1. NANOPARTICLES FOR DRUG DELIVERY: THE SMALLER, THE BETTER ? Gurny R., Pourtier M., Vargas A., Delie F. Department of Pharmaceutics and Biopharmaceutics School of Pharmaceutical Sciences, University of Geneva, University of Lausanne

2. Nanoparticles = NPs HOW SMALL ARE NANOPARTICLES?

3. CURRENTLY USED SMALL PARTICLES AS THERAPEUTIC VECTORS 0 100 200 300 400 500 600 700 800 900 1000 10000 nm MicroparticlesNanoparticles Several studies on particles < 1000 nm 100

4. NANOPARTICLES ARE EASY TO PREPARE 1  m Mean size: 300 nm Gurny et al., Drug Dev. Ind. Pharm (1981)

5. VITAL PARAMETERS FOR NPs FOR BIOMEDICAL APPLICATIONS  Loading  Biodistribution Precise control of the size  Release profile of the drug  Elimination of NPs Shakweh et al., Eur. J. Pharm. Biopharm. (2005) Mean size: 310 nm

6. SOME HISTORICAL ASPECTS 1  m 1970 1980 1990 2000 20102020 Microparticles Nanoparticles 1  m Picoparticles ?

7. RESEARCH ARTICLES ON NANOPARTICLES Source: SciFinder Scholar 350 300 250 200 150 100 50 0 Number of citations including the terms "nanoparticles and cancer" 1975- 1980 1981- 1985 1986- 1990 1991- 1995 1996- 2000 2001- 2005

8. SMALL PARTICLES ? WHY ? FOR WHAT ?  Minimize embolization in case of parenteral administration  Increase the release surface  Increase the uptake (passage across biological barriers)  Facilitate sterilization

9. % in class Size (nm) 50 100 200 600 800 1000 400 CHARACTERIZATION OF SIZE AND POLYDISPERSITY Size distribution % in class Size (nm) 50 100 200 4006008001000 Mean size: 600 nm Dispersity Monodisperse: PI < 0.1 600 Monodisperse Polydisperse 600 Polydisperse: PI > 0.1

10. AVAILABLE METHODS Static and dynamic light scattering (SLS, DLS)Static and dynamic light scattering (SLS, DLS) www.ibmb.uni.wroc.pl/ liplip/zeta.jpg http://www.icmse.cartuja.csic.es/servicios/sema.gif http://gene.concordia.ca/csfg/ images/equip/auc.jpg www.wyatt.com/solutions/hardware/eclipse.cfm Scanning electronic microscopy (SEM)Scanning electronic microscopy (SEM) Analytical ultracentrifugation (ANUC)Analytical ultracentrifugation (ANUC) Field flow fractionation (FFF)Field flow fractionation (FFF)

11. DATA OBTAINED SLS/DLSSEMANUCFFF Rapid All sizes >50 nm Direct Less influence of extremes Large sample Population Separation (DLS) Non adapted for very polydispersed samples > 2  m Time consuming Influence of the preparation Complexity of the conversion: Turbidity / size Difficult > 1  m Fractions collection Mild method

12. THE BEST METHOD ? Determination by light scattering: Size average: 313.7 nm Polydispersity: 0.093  0.017 … real size ? !

13. SAFETY AND CHARACTERIZATION CONCERNS … What are the critical physical and chemical properties, including residual solvents, processing variables, impurities and excipients? What are the critical physical and chemical properties, including residual solvents, processing variables, impurities and excipients? How do physical characteristics impact product quality and performance? How do physical characteristics impact product quality and performance? What are the standard tools used for this characterization? What are the standard tools used for this characterization? FDA Perspective on Nanomaterial-Containing Products Nanobusiness Conference, May 2005

14. DETERMINATION OF SIZE … real size ? !

15. ACCURATE DETERMINATION OF OTHER CHARACTERISTICS ! ? The contribution of these parameters is of great importance in life sciences Size Charge Charge Hydrophobicity Hydrophobicity

16. Adsorption of blood components on nanoparticles = f(time) NP size: 312 nm, 2-D PAGE Allémann et al; J. Biomed. Mater. Res. (1997)

