Novel targeting of PEGylated liposomes for codelivery of TGF-β1 siRNA and four antitubercular drugs to human macrophages for the treatment of mycobacterial infection: a quantitative proteomic study

Figures & data

Figure 1 Representative spectra of SM, PEG, SM-PEG, DMSO, and dd-H₂O.

Notes: Representative UV and chemical structure of SM (A), FL (B), and FTIR (C) spectra for SM, PEG, SM-PEG, DMSO, and dd-H₂O. FTIR shows SM-PEG formed by the −OH of SM connected with -NH₂ of PEG. Arrow 1 shows the spectral peaks of PEG, and arrow 2 points to the spectral peaks of SM.
Abbreviations: dd-H₂O, distilled deionized water; DMSO, dimethyl sulfoxide; FL, fluorescence; FTIR, Fourier transform infrared spectroscopy; PEG, polyethylene glycol; SM, streptomycin; UV, ultraviolet.

Figure 2 The morphology of newly synthesized NP-siRNA liposomes under SEM and TEM.

Notes: (A) SEM images showing the round shapes of newly synthesized NP-siRNA liposomes. (B) TEM images showing the spherical shapes of the newly synthesized NP-siRNA liposomes. (C) TEM images showing the irregular shapes of PEG. (D) TEM images showing the macrophages without exposure to the NP-siRNA liposomes. (E) TEM images showing the endocytosis of NP-siRNA liposomes by macrophages at 37°C. (F) TEM images showing the endocytosed NP-siRNA liposomes in macrophages at 37°C. (G) TEM images showing lack of endocytosis of NP-siRNA liposomes by macrophages at 4°C. Arrows point to NP-siRNA liposomal nanoparticles.
Abbreviations: NP, nanoparticle; PEG, polyethylene glycol; SEM, scanning electron microscopy; siRNA, small interfering RNA; TEM, transmission electron microscopy.

Figure 3 The size distribution of the novel NP-siRNA liposomes.

Notes: The size and the zeta potential of the novel NP-siRNA liposomes were measured using a Malvern Zeta Nanosizer ZS 90. The peak size of the novel NP-siRNA liposomes was 265.1 nm with a PDI of 0.290 and zeta potential was 31.8 mV.
Abbreviations: NP, nanoparticle; PDI, polydispersity index; siRNA, small interfering RNA.

Figure 4 Drug release profiles of NP-siRNA liposomes containing the anti-TB drugs and free antidrugs determined by a dialysis method.

Notes: (A) The drug release profile of liposomes and free INH over 72 hours. (B) The drug release profile of liposomes and free RIF over 72 hours. (C) The drug release profile of liposomes and free PZA over 72 hours. (D) The drug release profile of liposomes and free SM over 72 hours. Drug concentrations are measured by validated LC–MS methods. Data are the mean ± SD of three independent experiments. Data were analyzed by two-way ANOVA. Statistical probability is indicated as follows: **P<0.01; ***P<0.001 by two-way ANOVA.
Abbreviations: ANOVA, analysis of variance; INH, isoniazid; LC–MS, liquid chromatography mass spectrometry; NP, nanoparticle; PZA, pyrazinamide; RIF, rifampicin; SD, standard deviation; siRNA, small interfering RNA; SM, streptomycin; TB, tuberculosis.

Figure 5 Cytotoxicity of the novel NP-siRNA liposomes and free anti-TB drugs toward THP-1-derived macrophages determined by the MTT assay.

Notes: Cells were cultured and treated with NP-siRNA liposomes or the anti-TB drugs for 24 hours. The IC₅₀ values were determined and compared. (A) Survival of THP-1-derived macrophages treated with 2.5–1,000 μg/mL NP-siRNA liposomes for 24 hours. (B) Survival of THP-1-derived macrophages treated with 2.5–200 μg/mL combined anti-TB drugs including INH, RIF, PZA, and SM for 24 hours. (C) Survival of THP-1-derived macrophages treated with 0.1–100 μM INH for 24 hours. (D) Survival of THP-1-derived macrophages treated with 1–100 μM RIF for 24 hours. (E) Survival of THP-1-derived macrophages treated with 1–100 μM PZA for 24 hours. (F) Survival of THP-1-derived macrophages treated with 0.1–100 μM SM for 24 hours. Data are the mean ± SD of three independent experiments.
Abbreviations: INH, isoniazid; MTT, thiazolyl blue tetrazolium bromide; NP, nanoparticle; PZA, pyrazinamide; RIF, rifampicin; SD, standard deviation; siRNA, small interfering RNA; SM, streptomycin; TB, tuberculosis.

Figure 6 Effects of the novel NP-siRNA liposomes on cell cycle distribution, apoptosis, and autophagy in THP-1-derived macrophages.

