Plasmid
The pET30a-recombinant human protamine (PA) plasmid was generated based on PA amino acid sequences from NCBI (#NP_002752.1). Codons were optimized for expression in E. coli cells and gene synthesized from Abclon. pCMV-HIC1 was purchased from Sino Biological (#HG18546-UT).
Expression and purification of recombinant human protamine protein (PA)
For PA protein expression from E. coli, BL21 Star™ (DE3) (Thermo Fisher) was transformed with pET30a-PA. Transfected cells were cultured in terrific broth until the optical density at 600 nm was 0.4–0.5. After induction with 1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG), cells were incubated at 25°C for 18 h. For PA protein purification, cultured cells were lysed with cell lysis buffer (100 mM Tris-HCl, 1 M NaCl, 20 mM imidazole, and 0.5 mM PMSF, pH 7.5). The dissolved cell solution was probe-sonicated and centrifuged at 15,000 g for 20 min at 4°C. The supernatant was reacted with the HisPur™ Cobalt Resin (Thermo Fisher) and washed with a column wash buffer (100 mM Tris-HCl, 0.5 M NaCl, and 40–60 mM imidazole, pH 7.5) before PA protein elution (100 mM Tris-HCl, 0.5 M NaCl, and 300 mM imidazole, pH 7.5). Eluted protein was further purified on a Strong Cation Exchange Column (Thermo Fisher), dialyzed against a storage buffer (100 mM Tris-HCl, 0.2 M NaCl, 0.1 mM EDTA, 0.5 mM PMSF, and 20% glycerol, pH 7.5), and then stored at -80°C.
NK-92 culture and plasma membrane protein isolation
The human NK cell line (NK-92) was procured from the American-type culture collection (ATCC). According to the manufacturer's culture instructions, NK-92 cells were cultured with alpha MEM (without ribonucleosides and deoxyribonucleosides, with 2 mM L-glutamine and 1.5 g/mL sodium bicarbonate), added with supplement (12.5% horse serum, 12.5% fetal bovine serum, 1% penicillin/streptomycin, 0.2 mM Myo-inositol, 0.1 mM 2-mercaptoethanol, 0.02 mM folic acid, and 100 U/mL human recombinant IL-2).
For isolation of NK cell plasma membrane proteins, cultured cells were harvested by centrifugation (125 g, 10 min, 4°C). NK cell plasma membrane proteins were extracted with the Mem-PER™ plus membrane protein extraction kit (Thermo Scientific) according to the manufacturer's instructions and then stored at -80°C.
Cell culture
All cell lines were procured from ATCC. MDA-MB-231, MDA-MB-468, and HCC38 cells were cultured in ATCC-modified RPMI medium (Gibco) and supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S). MCF10A and MCF12A were cultured in DMEM/F12 medium (Gibco) and supplemented with 5% horse serum, 20 ng/mL human recombinant EGF protein, 0.01 mg/mL insulin, 500 ng/mL hydrocortisone and 1% P/S. MCF7 cells were cultured in DMEM medium and supplemented with 10% FBS and 1% P/S, and all cells tested negative for mycoplasma.
Synthesis and characterization of NK-LNP with pDNA
Lipid-nanoparticles (LNPs) were prepared using egg PC:cholesterol:C18-ceramide: DGS-NTA(Ni) (molar ratio of 1:0.2:1:0.05), dissolved in 1 mL chloroform with a thin-film hydration method. The evaporated lipid film was hydrated with 30 µM purified PA protein and 30.82 nM pCMV-HIC1 plasmid complex in 1 mL phosphate-buffered saline (PBS, pH 7.2). The LNP was formed with a hydrated lipid/PA/plasmid DNA (pDNA) mixture through a liquid nitrogen (LN2) freezing-thawing procedure. For NK-LNP formation, the assembled LNP was further frozen and thawed in the presence of isolated NK cell membrane proteins (total lipid: NK cell membrane protein = 1:5, w/w). Excess PA/pDNA/NK cell membrane proteins were removed with a CL-4B Sepharose size extraction column. The resultant NK-LNP with pDNA solution was filtered through a 0.2 µm syringe filter and then kept at 4°C. NK-LNP with pDNA morphology was observed using cryo-transmission electron microscopy (cryo-EM, Tecnai F20 G2, FEI). Hydrodynamic size and zeta potential of the NK-LNP with pDNA were characterized by a Zetasizer (Nano ZS, Malvern Instruments). Particle concentration was measured by a nanoparticle tracking analysis (NTA, Nanosight NS3000, Malvern Instruments). The fused NK cell membrane proteins and encapsulated PA/pDNA complexes of the NK-LNP were qualitatively analyzed by western blotting after being powdered with a freeze-dryer and lysed in a membrane protein extraction buffer. Encapsulated pDNA was quantified by electrophoresis before removing the non-conjugated pDNA with PA.
