Reducing the Cytotoxicity of Lipid Nanoparticles Associated with a

Subscriber access provided by UNIV OF NEW ENGLAND ARMIDALE

Reducing the cytotoxicity of lipid nanoparticles associated with a fusogenic-cationic lipid in a natural killer cell line by introducing a polycation based siRNA core Takashi Nakamura, Koharu Yamada, Yuki Fujiwara, Yusuke Sato, and Hideyoshi Harashima Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b01166 • Publication Date (Web): 18 Apr 2018 Downloaded from http://pubs.acs.org on April 19, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Molecular Pharmaceutics

Reducing the cytotoxicity of lipid nanoparticles associated with a fusogenic-cationic lipid in a natural killer cell line by introducing a polycation based siRNA core Takashi Nakamura, Koharu Yamada†, Yuki Fujiwara†, Yusuke Sato, and Hideyoshi Harashima* Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812, Japan †

Koharu Yamada and Yuki Fujiwara contributed equally to this study.

*Correspondence: Hideyoshi Harashima Faculty of Pharmaceutical Sciences, Hokkaido University Sapporo, Hokkaido 060-0812, Japan. Telephone: +81-11-706-3919, Fax: +81-11-706-3734 E-mail: [email protected]

Note The authors declare no competing financial interest.

ACS Paragon Plus Environment

Molecular Pharmaceutics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT Introducing siRNA into human immune cells by an artificial delivery system continues to be a challenging issue. We previously developed a multifunctional envelope-type nanodevice (MEND) containing YSK12-C4, a fusogenic-cationic lipid, (YSK12-MEND) and succeeded in the efficient delivery of siRNA into human immune cell lines. Significant cytotoxicity, however, was observed at siRNA doses needed for gene silencing in NK-92 cells. NK-92 cells, a unique natural killer (NK) cell line, would be applicable for use in clinical NK therapy. Thus, reducing the cytotoxicity of the YSK12-MEND in NK-92 cells would strengthen the efficacy of NK-92 cell based therapy. The amount of YSK12-C4 in the MEND needed to be reduced to reduce the cytotoxicity, because the cytotoxicity was directly associated with YSK12-C4. In the present study, we decreased the total amount of lipid, including YSK12-C4, by introducing a core formed by electrostatic interactions of siRNA with a polycation (protamine) (siRNA core), which led to a decrease in cytotoxicity in NK-92 cells. We prepared a YSK12-MEND containing a siRNA core (YSK12-MEND/core) at charge ratios (CR: YSK12-C4/siRNA) of 10, 5, 3, and 2.5, and compared the YSK12-MEND/core with that for a YSK12-MEND (CR16.9). Cell viability was increased by more than 2 times at a CR5 or less. On the other hand, the YSK12-MEND/core (CR5) maintained the same gene silencing efficiency (60%) as the YSK12-MEND. Interestingly, the cellular uptake efficiency and hemolytic activity of YSK12-MEND/core (CR5) was reduced compared to that for the YSK12-MEND. In calculating the silencing activity per cellular uptake efficiency and hemolytic activity, the value for the YSK12-MEND/core (CR5) was more than 2 times as high as that of YSK12-MEND. The fact indicates that after endosomal escape, the process can be enhanced by using a YSK12-MEND/core (CR5). Thus, introducing an siRNA core into lipid nanoparticles can be potent strategy for decreasing cytotoxicity without an appreciable loss of gene silencing activity in NK-92 cells.

KEYWORDS lipid nanoparticles, siRNA, fusogenic-cationic lipids, siRNA core, NK cells, NK-92


Page 2 of 33

Page 3 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


INTRODUCTION Adaptive cell therapy is now a leading component of cancer immunotherapy and has revolutionized cancer therapy alongside immune checkpoint inhibitors.1 A chimeric antigen receptor modified T cell (CAR-T) therapy against CD19 (CTL019) appears to be on the brink of approval as of August 2017. CAR-T therapy is a personalized treatment that involves infusing a patient’s genetically modified T cells that target tumor cells. The CTL019 procedure was reported to show a tremendous therapeutic efficiency against acute lymphoblastic leukemia (ALL).2 In addition to CAR-T therapy, natural killer (NK) cell therapy is also a type of potent adaptive cell therapy. NK cell therapy has several advantages over T cell therapy, in that the killing activity of NK cells is tightly regulated by only a balance between activating and inhibitory signals mediated by a multitude of receptors on the cell surface.3 This enables NK cells to kill tumor cells without being recognized by tumor antigens or clonal expansion, thus permitting the scanning for altered protein expression pattern on cells and allowing healthy cells to be distinguished from tumor cells with absolute accuracy. In addition, unlike T cells, NK cells do not cause a graft-versus-host disease (GVHD).4 Thus, NK cell therapy can be extended to allogenic adaptive cell transfer.5 However, NK cell therapy using primary NK cells from patients faces some serious challenges, such as individual differences in the performance of NK cells, clinical-scale enrichment, cost, and longer-term storage.6 Therefore, NK-92 cells, a human NK cell line, has attracted considerable interest as an off-the-shelf NK cell therapy.6 NK-92 cells have already been evaluated in clinical trials.7 Moreover, to enhance the therapeutic utility, CAR-engineering technology has also introduced to NK-92 cell therapy.8 Although CAR-engineering technology is an established strategy, it will be necessary to control the functions of immune cells at the gene level for the successful development of cell-based cancer immunotherapy. Small interfering RNA (siRNA) technology has become a powerful tool for controlling the functions of cells at the gene level, because the expression of various genes can be suppressed by merely changing the sequence of the siRNA.9 Such siRNA technology has been successfully used to control gene expression, in particular, in in vitro situations by the development of effective commercially available delivery systems. Thus, siRNA technology would also be expected to be applied to cell-based cancer immunotherapy. However, introducing siRNA into human immune cells by the currently



