Gemini surfactant-based systems for drug and gene delivery

CHAPTER

Gemini surfactant-based systems for drug and gene delivery

13

Amal Makhlouf, Istvan Hajdu and Ildiko Badea University of Saskatchewan, Saskatoon, SK, Canada

CHAPTER OUTLINE 13.1 Introduction ...................................................................................................561 13.2 Structural Modifications in Gemini Surfactants ................................................562 13.2.1 Modifications in the Headgroup of the Gemini Surfactants .............562 13.2.2 Modifications in the Spacer of the Gemini Surfactants...................570 13.2.3 Modifications in the Alkyl Chain of the Gemini Surfactants ............571 13.2.4 Giant Gemini Surfactants ............................................................572 13.2.5 Dissymmetric Gemini Surfactants ................................................572 13.2.6 Counter-Ion Effect ......................................................................573 13.3 In Vitro Structure-Activity Relationship ............................................................574 13.4 Biologically Relevant Properties of the Gemini Surfactants ...............................578 13.4.1 Gemini Surfactants With Cleavable Spacer Groups ........................580 13.4.2 Gemini Surfactants With Heterocyclic Head Groups.......................582 13.4.3 Cyclodextrin-Gemini Surfactants ..................................................584 13.4.4 Amino Acid
Based Gemini Surfactants........................................587 13.4.5 Sugar-Based Gemini Surfactants..................................................589 13.5 In Vitro/In Vivo Correlation .............................................................................590 13.6 Conclusions...................................................................................................593 References .............................................................................................................594 Further Reading ......................................................................................................600

13.1 INTRODUCTION Gemini surfactants, N,N-bis(dimethylalkyl)-α,ω-alkanediammonium halide derivatives, are attracting increasing interest in the fields of drug and gene delivery. This arises from the flexibility in chemical modification of these compounds that can modulate their physicochemical properties. Modification of the length, degree of unsaturation, and substitution of different functional groups in the spacer and

Organic Materials as Smart Nanocarriers for Drug Delivery. DOI: http://dx.doi.org/10.1016/B978-0-12-813663-8.00013-0 © 2018 Elsevier Inc. All rights reserved.

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alkyl tails can provide opportunities to tailor their chemical structure for specific needs. Gemini surfactants are characterized by their ability to spontaneously aggregate (B1000-fold superiority over monomer surfactants) into complex structures, including micelles, inverted micelles, liposomes, and cubosomes, depending on the chemical composition of tails and head moieties. Their low critical micelle concentration (CMC), compared to their monomer counterparts, is advantageous for minimizing the amount of gemini surfactant used in the formulation process, which is essential for ensuring an optimal safety profile for the delivery system. Several generations of gemini nanoparticles have been created to deliver small and large molecules to tumor cells, including melanoma and cervical cancer. Table 13.1 summarizes the chemical modifications and uses of the gemini surfactants in the past decade. Gemini surfactants have demonstrated high gene delivery capacity, due to the ability to condense plasmid DNA and self-assemble into lipoplexes with a net positive charge, resulting in electrostatic attraction to the anionic cell surface. Structure-activity relationship studies were carried out to understand the molecular requirements for nanoparticle formation and cellular uptake. Animal studies have been performed to investigate the efficiency of gemini surfactantbased carriers. The aim of this chapter is to summarize and discuss the effect of structural modification on the performance of gemini surfactant formulations and correlate the in vitro characteristics to the in vivo results in drug and gene delivery.

13.2 STRUCTURAL MODIFICATIONS IN GEMINI SURFACTANTS The term gemini, meaning twin, surfactant was first used in the early 1990s (Menger and Littau, 1991) to describe bis-surfactants. The early gemini surfactants were synthesized by coupling two individual surfactant molecules by a rigid spacer. The individual surfactant monomers usually have a polar head group and a nonpolar hydrocarbon tail of different chain length (C8
C18). Over time, spacers of different length, flexibility, and chemical structure were used for gemini surfactants synthesis. Using the effective tool of synthesis, gemini surfactants have been synthesized and characterized, exhibiting special and enhanced properties (including vesicle formation) relative to the early counterparts. The effect of the constitutive elements: spacer, hydrocarbon tail and head groups, on the CMC and the ability to form supramolecular structures was thoroughly investigated and reported in the literature.

13.2.1 MODIFICATIONS IN THE HEADGROUP OF THE GEMINI SURFACTANTS The most widely studied gemini surfactants are cationic alkanediyl-α,ω-bis (alkyldimethylammonium) dibromide, which are referred to as CmCsCm(Me),

Table 13.1 Chemical Modifications and Rational of Design for Gemini Surfactants Rationale of Design

Reference

N,N-Bis(dimethylalkyl)-α,ω-alkanediammonium bromide

Nonviral gene delivery vector

Foldvari et al. (2006)

Phytanyl-substituted gemini surfactants (phy-3-m)

Improvement of transfection efficiency

Wang and Wettig (2011)

Gemini Name

Chemical Structure

m-7NH-m series

Donkuru et al. (2012)

Glycyl-lysine substituted gemini surfactant (12-7NGK-12)

Singh et al. (2012)

Pyridinium cationic gemini surfactant

Sharma and Ilies (2014)

(Continued)

Table 13.1 Chemical Modifications and Rational of Design for Gemini Surfactants Continued Gemini Name

Chemical Structure

Rationale of Design

Reference

Fluorinated gemini bis-pyridinium surfactan

Fisicaro et al. (2017)

a. Amide (12Ser)2CON5,

Cardoso et al. (2015b)

b. Ester (12Ser)2COO5

Cholesterol-based diquaternary ammonium gemini surfactant

C12(azo)C12

Kim et al. (2011)

Improvement of physicoelastic properties

Song et al. (2010)

PS-(APOSS)2-PS-i

Wang et al. (2013)

PSn-(XPOSS)-(YPOSS)-PSm

Su et al. (2014)

(Continued)

Table 13.1 Chemical Modifications and Rational of Design for Gemini Surfactants Continued Gemini Name

Chemical Structure

Rationale of Design

Reference

Dissymmetric gemini surfactant with different alkyl chain length

Xu et al. (2011)

(Oligooxa)-α,ω-bis(m-alkylbenzene sulfonate)

Liu et al. (2010)

Sulfonate containing gemini surfactant

Zhu et al. (2012)

((C18)2-Lys-CysmPEG)2

Dodecyl esterquat gemini surfactant

Dodecyl betainate gemini surfactant

Improvement of biodegradability

Kim et al. (2014)

Tehrani-Bagha et al. (2015)

Gemini piperidinium surfactants

Bhadani et al. (2016)

(C12Cys)2

Branco et al. (2015)

(PEG)2-Cys-(PLA)2

Kim et al. (2015)

Cationic gemini surfactat drived from lysine

Colomer et al. (2011)

Ester-based gemini surfactant; 2,20 -[ethane1,2-diylbis(oxy)]bis(N-tetradecyl-N,N-dimethyl2-oxoethanaminium) dibro-mide [C14-(MEG)-C14]2Br N-Dodecyl-N-[2-(N0 -dodecyl-N0 ,N0 dimethylammonio)acetyloxyethyl]-N,Ndimethylammonium dibromide (12Q2OCO1Q12)

Bhadani et al. (2014)

Tehrani-Bagha et al. (2015)

(Continued)

Table 13.1 Chemical Modifications and Rational of Design for Gemini Surfactants Continued Gemini Name

Chemical Structure

Rationale of Design

Reference

Diester cationic gemini srfactant

Fatma et al. (2016)

Gemini alkyl O-glucoside surfactants

Liu et al. (2013)

