Challenges in Method Development and Particle Analysis of Biomaterials-Zhibin.pdf

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Challenges in Method Development and Particle Analysis of Biomaterials
Zhibin Guo
Senior Application Engineer
Bettersize Instruments Ltd.

www. bettersizeinstruments.com info@bettersize.com 2 / 20
I. Dynamic Light Scattering (DLS)
 Measuring Principle
 Biomaterial Example: AuNPs, Lysozyme, Latexes Coupled With Antibodies
II. Electrophoretic Light Scattering (ELS)
 Biomaterial Example: Bio-MOF, Calcium Silicate Nanocomposites
III. Static Light Scattering (SLS)
 Biomaterial Example: BSA
Light Scattering Techniques for Biomaterials

Light Scattering Techniques
Vibrational Energy States
Virtual Energy States
0
1
2
3
4
Excitation
Energy
Stokes
Raman
Scattering
Anti-Stokes
Raman
Scattering
Rayleigh
Scattering
Rayleigh Scattering
Stokes Raman Scattering
Anti-Stokes Raman Scattering
Laser
Particle Interaction
Scattered
light
Size Zeta Potential
Molecular Weight

Dynamic Light Scattering (DLS)
100 nm Particles
600 nm Particles
• Faster movement
• Rapid decay
• Smaller particles
• Slower movement
• Slower decay
• Larger particles

https://doi.org/10.1016/j.talanta.2022.124047
Binding of DNA sequences of Omicron (SARS-CoV-2) N501Y variant
to AuNPs detection probes leads to AuNPs aggregation.
The AuNPs aggregates will generate a strong PA signal on a bio-sensor based
on photoacoustic (PA) effect, thus enabling a fast DNA detection function.
Biomaterial Example: AuNPs
• DLS can sensitively monitor the size change of AuNPs
when adding 0.1-0.7mM MgSO4 to 0.1nM AuNP solution

Biomaterial Example: Lysozyme
Correlation functions of 10 mg/mL lysozyme
Correlation functions of 5 mg/mL lysozyme
Size distributions of 10 mg/mL lysozyme
Size distributions of 5mg/mL lysozyme
Concentration Z-ave (nm) Calculated Mw
(kDa)
10 mg/mL 3.47±0.31 10.3
5 mg/mL 3.79±0.90 12.4
D = K � 𝑀𝑀−𝛼𝛼
Mark-Houwink Equation • Perform measurements at several
concentrations to see if you get the same
Z-ave to confirm your results are valid.

Biomaterial Example: Latexes Coupled with Antibodies
Overlay of size distributions
Sample Z-ave (nm) RSD% PDI
A 200.47±1.73 0.86 0.027
B 99.52±0.41 0.42 0.028
C 411.59±4.05 0.98 0.029
Measured Z-average size of different antibodies
Latex spheres combined with monoclonal antibodies
• Size and size distribution of latexes are critical factors that
determine the response and sensitivity of turbidity assays.
• DLS can efficiently examine the variations in particle size.

Electrophoretic Light Scattering (ELS)
Stern layer
Shear layer
Surface potential
Stern potential
Zeta potential
mV
Positively
Charged
Particle
Distance
PC
PZT
𝝁𝝁 =
𝜆𝜆
𝑛𝑛 𝑠𝑠𝑠𝑠𝑠𝑠 𝜃𝜃
�
1
𝐸𝐸
� ∆𝒇𝒇
𝜻𝜻 =
2𝜀𝜀𝑟𝑟𝜀𝜀0
3𝜂𝜂𝝁𝝁
𝑓𝑓 𝜅𝜅𝜅𝜅
Henry’s Equation
• High repulsion force of particles
• High zeta potential
😊😊 Stable particle system
• Flocculation, aggregation
• Low or zero zeta potential
😒😒 Unstable particle system

Autotitrator with ELS
BeNano Nanoparticle Analyzer
BAT-1 Autotitrator
Circulation Tubes
• Multi-step pH titration can be performed by the BAT-1 Autotitrator from pH 1 to 13 automatically with the BeNano.

Biomaterial Example: Bio-MOF
https://doi.org/10.1016/j.ijpharm.2022.121912
-14.18 mV
-4.13 mV
-20.48 mV
-8.86 mV
4.20 mV
Polydopamine
pH = 8.5
Bio-MOF MP MPD
DOX⋅HCl
• A change in the zeta
potential can indicate
whether the drug is
loaded or separated
from the carrier.

Particle size change in PBS solution
left for 72 hours
Particle size change in FBS solution
placed for 72 hours
Polydopamine
pH = 8.5
Bio-MOF MP
DOX
MPDH MPD
HA
Targeted therapeutic nano-delivery system with multi-functionality
https://doi.org/10.1016/j.ijpharm.2022.121912
Biomaterial Example: Bio-MOF
• The drug delivery system remains highly stable as evidenced
by its minimal size variation over time, unaffected by salt ions
and proteins.

https://doi.org/10.1016/j.jconrel.2021.11.044
Biomaterial Example: Calcium Silicate Nanocomposites
Particle size distribution before and after loading with indocyanine
green (ICG) and mannose-based hyaluronic acid (Man-HA)
Z-ave=143 nm
Z-ave=118.5 nm
Average zeta potential before and after loading
indocyanine green (ICG), mannose-based
hyaluronic acid (Man-HA)
-13.6 mV
-8.4 mV
• Both the size change and the zeta
potential change demonstrate an
interaction between loaded drug ICG
and drug carrier MCNs.
Dual-targeted multifunctional
nanoplatform based on mesoporous
calcium silicate nanocomposites (MCNs)
ICG ALE/Man-g-HA
Calcium Silicate
Hydrogen
bonding

Static Light Scattering (SLS)
Toluene Solvent
Solution with the
concentration of C1
Solution with the
concentration of C2
Solution with the
concentration of C3
Solution with the
concentration of C4
Intercept Mw
Slope A2
𝐾𝐾𝑐𝑐
𝑅𝑅𝜃𝜃
=
1
𝑀𝑀𝑤𝑤
+ 2𝐴𝐴2𝑐𝑐
An example of the dependence of mechanical strength
on the molecular weight of polymers

 The monomeric molecular weight of BSA is
approximately 67 kDa.
Solvent Mw (kDa) A2 (mL mol/g2)
10mM NaCl 96.15 1.5E-7
100mM NaCl 101.01 1E-6
10mM PBS 91.74 4E-7
Biomaterial Example: Bovine Serum Albumin (BSA)
• There are small amounts of oligomers or aggregates
present in the BSA protein solutions across different
solvents.

Formulation 1
Formulation 2
Concentration (mg/mL)
D
app
/D
0
SLS - Second Virial Coefficient (B22) DLS – Diffusion interaction parameter (kD)
+B22 =😊😊
- B22 =😒😒
+KD =😊😊
- KD =😒😒

Sample Size (nm) Zeta potential (mV) Tagg
1# 8.47±0.11 -10.3±1.15 68℃
2# 8.38±0.15 0.51±0.53 52℃
3# 8.21±0.05 -10.3±0.69 48℃
Measured size, zeta potential and aggregation temperature
Sample Concentration Dispersant
1# 10 mg/mL BSA PBS buffer
2# 10 mg/mL BSA PBS buffer + DTT
3# 10 mg/mL BSA PBS buffer + DTT + NaOH, pH=8.3
Sample preparation
• The temperature denaturation points of formulations 1#,
2#, and 3# decrease in sequence, indicating a
decreasing trend in the stability of their protein solutions.

Free Sample Testing
Costa Mesa, California
Bettersize Instruments US Facility

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