A mannan-based probe for multimodal imaging of cancer and immune cells
Daniel Jirak1, Andrea Galisova1, Marketa Jiratova1, Mariia Rabyk2, Martin Hruby2, and Milan Hajek1

1IKEM, Prague, Czech Republic, 2UMCH, Prague, Czech Republic


Presented mannan-based polymers have promising properties for tumor and metastasis imaging due to their biocompatibility, nanosize and specificity for the immune cells. In this study, two mannan-based polymers were tested by multimodal imaging (MRI and fluorescence). The polymers showed superior imaging properties compared to a commercially available contrast agent. FLI signal at the liver and higher signal at the injection site in the mouse with MN-Ox suggested slower elimination process due to addition of polyoxazoline chains in its structure. Both probes were visualized by MR and optical imaging modality at the injection sites and in the lymph nodes of the experimental mice suggesting their promising properties for cancer diagnosis.


Mannan can be prepared in the form of nanoparticles allowing its use as a drug delivery carrier into solid tumors via Enhanced Permeability and Retention (EPR) effect. Moreover, due to its preferential uptake by immune cells (macrophages and dendritic cells) via DC-SIGN receptors1, mannan is preferably accumulated at the site of inflammation and in the lymph nodes. Therefore, mannan can serve as a multifunctional probe for diagnosis of tumors, metastasis and sentinel lymph nodes, as well as inflammation processes. In this study, mannan core was modified by a chelate containing gadolinium for magnetic resonance imaging (MRI) and by a near-infrared fluorescent dye for fluorescence imaging (FLI). Moreover, the probe was grafted with poly(2-methyl-2-oxazoline) for increasing of probability of accumulation in the tutor tissue. Mannan without (MN) and with oxazoline (MN-Ox) were characterised by MRI and FLI. Accumulation of the probes was assessed on the tumor-bearing mice.


Mannan was prepared by allylation with allyl bromide and cysteamine was added by thiol-click addition; then DOTA-Gd(III) complex, and a fluorescent dye IR800CW were added via the reaction with the corresponding succinimidyl esters and Gd+ chelation. To characterize the physicochemical properties of prepared conjugates, dynamic light scattering (DLS) measurements were conducted. The MR properties of the probes were assessed by r1 (0.5T, saturation recovery sequence, recycle delay 12 s or 5 s) and r2 relaxometry (0.5T, CPMG sequence, recycle delay 10 s, interpulse delay 1 ms, 5000 points), MR imaging (4.7T, T1-weighted images, turbo spin echo sequence, TR=112 ms, TE=11ms, TF=2, resolution 0.27x0.27x1.5 mm3, scan time 6.5 min) and fluorescence imaging (10 s exposure, excitation 745 nm, emission 810-875 nm). For in vivo monitoring, 50 µL of MN and MN-Ox was administered into the calf muscle of the right hind leg of Balb/c mice with induced xenograft tumors (application of 250 000 4T1 cells into the 5th mammary gland). The agents were dissolved in saline solution reaching 18 mmol/L concentration of Gd3+. T1-weighted MR images of the animals were acquired by a turbo spin echo sequence with the parameters: TR=339 ms, TE=12 ms, TF=2, resolution 0.14x0.14x0.70 mm3, scan time = 4.5 min. In vivo FLI images of mice and ex vivo images of the harvested organs were acquired at various time points (0-7 days) after the agent injection using the same adjustments as for the phantoms. As a reference contrast agent, a commercially available contrast agent gadoterate meglumine (GM) was used for comparison.


In the conjugates, the hydrodynamic radius of mannan had increased from 1.5 nm (neat mannan) to 3.3 nm (MN) and 3.7 nm (MN-Ox). Both MN-based agents have higher r1 and r2 relaxivites than GM (Table 1). Similarly, MR signal and corresponding CNR values of MN-based probes were comparable compared to GM (Fig. 1A). Mannan agents showed a strong fluorescent signal; fluorescence of GM was in the range of background (Fig. 1B). After intramuscular administration in mice, both MN and MN-Ox agents were visualized at the injection sites by MRI and FLI for 7 days. Both MRI and FLI signal of lymph nodes increased after probes administration with maximum between 6-24 hours; the highest signal was found in the sentinel lymph nodes (Fig. 2). In vivo FLI signal from the liver was higher in case of MN compared to MN-Ox (Fig 3). Ex vivo FLI revealed higher accumulation of MN in the tumors compared to MN-Ox (Fig. 4).


Accumulation of the mannan-based nanoprobes in the tumor tissue and the lymph nodes confirmed their targeting to immune cells and solid tumors. The mannan-based probes modified with were present at the injection sites for 7 days indicating slow biodegradation with the maximum of MRI and FLI signal at the first day following agents administration. Slower elimination rate of MN-Ox compared to MN was manifested by lower signal in the liver; however there was a lower FLI signal of MN-Ox in the tumor compared to MN probably due to refuse of oxazoline by the tumor-associated macrophages.


Here, we present a novel multimodal sensitive mannan-based nanoprobes which target lymph nodes and tumor tissue. Easily modified chemical structure allows incorporation of drugs and makes this platform a suitable drug delivery system for theranostics of cancer, metastasis and inflammation.


Supported by MH CR-DRO (Institute for Clinical and Experimental Medicine IKEM, IN00023001) and the Ministry of Health, Czech Republic (grant #15-25781A).


[1] Cui Z. et al. Drug Dev Ind Pharm, 29(6), 2003


Characterization of the probes. MRI (left) and FLI (right) signals of the MN, MN-Ox and GM. Average radiance was measured from the area covering one well with each agent concentration. MR mages of the probes with different Gd3+ concentration - the numbers represent Gd3+ concentration expressed in mM. FLI images of the probes with different dye concentration – the numbers represent concentration of a fluorescent dye expressed in µg/mL.

In vivo MR images. Representative MR images of the mice with injected MN-Ox into the muscle. T - tumor, LN - sentinel lymph node.

In vivo fluorescence imaging. The fluorescent images of the mouse with injected MN-Ox (A) and with MN (B) at 7 days following probes injection.

Ex vivo fluorescence imaging of harvested organs at day 1 from mice after injection MN and MN-Ox probes.

Table 1: Relaxivities of MN, MN_OX and GM measured at 0.5T.

Proc. Intl. Soc. Mag. Reson. Med. 26 (2018)