13.2 Structural Modifications in Gemini Surfactants

where m and s stand for the carbon atom number in the tail alkyl chain and in the methylene spacer, respectively. Previous studies had shown that the variation of the spacer and the alkyl chain usually affects the aggregation behavior of gemini surfactants. It has been found that, in the case of 4 # s # 12 and 12 # m # 16, CmCsCm(Me), gemini surfactants tend to form higher-curvature aggregates in aqueous solutions, such as spherical or elongated micelles (Han and Wang, 2011). Interestingly, when the dimethylammonium headgroups were replaced by diethylammonium headgroups (C12CsC12(Et), s 5 4, 6, 8, 10, 12), larger number of vesicles were observed in the aqueous solutions. These vesicles are relatively stable over time and possess superior thermal stability, which means that the hydrocarbon parts of the polar headgroups of gemini surfactants strongly affect the aggregation behavior (Lu et al., 2007). In this regard, a series of cationic gemini surfactants with diethylammonium headgroups and a diamido spacer were synthesized; their surface and bulk properties were investigated utilizing surface tension, electrical conductivity, viscosity, dynamic light scattering (DLS), and transmission electron microscopy (TEM) measurements. It was found that the surface tension declined upon increasing concentration above the CMC of these gemini surfactants. This surface tension behavior could be explained by the rapid increase in the counter ion activity in the bulk phase. The vesicles formed larger aggregates (sponge-like structures) at high surfactant concentration (Zhang et al., 2012). Among the polar head groups used in the synthesis of gemini surfactants, the sulfonate group has attracted great attention, since it increases the surfactant solubility in the presence of different ions and in a wide pH range. Four anionic sulfonate-containing gemini surfactants, C7
Mx
C7, where x 5 0, 1, 2 and 3, with rigid or semi-rigid spacers were synthesized (Zhu et al., 2012). A fully rigid spacer of C7
M0
C7 was composed of two benzene rings and two carbonyl groups. The basic surface properties of these gemini surfactants were compared to those of the conventional single-chain sodium dodecylbenzenesulfonate (SDSB). The plots of surface tension versus logarithm of concentration of these surfactants were unlike the traditional plot, exemplified by SDBS. Above the CMC, the surface tension of gemini surfactant aqueous solutions did not become steady, but still decreased continuously with the increase in concentration. However, the decrease is not so steep as that below the CMC. Gemini surfactants with semi-rigid spacers exhibited superior surface activity compared, to that with fully rigid spacer. The aggregation behavior of C7
Mx
C7 in water was investigated using DLS and TEM. It was found that gemini surfactants with a rigid spacer preferred to form vesicles, whereas the ones with semi-rigid spacers self-assembled into a mixture of micelles and vesicles at low surfactant concentration and formed vesicles at high surfactant concentration (Zhu et al., 2012). A series of anionic gemini surfactants called (oligooxa)-α, ω-bis(malkylbenzene sulfonate) with different length in spacer and in hydrophobic

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chain, C8ExC8 (x 5 1, 2, 3, 4, 8) and CnE4Cn (n 5 8, 10, 12), have been synthesized from 4-n-alkylphenol and their basic physicochemical properties were investigated. As the number of oxyethylene segments increased, the CMC decreased initially then increased; the surface area per molecule decreased initially and then reached a constant value of approximate 1.30 nm2. It was confirmed that the hydrophilic spacer with oxyethylene moieties was not fully extended at the airwater interface. With increasing spacer length, the spacer became sufficiently flexible to adopt a particular conformation with a loop at the water side of the air-water interface. Fluorescence data showed that the micropolarity increased little from C8E1C8 to C8E4C8 and decreased obviously when x changes from 4 to 8. In addition, the plot of the logarithm of the CMC against the alkyl chain length for CnE4Cn series showed a linear decrease with the increase in the chain length. The micropolarity of the gemini surfactants CnE4Cn decreased with the increase in the alkyl chain length (n from 8 to 12) (Liu et al., 2010).

13.2.2 MODIFICATIONS IN THE SPACER OF THE GEMINI SURFACTANTS Many studies have shown that the spacer length and nature play a major rule in the aggregation behavior of gemini surfactants, which has been attributed to the conformational changes of gemini surfactant molecules and to the changes in the spacer location in the micelles (Zana, 2002). The effect of the spacer length on the electrostatic interactions of cationic gemini surfactant micelles with trianionic curcumin has been studied by Ke et al. The UV-absorption and fluorescence measurements have been used to investigate the interactions between cationic gemini surfactant micelles of (C12CsC12 Br2, where s 5 2, 3, 4, 6, and 12, indicating the number of carbons in the spacer) with trianionic curcumin (Cur32) at pH 13. The electrostatic interactions of gemini surfactants with curcumin were significantly regulated by the spacer length. Among five gemini surfactant micelles, the maximum intensities of absorption and fluorescence spectra of Cur32 were observed with C12C6C12 Br2 micelle with the minimum alkaline degradation at pH 13. These findings were explained by the optimum matching of the positive charge distribution in C12C6C12 Br2 micelle to the negative charges in Cur32. Accordingly, gemini surfactants with diverse structures may give great possibilities to design new effective systems, as well as to control the physicochemical properties and the bioactivities of natural drugs like curcumin (Ke et al., 2013). The effect of the spacer group of cationic gemini surfactants on microemulsion phase behavior was thoroughly examined. The phase behavior of a system of n-butanol/n-octane/water/cationic gemini surfactant, alkanediylα,ω-(dimethydodecyl-ammonium bromide) (12-n-12, n 5 3, 4, 6 n 5 3, 4, 6), was studied by determining the pseudo-ternary phase diagrams. The results showed that the spacer group of gemini surfactants has a great effect on the phase

13.2 Structural Modifications in Gemini Surfactants

behavior. The longer the spacer group, the more similar the gemini surfactant properties to monomeric surfactants. The content of surfactant and cosurfactant needed for microemulsion formation increased by increasing the length of the gemini surfactant spacer group. The study has also shown that the shorter spacer group of gemini surfactant was favorable for the formation of higher ordered surfactant aggregates, such as liquid crystals. The microstructures of each region for the studied systems have been investigated by electrical conductivity measurements, UV-visible absorbance spectra of pyrene probe, and DLS. The spherical and network structures of microemulsions were further verified by freezingetching TEM (Chen et al., 2006).

13.2.3 MODIFICATIONS IN THE ALKYL CHAIN OF THE GEMINI SURFACTANTS The micellization properties of cationic gemini surfactants CmC6CmBr2, with m 5 7, 8, 9, 10, 11, 12, and 16 has been investigated by isothermal titration microcalorimetry (Li et al., 2005). The CMC and enthalpy of micellization (ΔHmic) were determined from calorimetric titration curves. The linear decrease of log CMC with increasing the length of the hydrophobic chain was consistent with the increase in the hydrophobicity of the alkyl chain. Interestingly, by increasing the length of the alkyl chain, the ΔHmic values of the surfactants with even numbered alkyl chains varied from endothermic to exothermic, whereas the ΔHmic values of the surfactants with odd-numbered alkyl chains were all endothermic. The observation might be related to the different orientation of the C
C bond attached to the quaternary ammonium group between the even- and the odd-numbered alkyl chains (Li et al., 2005). The effect of alkyl chain and spacer lengths on the aggregation behavior, micellar surface charge density, and the phase transition between spherical and rod geometries of the cationic gemini surfactants CmH2m11N(CH3)2(CH2)S (CH3)2 N CmH2m11,2Br2 with m 5 12, 14 and s 5 2, 4 were studied by performing surface tension, electrical conductivity, pulsed field gradient nuclear magnetic resonance (PFG-NMR), and cyclic voltammetry measurements over the temperature range 298 to 323K. The CMC, surface excess, mean molecular surface area, degree of counter ion dissociation, and thermodynamic parameters of micellization were determined from the surface tension and conductance measurements. All enthalpy-entropy plot compensation exhibited excellent linearity. The micellar self-diffusion coefficients and intermicellar interaction parameters, obtained from the PFG-NMR and cyclic voltammetry measurements, were found to decrease slightly with increasing temperature, which suggested that the micellar surface charge decreased with increasing temperature (Alimohammadi et al., 2012).

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13.2.4 GIANT GEMINI SURFACTANTS A unique type of amphiphile, called “giant surfactant”, has been designed and synthesized with a distinct self-assembly behavior. It can form diverse supramolecular structures, based on minute differences in the shape and symmetry of the nanoparticle heads, as well as the geometry and topology of the polymer tails. Giant gemini surfactants consist of two acid-functionalized polyhedral oligomeric silsesquioxane (APOSS) heads and two polystyrene (PS) tails, covalently linked via a rigid spacer (p-phenylene or biphenylene) (PS
(APOSS)2
PS) (Wang et al., 2013). The study of their self-assembly in solution revealed a morphological transition from vesicles to worm-like cylinders and further to spheres, as the degree of ionization of the carboxylic acid groups on POSS heads increased. It was found that the PS tails are generally less stretched in the micellar cores of these giant gemini surfactants than those of the corresponding single-tailed (APOSS
PS) giant surfactants. It was further observed that the PS tail conformation in the micelles was also affected by the length of the rigid spacers, where a longer spacer exhibited even more stretched PS tail conformation. This constraint effectively increased the local charge density and led to an anisotropic head shape that required a proper redistribution of the APOSS heads on the micellar surface to minimize the total electrostatic repulsive free energy. These results expanded the scope of giant gemini surfactants and helped to understand their solution selfassembly behaviors. The rational design and tandem synthesis of asymmetric giant gemini surfactants, based on polyhedral oligomeric silsesquioxane (POSS), has been described (Su et al., 2014). In two cascading processes (typically within 5 h), asymmetric giant gemini surfactants could be synthesized, where the length of the two polymer tails and the identity of the two POSS heads can be independently controlled and systematically varied. It represented a convenient, efficient, and modular way to prepare giant molecules with rigorous structural precision in few steps. This study can expand the scope of giant gemini surfactants and facilitate further structural modifications toward pharmaceutically feasible macromolecules (Wang et al., 2013).

