CHARACTERIZATION OF NANO CARRIERS FOR DRUG DELIVERY SYSTEMS: THE LIPIDOTS NanoSafe 2016 Amandine Arnould 07-11/11/2016

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1 CHARACTERIZATION OF NANO CARRIERS FOR DRUG DELIVERY SYSTEMS: THE LIPIDOTS NanoSafe 2016 Amandine Arnould 07-11/11/2016

2 CONTEXT : NANOPARTICLES IN SENSITIVE MEDIA Nano-safety issue Environment Medicine Agribusiness Cosmetic - Air decontamination - Water treatment - Bactericide - Drug carriers - Food colouring - Anticaking - UV filter - Colouring Fe, Al Ag, Liposomes TiO 2, SiO 2 Core shell TiO 2 Robust characterization on organic nanoparticles Stability of the nanoparticles (aging) Interaction with biological media (toxicity) 2

3 GLOBAL INFORMATIONS ABOUT LIPIDOTS Ultrasonication process Targeting agent Wax & soybean oil PEG chains Lecithin Lipophilic drug or contrast agent Tunable diameter (lipid ratio) (30 to 120 nm) Completely biodegradable & biocompatible Lipophilic drug or contrast agent Amorphous particles 3

4 TEM OBSERVATIONS Transmission Electron Microscopy Size Agglomeration, dispersion Ø Drying process Negative staining + drying process Plunge freezing (cryo-tem) In-situ liquid Well adapted for metallic particles Optimizations for organic particles 4

5 TEM OBSERVATIONS Drying process: concentration adjustment Without staining: Suspension droplet Pipette Carbon film Drying With negative staining: Pipette Mica Staining Uranyl acetate Grid Carbon film Drying Shadow Quick methods 5

6 TEM OBSERVATIONS L m = 70 nm W m = 45 nm Without staining With negative staining D m = 37 nm Particle dehydration Poor contrast Concentration on the lacey carbon Not appropriate Real shape or artefact? Agglomeration Interior of the particle not visible 6

7 TEM OBSERVATIONS Cryogenics: preservation and 3D reconstruction Pipette Blotting papers Suspension Freeze Carbon film Amorphous & homogenous ice: optimizations 7

8 Ice thickness «t» (nm) TEM OBSERVATIONS Cryogenics: ice thickness optimizations Blot: 3s Blot: 1s ,0 0,5 1,0 1,5 Position (µm) µm µm t < D min D Lipidot 20 nm < D < 80 nm D min < t < D max D min < t < D max Homogenous repartition of the particles on the grid 8

9 TEM OBSERVATIONS D m = 32 nm Quite good contrast Ice contaminations Round shape Native state cryofixed 9

10 TEM OBSERVATIONS In-situ liquid: native state check-in for particles in suspension Amorphous SiN window (50 nm) Large E-chip (Si) 0 to 5 µm Suspension Small E-chip (Si) Fixing screw Lid Large e-chip Small e-chip O-ring Holder extremity Suspension input Suspension output 10

11 TEM OBSERVATIONS D m = 37 nm Lipidots Lipidots Contaminations Poor contrast Lack of statistic Clean the holder & retry with smaller spacer (50 nm) 11

12 CONCLUSION Comparison of the different sample preparation methods Drying process poor contrast Negative staining + drying no special equipment required Preserved shape quick dehydration L m = 70 nm W m = 45 nm DLS D m = 37 nm overstatement of the diameter flattening concentration on the lacey carbon D h = 71,8 nm agglomeration Interior of the particle non visible Plunge freezing In-situ liquid lots of particles D m = 32 nm no preparation artefact good contrast D m = 37 nm poor contrast ice thickness ice contamination native state time consuming 12

13 Hydrodynamic diameter (nm) Corrected absorption at 350 nm PERSPECTIVES Stability of reference particles in PBS [1] Stability in biological media F120 F100 F80 F50 0,03 1h 3h 5h 24h , ,01 30 DLS Time (days) 0 Turbidity PBS DMEM BSA DMEM+FBS Media NC75 (75% wax, 25% soybean oil), 4 C F50 NC75, 0,2 % m/m, 37 C High stability (> 13 months) DMEM PBS ; stable over 24h PBS (Phosphate Buffered Saline): Buffer solution DMEM (Dulbecco s Modified Eagle Medium): ~ plasma without protein BSA (Bovine Serum Albumin): ~ liver proteins FBS (Foetal Bovine Serum): ~ plasma with plasma proteins & biomolecules BSA: quite stable over 24h ; increased of the particle diameter compared to PBS? DMEM + FBS: destabilization of the particles? Adsorption of proteins at the surface of the particles? [1] Delmas, T. (2011). Caractérisation physicochimique et compréhension des propriétés de vectorisation des nanoparticules lipidots pour les applications biomédicales (PhD thesis). 13

14 ACKNOWLEDGMENTS Thank you for your attention Maria Bacia (IBS) Fanny Caputo (CEA) Anne-Claude Couffin (CEA) Isabelle Texier-Nogues (CEA) Constantin Mattei (CEA) Benoit Gallet (IBS) Jean-François Damlencourt (CEA) Romain Soulas (CEA) Delphine Boutry (CEA) Stéphane Aguy (Eden Instrument) 14

15 TEM OBSERVATIONS D m = 37 nm [2] Liposome Liposome [2] Hoppe, S. M., Sasaki, D. Y., Kinghorn, A. N., & Hattar, K. (2013). In-situ transmission electron microscopy of liposomes in an aqueous environment.langmuir, 29(32),

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