Is Tumor Cell Specificity Distinct from Tumor Selectivity In Vivo?: A Quantitative NIR Molecular Imaging Analysis of Nanoliposome Targeting

Abstract

The significance and ability for receptor targeted nanoliposomes (tNLs) to bind to their molecular targets in solid tumors in vivo has been questioned, particularly as the efficiency of their tumor accumulation and selectivity is not always predictive of their efficacy or molecular specificity. This study presents, for the first time, in situ NIR molecular imaging-based quantitation of the in vivo specificity of tNLs for their target receptors, as opposed to tumor selectivity, which includes influences of enhanced tumor permeability and retention. Results show that neither tumor delivery nor selectivity (tumor-to-normal ratio) of cetuximab and IRDye conjugated tNLs correlate with EGFR expression in U251, U87 and 9L tumors, and in fact underrepresent their imaging-derived molecular specificity by up to 94.2%. Conversely, their in vivo specificity, which we quantify as the concentration of tNL-reported tumor EGFR provided by NIR molecular imaging, correlates positively with EGFR expression levels in vitro and ex vivo (Pearson's r= 0.92 and 0.96, respectively). This study provides a unique opportunity to address the problematic disconnect between tNL synthesis and in vivo specificity. The findings encourage their continued adoption as platforms for precision medicine, and facilitates intelligent synthesis and patient customization in order to improve safety profiles and therapeutic outcomes.

Keywords: Cancer; Molecular Recognition; Nanoparticles; Receptors; Specificity.

Figure 1.

a) Modular synthesis of NIRA-tNLs starting with amide coupling of IRDye-680RD to nanoliposomal PEG-NH₂, followed by strain-promoted cycloaddition through copper-free click chemistry conjugation of nanoliposomal PEG-DBCO to azido-derivatized Cet. The sham mimetic is fabricated using the same strategy with a non-specific human IgG molecules and IRDye 800CW. b) Absorption spectra of the nano-Cet-680 NIRA-tNL, the nano-IgG-800 sham mimetic, and an equimolar mix of both constructs in PBS (1 μM dye equivalent). Nano-Cet-Rhod tNLs exhibited a greater degree of binding to U87 cells than the nano-IgG-Rhod sham mimetic (c) with up to 14-fold selectivity at lipid equivalent concentrations of 25 μM and 50 μM (d). (e) The shelf life of both the NIRA-tNL and the sham mimetic extends beyond 6 weeks in storage at 4°C. (Mean ± S.E.M., n = 3 biological replicates; Analysis Of Variance (ANOVA) with a Tukey post-test, *** = P<0.0001).

Figure 2.

a) Representative images of mice bearing U251 (high EGFR), U87 (medium EGFR) and 9L (EGFR-null) tumors. White light images are matched with optically-corrected NIRA-tNL fluorescence images that represent tumor delivery, and NIRA-tNL binding potential contrast images which correspond to specificity for EGFR (concentration of tNL-reported EGFR). Comparative images split between the NIRA-tNL contrast alone and NIRA-tNL EGFR specificity emphasize that imaging of the tumor accumulation of NIRA-tNLs alone cannot not inform of their in vivo specificity. b) Corresponding quantitation of the in vivo specificity of the NIRA-tNLs in the U251, U87 and 9L tumors, and the combined skin at 24, 48h and 72 following intravenous administration. (Mean ± S.E.M., biological replicates n = 4 (U251), 3 (U87) and 5 (9L); ANOVA with a Tukey post-test; * = P< 0.05, ** = P<0.01, *** = P<0.0005, **** = P< 0.0001.)

Figure 3.

a) The imaging-derived in vivo specificity of the NIRA-tNLs (concentration of reported EGFR) in U251, U87, and 9L tumors correlates positively with the EGFR expression of each individual tumor line at the cellular level in vitro (EGFR/cell) and with the relative tumor EGFR expression levels from ex vivo immunohistochemistry analyses (Pearson’s r =0.9225*** and 0.9591****, respectively). However, neither the in vivo tumor delivery (% I.D./g tumor; b) nor tumor selectivity of the NIRA-tNLs (tumor-to-normal brain ratio; c) at 24 h correlated with EGFR expression ex vivo. While the NIRA-tNL specificity at 24h normalized to that of 9L EGFR-null tumors exhibited a linear trend, both the raw NIRA-tNLs tumor delivery (% I.D./g tumor) normalized to 9L tumors (d) and the NIRA-tNLs tumor selectivity (tumor-to-normal ratio) normalized to 9L tumors (e) grossly underrepresented specificity in U87 and U251 tumors. (Values are mean ± S.E.M., statistical significance was calculated using ANOVA with a Tukey post-test; n = 4 biological replicates (U251), 3 biological replicates (U87) and 5 biological replicates (9L); * = P< 0.05, *** = P<0.0005, **** = P< 0.0001.)

Figure 4.

Conceptual overview of our proposed paradigm shift in the informed and iterative synthesis of receptor targeted nanoliposomes. This paradigm shift combines the multifaceted modulation of key and emerging parameters that are important for NIRA-tNL fabrication, with quantitation of in vivo molecular specificity of the constructs. Iterative engineering processes will ultimately implement rigorous pre-clinical development routines that enable the intelligent and informed development of NIRA-tNLs, minimizing failures and providing valuable modular insights for achieving in vivo molecular specificity.

Scheme 1.

Conceptual representation of how the in vivo specificity of NIR-active targeted nanoliposomes (nano-Cet-680; NIRA-tNLs) for solid tumor receptors is measured using NIR molecular imaging. IRDye 680RD and Cetuximab functionalized NIRA-tNLs are intravenously administered to mice bearing tumors overexpressing EGFR. A non-specific spectrally distinct nanoconstruct functionalized with IRDye 800CW and IgG (nano-IgG-800) is co-administered as a sham NIRA-tNL-mimetic reference for in situ quantitative NIR molecular imaging and binding potential derivation. The in situ specificity of the NIRA-tNL for EGFR is calculated from the derived binding potentials and presented as the concentration of EGFR it reports in vivo.