Cytotoxicity assessment,inflammatory properties,and cellular uptake of Neutraplex lipid-based nanoparticles in THP-1 monocyte-derived macrophages

Materials

Dioctadecylamidoglycylspermine (DOGS) was purchased from PolyPeptide Laboratories (Strasbourg, France), dioleoylphosphatidylethanolamine (DOPE), fluorescein-labeled DOPE, and cardiolipin from Sigma-Aldrich (Oakville, Ontario, Canada). SiRNAs and RNase-free water were purchased from Applied Biosystems (Foster City, California, USA). THP-1 monocyte/macrophage cell line was obtained from the American Type Culture Collection. ^32,33 The human epithelial cancer cervical cell line TZM-bl was obtained through the US National Institutes of Health AIDS Research and Reference Reagent Program from Dr John C Kappes, Dr Xiaoyun Wu, and Tranzyme Inc (Durham, North Carolina, USA). ^34,35 Roswell Park Memorial Institute (RPMI)-1640 and Dulbecco’s minimum essential medium (DMEM) cell culture medium, phosphate-buffered saline (PBS), Hank’s balanced salt solution (HBSS), phorbol myristate acetate (PMA), trypsin–ethylenediaminetetraacetic acid (EDTA) solution, dimethyl sulfoxide (DMSO), LysoTracker® Blue DND-2, and Texas Red® wheat germ agglutinin (WGA) were all purchased from Life Technologies (Burlington, Ontario, Canada). CellTiter-Blue® (CTB) Cell Viability, CytoTox-96® (lactate dehydrogenase (LDH)), and Beta-Glo® assay kits were obtained from Promega (Madison, Wisconsin, USA). Capture beads were purchased from Luminex (Austin, Texas, USA). Cytokine antibody pairs and standards were obtained from R & D Systems Inc. (Minneapolis, Minnesota, USA) (interleukines IL-1β, IL-8) and interleukins IL-6, IL-10, tumor necrosis factor alpha TNF-α, and streptavidin-phycoerythrin (streptavidin-PE) was obtained from Thermo Fisher Scientific, Waltham, Massachusetts, USA. Milli-Q and deionized water were produced by a Millipore water purification system (EMD Millipore, Etobicoke, Ontario, Canada). Analytical grade solvents and reagents were purchased from Sigma-Aldrich unless otherwise specified.

SiRNA

Synthetic double-stranded siRNA sequences were dissolved in sterile RNase-free water at a concentration of 50 μM and stored as indicated by the manufacturer. SiRNAs used in this study include siCCR5 targeting C–C chemokine receptor type 5 (CCR5) labeled at the 3′ end with Cy3 (sense strand: 5′-GGGCUCUAUUUUAUAGGCUdtdt-3′), siCXCR4 targeting chemokine C–X–C motif receptor 4 (CXCR4; sense strand: 5′-GGAAGCUGUUGGCUGAAAAdtdt-3′), and a scrambled siRNA that bears no homology to the human genome which was used as a nontargeting random control siRNA (sense strand: 5′-AGUACUGCUUACGAUACGGdTdT-3′).

Preparation of lipidic NPs and nanolipoplexes

Nx lipid-based NPs were prepared as previously described. ²⁸ Briefly, an ethanol solution of a lipid mixture containing 1-mg DOGS, 1-mg of DOPE, and 0.5-mg cardiolipin was injected in an aqueous solution to form small unilamellar vesicles (SUVs) with a final concentration of 6.25-mg/mL lipids. This preparation can be kept for over 9 months at 4°C. ³⁶ To prepare fluorescein-labeled Nx (fluorescein isothiocyanate (FITC)-Nx) NPs, FITC-DOPE was used. Nanolipoplexes (Nx/siRNA) were prepared as previously described ²⁸ by mixing the SUV suspension with siRNA in sterile RNase-free water at two different lipids: nucleic acid (NA) weight ratios to obtain cationic (Nx+12/siRNA) or anionic (Nx-40/siRNA) nanolipoplexes depending on the NA load charge in the formulation. After incubating for 30 min at room temperature, the mixture was slightly mixed and stored at 4°C until their use. Nanolipoplexes were used within 2 weeks from preparation.

