Liver-Targeted Near-Infrared Fluorescence/Photoacoustic Dual-Modal Probe for Real-Time Imaging of <i>In Situ</i> Hepatic Inflammation

Early diagnosis of hepatic inflammation is the key to timely treatment and avoid the worsening of liver inflammation. Near-infrared fluorescence (NIRF) probes have high sensitivity but low spatial resolution in lesion imaging, while photoacoustic (PA) imaging has good spatial location information. Therefore, the development of a NIRF/PA dual-modal probe integrated with high sensitivity and spatial location feedback can achieve an accurate early diagnosis of hepatic inflammation. Here, we report an activatable NIRF/PA dual-modal probe (hCy-Tf-CA) for the detection of the superoxide anion (O2·–) in early hepatic inflammation. hCy-Tf-CA showed high selectivity and sensitivity for detecting O2·– fluctuation in vitro. More importantly, by introducing hepatocyte-targeting cholic acid (CA), the probe successfully achieved accurate in situ imaging of acute inflammatory liver injury (AILI) and autoimmune hepatitis (AIH) in vivo. The introduced CA not only promotes the hepatic targeting accumulation of probes but also improves the performance of low background dual-modal imaging in vivo. Therefore, hCy-Tf-CA provides an effective strategy for significantly improving in situ imaging performance and holds great potential for early, sensitive, and accurate diagnosis of hepatic inflammation

Subtype analysis of the HBV antigen-specific IgGs in sera of mice immunised with different vaccine combinations.

<p>Antigen specific IgG1 and IgG2a or IgG2b levels were determined using an IgG isotyping ELISA, as described in Materials and Methods. Sera were collected 2 weeks after the last immunization and diluted 1∶100. Bars indicate the average OD value at 450 nm (OD₄₅₀) of each group.</p

ICS analyse of HBV PreS1- or S-specific CD4+ cells producing interferon-γ (IFN-γ), tumour necrosis factor-α (TNF-α) and interleukin-4 (IL-4) induced by heterologous rTTV and recombinant protein prime/boost immunization.

<p>Splenocytes from four mice per group were isolated 14 days after last immunization. The splenocytes were exposed to HBV PreS1 peptide(S1–9) or S peptides pool and cytokine production was measured by monoclonal antibody staining and flow cytometric analysis. (a) Flow cytometer plot of results obtained from one representative individual mouse from each group. (b), Average (± SEM) of the percentage of IFN-γ-producing, TNF-α-producing and IL-4-producing CD4+ T cells obtained from four mice per group following stimulation with HBV PreS1 peptide (S1–9) or S peptides pool. Compared with RVJSS1 prime/HBSS1 boost, mice receiving HBSS1+Al(OH)3 prime/RVJSS1 boost generated markedly higher PreS1-specific CD4 T cell responses for two cytokines (IFN-γ and TNF-α, <i>P</i><0.05) and S- specific CD4 T cell responses for TNF-α(<i>P</i><0.05), mice receiving HBSS1 prime/RVJSS1 boost also generated markedly higher PreS1-specific CD4 T cell responses for TNF-α(<i>P</i><0.05).</p

Characterization of recombinant vaccinia virus RVJSS1 and HBV particle-like subunit vaccine HBVSS1.

<p>(<b>A</b>) Schematic diagram of recombinant vaccinia virus RVJSS1, which contains two expression cassettes in a back-to-back orientation, flanked by vaccinia virus TK region sequences. Virus RVJSS1 was a Tiantan strain vaccinia virus with a <i>lacZ</i> gene led by a p11 promoter inserted into the TK region; the expression cassette on the right consists of the SS1 fusion protein led by the 7.5 K promoter. (<b>B</b>) Immunofluorescence assay to confirm SS1 fusion protein expression. CEF cells were infected by the purified RVJSS1 virus and fixed, permeabilised, stained with rabbit anti-PreS1 antibody and fluorescein isothiocyanate (FITC) conjugated to a secondary antibody, and then visualised using fluorescence microscopy. (<b>C</b>) Western blot analyses to detect expression of SS1 fusion proteins in CEFs infected with RVJSS1 using specific antibodies. The expression bands of the SS1 proteins are indicated by arrowheads. (<b>D</b>) Negative staining of purified HBVSS1 particles vaccine using electron microscopy.</p

HBV S epitopes screening and ELISpot analysis of IFN-γ secretion in mouse splenocytes of each immunization group.

<p>(<b>A</b>) HBV S epitopes were screened by IFN-γ ELISpot analysis. Actual sample wells of HBS-specific ASC spots. (<b>B</b>–<b>D</b>) HBV peptide-specific ASC frequency in each group. Data represent the average of spot-forming cells (SFCs) per million splenocytes from six mice/group plus the standard error. <b>B</b>:Splenocytes were collected 10 days after the first immunization and the number of IFN-γ secreting cells generated in response to S peptides; <b>C–D</b>: Splenocytes were collected 2 weeks after the last immunization and the number of IFN-γ secreting cells generated in response to PreS1 and S peptide pool stimuli respectively. Statistical differences between groups were determined, and differences are shown as *<i>p</i><0.05 and **<i>p</i><0.01. (<b>E</b>) Sample wells with spots with mock or HBV peptide stimulation.</p

ICS analyse of HBV PreS1- or S-specific CD8+ cells producing interferon-γ (IFN-γ), tumour necrosis factor-α (TNF-α) and interleukin-4 (IL-4) induced by heterologous rTTV and recombinant protein prime/boost immunization.

<p>Splenocytes from four mice per group were isolated 14 days after last immunization. The splenocytes were exposed to HBV PreS1 peptide(S1–9) or S peptides pool and cytokine production was measured by monoclonal antibody staining and flow cytometric analysis. (a) Flow cytometer plot of results obtained from one representative individual mouse from each group. (b), Average (± SEM) of the percentage of IFN-γ-producing, TNF-α-producing and IL-4-producing CD8+ T cells obtained from four mice per group following stimulation with HBV PreS1 peptide (S1–9) or S peptides pool. Compared with RVJSS1 prime/HBSS1 boost, mice receiving HBSS1+Al(OH)3 prime/RVJSS1 boost generated markedly higher PreS1-specific CD8 T cell responses for IFN-γ(<i>P</i><0.05) and S- specific CD8 T cell responses for TNF-α(<i>P</i><0.05), mice receiving HBSS1 prime/RVJSS1 boost generated markedly higher PreS1- and S-specific CD8 T cell responses for TNF-α(<i>P</i><0.05).</p

Total antibody positivity rate after single or double immunization.

<p>Note: Mice were randomly assigned to five groups (8/group). Except for the NS and RVJ mock groups, mice received different prime–boost vaccination regimens. Sera were collected 2 weeks after each immunization, and HBV specific antibody was detected by ELISA. Seroconversion is indicated in the table.</p><p>NS: Normal saline.</p><p>RVJ: recombinant vaccinia virus.</p

The anti-PreS1 and S antibody responses elicited by different regimens detected after first immunization (A–B) or second immunization(C–D).

<p>Each group received a different prime–boost vaccination, and antiserum was collected 2 weeks after the each immunization. Total HBV antigen-specific IgG titres were determined by ELISA. The symbols represent the titers of the sera from the individual mice. The horizontal lines represent the means (n = 6). The statistic significance of the results was analyzed and indicated as *<i>p</i><0.05.</p