Liposome-peptide constructs

Besides drug delivery, liposomes have also gained wide acceptance in other fields such as diagnostic imaging (4,5) and vaccines. In this chapter we will focus our attention specifically on the conjugation of peptides to liposomes. We will outline the major techniques involved, present some applications of liposomes-peptides constructs, and mainly discuss their use as vaccines. 

The two examples from our work we are going to describe below are the design and study of liposomal diepitope constructs combining either (i) B and T-helper (Th) peptide epitopes, which induced particularly powerful humoral responses (21) (Fig. 3) or (ii) CTL and Th epitopes, which provided a powerful antitumor vaccine (74) (Fig. 4). For the production of these constructs we have conjugated peptides that contain a cysteine residue either at the N- or C-terminus, to the surface of preformed liposomes by reaction with thiol reactive functionalized phospholipids and/or PamaCys lipopeptide anchors (Fig. 2). To that end, we have developed strategies that give, in aqueous media, high... 

TAT liposomes remain intact within one hour of translocation and slowly migrate through the cell, bypassing the endocytic pathway, to the perinuclear zone where they disintegrate (95). The mechanism utilized by TAT to migrate across the membrane was thought to be energy independent because it operates at similar rates at both 4°C and 37°C (95,96). Cell entry by TAT is also unhindered by metabolic inhibitors such as sodium azide or iodoacetamide (97). Peptides constructed of both the d and l amino acids of Antp can be detected intracellularly, the inference of which is that no specific receptor was required because both isomers had equal potential (98,99). 

The mode of association of peptides to liposome carriers might also be critical to induce a preferential immune response either humoral or cell mediated. For example, using a human mucin MUCl 20-mer peptide, it was found that only the physical association of the peptide to liposomes (either encapsulated or surface exposed after anchoring) was necessary to observe a cell-mediated response (34). In line with this observation, it was recently shown that a soluble peptide, representing a Melan-A/MART-1 tumor-associated antigen, when encapsulated into sterically stabilized liposomes, was able to stimulate a CTL response and this construct represented a suitable formulation for a specific tumor immunotherapy (69). In contrast, and in agreement with other studies (16), only the liposome surface exposed... 

Frisch B, Roth A, Schuber F. Synthetic peptide-based highly immunogenic liposomal constructs. Meth Enzymol 2003 373 51. 

Boeckler C, et al. Design of highly immunogenic liposomal constructs combining structurally independent B cell and T helper cell peptide epitopes. Eur J Immunol 1999 29 2297. 

Roth A, et al. Induction of effective and antigen-specific antitumor immunity by a liposomal ErbB2/HER2 peptide-based vaccination construct. Br J Cancer. In press. 

Construction of a Liposomal Carrier for Tyrosine-Related Protein-2 Peptide at Lab Scale... 

Preparation and characterization of liposomes formed with natural phospholipids were well established. However, in using liposomes for simulation of enzymatic functions, especially in acid-base catalysis, difficulties would be encountered due to their chemicai and morphological instabilities. Thus, bilayer membranes composed of synthetic amphiphiles are more favorable candidates for enzyme mimics. For example, artificial vitamin Bg-dependent enzymes were constructed from catalytic bilayer membranes in combination with a bilayer-forming peptide lipid (10), a hydrophobic vitamin derivative (11), and metal ions (Fig. 5). The catalyst acts as an artificial aminotransferase, showing marked substrate specificity, high enantioselectivity, and turnover behavior for the transamination of a-amino acid with a-keto acids. In addition, the reaction fields provided by the catalytic bilayer membranes are suitable to establish multienzyme systems through functional ahgnments of artificial enzymes and natural ones in a sequential manner. 

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