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Liver-targeted cyclosporine A-encapsulated poly (lactic-co-glycolic) acid nanoparticles inhibit hepatitis C virus replication.

Jyothi KR, Beloor J, Jo A, Nguyen MN, Choi TG, Kim JH, Akter S, Lee SK, Maeng CH, Baik HH, Kang I, Ha J, Kim SS - Int J Nanomedicine (2015)

Bottom Line: Furthermore, our delivery system exhibited high specificity to liver, thus contributing to the reduced immunosuppressive effect and toxicity profile of CsA.Finally, targeted nanoparticles were able to effectively inhibit viral replication in vitro and in an HCV mouse model.As a proof of principle, we herein show that our delivery system is able to negate the adverse effects of CsA and produce therapeutic effects in an HCV mouse model.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, School of Medicine, Kyung Hee University, Seoul, Republic of Korea.

ABSTRACT
Therapeutic options for hepatitis C virus (HCV) infection have been limited by drug resistance and adverse side effects. Targeting the host factor cyclophilin A (CypA), which is essential for HCV replication, offers a promising strategy for antiviral therapy. However, due to its immunosuppressive activity and severe side effects, clinical application of cyclosporine A (CsA) has been limited as an antiviral agent. To overcome these drawbacks, we have successfully developed a liver-specific, sustained drug delivery system by conjugating the liver-targeting peptide (LTP) to PEGylated CsA-encapsulated poly (lactic-co-glycolic) acid (PLGA) nanoparticles. Furthermore, our delivery system exhibited high specificity to liver, thus contributing to the reduced immunosuppressive effect and toxicity profile of CsA. Finally, targeted nanoparticles were able to effectively inhibit viral replication in vitro and in an HCV mouse model. As a proof of principle, we herein show that our delivery system is able to negate the adverse effects of CsA and produce therapeutic effects in an HCV mouse model.

No MeSH data available.


Related in: MedlinePlus

Antiviral effect of CsA-encapsulated poly (glycolic-co-lactic) acid nanoparticles in vitro.Notes: (A) HCV replicon cells were treated with 2.5 μg of CsA, CsANP, or CsANP-LTP for different time intervals, and luciferase activity was determined. The data are shown as the mean ± SD. (B) HCV NS5A expression levels were determined by Western blot analysis after HCV replicon cells were incubated with 2.5 μg of CsA, CsANP, or CsANP-LTP for different time intervals. All data are representative of three individual experiments. The sustained anti-HCV effect of CsA from targeted nanoparticles was determined by treatment-withdrawal experiments in HCV replicon cells. After 12 hours of treatment with 2.5 μg of CsA, CsANP, or CsANP-LTP, the cells were replaced with fresh culture medium followed by culturing for different time points. (C) Luciferase assay. The data are shown as the mean ± SD. **P<0.01 versus CsA treatment. (D) Western blot analysis of HCV NS5A expression levels. Actin was used as loading control. The density values of HCV NS5A relative to actin are expressed as mean ± SD of three independent experiments. *P<0.05, **P<0.01 versus CsA treatment.Abbreviations: Con, control group; CsA, cyclosporine A; CsANP, cyclosporine A nanoparticles; CsANP-LTP, CsANP conjugated with liver-targeting peptide; HCV, hepatitis C virus; RLU, relative light units; SD, standard deviation.
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f3-ijn-10-903: Antiviral effect of CsA-encapsulated poly (glycolic-co-lactic) acid nanoparticles in vitro.Notes: (A) HCV replicon cells were treated with 2.5 μg of CsA, CsANP, or CsANP-LTP for different time intervals, and luciferase activity was determined. The data are shown as the mean ± SD. (B) HCV NS5A expression levels were determined by Western blot analysis after HCV replicon cells were incubated with 2.5 μg of CsA, CsANP, or CsANP-LTP for different time intervals. All data are representative of three individual experiments. The sustained anti-HCV effect of CsA from targeted nanoparticles was determined by treatment-withdrawal experiments in HCV replicon cells. After 12 hours of treatment with 2.5 μg of CsA, CsANP, or CsANP-LTP, the cells were replaced with fresh culture medium followed by culturing for different time points. (C) Luciferase assay. The data are shown as the mean ± SD. **P<0.01 versus CsA treatment. (D) Western blot analysis of HCV NS5A expression levels. Actin was used as loading control. The density values of HCV NS5A relative to actin are expressed as mean ± SD of three independent experiments. *P<0.05, **P<0.01 versus CsA treatment.Abbreviations: Con, control group; CsA, cyclosporine A; CsANP, cyclosporine A nanoparticles; CsANP-LTP, CsANP conjugated with liver-targeting peptide; HCV, hepatitis C virus; RLU, relative light units; SD, standard deviation.

