Investigating the Cellular Specificity in Tumors of a Surface-Converting Nanoparticle by Multimodal Imaging

Abstract

Active targeting of nanoparticles through surface functionalization is a common strategy to enhance tumor delivery specificity. However, active targeting strategies tend to work against long polyethylene glycol's shielding effectiveness and associated favorable pharmacokinetics. To overcome these limitations, we developed a matrix metalloproteinase-2 sensitive surface-converting polyethylene glycol coating. This coating prevents nanoparticle-cell interaction in the bloodstream, but, once exposed to matrix metalloproteinase-2, i.e., when the nanoparticles accumulate within the tumor interstitium, the converting polyethylene glycol coating is cleaved, and targeting ligands become available for binding to tumor cells. In this study, we applied a comprehensive multimodal imaging strategy involving optical, nuclear, and magnetic resonance imaging methods to evaluate this coating approach in a breast tumor mouse model. The data obtained revealed that this surface-converting coating enhances the nanoparticle's blood half-life and tumor accumulation and ultimately results in improved tumor-cell targeting. Our results show that this enzyme-specific surface-converting coating ensures a high cell-targeting specificity without compromising favorable nanoparticle pharmacokinetics.

Figure 1

Physical characterization of the surface-converting nanoparticles. Design of the surface-converting polymer–lipid platform. (A) Schematic of the nanoparticle structure. The nanoparticle platform is made of a PLGA polymeric core shielded by a phoshopholipid corona. The surface-converting capacity was obtained by introducing MMP2-cleavable mPEG–MMP2p-DSPE (with mPEG being mPEG-2000) in the corona. (B) Dynamic light scattering (DLS) hydrodynamic size and polydispersity measurements of the different nanoparticle platforms (n = 3). (C) Negative staining transmission electron microscopy of: (i) untargeted-NP, (ii) RGD-NP, (iii) PEG-3000/RGD-NP, and (iv) PEG-MMP2p/RGD-NP (scale bar: 100 nm). The discrepancy between the nanoparticle mean hydrodynamic size in solution measured by DLS and the transmission electron microscopy images can be explained by the sample dehydration process required for the latter technique.

Figure 2

Multimodal imaging capacity of the nanoparticles. (A) Schematic depiction of the integration of multiple contrast agents within the nanoparticles. Fluorescent cyanine-based molecules were integrated in the polymeric core by using cyanine conjugated PLGA. Magnetic resonance contrast agent gadolinium was introduced by incorporating Gd–DTPA–DSA at the expense of PEG350–DSPE in the phospholipid corona. Nanoparticles were radiolabeled by introducing the hydrophobic chelator C₃₄-DFO during the formulation followed by complexion with ⁸⁹Zr prior to use. (B) Size-exclusion chromatograms showing fluorescent (green) and radioactive (orange) traces of ⁸⁹Zr-labeled PEG-3000/RGD-NP. The radioactive trace of free ⁸⁹Zr (black) is shown as control. (C) Dynamic light scattering hydrodynamic diameter, polydispersity index, r₁ measurements, and MR images of the nanoparticles labeled with different amounts of Gd–DTPA–DSA. The T₁-weighted MR images were obtained at a field of 7T, and r₁ was measured at a field of 1.41T. Size and polydispersity index: mean (n = 3). (D) Fluorescence emission spectra of nanoparticles labeled with Cy3.5 and Cy5 after excitation at 488 (i) and 633 (ii) nm. The excitations and filter pass-bands correspond to the flow cytometry channels used in the study.

Figure 3

Proof of principle of the surface-converting approach. (A) Avidin-induced aggregation of biotin-functionalized nanoparticles. Nanoparticle relative size was measured by DLS before and after incubation with avidin. (n = 3). (B) Binding of biotin-functionalized, Cy5.5-labeled nanoparticles on a streptavidin-coated plate. (n = 3); *, P < 0.05; ***, P < 0.001.

Figure 4

In vitro characterization of our surface-converting nano-articles. Fluorescence microscopy analysis of α_vβ₃ integrin-expressing (A) endothelial cells (HUVEC) and (B) breast tumor cells (MDA-MB-231) incubated with Cy5.5-labeled nanoparticles: (i) untreated cells (control), (ii) Untargeted-NP, (iii) PEG-3000/RGD-NP, and (iv) RGD-NP. Staining: blue, DAPI and red, Cy5.5. (v) Same field of view as (Biv) imaged with the green channel (GFP). Scale bar: 50 μm. Flow cytometry analysis of HUVEC (C) and MDA-MB-231 (D) cells incubated with Cy5.5-labeled nanoparticles. n = 4; ***, P < 0.001.

Figure 5

Pharmacokinetics and biodistribution of the surface-converting nanoparticles. Nanoparticles were injected intravenously in nude mice bearing orthotopic MDA-MB-231 GFP breast cancer tumors. (A) Blood time–activity curve for the different ⁸⁹Zr-labeled nanoparticles (n = 3 per nanoparticle type; ID: injected dose). (B) Ex vivo autoradiography of selected tissues 24 h after the administration of ⁸⁹Zr-labeled nanoparticles. Organs are Liv: liver, Kid: kidneys, Lun: lung, Spl: spleen, Tum: tumor, Mus: muscle, Bra: brain, Hea: heart. (C) Radioactivity distribution in selected tissues of ⁸⁹Zr-labeled nanoparticles expressed as percentage of injected dose per gram of tissue (%ID/g). (n = 3 per nanoparticle type). (D) Radioactivity concentration for tumor-to-muscle ratio. n = 3 per nanoparticle type; vs RGD-NP; *, P < 0.05.

Figure 6

Tumor cell targeting by surface-converting nanoparticles. (A) Schematic of the experiment: mice bearing orthotopic breast cancer tumors were co-injected with a mixture of two nanoparticle formulations, either Cy3.5-labeled PEG-MMP2p/RGD-NP and Cy5-labeled RGD-NP or Cy3.5-labeled PEG-MMP2p/RGD-NP and Cy5-labeled PEG-3000/RGD-NP. 24 h after injection, the animals were sacrificed, and the fluorescence in the different cell types (tumor cells, leukocytes, and endothelial cells) was measured by flow cytometry. (B) Percentages of PEG-MMP2p/RGD-NP-positive (red) and RGD-NP-positive (blue) cells in each animal for tumor cells (Tum), leukocytes (Leu), and endothelial cells (End), respectively. n = 5; *, P < 0.05. (C) Ratio between percentages of PEG-MMP2p/RGD-NP-positive cells and RGD-NP-positive cells for each cell type (n = 5). (D) Percentages of PEG-MMP2p/RGD-NP-positive (green) and PEG-3000/RGD-NP-positive (blue) cells in each animal for tumor cells (Tum), leukocytes (Leu), and endothelial cells (End), respectively (n = 5). (E) Ratio between percentages of PEG-MMP2p/RGD-NP-positive cells and PEG-3000/RGD-NP-positive cells for each cell type (n = 5).