Targeting and transfection efficiency of NK-LNP
MCF12A, MDA-MB-231, and MDA-MB-468 cells were seeded into 6-well cell culture slides (0.35 × 106 cells) at 37°C under 5% CO2. Depending on experimental conditions, cells were incubated with the NK-LNP(RITC-labelled PA/pDNA) (6.57 x 1010 ± 3.12 x 109 particles in 1 mL opti-MEM). First, to determine the TNBC targeting efficacy of the NKG2D receptor, cells were incubated with an NKG2D ligand blocker (anti-ULBP2/5/6, R&D Systems). After fixation, cells were treated with particles under 37 ℃ for 1 h and analyzed by flow cytometry (Accuri C6 Plus, BD). To prove receptor-mediated endocytosis of NK-LNP(PA/pDNA), TNBC was added to genistein (10 µg/mL), chlorpromazine (10 µg/mL), nocodazole (1.5 µg/mL), and cytochalasin B (2.4 µg/mL). NK-LNP(PA/pDNA) treated cells were then incubated at 37°C for 24 h (except for samples at 4°C) and analyzed by flow cytometry. To verify transfection efficiency and nucleus delivery of PA/pDNA, particles-treated cells were fixed and incubated with permeabilization (0.01% Triton X-100) and then in a blocking solution (3% BSA in TBST). Following the reaction at 4°C with an anti-CD56 antibody (Cell signaling), cells were incubated with fluorescence-conjugated secondary antibodies. These cells were observed under a FACS and cell automated microscope (Lionheart FX, BioTek). Endosome escape was also performed according to the manufacturer’s instructions (Invitrogen).
Western blot analysis
MDA-MB-468 cells were seeded in 60-mm2 cell culture plates (1.3 × 106 cells) at 37°C under 5% CO2, followed by incubation with NK-LNP(PA/pDNA) (2.44 x 1011 ± 1.19 x 1010 particles in 2.5 mL opti-MEM) for 6 h at 37°C. These cells were lysed in RIPA buffer with a protease and phosphatase inhibitor (Sigma). Whole-cell lysates were quantified by a BCA protein assay, loaded onto 12% SDS-PAGE gels under denaturing conditions, and transferred onto PVDF membranes (iBlot2 Dry Blotting System, Thermo Fisher Scientific). Membranes were blocked with 5% skim milk and incubated overnight at 4°C with the following primary antibodies: anti-Na-K ATPase, p21CIP, Cyclin D1 (Abcam), anti-HIC1, NKG2D (Anova), anti-CD56, phosphor-NFκB, cleaved caspase3 (Cell Signaling), anti-CD45, NP62, SIRT1, PCNA, BCL2, BAX, and actin (Santa Cruz). Membranes were incubated with HRP-conjugated secondary antibodies and developed with Western ECL substrate. Luminescent images were analyzed using a LAS500 (GE Healthcare).
Cell viability, migration, invasion, and apoptosis assay
MDA-MB-468 cells and MCF12A were seeded in 96 well plates (1.5 x 104 cells/well) and treated with NK-LNP (PA/pDNA) synthesized under various conditions. The cells were incubated with WST-8 reagent (Dojindo) for 1 h, and the absorbance was measured using a microplate reader (BioTek) at 450 nm every 24 hours. To estimate cell migration and invasion capability of NK-LNP(PA/pDNA), MDA-MB-468 cells were seeded in 12 well plates (0.15 x 106 cells/well), treated with NK-LNP(PA/pDNA) without C18-ceramide, and analyzed following the manufacturer’s instructions (Komabiotech). To evaluate cellular apoptosis depending on NK-LNP(PA/pDNA) with or without C18-ceramide, MDA-MB-468 cells were treated under various experimental conditions for 48 h. They were then stained with an EzWay Annexin V-FITC Apoptosis Detection Kit (Komabiotech).
THP1 immune cell real-time PCR analysis and cytokine detection
THP-1 cells (human monocytes) were obtained from the Korean Cell Line Bank (KCLB). These THP-1 cells were seeded (1.3 x 106 cells) and differentiated into M0-macrophage by phorbol 12-myristate 13-acetate (PMA, 65 ng/mL) at 37 ℃ for 24 h [25, 26]. After that, M0-macrophages were treated with NK-LNP(PA/pDNA) for 48 h (Fig. S11A). THP-1 cells were also seeded (1.3 x 106 cells) and differentiated into immature dendritic cells in the presence of human recombinant IL-4 protein (0.65 µg/mL) and GM-CSF (0.65 µg/mL) at 37 ℃ for 5 days [27]. After that, immature dendritic cells were co-cultured with NK-LNP(PA/pDNA) and treated with TNBC (Fig. S12). After 48 h, THP-1 cells were harvested, and total RNA was extracted using TRIzol (Invitrogen). Real-time PCR was carried out in Quantstudio3 (Applied Biosystems) using SYBR Green premix (Thermo). Real-time PCR primer sequences are listed in Table S1. Secreted cytokines were quantified using ELISA (human TNFα, IL-6, IL-10, and IL-12 ELISA kit; Abfrontier) according to the manufacturer’s assay procedure.
Statistical analysis
All experiment data are expressed as mean ± standard error of the mean (SEM). Statistical results were analyzed using an unpaired two-tailed Student’s t-test. All analyses were conducted using GraphPad Prism 8. Statistical details are described in figure legends.