13.2.5 DISSYMMETRIC GEMINI SURFACTANTS Dissymmetric gemini surfactants, formed of two identical head groups and two hydrocarbon chains with different lengths or nature, are gaining increasing attention due to their interesting physicochemical properties. A series of dissymmetric gemini cationic surfactants was synthesized to contain a dodecanoic acid dimethyl ethylamine ester as the constant cationic part on one side of the hydroxypropyl center, and a similar cationic counterpart, but with a different acid length (from octanoic to palmitic) on the other side of the center (Xu et al., 2011). The surface tension measurements of dissymmetric gemini surfactants showed low CMC and strong adsorption at the air-water interface. The CMC was observed to increase

13.2 Structural Modifications in Gemini Surfactants

with increasing the length of the alkyl group. They also showed good foaming properties and wetting capabilities (Xu et al., 2011). Phytanyl-substituted gemini surfactants, phy-3-m (m 5 12, 16, and 18), were synthesized, characterized, and their aggregation properties were determined, with the aim of developing an efficient nonviral gene delivery vectors (Wang and Wettig, 2011). Kraftt temperatures were observed to increase while CMCs substantially decreased for phy-3-m surfactants compared to the symmetric m-3-m surfactants. Packing parameters calculated for the phy-3-m surfactants indicated the formation of vesicles rather than spherical or cylindrical micelles consistent with experimental determinations of particle diameters and the morphology images obtained by TEM. Preliminary transfection assays in vitro demonstrated that the phytanyl substitution resulted in increased transfection efficiencies, compared to the symmetric 16-3-16 surfactant.

13.2.6 COUNTER-ION EFFECT The effect of inorganic and organic salts on the aggregation behavior of cationic gemini surfactants has been studied The salts studied effectively reduced CMC values of the cationic gemini surfactants. The ability to promote the surfactant aggregation decreased in the order of C6H5COONa . p-C6H4(COONa)2 . Na2SO4 . NaCl. For 12-4-12 solution, the penetration of C6H5COO2 anions and charge neutralization induced morphological change from micelles to vesicles, whereas the other salts only slightly increased the size of micelles. The 12-4 (OH)2-12 solution changed from the micelle-vesicle coexistence to vesicles, with the addition of C6H5COONa, whereas the other salts transfer 12-4(OH)2-12 solution from the micelle-vesicle coexistence to micelles. Compared with 12-4-12, the two hydroxyls in the spacer of 12-4(OH)2-12 promoted the micellization of 12-4 (OH)2-12 and reduced the amounts of C6H5COONa required to induce the micelle-to-vesicle transition (Yu et al., 2010). 2 The effect of inorganic (Br2, NO2 3 , SCN ) and organic (benzoate and salicylate) salts on the characteristic solution properties of bis(quaternary ammonium) gemini surfactant, butanediyl-1,4-bis(dimethyldodecylammonium bromide) (12-4-12), was explored. The results revealed that counterions induced synergistic effects and greatly enhanced the efficiency of gemini surfactants in surface tension reduction, as well as the ability of micellization. The CMC of the gemini surfactants decreased with increasing the salt concentration. 1H NMR studies revealed that this was the result of a reduction in charge density per surface area of the micelles, which led to lowering of Coulombic repulsions between the head groups, due to the strong binding of counterions with surfactant head groups. The ability to promote aggregation decreased in the order: Na Salicylate . Na Benzoate . KSCN . KNO3 . KBr. The effect on the CMC parallels the anion radius were such that, the greater the anion radius, the greater the polarizability, and lower the heat of dehydration. These factors will enhance the attraction

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between gemini cation and the added anion, and will determine the decrease in CMC. A similar result was observed for the effect of salts on the micellar structural transition at a concentration range above the CMC. Furthermore, combinations of salt anions and gemini surfactants exhibited thickening of their aqueous solutions. The anions of organic salts also promoted the hydrophobic interaction between the alkyl tails of gemini surfactant (Khan et al., 2012). This body of work in the past decade, regarding changes induced in the selfassembly behavior of the gemini surfactants by structural modifications of the constitutive elements: head group, spacer, and tail; highlights the versatility of these molecules and the opportunity to design and synthesize various molecules to suit specific drug delivery needs.

13.3 IN VITRO STRUCTURE-ACTIVITY RELATIONSHIP In order to drive rational design of gemini surfactants as drug delivery systems, structure-activity relationships of a series of molecules need to be established in a systematic manner. The effect of the tail length of cationic gemini surfactants on dipalmitoyl phosphatidylcholine (DPPC) lipid membranes was investigated by Almeida et al. Differential scanning calorimetry results indicated that surfactants presenting shorter tails (12 carbons) induced a decrease in the overall order of the bilayer, while those with longer tails (16 and 18 carbons) led to the formation of more ordered structures. It was also observed, by a detailed fluorescence anisotropy, that the shorter tail surfactants (6 and 10 carbons) were responsible for a more disruptive effect upon the membrane (Almeida et al., 2010). Lipoplexes of gemini compounds, where m 5 12, 18:1 and s 5 2, 3 and 6 with DNA, have been investigated by Wang et al, using isothermal titration calorimetry (ITC), DLS, zeta potential, atomic force microscopy (AFM) and circular dichroism (CD) techniques. The results showed that the properties of lipoplexes were dependent on the structure of gemini surfactants, the presence of dioleoylphosphatidylethanolamine (DOPE), and the titration sequence, i.e., addition of increasing concentrations of DNA to the gemini surfactants or vice versa. ITC data showed that the interaction between DNA and gemini surfactants was endothermic and the observed enthalpy versus charge ratio profile depended upon the titration sequence. DLS data indicated that DNA was condensed into lipoplexes of 100
200 nm and AFM images suggested that lipoplex morphology varied from isolated globular-like aggregated particles to larger-size aggregates, with great diversity in morphology (Wang et al., 2007). The structural and physicochemical properties of cationic lipid-based DNA complexes have been investigated for the purpose of designing nanoscale selfassembling delivery systems for cutaneous gene therapy. DNA/gemini surfactant (spacer n 5 3
16; chain m 5 12 or 16) complexes (1:10 charge ratio), with or

13.3 In Vitro Structure-Activity Relationship

without DOPE, designed for cellular transfection, were generally in the range of 100
200 nm, as demonstrated by AFM and particle size analysis It was demonstrated that the gemini surfactants with a 3-carbon spacer showed higher gene delivery efficiency, compared to 4
12 carbon gemini surfactants. The transfection efficiency correlated with the ability of the gemini surfactants to compact DNA, indicated by the changes in their CD spectra (Badea et al., 2005). Smallangle X-ray scattering (SAXS) measurements indicated that the DNA/gemini surfactant complexes lacked long-range order, whereas DNA/gemini/DOPE complexes exhibited lamellar and polymorphic phases other than hexagonal (Foldvari et al., 2006). Correlation studies using transfection efficiency data in PAM212 keratinocytes and in vitro skin absorption indicated that formulations containing gemini surfactants with the ability to induce structures other than lamellar in the resulting complexes, generally exhibited greater transfection activity and cutaneous absorption (Badea et al., 2007). The transfection properties of amine-substituted gemini surfactant-based nanoparticles formulated from gemini surfactants, plasmid DNA and the helper lipid, DOPE, was elucidated using a luciferase assay. The incorporation of a pH-active imino group within the spacer of the gemini surfactant resulted in a significant increase in the transfection efficiency that could be related to both pH-induced changes in nanoparticle structure and the formation of multiple phases that more readily allow for membrane fusion to facilitate DNA release (Wettig et al., 2007). In the same context, pH-sensitive gemini surfactant nanoparticles were hypothesized to stage endosomal release of DNA (Donkuru et al., 2012). The surfactant component was either m-7-m unsubstituted base structure or the pH-sensitive 1,9-bis(alkyl)-1,1,9,9-tetramethyl-5-amino-1,9-nonanediammonium dibromide surfactants (m-7NH-m), where m 5 12, 16, 18 and 18:1. Analytical and physicochemical characterization of the gemini surfactants included purity, aggregation properties under pH 2.5
10.5, CMC and pKa. Gemini surfactant nanoparticles formulated from the m-7NH-m gemini surfactants at a surfactant:DNA charge ratio of 10:1 showed higher transfection efficiency, compared to the unsubstituted compounds; the transfection efficiency increased with increasing tail length. Morphological studies of the nanoparticles by TEM showed fusogenic changes at pH 5 5. The incorporation of a pH-active amine group within the spacer of the gemini surfactants significantly enhanced transfection efficiency in keratinocytes. This might be attributed to optimal interactions between DNA phosphate groups and the m-7NH-m gemini surfactants, owing to their
NH
groups, trimethylene spacing between nitrogen centers, and the acidic pH-induced polymorphic changes, leading to endosomal release of the plasmid (Wettig et al., 2007). Such results highlighted the amino-substituted gemini surfactants as potential components for developing nonviral nanoparticles with enhanced gene delivery for targeting diseases affecting the skin. Examining DNA condensation by polyamines, novel gemini surfactants have been designed that incorporated aza or imino substituents within the spacer group

