The oral bioavailability of a drug is limited by factors, such as membrane permeability, solubility, and dissolution rate ^[1][2][1,2]. In general, miniaturization of compound crystals and solid dispersions are known to improve the solubility of poorly water-soluble compounds ^[3][4][5][6][3,4,5,6] and is advantageous by increasing dissolution rate due to increased surface area. According to the Noyes-Whitney equation, an increase in the surface area results in fast rates of drug dissolution [7]. For poorly water-soluble drugs, such as sirolimus [8], aprepitant [9], and fenofibrate [10], micronization technology has been applied to improve the solubility of compounds. There are two types of miniaturization methods: top-down ^[11][12][13][11,12,13] and the bottom-up. The bottom-up method can produce nano-sized particles by crystallization from a solution or coacervation with a sharp size distribution. PWe have previously it is prepared nanocrystal suspensions of poorly water-soluble drugs using a continuous crystallizer (PureNano^TM) [14]. In the PureNano^TM system, crystallization is immediately performed by a high shearing force, which is generated after the rapid feeding of aqueous and organic solutions to the collision jet field. Therefore, well-size-controlled particles can be prepared using PureNano^TM, compared with the usual crystallization method. However, nanoparticles gradually aggregate and induce crystal growth because of the high surface energy of nanoparticles, which is concerning [15]. To overcome these concerns, a dispersion stabilizer is necessary to avoid particle aggregation and crystal growth.

Silymarin (SLM) is an ingredient extracted from milk thistle [16], which contains the most effective silybin A and silybin B, as well as is silydianin, silychristin, taxifolin, and quercetin ^[17][18][17,18]. SLM has beneficial pharmacological activities such as immunosuppressive action, antitumor action [16], and hepatoprotective action [19]. However, SLM is poorly water-soluble, which limits its absorption after oral administration. Methods to improve the absorption of SLM after oral administration have been reported, including micronization, such as nanocrystals, nanosuspensions, and solid dispersions, and the use of carriers, such as cyclodextrins, liposomes, and lipid nanoparticles ^{[20][21][22][23]}[20,21,22,23]. It has been suggested that improving the solubility of SLM in either technique improves the absorbability after oral administration. However, there are few reports on the effect of improving the solubility of SLM using both miniaturization and carriers.

Cyclodextrins (CyDs) are cyclic oligosaccharides consisting of six to eight glucose units linked by α-1,4-glycosidic linkages. The potential use of natural CyDs and their synthetic derivatives has been extensively studied to improve certain properties of drugs, such as solubility, stability, and bioavailability. One of the most useful applications of CyDs in dosage form design is to enhance the solubility of poorly water-soluble drugs by complex formation ^[24][25][24,25]. Among them, β-CyD is widely used as a pharmaceutical additive for inclusion, but its low solubility may be a problem. Therefore, water-soluble CyD derivatives have been developed to improve their functionality. Hydroxypropyl-β-CyD (HP-β-CyD), a hydroxypropylated product, is used as a solubilizer for oral syrups and injections of itraconazole [26]. Recently, it has been reported that CyD is utilized as additives to prepare the submicron-sized drug particles by supercritical treatment or co-milling ^[27][28][27,28].

2. Preparation of Silymarin (SLM) Nanoparticles (NPs)

2.1. Preparation of SLM NPs Using PureNano^TM

SLM was dissolved in ethanol and distilled water and placed in PureNano^TM to prepare an SLM suspension. The particle size of the SLM suspension was measured immediately. The organic solvent in the SLM suspension was distilled off using a rotary evaporator, and particle size was measured. The suspension was powdered by freeze-drying (FD) to obtain SLM NPs. The redispersibility of the SLM NPs (FD product) was evaluated by measuring the particle size after redispersion in distilled water. Table 1 shows the particle size and polydispersity index (PDI) of the SLM suspensions after PureNano^TM or evaporation of the organic solvent, and SLM NPs after FD. The particle size and PDI of SLM suspensions just after nano-crystallization by PureNano^TM was 188.6 nm and 0.138, respectively. Meanwhile, the particle size of the SLM suspension after evaporation was slightly increased due to Ostwald ripening, because PDI of the nanosuspension did not change ^[29][30][30,31]. Moreover, the size of SLM NPs after FD was more than 1000 nm, probably due to agglomeration, suggesting poor redispersibility in water (Table 1). These data indicate the importance of water-soluble dispersion stabilizers during the FD process to obtain SLM NPs with high redispersibility.

Table 1.

Particle sizes of silymarin (SLM) suspensions and freeze-dried (FD) powder of SLM.

Condition	Particle Size (nm)	PDI

PureNano	TM	188.6	0.138
Evaporation	224.8	0.166
Freeze Dry (FD)	>1000	Not detected

2.2. Effect of Dispersion Stabilizer on Preparation of SLM NPs

SLM NPs, SLM/HPMC, and SLM/S-1670 with sub-micron sizes and narrow size distributions were successfully formulated. After crystallization, particle size of the SLM NPs was approximately 300 nm, regardless of the type of additive. In contrast, the particle size of SLM/HPMC NPs after FD was increased to 677.7 nm, but smaller than that of SLM/S-1670 NPs after FD (Table 2). This might be because the weight ratio of HPMC was higher than that of S-1670, due to steric hindrance through aggregation. Moreover, the particle size of SLM/HPMC NPs after one week was slightly increased; up to 697.1 nm. In contrast, SLM/S-1670 NPs aggregated after one week, and their particle size was more than 1000 nm. Because of the interparticle interaction in SLM/S-1670 NPs was stronger than that in SLM/HPMC NPs (HLB: hydrophilic-lipophilic balance value of S-1670 was 16). Therefore, HPMC wase selected HPMC as a dispersion stabilizer rather than S-1670 in subsequent experiments.

