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. 2021 Mar 1;16(3):e0247673.
doi: 10.1371/journal.pone.0247673. eCollection 2021.

Maltohexaose-indocyanine green (MH-ICG) for near infrared imaging of endocarditis

Kiyoko Takemiya  1 , Joachim J Røise  2   3 , Maomao He  2 , Chung Taing  3 , Alexander G Rodriguez  1 , Niren Murthy  2 , Mark M Goodman  4 , W Robert Taylor  1   5   6
Affiliations

Maltohexaose-indocyanine green (MH-ICG) for near infrared imaging of endocarditis

Kiyoko Takemiya et al. PLoS One. .

Abstract

Infectious endocarditis is a life-threatening disease, and diagnostics are urgently needed to accurately diagnose this disease especially in the case of prosthetic valve endocarditis. We show here that maltohexaose conjugated to indocyanine green (MH-ICG) can detect Staphylococcus aureus (S. aureus) infection in a rat model of infective endocarditis. The affinity of MH-ICG to S. aureus was determined and had a Km and Vmax of 5.4 μM and 3.0 X 10-6 μmol/minutes/108 CFU, respectively. MH-ICG had no detectable toxicity to mammalian cells at concentrations as high as 100 μM. The in vivo efficiency of MH-ICG in rats was evaluated using a right heart endocarditis model, and the accumulation of MH-ICG in the bacterial vegetations was 2.5 ± 0.2 times higher than that in the control left ventricular wall. The biological half-life of MH-ICG in healthy rats was 14.0 ± 1.3 minutes, and approximately 50% of injected MH-ICG was excreted into the feces after 24 hours. These data demonstrate that MH-ICG was internalized by bacteria with high specificity and that MH-ICG specifically accumulated in bacterial vegetations in a rat model of endocarditis. These results demonstrate the potential efficacy of this agent in the detection of infective endocarditis.

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Conflict of interest statement

Kiyoko Takemiya, W. Robert Taylor, Niren Murthy, and Mark M. Goodman have equity interest in Microbial Medical, Inc which is related to this study. Joachim J. Røise, Maomao He, Chung Taing, and Alexander G. Rodriguez have no conflict of interest. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Figures

Fig 1
Fig 1. Synthesis of MH-ICG.
a) Maltohexaose-azide was conjugated with ICG-alkyne through a copper-catalyzed click reaction. b) Analytical HPLC confirmed the purity of MH-ICG. Purified MH-ICG was injected onto an analytical Xbridge C18 5 μm 4.6 x 150 mm column. (Flow rate: 1 ml/min. Solvent system: MeCN/H2O + 0.1% TFA, 0–20 min: 40% MeCN, 20–40 min: gradient to 100% MeCN.) Product eluted at 27.2 min. c) Fluorescence spectrum of MH-ICG. The absorbance and fluorescence intensities are indicated as solid and broken lines, respectively. The emission spectrum was generated with an excitation wavelength of 760 nm.
Fig 2
Fig 2. Basic characteristics of MH-ICG in vitro.
To evaluate the uptake of MH-ICG in bacteria, S. aureus, E. coli, and LamB mutant E. coli were cultured with MH-ICG at various concentrations (n = 3 /each concentration). The internalized amount of MH-ICG by S. aureus (a) and E. coli (b) followed Michaelis-Menten kinetics. The Km and Vmax calculated with a Lineweaver-Burk plot were 5.4 μM and 3.0 X 10−6 μmol/minutes/108 CFU for S aureus and 6.9 μM and 6.3 X 10−7 μmol/minutes/108 CFU for E. coli, respectively. LamB mutant E. coli internalized a small amount of MH-ICG at the level of detection. c) Cytotoxicity of MH-ICG was evaluated in CHO-K1 cells. The cell viability was evaluated for 24 hours (i) and 72 hours (ii) after loading of MH-ICG. The cells were incubated with 0 to 100 μM of MH-ICG (n = 4 /each concentration), and no reduction in cell viability was observed up to 100 μM of MH-ICG at either time points.
Fig 3
Fig 3. Accumulation of MH-ICG in vegetation.
a) Right heart endocarditis was established by catheterizing in the right ventricle followed by injection of S. aureus. The vegetation was found around the catheter in the right ventricle (the arrow). b) i) Whole image of the specimen. Arrow: vegetation, *: the catheter in right ventricle, RV: right ventricle, LV: left ventricle. Bar: 2 mm. ii) Low power field of vegetation around the catheter. In the vegetation, spreading and accumulation of Gram-positive bacteria are observed in the fibrous tissue. Bar: 250 μm. iii) High-power field of vegetation. Gram-positive cocci are observed among large inflammatory cell infiltrates. Bar: 25 μm. iv) High-power field of vegetation with accumulation of bacteria. The deep purple spot is composed of Gram-positive cocci. Bar: 25 μm. c) Near infrared imaging with MH-ICG in the right heart endocarditis model in rats. Accumulation of MH-ICG was only observed in the vegetation. The hearts from the control rats had a very low fluorescent signal. d) Accumulation of MH-ICG in vegetation was quantified as an intensity ratio defined as [fluorescent intensity in vegetation or the right ventricle]/[fluorescent intensity in the left ventricle free wall]. The intensity ratio in IE rats was 2.5 ± 0.2, which was significantly increased versus control rats (n = 5 /group).
Fig 4
Fig 4. Distribution of MH-ICG in vivo.
Healthy rats were injected with MH-ICG. a) The plasma concentration of MH-ICG was monitored for six hours. The plasma concentration was reduced very rapidly in two hours, and the biological half-life of MH-ICG in the initial phase was calculated as 14.0 ± 1.3 minutes (n = 4). Note that the concentration of MH-ICG is on a logarithmic scale, and some standard error bars overlap the average circles. b) Metabolism and distribution of MH-ICG were also evaluated: After 24 hours, about 50% of injected MH-ICG was excreted into feces. The MH-ICG that remained in the liver and in the kidneys were 11.7 ± 3.5% and 1.5 ± 0.2%, respectively (n = 4).
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