Particle size and surface charge

Particle size and zeta potential were measured by dynamic light scattering (DLS) using a Zetasizer Nano ZS (Malvern Instruments Limited, Worcestershire, UK) with 633-nm laser wavelength and a measurement angle of 173° (backscatter detection) at 25°C in water (pH 7.0). Zeta (ζ) potential was employed to evaluate surface charge density of the nanoformulations. The width of the DLS hydrodynamic diameter distributions is indicated by the polydispersity index. Nanoformulations were vortexed before analysis and each sample was measured in triplicate.

Cell culture

The TZM-bl indicator cell line was generated from a clone of HeLa cells (JC.53, clone 13). TZM-bl cells express human CD4 and the human chemokine receptors CXCR4 and CCR5 and are highly sensitive to infection with diverse isolates of HIV-1. TZM-bl cells express ß-galactosidase and luciferase under the control of HIV-1 promoter, which is transactivated by the Trans-Activator of Transcription (HIV-1 Tat) protein in relation to the level of virus replication. ^34,35 THP-1 cells were maintained in RPMI 1640 and TZM-bl cells in DMEM, supplemented with 10% heat-inactivated fetal bovine serum, 50-μg/mL gentamicin, and 2-mM L-glutamine at 37°C, 5% carbon dioxide (CO₂). THP-1 MDMs were obtained by adding PMA to the culture medium at a final concentration of 10 ng/mL. THP-1 cells were incubated for 3–5 days to allow the monocytes to differentiate into macrophages. MDMs will be referred to in the text and figures hereafter as macrophages. All cells were cultured in 75-cm² culture flasks at 37°C in a humidified atmosphere of 5% CO₂ and were harvested when they reached about 80–85% confluence using 0.25% trypsin-EDTA solution diluted in PBS.

Cytotoxicity assays

Pro-inflammatory assays

Confocal studies

THP-1 cells were seeded in a glass flat-bottomed 96-well tissue culture plates for confocal microscopy (MatTek, Ashland, Massachusetts, USA) at a density of 2 × 10⁴ cells per well with culture medium containing PMA to allow differentiation. After 5 days of incubation, differentiated THP-1 cells were washed with PBS and fresh culture medium containing FITC-labeled Nx NPs at 4.4 µg/mL or nanolipoplexes (Cy3-labeled anti-CCR5 siRNA complexed to nonfluorescent Nx NPs at a final concentration of 50 nM, corresponding to 8.8 µg/mL NPs for Nx+12/Cy3-siCCR5 or 2.6 µg/mL NPs for Nx-40/Cy3-siCCR5) was added to the wells. In control wells, only culture medium was added to the cells. Cells were incubated at 37°C for 30 min to 24 h, then washed with PBS and fresh culture medium without NPs was added to each well. Cellular uptake and internalization of FITC-Nx NPs or Nx/Cy3-siCCR5 nanolipoplexes were visualized using confocal laser scanning microscopy (LSM700; Carl Zeiss Canada, Toronto, Ontario, Canada). For localization studies, cells were stained for late endosomes/lysosomes using the blue fluorescent dye LysoTracker and for cellular membrane using the red fluorescent dye WGA as per the manufacturer protocols as described earlier. ²⁶ Briefly, live cells were stained with 75 nM LysoTracker Blue DND-22 for 30 min prior to microscopy observation. For membrane staining, cells were fixed with cold methanol for 5 min at –20°C, then washed three times with 2-mL HBSS without phenol red and incubated with WGA conjugated with Texas Red for 10 min at room temperature.

HIV inhibition assay

TZM-bl cells were seeded at 6 × 10³ cells per well in a 96-well microtiter plate and allowed to adhere overnight in a humidified CO₂ incubator at 37°C. Then, supernatants were removed and replaced by fresh complete DMEM culture medium containing various concentrations of cationic or anionic nanolipoplexes (Nx+12/ or Nx-40/siCXCR4 or Nx+12/ or Nx-40/random siRNA sequence) and cells were incubated for 24 h at 37°C. Following incubation, cells were rinsed to remove the nanolipoplexes suspension and were infected with HIV-1-IIIB at multiplicity of infection 0.1 for 2 h at 37°C. After infection, cells were rinsed to eliminate unbound viruses and fresh complete culture medium was added. Then, cells were incubated for an additional 24vh at 37°C. After that time, HIV replication was evaluated by measuring ß-galactosidase activity using the Beta-Glo assay accordingly to the manufacturer instructions. Briefly, 100 µL of Beta-Glo reagent was added to each well and plates were incubated for 30 min at room temperature. Light intensity of each well was measured using a BioTek Synergy 2 multimode instrument (Thermo Fisher Scientific, Nepean, Ontario, Canada). Various control wells were added to each plate: wells without cells (background controls), uninfected cells without nanolipoplexes (negative controls), infected cells without nanolipoplexes (positive controls), and infected cells treated with nanolipoplexes containing a nontargeting scrambled siRNA sequence (random controls).

