Carbon Chemist

Tag: Nanostructures

  • New insights into gas vesicle clustering in bacteria open doors for biomedical applications

    New insights into gas vesicle clustering in bacteria open doors for biomedical applications

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    Gas vesicles are hollow structures made of protein found in the cells of certain microorganisms, and researchers at Rice University believe they can be programmed for use in biomedical applications.

    Inside cells, gas vesicles are packed in a beautiful honeycomb pattern. How this pattern is formed has never been thoroughly understood. We are presenting the first identification of a protein that can regulate this patterning, and we believe this will be a milestone in molecular microbiology.”


    George Lu, assistant professor of bioengineering and a Cancer Prevention and Research Institute of Texas scholar

    Lu and colleagues have published their findings in a paper published in Nature Microbiology. The lead author is Zongru Li, a fourth-year bioengineering doctoral student in Lu’s Laboratory for Synthetic Macromolecular Assemblies.

    “Gas vesicles are cylindrical tubes closed by conical end caps,” Li said. “They provide buoyancy within the cells of their native hosts.”

    The vesicles are found naturally in five phyla of bacteria and two groups of the archaea (single-cell organisms). Most are restricted to planktonic microorganisms often found in fresh-water ponds. The recent engineering of vesicles has led to several applications, including reporter gene imaging, acoustic control and payload delivery.

    Co-author Yifan Dai, assistant professor of biomedical engineering at McKelvey School of Engineering at Washington University in Saint Louis, said they were drawn to the research with the question of why the vesicles can form in the honeycomb pattern.

    With help from his WashU colleague Alex Holehouse and colleagues from Duke University, Ashutosh Chilkoti and Lingchong You, the team of researchers found that this pattern is the most efficient use of space and the cluster form plays a part in how it functions. Most notably, these protein clusters formed in subsaturated solution, a previously identified new form of biological structure, and that drives the organization of these vesicles. Bottom line, they found the function behind this mysterious new form.

    “These teams led by Lu lab found that a unique form of protein clusters exclusively assembled in subsaturated solution drives the clustering behaviors,” said Dai. This adds to the line of evidence on how phase transition affects cellular organization and cellular functions, he added.

    Lu and his team, using genetic, biochemical and imaging approaches, are exploring the protein nanostructures. Gas vesicles stabilize the air bubbles inside the bacterial cytosol ⎯ the fluids inside the cells ⎯ and provide a liquid-gas interface which can be used for ultrasound or MRI contrast.

    “In our lab, we are leveraging the power of synthetic biology to expand the applications of these protein nanostructures,” Li said. “By engineering genes and cells, we aim to build gas vesicles that perform even more efficiently in biotechnological and biomedical applications.”

    Li earned his B.S. in chemical engineering from the University of Rochester in 2018 and his M.S. in biotechnology from Northwestern University in 2020.

    Co-authors of the paper are Andrew Anderson, Manuel Iburg, Qionghua Shen, postdoctoral researchers in BIOE at Rice; Richard Lin ’23 BIOE, sustainable solutions and innovation analyst, NRG; Brandon Zimmer ’23 BIOE; Matthew Meyer, Rice electron microscopy research scientist; Yifan Dai and Alex Holehouse, assistant professors of biomedical engineering at WashU; Lingchong You, James L. Meriam Distinguished Professor of Biomedical Engineering at Duke; Ashutosh Chilkoti, the Alan L. Kaganov Distinguished Professor of Biomedical Engineering at Duke; and Emery Usher, postdoctoral researcher in biochemistry and molecular biology at WashU.

    The research was supported by the Cancer Prevention and Research Institute of Texas, the National Institutes of Health (R00 EB024600, R21 EB033607), the Welch Foundation, G. Harold and Leila Y. Mathers Foundation, Hearing Health Foundation, John S. Dunn Foundation, German Research Foundation, W.M. Keck Foundation, the Institute of Biosciences and Bioengineering at Rice and the Air Force Office of Scientific Research (FA9550-20-1-0241).

