characterizing nanoparticles used in bio applications€¦ · microsoft powerpoint - july 7 2011...
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© 2011 HORIBA, Ltd. All rights reserved.
Characterizing Nanoparticles Used in Bio Applications
Mark [email protected]
© 2011 HORIBA, Ltd. All rights reserved.
Outline
Define nanoparticleParticle size analysis techniquesMaking nanoparticlesApplicationsMicelles, liposomes, engineered
nanoparticles for drug deliveryOther analytical techniquesFluorescence
Zeta potential
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What is a Nanoparticle?
Size range from approximately from 1- 100 nm
Particle size answer: “it depends..”
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Size Measurements
Dynamic Light Scattering (DLS)
Particle size 0.3 nm –several µmSuspensions only
Zeta potential MW, A2
Laser diffraction Particle size 30 nm –
3000 µmSuspensionsPowders
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DLS Optics
Particles
Zeta potential
Backscatter (173°)(High conc.)
90° for size and MW, A2
Laser
Modulator
PDFor T%532nm, 10mW
Attenuator
Attenuator
Particles movingdue to Brownianmotion
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DLS Measurement Principle.
D6tt 2
ts
n(1)=2n(2)=1 n(4)=3
n(3)=1 n(5)=2
n(i)
j
<n(i)n(i+j)>
0 1 53 42
<n(i)>2
<n2(i)>
g(q、r)
0 1 53 42 j
τts0 4ts 5ts2ts 3ts
a6Tk
q1D B
R2
Particle’s moving distance
Diffusion constant
Particle radius
Relaxation time
q: Scattering vector η: Viscosity kB: Boltzmann constant
Autocorrelation Function
r,qglnlim10
R
Relaxation time
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Laser Diffraction
•Converts scattered light to particle size distribution
•Quick, repeatable•Most common technique•Low end: 30 nm
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Making Nanoparticles
Top DownMake particles smaller
Bottom UpBuild from atomic or
molecular level up
Self assembly of micelles
10 nm
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Top Down: Elan NanoCrystal® Technology
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Top Down: Elan NanoMill
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Size Reduction Measured on LA-950
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Top Down: Microfluidizer*
* See http://www.microfluidicscorp.com/
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Ceria: Before, After Processing
Laser diffraction required for before sample
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Bottom-up Self Assembly: Micelles
Hydrophobic tail Hydrophilic head
R +/-non polar polar-c-h-c-h-c-h-
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Critical Micelle Concentration
Triton X-100
CMC
Experiment1. Determine weight a drop from pipette2. 10 mMol NaCl soln prepared in beaker w/stir bar 3. Drops Triton X-100 were added, mixed 10 minutes4. Remove small amount, measure by DLS
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Curcumin- Casein Micelles*
*Sahu,et el, Fluorescence Study of the Curcumin-Casein Micelle Complexation and Its Application as a Drug NanocarrierTo Cancer Cells, Biomacromolecules 2008, 9, 2905–2912
curcumin casein micelle
Particle size by DLS (insert), SEM (left), and AFM (right)
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Curcumin- Casein Micelles*
Binding constant kb = 1.48 x 104 M-1
*Sahu,et el, Fluorescence Study of the Curcumin-Casein Micelle Complexation and Its Application as a Drug NanocarrierTo Cancer Cells, Biomacromolecules 2008, 9, 2905–2912
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Liposomes
100 nm
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Liposome Size Reduction: Filter Membrane
Liposome particle size after 5 passes through a 100 mm membrane ~ 250 nm
Liposome particle size after 20 passes through a 100 mm membrane ~ 150 nm
Size reduced by passingthrough 100 nm filter membraneMeasured by DLS
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Liposome Size Reduction: Microfluidizer
Unprocessed
Laser diffraction
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Liposome Size Reduction: Microfluidizer
Processed
Laser diffraction
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Liposome: Before, After
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Nanoparticles for Drug Delivery: Bottom Up
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Nanoparticles for Drug Delivery
Targeting ligand provides recognition, enabling targeted nanoparticles to identify and bind to their intended target site.Surface functionalization shields targeted nanoparticles from the immune system.Polymer matrix encapsulates payload molecules in a matrix of biodegradable polymers .Therapeutic payloads include small molecules, peptides, proteins, etc.
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Nanoparticles for Drug Delivery
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Laser Diffraction Results: D10, D50, D90
Size
Freq
uenc
y
D10 D50 D90 D100
10%below
90%below
50%below
100%below
Never use D100 from laser diffraction
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Diffraction Results: D10, D50, D90
Size
Freq
uenc
y
D10 D50 D90D10 D90
Central value the samebut D10 & D90 significantlydifferent
Symmetric distribution: mean = median = mode
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Asymmetric Distribution
Mode
Median
Mean
D4,3
Freq
uenc
y
Size
Mode<Median<Meanif skewed to larger sizes
Note: D4,3 sensitive to large particles
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Volume Mean Diameter
• D[4,3] which is often referred to as the Volume Mean Diameter [ VMD ]
D [ 4,D [ 4, 3 ]3 ]
i i
i i
D nD n
4
3
Setting a D [4,3] specification will emphasize the presence of large particles
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PLA Nanoparticles for Drug Delivery
9 fold increase
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PLA Nanoparticles for Drug DeliveryPure Spiked with 1 µm PSL
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ProteinsMolecular surface of several proteins showing their comparative sizes.