Statistics

Statistical analysis was performed using SigmaPlot (version 12.5). A p value of less than 0.05 was considered as statistically significant.

Physicochemical characteristics of the nanoformulations

Particle size analysis and surface charge of Nx NPs and Nx/random siRNA nanolipoplexes were determined by DLS in water and summarized in Table 1. Small particles with a diameter under 100 nm were formed. The size of both the cationic Nx+12/random siRNA (92.2 ± 5.6 nm) and the anionic Nx-40/random siRNA (93.5 ± 17 nm) nanolipoplexes was slightly higher compared to Nx only NPs (83.4 ± 12.5 nm), indicating the presence of the siRNAs in the nanolipoplex formulations. Homogenous lipid NPs and nanolipoplexes were obtained with polydispersity varying from 0.192 to 0.327 depending on the nanoformulations (Table 1). Zeta potential varied depending on the ratio lipid: NA as expected being less positive when higher amount of NA was used in the preparation of the nanolipoplexes. Nx NPs containing no NA formed the highest positively charged particles (+50.7 ± 2 mV) while Nx+12 nanolipoplexes containing NA at a ratio of lipid: NA of 13 formed less positively charged particles (+37 ± 5.5 mV) and the Nx-40 nanolipoplexes containing the highest amount of NA with a lipid: NA ratio of 3.9 formed highly negatively charged particles (−55.9 ± 3.3 mV) compared to Nx NPs only. The DLS results indicated a narrow size distribution and a good colloidal stability of the NPs (Online Supplementary Figure S1). The results were reproducible in multiple batches.

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Table 1.

Physicochemical characterization of the lipoplexes used in this study.^a

Lipoplexe	Ratio lipid–NA	Size (nm)	Polydispersity	Zeta potential (mV)
Nx only		83.4 ± 12.5	0.327 ± 0.035	+50.7 ± 2.0
Nx+12	13	92.2 ± 5.6	0.192 ± 0.016	+37.0 ± 5.5
Nx-40	3.9	93.5 ± 17.0	0.229 ± 0.043	−55.9 ± 3.3

Nx: Neutraplex; Nx+: cationic Nx lipoplexes; Nx−: anionic Nx lipoplexes; NA: nucleic acid.

^aParticles mean diameter was measured by dynamic light scattering and surface charge by zeta potential. Nx lipoplexes were prepared as described in the Materials and methods using the control scrambled siRNA sequence. Values represent mean ± standard deviation of four different experiments (n = 4).

