Lipid nanoparticles fuse with cell membranes of immune cells at low temperatures leading to the loss of transfection activity

https://doi.org/10.1016/j.ijpharm.2020.119652Get rights and content

Abstract

Delivering nucleic acid using a non-viral vector is a potent strategy for gene modification and controlling gene expression in immune cell therapy. Since the low-temperature storage (0–4 °C) or cryopreservation of cells are indispensable for performing immune cell therapy, we investigated the interactions between an siRNA-loaded lipid nanoparticle (LNP), a multifunctional envelope-type nanodevice (MEND) containing YSK12-C4 (YSK12-MEND), and human immune cell lines (NK-92 and Jurkat) at low-temperature and its effect on transfection activity. The YSK12-MEND readily bound to the cell membrane of NK-92 cells at low-temperature, but no internalization of the YSK12-MEND by cells was observed, even after returning the temperature to 37 °C. Gene silencing activity was completely impaired. The cause of this inhibition appears to be membrane fusion between the YSK12-MEND and cell membrane at the low-temperature. Collectively, our results suggest that the exposure of siRNA-loaded LNPs to cells at low-temperature should be avoided in defining transfection protocols in immune cell therapy.

Introduction

The success of immune checkpoint inhibitors created the present tide of cancer immunotherapy. Immune checkpoint inhibitors are now used as the first line treatment for cancer (Pai-Scherf et al., 2017). Chimeric antigen receptor (CAR) T cell therapy targeting CD19, a type of adoptive cell therapy (ACT), has also been approved for the treatment of refractory, high-grade B cell malignancies. A feature of ACT typically includes a high antitumor activity, because killer immune cells such as killer T cells and natural killer (NK) cells that directly attack cancer cells proliferate when ACT therapy is administered. An analysis in which the number of drug development pipelines in cancer immunotherapy were compared between 2017 and 2019 showed that the use of ACT has tripled and that the growth ratio and number uses are the highest, indicating high expectations for its use (Xin Yu et al., 2019a). CAR T cells are prepared by introducing a gene coding single chain antibody (scFv, antigen-binding domain), transmembrane domain, CD3-ζ domain and costimulatory domain to apheresis T cells from cancer patients (Majzner and Mackall, 2019). NK cell therapy is following CAR T cell therapy (Xin Yu et al., 2019b). Autologous NK cells, allogeneic NK cells and NK cell lines are used (Cheng et al., 2012). Unlike T cells, the risk of graft-versus-host (GVH) reactions is low. In addition, the use of cell lines has led to the success of off-the-shelf cell therapy. In any type of cell therapy, technologies for genetic modification and controlling gene expression are indispensable for development of ACT.

In clinical trials and clinical developments regarding CAR T and NK cell therapies, viral vectors such as the lentivirus and retrovirus are employed to introduce genes. However, there are profound and persistent concerns regarding the safety of using such viral vectors (Hacein-Bey-Abina et al., 2003, Field et al., 2013). Furthermore, the effort, cost and regulation associated with the manufacturing of viral vectors for clinical has resulted in high drug price and has limited the availability of such drugs to patients on a global scale. Therefore, the use of non-viral vectors would be predicted to overcome these problems. Various studies have been conducted in this area and the technology associated with the production of non-viral vectors is increasing greatly (Kebriaei et al., 2017, Ramishetti and Peer, 2019). In particular, technology involving applications of lipid nanoparticles (LNPs) which are capable of efficiently delivering cargoes with highly fusogenic properties to the cytosol of cells has been applied to various modalities including plasmid DNA, mRNA, genome editing and adjuvants (Cullis and Hope, 2017, Kulkarni et al., 2017, Kawai et al., 2018, Pardi et al., 2017, Finn et al., 2018, Miyabe et al., 2014, Nakamura et al., 2015). LNPs loaded with siRNA have now been approved for use. We also proposed the use of a multifunctional envelope-type nanodevice (MEND), a LNP type of non-viral vector for nucleic acids (Kogure et al., 2008, Nakamura et al., 2012, Nakamura et al., 2019). We synthesized a fusogenic-cationic lipid, YSK12-C4, and confirmed that it efficiently delivered siRNA to mouse and human immune cells (Warashina et al., 2016, Nakamura et al., 2016, Nakamura et al., 2018, Endo et al., 2019). YSK12-C4 contains unsaturated carbon chains and efficiently facilitates the cytosolic delivery of siRNA. A MEND containing YSK12-C4 (YSK12-MEND) showed a 75% silencing activity in NK-92 cells (Nakamura et al., 2016). NK-92 cells, a human NK cell line, are now considered to be an attractive candidate for NK cell therapy (Suck et al., 2016). Thus, the YSK12-MEND is expected to contribute to controlling gene expression for enhancing ACT, especially in NK-92 cell therapy.

