The challenging nature of primary T lymphocytes for transfection: Effect of protamine sulfate on the transfection efficiency of chemical transfection reagents.
Gene transfer techniques
Protamine sulfate
T-Lymphocytes
Transfection
Journal
Research in pharmaceutical sciences
ISSN: 1735-5362
Titre abrégé: Res Pharm Sci
Pays: Iran
ID NLM: 101516968
Informations de publication
Date de publication:
Oct 2020
Oct 2020
Historique:
received:
15
07
2020
revised:
11
08
2020
accepted:
13
09
2020
entrez:
25
2
2021
pubmed:
26
2
2021
medline:
26
2
2021
Statut:
epublish
Résumé
The optimization of an effective non-viral gene delivery method for genetic manipulation of primary human T cells has been a major challenge in immunotherapy researches. Due to the poor transfection efficiency of conventional methods in T cells, there has been an effort to increase the transfection rate in these cells. Protamine is an FDA-approved compound with a documented safety profile that enhances DNA condensation for gene delivery. In this study, the effect of protamine sulfate on the transfection efficiency of standard transfection reagents, was evaluated to transfect primary human T cells. In this regard, pre-condensation of DNA was applied using protamine, and the value of the zeta potential of DNA/protamine/cargo complexes was determined. T cells were transfected with DNA/protamine/cargo complexes. The transfection efficiency rate was evaluated by flow cytometry. Also, the green fluorescent protein expression level and cytotoxicity of each complex were identified using real-time polymerase chain reaction and MTT assay, respectively. Our results demonstrated that protamine efficiently increases the positive charge of DNA/cargo complex without any cytotoxic effect on the primary human T cells. We observed that the transfection efficiency in DNA/protamine/ Lipofectamine Protamine sulfate enhanced the transfection rate of T cells; and could be a promising non-viral gene delivery method to achieve a safe, rapid, cost-effective, and efficient system which will be further applied in gene therapy and T cells manipulation methods.
Sections du résumé
BACKGROUND AND PURPOSE
OBJECTIVE
The optimization of an effective non-viral gene delivery method for genetic manipulation of primary human T cells has been a major challenge in immunotherapy researches. Due to the poor transfection efficiency of conventional methods in T cells, there has been an effort to increase the transfection rate in these cells. Protamine is an FDA-approved compound with a documented safety profile that enhances DNA condensation for gene delivery.
EXPERIMENTAL APPROACH
METHODS
In this study, the effect of protamine sulfate on the transfection efficiency of standard transfection reagents, was evaluated to transfect primary human T cells. In this regard, pre-condensation of DNA was applied using protamine, and the value of the zeta potential of DNA/protamine/cargo complexes was determined. T cells were transfected with DNA/protamine/cargo complexes. The transfection efficiency rate was evaluated by flow cytometry. Also, the green fluorescent protein expression level and cytotoxicity of each complex were identified using real-time polymerase chain reaction and MTT assay, respectively.
FINDINGS/RESULTS
RESULTS
Our results demonstrated that protamine efficiently increases the positive charge of DNA/cargo complex without any cytotoxic effect on the primary human T cells. We observed that the transfection efficiency in DNA/protamine/ Lipofectamine
CONCLUSION AND IMPLICATIONS
CONCLUSIONS
Protamine sulfate enhanced the transfection rate of T cells; and could be a promising non-viral gene delivery method to achieve a safe, rapid, cost-effective, and efficient system which will be further applied in gene therapy and T cells manipulation methods.
Identifiants
pubmed: 33628285
doi: 10.4103/1735-5362.297846
pii: RPS-15-437
pmc: PMC7879792
doi:
Types de publication
Journal Article
Langues
eng
Pagination
437-446Informations de copyright
Copyright: © 2020 Research in Pharmaceutical Sciences.
Déclaration de conflit d'intérêts
The authors declare that no conflict of interest in this study.
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