University Hospital Basel

Medical Intensive Care Unit, University Hospital Basel
National Centre of Competence in Nanoscale Science, University of Basel
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Polymer nanocontainers as intelligent nanometer-sized bioreactors



 

What is the background of this project?
Nanotechnology is believed by many to be one of the most important scientific topics of the next decade with a huge impact on biology, pharmacology, and medicine. In particular, medicine with its reliance on safe, potent, user-friendly, and target-specific therapies for important diseases such as arteriosclerosis, cancer, infections or autoimmune disorders will profit from nanometer-sized devices for diagnostic and therapeutic applications.
Our group investigates the use of nanometer-sized polymer-based vesicles (called nanocontainers) made of amphiphilic triblock (hydrophilic-hydrophobic-hydrophilic) copolymer building blocks for targeted delivery of drugs or contrast agents in humans. In aqueous solution, the used polymers form unilamellar vesicles with specific diameters, allowing the encapsulation of water-soluble substances such as drugs, enzymes, nucleotides, radioisotopes, or contrast media into the self-assembled nanostructures.
 

 

What are the main results of this nanoreactor project?

 

The goal of this subproject is the development of an intelligent sensor-effector reactivity within polymer nanocontainers. This would enable a controlled activation or release of encapsulated content inside the target cell or would even allow the creation of nanometer-sized simple bioreactors for target-specific therapeutic applications in medicine.
The amphiphilic copolymer membrane was functionalized with integrated bacterial pore proteins, which build size-selective pores for passive diffusion (Cut-off 600 Dalton) of polar or charged molecules. Furthermore, we encapsulated a pH-sensitive enzyme (activity maximum at pH 5) into the now permeable nanocontainers. As soon as the system is in a low pH environment (e.g. site of infection, inflammation and certain tumors), the enzyme is switched on and can activate an inert prodrug into a potent drug by dephosphorylation. We tested the pH- and substrate-dependency of such a prototype enzyme nanoreactor system by converting a non-fluorescent precursor substrate into a insoluble, highly fluorescent product. In fluorescence and confocal microscopy, we confirmed the functionality of the designed system and tested the various subsystems such as pores, substrate, or enzyme. Results: 1) Bacterial pore proteins integrate into the polymer membrane and remain functional. 2) The activity of the system is limited to pH 4-6.5 and can be switched on and off. 3) The system is time- and substrate dependent. 4) The encapsulated enzyme remains functional and is protected inside the nanocontainer.

 

 

Two-dimensional outline of the nanoreactor system: The nanoreactors are based on a synthetic triblock copolymer membrane functionalized with bacterial ompF pore proteins that make intact, size-selective channels for passive diffusion across the membrane. Encapsulated acid phosphatase enzyme processes a non-fluorescent substrate into an insoluble, fluorescent reaction product at pH 4-6.5.

 

Channel- and pH-dependent nanoreactor activity: Active nanoreactors with ompF pores in the upper left panel show strong fluorescent activity at pH 5, whereas controls at pH 7.5 and without the ompF pores show no significant fluorescent activity in confocal microscopy after 3 hours. Substrate concentration in this experiment was 75 µM.

 

Nanoreactor activity in acidic, neutral and basic environment: Active nanoreactors with ompF pores were incubated with 75 µM phosphatase substrate at changing pH conditions. The resulting fluorescent activity was measured and visualised after 3 hours reaction time by fluorescence microscopy. Strong green-yellowish fluorescence was observed at pH 5, weaker fluorescence could be observed in the pH range between 6.5 and 4. At highly acidic, neutral and basic conditions, the nanoreactor showed no enzyme activity.