Lymphatic biodistribution of polylactide nanoparticles

Abstract

Tumor metastases occur through both the cardiovascular and lymphatic circulations. However, the majority of nanoparticle biodistribution studies have been focused on the cardiovascular circulation. In this study, we report the formulation of Cy5-labeled polylactide (Cy5-PLA) nanoparticles with controlled size and surface features and the subsequent evaluation of their lymphatic biodistribution. Cy5-PLA nanoparticles were formulated through Cy5/(BDI)ZnN(TMS)2-mediated [(BDI) = 2-((2,6-diisopropylphenyl)amido)-4-((2,6-diisopropylphenyl)-imino)-2-pentene] ring-opening polymerization of lactide followed by nanoprecipitation. Their lymphatic biodistribution was evaluated by using whole-body fluorescence imaging of nude mice and ex vivo fluorescence imaging of the resected organs. This technique has the potential for providing optical contrast and drug delivery through the lymphatic circulation for the treatment of metastatic cancer.

Figure 1

Preparation of poly(ethylene glycol)ated (PEGylated) Cy5-PLA nanoparticles for lymphatic system tracking by means of Cy5-initiated LA polymerization in the presence of (BDI)ZnN(TMS)₂, followed by co-nanoprecipitation with polylactide-b-methoxylated PEG (PLA-mPEG_5K).

Figure 2

Polymer and nanoparticle properties. A, Excitation and emission of the Cy5-PLA polymer, with the maximum absorption at 656 nm and the maximum emission at 673 nm. B, Plot of a single distribution of Cy5-PLA/PLA-mPEG_5k nanoparticles analyzed by dynamic light scattering. Transmission electron microscopy (inset) demonstrates uniform particle size.

Figure 3

Imaging of Cy5-PNP biodistribution in nude mice. A, In vivo fluorescence images taken immediately (0 hours) and 24 hours after PNP injection into the left footpad. The left column shows fluorescence from the Cy5-PNP biodistribution and the right column shows overlay with brightfield images. Yellow arrows show the site of injection; white arrows show distributing PNPs. B, Representative organs from two PNP-injected nude mice (#1 and #2) and one noninjected control nude mouse (CON). The left columns show the fluorescence images and the right columns show the overlays with brightfield images. No fluorescence is seen in any of the control organs. C, Cy5-PNP biodistribution in the left (C1–C4) and right (C5–C8) popliteal lymph nodes. Images C1, C2, C5, and C6 are from two PNP-injected animals and C3, C4, C7, and C8 are from one noninjected control animal. Fluorescence is seen only in the images from the injected animals, with the strongest signal coming from the left popliteal lymph node (injected side) compared to the right popliteal lymph node (noninjected side).

Figure 4

Average fluorescence signal intensities from organs following footpad and tail vein routes of administration. Cy5-PLA PNP were injected into either the lymphatic system via the footpad or the cardiovascular system via the tail vein. Colored bars represent the average signal intensity per organ, and error bars represent the standard deviation between measured areas of interest within each organ. Statistical analysis confirms lower fluorescence from the left popliteal lymph node and greater fluorescence from the right popliteal lymph node following tail vein injection compared to footpad injection. The statistical analysis also confirms greater fluorescence from the kidney following cardiovascular system delivery compared to delivery via the lymphatic system. *Statistically significant (p < .05) compared to the footpad-injected route.

Figure 5

Average fluorescence signal intensity from the brains of footpad-injected animals after 24-hour circulation. One animal was injected with Cy5-PLA PNPs and the other was injected with an equal concentration of free Cy5 dye. Approximately half of the Cy5 dye remains encapsulated inside the PNPs. There was no fluorescence signal detected from the brain of the control animal. *Statistically significant (p < .05) compared to the control. **Statistically significant (p < .05) compared to the Cy5-PNP complex.