**4.3 Nanoparticles**

350 Biomedical Science, Engineering and Technology

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

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

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).

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.,

Rhamnolipids and sophorolipids have also been mixed with lecithins to prepare biocompatible microemulsions in which the phase behavior was unaffected by changes in

**4.2 Biosurfactants potential in drug delivery** 

Kitamoto et al., (2009).

subsequent DNA release.

2008).

Another interesting application for natural surfactant is the the synthesis of metal-bound nanoparticles as an alternative environmentally friendly technology (Sharma et al., 2009). Nanomaterials synthesis and use has been an active research area due to interesting properties of the nanomaterials as compared to bulk material use (Palanisamy & Raichur, 2009). Metal nanoparticles have beening explored in various fields such as catalysis, mechano- and electrical applications and biomedical uses (Van Bogaert & Soetaert, 2010). The reduction in size gives rise to size dependent effects such as high surface to volume ratio, lower melting point, changes in electronic structure and changes in lattice structure and interatomic distances which in turn affect the processing parameters (Liveri, 2006, as cited in Palanisamy & Raichur, 2009).

The use of gold nanoparticles, in particular, is currently undergoing a dramatic expansion in the field of drug and gene delivery, targeted therapy and imaging technologies (Boisselier & Astruc, 2009; Pissuwan et al., 2009, 2011). Potential therapeutic applications of gold compound and gold nanoparticles also include anti-HIV activity, anti-angiogenesis, antimalarial activity, anti-arthritic activity and biohydrogen production (Kalishwaralal et al., 2010). Silver nanoparticles are also been reported to possess anti-fungal activity, antiinflammatory effect, anti-viral, anti-angiogenesis and anti-platelet activity (Kalishwaralal et al., 2010).

Reddy et al., (2009) successfully synthesized surfactin-mediated gold nanoparticles and investigated the effects of proton concentrations and temperature on the morphology of the obtained nanoparticles. It was demonstrated that the nanoparticles synthesized at pH 7 and 9 remained stable for 2 months, while aggregates were observed at pH 5 within 24 h. Moreover, the nanoparticles formed at pH 7 were uniform in shape and size and were polydispersed and anisotropic at pH 5 and 9. The nanoparticles synthesized produced at room temperature were monodispersed and were more uniform when compared to those formed at 4°C. More recently they also carried out a biological synthesis of gold and silver nanoparticles using the bacteria *Bacillus subtilis.* Gold nanoparticles were synthesized both intra- and extracellularly, while silver nanoparticles were exclusively formed extracellularly (Reddy et al., 2010). According to the Authors the nanoparticles were stabilized by the surface-active molecules i.e., surfactin or other biomolecules released into the solution by *B. subtilis*.

Surfactin produced by *Bacillus amyloliquefaciens* KSU-109 was also used for the synthesis of cadmium sulfide nanoparticles which remained stable up to six months without compromising their functionality (Singh et al., 2011). This kind of nanoparticles works as semiconductors with unique optical properties and tunable photo-luminescence allowing potential applications in solar energy conversion, nonlinear optical, photoelectrochemical cells and heterogeneous photocatalysis (Singh et al., 2011). In addition, surfactin produced by strain KSU-109 was easily extracted and used without further purification for nanoparticles stabilization under ambient conditions (Singh et al., 2011). Such simple, inexpensive and environmental friendly procedure of obtaining surfactin offers a further advantage of use in nanobiotechnology for the large-scale production of highly stable metal nanoparticles.

Both rhamnolipids and sophorolipids have also been successfully used for the synthesis and stabilization of metal-bound nanoparticles. Purified rhamnolipids from *P. aeruginosa* strain BS-161R were used to synthesize silver nanoparticles which exhibited good antibiotic activity against both Gram-positive and Gram-negative pathogens and *Candida albicans*, suggesting their broad spectrum antimicrobial activity (Kumar et al., 2010). In another work, a glycolipid biosurfactant produced from sponge-associated marine bacteria *Brevibacterium casei* MSA19, using agro-industrial and industrial waste as substrate, were used as a "green" stabilizer for the synthesis of stable and uniform silver nanoparticles (Kiran et al., 2010b). The biosurfactant acted as stabilization agent and prevented the formation of aggregates.

Palanisamy & Raichur, (2009) also described a simple and eco-friendly method for synthesizing spherical nickel oxide nanoparticles by microemulsion technique using rhamnolipids as alternative surfactant. The synthesized nanoparticles were found to be fully crystalline and spherical in shape with uniform distribution and increasing the pH of the solution decreased the size of the nanoparticles. Xie et al., (2006) were also able to synthesize silver nanoparticles in rhamnolipid reverse micelles while in another study rhamnolipids were used as capping agents for the synthesis of ZnS nanoparticles in aqueous medium (Narayanan et al., 2010).

Sophorolipids were also tested for use in nanoparticles synthesis and reported to be good reducing and capping agents for cobalt and silver particles (Kasture et al., 2007, 2008, as cited in Van Bogaert & Soetaert, 2010). Singh et al., (2009) demonstrated the antibacterial activity of sophorolipid-coated silver and gold nanoparticles against both Gram-positive and –negative bacteria. They also verified that sophorolipid-coated gold nanoparticles were more cyto and geno-compatible with respect to silver nanoparticles (Singh et al., 2010). They also plan to investigate these nanoparticles suitability for medical and diagnostic applications.

Recently, methodologies for the biological synthesis of metal nanoparticles using microbes have also been described (Narayanan & Sakthivel, 2010; Kalishwaralal et al., 2010; Reddy et al., 2010). In addition Smyth et al., (2010b) reported on the production of selectively deuterated rhamnolipids and sophorolipids using deuterated substrates. The production of such deuterated biosurfactants, in particular, or other bioactive microbial products in general, in which distinct pattern of labeling could be achieved resulting in varing molecular weight products and or stereochemistry unrecognised by existing degradative enzymes is very improtant. Such molecules would have great future implications with regards to efficacy and/or persistence or the development of resistance for some bioactives particularly in biomedical related applications.
