Particle size is an important detection index for nano-pharmaceutical preparations. All nano-pharmaceutical preparations must be tested for particle size and particle size distribution, including nano-emulsion, nano-crystals, nano-particles, and nanoplex. There are many particle size measuring instruments, including nanometer laser particle size measuring instruments and microscopes (such as transmission electron microscope and scanning electron microscope) based on dynamic light scattering technology.

Electron microscope technology

Transmission electron microscope (TEM) and scanning electron microscope (SEM) are versatile electronic microscopy instruments. They are intuitive methods for particle size observation and measurement, and have high reliability. The size and shape of the nanometer drug delivery system can be observed with an electron microscope, the particle thickness can be estimated based on the contrast of the image, and statistics can be combined with image analysis to give a particle size distribution. If the particles are embedded and sliced to make thin samples, the microstructure inside the particles can also be analyzed.

In the electron microscope measurement, it should be noted that: 1)The measured particle size may be the size of the aggregates, so when preparing the nanoparticle SEM sample, it should be fully dispersed; 2) The measurement result is not statistical because the amount of the electron microscope sample is very small. As a result, the particles in the observation range are not representative; 3) The result observed by electron microscope is particle size rather than grain size.

Dynamic laser scattering

Dynamic laser scattering (DLS), also known as photon correlation spectroscopy (PCS), is the most widely used method for analyzing particle size of nanoparticles. This method obtains particle size information by measuring the diffusion coefficient of nanoparticles in a liquid. When nanoparticles are dispersed in a solvent, the particles diffuse in the solvent due to the Brownian motion of the nanoparticles. The velocity of the Brown moving particles is related to the particle size, which is consistent with the Stokes-Einstein equation: d (H) = kT / 3 πηD. In the formula, d (H) is the particle size; k is the Boltzmann constant; T is the thermodynamic temperature; η is the viscosity; D is the diffusion coefficient.

According to the viscosity η of the solvent (dispersion medium) and the dispersion temperature T, the particle diameter d can be obtained by measuring the diffusion coefficient D of the nanoparticles in the dispersion. The laser diffraction particle size analyzer is more accurate for samples with a particle size of more than 5 μm; the dynamic light scattering particle size analyzer is accurate for nano and sub-micron particle samples with a particle size of less than 5 μm. In this method, it should be noted that the best particles are spherical and monodisperse. In fact, the measured particles are mostly irregular and polydisperse. The particle shape and particle size distribution characteristics have a greater impact on the particle size analysis results, and the more irregular the particle shape and the wider the particle size distribution, the larger the error in the particle size analysis results. Laser particle size analysis has the advantages of small sample consumption, high degree of automation, fastness, good repeatability, and online analysis. Its disadvantage is that it limits the concentration of the sample and makes it difficult to analyze the particle size and particle size distribution of the high concentration system. At present, there are advanced instruments to relax the concentration range, but the sample with lower concentration is still more accurate than the sample with higher concentration due to the small interparticle interference. When using a laser particle size analyzer, you must have an understanding of the system particle size range, otherwise the results may be biased.

Small-angle X-ray scattering method

Small-angle scattering refers to the phenomenon of coherent scattering near the reciprocal lattice origin (000) node in X-ray diffraction. Small-angle X-ray scattering (SAXS) technology can study a variety of particles in the range of several nanometers to hundreds of nanometers. Analyzing the small-angle scattering pattern can obtain information on the long-period structure of the substance, or the shape, scale, or mass information of the submicron particles (or pores). Small-angle scattering is an extremely powerful technique or tool for analyzing the spatial correlation of diffuse objects, such as polymer chains and macromolecules in solution.

Specific surface area method

According to the specific surface area Sw of the unit mass powder, the diameter of the particles in the nanopowder can be calculated (assuming that the particles are spherical). The general measurement method of Sw is the BET multilayer gas adsorption method. Nitrogen is the adsorbent most commonly used by the specific surface area (BET) method, and the specific surface area ranges from 0.1 to 1000m2/g. The advantage of this method is that the equipment is simple and the test speed is fast, but it is only the specific surface area information of the nano-powder. After conversion, the average particle size is obtained, but the particle size distribution cannot be understood.

Atomic force microscopy

Atomic force microscopy (AFM) scans the surface of a sample by a tiny probe to convert the interaction between the probe and the surface of the sample into a surface topography and characteristic image. Its advantage is that it can provide a three-dimensional and high-resolution image of the surface, and has a high horizontal and vertical resolution. In addition to measuring the particle size, it can also describe the sample shape. Its disadvantages are small sample observation time and time consuming. Similar technologies to AFM include scanning transmission microscopy (STEM) and scanning transmission x-ray microscopy (STXM).

Selection of particle size measurement techniques

Nano drug delivery systems consist of a series of particles of varying sizes. Depending on the purpose of the measurement, the overall particle size distribution can be expressed by the number or volume diameter. The particle size distribution is determined not only by the average particle diameter, but also by the method of evaluating the average particle diameter. In addition, the particle distribution shape greatly affects the result of particle distribution. The influence of large particles on the calculation results in DLS measurement is very significant, and the scattered light of core-shell double-layer particles and single-layer particles with the same particle size distribution is different at different wavelengths. In practical applications, generally based on the actual particle size range, the detection method is reasonably selected, and two or more methods can be used to separately measure and verify each other. The triptolide nanoparticles were characterized by TEM and AFM at the same time. It was found that the structure of the nanoparticles was round and the surface was smooth.

Author's Bio: 

CD Bioparticles is a leading manufacturer of magnetic particles and related products for immunoassay development. It provides a comprehensive list of immunomagnetic bead products conjugated with different coating materials and functional groups in multiple sizes for research and industrial prospect development. Its Absolute Mag™ platform is dedicated to developing magnetic microsphere for life science applications, such as immunoprecipitation, cell isolation, RNA/DNA extraction, and protein purification.