Liposomes on self-service

A prototype of a cell capable of synthesizing components of its own membrane has been created

Anna Guseva, "Elements"

Fig. 1. An artificial cell is a synthetic biopolymer capsule containing molecules that perform certain functions. The minimal set of components of an artificial cell includes a cell membrane, information carrier molecules (DNA or RNA) and a system that allows the cell to produce proteins. Although full-fledged artificial cells have not yet been created, scientists are actively working in this direction. Drawing from the website cell-free.org .

Living cells have been successfully used to solve many biotechnological problems for a long time. But the cell is a very complex system. Its behavior depends on external conditions, so it does not always obey the instructions of scientists who are trying to turn a cell, for example, into a factory for the production of medicines. Therefore, biotechnologists are trying to develop artificial cell analogues containing the minimum possible set of genes serving a specific purpose. Scientists from the Netherlands have taken an important step towards solving this problem: they created a prototype of a cell synthesizing lipids, which make up its membrane.

The term "biotechnology" appeared only in the XX century, but it can be assumed that the development of this discipline began in ancient times, when people first began to cook bread, wine, cheese and other products that use microorganisms. A real breakthrough in the development of biotechnology occurred in the 70s of the XX century, when scientists learned to directly change the DNA of living organisms. Since then, genetically modified cells of bacteria, plants and animals have found many useful applications. As successful examples, we can cite organisms that synthesize drugs and biofuels. Thus, the bacterium Escherichia coli, "programmed" for the synthesis of insulin, helped to establish a wide industrial production of this hormone, providing diabetic patients with the necessary medicine at an affordable price. And acetogenic bacteria (microorganisms that secrete acetate during anaerobic respiration) have been adapted for the production of ethanol, acetone and butanol, which are used as fuel components.

But not all attempts to turn cells into living factories or adapt them for other applications have been so successful. The fact is that in addition to executing the genetic program that scientists add to the cell (for example, the instructions "Synthesize insulin!"), the cell simultaneously follows hundreds of other instructions from its own genetic apparatus ("Multiply!", "Look for food!", "Protect yourself from danger!"). These parallel processes may prevent her from doing what scientists need. Moreover, during its lifetime, a cell can mutate or change our instructions in unexpected ways. And for the use of such cellular machines, for example, in medicine, high reliability and a guarantee against any surprises are required.

An alternative to using living cells is to create simplified analogues of them, which instead of a complete genome contain only a small collection of necessary genes. One of the first examples of this approach was cell-free systems. They are extracts containing everything necessary for protein synthesis: ribosomes, polymerases and other components of transcription and translation (Y. Lu, 2017. Cell-free synthetic biology: Engineering in an open world). The components of cell-free systems can be stored in solution or in freeze-dried (frozen and dried) form. If you mix the solutions in certain proportions or add water to the dried components, and then add DNA, then the purified proteins will begin to follow the instructions encoded in the genes.

Cell–free systems are often used to study the regulation of gene activity, to synthesize modified proteins, and to create biosensors - biological systems that can, for example, detect toxic substances in water or measure the level of certain trace elements in a blood sample (A. Tinafar et al., 2019. Synthetic Biology Goes Cell-Free). But the disadvantage of cell-free systems is that they cannot grow and reproduce independently and do not have a barrier separating and protecting them from the external environment. This means that cell-free systems are difficult to use outside the laboratory: unlike living cells, they cannot regenerate and quickly fail under adverse conditions.

To make a cell-free system more like a cell, you can surround it with a membrane. The cell membrane consists of proteins and lipids – molecules that do not dissolve in water, but are grouped together, forming a double layer that protects the contents of the cell from the external environment. Some researchers have already managed to create membrane capsules containing cell-free systems. In one of the first papers on this topic, scientists from Princeton University managed to enclose components for the production of green fluorescent protein in a lipid envelope (V. Noireaux, A. Libchaber, 2004. A vesicle bioreactor as a step towards an artificial cell assembly). In another study, lipid capsules containing genes for the production of proteins capable of destroying cancer cells were created (N. Krinsky et al., 2018. Synthetic Cells Synthesize Therapeutic Proteins inside Tumors). These results may have interesting applications – for example, such capsules can be injected into the tumor area so that they locally produce the drug. But how to make artificial cells regenerate – increase in volume, and subsequently divide, creating their own copies? Researchers from Delft Technical University in the Netherlands tried to answer this question.

The authors of an article published recently in the journal Nature Communications decided to create an artificial cell that can synthesize two types of lipids often found in bacterial membranes: phosphatidylethanolamine and phosphatidylglycerol. They used glycerol-3-phosphate and acetyl-CoA molecules as initial components for lipid synthesis.

The conversion of the initial synthesis components into phospholipids occurs in several stages. A special enzyme is responsible for each of them. The researchers created a mini-genome with the genes necessary for the production of these enzymes (Fig. 2).

Fig. 2. Synthesis of phospholipids using enzymes encoded in the mini-genome of an artificial cell. The mini-genome contains seven genes necessary for the synthesis of phosphatidylethanoamine (PG) and phosphatidylglycerol (PE). The enzymes are embedded in the liposome membrane, where the synthesis of new lipids takes place. The main reaction products are highlighted in bold, and the names of enzymes are highlighted in color. Enzymes involved in the reaction: glycerol-3-phosphate-acyltransferase (PlsB), lysophosphatidic acid-acyltransferase (PlsC), CdsA integral membrane protein, CDP-diacylglycerol-glycerol-3-phosphate-3-phosphatidyl transferase (PgsA), phosphatidylglycerol phosphatases A, B, C (PgpA, PgpB, PgpC). A drawing from the article under discussion in Nature Communications.