    Source:

    Journal reference:

    Li, Z., et al. (2024). Phase transition of GvpU regulates gas vesicle clustering in bacteria. Nature Microbiology. doi.org/10.1038/s41564-024-01648-3.

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  • DNA origami vaccine DoriVac paves way for personalized cancer immunotherapy

    DNA origami vaccine DoriVac paves way for personalized cancer immunotherapy

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    Therapeutic cancer vaccines are a form of immunotherapy in the making that could not only destroy cancer cells in patients, but keep a cancer from coming back and spreading. Multiple therapeutic cancer vaccines are being studied in clinical trials, but despite their promise, they are not routinely used yet by clinical oncologists to treat their patients. 

    The central ingredient of therapeutic cancer vaccines is antigens, which are preferentially produced or newly produced (neoantigens) by tumor cells and enable a patient’s immune system to search and destroy the cancerous cells. In most cases, those antigens cannot act alone and need the help of adjuvant molecules that trigger a general alarm signal in immune cells known as antigen-presenting cells (APCs). APCs internalize both antigen and adjuvant molecules and present the antigens to different types of T cells. Those T cells then launch an immediate attack against the tumor, or preserve a longer-lasting memory of the tumor for future defense.

    A cancer vaccine’s effectiveness depends on the level and duration of the “alarm” its adjuvants can ring in APCs. Previously, researchers found that delivering adjuvant and antigen molecules to APCs simultaneously using nanostructures like DNA origami can increase APC activation. However, none of these approaches systematically investigated how the number and nanoscale arrangement of adjuvant molecules affect downstream tumor-directed immunity. 

    Now, a research team at the Wyss Institute at Harvard University, Dana-Farber Cancer Institute (DFCI), Harvard Medical School (HMS), and Korea Institute of Science and Technology (KIST) has created a DNA origami platform called DoriVac, whose core component is a self-assembling square block-shaped nanostructure. To one face of the square block, defined numbers of adjuvant molecules can be attached in highly tunable, nanoprecise patterns, while the opposite face can bind tumor antigens. The study found that molecules of an adjuvant known as CpG spaced exactly 3.5 nanometers apart from each other resulted in the most beneficial stimulation of APCs that induced a highly-desirable profile of T cells, including those that kill cancer cells (cytotoxic T cells), those that cause beneficial inflammation (Th-1 polarized T cells), and those that provide a long-term immune memory of the tumor (memory T cells). DoriVac vaccines enabled tumor-bearing mice to better control the growth of tumors and to survive significantly longer than control mice. Importantly, the effects of DoriVac also synergized with those of immune checkpoint inhibitors, which are a highly successful immunotherapy that is already widely used in the clinic. The findings are published in Nature Nanotechnology.

    “DoriVac’s DNA origami vaccine technology merges different nanotechnological capabilities that we have developed over the years with an ever-deepening knowledge about cancer-suppressing immune processes,” said Wyss Core Faculty member William Shih, Ph.D., who led the Wyss Institute team together with first-author Yang (Claire) Zeng, M.D., Ph.D. “We envision that in the future, antigens identified in patients with different types of tumors could be quickly loaded onto prefabricated, adjuvant-containing DNA origami to enable highly effective personalized cancer vaccines that can be paired with FDA-approved checkpoint inhibitors in combination therapies.”

    Shih is also a Professor at HMS and DFCI’s Department of Cancer Biology and, as some of the other authors, a member of the NIH-funded cross-institutional “Immuno-engineering to Improve Immunotherapy” (i3) Center based at the Wyss. 

    DNA origami rationale

    The CpG adjuvant is a synthetic strand of DNA made up of repeated CpG nucleotide motifs that mimic the genetic material from immune cell-invading bacterial and viral pathogens. Like its natural counterparts, CpG adjuvants bind to a “danger receptor” called TLR9 in immune cells, which in turn induces an inflammatory (innate) immune response that works in concert with the antigen-induced (adaptive) immune response. 

