To adsorb or not to adsorb? That’s the question
Prolonged exposure to antibiotics allows bacteria to gain capacity to overcome drugs designed to fight them. So, if such antibiotic resistant bacteria cause the infection, the only chance to use a specialized virus called phage infecting specific bacterial species. It is a powerful weapon against deadly diseases. At the same time, the effective treatment depends on factors that would not be suspected for years to have an impact on the success of the treatment. Recently, researchers from the Institute of Physical Chemistry of the Polish Academy of Sciences headed by dr. Jan Paczesny and Professor Robert Holyst explained why in some cases phage therapies can fail. They revealed that the material used to store phages has a huge effect on their numbers in the formulation used for treatment.
Antibiotics have been used for many decades to treat various bacterial infections. Thanks to them, many diseases are easier to fight. Unfortunately, as long as antibiotics are widely applied in medical treatment, many bacteria develop resistance to antibiotics. As a result, some of them can defeat all the antibiotics currently available in the pharmaceutical market. What if a person infected with highly resistant bacteria and even the strongest antibiotic – vancomycin, doesn’t work? Still, there is a chance of winning the mortal combat. The solution is based on the application of bacteriophages, also called phages. These are specialized viruses that can infect and kill bacteria without harmful effects on humans. This therapy is called last-ditch viral therapy and can be used against chronic diseases, for example, inflammation of the urinary tract. Unfortunately, despite beneficial treatment and numerous trials, the number of active bacteriophages in the injectable solution can drastically decrease, rendering the treatment ineffective. Recently, scientists from the Institute of Physical Chemistry of the Polish Academy of Scientists headed by dr. Jan Paczesny and Professor Robert Holyst discovered that the drop in the concentration of phage in a formulation depends on the properties of the container used to store them. Surprisingly, depending on the polymer used to make the container, the phage may or may not “sit” on the surface of the polymers. This is caused by hydrophobicity, which is one of the most critical factors governing the adsorption of phages to surfaces. As a result, even the same sets of containers purchased from different suppliers and manufacturers may have other surface properties. Then the inner walls can trap all phages in the mass, leading to a reduced concentration in the formulation.
Thus, the number of active phages can drop dramatically, even by several orders of magnitude, depending on the type of container used for phage storage. These differences can be caused by additives used in the manufacture of polymers, such as plasticizers or slip agents. For this reason, the researchers tested several types of polypropylene-based containers dedicated to laboratory use.
Dr Paczesny remarks: “We checked whether the leachables are responsible for the observed effect of a decreasing number of active phages in the polypropylene containers. We considered the possibility that leachables released from “unsafe” tubes could deactivate phages or that leachables from “safe” tubes could protect phages against external factors (eg temperature). Next, we examined the adsorption of virions on the walls of polypropylene containers as a mechanism responsible for the observed effect. The uncontrolled adsorption of phages and their subsequent disappearance from solution can cause serious errors and non-reproducible results. This is extremely important for phage therapy.
The researchers tested various containers with standard operations such as mixing, heating or prolonged storage of active phages. They presented that the decrease in the number of active phages in mass depends mainly only on plastic properties such as wettability. They also studied the effect of the physical structure of phages, including the electrostatic properties of the phage surface. Yet their results clearly indicate that the type of plastic has enormous significance when comparing the surface potential of phages when it comes to phages that settle on the walls of containers. In this work, the researchers proposed particular solutions for containers having specific properties such as a treatment with a particular surfactant limiting the adsorption of the phage on the surface of the container. In detailed studies, it has been shown that their agglomeration causes the number of active phages to drop in bulk on the walls of the containers. The researchers explained why phages tend to adsorb to more hydrophobic surfaces. When the aqueous solution with the phages is stored in the container made of a hydrophobic material such as PP, the aggregation of the phages on the walls is more energy efficient than their stay in the solution. It is caused by the properties of water which have limited contact with hydrophobic walls when coated with phages.
“Our results are critically important not only for phage-related studies. The lack of reproducibility of measurements becomes the most important threat for the scientific community. Scientists may not even know that the PP tubing supplier can apparently provide the same product which differs significantly from batch to batch. Sometimes this can lead to wrong conclusions. Or, as in the case of phages, it may preclude the development of essential technologies due to low efficiency caused only by inappropriate containers. – claims Dr. Paczesny
The studies presented in the scientific reports of April 1, 2021 present a significant problem with the effectiveness of Last Change viral phage therapy. In case of sudden need for its application, the choice of container for phage storage seems to be the least important. However, as we can see here, such a factor has a huge impact on the number of active phages in the solution determining the success of a therapy.
Such a small step for phages can be a big step for a patient struggling with bacteria that can only be killed with phages. The data presented and the proposed solutions solve non-reproducible results in the treatment of infections with phages, but, without doubt, will improve any phage-based projects.