Self-assembling, pH-responsive nanoflowers for inhibiting PAD4 and neutrophil extracellular trap formation and improving the tumor immune microenvironment

Di Zhu; Yu Lu; Lin Gui; Wenjing Wang; Xi Hu; Su Chen; Yanming Wang; Yuji Wang

doi:10.1016/j.apsb.2021.11.006

Self-assembling, pH-responsive nanoflowers for inhibiting PAD4 and neutrophil extracellular trap formation and improving the tumor immune microenvironment

Acta Pharm Sin B. 2022 May;12(5):2592-2608. doi: 10.1016/j.apsb.2021.11.006. Epub 2021 Nov 12.

Authors

Di Zhu^{1

2}, Yu Lu^{1

2}, Lin Gui^{1

2}, Wenjing Wang³, Xi Hu⁴, Su Chen⁵, Yanming Wang⁶, Yuji Wang^{1

2}

Affiliations

¹ Department of Medicinal Chemistry, College of Pharmaceutical Sciences of Capital Medical University, Beijing 100069, China.
² Beijing Area Major Laboratory of Peptide and Small Molecular Drugs, Engineering Research Center of Endogenous Prophylactic of Ministry of Education of China, Beijing Laboratory of Biomedical Materials, Beijing 100069, China.
³ Beijing Institute of Hepatology, Beijing Youan Hospital, Capital Medical University, Beijing 100069, China.
⁴ Quantum Design China Ltd., Universal Business Park, Beijing 100015, China.
⁵ Laboratory of Biomaterials and Biomechanics, Beijing Key Laboratory of Tooth Regeneration and Function Reconstruction, School of Stomatology, Capital Medical University, Beijing 100050, China.
⁶ School of Life Sciences, Henan University, Kaifeng 475004, China.

Abstract

Self-assembling carrier-free nanodrugs are attractive agents because they accumulate at tumor by an enhanced permeability and retention (EPR) effect without introduction of inactive substances, and some nanodrugs can alter the immune environment. We synthesized a peptidyl arginine deiminase 4 (PAD4) molecular inhibitor, ZD-E-1M. It could self-assembled into nanodrug ZD-E-1. Using confocal laser scanning microscopy, we observed its cellular colocalization, PAD4 activity and neutrophil extracellular traps (NETs) formation. The populations of immune cells and expression of immune-related proteins were determined by single-cell mass cytometry. ZD-E-1 formed nanoflowers in an acidic environment, whereas it formed nanospheres at pH 7.4. Accumulation of ZD-E-1 at tumor was pH-responsive because of its pH-dependent differences in the size and shape. It could enter the nucleus and bind to PAD4 to prolong the intracellular retention time. In mice, ZD-E-1 inhibited tumor growth and metastasis by inhibiting PAD4 activity and NETs formation. Besides, ZD-E-1 could regulate the ratio of immune cells in LLC tumor-bearing mice. Immunosuppressive proteins like LAG3 were suppressed, while IFN-γ and TNF-α as stimulators of tumor immune response were upregulated. Overall, ZD-E-1 is a self-assembling carrier-free nanodrug that responds to pH, inhibits PAD4 activity, blocks neutrophil extracellular traps formation, and improves the tumor immune microenvironment.

Keywords: Carrier-free; Lymphocyte activation gene-3; Nanoflowers; Neutrophil extracellular traps; Self-assembly; Single-cell mass cytometry; Tumor immune microenvironment; pH-Response.