habib-Jiwan JL, Rakotoaly RH. Synthesis and prop-erties of cationic oligomeric surfactants. Langmuir 2005;21(16):7170–7179.

99. Almeida JA, Marques EF, Jurado AS, Pais AA. The effect of cationic gemini surfactants upon lipidmembranes. An experimental and molecular dynamics simulation study. Phys Chem Chem Phys2010;12(43):14462–14476.

100. Almeida JA, Faneca H, Carvalho RA, Marques EF, Pais AA. Dicationic alkylammonium bromidegemini surfactants. Membrane perturbation and skin irritation. PLoS One 2011;6(11):e26965.

101. Almgren M. Mixed micelles and other structures in the solubilization of bilayer lipid membranes bysurfactants. Biochim Biophys Acta 2000;1508(1-2):146–163.

102. Attwood D, Florence AT. Interaction of surfactants with membranes and membrane components.Surfactant Systems: Their Chemistry, Pharmacy and Biology, 1st edition. New York: Chapman andHall; 1983. p 619.



103. Poole AR, Howell JI, Lucy JA. Lysolecithin and cell fusion. Nature 1970;227(5260):810–814.

104. Ferguson TM, Prottey C. Simple and rapid method for assaying cytotoxicity. Food and CosmeticsToxicol 1974;12(3):359–366.

105. Kirby AJ, Camilleri P, Engberts JBFN, Feiters MC, Nolte RJM, Soderman O, Bergsma M, BellPC, Fielden ML, Rodriguez CLG, Guedat P, Kremer A, McGregor C, Perrin C, Ronsin G, vanEijk MCP. Gemini surfactants: New synthetic vectors for gene transfection. Angew Chem Int EdEngl 2003;42(13):1448–1457.

106. Felgner PL, Barenholz Y, Behr JP, Cheng SH, Cullis P, Huang L, Jessee JA, Seymour L, Szoka F,Thierry AR, Wagner E, Wu G. Nomenclature for synthetic gene delivery systems. Hum Gene Ther1997;8(5):511–512.

107. Zabner J, Fasbender AJ, Moninger T, Poellinger KA, Welsh MJ. Cellular and molecular barriersto gene transfer by a cationic lipid. J Biol Chem 1995;270(32):18997–19007.

108. Xu Y, Szoka FC, Jr. Mechanism of DNA release from cationic liposome/DNA complexes used incell transfection. Biochemistry 1996;35(18):5616–5623.

109. Lechardeur D, Sohn KJ, Haardt M, Joshi PB, Monck M, Graham RW, Beatty B, Squire J,O’Brodovich H, Lukacs GL. Metabolic instability of plasmid DNA in the cytosol: a potentialbarrier to gene transfer. Gene Ther 1999;6(4):482–497.

110. Zanta MA, Belguise-Valladier P, Behr JP. Gene delivery: a single nuclear localization signal peptideis sufficient to carry DNA to the cell nucleus. Proc Natl Acad Sci USA 1999;96(1):91–96.

111. Rosenzweig HS, Rakhmanova VA, MacDonald RC. Diquaternary ammonium compounds as trans-fection agents. Bioconj Chem 2001;12(2):258–263.

112. Cardoso AM, Faneca H, Almeida JA, Pais AA, Marques EF, de Lima MC, Jurado AS. Geminisurfactant dimethylene-1,2-bis(tetradecyldimethylammonium bromide)-based gene vectors: a bio-physical approach to transfection efficiency. Biochim Biophys Acta 2011;1808(1):341–351.

113. Vlachy N, Jagoda-Cwiklik B, Vacha R, Touraud D, Jungwirth P, Kunz W. Hofmeister series andspecific interactions of charged headgroups with aqueous ions. Adv Colloid Interface Sci 2009;146(1-2):42–47.

114. Collins KD, Neilson GW, Enderby JE. Ions in water: characterizing the forces that control chemicalprocesses and biological structure. Biophys Chem 2007;128(2-3):95–104.

