The important parameters which need to be evaluated for the SLNs are:

• particle size
• size distribution kinetics (zeta potential)
• degree of crystallinity
• lipid modification (polymorphism)
• coexistence of additional colloidal structures (micelles, liposome, super cooled, melts, drug nanoparticles)
• time scale of distribution processes
• drug content
• in vitro drug release
• surface morphology

The particle size/size-distribution may be studied using :
  • photon correlation spectroscopy (PCS)
  • transmission electron microscopy (TEM)
  • scanning electron microscopy (SEM)
  • atomic force microscopy (AFM)
  • scanning tunneling microscopy (STM)
  • freeze fracture electron microscopy (FFEM)




Measurement of particle size and zeta potential

PCS is a good tool to characterize nanoparticles, but it is not able to detect larger microparticles. To detect larger microparticle can be used LD measurements. This method is based on the dependence of the diffraction angle on the particle radius (Fraunhofer spectra). Smaller particles cause more intense scattering at high angles compared to the larger ones. LD can coverage broad size range from the nanometer to the lower millimeter range.

It is highly recommended to use PCS and LD simultaneously.They detect light scattering effects which are used to calculate particle size. Platelet structures commonly occur during lipid crystallization and have also been suggested in the SLN.

Difficulties may arise both in PCS and LD measurements for samples which contain several populations of different size. Therefore, additional techniques might be useful. For example, light microscopy is recommended, although it is not sensitive to the nanometer size range. It gives a fast indication of the presence and character of microparticles.

Electron microscopy provides, in contrast to PCS and LD, direct information on the particle shape. Solvent removal may cause modifications which will influence the particle shape. Zeta potential is an important product characteristic of SLNs since its high value is expected to lead to deaggregation of particles in the absence of other complicating factors such as steric stabilizers or hydrophilic surface appendages. It is usually measured by zetameter.


Dynamic light scattering (DLS)

Dynamic Light Scattering (DLS), sometimes called Photon Correlation Spectroscopy (PCS) or Quasi-Elastic Light Scattering (QELS) records the variation in the intensity of scattered light on the microsecond time scale. DLS is the only technique able to measure particles in a solution or dispersion in a fast, routine manner with little or no sample preparation. The minimal or entire lack of sample preparation ensures that the sample is measured as it actually is.The preparation required for other techniques can change the properties of the particles, for example aggregates can be created or destroyed. The advantages of the method are the speed of analysis, lack of required calibration, and sensitivity to submicrometer particles. Size range from a few nanometers to about 3 microns

DLS Mechanism of Action :Photon Correlation Spectroscopy (PCS) is based on Dynamic Light Scattering. The time decay of the near-order of the particles caused by the Brownian motion is used to evaluate the size of nanoparticles via the Stokes-Einstein relation. At constant temperature T the method only requires the knowledge of the viscosity  of the suspending fluid for an estimation of the average particle size and its distribution function (and for volume fractions the refractive index n).


Static light scattering/Fraunhofer diffraction
Static light scattering (SLS) is an ensemble method in which the pattern of light scattered from a solution of particles is collected and fit to fundamental electromagnetic equations in which size is the primary variable. The method is fast and rugged, but requires more cleanliness than DLS, and advance knowledge of the particles' optical qualities. Static light scattering is a technique in physical chemistry that measures the intensity of the scattered light to obtain the average molecular weight Mw of a macromolecule like a polymer or a protein. Measurement of the scattering intensity at many angles allows calculation of the root mean square radius, also called the radius of gyration Rg. By measuring the scattering intensity for many samples of various concentrations, the second virial coefficient A2, can be calculated

Acoustic methods
Another ensemble approach, acoustic spectroscopy, measures the attenuation of sound waves as a means of determining size through the fitting of physically relevant equations. In addition, the oscillating electric field generated by the movement of charged particles under the influence of acoustic energy can be detected to provide information on surface charge.

Nuclear magnetic resonance (NMR)
NMR can be used to determine both the size and the qualitative nature of nanoparticles. The selectivity afforded by chemical shift complements the sensitivity to molecular mobility to provide information on the physicochemical status of components within the nanoparticle.

NMR is an effect whereby magnetic nuclei in a magnetic field absorb and re-emit electromagnetic (EM) energy. This energy is at a specific resonance frequency which depends on the strength of the magnetic field and other factors. This allows the observation of specific quantum mechanical magnetic properties of an atomic nucleus.

Electron microscopy
SEM and TEM provide a way to directly observe nanoparticles, physical characterization of nanoparticles with the former method being better for morphological examination. TEM has a smaller size limit of detection, is a good validation for other methods, and affords structural required, and one must be cognizant of the statistically small sample size and the effect that vacuum can have on the particles.

An electron microscope is a type of microscope that uses a particle beam of electrons to illuminate the specimen and produce a magnified image. Electron microscopes (EM) have a greater resolving power than a light-powered optical microscope, because electrons have wavelengths about 100,000 times shorter than visible light (photons), and can achieve better than 50 pm resolution[1] and magnifications of up to about 10,000,000x, whereas ordinary, non-confocal light microscopes are limited by diffraction to about 200 nm resolution and useful magnifications below 2000x.


Atomic force microscopy (AFM)
There are a number of related techniques such as Scanning Tunnelling Microscopy (STM)that all require a well trained technician to prepare the sample in a suitable manner for scanning with a probe.

In this technique, a probe tip with atomic scale sharpness is rastered across a sample to produce a topological map based on the forces at play between the tip and the surface. The probe can be dragged across the sample (contact mode), or allowed to hover just above (noncontact mode), with the exact nature of the particular force employed serving to distinguish among the subtechniques. That ultrahigh resolution is obtainable with this approach, which along with the ability to map a sample according to properties in addition to size, e.g., colloidal attraction or resistance to deformation, makes AFM a valuable tool. The technique is only suitable for ‘hard’ materials, i.e. those not affected by the preparation technique and is poor from a statistical p oint of view as only tens or hundreds of particles are measured.

X-ray diffraction (powder X-ray diffraction) and differential scanning calorimetry (DSC)
The geometric scattering of radiation from crystal planes within a solid allow the presence or absence of the former to be determined thus permitting the degree of crystallinity to be assessed. Another method that is a little different from its implementation with bulk materials, DSC can be used to determine the nature and speciation of crystallinity within nanoparticles through the measurement of glass and melting point temperatures and their associated enthalpies.

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