3.1.3. Structural Characterization

The crystalline form is also a quality factor of nanocrystal that needs to be noted. During the process of nanosizing, the crystalline state of drug particles may change, such as from crystalline state to amorphous state, which has a significant difference in physical properties, such as density, hardness, solubility, and stability. Therefore, the structure characterizations of nanocrystal need to be carefully considered.

Differential scanning calorimetry (DSC) is a common method for thermal analysis of nanocrystals. DSC measures the crystallinity of drug nanoparticles by detecting the glass transition temperature, melting point, and associated enthalpy. DSC also can be used to determine the interactions between excipients and drugs in nanocrystals as well as numerous thermodynamic and kinetic parameters [2]. The method has a wide temperature range and high resolution so that it has an indestructible place in structural characterization. In addition, thermal analysis can be investigated by thermogravimetric measurements or differential thermal analysis (DTA). Thermogravimetric analysis is a technique to study the thermal stability, heat flow, and structural deformation of particles in the inert environment [82]. DTA can be used for phase changes and other thermal processes, such as the determination of melting points [83]. In combination, thermogravimetric and differential thermal analysis (TG-DTA) can characterize multiple thermal properties of a nanocrystal sample at the same time. It is valuable for the investigation of thermal stability as well as for the determination of volatile content and other compositional analyses [2]. X-ray powder diffraction (XPRD) analysis based on constructive interference between monochromatic Xrays and samples, is another tool for characterizing the crystallinity of nanocrystals [52]. It can be used to determine the structural parameters and crystal structure of some crystalline substances, as well as amorphous substances.

Fourier transform infrared spectroscopy (FTIR) is a method of measuring interferograms and performing Fourier transform on the interferograms to measure infrared spectra with high resolution. FTIR can study the interaction between drugs and excipients in nanocrystal formulations at the molecular level. When the two components interact, FTIR spectroscopy shows the interaction by changing the vibrational frequency of the molecules [84].

#### 3.1.4. Surface Property

The particle surface charge situation is closely related to the stability of drug nanocrystals [85], which is generally reflected by the zeta potential (ZP) values. Currently, the measurement methods include electrophoresis, electroosmosis, flow potential, and ultrasound methods, among which laser Doppler electrophoresis is commonly used for measurement. The higher the ZP values, the greater the electrostatic repulsion among the particles, and the more stable the system. Usually, when the particles have sufficient ZP values, it will provide effective charge repulsion to prevent particle aggregation. In general, an absolute ZP value above 30 mV provides great stability and about 20 mV provides only short-term stability. However, for larger molecular weights of APIs, the stabilizer acts mainly through steric stabilization. In this case, a ZP value of only 20 mV or even less can provide sufficient stabilization [86].

#### *3.2. Performance Evaluation*

#### 3.2.1. Stability

The stability of nanoparticles is essential for the generation of nanosized effects. The stability of nanocrystals includes chemical stability (degradation and spoilage) and physical stability (sedimentation, agglomeration, crystal growth, and crystalline state) [87]. The large specific surface area of nanocrystals results in high free energy, which may result in physical instability, such as aggregation or agglomeration [88]. Therefore, surfactants/polymers are necessary to stabilize the nanocrystals. The choices of stabilizers can also affect the performances of drug nanocrystals *in vivo* and further formulations.

Stabilizers can mainly be divided into ionic and nonionic stabilizers (Table 1). One mechanism by which stabilizers work is to reduce the surface tension at the interphase interface, where electrostatic repulsion from ionic surfactants stabilizes the nanosuspension. The other mechanism is steric stabilization, where polymers cover the particles to prevent particle-to-particle aggregation [89]. The basic principles that must be followed in the selection of stabilizers are that the addition dose must be acceptable and safe to humans. For instance, stabilizers will not cause any allergic or immune response [90]. The current screening of surfactants/stabilizers is primarily based on reported references and experience. Experience indicates that binary or ternary mixtures of electrostatic stabilizers typically generate enhanced stabilization [2]. The stability is generally divided into long-term and short-term stability, which can be measured by the size, ZP values, and morphology of the nanocrystals [91].