115. Zuidam NJ, Barenholz Y. Electrostatic and structural properties of complexes involving plasmidDNA and cationic lipids commonly used for gene delivery. Biochim Biophys Acta 1998;1368(1):115–128.

116. Wagner K, Harries D, May S, Kahl V, Radler JO, Ben-Shaul A. Direct evidence for counterionrelease upon cationic lipid-DNA condensation. Langmuir 2000;16:303–306.

117. Kikuchi IS, Carmona-Ribeiro AM. Interactions between DNA and synthetic cationic liposomes. JPhys Chem B 2000;104:2829–2835.

118. Foldvari M, Wettig S, Badea I, Verrall R, Bagonluri M. Dicationic gemini surfactant gene deliv-ery complexes contain cubic-lamellar mixed polymorphic phase NSTI Nanotech 2006. Volume 2.Boston, MA; 2006. p 400–403.

119. Foldvari M, Badea I, Wettig S, Verrall R, Bagonluri M. Structural characterization of novel gemininon-viral DNA delivery systems for cutaneous gene therapy. J Exp Nanosci 2006;1(2):165–176.

120. Bell PC, Bergsma M, Dolbnya IP, Bras W, Stuart MCA, Rowan AE, Feiters MC, Engberts JBFN.Transfection mediated by gemini surfactants: Engineered escape from the endosomal compartment.J Am Chem Soc 2003;125(6):1551–1558.

121. Jiang N, Wang JB, Wang YL, Yan HK, Thomas RK. Microcalorimetric study on the interaction ofdissymmetric gemini surfactants with DNA. J Coll Interface Sci 2005;284(2):759–764.

122. Wang HT, Wettig SD. Synthesis and aggregation properties of dissymmetric phytanyl-gemini sur-factants for use as improved DNA transfection vectors. Phys Chem Chem Phys 2011;13(2):637–642.

123. Badea I, Wettig S, Verrall R, Foldvari M. Topical non-invasive gene delivery using gemini nanopar-ticles in interferon-gamma-deficient mice. Eur J Pharm Biopharm 2007;65(3):414–422.



124. Badea I, Verrall R, Baca-Estrada M, Tikoo S, Rosenberg A, Kumar P, Foldvari M. In vivo cutaneousinterferon-gamma gene delivery using novel dicationic (gemini) surfactant-plasmid complexes. JGene Med 2005;7(9):1200–1214.

125. Bajaj A, Kondiah P, Bhattacharya S. Design, synthesis, and in vitro gene delivery efficacies ofnovel cholesterol-based gemini cationic lipids and their serum compatibility: A structure-activityinvestigation. J Med Chem 2007;50(10):2432–2442.

126. Kim BK, Doh KO, Bae YU, Seu YB. Synthesis and Optimization of Cholesterol-Based Diqua-ternary Ammonium Gemini Surfactant (Chol-GS) as a New Gene Delivery Vector. J MicrobiolBiotechnol 2011;21(1):93–99.

127. Fisicaro E, Compari C, Duce E, Donofrio G, Rozycka-Roszak B, Wozniak E. Biologically activebisquaternary ammonium chlorides: Physico-chemical properties of long chain amphiphiles andtheir evaluation as non-viral vectors for gene delivery. Biochim Biophys Acta 2005;1722(2):224–233.

128. Rashin AA. Aspects of protein energetics and dynamics. Progress in Biophys Mol Biol 1993;60(2):73–200.

129. Schneider B, Patel K, Berman HM. Hydration of the phosphate group in double-helical DNA.Biophys J 1998;75(5):2422–2434.

130. Leikin S, Parsegian VA, Rau DC, Rand RP. Hydration Forces. Annual Rev Phys Chem1993;44(1):369–395.

131. Bajaj A, Paul B, Indi SS, Kondaiah P, Bhattacharya S. Effect of the hydrocarbon chain andpolymethylene spacer lengths on gene transfection efficacies of gemini lipids based on aromaticbackbone. Bioconj Chem 2007;18(6):2144–2158.

