An Orthogonally Clickable and Stimuli-Responsive Poly(β-amino ester) for the Co-delivery of Doxorubicin and BCL‑2 siRNA

Abstract

A potent drug delivery system (DDS) based on poly-(β-amino ester)-s (pBAEs) to tackle multidrug resistance (MDR) in lung cancer by codelivering siRNA targeting antiapoptotic BCL-2 and doxorubicin (DOX) has been prepared. Engineered via strain-promoted azide-alkyne cycloaddition (SPAAC) to attach a tripeptide end-chain moiety and thiol-disulfide exchange to conjugate DOX, the system employs a hydrazone linker for dual pH- and redox-responsive release. This ensures precise tumor targeting with minimal leakage in the circulation. In multidrug-resistant lung cancer cells (GLC-4/ADR), it sharply downregulates BCL-2 expression, amplifying DOX's therapeutic impact.

Keywords: combination therapy; drug delivery; multidrug-resistance; poly(β-amino ester); siRNA; stimuli-responsive; strain-promoted azide−alkyne cycloaddition (SPAAC); thiol−disulfide exchange.

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Schematic representation of bifunctionalized pBAE-CR3-D synthesis, polyplex self-assembly, and codelivery of siBCL-2 and DOX to cancerous cells.

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Scheme for the synthesis of pBAE-CR3-PD.

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(A) Gel retardation assay of pBAE-CR3-D/siBCL-2 nanocomplexes. Polymer/siRNA nanocomplexes were prepared at different ratios ranging from 1:5 to 1:45 (w/w). Polyplexes were freshly prepared prior to the assay and loaded into 1% agarose gel containing EtBr. The electrophoresis was run for 30 min at 80 V. (B) In vitro DOX release profiles of pBAE-CR3-D/siBCL-2 polyplexes at different pH values and 10 mM glutathione (GSH). Data are represented as means ± SD (n = 3).

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Cell viability of (A) GLC-4 and (B) GLC-4/ADR cells evaluated by the MTS assay after incubation with different polyplexes and free DOX for 48 h. Data are represented as means ± SD, *p < 0.05 and **p < 0.01. (5 DOX molecules per polymer, polymer length n = 9).

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Quantitative analysis of apoptotic GLC-4/ADR cells using the Annexin V-FITC assay after treatment with different samples: (a) control group, (b) control group treated with DOX (nonstained), (c) pBAE-CR3/SCR, (d) free DOX, (e) pBAE-CR3-D/SCR, and (f) pBAE-CR3-D/siBCL-2. Dose: 113 μM siRNA and 28.5 μM DOX. Incubation time: 48 h. (A) Quadrant analysis of Annexin V-FITC/DAPI. (B) Apoptosis rate (early and late apoptosis) graph. Data are represented as means ± SD (n = 3), ***p < 0.001.

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(A) Confocal laser scanning microscopy (CLSM) images of GLC-4/ADR cells treated with polyplex nanoparticles pBAE-CR3-D/FAM-siRNA. The nuclei were stained with DAPI (blue), red fluorescence indicates DOX and green fluorescence indicates SCR siRNA labeled with 6-FAM (FAM-siRNA). Dose: 452 μM FAM-siRNA and 114 μM DOX. The scale bars represent 10 μm. (B) Effectiveness of BCL-2 gene suppression in GLC-4/ADR cells. Relative BCL-2/GAPDH mRNA expression evaluated by RT-PCR. (C) Protein expression of BCL-2 evaluated by western blot. (D) Western blot of GAPDH and BCL-2 proteins quantified by ImageJ. Cells for RT-PCR and western blot assays were treated with the DDS for 48 h. Dose: 113 μM siRNA and 28.5 μM DOX. Data are represented as means ± SD (n = 3), *p < 0.05.