17. IN VITRO RELEASE Allémann et al; Pharm. Res. (1993) 671 nm (  ) 274 nm (  ) 303 nm (  )

18. BIOLOGICAL ACTIVITY ON ISOLATED TUMOUR CELLS =f (SIZE) Konan-Kouakou et al; J. Control. Rel. (2005) 370 nm 167 nm Free

19. Biodegradable particles with well defined sizes 0 100 200 300 400 500 600 700 800 900 1000 2000 nm SEM: Magnification: x 10 000 (5 to 15 KV, 5 to 39 mm) 1  m Polymer: poly(D,L-lactide-co-glycolide) acid (PLGA) Pourtier M. & al., unpublished data

20. Biological response = f(size, surface properties) ?

21. Interaction of NPs with biological surfaces Caco-2 = human colon carcinoma cell line Culture in plates Fluorescent particles: different sizes + Uptake ? Quantitative Fluorescence spectroscopy Qualitative Confocal microscopy Incubation

22. BIODISTRIBUTION=f(SIZE) ? I.V. administration PHAGOCYTOSIS MECHANICAL FILTRATION Macrophage Blood vessel Interstitial space

23. I…....I.....I....I....I...I...I..I.I.I…....I.....I....I....I...I...I..I.I.I…....I.....I....I....I...I...I..I.I.I…....I.....I....I. 1…………………………10………………………..100………………………1000………………………… nm FENESTRAE IN BLOOD CAPILLARY WALLS No fenestration 400 to 800 nm Tumours 80 to 1400 nm Inflammation Skeletal and cardiac muscles Lung Skin Kidney Small intestine Salivary glands Liver Spleen Bone marrow Blood brain barrier 1.8 to 2.0 nm 40 to 60 nm Up to 150 nm

24. BIODISTRIBUTION OF BIODEGRADABLE NP Data taken from Fang et al; Eur. J. Pharm. Sci. (2006) Cyanoacrylate- MePEG (5000) NP Biodistribution in mice 1 hour after administration

25. Internalisation of anti-HER2 nanoparticles in SKOV-3 cells

26. DDDD Normal vessels D = Drug PARENTERAL ADMINISTRATION OF NANOPARTICLES = Nanoparticle D D D D D D D D D D D = Drug = Nanoparticle D D D D D D D D D D D D D D = Drug = Nanoparticle Fenestrated vessels

27. Solid tumours Folkman, J. Scientific American 1996, 275 (3):150-154 NANOPARTICLES AND CANCER  Small nanoparticles can get into tumoural tissues

28. Data taken from Fang et al; Eur. J. Pharm. Sci. (2006) Biodistribution in S-180 tumour- bearing mice 6h after I.V administration NANOPARTICLES AND CANCER Poly methoxypolyethyleneglycol cyanoacrylate-co-n-hexadecyl cyanoacrylate NPs

29. SO, NP SIZE INFLUENCES BOTH THE CLEAREANCE AND THE BIODISTRIBUTION, BUT… Precision in size measurements ? Precision in size measurements ? Polydispersity of batches? Polydispersity of batches? Some case studies…

30. From the abstract We have prepared hydrogel nanoparticles of polyvinylpyrrolidone of a size less than 100 nm diameter with precise size distribution… …… Increasing the surface hydrophobicity as well as particle size can increase the RES uptake of these particles.

31. SIZE DETERMINATION BY QUASI-ELASTIC LIGHT SCATTERING …all formulations appeared to be very homogenous irrespective of their composition …… ….. their narrow size distribution.

32. From the abstract …. We found that small diameter (18 nm) … showed the most favorable biological behavior … … and diameter of the nanoparticle play important roles ….

33. Size determined by hydrodynamic light scattering

34. From the abstract … the biodistribution properties of the PLGA/PLGA-PEG nanoparticles are also influenced by the size of the nanoparticles …

35. ACKNOWLEDGMENTS Angélica Vargas Angélica Vargas Marie Pourtier Marie Pourtier Florence Delie Florence Delie