Lipoaminoacids Enzyme-Based Production and Application as Gene Delivery Vectors

Abstract

Biosurfactant compounds have been studied in many applications, including biomedical, food, cosmetic, agriculture, and bioremediation areas, mainly due to their low toxicity, high biodegradability, and multifunctionality. Among biosurfactants, the lipoplexes of lipoaminoacids play a key role in medical and pharmaceutical fields. Lipoaminoacids (LAAs) are amino acid-based surfactants that are obtained from the condensation reaction of natural origin amino acids with fatty acids or fatty acid derivatives. LAA can be produced by biocatalysis as an alternative to chemical synthesis and thus become very attractive from both the biomedical and the environmental perspectives. Gemini LAAs, which are made of two hydrophobic chains and two amino acid head groups per molecule and linked by a spacer at the level of the amino acid residues, are promising candidates as both drug and gene delivery and protein disassembly agents. Gemini LAA usually show lower critical micelle concentration, interact more efficiently with proteins, and are better solubilising agents for hydrophobic drugs when compared to their monomeric counterparts due to their dimeric structure. A clinically relevant human gene therapy vector must overcome or avoid detect and silence foreign or misplaced DNA whilst delivering sustained levels of therapeutic gene product. Many non-viral DNA vectors trigger these defence mechanisms, being subsequently destroyed or rendered silent. The development of safe and persistently expressing DNA vectors is a crucial prerequisite for a successful clinical application, and it one of the main strategic tasks of non-viral gene therapy research.

1. Introduction

Cationic lipids or cationic surfactants are amphiphilic molecules that contain a positively charged hydrophilic moiety (headgroup) linked to a hydrophobic domain, usually one or two long alkyl chains (tails) or a sterol motif.

Lipoaminoacids (LAAs) are amino acid-based surfactants obtained from the condensation reaction of natural amino acids with fatty acids or fatty acid derivatives that meet the requirements of both biological and ecological compatibility. Besides the detergent-like characteristics, recent studies have associated modifications on biosurfactants structures to the improvement of drug and gene delivery, highlighting the possible solutions for biologic therapies (e.g., gene therapy, immunotherapy). Many LAAs are known to have antimicrobial, antiviral [1] or antitumor activity, due to the ability to modulate the immune system or inhibit some enzymes and toxins [2,3]. LAAs offer the additional advantage of being amenable to production by biocatalysis in alternative to chemical synthesis and have, thus, become very attractive from both biomedical and the environmental perspectives.

Recently, cationic gemini surfactants have attracted special attention as efficient transfection agents in vitro and promising alternatives to viral vectors in gene therapy [4,5,6]. Successful gene therapy crucially depends on the development of effective vectors, especially for the safe introduction of the selected gene into living cells. Commonly used viral vectors present some drawbacks that include in some cases of residual infectivity and immunogenic and inflammatory responses [7,8]. Cationic lipids that bind and condense the negatively charged phosphate backbone of DNA into nanosized cationic lipid-DNA complexes (lipoplexes) are attractive synthetic alternatives to viral vectors due to their ease of preparation, higher loading capacity, lower cytotoxicity, and safer immunogenic profile [7,8]. Cationic LAAs are thus promising transfection agents that meet the requirements of physiological and ecological compatibility.

Nowadays, gene therapy has emerged as an effective approach in the treatment or prevention of genetic, acquired diseases, or as an alternative method to chemotherapy in cancer treatment [7,8]. Moreover, in some situations, there are several hurdles for overcoming in developing effective gene-based therapies, which include cellular uptake, endosomal escape avoiding DNA degradation, and nuclear localization (Scheme 1) [7,8,9,10].

This review focuses on several aspects that are related to the enzyme-based production of lipoaminoacids, their biological applications as gene delivery vectors, and future trends.

2. Cationic Lipoaminoacids

2.1. Structure and Characterization

Cationic gemini surfactants are dimeric structures that consist of two hydrophobic alkyl chains that are linked by a spacer chain to two cationic headgroups that have shown high transfection efficiency [7,8]. They are able to compact DNA into nanosized particles with positive surface charge, which enables interaction with the cell surface and endocytosis. Moreover, cationic gemini surfactants have several advantages as compared to their monomeric counterparts, such as lower cytotoxicity, lower CMC (critical micellar concentration), and polymorphic phase behavior [7,8,11].

The positive charge of cationic lipids is generally associated with amine groups with different degrees of substitution, such as primary, secondary, and tertiary amines, and quaternary ammonium salts, but other functions, like amidine, guanidine, pyridine, imidazole, and amino acids, have been used [12,13,14].

Lipoaminoacids, which are condensation products of amino acids with fatty acids or fatty acid derivatives (Figure 1), usually have good biocompatibility and biodegradability [15,16]. The polar headgroup of LAAs, which contains both donor and acceptor hydrogen bonding groups that are capable of intra- and intermolecular interactions, adds further complexity to their self-assembly behavior. LAAs can form a wide variety of self-assembled nanostrucures, from tubules and helical ribbons to micelles and vesicles [16,17,18]. On the other hand, the constrained cyclic pyrrolidine structure of proline (a β-sheet breaker) hinders intermolecular hydrogen bonding [19] and the bulkier headgroup of proline-based cationic LAAs usually leads to less efficient DNA binding [20]. Some cationic LAAs also show interesting biologic properties, such as in vitro antimicrobial and antibiofilm activities and promising anticancer activity (IC₅₀ 15.3–22.4 μmol L⁻¹) in several human cancer cell lines [21,22,23].