Table 2. Effects of hypromellose (HPMC) and S-1670 on particle sizes of suspension prepared by PureNano^TM, evaporated samples, freeze drying samples (FD) and FD samples stored for one week.

Compound	Stabilizer	Condition	Particle Size (nm)	PDI

		PureNano	TM	337.6	0.045
	HPMC	Evaporation	339.0	0.165
		FD	677.7	0.463

^32][32,33]. This is in agreement with the findings of Kayaert et al. ^[31][32][32,33]. Therefore, it is presumed that the increase in the particle size of the SLM/HPMC suspensions depending on the HPMC concentration was likely due to the adsorption of HPMC on the surface of silymarin and the formation of HPMC layers. Moreover, it was clarified that the particle size after FD maintained the nanoparticles only when the weight ratio of SLM to HPMC was 1:1. In contrast, particle agglutination was observed at weight ratios of SLM to HPMC of 1:0.5 and 1:3. This could be because when the HPMC concentration was low, it was presumed that the amount of HPMC adsorbed on the SLM fine particles was insufficient. The SLM/HPMC suspension was concentrated during the FD process, and contact between the particles could not be suppressed. In contrast, when the HPMC concentration was high, the viscosity of the SLM/HPMC suspension increased and the molecular motility of the particles was suppressed, resulting in agglomeration of the particles. In addition, HPMC was considered to adsorb on SLM caused an interaction between particles and aggregation. These results indicate that selecting the optimal concentration of HPMC due to the preparation of SLM/HPMC nanoparticles is important.

Table 3. Effect of hypromellose (HPMC) content on particle sizes of silymarin (SLM) suspensions prepared by PureNano^TM and their freeze-dried (FD) samples.

Compound	Condition	Ratio (w/w)	Particle Size (nm)	PDI

Table 5. Silymarin (SLM) contents in silymarin/hypromellose nanoparticles (SLM/HPMC NPs) in the presence of erythritol (Ery), α-cyclodextrins (α-CyD), and hydroxypropyl (HP)-β-CyD.

Sample	Content (%)	Theoretical Content (%)

1/0.5

227.5

0.045

SLM/HPMC/Ery FD	32.25 ± 0.01
	PureNano	TM	1/1	387.6	0.165
SLM/HPMC/α-CyD FD	SLM/HPMC		1/3	1240.9
SLM		FD after 1 week	697.1	0.478
		PureNano	TM	281.5	0.115
	S-1670	Evaporation	329.5	0.221
		FD	912.1	0.496
		FD after 1 week	>1000	Not detected

Next, to examine the effect of HPMC concentration on particle size of SLM/HPMC NPs, we prepared SLM/HPMC NPs was prepared with various HPMC concentration (0.5–3%, w/w) (Table 3). The particle size of SLM/HPMC NPs increased as the HPMC content increased. Van den Mooter et al. have reported that HPMC adsorbs to the crystal surface of a drug and forms intramolecular hydrophobic interactions via the hydrophilic and hydrophobic groups of HPMC ^[31][

0.463

	1/0.5	>1000	0.115
	FD	1/1	617.0	0.221
		1/3	>1000	0.496

2.3. Effect of Cryoprotectants on Preparation of SLM NPs

In general, it is known that particles aggregate into coarse particles during the drying process. Therefore, cryoprotectants have been used to suppress particle aggregation ^[33][34][34,35]. Next, we evaluated the effect of various cryoprotectants on the aggregation of FD powder of SLM/HPMC NPs was evaluated. As shown in Table 4, the particle sizes of SLM/HPMC in the presence of cryoprotectants such as erythritol (Ery), mannitol, trehalose, α-CyD, and HP-β-CyD were smaller than those without cryoprotectants (particle size: 617 nm, Table 3), suggesting that each cryoprotectant suppressed the aggregation of SLM/HPMC NPs. It is assumed that these sugar cryoprotectants dissolve in water and suppress aggregation by interposing between the SLM/HPMC NPs. These results suggest that sugar structures have little effect on the suppression of aggregation. In addition, SLM contents in the SLM/HPMC/Ery NP, SLM/HPMC/α-CyD NP and SLM/HPMC/HP-β-CyD NP was 32.25 ± 0.01%, 31.30 ± 0.64% and 31.85 ± 0.41%, respectively, and were close to the theoretical SLM content of 33.3% (Table 5). These results imply that SLM does not disappear or decompose during particle preparation. However, in drug formulation, it is important to assess the physical and chemical stability of the formulation. Further elaborate researchtudy on nanoparticle stability is necessary.

Table 4. Effect of cryoprotectants on particle sizes of silymarin/hypromellose nanoparticles (SLM/HPMC NPs) in the presence of various cryoprotectants.

Compound	Cryoprotectant	Ratio (w/w/w)	Particle Size (nm)	PDI

	Erythritol	1/1/1	253.8	0.108
	Mannitol	31.30 ± 0.641/1/1	271.8	0.170
33.33	SLM/HPMC	Trehalose	1/1/1	265.2	0.160
SLM/HPMC/HP-β-CyD FD	31.85 ± 0.41	α-CyD	1/1/1	249.1	0.149
	HP-β-CyD	1/1/1	254.8	0.225