Effective transvascular delivery of nanoparticles across the blood-brain tumor barrier into malignant glioma cells - PubMed (original) (raw)

doi: 10.1186/1479-5876-6-80.

Ariel S Kanevsky, Haitao Wu, Kyle R Brimacombe, Steve H Fung, Alioscka A Sousa, Sungyoung Auh, Colin M Wilson, Kamal Sharma, Maria A Aronova, Richard D Leapman, Gary L Griffiths, Matthew D Hall

Affiliations

Effective transvascular delivery of nanoparticles across the blood-brain tumor barrier into malignant glioma cells

Hemant Sarin et al. J Transl Med. 2008.

Abstract

Background: Effective transvascular delivery of nanoparticle-based chemotherapeutics across the blood-brain tumor barrier of malignant gliomas remains a challenge. This is due to our limited understanding of nanoparticle properties in relation to the physiologic size of pores within the blood-brain tumor barrier. Polyamidoamine dendrimers are particularly small multigenerational nanoparticles with uniform sizes within each generation. Dendrimer sizes increase by only 1 to 2 nm with each successive generation. Using functionalized polyamidoamine dendrimer generations 1 through 8, we investigated how nanoparticle size influences particle accumulation within malignant glioma cells.

Methods: Magnetic resonance and fluorescence imaging probes were conjugated to the dendrimer terminal amines. Functionalized dendrimers were administered intravenously to rodents with orthotopically grown malignant gliomas. Transvascular transport and accumulation of the nanoparticles in brain tumor tissue was measured in vivo with dynamic contrast-enhanced magnetic resonance imaging. Localization of the nanoparticles within glioma cells was confirmed ex vivo with fluorescence imaging.

Results: We found that the intravenously administered functionalized dendrimers less than approximately 11.7 to 11.9 nm in diameter were able to traverse pores of the blood-brain tumor barrier of RG-2 malignant gliomas, while larger ones could not. Of the permeable functionalized dendrimer generations, those that possessed long blood half-lives could accumulate within glioma cells.

Conclusion: The therapeutically relevant upper limit of blood-brain tumor barrier pore size is approximately 11.7 to 11.9 nm. Therefore, effective transvascular drug delivery into malignant glioma cells can be accomplished by using nanoparticles that are smaller than 11.7 to 11.9 nm in diameter and possess long blood half-lives.

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Figures

Figure 1

Figure 1

Synthesis of Gd-dendrimers and transmission electron microscopy of higher generation Gd-dendrimers. A) A two-dimensional representation of naked polyamidoamine dendrimers up until generation 3 showing ethylenediamine core. B) The naked dendrimer has a cationic exterior. Functionalizing the terminal amine groups with Gd-diethyltriaminepentaacetic acid (charge -2) neutralizes the positive charge on the dendrimer exterior. C) Annular dark-field scanning transmission electron microscopy images of Gd-G5, Gd-G6, Gd-G7, and Gd-G8 dendrimers adsorbed onto an ultrathin carbon support film. Scale bar = 20 nm.

Figure 2

Figure 2

Gd concentration within blood and glioma tissue over time following intravenous Gd-dendrimer infusions at doses of 0.03 mmol Gd/kg bw and 0.09 mmol Gd/kg bw. A) Blood concentrations of Gd-dendrimers measured in the superior sagittal sinus following 0.03 mmol Gd/kg bw infusion. Gd-G1 (n=6), Gd-G2 (n=5), Gd-G3 (n=5), and lowly conjugated Gd-G4 (n=5) dendirmers imaged for 1 hour. Standard Gd-G4 (n=6), Gd-G5 (n=6), Gd-G6 (n=5), Gd-G7 (n=6), and Gd-G8 (n=5) dendrimers imaged for 2 hours. Error bars represent standard deviations. B) Blood concentrations of Gd-dendrimers measured in the superior sagittal sinus following 0.09 mmol Gd/kg bw infusion. Gd-G1 (n=4), Gd-G2 (n=6), Gd-G3 (n=6), lowly conjugated Gd-G4 (n=4), standard Gd-G4 (n=6), Gd-G5 (n=6), Gd-G6 (n=5), Gd-G7 (n=5), and Gd-G8 (n=6). Blood concentrations of Gd-G6, Gd-G7, and Gd-G8 dendrimers not shown for clarity. C) At both doses, lowly conjugated Gd-G4 dendrimers (molecular weight 24.4 kD) remain for a short period of time within the extravascular tumor space. 0.03 mmol Gd/kg bw dose n=5, 0.09 mmol Gd/kg bw dose n=4. D) At both doses, standard Gd-G4 dendrimers (molecular weight 39.8 kD) remain for longer within the extravascular tumor space. 0.03 mmol Gd/kg bw dose n=6, 0.09 mmol Gd/kg bw dose n=6. E) At both doses, Gd-G5 dendrimers accumulate within the extravascular tumor space. 0.03 mmol Gd/kg bw dose n=6, 0.09 mmol Gd/kg bw dose n=6. F) At the 0.03 mmol Gd/kg bw dose (n=5), Gd-G6 dendrimers do not extravasate out of tumor microvasculature. At the 0.09 mmol Gd/kg bw dose (n=5), Gd-G6 dendrimers extravasate. G) At the 0.03 mmol Gd/kg bw dose (n=6), Gd-G7 dendrimers do not extravasate. At the 0.09 mmol Gd/kg bw dose (n=5), Gd-G7 dendrimers extravasate. H) Irrespective of dose, Gd-G8 dendrimers do not extravasate out of brain tumor microvasculature. 0.03 mmol Gd/kg bw dose n=5, 0.09 mmol Gd/kg bw dose n=6. In panels C through H, Gd tumor concentrations and standard deviations shown are weighted for total tumor volume.

