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Fluorescence Tomographic Imaging of Sentinel Lymph Node Using Near-Infrared Emitting Bioreducible Dextran Nanogels

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Fluorescence Tomographic Imaging of Sentinel Lymph Node Using Near-Infrared Emitting Bioreducible Dextran Nanogels

Jiejing Li et al. Int J Nanomedicine.

Abstract

Sentinel lymph node (SLN) mapping is a critical procedure for SLN biopsy and its diagnosis as tumor metastasis in clinical practice. However, SLN mapping agents used in the clinic frequently cause side effects and complications in the patients. Here, we report the development of a near-infrared (NIR) emitting polymeric nanogel with hydrodynamic diameter of ~28 nm - which is the optimal size for SLN uptake - for noninvasive fluorescence mapping of SLN in a mouse. This polymeric nanogel was obtained by coupling Cy7, an NIR dye, to the self-assembled nanogel from disulfide-linked dextran-deoxycholic acid conjugate with the dextran of 10 kDa, denoted as Dex-Cy7. Fluorescence imaging analysis showed that Dex-Cy7 nanogels had an enhanced photostability when compared to Cy7 alone. After intradermal injection of Dex-Cy7 nanogel into the front paw of a mouse, the nanogels were able to migrate into the mouse's axillary lymph node, exhibiting longer retention time and higher fluorescence intensity in the node when compared to Cy7 alone. An immunohistofluorescence assay revealed that the nanogels were localized in the central region of lymph node and that the uptake was largely by the macrophages. In vitro and in vivo toxicity results indicated that the dextran-based nanogels were of low cytotoxicity at a polymer concentration up to 1,000 μg/mL and harmless to normal liver and kidney organs in mice at an intravenous dose of 1.25 mg/kg. The results of this study suggest that NIR-emitting polymeric nanogels based on bioreducible dextran-deoxycholic acid conjugates show high potential as fluorescence nanoprobes for safe and noninvasive SLN mapping.

Keywords: dextran; disulfide; lymph node; nanogel; tomographic imaging.

Figures

Figure 1
Figure 1
Synthesis of dextran-deoxycholic acid conjugates linked by disulfide bond (Dex–SS–DCA). Note: Reaction steps are denoted by i–iv. Abbreviations: PDA, 2-(pyridyldithio)-ethylamine; Dex–PNC, p-nitrophenyl chloroformate-coupled dextran; Dex–SH, thiolated dextran; Dex–SS–NH2, dextran-cysteamine conjugate; Dex–SS–DCA, disulfide-linked dextran-deoxycholic acid conjugate.
Figure 2
Figure 2
1H NMR spectrum of (A) Dex–SS–NH2 and (B) Dex10k–SS–DCA. Abbreviations: Dex–SS–NH2, dextran-cysteamine conjugate; Dex–SS–DCA, disulfide-linked dextran-deoxycholic acid conjugate; DMSO, dimethyl sulfoxide; ppm, parts per million; D2O, deuterium oxide.
Figure 3
Figure 3
Characterization of bioreducible dextran-based nanogels. Notes: (A) Size distribution of Dex10k–SS–DCA nanogels (the insert is the TEM image of nanogel, scale bar: 50 nm). (B) UV–vis spectrum of Dex–Cy7. (C) Imaging of Cy7 alone or Dex–Cy7 nanogel solution placed in a cuvette at the same amount of Cy7. (D) Fluorescence spectrum of Dex–Cy7 and Cy7 at the same Cy7 concentration of 1.5 nmol/mL at an excitation wavelength of 550 nm. (E) Imaging of Cy7 or Dex–Cy7 nanogel solution in a tube at different time intervals in the range from 0 (as a control) to 24 hours. (F) Photostability analysis of Cy7 and Dex–Cy7 solutions by plotting fluorescence intensity as a function of time. Abbreviations: Abs, absorbance; Conc, concentration; Ex, excitation; Em, emission; Dex–SS–DCA, dextran-deoxycholic acid conjugate; TEM, transmission electron microscopy.
Figure 4
Figure 4
In vitro and in vivo toxicity test of Dex-Cy7 nanogels. Notes: Cytotoxicity of Dex–Cy7 nanogels by testing cell viability of (A) DC 2.4 and (B) MCF-7 cells after they are exposed in different polymer concentrations. H&E staining of (C) liver or (D) kidney tissue 3 days after intravenous injection of Dex–Cy7 nanogel solution into a mouse at a dose of 1.25 mg/kg. Abbreviation: H&E, hematoxylin and eosin.
Figure 5
Figure 5
Three-dimensional fluorescence tomographic imaging of lymph node in a mouse using Dex-Cy7 nanogel. Notes: (A) Typical in vivo fluorescence imaging of lymph node (LN) after intradermal injection of Dex–Cy7 solution into the front left paw of a mouse. (B) Ex vivo imaging of isolated left and right (control) LN. (C) In situ injection of Dex–Cy7 solution in the left LN after removal of mouse skin. (D) In vivo and ex vivo imaging of left and right LN after intradermal injection of Dex–Cy7 solution into the front right paw of a mouse. (E) Typical tomographic imaging of LN at transverse (z) and axial (x, y) cross-sections and their image combinations 2 hours after intradermal injection. Abbreviations: L, left; R, right.
Figure 6
Figure 6
Comparison study between fluorescence imaging and visualization of lymph node in a mouse. Notes: In vivo imaging (A) and visualization (B) of lymph node (LN) 2 hours after intradermal injection of both Dex–Cy7 and methylene blue solution into the front left paw of a mouse. (C) Ex vivo observation and fluorescence imaging of left LN (L), fat, and right LN (R).
Figure 7
Figure 7
Accumulation kinetics study of Dex-Cy7 nanogel in lymph node in a mouse. Notes: (A) Accumulation kinetics of Dex–Cy7 in the left lymph node (LN) after intradermal injection and (B) quantified fluorescence signals in the LN for Dex–Cy7 and Cy7 alone (control). (C) Ex vivo imaging of the organs 120 minutes or 3 days after intradermal injection of Dex–Cy7. Abbreviation: LN, lymph node.
Figure 8
Figure 8
Immunohistofluorescence images of lymph node sections of a mouse 2 hours after intradermal injection of Dex–RB solution. Notes: The sections were stained with anti-CD68 (A) and anti-CD205 (B) and observed at 10× and 60× magnification. Abbreviation: DAPI, 4′,6-diamidino-2-phenylindole dihydrochloride; Dex-RB, rhodamine B-labeled Dex–SS–DCA nanogel.
Figure 9
Figure 9
In vitro fluorescence images of Dex–RB accumulated in DC 2.4 cells at different time intervals. Notes: The lysosomes (in green) were stained with lysotracker. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole dihydrochloride; h, hours; Dex-RB, rhodamine B-labeled Dex–SS–DCA nanogel.

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