LAAs have also been studied as drug delivery agents for hydrophobic anticancer [24,25] and antimicrobial [26,27,28] drugs to enhance drug solubilization and bioavailability. LAAs improved the solubility of an investigational anticancer drug from <0.15 μg mL⁻¹ to 8.62 mg mL⁻¹, being more efficient than nonionic polymeric surfactant polysorbate 80 being used as a model solubilizer, which only achieved 3.16 mg mL⁻¹ drug solubilization [24]. Self-assembled vesicles (bolasomes) that were formed by asymmetric Lys-His bola-type LAAs were also shown to encapsulate doxorubicin with 97.5% encapsulation efficiency [18].

The micelles that were formed by a gemini LAA derived from cysteine, (C₈Cys)₂, under biomimetic conditions (phosphate buffered-saline (PBS), pH 7.4) were able to solubilize antifungal agent amphotericin B in its monomeric and less toxic form, which increased the molar solubilization ratio of the hydrophobic drug to 0.072 as compared to 0.036 in the commercial deoxycholate micellar formulation [26,27]. Cationic vesicles that formed by the lysine gemini LAA, C₆(LL)₂ (Figure 1) and cholesterol showed sustained and controlled release of methotrexate in vitro. A cumulative percentage release of 91% was achieved within 24 h when compared to 100% drug release within 6 h in free methotrexate control assay [25]. The cationic vesicles also behaved as percutaneous permeation enhancers for tetracycline in ex-vivo skin permeation assays, attaining 52% drug cumulative permeation after 24 h when compared to 36% for tetracycline solution [25].

The hemolytic activity of cationic LAAs is also lower than that of correspondent quaternary ammonium surfactants (e.g., HC₅₀ 24 μmol L⁻¹ for C₁₆His (Figure 1) vs. 45 μmol L⁻¹ for cetyltrimethylammonium bromide, CTAB), increasing with alkyl chain length due to stronger interactions between the hydrophobic chain and the lipid core of red blood cells (RBCs) [28]. Toxicity evaluation of LAAs in vitro showed improved biocompatibility when compared to marketed surfactants Triton X-100 and Tween 80 in hemolytic and cell culture assays, with LAAs leading to higher than 80% cell viability in the MTT assay performed in MCF-7 and HEK cell lines [29]. The results were corroborated in vivo according to aspartate transaminase (AST), alanine transaminase (ALT), blood urea nitrogen (BUN), and creatinine serum levels, as well as histological examination of spleen, liver, and kidney after intravenous LAA administration to mice (10 mg/kg body weight) [29].

2.2. Biocatalytic Production

In the context of green and sustainable chemistry, biocatalysis is an interesting alternative, due to the mild reaction conditions (e.g., room temperature and pressure, physiological pH), high activities, chemo-, regio-, and stereo-selectivities of catalysts, generally shorter reactions, with less energy consumption and lower waste generation than conventional organic synthesis [30].

LAAs that were obtained from naturally renewable sources could be produced by green-chemistry approaches, including biotechnological procedures, such as fermentation or enzymatic catalysis [31,32,33,34,35]. Protein hydrolysates from bioindustrial waste can also be used as a source of natural amino acids, allowing for the conversion of secondary products into high added-value compounds [36,37].

2.2.1. Enzymes

Lipases and proteases are naturally involved, respectively, in lipid and protein metabolism and they are one of the most widely used enzymes in biotechnology [38,39].

Lipases (EC 3.1.1.3, triacylglycerol lipase) are water-soluble hydrolases, which can be found in animals, plants, and microorganisms [40]. Fungi, like Mucor, Rhizopus, Geotrichum, Rhizomucor, Aspergillus, Candida, and Penicillium, are microrganisms producers of lipases and the production and purification fine tuning methods have intensively been developed in the last decade [10,41,42].

Lipases catalyse hydrolysis with water being a reactant in the system. If water is removed from the system, the equilibrium shifts and the lipases perform esterification, transesterification, and interesterification reactions (

Table 1. Reactions catalysed by lipases.

Reactions
Hydrolysis	R1COOR2 + H2O ↔ R1COOH + R2OH
Esterification	R1COOH + R2OH ↔ R1COOR2 + H2O
	(a) Acidolysis
	R1COOR2 + R3COOH ↔ R3COOR2 + R1COOH
Transesterification	(b) Alcoholysis
	R1COOR2 + R3OH ↔ R1COOR3 + R2OH
	(c) Aminolysis
	R1COOR2 + R3NH2 ↔ R1CONHR3 + R2OH

) [40]. The reactions carried out by lipases usually occur at the interface.

The typical substrates of lipases are apolar, like fats and oils, have long hydrocarbon-like groups that are mostly flexible, which can be accommodated in the active site, resulting in a wide range of substrate specificity [43]. This specificity usually differs in the preference of acyl and alkyl group size. Some lipases show a strong or a weak stereospecificity, examples are, respectively, Rizhomucor miehei lipase (RML) and porcine pancreatic lipase (PPL) [27]. The specificity of lipases is categorized by the position of ester bonds (hydrolysed or formed) in the substrate molecules (regioselectivity), the class of substrate accepted (chemo-selectivity), and stereoselectivity [44].

Proteases are enzymes that are used in numerous applications, predominantly in protein peptide bonds hydrolysis. They also promote acyl-transfer reactions, which may influence the enzyme selectivity: to decrease the water activity in the reaction medium and increase the substrate concentration [45].

