2. H42 and H96

H42 and H96 are combined with spray drying, freeze drying, and HPH techniques, respectively. The solution of insoluble drug and stabilizer is pretreated by spray/freeze drying, uniformly dispersed in the stabilizer skeleton, then redispersed into the water by HPH to prepare drug nanocrystals. The combination reduces particle aggregation and improves processing efficiency. It is suitable for large-scale production. Möschwitzer et al. [57] used poloxamer 188 as a stabilizer to prepare hydrocortisone acetate powder by H42. More uniform nanosuspensions exhibiting good long-term storage stability were obtained. Yu [58] prepared meloxicam and naproxen drug nanoamorphous using the H96 technique.

## 3. CT

CT is a combination of top-down technology. The two most common types of wet bead milling methods used are rotor-stator and mills [59]. Taking the former as an example, artcrystals is a combined rotor-stator high-speed shear and HPH technology. Firstly, the drug suspension is pretreated by shearing in a rotor-stator high-speed shear, then the nanocrystals are homogenized at high pressure to obtain stable and homogeneous suspensions. Wadhawan et al. [60] obtained crystalline acyclovir nanocrystals with a mean particle size of 400–500 nm using high pressure homogenizer and hydroxypropyl cellulose-LF as a stabilizer followed by wet bead milling. The saturation solubility of the nanocrystals was 1.6 times higher than that of micronized acyclovir. Martena et al. [61] prepared nicergoline nanocrystals in aqueous solutions of polysorbate 80. Four different techniques, HPH, bead milling (BM), and combined techniques (HPH + BM, BM + HPH) were explored in his work. The combined technique was found to be superior, but HPH + BM produced nanocrystals with a smaller mean particle size than BM + HPH. Particle solubility increased for all nanocrystals, especially for HPH and the combination technique, which obtained nanocrystals showing a higher dissolution rate.

#### 2.3.3. Other New Combinative Technology

1. Precipitation-lyophilization-homogenization (PLH) method

PLH is a combination of precipitation-lyophilization-homogenization method. Morakul et al. [62] obtained clarithromycin nanocrystals by this method, using poloxamer 407 and sodium dodecyl sulfate (SDS) as co-stabilizers. The obtained clarithromycin nanocrystals were cubic particles, about 400 nm, in a crystalline or partially amorphous state. It had high solubility and permeability.

#### 2. High gravity antisolvent precipitation process (HGAP)

HGCP technology is merged with antisolvent precipitation process to form HGAP. The benefits of the HGCP are retained while the disadvantages of impurities in the product are eliminated [12]. Zhao et al. [63] prepared danazol nanocrystals with uniform size distribution by the HGAP process. The average particle size was 190 nm. The molecular state and crystalline form of Danazol nanoparticles were maintained. The nanoparticles were highly evaluated by the industry for its high recovery rate and continuous production capacity.

#### 3. Microjet reactor technology (MRT)

MRT is similar to HPH. The drug solution is mixed in the high pressure chamber through the micro-hole of the nozzle to form a high-speed fluid sprayed into the reaction chamber, and convective shear in the reaction chamber to form turbulence. At the same time, there is cavitation, impact, and shear effect to reduce the product particle size. The influencing factors of MRT include the mixing ratio of solution and antisolvent, jet strength, stabilizer dosage, temperature, etc. This method can realize continuous large-scale production. However, the energy consumption and path clogging [37] cannot be ignored. Chen et al. [64] prepared albendazole nanocrystals by MRT with a mean particle size of 367.34 ± 0.68 nm under the optimal preparation process. The nanocrystals can significantly improve the dissolution performance of albendazole and facilitate the improvement of oral absorption of the drug.

4. Evaporative precipitation into aqueous solution (EPAS)

The EPAS method dissolves the API in the low-boiling-point solvent and heats above its boiling point. Thereafter, the heated solution is sprayed into heated aqueous solutions containing stabilizers [12]. Chen et al. [65] produced amorphous nanoparticle suspensions of cyclosporine A by EPAS. Due to the low crystallinity, small particle size of nanoparticles, and hydrophilic stabilizers, it has shown a high dissolution rate.

5. Antisolvent precipitation-high pressure homogenization method

Huang et al. [66] combined the antisolvent precipitation method and HPH method to prepare celecoxib nanocrystal suspensions with a particle size of 283.67 ± 20.84 nm, using polyvinylpyrrolidone K30 (PVP K30) and SDS as crystal stabilizers. The solubility of celecoxib nanocrystals was obviously higher than that of the raw celecoxib and the physical mixture. The product remained quite stable under high temperature and high moisture conditions for 10 days of storage.

6. Ultrasound probe-high pressure homogenization method

Jin et al. [67] used an ultrasound probe combined with HPH and fluidized drying process to prepare baicalin nanocrystals with an average particle size of 248 ± 6 nm and PDI 0.181 ± 0.065 by selecting mixed surfactant poloxamer 188 as a steric stabilizer and SDS as an electrostatic stabilizer. The results of pharmacokinetic experiments in rats showed that the drug bioavailability *in vivo* was significantly improved.

7. Rotary evaporation method-high pressure homogenization method

Zuo [68] prepared curcumin-artemisinin cocrystal nanomedicine by the rotary evaporation-HPH method. The particle size of nanomedicine was 234.6 nm after optimization. Curcumin-artemisinin cocrystal nanomedicine showed obvious solubility advantages and excellent stability compared with that of raw curcumin, curcumin-artemisinin cocrystals, and curcumin nanocrystals. Yu [58] also obtained quercetin drug nanoamorphous using the rotary evaporation method assisted by the HPH method.

8. Melt quench-high pressure homogenization method

Yu [58] also used the combined melt quench-high pressure homogenization technique to prepare nanoamorphous indomethacin. The particle size of the prepared suspension was 245 nm. The solubility of the nanosuspensions was significantly enhanced. However, the stability of the nanoamorphous was poor. The particle size started to increase significantly within 7 days and even reached 890 nm after 30 days due to the presence of water and the occurrence of recrystallization.

9. Antisolvent precipitation-ultrasound method

Zhang et al. [69] obtained fenofibrate nanocrystals using the ultrasound probeprecipitation method. However, one of the disadvantages of ultrasonic probes is that they can leave metal particles and thus are not suitable for industrial production. Liu et al. [70] used alpha tocopherol succinate as an auxiliary stabilizer in the organic phase to prepare carvedilol nanosuspensions by this method. The mean particle size of the nanoparticles was 212 nm and it was stable at 25 ◦C for 1 week. The dissolution rate of the nanosuspension was significantly increased. *In vivo* tests indicated that the nanosuspensions showed approximately two-fold increase in each index compared with commercial tablets. Additionally, the method is fast, inexpensive, and easy to control. Paclitaxel nanocrystals [71], zaleplon nanocrystals [72], and nintedanib nanocrystals [73] are also prepared using this method.

#### **3. Characterization and Evaluation**

After obtaining nanocrystal products, characterization and performance evaluation are crucial to the further application of the products. On the one hand, the purpose of characterization is to understand the properties of the sample such as size, morphology, and crystalline form. Based on this, the performance of the product can be quantitatively

controlled on different quality characteristics, and then the quality controllability of the product can be achieved [19]. On the other hand, evaluation is to obtain the nanocrystal performance index (stability, cytotoxicity, dissolution rate) to explore more effective drug delivery strategies.
