Limits...
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: Developing nanomaterials that are effective, safe, and selective for gene transfer applications is challenging.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 the hybrid phage/polymer cell surface accessibility. Evaluation of the phage/polymer cell surface accessibility by quantifying the free cell-unbound phage in the external fluid phase above the adherent cell layer by infection of host bacteria. (a) Representative plates showing bacterial colonies generated by phage recovered from the supernatant of transduced cells. (b) Quantitative analysis of the recovered phage following bacterial colony counting. (c) Confocal fluorescent microscopic images of 9L cells following treatment with different vectors. Cells were first immunofluorescent stained for extracellular phage using an anti-phage primary and goat anti-rabbit AlexaFluor-594 secondary (red) antibodies prior to permeabilization and staining for intracellular phage using the same primary and goat anti-rabbit AlexaFluor-488 secondary (green) antibodies.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4221597&req=5

fig4: Characterization of the hybrid phage/polymer cell surface accessibility. Evaluation of the phage/polymer cell surface accessibility by quantifying the free cell-unbound phage in the external fluid phase above the adherent cell layer by infection of host bacteria. (a) Representative plates showing bacterial colonies generated by phage recovered from the supernatant of transduced cells. (b) Quantitative analysis of the recovered phage following bacterial colony counting. (c) Confocal fluorescent microscopic images of 9L cells following treatment with different vectors. Cells were first immunofluorescent stained for extracellular phage using an anti-phage primary and goat anti-rabbit AlexaFluor-594 secondary (red) antibodies prior to permeabilization and staining for intracellular phage using the same primary and goat anti-rabbit AlexaFluor-488 secondary (green) antibodies.

Mentions: We also investigated the phage/polymer cell surface accessibility to determine whether gene delivery efficiency by the RGD4C-phage is limited by inefficient access to the negatively charged cell surface. We therefore carried out a supernatant-depletion assay, where the free cell-unbound phage in the external fluid phase above the adherent cell layer was quantified by infection of host bacteria followed by colony counting (Figure 4a). A large amount of free phage particles (90% of input phage particles) were recovered from the supernatant of cells treated with the RGD4C-phage vector (Figure 4b), showing that only a small fraction (10% of input phage) was bound to the cell surface. By contrast, very little phage (6%) was recovered from the supernatant of cells incubated with RGD4C-PDL and RGD4C-DEAE.DEX phage/polymer complexes, indicating that most of the phage (94%) was bound to the surface of cells (Figure 4b). No phage depletion was observed in the supernatant of cells treated with the control NT phage (Figure 4b). Confocal microscopic imaging following immunofluorescence with an anti-phage antibody revealed greater cell surface localization of the RGD4C-phage/polymer aggregates than the uncomplexed RGD4C-phage alone (Figure 4c). No phage was observed on cells incubated with the control NT phage (Figure 4c). These data strongly suggest that incorporation of phage into a cationic complex increases phage accessibility to the cell surface.


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 the hybrid phage/polymer cell surface accessibility. Evaluation of the phage/polymer cell surface accessibility by quantifying the free cell-unbound phage in the external fluid phase above the adherent cell layer by infection of host bacteria. (a) Representative plates showing bacterial colonies generated by phage recovered from the supernatant of transduced cells. (b) Quantitative analysis of the recovered phage following bacterial colony counting. (c) Confocal fluorescent microscopic images of 9L cells following treatment with different vectors. Cells were first immunofluorescent stained for extracellular phage using an anti-phage primary and goat anti-rabbit AlexaFluor-594 secondary (red) antibodies prior to permeabilization and staining for intracellular phage using the same primary and goat anti-rabbit AlexaFluor-488 secondary (green) antibodies.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig4: Characterization of the hybrid phage/polymer cell surface accessibility. Evaluation of the phage/polymer cell surface accessibility by quantifying the free cell-unbound phage in the external fluid phase above the adherent cell layer by infection of host bacteria. (a) Representative plates showing bacterial colonies generated by phage recovered from the supernatant of transduced cells. (b) Quantitative analysis of the recovered phage following bacterial colony counting. (c) Confocal fluorescent microscopic images of 9L cells following treatment with different vectors. Cells were first immunofluorescent stained for extracellular phage using an anti-phage primary and goat anti-rabbit AlexaFluor-594 secondary (red) antibodies prior to permeabilization and staining for intracellular phage using the same primary and goat anti-rabbit AlexaFluor-488 secondary (green) antibodies.
Mentions: We also investigated the phage/polymer cell surface accessibility to determine whether gene delivery efficiency by the RGD4C-phage is limited by inefficient access to the negatively charged cell surface. We therefore carried out a supernatant-depletion assay, where the free cell-unbound phage in the external fluid phase above the adherent cell layer was quantified by infection of host bacteria followed by colony counting (Figure 4a). A large amount of free phage particles (90% of input phage particles) were recovered from the supernatant of cells treated with the RGD4C-phage vector (Figure 4b), showing that only a small fraction (10% of input phage) was bound to the cell surface. By contrast, very little phage (6%) was recovered from the supernatant of cells incubated with RGD4C-PDL and RGD4C-DEAE.DEX phage/polymer complexes, indicating that most of the phage (94%) was bound to the surface of cells (Figure 4b). No phage depletion was observed in the supernatant of cells treated with the control NT phage (Figure 4b). Confocal microscopic imaging following immunofluorescence with an anti-phage antibody revealed greater cell surface localization of the RGD4C-phage/polymer aggregates than the uncomplexed RGD4C-phage alone (Figure 4c). No phage was observed on cells incubated with the control NT phage (Figure 4c). These data strongly suggest that incorporation of phage into a cationic complex increases phage accessibility to the cell surface.

Bottom Line: Developing nanomaterials that are effective, safe, and selective for gene transfer applications is challenging.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