Biointerfacial Characterization

(125:583)

Nanoparticles
L. Anthony and P. Moghe
Lectures:
Nov. 16th: Part I: Nanoparticle
Characterization**
Dec. 4th: Part II: Biological
Characterization
(Nanoparticles at
Interfaces)

Lab Demo:
November 20th:
** Slides in thisParticle
set are forSize by DLS;
November 16th Zeta
Potential
[8:40 - 9:20 AM; Wright-Rieman
 Outline for Nanoparticle Characterization: Part I

 Introduction/context:
Particles at biointerfaces
Properties of particles in dispersions/emulsions
 Particle Size and Particle Size Distribution
 Surface Charge: Zeta Potential, Isoelectric Point, Electrophoretic Mobility

Assigned papers

Nanoscale anionic macromolecules for selective retention of low-density lipoproteins

Chnari E, Lari HB, Tian L, Uhrich KE, Moghe PV
BIOMATERIALS 26 (17): 3749-3758 JUN 2005

Optimization of the preparation process for human serum albumin (HSA) nanoparticles
K. Langer, S. Balthasar V. Vogel, N. Dinauer H. von Briesen D. Schubert
INT. J. PHARMACEUTICS 257 (2003) 169-18
Other papers/information posted on class website:

Albumin-derived nanocarriers: Substrates for enhanced cell adhesive ligand display and cell motility
Sharma RI, Pereira M, Schwarzbauer JE, Moghe PV
BIOMATERIALS 27 (19): 3589-3598 JUL 2006

Quantum dot bioconjugates for imaging, labeling, and sensing

Medintz, IL, Uyeda, HT, Goldman, ER, Mattoussi, H
NATURE MATERIALS, 4, 435-446 (2005)

A Nanoparticle-Based Model Delivery System To Guide the Rational Design of Gene Delivery
to the Liver. 1. Synthesis and Characterization
Stephen R. Popielarski, Suzie H. Pun,† and Mark E. Davis*
BIOCONJUGATE CHEM. 1063 2005, 16, 1063-1070

Vesicle Size Distributions Measured by Flow Field-Flow Fractionation Coupled with Multiangle
Light Scattering
Brian A. Korgel, John van Zanten, Harold Monbouquette
BIOPHYSICAL JOURNAL 74 June 1998 3264–3272

Short Monographs by Vendors:

Basic Principles of Particle Size Analysis
Zeta Potential, a Complete Course in Five Minutes
The Importance of Sample Viscosity in Dynamic Light Scattering Measurements
A Guide to Choosing a Particle Sizer
 Outline for Part I: Nanoparticle Characterization

 Introduction/context:
Particles at biointerfaces
Properties of particles in dispersions/emulsions

 Particle Size and Particle Size Distribution

 Surface Charge: Zeta Potential, Isoelectric Point, Electrophoretic Mobility

 Introduction/Context: Particles at biointerfaces

Some reasons for using engineered nanoparticles in

biointerfacial research:
 to study/effect a cellular process
Example: Albumin-derived nanocarriers: Substrates for
enhanced cell adhesive ligand display and cell motility
Sharma RI, Pereira M, Schwarzbauer JE, Moghe PV
BIOMATERIALS 27 (19): 3589-3598 JUL 2006
 to transport drugs/biomaterials or biomolecules
***Example: Nanoscale anionic macromolecules for
selective retention of low-density lipoproteins
Chnari E, Lari HB, Tian L, Uhrich KE, Moghe PV
BIOMATERIALS 26 (17): 3749-3758 JUN 2005
 to visualize phenomena such as transport, sequestration, etc
Example: Quantum dot bioconjugates for
imaging, labeling, and sensing
Medintz, IL, Uyeda, HT, Goldman, ER, Mattoussi, H
NATURE MATERIALS, 4, 435-446 (2005)

*** Paper for discussion; examples given later in talk

 Introduction/Context: Particles at biointerfaces

Generic view of experiments with particles at biointerfaces:

tissue, cells or sub-cellular

system under study

Make (or purchase) Further modify/engineer Introduce particles into

the nanoparticles particles/dispersion tissue/cellular/subcellular
and/or the dispersion >Conjugated molecules system
>Buffers/salts
>etc
and
Study the particles and/or
the effects of the particles
 Introduction/Context: Properties of particles in dispersions/emulsions

Note: Properties of the particle at the biointerface

depend on:
Intrinsic properties of particles AND
Mediating properties of the liquid phase(s)

tissue, cells or sub-cellular

system under study

Make (or purchase) Further modify/engineer Introduce particles into

the nanoparticles particles/dispersion tissue/cellular/subcellular
and/or dispersion >Conjugated molecules system (in vitro usually)
>Buffers/salts
>etc
Study effect of particles

Note: cellular milieu is a very complex dispersant!

