Lipids for vesicular nanobots

Hello there, this is Michael! In my previous blog posts, I have given you sneak peeks into my day-to-day activities here at the Dietz lab in Munich. To contribute to our online lecture series, I would like to give you a deeper and more detailed view into the science and logics behind my work. What are lipid vesicles? What are lipids? What’s that got to do with DNA? And why does it matter?

I am using DNA origami nanostructures to pinch off small vesicles from giant ones. The resulting structures resemble actual virus particles in shape and size, and the general mechanism by which they are created is very similar, too! Viruses are genetic material in the form of DNA or RNA encapsulated within a protein shell, which in some viruses is itself encapsulated within a so-called lipid vesicle. DNA Origami is a powerful technique to construct defined shapes on the nano scale, but it does have limitations. Just how modern houses are composed of more than one building material, depending on its intended use it may also be beneficial to construct nanomaterials from more materials than just DNA. This is where lipids come into play.

Lipids are so called amphiphilic molecules. In the biosciences, a very important property of a chemical substance is its solubility in water. Some substances like glucose or table salt are highly soluble in water, whereas other substances hardly dissolve in water at all. The same applies to liquids. Drinking alcohol (chemically known as ethanol) will easily mix with water allowing us to mix alcoholic beverages, but other liquids like oils form separate liquid phases. The latter is a common problem in the kitchen when preparing salad dressings. A vinaigrette, for example, is not just a mixture of oil and vinegar, but recipes also include seemingly random ingredients such as egg yolk or mustard. While both of these ingredients surely add to the taste, the more chemical reasoning is that both, egg yolk and mustard, contain so called emulsifiers. Emulsifiers are substances belonging to the group of “surfactants” which make it possible to stably mix normally immiscible liquids like oil and water. To understand these weird mixing behaviours, we have to look at the molecular structures and properties of the participating chemical compounds.

Water, that is H2O, is made up of two hydrogen atoms bound to a central oxygen atom. To better understand its properties, we can check the periodic table for the so called “electronegativity” of its components. Electronegativity is the property of each given atom that describes its pulling force on the electrons around it. The higher the electronegativity, the greedier for electrons an atom is. Hydrogen has a very average electronegativity of 2.2, whereas oxygen at 3.4 has the second highest value in the periodic table. This means that oxygen is pulling much harder on the electrons it shares with its two hydrogen atoms, resulting in an electron distribution within the H2O molecule that is skewed towards oxygen. In other words: the electrons of the hydrogens will on average be closer to the oxygen, so that it becomes slightly negatively charged, while the two hydrogen atoms carry a slight positive charge. The net charge of water is still neutral, but this skewed charge distribution within the molecule makes water “polar”. The opposite case is when the difference in electronegativity of the constituent atoms of a chemical compound is very small, leading to a more even charge distribution and thus a “non-polar” molecule. By knowing the polarity of a molecule, one can try to predict its solubility or miscibility in different solvents. Polar solvents like water will readily dissolve polar molecules like sugar, while non-polar solvents preferably dissolve non-polar molecules. Other words to describe this behaviour are hydrophilicity (ancient greek for “love for water”) and hydrophobicity (ancient Greek for “fear of water”). Hydrophilic substances will dissolve in water while hydrophobic substances will not. However, in practice polarity is not a label stating whether a molecule will either dissolve in water or not, but it is more like a gradual scale where non-polar molecules may also dissolve in water, albeit to a much lesser extent than polar molecules. The greater structure of a molecule must also be taken into account. Let me give you an example: The acid in vinegar, acetic acid, has a highly polar -COOH group coupled to a (mostly) non-polar -CH3 group. Still, vinegar is miscible with water. However, if one extends the molecule by inserting a chain of 14 non-polar -CH2– groups between the CH3– and -COOH groups, one gets palmitic acid, a very common component of oils that is generally immiscible with water (Figure 1). In this case, the non-polar hydrocarbon chain dominates the molecule and defines its dissolution behaviour more than the polar -COOH group.

Figure 1: Chemical structures of acetic acid and palmitic acid.

