Drug delivery to the brain has been a longstanding challenge in medicine. One option is focused ultrasound (FUS), which can safely disrupt the blood–brain barrier for drug delivery. But how much of a drug gets into the brain depends heavily on its size. A long-standing assumption is that delivery decreases as drug size increases. But by using a clever spin on MRI physics, researcher Matthew Hoch and colleagues from the University of Virginia have shown that “the smaller the better” might not be entirely correct.

The blood–brain barrier is one of biology’s toughest engineering problems. Composed of a dense, tightly sealed layer of cells lining every blood vessel in the brain, the blood–brain barrier only lets nutrients and oxygen through and protects the central nervous system by blocking bloodborne pathogens. In doing so, however, it also blocks the delivery of drugs to the brain. Brain tumours compound this with their own blood–tumour barrier, further hindering drug delivery. For years, researchers have pondered how therapeutics could be delivered past these checkpoints; the answer may simply lie in the use of sound.

The process begins with the injection of gas-filled microbubbles into the bloodstream, where they circulate freely. Exposure to FUS then expands and contracts the volume of these microbubbles, creating a temporary, localized gap that pries the barrier open just long enough for drugs to slip through. Although the technique works, it is unclear how much of a drug actually comes through. In this new study, the team investigate how the size of the drug influences FUS-mediated drug delivery and introduce a novel technique to measure precisely how much drug is delivered.

The measurement problem

Many neurotherapies, with a range of sizes, could benefit from this drug delivery method. But measuring how much of the drug gets past the blood–brain barrier is challenging with current imaging methods. For example, fluorescence imaging is limited by its semi-quantitative nature, while PET scanning suffers from poor spatial resolution.

Standard MRI methods are not only time-consuming, but, in the form of T1-mapping, are typically limited by the size of commercially available contrast agents, which are small in comparison to larger neurotherapeutics used for immunotherapy and gene delivery. Other standard MRI methods sensitive to the presence of iron (T2-mapping) provide an alternative avenue to measure larger agents, but can again be limited by scan time for 3D visualization. Both approaches also have potential to suffer from low sensitivity at low concentrations.

Hoch and his team solve this measurement problem by introducing quantitative susceptibility mapping (QSM), an MRI technique with diverse applications in neuroimaging including evaluation of haemorrhage, iron deposition and calcification. QSM works by exploiting the distortion of local magnetic fields by different materials, a property called magnetic susceptibility. By injecting iron-based nanoparticles that induce measurable, spatially localized shifts in the MRI signal’s phase, QSM can generate a 3D map of magnetic susceptibility. This can then be used to calculate the actual concentration of iron-based nanoparticles within tissues.

By transforming an MRI scanner into a sensitive nanoparticle detector, the researchers present a novel way to quantify drug delivery down to fractions of a percent of the injected dose.

What did they find?

The team tested this approach using four different particles, with a 20-fold range in size. The first was a small gadolinium-based contrast agent, MultiHance (a proxy for small molecule therapeutics), at around 2.3 nm; the other three were iron oxide nanoparticles (IONPs) of 15, 23 and 45 nm, sizes that mirror the dimensions of therapeutic candidates used for immunotherapy and gene therapy.

Using healthy and glioma brain tumour-carrying mice, the researchers used FUS treatment to open the blood–brain barrier and deliver particles of varying sizes. They recorded MRI scans before and after delivery and used QSM to generate a precise measurement of how much of each particle made it into brain tissue.

The results were unexpected. Drug delivery followed a bell-shaped curve in healthy mice. From the smallest (2.3 nm) particle to the 15 nm IONP, delivery increased 2.6-fold, then at 23 nm, remained steady. Interestingly, at 45 nm, delivery dropped by 2.5-fold compared with the 23 nm nanoparticle, comparable to that of the smallest agent.

This sweet spot, ranging from 15 to 23 nm, demonstrates that smaller isn’t always better. The researchers suspect that this trend in drug delivery is due to a balance between how easily a particle can squeeze through the barrier (so favouring smaller particles) and how long a particle stays in circulation, giving it more time to travel across the barrier (favouring larger particles).