Cytotoxicity assessment

First we evaluated the effects of the Nx NPs on THP-1 macrophages cell viability using the CTB assay and on cell integrity by measuring the amount of LDH released from the cells after 48 h of exposure to increased concentration of NPs from µg/mL 2.19 to 35 µg/mL. As illustrated in Figure 1(a), Nx NPs did not affect macrophages cell viability or cytoplasmic membrane integrity up to 17.5 µg/mL. When the cells were treated with 35 µg/mL, cell viability decreased to 84.5% and release of LDH increased by 18% when compared to untreated control cells (p < 0.001). Next, we wanted to investigate the effects of NPs on THP-1 macrophages cell viability and integrity over time (Figure 1(b)) looking at times earlier than 48 h to confirm we have not missed any possible transient cytotoxic effect that might have been observed within 24 h of exposure. Since we found that exposure to 35 µg/mL of NPs for 48 h did not potently affect the cells (Figure 1(a)), for this next series of experiments, we only used the two highest concentrations tested in the previous assay (17.5 and 35 µg/mL) and added a higher concentration (70 µg/mL) to further evaluate the cytotoxicity of the NPs (Figure 1(b)). We observed that the maximal effect on macrophages cell viability was already obtained after 4 h of incubation, 73 and 79% for 70 and 35µg/mL, respectively, but was not found statistically different from 48 h (for the 35 µg/mL concentration; 84.5%, Figure 1(a)). The difference in cell viability over time was only found to be statistically significant at 35 µg/mL between 4-h and 24-h post-incubation (p < 0.05). The rate of CTB reduction within each time of incubation was not found to be concentration-dependent between 2 h and 24 h, although it was previously found statistically significant at 48 h (17.5 vs. 35 µg/mL, p < 0.05; Figure 1(a)). To note, NPs did not interfere with the CTB reduction assay as shown in Online Supplementary Figure S2. Maximum release of LDH (122%) was observed at 16 h in cells treated with 70 µg/mL (Figure 1(c)) but was only found significantly higher when compared with cells incubated for 2 h (2 vs. 16h, p < 0.01) and was not found to be statistically different from LDH release at 48 h (119%; data not shown). At 35 µg/mL, LDH release was not found significantly higher at any time point between 2 h and 24 h than at 48 h (118%; Figure 1(a)). A concentration-dependent effect on LDH release was only observed at 16-h post-incubation (Figure 1(c)) between 35 µg/mL and 70 µg/mL (p < 0.05). At 48 h, we have also observed a significant difference between 17.5 µg/mL and 35 µg/mL (p < 0.05; Figure 1(a)). THP-1 macrophages treated with 10% DMSO served as positive controls in these experiments and the results are shown in Online Supplementary Figure S3. At this concentration of DMSO, macrophages cell viability was already dramatically reduced at 2 h, while LDH release increased over time to be maximal at 24 h. These results validate the CTB and LDH assays performed.

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Figure 1.

Cytotoxicity assays: Concentration-dependent effects and time-course of Nx nanoparticles. Macrophages were exposed to nanoparticles for A) 48h or B-C) the indicated time, then reduction of CellTiter-Blue and LDH release in culture supernatant were measured using the respective cytotoxicity assay as described in Materials and Methods. Data represent the means ± SEM of A) three separate experiments done in triplicates or B-C) at least two independent experiments done in triplicates. B-C) Dark grey column: 17.5µg/mL; Light grey column: 35µg/mL; Black column: 70µg/mL. Statistically significant based on a two-way ANOVA analysis A) Concentration, Assays or B-C) Concentration, Time. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001.

Cellular uptake and intracellular localization of NPs

Next, we wanted to evaluate the ability of human macrophages to internalize the Nx NPs. First, we performed a time course study by exposing THP-1 macrophage cells to 4.4 µg/mL of FITC-labeled Nx NPs for 30 min to 24 h. Uptake and localization of the FITC-Nx NPs were visualized using confocal microscopy and by staining the cells for late endosomes/lysosomes (LysoTracker Blue) and cellular membrane (WGA; Red) markers. Results of the time course study are shown in Figure 3. After 30 min exposure, NPs were already found inside the cells (Figure 2(b)) and cell uptake increased over time significantly up to 24 h (Figure 2(b) and (d) to (f)). NPs seemed to be endocytosed by the macrophages since they were found to co-localize with the late endosome/lysosome marker at 30 min and 1.5 h (Figure 2(b) and (d), respectively). As early as 1.5-h post-exposure, fluorescent NPs showed a diffuse pattern in the cytoplasm of some cells indicating that they were able to escape the late endosomes/lysosomes compartment. After longer time of exposure (3 and 24 h), NPs were found mainly free in the cytoplasm of live cells (Figure 2(e) and (f), respectively). At the low concentration tested (4.4 µg/mL of NPs), after 24-h post-exposure, fluorescence in live cells was very bright and diffuse throughout the cytoplasm (Figure 2(f)). Next, we wanted to investigate whether the NPs could deliver siRNAs into macrophages. For these experiments, 50 nM Cy3-labeled siCCR5 (red fluorescence) was complexed with Nx NPs at two different lipids: NA ratios as described earlier and cellular uptake of nanolipoplexes was visualized 24 h after exposure in THP-1 macrophages stained for late endosomes/lysosomes (blue fluorescence). Results are shown in Figure 3. In control cells, no red fluorescence was observed (Figure 3(a) to (d)). In macrophages exposed to cationic nanolipoplexes Nx+12/Cy3-siCCR5 (Figure 3(e) to (h)), red fluorescence showed a punctate pattern and was found mainly inside the cells and not co-localized with late endosomes/lysosomes (Figure 3(g) and (h)). In some cells, fluorescent siRNAs were observed close to the cellular membrane still in a punctate pattern but not in the endosomal pathway and they were also found co-localized with the late endosomes/lysosomes marker. Anionic nanolipoplexes Nx-40/Cy3-siCCR5 were also efficiently taken up by macrophages (Figure 3(i) to (l)). At 24-h post-exposure, the red fluorescence showed also a punctate pattern but was not as bright as in the cells exposed to the cationic Nx+12/Cy3-siCCR5 nanolipoplexes (Figure 3(k) and (l)) and was found mainly co-localized with the late endosomes/lysosomes marker (Figure 3(l)). However, when incubation was prolonged to 48 h, Cy3-siCCR5 uptake and delivery inside the cells was similar for both nanolipoplex formulations with very bright fluorescence mainly distributed throughout the cytoplasm and still showing a punctate pattern (Online Supplementary Figure S4). To note, cell morphology was not found different in control cultures than in cells exposed to either NPs alone or nanolipoplexes. Finally, Z-stacks analysis confirmed that the NPs were indeed internalized and not just adhering to the surface of the cells (data not shown).

