Researchers Discover the Mechanism of Nanomaterial-Protein Interaction for Regulation of Tumor Immune Microenvironment

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A research team led by Prof. Chen Chunying from the National Center for Nanoscience and Technology (NCNST) of the Chinese Academy of Sciences (CAS) reported the mechanism of graphyne-intracellular protein interaction for regulation of the phenotype of macrophages. The related result "The Underlying Function and Structural Organization of the Intracellular Protein Corona on Graphdiyne Oxide Nanosheet for Local Immunomodulation" was published in Nano Letters (2021, 21, 14, 6005-6013). The researchers used isotope technique to analysis the interaction between graphyne oxide and its highly enriched signal transduction and activator of transcription (STAT3) protein in the intracellular protein corona, which affects the phenotype of tumor-associated macrophages, and improves the tumor immunosuppressive microenvironment.
After entering the body, nanomaterials/nano drugs will face a complex physiological environment and interact with the surrounding biological fluids or biological molecules (proteins, DNA and lipids). The interaction of the nanomaterial-biological interface is the core step that determines its biological effects.
Tumor-associated macrophage is the most numerous inflammatory cell group in a variety of tumors. It promotes tumor growth, metastasis and recurrence, induces immunosuppression, and is associated with poor prognosis of solid tumors. There are three treatment methods for tumor-associated macrophages: inhibition of the recruitment of macrophages, direct killing of TAM and TAM reprogramming. At present, a variety of small molecule drugs have been used in clinical trials, but small molecules lack targeting ability. Due to diverse physical and chemical properties, many nanomaterials can be used as delivery vehicles and immunomodulators to improve the tumor immunosuppressive microenvironment. 
Graphyne is an emerging two-dimensional carbon material and it has shown broad application prospects in catalysis, energy, biomedicine and other fields. Researchers used the isotope 13C labeling of GDYO and proteomics methods to find for the first time that a unique protein corona is formed on the surface of GDYO in macrophages, which is highly enriched in STAT3 protein. STAT3 is an important signal transduction protein and transcription factor in cells. It is closely related to the occurrence and development of tumors. By inhibiting its signal pathway, it can inhibit tumor growth and metastasis. By inhibiting the activation of STAT3 protein, GDYO reverses immunosuppressive M2 macrophages into pro-inflammatory M1 macrophages, improves the immunosuppression caused by TAMs, increase the infiltration and activation of killer T cells, and improve the efficacy of PD-L1 antibodies. 
This article uses the isotope 13C to label GDYO for the first time, quantitatively analyzes the interaction ratio between GDYO and intracellular proteins. At the same time, the distribution and metabolism of GDYO in peritoneal macrophages and tumors after administration are analyzed, which provides a direct, accurate and quantitative analysis method for the assessment of the biological behavior of carbon nanomaterials in vivo.
GDYO comprises of hybridized sp and sp2 carbon atom and the surface consists of well-arranged C=O and C-OH groups, making GDYO behave in an unpredictable manner at the nano-bio interface. The interaction at the GDYO–STAT3 interface, driven by structure matching, hydrogen bonding and salt bridges, simultaneously triggers the immune response in the tumor microenvironment. The distance between two adjacent C=O and/or C-OH groups is 5.5 Angstroms. The pitch of α-helix in the N-terminal domain of STAT3 protein is 5.4 Angstroms, which matches the distance of oxygen-containing groups. Furthermore, C=O and C-OH groups form hydrogen bonds or salt bridges with amino acid residues in the STAT3 protein. These results explain the molecular mechanism of the unique strong interaction of GDYO-STAT3. The research comprehensively uses proteomics, theoretical calculations, and isotope quantification technology to reveal for the first time the interaction mechanism of nanoparticle-protein interface in macrophages, which is useful for in-depth understanding of nano-biological interface regulation of complex biology.
Chen Chunying's research group has been committed to the research of nanomaterials-biological interface for a long time, and has made a series of progress. In 2011, they discovered for the first time that carbon tubes can rapidly adsorb proteins in the blood to form a corona to reduce biological toxicity (Proc. Natl. Acad. Sci. U.S.A. 2011, 108, 16968-16973). In 2019, it was discovered that Gd@C82(OH)22 nanoparticles specifically bind to the complement component C1q protein in the blood of lung cancer patients, and activate the innate immune response by changing the structure of the C1q molecule (Nano Lett. 2019, 19, 7, 4692-4701). In 2021, the unique in vivo transportation, metabolism and bioavailability of molybdenum disulfide nanomaterials mediated by protein corona was reported for the first time (Nat. Nanotechnol. 2021, 16, 708-716). These studies provide key and cutting-edge analytical methods for nano-biological effects and nano-medicine research, and vigorously promote the development of nano-biomedicine.
Guo Mengyu, Special Research Assistant of the National Center for Nanoscience and Technology, Researcher Zhao Lina of the Institute of High Energy Physics of the Chinese Academy of Sciences, and Researcher Liu Jing of the of the National Center for Nanoscience and Technology are the first authors of this article. Associate researcher Cai Rong and Researcher Chen Chunying are the corresponding authors. This work was financially supported by the National Key Research and Development Program, the National Natural Science Foundation of China, the Strategic Priority Research B Program of the Chinese Academy of Sciences, and the research and development projects in key areas of Guangdong Province.
Figure 1. Graphyne oxide improves tumor immunosuppressive microenvironment through interaction with its intracellular protein corona.

CHEN Chunying

National Center for Nanoscience and Technology



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