RALA-mediated delivery of FKBPL nucleic acid therapeutics

Abstract

Aims: RALA is a novel 30 mer bioinspired amphipathic peptide that is showing promise for gene delivery. Here, we used RALA to deliver the FK506-binding protein like - FKBPL gene (pFKBPL) - a novel member of the immunophilin protein family. FKBPL is a secreted protein, with overexpression shown to inhibit angiogenesis, tumor growth and stemness, through a variety of intra- and extracellular signaling mechanisms. We also elucidated proangiogenic activity and stemness after utilizing RALA to deliver siRNA (siFKBPL).

Materials & methods: The RALA/pFKBPL and RALA/siFKBPL nanoparticles were characterized in terms of size, charge, stability and toxicity. Overexpression and knockdown of FKBPL was assessed in vitro and in vivo.

Results: RALA delivered both pFKBPL and siFKBPL with less cytotoxicity than commercially available counterparts. In vivo, RALA/pFKBPL delivery retarded tumor growth, and prolonged survival with an associated decrease in angiogenesis, while RALA/siFKBPL had no effect on tumor growth rate or survival, but resulted in an increase in angiogenesis and stemness.

Conclusion: RALA is an effective delivery system for both FKBPL DNA and RNAi and highlights an alternative therapeutic approach to harnessing FKBPL's antiangiogenic and antistemness activity.

Keywords: FKBPL DNA; RALA peptide; RNAi; antiangiogenic; nanoparticle.

Figure 1.. Biophysical characterization of RALA-pFKBPL nanoparticles and RALA-siFKBPL nanoparticles.

(A) Gel retardation assay of RALA-pFKBPL nanoparticles from N:P ratio 0–15. (B) Gel retardation assay of RALA-siFKBPL nanoparticles from N:P ratio 0–15; each lane (L–R) represents an increase in N:P ratio from 0 to 15. (C) Particle size and zeta potential of RALA-pFKBPL nanoparticles (bars = size, line = charge). (D) Particle size and zeta potential of RALA-siFKBPL nanoparticles from N:P ratio 6–15 (bars = size, line = charge). (E) Serum stability of RALA -pFKBPL nanoparticles for 4 h at 37°C. (F) Dynamic light scattering of RALA-pFKBPL (red) and RALA-siFKBPL (green) at N:P10 with PDI and (G) transmission electron microscope image of RALA-siFKBPL complexes at N:P 10 with RALA-siFKBPL nanoparticles indicated by arrows. pFKBPL: FK506-binding protein like – FKBPL gene, delivered using RALA.

Figure 2.. N:P 10 RALA-siFKBPL and RALA-pFKBPL nanoparticles in vitro.

(A) Western blot for FKBPL expression in ZR-75–1 cells following transfection for 72 h with RALA-siFKBPL or RALA-siNT N:P ratio 10 complexes, and also with Oligofectamine as a positive control. (B) Western blot for FKBPL expression in ZR-75–1 cells following transfection for 72 h with RALA-pFKBPL or RALA-pEV N:P ratio 10, and also with Lipofectamine 2000 as a positive control. (C) Densitometric analysis of RALA-siFKBPL knockdown. n = 3 ± SEM. Statistical analysis was by one-way ANOVA, where *p < 0.05; **p < 0.01 and ***p < 0.001. (D) Relative cell viability of ZR-75–1 cells 72 h post-transfection with RALA-siFKBPL and RALA-pFKBPL N:P ratio 10 complexes determined via WST-1 assay. n = 3 ± SEM. Statistical analysis was by t-test, where *p < 0.05. pEV: Empty vector plasmid; pFKBPL: FK506-binding protein like – FKBPL gene, delivered using RALA; siFKBPL: FKBPL siRNA; siNT: Nontargeting siRNA.

Figure 3.. RALA is a suitable delivery vehicle for FKBPL overexpressing plasmid and FKBPL siRNA in vitro and in vivo.

(Ai) In vitro FKBPL knockdown was confirmed by western blot, representative images of which are shown. Twice weekly in vivo ZR-75–1 intratumoral administration of RALA-pFKBPL or of RALA-siFKBPL resulted in enhanced (ii) or reduced (iii) immunohistochemistry staining for FKBPL respectively, and for RALA-pFKBPL, revealed areas of necrosis at the experimental end point ([i]; bottom panel). Prior to intratumoral injection, 2 × 10⁶ ZR-75–1 cells were transfected with a sample of the in vivo nanoparticle preparation, and FKBPL knockdown. (B) Administration of RALA-pFKBPL significantly prolonged survival (time to tumor tripling) (i) and reduced overall tumor growth (ii) compared with administration of RALA-pEV. However, administration of RALA-siFKBPL showed no difference in tumor growth rate (iii). pEV: Empty vector plasmid; pFKBPL: FK506-binding protein like – FKBPL gene, delivered using RALA; siFKBPL: FKBPL siRNA; siNT: Nontargeting siRNA.

Figure 4.. In vivo ZR-75–1 intratumoral administration of RALA-pFKBPL or RALA-siFKBPL.

(A) Representative images of stained tumor sections following delivery of RALA-siFKBPL or RALA-pFKBPL at 4× and 40× are shown, with vessels marked with arrows. (B) Manual quantification of CD34⁺ blood vessels. Results were taken from a minimum of 27 fields of view and were statistically analyzed by unpaired t-test with Welch's correction, where **p < 0.01 and ***p < 0.001. (C) A subsequent cell count and mammosphere assay of cells extracted from excised tumors at experimental end point showed a significant increase in cell count and MFE after delivery of siFKBPL compared with delivery of siNT, and a significant decrease in cell count after delivery of pFKBPL compared with delivery of pEV; MFE was unable to be determined for pFKBPL due to insufficient cell numbers. pEV: Empty vector plasmid; pFKBPL: FK506-binding protein like – FKBPL gene, delivered using RALA; SD: Standard deviation; SE: Standard error; siFKBPL: FKBPL siRNA; siNT: Nontargeting siRNA. (C) Data taken from [23].