**Table 1.** The types and usage of commonly used stabilizers.

Guo et al. [92] prepared nitrendipine nanocrystals (NTD-NCs) using a media milling method. Rectangular nanocrystals with 1.25% (*w*/*v*) HPMC-E5 and 0.4% (*w*/*v*) SDS as stabilizers were obtained with a particle size of 256.5 ± 6.6 nm. The size of the nanocrystals was monitored for 30 days (at 4 ◦C, 25 ◦C, and 40 ◦C). As shown in Figure 5, there was no change in monitoring results after 30 days of storing at 25 ± 2 ◦C (*p* > 0.05). When the product was stored at 4 ± 2 ◦C, it was noticed that there was a slight increase in particle size within 20 days. It is speculated to be due to recrystallization of free drug molecules from the existing larger crystal surfaces. When the product was stored at 4 ± 2 ◦C, a significant increase in particle size occurred. It is speculated that the Ostwald ripening phenomenon occurred. The particle sizes of the freshly prepared nanocrystals were monitored at 4 ◦C, 25 ◦C, and 40 ◦C for 30 days. As shown in Figure 5, the particle size remained constant after storing at 25 ± 2 ◦C for 30 days (*p* > 0.05). When stored at 4 ± 2 ◦C, a slight increase of particle size was observed from 268 nm to 300 nm for 20 days. This may be due to the recrystallization of free drug molecules on the surface of existing larger crystals. A significant increase in particle size was found at 40 ± 2 ◦C, where the Ostwald ripening phenomenon may have occurred. This indicates that the nanocrystals are unstable at higher temperatures. Therefore, appropriate temperature conditions are critical for the storage of NTD-NCs.

**Figure 5.** Particle size monitoring data of NTD nanocrystals stored at different temperature conditions for 30 days. Figure 5 was reprinted from ref. [92] with permission from Elsevier, Copyright® 2019.

Al Shaal et al. [93] prepared apigenin nanocrystals and assessed the long-term physical stability. The nanocrystals were stored under different conditions (refrigerated, room temperature, and 40 ◦C) for 6 months. According to PCS and LD data, neither significant increase in particle size nor change in particle size distribution can be found. No clearly visible aggregation was seen for the formulations on the day of production and after 6 months of storage at room temperature.

Luo et al. [94] prepared silymarin nanocrystal micro pills with PVP K30 and SDS as stabilizers. The produced nanocrystal micro pills were placed in a weighing open bottle for 10 days under high temperature/humidity/light conditions. The samples were taken on days 0, 5, and 10, respectively. The results showed that no significant changes occurred but the samples should be stored in a dry environment due to slight moisture absorption under high humidity conditions.

#### 3.2.2. Cytotoxicity

Safety is the primary concern for pharmaceuticals, so toxicity assessment is a priority for new drug registration [90]. Cytotoxicity assays are assessed by measuring the number or growth of cells before and after exposure to the sample [95]. It can be divided into leachate test, direct contact test, and indirect contact test. Cell viability is often used as an important evaluation indicator. Cytotoxicity experiments can provide the prerequisites for *in vivo* animal experiments, so it plays a vital role in the evaluation of drugs. Additionally, embryo toxicity assessment is more rapid than whole organism methods (<96 h).

Sheng et al. [96] prepared oridonin nanocrystals (ORI-NCs) and studied the *in vitro* cytotoxicity of ORI-NCs on Madin-Darby canine kidney (MDCK) cells. Both ORI and ORI-NCs exhibited an effect on inhibiting MDCK cell proliferation. The decrease in cell viability with increasing concentration indicates that ORI-NCs and ORI drugs exert concentrationdependent effects on MDCK cells. With increasing concentration, the cell viability of ORI-NCs decreased more significantly than that of ORI.

Choi [97] explored the cytotoxicity characteristics of cilostazol nanocrystals (CLT-NCs) in the Caco-2 cells. CLT-NC was successfully prepared by the ultrasonic probe method with a mean size of 500–600 nm. Cytotoxicity of CLT-NC and CLT was assessed in Caco-2 cells. The experimental results demonstrated that all CLT concentrations tested (0.02–20.0 μg/mL) did not exhibit toxicity (Figure 6a). Therefore, CLT-NC is safe for cells at the related concentrations.

**Figure 6.** (**a**) Cell viability was assessed in Caco-2 cells after induction of CLT and CLT-NC; (**b**) Zebrafish embryo morphology at the indicated hpf following GB-NCs treatment (Scale bar stands for 250 μm); The survival rate (**c**), hatching rate (**d**), heart rate (**e**) and body length (**f**) of zebrafish larvae at 96 hpf in the presence of GB-NCs. Figure 6a was reprinted from ref. [97] with permission from Springer Nature, Copyright® 2020. Figure 6b–f were reprinted from ref. [98] with permission from Elsevier, Copyright® 2020.

