Targeted drug delivery is a powerful and promising area of medicine. Therapies that pinpoint the exact areas of the body where they’re needed — and nowhere they’re not — can reduce the medicine dosage and avoid potentially harmful “off target” effects elsewhere in the body. A targeted immunotherapy, for example, might seek out cancerous tissues and activate immune cells to fight the disease only in those tissues.

The tricky part is making a therapy truly “smart,” where the medicine can move freely through the body and decide which areas to target.

Researchers at the 91̽�� took a significant step toward that goal by designing proteins with autonomous decision-making capabilities. In a proof-of-principles study in Nature Chemical Biology, researchers demonstrated that by adding smart tail structures to therapeutic proteins, they could control the proteins’ localization based on the presence of specific environmental cues. These protein tails fold themselves into preprogrammed shapes that define how they react to different combinations of cues. In addition, the experiment showed that the smart protein tails could be attached to a carrier material for delivery to living cells.

Advances in synthetic biology also allowed the researchers to manufacture these proteins cheaply and in a matter of days instead of months.

“We’ve been thinking about these concepts for some time but have struggled with ways to increase and automate production,” said senior author , a 91̽��professor of chemical engineering and bioengineering. “We’ve now finally figured out how to produce these systems faster, at scale and with dramatically enhanced logical complexity. We are excited about how these will lead to more sophisticated and scalable disease-honing therapies.”

The concept of programmable biomaterials isn’t new. Scientists have developed numerous strategies to make systems responsive to individual cues — such as pH levels or the presence of specific enzymes — that are associated with a particular disease or area of the body. But it’s rare to find one cue, or “biomarker,” that’s unique to one spot, so a material that hones in on just one biomarker might act on a few unintended places in addition to the target.

One solution to this problem is to seek out a combination of biomarkers. There might be many areas of the body with particular enzyme or pH levels, but there are likely fewer areas with both of those factors. In theory, the more biomarkers a material can identify, the more finely targeted drug delivery can be.

In 2018, DeForest’s lab created a new class of materials that responded to multiple biomarkers using Boolean logic, a concept traditionally used in computer programming.

“We realized that we could program how therapeutics were released based simply on how they were connected to a carrier material,” DeForest said. “For example, if we linked a therapeutic cargo to a material via two degradable groups connected in series — that is, each after the other — it would be released if either group was degraded, acting as an OR gate. When the degradable groups were instead connected in parallel — that is, each on a different half of a cycle — both groups had to be degraded for cargo release, functioning as an AND gate. Excitingly, by combining these basic gates we could readily create advanced logical circuits.”

It was a big step forward, but it wasn’t scalable — the team built these large and complex logic-responsive materials manually through traditional organic chemistry.

But over the next several years, the related field of synthetic biology advanced by leaps and bounds.

“The field has developed exciting new protein-based tools that can allow researchers to form permanent bonds between proteins,” said co-first author , a 91̽��doctoral student of bioengineering. “It opened doors for new protein structures that were previously unachievable, which made more complex logical operations possible.”

Additionally, it became practical to use living cells as factories to produce these complex proteins, allowing scientists to design custom DNA blueprints for new proteins, insert the DNA into bacteria or other host cells, and then collect the proteins with the desired structure directly from the cells.

With these new tools, DeForest and his team streamlined and improved many steps of the process at once. They designed and produced proteins with tails that spontaneously fold into more bespoke shapes, creating complex “circuits” that can respond to up to five different biomarkers. These new proteins can attach to various carriers — hydrogels, tiny beads or living cells — for delivery to a cell, or theoretically a disease site. The team even loaded up one carrier with three different proteins, each programmed to deliver their unique cargo based on different sets of environmental cues.

“We were so excited about the results,” DeForest said. “Using the old process, it would take months to synthesize just a few milligrams of each of these materials. Now it takes us a couple of weeks to go from construct design to product. It’s been a complete game changer for us.”