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in order to increase interactions with DNA and potentially improve their DNA transfection ability (Vijayanathan et al., 2001). Enhanced gene expression was demonstrated in epithelial cells transfected with amino acid-substituted gemini surfactant nanoparticles. Amino acid (glycine and lysine) and dipeptide (glycyl-lysine and lysyl-lysine)-substituted spacers of gemini surfactants were synthesized, and their efficiency in gene delivery was assessed in three epithelial cell lines: African green monkey kidney fibroblast-like cell line, mouse keratinocytes, and cottontail rabbit epithelial cell lines. The cells were transfected with plasmid DNA encoding for interferon gamma (IFNγ) and green fluorescent protein, complexed with gemini compounds in the presence of DOPE as a helper lipid. Size, zeta potential, and CD measurements were used to characterize the plasmid/gemini and plasmid/gemini/helper lipid complexes; gene expression was quantified by enzyme-linked immunosorbent assay (ELISA). Gene expression was found to increase up to 72 h and then declined by the seventh day. In general, the glycine-substituted surfactant showed consistently high gene expression in all three cell lines. Results of physicochemical and spectroscopic studies of the complexes indicated that substitution of the gemini spacer does not interfere with compaction of the DNA. The superior performance of these spacer-substituted gemini surfactants might be attributed to their better biocompatibility, compared to the surfactants possessing unsubstituted spacers (Yang et al., 2010). The cytotoxicity of amino acid
substituted gemini surfactant-based DNA nanoparticles was evaluated and the relationship between transfection efficiency, toxicity and their physicochemical characteristics (such as size, binding properties, etc.) was explored (Singh et al., 2011). An overall low toxicity was observed for all complexes with no significant difference among substituted and unsubstituted compounds, suggesting a more balanced protection of the DNA by the glycine and glycyl-lysine-substituted compound. The cellular uptake and intracellular trafficking for amino acid
substituted gemini surfactant-based DNA nanoparticles have been evaluated in cotton tail rabbit epithelial cells (Singh et al., 2012). Clathrin- and caveolae-mediated uptake was found to be equally contributing to cellular internalization of both 12-7NH-12 (parent gemini surfactant) and 127NGK-12 (glycine-lysine-substituted gemini surfactant) nanoparticles. TEM images showed that the 12-7NGK-12-based nanoparticles were cylindrical whereas the 12-7NH-12-based nanoparticles were spherical, which may also influence the cellular uptake mechanism of these particles. A high buffering capacity, pH-dependent increase in particle size, and balanced DNA binding properties may contribute to a more efficient endosomal escape of 12-7NGK-12, compared to 12-7NH-12 nanoparticles, leading to higher gene expression (Singh et al., 2012). The structure-activity relationship of peptide-modified gemini surfactant-based carriers has been elucidated (Al-Dulaymi et al., 2016). Glycyl-lysine-modified gemini surfactants that differ in the length and degree of unsaturation of their alkyl tail were used to engineer DNA nanoassemblies. The highest activity of

13.3 In Vitro Structure-Activity Relationship

glycyl-lysine-modified gemini surfactants was observed with the 16-carbon tail compound at 2.5 nitrogen to phosphate ratio, showing a 5- to 10-fold increase in the level of reporter protein, compared to the 12 and 18:1 carbon tail compounds. This ratio was significantly lower, compared to the previously studied gemini surfactants with alkyl or amino- spacers and was associated with the highest cell viability (85%). This high efficiency and low toxicity was attributed to the lowest CMC of the 16 tail gemini surfactant and a balanced packing of the nanoparticles, by mixing a saturated and unsaturated lipid together, studied by SAXS and Langmuir Blodgett trough. The results showed that the length and nature of the tail of the gemini surfactants play an important role in determining the transfection efficiency of the delivery system (Al-Dulaymi et al., 2016). Among the factors that restrict the clinical applications of cationic lipid/DNA lipoplexes is the instability in aqueous formulations. The influence of lyophilisation on the essential physiochemical properties and in vitro transfection efficiency of gemini surfactant lipoplexes was investigated (Mohammed-Saeid et al., 2012). Gemini surfactant (12-7NH-12) lipoplexes were stabilized with sucrose and trehalose. Lyophilisation significantly improved the physical stability of gemini surfactant-based lipoplexes, compared to liquid formulations. The transfection efficiency of the lipoplexes increased two- to threefold, compared to fresh formulations upon lyophilisation. This effect can be attributed to the improvement of DNA compaction and changes in the lipoplex morphology, due to the lyophilization-rehydration cycles. All lyophilized formulations showed a significant loss of gene transfection activity after 3 months of storage. This could be attributed to the conformational changes in the supramolecular structure of the lipoplexes as a function of time and temperature, rather than to DNA degradation (Mohammed-Saeid et al., 2012). Gemini surfactants with hexadecyl tails and hydroxyethylated head groups, bridged with tetramethylene (G4), hexamethylene (G6) and dodecamethylene (G12) spacers, were shown to self-assemble at the lower CMC, compared to their conventional m-s-m analogs (Sharma et al., 2004). The lipoplex formation and the plasmid DNA transfer into different kinds of host cells was studied. A high transfection efficacy has been demonstrated for DNA/gemini complexes in eukaryotic cells, which increased as follows: G6 , G4 , G12. Different activity series, i.e., G6 . G4 . G12 was obtained in the case of transformation of bacterial cells with plasmid DNA/gemini complexes, mediated by electroporation technique. Only G6 showed transformation efficacy exceeding the uncomplexed DNA (Zakharova et al., 2016). In other research, the nonviral gene delivery by a series of dicationic gemini surfactant
phospholipid nanoparticles was evaluated, and the mechanism of interaction with cellular membranes of murine PAM212 epidermal keratinocytes was explored using flowcytometry (Gharagozloo et al., 2015). Nanoparticles were prepared using 12 different gemini surfactants (m-s-m, with m 5 12, 16 and 18C alkyl tail and s 5 3 and 7-carbon polymethylene spacer group and 7C substituted

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spacers with 7NH and 7NCH3) in the presence of DOPE as helper lipid. No significant loss of viability was detected in cells transfected with 18 carbon tail series nanoparticles, whereas a significant reduction of viability was detected in cells treated with 12 carbon tail nanoparticles. It was found that 18-3-18 gemini surfactant nanoparticles had higher transfection efficiency and comparable viability profile to the commercial Lipofectamine. The interaction of 18-3-18 gemini surfactant nanoparticles with PAM212 cell membranes involved an increase in permeability, possibly through the formation of nanoscale pores, which could explain efficient gene delivery (Gharagozloo et al., 2015). The effect of DOPE and cholesterol as helper lipids on the structure and cellular uptake of gemini surfactant [CmH2m11(CH3)2N 1 (CH2)sN 1 (CH3)2CmH2m11]2Br21 nanoparticles was investigated. The most efficient complexes were those containing helper lipids, which displayed a morphologically labile architecture, implicated in the efficient DNA uptake by Hela cells. Complexes lacking helper lipids were translocated directly across the lipid bilayer, while those containing helper lipids were taken up by cells by macropinocytosis (Cardoso et al., 2014). In another study, a cholesterol-based diquaternary ammonium gemini surfactant was synthesized and assessed as a nonviral gene delivery vector (Kim et al., 2011). The optimal efficiency was found to be at a weight ratio of 1:4 of lipid:DOPE, and at a ratio between 10:1B15:1 of liposome:DNA. The transfection efficiency was compared with commercial liposomes and with Lipofectamine, 1,2-dimyristyloxypropyl-3-dimethylhydroxyethylammonium bromide (DMRIE-C), and N-[1-(2,3-dioleoyloxy)propyl]N,N, N-trimethylammonium chloride (DOTAP). The results indicated that efficiency of cholesterol-based gemini surfactant was greater than that of all the tested commercial liposomes in COS7 and Huh7 cells, and higher than DOTAP and Lipofectamine in A549 cells. Confirmation of these findings was observed using green fluorescent protein expression. Furthermore, cholesterol-based gemini surfactant exhibited a moderate level of cytotoxicity, indicating potential use as a nonviral vector for gene delivery (Kim et al., 2011).