Components of the cell-free system and pre-prepared lipids were added to the mixture containing copies of the genome. They form lipid vesicles (liposomes) – scaffolds to which new lipids created by enzymes will be attached. As a result, lipid bubbles containing a mini-genome and everything necessary for gene expression were formed in the mixture (Fig. 3). It is worth noting that copies of the genome are randomly distributed in solution. Some of them are inside the bubbles, and some are outside. But since the bubbles occupy a significant part of the volume of the solution, many of them turn out to be filled with all the necessary components.

Fig. 3. The scheme of the experiment on the creation of artificial cells synthesizing lipids – components of membranes. Copies of the genome, lipids, components of the cell-free system and initial molecules for lipid synthesis are added to the mixture. As a result, liposomes are formed containing enzymes that produce new lipids (blue). Due to the random distribution of components, some reactions may take place outside of liposomes.

To test whether their idea works, the authors replaced one of the carbon atoms in the original molecules with a heavier isotope of this element. This helped them distinguish new lipids from those already present in liposomal scaffolds. Then they applied the method of mass spectrometry, which allows the identification of molecules based on information about their weight and charge. The results of this experiment showed that new lipids were indeed formed in the mixture.

But the question of the effectiveness of artificial cells remained open. In the experiment described above, the total level of new lipids in the mixture was measured and it was not taken into account which proportion of all reactions occurred inside liposomes and which outside. After the liposomes were formed, the authors added the enzymes protease and DNase to the mixture, which destroy proteins and DNA that are not protected by liposomes. This helps to avoid lipid synthesis outside of liposomes.

To confirm that the proteins are now only inside artificial cells, the researchers labeled their membranes with a red fluorescent protein and added DNA of a yellow fluorescent protein (Fig. 4). Like copies of the mini-genome, these DNA were randomly distributed in the mix. The yellow signal indicated the protein localization areas, helping to make sure that the proteins outside the liposomes were destroyed. Measurements of the concentration of new lipids made exclusively in liposomes showed that the synthesis has a high efficiency: 40% of the initial products were successfully converted into lipids.

Fig. 4. Image of liposomes obtained by fluorescence microscopy. Liposome membranes are marked with a red fluorescent protein (they look like purple rings). The DNA of a yellow fluorescent protein was added to the reaction. Without the addition of protease and DNase, gene expression occurs both inside and outside liposomes (left). In the presence of protease (in the center) or DNase (on the right), gene expression is localized inside liposomes. A drawing from the article under discussion in Nature Communications.

At the next stage, the authors tried to answer several important questions: what percentage of liposomes produce new lipids? How fast is the synthesis? How do liposomes differ from each other? To track the synthesis of new lipids, the researchers used a green fluorescent protein. With its help, they created a marker that was attached to the liposome membrane at the place where a new lipid appeared (Fig. 5). As new lipids were synthesized, the liposome membranes began to fluoresce green.

Fig. 5. Identification of liposomes synthesizing new lipids. The fluorescent sample is attached to the membrane at the place where the new lipids are embedded. As they are synthesized, the fluorescence brightness increases, allowing the identification of "active" liposomes. A drawing from the article under discussion in Nature Communications.

The authors traced how the fluorescence of individual liposomes changes over time. They noticed that the brightness gradually increases and reaches its maximum value after 16 hours (Fig. 6, left). But it turned out that only about half of the liposomes produced new lipids (Fig. 6, right). This is because the components needed for synthesis did not get into some liposomes. Although 50% is a good result for the first attempt, in the future the authors hope to increase the efficiency of their technology.

Fig. 6. On the left – dynamics of phospholipid synthesis and addition of a fluorescent sample. Observations using fluorescence microscopy allow you to track the synthesis of lipids in real time. The fluorescent sample is attached to the membrane at the place where the new lipids are embedded. An increase in the brightness of the green fluorescent protein corresponds to an increase in the concentration of new lipids in the membrane. On the right is the distribution of liposomes producing new lipids. Analysis of the full image allows you to determine the percentage of liposomes that synthesize new lipids. Images from the discussed article in Nature Communications.

At the next stage, researchers will need to find a way to create an artificial cell that can significantly increase in volume and divide. To do this, they propose to improve the cell-free system and increase the concentration of enzymes that produce lipids. And when the cell reaches the desired volume, division will occur automatically (some bacteria have such a rare mechanism of division, see R. Mercier et al., 2013. Excess Membrane Synthesis Drives a Primitive Mode of Cell Proliferation). This process can be regulated with the help of temperature fluctuations that provoke deformation of the membrane.

The results described in the article under discussion are an important step in the work on creating a full–fledged artificial cell. They show that the entire chain of reactions necessary for the synthesis of membrane lipids can be placed inside liposomes. In addition, the artificial cell design created by the authors can be used as a platform for the production of various types of lipids useful in pharmaceuticals and industry: components of ointments, emulsions, coatings and paints.

It is hoped that advances in the creation of artificial cells in the future will help to build reliable bioreactors for the cheap production of complex chemical compounds. Artificial cells can serve as the basis for biosensors that work in conditions incompatible with the survival of ordinary cells, and will also be useful for creating new methods of treatment and diagnosis of diseases – for example, as a tool for local synthesis and targeted drug delivery.

A source:
Blanken et al., Genetically controlled membrane synthesis in liposomes // Nature Communications, 2020.

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