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Targeting cyclin B1 through peptide-based delivery of siRNA prevents tumour growth.

Crombez L, Morris MC, Dufort S, Aldrian-Herrada G, Nguyen Q, Mc Master G, Coll JL, Heitz F, Divita G - Nucleic Acids Res. (2009)

Bottom Line: In this study, we report a novel peptide-based approach, MPG-8 an improved variant of the amphipathic peptide carrier MPG, that forms nanoparticles with siRNA and promotes their efficient delivery into primary cell lines and in vivo upon intra-tumoral injection.We have validated the therapeutic potential of this strategy for cancer treatment by targeting cyclin B1 in mouse tumour models, and demonstrate that tumour growth is compromised.The robustness of the biological response achieved through this approach, infers that MPG 8-based technology holds a strong promise for therapeutic administration of siRNA.

View Article: PubMed Central - PubMed

Affiliation: Centre de Recherches de Biochimie Macromoléculaire, Department of Molecular Biophysics and Therapeutic, UMR-5237 CNRS-UM2-UM1, 1919 Route de Mende, 34293 Montpellier, France.

ABSTRACT
The development of short interfering RNA (siRNA), has provided great hope for therapeutic targeting of specific genes responsible for pathological disorders. However, the poor cellular uptake and bioavailability of siRNA remain a major obstacle to their clinical development and most strategies that propose to improve siRNA delivery remain limited for in vivo applications. In this study, we report a novel peptide-based approach, MPG-8 an improved variant of the amphipathic peptide carrier MPG, that forms nanoparticles with siRNA and promotes their efficient delivery into primary cell lines and in vivo upon intra-tumoral injection. Moreover, we show that functionalization of this carrier with cholesterol significantly improves tissue distribution and stability of siRNA in vivo, thereby enhancing the efficiency of this technology for systemic administration following intravenous injection without triggering any non-specific inflammatory response. We have validated the therapeutic potential of this strategy for cancer treatment by targeting cyclin B1 in mouse tumour models, and demonstrate that tumour growth is compromised. The robustness of the biological response achieved through this approach, infers that MPG 8-based technology holds a strong promise for therapeutic administration of siRNA.