Proteases are reported in plants, animals, and microorganism. Examples of plant proteases are papain, bromelain, keratinases; of animal origin are pancreatic trypsin, chymotrypsin, pepsin, rennin, among others; among microorganisms, bacteria and fungi are the most prominent [45]. Aspergillus oryzae produces three proteases; acid, neutral, and alkaline. Among bacteria, Bacillus spp. are attractive industrial tools for a source of proteases. Microorganisms represent an excellent source of enzymes due to their biochemical diversity and susceptibility to genetic manipulation. Microbial proteases represent one of the largest groups of industrial enzymes and they account for approximately 60% of the total industrial enzyme sales in the world [45].

2.2.2. Solvents

One of the main problems in using enzymes, as lipases, for LAAs production is the fact that these compounds are made of two substrates with different polarities. In fact, amino acid is hydrophilic and the tail is hydrophobic, the global reaction depends on the choice of a solvent suitable for the two components. Studies suggest that solvents with a log P lower than 3 are preferable to the bioreaction with lipases, like acetonitrile, terbutanol, 2-methyl 2-butanol, or ethyl methylketone [35]. The use of tertiary alcohols as solvent is also a good choice. Tertiary alcohols have intermediate polarity and solubilize partially the substrates of LAAs; additionally, they do not interfere in the reaction by inactivating the enzyme [35] and are relatively simple to remove from the final product. The purification can be carried out by precipitation, by exchange chromatography.

The use of ionic liquids [42], like (MeOSO3) and (PF6) [46], has shown good results in this type of bioreaction, although they are still an expensive solution to an industrial scale process [35].

Another alternative is the use of eutectic mixtures. They consist on the combination of a solid organic salt, like sodium chloride, and a complexing agent, like urea or glycol [47]. Eutectic liquids have similar costs as organic solvents and, because the purification processes for salt removal can be discarded, the process can became even cheaper [46]. This new solvent was appropriate for enzymatic reactions maintaining enzyme stability with increased activity, and it has already been tested for bioprocesses involving either lipases or proteases biocatalysis [47]. A high stability of the enzyme in the bioreaction solvent medium might allow for temperature rising, with an increase in the biocatalysis rate [46].

Solvent free systems are very interesting for the synthesis of lipoamino acids due to the difficulties in finding a suitable solvent for both the polar head (amino acid) and hydrophobic tail (long aliphatic chain) [48]. In some situations, the hydrophobic moiety (e.g., dodecylamine) is liquid at room temperature or it melts at a temperature that is compatible with the enzymatic activity (e.g., 45 °C); therefore, the substrate creates the necessary liquid phase where the reaction takes place. Additionally, the liquid phase can be formed by adding a third liquid compound or in a eutectic system with no further liquid added [49].

2.2.3. Immobilization

The main disadvantages in the use of biocatalytic systems are the cost of enzymes and the separation from the final products in the bioreaction medium, slow reaction rates, and the absence of optimized reactors for biphasic media catalysis [50].

Industrial enzymes application is often hampered by the lack of operational stability, coupled with relatively high price. One way to overcome these drawbacks is immobilization, affording enzymes improved operational stability and providing easier separation and reuse. When enzymes are trapped in a solid matrix, its solubilisation improves along with thermostability and they become easily recovered after the reaction [18]; the reusability significantly reduces the costs that are associated with these enzyme catalyzed reactions [50].

The use of nanobiocatalysts, combining nanotechnology and biocatalysis, is considered to be an exciting and rapidly emerging area [13]. Thus, nanobiocatalysis, as a new frontier in biotechnology, is a new innovative sub-field of biocatalyst, which explores advanced materials as enzyme carriers, as well as provides robust nanostructured materials with properties that are tailored to their applications as enzyme scaffolds. Limitations of solid phase biocatalysis are due to the poor mobility of the catalytic centers resulting in a net loss of biocatalytic activity as compared with soluble enzymes that can more readily reach the surface of insoluble substrates and adjust spatial orientation at the phase boundary [49].

Lipases activity has the tendency to increase as more hydrophobic immobilization materials are used. The higher activity is associated with structural alterations upon exposure of the active site, because the surface is recognized in a similar way to that of the interfacial activation [35,50,51,52,53].

The main forms of commercial immobilized lipases are adsorption onto polymer based matrices; nonetheless, Novozymes has also developed a new and less expensive method by combining adsorption and encapsulation in silica granules. Additionally, when the immobilized particles are also magnetic, the enzyme becomes especially easy to recover and the overall process becomes even more productive. Another critical point concerning the reaction, specifically involving lipase, is its conformational changes. Lipase has two different conformations, closed (inactive) and open (active), so it is very important to make sure that the enzyme is immobilized in its active form to guarantee good reaction rates [52,53].

One of the applications of LAAs is in the food industry mainly due to the currently trends in the consumption of functional and natural ingredients. Bernal [54] carried out the biosynthesis of lauroyl glycine lipoaminoacid with a lipase from Pseudomonas stutzeri and a protease from Bacillus subtilis, immobilized in octyl-glyoxyl silica and glyoxyl-silica supports, respectively. The catalytic performances of both enzymes were compared with respect to the characteristics of the immobilized biocatalysts and synthesis conditions. Additionally, the reaction media was tested with three solvents to evaluate the expressed activity, stability, and catalytic behavior during biosynthesis. That research indicated that lipase and protease favor lauroyl glycine synthesis. Moreover the immobilized protease, after 96 h of synthesis, at 45 ºC and acetone as solvent, addressed the best balance between selectivity and yield, 40% yield for lauroyl glycine, and less than 5% for dipeptide [54].