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Figure 2.

Time course of uptake and internalization of Nx nanoparticles in macrophages. Cells were exposed to FITC-labelled NPs (green fluorescence) for B) 30min, D) 1.5h, E) 3h, F) 24h at a final concentration of 4.4µg/mL. A and C) Untreated control cultures incubated without NPs for A) 30min or C) 1.5h. Cells were stained for A-E) late endosomes/lysosomes using Lysotracker (blue fluorescence) and A-D) cellular membrane using Wheat Germ Agglutinin (red fluorescence). E and F) Live cells. After incubation cells were washed and visualized by confocal microscopy. Multiple fields were analyzed and images are representative of two experiments.

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Figure 3.

Uptake of nanolipoplexes in live macrophages. A-D) Untreated control cells incubated for 24h without nanolipoplexes, E-L) Cells exposed to nanolipoplexes (red fluorescence) at a final concentration of 50 nM for 24h: E-H) Nx+12/Cy3-siCCR5 (corresponding to 8.8 µg/mL of NPs) or I-L) Nx-40/Cy3-siCCR5 (corresponding to 2.6 µg/mL of NPs). After incubation cells were stained for late endosomes/lysosomes using Lysotracker (blue fluorescence), washed, and visualized by confocal microscopy. Multiple fields were analyzed and images are representative of two experiments.

Pro-inflammatory effects of NPs

The pro-inflammatory effects of Nx NPs were investigated by first evaluating whether the NPs could alter neutrophil apoptosis rates measured by cytology and their ability to spread onto glass. As shown in Figure 4(a), after 24 h of culture, control neutrophils were approximately 40% apoptotic as measured by cytology. As expected, GM-CSF delayed apoptosis (22.5%), whereas ATO induced neutrophil apoptosis (70%). In this assay, Nx NPs were not found to modulate neutrophil apoptosis (46%) at the concentration tested (100 µg/mL). To note, due to the limited number of donors (2–3 donors), these responses were not found to be statistically significant. In addition, because it was previously found that apoptotic neutrophils lose their ability to spread onto glass, ^42,43 we investigated whether Nx NPs could regulate this response. The results shown in Figure 4(b) are in agreement with the cytology data showing that Nx NPs did not alter the neutrophil apoptotic rate. After 12 h of culture, the number of spread cells remained stable after exposure to 100-µg/mL Nx NPs (25%) and was similar to the number of spread cells found in cells treated with water (17%). In contrast, cultures treated with GM-CSF, fMLP, or TiO₂ significantly increased the number of spread cells showing their ability to modify apoptotic rate in human neutrophils as we have previously reported. ^44,45 Furthermore, Nx NPs were tested for their capacity to alter the production of pro-inflammatory cytokines by human macrophages using the THP-1 cell model. Since in the confocal studies we found that with only 4.4 µg/mL, THP-1 cells were fully exposed to the Nx NPs, for this series of experiments we reasoned that exposure to the highest concentrations (35 and 70 µg/mL) will not be necessary. We evaluated three concentrations (2.2, 8.8, and 17.5 µg/mL). As illustrated in Figure 4(c), Nx NPs did not significantly induce pro-inflammatory cytokine secretion when compared to control cultures up to the highest concentration tested (17.5 µg/mL).

