The Challenging Nature of Primary T Lymphocytes for Transfection: Effect of Protamine Sulfate on the Transfection Efficiency of Chemical Transfection Reagents Publisher



Rahimmanesh I¹ ; Totonchi M² ; Khanahmad H^{1, 3} Show Affiliations
Source: Research in Pharmaceutical Sciences Published:2020

Abstract

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: 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: 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 2000 and DNA/protamine/TurboFectTM was 87.2% and 78.9%, respectively, while transfection of T cells by Lipofectamine 2000 and TurboFectTM would not result in sufficient transfection. Conclusion and implications: 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. © 2020 Wolters Kluwer Medknow Publications. All rights reserved.