Mentions: To evaluate the anti-HCV activity of the formulated nanoparticles, HCV replicon cells expressing luciferase were used. The luciferase activity of HCV replicon cells directly correlates with the level of HCV RNA synthesis.34 CsA is known to effectively reduce HCV RNA levels by blocking the interaction of CypA with viral proteins.15,35 Both the luciferase assay (Figure 3A) and Western blot analysis (Figure 3B) showed that CsANP or CsANP-LTP can decrease HCV replication levels as efficiently as CsA for up to 72 hours. CsA treatment resulted in significantly higher levels of toxicity in HCV replicon cells, while CsANP or CsANP-LTP treatment did not affect the cell viability in time- and concentration-dependent experiments (Figure S1). Next, the prolonged antiviral effect of CsANP-LTP under sustained release condition was investigated via treatment-withdrawal experiments. After 12 hours’ incubation with CsA or nanoparticle formulations, the treatment was withdrawn and the cells were replaced with new media followed by culturing for the different time periods. Despite the withdrawal of treatment, CsANP-LTP showed significant sustained reduction in the replication of HCV replicon RNA and NS5A protein levels even up to 5 days (Figure 3C and D). CsANP-LTP decreased the NS5A levels more efficiently than free CsA or CsANP. This enhanced antiviral effect of CsANP-LTP was significant compared with free CsA. Cellular uptake of targeted PLGA nanoparticles showed that CsANP-LTP is specifically targeted into liver cells (Figure S2). Furthermore, we could not detect significant uptake of targeted PLGA nanoparticles in other cell lines such as A549 cells (human lung cancer) and mouse splenocytes (Figure S3 and S4). Moreover, attachment of a PEG moiety between the nanoparticle and LTP improved the anti-HCV effect of CsANP-LTP (Figure S5). In summary, these results suggest that CsANP-LTP effectively enhances the inhibition of HCV replication.


Liver-targeted cyclosporine A-encapsulated poly (lactic-co-glycolic) acid nanoparticles inhibit hepatitis C virus replication.

Jyothi KR, Beloor J, Jo A, Nguyen MN, Choi TG, Kim JH, Akter S, Lee SK, Maeng CH, Baik HH, Kang I, Ha J, Kim SS - Int J Nanomedicine (2015)

Antiviral effect of CsA-encapsulated poly (glycolic-co-lactic) acid nanoparticles in vitro.Notes: (A) HCV replicon cells were treated with 2.5 μg of CsA, CsANP, or CsANP-LTP for different time intervals, and luciferase activity was determined. The data are shown as the mean ± SD. (B) HCV NS5A expression levels were determined by Western blot analysis after HCV replicon cells were incubated with 2.5 μg of CsA, CsANP, or CsANP-LTP for different time intervals. All data are representative of three individual experiments. The sustained anti-HCV effect of CsA from targeted nanoparticles was determined by treatment-withdrawal experiments in HCV replicon cells. After 12 hours of treatment with 2.5 μg of CsA, CsANP, or CsANP-LTP, the cells were replaced with fresh culture medium followed by culturing for different time points. (C) Luciferase assay. The data are shown as the mean ± SD. **P<0.01 versus CsA treatment. (D) Western blot analysis of HCV NS5A expression levels. Actin was used as loading control. The density values of HCV NS5A relative to actin are expressed as mean ± SD of three independent experiments. *P<0.05, **P<0.01 versus CsA treatment.Abbreviations: Con, control group; CsA, cyclosporine A; CsANP, cyclosporine A nanoparticles; CsANP-LTP, CsANP conjugated with liver-targeting peptide; HCV, hepatitis C virus; RLU, relative light units; SD, standard deviation.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4321639&req=5