The transfection efficiency and cytotoxicity for in vitro transfection with non-viral vectors are influenced by the capability of the non-vial vector itself, the dose being used, exposure time, the absence or presence of serum, culture medium, temperature, etc. Naturally, most studies have focused on the capability of the non-vial vector itself. In the studies, the transfection protocols such as the dose, exposure time, the presence or absence of serum, culture medium were optimized for the non-viral vectors used in these studies. Nevertheless, the temperature used in these studies is generally 37 °C. It should be noted here that endocytosis pathways do not functions at low temperature such as 4 °C. On the other hand, cells are often stored at low temperatures (0–4 °C) and the cryopreservation of cells is a widely used method for maintaining the function of cells, and are indispensable for performing ACT. Therefore, investigating the effect of low-temperature (0–4°C) conditions on the transfection activity by non-viral vectors could provide important insights into setting transfection protocols in ACT.

In this study, we assessed the interactions between the YSK12-MEND and human immune cell lines at low-temperature (0–4°C) conditions (defined as 4 °C), and the influence of this on siRNA transfection efficiency. Unexpectedly, the YSK12-MEND was able to bind to cell membranes at 4 °C, but was not taken up by the cells, even after returning the temperature to 37 °C. In this case, the gene silencing activity of the YSK12-MEND was completely inhibited. The cause of the inhibition of cellular internalization would be predicted to be membrane fusion between the YSK12-MEND and the cell membrane at 4 °C. Collectively, our results suggest that exposing siRNA-loaded LNPs to cells at 4 °C should be avoided, because the cellular internalization of LNPs is inhibited by membrane fusion once the LNPs bind to the cell membrane at this low temperature. These observations will be useful for setting transfection protocols of siRNA to immune cells that involve delivery by lipid-based non-viral vectors.

Section snippets

Reagents

YSK12-C4, (6Z, 9Z, 28Z, 31Z)-19-(4-(dimethylamino)butyl) heptatriaconta-6,9,28,31-tetraen-19-ol, was synthesized as previously described (Warashina et al., 2016). Cholesterol and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) were purchased from Avanti Polar Lipids Inc. (Alabaster, AL). 1,2-Dimirystoyl-sn-glycerol methoxyethyleneglycol 2000 ether (PEG2000-DMG) and 1,2-Distearoyl-sn-glycero-3- phosphocholine (DSPC) were obtained from the NOF Corporation (Tokyo, Japan). Silencer® GAPDH siRNA as

Effect of 4 °C conditions on gene silencing activity and cytotoxicity by the YSK12-MEND in NK-92 cells

The transfection activity of the YSK12-MEND is largely inhibited in the presence of serum. Thus, the cells were exposed to the YSK12-MEND in medium-free serum (Opti-MEM) (Warashina et al., 2016, Nakamura et al., 2016). After a 2 h exposure of the YSK12-MEND in Opti-MEM, medium containing serum (culture medium) was then added to the cells without washing out the extra YSK12-MEND. We first investigated the effect of a temperature of 4 °C on gene silencing activity and cytotoxicity by the

Discussion

In this study, we investigated the interaction between the YSK12-MEND and human immune cell lines at a temperature of 4 °C (4 °C condition), and the influence of this condition on the siRNA transfection efficiency. Generally, nanoparticles that are taken up by cells via electrostatic interactions or receptor-mediated interaction bind to the cell membrane and no subsequent internalization occurred in the 4 °C condition (Pires et al., 1999, Suesca et al., 2013). The nanoparticles that were bound

Conclusions

In conclusion, we report herein that once the YSK12-MEND becomes bound to the cell membrane at low-temperature (0–4°C), the cellular internalization of YSK12-MEND is inhibited, and gene silencing activity was completely impaired even when the temperature is returned to 37 °C. The cause of the inhibition of cellular internalization appears to be membrane fusion between the YSK12-MEND and the cell membrane at the 4 °C condition. Collectively, our results suggest that the exposure of siRNA-loaded

CRediT authorship contribution statement

Takashi Nakamura: Conceptualization, Methodology, Formal analysis, Data curation, Writing - original draft, Visualization, Supervision, Project administration, Funding acquisition. Koharu Yamada: Methodology, Formal analysis, Validation, Investigation, Writing - review & editing, Visualization. Yusuke Sato: Resources, Writing - review & editing. Hideyoshi Harashima: Conceptualization, Methodology, Writing - review & editing, Supervision, Funding acquisition.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This work was supported, in part, by JSPS KAKENHI (Grant Number 17H03974) (T.N.) and the Special Education and Research Expenses from the Ministry of Education, Culture, Sports, Science and Technology (H.H.). We also appreciate the helpful advice of Dr. Milton S. Feather for examining the English in this manuscript.

References (41)

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