Enhanced apoptosis of ovarian cancer cells via nanocarrier-mediated codelivery of siRNA and doxorubicin

Abstract

A folate conjugated ternary copolymer, FA-PEG-PEI-PCL, of poly(ethylene glycol) (PEG), poly(ethylene imine) (PEI), and poly(ɛ-caprolactone) (PCL) was synthesized. The copolymer self-assembled into cationic micelles capable of co-delivering siRNA and the anticancer drug doxorubicin (DOX). This dual functional nanocarrier demonstrated low cytotoxicity and high performance in drug/siRNA delivery. Upon the codelivery of siRNA, targeting the Bcl-2 gene, and DOX, using the folate-targeted nanocarrier, DOX-induced apoptosis in the skov-3 cells overexpressing folate receptor was significantly enhanced through a mechanism of downregulating the antiapoptotic protein Bcl-2, while simultaneously upregulating the proapoptotic protein Bax. This work suggested that the combination of Bcl-2 siRNA and DOX therapies is feasible, based on our dual functional nanocarrier, which set up a good basis for a future in vivo test.

Keywords: apoptosis; chemotherapy; codelivery; gene silencing; tumor targeting.

Figure 1

Synthesis of folate-conjugated triblock copolymer FA–PEG–PEI–PCL. Abbreviations: FA–PEG–NHS, folate-poly(ethylene glycol)-N-hydroxysuccinimide; lPEI–PCL, linear poly(ethylene imine)-poly(ɛ-caprolactone); FA–PEG–PEI–PCL, folate-poly(ethylene glycol)-poly(ethylene imine)-poly(ɛ-caprolactone).

Figure 2

¹H NMR spectra (300 MHz) of (A) FA–PEG–COOH in D₂O and (B) FA–PEG–PEI–PCL in CDCl₃. Abbreviations: FA–PEG–COOH, folate-poly(ethylene glycol)-COOH; FA–PEG– PEI–PCL, folate–poly(ethylene glycol)–poly(ethylene imine)–poly(ɛ-caprolactone).

Figure 3

Preparation of DOX and siRNA coencapsulated nanocomplexes with folate receptor-targeting function. Abbreviations: DOX, doxorubicin; FA, folate; siRNA, small interference RNA; PEG–PEI–PCL, poly(ethylene glycol)–poly(ethylene imine)–poly(ɛ-caprolactone).

Figure 4

Particle sizes and zeta potentials of siRNA and DOX-loaded nanocomplexes (A) D-FPPP/Bcl-2 and (B) D-NPPP/Bcl-2 formed at different N/P ratios (n = 5), as determined by DLS measurements. Note: DOX loading contents: 4.56% in D-FPPP and 5.14% in D-NPPP. Abbreviations: DOX, doxorubicin; D-FPPP, DOX-loaded folate–poly(ethylene glycol)–poly(ethylene imine)–poly(ɛ-caprolactone) micelle; D-NPPP, DOX-loaded nontargeted poly(ethylene glycol)–poly(ethylene imine)–poly(ɛ-caprolactone) micelle; DLS, dynamic light scattering; D-FPPP/Bcl-2, Bcl-2 siRNA and DOX loaded folate–poly(ethylene glycol)–poly(ethylene imine)–poly(ɛ-caprolactone) micelle; D-NPPP/Bcl-2, Bcl-2 siRNA and DOX loaded nontargeted poly(ethylene glycol)– poly(ethylene imine)–poly(ɛ-caprolactone) micelle.

Figure 5

Electrophoretic mobility of siRNA in agarose gel after complexing with DOX-loaded FA–PEG–PEI–PCL micelle (D-FPPP) and DOX-loaded PEG–PEI–PCL micelle (D-NPPP) at various N/P ratios. Abbreviations: DOX, doxorubicin; siRNA, small interference RNA; D-FPPP, DOX loaded folate–poly(ethylene glycol)–poly(ethylene imine)–poly(ɛ-caprolactone) micelle; D-NPPP, DOX loaded nontargeted poly(ethylene glycol)–poly(ethylene imine)–poly(ɛ-caprolactone) micelle.

Figure 6

Scanning electron microscopy (SEM) images of the (A) DOX-loaded micelle D-NPPP and (B) DOX-SCR-loaded nanocomplex D-NPPP/SCR (N/P = 30). Abbreviations: DOX, doxorubicin; D-NPPP, DOX loaded nontargeted poly(ethylene glycol)–poly(ethylene imine)–poly(ɛ-caprolactone) micelle; D-NPPP/ SCR, scrambled siRNA and DOX loaded nontargeted poly(ethylene glycol)– poly(ethylene imine)–poly(ɛ-caprolactone) micelle.

