Page 231 - Haematologica March 2020

CXCR4-targeted nanocarrier to DLBCL cells
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Figure 3. T22-GFP-H6 pharmacokinetics in plasma and clearance in CXCR4+ SC Toledo mouse model. (A) Representative images of fluorescence intensity (FLI) reg- istered in plasma (250 mL) of mice treated with buffer or 200 mg T22-GFP-H6, 10 minutes (min), 30 min, 2 hour (h), 5h, 15h, 24h and 48h after its administration. (B) Graphical representation of FLI quantification in plasma over time. (C and D) Western blot analysis of the fate of the T22-GFP-H6 protein in hepatic and renal tis- sue in mice treated with buffer or 10 min, 30 min, 2h, 5h, 15h, 24h and 48h after nanocarrier administration. (E) Western blot analysis of the fate and processing of the T22-GFP-H6 protein in subcutaneous (SC) Toledo tumors in mice treated with buffer or 10min, 30min, 2h, 5h, 15h, 24h and 48h after nanocarrier adminis- tration. Note the almost complete proteolysis of the full-length protein nanocarrier in tumor tissue, and the absence of proteolysis in liver and kidney. GAPDH was used as a loading control. T22-GFP-H6 was detected with an anti-GFP antibody.
Highly selective T22-GFP-H6 tumor uptake in mice bearing subcutaneous CXCR4+ diffuse large B-cell lymphoma tumors without toxicity
We evaluated T22-GFP-H6 biodistribution in the CXCR4+ SC Toledo mouse model, measuring the fluores- cence emitted by the nanocarrier GFP domain, after a sin- gle 200 mg IV dose. T22-GFP-H6 accumulated in CXCR4+ SC tumors 2h after injection, reaching a FLI peak 5h post injection, and decreasing considerably after 24h (Figure 2A and B). Tumor uptake 5h post injection was 35.85 times higher than lung uptake, which was taken as a reference among the normal mouse organs because, although almost negligible, it did show the most sustained FLI emis- sion over time (Figure 2B and Online Supplementary Table S1). Similar observations were made in all non-tumor organs analyzed (Figure 2B and C). Moreover, we did not observe any histological alteration in Hematoxylin & Eosin (H&E) stained normal organs (Figure 2D).
The quantification of the AUC of emitted FLI over the study period (Figure 2E and Online Supplementary Table S2) showed that tumor tissue accumulated 86.13±4.04% of the total FLI detected in all organs, including tumor and non-tumor tissues. In contrast, the liver, which was the non-tumor organ with higher AUC, reached only 5.96±2.83% (Figure 2F). Therefore, T22-GFP-H6 displayed a specific targeting of CXCR4+ SC DLBCL tumors with negligible nanocarrier accumulation in non-tumor bearing organs, which supports a highly selective tumor uptake as compared to normal cells.
After a single T22-GFP-H6 IV administration, measure- ment of circulating nanocarrier showed a fast biodistribu- tion half-life (t1/2≈20min) in the blood compartment, fol- lowed by a slower elimination phase (t1/2≈75min), becom- ing undetectable in plasma at 2h (Figure 3A and B).
The highly unusual T22-GFP-H6 tumor uptake and its
low accumulation in the expected non-tumor drug clear- ance organs (i.e. liver and kidney) triggered the analysis of the nanocarrier fate in these organs by western blot. After a single T22-GFP-H6 dose, we observed the full-length protein (≈30kDa) present in liver and kidney 10min post administration (Figure 3C and D), becoming undetectable over a period which ranged from 30min to 48h. In sharp contrast, we detected full-length T22-GFP-H6 protein in Toledo SC tumors at 10min, 30min, 2h and 5h. Interestingly, faint proteolytic bands appeared over a peri- od which ranged from 30min to 2h, which became more intense at 5h. Over a period which ranged from 15h to 48h, the full-length protein decreased dramatically and the nananocarrier was mostly proteolyzed (Figure 3E). These results, together with the observed FLI AUC in tumor and normal organs, suggest that the proteolytic activity observed in the tumor makes it the main nanocarrier clear- ance organ.
T22-GFP-H6 and CXCR4 receptor co-localization in the cell membrane followed by its internalization in CXCR4+ diffuse large B-cell lymphoma cells
At the FLI peak (5h) after a single 200μg injection, we observed nanocarrier internalization in 56.2±12.0% of the Toledo cells (green staining with anti-GFP IF) in tumors, whereas all (100%) tumor cells over-expressed CXCR4 (red staining with anti-CXCR4 IF). In buffer-treated tumors, the CXCR4 receptor localized mainly at Toledo cell membrane, while a dot-like staining inside the cell cytosol was observed in the nanocarrier-treated-tumors; a finding consistent with receptor internalization within endocytic vesicles. Merged (yellow) images showed nanocarrier and CXCR4 co-localization in the membrane of Toledo cells in T22-GFP-H6-treated tumors. Once into the cytosol, the CXCR4 and T22-GFP-H6 stained endoso-
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