Characterizing Nanoparticles Used in Bio … Nanoparticles Used in Bio Applications ... Applications...

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Characterizing Nanoparticles Used in Bio Applications

Mark [email protected]


Outline

Define nanoparticleParticle size analysis techniquesMaking nanoparticlesApplicationsMicelles, liposomes, engineered

nanoparticles for drug deliveryOther analytical techniquesFluorescence

Zeta potential


What is a Nanoparticle?

Size range from approximately from 1- 100 nm

Particle size answer: “it depends..”


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


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


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


Laser Diffraction

•Converts scattered light to particle size distribution

•Quick, repeatable•Most common technique•Low end: 30 nm


Making Nanoparticles

Top DownMake particles smaller

Bottom UpBuild from atomic or

molecular level up

Self assembly of micelles

10 nm


Top Down: Elan NanoCrystal® Technology


Top Down: Elan NanoMill


Size Reduction Measured on LA-950


Top Down: Microfluidizer*

* See http://www.microfluidicscorp.com/


Ceria: Before, After Processing

Laser diffraction required for before sample


Bottom-up Self Assembly: Micelles

Hydrophobic tail Hydrophilic head

R +/-non polar polar-c-h-c-h-c-h-


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


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)


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


Liposomes

100 nm


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


Liposome Size Reduction: Microfluidizer

Unprocessed

Laser diffraction


Liposome Size Reduction: Microfluidizer

Processed

Laser diffraction


Liposome: Before, After


Nanoparticles for Drug Delivery: Bottom Up


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.


Nanoparticles for Drug Delivery


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


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


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


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


PLA Nanoparticles for Drug Delivery

9 fold increase


PLA Nanoparticles for Drug DeliveryPure Spiked with 1 µm PSL


ProteinsMolecular surface of several proteins showing their comparative sizes.

Immunoglobulin G (IgG)

hemoglobin

insulin

adenylate kinase

glutamine synthetase


●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


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


Protein Reproducibility


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


1％ Sodium-Acetate bufferSample Preparation

InfluenzavirusVirus

138.1Z ave. (nm)

Results

Virus


Dendrimers

Repeatedly branched, roughly spherical large molecules

CoreInner shellOuter shell


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


●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


●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


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


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


MW Data


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


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


-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


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


Acknowledgements

Microfluidics

Elan

HORIBA Applications Lab Irvine, CA HORIBA Applications Lab, Kyoto, Japan HORIBA Applications Lab, Longjumeau,

France Unnamed (at their request) customers


Thank youThank youThank you


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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