Figure 7

Four upper panels: laser confocal microscopic images of cells incubated with D-FPPP/FITC (targeting), D-NPPP/FITC (nontargeting), and D-FPPP/FITC in the presence of FA (1 mg/L) (targeting + FA). Notes: Bottom panel: flow cytometric measurement of DOX and/or FITC positive cells. Nuclei: stained blue with Hoechest 33342 (Aldrich); Red florescence: DOX; Green fluorescence: SCR-FITC. Incubation time: 4 hours. Dose: 50 nM siRNA. N/P = 30. DOX loading contents: 4.56% in D-FPPP and 5.14% in D-NPPP. Q1: DOX florescent cells; Q2: DOX and FITC florescent cells; Q3: nonflorescent cells; Q4: FITC florescent cells. Abbreviations: DOX, doxorubicin; FA, folate; FITC, fluorescein isothiocyanate; D-FPPP/FITC, DOX and FITC labeled siRNA loaded folate–poly(ethylene glycol)– poly(ethylene imine)–poly(ɛ-caprolactone) micelle; D-NPPP/FITC, DOX and FITC labeled siRNA loaded nontargeted poly(ethylene glycol)–poly(ethylene imine)– poly(ɛ-caprolactone) micelle.

Figure 8

Efficacy of nanocomplexes in suppressing the Bcl-2 mRNA expression in skov-3 cells, as quantified by real-time PCR analysis (n = 3). Notes: For the nanocomplex pairs shown under the X-axis, the blank and grey bars show data for nanocomplexes delivering siRNA alone and nanocomplexes codelivering siRNA and DOX, respectively. Incubation time: 96 hours; N/P = 30; Dose: 25 nM siRNA per well. *P < 0.05, compared with D-FPPP/Bcl-2; ^#P < 0.05, compared with B-FPPP/Bcl-2 group; ^&P < 0.05, compared with D-FPPP/SCR; ^§P < 0.05, compared with D-NPPP/SCR. Abbreviations: DOX, doxorubicin; D-PPP, DOX loaded poly(ethylene glycol)– poly(ethylene imine)–poly(ɛ-caprolactone) micelle; B-PPP, blank poly(ethylene glycol)–poly(ethylene imine)–poly(ɛ-caprolactone) micelle; FPPP/Bcl-2, Bcl-2 siRNA loaded folate–poly(ethylene glycol)–poly(ethylene imine)–poly(ɛ-caprolactone) micelle; NPPP/Bcl-2, Bcl-2 siRNA loaded nontargeted poly(ethylene glycol)– poly(ethylene imine)–poly(ɛ-caprolactone) micelle; FPPP/SCR, scrambled siRNA loaded folate–poly(ethylene glycol)–poly(ethylene imine)–poly(ɛ–caprolactone) micelle; NPPP/SCR, scrambled siRNA loaded nontargeted poly(ethylene glycol)– poly(ethylene imine)–poly(ɛ-caprolactone) micelle.

Figure 9

Protein expression levels of (A) Bcl-2 gene and (B) Bax gene determined by ELISA (n = 6). Notes: Incubation time: 96 hours; Dose: 50 nM siRNA per well; N/P = 30. Statistics in A: *P < 0.05 compared with D-FPPP/Bcl-2; ^#P < 0.05 compared with B-FPPP/ Bcl-2; ^&P < 0.05 compared with D-FPPP/SCR; ^§P < 0.05 compared with D-NPPP/ SCR. Statistics in B: *P < 0.05 compared with D-FPPP/Bcl-2; ^&P < 0.05 compared with D-FPPP/SCR; ^§P < 0.05 compared with D-NPPP/SCR. Abbreviations: DOX, doxorubicin; D-PPP, DOX loaded poly(ethylene glycol)– poly(ethylene imine)–poly(ɛ-caprolactone) micelle; B-PPP, blank–poly(ethylene glycol)–poly(ethylene imine)–poly(ɛ-caprolactone) micelle; FPPP/Bcl-2, Bcl-2 siRNA loaded folate–poly(ethylene glycol)–poly(ethylene imine)–poly(ɛ-caprolactone) micelle; NPPP/Bcl-2, Bcl-2 siRNA loaded nontargeted poly(ethylene glycol)– poly(ethylene imine)–poly(ɛ-caprolactone) micelle; FPPP/SCR, scrambled siRNA loaded folate–poly(ethylene glycol)–poly(ethylene imine)–poly(ɛ–caprolactone) micelle; NPPP/SCR, scrambled siRNA loaded nontargeted poly(ethylene glycol)– poly(ethylene imine)–poly(ɛ-caprolactone) micelle.