Notes: Cells were treated with the novel NP-siRNA liposomes at 2.5, 12.5, and 62.5 μg/mL for 24 hours, and the effects on cell cycle distribution, apoptosis, and autophagy in macrophages were examined using flow cytometry and confocal microscopy. (A) Representative DNA fluorescence histograms show the effect of treatment with NP-siRNA liposomes on cell cycle distribution. (B) Bar graphs show the percentage of macrophages in G₁, S, and G₂/M phases. (C) Representative flow cytometric dot plots show the distribution of macrophages undergoing early or late apoptosis when treated with the NP-siRNA liposomes at 2.5, 12.5, and 62.5 μg/mL for 24 hours. (D) Bar graphs show the percentage of macrophages undergoing early or late apoptosis when treated with the NP-siRNA liposomes at 2.5, 12.5, and 62.5 μg/mL for 24 hours. (E) Representative flow cytometric dot plots show the autophagic cells when treated with the NP-siRNA liposomes at 2.5, 12.5, and 62.5 μg/mL for 24 hours. (F) Bar graphs show the percentage of autophagic macrophages when the cells were treated with the NP-siRNA liposomes at 2.5, 12.5, and 62.5 μg/mL for 24 hours. (G) The confocal microscopic images show the Cyto-ID^® stained autophagic cells with green fluorescence when the cells were treated with the NP-siRNA liposomes at 2.5, 12.5, and 62.5 μg/mL for 24 hours. (H) Bar graphs show the fluorescence level, reflecting the level of autophagic macrophages when the cells were treated with the NP-siRNA liposomes at 2.5, 12.5, and 62.5 μg/mL for 24 hours. Data are present as the mean ± SD of three independent experiments. ***P<0.001 by one-way ANOVA followed by Tukey’s post hoc test.
Abbreviations: ANOVA, analysis of variance; NP, nanoparticle; SD, standard deviation; siRNA, small interfering RNA.

Figure 7 Effects of the novel NP-siRNA liposomes on the expression levels of pro- and antiautophagic proteins in THP-1-derived macrophages determined by Western blotting assay.

Notes: (A) Representive blots of p-PI3K, p-mTOR, mTOR, p-p38 MAPK, p38 MAPK, PTEN, beclin 1, and LC3 in macrophages treated with the NP-siRNA liposomes at 2.5, 12.5, and 62.5 μg/mL for 24 hours. (B) Bar graphs show the effect of NP-siRNA liposomes on the levels of p-PI3K, p-mTOR, mTOR, p-p38 MAPK, p38 MAPK, PTEN, beclin 1, and LC3 in macrophages. β-actin was used as the internal control. Data are the mean ± SD of three independent experiments. *P<0.05 by one-way ANOVA followed by Tukey’s post hoc test.
Abbreviations: ANOVA, analysis of variance; LC3, microtube-associated protein 1A/1B light chain 3; MAPK, mitogen-activated protein kinase; mTOR, mammalian target of rapamycin; NP, nanoparticle; PI3K, phosphoinositide 3-kinase; PTEN, phosphatase and tensin homologue; SD, standard deviation; siRNA, small interfering RNA.

Figure 8 Effects of the novel NP-siRNA liposomes on the expression levels of TGF-β1, IL-6, IL-8, TNF-α, and IFN-γ in THP-1-derived macrophages determined by ELISA.

Notes: Macrophages were cultured and treated with 12.5 μg/mL NP-siRNA liposomes, DMSO, NP-siRNA-control or medium for 24 hours. The levels of cytokines were determined using ELISA. LPS was included as the positive control. Data are the mean ± SD of three independent experiments. *P<0.05; **P<0.01; and ***P<0.001 by one-way ANOVA followed by Tukey’s post hoc test.
Abbreviations: ANOVA, analysis of variance; DMSO, dimethyl sulfoxide; IFN, interferon; IL, interleukin; ELISA, enzyme-linked immunosorbent assay; LPS, liposaccharide; NP, nanoparticle; SD, standard deviation; siRNA, small interfering RNA; TGF-β1, transforming growth factor-β1; TNF-α, tumor necrosis factor-α.

Figure 9 Effects of the novel NP-siRNA liposomes on the mRNA expression levels of TGF-β1, IL-6, IL-8, TNF-α, and IFN-γ in THP-1-derived macrophages determined by RT-PCR assay.

Notes: THP-1-derived macrophages were cultured and treated with 12.5 μg/mL NP-siRNA liposomes, DMSO, NP-siRNA-control or medium for 6 hours. The mRNA levels of cytokines were determined using RT-PCR assay. Data are the mean ± SD of three independent experiments. *P<0.05 and **P<0.01 by one-way ANOVA followed by Tukey’s post hoc test.
Abbreviations: ANOVA, analysis of variance; DMSO, dimethyl sulfoxide; IFN, interferon; IL, interleukin; NP, nanoparticle; RT-PCR, real-time polymerase chain reaction; SD, standard deviation; siRNA, small interfering RNA; TGF-β1, transforming growth factor-β1; TNF-α, tumor necrosis factor-α.