13.4 BIOLOGICALLY RELEVANT PROPERTIES OF THE GEMINI SURFACTANTS The interaction of gemini surfactants with living cells is of a significant importance for pharmaceutical and biotechnological applications. Due to their amphiphilic nature and aggregation properties, gemini surfactants can perturb cellular components. These effects can be advantageous, leading to enhanced drug delivery into specific tissue and cells, but can also trigger toxic effect. A major impact can be noticed at the level of external and internal membranes, involving membrane disruption and temporary membrane denaturation. These processes can induce metabolic inhibition, alter homeostasis, and ultimately

13.4 Biologically Relevant Properties of the Gemini Surfactants

cause cell death (Sharma and Ilies, 2014). The mass transfer between the host bilayer and the foreign amphiphile-based assembly can occur immediately or it can be delayed via processes such as endocytosis (Almeida et al., 2011). The concentration of gemini surfactant, temperature, nature of the formulation, and pH are several important factors defining this interaction and its outcome. Due to the membrane destabilization properties, gemini surfactants are used as antimicrobials and biocides. In this regard, cationic surfactants based on lysine were synthesized and evaluated for their antimicrobial and hemolytic activities. The CMC was dependent on the hydrophobic character of the molecules. Nevertheless, the antimicrobial and hemolytic activities were related to the structure of the compounds as well as the type of cationic charges. The most active surfactants against the bacteria were those with a cationic charge on the trimethylated amino group, whereas all tested surfactants showed low hemolytic character. Thereafter, gemini surfactants were adequate for applications that need nontoxic cationic active surfactants that aggregate at very low concentrations and have antimicrobial properties (Colomer et al., 2011). It must be emphasized that the route of administration also governs the observed cytotoxic effect of gemini surfactants. For example, when administered orally, cationic surfactants are poorly absorbed from the intestinal tract, thus exhibiting low cytotoxicity (Almgren, 2000). Their toxicity increases 10
100 times when administered intravenously, due to the interaction with blood components such as blood cells, albumin, etc. Topical administration of positively charged surfactants alter the skin permeability, water-binding capacity, and can cause denaturation of proteins in the stratum corneum (Almeida et al., 2011). Consequently, gemini surfactants were proposed as skin permeation enhancers, and structure-activity relationship studies have been conducted to quantify their benefits and unwanted effects, such as irritation and long-term skin damage. In this regard, the structure-activity relationship of alkylammonium gemini surfactants (C12-m-C12, where m 5 2, 6, and 10) as dermal permeation enhancers for three model drugs, namely lidocaine HCl, caffeine, and ketoprofen has been studied (Silva et al., 2013). In vitro permeation studies across dermatomed porcine skin were performed and the maximal enhancing effect was achieved with the most hydrophilic and charged drug (lidocaine HCl). In this case, the gemini surfactant with the intermediate spacer length was the most effective (12-6-12). In contrast, for caffeine and ketoprofen, the gemini surfactant with the shortest spacer (12-2-12) was the most effective permeation enhancer. This suggested that this class of molecules promotes both stratum corneum perturbation and drug transport through the lipid barrier. It was also found that these compounds possess similar cytotoxic profiles to that of azone (1-dodecylazacycloheptan-2-one or laurocapram) and their direct application on the skin did not cause significant morphological changes. Therefore, they are promising candidates for permeation enhancers, especially in the case of hydrophilic ionized compounds that do not easily cross the stratum corneum (Silva et al., 2013).

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The biodegradability and aquatic toxicity of a series of C12 quaternary ammonium-based gemini surfactants have been studied. It was found that gemini surfactants are not biodegradable by the CO2 headspace test, a standard method for biodegradability assessment (OECD). The failure in the biodegradation tests can be attributed to the toxic effects of these compounds towards the aerobic microorganisms responsible for biodegradation, under the standard test conditions. The results of aquatic toxicity (Daphnia magna test) (OECD) revealed that gemini surfactants are less toxic to aquatic life than the monomeric ones. Furthermore, the aquatic toxicity can be reduced by increasing the hydrophilicity of the surfactant molecule, by including a heteroatom in the spacer or replacing a methyl by a hydroxyethyl group in the ammonium polar head group (Garcia et al., 2016). Environmental concerns and biodegradability issues have influenced the design of biocompatible surfactant molecules having functional groups that are biodegradable.

13.4.1 GEMINI SURFACTANTS WITH CLEAVABLE SPACER GROUPS Since cytotoxicity and environmental concerns limited the practical usage of gemini surfactants, it was necessary to develop surfactants which are biodegradable, eco-friendly, and biocompatible in nature. For instance, a series of quaternary ammonium gemini surfactants having different ethylene oxide (EO) units as spacer linked with ester functionality have been synthesized and characterized. Self-aggregation and thermodynamic properties of gemini surfactants have been investigated with respect to the effect of increasing EO spacer units. The CMC increased with the increase in EO spacer units. Micellar solutions of these surfactants demonstrated greater ability to solubilize a large amount of nonionic amphiphile monolaurin. A rheology study showed that the length of the polar EO spacer units affected the viscosity of gemini surfactants solutions. The maximum viscosity was achieved with gemini surfactant having single EO unit spacer and the viscosity decreased upon the increase in EO spacer units (Bhadani et al., 2014). In another study, an homolog of carboxylate gemini surfactants with an azobenzene spacer and different lengths of the alkyl tails, referred to as Cm(azo) Cm, has been synthesized. All the surfactants formed worm-like micelles at relatively low concentration. This was attributed to the long and rigid characteristic of the azobenzene spacer, which yielded the pseudo volume between the two tails and, accordingly, a columnar-like molecular geometry favorable for the formation of worm-like micelles. The results of rheology and freeze-fractured TEM (FF-TEM) showed that the length of the alkyl tails strongly affected the viscoelastic properties of the worm-like micelle solution. With the increase of the alkyl tail length, the solution changed gradually to a gel-like state (Song et al., 2010). Cationic gemini surfactants that have ester bonds between the hydrophobic tail and the cationic moiety have been synthesized, where the ester bonds were either with the ester carbonyl group away from the positive charge (esterquat-type

13.4 Biologically Relevant Properties of the Gemini Surfactants

arrangement) or facing the positive charge (betaine ester-type arrangement) (Tehrani-Bagha et al., 2007). The chemical hydrolysis of these surfactants was investigated and compared with the hydrolysis of the corresponding monomeric surfactants. The betaine ester type of surfactants was found to hydrolyze faster than the esterquat surfactants. It was also seen that, above the CMC, the gemini surfactants were much more susceptible to alkaline hydrolysis than the corresponding monomeric surfactants. The biodegradation of the gemini surfactant and the monomeric surfactants were also studied And it was found that the monomeric surfactants were rapidly degraded, whereas the two gemini surfactants were more resistant to biodegradation and could not be classified as readily biodegradable (Tehrani-Bagha et al., 2007). This observation might impair the applications of these surfactants in the medical field. In the same context, the aggregation behavior of ester-containing gemini surfactants, dodecyl esterquat, and dodecyl betainate gemini was investigated using tensiometry, conductometry, viscometry, DLS, TEM, and optical microscopy techniques in the absence and presence of NaBr electrolyte. The ester-containing gemini surfactants formed spherical aggregates at low concentration (1.1%wt). At higher concentration (B3.7%wt), the morphology was different, depending on the position of ester bond in alkyl chain and the spacer length. Dodecyl betainate gemini surfactant with short spacer (s 5 2) formed gel because of the formation of worm-like micelles in the aqueous solution. Dodecyl betainate gemini surfactant (s 5 3) formed large vesicles enclosing smaller ones, and dodecyl esterquat gemini surfactant (s 5 3) formed both cylindrical and spherical micelles. The salt addition induced the growth of micelles and, in the case of dodecyl betainate gemini surfactant (s 5 2), changed the morphology from worm-like micelles to lamellar phase (Javadian et al., 2013). Moreover, cationic gemini surfactant with dodecyl tails and a spacer that contains an ester bond has been studied for its chemical hydrolysis, biodegradation, and toxicity. Due to the proximity to the two quaternary ammonium groups, the ester bond was very stable on the acid side and very labile at slightly alkaline conditions. The hydrolysis products were two single-chain surfactants, which were less surface active than the intact gemini surfactant. These surfactants were found to be readily biodegradable, namely more than 60% biodegradation after 28 days. The gemini surfactant was found to be toxic to aquatic organisms (ErC50 value of 0.27 mg/L), although less toxic than the two hydrolysis products (Tehrani-Bagha et al., 2015). Mixed micelles formed of biodegradable dicationic ester based gemini surfactants with bile salts showed that the interaction of two bile salts (sodium cholate and sodium deoxycholate) with diester bonded gemini surfactants (m-E2-m) led to a decrease in the CMC values by increasing the mole fraction of gemini surfactant. Values of the interaction parameter came out to be negative at all molar ratios of the mixtures, indicating synergistic interactions. The sodium cholate generated stronger synergism with m-E2-m gemini surfactants, in comparison to sodium deoxycholate, which has been explained by the formation of more

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hydrogen bonds between gemini surfactants and sodium cholate, compared to sodium deoxycholate (Akram et al., 2014). Among the various degradable linkages, reducible disulfide bonds have been employed in the design of redox-responsive drug delivery systems for sitespecific drug release, since the concentration of glutathione (GSH), a thiolcontaining tripeptide produced in the cytoplasm, is known to be considerably higher in the cytosol and around some tumors than in plasma (Sun et al., 2006). Gemini surfactants with disulfide spacer have been found to undergo significant changes in their CMC and aggregation behaviors when degraded into monomeric surfactants in a reductive environment. In this regard, thiol-responsive micelles consisting of nonionic gemini surfactants with a cystine disulfide spacer, (C18-Cys-mPEG)2 and ((C18)2-Lys-Cys-mPEG)2, were developed and characterized (Kim et al., 2014). Both surfactants formed micelles with average diameters of 13 and 22 nm above the CMC. The micelles of ((C18)2-Lys-Cys-mPEG)2, containing more stearoyl groups, encapsulated more hydrophobic indomethacin with higher entrapment efficiency than those of (C18-Cys-mPEG)2. The micelles exhibited an accelerated release of encapsulated indomethacin with the increase in concentration of the reducing agent (GSH) whereas they were unaffected by the presence of reduced glutathione (GSSG). The 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)22-(4-sulfophenyl) 22H-tetrazolium (MTT) studies revealed the noncytotoxic nature of the these gemini surfactants. These results suggested the potential of disulfide-based gemini surfactants micelles as efficient drug delivery vehicles (Kim et al., 2014). In another study, thiol-responsive gemini surfactant micelles consisting of hydrophilic poly(ethylene glycol) and hydrophobic poly(lactide PLA) blocks with a cystine disulfide spacer were reported as effective intracellular nanocarriers for drugs. In the presence of cellular GSH as a reducing agent, gemini surfactant micelles gradually destabilize into monomeric micelles through cleavage of the cystine linkage. This destabilization of the gemini surfactant micelles changed their size distribution, with the appearance of small aggregates, and led to the enhanced release of encapsulated doxorubicin. The results of cellular uptake study, as well as cell viability measurements for anticancer efficacy, suggested the potential of disulfide-based gemini surfactant micelles as controlled drug delivery carriers (Kim et al., 2015).