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Figure 4.

Inflammatory effects of Neutraplex nanoparticles. A) Effects on neutrophil apoptosis. Freshly isolated human polymorphonuclear cells (PMNs) (10 × 10⁶ cells/mL) were incubated with water (Ctrl), 65ng/mL GM-CSF (GM), 5µM ATO, or 100µg/mL of Nx NPs for 24h, and apoptosis was assessed by cytology (Diff-Quick staining) as described in Materials and Methods. Results are means ± SEM (n≥2 different blood donors). B) Effects on neutrophil spreading. PMNs were incubated for 12h with water (Ctrl), 65ng/mL GM-CSF (GM), 10^-9M fMLP, 100µg /mL TiO2, or 100µg /mL Nx NPs and the spreading of cells onto glass was assessed as described in Materials and Methods. Results are means ± SEM (n=4 different blood donors). C) Effects on cytokine secretion. THP-1 macrophages were exposed to increasing concentrations of Nx NPs for 48h then supernatants were collected. Concentration of cytokines present in the sample was analysed using a multiplex bead assay. Results are means ± SD of duplicates. Data for IL-6 and TNF-alpha are below 1 pg/mL. *, P < 0.05 vs the control and ***, P < 0.001 vs the control.

SiRNA activity of nanolipoplexes

Next, we wanted to evaluate whether siRNAs complexed to Nx NPs could conserve their activity and be developed as anti-HIV therapeutics. To assess this, we used the TZM-bl reporter cell line which expresses CCR5, CXCR4, and CD4 molecules allowing the cells to be infected by HIV and previously validated anti-CXCR4 siRNAs for their RNAi activity. ²⁸ First, we verified by confocal microscopy, the ability of the nanolipoplexes to be internalized by TZM-bl cells. We found that both nanolipoplexes were efficiently taken up by the cells and were able to escape the endosomes after 3 h of exposure (data not shown). Then, we performed the HIV inhibition assays. Data are shown in Figure 5 in terms of percent reduction of ß-galactosidase activity compared to the nontargeting control siRNA sequence. In this assay, the highest level of inhibition of HIV replication observed was around 30% when using Nx-40/siCXCR4 nanolipoplexes at 12.5 and 25 nM when compared to Nx-40/random siRNA controls. However, although the results showed a concentration-dependent effect, it was not found statistically significant for both nanolipoplex formulations at concentrations higher than 12.5 nM (p ≤ 0.05). Anionic Nx-40 nanolipoplexes showed higher inhibitory effect than cationic Nx+12 nanolipoplexes at all concentrations tested except at 100 nM, but again the differences were not found statistically significant. We also evaluated the effects of the Nx NPs alone (no siRNAs) on the replication of HIV-1 (Online Supplementary Figure S5). We found that at concentrations of NPs of 8.8 µg/mL and higher, ß-galactosidase activity was reduced when compared to untreated control cultures (p < 0.001) indicating that the Nx NPs themselves can interfere with HIV-1 replication in this infectivity assay. In addition, the effect of Nx NPs alone on the viability of TZM-bl cells was evaluated using the CTB assay and no cytotoxicity was observed up to 35 µg/mL, the highest concentration tested (Online Supplementary Figure S6). Finally, Nx/siRNA nanolipoplexes did not cause cytotoxicity in TZM-bl cells up to the highest concentration tested: 111.05% ± 3.82% and 94.98% ± 5.01% of untreated control cells with 200 nM of Nx+12/siRNA and 400 nM of Nx-40/siRNA, respectively (data not shown).

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Figure 5.

Inhibition of HIV-1 replication by Nx/siCXCR4 nanolipoplexes. TZM-bl cells were first exposed to Nx nanolipoplexes at the indicated concentration then cells were infected with HIV-1 for 2h at MOI 0.1. After 2h, fresh culture medium was added and cells were further incubated for 24h. HIV replication was monitored using the β-Glo assay as described in Materials and Methods. Data represent mean ± SD of four replicates (for Nx-40/siCXCR4) or mean ± SEM of two experiments with at least four replicates (for Nx+12/siCXCR4) and are corrected for background signal. Numbers in brackets represent the amount of lipid in µg/mL in the nanolipoplex formulations.