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Stabilization of Poly (?-Amino Ester) Nanoparticles for the Efficient Intracellular Delivery of PiggyBac Transposon.

ABSTRACT: The administration of gene-editing tools has been proposed as a promising therapeutic approach for correcting mutations that cause diseases. Gene-editing tools, composed of relatively large plasmid DNA constructs that often need to be co-delivered with a guiding protein, are unable to spontaneously penetrate mammalian cells. Although viral vectors facilitate DNA delivery, they are restricted by the size of the plasmid to carry. In this work, we describe a strategy for the stable encapsulation of the gene-editing tool piggyBac transposon into Poly (?-amino ester) nanoparticles (NPs). We propose a non-covalent and a covalent strategy for stabilization of the nanoformulation to slow down release kinetics and enhance intracellular delivery. We found that the formulation prepared by covalently crosslinking Poly (?-amino ester) NPs are capable to translocate into the cytoplasm and nuclei of human glioblastoma (U87MG) cells within 1 h of co-culturing, without the need of a targeting moiety. Once internalized, the nanoformulation dissociates, delivering the plasmid presumably as a response to the intracellular acidic pH. Transfection efficiency is confirmed by green fluorescence protein (GFP) expression in U87MG cells. Covalently stabilized Poly (?-amino ester) NPs are able to transfect ~55% of cells causing non-cytotoxic effects. The strategy described in this work may serve for the efficient non-viral delivery of other gene-editing tools.

SUBMITTER: Rodgers T

PROVIDER: S-EPMC7909559 | biostudies-literature | 2021 Jan

REPOSITORIES: biostudies-literature

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Stabilization of Poly (β-Amino Ester) Nanoparticles for the Efficient Intracellular Delivery of PiggyBac Transposon.

Rodgers Tina T Muzzio Nicolas N Watson Caleb C Romero Gabriela G

Bioengineering (Basel, Switzerland) 20210120 2

The administration of gene-editing tools has been proposed as a promising therapeutic approach for correcting mutations that cause diseases. Gene-editing tools, composed of relatively large plasmid DNA constructs that often need to be co-delivered with a guiding protein, are unable to spontaneously penetrate mammalian cells. Although viral vectors facilitate DNA delivery, they are restricted by the size of the plasmid to carry. In this work, we describe a strategy for the stable encapsulation of ...[more]

PMID: 33498466

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Project description:Recently, biomaterials have been designed to contain redox-sensitive moieties, such as thiols and disulfides, to impart responsive degradation and/or controlled release. However, due to the high sensitivity of cellular redox-based systems which maintain free-radical homeostasis (e.g. glutathione/glutathione disulfide), if these biomaterials modify the cellular redox environment, they may inadvertently affect cellular compatibility and/or oxidative stress defenses. In this work, we hypothesize that the degradation products of a poly(β-amino ester) (PBAE) hydrogel formed with redox sensitive disulfide (cystamine) crosslinking could serve as a supplement to the environmental cellular antioxidant defenses. Upon introduction into a reducing environment, these disulfide-containing hydrogels cleave to present bound-thiol groups, yet remain in the bulk form at up to 66 mol% cystamine of the total amines. By controlling the molar fraction of cystamine, it was apparent that the thiol content varied human umbilical vein endothelial cell (HUVEC) viability IC50 values across an order of magnitude. Further, upon introduction of an enzymatic oxidative stress generator to the cell culture (HX/XO), pre-incubated thiolated hydrogel degradation products conferred cellular and mitochondrial protection from acute oxidative stress, whereas non-reduced disulfide-containing degradation products offered no protection. This polymer may be an advantageous implantable drug delivery system for use in acute oxidative stress prophylaxis and/or chronic oxidative stress cell therapies due to its solid/liquid reversibility in a redox environment, controlled thiolation, high loading capacity through covalent drug-addition, and simple post-synthesis modification which bound-thiols introduce. STATEMENT OF SIGNIFICANCE:In this work, we demonstrate a unique property of disulfide containing degradable biomaterials. By changing the redox state of the degradation products (from oxidized to reduced), it is possible to increase the IC50 of the material by an order of magnitude. This dramatic shift is linked directly to the oxidative stress response of the cells and suggests a possible mechanism by which one can tune the cellular response to degradable biomaterials.

Dataset Information

Stabilization of Poly (?-Amino Ester) Nanoparticles for the Efficient Intracellular Delivery of PiggyBac Transposon.

Publications

Stabilization of Poly (β-Amino Ester) Nanoparticles for the Efficient Intracellular Delivery of PiggyBac Transposon.

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