 Introduction/Context: Properties of particles in dispersions/emulsions

Cover up
tissue, cells or sub-cellular
system under study

Properties of Particles Properties of Dispersant (s) Properties of Particles in

Average size (diameter) Aqueous or organic? (or mix?) dispersion or cellular milieu
Size distribution Average size (diameter)
Shape Bulk composition
Size distribution
Bulk chemical composition Additives (what?, how much?) Shape
Crystalline/amorphous Ionic or non-charged Bulk chemical composition
Pore size/roughness Small molecules
Bio and macro molecules
Surface composition
covalently linked molecules
Surface composition adsorbed species
covalently linked molecules pH, conductivity surface charge (zeta potential)
adsorbed species Viscosity
Compliance/modulus, etc
Refractive index
Density Pore size/roughness
Compliance/modulus, etc Crystalline/amorphous
Refractive index Refractive index
Density Density
 Introduction/Context: Properties of particles in dispersions/emulsions

Cover up
tissue, cells or sub-cellular
system under study

Properties of Particles Properties of Dispersant (s) Properties of Particles in

Average size (diameter) Aqueous or organic? (or mix?) dispersion or cellular milieu
Size distribution Average size (diameter)
Shape Bulk composition
Size distribution
Bulk chemical composition Additives (what?, how much?) Shape
Crystalline/amorphous Ionic or non-charged Bulk chemical composition
Pore size/roughness Small molecules
Bio and macro molecules
Surface composition
covalently linked molecules
Surface composition adsorbed species
covalently linked molecules pH, conductivity surface charge (zeta potential)
adsorbed species Viscosity
Compliance/modulus, etc
Refractive index
Density Pore size/roughness
Compliance/modulus, etc Crystalline/amorphous
Refractive index Refractive index
Density Density
 Outline for Part I: Nanoparticle Characterization

 Introduction/context:
Particles at biointerfaces
Properties of particles in dispersions/emulsions

 Particle Size and Particle Size Distribution

 Surface Charge: Zeta Potential, Isoelectric Point, Electrophoretic Mobility

 Particle Size and Distribution: “Families” of Measurement
Principles
ensemble fractionation counting/”sorting”

All particles analyzed Particles separated/sorted in Particles separated (diluted)

simultaneously space/time but not sorted
Based on first principles Based on differential (zonal) Based on size-dependent
(optics, acoustics, etc) migration change in electronic
(V, I, R, C, L) or optical signal
Size obtained from curve Size obtained by comparison Size obtained from prior
fitting/matrix inversion with calibration standards calibration (instrumental)
Distribution data from further Distribution data from detector Distribution data from electronic
computations on data output (graphical trace) “binning” of detector signals

Dynamic Light Scattering Size Exclusion Chromatogr. Coulter & Elzone (r)
Static Light Scattering Capillary Hydrodynamic Flow Accusizer (r)
(& Laser Diffraction) Field Flow Fractionation
Acoustic Attenuation Electrophoresis Microscopy with image
[Sedimentation Velocity] analysis is analogous

Also: hyphenated methods: e.g. fractionation method

with ensemble-method detection (e.g. FFF-DLS).
 Ensemble Methods

Dynamic Light Scattering Brownian Motion of Particles; temporal

fluctuations in intensity of scattered light

Static Light Scattering Angular dependence of intensity of

(& Laser Diffraction) scattered light (Rayleigh/Mie)

Acoustic Attenuation (new, not widely used; not covered in

this lecture)

“Instrument as black box” approach:

Put sample in cuvette (diluting if necessary per mfgr.’s instructions)
Enter known constants or accept instrument defaults
(e.g. refractive index, viscosity, etc)
Choose adjustable parameters or accept instrument defaults
Press GO; come back when done; pick up print out

Now, let’s take a closer

look…
 Dynamic Light Scattering (DLS)
aka Photon Correlation Spectroscopy (PCS)
aka Quasielastic Light Scattering (QELS)

Step 1: Make the optical

measurement: Temporal
fluctuations in intensity
of scattered light (wavelength
and angle are set by instrument; RI is
input or assumed)

“speckle pattern”