f3-ijn-10-903: Antiviral effect of CsA-encapsulated poly (glycolic-co-lactic) acid nanoparticles in vitro.Notes: (A) HCV replicon cells were treated with 2.5 μg of CsA, CsANP, or CsANP-LTP for different time intervals, and luciferase activity was determined. The data are shown as the mean ± SD. (B) HCV NS5A expression levels were determined by Western blot analysis after HCV replicon cells were incubated with 2.5 μg of CsA, CsANP, or CsANP-LTP for different time intervals. All data are representative of three individual experiments. The sustained anti-HCV effect of CsA from targeted nanoparticles was determined by treatment-withdrawal experiments in HCV replicon cells. After 12 hours of treatment with 2.5 μg of CsA, CsANP, or CsANP-LTP, the cells were replaced with fresh culture medium followed by culturing for different time points. (C) Luciferase assay. The data are shown as the mean ± SD. **P<0.01 versus CsA treatment. (D) Western blot analysis of HCV NS5A expression levels. Actin was used as loading control. The density values of HCV NS5A relative to actin are expressed as mean ± SD of three independent experiments. *P<0.05, **P<0.01 versus CsA treatment.Abbreviations: Con, control group; CsA, cyclosporine A; CsANP, cyclosporine A nanoparticles; CsANP-LTP, CsANP conjugated with liver-targeting peptide; HCV, hepatitis C virus; RLU, relative light units; SD, standard deviation.
Mentions: To evaluate the anti-HCV activity of the formulated nanoparticles, HCV replicon cells expressing luciferase were used. The luciferase activity of HCV replicon cells directly correlates with the level of HCV RNA synthesis.34 CsA is known to effectively reduce HCV RNA levels by blocking the interaction of CypA with viral proteins.15,35 Both the luciferase assay (Figure 3A) and Western blot analysis (Figure 3B) showed that CsANP or CsANP-LTP can decrease HCV replication levels as efficiently as CsA for up to 72 hours. CsA treatment resulted in significantly higher levels of toxicity in HCV replicon cells, while CsANP or CsANP-LTP treatment did not affect the cell viability in time- and concentration-dependent experiments (Figure S1). Next, the prolonged antiviral effect of CsANP-LTP under sustained release condition was investigated via treatment-withdrawal experiments. After 12 hours’ incubation with CsA or nanoparticle formulations, the treatment was withdrawn and the cells were replaced with new media followed by culturing for the different time periods. Despite the withdrawal of treatment, CsANP-LTP showed significant sustained reduction in the replication of HCV replicon RNA and NS5A protein levels even up to 5 days (Figure 3C and D). CsANP-LTP decreased the NS5A levels more efficiently than free CsA or CsANP. This enhanced antiviral effect of CsANP-LTP was significant compared with free CsA. Cellular uptake of targeted PLGA nanoparticles showed that CsANP-LTP is specifically targeted into liver cells (Figure S2). Furthermore, we could not detect significant uptake of targeted PLGA nanoparticles in other cell lines such as A549 cells (human lung cancer) and mouse splenocytes (Figure S3 and S4). Moreover, attachment of a PEG moiety between the nanoparticle and LTP improved the anti-HCV effect of CsANP-LTP (Figure S5). In summary, these results suggest that CsANP-LTP effectively enhances the inhibition of HCV replication.

Bottom Line: Furthermore, our delivery system exhibited high specificity to liver, thus contributing to the reduced immunosuppressive effect and toxicity profile of CsA.Finally, targeted nanoparticles were able to effectively inhibit viral replication in vitro and in an HCV mouse model.As a proof of principle, we herein show that our delivery system is able to negate the adverse effects of CsA and produce therapeutic effects in an HCV mouse model.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, School of Medicine, Kyung Hee University, Seoul, Republic of Korea.

ABSTRACT
Therapeutic options for hepatitis C virus (HCV) infection have been limited by drug resistance and adverse side effects. Targeting the host factor cyclophilin A (CypA), which is essential for HCV replication, offers a promising strategy for antiviral therapy. However, due to its immunosuppressive activity and severe side effects, clinical application of cyclosporine A (CsA) has been limited as an antiviral agent. To overcome these drawbacks, we have successfully developed a liver-specific, sustained drug delivery system by conjugating the liver-targeting peptide (LTP) to PEGylated CsA-encapsulated poly (lactic-co-glycolic) acid (PLGA) nanoparticles. Furthermore, our delivery system exhibited high specificity to liver, thus contributing to the reduced immunosuppressive effect and toxicity profile of CsA. Finally, targeted nanoparticles were able to effectively inhibit viral replication in vitro and in an HCV mouse model. As a proof of principle, we herein show that our delivery system is able to negate the adverse effects of CsA and produce therapeutic effects in an HCV mouse model.

No MeSH data available.


Related in: MedlinePlus