Directive 2010/63/EU laid down restrictions on animal testing for the testing of cosmetics and their ingredients throughout the EU. Therefore, there is an intense search for alternatives to test the absorption and toxicity of nanoparticles from cosmetics such as sun creams. A team of researchers from Graz University of Technology (TU Graz) and the Vellore Institute of Technology (VIT) in India is working on the development of skin imitations that mimic the native three-layer tissue structure and biomechanics of human skin. Such imitations can be produced using 3D printing and consist of hydrogel formulations that are printed together with living cells.

Hydrogels in which skin cells survive and grow

“The hydrogels for our skin imitation from the 3D printer have to fulfil a number of requirements,” says Karin Stana Kleinschek from the Institute of Chemistry and Technology of Biobased Systems. “The hydrogels must be able to interact with living skin cells. These cells not only have to survive, but also have to be able to grow and multiply.” The starting point for stable and 3D-printable structures are hydrogel formulations developed at TU Graz. Hydrogels are characterised by their high-water content, which creates ideal conditions for the integration and growth of cells. However, the high-water content also requires methods for mechanical and chemical stabilisation of the 3D prints.

TU Graz is working intensively on cross-linking methods for stabilisation. Ideally, following nature’s example, the cross-linking takes place under very mild conditions and without the use of cytotoxic chemicals. After successful stabilisation, the cooperation partners in India test the resistance and toxicity of the 3D prints in cell culture. Only when skin cells in the hydrogel survive in cell culture for two to three weeks and develop skin tissue can we speak of a skin imitation. This skin imitation can then be used for further cell tests on cosmetics.

Successful tests

The first tests of 3D-printed hydrogels in cell culture were very successful. The cross-linked materials are non-cytotoxic and mechanically stable. “In the next step, the 3D-printed models (skin imitations) will be used to test nanoparticles,” says Karin Stana Kleinschek. “This is a success for the complementary research at TU Graz and VIT. Our many years of expertise in the field of material research for tissue imitations and VIT’s expertise in molecular and cell biology have complemented each other perfectly. We are now working together to further optimise the hydrogel formulations and validate their usefulness as a substitute for animal experiments.”

In a recent study published in the journal Proceedings of the Royal Society B, an international team of researchers tested a new hypothesis for why some Lepidoptera have very specific diets, feeding on only a few types of plants, while others are far less picky.

The new idea, called the Salient Aroma Hypothesis, suggests that the smells plants release play a crucial role in determining how specialized a butterfly or moth’s diet becomes. The researchers found that greater availability of plant aromas during the day provides more chemical information for day-active insects to use to locate and specialize on particular host plants, while the decrease in plant aromas at nighttime means night-active Lepidoptera have to take what they can get and have a more varied diet.

“This idea provides a new perspective on why some butterflies and moths are picky eaters while others are not,” said Po-An Lin, an assistant professor at the National Taiwan University who launched the research while earning his doctoral degree from Penn State and continued the work as a postdoc in Taiwan. “It also highlights the critical role of plant volatiles, or scents, in shaping insect-plant interactions and evolutionary adaptations.”

To determine whether plant scent may have driven adaptation, the researchers looked at the insects’ primary organs for smelling — the antennae — and compared the antennal size of 582 specimens from 94 species of butterflies and moths.

The Penn State team collaborated with a team at Harvard that found that female Lepidoptera that are active during the day tend to have larger antennae relative to their body size than those active at night.

This might suggest that having better “smelling” equipment is more beneficial when there are more smells to detect, explained Gary Felton, the Ralph O. Mumma Professor of Entomology at Penn State, co-author on the paper and Lin’s research adviser. Similarly, specialist female Lepidoptera — those that feed from only a few types of plants — often have larger antennae than generalist females, possibly because they need to be very good at detecting the specific aromas of their host plants.

“The relationship between antennal size and host plant breadth was very strong,” Felton said. “Larger antennal sizes have been associated with a greater number of sensilla, the sensory structures involved in the sense of smell, thereby increasing the surface area for sensory receptors. The enhanced capacity may be a key adaptation for how certain Lepidoptera have evolved to feed on a limited and specific range of plants.”

The findings suggest a potential link between the availability of plant aromas during the day and an evolutionary investment in olfactory structures in the insects, particularly in females that engage in host plant selection by laying their eggs on the plant, Lin explained.

“This finding demonstrates how the availability of chemical signals influences the evolution of sensory organs in insects,” he said. “It provides a fascinating example of how plants, through their chemical emissions, have played a direct role in shaping the evolution of the insects that rely on them.”

Lin and colleagues at Penn State used a combination of approaches to investigate the link between plant aromas and Lepidoptera diets. They first conducted a meta-analysis of existing scientific literature to confirm that plants generally release more diverse and abundant volatile organic compounds, or aromas, during the day versus the night. Then they studied the Lepidoptera family tree to analyze the relationship between the insects’ activity patterns — day or night active — and their preferred host plants, using statistical models that account for evolutionary relationships.

