Cryptotanshinone (CTS, Figure 1), also named cryptotanshinone or tanshinone C, is an important diterpene quinone isolated from the roots and rhizomes of the plant Salvia miltiorrhiza Bunge (Li H. et al., 2021). CTS has been reported to show a variety of pharmacological activities, such as neuroprotective, antineoplastic, anti-inflammatory, antioxidant, and radioprotective effects, to treat multiple diseases and disorders, including acute ischemic stroke, atherosclerosis, Alzheimer’s disease, diabetes, obesity, chronic hepatitis and cancer (Wu et al., 2020; Ashrafizadeh et al., 2021).

Despite these comprehensive advantages, no CTS preparation is currently available on the market because of its low solubility in water. Although the logP of CTS is 4.13 which indicates its good permeability, its 9.76 μg/ml solubility in water (Zhang et al., 2006) hinders the oral bioavailability of this BCS II compound (Bhalani et al., 2022), evidenced by 2.05% of the absolute oral bioavailability in rats (Zhang et al., 2006). Oral administration of 20 mg/kg CTS in pigs resulted in non-detectable drug in serum, while only 0.043 μg/ml CTS was detected 1 h after administration when the dose increased to 40 mg/kg (Xue et al., 1999). Researchers to date have tried to use solid lipid nanoparticles (Hu et al., 2010), lipid-polymer hybrid nanoparticles (Liu et al., 2020), solid dispersions (Zhai et al., 2017), cyclodextrin inclusions (Li et al., 2003), solid self-microemulsifying drug delivery systems (Bi et al., 2016) and other technologies to increase the solubility of CTS for oral administration (Cai et al., 2016). Nevertheless, the technologies above have strict requirements for the physical and chemical characteristics of drug molecules, resulting in low drug loading, low entrapment efficiency and premature drug release. The composition of the above preparations is complex, suitable carriers are needed, and toxic excipients such as Tween 80™ and sodium dehydrocholate (Hu et al., 2010) may be used. Moreover, the preparation process of these technologies is complicated and difficult to industrialization, which hinders their clinical translation.

In the past 3 decades, drug nanocrystals, as an emerging technology that could enhance the dissolution and bioavailability of compounds belonging to BCS II, have sprung up and attracted extensive attention from pharmaceutical researchers (Verma et al., 2009). Drug nanocrystals are insoluble drug particles that form inhomogeneous water dispersions with particle sizes of 1–1000 nm (Li J. et al., 2021). According to the Noyes–Whitney equation, a decrease in particle size will lead to an increase in the effective surface area in the diffusion layer, thus increasing the dissolution rate of drugs (Noyes and Whitney, 1897). Morover, drug nanocrystals can improve adhesiveness to the gut wall and diffusion to intestinal water layers (Imono et al., 2020). Different from other formulation strategies, drug nanocrystals do not require a carrier and are composed of almost 100% drug particles and a minimum amount of suitable surfactants and/or polymers as stabilizers (Hou et al., 2017). Meanwhile, the high drug loading of nanocrystals increases patient compliance (Li J. et al., 2021), eliminates food effects and improves efficacy and safety for oral administration (Wang et al., 2022). The production methods, including solvent-antisolvent precipitation, high-pressure homogenization and media milling, are universal and simple for large-scale production with excellent reproducibility (Chen et al., 2011). Although this technology was developed in the 1970s, more than 20 nanocrystal-based drug products have been approved for analgesia, schizophrenic, antifungal, antitumor and anti-inflammatory treatments, and several clinical trials are currently undergoing at different stages (Fan et al., 2018). The research revealed that the submissions received by the FDA for nanocrystal drug products comprise approximately 30% of all the applications for drug products containing nanomaterials (Chen et al., 2017), which fully confirms the broad prospects of nanocrystal preparations.

Herein, freeze-dried CTS nanocrystals were successfully developed by a combination approach with precipitation followed by high-pressure homogenization to improve the oral bioavailability of CTS. The physicochemical characteristics, including particle size, crystal morphology, crystalline state, thermodynamic properties, solubility, drug release profiles and stability, were determined. The pharmacokinetics of CTS nanocrystals were evaluated in rats.

