Tumor targeting RGD conjugated bio-reducible polymer for VEGF siRNA expressing plasmid delivery

Abstract

Targeted delivery of therapeutic genes to the tumor site is critical for successful and safe cancer gene therapy. The arginine grafted bio-reducible poly (cystamine bisacrylamide-diaminohexane, CBA-DAH) polymer (ABP) conjugated poly (amido amine) (PAMAM), PAM-ABP (PA) was designed previously as an efficient gene delivery carrier. To achieve high efficacy in cancer selective delivery, we developed the tumor targeting bio-reducible polymer, PA-PEG1k-RGD, by conjugating cyclic RGDfC (RGD) peptides, which bind αvβ3/5 integrins, to the PAM-ABP using polyethylene glycol (PEG, 1 kDa) as a spacer. Physical characterization showed nanocomplex formation with bio-reducible properties between PA-PEG1k-RGD and plasmid DNA (pDNA). In transfection assays, PA-PEG1k-RGD showed significantly higher transfection efficiency in comparison with PAM-ABP or PA-PEG1k-RAD in αvβ3/5 positive MCF7 breast cancer and PANC-1 pancreatic cancer cells. The targeting ability of PA-PEG1k-RGD was further established using a competition assay. To confirm the therapeutic effect, the VEGF siRNA expressing plasmid was constructed and then delivered into cancer cells using PA-PEG1k-RGD. PA-PEG1k-RGD showed 20-59% higher cellular uptake rate into MCF7 and PANC-1 than that of non-targeted polymers. In addition, MCF7 and PANC-1 cancer cells transfected with PA-PEG1k-RGD/pshVEGF complexes had significantly decreased VEGF gene expression (51-71%) and cancer cell viability (35-43%) compared with control. These results demonstrate that a tumor targeting bio-reducible polymer with an anti-angiogenic therapeutic gene could be used for efficient and safe cancer gene therapy.

Keywords: Bio-reducible polymer; Cancer gene therapy; RGD peptides; Targeted gene delivery; VEGF siRNA.

Fig. 1

(A) Structure of PA-PEG_1k-RGD Cyclic RGDfC peptides were conjugated to PAM-ABP using cross linker, Mal-PEG_1k-NHS. (B) ¹H NMR spectrum of PA-PEG_1kRGD

Fig. 2. Physical characterization of PA-PEG_1k-RGD/pDNA complexes

(A) Gel retardation assay Polymers/pDNA complexes were prepared at various weight ratios and analyzed by 1% agarose gel electrophoresis. (B) Agarose gel electrophoresis in the absence and in the presence of 5 mM DTT. PEI25k or PAM-ABP/pCMV-Luc complex was prepared at 1:1 or 1:5 weight ratio, respectively. PA-PEG_1k-RGD or PA-PEG_1k-RAD/pCMV-Luc complex was prepared 1:7 (pDNA: polymer) weight ratio condition. (C) Size and (D) zeta potential Complexes were prepared with various weight ratios polymers and pDNA. Data are expressed as mean values (±standard deviation) of triplicate experiments.

Fig. 3. Transfection efficiency of PA-PEG_1k-RGD

PA-PEG_1k–RGD/pDNA complex was prepared at various weight ratios and transfected into (A) PANC-1 pancreatic cancer cells. PEI25k, PAM-ABP, PA-PEG_1k-RGD or PA-PEG_1k-RAD/pCMV-Luc complex was transfected into (B) HUVEC α_vβ₃ positive and (C) 293 α_vβ_3/5 negative normal cells. Each polymer/pDNA complex was prepared under optimal condition. After 48 hrs of incubation, luciferase activity was measured. The data are expressed as mean values (±standard deviation) of triple experiments. *P<0.01 as compared with weight ratio of 1:5 (pDNA: PA-PEG_1k–RGD).**P<0.003 as compared with PAM-ABP.

Fig. 4. Green fluorescent protein expression levels by PA-PEG_1k-RGD

Polymers/pCMV-GFP complexes were transfected into MCF7 and PANC-1 cancer cells. After 48 h of incubation, GFP expression level was evaluated by (A and B) fluorescence microscopy and (C and D) fluorescence intensity measurement. The data are expressed as mean values (±standard deviation) of triplicate experiments. *P<0.003 as compared with PAM-ABP. **P<0.001 as compared with PEI25k.

Fig. 5. Cytotoxiciy of RGD peptides conjugated PAM-ABP

(A) Cell viability of PA-PEG_1k-RGD at various weight ratios PA-PEG_1k-RGD/pCMV-Luc complexes were transfected into MCF7 and PANC-1 cells with increasing weight ratios of PA-PEG_1k-RGD polymer. (B) Comparison of polymer cytotoxicity PEI25k, PAM-ABP, PA-PEG_1k-RGD and PA-PEG_1k-RAD were prepared with pDNA at optimal conditions for complex formation. After 48 hrs, cytotoxicity was evaluated by MTT assay. The complex ratio was presented by pDNA: polymer weight ratio. The data are expressed as mean values (±standard deviation) of quadruplicate experiments.

Fig. 6. Competition assay with free cyclic RGDfC peptides

PA-PEG_1k-RGD/pCMV-GFP or PA-PEG_1k-RAD/pCMV-GFP complex was prepared 1:7 (pDNA: polymer) weight ratio condition and then transfected into (A) PANC-1 and (B) 293 cells. The free cyclic RGDfC peptides were pretreated before 15 min of complex treatment. After 48 hrs incubation, expressed GFP intensity (OD (sample)/mg protein) was measured and presented by relative GFP activity (% of free RGD untreated sample in each polymer) *P<0.014 as compared with free RGD untreated (-) PA-PEG_1k-RGD.

Fig. 7. Cellular binding and uptake efficiency of PA-PEG1_1k-RGD/pshVEGF complex

(A) Structure of pshVEGF VEGF siRNA sequence containing hairpin structure was inserted into pSilencer plasmid to constructed VEGF siRNA expressing plasmid, pshVEGF. Polymers/YOYO-1 dye stained pshVEGF complexes were prepared at optimized weight ratio, respectively. The complexes were treated into (B) MCF7 and (C) PANC-1 cells for 4 hrs and then, cells were analyzed by FACS. a; PEI25k, b; PAM-ABP, c; PA-PEG1_1k-RGD and d; PA-PEG1_1k-RAD. Gray area indicated untreated cells.

Fig. 8. Down regulation of VEGF expression by pshVEGF delivery into cancer cells 48hrs incubated

(A) MCF7 and (B) PANC-1, 72 hrs incubated of (C) MCF7 and (D) PANC-1 cells. Polymers/pshVEGF complexes were trasnfected into MCF7 and PANC-1 cells. After 48 or 72 hrs incubation, VEGF expression level of each conditioned medium was measured by VEGF ELISA. Control;untreated cells, PA-PEG_1k-RGD/sc; scrambled shRNA expressing plasmid (pscRNA) was use for complex formation with PA-PEG_1k-RGD. The data are expressed as mean values (±standard deviation) of quadruplicate experiments. *P<0.003 as compared with PEI25k.

Fig. 9. Cell growth inhibition

Polymers/pshVEGF complexes were transfected into (A) MCF7, (B) PANC-1 (C) 293 cells. After 72 hrs of incubation, cell growth inhibition efficiency was indirectly measured by MTT assay. Scrambled shRNA (pscRNA) was used in PA-PEG_1k-RGD/sc complex. The data are expressed as mean values (±standard deviation) of triplicate experiments. *P<0.005 as compared with PEI25k.