13.4.2 GEMINI SURFACTANTS WITH HETEROCYCLIC HEAD GROUPS With the aim of increasing the biodegradability, a new category of gemini surfactants consisting of two positively charged heterocyclic headgroups attached to hydrophobic alkyl tail connected by a spacer, have demonstrated their effectiveness for drug and gene delivery. Among the different heterocyclic gemini surfactants, pyridinium (Chauhan et al., 2014), imidazolium (Ren et al., 2015), and pyrrolidinium gemini surfactants (Zou et al., 2015) have been widely investigated for their physicochemical properties. For instance, Sharma et al. investigated the

13.4 Biologically Relevant Properties of the Gemini Surfactants

use of pyridinium gemini surfactant in mixture with pyridinium lipid, both synthesized and characterized previously (Sharma et al., 2013), to combine the membrane-destabilization properties of the gemini surfactant, its high chargemass ratio and transfection properties with the good transfection and cytotoxic profile of pyridinium lipid-based formulations. The transfection efficiency of lipoplexes derived from cationic gemini surfactants and lipids was several times superior to that of lipoplexes formed of pyridinium lipid alone. The cationic gemini surfactant/lipid blends had a higher charge density than the corresponding pure cationic lipid-based formulations, while displaying reduced cytotoxicity. Consequently this synergistically combined the properties of the two classes of cationic amphiphiles for overcoming some of the intracellular delivery barriers (such as endosomal escape) against gene delivery (Sharma et al., 2014). The compaction and condensation of DNA, induced by cationic imidazolium gemini surfactants ([Cn-4-Cnim]Br2, n 5 10, 12, 14) at different charge ratios, has been thoroughly investigated (Zhou et al., 2012). Cationic imidazolium gemini surfactants can interact with DNA via attractive electrostatic interaction, strong hydrophobic forces, and π
π interaction. Upon the addition of imidazolium gemini surfactants, DNA molecules undergo the process of compaction to multimolecular condensation, accompanied by conformation change. The impact of the electrostatic interaction was elucidated from the charge density and charge polarity changes as noticed from zeta potential measurements. The stronger interaction between DNA and imidazolium gemini surfactants with longer tails indicated that hydrophobic interactions also played an important role in binding of [Cn-4-Cnim] Br2 to DNA. The π-π interaction between imidazolium groups of gemini surfactants and DNA also contributed in lipoplex formation, as elucidated from the EtBr exclusion assay. The demonstrated process for DNA compaction and multimolecular condensation could be beneficial for the application of gemini surfactants as gene delivery agents (Zhou et al., 2012). Biological and thermodynamic properties of a homologous series of highly fluorinated bis-pyridinium cationic gemini surfactants, differing in the length of the spacer bridging the pyridinium polar heads, were investigated. Gene delivery ability was dependent on the spacer length, because structural changes of gemini molecules occur when the spacer reaches certain length, proved by the trends of the apparent and partial molar enthalpies versus molality. All tested compounds (except that with the longest spacer), at different levels, could deliver DNA plasmid when co-formulated with DOPE. The compound with a spacer formed of eight carbon atoms gave rise to a gene delivery ability that was comparable to that of the commercial reagent. The compounds with the longest spacer compacted DNA in loosely condensed structures, which were not efficient for transfection (Fisicaro et al., 2017). Ester functionalized gemini piperidinium surfactants with different spacer and hydrophobic alkyl (i.e., dodecyl and tetredecyl) chains were synthesized, characterized and investigated for their dilute solution properties. The investigation of gemini piperidinium surfactants by SAXS technique revealed the ability of these

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Shell region of micelle consisting of hydrophilic headgroups and counterions

Core region of micelle consisting of hydrophobic tail

Counterions

rc rc+s

Headgroups

rc rc+s

Shell of micelle

Dmax

Dmax

(A)

(B)

FIGURE 13.1 (A) Model structure of core-shell type micelle. (B) Cross-sectional view of core-shell type micelle structure.

surfactants to form core-shell type micelles in aqueous solution (Fig. 13.1). The size and internal structure of the micelles depended on the methylene spacer units, as well as the hydrophobic alkyl chain length of gemini surfactants. Gemini surfactants containing tetradecyl alkyl chain demonstrated the ability to form premicellar aggregate at lower concentration, below their CMC values (Bhadani et al., 2016). These surfactants contain ester moiety as a part of the spacer, which is a biocompatible functional group, and the ease of synthesis combined with excellent surfactant properties makes these surfactants ideal for several medical and industrial applications. The effect of alkyl chain length on cytotoxicity and antimicrobial activity of a series of biocompatible ester-linked cationic gemini surfactants, ethane-1,2-diyl bis(N,N-dimethyl-N-alkylammoniumacetoxy) dichlorides (m-E2-m, m 5 12, 14, 16) was examined. Antimicrobial activity of m-E2-m, against various prokaryotic and eukaryotic microorganisms, was studied by measuring the diameter of inhibition zone, whereas cytotoxicity was evaluated using 3T3-L1 fibroblast cells. Cytotoxicity against these cells depended upon the type of the target microorganisms, nature of the cells, and hydrophobicity of the molecules. Antimicrobial activity of the gemini surfactant 16-E2-16 was lower than that of its corresponding single-chain counterpart. The gemini surfactants used for the present study (m-E2-m), which have excellent surface properties and much lower CMC values, showed low toxicity and significant antimicrobial activity (Fatma et al., 2016).

13.4.3 CYCLODEXTRIN-GEMINI SURFACTANTS A cyclodextrin-based gemini surfactant drug delivery system was introduced with the aim of improving the delivery of poorly water soluble drugs through a variety

13.4 Biologically Relevant Properties of the Gemini Surfactants

of routes of administration, including parenteral and topical application (Badea et al., 2015). ß-Cyclodextrin (CD) is a natural product with low toxicity. It has an ability to enhance the delivery of insoluble hydrophobic drugs through biological barriers (Yallapu et al., 2010). Cyclodextrin is a ring structure containing seven D-(1) glucopyranose units attached by a-(1,4) glucosidic bonds, which creates a relatively lipophilic inner cavity and hydrophilic outer surface. Hydrophobic drugs form noncovalent inclusion complexes, with the interior cavity of the CD in an aqueous environment. Based on the potential of the CD to incorporate insoluble agents, a delivery system comprising of CD attached to gemini surfactants was developed for enhanced drug absorption. A cyclodextrin-gemini surfactant (CD-gemini)-based delivery system to incorporate poorly soluble curcumin analog (NC 2067) for the treatment of melanoma was designed and evaluated (Michel et al., 2012) as a noninvasive therapy for in-transit melanoma metastasis that lacks adequate treatment to date (Fig. 13.2). The NC 2067-CDgemini formulation was highly efficient in inhibiting the growth of melanoma cells, with IC50 values significantly lower than melphalan, the drug currently used for the treatment of in-transit melanoma. CDgemini formulations showed excellent cellular selectivity, triggering apoptosis in the A375 cell line while showing no cytotoxicity to healthy human epidermal keratinocytes. The interaction between NC 2067 with CD or CDgemini was physicochemically characterized (Poorghorban et al., 2015a). Synchrotron-based small-and wide-angle X-ray scattering and size measurements were employed to assess the supramolecular morphology of the complex formed by curcumin analogue with CDgemini. Physical mixtures of NC 2067 and CD or CDgemini showed characteristics of the individual components, whereas the complex of NC 2067 and CD or CDgemini surfactant presented new structural features, supporting the formation of the host/guest complexes. Complexes of NC 2067 with CDgemini formed nanoparticles having sizes of 100
200 nm. NC 2067 retained its anticancer activity toward A375 melanoma cells in the complex with CDgemini for different drug-to-carrier mole ratios, with an IC50 value comparable to that for NC 2067 without the carrier (Poorghorban et al., 2015a). Therefore, CDgemini showed good potential to be used as a delivery system for poorly soluble anticancer agents. The host/guest complexes composed of native CD or CD-gemini with NC 2067 were examined using 1D/2D rotational Overhauser effect spectroscopy (ROESY) NMR methods. The results showed that the hydrocarbon domain of the gemini surfactant was self-included within the CD internal cavity. The stoichiometry of the CD/NC 2067 system was estimated to be a 2:1 complex, according to the continuous variation method (Job plot). 1D/2D ROESY spectra and molecular modeling indicated that NC 2067 was included by two CD molecules through its styryl and/or benzoyl groups. In contrast, the addition of NC 2067 to CD-gemini resulted in only slight measurable changes to the system, and that supported the probability of CD-gemini self-inclusion. The drug (NC 2067) was weakly bound to the CDgemini system and likely formed a ternary outer-sphere complex (Poorghorban et al., 2015b).