Liu et al. [98] obtained ginkgolide B nanocrystals (GB-NCs), a potent anti-parkinsonism compound. The toxicity assays were done in zebrafish embryos. The zebrafish embryos showed no morphological changes following GB-NCs treatment (Figure 6b), and no differences in hatching, heart rate, body length, or survival occurred at 96 h post-fertilization (hpf) in GB-NCs relative to control groups (Figure 6c–f). These data confirmed that the GB-NCs are non-toxic to zebrafish.

#### 3.2.3. *In Vitro* Dissolution

Dissolution or release of drugs is an important quality attribute of nanomedicines, which may have significant effects on drug absorption, *in vivo* safety and efficacy. *In vitro* dissolution or release can reflect the *in vivo* behavior of nanomedicines to some extent [41]. The flow cell method is often used to study the dissolution or release of drugs *in vitro*. A constant-flow pump is used to pump the release medium at the desired temperature into contact with the sample at the lower end of the flow cell at a suitable flow rate, and the release medium is filtered through the upper end of the flow cell to measure the concentration of the drug in the release medium at set time intervals [99]. Parameters such as dissolution rate and saturation solubility were determined to help predict the *in vivo* performance of drug nanocrystals. To a large extent, the dissolution rate depends on the size and surface area of the drug particles. The most important evaluation indicator is the dissolution curve, which can distinguish differences in product bioequivalence.

Zhu et al. [73] fabricated rod-shaped Nintedanib nanocrystals (BIBF-NCs) by the antisolvent precipitation-ultrasonic method with sodium carboxymethylcellulose as a stabilizer. The particle size was 325.30 ± 1.03 nm and the zeta potential was 32.70 ± 1.24 mV. Then, the *in vitro* dissolution of BIBF powder and BIBF-NCs in pH 6.8 PBS during 120 min were explored. Compared with the dissolution rate of BIBF powder at 10 min ((6.10 ± 1.55)%, *p* < 0.05), the BIBF-NCs was (73.74 ± 5.33)%. At 120 min both still exhibited large dissolution rate differences (Figure 7a). This phenomenon is attributed to smaller particle size reducing the diffusion layer thickness and increasing the surface area available for dissolution. Meanwhile, BIBF-NCs (rod-shape) have a larger surface area, resulting in higher dissolution rates. Additionally, the cumulative release of the drug in the gastrointestinal tract was simulated. The results showed that the cumulative release of BIBF-NCs was significantly higher than that of BIBF crude powder (Figure 7b).

**Figure 7.** (**a**) Dissolution curves of BIBF powder and BIBF-NCs in pH 6.8 PBS (containing 0.5% (*w*/*v*) Tween 80); (**b**) BIBF release profile of the BIBF powder and BIBF-NCs in gastric and intestinal simulated liquid media. Data represent the mean ± SD, n = 3. (\* *p* < 0.05, compared to BIBF crude). Figure 7a,b were reprinted from ref. [73] with permission from Elsevier, Copyright® 2022.

#### 3.2.4. *In Vivo* Dissolution

In order to observe the functional, metabolic, and morphological changes caused by drug nanocrystals, it is often necessary to deliver the drugs to animals. There are various ways of drug delivery with an emphasis on oral and injection. After the intervention with the drug, the corresponding tissues are taken for index testing. For example, the drug concentration in blood and certain organs is measured. Parameters such as dissolution rate, bioavailability, or relative bioavailability are calculated to study the *in vivo* effects of nanocrystals. Other qualitative and quantitative means such as detection of lesion sites can also be used. Further *in vivo* pharmacokinetic and pharmacodynamic investigations can be performed to calculate the key parameters of peak concentration, time to peak, and area under the curve (AUC).

Soroushnia et al. [100] prepared midazolam nanosuspensions with a particle size of 197 ± 7 nm and zeta potential of 31 ± 4 mV using an ultrasound technique. Then, *in vivo* tests were carried out in rabbits. Plasma concentration-time curves (Figure 8) and pharmacokinetic parameters (Table 2) were obtained for the midazolam nanosuspensions. In the *in vivo* evaluation, a higher Cmax (111.90% higher), a higher AUC0-t (275.08%), and a shorter Tmax (15 min) were observed for the midazolam nanosuspension, indicating that the midazolam nanosuspension is more readily absorbed.

**Figure 8.** The mean plasma concentration-time curve of the midazolam coarse suspension (-) and the midazolam nanosuspension (), (n = 3). Figure 8 was reprinted from ref. [100] with permission from Springer Nature, Copyright® 2021.

