The team of researchers at Saarland University, led by Professor of Condensed Matter Physics Karin Jacobs, initially had something quite different in mind. Originally, the team set out to research and describe the characteristics of hydrophobins – a group of naturally occurring proteins. ‘We noticed that the hydrophobins form colonies when they are placed in water. They immediately arrange themselves into tightly packed structures at the interface between water and glass or between water and air,’ explains Karin Jacobs. ‘There must therefore be an attractive force acting between the individual hydrophobin molecules, otherwise they would not organize themselves into colonies.’ But Professor Jacobs, research scientist Dr Hendrik Hähl and their team did not know how strong this force was.
This is where the neighbouring research group led by Professor Ralf Seemann got involved. One of Seemann’s research teams, which is headed by Dr Jean-Baptiste Fleury, studies processes that occur at the interfaces between two liquids. The research team set up a minute experimental arrangement with four tiny intersecting flow channels, like a crossroads, and allowed a stream of oil to flow continuously from one side of the crossing to the other. From the other two side channels they injected ‘fingers’ of water which protruded into the crossing zone. As the hydrophobins tended to gather at the interface of the carrier medium, they were in this case arranged at the water-oil interface at the front of the fingers. The physicists then ‘pushed’ the two fingers closer and closer together in order to see when the attractive force took effect. ‘At some point the two aqueous fingers suddenly coalesced to form a single stable interface consisting of two layers,’ says Ralph Seemann. ‘The weird thing is that it also functions the other way around, that is, when we use oil fingers to interrupt a continuous flow of water,’ he explains. This finding is quite new, as up until now other molecules have only exhibited this sort of behaviour in the one or the other scenario. Normally proteins will orient themselves so that either their hydrophilic (‘water loving’) sides are in contact with the aqueous medium, or their hydrophobic (‘water fearing’) side is in contact with an oily medium. That a type of molecule can form stable bilayers in both environments is something wholly new.
Encouraged by these findings, the researchers decided to undertake a third phase of experiments to find out whether the stable bilayer could be reconfigured to form a small membrane-bound transport sac – a vesicle. They attempted to inflate the stable membrane bilayer in a manner similar to creating a soap bubble, but using water rather than air. The experiment worked. The cell-like sphere with the outer bilayer of natural proteins was stable. ‘That’s something no one else has achieved,’ says Jean-Baptiste Fleury, who carried out the successful experiments. Up until now it had only been possible to create monolayer membranes or vesicles from specially synthesized macromolecules. Vesicles made from a bilayer of naturally occurring proteins that can also be tailored for use in an aqueous or an oil-based environment are something quite new.
In subsequent work, the research scientists have also demonstrated that ion channels can be incorporated into these vesicles, allowing charged particles (ions) to be transported through the bilayer of hydrophobins in a manner identical to the way ions pass through the lipid bilayers of natural cells.
As a result, the physicists now have a basis for further research work, such as examining the means of achieving more precisely targeted drug delivery. In one potential scenario, the vesicles could be used to transport water-soluble molecules through an aqueous milieu or fat-soluble molecules through an oily environment. Dr Hendrik Hähl describes the method as follows: ‘Essentially we are throwing a vesicle “cape” over the drug molecule. And because the “cape” is composed of naturally occurring molecules, vesicles such as these have the potential to be used in the human body.’
The results of this research work were a surprise. Originally, the goal was simply to measure the energy associated with the agglomeration of the hydrophobin molecules when they form colonies. But the discovery that hydrophobin bilayers could be formed in both orientations, opened the door to experiments designed to see whether vesicles could be formed. That one thing would lead to another in this way, offers an excellent example of the benefits of this type of basic, curiosity-driven research. ‘The “discovery” of these vesicles is archetypal of this kind of fundamental research. Or to put it another way, if someone had said to us at the beginning: “Create these structures from a natural bilayer,” we very probably wouldn’t have succeeded,’ says Professor Karin Jacobs in summary.
Researchers funded in part by NIBIB have recently shown that magnetic bacteria are a promising vehicle for more efficiently delivering tumor-fighting drugs. They reported their results in the August 2016 issue of Nature Nanotechnology.
One of the biggest challenges in cancer therapy is being able to sufficiently deliver chemotherapy drugs to tumors without exposing healthy tissues to their toxic effects. One way researchers have attempted to overcome this is by developing nanocarriers—extremely small particles packed with drugs. The nanocarriers are designed so they’re only taken up by cancer cells, thereby preventing the drugs from being absorbed by healthy tissues as they travel through the body’s circulation.
