Gelatin nanofiber mats with Lipofectamine/plasmid DNA complexes for in vitro genome editing

Furuno, Kotoko; Suzuki, Keiichiro; Sakai, Shinji

doi:10.1016/j.colsurfb.2022.112561

Cited by 8 publications

(20 citation statements)

References 36 publications

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“…The pDNA complexes were immobilized on the nanofibers through the electrostatic interaction of cationic pDNA complexes and anionic gelatin [ 37 , 38 ]. Additionally, previous research described that the pDNA complexes lost their ability to deliver pDNA into cells by incubation in a cell culture medium because of the complexation of the pDNA complexes and serum proteins [ 11 , 38 ]. However, immobilization of the pDNA complexes on the nanofibers achieved the sustained delivery of the pDNA complexes.…”

Section: Resultsmentioning

confidence: 99%

“…For the knock-in experiment, mutant GFP targeting gRNA expression plasmid (Δg1-gRNA) was generated as previously described [ 28 ]. For the knock-out experiment, the GFP target gRNA expression vector (gRNA-GFP) was generated as previously described [ 11 ].…”

Section: Methodsmentioning

confidence: 99%

“…Human embryonic kidney-derived HEK293 cells (American Type Culture Collection, Manassas, VA, USA), constitutive GFP-expressing HEK293 cells defined as HEK293EGIPneo [ 11 ], and HEK293 cells possessing a 35 bp deletion in the GFP sequence defined as HEK293EGIP*35 cells [ 28 ] were cultured in Dulbecco’s modified Eagle medium (FUJIFILM Wako Pure Chemical Co.) containing 10% ( v / v ) fetal bovine serum (FBS; Thermo Fisher Scientific Inc.), 1% ( v / v ) minimum essential medium non-essential amino acid solution (MEM NEAA; Thermo Fisher Scientific Inc.), and 1% ( v / v ) penicillin-streptomycin (Thermo Fisher Scientific, Inc.) in 5% CO 2 at 37 °C.…”

Section: Methodsmentioning

confidence: 99%

“…Lipofectamine/pDNA complexes were immobilized on the nanofibers by immersion in solutions containing the pDNA complexes, as previously described [ 11 ]. Briefly, the pDNA complexes were produced by mixing 1 μg of pDNA with 1 μL of P3000 reagent according to the protocol for Lipofectamine 3000.…”

Section: Methodsmentioning

confidence: 99%

“…Scaffolds, such as hydrogels [ 2 , 10 ] and nanofibers [ 11 , 12 , 13 , 14 ], with nucleic acids, could be used for gene delivery as an alternative method to eliminate the above issues. These systems are capable of direct and sustained delivery of nucleic acids to cells and tissues [ 10 ].…”

Section: Introductionmentioning

confidence: 99%

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Gelatin-Based Electrospun Nanofibers Cross-Linked Using Horseradish Peroxidase for Plasmid DNA Delivery

2022

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The delivery of nucleic acids is indispensable for tissue engineering and gene therapy. However, the current approaches involving DNA/RNA delivery by systemic and local injections face issues such as clearance, off-target distribution, and tissue damage. In this study, we report plasmid DNA (pDNA) delivery using gelatin electrospun nanofibers obtained through horseradish peroxidase (HRP)-mediated insolubilization. The nanofibers were obtained through the electrospinning of an aqueous solution containing gelatin possessing phenolic hydroxyl (Ph) moieties (Gelatin-Ph) and HRP with subsequent HRP-mediated cross-linking of the Ph moieties by exposure to air containing 16 ppm H2O2 for 30 min. Then, Lipofectamine/pDNA complexes were immobilized on the nanofibers through immersion in the solution containing the pDNA complexes, resulting in transfection and sustained delivery of pDNA. Cells cultured on the resultant nanofibers expressed genome-editing molecules including Cas9 protein and guide RNA (gRNA), resulting in targeted gene knock-in and knock-out. These results demonstrated the potential of Gelatin-Ph nanofibers obtained through electrospinning and subsequent HRP-mediated cross-linking for gene therapy and tissue regeneration by genome editing.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Gelatin-Based Electrospun Nanofibers Cross-Linked Using Horseradish Peroxidase for Plasmid DNA Delivery

2022

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Local delivery of adeno‐associated viral vectors with electrospun gelatin nanofiber mats

Furuno,

Elvitigala,

Suzuki

et al. 2023

J Biomed Mater Res

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Adeno‐associated viral (AAV) vectors play a significant role in gene therapy, yet the typical delivery methods, like systemic and local AAV injections, often lead to unintended off‐target distribution and tissue damage due to injection. In this study, we propose a localized delivery approach for AAV vectors utilizing electrospun gelatin nanofiber mats, which are cross‐linked with glutaraldehyde. The AAV vectors, which encoded a green fluorescent protein (GFP), were loaded onto the mats by immersing them in a solution containing the vectors. The amount of AAV vector loaded onto the mats increased as the vector concentration in the solution increased. The loaded AAV vector was steadily released into the cell culture medium over 3 days. The mats incubated for 3 days also showed the ability to transduce into the cells cultured on them. We evaluated the effectiveness of this delivery system by attaching the mats to mouse livers. GFP expression was visible on the surface of the liver beneath the attached mats, but not in areas in direct contact with the mats. These findings suggest that the attachment of AAV vector‐loaded electrospun gelatin nanofiber mats to a target site present a promising solution for localized gene delivery while reducing off‐target distribution.

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Multifunctional electrospun membranes with hydrophilic and hydrophobic gradients property for wound dressing

Luo

Shen

et al. 2023

Colloids and Surfaces B: Biointerfaces

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Gelatin nanofiber mats with Lipofectamine/plasmid DNA complexes for in vitro genome editing

Cited by 8 publications

References 36 publications

Gelatin-Based Electrospun Nanofibers Cross-Linked Using Horseradish Peroxidase for Plasmid DNA Delivery

Gelatin-Based Electrospun Nanofibers Cross-Linked Using Horseradish Peroxidase for Plasmid DNA Delivery

Local delivery of adeno‐associated viral vectors with electrospun gelatin nanofiber mats

Multifunctional electrospun membranes with hydrophilic and hydrophobic gradients property for wound dressing

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