available non-viral delivery systems remains a quite difficult task. Although a non-viral delivery system would be desirable in terms of ease, only a few reports have appeared on the efficient siRNA introduction by non-viral vectors.10-12 Therefore, the development of a non-viral delivery system for the efficient delivery of siRNA to human immune cells is currently an important issue. In a recent study, we proposed the use of a multifunctional envelope-type nanodevice (MEND) as a non-viral delivery system for nucleic acids.13 We recently synthesized a fusogenic-cationic lipid, YSK12-C4, and succeeded in delivering siRNA to mouse dendritic cells and human immune cell lines.14-15 The ionizable-cationic lipid structure of YSK12-C4 that contains unsaturated carbon chains facilitates the endosomal escape of the lipid nanoparticles. When the MEND containing YSK12-C4 (YSK12-MEND) was used to transfect siRNA to human immune cell lines, Jurkat (human T cells), THP-1 (human monocytes) and KG-1 (human macrophages), the gene silencing efficiencies at the mRNA level were in excess of 90% at an siRNA dose range of 3-30 nM.15 On the other hand, the corresponding range for Lipofectamine RNAiMAX, one of the more popular siRNA transfection reagents for in vitro gene silencing, was 19-58%.15 This fact clearly indicates that the YSK12-MEND represents a potentially valuable system for delivering siRNA to human immune cells. The YSK12-MEND also achieved a 75% gene silencing efficiency in NK-92 cells at an siRNA dose of 30 nM, accompanied by a significant decrease in cell viability.15 The cytotoxicity in NK-92 cells associated with the YSK12-MEND appears to have been caused by the YSK12-C4 lipid, because the YSK12-MEND had a positive charge and showed strong endosome disruption activity.14 To reduce the cytotoxicity associated with the YSY12-MEND, we attempted to decrease the amount of YSK12-C4, namely the total lipids, in the MEND by introducing a polycation based siRNA core. In the past design, when such siRNA-loaded lipid nanoparticles were prepared by an alcohol diluted method, the cationic lipids were found to electrostatically interact with the siRNA and the inverted micelle of the cationic lipid surrounding the siRNA.16-17 That is, YSK12-C4 functioned as a condenser of siRNA in addition to being an enhancer of cellular internalization and endosomal escape. Alternatively, a cationic polymer could also function as a condenser of siRNA instead of YSK12-C4. In fact, protamine, a well-known polycation, which is produced by the sperm of fish, has been used as a condenser for nucleic acids including siRNA.18 We also previously used protamine as a condenser for siRNA and packaged the siRNA/protamine


Page 4 of 33

Page 5 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


core into a MEND19, with more recent data demonstrating a reduction in the systemic toxicity of the siRNA-loaded MEND (Sato Y, et al., unpublished data). In the present study, we used a protamine molecule as a siRNA condenser, as well, and prepared a YSK12-MEND containing an siRNA core (YSK12-MEND/core) which made it possible to reduce the total amount of lipid in the YSK12-MEND. The cytotoxicity, gene silencing activity, cellular uptake and hemolytic activity of YKS12-MEND/core were compared with the corresponding values for the YSK12-MEND. The findings in this study indicate that the YSK12-MEND/core represents a potent system for delivering siRNA to NK-92 cells and provides a useful strategy for developing low-toxic, lipid nanoparticles based on the use of a fusogenic-cationic lipid.



