Turkish Team Discovers Bacteria-Based Biohybrid Microrobots for Cancer Treatment

On 15 July, a study published in Science Advances made its mark on the scientific world. Prof. Dr. Metin Sitti led scientists from Germany's Max Planck Institute, who announced a method using the bacterium E. coli as a microrobot. The team built fully mobile biohybrid microrobots carrying drug molecules, which could be magnetically guided to navigate 3-dimensional biological material and deliver their payloads on demand. The study showed the scientific world how bacterial biohybrid microrobots can be steered through a variety of biological matrices.

Max Planck Institute Intelligent Systems (MPI-IS) The Physical Intelligence department head Metin Sitti said of their work:

“Bacteria-based biohybrid microrobots with medical functionalities could one day fight cancer more effectively. This is a new treatment approach that is not too far removed from how we treat cancer today. Medical microrobots could be used to seek out and destroy tumor cells. Our work is an excellent example of basic science research aimed at benefiting our society.”

In their paper, the team concluded: “Overall, the bacterial biohybrid design presented here provides a systematic, high-throughput platform for multifunctional medical microrobots that can overcome biological barriers and perform stimuli-responsive active drug release.”

Bacteria tolerate and are drawn to chemical challenges such as low oxygen levels or high acidity, which are common near tumor tissue. Treating cancer by injecting bacteria nearby is known as bacteria-mediated tumor therapy. The microorganisms flow toward the tumor site and activate the patient's immune system along the way. Bacteria-mediated tumor therapy has in fact been tested for quite a long time, but this new study has brought fresh impetus to this research.

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Biohybrid microrobots, which combine artificial components (such as micro/nanocarriers) with a self-propelled organism (such as a bacterium or alga), have sensing and targeting mechanisms. Among the different types of microrobots, bacteria-powered biohybrids stand out. Bacterial biohybrids therefore become ideal candidates for medical microrobot applications, especially when designed with multiple functional units such as contrast agents, therapeutics and targeting moieties.

Adding artificial components to a bacterium, however, is not easy. Complex chemical reactions are involved. The researchers emphasize that many techniques used to design biohybrid microrobots can have unwanted effects, for example by changing bacterial movement or protein expression.

In the current study, the team reported the development of bacterial biohybrids that outperformed previously reported E. coli -based microrobots, retained their original motility, and demonstrated an ability to be guided through biological material and colonize tumor spheroids (spherical structures).

In their study, the researchers equipped bacteria with both liposomal (fat molecule particles) and magnetic particles with approximately 90% efficiency. To do this, the team first attached nanoliposomes (NL) to each bacterium. The nanoliposomes were designed and structured to encapsulate the water-soluble chemotherapy drug doxorubicin (DOX). Indocyanine green (ICG), a medical fluorescent dye that melts when illuminated with near-infrared light, was embedded in the nanoliposomes' phospholipid bilayer. “…we designed a photothermally active liposomal formulation with ICG embedded in the phospholipid bilayer, capable of absorbing near-infrared light and converting it into heat, which ultimately triggers structural changes in the lipid membrane and the release of intraliposomal contents, namely chemotherapy molecules,” the team said.

Figure – 1: Bacterial biohybrids carrying stimuli-responsive ICG-DOX NLs migrate to tumor spheroids and release their cargo upon NIR stimulation. The NLs convert light energy into heat, which then causes phospholipid breakdown and ultimately triggers the release of DOX molecules.

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The researchers also added magnetic nanoparticles to each bacterium. When exposed to a magnetic field, the iron oxide particles act as a high-level booster for this already highly mobile microorganism. This makes it easier to control the flow of bacteria — a design improved for in vivo application. The liposomes and magnetic particles were attached to the bacterium using a tough-to-break streptavidin and biotin complex developed several years earlier, which is useful in constructing biohybrid microrobots.

Figure – 2: Bacterial biohybrids carrying nanoliposomes (200 nm) and magnetic nanoparticles (100 nm). The nanoliposomes are loaded with chemotherapeutic DOX and photothermal agent ICG, and both carriers are attached through biotin-streptavidin interactions to E. coli bacteria 2 to 3 µm long.

By experimenting with bacterial biohybrid microrobots, the team showed that bacteria could be guided externally in different ways:

• First, through a narrow L-shaped channel with two compartments at either end, each containing a tumor spheroid.
• Second, through a much narrower structure resembling small blood vessels. The team added a magnet on one side and showed how precisely they could direct the drug-loaded microrobots toward tumor spheroids.
• Third, the team went a step further and guided the microrobots through a dense collagen gel (similar to tumor tissue) with three levels of stiffness and porosity, ranging from soft to medium and hard. The stiffer the collagen, the tighter the network of protein strands and the harder it is for bacteria to find a path through the matrix.

The researchers showed that, when they added a magnetic field, the bacteria managed to reach the other end of the gel because they had greater force. Thanks to constant alignment, the bacteria found a path between the fibers.

"The results reported here showed that bacterial biohybrids could penetrate and move through a confined, porous biological microenvironment under constant magnetic alignment."

After the microrobots were directed to accumulate in the tumor spheroid, a near-infrared laser was used to produce a beam that could be as hot as 55°C, triggering the melting of the liposome and release of the attached drugs. A low pH level, or acidic environment, also caused the nanoliposomes to open — so the drugs were automatically released near the tumor.

"With the biohybrid design presented here, we not only preserved the bacteria’s natural swimming speed and motility, but also actively guided bacterial biohybrids using various magnetic fields and flow conditions and demonstrated colonization of tumor spheroids, which could be an important feature for localized ‘molecular cargo’ delivery applications.”

MPI-IS The Physical Intellegence department member and the study's first author Birgül Akolpoglu said:

"Imagine injecting these kinds of bacteria-based microrobots into a cancer patient’s body. With a magnet, we could guide the particles precisely toward the tumor. Once enough microrobots surround it, we direct a laser at the tissue and thereby trigger drug release. At that point, the immune system is not just prompted to wake up; the additional drugs also help destroy the tumor."

One of the study's co-lead authors, Yunus Alapan, said:

"This process of delivering drugs to the tumor site (with bacteria-based microrobots) will be minimally invasive, painless and minimally toxic for the patient, and the drugs will act where needed (at the tumor site), rather than throughout the whole body."

As the study's conclusion, the authors stated:

“Our design strategy, together with

1. Maintaining bacterial biohybrids' motility after functionalization
2. Three-dimensional matrix adhesion capabilities
3. Stimuli-responsive molecular cargo delivery

, establishes an improved and optimized fabrication route for multifunctional bacteria-based biohybrid microrobots with outstanding performance. The biohybrid design reported here offers a highly efficient assembly route for a magnetically controlled biohybrid microrobotic system with much higher movement speeds than previous biohybrid designs containing E. coli.”