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Related in: MedlinePlus

Systemic administration of MPG-8/MPG-8chol/cyclin B1 siRNA blocks tumour growth in vivo. (A) Inhibition of PC3 tumour growth upon intravenous injection. Swiss nude nice (a cohort of N = 6 animals) were injected subcutaneously with 106 PC3 cells and tumour analysis was performed as described in Figure 4A. Animals were treated by intravenous tail vein injection, every 3 days, with a solution of 0.1 ml of either PBS (black), free Cyc-B1 siRNA (100 µg:blue), Cyc-B1 siRNA (10 µg) complexed with MPG-8 (purple), control siRNA Cyc-B3 (100 µg: green) or Cyc-B1 siRNA (5 µg: orange and 10 µg: red) complexed with MPG-8/chol-MPG-8 at a 1/20 molar ratio. Curves show the mean value of tumour size in a group of six animals. After 48 days, PC3 tumours were removed, and Cyclin B1 protein levels were evaluated by western blotting (insert) in control (lane a), 5 µg siRNA (lane b) and 10 µg siRNA (lane c) complexed with MPG-8/chol-MPG-8 at a 1/20 molar ratio. *P < 0.05 versus saline control and **P < 0.01 versus saline control. (B) Inhibition of SK-BR3 HER2 tumour growth upon intravenous injection. Swiss nude mice (a cohort of N = 10 animals) were injected subcutaneously with 106 SK-BR3 HER2 cells. Ten days after tumour implant, when tumour size reached 100 mm3, animals were treated by intravenous tail vein injection, every 3 days from D10 to D30, then every 10 days, with a solution of 0.1 ml of either PBS (green), Cyc-B1 siRNA (10 µg) complexed with MPG-8 (blue) and Cyc-B3 (100 µg: orange) Cyc-B1 siRNA (10 µg: red) complexed with MPG-8/Chol-MPG-8. Control mice treated by intravenous tail vein injection of (10 µg) Cyc-B1 siRNA complexed with MPG-8/Chol-MPG-8 (black) *P < 0.05 versus saline control and **P < 0.01 versus saline control. (C) Expression of IFN response genes: expression of INF-β and IL8 relative to GAPDH was analysed by quantitative RT–PCR. HeLa, MCF7 and SCK3-Her2 cells were treated with MPG-8 carrier alone (white) (20 µM), 20 nM of Cyc-B1 siRNA associated with MPG-8 (black) or MPG-8/Chol-MPG8 (light grey) particles. Poly(I:C) was used as a positive control to induce interferon response (grey). (D) MPG-8 formulation does not induce interferon response in vivo. MPG-8/siRNA, MPG-8/MPG-8-Chol/siRNA (0.5 mg/kg), and MPG-8 carrier were intravenously injected into mice and IL-6 (grey), TNF-α (white) and IFN-γ (black) induction were measured in the serum 6 h after injection by sandwich ELISA and expressed as pg/ml. (P versus saline controls *<0.05, **<0.01) each value is the mean of three separate experiments.
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Figure 7: Systemic administration of MPG-8/MPG-8chol/cyclin B1 siRNA blocks tumour growth in vivo. (A) Inhibition of PC3 tumour growth upon intravenous injection. Swiss nude nice (a cohort of N = 6 animals) were injected subcutaneously with 106 PC3 cells and tumour analysis was performed as described in Figure 4A. Animals were treated by intravenous tail vein injection, every 3 days, with a solution of 0.1 ml of either PBS (black), free Cyc-B1 siRNA (100 µg:blue), Cyc-B1 siRNA (10 µg) complexed with MPG-8 (purple), control siRNA Cyc-B3 (100 µg: green) or Cyc-B1 siRNA (5 µg: orange and 10 µg: red) complexed with MPG-8/chol-MPG-8 at a 1/20 molar ratio. Curves show the mean value of tumour size in a group of six animals. After 48 days, PC3 tumours were removed, and Cyclin B1 protein levels were evaluated by western blotting (insert) in control (lane a), 5 µg siRNA (lane b) and 10 µg siRNA (lane c) complexed with MPG-8/chol-MPG-8 at a 1/20 molar ratio. *P < 0.05 versus saline control and **P < 0.01 versus saline control. (B) Inhibition of SK-BR3 HER2 tumour growth upon intravenous injection. Swiss nude mice (a cohort of N = 10 animals) were injected subcutaneously with 106 SK-BR3 HER2 cells. Ten days after tumour implant, when tumour size reached 100 mm3, animals were treated by intravenous tail vein injection, every 3 days from D10 to D30, then every 10 days, with a solution of 0.1 ml of either PBS (green), Cyc-B1 siRNA (10 µg) complexed with MPG-8 (blue) and Cyc-B3 (100 µg: orange) Cyc-B1 siRNA (10 µg: red) complexed with MPG-8/Chol-MPG-8. Control mice treated by intravenous tail vein injection of (10 µg) Cyc-B1 siRNA complexed with MPG-8/Chol-MPG-8 (black) *P < 0.05 versus saline control and **P < 0.01 versus saline control. (C) Expression of IFN response genes: expression of INF-β and IL8 relative to GAPDH was analysed by quantitative RT–PCR. HeLa, MCF7 and SCK3-Her2 cells were treated with MPG-8 carrier alone (white) (20 µM), 20 nM of Cyc-B1 siRNA associated with MPG-8 (black) or MPG-8/Chol-MPG8 (light grey) particles. Poly(I:C) was used as a positive control to induce interferon response (grey). (D) MPG-8 formulation does not induce interferon response in vivo. MPG-8/siRNA, MPG-8/MPG-8-Chol/siRNA (0.5 mg/kg), and MPG-8 carrier were intravenously injected into mice and IL-6 (grey), TNF-α (white) and IFN-γ (black) induction were measured in the serum 6 h after injection by sandwich ELISA and expressed as pg/ml. (P versus saline controls *<0.05, **<0.01) each value is the mean of three separate experiments.

