**4. Biotechnological and nanotechnological applications of surface-active compounds**

Biosurfactants, have been increasingly attracting attention in the field of nanotechnology (Kitamoto et al., 2005, 2009). During the last decade, unique properties of biosurfactants, like versatile self-assembling and biochemical properties, which are not usually observed in conventional chemical surfactants, have been reported (Kitamoto et al., 2005, 2009). In recent years, the development of new functional structures and/or systems using selfassembly of amphiphilic molecules has evolved into a dynamic and rapidly growing area of nanotechnology (Ariga et al., 2007, Shimizu et al., 2005, as cited in Kitamoto et al., 2009) due to their ability to self-assemble into hierarchically ordered structures using hydrogen bonding, hydrophobic and van der Waals interactions as mentiond earlier.

Mannosylerythritol lipids (MELs) show the most interesting self-assembling properties and numerous related potential applications (Kitamoto et al., 2009). Konishi et al., (2007), Imura et al., (2007, 2008), and Ito et al., (2007), for example, developed and studied the kinetics of interactions in carbohydrate ligand systems composed of self-assembled monolayers of mannosylerythritol lipid-A (MEL-A) serving as a high-affinity, easy to handle and low-cost ligand system for immunoglobulin G and M and lectins..

Table 1 below lists the latest discoveries in the biotechnological and nanotechnological fields applicable to biosurfactants, and in particular the latest successful results of mannosylerythritol lipids (MELs) application in the enhancement of the gene transfection efficiency of cationic liposomes as well as some interesting applications of glycolipids and other biosurfactants in drug delivery and gene therapy. Biosurfactants use as a "green" alternative for high-performance nanomaterials production and, in particular, for the synthesis and stabilization of metal-bound nanoparticles will also be described.



Table 1. Examples of recent biosurfactant applications in the biotechnological and nanotechnological fields.

### **4.1 Liposomes and gene transfection**

348 Biomedical Science, Engineering and Technology

mediated gene transfection Inoh et al., (2009)

kinase gene therapy Maitani et al., (2006)

sophorolipids Smyth et al., (2010b) Cadmium sulfide nanoparticles Singh et al., (2011)

antibioticmicrobial activity Kumar et al., (2010)

Silver nanoparticles Xie et al., (2006)

ZnS nanoparticles Narayanan et al., (2010) Microemulsions Xie et al., (2005, 2007) Alcohol-free microemulsions Nguyen & Sabatini, (2009)

Silver nanoparticles Kasture et al., (2008)

of aciclovir Nicoli et al., (2010)

nanoparticles Reddy et al., (2009) Cadmium sulfide nanoparticles Singh et al., (2011)

Water-in-oil microemulsions Worakitkanchanakul et al.,

(2008)

(2008)

(2009)

Kitamoto et al., (2009)

Nguyen et al., (2010)

Palanisamy & Raichur,

Singh et al., (2009, 2010)

Nguyen et al., (2010)

Worakitkanchanakul et al.,

**Biosurfactant type Activity/application Study** 

*In vivo* promotion of liposome-

Herpes simplex virus thymidine

Increase membrane fluidity of monolayers composed of L-αdipalmitoylphosphatidylcholine

Deuterated rhamnolipids and

Nickel oxide nanoparticles by microemulsion technique

Sophorolipids Cobalt nanoparticles Kasture et al., (2007)

Sophorolipid-coated silver and gold nanoparticles with antibacterial

Biocompatible microemulsions of lecithin/rhamnolipid/sophorolipid

biosurfactant Silver nanoparticles Kiran et al., (2010b)

Enhancers for the skin accumulation

Table 1. Examples of recent biosurfactant applications in the biotechnological and

Biocompatible microemulsions of lecithin/rhamnolipid/sophorolipid

Self-assembling and vesicle-forming

(DPPC)

activity

biosurfactants

Rhamnolipids Silver nanoparticles with

activity

biosurfactants

Surfactin Surfactin-mediated synthesis of gold

Mannosylerythritol

Rhamnolipids and sophorolipids

lipids-B

Glycolipid

Fengycin and surfactin

nanotechnological fields.