It is the first attempt to develop lyophilized CTS nanocrystals and its enhanced oral bioavailability was also well evaluated. It is proved that optimized CTS nanocrystals had remarkable enhancement in solubility, dissolution rate and bioavailability. Our result indicated that nanocrystals are a suitable delivery approach for CTS oral administration.

The strategies for nanocrystal fabrication are divided into top-down, bottom-up methods, and combination techniques. In the case of top-down methods, milling and homogenization are currently utilized to reduce microsized drug crystal size with the aid of physical forces. Bottom-up methods such as solvent-antisolvent precipitation, supercritical fluids, and solvent evaporation are employed to prepare homogenous and small particles. The combination of both approaches, usually involving bottom-up and top-down processes in sequence, favors the production of more homogenous and smaller nanocrystals (Fontana et al., 2018). Herein, we first dissolved CTS in water-miscible acetone and then precipitated it by the addition of a stabilizer-containing antisolvent under rapid mixing to obtain fine crystalline CTS. Solvent-antisolvent precipitation refers to the nucleation and precipitation of API under supersaturation conditions. A more concentrated drug solution leads to a higher number of nuclei with smaller particles generated (Lonare and Patel, 2013). Therefore, the drug concentration in acetone was set as the saturated concentration. Other variables that impact drug crystals formed by precipitation are stabilizers (Matteucci et al., 2006). A suitable stabilizer is able to provide favorable surface covering and steric stabilization on the newly formed surface area of the nanosuspension (Sattar et al., 2017; Ullah et al., 2018). We found that poloxamer 407 was the best among these commonly used stabilizers. Poloxamers are non-ionic polymers consisting of hydrophilic ethylene oxide and relatively hydrophobic propylene oxide blocks with good biocompatibility and non-toxicity. In addition to the particle‒particle repulsion provided by hydrophilic chains from polymers HPMC and PVP, amphiphilic poloxamers can additionally offer adequate physical adsorption on the precipitated hydrophobic drug surface by their hydrophobic domains. The optimal stabilizer is mainly dependent on the similarity of hydrophobicity between the drug and stabilizer (Shegokar and Müller, 2010). Compared with poloxamer 188 (20%), poloxamer 407 has a higher weight ratio of polyoxypropylene chain segments (30%). It can diffuse to and adsorb readily on the CTS surface to reduce the high surface free energy, promote the nucleation rate, and impede crystal growth by coagulation and condensation (Matteucci et al., 2006). Subsequently, the concentration of poloxamer 407, homogenization pressure and number of cycles were optimized to achieve a desirable particle size. Smaller particles would be obtained with a higher concentration of stabilizer owing to the insufficiency of the poloxamer to arrest crystal growth. However, a further increase in the stabilizer resulted in larger crystals because the viscosity of the aqueous solution increased (Sharma and Mehta, 2019). We obtained CTS nanocrystals with an average particle size of 315.67 ± 11.02 nm and a polydispersity index of 0.20 ± 0.02 when 1% poloxamer 407 was used. A small particle size (∼300 nm) and narrow size distribution (PDI <0.25) are usually considered acceptable for nanocrystal production (Patravale et al., 2004). Homogenization disrupts drug particles to nanoscale dimensions with shear and impact forces. Obviously, high energy inputs, such as higher homogenization pressure and an increase in homogenization cycles, would reduce the average particle size (Figures 2B, C). However, further increases in the homogenization pressure and number of cycles did not decrease the particle size impeded by enhanced recoalescence.

Lyophilization is a suitable solidification technique to transform nanosuspensions into a stable and easily redispersible formulation. As the conventional cryoprotectant, CTS nanocrystals lyophilized with mannitol exhibited quick reconstitution in water (<30 s) without agglomerates (Table 2). Neither Ostwald ripening nor aggregation was observed up to 6 months of storage at 4°C, as evidenced by the unchanged particle size and PDI value. The characteristic peaks of CTS nanocrystals in the diffractogram showed that the preparation process, including precipitation, homogenization, and lyophilization, did not change the drug crystalline structure. However, the dramatic peak decrease in both DSC and PXRD patterns revealed the partial amorphization and reduced crystallinity of CTS in nanocrystals (Figures 4, 5), which was mainly attributed to the reduction in crystal lattice energy caused by the incorporation of CTS in the hydrophobic domain of poloxamer 407 micelles and the complete surface coverage of particles by the stabilizer (Fontana et al., 2018). Additionally, amorphization/partial amorphization of drugs has been observed after lyophilization because solutes that cannot crystallize are converted to an amorphous state during the cooling step (Gol et al., 2018). Mannitol is a crystalline material with a characteristic endothermic peak at approximately 170°C. However, it disappeared in CTS products owing to the total amount of mannitol transformed into an amorphous form, which was also observed in other studies (Gol et al., 2018; Sharma and Mehta, 2019).

