A Step-by-Step Approach to Improve Clinical Translation of Liposome-Based Nanomaterials, a Focus on Innate Immune and Inflammatory Responses
Abstract
:1. Introduction
2. Results
2.1. Physical-Chemical Characterization of Liposomes
2.2. AF4-DLS Analysis
2.3. AUC Confirmed Results Obtained with DLS and AF4-DLS
2.4. Bacterial and Endotoxin Contamination
2.5. Assessment of Liposome Cytotoxicity
2.6. Effects of Liposomes on Complement Activation
2.7. Effects of Liposomes on the Production of Inflammation-Related Cytokines by Human Blood Cells
3. Discussion
4. Conclusions
5. Materials and Methods
5.1. Selection of Liposomes
5.2. Physical-Chemical Characterization
5.3. Bacterial and Endotoxin Contamination
5.3.1. Assessment of Bacterial Contamination
5.3.2. Assessment of Endotoxin Contamination
5.4. Cytotoxicity Evaluation
5.4.1. Hep G2 Cells
5.4.2. PBMC
5.5. Complement Activation Assays
5.6. Activation of Human Blood Leukocytes
5.7. Statistical Analysis
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
AF4 | Asymmetrical Flow-field-flow-fractionation |
ANOVA | Analysis of variance |
AUC | Analytical Ultracentrifuge |
CHOL | Cholesterol |
DC-CHOL | 3b-(N-(N′,N′-dimethylaminoethane)-carbamoyl) cholesterol hydrochloride |
DLS | Dynamic Light scattering |
DOPE | 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine |
DOTAP | 1,2-dioleoyl-3-timethylamonium-propane |
DPPC | 1,2-dipalmitoyl-sn-glycero-3-phosphocholine |
DSPC | 1,2-distearoyl-sn-glycero-3-phosphocholine |
ELISA | Enzyme-linked immunosorbent assay |
ELS | Electrophoretic Light Scattering |
EMA | European Medicinal Agency |
EUNCL | European Nanomedicine Characterization Laboratories |
FDA | Food and Drug Administration |
Hep G2 | Human Hepatocarcinoma G2 cell line |
HSPC | Hydrogenated phosphatidylcholine |
ICH-S8 | Guideline S8 (Immunotoxicology studies for human pharmaceuticals) of the EMA International Council on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) |
ISO | International Standards Organization |
JRC | Joint Research Centre |
LAL | Limulus Amoebocyte Lysate |
LDH | Lactate dehydrogenase |
MTT | 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H tetrazolium bromide |
NM | Nanomaterial |
PBMC | Peripheral blood mononuclear cells |
PBS | Phosphate Buffer Saline |
PC | Phosphatidylcholine |
PDI | Polydispersity Index |
PSD | Particle Size Distribution |
SD | Standard Deviation |
TLR4 | Toll-like-receptor 4 |
USNCL | National Cancer Institute-Nanotechnology Characterization Laboratory |
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Liposome Description | Lipid Composition | Bulk Buffer Solution | Catalogue nr. | Lipid Stock Concentration (mg/mL) |
---|---|---|---|---|
Plain DPPC/CHOL liposomes | DPPC:CHOL (55:55 mol/mol) | 10% sucrose, 20 mM HEPES, <10% ethanol, pH 7.4 | F10102 | 34.6 |
Plain DSPC/CHOL liposomes | DSPC:CHOL (55:55 mol/mol) | 10% sucrose, 20 mM HEPES, pH 7.4 | F10103 | 36.5 |
