Lecture 10: Tools - 1 Techniques for Physicochemical Characterization of Nanomaterials

Lecture 10: Tools - 1

Techniques for Physicochemical Characterization of Nanomaterials

Outline

  • Nanomaterial characterization: Key aspects include:
      - Size
      - Surface properties
      - Shape
      - Composition
      - Stability

  • Overview of methods: Various techniques used to characterize nanomaterials includes:
      - Laser Dynamic light scattering (DLS)
      - Hydrodynamic diameter vs. particle diameter
      - Polydispersity index, Zeta potential
      - Wavelength and resolution
      - Transmission electron microscopy (TEM)

Quality Assurance in Medicine

  • Importance: Quality assurance is critical in medicine as it can be a matter of life and death.

  • Diagnostics:
      - National Association of Testing Authorities (NATA): An accreditation body recognized by the Australian Government.
      - Provides accreditation services for areas such as medical and veterinary testing, forensic science, medical imaging, and reference material production.

  • Therapeutics:
      - Good Manufacturing Practice (GMP): Ensures that products are produced and controlled consistently according to quality standards, minimizing risks that cannot be eliminated through final product testing.
      - Good Laboratory Practice (GLP): A quality management system for nonclinical laboratories to ensure reliability, consistency, and integrity of tests pertaining to chemical safety, including pharmaceuticals.

Techniques for Physicochemical Characterization of Nanomaterials

  • Size: A crucial factor influencing:
      - Circulation and navigation of nanomaterials in the bloodstream.
      - Ability to penetrate physiological drug barriers.
      - Site and cell-specific localization.
      - Induction of cellular responses (Yi Zhang and Tza-Huei Wang, Theranostics 2012; 2(7):631-654).

  • Surface Properties: Characteristics influenced by atomic/molecular composition and physical structure of surfaces:
      - Surface composition
      - Surface energy
      - Wettability
      - Surface charge and species absorbance/adhesion

  • Composition: Influences transport, delivery and biodistribution. Impurities can affect drug efficacy and side effects.
      - Purity determined by chemical composition analysis.
      - Requires purification processes to remove residuals and ensure absence of endotoxins.
      - Combining types of nanomaterials may form complexes such as chelates or conjugants which complicate analysis.

Shape

  • The shape of nanomaterials significantly impacts:
      - Drug delivery, degradation, transport, targeting, and internalization.
      - Efficiency of drug delivery carriers influenced by shapes (e.g., phagocytosis by macrophages).
      - Flow and adhesion in the circulatory system affected by shapes, impacting in vivo circulation time of nanomedicine.
      - Shape influences cellular uptake, biocompatibility, and tissue retention.

Stability

  • Defined as the ability to retain properties over time post-manufacture.

  • Impact on toxicity: Quantum dot cytotoxicity can arise from synthesis, storage, or degradation processes.

  • Factors affecting stability similar to conventional pharmaceuticals:
      - Temperature, moisture, solvents, pH, particle/molecular size, radiation exposure, enzymatic degradation, presence of excipients.

Overview of Methods Used

  • Various methods available for physicochemical characterization:
      - Dynamic light scattering (DLS)
      - Fluorescence correlation spectroscopy (FCS)
      - Raman scattering (RS), Surface Enhanced Raman (SERS)
      - Tip-enhanced Raman spectroscopy (TERS)
      - Near-field scanning optical microscopy (NSOM)
      - Circular dichroism (CD)
      - Mass spectroscopy (MS)
      - Infrared spectroscopy (IS)
      - Attenuated total reflection Fourier transform infrared (ATR–FTIR)
      - Scanning Electron Microscopy (SEM)
      - Environmental SEM (ESEM)
      - Transmission Electron Microscopy (TEM)
      - Scanning tunneling microscopy (STM)
      - Atomic force microscopy (AFM)
      - Nuclear magnetic resonance (NMR)
      - X-ray diffraction (XRD)
      - Small-angle X-ray scattering (SAXS)

Measuring Particle Size by Light Scattering

  • Determines area/volume diameter, polymers radius of gyration, or molecular mass.

  • Principle of operation:
      - Interaction of laser light causes scattering; intensity of scattered light measured.
      - Two principles: Static light scattering and Dynamic light scattering.

  • Size Range: 0.1 nm - 1000 µm.

  • Benefits: Well-established, easy to operate, yields reproducible data.

  • Drawbacks:
      - Changes in properties for diluted samples.
      - Tends to oversize large particles and overestimate small ones.

Static Light Scattering

  • Particle size information from intensity of scattering pattern at various angles.

  • Influenced by:
      - Wavelength of the light
      - Scattering angle
      - Particle size
      - Relative index of refraction (n) of the particle and the medium.

  • Particle size determined by correlating light intensity variations to Brownian movement.

Dynamic Light Scattering (DLS)

  • Lower detection limit: 0.5 nm to 5 nm, depending on the particle size.

