Biocompatibility assessment of functionalized magnetic mesoporous silica nanoparticles in human HepaRG cells

Figures & data

Figure 1. TEM characterization of M-MSNs. (A) As the pristine state, (B) covered with PEG, and (C) cryogenic TEM characterization of lipid bilayer DMPC M-MSNs.

Table 1. Physicochemical characterization of pristine M-MSNs and those covered with PEG or DMPC lipid bilayer.

Nanoparticles	Diameter by TEM andCryoTEM* (nm)	Hydrodynamic diameterby DLS (nm)	Polydispersityindex	Zeta potential atpH 7.4 (mV)
Pristine M-MSNs	117 (±2)	172 (±6)	0.17	−39.1 (±1.4)
PEG M-MSNs	123 (±3)	156 (±1)	0.18	−30.4 (±2.9)
DMPC M-MSNs*	132 (±4)	180 (±2)	0.12	−10.3 (±0.4)

Values are mean ± standard deviation (n = 3).

*Characterization by CryoTEM.

Figure 2. MTT cell viability assay of HepaRG cells exposed for 48 h to increase concentrations of pristine M-MSNs (blue curve), PEG M-MSNs (purple curve), DMPC M-MSNs (red curve), and chlorpromazine as the positive control (grey curve). Concentration equivalences: * = 1.6 µg/cm², ** = 16 µg/cm², and *** = 80 µg/cm². Results are expressed as mean percent viable cells ± SD (n = 3) compared with control cells.

Figure 3. Real-time impedance cell index (CI) monitoring of HepaRG cells (n = 3) exposed to (A) 16 µg/cm² and (B) 80 µg/cm² pristine M-MSNs (blue), PEG M-MSNs (purple), and DMPC M-MSNs (red) for 72 h. The black arrow represents the starting point of exposure. CI was normalized at this point to ensure inter-dose comparison and control cells were defined as baseline.

Figure 4. TEM imaging of HepaRG cells exposed to 16 µg/cm² for 6 h to (A) pristine M-MSNs, (B) PEG M-MSNs, (C) DMPC M-MSNs, and for 24 h to (D) pristine M-MSNs, (E) PEG M-MSNs, (F) DMPC M-MSNs. N indicates the nucleus, BC indicates bile canaliculi, and arrows indicate NPs.

Figure 5. Time- and dose-dependent effects of exposure to NPs on the number of significantly differentially expressed genes. HepaRG cells were exposed to 1.6, 16, and 80 µg/cm² pristine, PEG-, and DMPC-coated M-MSNs for 24 or 48 h. After extraction and labeling, RNA was hybridized to a human oligo microarray (6 × 60 k Agilent V3 SurePrint). Bars represent the number of differentially expressed transcripts after statistical analysis using Genespring GX13 software (Agilent), with a p value ≤0.05 and a fold-change ≥2.

Figure 6. Canonical pathways elicited by each M-MSN (80 µg/cm²). The percentage of modulated transcripts of our datasets belonging to six major altered canonical pathways after (A) 24 h and (B) 48 h exposure to 80 µg/cm² M-MSNs. These pathways were all significant according to a Fisher’s statistical test (p value <0.05), revealed with Ingenuity Pathway Analysis (IPA, QIAGEN).

Figure 7. Representation of M-MSN mode of action on HepaRG hepatocytes. HepaRG cultures (n = 3) were exposed for 24 and 48 h to three concentrations (1.6, 16, and 80 µg/cm²) of NPs including pristine (blue dot), PEG- (purple dot) and DMPC- (red dot)coated M-MSNs. N = nucleus. At 80 µg/cm², the hepatic cholestasis pathway is highlighted by the downregulation of most of its main effectors such as BSEP and NCTP, characterized by an intrahepatic accumulation of cytotoxic bile acids, which ultimately causes liver injury.

Table 2. Fold changes (FC) of main transcripts modulated by exposure to pristine, PEG, and DMPC M-MSNs (80 µg/cm² at 24 and 48 h) corresponding to hepatobiliary system disruption.