In the mice with brain tumours, where there is an addition of the tumour’s own leaky, disorganized vasculature, the blood–tumour barrier, drug delivery can differ from that to healthy brain. Specifically, this barrier is thought to make drug delivery more difficult than in healthy tissue due to higher tissue pressures and inconsistent blood flow. But the researchers unexpectedly found that FUS boosted delivery of both small and large agents into tumours, with no real difference to the drug delivery levels seen in healthy brain tissue, despite the added presence of the blood–tumour barrier.

“The outcome is exciting because it means that focused ultrasound delivery performance is not expected to diminish in brain tumours,” says senior author Richard J Price in a press statement. “In fact, it may even be enhanced for some types of therapeutics.”

FUS-mediated barrier opening is currently in clinical trials for use in Alzheimer’s disease and glioblastoma, and this research has implications in both drug engineering and treatment planning. By introducing QSM, Hoch and his colleagues have provided a universal, precision technique to quantify contrast agent delivery. Taking existing tools, used for a specific physics measurement problem, and developing them to answer a completely different biological question is no small feat. Here, the researchers present an ingenious intersection of physics and biology, and its potential application in medicine.

The researchers report their findings in Radiology.

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Islet transplantation, in which insulin-producing islet cells from a donor are implanted into a patient’s liver, is a promising therapy for type 1 diabetes. To prevent rejection of the donated cells, however, recipients need to continuously take immunosuppression drugs. Using a new type of cell encapsulation, researchers at The Pennsylvania State University demonstrated that diabetic mice could maintain normal blood glucose levels for 100 days, without requiring immunosuppressants. This represents a substantially longer time period than traditional cell therapies for diabetes.

The Penn State researchers developed their cell encapsulation process based on the molecular characteristics of the zona pellucida, a thin membrane that coats mammalian eggs. Once an egg is fertilized, biochemical reactions lead to hardening of the zona pellucida, which acts as a protective shield during embryo development. Here, biomimetic zona pellucida (BZP) encapsulation serves to hide the donor islets from the body’s immune system.

Pancreatic islets are clusters of five types of cells that work in tandem to regulate insulin and sugar levels in the body and prevent severe insulin deficiency. Islets harvested from healthy pancreases of deceased organ donors were first transplanted into Type 1 diabetes mellitus patients in 1974.

And in 2000, a landmark procedure increasing the mass of islets transferred and using steroid-free immunosuppression regimens was introduced by the University of Alberta in Edmonton, offering the hope of curing diabetes. But when implemented clinically, only 24% of recipients achieved insulin independence at 28 months with the Edmonton protocol.

Research on islet implantation continued unabated, particularly initiatives in islet encapsulation, using semi-permeable membranes to allow the entry of oxygen and nutrients and prevent the entrance of immune response cells. Most current work in this area uses large, 1.5 mm capsules. The Penn State team took an opposite tack: creating a thin 20-µm hydrogel capsule that spontaneously generates on the cell surface with 100% encapsulation efficiency.

Protection from immune attack

Principal investigator Yong Wang and colleagues spent eight years developing an ultrathin hydrogel layer that effectively lays against the curved edge of living cells or cell clusters without impacting their functionality. The islet encapsulation process takes place spontaneously in aqueous solutions under physiological conditions, without exposure to harsh physical, chemical or biological factors, ensuring that BZP development on the cell membrane does not cause any loss of cellular viability or function.

Writing in Nature Biomedical Engineering, the researchers explain that they emulated the biological processes that harden the zona pellucida on a fertilized egg (calcium ion-induced glycoprotein cleavage, assembly and crosslinking on the egg surface). In spite of its thinness, the BZP protects the cell as effectively as the much larger 1.5 mm diameter capsules.

The small capsule size significantly reduces the potential transplantation volume, improving an organ’s ability to receive and accommodate the large number of islets needed to be effective. When coating a 100-µm islet, for example, a 20-µm-thick BZP uses only 1/71 of the polymer volume of a 500-µm microcapsule. The researchers also suggest that the reduced polymer volume used for cell protection by the thin capsule may be a safety advantage of BZP compared to larger capsules.

In vivo assessment

The researchers first examined whether BZP could reduce the level of foreign body response to polystyrene microparticles injected into the peritoneal cavities of mice. Compared with uncoated microparticles, the BZP-coated particles induced a lower immune system response. They examined BZP synthesized using two types of alginate (involved in crosslinking reactions during BZP formation and hardening): chemically modified ultrapure alginate (BZP-U) and regular alginate (BZP-R). Tests in mice verified that BZP-U significantly mitigated the foreign body response compared with BZP-R encapsulation.