The main advantage of nanocrystals is the augmentation of the saturated solubility and subsequent dissolution rate of insoluble drugs. According to the Ostwald-Freundlich equation, the saturation solubility increases when the particle size is reduced below 1 µm (Buckton and Beezer, 1992). The tremendous rise in solubility at pH 1.2, pH 6.8, and water by CTS nanocrystals clearly confirmed the effect of particle size on drug solubility (Figure 6A). Meanwhile, nanocrystals can accelerate the dissolution rate as a consequence of the large interfacial area introduced by nanosized particles. The Noyes-Whitney equation precisely describes the relationship between the diffusion coefficient, surface area, dissolution volume, diffusion layer thickness and dissolution rate (Noyes and Whitney, 1897). The transformation of crystallinity to amorphous states, as we presented above in the PXRD and DSC spectra, contributed to rapid drug dissolution as well. Additionally, the surfactant poloxamer 407 not only acts as the crystal stabilizer, but also has low surface tension and small contact angle to promote the interaction of drug nanocrystals with the aqueous phase by wetting and dispersibility resulting in a quick dissolution profile (Meruva et al., 2020; Zhang et al., 2020). On the contrary, bulk CTS is hard to wetting because of its hydrophobicity. The release behavior of CTS nanocrystals in pH progressive media reflected the enhanced drug dissolution in vitro (Figure 6B), which endowed oral absorption in further in vivo studies.

Oral absorption is limited by the rate and extent of CTS dissolution. Therefore, improvement of CTS bioavailability (2.9-fold) was routinely observed once CTS nanocrystallization was achieved. Comparable CTS absorption was also reported to be enhanced by employing other delivery systems. Relative bioavailability of CTS loaded solid lipid nanoparticles was 186%–299% compared with CTS suspension depending on the lipid matrix and the amount of sodium dehydrocholate used in different formulations (Hu et al., 2010). Wheat germ agglutuinin-modified lipid-polymer hybrid nanoparticles could efficiently penetrate mucus layer and augment enterocyte uptake and transport via receptor-mediated endocytosis, resulting in 6.5-fold higher bioavailability than CTS suspension (Liu et al., 2020). Solid self-microemulsifying drug delivery system showed 499% relative bioavailability after oral administration in rats (Bi et al., 2016). The increased oral absorption was closely related to the substantial surfactants which were able to improve solubilization of CTS. Meanwhile, intestinal lipid bilayer membranes were also affected by these surfactants, resulting in the enhancement of drug permeability. The merit of CTS nanocrystals is simple composition, low surfactants comsuption, and easy preparation process. A high concentration gradient means fast diffusion of drug molecules across the intestinal membrane, which accounts for significantly higher C_max (Tab. 2) (Lv et al., 2022). In addition to the conventional mechanism, namely, enhanced solubility and dissolution rate, transepithelial transport of intact nanocrystals (Zhu et al., 2022), increased adhesiveness to the gastrointestnal tract wall with a large contact area of nanocrystals, along with enhanced permeability by poloxamer (Li et al., 2011), might also be involved in the enhancement of oral absorption. The T_max and MRT of raw CTS showed no obvious significant difference compared with those of the CTS nanocrystals.

CTS nanocrystals were successfully fabricated by an antisolvent precipitation-homogenization approach with a stabilizer of Poloxamer 407. Rational selection of the polymer concentration, homogenization pressure and number of cycles facilitated optimal control of the particle size. The freeze-dried product was stable for at least 6 months when stored at 4 °C. Optimized CTS nanocrystals had remarkable enhancement in solubility, dissolution rate and bioavailability over the raw material. Therefore, nanocrystals are a suitable delivery approach for CTS oral administration.