Figure 10

Detection of apoptotic skov-3 cells by TUNEL assay. (A) Percentage of apoptotic cells summarized from data of TUNEL assay (n = 6). (B) Images show cells incubated with: (a) D-FPPP/Bcl-2; (b) D-NPPP/Bcl-2; (c) D-FPPP/SCR; (d) D-NPPP/SCR; (e) B-FPPP/Bcl-2; (f) B-NPPP/Bcl-2; (g) B-FPPP/SCR; (h) B-NPPP/ SCR; (i) normal culture medium (control group). Notes: Incubation time: 96 hours; N/P = 30; Dose: 50 nM siRNA per well. *P < 0.05 compared with D-FPPP/Bcl-2; ^#P < 0.05 compared with B-FPPP/Bcl-2; ^&P < 0.05 compared with D-FPPP/SCR. Abbreviations: DOX, doxorubicin; siRNA, small interference RNA; D-PPP, DOX loaded poly(ethylene glycol)–poly(ethylene imine)–poly(ɛ-caprolactone) micelle; B-PPP, blank poly(ethylene glycol)–poly(ethylene imine)–poly(ɛ-caprolactone) micelle; FPPP/Bcl-2, Bcl-2 siRNA loaded folate–poly(ethylene glycol)–poly(ethylene imine)–poly(ɛ-caprolactone) micelle; NPPP/Bcl-2, Bcl-2 siRNA loaded nontargeted poly(ethylene glycol)–poly(ethylene imine)–poly(ɛ-caprolactone) micelle; FPPP/ SCR, scrambled siRNA loaded folate–poly(ethylene glycol)–poly(ethylene imine)– poly(ɛ–caprolactone) micelle; NPPP/SCR, scrambled siRNA loaded nontargeted poly(ethylene glycol)–poly(ethylene imine)–poly(ɛ-caprolactone) micelle.

Figure 11

Activation of caspase-3 determined by Caspase-3 Colorimetric Assay Kit (n = 6). Notes: Incubation time: 96 hours; N/P = 30; Dose: 50 nM siRNA per well. *P < 0.05 compared with D-FPPP/Bcl-2; ^&P < 0.05 compared with D-FPPP/SCR; ^§P < 0.05 compared with D-NPPP/SCR. Abbreviations: DOX, doxorubicin; siRNA, small interference RNA; D-PPP, DOX loaded poly(ethylene glycol)-poly(ethylene imine)-poly(ɛ-caprolactone) micelle; B-PPP, blank poly(ethylene glycol)-poly(ethylene imine)-poly(ɛ-caprolactone) micelle; FPPP/Bcl-2, Bcl-2 siRNA loaded folate-poly(ethylene glycol)-poly(ethylene imine)-poly(ɛ-caprolactone) micelle; NPPP/Bcl-2, Bcl-2 siRNA loaded nontargeted poly(ethylene glycol)-poly(ethylene imine)-poly(ɛ-caprolactone) micelle; FPPP/ SCR, scrambled siRNA loaded folate-poly(ethylene glycol)-poly(ethylene imine)- poly(ɛ-caprolactone) micelle; NPPP/SCR, scrambled siRNA loaded nontargeted poly(ethylene glycol)-poly(ethylene imine)-poly(ɛ-caprolactone) micelle.

Figure 12

(A) Cell viability in the presence of polymers (PEI or blank copolymer micelles) and their SCR-complexed nanocomplexes at various polymer concentrations. (B) Viability of cells in the presence of nanocomplexes at various DOX concentrations. Notes: Incubation time: 96 hours; N/P = 30 for all siRNA nanocomplexes; data are shown as mean ± SD (n = 3). Abbreviations: DOX, doxorubicin; siRNA, small interference RNA; B-FPPP, blank folate-poly( ethylene glycol)-poly(ethylene imine)-poly(ɛ-caprolactone) micelle; B-NPPP, blank-nontargeted poly(ethylene glycol)-poly(ethylene imine)-poly(ɛ-caprolactone) micelle; B-FPPP/SCR, scrambled siRNA-blank-folate-poly(ethylene glycol)-poly(ethylene imine)- poly(ɛ-caprolactone) micelle; B-NPPP/SCR, scrambled siRNA-blank-nontargeting-poly( ethylene glycol)-poly(ethylene imine)-poly(ɛ-caprolactone) micelle; PEI25k, scrambled siRNA complexed poly(ethylene imine) 25kDa.