DSPC/CHOL liposomes with ammonium sulfate gradient | DSPC:CHOL (55:55 mol/mol) | 10% sucrose, 20 mM NaPO4, pH 6.5 | F20103A | 42.2 |
HSPC/CHOL liposomes with ammonium sulfate gradient | HSPC:CHOL (55:55 mol/mol) | 10% sucrose, 20 mM NaPO4, pH 6.5 | F20104A | 36.3 |
DOTAP/DOPE cationic liposomes | DOTAP:DOPE (50:50 mol/mol) | Nuclease-free water, 10% ethanol, pH 5.5 | F50102 | 3.6 |
DC-CHOL/DOPE cationic liposomes | DC-CHOL:DOPE (30:70 mol/mol) | Nuclease-free water, pH 5.5 | F50105 | 3.4 |
Clophosome—high potency clodronate liposomes (neutral) | PC:CHOL (* mol/mol) | PBS (0.9% NaCl, 20 mM NaPO4), pH 7.5 | F70101C-NH | 20.0 |
Control empty liposomes for Clophosome—high potency (neutral) | PC:CHOL (* mol/mol) | PBS (0.9% NaCl, 20 mM NaPO4), pH 7.4 | F70101-NH | 20.0 |
Liposome | Diameter (nm) | PDI | Zeta-Potential (mV) | Mobility (µm cm/Vs) | ||
---|---|---|---|---|---|---|
Dh | Dh * | DS | ||||
F10102 | 98.9 (±26.2) | 92.2 (±8.8) | 96.5 | 0.05 | −5.90 (±1.92) | −0.40 (±0.14) |
F10103 | 104.2 (±28.0) | 93.4 (±10.0) | 93.9 | 0.06 | −11.90 (±2.20) | −0.90 (±0.16) |
F20103A | 115.1 (±31.0) | 105.6 (±12.2) | 90.1 | 0.06 | −16.60 (±6.35) | −1.20 (±0.07) |
F20104A | 103.7 (±27.4) | 96.4 (±9.8) | 93.9 | 0.05 | −11.80 (±2.09) | −0.80 (±0.15) |
F50102 | 98.2 (±50.1) | ND | ND | 0.36 | +4.10 (±0.39) | +0.30 (±0.03) |
F50105 | 929.2 (±119.3) | ND | ND | 0.49 | −8.10 (±3.40) | −0.60 (±0.26) |
F70101C-NH | 552.3 (±79.1) | ND | ND | 0.59 | −7.50 (±0.01) | −0.50 (±0.01) |
F70101-NH | 1154.4 (±334.2) | ND | ND | 0.54 | −7.20 (±0.72) | −0.50 (±0.05) |
Liposomes | Kinetic LAL Test (OD 405 nm) | End-Point LAL Test (OD 540 nm) | ||
---|---|---|---|---|
Recovery Rate (%) | Endotoxin (EU/mg) | Recovery Rate (%) | Endotoxin (EU/mg) | |
F10102 | 121 | 1.2 | 99 | 0.8 |
F10103 | 135 | 14.5 | 93 | 8.4 |
F20103A | NM | NM | 112 | 100.6 |
F20104A | 131 | 0.7 | 110 | 1.0 |
F50102 | NM | NM | NM | NM |
F50105 | NM | NM | NM | NM |
F70101C-NH | 111 | 660.0 | 107 | 273.3 |
F70101-NH | 97 | 4.7 | 87 | 6.0 |
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Della Camera, G.; Lipsa, D.; Mehn, D.; Italiani, P.; Boraschi, D.; Gioria, S. A Step-by-Step Approach to Improve Clinical Translation of Liposome-Based Nanomaterials, a Focus on Innate Immune and Inflammatory Responses. Int. J. Mol. Sci. 2021, 22, 820. https://doi.org/10.3390/ijms22020820
Della Camera G, Lipsa D, Mehn D, Italiani P, Boraschi D, Gioria S. A Step-by-Step Approach to Improve Clinical Translation of Liposome-Based Nanomaterials, a Focus on Innate Immune and Inflammatory Responses. International Journal of Molecular Sciences. 2021; 22(2):820. https://doi.org/10.3390/ijms22020820
Chicago/Turabian StyleDella Camera, Giacomo, Dorelia Lipsa, Dora Mehn, Paola Italiani, Diana Boraschi, and Sabrina Gioria. 2021. "A Step-by-Step Approach to Improve Clinical Translation of Liposome-Based Nanomaterials, a Focus on Innate Immune and Inflammatory Responses" International Journal of Molecular Sciences 22, no. 2: 820. https://doi.org/10.3390/ijms22020820