Figures from: http://www.science.uva.nl/~sprik/masterlaser/dlsexp/dls.html

 Dynamic Light Scattering (DLS)
aka Photon Correlation Spectroscopy (PCS)
aka Quasielastic Light Scattering (QELS)

Step 1: Make the optical

measurement: Temporal
fluctuations in intensity
of scattered light (wavelength
and angle are set by instrument; RI is
input or assumed)

**Step 2: Establish the

correlation function and
solve for Dr
**Step 3: Use the Stokes-Einstein
relationship to solve for Rh (Dh= 2Rh)
**Step 4: Further process autocorrelation
data to obtain distribution information

**Done by the instrument,

although user can adjust inputs
and processing options
 Dynamic Light Scattering (DLS)
aka Photon Correlation Spectroscopy (PCS)
aka Quasielastic Light Scattering (QELS)

Step 1: Make the optical

measurement: Temporal
fluctuations in intensity
of scattered light (wavelength
and angle are set by instrument; RI is
input or assumed)

Step 2: Establish the

correlation function and
determine Dr
Step 3: Use the Stokes-Einstein
relationship to solve for Rh (Dh= 2Rh)
Step 4: Further process autocorrelation
data to obtain distribution information

**Done by the instrument,

although user can adjust inputs
and processing options
 Ensemble Methods
Dynamic Light Scattering (DLS)
aka Photon Correlation Spectroscopy (PCS)
aka Quasielastic Light Scattering (QELS)

Step 1: Make the optical

measurement: Temporal
fluctuations in intensity
of scattered light (wavelength
and angle are set by instrument; RI is
input or assumed)

Step 2: Establish the

correlation function and
determine Dr
Step 3: Use the Stokes-Einstein
relationship to solve for Rh (Dh= 2Rh)
Step 4: Further process autocorrelation
data to obtain distribution information

The data reduction is done by

the instrument, although user
can adjust inputs and processing
options
 Ensemble Methods
Static Light Scattering (and Laser Diffraction)

Analogous to dynamic light scattering, but

different optical property measured:

Step 1: Measure the intensity of scattered light

as a function of scattering angle for a
dispersion of particles, usually at high
dilution

** Step 2: Process the data to obtain the mean

particle size and the distribution

The data reduction is done by

the instrument, although user
can adjust inputs and processing
options
Example: Sizing of Nanoparticles with Dynamic Light Scattering:

LDL Uncharged COOH functionalized

control nanocarrier control nanocarrier control

Uncharged Charged nanocarrers

nanocarrers + LDL + LDL

COOH functionalized
nanocarriers + LDL
 Fractionation Methods
Size Exclusion and other chromatography
Capillary Hydrodynamic Flow
Field Flow Fractionation (several variants)
Capillary Electophoresis
Sedimentation/Centrifugation

Instrument as black box approach:

Prepare sample if needed (diluted ; buffers and electrolytes added)
Prepare eluant and/or other media (gel, sucrose density gradient, etc)
Enter known constants or accept instrument defaults
Inject standard sample, start pump or rotor or turn on applied field
Run Calibration Standards,
Run Samples
Analyze data (graphically and mathematically)
m p o rt a nt, but
a i ls ar e not i is much
The det fr a ctionati o n
v e than
h at en s i
note t a nd labor int
e
more tim le methods.
mb
the ense ca l i br ation
Now, let’s take a closer mu s t e stablish
look… User
curve
 Fractionation Methods: Measurement Principles

Separation by differential (zonal) migration

Xyz
Mixture in -------> Peaks 0ut
Carrier fluid pumped through column, channel, etc
Analye velocity is a characteristic fraction of
carrier velocity, due to analyte interaction with
(i) stationary phase in column
or (ii) to an applied field
 Fractionation Methods: Measurement Principles

Adsorption, partition, size

Chromatography exclusion
Affinity, polarity,size

Electrophoretic mobility
Electrophore
Ionic size, charge
sis

Laminar flow profile

(velocity gradient)
Hydrodynamic flow
Size (big first)

Field-flow Laminar flow profile

Particle mass
fractionatio
Size (small first)
n
 Example: Capillary Hydrodynamic Flow

Comparison of CMP polishing slurries from

two vendors
Both slurrries nominally 50 nm particle
size
One is much more monodisperse than the
other