Immunocompetent diabetic mice treated with BZP-U-coated islets had their blood sugar restored to healthy levels within seven days. Additionally, the majority stayed diabetes free for over 100 days without continuously needing systemic immunosuppression. By comparison, mice treated with uncoated islets could not maintain healthy blood sugar levels for more than seven days.

In addition to effectively regulating blood glucose levels, one of the biggest advantages of the BZP approach is that it eliminates the need for long-term systemic immunosuppression. Lantidra, the only islet transplantation treatment for Type 1 diabetes cleared by the US Food and Drug Administration, requires continuous immunosuppressants to stop immune system attacks. Such long-term immunosuppressant use can lead to increased risk of infection and other serious health issues, including cancer.

Wang and colleagues plan to further study the BZP approach to better understand the specific duration of resistance each islet transplant may offer, as well as its effect in large animals.

“Large animal studies will be necessary to fully demonstrate their potential in cell transplantation,” the researchers write. “When large animals are used, one needs to examine not only glucose levels but also other key biomarkers. Moreover, the long-term immune responses to BZP and BZP-encapsulated cells warrant further investigation to yield deeper insights into BZP optimization and improvement.”

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Tissue engineering offers a means to restore, maintain or replace biological tissues. But it is difficult to grow blood vessels using conventional fabrication techniques like 3D printing, as they are typically not precise enough to print fine capillaries and veins. A team of engineers from the Massachusetts Institute of Technology (MIT) has now developed a new approach to do just that, by using mechanical forces. Their blood-vessel-on-a-chip could be the first step in the scalable production of vascular networks that could then be implanted in the body to replace tissue that has been damaged by disease or injury.

The researchers, led by Ritu Raman, made their chip by filling a Petri dish with a gel containing nutrients and cell-growth factors. They then embedded a small magnet into the gel. Finally, they introduced a thin, hollow tube into the gel and coated the inside of the tube with live endothelial cells – the cells that naturally line blood vessels in the body.

Once anchored onto the inner walls of the tube, the endothelial cells began sprouting new, capillary-like vessels in the gel. And when the researchers moved the embedded magnet back and forth in different directions and by varying degrees, they found that the mechanical forces created by the moving magnet could stretch the vessels and increase the number of new capillaries that grew, as well as their length.

‘Steering’ blood vessel growth

“We also showed that blood vessel growth, or angiogenesis, could be directionally patterned using the mechanical forces created by the magnet and that these directions could change dynamically over time,” explains Raman. “This allows us to ‘steer’ blood vessel growth in all three spatial directions (x, y, z) and make complex geometries (like L-shaped branches) that would not be possible to fabricate using conventional methods.”

The researchers had previously demonstrated that biological cells are highly responsive to mechanical forces in the body, but they did not know how different types of forces – for example, stretching in different directions and at different degrees and frequencies – impacted the 3D structure of blood vessels. The main obstacle lay in developing a coupled magnet system to precisely stretch blood vessels in different directions. To achieve this, they designed a method for mechanically stimulating tissues, dubbed “magnetic matrix actuation”.

In their new work, which is detailed in PNAS, they went a step further and developed a method to accurately yet non-invasively stretch 3D blood vessel tissues they had built in the laboratory.

To better understand the effect of mechanical forces on angiogenesis, the MIT team decided to look into the role of a gene known as PIEZO1, which is just one of the many mechanically sensitive genes in the body. They built on the work of molecular biologist Ardem Patapoutian, who received the 2021 Nobel Prize in Physiology or Medicine for discovering that ion channels in cells open and close in response to mechanical pressure. One such ion channel is PIEZO1, which is regulated by the PIEZO1 gene.

By suppressing this PIEZO1 gene in the endothelial cells they studied, the researchers indeed observed that significantly fewer new blood vessels grew in the gel. This, they say, provides evidence that the applied mechanical forces in their experiment may have been opening the PIEZO1 ion channel in the endothelial cells, so triggering the new blood vessel growth.

“My group has always been interested in engineering artificial tissues that can be implanted in the body to repair damage caused by disease or traumatic injury, but such artificial tissues require integrated networks of blood vessels in order to function,” says Raman. “We hope that our method for precisely fabricating complex networks of blood vessels within engineered tissues will enable the development of such implants,” she tells Physics World.

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