		Pristine M-MSNs		PEG M-MSNs		DMPC M-MSNs
Gene symbol	Entrez gene name	FC (24 h)	FC (48 h)	FC (24 h)	FC (48 h)	FC (24 h)	FC (48 h)	Pathways
ABCB11	ATP binding cassette subfamily B member 11	–	−4.0	–	–	–	−3.8	1, 2, 4
ABCB4	ATP binding cassette subfamily B member 4	−2.4	−2.2	–	−2.2	–	−3.5	4
ABCG5	ATP binding cassette subfamily G member 5	−6.3	−17.0	–	−7.0	−3.5	−19.8	1, 4
ABCG8	ATP binding cassette subfamily G member 8	−5.5	−9.1	–	−5.7	−3.3	−9.0	1, 4
ACOX2	Acyl-CoA oxidase 2	−2.5	−6.5	–	−3.8	−2.6	−7.6	1
AHRR	Aryl-hydrocarbon receptor repressor	–	−2.1	–	–	–	–	6
AKR1C4	Aldo-keto reductase family 1 member C4	−2.9	−3.7	–	−3.6	−2.6	−5.2	1
AKR1D1	Aldo-keto reductase family 1 member D1	−2.3	−6.1	–	−7.5	−2.7	−8.9	1
ALDH1A1	Aldehyde dehydrogenase 1 family member A1	−4.1	−3.0	–	−2.7	−2.8	−2.7	6
ALDH1A3	Aldehyde dehydrogenase 1 family member A3	–	−2.2	–	−2.0	–	−3.6	6
ALDH1L1	Aldehyde dehydrogenase 1 family member L1	−3.6	−6.4	–	−4.7	−2.8	−4.2	1
ALDH1L1	Aldehyde dehydrogenase 1 family member L1	−3.6	−6.4	–	−4.7	−2.8	−4.2	6
ALDH3A1	Aldehyde dehydrogenase 3 family member A1	−2.3	–	–	−3.0	–	–	6
ALDH5A1	Aldehyde dehydrogenase 5 family member A1	−2.1	−2.6	–	−2.2	–	−2.7	6
ALDH6A1	Aldehyde dehydrogenase 6 family member A1	−2.4	−3.9	–	−3.2	–	−3.9	6
ALDH7A1	Aldehyde dehydrogenase 7 family member A1	−4.2	−5.0	–	−4.6	–	−2.4	1, 6
CAT	Catalase	−2.3	−3.4	–	−2.4	–	−3.5	6
CHST11	Carbohydrate sulfotransferase 11	2.8	2.8	–	3.1	2.4	2.9	6
CHST13	Carbohydrate sulfotransferase 13	–	−2.5	–	–	–	−2.3	6
CHST15	Carbohydrate sulfotransferase 15	4.3	2.8	–	–	3.8	–	6
CHST3	Carbohydrate sulfotransferase 3	–	2.3	–	4.0	–	–	6
CITED2	Cbp/p300 interacting transactivator with Glu/Asp rich carboxy-terminal domain 2	–	−2.7	–	–	–	–	6
CRP	C-reactive protein	8.1	12.9	2.1	10.7	4.8	6.6	5
CSF2	Colony stimulating factor 2	6.2	3.0	–	3.0	4.2	–	4
CUL3	Cullin 3	3.0	–	–	–	–	–	6
CXCL8	C-X-C motif chemokine ligand 8	3.9	–	–	–	–	–	5
CYP1A1	Cytochrome P450 family 1 subfamily A member 1	–	–	–	–	−2.6	–	6
CYP1A2	Cytochrome P450 family 1 subfamily A member 2	−5.2	−7.5	–	−9.2	−6.4	−10.8	2, 6
CYP27A1	Cytochrome P450 family 27 subfamily A member 1	–	−3.3	–	−2.1	–	−2.4	4
CYP2A6 (includes others)	Cytochrome P450 family 2 subfamily A member 6	−6.1	−339.0	–	−22.0	−7.7	−89.2	1, 2
CYP2B6	Cytochrome P450 family 2 subfamily B member 6	−2.6	−37.9	–	−7.1	−2.4	−26.3	1, 2, 6
CYP2C19	Cytochrome P450 family 2 subfamily C member 19	−5.0	−13.8	–	−4.3	−2.7	−10.5	1, 2, 6
