contrast enhancement for lnp characterization …...comparison of 5 techniques : dls, af4-mals,...
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CONTRAST ENHANCEMENT FOR LNP CHARACTERIZATION USING TRANSMISSION ELECTRON MICROSCOPY
NanoSafe 2018 | Amandine Arnould | 05-09/11/2018
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GLOBAL INFORMATION ABOUT LIPID NANOPARTICLES
Completely biodegradable & biocompatible
Lipophilic drug or contrast agent
Amorphous particles
Commonly characterized by DLS (size)
F80 - NC75 (75% wax / 25% soybean oil): Dh = 80 nmUltrasonication process
Robust characterization required:
Targeting agent
PEG chains
Lecithin
Wax & soybean oil
Lipophilic drug or contrast agent
- Blood capillaries- EPR effect- Cell internalization
Drug internalizationAgglomerates or aggregates of LNP less effective
NanoSafe 2018 | Amandine Arnould | 05-09/11/2018
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CHARACTERIZATION METHODS : DLS, AF4-LS AND TEM
Light Scattering (LS)
DLS (batch mode)
AF4-LS(online mode)
SPECTROSCOPY
• Commonly used technique
• Fast and cheap
• Only for spherical and monodispersed samples
• Fractionation before detection
• Detectors: DLS, MALS, ICP-MS, UV-Vis, …
• MALS : shape based model Hydrodynamic diameter (nm)
Inte
nsity
(%
)
Transmission Electron Microscopy
(TEM)MICROSCOPY
• 2D and 3D imaging technique
• Suitable sample preparation required
Dry process
Rapid freezing
In-situ liquid
Diameter (nm)
Num
ber
(%)
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TEM SAMPLE PREPARATION AND RESULTS : DRYING
Without negative staining
Carbon film
Artefact on the carbon film or lack of contrast of LNPs ?
With negative staining
Carbon film
Staining
Good contrast but agglomeration and intern structure not visible
Dg = 31 nmσ = 11 nm
NanoSafe 2018 | Amandine Arnould | 05-09/11/2018
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TEM SAMPLE PREPARATION AND RESULTS: CRYO
Cryo-TEM
Carbon film
Hole filled with solution
Dg = 25 nm σ = 8 nm
Particle deformation (blot process), cryo-TEM not adapted
Vitrobot parameters: - Grid C-Flat-T- Blot force: -5- Blot time: 2s- Drain time: 0s
*Danino, D. (2012). Cryo-TEM of soft molecular assemblies. Current opinion in colloid & interface science, 17(6), 316-329. NanoSafe 2018 | Amandine Arnould | 05-09/11/2018
Blot process
Carbon film
Blotting papers
Blot artefact
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TEM SAMPLE PREPARATION AND RESULTS: IN-SITU
In-situ liquid TEM
Innovative technique
NanoSafe 2018 | Amandine Arnould | 05-09/11/2018
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TEM SAMPLE PREPARATION AND RESULTS: IN-SITU
In-situ liquid without grafting
Dcore = 28 nmDg = 58 nm
No contrast, hydrolysis
In-situ liquid with grafting
Contrast OK + Core/shell structure
Si chip
Au spacer(50 nm)
Si chip
Au spacer(50 nm)
SiN windowSiN window
NanoSafe 2018 | Amandine Arnould | 05-09/11/2018
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LIGHT SCATTERING RESULTS : DLS AND AF4-MALS
DLS (batch mode)
Smallest particles hidden by the largest ones
NanoSafe 2018 | Amandine Arnould | 05-09/11/2018
Hydrodynamic diameter (nm)
Inte
nsity
(%
)
AF4-MALS (online mode)
Analysis OK
Injection time (min)
Inte
nsity
(%
)
Geom
etric diameter (nm
)
Dh = 101 nm σ = 57 nm
Dg = 46 nm σ = 8 nm
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COMPARISON OF THE CHARACTERIZATION METHODS
NanoSafe 2018 | Amandine Arnould | 05-09/11/2018
Cryo-TEM : not adapted for LNPs (preparation artefact on the nanoparticle shape)
In-situ liquid : weak statistic, core@shell structure visible
AF4-MALS, in-situ TEM and negative staining well adapted for lipid nanoparticle characterization: REPEATABILITY, RELIABILITY
Log normal distribution for DLS, AF4-MALS and TEM (negativestaining)
DLS: not adapted because Lipidots are not monodisperse
Negative staining TEM: tangle of PEG chains due to the drying process
AF4-MALS: flattening of PEG chains in water
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CONCLUSION AND PERSPECTIVES
