Function-spacer-lipid Kode construct

Function-Spacer-Lipid Kode constructs are amphiphatic, water dispersible biosurface engineering constructs that can be used to engineer the surface of cells, viruses and organisms, or to modify solutions and non-biological surfaces with bioactives. FSL Kode constructs spontaneously and stably incorporate into cell membranes. FSL Kode constructs with all these aforementioned features are also known as Kode Constructs. The process of modifying surfaces with FSL Kode constructs is known as "koding" and the resultant "koded" cells, viruses and liposomes are respectively known as kodecytes, and kodevirions.

Technology description

All living surfaces are decorated with a diverse range of complex molecules, which are key modulators of chemical communications and other functions such as protection, adhesion, infectivity, apoptosis, etc. Functional-Spacer-Lipid Kode constructs can be synthesized to mimic the bioactive components present on biological surfaces, and then re-present them in novel ways.
The architecture of an FSL Kode construct, as implicit in the name, consists of three components - a functional head group, a spacer, and a lipid tail. This structure is analogous to a Lego minifigure in that, they have three structural components, with each component having a separate purpose. In the examples shown in all the figures, a Lego 'minifig' has been used for the analogy. However, it should be appreciated that this is merely a representation and the true structural similarity is significantly varied between Lego minifigures and FSL Kode constructs . The functional group of an FSL is equivalent to a Lego minifigure head, with both being at the extremity and carrying the character functional components. The spacer of the FSL is equivalent to the body of the Lego minifigure and the arms on the minifigure are representative of substitutions which may be engineered into the chemical makeup of the spacer. The lipid of the FSL anchors it to lipid membranes and gives the FSL construct its amphiphatic nature which can cause it to self-assemble. Because the lipid tail can act directly as an anchor it is analogous to the legs of a Lego minifigure.

Flexible design

The functional group, the spacer and the lipid tail components of the FSL Kode construct can each be individually designed resulting in FSL Kode constructs with specific biological functions. The functional head group is usually the bioactive component of the construct and the various spacers and lipids influence and effect its presentation, orientation and location on a surface. Critical to the definition of an FSL Kode construct is the requirement to be dispersible in water, and spontaneously and stably incorporate into cell membranes. Other lipid bioconjugates that include components similar to FSLs but do not have these features are not termed as Function-Spacer-Lipid Kode constructs.

Functional groups

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A large range of functional groups have already been made into FSL Kode constructs. These include:

Carbohydrates – ranging from monosaccharides to polysaccharides and including blood group antigens, hyaluronic acid oligomers and sialic acid residues
Peptide/protein – ranging from single amino acids to proteins as large as antibodies
Labels – including fluorophores, radioisotopes, biotin, etc.
Other – chemical moieties such as maleimide, click residues, PEG, charged compounds

Note 1: Multimeric – the presentation of the F residue can be as multimers with controlled spacing and be variable.
''Note 2: Mass – the mass that can be anchored by an FSL Kode constructs can range from 200 to >1 million Da''

Spacers

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The spacer is an integral part of the FSL Kode construct and gives it several important characteristics including water dispersibility.

Length – the spacer can be varied in length, for example 1.9 nm, 7.2 nm, 11.5 nm, allowing for enhanced presentation of Functional groups at the biosurface.
Optimizes 'F' presentation – The presentation of the bioactive on a spacer reduces steric hindrance and increases the bioactive surfaces exposed and available for interactions
Rigidity – the spacer can be modified to be either flexible or rigid depending upon desired characteristics
Substitutions – the spacer can be modified both in charge, and polarity.
Branches – usually the spacer is linear, but it can also be branched including specific spacing of the branches to optimize presentation and interaction of the F group.
Inert – important to the design of FSL Kode constructs is the biologically inert nature of the spacer. Importantly this feature means the S-L components of the constructs are unreactive with undiluted serum. Consequently, the constructs are compatible in vivo use, and can improve diagnostic assay sensitivity by allowing for the use of undiluted serum.
Lipids