Fait et al. [55] biosynthetized two novel arginine-based cationic surfactants while using papain as biocatalyst, an endopeptidase from Carica papaya latex, adsorbed onto polyamide. Yields that were higher than 90 and 80% were achieved for the synthesis of N-benzoyl-arginine decyl amide (Bz-Arg-NHC10) and N-benzoyl-arginine dodecyl amide (Bz-Arg-NHC12), respectively [55]. Additionally, the authors developed a purification process, using water and ethanol as the main separation solvents in a single cationic exchange chromatographic separation step, in order to make the process more sustainable. These LAAs showed antimicrobial activity against both Gram-positive and Gram-negative bacteria, revealing their potential use as effective disinfectants, since, after only 1 h of contact, they reduced 99% the initial bacterial population [55].

Ribeiro [56] reported the biosynthesis of three lipoaminoacids [(C₁₂Cys)₂, C₁₂Lys, C₁₂Phe] with lipases in a eutectic mixture. In this work, the substrates used were dodecylamine (C₁₂) and the aminoacids, cystine (Cys), lysine (Lys), and phenyalalanine (Phe). The lipase TLL was immobilized in sol@gel.

The overall biocatalytic process that was carried out with lipases and proteases, which do not need cofactors, eco-friendly, can be less expensive than conventional chemistry, as well as the costs associated with the time to industry production.

3. Cationic Lipoaminoacid-DNA Interactions

The DNA double helix structure is composed of two polynucleotide strands, with each nucleotide consisting of a purine—adenine (A) or guanine (G)—or pyrimidine base—thymine (T) or cytosine (C)—linked to a 2-deoxyribose sugar molecule and a phosphate group, which is negatively charged at physiological pH. In each strand, phosphodiester bonds between the third and fifth carbon atoms of adjacent sugar rings hold the nucleotides together. In double-stranded DNA (dsDNA), the two strands run antiparallel to each other and the nucleobases of the two separate strands are bound together by hydrogen bonds according to Watson-Crick base pairing rules (A with T and C with G) with additional base-stacking interactions between aromatic nucleobases helping to stabilize the structure. As a result of the duplex structure, the DNA molecule has two asymmetric grooves of different sizes, the major groove and the minor groove, which may provide binding sites.

The condensation of DNA can be achieved while using multivalent cations (e.g., cobalt hexamine, spermidine) or monovalent alkylammonium cations (cationic surfactants) with long aliphatic chains, usually of 12 carbon atom length or higher and other cationic lipids [7,8,45,57,58,59,60]. Cationic short-chain surfactants, like octyltrimethylammonium bromide, can lie down on the DNA surface (while long-chain surfactants cannot) and directly interact with the DNA grooves through hydrophobic interactions, thus hindering DNA condensation [59]. On the other hand, upon binding to DNA, the long alky chains of dodecyltrimethylammonium bromide (DTAB) and cetyltrimethylammonium bromide (CTAB) point away from the DNA surface, which allows for hydrophobic interactions between the aliphatic chains, thus inducing aggregation and DNA compaction [59].

Cationic surfactants interact with DNA through a cooperative mechanism that involves both electrostatic and hydrophobic interactions [60], inducing dsDNA transition from an extended coil state to a compact globule condition over a certain surfactant concentration range, as shown from fluorescence microscopy studies [61]. Potentiometric studies on the interaction of CTAB, tetradecyltrimethylammonium bromide (TTAB), DTAB, and dodecylamine (DDA) with DNA suggest that complex stoichiometry with molar ratio of DNA phosphate to surfactant close to 1:1 is favored [62,63,64,65]. Monte Carlo simulations have shown that DNA compaction was associated with the polycations promoting bridging between different sites of the polyanion [61]. Recently, it has been reported that cationic gemini surfactant 16-3-16 binds to plasmid DNA forming a surfactant-DNA complex that behaves as a single amphiphilic entity [66].

Cationic surfactant-DNA interactions have been extensively debated regarding their biological significance and potential biomedical relevance. Experimental and theoretical studies suggest that, at low surfactant concentrations, far below the critical micelle concentration (CMC) of the surfactant, the surfactant monomers bind DNA through electrostatic interactions between the positively charged headgroups of the cationic surfactant and the negatively charged phosphate groups of the DNA backbone [61,67,68,69]. A further increase in surfactant concentration leads to cooperative binding due to hydrophobic interaction between the alkyl chains of neighboring surfactant molecules with the formation of micelle-like clusters along the polynucleotide chain once critical aggregation concentration (CAC) is reached. The use of ultra-sensitive techniques and complementary methodologies, such as atomic force microscopy (AFM), optical or magnetic tweezers provided evidence of this mechanism [59]. The onset of DNA compaction occurs at CAC with the formation of surfactant-DNA complexes that grow in size with increasing surfactant concentration until saturation of the biopolymer. Further addition of the surfactant then leads to free micelle formation in the bulk solution. Usually, CAC is independent of DNA concentration (since DNA chains behave like a separate phase when in contact with the surfactant molecules), while CMC increases with DNA concentration due to the postponed appearance of free micelles in the bulk phase [60,70].