Set up of a characterization procedure for the study of LNPs
Comparison of 5 techniques : DLS, AF4-MALS, negative staining TEM, cryo-TEM, in-situ liquid TEM
AF4-MALS & negative staining TEM : best methods
In-situ liquid TEM : core/shell structure visible BUT work on contrast enhancement (patent)
Study of drug loaded nanoparticles, stability (aging, biological media) (article in review)
In-situ: interaction of nanoparticles with a protein-based media
NanoSafe 2018 | Amandine Arnould | 05-09/11/2018
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ACKNOWLEDGMENTS
Co-authors : F. Caputo1, M. Bacia2, I. Texier1, M. Escude1, G. Effantin2, A.C. Couffin1, R. Soulas3, J.F. Damlencourt3
Univ. Grenoble Alpes, F-38000 Grenoble, France
1CEA-LETI, MINATEC Campus, F-38054 Grenoble, France
2IBS, EPN Science Campus, F-38044 Grenoble, France
3CEA-LITEN, MINATEC Campus, F-38054 Grenoble, France
Thank you for your attention
NanoSafe 2018 | Amandine Arnould | 05-09/11/2018
Commissariat à l’énergie atomique et aux énergies alternatives17 rue des Martyrs | 38054 Grenoble Cedexwww-liten.cea.fr
Établissement public à caractère industriel et commercial | RCS Paris B 775 685 019
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Dh = 80 nm Dg = 46 nm
Dg = 31 nm Dg = 25 nm Dg = 58 nm
COMPARISON OF THE CHARACTERIZATION METHODS
NanoSafe 2018 | Amandine Arnould | 05-09/11/2018
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TEM SAMPLE PREPARATION AND RESULTS: CRYO
Cryo-TEM
Carbon film
Hole filled with solution
Dg = 25 nm σ = 8 nm
Vitrobot parameters: - Grid C-Flat-T- Blot force: -5- Blot time: 2s- Drain time: 0s
*Malis, T., Cheng, S. C., & Egerton, R. F. (1988). EELS log‐ratio technique for specimen‐thickness measurement in the TEM. Microscopy Research and Technique, 8(2), 193-200.
CBED: crystalline material
Contamination spot: incompatible with ice
EDX: Zeta factor (complex)
EELS: t/λ quick to set up
Thickness measurement techniques:
Problem with the particle distribution within the holes and with their morphology
Ice in the center of the holestoo thin ?
NanoSafe 2018 | Amandine Arnould | 05-09/11/2018
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TEM SAMPLE PREPARATION AND RESULTS: CRYO
Cryo-TEM
*Malis, T., Cheng, S. C., & Egerton, R. F. (1988). EELS log‐ratio technique for specimen‐thickness measurement in the TEM. Microscopy Research and Technique, 8(2), 193-200.
1 µm-60
-40
-20
0
20
40
60
0,0 0,5 1,0 1,5
-60
-40
-20
0
20
40
60
0,0 0,5 1,0 1,5Ic
e th
ickn
ess
”t”
(nm
)
D
Position within the hole (µm)
Lipidot20 nm < D < 80 nmt < Dmin
Dmin < t < Dmax
1 µm
Dmin < t < Dmax
NanoSafe 2018 | Amandine Arnould | 05-09/11/2018
Ice thickness measurement
Particle distribution within the holes linked to ice thickness
Particles still deformed
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CONTEXT : NANOCARRIERS FOR DRUG DELIVERY
NanoSafe 2018 | Amandine Arnould | 05-09/11/2018
Organic
Metallic
Semiconductor
Active
Passive (EPR effect)
Fewer side effects
Diagnostic
Drug delivery
Nanometer scale
Biocompatible
(Biodegradable)
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COMPARISON OF THE CHARACTERIZATION METHODS
DLS : not adapted because Lipidots are not monodisperse
Cryo-TEM : preparation artefact on the nanoparticle shape, distortion
Negative staining : high statistic, tangle of PEG chains due to the drying process
In-situ liquid : weak statistic, flattening of PEG chains, core@shell nanoparticles
AF4-MALS, in-situ TEM and even negative staining well adapted for lipid nanoparticle characterization: REPEATABILITY, RELIABILITY
Characterization method MeasureDiameter
(nm)Pros Cons
DLS Dh 80 Quick methodOnly for monodisperse
solutions
AF4-MALS Dg 46 Fractionation Time consuming
TEM
Negative staining
Dg
31 Quick method Mask of the particles
Cryo 25 Cryofixation Preparation artefact
In-situ liquid 58 No preparation artifact Contrast
NanoSafe 2018 | Amandine Arnould | 05-09/11/2018
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