Purpose

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.

Methods

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.

Results

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).

Discussion

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.

Conclusion

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.

Acknowledgements

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

References

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