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

Nanoparticles = NPs HOW SMALL ARE NANOPARTICLES?

CURRENTLY USED SMALL PARTICLES AS THERAPEUTIC VECTORS nm MicroparticlesNanoparticles Several studies on particles < 1000 nm 100

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

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

SOME HISTORICAL ASPECTS 1  m Microparticles Nanoparticles 1  m Picoparticles ?

RESEARCH ARTICLES ON NANOPARTICLES Source: SciFinder Scholar Number of citations including the terms "nanoparticles and cancer"

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

% in class Size (nm) CHARACTERIZATION OF SIZE AND POLYDISPERSITY Size distribution % in class Size (nm) Mean size: 600 nm Dispersity Monodisperse: PI < Monodisperse Polydisperse 600 Polydisperse: PI > 0.1

AVAILABLE METHODS Static and dynamic light scattering (SLS, DLS)Static and dynamic light scattering (SLS, DLS) liplip/zeta.jpg images/equip/auc.jpg Scanning electronic microscopy (SEM)Scanning electronic microscopy (SEM) Analytical ultracentrifugation (ANUC)Analytical ultracentrifugation (ANUC) Field flow fractionation (FFF)Field flow fractionation (FFF)

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

THE BEST METHOD ? Determination by light scattering: Size average: nm Polydispersity:  … real size ? !

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

DETERMINATION OF SIZE … real size ? !

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

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

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

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

Biodegradable particles with well defined sizes nm SEM: Magnification: x (5 to 15 KV, 5 to 39 mm) 1  m Polymer: poly(D,L-lactide-co-glycolide) acid (PLGA) Pourtier M. & al., unpublished data

Biological response = f(size, surface properties) ?

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

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

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

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

Internalisation of anti-HER2 nanoparticles in SKOV-3 cells

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

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

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

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…

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.

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

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

Size determined by hydrodynamic light scattering

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

ACKNOWLEDGMENTS Angélica Vargas Angélica Vargas Marie Pourtier Marie Pourtier Florence Delie Florence Delie