CYP2C8	Cytochrome P450 family 2 subfamily C member 8	−8.9	−20.7	–	−4.7	−4.9	−10.4	1, 2, 6
CYP2C9	Cytochrome P450 family 2 subfamily C member 9	−3.4	−12.3	–	−4.3	−2.7	−10.8	1, 2, 6
CYP3A4	Cytochrome P450 family 3 subfamily A member 4	−13.1	−70.3	–	−19.0	−4.8	−35.9	1, 2, 6
CYP3A5	Cytochrome P450 family 3 subfamily A member 5	−3.2	−2.5	–	−2.7	−2.6	−2.9	1, 2, 6
CYP3A7	Cytochrome P450 family 3 subfamily A member 7	−7.1	−9.8	–	−9.8	−3.7	−8.6	1, 2, 6
CYP4A11	Cytochrome P450 family 4 subfamily A member 11	−3.0	−19.8	–	−5.5	−4.3	−16.3	1
CYP7A1	Cytochrome P450 family 7 subfamily A member 1	−90.4	−83.1	−2.7	−48.8	−21.9	−42.3	1, 2, 4
CYP8B1	Cytochrome P450 family 8 subfamily B member 1	−2.1	−5.4	–	–	–	−6.4	1, 4
FMO1	Flavin containing monooxygenase 1	−5.7	−6.2	–	−3.6	−5.1	−6.4	6
FMO5	Flavin containing monooxygenase 5	−7.8	−12.7	–	−5.5	−3.5	−9.1	6
FOS	Fos proto-oncogene, AP-1 transcription factor subunit	4.1	3.6	2.8	3.2	3.0	2.4	5
G6PC	Glucose-6-phosphatase catalytic subunit	−8.9	−52.0	–	−8.5	−7.1	−44.3	2
GSTA2	Glutathione S-transferase alpha 2	−3.0	−6.5	–	−4.2	−2.6	−4.4	2, 6
GSTM1	Glutathione S-transferase mu 1	–	−3.3	–	−2.2	–	–	6
GSTM2	Glutathione S-transferase mu 2	–	−2.9	–	−2.6	–	−2.4	6
GSTM3	Glutathione S-transferase mu 3	−2.5	−2.8	–	–	–	–	6
HMGCS2	3-Hydroxy-3-methylglutaryl-CoA synthase 2	−4.2	−10.0	–	−5.0	−2.7	−5.0	2
HNF4A	Hepatocyte nuclear factor 4 alpha	−2.1	–	–	–	−2.3	–	2, 4
HSPB2	Heat shock protein family B (small) member 2	−2.9	−4.0	–	−3.7	–	–	5
IGFBP1	Insulin like growth factor binding protein 1	3.4	3.8	–	7.6	3.8	3.4	2
IKBKB	Inhibitor of nuclear factor kappa B kinase subunit beta	–	−2.4	–	–	–	–	4
IL1B	Interleukin 1 beta	3.3	–	–	3.2	3.8	–	1, 4, 5
IL1F10	Interleukin 1 family member 10 (theta)	2.7	–	–	–	–	–	5
IL1R2	Interleukin 1 receptor type 2	−2.4	−6.1	–	−3.5	–	–	1, 5
IL1RAP	Interleukin 1 receptor accessory protein	2.9	2.2	–	2.5	2.7	–	1, 4, 5
IL1RL1	Interleukin 1 receptor like 1	6.1	5.8	–	3.6	3.7	3.8	1, 4, 5
IL1RN	Interleukin 1 receptor antagonist	−2.2	−5.7	–	−3.3	–	−5.8	4, 5
IL6	Interleukin 6	55.1	13.0	7.1	9.2	21.6	5.9	2, 4, 5
IL6R	Interleukin 6 receptor	−2.1	–	–	–	–	–	5
IRAK2	Interleukin 1 receptor associated kinase 2	4.6	3.0	2.7	3.9	3.2	–	4
JUN	Jun proto-oncogene, AP-1 transcription factor subunit	2.5	2.2	2.3	2.6	2.3	–	4, 5
KLB	Klotho beta	−3.1	−3.6	–	−6.7	−2.0	−6.4	5
LBP	Lipopolysaccharide binding protein	–	2.1	–	–	–	–	4
LIF	Leukemia inhibitory factor	7.1	3.4	2.5	4.1	3.8	–	4
