Lipid cell surface

In this chapter we describe the basic principles involved in the controlled production and modification of two-dimensional protein crystals. These are synthesized in nature as the outermost cell surface layer (S-layer) of prokaryotic organisms and have been successfully applied as basic building blocks in a biomolecular construction kit. Most importantly, the constituent subunits of the S-layer lattices have the capability to recrystallize into iso-porous closed monolayers in suspension, at liquid-surface interfaces, on lipid films, on liposomes, and on solid supports (e.g., silicon wafers, metals, and polymers). The self-assembled monomolecular lattices have been utilized for the immobilization of functional biomolecules in an ordered fashion and for their controlled confinement in defined areas of nanometer dimension. Thus, S-layers fulfill key requirements for the development of new supramolecular materials and enable the design of a broad spectrum of nanoscale devices, as required in molecular nanotechnology, nanobiotechnology, and biomimetics [1-3]. 

Plasmid DNA can be complexed electrostatically with cationic polymers. These complexes can be used for gene transfer [241]. Like the complexes of DNA with cationic lipids these complexes adhere to the cell surface with their cationic surface charges. Thereafter, they are internalized, presumably by adsorptive endocytosis. 

Lipophorin acts as a reusable shuttle between the membrane-bound lipophorin receptors in tissues (Tsuchida and Wells 1990, Gopalapillai et al. 2006) and is not generally endocytosed in the cells (Law and Wells 1989, Arrese et al. 2001, Canavoso et al. 2001). Thus, the intracellular CBP alone seems not to be able to pick up carotenoid from the lipophorin that resides outside of the cell. Cell surface components are thought to be necessary to allow intracellular delivery of carotenoids (Figure 24.6, magnification) (Arrese et al. 2001). The lipid transfer particle (LTP) (Blacklock and Ryan 1994, Tsuchida et al. 1997) on the outer surface of membranes and unknown membrane-spanning factors that specifically transfer carotenoids might be candidates. 

The other major class of extracellular LBPs of mammals is the lipocalins (Flower, 1996). These are approximately 20 kDa, P-sheet-rich proteins, performing functions such as the transport of retinol in plasma or milk, the capture of odorants in olfaction, invertebrate coloration, dispersal of pheromones, and solubilizing the lipids in tears (Flower, 1996). The retinol-binding protein (RBP) of human plasma is found in association with a larger protein, transthyretin, the complex being larger than the kidney threshold and thus not excreted, although the RBP itself may dissociate from the complex to interact with cell surface receptors in the delivery of retinol (Papiz et al., 1986 Sundaram et al., 1998). 

Other systems like electroporation have no lipids that might help in membrane sealing or fusion for direct transfer of the nucleic acid across membranes they have to generate transient pores, a process where efficiency is usually directly correlated with membrane destruction and cytotoxicity. Alternatively, like for the majority of polymer-based polyplexes, cellular uptake proceeds by clathrin- or caveolin-dependent and related endocytic pathways [152-156]. The polyplexes end up inside endosomes, and the membrane disruption happens in intracellular vesicles. It is noteworthy that several observed uptake processes may not be functional in delivery of bioactive material. Subsequent intracellular obstacles may render a specific pathway into a dead end [151, 154, 156]. With time, endosomal vesicles become slightly acidic (pH 5-6) and finally fuse with and mature into lysosomes. Therefore, polyplexes have to escape into the cytosol to avoid the nucleic acid-degrading lysosomal environment, and to deliver the therapeutic nucleic acid to the active site. Either the carrier polymer or a conjugated endosomolytic domain has to mediate this process [157], which involves local lipid membrane perturbation. Such a lipid membrane interaction could be a toxic event if occurring at the cell surface or mitochondrial membrane. Thus, polymers that show an endosome-specific membrane activity are favorable. 

A highly stable and shielded polyplex should circulate in the blood stream without undesired interactions until it reaches the target cell. At that location, specific interactions with the cell surface should trigger intracellular uptake. While lipid membrane interaction is undesired at the cell surface, it should happen subsequently within the endosomal vesicle and mediate polyplex delivery into the cytosol. During or after intracellular transport to the site of action, the polyplex stability should be weakened to an extent that the nucleic acid is accessible to exert its function. 

Proteins can be immobilized on the cell surface with the use of a short, single stranded DNA (ssDNA) attached to the end of a PEG chain (ssDNA-PEG-lipid) [114—116, 119, 125]. First, an ssDNA-PEG-lipid is prepared by conjugating... 

Fig. 9 Surface modification of cells with ssDNA-PEG-lipid. (a) Real-time monitoring of PEG-lipid incorporation into a supported lipid membrane by SPR. (r) A suspension of small unilamellar vesicles (SUV) of egg yolk lecithin (70 pg/mL) was applied to a CH3-SAM surface. A PEG-lipid solution (100 pg/mL) was then applied, (ii) Three types of PEG-lipids were compared PEG-DMPE (C14), PEG-DPPE (C16), and PEG-DSPE (C18) with acyl chains of 14, 16, and 18 carbons, respectively, (b) Confocal laser scanning microscopic image of an CCRF-CEM cell displays immobilized FITC-oligo(dA)2o hybridized to membrane-incorporated oligo(dT)20-PEG-lipid. (c) SPR sensorigrams of interaction between oligo(dA)2o-urokinase and the oligo (dT)2o-PEG-lipid incorporated into the cell surface, (i) BSA solution was applied to block nonspecific sites on the oligo(dT)20-incorporated substrate, (ii) Oligo(dA)20-urokinase (solid line) or oligo(dT)20-urokinase (dotted line) was applied...

The ssDNA-PEG-lipid provides versatility in cell surface modifications. It enables the immobilization of a broad spectrum of proteins and low molecular... 

Inui O, Teramura Y, Iwata H (2010) Retention dynamics of amphiphilic polymers PEG-lipids and PVA-Alkyl on the cell surface. ACS Appl Mater Interfaces 2 1514—1520... 

An additional virus that has more recently gained some attention as a possible vector is that of the sindbis virus. A member of the alphavirus family, this ssRNA virus can infect a broad range of both insect and vertebrate cells. The mature virion particles consist of the RNA genome com-plexed with a capsid protein C. This, in turn, is enveloped by a lipid bilayer in which two additional viral proteins (El and E2) are embedded. The E2 polypeptide appears to mediate viral binding to the surface receptors of susceptible cells. The major mammalian cell surface receptor it targets appears to be the highly conserved, widely distributed laminin receptor. 

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