Optimization of Particle Size for 
PLGA Nanoparticles Prepared by 
the Ouzo Effect
Erik Rytting, Tobias Lebhardt, and Thomas Kissel
Philipps-Universit?tMarburg
Why Nanoparticles?
? Nanoparticles can be the right tool for the job
? Advantages
? Enhanced bioavailability and controlled release
? Penetration across biological barriers
? Increased drug targeting efficiency
Methods for Nanoparticle Production
Materials
? Polymers
? Metals
? Ceramics
? Biological materials
? Liposomes
? Peptides
? Polymeric Nanoparticles
? Nanoparticles formed using monomers as the starting point
? Nanoparticles prepared using preformed polymers as the starting 
point
Methods Employing Preformed Polymers
? Emulsion Diffusion 
? Interfacial Precipitation
? Emulsion Evaporation 
? Double Emulsion Evaporation
? Salting Out
? Solvent Displacement 
? Nanoprecipitation, Solvent Diffusion, Spontaneous 
Precipitation, Ouzo Effect
Solvent Displacement
? Polymer and drug are dissolved in a water-miscible 
organic solvent
Solvent Displacement
? Polymer and drug are dissolved in a water-miscible 
organic solvent
? This solution is added to an aqueous solution
Solvent Displacement
? Polymer and drug are dissolved in a water-miscible 
organic solvent
? This solution is added to an aqueous solution
? Rapid solvent diffusion leads to instantaneous 
nanoparticle formation
Solvent Displacement
? Polymer and drug are dissolved in a water-miscible 
organic solvent
? This solution is added to an aqueous solution
? Rapid solvent diffusion leads to instantaneous 
nanoparticle formation
? Solvent is removed by evaporation
Solvent Displacement
? Advantages
? Relatively simple process
? Nontoxic solvents can be used
? Does not require high shear stress
? Does not require surfactants or stabilizers
? Disadvantages
? Solvent evaporation can be time consuming
? Poor encapsulation of hydrophilic drugs
Nanoparticle Characterization
? Size
? Surface Characteristics
? Encapsulation efficiency
? Drug Release
? Degradation
? Biocompatibility
Nanoparticle Characterization
? Size
? Surface Characteristics
? Encapsulation efficiency
? Drug Release
? Degradation
? Biocompatibility
The Importance of Size
The Importance of Size
? Basketball
Average Player Height/40 minutes
1.94
1.95
1.96
1.97
1.98
Air Force Kansas Florida
m e
t e
r s
2005-2006 Season
The Importance of Size
22/32620/326281/326Blocked Shots
59/32620/326296/326Rebound Margin
FloridaKansasAir ForceNCAA Div. I Rank
The Importance of Size
? Nanoparticle Size
? Endothelial Permeability
? Particle escape from vasculature
? Liver sinus endothelium (up to 150 nm)
? Tumor capillaries (up to 300 nm)
? Blood-brain barrier
? SplenicFiltration
? Macrophage Clearance
? Cellular Uptake
? Particle Degradation and Drug Release Rates
Ouzo Effect
? Solvent displacement is also referred to as the Ouzo 
effect
? Ouzo is a mixture of anethol, ethanol, and water (from 
anis seeds)
? Additional water leads to micro/nanoparticle formation 
? Solution proportions fall into a thermodynamically 
metastableregion
? Homogeneous nucleation occurs 
? Leads to a uniform dispersion (milliseconds) followed by 
Ostwaldripening (seconds)
Ouzo Region
From Vitale and Katz, Langmuir 2003, 19, 4105-4110.
Ouzo Region for RG502H/Acetone
0
0.2
0.4
0.6
0.8
1
1E-06 0.00001 0.0001 0.001 0.01 0.1 1
weight fraction polymer
w e
i g h
t  f
r a
c t i
o n
 a c
e t o
n e Ouzo region
outside Ouzo region
Ouzo Region for RG502H/Acetone
0
0.2
0.4
0.6
0.8
1
1E-06 0.00001 0.0001 0.001 0.01 0.1 1
weight fraction polymer
w e
i g h
t  f
r a
c t i
o n
 a c
e t o
n e Ouzo region
outside Ouzo region
No Particles
Stable 
Nanoparticles
Unstable Particles
Particle Size Distribution
0
5
10
15
20
25
1 10 100 1000 10000 100000
Particle Size (nm)
I n
t e
n s
i t y
 ( %
)
Surfactants & Polymer Concentration
80
90
100
110
120
130
140
150
160
0 0.005 0.01 0.015 0.02 0.025
mg RG502H/mg acetone
Na
n o
p a
r t i
c l e
 S
i z e
 ( n
m
)
0.005% Alveofact
0.1% Poloxamer
Water
Surfactant Concentrations
0
20
40
60
80
100
120
140
160
0.00 0.03 0.10 0.30
Poloxamer Concentration (%)
N a
n o
p a
r t i
c l e
 S
i z e
 ( n
m )
Different Solvents
0
40
80
120
160
200
Acetone Acetonitrile THF
P a
r t i
c l e
 S
i z e
 ( n
m )
Different Solvents
0
40
80
120
160
200
Acetone Acetonitrile THF
P a
r t i
c l e
 S
i z e
 ( n
m )
0
0.1
0.2
0.3
0.4
0.5
Acetone Acetonitrile THF
S o
l v e
n t
 V
i s c
o s
i t y
 ( c
P )
PLGAsof Increasing MW
0.3
0.35
0.4
0.45
0.5
0.55
0.6
0 2 4 6 8 10 12
[Polymer] (mg/mL)
K i
n e
m
a t
i c  
V i
s c
o s
i t y
 ( m
m
? / s
)
RG502H
RG503H
RG504H
48RG504H
23RG503H
12RG502H
Mn (kDa)Polymer
Viscosity
80
90
100
110
120
130
140
150
160
170
0 5 10 15 20
[Polymer in Acetone] (mg/mL)
P a
r t i
c l e
 S
i z e
 ( n
m )
RG502H (RV = 1.19)
RG503H (RV = 1.30)
RG504H (RV = 1.41)
Relative Viscosity
          Viscosity of 10 mg/mL polymer
RV = 
           Viscosity of acetone alone
Injection Rate
100
105
110
115
120
125
130
135
140
145
150
2 4 6 8 10 12
Injection Rate (mL/min)
N a
n o
p a
r t i
c l e
 S
i z e
 ( n
m
)
4.0 mg/mL RG 502 H in acetone added to 0.1% Poloxamer.
