Generalizable framework for Implantable Living Materials composed of highly engineered hydrogels and synthetically engineered bacteria opens diverse novel therapeutic avenues
By Benjamin Boettner
(BOSTON) — Patient recovery from many debilitating conditions and diseases could be sped up significantly and be more effective if drugs and therapeutic molecules were delivered right to where they are needed in the body, over the entire regenerative process, and in doses finely tuned to therapeutic needs. An intriguing way to achieve this is the use of implantable, synthetically engineered, living cells that can sense injury or disease-associated conditions in their environment and flexibly respond by producing the right amount of a therapeutic molecule.
Bacteria, in particular, are promising in this regard as they can thrive in harsh physiological environments within the body, such as within infected or inflamed tissues, tissues undergoing mechanical movements, and tumors. Some of these microbial therapies have even advanced into clinical trials to treat certain cancers, metabolic disorders, and the progression of kidney stones. However, thus far, such trials have failed, and microbes are feared to also pose significant safety risks because they cannot be contained at specific sites in the body.
Now, a research team at Harvard’s Wyss Institute and John A. Paulson School of Engineering and Applied Sciences (SEAS) led by Wyss Founding Core Faculty member David Mooney, Ph.D., has developed an “Implantable Living Materials” (ILM) platform that offers a compelling solution to this problem. By encapsulating a genetically engineered, therapeutic strain of E. coli bacteria within a biomaterial made from a hydrogel that was specifically designed to regulate bacterial growth and resist mechanical stresses, like those present at physically active sites in the body, the bacteria could be confined for over six months.
The E. coli bacteria were equipped with a synthetic gene circuit that allowed them to sense pathogenic Pseudomonas aeruginosa bacteria causing infections and then respond by releasing a therapeutic molecule that killed the nearby residing pathogens. Implanted into the joints of mice next to a specialized orthopedic implant designed to help heal femoral injuries, the ILM autonomously and effectively treated infections with P. aeruginosa, a common cause of often debilitating orthopedic device infections. The findings were published in Science.
“With this new strategy combining both an engineered material with designed mechanical features and genetically engineered microbes that produce therapeutic payloads on demand, we provide a generalizable framework for deploying future microbial medicines,” said Mooney. “The precision, safety, and therapeutic durability afforded by this ILM strategy could be a potential solution for treating a wider range of diseases and infections, enabling therapeutic efficacies that might surpass those of other drug delivery strategies.” Mooney is also the Robert P. Pinkas Professor of Bioengineering at SEAS.
The precision, safety, and therapeutic durability afforded by this ILM strategy could be a potential solution for treating a wider range of diseases and infections, enabling therapeutic efficacies that might surpass those of other drug delivery strategies.
Breathing life into therapeutic materials
“In the beginning, we asked the seemingly simple question, what if we could design a material that safely encapsulates drug-delivering bacteria inside and allows therapeutic drugs to pass through to where they are needed,” said first-author Tesuhiro Harimoto, Ph.D., who spearheaded the project as a postdoctoral fellow in Mooney’s group. Although scientists have extensively studied how physical parameters of synthetic materials change with tweaks made to their composition and chemical connections, “this was a big ask since the encapsulating material had to reconcile two often contradictory features: it needed to be sufficiently ‘stiff’ so that bacteria pushing against it from the inside can’t break it apart, and sufficiently ‘tough’ to provide a enclosure that protects against external physical stresses in mechanically active tissues.”
An expanding bacterial colony can exert pressures that are multiple orders of magnitude higher than those produced by the mammalian cells. Also, the type of stresses produced by the body’s various mechanical forces like, for example, generated by tension in muscles or compression on joints, can fatigue a material over time and disrupt it from the outside. However, introducing too much stiffness can often make a material too brittle, which means that cracks can quickly propagate through it; and a high toughness, which, in principle, allows a material to resist fracturing, often makes it soft.