MATERIALS AND METHODS Materials YSK12-C4 (6Z, 9Z, 28Z, 31Z)-19-(4-(dimethylamino)butyl) heptatriaconta-6,9,28,31-tetraen-19-ol was synthesized as previously described.14 Cholesterol was purchased from Avanti Polar Lipids Inc. (Alabaster, AL). 1,2-Dimirystoyl-sn-glycerol methoxyethyleneglycol 2000 ether (PEG2000-DMG) and 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) were purchased from the NOF Corporation (Tokyo, Japan). Anti-human GAPDH siRNA (Silencer® GAPDH siRNA) was obtained from Thermo Fisher Scientific. Cy5-labeled siRNA (Cy5-siRNA, sense: 5 ′ -AcAuGAAGcAGcACGACuU(dT*dT)-3 ′ ; antisense: 5 ′ -AAGUCGUGCUGCUUC AUGU(dTdT) Cy5-3′ , 2′ -OMe are denoted in lower case letters and phoshorothioate linkages are represented by asterisks) was synthesized by BIONEER (Daejeon, Korea). Protamine was purchased from Merck (Darmstadt, Germany). Chloroquine was obtained from Wako Chemicals (Osaka, Japan). Cell line NK-92 cells were purchased from the American Type Culture Collection (Manassas, VA) and cultured in α-MEM containing 0.2 mM inositol, 0.1 mM 2-mercaptoethanol, 0.02 mM folic acid, 200 U/mL interlukin-2 (IL-2) (Peprotech, Rocky Hill, NJ), 12.5% horse serum and 12.5% fetal bovine serum (FBS) (culture medium). Preparation of YSK12-MEND and YSK12-MEND/core YSK12-MEND was prepared by the t-BuOH dilution procedure as previously reported.14 The YSK12-MEND was composed of YSK12-C4, cholesterol and PEG2000-DMG (85/15/1 mol ratio). Briefly, a 600 pmol siRNA (siGAPDH) solution (200 µL) was gradually added to 90% (v/v) t-BuOH solution (400 µL) containing 425 nmol of YSK12-C4, 75 nmol of cholesterol, and 5 nmol of PEG2000-DMG with vortexing. The mixture was then rapidly diluted with 2 mL of 20 mM citrate buffer (pH 6.0) to a final concentration of < 20% t-BuOH. The residual t-BuOH was replaced with PBS (pH 7.4) by ultrafiltration. The YSK12-MEND/core was also prepared by the t-BuOH dilution procedure, as previously reported, with minor modifications.14 It was composed of YSK12-C4, cholesterol, POPE, and PEG2000-DMG (85/10/5/1 mol ratio). A 5.3 mg/mL aliquot of a


Page 6 of 33

Page 7 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


protamine solution (100 µL, 1 mM citrate buffer, pH4.5) was gradually added to 6 µM siRNA (siGAPDH) solution (100 µL, 1 mM citrate buffer, pH4.5) with vortexing to form the siRNA core. The charge ratio of protamine/siRNA was 1:1. The siRNA core solution was gradually added to 90% (v/v) t-BuOH solution (200 µL) containing YSK12-C4, cholesterol, POPE, and PEG2000-DMG with vortexing. The total amount of lipid in the 90% (v/v) t-BuOH solution was determined based on a charge ratio (CR) of YSK12-C4/siRNA. The total amount of lipid for CR10, 5, 3, and 2.5 were 300, 150, 90, and 75 nmol, respectively. The mixture was then rapidly diluted with 1 ml of 1 mM citrate buffer (pH 4.5) and was stirred immediately. The residual t-BuOH was replaced with PBS (pH 7.4) by ultrafiltration. The diameters and zeta potentials of the MENDs were measured by means of a ZETASIZER Nano (ZEN3600, Malvern Instruments Ltd., Malvern, WR, UK). The diameter, polydispersity index (PDI) and zeta-potential of the MENDs were measured in 10 mM HEPES buffer (pH 7.4). The siRNA encapsulation efficiency and total concentration of siRNA were determined by a Ribogreen assay as described previously.14 Analysis of cell viability The viability of NK-92 cells was evaluated by a Premix WST-1 Cell proliferation Assay System (Takara Bio Inc., Shiga, Japan) as reported previously.15 NK-92 cells (8×105 cells/mL) were mixed with YSK12-MEND or YSK12-MEND/core at a siRNA dose range of 10–150 nM in serum-free OPTI-MEM I containing 200 U/mL IL-2 in a microtube (total volume: 150 µL). Samples (50 µL) of the mixtures were transferred to 96 well plates (duplicate) and then incubated for 2 h at 37°C, 5% CO2. After the incubation, 50 µL of culture medium was added to the wells, followed by incubation at 37°C, in an atmosphere of 5% CO2 for 22 h. A 10 µL aliquot of WST-1 assay reagent was then added to the wells and the cells were incubated for 1 h at 37°C, 5% CO2. The absorbance at 450 nm was measured using a microplate reader, and cell viability was calculated (the absorbance of non-treatment was set at 100%). For the analysis of cell viability in the presence of chloroquine, NK-92 cells 5 (8×10 cells/mL) were pre-incubated in serum-free OPTI-MEM I containing 200 µM chloroquine and 200 U/mL IL-2 in a microtube for 1 h at 37°C, 5% CO2. NK-92 cells were mixed with YSK12-MEND at a siRNA dose range of 10–60 nM in serum-free OPTI-MEM I containing 200 U/mL IL-2 in a microtube (total volume: 150 µL). Aliquots (50 µL) of the



