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Hybrid Nanomaterial Complexes for Advanced Phage-guided Gene Delivery.

Yata T, Lee KY, Dharakul T, Songsivilai S, Bismarck A, Mintz PJ, Hajitou A - Mol Ther Nucleic Acids (2014)

Bottom Line: We demonstrate that the phage complex with cationic polymers generates positively charged phage and large aggregates that show enhanced cell surface attachment, buffering capacity, and improved transgene expression while retaining cell type specificity.Moreover, phage/polymer complexes carrying a therapeutic gene achieve greater cancer cell killing than phage alone.This new class of hybrid nanomaterial platform can advance targeted gene delivery applications by bacteriophage.

View Article: PubMed Central - PubMed

Affiliation: Phage Therapy Group, Department of Medicine, Imperial College London, London, UK.

ABSTRACT
Developing nanomaterials that are effective, safe, and selective for gene transfer applications is challenging. Bacteriophages (phage), viruses that infect bacteria only, have shown promise for targeted gene transfer applications. Unfortunately, limited progress has been achieved in improving their potential to overcome mammalian cellular barriers. We hypothesized that chemical modification of the bacteriophage capsid could be applied to improve targeted gene delivery by phage vectors into mammalian cells. Here, we introduce a novel hybrid system consisting of two classes of nanomaterial systems, cationic polymers and M13 bacteriophage virus particles genetically engineered to display a tumor-targeting ligand and carry a transgene cassette. We demonstrate that the phage complex with cationic polymers generates positively charged phage and large aggregates that show enhanced cell surface attachment, buffering capacity, and improved transgene expression while retaining cell type specificity. Moreover, phage/polymer complexes carrying a therapeutic gene achieve greater cancer cell killing than phage alone. This new class of hybrid nanomaterial platform can advance targeted gene delivery applications by bacteriophage.

No MeSH data available.


Related in: MedlinePlus

Characterization of tumor cell transduction by the hybrid phage/polymer. (a) Optimization of polymer types and concentrations. M21 and 9L cells were treated with RGD4C-phages carrying the Luc transgene premixed with increasing concentrations of poly-d-lysine (PDL) and DEAE.DEX and Luc gene expression was measured using the luciferase assay at day 3 post-transduction. (b) Cytotoxicity of the RGD4C-phage complexed with increasing concentrations of cationic polymers in both M21 and 9L cell lines. Cell viability was measured using the CellTiter-Glo cell viability assay, at 48-hour-post transduction. The cell viability rate (%) was calculated as percentage of control ([A]test/[A]control × 100) (n = 3). (c) Kinetics of Luc gene expression following transduction of M21 and 9L cells with the RGD4C-phage premixed with PDL or DEAE.DEX (RGD4C-PDL and RGD4C-DEAE.DEX, respectively), RGD4C-phage alone (RGD4C), or non-targeted phage (NT). The luciferase assay was performed daily over a time course of 5 days after vector transduction.
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fig2: Characterization of tumor cell transduction by the hybrid phage/polymer. (a) Optimization of polymer types and concentrations. M21 and 9L cells were treated with RGD4C-phages carrying the Luc transgene premixed with increasing concentrations of poly-d-lysine (PDL) and DEAE.DEX and Luc gene expression was measured using the luciferase assay at day 3 post-transduction. (b) Cytotoxicity of the RGD4C-phage complexed with increasing concentrations of cationic polymers in both M21 and 9L cell lines. Cell viability was measured using the CellTiter-Glo cell viability assay, at 48-hour-post transduction. The cell viability rate (%) was calculated as percentage of control ([A]test/[A]control × 100) (n = 3). (c) Kinetics of Luc gene expression following transduction of M21 and 9L cells with the RGD4C-phage premixed with PDL or DEAE.DEX (RGD4C-PDL and RGD4C-DEAE.DEX, respectively), RGD4C-phage alone (RGD4C), or non-targeted phage (NT). The luciferase assay was performed daily over a time course of 5 days after vector transduction.

Mentions: We sought to assess whether the efficiency of gene delivery by the RGD4C-phage to eukaryotic cells can be improved if phage viral particles are integrated with cationic polymers. We therefore studied the efficacy with which RGD4C-phage/polymer complexes transduce human M21 melanoma cells, which are known to express high levels of αv integrin receptors for the RGD4C ligand.17,21 To rule out the possibility that the observed effects are not cell or species specific, we also assessed the efficacy on the rat 9L glioblastoma cells, which have previously been shown to be transduced by the RGD4C-phage.19,22 We first sought to determine the optimal ratio of two cationic polymers (poly-d-lysine (PDL) and DEAE-DEX) and RGD4C-phage using RGD4C-phage vector carrying the firefly luciferase (Luc) reporter gene. Quantification of luciferase activity in 9L and M21 cells at 72-hour post-cell transduction showed that Luc gene expression by the RGD4C-phage dramatically improved with increased concentrations of PDL and DEAE.DEX polymers (Figure 2a), as compared with RGD4C-phage alone (0 μg/ml of polymer). Maximum gene transfer levels were achieved in both M21 and 9L cells at polymer/phage ratios of 30 ng/μg for PDL and 60 ng/μg for DEAE.DEX, respectively, after which a gradual decrease in Luc gene expression occurred (Figure 2a). To determine whether the decreased transgene expression at high amounts of cationic polymers was associated with PDL and DEAE.DEX cytotoxicity, we performed cell viability assays and showed that this range of polymer concentrations was not associated with any toxic effects (Figure 2b).