    “We knew from previous work that to trigger strong inflammatory responses, TLR9 receptors need to dimerize and aggregate into multimeric complexes binding to multiple CpG molecules. The nanoscale distances between the CpG-binding domains in effective TLR9 assemblies revealed by structural analysis fell right into the range of what we hypothesized we could mirror with DNA origami structures presenting precisely spaced CpG molecules,” explained Zeng, who was an Instructor in Medicine at the time of the study and now is a senior scientist at DFCI and Harvard Medical School (HMS). In addition to Shih, Zeng was also mentored on the project by senior authors Ju Hee Ryu, Ph.D., a Principal Researcher at KIST, and Wyss Founding Core Faculty member David Mooney, Ph.D., who also is Professor at Harvard John A. Paulson School of Engineering and Applied Sciences (SEAS), and one of the i3 Center’s Principal Investigators. 

    Zeng and the team fabricated DoriVac vaccines in which different numbers of CpG strands were spaced at 2.5, 3.5, 5, or 7 nanometers apart from each other on one face of the square block, and a model antigen was attached to the opposite face. They protected their structures from being degraded in the body using a chemical modification method that Shih’s group had developed earlier. When internalized by different types of APCs, including dendritic cells (DCs), which orchestrate tumor-directed T cell responses, the DoriVac vaccines improved the uptake of antigens compared to controls consisting of free antigen molecules. A CpG spacing of 3.5 nanometers produced the strongest and most beneficial responses in APCs, and significantly outperformed a control vaccine containing only free CpG molecules. “We were excited to find that the DoriVac vaccine preferentially induced an immune activation state that supports anti-tumor immunity, which is what researchers generally want to see in a good vaccine,” said Zeng. 

    Besides spacing, the numbers of CpG molecules in DoriVac vaccines also mattered. The team tested vaccines containing between 12 to 63 optimally spaced CpG molecules and found that 18 CpG molecules provided the best APC activation. This meant that their approach can also help limit the dosage of CpG molecules and thus minimize commonly observed toxic side effects observed with adjuvants.

    Gained in (tumor) translation

    Importantly, these in vitro trends translated to in vivo mouse tumor models. When prophylactically injected under the skin of mice, DoriVac vaccines accumulated in the closest lymph nodes where they stimulated DCs. A vaccine loaded with a melanoma antigen prevented the growth of subsequently injected aggressive melanoma cells. While all control animals had succumbed to the cancer by day 42 of the experiment, DoriVac-protected animals all were alive. DoriVac vaccines also inhibited tumor growth in mice in which the formation of melanoma tumors was already underway, with a 3.5 nanometer spacing of 18 CpG molecules again providing maximum effects on DC and T cells, and the strongest reduction in tumor growth.

    Next, the team asked whether DoriVac vaccines could also boost immune responses produced by small “neoantigens” emerging in melanoma tumors. Neoantigens are ideal targets because they are exclusively made by tumor cells. However, they often are not very immunogenic themselves, which make highly effective adjuvants an important component in neoantigen vaccines. A DoriVac vaccine customized with four neoantigens enabled the researchers to significantly suppress growth of the tumor in mice that produced the neoantigens.

    Finally, the researchers asked whether DoriVac could synergize with immune checkpoint therapy, which reactivates T cells that have been silenced in tumors. In mice, the two therapies combined resulted in the total regression of melanoma tumors, and prevented them from growing back when the animals were exposed to the same tumor cells again four months later. The animals had built up an immune memory of the tumor. The team obtained a similar vaccination efficiency in a mouse lymphoma model.

    We think that DoriVac’s value for determining a sweet spot in adjuvant delivery and enhancing the delivery and effects of coupled antigens can pave the way to more effective clinical cancer vaccines for use in patients with a variety of cancers.”


