Ratiometric in vivo auditioning of targeted silver nanoparticles

Abstract

Attaching affinity ligands to nanoparticles (NPs) increases selectivity for targeting cells and tissues, and can result in improved sensitivity and reduced off-target toxicity in diagnostic and therapeutic systems. The decision over key features - NP size, shape, coating strategies and targeting ligands for clinical translation is often hampered by a lack of quantitative in vivo NP homing assays. Sensitive, internally controlled assays are needed which allow for quantitative comparisons (auditions) among various formulations of targeted NPs. We recently reported the development of peptide-functionalized, isotopically-barcoded silver NPs (AgNPs) for ultrasensitive studies centered on measuring relative ratios of NP internalization into cultured cells. Here we evaluated the application of this technology for NP homing studies in live mice using peptides with previously described tissue tropism; one peptide that favors vascular beds of the normal lungs (RPARPAR; receptor neuropilin-1, or NRP-1) and another that is selective for central nervous system vessels (CAGALCY). Equimolar mixtures of the peptide-targeted Ag107-NPs and Ag109 control particles were mixed and injected intravenously. Distribution profiles of Ag107 and Ag109 in tissue extracts were determined simultaneously through inductively coupled plasma mass spectrometry (ICP-MS). Compared to non-targeted particles up to ∼9-fold increased lung accumulation was seen for RPARPAR-OH AgNPs (but not for AgNPs functionalized with RPARPAR-NH₂, which does not bind to NRP-1). Similarly, AgNPs functionalized with the brain-homing CAGALCY peptide were overrepresented in brain extracts. Spatial distribution (mapping) analysis by laser ablation ICP-MS (LA-ICP-MS) was used to determine the ratio Ag107/Ag109 in tissue cryosections. The mapping demonstrated preferential accumulation of the RPARPAR-AgNPs in the perivascular areas around pulmonary veins, and CAGALCY AgNPs accumulated in discrete areas of the brain (e.g. in the vessels of cerebellar fibrillary tracts). Based on these results, the internally controlled ratiometric AgNP system is suitable for quantitative studies of the effect of targeting ligands on NP biodistribution, at average tissue concentration and distribution at the microscopic level. The platform might be particularly relevant for target sites with high local variability in uptake, such as tumors.

Fig. 1

Systemic CF555-labeled RPARPAR-OH-AgNPs home to lung tissue. Balb/c mice were injected in the tail vein with CF555-labelled RPARPAR-OH and biotin-AgNPs in 200 μl PBS. After 1 h or 5 h post i.v. administration, the animals were perfused with 15 ml PBS to wash out blood and to remove unbound plasma AgNPs, and the organs of interest were snap-frozen. (A) Confocal imaging of lung cryosections from mice injected with CF555-AgNPs, 5 h post i.v. administration. Arrows point at areas of accumulation of CF555-positive RPARPAR-OH AgNPs. N = 3; scale bars = 50 μm. (B) Quantitation of the CF555 fluorescence in tissue sections. AgNP signal (red in panel A) expressed as percent of pixels with intensity above threshold. Five randomly chosen fields from 3 animals per group were analyzed. Data represent mean ± SD, ** p < 0.01.

Fig. 2

Biodistribution of systemically administered CF555-CAGALCY-AgNPs. (A) Confocal imaging of tissues from mice injected with CAGALCY-functionalized AgNPs labeled with CF555 fluorescent dye, 5 h post i.v. administration. Arrows point at areas of accumulation of CAGALCY AgNPs (red). Scale bars = 100 μm. (B) Quantitation of CF555 fluorescence in tissue sections. Ag pixels/field were plotted for each type of nanoparticle as fold over control. N = 3; 5 fields were analyzed per tissue. Data represent mean ± SD.

Fig. 3

ICP-MS analysis of tissue distribution of homing peptide-functionalized AgNPs. (A) Outline of the ICP-MS ratiometric homing experiments. Balb/c mice were injected in tail vein with an equimolar mix of peptide-AgNPs (Ag107) and biotin-AgNPs (Ag109) in 200 μl PBS. 1 h or 5 h post i.v. administration, the animals were perfused with 15 ml PBS to remove free plasma AgNPs, and the organs of interest were snap-frozen. Tissues were subjected to HNO₃/H₂O₂ extraction protocol for ICP-MS, or cryosectioned for laser ablation ICP-MS (LA-ICP-MS). (B) ICP-MS analysis of tissue distribution of RPARPAR-OH-AgNPs after 1 or 5 h post i.v. administration. Data represent mean values ± SD (n = 3). *** p < 0.001, ** p < 0.01, * p < 0.05. (C) ICP-MS-based analysis of tissue distribution of CAGALCY AgNPs. Balb/c mice were intravenously injected with an equimolar mixture of targeted CAGALCY AgNPs and biotin-AgNPs. 5 h post i.v. administration the animals were perfused with PBS, and the extracts of organs were subjected to ICP-MS to for relative quantitation of silver isotopes. Data represent mean ± SD (n = 3), *** p < 0.001, ** p < 0.01, * p < 0.05.

Fig. 4

Ratiometric LA-ICP-MS profiling on sections of lung. (A) Balb/c mice were injected i.v. with 200 μL of a mixture of RPARPAR-OH-Ag107NPs and control biotin-Ag109NPs. At 5 h time point mice were perfused and organs collected. Tissues were snap-frozen, sectioned at 30 μm, and laser ablation line scans using 40 μm spot were performed. Hematoxylin–eosin staining of a lung cryosection used for LA-ICP-MS. The laser ablation path is indicated by arrow. Note the microanatomical heterogenity of the lung section. The region of the graph indicated by the dashed box and pink shading correspond to the inset showing the laser ablation path in panel B. (B) Areas along the ablation path with Ag107/Ag109 > 9 are indicated in red. Elevated Ag107/Ag109 ratios are associated with vascular and bronchial structures. Data are representative of 16 laser ablation paths. Scale bars: (A) 200 μm; (C) 100 μm.