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Use of human peripheral blood mononuclear cells to define immunological properties of nucleic acid nanoparticles


This protocol assesses proinflammatory properties of nucleic acid nanoparticles (NANPs) using a validated preclinical model, peripheral blood mononuclear cells (PBMCs), that is highly predictive of cytokine responses. The experimental procedure details the preparation of pyrogen-free NANPs, isolation of PBMCs from freshly collected human blood, and analysis of characteristic biomarkers (type I and III interferons) produced by PBMCs transfected with NANPs. Although representative NANPs with high and low immunostimulatory potential are used as standards throughout the procedure, this protocol can be adapted to any NANPs or therapeutic nucleic acids, irrespective of whether they are carrier based or carrier free; additional cytokine biomarkers can also be included. We test several commercial platforms and controls broadly accessible to the research community to quantify all biomarkers in either single- or multiplex format. The continuous execution of this protocol takes <48 h; when immediate analysis is not feasible, single-use aliquots of the supernatants can be frozen and stored (−20 °C; 12 months).

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Fig. 1: The main strategies for NANP design.
Fig. 2: Schematic representation of the experimental design required to assess the immunological properties of NANPs and their interactions with PBMCs.
Fig. 3: Verification that NANPs retain structural integrity upon complexation with Lipofectamine 2000, and of their cellular uptake.

Data availability

The data presented in this article have been published before17,23,25,26,31,33 and are available to users without restrictions other than the copyright.


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This study was supported in part by federal funds from the National Cancer Institute, National Institutes of Health, under contracts HHSN261200800001E and 75N91019D00024 (to M.A.D.). The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government. Research reported in this publication was also supported by the National Institute of General Medical Sciences of the National Institutes of Health under award no. R01GM120487 (to K.A.A.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The authors thank J. Halman and M. Chandler of the University of North Carolina at Charlotte and E. Hong and E. Cedrone of the Nanotechnology Characterization Lab, National Cancer Institute, for excellent technical assistance.

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M.A.D. and K.A.A. conceived the study, developed and validated the protocol and co-wrote the manuscript.

Corresponding authors

Correspondence to Marina A. Dobrovolskaia or Kirill A. Afonin.

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The authors declare no competing interests.

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Peer review information Nature Protocols thanks Remi Creusot, Chunhai Fan and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Key references using this protocol

Hong, E. et al. Nano Lett. 18, 4309–4321 (2018):

Rackley, L. et al. Adv. Funct. Mater. 28, 1805959 (2018):

Hong, E. et al. Molecules 24, 1094 (2019):

Afonin, K. A. et al. Nat. Protoc. 6, 2022–2034 (2011):

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Dobrovolskaia, M.A., Afonin, K.A. Use of human peripheral blood mononuclear cells to define immunological properties of nucleic acid nanoparticles. Nat Protoc 15, 3678–3698 (2020).

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