    Yang (Claire) Zeng, M.D., Ph.D., First Author

    The team is currently translating the DoriVac platform toward its clinical application, which is supported by the study’s assessment of vaccine distribution and vaccine-directed antibodies in mice, as well as cytokines produced by immune cells in response to the vaccines in vivo. 

    “The DoriVac platform is our first example of how our pursuit of what we call Molecular Robotics – synthetic bioinspired molecules that have programmable shape and function – can lead to entirely new and powerful therapeutics. This technology opens an entirely new path for development of designer vaccines with properties tailored to meet specific clinical challenges. We hope to see its rapid translation into the clinic,” said Wyss Institute Founding Director Donald Ingber, M.D., Ph.D., who is also the Judah Folkman Professor of Vascular Biology at HMS and Boston Children’s Hospital, and the Hansjörg Wyss Professor of Bioinspired Engineering at SEAS.

    Other authors on the study are Olivia Young, Christopher Wintersinger, Frances Anastassacos, James MacDonald, Giorgia Isinelli, Maxence Dellacherie, Miguel Sobral, Haiqing Bai, Amanda Graveline, Andyna Vernet, Melinda Sanchez, Kathleen Mulligan, Youngjin Choi, Thomas Ferrante, Derin Keskin, Geoffrey Fell, Donna Neuberg, Cathrine Wu, and Ick Chan Kwon. The study was funded by the Wyss Institute’s Validation Project and Institute Project programs, Claudia Adams Barr Program at DFCI, Korean Fund for Regenerative Medicine (award #21A0504L1), Intramural Research Program of KIST (award #2E30840), and National Institutes of Health (under the i3 Center supporting U54 grant (award #CA244726-01).

    Source:

    Wyss Institute at Harvard University

    Journal reference:

    Zeng, Y. C., et al. (2024). Fine tuning of CpG spatial distribution with DNA origami for improved cancer vaccination. Nature Nanotechnology. doi.org/10.1038/s41565-024-01615-3.

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  • New microscopy technology unveils detailed images of brain cancer tissue

    New microscopy technology unveils detailed images of brain cancer tissue

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    Brigham and MIT researchers uncovered never-before-seen details in human brain tissue with new, inexpensive microscopy technology.

    Key takeaways:

    • Researchers have developed a new microscopy technology called decrowding expansion pathology (dExPath) to analyze brain tissue.

    • By pulling proteins apart with dExPath, researchers can stain proteins in tissue that could not be accessed before, highlighting nanometer sized structures or even cell populations that were previously hidden.

    • This “super-resolution imaging” technology has the potential to provide insights that could improve diagnostic strategies and patient outcomes.

    Researchers from Brigham and Women’s Hospital, a founding member of Mass General Brigham, and the Massachusetts Institute of Technology (MIT) have unveiled unprecedentedly detailed images of brain cancer tissue through the use of a new microscopy technology called decrowding expansion pathology (dExPath).Their findings, published in Science Translational Medicine, provide novel insights into brain cancer development, with potential implications for advancing the diagnosis and treatment of aggressive neurological diseases.

    “In the past, we have relied on expensive, super-resolution microscopes that only very well-funded labs could afford, required specialized training to use, and are often impractical for high-throughput analyses of brain tissues at the molecular level,” said Pablo Valdes, MD, PhD, a neurosurgery resident alumnus at the Brigham and lead author of the study. “This technology brings reliable, super-resolution imaging to the clinic, enabling scientists to study neurological diseases at a never-before-achieved nanoscale level on conventional clinical samples with conventional microscopes.”

    Researchers previously relied on costly, super-high-resolution microscopes to image nanoscale structures in cells and brain tissue, and, even with the most advanced technology, they often struggled to effectively capture these structures at the nanoscale level.