Figure 3

Figure 3

Gd concentration maps showing Gd-dendrimer distribution within the largest and smallest gliomas of each generation over time. A) Gd-G5, Gd-G6, and Gd-G7 dendrimers slowly accumulate within the extravascular tumor space of the largest RG-2 gliomas within the size range of tumors in the study. Gd-G8 dendrimers remain intravascular. The volume, in mm3, for each tumor shown is 104 (Gd-G1), 94 (Gd-G2), 94 (Gd-G3), 162 (lowly conjugated Gd-G4), 200 (standard Gd-G4), 230 (Gd-G5), 201 (Gd-G6), 170 (Gd-G7), and 289 (Gd-G8). B) Gd-G5 and G6 dendrimers still slowly accumulate within tumor tissue of the smallest RG-2 gliomas, which have a minimally compromised blood-brain tumor barrier. Gd-G7 dendrimers are impermeable to the BBTB of the smallest RG-2 gliomas and remain intravascular. Gd-G8 dendrimers continue to be impermeable to the blood-brain tumor barrier of the smallest RG-2 gliomas. The volume, in mm3, for each tumor shown is 27 (Gd-G1), 28 (Gd-G2), 19 (Gd-G3), 24 (lowly conjugated Gd-G4), 17 (standard Gd-G4), 18 (Gd-G5), 22 (Gd-G6), 24 (Gd-G6), and 107 (Gd-G8). Each animal received an intravenous 0.09 mmol Gd/kg bw.

Figure 4

Figure 4

Modeled pharmacokinetic parameters of lower generation Gd-dendrimers. A) The increase in Gd-dendrimer generation and size from that of Gd-G1 to that of lowly conjugated Gd-G4 results in a decrease in particle transvascular flow rate (Ktrans). Large tumors have higher Ktrans values. B) Lowly conjugated Gd-G4 dendrimer distribution within the glioma extravascular extracellular space (ve) is influenced to the greatest extent by the decrease in Ktrans. Large tumors have higher ve values. C) Fractional plasma volume (vp) within glioma vasculature is maintained across dendrimer generations. Large tumors have higher vp values. Large circles (Gd-G1 n= 4, Gd-G2 n=6, Gd-G3 n=7, and Gd-G4 n=2) represent large tumors (> 50 mm3), small circles (Gd-G1 n=4, Gd-G2 n=6, Gd-G3 n=5, and Gd-G4 n=6) represent small tumors (< 50 mm3), horizontal bars represent mean of observations weighted with respect to individual tumor volumes. Shown are Bonferroni corrected p-values from the nine post hoc comparisons for the three parameters, NS = not significant. D) There a more widespread distribution of Gd-G1 particles within the extravascular extracellular tumor space as shown by the greater range of ve values; whereas, there is a more focal distribution of lowly conjugated Gd-G4 dendrimers as shown by the lower range of ve values. Shown are voxels surviving censorship. Tumor volumes, in mm3, for tumors shown are 104 (Gd-G1) and 162 (lowly conjugated Gd-G4).

Figure 5

Figure 5

Fluorescence microscopy of glioma cell uptake of rhodamine B labeled Gd-dendrimer generations in vivo versus ex vivo. A) Synthetic scheme for production of rhodamine B (RB) labeled Gd-polyamidoamine dendrimers. The naked polyamidoamine dendrimer is first reacted with rhodamine B and then with Gd-DTPA. B) As shown by fluorescence microscopy in vitro, rhodamine B Gd-G2, rhodamine B Gd-G5, and rhodamine B Gd-G8 accumulate in glioma cells. Rhodamine B Gd-G2 dendrimers enter RG-2 glioma cells, and in some cases, the nucleus (left). Rhodamine B Gd-G5 dendrimers enter the cytoplasm of RG-2 glioma cells, but do not localize within the nucleus (middle). Rhodamine B Gd-G8 dendrimers enter RG-2 glioma cells in vitro (right). Shown are merged confocal images of blue fluorescence from DAPI-Vectashield nuclear (DNA) stain and red fluorescence from rhodamine B labeled Gd-dendrimers. Scale bars = 20 µm. C) At 2 hours dynamic contrast-enhanced MRI shows substantial extravasation of rhodamine B Gd-G5 dendrimers and some extravasation of rhodamine B Gd-G8 dendrimers. Rhodamine B Gd-G5 n=6, rhodamine B Gd-G8 n=2. D) Low power fluorescence microscopy ex vivo of brain tumor and normal brain surrounding tumor shows that there is substantial accumulation of rhodamine B Gd-G5 dendrimers within tumor tissue (left, T = tumor, N = normal, scale bar = 100 µm). High power shows subcellular localization within malignant glioma cells (upper right, scale bar = 20 µm). Hemotoxylin and Eosin stain of tumor and surrounding brain (lower right, scale bar = 100 µm). Tumor volume is 31 mm3. E) Also shown by low power fluorescence microscopy ex vivo is some accumulation of rhodamine B Gd-G8 dendrimers within brain tumor tissue (left, T = tumor, N = normal, scale bar = 100 µm). High power confirms minimal subcellular localization within glioma cells (upper right, scale bar = 20 µm). Hematoxylin and Eosin stain of tumor and surrounding brain (lower right, scale bar = 100 µm). Tumor volume is 30 mm3.

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