According to isothermal titration calorimetry (ITC), studies on cationic surfactant binding to DNA, lipid binding, counterion displacement, and DNA condensation are highly cooperative processes that are driven by a large increase in entropy [69]. The strong associative behavior of cationic surfactant-DNA systems, similar to other polyelectrolyte-oppositely charged surfactant systems [71,72,73], results in a pronounced decrease of the CAC relative to the CMC, usually by several orders of magnitude [69]. Moreover, phase separation by coacervation (liquid-liquid phase separation) or precipitation (solid-liquid phase separation) can occur near the neutralization point, mainly depending on DNA concentration, pH, ionic strength, and temperature of the solution [68,70]. The decrease in the superficial charge of the complexes due to partial charge neutralization with increasing surfactant concentration leads to reduced electrostatic repulsions that promote the aggregation and coagulation of the complexes. Thus, viscous liquids, gels, liquid crystalline phases, coacervates, or precipitates often form at CAC or higher surfactant concentrations [68,70]. Further addition of surfactant can lead to redissolution of the complexes due to acquired net positive charge or solubilization by free micelles once CMC is reached [70].

Electrolyte addition screens both the repulsion between the surfactant headgroups and the electrostatic attraction between the cationic surfactant and the negatively charged phosphate groups of DNA, which thus weakens the binding of surfactant to biopolymer while also enhancing the formation of free micelles in the bulk phase [63]. Therefore, an increase in the ionic strength of the solution usually increases CAC, but decreases the CMC and these opposite effects lead to a critical ionic strength after which DNA does not form complexes with the surfactant due to the complete salt screening of the electrostatic attractions between the two species [63]. However, a salt-enhancing effect on complex formation has also been observed upon the addition of micellar solutions to DNA due to micellar growth promoted by added salt, which results in larger micelles with higher surface charge density and increased electrostatic interaction with the polyelectrolyte [63].

4. Applications of Cationic Gemini Surfactants as Transfection Agents

The morphology of the nanocomplexes that formed between DNA and cationic surfactants depends on the molecular structure of the surfactant and on the flexibility of the polynucleotide chain [7,8]. According to synchrotron small-angle X-ray scattering (SAXS) studies, double-tailed surfactant didodecyldimethylammonium bromide (DDAB) formed bilayer lamellar structures with dsDNA, while single-tailed surfactant CTAB preferentially formed bidimensional hexagonal structures with dsDNA and a cubic bicontinuous phase with the more flexible single stranded DNA (ssDNA) [14]. Non-lamellar phases, such as inverted hexagonal (H_II phase) or cubic phases, have been implicated in bilayer destabilization and membrane fusion. Therefore, a helper lipid that favors the formation of an inverted hexagonal (H_II) phase, such as dioleoylphosphatidylethanolamine (DOPE), is often added to cationic lipid formulations for gene delivery to promote the fusion of the complexes with the endosomal membrane and facilitate DNA release into the cytoplasm [7,8]. Cholesterol is also frequently included as a helper lipid for providing stabilization in vivo and avoid transfection inhibition by serum. Serum is a major obstacle in cationic liposome-mediated gene transfection, since the negatively charged serum proteins can adsorb on the cationic liposomes, leading to DNA release or complex precipitation [7,8].

Bis-quaternary ammonium gemini surfactants (BisQuats) of the type m-s-m, where m and s are the lengths of the hydrocarbon tails and spacer, respectively, have been widely investigated and have shown high affinity towards DNA [8,66,74,75]. A comparison between gemini 12-3-12 and its monomeric counterpart 12-3-1 showed that the gemini was more efficient in compacting DNA, thus supporting the importance of the additional hydrophobic tail in the compaction process [75]. Furthermore, surfactant architecture correlates with transfection efficiency, which depends on the length and flexibility of alkyl tails and spacer, nature of the headgroup, and type of counterion [74]. However, structure-activity relationships are difficult to establish, since minor changes in the surfactant structure can have major effects on the physicochemical and biological activity, often resulting in no correlation between in vitro and in vivo transfection efficiency.

Increasing the surfactant tail length increases the hydrophobicity and promotes aggregation, which usually results in higher transfection efficiency following the order C₁₂ < C₁₄ < C₁₆ < C₁₈, while longer alkyl chains lead to insoluble surfactants [7,8,74]. The oleyl chain is often associated with high transfection efficiency, since unsaturation reduces the gel-to-liquid crystalline phase transition temperature to below the physiological temperature, thus enhancing membrane fluidity [1,2]. Asymmetric dimeric surfactants (heterogeminis, m-s-n) incorporating two alkyl chains of different lengths can improve transfection by promoting membrane fusion [74,76]. However, increasing the dissymmetry of m-s-n heterogemini surfactants weakens the interaction with DNA due to the disruption of the intermolecular hydrophobic interactions between the alkyl chains that occur at higher m/n [74,76].