    Ed Boyden, PhD, the Y. Eva Tan Professor in Neurotechnology at MIT and co-senior author on this study, began addressing this problem by labeling tissues, and then chemically modifying them to enable uniform physical expansion of tissues. However, this expansion technology was far from perfect. Relying on enzymes known as proteases to break up tissue, scientists found that this chemical treatment with enzymes destroyed proteins before they could analyze them, leaving behind only a skeleton of the original structure, retaining only the labels.

    Working together, Boyden and E. Antonio Chiocca, MD, PhD, Neurosurgery Chair at Brigham and Women’s Hospital and co-senior author on this study, mentored Valdes during his training as a neurosurgeon-scientist, to develop novel chemistries with dExPath to address the limitations of the original expansion technology.

    Their new technology chemically modifies tissues by embedding them in a gel and ‘softening’ the tissues with a special chemical treatment that separates protein structures without destroying them and which allows tissues to expand. This provided exciting findings to the MIT and Brigham researchers, who routinely use commercially available antibodies to bind to and illuminate biomarkers in a sample. Antibodies, however, are large and many times cannot easily penetrate cell structures to reach their target. Now, by pulling proteins apart with dExPath, these same antibodies used for staining can penetrate spaces to bind proteins in tissue that could not be accessed before expansion, highlighting nanometer sized structures or even cell populations that were previously hidden.

    The human brain has several stop guards in place to protect itself from pathogens and environmental toxins. But these elements make studying brain activity challenging. It can be a bit like driving a car through mud and ditches. We cannot access certain cell structures in the brain because of barriers that stand in the way. That is just is one of the reasons that this new technology could be so practice changing. If we can take more detailed and accurate images of brain tissue, we can identify more biomarkers and be better equipped to diagnose and treat aggressive brain diseases.”


    E. Antonio Chiocca, MD, PhD, Chair of the Department of Neurosurgery, Brigham and Women’s Hospital

    To validate the effectiveness of dExPath, Boyden and Chiocca’s team applied the technology to healthy human brain tissue, high and low-grade brain cancer tissues, and brain tissues affected by neurodegenerative diseases including Alzheimer’s and Parkinson’s diseases. Investigators stained tissue for brain and disease specific biomarkers and captured images before and after expanding samples with dExPath.

    The results revealed uniform and consistent expansion of the tissue without distortion, enabling accurate analysis of protein structures. Additionally, dExPath effectively eliminated fluorescent signals in brain tissue called lipofuscin, which makes imaging of subcellular structures in brain tissues very difficult, further enhancing image quality. Further, dExPath provided stronger fluorescent signals for improved labeling as well as simultaneous labeling of up to 16 biomarkers in the same tissue specimen. Notably, dExPath imaging revealed that tumors previously classified as “low-grade” contained more aggressive features and cell populations, suggesting the tumor could become far more dangerous than anticipated.

    While promising, dExPath requires validation on larger sample sizes before it can contribute to the diagnosis of neurological conditions such as brain cancer. Valdes underscores that, although still in its early stages, his team aspires for this technology to eventually serve as a diagnostic tool, ultimately enhancing patient outcomes.

    “We hope that with this technology, we can better understand at the nanoscale levels the intricate workings of brain tumors and their interactions with the nervous system without depending on exorbitantly expensive lab equipment,” said Valdes who is now an assistant professor of neurosurgery and Jennie Sealy Distinguished Chair in Neuroscience at the University of Texas Medical Branch. “The accessibility of dExPath will bring enable super-resolution imaging to understand biological processing at the nanometer level in human tissue in neuro-oncology and in neurological disease such as Alzheimer’s and Parkinson’s, and one day, could even improve diagnostic strategies and patient outcomes.”

    Source:

    Brigham and Women’s Hospital

    Journal reference:

    Valdes, P. A., et al. (2024) Improved immunostaining of nanostructures and cells in human brain specimens through expansion-mediated protein decrowding. Science Translational Medicine. doi.org/10.1126/scitranslmed.abo0049.

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