Drug delivery, biodistribution and anti-EGFR activity: theragnostic nanoparticles for simultaneous in vivo delivery of tyrosine kinase inhibitors and kinase activity biosensors

Abstract

In vivo delivery of small molecule therapeutics to cancer cells, assessment of the selectivity of administration, and measuring the efficacity of the drug in question at the molecule level, are important ongoing challenges in developing new classes of cancer chemotherapeutics. One approach that has the potential to provide targeted delivery, tracking of biodistribution and readout of efficacy, is to use multimodal theragnostic nanoparticles to deliver the small molecule therapeutic. In this paper, we report the development of targeted theragnostic lipid/peptide/DNA lipopolyplexes. These simultaneously deliver an inhibitor of the EGFR tyrosine kinase, and plasmid DNA coding for a Crk-based biosensor, Picchu-X, which when expressed in the target cells can be used to quantify the inhibition of EGFR in vivo in a mouse colorectal cancer xenograft model. Reversible bioconjugation of a known analogue of the tyrosine kinase inhibitor Mo-IPQA to a cationic peptide, and co-formulation with peptides containing both EGFR-binding and cationic sequences, allowed for good levels of inhibitor encapsulation with targeted delivery to LIM1215 colon cancer cells. Furthermore, high levels of expression of the Picchu-X biosensor in the LIM1215 cells in vivo allowed us to demonstrate, using fluorescence lifetime microscopy (FLIM)-based biosensing, that EGFR activity can be successfully suppressed by the tyrosine kinase inhibitor, released from the lipopolyplexes. Finally, we measured the biodistribution of lipopolyplexes containing ¹²⁵I-labelled inhibitors and were able to demonstrate that the lipopolyplexes gave significantly higher drug delivery to the tumors compared with free drug.

Fig. 1. (a) Structures of the previously reported ¹⁸F PET radiotracer F-[PEG₆-IPQA] and the EGFR TKI Mo-IPQA; (b) peptide, lipid and plasmid DNA components of the lipopolyplex nanoparticles used in this work.

Scheme 1. Reagents and conditions: (i) Ph₃CSH, NaH, THF, rt, 2 h (82%); (ii) SnCl₂·H₂O, THF, reflux, 3 h (61%); (iii) isobutyl chloroformate, acrylic acid, Et₃N, −40 °C, 35 min (57%); (iv) DTNP, CH₂Cl₂, CF₃COOH, rt, 2 h (48%); (v) CysLys₁₆7, MeOH, rt, 1 h (59%).

Fig. 2. Effect of different lipopolyplex formulations and drug loading on the expression of EGFR in targeted cells and inhibition of its activity upon stimulation with EGF. LIM1215 cells transfected with different lipopolyplexes without inhibitor (CLA1–4, NAE1–4, NGE1–4) (a) or with increasing amount of inhibitor per particle (F1LA1–3, F2LA1–3) (b) 24 h after seeding. High resolution images of 2 × 2 mm area used to quantify transfection efficiency (number of cells expressing biosensor, GFP channel, over total number of cells in field of view, UV channel. (c) FRET-FLIM analysis of the inhibition of EGF-induced EGFR activity in cells by free K₁₆Cys-S-S-[PEG₆-IPQA] 1 (10 mM). *P = 0.03, the difference is statistically significant between the groups. (d) Time course of the inhibitory effect of K₁₆Cys-S-S-[PEG₆-IPQA] 1 delivered to the cells by lipopolyplex or as a free agent.

Scheme 2. Reagents and conditions: (i) TBDPSCl, imidazole, CH₂Cl₂, 0 °C to rt, 3 h (98%); (ii) SnCl₂·2H₂O, THF, 60 °C, 3.5 h, (65%); (iii) Pd(PPh₃)₄, Sn₂Me₆, dioxane, reflux, 2.5 h, (95%); (iv) isobutyl chloroformate, acrylic acid, Et₃N, −40 °C, 30 min (53%); (v) TBAF, THF, 0 °C to rt, 1 h (95%); (vi) N-maleoyl-β-alanine, DCC, DMAP, CH₂Cl₂, rt, 2 h, (89%); (vii) CysLys₁₆7, NaHCO₃, rt, 1 h (55%); (viii) sodium [¹²⁵I]iodide, iodogen, 30 min rt, 28% radiochemical yield.

Fig. 3. In vivo radioactive organ biodistribution data presented in % Injected Dose per gram (%ID g⁻¹) for MeO-[PEG₆-[^125I]-IPQA] 8 (black columns) and F1LA1 K₁₆Cys-SMal-[PEG₃-[¹²⁵I]-IPQA] 9 lipopolyplex (grey columns) 3, 6 and 24 h after injection. The tumor %ID g⁻¹ at 3, 6 and 24 h for both tracers is represented on a separate graph.

Fig. 4. The effect of the inhibitor on EGFR activity measured by Picchu-X biosensor delivered by lipopolyplexes to tumor cells in vivo. Fresh frozen LIM1215 xenografts from animals injected with CLA1 lipopolyplex with pDNA encoding for Picchu-X biosensor alone, or F1LA1 K₁₆Cys-SMal-[PEG₃-[¹²⁵I]-IPQA]-lipopolyplex (additionally loaded with K₁₆Cys-SMal-[PEG₃-[¹²⁵I]-IPQA]) were cut and lifetime images were taken (see Material and methods section). Tumors from control group (injected with lipopolyplex without inhibitor) exhibited low lifetime of GFP in the biosensor (yellow-red colors in pseudocolor map) indicating high level of EGFR activation in the cells. In tumors from animals injected with lipopolyplex loaded with EGFR inhibitor we found high lifetime of GFP (blue color on pseudocolor map) indicating inhibition of EGFR activity in cells. *P = 0.004 (N = 8 images per group), the difference is statistically significant between the groups.