The unsaturation degree of the hydrophobic fatty acid-derived chains in the surfactant structure influence phase transition temperature of the LAA and the fluidity of the bilayer in the supramolecular aggregates. In LAAs (and non-LAA surfactants), increased unsaturation decreases the lamellar (L_α) to reversed hexagonal (H_II) phase transition temperature, increasing fusogenicity. The presence of cis double bonds increases bilayer lipid fluidity in the lipoplexes, which promotes DNA release and thus intracellular delivery of the encapsulated nucleic acids [77]. Structure-delivery relationships of cationic lysine-based gemini surfactants and their lipoplexes revealed Ol-Lys-H-6 (Figure 1) with oleoyl (C_18:1 cis double bond) chain as the best hydrophobic tail for transfection efficiency when compared to elaidoyl (C_18:1 trans double bound) or saturated stearoyl (C_18:0) chains due to improved membrane fluidity [78]. The CMC is also higher for unsaturated LAAs than for the saturated isomers due to packing constraints in the micellar core that arise from the presence of double bonds in the hydrophobic tails, which hinders the micellization process, and often results in decreased toxicity [24]. The unsaturated derivative showed decreased in vitro toxicity against HUVEC cells in the MTT assay when compared to the correspondent saturated Lys-based LAA (IC₅₀ 0.32 vs. 0.02 mg mL⁻¹), reduced membrane damage in the LDH assay (IC₅₀ 0.68 vs. 0.20 mg mL⁻¹), and lower hemolytic activity (HC₅₀ 3.71 vs. 0.15 mg mL⁻¹) against rat RBCs [24]. Furthermore, the stereochemistry of diastereomeric cyclobutane β-amino acid-based cationic LAAs, developed as potential candidates for gene therapy, also influences self-assembly behavior, with cis-isomers preferentially forming micelles or vesicles, while trans-isomers predominantly formed fibers [79].

The spacer chain length can also influence the type of supramolecular aggregate formed in solution, and cationic gemini surfactants with unsaturated chains 18:1-s-18:1 and short spacers (s = 2, 3) tend to form vesicles, while those with a longer spacer (s = 6) preferentially formed micelles [80]. The type of aggregate formed is correlated with transfection efficiency, with gemini surfactants having vesicle structures that perform better than those with micellar structure, due to the preference of the former to adopt non-lamellar structures that promote DNA release and escape from the endosome [80]. However, in the presence of helper lipid DOPE, the micelle systems can change their morphology to inverted hexagonal (H_II) structures with increased membrane fluidity [80].

The effect of the spacer chain length on the binding of saturated 12-s-12 dimeric surfactants to calf thymus DNA has also been studied. High transfection efficiency was obtained for gemini surfactants with either short (s < 4) or long (s > 10) spacers, while the spacers of intermediate lengths were less efficient as a result of competition between entropy loss and enthalpy gain [81,82]. Similar behavior was found for the compaction of bacteriophage T4 DNA by a series of gemini surfactants based on cationic peptides that were connected at their carboxylic acid groups by an α,ω-diamino alkyl spacer upon changing the spacer length [75]. Increasing the hydration level of the spacer through the introduction of hydrophilic substituents (imino, amino acid, dipeptide), although increasing the CMC of the gemini surfactants also increased transfection efficiency, which was attributed to the higher biocompatibility and flexibility of the spacer [65].

Regarding the polar headgroups, higher valency leads to higher DNA compaction due to increased electrostatic attractions [74,83]. The size of the headgroup is also important due to the entropy loss when the gemini surfactants bind to DNA requiring compensation by the hydrophobic interactions upon DNA-induced surfactant self-assembly. Surfactants with larger headgroups form smaller micelles that are less efficient in DNA compaction [84]. The binding of cationic surfactants to DNA is strongly dominated by the positive entropy gain on the release of the small counterions from the surfactants and from DNA [83]. The nature of the counterion strongly influences the surfactant aggregation process in both the presence and in absence of DNA, as shown from isothermal titration calorimetry (ITC) and conductivity measurements for cationic gemini surfactants 12-6-12 with different counterions (F⁻, Cl⁻, Br⁻, CH₃CO₂⁻, NO₃⁻, and SO₄²⁻) [83]. Counterion effects generally followed the Hofmeister or lyotropic series due to ion specific effects and changes in water structure that were associated with the level of headgroup (de)hydration [83]. For cationic surfactants with monovalent counterions, changing the counterion from chloride to iodide is associated with enhanced binding to the DNA due to decreasing hydration of the counterion [83].

Different counterions can also produce different aggregation behavior, which can influence DNA compaction and transfection efficiency. Isothermal titration calorimetry (ITC), dynamic light scattering (DLS), and atomic force microscopy (AFM) have been used to study the interaction of salmon sperm DNA with micelles, vesicles, and mixed surfactant-lipid liposomes [58]. Different modes of interaction were observed, depending upon the aggregation state of the surfactant. Cationic vesicles have been associated with higher transfection efficiency when compared to cationic micelles due to packing constraints [80].

The divalent LAA arginine-N-lauroyl amide dihydrochloride (ALA, Figure 1) has been used as nontoxic cationic component in the construction of biocompatible catanionic vesicles for DNA encapsulation [85], which were spontaneously formed in aqueous mixtures of anionic surfactant sodium cetylsulfate (SCS) or sodium octylsulfate (SOS) and excess of cationic ALA. The addition of DNA to the vesicles led to associative phase separation at very low DNA concentrations. A lamellar structure with DNA arranged between the amphiphile bilayers was inferred from cryo-transmission electron microscopy (TEM) and SAXS studies, with an increase in the repeat distance of the complexes from 4.7 to 5.8 nm upon substitution of short chain SOS by long chain SCS [86]. DNA pre-condensation with ALA improved the transfection efficiency of cationic liposomal formulations [87].

