Biodistribution studies of protein cage nanoparticles demonstrate broad tissue distribution and rapid clearance in vivo

Abstract

Protein cage nanoparticles have the potential to serve as multifunctional cell targeted, imaging and therapeutic platforms for broad applications in medicine. However, before they find applications in medicine, their biocompatibility in vivo needs to be demonstrated. We provide here baseline biodistribution information of two different spherical protein cage nanoplatforms, the 28 nm viral Cowpea chlorotic mottle virus (CCMV) and the 12 nm heat shock protein (Hsp) cage. In naive and immunized mice both nanoplatforms show similar broad distribution and movement throughout most tissues and organs, rapid excretion, the absence of long-term persistence within mice tissue and organs, and no overt toxicity after a single injection. These results suggest that protein cage based nanoparticles may serve as safe, biocompatible, nanoplatforms for applications in medicine.

Figure 1

Epifluorescence microscopy images exhibiting Texas Red-Labeled HspG41C in various mouse tissue vibratome sections after IV instillation of 50–100 μg protein. (a) Lymphatic duct filled with particles surrounding an alveolar airway at 1 hour (200x original magnification). (b) Pulmonary arteriole at 1 hour post injection (50 μg Protein) with specific fluorescence visible on the endothelial surface and perivascular areas of the interstitium (note arrows). (c) Pulmonary airway at 1-hour – pointing to particles situated on the endothelial surface and intersititially (100x original magnification). (d) Hepatic lobule at 30 minutes after instillation – terminal hepatic venule with radiating sinusoids and HspG41C-Texas Red fluorescene most likely associated with Kupffer cells (note arrows; 200x original magnification). (e) Frontal sections of the kidney at 24 hours post injection – interlobular capillary and cortical lymphastics satiated with fluorescent labeled cage (100x original magnification). (f) Kidney frontal sections at 1 hour demonstrating fluorescence in the convoluted tubules and interlobular connective tissue (100x original magnification).

Figure 2

Fluorescence microscopy displaying Texas Red-labeled CCMV cage in mouse liver and kidney at 1 hour post IV instillation of 50μg protein. (a) Hepatic lobule with terminal hepatic venule and radiating sinusoids filled with CCMV Texas Red-labeled particles most likely associated with Kupffer cells (note arrows; 100x original magnification). (b) Kidney frontal sections at 1 hour exhibiting Texas Red labeled CCMV cage in the convoluted tubules (200x original magnification).

Figure 3

Selected organ distribution of ¹²⁵I-labeled cages on a percent injected dose/gm of tissue basis. (A) Naïve mice receiving 50 μg protein IV injection of either ¹²⁵I-HspG41C or ¹²⁵I-CCMV cage at 1 and 24 hours. (B) Immunized mice receiving 50 μg protein IV injection of either ¹²⁵I-HspG41C or ¹²⁵I-CCMV cage at 1 and 24 hours.

Figure 4

Percentage of dose per organ in selected tissues. (A) ¹²⁵I-labeled CCMV and Hsp cage (50 μg protein) tail injected into naïve mice at 1 and 24 hours post injection (B) ¹²⁵I-labeled CCMV and Hsp cage (50 μg protein) tail injected into immunized mice at 1 and 24 hours post injection.

Figure 5

Comparison of selected organs from mice receiving IV free ¹²⁵I versus mice receiving either ¹²⁵I-CCMV or ¹²⁵I-HspG41C cage. (A) Percent of dose/g of lung, spleen and liver tissue at 1 and 24 hours. Naïve (N), immunized (I), and free ¹²⁵I (Free). (B) Percent of dose excreted in the urine and feces together at 24 hours.