Hybrid Nanomaterial Complexes for Advanced Phage-guided Gene Delivery.

Yata T, Lee KY, Dharakul T, Songsivilai S, Bismarck A, Mintz PJ, Hajitou A - Mol Ther Nucleic Acids (2014)

Characterization of tumor cell transduction by the hybrid phage/polymer. (a) Optimization of polymer types and concentrations. M21 and 9L cells were treated with RGD4C-phages carrying the Luc transgene premixed with increasing concentrations of poly-d-lysine (PDL) and DEAE.DEX and Luc gene expression was measured using the luciferase assay at day 3 post-transduction. (b) Cytotoxicity of the RGD4C-phage complexed with increasing concentrations of cationic polymers in both M21 and 9L cell lines. Cell viability was measured using the CellTiter-Glo cell viability assay, at 48-hour-post transduction. The cell viability rate (%) was calculated as percentage of control ([A]test/[A]control × 100) (n = 3). (c) Kinetics of Luc gene expression following transduction of M21 and 9L cells with the RGD4C-phage premixed with PDL or DEAE.DEX (RGD4C-PDL and RGD4C-DEAE.DEX, respectively), RGD4C-phage alone (RGD4C), or non-targeted phage (NT). The luciferase assay was performed daily over a time course of 5 days after vector transduction.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4221597&req=5

fig2: Characterization of tumor cell transduction by the hybrid phage/polymer. (a) Optimization of polymer types and concentrations. M21 and 9L cells were treated with RGD4C-phages carrying the Luc transgene premixed with increasing concentrations of poly-d-lysine (PDL) and DEAE.DEX and Luc gene expression was measured using the luciferase assay at day 3 post-transduction. (b) Cytotoxicity of the RGD4C-phage complexed with increasing concentrations of cationic polymers in both M21 and 9L cell lines. Cell viability was measured using the CellTiter-Glo cell viability assay, at 48-hour-post transduction. The cell viability rate (%) was calculated as percentage of control ([A]test/[A]control × 100) (n = 3). (c) Kinetics of Luc gene expression following transduction of M21 and 9L cells with the RGD4C-phage premixed with PDL or DEAE.DEX (RGD4C-PDL and RGD4C-DEAE.DEX, respectively), RGD4C-phage alone (RGD4C), or non-targeted phage (NT). The luciferase assay was performed daily over a time course of 5 days after vector transduction.
Mentions: We sought to assess whether the efficiency of gene delivery by the RGD4C-phage to eukaryotic cells can be improved if phage viral particles are integrated with cationic polymers. We therefore studied the efficacy with which RGD4C-phage/polymer complexes transduce human M21 melanoma cells, which are known to express high levels of αv integrin receptors for the RGD4C ligand.17,21 To rule out the possibility that the observed effects are not cell or species specific, we also assessed the efficacy on the rat 9L glioblastoma cells, which have previously been shown to be transduced by the RGD4C-phage.19,22 We first sought to determine the optimal ratio of two cationic polymers (poly-d-lysine (PDL) and DEAE-DEX) and RGD4C-phage using RGD4C-phage vector carrying the firefly luciferase (Luc) reporter gene. Quantification of luciferase activity in 9L and M21 cells at 72-hour post-cell transduction showed that Luc gene expression by the RGD4C-phage dramatically improved with increased concentrations of PDL and DEAE.DEX polymers (Figure 2a), as compared with RGD4C-phage alone (0 μg/ml of polymer). Maximum gene transfer levels were achieved in both M21 and 9L cells at polymer/phage ratios of 30 ng/μg for PDL and 60 ng/μg for DEAE.DEX, respectively, after which a gradual decrease in Luc gene expression occurred (Figure 2a). To determine whether the decreased transgene expression at high amounts of cationic polymers was associated with PDL and DEAE.DEX cytotoxicity, we performed cell viability assays and showed that this range of polymer concentrations was not associated with any toxic effects (Figure 2b).

Bottom Line: We demonstrate that the phage complex with cationic polymers generates positively charged phage and large aggregates that show enhanced cell surface attachment, buffering capacity, and improved transgene expression while retaining cell type specificity.Moreover, phage/polymer complexes carrying a therapeutic gene achieve greater cancer cell killing than phage alone.This new class of hybrid nanomaterial platform can advance targeted gene delivery applications by bacteriophage.

View Article: PubMed Central - PubMed

Affiliation: Phage Therapy Group, Department of Medicine, Imperial College London, London, UK.

ABSTRACT
Developing nanomaterials that are effective, safe, and selective for gene transfer applications is challenging. Bacteriophages (phage), viruses that infect bacteria only, have shown promise for targeted gene transfer applications. Unfortunately, limited progress has been achieved in improving their potential to overcome mammalian cellular barriers. We hypothesized that chemical modification of the bacteriophage capsid could be applied to improve targeted gene delivery by phage vectors into mammalian cells. Here, we introduce a novel hybrid system consisting of two classes of nanomaterial systems, cationic polymers and M13 bacteriophage virus particles genetically engineered to display a tumor-targeting ligand and carry a transgene cassette. We demonstrate that the phage complex with cationic polymers generates positively charged phage and large aggregates that show enhanced cell surface attachment, buffering capacity, and improved transgene expression while retaining cell type specificity. Moreover, phage/polymer complexes carrying a therapeutic gene achieve greater cancer cell killing than phage alone. This new class of hybrid nanomaterial platform can advance targeted gene delivery applications by bacteriophage.

No MeSH data available.


Related in: MedlinePlus