Mentions: The stability of drug-carrier formulations in vivo and in the blood circulation is a major issue for systemic administration of therapeutics. Despite its demonstrated potency in cellulo, intravenous injection of MPG-8/siRNA formulations (0.5 mg/kg) only produces an anti-tumoural response of about 12% (Figures 4A and 7A). Cholesterol modification of siRNA has been reported to enhance their potency and stability in vivo by maintaining the siRNA in the circulation for longer periods of time (30,31). Hence, in order to improve the bioavailability and stability of the MPG-8/siRNA particles, thereby rendering them more suitable for systemic administration, the surface layer of MPG-8/siRNA particles was functionalized with a cholesterol-moiety at the N- terminus of MPG-8 (Chol-MPG-8), through activation of the N-terminal beta alanine amino group. Cholesterol-functionalized MPG-8/siRNA particles were obtained stepwise by complexing siRNA molecules with MPG-8 at a molar ratio of 20/1, followed by coating of particles with a second layer of Chol-MPG-8. The optimal ratio of Chol-MPG-8 required was determined experimentally, by assessing the ability of the different particles containing between 5 and 50% Chol-MPG-8 to deliver Cyc-B1 siRNA. Below a ratio of 15% Chol-MPG-8/MPG-8, no significant difference in the efficiency of cyclin B1 silencing, with 85–92% knockdown of protein or mRNA levels (Figure 5A), nor of the associated G2 cell arrest (Figure 5B) were observed. In these conditions, an IC50 of 1.1 ± 0.2 nM was obtained for HS68 cells, similar to the value obtained for non-functionalized MPG-8 particles. Functionalized-MPG-8/siRNA particles with 15% of Chol-MPG-8 measure 180 ± 45 nm in diameter and are characterized by a zeta potential of 14 ± 2 v. These results indicate that functionalization of the MPG-8/siRNA particles with 15% of cholesterol alters neither their physicochemical parameters nor their efficiency to deliver siRNA. We next investigated to what extent cholesterol functionalization of MPG-8 influenced the in vivo bio-distribution of MPG-8/siRNA particles. Mice were injected intravenously with 10 µg of Alexa700 labelled-siRNA either naked or complexed with MPG-8 or MPG-8Chol/MPG-8. Kinetics of siRNA biodistribution were measured during the first 15 min (Figure 6A), then every hour for 5 h (Figure 6B) and fluorescence was quantified in the different organs 24 h after injection (Figure 6C). Naked siRNA was reported to be rapidly degraded, with a half-life of a few hours in vivo. Therefore, as expected, the control experiment performed with naked siRNA revealed that it rapidly accumulated in the bladder and in the liver over the first hours and was barely distributed throughout the rest of the body (Figure 6A, panel 1). In contrast, MPG-8/siRNA (Figure 6A, panel 2) and MPG-8/MPG-8-Chol/siRNA (Figure 6A, panel 3) formulations favoured the rapid distribution of siRNA throughout the body within the first 15 min following injection, more prominently for the cholesterol functionalized-particles (Figure 6A, panel 2). In both cases, MPG-8/siRNA (Figure 6B, top panel) and MPG-8/MPG-8-Chol/siRNA (Figure 6B, bottom panel) were found to access all tissues and siRNA distribution was optimal at 5 h, accumulating mainly in the lung, liver, plasma, skin and kidney, adrenal gland and spleen. Although we cannot exclude that fluorescence is due to degradation of the siRNA, the fact that siRNA remains in the plasma and in most of the tissues 24 h after injection (Figure 6C), and also to a certain extent in the brain, ovary and uterus, confirms the high stability of MPG-8/siRNA and MPG-8/MPG-8chol/siRNA particles. No major differences were obtained between MPG-8/siRNA and MPG-8/MPG-8chol/siRNA particles in terms of tissue targeting. However, cholesterol-functionalization of the particles significantly increased the distribution kinetics of siRNA within the first 15 min and maintained a higher level of siRNA in the plasma even after 24 h, in comparison to MPG-8/siRNA particles, suggesting that it may limit siRNA clearance, thereby further favouring delivery of siRNA into the tumour.Figure 5.