Gene transfection into the cells is a fundamental technology not only for molecular and cellular biology processes but also a clinical gene therapy (Ueno et al., 2007b). Although several methods for gene transfection have been investigated (Felgner et al., 1989, Fujiwara, 2000, Gao & Huang, 1991, Hatakeyama et al., 2007, Nishiyama et al., 2005, Ueno et al., 2007b), more efficient and safe systems are still needed (Ueno et al., 2007b). Among the various methods, lipofection using cationic liposomes is considered to be a promising method for introducing foreign gene to the targeted cells due to their high transfection efficiency, low toxicity and immunogenicity, ease of preparation and targeted application (Farhood et al., 1992, Felgner et al., 1989, Kogure et al., 2007, Lasic, 1998, Nakanishi, 2003, Inoh et al., 2010). The physicochemical properties of cationic liposomes, such as lipid packing density, shape, and zeta-potential, have a significant effect on gene transfection efficiency (Lin et al., 2003, Takeuchi et al., 1996, Wittenberg et al., 2008, Xu et al., 1999, as cited by Inoh et al., 2010).

Inoh et al., (2001) reported that MEL-A promoted DNA transfection efficiency mediated by cationic liposomes. Confocal laser scanning microscopic analysis showed the distribution of lipids and oligonucleotide DNA in MEL-A-containing liposome–DNA complex in the plasma membrane and the nucleus of target cells at 1 h after the addition of complex (Inoh et al., 2004). This suggests that MEL-A induces the membrane fusion between the target cells and the cationic liposomes, accelerating the efficiency of gene transfection significantly. Similarly, Igarashi et al., (2006) reported that MEL-A significantly increased the cellular association and the efficiency of gene transfection mediated by cationic liposomes in human cervix carcinoma Hela cells. Analysis of flow cytometric profiles clearly indicated that the amount of DNA associated with the cells was rapidly increased and sustained by addition of MEL-A to the liposome. Confocal microscopic observation also indicated that the MELlipoplex distributed widely in the cytoplasm and DNA presence was intensely detected in cytoplasm around the nucleus.

The above results suggested that MEL-A increased gene expression by enhancing the association of the lipoplexes with the cells in serum and, thus, MEL-liposome may prove a significant nonviral vector for gene transfection and gene therapy.

In an attempt to explain how MEL-A-containing liposomes could accelerate gene transfection, Ueno et al., (2007a) examined MEL-containing liposomes properties such as their activity for DNA capsulation and membrane fusion abilities of cationic liposomes with artificial anionic liposomes. They observed that MEL-A-containing liposomes exhibited high activity in DNA incapsulation and membrane fusion with anionic liposomes, which are important properties for gene transfection. On the other hand, MEL-B- and MEL-Ccontaining liposomes only increased either the incapsulation or the membrane fusion. Ueno et al., (2007b) further examined the mechanism of the transfection mediated by cationic liposomes with NBD-conjugated MEL-A and reported that MEL-A distributed on the intracellular membranes through the plasma membranes of target cells, while the cationic liposomes with MEL-A fused to the plasma membranes within 20–35 min. Thereafter, they noted that the oligonucleotide released from the vesicles was immediately transferred to the nucleus. They therefore suggested that MEL-A was capable of promoting the transfection efficiency of target cells by inducing membrane fusion between liposomes and the plasma membrane of these cells.

Recently Kitamoto et al., (2009) demonstrated that monolayers composed of L-αdipalmitoylphosphatidylcholine (DPPC) containing MEL-A had greater membrane fluidity than those containing only DPPC. It was also reported that unsaturated fatty acids in MEL-A significantly influenced surface pressure and packing density in the monolayer and thus the physicochemical properties of MEL-A and MEL-A/lipids (Imura et al., 2008). Transfection efficiency of nano vectors with MEL-A was investigated *in vivo* on tumor cells in the mouse abdominal cavity (Inoh et al., 2009). When a complex of the nano vectors with MEL-A and plasmid DNA was injected intraperitoneally into C57BL/6J mice bearing B16/BL6 tumors, the biosurfactant significantly increased liposome-mediated gene transfection to the mouse tumor cells. The transfection efficiency of the plasmids into the solid tumors by the cationic liposomes of cholesteryl-3beta-carboxyamidoethylene-Nhydroxyethylamine (OH-Chol) with MEL-A increased by approximatley 100-fold compared to that by the commercially available DC-Chol cationic liposomes without MEL-A. This suggests that nonviral vectors with MEL-A are very useful for gene transfection *in vivo*. The mechanisms of gene delivery by nano vectors with MEL-A and the numerous biological activities of these biosurfactants have been described by Nakanishi et al., (2009) and Kitamoto et al., (2009).