L.J. Anthony et al., Lucent Technologies, Bell Laboratories, unpublished work

 Example: Field Flow Fractionation

Same system as in DLS example: nanocarriers for LDL

sequestration
(Data below are unpublished work; Chnari, Moghe et
Note: this is also an example al. ) of Hyphenated Methods:
Field Flow Fractionation with Static Light
Scattering Detection
Particles separated in time/space for “real”
distribution;
Accurate
Using AFFF, unbound size data without calibration of FFF,
nanocarriers 80
Hydrodynamic Radius vs. Time micelle2a_Fx3Ramp15_01
LDL1a_Fx3Ramp15_01
Complex1a_Fx3Ramp15_01

Hydrodynamic Radius (nm)

and LDL just from25nm
of a round the as
DLS
well as Nanocarrier + LDL
complexes of 75nm were detected 60
Nanocarrier
(Fig. 8). The difference in elution times
Hydrodynamic Radius (nm)

between the unbound components

and the complexes is substantial
40
LDL
without peak overlap. In addition to
the near-ideal peak separation, the 20

method was proven to be non

destructive for the complexes. 0
12.0 14.0 16.0 18.0 20.0 22.0

Blue represents the unbound LDL, red the Time (Minutes)

Time (min)

unbound nanocarriers (M12P5 m icelles) and

green the complexes. Fig. 8: Isolation of LDL-Nanocarrier Complexes via AFFF
 Example: Sedimentation Velocity Analysis

Optimizing a
process for
preparing Assessing the
human serum process steps in
albumin particle synthesis
nanoparticles and purification
(from the assigned (note removal of
paper, K.Langer et
al.) fines)

Assessing
reproducibility of
three identical
runs
Caveat with all fractionation methods: Dispersion

Band-brodening in
time:
later eluting
zones are
inherently
broader
does not in
itself reflect
more
polydispersity;
effects need to
be deconvoluted
to determine
polydispersity
 Counting/”Sorting” Methods

Coulter(r) and Elzone(r) AccuSizer (r)

Particle displaces electrolyte in Particle obscures a light bean
sampling cell; changes the signal transmitted through the cell; changes
across the electrodes the light intensity
Mature method, widely used Newer method; excellent
- for cells and other “big” down to
particles ~ 300 nm, but true “nano”
range still being optimized
“Instrument as black box” approach:
Put sample in reservoir (diluting if necessary per mfg. instructions
Enter known constants or accept instrument defaults (refractive index,
viscosity, etc)
Choose adjustable parameters or accept instrument defaults
Press GO; come back when done; pick up print out

Now, let’s take a closer look…

http://openchemist.net/chemistry/coulter/node3.html
 Counting/”Sorting” Methods: Measurement Principles

After integration over the complete particle (i.e.

Measurement is over all the elements that contain the particle) we
based on an find that the instrument response is proportional to
experimentally the volume ν of a spherical particle, modified by a
determined function F:
relationship
between
“instrument
response” and Function F can be found from the integration over
particle size the particle but other approaches, including a best
fit from experimental results, are possible to find a
proper equation for F. The following equation was
found by De Blois and Bean using the
experimental (best fit) approach:

where d and D stand for the diameter of particle

and orifice.

http://openchemist.net/chemistry/coulter/node3.html
 Examples: Counting/”Sorting” submicron particles
(NiComp-PSS Accusizer)

Vendor’s data: “Real world” data:

Particles are ~245 and 380 nm Oversized (> 1 um) particles in
chemical mechanical polishing
process steps
(silica slurry, 50 nm nominal
particle size)
As-received

After polisher
set-up

After normal
polishing run
http://www.shjnj.cn/CPJS-2.htm

After wafer
L.J. Anthony et al: Proc. 2nd Int. Symp. Chemical Planarization in Integrated Circuit Device
broke on pad
Manufacturing, pp 181-196, The Electrochemical Society, 1998
 Outline for Part I: Nanoparticle Characterization

 Introduction/context:
Particles at biointerfaces
Properties of particles in dispersions/emulsions

 Particle Size and Particle Size Distribution

 Surface Charge:
Zeta Potential, Isoelectric Point, Electrophoretic Mobility
 Charged Species on Surfaces

 Origins of Surface Charge

 Characteristics of Surface Charge:
Definitions
 Zeta Potential and Electrophoretic
Mobility
 Determination of Zeta Potential
 Zeta Potential vs pH
 Assigned paper and other examples
 Origins of Surface Charge
1) Ionization of surface functional groups
Organic/molecular:
e.g. RCOOH <--> RCOO-, RNH2<--> RNH3+,
etc
As in protein/peptide C-terminus, N-
terminus,
certain side groups (aspartic acid, etc.)
Note: can be intrinsic to the particle
and/or surface-
functionalized/derivatized (biotin, etc.)

