**4. Metallochelating bond and its application for construction of proteoliposomes**

With respect to a potential application for the construction of vaccines, the question of *in vitro* and especially *in vivo* stability is of great importance. This problem could be divided into two fields. First, the stability of the liposomes themselves and second, the effect of the components presented in biological fluids (e.g., proteins and ions) on the stability of the metallochelating bond. It is beyond the scope of this chapter to address thoroughly this particular question. However, the GPC data indicate a good *in vitro* stability of the proteoliposomes containing recombinant His-tagged Outer surface protein C from *Borrelia burgdorferi* (rOspC) designated rOspC–HisTag during the chromatographic process, within which they experience a shear stress and dilution.

Also, the data on the incubation of rOspC-HisTag proteoliposomes in serum at 37 °C demonstrated the stability of the metallochelating bond linking the protein to the liposomal surface. In fact, *in vivo* fate of liposomes after the intradermal administration is different than that following an intravenous injection. First, dilution of proteoliposomes is not so rapid and second, the ratio of tissue fluid proteins to proteoliposomes is more favourable to proteoliposomes owing to their relatively high concentration at the site of application. Moreover, the flow rate of the tissue fluid within intradermal extracellular

Fig. 10. Transformation of micelles into liposomes during ultraltration monitored by DLS. The dashed vertical line indicates the ultraltrate volume, when the CMC of sodium cholate

With respect to a potential application for the construction of vaccines, the question of *in vitro* and especially *in vivo* stability is of great importance. This problem could be divided into two fields. First, the stability of the liposomes themselves and second, the effect of the components presented in biological fluids (e.g., proteins and ions) on the stability of the metallochelating bond. It is beyond the scope of this chapter to address thoroughly this particular question. However, the GPC data indicate a good *in vitro* stability of the proteoliposomes containing recombinant His-tagged Outer surface protein C from *Borrelia burgdorferi* (rOspC) designated rOspC–HisTag during the chromatographic process, within

Also, the data on the incubation of rOspC-HisTag proteoliposomes in serum at 37 °C demonstrated the stability of the metallochelating bond linking the protein to the liposomal surface. In fact, *in vivo* fate of liposomes after the intradermal administration is different than that following an intravenous injection. First, dilution of proteoliposomes is not so rapid and second, the ratio of tissue fluid proteins to proteoliposomes is more favourable to proteoliposomes owing to their relatively high concentration at the site of application. Moreover, the flow rate of the tissue fluid within intradermal extracellular

was reached. This line divides the ow-through volume axis into the leftpart, where micelles do predominantly exist and are transformed into liposomes, and the right part, where liposomes represent the main lipid form, while residual detergent and ethanol/THF

**4. Metallochelating bond and its application for construction of** 

are continuously removed by the process of ultraltration.

which they experience a shear stress and dilution.

**proteoliposomes** 

matrix is considerably lower than that of the muscle tissue or blood vessels. This fact is often overlooked. The stability of metallochelating bond probably depends also on the character of a particular protein. The study by Ruger shows that the single-chain Fv Ni-NTA-DOGS liposomes are unstable in human plasma and the majority of single-chain Fv fragments (anti CD 105) are released from the liposomal surface, which results in a lost of the specific targeting performance to the cells expressing a surface protein endoglin (CD 105) (Ruger et al., 2006). On the other hand, Ni-NTA3 –DTDA liposomes with single-chain Fv fragments (anti CD11c) bound onto the liposomal surface were able to target dendritic cells *in vitro* as well as *in vivo*. The application of the three-functional chelating lipid Ni-NTA3 –DTDA probably endows the metallochelating bond with a higher *in vivo* stability (van Broekhoven et al., 2004). Application of Ni-NTA-DOGS liposomes for the construction of experimental vaccine against sytemic *Candida* infection based on *Candida* Heat shock protein 90 kDa (rHSP90-HisTag) showed good stability in serum as well as strong immune response against recobinant rHSP90-HisTag antigen in mice (Masek et al., 2011b).

*In vivo* activity (immunogenicity) was also demonstrated for antigens associated with ISCOM particles via metallochelating lipid dipalmitoyliminodiacetic acid (Malliaros et al., 2004) and a peptide antigen associated with liposomes via Ni-NTA-DOGS (Chikh et al., 2002). Generally, metal ions, physico-chemical character of the metallochelating lipids and their surface density on the particles belong to the factors that could be optimized to get a required *in vivo* stability and, therefore, a strong immune response. The design and synthesis of new metallochelating lipids might accelerate a development and application of metallochelating liposomes for the construction of drug delivery systems and vaccines. Besides Ni2+, other divalent ions such as Zn2+, Co2+, Fe2+, and Cu2+, have to be considered and experimentally tested as well.

Fig. 11. Schematic illustration of recombinant His-Tagged protein bound onto the surface of metallochelating liposome (A) and formulae of metallochelating lipids NTA-DOGS (B), NTA–DTDA (C), trivalent NTA3 –DTDA (D).