Inoh et al., (2010) further investigated the effects of unsaturated fatty acid ratio within the MEL-A compound on the physicochemical properties and gene delivery into cells of cationic liposomes using MEL-A with three different unsaturated fatty acid (USF) component ratios. Gene transfer efficiency of cationic liposomes containing MEL-A (containing 21.5% USF) was much higher than that of those containing MEL-A (containing 9.1%USF) and MEL-A (containing 46.3%USF). In particular, MEL-A (21.5% USF)-containing cationic liposomes induced highly efficient membrane fusion after addition of anionic liposomes and led to subsequent DNA release.

Imaging analysis revealed that MEL-A (21.5% USF)-containing liposomes fused with the plasma membrane and delivered DNA into the nucleus of NIH-3T3 cells, MEL-A (46.3% USF)-containing liposomes fused with the plasma membrane did not deliver DNA into the nucleus, and MEL-A (9.1% USF)-containing liposomes neither fused with the plasma membrane nor delivered DNA into the nucleus. These results suggest that the MEL-A unsaturated fatty acid ratio significantly affects transfection efficiency due to changes in membrane fusion activity and the efficiency of DNA release from the liposomes.

Mannosylerythritol lipid-B (MEL-B) with a different configuration of the erythritol moiety was found to self-assemble into a lamellar phase over remarkably wide concentration and temperature ranges; furthermore it showed great potential as a vesicle-forming lipid, suggesting its potential application in drug and gene delivery as well as in transdermal delivery systems (Worakitkanchanakul et al., 2008). In another work, a liposome vector containing betasitosterol beta-D-glucoside biosurfactant-complexed DNA was successfully used for herpes simplex virus thymidine kinase gene therapy (Maitani et al., 2006).

### **4.2 Biosurfactants potential in drug delivery**

Properties such as detergency, emulsification, foaming and dispersion make biosurfactants interesting molecules with potential application in the field of drug delivery (Faivre & Rosilio, 2010). MEL-A for example has much higher emulsifying activity with soybean oil and tetradecane than polysorbate 80 (Kitamoto et al., 2009) and is able to form stable waterin-oil microemulsions without addition of co-surfactant or salt (Worakitkanchanakul et al., 2008).

Rhamnolipids and sophorolipids have also been mixed with lecithins to prepare biocompatible microemulsions in which the phase behavior was unaffected by changes in temperature and electrolyte concentration, making them desirable for cosmetic and drug delivery applications (Nguyen et al., 2010). In 1988, rhamnolipid liposomes were patented as drug delivery systems, useful as microcapsules for drugs, proteins, nucleic acids, dyes and other compounds, as biomimetic models for biological membranes and as sensors for detecting pH variations. These novel liposomes were described as safe and biologically decomposable, with suitable affinity for biological organisms, stable and with long service and shelf life.

The potential of lipopeptides, fengycin and surfactin to act as enhancers for the transdermal penetration and skin accumulation of aciclovir was also recently investigated (Nicoli et al., 2010) to elucidate any possible synergistic effect between surfactin and fengycin associated with anodal iontophoresis. It was demonstrated that these lipopeptides did not enhance aciclovir transport across the skin (not even when associated with iontophoresis) although they increased aciclovir concentration in the epidermis by a factor of 2 (Nicoli et al., 2010).

Microemulsion produced using biosurfactant are thermodynamically stable and their isotropic systems that form spontaneously-consisting of microdomains of oil or water stabilized by an interfacial film - in addition to their long-term stability, easy preparation and high solubilization capacity are considered to be very promising liquid vehicles for future drug delivery systems (Date et al., 2008, as cited in Faivre & Rosilio, 2010).
