Co-delivery of VEGF siRNA and Etoposide for Enhanced Anti-angiogenesis and Anti-proliferation Effect via Multi-functional Nanoparticles for Orthotopic Non-Small Cell Lung Cancer Treatment

Abstract

Targeting tumor angiogenesis pathway via VEGF siRNA (siVEGF) has shown great potential in treating highly malignant and metastatic non-small cell lung cancer (NSCLC). However, anti-angiogenic monotherapy lacked sufficient antitumor efficacy which suffered from malignant tumor proliferation. Therefore, the combined application of siVEGF and chemotherapeutic agents for simultaneous targeting of tumor proliferation and angiogenesis has been a research hotspot to explore a promising NSCLC therapy regimen. Methods: We designed, for the first time, a rational therapy strategy via intelligently co-delivering siVEGF and chemotherapeutics etoposide (ETO) by multi-functional nanoparticles (NPs) directed against the orthotopic NSCLC. These NPs consisted of cationic liposomes loaded with siVEGF and ETO and then coated with versatile polymer PEGylated histidine-grafted chitosan-lipoic acid (PHCL). We then comprehensively evaluated the anti-angiogenic and anti-proliferation efficiency in the in vitro tumor cell model and in bioluminescent orthotopic lung tumor bearing mice model. Results: The NPs co-delivering siVEGF and ETO exhibited tailor-made surface charge reversal features in mimicking tumor extracellular environment with improved internal tumor penetration capacity and higher cellular internalization. Furthermore, these NPs with flexible particles size triggered by intracellular acidic environment and redox environment showed pinpointed and sharp intracellular cargo release guaranteeing adequate active drug concentration in tumor cells. Enhanced VEGF gene expression silencing efficacy and improved tumor cell anti-proliferation effect were demonstrated in vitro. In addition, the PHCL layer improved the stability of these NPs in neutral environment allowing enhanced orthotopic lung tumor targeting efficiency in vivo. The combined therapy by siVEGF and ETO co-delivered NPs for orthotopic NSCLC simultaneously inhibited tumor proliferation and tumor angiogenesis resulting in more significant suppression of tumor growth and metastasis than monotherapy. Conclusion: Combined application of siVEGF and ETO by the multi-functional NPs with excellent and on-demand properties exhibited the desired antitumor effect on the orthotopic lung tumor. Our work has significant potential in promoting combined anti-angiogenesis therapy and chemotherapy regimen for clinical NSCLC treatment.

Keywords: VEGF siRNA; anti-angiogenesis; co-delivery; combined therapy; etoposide; multi-functional nanoparticles.

Scheme 1

Schematic Illustration of Co-delivery of VEGF siRNA and Etoposide for Enhanced Anti-angiogenesis and Anti-proliferation via Multi-functional Nanoparticles in Orthotopic Non-Small Cell Lung Cancer Treatment in vivo.

Figure 1

(A) Zeta potential and (B) particle size of the blank Lip, PHCL NPs and PHCL-Lip; (C) Zeta potential of PHCL-Lip and P-Lip at different pH; (D-E) The size distribution and TEM images of PHCL-Lip under different pH and redox conditions; (F) In vitro ETO release profiles from PHCL-Lip/ETO and ETO injection when exposed to different pH and redox stimuli.

Figure 2

(A) Cellular uptake and intracellular distribution of different formulations. CLSM images of A549 cells treated with (A) P-Lip/NR-siRNA^FAM or PHCL-Lip/NR-siRNA^FAM at pH 7.4 or 6.5 for 2 or 6 h and (B) P-Lip/siRNA^FAM or PHCL-Lip/siRNA^FAM at pH 6.5 for 2 or 6 h.

Figure 3

(A) VEGF mRNA level of A549 cells by RT-PCR analysis and (B) VEGF protein expression level in the culture medium of A549 cells by Western blot after treated with various siVEGF formulations.

Figure 4

(A-B) Cytotoxicity investigation of various ETO or siVEGF formulations against A549 cells by MTT assay (n=5), wherein “*” indicated comparison between PHCL-Lip/ETO at pH 7.4 and 6.5.

Figure 5

Penetration evaluation: (A) Scanned fluorescence distribution of the A549 spheroids after treated with PHCL-Lip/NR or P-Lip/NR at pH 7.4 or pH 6.5 conditions by CLSM. The concentration of NR was 5 μg/mL and bar was represented as 100 μm. (B) Semi-quantitative fluorescence intensity of different sections of A549 spheroids. (C) Semi-quantitated fluorescence intensity of center region of A549 spheroids at 60 μm section.

Figure 6

In vivo targeting evaluation and biodistribution of co-delivery formulations. (A) In vivo fluorescent and bioluminescent images of orthotopic A549 nude mice model after administrated with P-Lip/Dir or PHCL-Lip/Dir for 12 or 24 h; (B) Relative fluorescence intensity of the lung (tumor) of nude mice at 12 , 24 h or excised section; (C) Biodistribution of P-Lip/NR or PHCL-Lip/NR in orthotopic A549 nude mice after administration.

Figure 7

Tumor growth inhibition evaluations in orthotopic A549 tumor models. (A) Bioluminescence images and intensity (B) of mice treated with different formulations at predesigned time points; (C) Relative body weight of mice in different groups monitored every other day; (D) Representative H&E images of orthotopic A549 lung tumors after different formulations treatment. Different formulations represented a. Saline, b. ETO injection, c. P-Lip/ETO-siVEGF, d. PHCL-Lip/siVEGF, e. PHCL-Lip/ETO, f. PHCL-Lip/ETO- siVEGF. Data was represented as mean ± SD (n=5).

Figure 8

(A) Bioluminescence images and (B) intensity of excised major organs after treated with different formulations; (C) Representative H&E images of heart and liver after different formulations treatment; (D) Representative immunofluorescence images of vascular stained with blood vessel-specific-marker CD34 antibody in the orthotopic lung tumors of different groups. Different formulations represented a. Saline, b. ETO injection, c. P-Lip/ETO-siVEGF, d. PHCL-Lip/siVEGF, e. PHCL-Lip/ETO, f. PHCL-Lip/ETO- siVEGF. Data was represented as mean ± SD (n=5).