Optimal Size of the Agents

In order to determine the optimal size of Gd-dendrimer for lymphatic vessel and lymph node imaging, each agent was tested in vivo. Intracutaneous injection at the phalanges revealed that larger nano-sized agents worked better for visualizing the lymphatic vessels than smaller agents. MR lymphangiograms with the largest agent studied (G8; ~13 nm) showed the best depiction of the lymphatic system among all the tested agents (Figure 2.2). The relatively large size of these molecules meant that once the agent was absorbed by the lymphatic system it tended to remain within it. In comparison, low molecular weight Gd-DTPA only faintly demonstrated the distal large lymph nodes such as the axillary and external iliac lymph nodes early after injection. In order to detect sentinel lymph nodes, various size dendrimers were tested after mammary pad injection. Among the agents tested, a G6 agent of ~10 nm in diameter proved to be the most effective (Figure 2.3). In comparison, the larger G8 dendrimer showed slower

PAMAM-G8 DAB-G5 PAMAM-G4 Gadomer-17 Gd-DTPA

Figure 2.2. Whole body dynamic 3D-micro-MR lymphangiography of mice injected intracutaneously into all four middle phalanges with 0.005 mmol Gd/kg of PAMAM-G8, DAB-G5, PAMAM-G4, Gadomer-17, or Gd-DTPA are shown. The images obtained at 45 min post-injection are shown for all contrast agents tested. Maximum intensity projections are illustrated.

alei nl thoracic LN

Figure 2.3. A series of 3D dynamic MR lymphangiograms obtained 12 min after the injection of the five contrast agents including three Gd-dendrimers. The G6 contrast agent of ~9 nm in diameter was taken up by both lymph nodes and lymphatic vessels and achieved the largest enhancement ratio among all agents examined.

enhancement of the axillary lymph node (Kobayashi et al., 2006). Smaller nano-sized agents were not as good as G6 or G8 in depicting the lymphatics and SLN. Among the smaller agents, DAB-G5 and Gadomer-17 were acceptable to visualize lymph nodes probably because of their less hydrophilic properties than PAMAM series of contrast agents (Kobayashi et al., 2006).

The ideal lymphatic imaging agent needs to be small enough to enter the lymphatic vessels, yet large enough to be retained within the lymphatics and not leak from the capillary vessels. Lymphographic contrast agents must be at least 4 nm in diameter to enable efficient retention within the lymphatics (Alazraki et al., 2001; Kobayashi et al., 2003a). Molecules smaller than 4 nm in diameter tend to diffuse into the surrounding tissue, resulting in poor signal-to-background ratios. Larger molecules, on the other hand, diffuse more slowly from the interstitial space and, thus, accumulate more slowly in the sentinel node, requiring a longer imaging window for visualizing nodes. The larger diameter G8 agent (~12 nm), used in our previous report for deep lymphatic imaging studies (Kobayashi et al., 2003c), is too large for rapid uptake by lymphatic vessels arising from the breast, although it is suitable for imaging lymphatics of the extremities. In contrast, the G6 contrast agent is retained in the lymphatic vessels, resulting in efficient enhancement of lymphatic vessels and lymph nodes. Both G2 and G4 agents were too small to stay within the lymphatic vessels, resulting in convection away from the injection site and only minimal enhancement in the lymph nodes (Figure 2.4).

Figure 2.4. Top: G6 dendrimers are taken up by lymphatics and retained within the lymphatics resulting in opacification of the lymph nodes (black). Lower molecular weight contrast agents (bottom) are absorbed by the lymphatics, but leak out from them resulted in lower lymph node concentrations (gray).

Figure 2.4. Top: G6 dendrimers are taken up by lymphatics and retained within the lymphatics resulting in opacification of the lymph nodes (black). Lower molecular weight contrast agents (bottom) are absorbed by the lymphatics, but leak out from them resulted in lower lymph node concentrations (gray).

The use of agents with appropriate sizes enables us to clearly depict systemic or local lymphatic vessels and lymph nodes in normal mice.

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