Cytoplasm Hydrogelation-Mediated Cardiomyocyte Sponge Alleviated Coxsackievirus B3 Infection

Figures

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  Figure 1. Schematic illustration of GCs inhibiting CVB3 infectivity. GCs were prepared through intracellular hydrogelation to preserve the intact cellular morphology and membrane structure. Benefiting from surface crucial antigens, such as CAR and CD55 receptors, GCs were disguised as cardiomyocyte-like cells and competitively bound CVB3 to immobilize the virus and prevent further infection of host cells. GCs acted as virus decoys to inhibit in vivo CVB3 infectivity, and the technology of intracellular gelation on the host cell provided a new insight into neutralizing the corresponding virus without screening the specific neutralizing antibody.

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  Figure 2. Engineering and characterization of GCs. (A) Representative microscopic images of the Live group, NGD group, PEG-DA group, I2959 group, and GC group. Each group of cells, except for the Live group, was incubated in PBS for 24 h. (B) Scanning electron microscopy (SEM) images of Live cells, NGDs, and GCs. (C) Transmission electron microscopy (TEM) images of Live cells, NGDs, and GCs. (D, E) Stability of GCs in 1 × PBS over a span of 15 days at room temperature (D) and through repeated freezing–thaw cycles (E), determined by flow cytometry. NS = nonsignificant, **P ≤ 0.01 and ***P ≤ 0.001.

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  Figure 3. GCs inhibit CVB3 infection in normal host cells. (A) Coomassie blue analysis using SDS-PAGE in Live cells, NGDs, and GCs. (B) Immunofluorescent staining for α-actin. (C) Proliferation curves of HL-1 cells and GCs assessed by a CCK8 assay at 24 h. (D) Cell death detected by Annexin V/PI double staining and flow cytometry. (E) Effect of different doses of GCs (number ratio between GC and HL-1 cell, 1:10, 1:1, and 10:1) on HL-1 cell proliferation assessed by the CCK8 assay at 0, 24, and 48 h. (F) RT-PCR detection of CVB3 RNA levels in HeLa cells with or without the addition of NGDs and GCs. NC: Negative control. PC: Positive control with CVB3. (G) Plaque morphology of CVB3 on HeLa cells with or without the addition of NGDs and GCs. PFU: Plaque-forming units. NS = not significant, **P ≤ 0.01 and ***P ≤ 0.001.

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  Figure 4. In vivo safety assessment of GCs. (A) Balb/c mice were intravenously injected with 1 × 106 GCs every 2 days with a total of four doses (n = 5), and the body weight was recorded at experimental end points (day 7). (B) Comprehensive serum chemistry analysis was performed at day 7. Measurement parameters included TB (total bilirubin), ALB (albumin), AST (aspartate aminotransferase), ALT (alanine aminotransferase), CREA (creatinine), UREA (blood urea nitrogen), GLU (glucose), TBIL (total bilirubin), ALP (alkaline phosphatase), and IgG (immunoglobulin G). (C) Lymphocyte (LYM), monocyte (MON), and granulocyte (GRA) counts were detected at day 7. (D) Complete blood count collected from GC group mice was analyzed at day 7. The detected items included HGB (hemoglobin), RBC (red blood cells), HCT (hematocrit), MCV (mean corpuscular volume), MCH (mean corpuscular hemoglobin), MCHC (mean corpuscular hemoglobin concentration), RDW (red cell distribution width), PLT (platelets), PCT (platelet crit), MPV (mean platelet volume), PDW (platelet distribution width), and PLCR (platelet crit-to-large cell ratio). (E) Histopathological analysis of the heart, liver, spleen, lung, and kidney using…

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  Figure 5. In vivo therapy of GCs against CVB3 infection. (A) Survival rates of CVB3-infected mice after iv injection with NGDs and GCs (n = 5). (B) CVB3-infected mice were iv administered with 100 μL of PBS (100 μL per mouse), NS (prepared from 1 × 107 cardiomyocytes), GCs (1 × 106), and Ribavirin (50 mg/kg), every other day for a total of 7 injections, and body weight change curves of mice were created (n = 7). (C) RT-PCR detection of CVB3 RNA levels in the heart. (D–F) RT-PCR detection of IFN-β, IFN-γ, and Fas mRNA levels in the heart. (G) ELISA detects serum cTnI levels. (H–K) Serum CK, CK-MB, LDH, and LDH-L analyses were performed on day 7. (L) Histopathological analysis of the heart, liver, spleen, lung, and kidney using H&E-stained tissue sections on day 3, day 7, and day 14. *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001.