Phew, so what is the deal with emulsifiers? Why can they create stable mixtures (“emulsions”) of water and oil in a vinaigrette? Emulsifiers contain both: a non-polar part and a polar part. Unlike fatty acids such as palmitic acid, a surfactant is generally composed of a significant hydrophobic and a significant hydrophilic part. As such, surfactants are soluble in both, water and oil, and are therefore also called “amphiphiles” (ancient Greek for “love for both”). When preparing a vinaigrette, the hydrophilic portion of the emulsifier will orient itself towards the vinegar, while the hydrophobic part will orient towards the oil phase. The result is a water-in-oil emulsion, which is oil with a large number of tiny water droplets in it that are stabilised by a coat of emulsifiers. In absence of an emulsifier, the water droplets would just separate from the oil according to the density difference between both components and merge to form another aqueous vinegar phase.

In similarity of emulsifiers, lipids are hydrophobic or amphiphilic biomolecules playing many roles in an organism. The type of lipid I am working with is phospholipids, which are the main constituents of cell membranes. Phospholipids are amphiphile molecules that are composed of two fatty acids (like palmitic acid we discussed above) chemically linked to a phosphorus containing hydrophilic head group. This amphiphilic property of phospholipids means that, when exposed to water, the fatty acid chains of different phospholipid molecules will aggregate to minimise their contact with water. The result ranges from micelles (tiny balls made up entirely of phospholipids) to liposomes (hollow spheres of phospholipids where the fatty acid chains of opposing phospholipids point at one another, creating a so-called lipid bilayer) (Figure 2).

Figure 2: Schematic illustration of lipid bilayer.

If a liposome is truly empty and consists only out of a single bilayer lining it, it is termed “unilamellar”. An oversimplified description of a cell could be that of a giant unilamellar vesicle – or GUV for short. Of course, a cell is not that simple, and because of its membrane bound organelles, it is also not unilamellar. Nevertheless, GUVs are interesting model systems in synthetic biology and biophysics. The definition of a GUV is that of a lipid vesicle with a diameter of 1 µm or larger that does not contain smaller lipid vesicles on its inside. GUVs are differentiated from small unilamellar vesicles (SUVs), which are smaller than 0.1 µm and large unilamellar vesicles (LUVs), which bridge SUVs and GUVs in size (Figure 3).

Figure 3: Schematic illustrations of unilamellar vesicles and multilamellar vesicles including indications of size ranges in nm.

Size-wise, GUVs are comparable to biological cells, whereas SUVs are comparable to virus particles. Many virus particles, or virions, are created by pinching off a small lipid vesicle from the cell membrane. Viruses do this all the time, including the most famous SARS-CoV-2 that dominated our lives this year, but we have yet to construct a fully synthetic system that is able bud off SUVs from GUVs like a natural virus. This is what I have been working on for the last 2.5 years now, using DNA origami as a tool (Figure4). In this synthetic approach, the DNA origami nanostructures take over the role of proteins in natural viruses, which define the general shape of the virus and also trigger the budding process. While viruses aim to insert their genetic material into host cells, one could use nanobots made from DNA to introduce therapeutics into a cell. The problem here is that a DNA origami container is comparable to sieve: Large particles can be sealed into the DNA origami container, but small ones will be able to diffuse out of it.

Figure 4: Programmed folding of DNA into defined shapes and structures.

This limits the potential use of drug delivery systems made out of DNA to large compounds although many interesting therapeutics are, in fact, very small. This is where lipids step in. A lipid bilayer acts as a more or less tight barrier to many molecules but is mostly passive by itself. In biology, proteins that are integrated into the lipid bilayer give it a diverse array of functions. It is more feasible to use lipid vesicles as cargo carriers, and DNA origami structures for more complex functions such as sensing or actuation. By coupling DNA strands to hydrophobic molecules such as cholesterol, it is possible to tether origami structures onto lipid vesicles. We are not only interested in producing cargo carriers, but we are also interested in copying the budding mechanism we often see in nature. This would not only allow us to understand these processes better on a biophysical level, but at the same time we can demonstrate the versatility of DNA nanotechnology as a tool in synthetic biology. The goal could be an intelligent nanobot, whose functions are carried out by individual origami structures. An artificial budding system would be an exciting puzzle piece that would further expand the possible future applications of DNA nanotechnology.

I hope you enjoyed my article. If everything goes well, my next article might be a research paper. Keep your fingers crossed for me! 🙂

Michael

This online Lecture was written by Michael Pinner from Dietz group at TUM Munich.

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