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FIGURE 13.2 Schematic representation of the assembly of gemini nanoparticles with poorly soluble curcumin analog drug designed for the treatment of melanoma.

CDgemini delivery system was used to encapsulate melphalan, requiring a solvent system for solubilization leading to poor chemical stability, in order to improve its physicochemical and biological behavior. Melphalan-CDgemini nanoparticles showed optimal particle size for endocytosis, in the range of 200
250 nm and induced significantly higher cell death, compared to melphalan (IC50 is 36 μM for the nanoparticles vs 82 μM for melphalan) in melphalanresistant melanoma cells. The CDgemini delivery system did not alter the pathway

13.4 Biologically Relevant Properties of the Gemini Surfactants

of the cellular death triggered by melphalan and caused no intrinsic toxicity to the cells (Michel et al., 2016). These findings demonstrated, in principle, the applicability of a CDgemini delivery system as safe and efficient alternative to the current melanoma therapy, especially in chemoresistant cases.

13.4.4 AMINO ACID
BASED GEMINI SURFACTANTS With the aim to develop biocompatible surfactants with multifunctional properties, a considerable number of gemini surfactants have been synthesized with two polar heads (i.e., two amino acids) and two hydrophobic tails per molecule, separated by a covalently bound spacer structure of different ionic character and polarity (Pe´rez et al., 2014). The introduction of amino acids into the structure of the new surfactant molecules resulted in remarkable biocompatible properties and a large variety of chemical functionalities, resulting in new surfactants which are water soluble, nontoxic if orally administered, nonirritating, biodegradable, and with a minimal aquatic impact. All these properties have motivated the commercial development of these surfactants in food and cosmetic sectors, and highlighted their potential for biomedical applications (Pe´rez et al., 2005). In this regard, serine-derived gemini surfactants, varying in alkyl chain length and in the linker group, bridging the spacer to the head groups (amine, amide and ester), were evaluated for their ability to mediate gene delivery, either alone or in combination with helper lipids (Cardoso et al., 2015b). The most efficient complexes were those prepared with C12 surfactants and helper lipids, reaching 50% of transfected HeLa cells without causing cytotoxicity at a very low surfactant:DNA charge ratios (1:1 to 2:1). The most efficient complexes presented particle size suitable for intravenous administration and negative surface charge. The negative surface charge increases the likelihood of reaching the desired target cells and mediating transfection, even in the presence of serum. The most efficient and least cytotoxic complexes were produced from the biodegradable ester derivative, which proved that cleavable chemical bonds would facilitate the release of DNA inside the cells (Cardoso et al., 2015b). Two different families of cationic gemini surfactants (bis-quat conventional and serine-derived) were tested regarding their efficiency to deliver small interfering RNAs (siRNAs) in a human glioblastoma cell line (U87), in order to select an effective siRNA antisurvivin carrier. Cationic gemini surfactants were capable of efficiently delivering siRNA into the cell cytoplasm, even in the presence of serum. Gemini surfactant-based complexes efficiently suppressed the expression of the antiapoptotic protein, survivin, which resulted in a synergistic effect in terms of cytotoxicity induced by the chemotherapeutic drugs, temozolomide or etoposide (Cruz et al., 2016). These findings revealed the potential to improve the therapeutic efficacy of anticancer drugs and to allow the reduction in chemotherapeutic doses currently used to treat glioblastoma, consequently minimizing the side effects associated with cancer treatment.

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Gene delivery-targeting mitochondria has the potential to transform the therapeutic field of mitochondrial genetic diseases. A plasmid DNA construct, able to be specifically expressed in these organelles, was designed by including a codon, which codes for an amino acid, only if read by the mitochondrial ribosomes. Gemini surfactants, conventional bis-quat gemini surfactant 14 2 2 2 14 and serine-derived bis-quat gemini surfactants (nSer)2N5 (n 5 14 and 16), were shown to successfully deliver plasmid DNA to mitochondria. Gemini surfactant-based DNA complexes were taken up by cells through a variety of routes, including endocytic pathways, and showed a propensity for inducing membrane destabilization under acidic conditions, thus facilitating cytoplasmic release of DNA. Furthermore, the complexes interacted extensively with lipid membrane models, mimicking the composition of the mitochondrial membrane, which predicted a favored interaction of the complexes with mitochondria in the intracellular environment (Cardoso et al., 2015a). Due to the membrane destabilizing effect of gemini surfactants, they were proposed as absorption enhancers for small hydrophilic molecules and macromolecules such as peptides, which are poorly absorbed from the gastrointestinal tract. In this regard, the biocompatible amino acid
based gemini surfactant, dilauramidoglutamide lysine (SLG-30), was examined as an intestinal absorption enhancer for 5(6)-carboxyfluorescein and fluorescein isothiocyanate-dextrans. The intestinal absorption of both drugs was significantly enhanced by SL-30, as found by the in situ closed-loop method in rats. Furthermore, the calcium levels in plasma significantly decreased when calcitonin was coadministered with SLG-30, indicating the increased intestinal absorption of the model peptide, calcitonin. In addition, no significant increase in lactate dehydrogenase activity or in protein release from the intestinal epithelium was observed in the presence of SLG-30, suggesting the safety of this compound (Alama et al., 2016). These findings indicated that biocompatible gemini surfactants are promising absorption-enhancers for improving the intestinal absorption of poorly absorbed drugs, without causing serious damage to the intestinal epithelium. The role of aggregate size in the hemolytic and antimicrobial activity of colloidal solutions, based on single and gemini surfactants derived from arginine has been elucidated (Tavano et al., 2013). The physicochemical properties of colloidal systems composed of arginine-based surfactants (single or gemini structures), with or without additive compounds such as dilauroylphosphatidylcholine (DLPC) or cholesterol, have been characterized. The monocaternary surfactant and the gemini surfactant with the shortest spacer chain (6 carbons) formed micelles, while aqueous solutions of pure gemini surfactants with longer spacers (9 and 12 carbons) formed large aggregates. The addition of phospholipids or cholesterol changed the aggregation behavior of these surfactants. In the case of single-chain surfactants and gemini surfactant with C6 spacer, the incorporation of additives gave rise to the formation of cationic vesicles. For gemini surfactants with a longer spacer, this type of additive promoted the formation of smaller

13.4 Biologically Relevant Properties of the Gemini Surfactants

aggregates. The capability of disrupting erythrocyte membranes, as well as the antimicrobial activity, depended on the hydrophobicity of the molecules and the size of aggregates in the solution. For instance, gemini surfactants with short spacer chains were more hemolytic than their single-chain homolog, while gemini surfactants with long spacers were much less hemolytic than their single-chain counterpart (Tavano et al., 2013). Consequently, the physicochemical and biological characterization might be important for the potential pharmaceutical applications of these systems.

13.4.5 SUGAR-BASED GEMINI SURFACTANTS The development of sugar-based nonionic gemini alkyl glycoside surfactants is attractive in terms of sustainability and environmental safety. Alkyl O-glycosides are nonionic surfactants prepared from long-chain alcohols and carbohydrates (Balzer and Luders, 2000). Their excellent biodegradability, nontoxicity, and availability from renewable resources render them more attractive than other nonionic surfactants for pharmaceutical applications. A novel type of nonionic gemini alkyl O-glucoside surfactant has been designed and evaluated (Liu et al., 2013). The gemini glucosides have been readily prepared by glycosylation of the gemini alkyl chains that are synthesized from regioselective ring-opening of ethylene glycol epoxides by the alkyl alcohols. The new gemini alkyl glucosides exhibited significantly better surface activity than the known class of alkyl glucosides and interesting self-assembly behavior. These molecules have great potential as nanocarriers in drug and gene delivery. In another study, the interaction of a series of cationic gemini surfactants with a sugar-based surfactant, octyl-β-D-glucopyranoside (β-C8G), in the presence of 5 mmol/dm3 NaBr at 30 C has been investigated (Siddiqui et al., 2013). The CMCs of pure surfactants, as well as mixed systems, were determined. The interaction parameters, minimum surface area per molecule, surface excess, mixed micelle composition, free energies of micellization, and activity coefficients were determined. The results illustrated the effects of steric and electrical factors on mixed micelle formation and surfactant-surfactant interactions. Viscometric studies showed that the morphological behavior of the mixed micelles was dictated by the length of the spacer and alkyl chain of gemini surfactants. Short spacers and long alkyl chain gemini surfactants were found to have stronger ability to form larger assemblies (Siddiqui et al., 2013). The studied biodegradable sugar-based and gemini surfactant mixtures are promising options to develop environmental friendly surfactant systems with low intrinsic toxicity. The interaction of gemini surfactants with plasma protein may have an important impact on the applications of such compounds in the biomedical field. In this regard, the interaction of imidazole-based gemini surfactants with bovine serum albumin (BSA) was studied using synchrotron radiation scattering (SR-SAXS), CD, and NMR (Gospodarczyk et al., 2014). It was found that the binding of

