Making therapeutic proteins last longer

Happy TRAILs to you: PEGylation of proteins through complementary interactions between a His-tag and a Ni2+ complex of nitrilotriacetic acid (NTA, see picture), a well-established practice in protein research, was used to improve the half-life of therapeutic proteins in the blood following systemic administration in vivo. Animal models show that this site-specific modification improves the efficacy of modified TRAIL proteins.

Happy TRAILs to you: PEGylation of proteins through complementary interactions between a His-tag and a Ni2+ complex of nitrilotriacetic acid (NTA, see picture), a well-established practice in protein research, was used to improve the half-life of therapeutic proteins in the blood following systemic administration in vivo. Animal models show that this site-specific modification improves the efficacy of modified TRAIL proteins.

Proteins are responsible for pretty much everything in the human body. When there is a problem with the proteins, it usually leads to disease.

Protein therapy shows enormous potential for treating disease. But sometimes the proteins in a therapeutic treatment break down or are metabolized before they ever reach their target destination.

In a recent paper published in Angewandte Chemie, researchers from the laboratories of Martin Pomper (radiology oncology) and Seulki Lee (radiology, Center for Nanomedicine) at the Johns Hopkins School of Medicine and developed a simple method to validate protein drugs in animal models, said Lee. An illustration related to the paper appeared on the cover of the journal.

“We show that we can extend the half-life, that is, the amount of time the drug stays in the blood, while maintaining the activity of the model protein drug, TRAIL,” said one of the lead authors Maggie Swierczewska. “This has great implications for drug screening and validation methods, especially for the growing protein drug market.”

According to the paper, by attaching a molecule of  polyethylene glycol (PEG) to certain sites on the TRAIL protein drugs through an already well known method, the half-life of the drug could be extended without affecting its beneficial activity.

Authors on this paper include Tae Hyung Kim, Magdalena Swierczewska, Yumin Oh, AeRyon Kim, Dong Gyu Jo, Jae Hyung Park,  Youngro Byun, Scheherazade Sadegh-Nasseri, Martin G. Pomper, Kang Choon Lee, Seulki Lee. Author affiliations include the departments of Radiology and Pathology at the Johns Hopkins School of Medicine, the Johns Hopkins Center of Cancer Nanotechnology Excellence, the Johns Hopkins Institute for NanoBioTechnology, Center for Nanomedicine and collaborators at Sungkyunkwan University and Seoul National University, both in Korea.

Reference: Kim, T. H., Swierczewska, M., Oh, Y., Kim, A., Jo, D. G., Park, J. H., Byun, Y., Sadegh-Nasseri, S., Pomper, M. G., Lee, K. C. and Lee, S. (2013), Mix to Validate: A Facile, Reversible PEGylation for Fast Screening of Potential Therapeutic Proteins In Vivo. Angew. Chem. Int. Ed.. Vol. 52, Issue 27, pages 6880-6884, doi: 10.1002/anie.201302181

In vivo visualization of angiogenesis during wound healing featured on journal cover

Laser speckle contrast images showing (l-r) sequential images of the in vivo blood flow changes that occur on days 0, 3, 5, 7, 10 and 12 after wound creation.

Innovative ways of imaging wound healing can reveal much about blood vessel remodeling and blood flow following an injury. Researchers in the Russell H. Morgan Department of Radiology and Radiological Science and Department of Biomedical Engineering at the Johns Hopkins University School of Medicine have developed a method for using laser speckle contrast imaging (LSCI) to elucidate the changes that occur in the microvasculature over time as a wound heals. Researchers in the laboratory of Arvind P. Pathak have visualized the wound healing process in a mouse ear model by capturing images of angiogenesis—or the development of new blood vessels—over a 12-day period.

“LSCI is a powerful tool for observing the architecture and remodeling of microvasculature as well as the hemodynamic changes (blood flow) during angiogenesis,” said Pathak, an assistant professor of radiology and oncology and principal investigator on the study. “Being able to watch this process occur in a living animal helps us better understand the role of the vasculature during various phases of the wound healing process.”

Stunning images obtained from their experiments were featured on the cover of the March issue of the journal Angiogenesis. The LSCI images shown on the cover from left to right are sequential images of the in vivo blood flow changes that occur on days 0, 3, 5, 7, 10 and 12 after wound creation. The “hotter” colors indicate higher blood flow. The background image is a grayscale LSCI image from an uninjured mouse ear.

Wound healing typically proceeds in three phases, Pathak explained: inflammation (which initiates the immune response and recruits immune cells and molecules to the injury), proliferation (the formation of new blood vessels and epithelium) and remodeling (which removes the vascular scar created during blood vessel formation). LSCI is ideal for imaging the progression of each phase because it can monitor in vivo changes in microvascular architecture and hemodynamics at the same time, he said.

LSCI images are created when tissue illuminated by a laser is photographed through a small aperture, explained Pathak. “The resulting images exhibit a random interference pattern, also called a ‘speckle’ pattern. In blood vessels, this speckle pattern shifts due to the orderly motion of red blood cells, causing a blur over the exposure time of the camera. The degree of blurring in the LSCI image is proportional to the velocity of blood in the vessels and constitutes the biophysical basis of LSCI. Therefore, LSCI can distinguish blood vessels in tissue without any fluorescent dye or contrast agent.”

In this way, Pathak added, LSCI is capable of “wide area mapping” of the tissue, allowing us to measure not only the length and perfusion of blood vessels but their tortuosity (twistiness) and the overall flow of blood to the wound site as healing progresses.

In addition to angiogenesis research, the imaging method has practical applications in drug testing, Pathak said. “Using LSCI alongside a drug study would provide better insight into the efficacy of drug delivery and therapeutic outcome,” he said.

The lead author of the paper was Abhishek Rege, a graduate student in biomedical engineering co-mentored by Pathak and Nitish V. Thakor, professor of biomedical engineering in whose neuroengineering laboratory LSCI was developed. Kevin Rhie, a research technician in Pathak’s laboratory was the other author on this study.

This work was supported jointly by a Johns Hopkins Institute for NanoBiotechnology (INBT) Junior Faculty Pilot Award to Pathak, and grants from the National Institute of Aging and the Department of Health and Human Services to Thakor.

Story by Mary Spiro