Targeting cyclin B1 through peptide-based delivery of siRNA prevents tumour growth.

Crombez L, Morris MC, Dufort S, Aldrian-Herrada G, Nguyen Q, Mc Master G, Coll JL, Heitz F, Divita G - Nucleic Acids Res. (2009)

Systemic administration of MPG-8/MPG-8chol/cyclin B1 siRNA blocks tumour growth in vivo. (A) Inhibition of PC3 tumour growth upon intravenous injection. Swiss nude nice (a cohort of N = 6 animals) were injected subcutaneously with 106 PC3 cells and tumour analysis was performed as described in Figure 4A. Animals were treated by intravenous tail vein injection, every 3 days, with a solution of 0.1 ml of either PBS (black), free Cyc-B1 siRNA (100 µg:blue), Cyc-B1 siRNA (10 µg) complexed with MPG-8 (purple), control siRNA Cyc-B3 (100 µg: green) or Cyc-B1 siRNA (5 µg: orange and 10 µg: red) complexed with MPG-8/chol-MPG-8 at a 1/20 molar ratio. Curves show the mean value of tumour size in a group of six animals. After 48 days, PC3 tumours were removed, and Cyclin B1 protein levels were evaluated by western blotting (insert) in control (lane a), 5 µg siRNA (lane b) and 10 µg siRNA (lane c) complexed with MPG-8/chol-MPG-8 at a 1/20 molar ratio. *P < 0.05 versus saline control and **P < 0.01 versus saline control. (B) Inhibition of SK-BR3 HER2 tumour growth upon intravenous injection. Swiss nude mice (a cohort of N = 10 animals) were injected subcutaneously with 106 SK-BR3 HER2 cells. Ten days after tumour implant, when tumour size reached 100 mm3, animals were treated by intravenous tail vein injection, every 3 days from D10 to D30, then every 10 days, with a solution of 0.1 ml of either PBS (green), Cyc-B1 siRNA (10 µg) complexed with MPG-8 (blue) and Cyc-B3 (100 µg: orange) Cyc-B1 siRNA (10 µg: red) complexed with MPG-8/Chol-MPG-8. Control mice treated by intravenous tail vein injection of (10 µg) Cyc-B1 siRNA complexed with MPG-8/Chol-MPG-8 (black) *P < 0.05 versus saline control and **P < 0.01 versus saline control. (C) Expression of IFN response genes: expression of INF-β and IL8 relative to GAPDH was analysed by quantitative RT–PCR. HeLa, MCF7 and SCK3-Her2 cells were treated with MPG-8 carrier alone (white) (20 µM), 20 nM of Cyc-B1 siRNA associated with MPG-8 (black) or MPG-8/Chol-MPG8 (light grey) particles. Poly(I:C) was used as a positive control to induce interferon response (grey). (D) MPG-8 formulation does not induce interferon response in vivo. MPG-8/siRNA, MPG-8/MPG-8-Chol/siRNA (0.5 mg/kg), and MPG-8 carrier were intravenously injected into mice and IL-6 (grey), TNF-α (white) and IFN-γ (black) induction were measured in the serum 6 h after injection by sandwich ELISA and expressed as pg/ml. (P versus saline controls *<0.05, **<0.01) each value is the mean of three separate experiments.
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Figure 7: Systemic administration of MPG-8/MPG-8chol/cyclin B1 siRNA blocks tumour growth in vivo. (A) Inhibition of PC3 tumour growth upon intravenous injection. Swiss nude nice (a cohort of N = 6 animals) were injected subcutaneously with 106 PC3 cells and tumour analysis was performed as described in Figure 4A. Animals were treated by intravenous tail vein injection, every 3 days, with a solution of 0.1 ml of either PBS (black), free Cyc-B1 siRNA (100 µg:blue), Cyc-B1 siRNA (10 µg) complexed with MPG-8 (purple), control siRNA Cyc-B3 (100 µg: green) or Cyc-B1 siRNA (5 µg: orange and 10 µg: red) complexed with MPG-8/chol-MPG-8 at a 1/20 molar ratio. Curves show the mean value of tumour size in a group of six animals. After 48 days, PC3 tumours were removed, and Cyclin B1 protein levels were evaluated by western blotting (insert) in control (lane a), 5 µg siRNA (lane b) and 10 µg siRNA (lane c) complexed with MPG-8/chol-MPG-8 at a 1/20 molar ratio. *P < 0.05 versus saline control and **P < 0.01 versus saline control. (B) Inhibition of SK-BR3 HER2 tumour growth upon intravenous injection. Swiss nude mice (a cohort of N = 10 animals) were injected subcutaneously with 106 SK-BR3 HER2 cells. Ten days after tumour implant, when tumour size reached 100 mm3, animals were treated by intravenous tail vein injection, every 3 days from D10 to D30, then every 10 days, with a solution of 0.1 ml of either PBS (green), Cyc-B1 siRNA (10 µg) complexed with MPG-8 (blue) and Cyc-B3 (100 µg: orange) Cyc-B1 siRNA (10 µg: red) complexed with MPG-8/Chol-MPG-8. Control mice treated by intravenous tail vein injection of (10 µg) Cyc-B1 siRNA complexed with MPG-8/Chol-MPG-8 (black) *P < 0.05 versus saline control and **P < 0.01 versus saline control. (C) Expression of IFN response genes: expression of INF-β and IL8 relative to GAPDH was analysed by quantitative RT–PCR. HeLa, MCF7 and SCK3-Her2 cells were treated with MPG-8 carrier alone (white) (20 µM), 20 nM of Cyc-B1 siRNA associated with MPG-8 (black) or MPG-8/Chol-MPG8 (light grey) particles. Poly(I:C) was used as a positive control to induce interferon response (grey). (D) MPG-8 formulation does not induce interferon response in vivo. MPG-8/siRNA, MPG-8/MPG-8-Chol/siRNA (0.5 mg/kg), and MPG-8 carrier were intravenously injected into mice and IL-6 (grey), TNF-α (white) and IFN-γ (black) induction were measured in the serum 6 h after injection by sandwich ELISA and expressed as pg/ml. (P versus saline controls *<0.05, **<0.01) each value is the mean of three separate experiments.