Yet while nanocarriers do a good job protecting healthy tissues, the amount of drug successfully delivered to tumors remains low. The main reasons for this shortcoming are that nanocarriers rely on the circulation system to carry them to the tumor, so a large percentage are filtered out of the body before ever reaching their destination. In addition, differences in pressure between the tumor and its surrounding tissue prevent nanocarriers from penetrating deep inside the tumor. As a result, nanocarriers aren’t able to reach the tumor’s hypoxic zones, which are regions of active cell division that are characterized by low oxygen content.
“Only a very small proportion of drugs reach the hypoxic zones, which are believed to be the source of metastasis. Therefore, targeting the low-oxygen regions will most likely decrease the rate of metastasis while maximizing the effect of a therapy,” says Sylvain Martel, Ph.D., Director of the Polytechnique Montréal NanoRobotics Laboratory and lead researcher of the study.
Martel and his research team were attempting to develop robotic nanocarriers that would travel to hypoxic zones when they realized nature may have already created one in the form of a bacteria called magnetococcus marinus or MC-1. MC-1 cells thrive in deep waters where oxygen is sparse. In order to find these areas, the bacteria rely on a two-part navigation system. The first part involves a chain of magnetic nanocrystals within MC-1 that acts like a compass needle and causes the bacteria to swim in a north direction when in the Northern Hemisphere. The second part consists of sensors that allow the bacteria to detect changes in oxygen levels. This unique navigation system helps the bacteria migrate to and maintain their position at areas of low oxygen.
With funding support from NIBIB and others, Martel’s research team conducted a series of experiments to show that the bacteria’s unique navigation system could be exploited to more efficiently deliver drugs to tumors.
In an initial experiment, mice that had been given human colorectal tumors were injected with either live MC-1 cells, dead MC-1 cells, or as a control group, non-magnetic beads (roughly the same size as the bacteria). The injection was made into the tissue directly adjacent to the tumors after which the mice were exposed to a computer-programmed magnetic field, meant to direct the cells or beads into the tumor. Upon examination of the tumors, the researchers found minimal penetration of the dead bacterial cells and the beads into the tumor, whereas the live bacterial cells were found deep within the tumor and especially in regions with low oxygen content.
“When they get inside the tumor, we switch off the magnetic field and the bacteria automatically rely on the oxygen sensors to seek out the hypoxic areas,” says Martel. “We constrain them to the tumor and then let nature do the rest.”
Next, the researchers wanted to see whether attaching vesicles loaded with drugs to the cells would affect their movement into the tumors. They attached approximately 70 drug-containing vesicles to each bacterial cell. The cells were then injected into another set of mice with colorectal tumors and exposed to the magnet. After examining the tumors of those mice, the researchers estimated that on average, 55% of the injected bacterial cells with attached vesicles made it into the tumor. For comparison, some researchers estimate that only approximately 2% of drugs delivered via current nanocarriers make it into tumors.
“This proof-of-concept work shows the potential to tap into the intricate and optimized cell machinery of single celled organisms such as bacteria,” said Richard Conroy, Ph.D., director of the Division of Applied Sciences and Technology at NIBIB. “The ability to actively and precisely target drug delivery to a tumor will help reduce side effects and potentially improve the efficacy of treatments.”
The next step for Martel’s team is to determine the effects of the drug-loaded bacterial cells on reducing tumor size. They would also like to test whether the bacteria can be used to deliver other types of cancer-killing medicines such as molecules that instruct the immune system to attack tumors.
In addition, the team is working to expand the types of tumors the bacteria could be used for. Currently, the bacteria have to be injected very close to the tumor because, if injected into arteries, the excessive blood flow and the distance needed to travel would impact the number of bacteria that reach the tumor. This limits the drug delivery approach to cancers that are easily accessible such as colorectal, prostate, and potentially breast cancer. However, Martel’s team has shown in animals that they can transport the bacteria through arteries and sufficiently close to the tumor by first encapsulating them in magnetic carriers and propelling them by the magnetic field of an MRI scanner. The bacteria can then be released from the carriers, like torpedoes from a submarine, once close to the tumor. This multi-step approach could potentially open the door for using the bacteria to deliver drugs to tumors deeper in the body.
Martel says that preliminary test results of the bacteria in mice and rats and the fact that the bacteria die within 30 minutes of being injected, suggest that they could potentially be safe in humans.