“The sky’s the limit. You can create delayed and independent delivery of many different components in one treatment,” Ross said. “And I think we could create much, much larger logical circuits that a protein can be responsive to. We’re at the point now that the technology is outpacing what we’ve seriously considered in terms of applications, which is a great place to be.”

The researchers will now continue searching for more biomarkers that proteins could target. They also hope to start collaborating with other labs at the 91̽��and beyond to build and deploy real-world therapies.

The team outlined other uses for the technology as well. The same tools could manufacture therapies within a single cell and direct them to specific regions, a sort of microcosm of how the process works in the body. DeForest also envisions diagnostic tools like blood tests that could, say, turn a certain color when a complex set of cues within the blood sample are present.

DeForest thinks the first practical applications are likely to be cancer treatments, but with more research, the possibilities feel endless.

“The dream is to be able to pick any arbitrary location inside of the body — down to individual cells — and program a material to go and act there,” he said. “That’s a tall order, but with these technologies we’re getting closer. With the right combination of biomarkers, these materials will just get more and more precise.”

Co-authors include , a former 91̽��undergraduate student of chemical engineering; , a 91̽��undergraduate student of bioengineering; and , a 91̽��doctoral student of chemical engineering.

This research was funded by the National Science Foundation and the National Institutes of Health.

For more information, contact DeForest at profcole@uw.edu.��

When researchers want to study how COVID makes us sick, or what diseases such as Alzheimer’s do to the body, one approach is to look at what’s happening inside individual cells.

Researchers sometimes grow the cells in a 3D scaffold called a “hydrogel.” This network of proteins or molecules mimics the environment the cells would live in inside the body.

New research led by the 91̽�� demonstrates a new class of hydrogels that can form not just outside cells, but also inside of them. The team created these hydrogels from protein building blocks designed using a computer to form a specific structure. These hydrogels exhibited similar mechanical properties both inside and outside of cells, providing researchers with a new tool to group proteins together inside of cells.

The team Jan. 30 in the Proceedings of the National Academy of Sciences.

“In the past 10 years, there’s been a shift in the world of cell biology,” said co-senior author , a 91̽��associate professor of chemical engineering and of bioengineering. “Classically, folks have attributed much of the cell’s interior organization to membrane-bound organelles, such as mitochondria or the nucleus. But now scientists are realizing that the cell actually has other ways to locally concentrate certain molecules or proteins without using membranes, for example, by protein-protein interactions. This concentrating allows the cell to turn on or off specific functions that can be helpful or ultimately lead to disease.”

DeForest continued: “What I think is pretty exciting here is that we have good mechanical control of our hydrogels — even when they are made inside human cells. This means we can tune them to essentially function as a synthetic version of whatever sequestering phenomenon we want to study, such as how protein aggregation can lead to Alzheimer’s.”

One key element of this research was that the protein building blocks were designed from scratch — they don’t exist anywhere in nature — using computers.

“You can imagine a protein as a string of subunits called amino acids. That string folds up to form a three-dimensional structure. There are 20 different amino acids, and a typical protein is made up of 100 to 200 of them. That makes the system very complex, because how do you know how it’s going to fold?” said co-lead author , who completed this research as a 91̽��postdoctoral researcher at the and is now a research fellow at Harvard Medical School and Boston Children’s Hospital. “That’s where the computer comes into play — it does calculations to estimate the most likely three-dimensional shape. And similarly, you can tell it what shape you want and it tells you what sequence you need to build the protein.”

To make a variety of hydrogels with different properties, the team used computational design to control how floppy or rigid the protein building blocks were and how the building blocks organized and connected to create the hydrogel. The researchers also used two different methods to link the building blocks together: One linked them irreversibly and the other allowed the proteins to disconnect and reconnect.

“Irreversibly crosslinked systems are going to be intrinsically more stable, making them better for long-term cell culture and functional tissue engineering,” said DeForest, who is also a faculty member with the 91̽�� and the 91̽��. “But the reversibly crosslinked systems are more fluid, which may be better for driving specific protein-protein interactions within living cells.”