The binding of amino acid-based surfactants to calf thymus DNA was found to occur in two consecutive steps, according to stopped-flow fluorescence spectroscopy data [88]. The fast step was attributed to the surfactant binding to DNA and micelle formation in its vicinity and the slower step was attributed to DNA condensation and the possible rearrangement of the surfactant aggregates [88]. A recent study on the interaction of a novel surfactant derived from lysine, (S)-5-acetamido-6-(dodecylamino)-N,N,N-trimethyl-6-oxohexan-1-ammonium chloride (LYCl, Figure 1) with dsDNA from calf thymus showed that association mainly occurred through groove binding and electrostatic interactions, leading to DNA compaction [89]. The interactions were found to be reversible upon the addition of β-cyclodextrin (β-CD), resulting in the decompaction of dsDNA due to stronger and more specific β-CD-LYCl hydrophobic interactions [89].

Studies concerning the interaction between DNA and amino acid-based surfactants have shown that the strength of the interaction is highly dependent on the surfactant linker group connecting the hydrophobic tail(s) to the amino acid residue [90]. LAAs with amide groups were found to interact with oligonucleotides much more strongly than LAAs with ester groups [64], being able to displace up to 75% of ethidium bromide (EB) molecules bound to the DNA. On the other hand, LAAs with bulkier hydrophobic headgroups (e.g., phenylalanine versus alanine) were found to more strongly interact with DNA, but less efficiently excluded EB [90,91,92].

The nature of the spacer (length and hydrophobicity) in 1,5-dihexadecyl-N-lysyl-N-alkyl-L-glutamate LAAs strongly influenced the fusogenic potential toward biomembranes, which is an important parameter in intracellular DNA delivery [66]. Increased fusion potential was found to correlate with enhanced gene expression efficiency [66]. Enhanced gene expression has been observed in epithelial cells that were transfected with amino acid-substituted gemini nanoparticles for topical cutaneous and mucosal applications [9,65]. The cationic gemini surfactants contained amino acid (glycine or lysine) and dipeptide (glycyl-lysine or lysyl-lysine) substituted spacers showed high buffering capacity and pH-dependent increase in particle size, which contributed to more efficient endosomal escape leading to high transfection efficiency [10]. Moreover, cationic liposomes that were composed of 1,5-hexadecyl N-arginyl-L-glutamate (Arg-Glu2C₁₆, Figure 1) showed high transfection efficiency and low cytotoxicity in the neuronal SH-SY5Y cell line [93].

Substitution of aliphatic chains by cholesterol as the hydrophobic domain in cationic LAA by esterification of lysine, histidine, or arginine, improved the serum stability and cellular uptake of the DNA-lipid complexes by providing compact packing of DNA and enhancing membrane permeability [93]. Serine-derived gemini LAAs also formed serum-resistant complexes with the DNA of appropriate size for intravenous (i.v.) administration [94]. The most efficient and less cytotoxic complexes were prepared from serine gemini LAA with dodecyl chains and an ester linker between the methylene spacer and the serine headgroups (e.g., (12Ser)₂COO5, Figure 1) [94]. The cleavable ester bond facilitated intracellular DNA release while also generating a soft amphoteric surfactant, N-dodecyl-N,N-dimethylserine upon breakdown [94].

The ionization state of the headgroup, as well as the nature of the counterion, can also influence the self-assembly behavior and transfection efficiency of cationic LAAs [83]. Lysine-based cationic lipids with chloride counterions formed micelles, while those with trifluoracetate (TFA) counterions formed vesicles [95]. The transfection of COS-7 cells revealed that gene expression efficiency in relation to the ionization states of the hydrophilic headgroups decreased in the order: –NH₃⁺TFA⁻ > –NH₃⁺Cl⁻ > –NH₂ due to increased gel-to-liquid crystalline phase transition temperature of the cationic assemblies [95]. Higher phase transition temperatures are associated with increased membrane rigidity, and thus lower membrane fusion potential and decreased transfection efficiency. The lower phase transition temperature was attributed to the reduced hydration of the headgroup leading to smaller headgroup volume [95]. Smaller lipid headgroups lead to a preference for a fusogenic H_II phase relative to lamellar L_α and to a decrease of the L_α-to-H_II transition temperature [95].

5. Advantages of Cationic Lipoamino Acids in Gene Delivery

Cationic gemini LAA binding to DNA offers several advantages over conventional cationic surfactants, including lower CMC, higher tendency to self-assembly, and forming inverted micellar and cubic structures, which are associated with increased transfection efficiency [7,8,9,13,15,16]. The presence of hydrogen bonding donor and/or acceptor groups and ionizable amino groups in cationic LAAs promote DNA compaction via electrostatic and hydrogen bonding interactions. The cationic amino acid residue also improves the buffer capacity and promotes pH-dependent lamellar-to-hexagonal phase transition that facilitates DNA escape in the acidic milieu of the endosomal compartment, further promoted by the addition of a helper lipid, such as DOPE [9,13,15,16,96]. Moreover, the natural amino acid residues can also provide improved biocompatibility, biodegradability and hydrophilicity, lower hemolytic activity, and decreased cytotoxicity when compared to conventional cationic surfactants [7,13,15,16,96]. The modification of cationic gemini surfactants by introduction of amino acids in the spacer chain, e.g., 12-7N(Gly)-12 (Figure 1) was also shown to improve transfection efficiency in epithelial cells, which was attributed to the enhanced flexibility and improved biocompatibility of the gemini LAAs provided by the amino acid residue [97].