Inorganic/ionic:
2) Adsorption of charged species
e.g. SiOH <> SiO-)
Charged/ionizable molecules:
(For example, glass beads, hydroxyapatite)
e.g. surfactants, phospholipids
(For example: SDS, constituents of ECM)
Small ions:
e.g. Ca++, Mg++, etc.
(For example in certain physiological
processes)
Characteristics of Surface Charge: Definitions

Particle surface

Stern Layer: Rigid layer of ion

tightly bound to particle; ions
with the particle

Plane of hydrodynamic
shear: Also called
Slipping Plane: Boundary
of the Stern layer:
ions beyond the shear
plane do not travel with
the particle
Diffuse Layer:
Also called Electrical
Double Layer: Ionic
concentration not the same
as in bulk; there is a
gradient in concentration
of ions outward from the
particle until it matches
the bulk
Characteristics of Surface Charge: Definitions

Zeta
potential:
The
electrical
potential
that exists
at the
slipping
plane
The magnitude of the zeta potential gives an
indication of the potential stability of the colloidal
system
* If all the particles have a large zeta potential they will
repel each other
and there is dispersion stability
* If the particles have low zeta potential values then there
is no force to
prevent the particles coming together and there is
dispersion instability
 Zeta Potential and Electrophoretic
Mobility
In an applied electric field, charged
particles travel toward the electrode
of opposite charge. +
+ -
When attractive force of the electric -
field is balanced by the viscous drag
on the particle, the particle travels
This velocity
with constant is the partlcle’s
velocity.
electrophoretic mobility, UE
Note
relationship of UE = 2  z f(Ka)/3 
zeta potential
and z = Zeta potential
electrophoretic =dielectric constant (of electrolyte)
mobility; =dielectric constant (of electrolyte)
therefore… f(Ka) = Henry’s function
= ~1.5 (Smoluchowski approximation)
Zeta potential for particles >~ 200 nm and electrolyte ~> 1 x 10-3 M
can be = ~1.0 (Huckel approximation)
for smaller particles and/or dilute/non-aqueous dispersions
determined
 Determination of Zeta Potential

 Measure the Electrophoretic

Mobility, UE
(and know viscosity, dielectric constant; and
choose a Henry function)

 Solve Smoluchowski/Huckel Equation

for
Predominant Methods:
Zeta Potential
 Laser Doppler Velocimetry
 Phase Analysis Light
Scattering (PALS)
Method for particles with lower
mobilities
 Determination of Zeta Potential

Principles of PALS:
Similar to particle sizing by dynamic
light scattering
I.e. what is measured is temporal fluctuations
in intensity of light scattered by the particles
In
in light scattering, the fluctuations are
the dispersion.
related to Brownian motion of particles.
In PALS for ZP, the fluctuations are
related to the movement of the particle
in the applied field, i.e. to UE;
The ZP is then calculated from the UE
that is determined by the PALS
measurement.
(As in light scattering, the instrument’s
autocorrelator and software take care of
the data reduction.)
 Zeta Potential vs pH

Typical plot of Zeta pH dependency of

ZP is very
Potential vs pH.
important!
Zeta Potential,

Remember,
dispersion
stability (or
conversely,
ability of
mV

particles to
approach each
other) is
determined by ZP,
pH with ~ 30 mV being
the approximate
cutoff.
At ZP=0, net charge on
particle is 0. [In this example,
This is called the the dispersion is
stable below pH ~4
Zeta Potential and Electrolyte
Concentration
Zeta potential also depends on electrolyte
concentration! Remember that the ionic
environment of the particle exists as a
gradient that that eventually equilibrates
with the bulk solution.
Too few ions: not enough charge to
stabilize the particles
Too many ions: the double layer is
compressed and the particles can
approach (“salting out”)
Example: Zeta Potential Measurements

Optimizing a
Zeta potential Particle diameter process for
preparing
human serum
albumin
nanoparticles
(from the assigned
paper, K.Langer et
al.)
At low values
of Zeta
potential
(near pH 6),
the
dispersion
de-stabilizes
and the
particles
 Biointerfacial Characterization
(125:583)

Nanoparticles
L. Anthony and P. Moghe
Lectures:
Nov. 16th: Part I: Nanoparticle
Coming Characterization**
attractions! Dec. 4th: Part II: Biological
Characterization
(Nanoparticles at
Interfaces)

Lab Demo:
November 20th: Particle Size by DLS; Zeta
Potential
[8:40 - 9:20 AM; Wright-