To determine if the hydrogels in cells had similar characteristics compared to their extracellular counterparts, the researchers examined whether building blocks within the hydrogels could move around. A stiffer hydrogel would be more likely to trap the proteins in one position compared to a more fluid gel. The mechanical properties of each type of hydrogel remained even when inside a cell.

The team plans to further explore this system, including being able to better control how hydrogels form and localize within cells.

The most crucial part of this project, the researchers said, was the collaboration between protein designers and chemical and biological engineers.

“Our cross-disciplinary collaboration with Cole’s group has been very exciting, and has opened up routes to new classes of biomaterials with a wide range of applications,” said co-senior author , the director of the Institute for Protein Design and a professor of biochemistry in the 91̽��School of Medicine.

, a 91̽��doctoral student in bioengineering, is co-lead author on this paper. Additional co-authors are , a 91̽��research scientist in the Institute for Protein Design; , an assistant professor at Pohang University of Science and Technology who completed this research as a 91̽��postdoc at the Institute for Protein Design; , a 91̽��doctoral student in bioengineering; , a 91̽��doctoral student in molecular and cellular biology; , a 91̽��doctoral student in biological physics, structure and design; , a 91̽��research scientist at the Institute for Protein Design; , a group leader at the Hubrecht Institute who completed this research as a postdoc at the Institute for Protein Design; , an acting instructor at the Institute for Protein Design; Alee Sharma, an undergraduate student at Northeastern University; and , an associate professor at the Johns Hopkins School of Engineering. Mout and Sahtoe were part of the . This research was funded by the National Science Foundation, the Audacious Project, the Open Philanthropy Project, the Wu Tsai Translational Investigator Fund, the Center for the Science of Synthesis Across Scales and the National Institutes of Health.

For more information, contact DeForest at profcole@uw.edu, Mout at rubul.mout@childrens.harvard.edu and Baker at dabaker@uw.edu.

There are two major hurdles to overcome on the road to laboratory-grown organs and tissues. The first is to use a biologically compatible 3D scaffold in which cells can grow. The second is to decorate that scaffold with biochemical messages in the correct configuration to trigger the formation of the desired organ or tissue.

In a major step toward transforming this hope into reality, researchers at the 91̽�� have developed a technique to modify naturally occurring biological polymers with protein-based biochemical messages that affect cell behavior. Their approach, published the week of Jan. 18 in the Proceedings of the National Academy of Sciences, uses a near-infrared laser to trigger chemical adhesion of protein messages to a scaffold made from biological polymers such as collagen, a connective tissue found throughout our bodies.

Mammalian cells responded as expected to the adhered protein signals within the 3D scaffold, according to senior author , a 91̽��associate professor of chemical engineering and of bioengineering. The proteins on these biological scaffolds triggered changes to messaging pathways within the cells that affect cell growth, signaling and other behaviors.

These methods could form the basis of biologically based scaffolds that might one day make functional laboratory-grown tissues a reality, said DeForest, who is also a faculty member with the 91̽�� and the 91̽��.

“This approach provides us with the opportunities we’ve been waiting for to exert greater control over cell function and fate in naturally derived biomaterials — not just in three-dimensional space but also over time,” said DeForest. “Moreover, it makes use of exceptionally precise photochemistries that can be controlled in 4D while uniquely preserving protein function and bioactivity.”

DeForest’s colleagues on this project are lead author Ivan Batalov, a former 91̽��postdoctoral researcher in chemical engineering and bioengineering, and co-author , a 91̽��assistant professor of bioengineering and of laboratory medicine and pathology.

Their method is a first for the field, spatially controlling cell function inside naturally occurring biological materials as opposed to those that are synthetically derived. Several research groups, including DeForest’s, have developed light-based methods to modify synthetic scaffolds with protein signals. But natural biological polymers can be a more attractive scaffold for tissue engineering because they innately possess biochemical characteristics that cells rely on for structure, communication and other purposes.

“A natural biomaterial like collagen inherently includes many of the same signaling cues as those found in native tissue,” said DeForest. “In many cases, these types of materials keep cells ‘happier’ by providing them with similar signals to those they would encounter in the body.”