“Our analyses showed a significant correlation between being active during the day or night and the diversity of host plant species that Lepidoptera consume,” said Naomi Pierce, professor of biology at Harvard University and co-author on the paper.

The researchers found that day-active Lepidoptera, like monarch butterflies, have more opportunities and more specialized organs to detect plant aromas and, as such, have evolved to be picky eaters. On the other hand, night-active species, like the Polyphemus Moth, encounter fewer and less diverse plant aromas. With less clear chemical information available, it might be harder for them to be so selective, potentially leading them to have more generalized diets, feeding on a wider range of plants.

“Insect herbivores, such as butterflies and moths, must find the right plants to feed on and, in the case of females, to lay their eggs,” Lin said. “This is a crucial decision because caterpillars depend entirely on the selected plant for survival. Unlike humans, who eat a wide variety of foods to stay healthy, many insect herbivores specialize in feeding on only a few plant species. The Salient Aroma Hypothesis helps explain why some insects are highly specialized while others are more flexible in their diet.”

The other authors on the paper are Wei-Ping Chan and Even Dankowicz of Harvard University; Liming Cai of the University of Texas Austin; Yun Hsiao of National Taiwan University; and Kadeem Gilbert of Michigan State University.

The U.S. National Science Foundation, Taiwan’s National Science and Technology Council and the Yushan Fellowship Program from the Ministry of Education of Taiwan funded this work.

The aptamers — short single-strand snippets of DNA that can target molecules like larger antibodies do — not only deliver cancer-fighting drugs, but also are themselves toxic to the cancer stem cells, the researchers said.

Led by Xing Wang, a U. of I. professor of bioengineering and of chemistry, the researchers documented their findings in the journal Advanced Functional Materials.

“This work demonstrates a way to get to the root of leukemia,” Wang said. “Targeted cancer treatments often have problems with toxicity or efficacy. Our aptamers seek out these stem cells specifically and kill them effectively.”

Leukemia and other cancers of the blood are more difficult to target than cancers that produce localized tumors because the cancerous cells circulate throughout the body and can’t be surgically removed, said postdoctoral researcher Abhisek Dwivedy, first author of the paper. Leukemia has a high rate of relapse due to its evasive stem cells. Though they make up a tiny fraction of cancerous cells, leukemia stem cells have the ability to evade chemotherapy by retreating to the bone marrow, since they share markers and properties, Dwivedy said. The cancerous cells can lurk, sometimes for years, and later proliferate and migrate.

“It’s important in leukemia, lymphoma or other blood cancers that we actually target and eliminate these stem cells, because as long as any are remaining, they can cause relapse and secondary cancers,” Dwivedy said.

The researchers began by finding DNA aptamers that seek out markers found on the surface of acute myeloid leukemia stem cells. They wanted to target not just the cancer, but the stem cells specifically.

“A big thing we showed in this study is that having two targets is better than one in terms of selectivity,” Wang said. “There are known antibody-drug conjugates for blood cancers that target one marker, but that marker is also found on a lot of healthy cells. So there is a lot of toxicity associated with antibody conjugates. But we used two targets: a combination often found in leukemia cancer cells and leukemia stem cells. The two together give a very specific target.”

The researchers then paired their aptamers with the leukemia-fighting drug daunorubicin. The drug-laden aptamers carry the drug to their target, then release the drug once inside the cell so the drug can act.

“This is especially important for drugs like daunorubicin, because the drug on its own cannot cross the cell membrane easily. But aptamers can carry it in,” Dwivedy said.

The researchers tested the drug-delivering aptamers in leukemia cell cultures as well as in live mice with leukemia.

After 72 hours, the aptamer alone had reduced the cancer cells in culture by 40 percent, demonstrating the aptamer’s toxicity to the cancer, the researchers report. When the aptamers carried the leukemia-fighting drug, the cells were wiped out with a dose 500 times smaller than the standard dosage of the drug. In mice with leukemia, delivering the drug via aptamer yielded the same efficacy at a dose 10 times smaller than the clinical standard, showing that the one-two punch of the aptamer and drug is more effective than either alone.

“This was exciting to us, because in cancer research, what we see in vitro is not always what we see in the body. Yet we saw excellent survivability and tumor reduction in the mice treated with our aptamer-drug conjugates, at one-tenth of the therapeutic dose, and no off-target effects,” Wang said.

The researchers said they hope to expand their suite of drug-delivering aptamers by identifying key marker combinations for other cancers, as well as coupling the aptamers with other drugs.

“Every cancer cell has a signature in its surface biomarkers. If we can find markers that are present uniquely in cancer cells, we can target other cancer types as well. Also, in my experience, it’s much easier to pair a drug with the DNA molecules than proteins, so that opens possibilities for delivering more drugs this way,” Dwivedy said.