Surfactants ?Fast Injection
0
20
40
60
80
100
120
140
160
0.00 0.03 0.10 0.30
Poloxamer Concentration (%)
N a
n o
p a
r t i
c l e
 S
i z e
 ( n
m )
Surfactants ?Slow Injection
0
20
40
60
80
100
120
140
0 0.03 0.1 0.3
Poloxamer Concentration (%)
N a
n o
p a
r t i
c l e
 S
i z e
 ( n
m
)
Viscosity ?Fast Injection
80
90
100
110
120
130
140
150
160
170
0 5 10 15 20
[Polymer in Acetone] (mg/mL)
P a
r t i
c l e
 S
i z e
 ( n
m )
RG502H (RV = 1.19)
RG503H (RV = 1.30)
RG504H (RV = 1.41)
Relative Viscosity
          Viscosity of 10 mg/mL polymer
RV = 
           Viscosity of acetone alone
Viscosity ?Slow Injection
80
100
120
140
160
180
200
0 5 10 15 20
[Polymer in Acetone] (mg/mL)
P a
r t i
c l e
 S
i z e
 ( n
m
)
RG502H
RG504H
Importance of Size
33-625-824-7Win-Loss Record 
FloridaKansasAir Force
Importance of Size
22/32642/326267/326Scoring Offense
21/3261/326132/326Field Goal % Defense
50/32633/3261/326Scoring Defense
33-625-824-7Win-Loss Record 
FloridaKansasAir Force
Importance of Size
15/32644/3264/3263-Point Shooting %
22/32642/326267/326Scoring Offense
29/326140/32613/326Personal Fouls/Game
70/326173/32612/3263-Pointers Made/Game
34/326226/32629/326Free Throw Shooting %
21/3261/326132/326Field Goal % Defense
50/32633/3261/326Scoring Defense
33-625-824-7Win-Loss Record 
FloridaKansasAir Force
Improved Drug Delivery
? Nanoparticle Size
Improved Drug Delivery
? Nanoparticle Size
? Precision/Accuracy
Improved Drug Delivery
? Nanoparticle Size
? Precision/Accuracy/Targeting
Improved Drug Delivery
? Nanoparticle Size
? Precision/Accuracy/Targeting
? Other Strategies
Summary
Summary
? How can one control Basketball player size?
? Nutrition
? Exercise
? Genetics
Summary
? How can one control Nanoparticle size?
? Solvent viscosity
? Injection speed
? Polymer concentration
Acknowledgments
? Co-authors: Tobias Lebhardt& Thomas Kissel
? Others
? Claudia Packh?user
? Theresa Haas
? Ulrike Nierste
References
? Asteteand Sabliov, J BiomaterSciPolymer Edn, 2006, 17, 247-289.
? Avgoustakis, K. Current Drug Delivery, 2004, 1, 321-333.
? Balaet al., Critical Rev TherDrug Carrier Sys, 2004, 21, 387-422.
? Desai et al., PharmRes, 1997, 14, 1568-1573.
? Fessiet al., IntJ Pharm, 1989, 55, R1-R4.
? Ganachaudand Katz, ChemPhysChem, 2005, 6, 209-216.
? Gaoand Jiang, IntJ Pharm, 2006, 213-219.
? Hughes, Nanomedicine, 2005, 1, 22-30.
? Mayer, IntJ ArtifOrgans, 2005, 28, 1163-1171.
? Moghimiet al., FASEB J, 2005, 19,311-330.
? Molpecereset al., J PharmSci, 1996, 85, 206-213.
? Panyamand Labhasetwar, Adv Drug Del Rev, 2003, 329-347.
? Sahooand Labhasetwar, Drug Discovery Today, 2003, 8, 1112-1120.
? Vitale and Katz, Langmuir2003, 19, 4105-4110.
? Yihand Al-Fandi, J Cell Biochem, 2006, 97, 1184-1190.
Poloxamer 188 NF (PluronicF68)
From Walsh et al., Ann. N.Y. Acad. Sci. 1066: 321?327 (2005).
Surface Charge
0
20
40
60
80
100
120
140
160
0 0.03 0.1 0.3 1
Poloxamer Concentration (%)
N a
n o
p a
r t i
c l e
 S
i z e
 ( n
m
)
-40
-35
-30
-25
-20
-15
-10
-5
0
0 0.03 0.1 0.3 1
Poloxamer Concentration (%)
Z e
t a
 P
o t
e n
t i a
l  ( m
V )
Fluorescent Nanoparticles
Nanoparticles prepared with 10 ?g Coumarin-6 per 10 mg RG502H in 1 mL acetone, Zave = 117 ? 3 nm.  Fluorescence 
Microscopy settings:  ?ex = 488 nm, ?em = 515 nm.  Suspension of particles in water after washing in a Sephadex G-50 column.