A dendritic, redox-responsive, supramolecular (Dr.S) system for lysis-triggered delivery for drug-resistant renal cancer

Abstract

Purpose: Aiming to improve the drug loading capacity of dendritic nanoparticles and enhance delivery efficacy in drug-resistant cancer, we developed and optimized a more advanced dendritic, redox-responsive, supramolecular (Dr.S) system for intravenous RAD001 administration. Materials and methods: The Dr.S system was engineered by linking 3^rd generation polyamidoamine dendrimers (G3 PAMAM) with 8-arm polyethylene glycol (PEG) to encapsulate a molecular targeted agent RAD001. The drug-loading capacity was measured by ultraviolet-visible spectrophotometry. In vitro release behavior was determined with a two-compartment model, and the in vivo distribution pattern was tracked by Cy5.5 fluorescence. The therapeutic effect of Dr.S/RAD001 was evaluated in RAD001-resistant cancer cells and tumor-bearing nude mice, respectively. Results: The Dr.S system encapsulating RAD001 with a loading efficiency of 10.6% formed a core-shell structure, by shifting hydrophobic PAMAM/RAD001 components towards inner space and exposing the hydrophilic PEG on the surface. The Dr.S/RAD001 system could respond to a lysis-mimicking reduction stimulus, and functionally release cargoes to facilitate tumor accumulation and cellular internalization. These features contributed to the enhanced anti-tumor activity of RAD001 in renal cancers in vitro and in vivo. The Dr.S/RAD001 system also reversed acquired RAD001-resistance by a 60-fold increase in tumor accumulation of the therapeutics. Conclusion: The functional Dr.S/RAD001 system enables lysis-triggered release of RAD001 to achieve better tumor accumulation, which helps overcome acquired drug resistance in renal cancers.

Fig. 1. Characterization of the Dr.S system. (a) ¹H NMR spectra of the Dr.S vector and its components, namely 8-arm polyethylene glycol (bPEG), 8-arm PEG with disulfide bond (bPEG-SS-PEG), 3^rd generation polyamidoamine (G3 PAMAM). (b) RAD001 loading process by the Dr.S system with nucleotide condensation. The corresponding transmission electron microscopy (TEM) images were shown. The bar represents 200 nm. (c) Ultraviolet-visible spectra of the Dr.S vector, free RAD001, and the Dr.S/RAD001 system.

Fig. 2. Redox-responsiveness of the Dr.S/RAD001 system. (a) The patterns of particle size (orange) and ζ-potential (purple) at different N : P ratios. (b and c) The time-dependent change of particle size (b) and ζ-potential (c) with DTT stimulation (yellow) or without any stimulation (red). *p < 0.05, vs. no stimulation, two-way ANOVA followed by post-hoc Bonferroni test. (d) The time-dependent release patterns of the Dr.S/RAD001 system with DTT stimulation (yellow) or without any stimulation (red).

Fig. 3. Enhanced tumor retaining and cellular internalization. (a) Fluorescence distribution in mice treated with tail-vein injection of PBS, nc-siRNA^Cy5.5+, or Dr.S^Cy5.5+/RAD001. The tumors and major organs were harvested to show fluorescence intensity. (b) The time-dependent internalization process of Dr.S^FITC+/RAD001 (green) visualized by confocal microscopy. The cytoplasm was stained with Alexa Fluor 594 (red) and the nucleus was stained with DAPI (blue).

Fig. 4. Therapeutic effect of the Dr.S/RAD001 system. (a) Dose-dependent antiproliferation of the Dr.S/RAD001 system (light red) in comparison with the Dr.S vector (dark red) and free RAD001 (rose). Best fit curves by non-linear regression models were shown in dashed lines. (b) Tumor growth patterns in mice treated with PBS (gray), the Dr.S vector (dark red), oral RAD001 (rose), and intravenous Dr.S/RAD001 (light red). *p < 0.05, vs. p.o. RAD001; two-way ANOVA followed by Bonferroni test. (c) The white-field photo of the xenograft tumors treated with PBS, the Dr.S vector, oral RAD001, and intravenous Dr.S/RAD001. (d) Attenuation of phosphorylated Akt (p-Akt) and mTOR (p-mTOR) expression by intravenous Dr.S/RAD001. (e) The intratumoral characterization of apoptotic cells (green) by the TUNEL assays and mTOR positive expression (dark brown), with histology analysis by H&E staining.

Fig. 5. Therapeutic effect of the Dr.S/RAD001 system in RAD001-resistant RCC cells. (a) Dose-dependent antiproliferation of the Dr.S/RAD001 system (light red) in comparison with free RAD001 (rose) in regular (ACHN) and RAD001-resistant (ACHN/R) cells. Best fit curves by non-linear regression models were shown in dashed (ACHN) and solid (ACHN/R) lines. (b) Growth patterns of the xenograft RAD001-resistant tumors treated with PBS (gray), oral RAD001 (rose), and intravenous Dr.S/RAD001 (light red). *p < 0.05, vs. p.o. RAD001; two-way ANOVA followed by Bonferroni test. (c) The white-field photo of the drug-resistant xenograft tumors treated with PBS, oral RAD001, and intravenous Dr.S/RAD001. (d) Intratumoral RAD001 dose normalized to the control oral RAD001 group.