They worked with two types of biological polymers: collagen and fibrin, a protein involved in blood clotting. They assembled each into fluid-filled scaffolds known as hydrogels.

The signals that the team added to the hydrogels are proteins, one of the main messengers for cells. Proteins come in many forms, all with their own unique chemical properties. As a result, the researchers designed their system to employ a universal mechanism to attach proteins to a hydrogel — the binding between two chemical groups, an alkoxyamine and an aldehyde. Prior to hydrogel assembly, they decorated the collagen or fibrin precursors with alkoxyamine groups, all physically blocked with a “cage” to prevent the alkoxyamines from reacting prematurely. The cage can be removed with ultraviolet light or a near-infrared laser.

Using methods previously developed in DeForest’s laboratory, the researchers also installed aldehyde groups to one end of the proteins they wanted to attach to the hydrogels. They then combined the aldehyde-bearing proteins with the alkoxyamine-coated hydrogels, and used a brief pulse of light to remove the cage covering the alkoxyamine. The exposed alkoxyamine readily reacted with the aldehyde group on the proteins, tethering them within the hydrogel. The team used masks with patterns cut into them, as well as changes to the laser scan geometries, to create intricate patterns of protein arrangements in the hydrogel — including an old 91̽��logo, Seattle’s Space Needle, a monster and the 3D layout of the human heart.

The tethered proteins were fully functional, delivering desired signals to cells. Rat liver cells — when loaded onto collagen hydrogels bearing a protein called EGF, which promotes cell growth — showed signs of DNA replication and cell division. In a separate experiment, the researchers decorated a fibrin hydrogel with patterns of a protein called Delta-1, which activates a specific pathway in cells called Notch signaling. When they introduced human bone cancer cells into the hydrogel, cells in the Delta-1-patterned regions activated Notch signaling, while cells in areas without Delta-1 did not.

These experiments with multiple biological scaffolds and protein signals indicate that their approach could work for almost any type of protein signal and biomaterial system, DeForest said.

“Now we can start to create hydrogel scaffolds with many different signals, utilizing our understanding of cell signaling in response to specific protein combinations to modulate critical biological function in time and space,” he added.

With more-complex signals loaded on to hydrogels, scientists could then try to control stem cell differentiation, a key step in turning science fiction into science fact.

The research was funded by the National Science Foundation, the National Institutes of Health and Gree Real Estate.

For more information, contact DeForest at profcole@uw.edu.

Proteins are key to this future. In our bodies, protein signals tell cells where to go, when to divide and what to do. In the lab, scientists use proteins for the same purpose — placing proteins at specific points on or within engineered scaffolds, and then using these protein signals to control cell migration, division and differentiation.

But proteins in these settings are also fragile. To get them to stick to the scaffolds, researchers have traditionally modified proteins using chemistries that kill off more than 90% of their function. In a published May 20 in the journal , a team of researchers from the 91̽�� unveiled a new strategy to keep proteins intact and functional by modifying them at a specific point so that they can be chemically tethered to the scaffold using light. Since the tether can also be cut by laser light, this method can create evolving patterns of signal proteins throughout a biomaterial scaffold to grow tissues made up of different types of cells.

“Proteins are the ultimate communicators of biological information,” said corresponding author , a 91̽��assistant professor of chemical engineering and bioengineering, as well as an affiliate investigator with the 91̽��Institute for Stem Cell & Regenerative Medicine. “They drive virtually all changes in cell function — differentiation, movement, growth, death.”

For that reason, scientists have long employed proteins to control cell growth and differentiation in tissue engineering.

“But the chemistries most commonly used by the community to bind proteins to materials, including scaffolds for tissue engineering, destroy the overwhelming majority of their function,” said DeForest, who is also a faculty member in the 91̽��. “Historically, researchers have tried to compensate for this by simply overloading the scaffold with proteins, knowing that most of them will be inactive. Here, we’ve come up with a generalizable way to functionalize biomaterials reversibly with proteins while preserving their full activity.”