To realize ILMs, the team started with polyvinyl alcohol (PVA), which is already used clinically, and processed it to form nanoscale interactive crystalline domains. The resulting scaffolds are simultaneously highly stiff and tough. “Finding out how to fabricate optimal hydrogels from PVA that are crosslinked through dense crystalline domains, and how to do this in a way that keeps the enclosed bacteria alive and active, was a big part of our study,” said Harimoto. The researchers included the bacteria in their fabrication process within tiny droplets of gelatin that protected them against desiccation and selective chemical manipulations. This strategy allowed them to fabricate an ideally stiff and tough material scaffold around the bacteria, using a combination of tolerable freeze-thaw cycles, salt conditions, and chemical treatment times. Late in the process, via a slight shift in temperature, the gelatin microgel could be dissolved to create internal voids for the bacteria to thrive in. Due to the tiny pore sizes within the PVA material, the bacteria remain constrained while soluble molecules they produce can travel to other sites in the body. The resulting ILM safely contained the bacteria over extended time intervals of up to six months and was resistant to repeated mechanical stresses.
Building in sense-and-response behavior
To provide proof-of-concept for ILMs, the team homed in on the infection of implanted periprosthetic devices designed to treat fractures or bone loss around existing artificial joint replacements by pathogenic P. aeruginosa strains. These are often difficult to diagnose in orthopedic patients and would greatly benefit from an autonomously functioning pathogen-responsive therapeutic delivery system—many treatments with periprosthetic devices fail due to infection, which goes along with inflammation and the spread of antibiotic resistance.
To effectively treat this and other types of infection, the therapy-delivering bacteria within the ILM needed to be genetically engineered to function as a drug depot with autonomous “sense-and-respond” capabilities. To achieve this, the team installed a synthetic gene circuit in the E. coli strain that enabled the bacteria to sense a small diffusible metabolite produced by P. aeruginosa, known as N-acyl homoserine lactone (AHL), and, in response, activate a self-destruction gene. The self-destruction process, or lysis, as biologists call it, was triggered in a fraction of ILM bacteria, which caused a synthetic P. aeruginosa-killing protein called chimeric pyocin (ChPy) that the bacteria produce continuously, to be released from the ILM. ChPy is toxic to P. aeruginosa, erasing the pathogen in the local ILM environment.
“When we tethered a therapeutic ILM to a stainless steel periprosthetic device that was infected with a pathogenic P. aeruginosa strain isolated from a patient’s wound and implanted next to the femur bone of mice, it significantly reduced the pathogen burden while safely containing its engineered bacteria over a three-day treatment course,” said Harimoto. “In contrast, in mice that we treated with a non-therapeutic control ILM that did not produce ChPy, the numbers of P. aeruginosa bacteria continued to rise over the same time interval. This demonstrated the ability of therapeutic ILMs to autonomously sense and treat periprosthetic infection in vivo.”
When we tethered a therapeutic ILM to a stainless steel periprosthetic device that was infected with a pathogenic P. aeruginosa strain isolated from a patient’s wound and implanted next to the femur bone of mice, it significantly reduced the pathogen burden while safely containing its engineered bacteria.
The researchers think that specifically engineered ILMs, as a novel class of therapeutics with excellent safety features and locally targeted drug release capabilities, have broad potential, ranging from tissue regeneration to immune modulation in a variety of disease settings. A patent application describing the use of ILMs for drug delivery has been filed.
“As an intriguing and potentially highly efficient drug delivery approach, ‘living therapeutics’ in the form of synthetically engineered microbial cells have been on the mind of researchers for a while, but have not been effectively translated into clinical products. The ILM platform developed by Dave’s group, through its ingenious use of engineered materials, is removing a central safety bottleneck that should lead to important advances in this field that will greatly benefit patients,” said Wyss Founding Director Donald Ingber, M.D., Ph.D. who is also the Judah Folkman Professor of Vascular Biology at Harvard Medical School and Boston Children’s Hospital, and the Hansjörg Wyss Professor of Biologically Inspired Engineering at Harvard John A. Paulson School of Engineering and Applied Sciences.
Other authors on the study are Fernando Herrero Quevedo, Janis Zillig, Sanjay Schreiber, Yi Wu, Christine Heera Ahn, Tania To, Rohan Thakur, Alexander Tatara, Shawn Kang, Zheqi Chen, Shanda Lightbown, and David Weitz. The study was supported by the Wyss Institute at Harvard University; Harvard Materials Research Science and Engineering Center (award #DMR-2011754); National Cancer Institute, National Institutes of Health (NIH; awards # K00 CA253756 and K99 CA300498); National Cancer Institute and National Institute on Aging, NIH (award #U54CA244726); National Institute of Allergy and Infectious Diseases, NIH (award #K08 AI180362); the National Human Genome Research Institute, NIH (award #F31 HG013052); and National Science Foundation (awards #DGE 2140743 and DGE1745303).