Mentions: The stability of drug-carrier formulations in vivo and in the blood circulation is a major issue for systemic administration of therapeutics. Despite its demonstrated potency in cellulo, intravenous injection of MPG-8/siRNA formulations (0.5 mg/kg) only produces an anti-tumoural response of about 12% (Figures 4A and 7A). Cholesterol modification of siRNA has been reported to enhance their potency and stability in vivo by maintaining the siRNA in the circulation for longer periods of time (30,31). Hence, in order to improve the bioavailability and stability of the MPG-8/siRNA particles, thereby rendering them more suitable for systemic administration, the surface layer of MPG-8/siRNA particles was functionalized with a cholesterol-moiety at the N- terminus of MPG-8 (Chol-MPG-8), through activation of the N-terminal beta alanine amino group. Cholesterol-functionalized MPG-8/siRNA particles were obtained stepwise by complexing siRNA molecules with MPG-8 at a molar ratio of 20/1, followed by coating of particles with a second layer of Chol-MPG-8. The optimal ratio of Chol-MPG-8 required was determined experimentally, by assessing the ability of the different particles containing between 5 and 50% Chol-MPG-8 to deliver Cyc-B1 siRNA. Below a ratio of 15% Chol-MPG-8/MPG-8, no significant difference in the efficiency of cyclin B1 silencing, with 85–92% knockdown of protein or mRNA levels (Figure 5A), nor of the associated G2 cell arrest (Figure 5B) were observed. In these conditions, an IC50 of 1.1 ± 0.2 nM was obtained for HS68 cells, similar to the value obtained for non-functionalized MPG-8 particles. Functionalized-MPG-8/siRNA particles with 15% of Chol-MPG-8 measure 180 ± 45 nm in diameter and are characterized by a zeta potential of 14 ± 2 v. These results indicate that functionalization of the MPG-8/siRNA particles with 15% of cholesterol alters neither their physicochemical parameters nor their efficiency to deliver siRNA. We next investigated to what extent cholesterol functionalization of MPG-8 influenced the in vivo bio-distribution of MPG-8/siRNA particles. Mice were injected intravenously with 10 µg of Alexa700 labelled-siRNA either naked or complexed with MPG-8 or MPG-8Chol/MPG-8. Kinetics of siRNA biodistribution were measured during the first 15 min (Figure 6A), then every hour for 5 h (Figure 6B) and fluorescence was quantified in the different organs 24 h after injection (Figure 6C). Naked siRNA was reported to be rapidly degraded, with a half-life of a few hours in vivo. Therefore, as expected, the control experiment performed with naked siRNA revealed that it rapidly accumulated in the bladder and in the liver over the first hours and was barely distributed throughout the rest of the body (Figure 6A, panel 1). In contrast, MPG-8/siRNA (Figure 6A, panel 2) and MPG-8/MPG-8-Chol/siRNA (Figure 6A, panel 3) formulations favoured the rapid distribution of siRNA throughout the body within the first 15 min following injection, more prominently for the cholesterol functionalized-particles (Figure 6A, panel 2). In both cases, MPG-8/siRNA (Figure 6B, top panel) and MPG-8/MPG-8-Chol/siRNA (Figure 6B, bottom panel) were found to access all tissues and siRNA distribution was optimal at 5 h, accumulating mainly in the lung, liver, plasma, skin and kidney, adrenal gland and spleen. Although we cannot exclude that fluorescence is due to degradation of the siRNA, the fact that siRNA remains in the plasma and in most of the tissues 24 h after injection (Figure 6C), and also to a certain extent in the brain, ovary and uterus, confirms the high stability of MPG-8/siRNA and MPG-8/MPG-8chol/siRNA particles. No major differences were obtained between MPG-8/siRNA and MPG-8/MPG-8chol/siRNA particles in terms of tissue targeting. However, cholesterol-functionalization of the particles significantly increased the distribution kinetics of siRNA within the first 15 min and maintained a higher level of siRNA in the plasma even after 24 h, in comparison to MPG-8/siRNA particles, suggesting that it may limit siRNA clearance, thereby further favouring delivery of siRNA into the tumour.Figure 5.