  • Determining size:
      - Measures diffusion constant rather than direct particle size.
      - Scattering intensity variations measured at a fixed angle (typically 90°) to assess size distribution and hydrodynamic radius.

DLS Instrumentation

  • Measures intensity of light scattered as a function of time.

  • Molecules in solution exhibit Brownian motion causing changes in light intensity.

  • Factors controlling diffusion:
      - Temperature: Increased temperature leads to faster motion.
      - Solvent viscosity: Higher viscosity slows molecules.
      - Size of molecules: Larger molecules move slower.

  • Constant conditions allow intensity variation to be related to the hydrodynamic radius (Rh).

Hydrodynamic Diameter vs. Particle Diameter

  • Hydrodynamic Radius: Defined by the molecule rotating in all directions plus the hydration layer.
      - Measure of the ease of moving the molecule through solvent.

Polydispersity Index

  • Measures the distribution of molecular mass/size in a sample.

  • Describes the degree of non-uniformity in the size distribution.

Zeta Potential

  • Key stability indicator of colloidal dispersions.

  • Magnitude indicates electrostatic repulsion between similarly charged particles:
      - High zeta potential = stable; low zeta = potential flocculation.

  • Defined as the potential difference between solid particle surface and the bulk of the liquid.

Zeta Potential Values and Stability Behavior

  • Zeta potential significance:
      - 0 to ±5 mV: Rapid coagulation/flocculation.
      - ±10 to ±30 mV: Incipient instability.
      - ±30 to ±40 mV: Moderate stability.
      - ±40 to ±60 mV: Good stability.
      - > ±61 mV: Excellent stability.

Strengths and Limitations of DLS

  • Strengths:
      - Non-destructive, rapid, reproducible measurements.
      - Can measure in any liquid media.

  • Limitations:
      - Sensitivity to small sample concentrations;
      - Oversizing large particles;
      - Limited resolution for polydisperse samples;
      - Assumes spherical shape; does not measure particle concentration.

Nanoparticle Tracking Analysis (NTA)

  • Analyzes particles in liquids, relating Brownian motion rate to size.

  • Size distribution determined from 10-2000 nm in liquid suspension.

  • Utilizes an ultramicroscope and laser illumination for visualizing particles.

  • Process:
      - Light scattering captured using CCD/EMCCD cameras and analyzed using software.
      - Allows visualization of real-time events like aggregation/dissolution.
      - Minimal sample preparation improves processing time.

Maximum Resolution of Tools

  • Lateral (x,y) resolution vs. axial (z) resolution (~2-2.5-fold lower):
      - Human Eye: ~100 µm (0.1 mm)
      - Light Microscope: 200 nm (x,y), 500 nm (z)
      - Diffraction Limit: Ability of light microscopy to distinguish structures smaller than half the wavelength, about 200 nm (described by Ernst Abbe, 1873).

  • Scanning Electron Microscope (SEM): 3 nm.

  • Transmission Electron Microscope (TEM): 0.2 nm.

  • Super Resolution Microscopy: ~20 nm (2014 Nobel Prize).

Transmission Electron Microscopy (TEM)

  • History: First built by Max Knoll and Ernst Ruska in 1931; commercial version in 1939.

  • Standard grid sizes: 3.05 mm diameter ring; sample thickness of 50-100 nm is optimal.

Applications of TEM

  • Used for:
      - Size and size distribution
      - Shape heterogeneity
      - Aggregation and dispersion

  • Example: TEM images of foamy carbon-coated silver nanoparticles demonstrate shape variations and agglomeration.

Sample Preparation for TEM

  1. Fixation: Chemical fixation (e.g., glutaraldehyde).

  2. Rinsing and Staining: Heavy metal compounds for treatment.

  3. Dehydration: Washing with increasing ethanol, followed by a polar substance.

  4. Embedding: Material infiltrated with resin.

  5. Trimming and Sectioning: Sections cut to ~70 nm with glass knives using an ultra-microtome.

  6. Collection on Grid.

TEM: Pros and Cons

  • Strengths:
      - Direct measurement of size and shape.
      - Higher spatial resolution than SEM.
      - Several analytical methods can be coupled with TEM.

  • Limitations:
      - Ultrathin samples are mandatory (approx. 70 nm thick).
      - Electron-dense stains applied for contrast.
      - Risk of sample damage or alteration.
      - Considerations of sample thickness for resolution.
      - Expensive equipment.

Learning Outcomes

  1. Understand how NP size, shape, and composition affect in vivo applications.

  2. Understand the relationship between NP stability and in vivo behavior/toxicity.

  3. Explain principles of dynamic light scattering (DLS) and its use in NP size determination.

  4. Explain polydispersity index use in NP characterization.

  5. Understand Zeta Potential influence on colloid stability.

  6. Recognize strengths and limitations of DLS technique.

  7. Understand wavelength-resolution relationship.

  8. Appreciate microscopy limitations.

  9. Describe principles of TEM and its applications, advantages, and limitations.