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gemini surfactants with BSA decreased its overall negative charge, therefore, the steric stabilization was weakened due to weaker repulsion forces. This resulted in BSA aggregation and increased gyration and hydrodynamic radii without a pronounced change in α-helix content. During initial aggregation, BSA was preserved at its native normal form at pH 7.7. Then, for higher surfactant concentrations, the interaction of α-helices with surfactants induced the unfolding and a dramatic decrease of α-helix content. In the same context, a cationic amino acid
based gemini surfactant derived from cysteine, (C12Cys)2, has been synthesized and its supramolecular behavior and its interaction with BSA have been characterized (Branco et al., 2015). Surface tension measurements were used to obtain important system parameters, such as CMC, critical aggregation concentration, protein saturation point, maximum surface excess concentration, minimum surface area per molecule at the air-solution interface, and the degree of surfactant binding to protein. Formation of a protein/surfactant complex was confirmed by UV-vis and fluorescence spectroscopy. The strength of the interaction was relatively weak, as revealed by the association constant obtained from fluorescence quenching data, which is in the order of 104 L/mol. The conformational changes observed in the protein by fluorescence spectroscopy suggested unfolding, with increasing surfactant concentrations up to saturation of the protein backbone at a surfactant concentration of 0.70 g/L, after which micelle formation occurs. Fluorescence quenching measurements allowed determination of surfactantprotein binding constant and the number of binding sites (Lakowicz, 2013). The interaction of gemini surfactants with plasma proteins may put some restrictions on the pharmaceutical applications of such unique molecules.

13.5 IN VITRO/IN VIVO CORRELATION Several animal models have been used to test the efficiency of gemini surfactantbased systems in vivo. In this regard, the effect of topical IFNγ gene therapy using gemini nanoparticles on pathophysiological markers of cutaneous scleroderma was tested in Tsk/ 1 mice. Cutaneous IFNγ gene delivery, using a series of gemini surfactants (spacer length n 5 2
16;alkyl chain m 5 12 or 16)/DNA complexes, was investigated (Badea et al., 2005). The complexes were formulated and characterized using CD and AFM to select gemini analogues with the highest transfection efficiency. Transfection and cellular expression of IFNγ from the bicistronic pGTmCMV IFN-GFP plasmid were evaluated in PAM212 keratinocyte culture by ELISA and fluorescence microscopy. Topical delivery of plasmid using liposomal and nanoemulsion systems, based on gemini surfactant 16-3-16, was evaluated in mice by IFNγ expression analysis. The in vitro transfection efficiency was found to be dependent on the spacer length of the gemini surfactant, with the C3 spacer showing the highest activity (both 12-3-12 and 16-3-16). Both

13.5 In Vitro/In Vivo Correlation

gemini cationic liposomes and gemini nanoemulsion produced significantly higher levels of IFNγ in the skin and lymph nodes, compared to naked DNA or a liposomal Dc-chol (cholesteryl 3β-(N-[dimethylaminoethyl]carbamate) formulation (Badea et al., 2005). Furthermore, the topical noninvasive gene delivery using gemini surfactant nanoparticles in IFNγ-deficient mice was conducted. Nanoparticles based on the gemini surfactant 16-3-16 (NP16-DNA) and Dc-chol (NPDc-DNA) were prepared and characterized. CD studies showed that the gemini surfactant compacted the plasmid more efficiently, compared to the Dc-chol. Structural polymorphism was observed in NP16-DNA with lamellar and Fd3m cubic phases present, whereas, for NPDc-DNA, two lamellar phases could be distinguished. In vivo, both topically applied nanoparticles induced higher gene expression, compared to untreated control and naked DNA. However, treatment with NPDc-DNA caused irritation and skin damage, whereas NP16-DNA showed no skin toxicity. Therefore, it was demonstrated that topical cutaneous gene delivery using gemini surfactant-based nanoparticles in IFNγ-deficient mice was safe and may provide increased gene expression in the skin, due to structural complexity of gemini surfactant-based nanoparticles (lamellar
cubic phases) (Badea et al., 2007). Later on, the effect of topical IFNγ gene therapy using gemini nanoparticles on pathophysiological markers of cutaneous scleroderma in Tsk/ 1 mice was assessed. The intradermally injected and topical noninvasive treatment with IFNγ-coding plasmid-nanoparticle decreased collagen synthesis in the Tsk/ 1 (tight-skin scleroderma) mouse model and reduced the clinical presentation of the disease. Topical administration of the IFNγ-coding plasmid nanoparticles was effective in expressing IFNγ levels after a 20-day treatment regimen, without increase in toxicity (Badea et al., 2012). These results demonstrated efficient in vivo transfection using a gemini surfactant-based delivery system able to modulate excessive collagen synthesis in scleroderma-affected skin. The in vitro and in vivo transfection capacity of pH-sensitive, sugar-based gemini surfactants was investigated by Wasungu et al. In an aqueous environment at physiological pH, these compounds formed bilayer vesicles, but underwent a lamellar-to-micellar phase transition in the endosomal pH range as a consequence of an increased protonation state. Because of their lamellar organization, these lipoplexes exhibited good colloidal stability in salt and in serum at physiological pH, compatible with prolonged stability in vivo. When injected intravenously in mice, these lipoplexes did not substantially accumulate, based on the observation that transfection in the lungs was not detectable as observed by in vivo bioluminescence (Wasungu et al., 2006). The potential of avoidance of the “preliminary capture” in the lungs may be utilized in developing clinically feasible gemini lipoplexes. Gemini surfactant
phospholipid nanoparticles were developed and characterized with the goal of establishing a gene delivery system as a noninvasive therapy for glaucoma (Fig. 13.3).

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FIGURE 13.3 Assembly and characterization of gemini nanoparticles for ocular administration. (A) The process of assembly of DOPE and DOPE/DPPC based gemini NPs. (B) Electron micrographs of the 12-7NH-12 NPs assembled with DOPE and DOPE/DPPC co-lipids.

The ocular biodistribution of gemini surfactant
DNA nanoparticles after intravitreal and topical administration was studied. Optimized nanoparticles (size range 150
180 nm) were biocompatible with rat retinal ganglion (RGC-5) cells, with N95% viability by PrestoBlue assay. After intravitreal injection in mice, the nanoparticles localized within the nerve fiber layer of the retina, whereas after topical application, nanoparticles were located in several anterior chamber tissues, including the limbus, iris, and conjunctiva (Fig. 13.4). Gemini surfactant nanoparticles were thermodynamically stable in the vitreous and tear fluid and were trafficked as single, nonaggregated particles after both types of administration (Alqawlaq et al., 2014). Amino acid
substituted gemini surfactant-based mucoadhesive gene delivery systems for potential use as noninvasive vaginal genetic vaccination has been developed. Poloxamer in diethylene glycol monoethyl ether aqueous solution produced dispersions that gelled near body temperature and had a high yield value, preventing leakage of the formulation from the vaginal cavity. Intravaginal administration in rabbits showed that the glycyl-lysinesubstituted gemini surfactant led to higher gene expression, compared to the parent unsubstituted gemini surfactant (Singh et al., 2015). This provided a proof-of-concept that amino acid
substituted gemini surfactants can be used as noninvasive mucosal (vaginal) gene delivery systems to treat diseases associated with mucosal epithelia.

13.6 Conclusions

FIGURE 13.4 Biodistribution pattern of plasmid gemini lipid DOPE-N NPs 4 h after topical application. Animals were treated with double-labeled NBD-PE (green) lipid and Cy5 (red) labeled pDNA 10:1 ρ 6 plasmid gemini lipid nanoparticles (n 5 4). Cy5-pDNA DOPE-N (red) was localized around the conjunctiva, the limbus and the iris (I, III), while absent from the cornea and retina (II, IV). These tissue samples were stained using WGA Alexa Fluor 488 conjugate (green).

13.6 CONCLUSIONS • • •

Gemini surfactants are promising drug/gene delivery agents because of their structural versatility demonstrated by numerous studies; Current efforts are in increasing safety by reducing toxicity and environmental impact; In vitro/in vivo characterizations and correlations are limited, so more work is needed in this area to improve rational design of gemini surfactant-based delivery systems.

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FURTHER READING OECD Test No. 211: Daphnia magna Reproduction Test. OECD Publishing. OECD Test No. 310: Ready Biodegradability—CO2 in Sealed Vessels (Headspace Test). OECD Publishing.