Impact of tumor-specific targeting on the biodistribution and efficacy of siRNA nanoparticles measured by multimodality in vivo imaging

Abstract

Targeted delivery represents a promising approach for the development of safer and more effective therapeutics for oncology applications. Although macromolecules accumulate nonspecifically in tumors through the enhanced permeability and retention (EPR) effect, previous studies using nanoparticles to deliver chemotherapeutics or siRNA demonstrated that attachment of cell-specific targeting ligands to the surface of nanoparticles leads to enhanced potency relative to nontargeted formulations. Here, we use positron emission tomography (PET) and bioluminescent imaging to quantify the in vivo biodistribution and function of nanoparticles formed with cyclodextrin-containing polycations and siRNA. Conjugation of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid to the 5' end of the siRNA molecules allows labeling with (64)Cu for PET imaging. Bioluminescent imaging of mice bearing luciferase-expressing Neuro2A s.c. tumors before and after PET imaging enables correlation of functional efficacy with biodistribution data. Although both nontargeted and transferrin-targeted siRNA nanoparticles exhibit similar biodistribution and tumor localization by PET, transferrin-targeted siRNA nanoparticles reduce tumor luciferase activity by approximately 50% relative to nontargeted siRNA nanoparticles 1 d after injection. Compartmental modeling is used to show that the primary advantage of targeted nanoparticles is associated with processes involved in cellular uptake in tumor cells rather than overall tumor localization. Optimization of internalization may therefore be key for the development of effective nanoparticle-based targeted therapeutics.

Fig. 1.

Average time–activity curves for the first 60 min after injection. Data are shown for mice injected with ⁶⁴Cu (A), ⁶⁴Cu-labeled DOTA (B), ⁶⁴Cu-labeled DOTA-siRNA (C), and Tf-targeted nanoparticles containing ⁶⁴Cu-labeled DOTA-siRNA (D). Error bars indicate SE.

Fig. 2.

Tissue distribution of ⁶⁴Cu-DOTA-siRNA delivered by targeted (Tf) and nontargeted (PEG) nanoparticles. (A) Fused micro-PET/CT images of mice at 1, 10, and 60 min after injection. (B) Blood clearance and tumor localization of Tf-targeted and nontargeted siRNA nanoparticles.

Fig. 3.

Multimodality in vivo imaging of siRNA nanoparticle delivery and function, using micro-PET/CT and BLI. (A) Fused micro-PET/CT images showing tumor-associated (arrow) activity 1 d after injection of targeted (Tf) and nontargeted (PEG) nanoparticles containing ⁶⁴Cu-DOTA-siRNA. (B) BLI of the same mice shown in A before injection and 1 d after injection. (C) Relative change in luciferase expression 1 d after injection of Tf-targeted (Tf, n = 7) and nontargeted (PEG, n = 4) nanoparticles containing ⁶⁴Cu-DOTA-siRNA for simultaneous PET imaging. Error bars indicate SE.

Fig. 4.

Analysis of the interplay between the EPR effect and tumor-specific targeting, using a three-compartment model. (A) Model schematic and governing equations to estimate tumor uptake. (B) Model fit for total tumor uptake (Total) from experimental data (Exp) in Fig. 2, using k₂₁ = k₂₃ = 0. C₂ is the concentration in compartment 2. (C) Effect of tumor clearance (k₂₁) and tumor-specific binding/uptake (k₂₃) on tumor accumulation 1 d after injection.