Take a shot

29 October 2018

Injectable bandages are hugely promising for quickly administering materials to prevent fatality from excessive blood loss. Dr Akhilesh K Gaharwar, assistant professor in the Department of Biomedical Engineering at Texas A&M University, talks to Emma Green about this potentially transformative advancement in wound care.

There are a number of materials that can stop bleeding, but these can generally only be used for surface wounds and require pressure to promote clotting, which makes them unsuitable for high-risk situations, such as internal wounds. Many of these products also have further limitations, such as the need for pre-processing, batch variability and a lack of shear thinning properties. In addition, none of the current materials are biofunctional, meaning that they cannot interact with the body to produce the desired clotting and healing outcomes.

One of the most serious types of injuries that require quick and effective treatments are those obtained from shrapnel by soldiers on the battlefield. Statistics have shown that soldiers critically wounded in combat often pass away within 60 minutes, often referred to as the ‘golden hour’, In order to prevent fatalities from excessive blood loss, materials that can be self-administered are vital. Similarly, it is essential to be able to stop uncontrolled bleeding during surgical procedures in order to improve patient outcomes.

We were trying to build an injectable bandage that can stop bleeding without applying pressure, so that we can use it for internal wounds.

Against the flow

A number of researchers have begun to investigate the benefits of injectable bandages for these purposes. As part of a 2014 project commissioned by the US military, a team of researchers developed a syringe that could create a sealed barrier made up of small pillsized sponges after being injected into a large wound. The antimicrobial clotting agent chitosan was used to create the material. This allowed the sponges to expand by soaking up excess blood while simultaneously speeding up the clotting process.

During testing, it was found that it took only 15 seconds to stop all excess blood flow. Although such technology is a big step forward for wound care, it has been challenging to create materials that can aid healing in addition to stopping bleeding. Injectable hydrogels are promising materials for promoting healing and halting blood flow, as these biomaterials can be introduced into a wound site using minimally invasive approaches. Hydrogels are a 3D water-swollen polymer network, similar to gelatine, which are able to simulate the structure of human tissues. Ideally, an injectable hydrogel should solidify after injection in the wound area and promote the natural clotting cascade.

An emerging approach to improve the functionality of hydrogel networks is to incorporate bioactive nanoparticles. A range of synthetic nanoparticles have been used for this purpose. Two-dimensional nanomaterials are a recent development that have unique structural and surface characteristics. These nanoengineered ultrathin materials, with sheet or disc-like morphology, could generate major therapeutic advances in the field of regenerative medicine and biomolecule delivery.

Food for thought

An injectable hydrogel has been created by Dr Akhilesh K Gaharwar and researchers from Texas A&M University using kappa-carrageenan, a common thickening agent for pastries, and synthetic two-dimensional nanosilicates. “We were trying to build an injectable bandage that can stop bleeding without applying pressure, so that we can use it for internal wounds,” explains Gaharwar. “We also wanted to initiate wound healing and inject a material than can degrade over time to have a therapeutic effect.”

When kappa-carrageenan is mixed with clay-based nanoparticles, this forms an injectable gelatine. The hydrogel is biofunctional because it is made from materials that we consume or are already found in our bodies. It is also particularly valuable because the components are readily available and affordable, and can be stored at room temperature for long periods of time.

Combining these materials creates a hydrogel that changes form depending on the pressure applied. “By combining kappa-carrageenan and nanosilicates, we can make injectable materials, with a texture similar to toothpaste,” explains Gaharwar. “When you apply pressure they flow through, but as soon as you remove the pressure they solidify to stop bleeding.”

Researchers found that clotting time using the hydrogel was less than three minutes. This can literally be the difference between life and death. The clotting ability was the result of the negative charge of the nanoparticles in the hydrogel. However, achieving this outcome was not without challenges. “It was difficult to find materials that could stimulate clotting,” says Gaharwar. “We tried different materials, polymers like hyaluronic acid, but because they did not have a negative charge we were not able to achieve those properties. We screened all of these materials until we got this combination.”

In addition to the rapid clotting mechanism of the hydrogel, researchers took advantage of special properties of the nanoparticle component. They used the electric charge of the nanoparticles to add growth factors that adhered to the particles to begin the body’s natural healing process. This is particularly valuable as other approaches rely on the body’s own ability to heal.