mixtures were transferred to a 96 well plate (duplicate) and then incubated for 2 h at 37°C, 5% CO2. Aliquots (50 µL) of culture medium and 10 µL of a WST-1 assay reagent were then added to the wells, and the cells were incubated for 1 h at 37°C, 5% CO2. The absorbance at 450 nm was measured using a microplate reader, and cell viability was calculated (the absorbance of the non-treatment sample was set to 100%). Evaluation of gene silencing activity at the mRNA level The evaluation of gene silencing at the mRNA level was performed as reported previously.15 NK-92 cells (4×105 cells) were seeded in a 12 well plate and then mixed with the YSK12-MEND or the YSK12-MEND/core at siRNA doses of 10, 30, 60 and 90 nM in 0.5 ml of serum-free OPTI-MEM I containing 200 U/mL IL-2. After a 2 h incubation, 0.5 ml of culture medium was added to the cells, followed by a further incubation for 22 h. The cells were then collected and the RNA isolated with a RNeasy Mini Kit (QIAGEN, Hilden, Germany) according the manufacturer’s instructions. To eliminate DNA contamination, the total RNA was treated with DNase I (Takara Bio Inc., Shiga, Japan). The total RNA (500 ng for each sample) was then reverse transcribed using a PrimeScript reverse transcription (RT) reagent Kit (Takara Bio Inc.) with oligo-dT primer. Quantitative polymerase chain reactions (PCR) were carried out on a Light Cycler 480 System (Roche Diagnostics, Basel, Switzerland) in a reaction mixture containing cDNA, with appropriate pairs of primers and THUNDERBIRD SYBR qPCR Mix (TOYOBO Co., Osaka, Japan). GAPDH levels were calculated by the comparative CT method using beta actin as endogenous housekeeping genes. The value for non-treated cells was set to 1. The following primer pairs were used: GAPDH: 5′-CCTCTGACTTCAACAGCGAC-3′ (forward); 5′-CGTTGTCATACCAGGAAATGAG-3′ (reverse); beta actin: 5′ -CACTCTTCCAGCCTTCCTTC-3′ (forward); 5′ -TACAGGTCTTTGCGCATGTC-3′ (reverse). Evaluation of cellular uptake The cellular uptake by NK-92 cells was evaluated, as reported previously.15 Samples of the Cy5-siRNA loaded YSK12-MEND or the YSK12-MEND/core were treated to NK-92 cells (4.0×105 cells) at siRNA doses of 10, 30, and 60 nM for 2 h at 37°C, 5% CO2 in 0.5 mL of serum-free OPTI-MEM I in 12 well plates. The Cy5-siRNA loaded YSK12-MEND and YSK12-MEND/core were prepared with a GAPDH siRNA solution


Page 8 of 33

Page 9 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


containing 15% Cy5-siRNA. After collecting the cells, they were washed with a 20 U/mL solution of heparin in PBS and then suspended in FACS buffer (PBS containing 0.1% NaN3 and 0.5% bovine serum albumin). The cells were analyzed by flow cytometry (Gallios, Beckman Coulter, Indianapolis, IN). The data were analyzed by the Kaluza software (Beckman Coulter). The value of the geometric mean (GeoMean) of fluorescence intensity (FI) was normalized by that for non-treated cells. Hemolysis assay Hemolysis assays were performed as reported previously.14 Fresh red blood cells (RBCs) were collected from a C57BL/6J mouse and suspended in PBS (pH 5.5, pH 6.5, and pH 7.4). The YSK12-MEND or the YSK12-MEND/core were added to the RBCs solution at siRNA doses of 10, 30, 60, and 120 nM in a microtube. The mixtures were then incubated at 37°C, 900 rpm for 45 min, after which the absorbance at 545 nm of the supernatant was measured. The samples incubated with 0.02 w/v% Triton X-100 were used as a positive control and without MENDs as a negative control. The absorbance of the negative control was set to 0% of hemolysis activity, whereas the absorbance of positive control was set to 100%. Statistical analysis Comparisons between the two treatments were performed by an unpaired t-test. Comparisons between multiple treatments were made using one-way or two-way ANOVA, followed by Tukey-Kramer test. A P-value of

Reducing the Cytotoxicity of Lipid Nanoparticles Associated with a

Recommend Documents