The epidermal growth factor receptor (EGFR) is an important anti-tumor target. The development of novel molecular-targeted anti-tumor drugs that can target the interior of tumor cells and specifically silence EGFR expression is valuable and promising. In this work, a promising anti-tumor conjugate comprising methoxy-modified EGFR siRNA and cyclic arginineglycine-aspartic acid (cRGD) peptides, which selectively bind to avb3 integrins, was synthesized and examined. To prepare cRGD-EGFR siRNA (cRGD-siEGFR), cRGD was covalently conjugated to the 5 0 -end of an siRNA sense strand using a thiol-maleimide linker. The cellular uptake and cytotoxicity of cRGD-siEGFR in vitro were tested using an avb3-positive U87MG cell line. In vivo bio-distribution, anti-tumor activity, immunogenicity and toxicity were investigated in a nude mouse tumor model through repeated i.v. administration of cRGD-siEGFR (7 times over a 48 h interval). Analyses of in vitro data showed that cRGD-siEGFR silenced EGFR expression effectively, with high tumor targeting ability. Administration of cRGD-siEGFR to tumor-bearing nude mice led to significant inhibition of tumor growth, obvious reduction of EGFR expression and down-regulation of EGFR mRNA and protein in tumor tissue. Furthermore, serum biochemistry and pathological section evaluation did not indicate any serious toxicity of cRGDsiEGFR in vivo. cRGD-siEGFR is likely a promising candidate with high targeting ability, substantial anti-tumor effects and low toxicity in vitro and in vivo.

The vascular endothelial growth factor receptor 2 (VEGFR2) is considered to be a pivotal target for anti-tumor therapy against angiogenesis of non-small cell lung cancer (NSCLC). However, effective and low-toxicity targeted therapies to inhibit VEGFR2 are still lacking. Here, biRGD-siVEGFR2 conjugate comprising murine VEGFR2 siRNA and [cyclo(Arg-Gly-Asp-D-Phe-Lys)-Ahx] 2 -Glu-PEG-MAL (biRGD) peptide which selectively binds to integrin avb3 receptors expressing on neovascularization endothelial cell was synthesized. The anti-tumor activity and renal toxicity of biRGD-siVEGFR2 or its combination therapy with low-dose apatinib were investigated on NSCLC xenografts. The immunogenicity of biRGD-siVEGFR2 was also evaluated in C57BL/6J mice. In vivo, intravenously injected biRGD-siVEGFR2 substantially inhibited NSCLC growth with a marked reduction of vessels and a down-regulation of VEGFR2 in tumor tissue. Furthermore, biRGD-siVEGFR2 in combination with low-dose apatinib achieved powerful anti-tumor effect with less nephrotoxicity compared with the regular dose of apatinib. Besides, no obvious immunogenicity of biRGD-siVEGFR2 was found. These findings demonstrate that biRGD-siVEGFR2 conjugate can be used as a new candidate for the treatment of NSCLC and its combination therapy with apatinib may also provide a novel strategy for cancer treatment in clinic.

Based on successful targeting to the αvβ3 integrin of cyclic arginine–glycine–aspartic acid (cRGD), cRGD-conjugated small interfering RNA (siRNA) exhibits tumor targeting and has become a new treatment strategy for solid tumors. However, the nephrotoxicity caused by its renal retention limits its clinical application. Here, we evaluated the protective effect of Gelofusine against cRGD-conjugated siRNA-induced nephrotoxicity in mice. Male Kunming mice (six per group) were either co-injected with Gelofusine and cRGD-siRNA or injected with cRGD-siRNA alone. After administration of these treatments five times, creatinine and blood urea nitrogen (BUN) levels were determined. Hematoxylin–eosin staining (HE staining) and transferase dUTP nick end labeling (TUNEL) analysis were used to compare the difference in renal damage between the groups. Additionally, fluorescence imaging was used to observe the distribution of cRGD-siRNA in vivo. The group co-injected with Gelofusine and cRGD-siRNA displayed lower creatinine and BUN levels than the cRGD-siRNA-alone group and showed less renal damage upon HE staining and TUNEL analysis. Gelofusine decreased the retention time and accelerated the elimination of cRGD-siRNA from the organs, as observed in the fluorescence images. These data indicate that Gelofusine significantly increased the excretion of cRGD-conjugated siRNA and reduced the associated renal damage.