*3.1. Characterization*

#### 3.1.1. Particle Size and Distribution

The particle size range of drug nanocrystals has a certain size requirement, which is generally less than 1 μm. Therefore, it is very important for drug nanocrystals to precisely control the particle size of the drug to obtain narrow and uniform particle size distribution. The particle size not only affects the drug loading and release behavior of API, but also is closely related to pharmacokinetics, biodistribution, and even the delivery mechanism of drug nanocrystals. To summarize, particle size and distribution are considered to be the most important index parameters of nanocrystals, which is one of the key elements in the development and control quality of nanocrystals [41].

The particle size and distribution can be evaluated with the help of offline or online evaluation tools. The measurement is usually performed by dynamic light scattering (DLS), also known as photon correlation spectroscopy (PCS). It observes Brownian motion using laser irradiation of particles and relates Brownian motion to particle size by analyzing the light intensity fluctuations of the scattered light. The measurement result is the hydrodynamic particle size, and the particle size distribution is generally expressed using PDI. In general, a PDI value of 0.1–0.25 represents a narrow particle size distribution and also indicates that the nanocrystal system is stable [74]. DLS can obtain accurate and statistical particle size distributions, but it is necessary to make nanocrystals into well-dispersed suspensions. From the variation of Z-average and PDI values, small increases in drug nanocrystal size can also be assessed by DLS. Therefore, DLS is considered effective for measuring the particle size of submicron and nanoparticles.

Laser Diffraction (LD) analyzers are designed based on the phenomenon of light diffraction, which occurs when light passes through particles and the angle of the diffracted light is inversely proportional to the size of the particles. Typical characterization parameters of LD are 50%, 90%, and 99% of the diameter, expressed as D50, D90, and D99, respectively (i.e., D50 means that 50% of the particle volume is below a given size). The measurement range of LD and PCS varies, where LD is generally used to measure particle sizes larger than 0.05 μm, while PCS usually gives the practical size in the range from 3 nm to 3 μm [75]. Furthermore, it is important to note that the data of particle size acquired by LD and PCS for nanosuspensions are different because the data from LD is volume-based, while PCS gives the average particle size based on light intensity-weighted particle size [2].

Small-angle X-ray scattering (SAXS) uses the X-ray small-angle scattering effect to measure the particle size distribution of nanocrystals. This is a simple method with high accuracy, but it is also relatively expensive. Typically, it can measure the size distribution of particles in the range of 1–300 nm.

#### 3.1.2. Morphological Characterization

Both the size and morphological state of nanoparticles can influence the structural properties of nanocrystals. Scanning electron microscopy (SEM), transmission electron microscopy (TEM), and atomic force microscopy (AFM) are frequently applied to characterize the morphological appearance of drug nanocrystals. It should be mentioned that some people use electron microscopy images supplemented with statistics to obtain the mean value of the particle size of nanocrystals, which is the actual particle size. In contrast, the previous section obtained by DLS or LD is only the hydrodynamic size of the nanoparticles.

SEM uses narrow-focused beams of high energy electrons to scan the sample for the purpose of characterizing the microscopic morphology of the substance. The imaging stereo effect is good, and the resolution can reach 1 nm. Therefore, it is now widely used to observe nanomaterials. In particular, when formulated nanosuspensions are converted

into dry powders, SEM analysis is critical to monitor changes in particle shape and size. SEM images of different nanocrystals obtained in the literature are as follows (Figure 3).

**Figure 3.** (**a**,**b**) SEM micrographs of clarithromycin nanocrystals obtained by media milling method; (**c**) SEM micrographs of carvedilol nanosuspensions using the antisolvent precipitationultrasonication method; (**d**) Transform Breviscapine (BVC) nanocrystals into BVC nanocrystals embedded microparticles (BVC-NEP) via spray-drying, adding matrix formers Maltodextrin. Figure 3a,b were reprinted from ref. [76] with permission from Elsevier, Copyright® 2018. Figure 3c was reprinted from ref. [70] with permission from Springer Nature, Copyright® 2012. Figure 3d was reprinted from ref. [77] with permission from Elsevier, Copyright® 2021.

TEM uses an electron beam as the light source to project accelerated and aggregated electron beams onto the very thin sample. In order for the electron beam to penetrate, the thickness of the sample should be less than 100 nm. TEM requires an appropriate concentration of the wet sample. The current resolution is up to 0.2 nm. TEM images of different nanocrystals obtained in the literature are as follows (Figure 4).

AFM is a new generation of scanning probe microscopy. It is capable of imaging in any environment (including liquids), and the low force of the needle tip on the sample surface prevents damage to the sample. It does not require the sample to be electrically conductive. The sample can be directly observed at the nanoscale without special treatment. A three-dimensional image of the sample surface can be obtained by collecting feedback signals from the force applied to the sample by the probe [80]. AFM can also provide information on the shape and structure of nanocrystals that cannot be accessed by other methods [81]. Overall, AFM has become an important tool for conducting real-time observations at the nanoscale.

**Figure 4.** Breviscapine (BVC) nanocrystals modified by d-Tocopherol acid polyethylene glycol 1000 succinate (TPGS) at different concentrations. (**a**) BVC-NC/@5%TPGS, (**b**) BVC-NC/@10%TPGS, (**c**) BVC-NC/@20%TPGS, (**d**) BVC-NC/@30%TPGS; (**e**,**f**) 10-Hydroxycamptothecin nanocrystals; (**g**,**h**) TEM of the resveratrol nanocrystals. Figure 4a–d were reprinted from ref. [77] with permission from Elsevier, Copyright® 2021. Figure 4e,f were reprinted from ref. [78] with permission from Elsevier, Copyright® 2021. Figure 4g,h were reprinted from ref. [79] with permission from Elsevier, Copyright® 2022.