Histidine-mediated membrane fusion at acidic pH via protonation of the imidazole side chain (pK_a 6.04) and disruption of the endosomes contributes to the high transfection efficiency usually associated with histidine-based LAAs [16,98,99], a trend also shown by non-LAA surfactants that contain imidazole as headgroup [8]. Cationic gemini histidine-based surfactants with very low CMC showed excellent DNA binding ability in the EB exclusion assay [99]. The cationic gemini LAAs with the longer alkyl chains (C₁₂, C₁₄, and C₁₆) completely displaced EB at cationic surfactant/DNA charge ratio of 1.75–2 [99]. The cationic histidine gemini LAA, C₃(C₁₆His)₂ (Figure 1), efficiently compacted plasmid DNA (pDNA) when mixed with helper lipid DOPE, which formed nanoaggregates with sizes of 120–290 nm that protected pDNA from enzymatic degradation [98]. The nanoaggregates showed high transfection efficiency in COS-7 and HeLa cells and low cytotoxicity in the alamar Blue assay, exceeding 80% cell viability, when considering the threshold for cell safety [98].

Reducible cationic LAAs with different amino acid polar headgroups containing α-tocopherol or lipoic acid moieties and a biodegradable disulfide bond that can be reduced under reductive conditions to release the encapsulated DNA showed serum stability and demonstrated high efficiency as gene vectors, which was superior to that of branched poly(ethylene imine) (PEI), considered to be the gold standard cationic polymer vector for gene delivery [100,101]. The transfection efficiency of lipoplexes from His-based and Arg-based reducible LAAs in the presence of 10% serum were 4.3 and 2.5 times higher, respectively, than that of PEI polyplexes [100]. Another reducible cationic LAA with promising potential as gene delivery vector is (C₁₂Cys)₂ (Figure 1), a gemini LAA that is derived from cysteine currently being studied by our research group [34].

The self-assembly of cationic LAAs containing a dioleoyl glutamate hydrophobic tail and a twin polar head of lysine, arginine, or histidine self-assemble in aqueous solution leads to nanosized aggregates having strong buffering capacity due to their residual amino-, guanidine-, or imidazole-rich periphery, depending on the amino acid side chain [102]. The assemblies with guanidine-rich periphery enhanced in vitro gene transfection in the presence of serum up to 190-fold as compared to commercial PEI_25k and led to a seven-fold enhanced expression in vivo in mice with HepG2 tumor xenografts, which was attributed to the formation of bidentate hydrogen bonds between the guanidine moiety and phosphate groups in the lipid membrane, which leads to improved cell-penetrating ability [102].

The toxicity, poor endosomal escape, and inefficient nuclear uptake of cationic polymers, which constitute major barriers for DNA delivery, can be overcome by cationic LAAs due to their improved biocompatibility, low cytotoxicity, and pH-dependent behavior that promotes structural changes from lamellar to inverted hexagonal phases at the acidic endosomal compartment facilitating DNA release [7,8,9,16,96]. Recently, intranuclear DNA delivery by cationic LAAs was further improved while using arginine-rich cross-linking peptides with different SV40 nuclear localization signal (NLS) content [103]. The nuclear import pathway is the classical transport between the cytoplasm and the nucleus, and the Arg-rich peptides modified with 90 mol% of SV40 NLS provided efficient intranuclear DNA delivery vehicles [103]. Thus, LAAs are efficient and safer potential alternatives to both conventional cationic surfactants and cationic polymers as non-viral gene delivery vectors.

6. Conclusions and Perspectives

Lipoaminoacids, amino acid-based surfactants, are a group of compounds based on amino acids and fatty acids or their derivatives. This interesting class of biocompatible compounds contains an amino acid as the hydrophilic part and an alkyl chain as the hydrophobic part, which confer surfactant behavior. LAAs have attracted significant attention as candidate drug and gene delivery vehicles due to their low toxicity and biodegradability. Applications, such as medical and cosmetic preservatives, food additives, mineral flotation, and pesticides, are among other applications.

Mainly for its promising applications in health, LAAs are an important area of further studies and manipulation to achieve better quality products. Biotechnology offered solutions for production concerns. Chemically processes produce considerable waste, while bioprocesses can be conducted in a cleaner and environmentally friendly manner. Additionally, biocatalysis production allows for specific LAAs, increasing the productivity rates.

The cationic gemini biosurfactants display complex formation due to affinity to nucleic acid molecules. Being a dimer, gemini LAAs have a connector spacer. Supramolecular arrangements are affected if this structural characteristic is disrupted.

Mainly for its promising applications in health, LAAs are an important area of further studies and manipulation to achieve better quality products. Further research areas appear to be important: (1) Develop suite bioengineered biocatalysts to perform LAAs biosynthesis; (2) Protein engineering strategies can be applied to create novel biocatalysts; (3) Develop and optimize immobilization strategies for new biocatalysts; (4) Since the gemini and the substrates have common parts and polarities the purification process through a quick an inexpensive process will be challenging; (5) Understanding the interaction between LAAs and DNA for better product selectivity; (6) Test the efficacy and safety of LAAs as gene and drug delivery systems; and, (7) These tests must include (i) cytotoxicity evaluation, (ii) characterization of the interaction of the gemini LAAs with bioactive molecules, like nucleic acids and peptides, (iii) its action on the target cells, and (iv) optimization of the quantities and proportions of LAAs.

This review highlights the potential of lipoaminoacids as transfection agents towards gene delivery in therapy application.