Scientists at the Max Planck Institute for Intelligent Systems (MPI-IS) combined robotics with biology to equip E. coli with artificial components, enabling them to construct fully motile biohybrid microrobots that carry drug molecules, and can be guided magnetically to navigate through 3D biological material and deliver their payloads on demand. The team reported on an in vitro study demonstrating how the bacterial biohybrid microrobots could be steered through various courses of biological matrices, and deliver anticancer drugs directly to target tumor spheroids.

“Bacteria-based biohybrid microrobots with medical functionalities could one day battle cancer more effectively,” suggested Metin Sitti, PhD, who leads the physical intelligence department and is co-author of the team’s study in Science Advances. “It is a new therapeutic approach not too far away from how we treat cancer today. The therapeutic effects of medical microrobots in seeking and destroying tumor cells could be substantial. Our work is a great example of basic research that aims to benefit our society.”

Sitti and colleagues described their development in a paper titled, “Magnetically steerable bacterial microrobots moving in 3D biological matrices for stimuli-responsive cargo delivery.” In their report they concluded, “Overall, the bacterial biohybrid design presented here provides a systematic and high-throughput platform for multifunctional medical microrobots that can overcome biological barriers and perform stimuli-responsive active therapeutic release.”

Bacteria are drawn to chemical gradients such as low oxygen levels or high acidity—both prevalent near tumor tissue. Treating cancer by injecting bacteria in proximity is known as bacteria-mediated tumor therapy. The microorganisms flow to where the tumor is located, grow there, and in this way activate the immune system of patients. Bacteria-mediated tumor therapy has been a therapeutic approach for more than a century.

E. coli are fast-swimming, versatile bacteria that can navigate through materials ranging from liquids to highly viscous tissues. The organisms also have highly advanced sensing capabilities. For the past few decades, scientists have looked for ways to increase the capabilities of this microorganism even further, by equipping bacteria with extra components to help fight the battle. “Biohybrid microrobots, which combine a motile microorganism (e.g., bacteria or algae) with an artificial component (e.g., micro/nanocarriers), are self-powered micromachines with intrinsic propulsion, sensing, and targeting mechanisms,” the authors explained. And among different types of microrobots, bacteria-driven biohybrids “stand out from the rest … Thus, bacterial biohybrids, particularly when decorated with multiple functional units, such as contrast agents, therapeutics, and targeting moieties, become ideal candidates for medical microrobot applications.”

However, adding artificial components to bacteria is no easy task. Complex chemical reactions are at play, and the density rate of particles loaded onto the bacteria matters to avoid dilution. Most of the techniques used to design biohybrid microrobots may also have unwanted effects and interfere with, for example, how the bacteria move, or alter protein expression, the investigators suggested. “ … current bacterial biohybrid designs lack high-throughput and facile construction with favorable cargoes, thus underperforming in terms of propulsion, payload efficiency, tissue penetration, and spatiotemporal operation.”

The team in Stuttgart has now reported the development of bacterial biohybrids that outperform previously reported E. coli-based microrobots, retaining their original motility, and exhibiting the ability to be steered through biological material and colonize tumor spheroids, where they can then release anticancer payloads on demand.

In their study, the researchers managed to equip bacteria with both liposomes and magnetic particles, with approximately 90% efficiency. To do this the team first attached several nanoliposomes (NLs) to each bacterium. The nanoliposomes were designed and constructed to encapsulate the water-soluble chemotherapeutic drug doxorubicin (DOX). Indocyanine green (ICG), a medical fluorescent dye that melts when illuminated by near-infrared light, was embedded in the NL phospholipid bilayer. “… we designed a photothermally active liposomal formulation with ICG embedded in the phospholipid bilayer that can absorb NIR light and convert it into heat, which ultimately triggers structural changes in the lipid membrane and the release of the intraliposomal content, i.e., chemotherapeutic DOX molecules,” the team noted.

Bacterial biohybrids carrying nanoliposomes (200 nm) and magnetic nanoparticles (100 nm). Nanoliposomes are loaded with chemotherapeutic DOX and photothermal agent ICG, and both cargoes are conjugated to E. coli bacteria (2 to 3 µm in length) via biotin-streptavidin interactions. [Akolpoglu et al., Sci. Adv. 8, eabo6163 (2022)]
The researchers also attached magnetic nanoparticles to each bacterium. When exposed to a magnetic field, the iron oxide particles serve as an on-top booster to this already highly motile microorganism. In this way, it is easier to control the swimming of bacteria—an improved design toward an in vivo application. The liposomes and magnetic particles were bound to the bacterium using a hard-to-break streptavidin and biotin complex, which had been developed a few years prior, and which comes in useful when constructing biohybrid microrobots.

The scientists’ carried out experiments with the bacterial biohybrid microrobots, demonstrating that they could be externally steered through different courses. First, through an L-shaped narrow channel with two compartments on each end, with one tumor spheroid in each. Second, through an even narrower set-up resembling tiny blood vessels. The team added an extra permanent magnet on one side and showed how they could precisely control the drug-loaded microrobots towards tumor spheroids.

And third—going one step further—the team steered the microrobots through a viscous collagen gel (resembling tumor tissue) with three levels of stiffness and porosity, ranging from soft to medium to stiff. The stiffer the collagen, the tighter the web of protein strings, and the more difficult it becomes for the bacteria to find a way through the matrix. The investigators showed that when they added a magnetic field, the bacteria managed to navigate all the way to the other end of the gel as had a higher force. Because of constant alignment, the bacteria found a way through the fibers. “The results reported here demonstrated that bacterial biohybrids could penetrate and move inside a confined and porous biological microenvironment under constant magnetic alignment,” the authors commented.

Once the microrobots were steered to accumulate at the tumor spheroid a near-infrared laser was used to generate rays with temperatures of up to 55°C, triggering a melting process of the liposome and a release of the enclosed drugs. A low pH level or acidic environment also caused the nanoliposomes to break open—hence the drugs were released near a tumor automatically. “With the biohybrid design presented here, we were not only able to preserve the inherent swimming velocity and motility of bacteria, but we also actively guided bacterial biohybrids using various forms of magnetic fields and flow conditions and showed the colonization of tumor spheroids, which might prove to be an essential aspect for localized cargo delivery applications,” the investigators wrote.

“Imagine we would inject such bacteria-based microrobots into a cancer patient’s body. With a magnet, we could precisely steer the particles towards the tumor,” suggested Birgül Akolpoglu, a PhD student in the physical intelligence department at MPI-IS. “Once enough microrobots surround the tumor, we point a laser at the tissue and by that trigger the drug release. Now, not only is the immune system triggered to wake up, but the additional drugs also help destroy the tumor.” Co-lead researcher and former postdoctoral researcher in the physical intelligence department, Yunus Alapan, PhD, added: “This on-the-spot delivery would be minimally invasive for the patient, painless, bear minimal toxicity and the drugs would develop their effect where needed and not inside the entire body.”

The authors concluded, “Our design strategy establishes an advanced and optimized fabrication route for bacteria-based biohybrid microrobots with exceptional performance and multifunctionality, together with (i) maintained motility of bacterial biohybrids after functionalization, (ii) 3D matrix penetration capabilities, and (iii) stimuli-responsive cargo delivery. The biohybrid design reported here presents a highly efficient assembly route for a magnetically controlled biohybrid microrobotic system with swimming velocities far higher than previous biohybrid designs incorporating E. coli.”

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