Immunoglobulin G (IgG)
hemoglobin
insulin
adenylate kinase
glutamine synthetase
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●Protein; Lysozyme
pH 4.3, 0.1 mg / mL, 0.1 M Sodium-Acetate bufferSample Preparation
from egg white, Molecular weight; 14,000Lysozyme
4.5Mean Dia. (nm)Results
Protein: Lysozyme
ConditionsTemperature; 25 C degreeSolvent; WaterRefractive Index; 1.333Distribution base; Mass
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10 %, pH 6.0Sample Preparation
FerritinProtein
19.7Z ave. (nm)
Results
Ferritin
φ7nm
φ12nm
φ7nmφ7nm
φ12nmφ12nm
ConditionsTemperature; 25 C degreeSolvent; WaterRefractive Index; 1.333Distribution base; Mass
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Protein Reproducibility
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Protein Size
Temperature (C)
Z av
erag
e
30 35 40 45 50 55 60
6050403020100
Melting Point
Tm0
20406080
100120140160180200
0 100 200 300 400
HoursSi
ze in
nm
Lysozyme Aggregation
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1% Sodium-Acetate bufferSample Preparation
InfluenzavirusVirus
138.1Z ave. (nm)
Results
Virus
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Dendrimers
Repeatedly branched, roughly spherical large molecules
CoreInner shellOuter shell
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Dendrimers
Applications: typically involve conjugating other chemical species to the dendrimer surface that can function as detecting agents (such as a dyemolecule), affinity ligands, targeting components, imaging agents, or pharmaceutically active compoundsDrug & gene deliverySensors
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●DDS materials; DendrimerDNT-107(7.2 nm); 1,4-Diaminobutan Core, Amidoamine surface, Gen 6.0,10% DNT-189 (9 nm) ; 1,4-Diaminobutan Core, Amidoethanol surface, Gen 6.0,10%
PAMAM DendrimerIn Methanol
7.6DNT-107
8.6DNT-189
Z ave. (nm)
DNT-107
DNT-189
Dendrimers
ConditionsTemperature; 25 C degreeSolvent; MethanolRefractive Index; 1.329Distribution base; Scattering light
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●Bio-sample; Vitamin B1
300 mg / mLSample Preparation
Vitamin B1 Molecular weight; 337.27Thiamine
0.4Mean Dia. (nm)
Results
Detection Limit
ConditionsTemperature; 25 C degreeSolvent; WaterRefractive Index; 1.333Viscosity; 1.68Distribution base; Mass
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Molecular Weight Measurement Principle
VIrIR
0
2
Laser
Detector: PMT
Scattered Light
Intercept = 1/ Mw
cn
Nn4K 4
0A
22πKC/Rθ parameter is calculated by scattered light and sample concentration.
C = Concentration
Multi angle scattered light intensity measurement preparing diluted solutions of known concentration.
θ135°
θ35°
θ60°θ90°
KC/Rθ
sinθ+kC
e.g. Polystyrene sample (Mw=5x105)
Optical ConstantReduced Scattered light Intensity
Mw =6.35 x105
・・・232
0CA3CA2
M1
RKClim
M: Molecular weightA2: Second virial coefficientA3: Third virial coefficient
second virial coefficient
Radius of gyration
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MW and A2 using One Angle
Light scatteringindependent of angle at very smallsizes (< 60-100 nm)
Measure severalconcentrations atone angle to createDebye plot
MW A2
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MW Data
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Zeta Potential
)(32 rf
o
)2
sin()2
sin(4
0
nE
Processor
laser
optical
modulator
attenuator
sample cell
photoelectricdetector
reference beam
beam stop
beam splitter
scattered/referencelight
Measure mobility Calculate zeta potential
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Zeta Potential: Emulsion
-60
-50
-40
-30
-20
-10
0
10
20
30
40
50
60
2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0
pH
Zeta potential/mV
Isoelectric point:pH where zetapotential = 0
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-10
-5
0
5
10
15
20
0 2 4 6 8 10 12 14
pH
zeta
pot
entia
l
Zeta Potential: Lysozyme (4 nm, 10 mg/mL)
Isoelectric point:pH where zetapotential = 0
© 2011 HORIBA, Ltd. All rights reserved.
SummaryBoth laser diffraction (LA-950) and DLS
can measure nanoparticlesUse diffraction when some samples > 1 m, but all > 30 nmCheck lower limit of system used
Use DLS when all < 1 mDLS + zeta potential + moleculer wt +
A2
© 2011 HORIBA, Ltd. All rights reserved.
Acknowledgements
Microfluidics
Elan
HORIBA Applications Lab Irvine, CA HORIBA Applications Lab, Kyoto, Japan HORIBA Applications Lab, Longjumeau,
France Unnamed (at their request) customers
© 2011 HORIBA, Ltd. All rights reserved.
Thank youThank youThank you
© 2011 HORIBA, Ltd. All rights reserved.
Gracias
謝謝
شكرا
Dank u
Спасибо תודה
谢谢
Danke
A dank
ध यवाद
Blagodaria
감사합니다
La ringrazio
Merci
DziękujęShukriya
Ngiyabonga
Tack
Trugéré ありがとう
ευχαριστώ
Obrigada
[email protected]@horiba.com
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