132. Bajaj A, Kondaiah P, Bhattacharya S. Effect of the nature of the spacer on gene transfer efficacies ofnovel thiocholesterol derived gemini lipids in different cell lines: A structure-activity investigation.J Med Chem 2008;51(8):2533–2540.

133. Bajaj A, Kondaiah P, Bhattacharya S. Gene transfection efficacies of novel cationic gemini lipidspossessing aromatic backbone and oxyethylene spacers. Biomacromolecules 2008;9(3):991–999.

134. Sharma VD, Aifuwa EO, Ilies MA. ACS Coll Surf Sci 2011;242.

135. Wettig SD, Verrall RE, Foldvari M. Gemini surfactants: A new family of building blocks for non-viral gene delivery systems. Curr Gene Ther 2008;8(1):9–23.

136. Wettig SD, Badea I, Donkuru M, Verrall RE, Foldvari M. Structural and transfection properties ofamine-substituted gemini surfactant-based nanoparticles. J Gene Med 2007;9(8):649–658.

137. Wettig SD, Wang C, Verrall RE, Foldvari M. Thermodynamic and aggregation properties ofaza- and imino-substituted gemini surfactants designed for gene delivery. Phys Chem Chem Phys2007;9(7):871–877.

Vishnu Dutt Sharma was born and educated in India, receiving his B. Pharm. degree in 2010,from Delhi University of Pharmaceutical Sciences and Research. In 2010, he joined Dr. Ilies’sgroup at Temple University School of Pharmacy in Philadelphia, for Ph.D. studies. His researchinterests are focused on understanding and manipulating the interfacial properties of amphiphiliccompounds for generation of novel drug and gene delivery systems with improved efficiency. Besidesthe present work he co-authored a book chapter and presented several communications at nationaland international conferences.

Dr. Marc A. Ilies was born and educated in Romania, receiving his B.Sc. degree from Universityof Bucharest in 1995 and M.Sc. degree from the same university in 1996, after a research stage atENSC Lille, France, in the framework of the Francophone Teaching Module. In 1996, he joinedProfessor Alexandru T. Balaban’s group at the Polytechnic University of Bucharest, receiving



his Ph.D. in 2001. From 1997, he was also a faculty in the Department of Chemistry, Schoolof Biotechnologies, University of Agricultural Sciences and Veterinary Medicine in Bucharest.In 2001, he followed Dr. Balaban at Texas A&M University in Galveston, TX, for postdoctoralstudies aimed to develop new pyridinium cationic lipids for gene delivery, in close collaboration withthe groups of Drs. E. Brad Thompson and Robert Garfield from UTMB Galveston, where he was avisiting scientist. In 2004, Dr. Ilies relocated to University of Pennsylvania for postdoctoral studiestoward self-assembling amphiphilic dendrons (with Dr. Virgil Percec), amphiphilic polymers, andtheir supramolecular assemblies as drug-delivery systems in cancer pharmacology (with Drs.Vladimir Muzykantov and Ian Blair). Since 2007, he is an Assistant Professor in the Departmentof Pharmaceutical Sciences, School of Pharmacy, Temple University. His research interests fitwithin the broadly defined bio-organic and medicinal chemistry at membrane interfaces usingpyridinium compounds, being focused on the development of chemical solutions for the deliveryof drugs and genes. His group is currently pursuing selective carbonic anhydrase inhibitors andactivators and novel drug and gene delivery systems. Dr. Ilies has published over 45 peer-reviewedscientific papers, reviews, and book chapters, and has co-authored one US patent. His publicationswere cited over 800 times, with an H-index of 18 (2011). He also serves in the editorial boards ofthree medicinal chemistry journals.


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