Their approach uses an enzyme called sortase, which is found in many bacteria, to add a short synthetic peptide to each signal protein at a specific location: the C-terminus, a site present on every protein. The team designs that peptide such that it will tether the signal protein to specific locations within a fluid-filled biomaterial scaffold common in tissue engineering, known as a hydrogel.

Targeting a single site on the signal protein is what sets the 91̽��team’s approach apart. Other methods modify signal proteins by attaching chemical groups to random locations, which often disrupts the protein’s function. Modifying just the C-terminus of the protein is much less likely to disrupt its function, according to DeForest. The team tested the approach on more than half a dozen different types of proteins. Results show that modifying the C-terminus has no significant effect on protein function, and successfully tethers the proteins throughout the hydrogel.

Their approach is analogous to hanging a piece of framed art on a wall. Instead of hammering nails randomly through the glass, canvas and frame, they string a single wire across the back of each frame to hang it on the wall.

In addition, the tethers can be cut by exposure to focused laser light, causing “photorelease” of the proteins. Using this scientific light saber allows the researchers to load a hydrogel with many different types of protein signals, and then expose the hydrogel to laser light to untether proteins from certain sections of the hydrogel. By selectively exposing only portions of the materials to the laser light, the team controlled where protein signals would stay tethered to the hydrogel.

Untethering proteins is useful in hydrogels because cells could then take up those signals, bringing them into the cell’s interior where they can affect processes like gene expression.

DeForest’s team tested the photorelease process using a hydrogel loaded with epidermal growth factor, a type of protein signal. They introduced a human cell line into the hydrogel and observed the growth factors binding to the cell membranes. The team used a beam of laser light to untether the protein signals on one side of an individual cell, but not the other side. On the tethered side of the cell, the proteins stayed on the outside of the cell since they were still stuck to the hydrogel. On the untethered side, the protein signals were internalized by the cell.

“Based on how we target the laser light, we can ensure that different cells — or even different parts of single cells — are receiving different environmental signals,” said DeForest.

This unique level of precision within a single cell not only helps with tissue engineering, but with basic research in cell biology, added DeForest. Researchers could use this platform to study how living cells respond to multiple combinations of protein signals, for example.�� This line of research would help scientists understand how protein signals work together to control cell differentiation, heal diseased tissue and promote human development.

“This platform allows us to precisely control when and where bioactive protein signals are presented to cells within materials,” said DeForest. “That opens the door to many exciting applications in tissue engineering and therapeutics research.”

Lead author on the paper is , a 91̽��doctoral student in chemical engineering. Co-author is , a 91̽��undergraduate alumna who is currently an analyst for Point B Consulting. The research was funded by the National Science Foundation and the 91̽��.

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For more information, contact DeForest at profcole@uw.edu.

Grant number: DMR-1652141.

Drug treatments can save lives, but sometimes they also carry unintended costs. After all, the same therapeutics that target pathogens and tumors can also harm healthy cells.

To reduce this collateral damage, scientists have long sought specificity in drug delivery systems: A package that can encase a therapeutic and will not disgorge its toxic cargo until it reaches the site of treatment — be it a tumor, a diseased organ or a site of infection.

In a published Jan. 15 in the journal , scientists at the 91̽�� announced that they have built and tested a new biomaterial-based delivery system — known as a hydrogel — that will encase a desired cargo and dissolve to release its freight only when specific physiological conditions are met. These environmental cues could include the presence of an enzyme or even the acidic conditions that could be found in a tumor microenvironment. Critically, the triggers that cause dissolution of the hydrogel can be switched out easily in the synthesis process, allowing researchers to create many different packages that open up in response to unique combinations of environmental cues.

The team, led by 91̽��chemical engineering assistant professor , designed this hydrogel using the same principles behind simple mathematical logic statements — those at the heart of basic programming commands in computer science.