**Table 2.** Main pharmacokinetic parameters of midazolam coarse suspension and nanosuspension in rabbits after buccal administration (n = 5). Table 2 was reprinted from ref. [100] with permission from Springer Nature, Copyright® 2021.


#### **4. Applications**

Nanocrystal formulations can be administered by various routes (Figure 9), where oral routes of drug delivery account for more than 60% of current drug nanocrystal delivery routes [101,102]. Nanocrystal-based formulations (Table 3) are widely used to treat cancer, inflammatory, cardiovascular, depression, and other diseases [35].

**Figure 9.** A schematic diagram of the nanocrystal drug delivery route and therapeutic response. Figure 9 was reprinted from ref. [102] with permission from Springer Nature, Copyright® 2020.

The oral route is considered to be one of the most appropriate, safe, and preferred routes [103]. During oral administration, dissolution of the nanocrystals begins in the intestine, where the drug particles are slightly absorbed by the cells. In contrast, cellular interactions and drug uptake play a key role in nanocrystals administered via parenteral routes. It can enhance the bioavailability, reduce toxicity, and release in specific targeting sites, such as the brain, lung, liver, kidney, or colon [4]. For pulmonary administration, the nebulized nanosuspension had a significantly higher inhalable fraction and also showed stronger mucus adhesion. Ocularly administered nanodropable dosage forms also have high drug loads and long-lasting drug effects [35]. In conclusion, the application and combination of various routes of drug administration will provide the basis for nanocrystals to play an increasingly important role in disease treatment.


**Table 3.** Examples of existing commercial products for drug nanocrystals.

Note: \* only represents the brand name of the drug.

#### **5. Conclusions and Perspective**

In this work, we reviewed the preparation method for drug nanocrystals, containing top-down (e.g., WBM, HPH), bottom-up (e.g., LAS, SCF), and combinative (e.g., Nanoedge, SmartCrystal) technology. After that, characterization methods and application scope of nanocrystals including DLS, SEM, XPRD, etc. were discussed. Next, evaluation paths and examples on stability, cytotoxicity, and *in vitro*/*in vivo* dissolution rate were introduced, which can reflect the practical performance of drug nanocrystals. Lastly, the applicability of drug nanocrystals was broadly demonstrated by various routes (e.g., oral, parenteral).

Although great progress has been made in nanocrystal technology, there are still many difficulties to be faced. For drug nanocrystal preparation technology, it is necessary to continuously improve the productive efficiency and stability of nanocrystals. For this purpose, the continuous strengthening of basic research on nanocrystal preparation, such as the optimization of nanomedicine formulations and process parameters is required. Furthermore, on industrial production of nanocrystals, the transferability from laboratory scale to industrial scale should be seriously considered.

What follows is the characterization and evaluation of nanocrystal products including the properties of the nanoparticles themselves and *in vivo*/*in vitro* behaviors. Until now, the release of nanocrystals is not yet fully understood. More detailed preclinical *in vivo* experiments are needed to explain the mechanism of transportation and absorption of nanocrystals *in vivo*, both for efficacy and safety. Additionally, more robust and in-depth *in vitro*/*in vivo* correlation evaluations are also required.

In addition, for specific applications area of nanocrystals, the high drug-carrying capacity of nanocrystals makes them a hot spot for the study of targeted insoluble drug formulations. However, further research is still needed on targeted drug nanocrystal delivery formulations, rates, and mechanisms.

Overall, nanocrystal technology has the potential for industrial applications and the ability to take on insolubility problems. This makes it a prospering technology for optimizing the bioavailability of insoluble drugs. Therefore, continuous efforts are needed to introduce drug nanocrystals into the pharmaceutical market.

**Author Contributions:** Investigation, Q.R., M.W. and J.O.; writing—original draft preparation, Q.R.; writing—review, D.H. and Z.G.; writing—modifications, Q.R., M.W. and W.K.; supervision, J.O. and J.G. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was financially supported by Shandong Provincial Key R&D Program (Major Key Technology Project) 2021CXGC01051 and Academic and technical leader training program for major disciplines in Jiangxi Province (20212BCJ23001).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Acknowledgments:** This work was financially supported by Shandong Provincial Key R&D Program (Major Key Technology Project) 2021CXGC01051 and Academic and technical leader training program for major disciplines in Jiangxi Province (20212BCJ23001). The financial support of Haihe Laboratory of Sustainable Chemical Transformations and the Institute of Shaoxing, Tianjin University.

**Conflicts of Interest:** The authors declare no conflict of interest.