Table 1 The top five canonical signaling pathways regulated by the novel NP-siRNA liposomes in THP-1-derived in macrophages

Pathway	P-value	Ratio
EIF2 signaling	2.93×10^−9	9/201 (0.045)
Actin cytoskeleton signaling	1.90×10^−7	8/242 (0.033)
Remodeling of epithelial adherens junctions	1.61×10^−6	5/70 (0.071)
Epithelial adherens junction signaling	4.19×10^−6	6/154 (0.039)
RhoGDI signaling	1.15×10^−5	6/202 (0.030)

Note: Data in the parenthesis shows the ratio of regulated proteins over the total proteins in the targeted signaling pathway.

Abbreviations: EIF2, eukaryotic initiation factor 2; RhoGDI, Rho GDP-dissociation inhibitor.

Table 2 The top eleven upstream proteins regulated by the novel NP-siRNA liposomes in THP-1-derived macrophages

Upstream regulator	P-value of overlap	Predicted activation state
APP	5.87×10^−17
CD3	4.90×10^−3	Inhibited
CD437	1.50×10^−5	Inhibited
HSF1	3.89×10^−6	Inhibited
MAPT	1.90×10^−18
MYC	2.30×10^−12	Activated
MYCN	1.16×10^−12	Activated
PSEN1	1.40×10^−15
Sirolimus	3.77×10^−11	Inhibited
TGF-β1	4.82×10^−5
TNF-α	1.27×10^−4

Abbreviations: APP, β-amyloid (A4) precursor protein; CD3, T-cell surface glyco-protein; CD437, a cell-permeable, selective agonist of retinoic acid receptor; HSF1, heat shock transcription factor 1; MAPT, microtubule-associated protein tau; MYC, v-myc avian myelocytomatosis viral oncogene homologue; MYCN, v-myc avian myelocytomatosis viral oncogene neuroblastoma derived homologue; PSEN1, presenilin 1; TGF-β1, transforming growth factor-β1; TNF-α, tumor necrosis factor-α.

Table 3 The upregulated and downregulated proteins involved in TGF-β1-mediated signaling pathway in THP-1-derived macrophages treated with the novel NP-siRNA liposomes at 12.5 μg/mL for 24 hours

ID	Gene in dataset	Prediction (based on expression direction)	Fold changea	Findingsb
P68104	EEF1A1	Affected	1.676	Regulated (1)
Q13509	TUBB3	Activated	1.638	Upregulated (3)
B7Z6Z4	MYL6	Activated	1.465	Upregulated (1)
Q01469	FABP5	Activated	1.402	Upregulated (1)
P06753	TPM3	Activated	1.392	Upregulated (9)
P28482	MAPK1	Activated	1.310	Upregulated (4)
P00558	PGK1	Inhibited	1.262	Downregulated (1)
P16070	CD44	Inhibited	1.261	Downregulated (3)
E9PK25	CFL1	Activated	1.155	Upregulated (1)
P06396-2	GSN	Inhibited	1.142	Downregulated (2)
Q01518	CAP1	Activated	1.072	Upregulated (2)
P50552	VASP	Activated	1.031	Upregulated (2)
P26038	MSN	Activated	1.012	Upregulated (1)
P07900-2	HSP90AA1	Activated	1.005	Upregulated (1)

Notes:

a The value indicates the ratio of heavy over light signal.

b The number in the brackets indicates the number of proteins involved.

Abbreviations: CAP1, CAP, adenylate cyclase-associated protein 1 (yeast); CD44, CD44 molecule (Indian blood group); CFL1, cofilin 1 (nonmuscle); EEF1A1, eukaryotic translation elongation factor 1 α1; FABP5, fatty acid binding protein 5 (psoriasis-associated); GSN, gelsolin; HSP90AA1, heat shock protein 90 kDa α (cytosolic), class A member 1; MAPK1, mitogen-activated protein kinase 1; MSN, moesin; MYL6, myosin, light chain 6, alkali, smooth muscle and nonmuscle; PGK1, phosphoglycerate kinase 1; TPM3, tropomyosin 3; TUBB3, tubulin, β3 class III; VASP, vasodilator-stimulated phosphoprotein.

Figure 10 The proteomic profiling of human macrophages treated with the novel NP-siRNA liposomes at 12.5 μg/mL for 24 hours determined using SILAC.