“The modular strategy that we have developed permits biomaterials to act like autonomous computers,” said DeForest, who is also a member of both the and the . “These hydrogels can be programmed to perform complex computations based on inputs provided exclusively by their local environment. Such advanced logic-based operations are unprecedented, and should yield exciting new directions in precision medicine.”

Hydrogels are more than 90 percent water; the remainder consists of networks of biochemical polymers. Hydrogels can be engineered to ferry a variety of therapeutics, such as pharmaceutical products, special cells or signaling molecules, for purposes including drug delivery or even 3-D tissue engineering for transplantation into patients.

The key to the team’s innovation lies in the way the hydrogels were synthesized. When researchers assembled the polymer network that comprises the biomaterial, they incorporated chemical “cross-link” gates that are designed to open and release the hydrogel’s contents in response to user-specified cues — much like how the locked gates in a fence will only “respond,” or open with a specific set of keys.

“Our ‘gates’ consist of chemical chains that could — for example — be cleaved only by an enzyme that is uniquely produced in certain tissues of the body; or be opened only in response to a particular temperature or specific acidic conditions,” said DeForest. “With this specificity, we realized we could more generally design hydrogels with gates that would open if only certain chemical conditions — or logic statements — were met.”

DeForest and his team built these hydrogel gates using simple principles of logic, which centers on inputs to simple binary commands: “YES,” “AND” or “OR.” The researchers started out by building three types of hydrogels, each with a different “YES” gate. They would only open and release their test cargo — fluorescent dye molecules — in response to their specific environmental cue.

One of the “YES” gates they designed is a short peptide — one of the constituent parts of cellular proteins. This peptide gate can be cleaved by an enzyme known as matrix metalloprotease (MMP). If MMP is absent, the gate and hydrogel remain intact. But if the enzyme is present in a cell or tissue, then MMP will slice the peptide gate and the hydrogel will burst open, releasing its contents. A second “YES” gate that the researchers designed consists of a synthetic chemical group called an ortho-nitrobenzyl ester (oNB). This chemical gate is immune to MMP, but it can be cleaved by light. A third “YES” gate contains a disulfide bond, which breaks upon reaction with chemical reductants but not in response to light or MMP. A hydrogel containing one of these types of “YES” gates is essentially “programmed” to respond to its physiological surroundings using the Boolean logic of its cross-link gate. A hydrogel with an oNB gate, for example, will open and release its contents in the presence of light, but not any of the other cues like the MMP enzyme or a chemically reductive environment.

They also created and tested hydrogels with multiple types of “YES” gates, essentially creating hydrogels with gates that would open and release their cargo in response to multiple combinations of environmental cues, not just one cue: light AND enzyme; reductant OR light; enzyme AND light AND reductant. Hydrogels with these more complex types of gates could still carry cargo, either fluorescent dyes or living cells, and release it only in response to the particular gate’s unique combination of environmental triggers.

The team even tested how well a hydrogel with an “AND” gate — reductant and the enzyme MMP — could ferry the chemotherapy drug doxorubicin. The doxorubicin-containing hydrogel was mixed with cultures of tumor-derived , which doxorubicin should kill easily. But the hydrogel remained intact, and the HeLa cancer cells remained alive unless the researchers added both triggers for the “AND” gate: MMP and reductant. One cue alone was insufficient to cause HeLa cell demise.

DeForest and his team are building on these results to pursue even more complex gates. After all, specificity is the goal, both in medicine and tissue engineering.

“Our hope is that, by applying Boolean principles to hydrogel design, we can create a class of truly smart therapeutic delivery systems and tissue engineering tools with ever-greater specificity for organs, tissues or even disease states such as tumor environments,” said DeForest. “Using these design principles, the only limits could be our imagination.”

Lead author on the paper is 91̽��doctoral student Barry Badeau. Co-authors are master’s alumnus Michael Comerford, student Christopher Arakawa and doctoral student Jared Shadish. The research was funded by the National Science Foundation and the 91̽��.

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For more information, contact DeForest at 206-543-5961, profcole@uw.edu or on Twitter .

Grant number: DMR 1652141.