Bottom Line: In this study, we report a novel peptide-based approach, MPG-8 an improved variant of the amphipathic peptide carrier MPG, that forms nanoparticles with siRNA and promotes their efficient delivery into primary cell lines and in vivo upon intra-tumoral injection.We have validated the therapeutic potential of this strategy for cancer treatment by targeting cyclin B1 in mouse tumour models, and demonstrate that tumour growth is compromised.The robustness of the biological response achieved through this approach, infers that MPG 8-based technology holds a strong promise for therapeutic administration of siRNA.

View Article: PubMed Central - PubMed

Affiliation: Centre de Recherches de Biochimie Macromoléculaire, Department of Molecular Biophysics and Therapeutic, UMR-5237 CNRS-UM2-UM1, 1919 Route de Mende, 34293 Montpellier, France.

ABSTRACT
The development of short interfering RNA (siRNA), has provided great hope for therapeutic targeting of specific genes responsible for pathological disorders. However, the poor cellular uptake and bioavailability of siRNA remain a major obstacle to their clinical development and most strategies that propose to improve siRNA delivery remain limited for in vivo applications. In this study, we report a novel peptide-based approach, MPG-8 an improved variant of the amphipathic peptide carrier MPG, that forms nanoparticles with siRNA and promotes their efficient delivery into primary cell lines and in vivo upon intra-tumoral injection. Moreover, we show that functionalization of this carrier with cholesterol significantly improves tissue distribution and stability of siRNA in vivo, thereby enhancing the efficiency of this technology for systemic administration following intravenous injection without triggering any non-specific inflammatory response. We have validated the therapeutic potential of this strategy for cancer treatment by targeting cyclin B1 in mouse tumour models, and demonstrate that tumour growth is compromised. The robustness of the biological response achieved through this approach, infers that MPG 8-based technology holds a strong promise for therapeutic administration of siRNA.

Show MeSH
Related in: MedlinePlus