Multimodal silica nanoparticles are effective cancer-targeted probes in a model of human melanoma

Abstract

Nanoparticle-based materials, such as drug delivery vehicles and diagnostic probes, currently under evaluation in oncology clinical trials are largely not tumor selective. To be clinically successful, the next generation of nanoparticle agents should be tumor selective, nontoxic, and exhibit favorable targeting and clearance profiles. Developing probes meeting these criteria is challenging, requiring comprehensive in vivo evaluations. Here, we describe our full characterization of an approximately 7-nm diameter multimodal silica nanoparticle, exhibiting what we believe to be a unique combination of structural, optical, and biological properties. This ultrasmall cancer-selective silica particle was recently approved for a first-in-human clinical trial. Optimized for efficient renal clearance, it concurrently achieved specific tumor targeting. Dye-encapsulating particles, surface functionalized with cyclic arginine-glycine-aspartic acid peptide ligands and radioiodine, exhibited high-affinity/avidity binding, favorable tumor-to-blood residence time ratios, and enhanced tumor-selective accumulation in αvβ3 integrin-expressing melanoma xenografts in mice. Further, the sensitive, real-time detection and imaging of lymphatic drainage patterns, particle clearance rates, nodal metastases, and differential tumor burden in a large-animal model of melanoma highlighted the distinct potential advantage of this multimodal platform for staging metastatic disease in the clinical setting.

Figure 1. Multimodal C dot design for α_νβ₃ integrin targeting and characterization.

(A) Schematic representation of the ¹²⁴I-cRGDY-PEG-ylated core-shell silica nanoparticle with surface-bearing radiolabels and peptides and core-containing reactive dye molecules (insets). (B) FCS results and single exponential fits for measurements of Cy5 dyes in solution (black) and PEG-coated (PEG-dot, red) and PEG-coated, cRGDY-labeled dots (blue, underneath red data set) showing diffusion time differences as a result of varying hydrodynamic sizes.

Figure 2. Competitive integrin receptor binding studies with ¹²⁴I-cRGDY-PEG-dots, cRGDY peptide, and anti–α_νβ₃ antibody using 2 cell types.

(A) High-affinity and specific binding of ¹²⁴I-cRGDY-PEG-dots to M21 cells by γ-counting. The inset shows Scatchard analysis of binding data, plotting the ratio of the concentration of receptor-bound (B) to unbound (or free [F]) radioligand or the bound-to-free ratio (B/F) versus the receptor-bound receptor concentration; the slope corresponds to the dissociation constant, K_d. (B) α_νβ₃ Integrin receptor blocking of M21 cells using flow cytometry and excess unradiolabeled cRGD or anti–α_νβ₃ antibody prior to incubation with cRGDY-PEG-dots and nonspecific binding with controls (RAD-PEG-dots, PEG-dots). (C) Specific binding of cRGDY-PEG-dots to M21 cells as against M21L cells lacking surface integrin expression using flow cytometry. Anti–α_νβ₃ integrin receptor antibody concentrations were used at 100 times (i.e., 100x) and 250 times (i.e., 250x) the particle (i.e., ¹²⁴I-cRGDY-PEG-dot) concentration. (D) Specific binding of cRGDY-PEG-dots to HUVECs by flow cytometry. Each bar represents mean ± SD of 3 replicates.

Figure 3. Pharmacokinetics and excretion profiles of the targeted and nontargeted particle probes.

(A) Biodistribution of ¹²⁴I-cRGDY-PEG-dots (targeted) in M21 tumor-bearing mice at various times from 4- to 168-hours p.i. The inset shows a representative plot of these data for blood to determine the T_1/2. Sm. int., small intestine; Lg. int., large intestine; conc., concentration. (B) Biodistribution of ¹²⁴I-PEG-dots (untargeted) from 4- to 96-hours p.i. (C) Clearance profile of urine samples collected up to 168-hours p.i. of unradiolabeled cRGDY-PEG-dots (n = 3 mice, mean ± SD). The inset shows a strong correlation between the percentage of the injected particle dose excreted and the corresponding measured fluorescence signal. (D) Corresponding cumulative %ID/g for feces at intervals up to 168-hours p.i. (n = 4 mice). For biodistribution studies, bars represent mean ± SD.