LY96	Lymphocyte antigen 96	–	3.4	2.2	3.0	2.2	2.8	4
MAP2K6	Mitogen-activated protein kinase kinase 6	−3.1	–	–	−2.6	−2.1	–	5
NDST4	N-deacetylase and N-sulfotransferase 4	−3.0	−5.3	–	−3.0	−2.0	−2.3	1
NFKB1	Nuclear factor kappa B subunit 1	2.0	–	–	–	–	–	5, 6
NFKB2	Nuclear factor kappa B subunit 2	2.1	–	–	2.2	–	–	5, 6
NFKBIB	NFKB inhibitor beta	–	2.1	–	–	–	–	4
NR0B2	Nuclear receptor subfamily 0 group B member 2	−7.2	−10.7	−3.6	−5.6	−5.0	–	1, 2, 4
NR1I2	Nuclear receptor subfamily 1 group I member 2	−2.9	−2.6	–	–	−2.1	−2.9	1, 2, 4, 6
NR1I3	Nuclear receptor subfamily 1 group I member 3	−4.1	−17.1	–	−5.7	−2.6	−18.9	1, 2, 6
PIK3R1	Phosphoinositide-3-kinase regulatory subunit 1	−2.8	−2.8	–	−2.1	−2.1	−2.2	5
PPARA	Peroxisome proliferator activated receptor alpha	−2.5	−2.0	–	–	–	–	2, 4
PPRC1	Peroxisome proliferator-activated receptor gamma, coactivator-related 1	3.0	2.8	–	2.5	2.3	–	4
PRKAR1B	Protein kinase cAMP-dependent type I regulatory subunit beta	–	2.1	–	–	–	–	4
PRKAR2B	Protein kinase cAMP-dependent type II regulatory subunit beta	−9.6	−11.4	−4.3	−20.6	−9.3	−6.7	2, 4
SLC10A1	Solute carrier family 10 member 1	−5.3	−16.4	–	−9.1	−3.8	−12.4	1, 4
SLC22A7	Solute carrier family 22 member 7	−2.5	−8.9	–	−2.8	−2.3	−9.6	4
SLC27A2	Solute carrier family 27 member 2	–	–	–	−2.1	–	−2.3	1
SLC27A5	Solute carrier family 27 member 5	−2.7	−15.3	–	−5.7	−3.0	−19.6	1
SLCO1B1	Solute carrier organic anion transporter family member 1B1	–	−3.9	–	–	–	−4.9	4
SLCO1B3	Solute carrier organic anion transporter family member 1B3	−3.6	–	–	–	–	–	1
SOCS1	Suppressor of cytokine signaling 1	24.2	13.5	4.2	9.4	9.2	5.2	5
SOCS3	Suppressor of cytokine signaling 3	8.9	7.3	–	2.8	3.1	2.5	5
SULT2A1	Sulfotransferase family 2A member 1	−4.7	−6.9	–	−4.8	−2.4	−4.4	1, 2, 6
UGT2B10	UDP glucuronosyltransferase family 2 member B10	−2.2	−3.5	–	−2.7	–	−5.1	6
UGT2B11	UDP glucuronosyltransferase family 2 member B11	−2.6	−4.0	–	−2.7	−2.4	−4.8	6
UGT2B15	UDP glucuronosyltransferase family 2 member B15	−2.4	−2.5	–	−2.6	−2.1	−4.0	6
UGT2B4	UDP glucuronosyltransferase family 2 member B4	−2.7	−4.1	–	−3.1	−2.5	−5.6	6
UGT2B7	UDP glucuronosyltransferase family 2 member B7	2.0	−2.1	–	–	–	−3.2	6

The exhaustive list with fold‐changes and p values is provided in ESI Table S4. Pathway 1, 2, 3, 4, 5, 6 are ‘Bile acid Biosynthesis’, ‘PXR/RXR’, ‘LPS/IL‐1 mediated inhibition of RXR function’, ‘Hepatic cholestasis’, ‘IL‐6 signaling’, and ‘Xenobiotic metabolism signaling’, respectively.