“These bacteria are really the perfect machine. They replicate, they’re cheap, and we can inject hundreds of millions or more at a time,” says Martel.
Beads, disks, bowls and rods: scientists at Radboud University have demonstrated the first methodological approach to control the shapes of nanovesicles. This opens doors for the use of nanovesicles in biomedical applications, such as drug delivery in the body.
The shape of nanovesicles – called ‘polymersomes’ in jargon – in a solution varies at different compositions of that solution, scientist Roger Rikken and his colleagues at Radboud University discovered. “Besides the spherical shapes, we can create disks, rods, and bowl shaped stomatocytes by varying the ratio of the solvent. This regulates the osmotic pressure and permeability of the vesicles, controlling their deflation and subsequent re-inflation,” Rikken explains.
For the first time, the shape of the nanovesicles is now fully controllable and predictable. This offers possibilities to transform and mould the vesicles into nanocontainers or nanorockets, which are highly desirable, e.g. for drug delivery in the body. The shape of the polymersomes also affects their flow properties, as is also believed to be the case for red blood cells. It is therefore of great importance to obtain full control over shape transformations to utilise vesicles in drug transport via the blood stream.
By using the magnets of the High Field Magnet Laboratory, Rikken was able to determine the exact shape of the vesicles at every solvent ratio. Subsequently, he studied the variety of shapes with electron microscopy and described them mathematically. In this way, he discovered that the shape transformation follows the path of the lowest energy. “Nature is always trying to stay in balance. The four shapes that we found turn out to be located exactly at the energy minima in an existing model. The basic idea behind our discovery is actually very logical, but it was never described before.”
Viruses are able to redirect the functioning of cells in order to infect them. Inspired by their mode of action, scientists from the CNRS and Université de Strasbourg have designed a “chemical virus” that can cross the double lipid layer that surrounds cells, and then disintegrate in the intracellular medium in order to release active compounds.
To achieve this, the team used two polymers they had designed, which notably can self-assemble or dissociate, depending on the conditions. This work, the result of collaborative efforts by chemists, biologists and biophysicists, is published in the 1st September issue of Angewandte Chemie International Edition.
Biotechnological advances have offered access to a wealth of compounds with therapeutic potential. Many of these compounds are only active inside human cells but remain unusable because the lipid membrane surrounding these cells is a barrier they cannot cross. The challenge is therefore to find transfer solutions that can cross this barrier.
By imitating the ability of viruses to penetrate into cells, chemists in the Laboratoire de Conception et Application de Molécules Bioactives (CNRS/Université de Strasbourg) sought to design particles capable of releasing macromolecules that are only active inside cells. To achieve this, these particles must comply with several, often contradictory, constraints. They must remain stable in the extracellular medium, they must be able to bind to the cells so that they be internalized, but they must be more fragile inside the cells so that they can release their content.
Using two polymers designed by the team, the scientists succeeded in creating a “chemical virus” that meets the conditions necessary for the direct delivery of active proteins into cells.
In practice, the first polymer (pGi-Ni2+) serves as a substrate for the proteins that bind to it. The second, recently patented polymer (?PEI), encapsulates this assembly thanks to its positive charges, which bind to the negative charges of pGi-Ni2+. The particles obtained (30-40 nanometers in diameter) are able to recognize the cell membrane and bind to it. This binding activates a cellular response: the nanoparticle is surrounded by a membrane fragment and enters the intracellular compartment, called the endosome. Although they remain stable outside the cell, the assemblies are attacked by the acidity that prevails within this new environment. Furthermore, this drop in pH allows the ?PEI to burst the endosome, releasing its content of active compounds.
Thanks to this assembly, the scientists were able to concentrate enough active proteins within the cells to achieve a notable biological effect. Thus by delivering a protein called caspase 3 into cancer cell lines, they succeeded in inducing 80% cell death1.
The in vitro results are encouraging, particularly since this “chemical virus” only becomes toxic at a dose ten times higher than that used during the study. Furthermore, preliminary results in the mouse have not revealed any excess mortality. However, elimination by the body of the two polymers remains an open question. The next stage will consist in testing this method in-depth and in vivo, in animals. In the short term, this system will serve as a research tool to vectorize2 recombinant and/or chemically modified proteins into cells. In the longer term, this work could make it possible to apply pharmaceutical proteins to intracellular targets and contribute to the development of innovative drugs.
Read more: Imitating viruses to deliver drugs to cells