Figure 4. Serial in vivo PET imaging of tumor-selective targeting.

(A) Representative whole-body coronal microPET images at 4-hours p.i., demonstrating M21 (left, arrow) and M21L (middle, arrow) tumor uptakes of 3.6 and 0.7 %ID/g, respectively, and enhanced M21 tumor contrast at 24 hours (right). (B) Representative 24-hour fluorescence image of the tumor. (C) In vivo uptake of ¹²⁴I-cRGDY-PEG-dots in α_vβ₃ integrin–overexpressing M21 (black, n = 7 mice) and nonexpressing M21L (medium dark gray, n = 5 mice) tumors, ¹²⁴I-PEG-dots in M21 tumors (dark gray, n = 5), and ¹²⁴I-cRAD-PEG-dots in M21 tumors (light gray, n = 3). (D) M21 tumor-to-muscle ratios for ¹²⁴I-cRGDY-PEG-dots (black) and ¹²⁴I-PEG-dots (gray). (E) Correlation of in vivo and ex vivo M21 tumor uptakes of cRGDY-labeled and unlabeled probes. Each bar represents mean ± SD.

Figure 5. Nodal mapping using multiscale near-infrared optical fluorescence imaging.

(A) Whole-body fluorescence imaging of the tumor site (T) and draining inguinal (ILN) and axillary (ALN) nodes and communicating lymphatics channels (LCs; bar) 1-hour p.i. in a surgically exposed living animal. (B) Corresponding coregistered white-light and high-resolution fluorescence images (top row) and fluorescence images only (bottom row), revealing nodal infrastructure of local and distant nodes, including high endothelial venules (HEVs). (C) Whole-body fluorescence image of the tumor site 10 minutes after subdermal PEG-dot injection. (D) Delayed whole-body fluorescence image of the tumor site 1 hour after PEG-dot injection. (E) Percentage increase in the area of fluorescence (fluor) relative to that measured at 10-minutes p.i. for targeted and nontargeted probes. Scale bars: 1.0 cm (A); 500 microns (B); 3 mm (C and D).

Figure 6. Imaging of metastatic disease in a spontaneous melanoma miniswine model.

(A) Whole-body dynamic ¹⁸F-FDG PET-CT sagittal and axial views, demonstrating primary tumor (green arrow) and single SLN (white arrow) posteriorly within the right (Rt) neck after i.v. injection. ant, anterior. (B) High-resolution dynamic PET-CT scan 1 hour after subdermal, 4-quadrant, peritumoral injection of ¹²⁴I-RGD-PEG-dots (SLN, arrow; left-sided node, arrowhead). (C) Whole-body Cy5 fluorescence image of the excised SLN. (D) Gross image of the cut surface of the black-pigmented SLN (asterisk), which measured 1.3 × 1.0 × 1.5 cm³, and annotated γ counted activity. (E) Low-power view of H&E-stained SLN, demonstrating scattered melanomatous clusters. (F) Corresponding high-power view of H&E-stained SLN, revealing melanoma cells (yellow arrowheads) and melanophages (white arrowhead). (G) Low-power image of a melanoma-specific marker, HMB-45, in representative SLN tissue. (H) High-power image of HMB-45–stained SLN tissue. (I) Low-power image of representative normal porcine nodal tissue. (J) High-power image of representative normal porcine nodal tissue. (K) Low-power view of H&E-stained contralateral hypermetabolic lymph node, demonstrating scattered melanomatous clusters (arrowhead). Tumor burden in this smaller node (1.0 × 0.6 × 1.0 cm³) was estimated to be 10- to 20-fold less than that in the SLN by pathological analysis. Scale bars: 1 mm (E, G, I, and K); 20 μm (F, H, and J).