The
study aims to investigate the in vivo distribution,
antitumor effect, and safety of cell membrane-penetrating peptide-modified
disulfide bond copolymer nanoparticles loaded with small-interfering
RNA (siRNA) targeting epidermal growth factor receptor (EGFR) and
bromodomain-containing protein 4 (BRD4) in triple-negative breast
cancer (TNBC). Polyethylene glycol disulfide bond-linked polyethylenimine
(PEG-SS-PEI) was modified with peptides GALA and CREKA and used as
vectors to prepare siRNA nanoparticles. The GALA- and CREKA-modified
PEG-SS-PEI nanoparticles (GC-NPs) were prepared by mixing siEGFR and
siBRD4 (1:1) with GALA-PEG-SS-PEI and CREKA-PEG-SS-PEI (1:1) in an
aqueous solution at an N/P ratio of 30:1. Nanoparticles loaded with
scrambled siRNA were prepared with the same method. The gene silencing
effect on EGFR and BRD4 in vitro was evaluated by
Western blotting analysis. TNBC xenograft models were established
by subcutaneous injection of MDA-MB-231 cells into female nude mice.
At 1, 3, 6, 12, and 24 h after administration of five formulations
of Cy5-siRNA (133 μg/10 g) via the tail vein, the mice were
observed and imaged for a biodistribution study using an in
vivo imaging system. In the pharmacodynamics experiment,
tumor-bearing mice were treated with respective siRNA preparations
at a dose of 133 μg/10 g for 18 days, and the body weight and
tumor size were recorded every other day. The protein expression levels
of EGFR, p-EGFR, PI3K, p-PI3K, Akt, p-Akt, BRD4, and c-Myc were determined
using Western blotting analysis. Hematological and serum biochemical
parameters, organ indices, and HE staining results for the heart,
liver, spleen, lung, and kidney were analyzed to evaluate the safety
of the nanoparticles. GC-NPs loaded with siEGFR and siBRD4 significantly
inhibited the expression of EGFR and BRD4 in vitro. The strongest fluorescence signals were observed in the GC-NP group,
especially in tumors, indicating the excellent tumor-targeted delivery
of GC-NPs we constructed. Tumor growth was significantly inhibited
in the GC-NP-treated group, and the expression of EGFR, p-EGFR, PI3K,
p-PI3K, Akt, p-Akt, BRD4, and c-Myc in the tumors decreased by 71%,
68%, 61%, 68%, 48%, 58%, 59%, and 74% compared to the control group,
respectively. There was no significant change in hematological parameters,
biochemical indices, or tissue morphology in GC-NP-treated mice. SiRNA
cotargeting EGFR and BRD4 delivered by GALA- and CREKA-modified PEG-SS-PEI
had favorable antitumor effects in vivo toward TNBC
with tumor-targeting efficacy and good biocompatibility.