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The lipid tail is essential for enabling lipid membrane insertion and retention but also for giving the construct amphiphilic characteristics that enable hydrophilic surface coating. Different membrane lipids that can be used to create FSLs have different membrane physiochemical characteristics and thus can affect biological function of the FSL. Lipids in FSL Kode constructs include:

Diacyl/diakyl e.g. DOPE
Sterols e.g. cholesterol
'''Ceramides'''
Optimising functional group (F) presentation

One of the important functions of an FSL construct is that it can optimise the presentation of antigens, both on cell surfaces and solid-phase membranes. This optimisation is achieved primarily by the spacer, and secondarily by the lipid tail. In a typical immunoassay, the antigen is deposited directly onto the microplate surface and binds to the surface either in a random fashion, or in a preferred orientation depending on the residues present on the surface of this antigen. Usually this deposition process is uncontrolled. In contrast, the FSL Kode construct bound to a microplate presents the antigen away from the surface in an orientation with a high level of exposure to the environment. Furthermore, typical immunoassays use recombinant peptides rather than discrete peptide antigens. As the recombinant peptide is many times bigger than the epitope of interest, a lot of undesired and unwanted peptide sequences are also represented on the microplate. These additional sequences may include unwanted microbial related sequences that can cause issues of low level cross-reactivity. Often the mechanism by which an immunoassay is able to overcome this low level activity is to dilute the serum so that the low level microbial reactive antibodies are not seen, and only high-level specific antibodies result in an interpretable result. In contrast, FSL Kode constructs usually use specifically selected peptide fragments, thereby overcoming cross-reactivity with microbial sequences, and allowing for the use of undiluted serum.
The F component can be further enhanced by presentation of it in multimeric formats and with specific spacing. The four types of multimeric format include linear repeating units, linear repeating units with spacing, clusters, and branching .

Mechanisms of interaction

Amphiphilic FSL Kode construct

The FSL Kode construct by nature of its composition in possessing both hydrophobic and hydrophilic regions are amphiphilic. This characteristic determines the way in which the construct will interact with surfaces. When present in a solution they may form simple micelles or adopt more complex bilayer structures with two simplistic examples shown in Fig. 5a. More complex structures are expected. The actual nature of FSL micelles has not been determined. However, based on normal structural function of micelles, it is expected that it will be determined in part by the combination of functional group, spacer and lipid together with temperature, concentration, size and hydrophobicity/hydrophilicity for each FSL Kode construct type.
Surface coatings will occur via two theoretical mechanisms, the first being direct hydrophobic interaction of the lipid tail with a hydrophobic surface resulting in a monolayer of FSL at the surface '. Hydrophobic binding of the FSL will be via its hydrophobic lipid tail interacting directly with the hydrophobic surface. The second surface coating will be through the formation of bilayers as the lipid tail is unable to react with the hydrophilic surface. In this case the lipids will induce the formation of a bilayer, the surface of which will be hydrophilic. This hydrophilic membrane will then interact directly with the hydrophilic surface and will probably encapsulate fibres. This hydrophilic bilayer binding is the expected mechanism by which FSLs are able to bind to fibrous membranes such as paper and glass fibres ' and .

Lipid membrane modification

After labeling of the surface with the selected F bioactives, the constructs will be present and oriented at the membrane surface. It is expected that the FSL will be highly mobile within the membrane and the choice of lipid tail will effect is relative partitioning within the membrane. The construct unless it has flip-flop behavior is expected to remain surface presented. However, the modification is not permanent in living cells and constructs will be lost at a rate proportional to the activity at the membrane and division rate of the cell. Additionally, when present in vivo with serum lipids FSLs will elute from the membrane into the plasma at a rate of about 1% per hour. In fixed cells or inactive cells stored in serum free media the constructs are retained normally.
Liposomes are easy koded by simply adding FSL Kode constructs into the preparation. Contacting koded liposomes with microplates or other surfaces can cause the labeling of the microplate surface.