A Novel Engineering Platelet Platform Target the Macrophages to Inhibit Cytokine Storm in Hemophagocytic Lymphohistiocytosis

Introduction:

Hemophagocytic lymphohistiocytosis (HLH) is a severe hematologic disorder characterized by abnormally increased macrophage activity due to abnormal immunoregulation. Etoposide (VP16) is the first-line drug for treating HLH, but its therapeutic effect is still unsatisfactory. The team's previous experiments showed that platelets (PLTs) with surface-associated anti-CD41 can be efficiently recognized and phagocytosed by macrophages. Therefore, we proposed to design and construct anti-CD41-PLT-VP16, a novel engineering platelet platform that can precisely target the abnormal macrophages, and congregate drugs in the specific site, which provided a new strategy for the immunotherapy of HLH.

Methods:

Anti-CD41-PLT-VP16 was constructed by antigen-antibody cross-linking and co-polymerization, which was characterized by electron microscopy, dynamic light scattering (DLS), fluorescent dye labeling technique, laser confocal microscopy, and Western Blot (WB). High-performance liquid chromatography (HPLC) determined the optimal encapsulation rate. In vitro, we evaluated the targeting ability and apoptosis effect of anti-CD41-PLT-VP16 on macrophages using laser confocal microscopy, CCK-8, and flow cytometry. The impact of anti-CD41-PLT-VP16 on macrophage activity and its degree of polarization was determined by WB and reactive oxygen species (ROS). In vivo, we constructed the humanized HLH mouse model and evaluated the efficacy of anti-CD41-PLT-VP16 by mouse blood routine examination, immunohistochemistry (IHC), and bone marrow smear. Meanwhile, we applied living animal imaging technology and fluorescent labeling to assess the targeting ability of anti-CD41-PLT-VP16 to macrophages in the liver and spleen. The toxicity of anti-CD41-PLT-VP16 was evaluated by H&E staining, liver and kidney routine, and body weight.

Results:

In this study, anti-CD41-PLT-VP16 was successfully constructed, its maximum encapsulation rate was 87.9%, and the built carrier had no significant effect on the physical and biological properties of PLTs. In vitro, the targeting efficiency of anti-CD41-PLT-VP16 was significantly improved compared to VP16. Macrophage apoptosis in the anti-CD41-PLT-VP16 group was 62.3%, 2.23 times greater than VP16. The macrophages' ROS levels were reduced 2.28 times in the anti-CD41-PLT-VP16 group compared with the PLT-VP16 groups. The proportion of M2 macrophages was 94.2% in the anti-CD41-PLT-VP16-treated group, which was higher than the control and VP16 group. In vivo, the greater therapeutic effect of anti-CD41-PLT-VP16 was shown by significant improvements in blood and cytokine data, and bone marrow smears showed the disappearance of bone marrow hemophagocytes. In addition, the macrophage activity in mice's liver and spleen in the anti-CD41-PLT-VP16 group decreased by 2.8 times than PLT-VP16. Meanwhile, the cardiopulmonary and renal toxicity was significantly less than VP16 alone.

Conclusion:

This study proposed to design anti-CD41-PLT-VP16 to achieve highly targeted and low-toxicity immune-targeted therapy for HLH. Results demonstrated that it could target the abnormally activated macrophages and lead apoptosis effect while significantly ameliorating the cytokine storm. In the clinic, this study is expected to provide a new idea for highly targeted immunotherapy of HLH. Benefit from the simple and safe method to construct this platform, this technology is convenient for industrial production and may benefit the patients.