Researchers attached vascular endothelial growth factor (VEGF) to the nanoparticles and then tested this combination in a cell culture test to mimic the healing process. The test uses a petri dish with a layer of endothelial cells on the surface that create a solid skin-like sheet. This is scratched down the centre, creating a hole in the sheet resembling a wound. When the hydrogel was added to the damaged endothelial cell wound, the cells were induced to grow back and fill in the scratched region, essentially mimicking the healing of a wound in the body.

During the transportation, they will lose a lot of blood, and because of that loss of blood they will die. We are hoping this technology can stop bleeding on the spot, before getting these soldiers to a healthcare centre.

War against wounds

Gaharwar envisions two key uses of this technology. The first of these is on the battlefield, where effective and quick treatments are needed to treat wounds, because soldiers are typically a long distance away from healthcare centres. One of the valuable aspects of this technology is the ability for the injection to be self-administered, so soldiers can kick-start the wound treatment themselves before their journey to the hospital. “During the transportation, they will lose a lot of blood, and because of that loss of blood they will die,” says Gaharwar. “We are hoping this technology can stop bleeding on the spot, before getting these soldiers to a healthcare centre.”

The second implication of this technology is the operating room. Researchers are currently developing new technologies that are able to stop bleeding and promote healing in realworld clinical settings. “Right now, we are trying to develop secondgeneration materials that are adhesive to tissue,” says Gaharwar. “When a surgeon is conducting any procedure, if they see any uncontrolled bleeding, they can use this material to seal tissue and stop bleeding. This material will also stimulate wound healing because it will be bioavailable.”

Although these implications are exciting, the current technology does have limitations. “We cannot release growth factors at different rates,” explains Gaharwar. “Now we can release growth factors for three to four weeks but if you want to add other growth factors to stimulate wound healing, it's not easy.” Overcoming this barrier will be a key focus of Gaharwar’s future research.

While researchers were encouraged by these results, they note that more testing is required. Gaharwar and his team are planning to conduct preclinical trials of the injectable bandage before proceeding to clinical use on human patients. The outcome of these trials is a little uncertain because of the higher blood pressure in humans, which will really test the strength of the hydrogel. If these tests go well, Gaharwar hopes the material will soon be able to be used in hospitals.

Researchers are also keen to embed other biologics into the hydrogel to speed up the overall healing process. Other experimental materials delivering drugs to wounds release a burst of medicine, requiring multiple injections, whereas this hydrogel can achieve a much slower release. The team believe they can include almost any type of small molecule drug or large molecule protein for sustained release from the gel. It is clear that hydrogels have immense potential, for wound care and beyond.

Carrageenans: not just a food additive

Carrageenans are a group of similar sulphated polysaccharides that have been used around the world for centuries as a gelatine and a home remedy. They have been made at home since 600BC but their commercial production did not begin until the 1930s. Carrageenans are used for a variety of purposes in the food industry, including as a stabilizer, thickener and emulsifier. They mimic the texture of fat in low-fat dairy products and help prevent non-dairy milk alternatives from separating. Carrageenans are also found in some packaged foods, luncheon meats, nutritional supplements and medications.

Edible red seaweed is the main source of carrageenan. The seaweed is harvested, dried and baked before being ground, sifted and washed to remove impurities like sand. It is then soaked in an alkaline solution, such as potassium hydroxide, and heated to remove the carrageenan. Filtration and centrifugation are used to remove cellulose mechanically. The remaining solution is evaporated to remove the water, and the powdered carrageenan is ground to meet final specifications.

Carrageenans are classified into three categories, depending on the amount of sulphation. The disaccharides in kappa-carrageenan have one sulphate, the disaccharides in iota-carrageenan have two, and the disaccharides in lambda-carrageenan have three. There have been some health scares about carrageenan, due to proposed links to problems such as chronic inflammation, insulin resistance and gastrointestinal issues. However, many of these studies used poligeenan, a form of degraded carrageenan, which is molecularly very different to the form of carrageenan typically used as an additive. Food-grade carrageenan has been declared safe by the Food and Drug Administration, The European Food Safety Authority and the World Health Organization.

Materials that can stop bleeding only work on surface wounds and require pressure to promote clotting.
Injectable hydrogels are meant to solidify in the wound area and promote natural coagulation.

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