Notes: The proteomic responses of macrophages to NP-siRNA were examined using SILAC, and the pathways were analzyed by IPA. This figure shows the top pathways from the proteomic study with the threshold of 1.2 of −log(P-value) (A and B).
Abbreviations: IPA, ingenuity pathway analysis; NP, nanoparticle; SILAC, stable isotope labeling with amino acids in cell culture; siRNA, small interfering RNA.

Figure 11 EIF2 signaling pathway was regulated when THP-1-derived macrophages were treated with the novel NP-siRNA liposomes at 12.5 μg/mL for 24 hours.

Notes: The proteomic response was determined by SILAC, and the pathways were analyzed using IPA. The figure shows that ERK1/2 was upregulated in the EIF2 signaling pathway. Red indicates an upregulation; green indicates a downregulation. The intensity of green and red colors indicates the degree of down- or upregulation. Solid arrow indicates direct interaction, and dashed arrow indicates indirect interaction.
Abbreviations: EIF2, eukaryotic initiation factor 2; ER, endoplasmic reticulum; ERK, extracellular signal-regulated kinase; IPA, ingenuity pathway analysis; NP, nanoparticle; SILAC, stable isotope labeling with amino acids in cell culture; siRNA, small interfering RNA.

Figure 12 The ERK/MAPK signaling pathway was regulated when THP-1-derived macrophages were treated with the novel NP-siRNA liposomes at 12.5 μg/mL for 24 hours.

Notes: The proteomic response was determined by SILAC, and pathways were analyzed using IPA. Red indicates an upregulation; green indicates a downregulation. The intensity of green and red colors indicates the degree of down- or upregulation. Solid arrow indicates direct interaction, and dashed arrow indicates indirect interaction.
Abbreviations: ERK, extracellular signal-regulated kinase; IPA, ingenuity pathway analysis; MAPK, mitogen-activated protein kinase; NP, nanoparticle; SILAC, stable isotope labeling with amino acids in cell culture; siRNA, small interfering RNA.

Figure 13 The NF-κB signaling pathway was regulated via ERK1/2 when THP-1-derived macrophages were treated with the novel NP-siRNA liposomes at 12.5 μg/mL for 24 hours.

Notes: The proteomic response was determined by SILAC, and pathways were analyzed using IPA. Red indicates an upregulation; green indicates a downregulation. The intensity of green and red colors indicates the degree of down- or upregulation. Solid arrow indicates direct interaction.
Abbreviations: ERK, extracellular signal-regulated kinase; IPA, ingenuity pathway analysis; NF-κB, nuclear factor-κB; NP, nanoparticle; SILAC, stable isotope labeling with amino acids in cell culture; siRNA, small interfering RNA.

Figure 14 The PI3K/AKT and HSP90 interacted with ERK1/2 when THP-1-derived macrophages were treated with the novel NP-siRNA liposomes at 12.5 μg/mL for 24 hours.

Notes: The proteomic response was determined by SILAC, and pathways were analyzed using IPA. Red indicates an upregulation; green indicates a downregulation. The intensity of green and red colors indicates the degree of down- or upregulation. Solid arrow indicates direct interaction, and dashed arrow indicates indirect interaction.
Abbreviations: AKT, protein kinase B; ERK, extracellular signal-regulated kinase; HSP90, heat shock protein 90; IPA, ingenuity pathway analysis; NF-κB, nuclear factor-κB; NP, nanoparticle; PI3K, phosphoinositide 3-kinase; SILAC, stable isotope labeling with amino acids in cell culture; siRNA, small interfering RNA.

Figure 15 Effect of the novel NP-siRNA liposomes on the expression levels of p-ERK, ERK, NF-κB, and Nrf2 in THP-1-derived macrophages determined using Western blotting analysis.

Notes: (A) Representative blots for p-ERK, ERK, NF-κB, and Nrf2 in macrophages when treated with DMSO, NP, NP-siRNA-control, and NP-siRNA at 12.5 μg/mL for 24 hours. (B) Bar graphs show the relative expression levels of the proteins in macrophages. (C) Representative blot of NF-κB when macrophages were treated with the novel NP-siRNA liposomes at 2.5, 12.5, and 62.5 μg/mL for 24 hours. (D) Bar graphs show the relative expression level of NF-κB in macrophages. The β-actin was used as the internal control. Data are the mean ± SD of three in dependent experiments. *P<0.05; **P<0.01; and ***P<0.001 by one-way ANOVA followed by Tukey’s post hoc test.
Abbreviations: ANOVA, analysis of variance; DMSO, dimethyl sulfoxide; ERK, extracellular signal-regulated kinase; NF-κB, nuclear factor-κB; NP, nanoparticle; Nrf2, nuclear factor (erythroid-derived 2)-like 2; SD, standard deviation; siRNA, small interfering RNA.