Enhanced Water Solubility and Oral Bioavailability of Paclitaxel Crystal

pharmaceutics

Article Enhanced Water Solubility and Oral Bioavailability of Paclitaxel Crystal Powders through an Innovative Antisolvent Precipitation Process: Antisolvent Crystallization Using Ionic Liquids as Solvent

1,2,3, 1,2,3, 1,2,3 1,2,3 1,2,3 Qilei Yang †, Chang Zu †, Wengang Li , Weiwei Wu , Yunlong Ge , Lingling Wang 1,2,3, Li Wang 1,2,3, Yong Li 1,2,3 and Xiuhua Zhao 1,2,3,*

1 College of Chemistry, Chemical Engineering and Resource Utilization, Northeast Forestry University, Harbin 150040, China; [email protected] (Q.Y.); [email protected] (C.Z.); [email protected] (W.L.); [email protected] (W.W.); [email protected] (Y.G.); [email protected] (L.W.); [email protected] (L.W.); [email protected] (Y.L.) 2 Key Laboratory of Forest Plant Ecology, Northeast Forestry University, Ministry of Education, Harbin 150040, China 3 Engineering Research Center of Forest Bio-preparation, Ministry of Education, Northeast Forestry University, Harbin 150040, China * Correspondence: [email protected]; Tel.: +86-451-82191517; Fax: +86-451-8210-2082 These authors contributed equally to this work and should be considered co-ﬁrst authors. † Received: 22 September 2020; Accepted: 18 October 2020; Published: 22 October 2020

Abstract: Paclitaxel (PTX) is a poor water-soluble antineoplastic drug with significant antitumor activity. However, its low bioavailability is a major obstacle for its biomedical applications. Thus, this experiment is designed to prepare PTX crystal powders through an antisolvent precipitation process using 1-hexyl-3-methylimidazolium bromide (HMImBr) as solvent and water as an antisolvent. The factors influencing saturation solubility of PTX crystal powders in water in water were optimized using a single-factor design. The optimum conditions for the antisolvent precipitation process were as follows: 50 mg/mL concentration of the PTX solution, 25 ◦C temperature, and 1:7 solvent-to-antisolvent ratio. The PTX crystal powders were characterized via scanning electron microscopy, Fourier transform infrared spectroscopy, high-performance liquid chromatography–mass spectrometry, X-ray diffraction, differential scanning calorimetry, thermogravimetric analysis, Raman spectroscopy, solid-state nuclear magnetic resonance, and dissolution and oral bioavailability studies. Results showed that the chemical structure of PTX crystal powders were unchanged; however, precipitation of the crystalline structure changed. The dissolution test showed that the dissolution rate and solubility of PTX crystal powders were nearly 3.21-folds higher compared to raw PTX in water, and 1.27 times higher in artificial gastric juice. Meanwhile, the bioavailability of PTX crystal increased 10.88 times than raw PTX. These results suggested that PTX crystal powders might have potential value to become a new oral PTX formulation with high bioavailability.

Keywords: paclitaxel; antisolvent precipitation process; ionic liquids; bioavailability

1. Introduction Polymorphisms refer to the variety of crystal structures created by diﬀerent interactions between the same molecules in a solid state, which decides the physiochemical properties of crystals. Polymorph design of active pharmaceutical ingredients is normally regarded as one of the most important factors in drug development and production, as it determines the bioavailability of the

Pharmaceutics 2020, 12, 1008; doi:10.3390/pharmaceutics12111008 www.mdpi.com/journal/pharmaceutics Pharmaceutics 2020, 12, 1008 2 of 16 drug and its stability [1]. Determination of distinct crystal habits is significantly influenced by solvent recrystallization because the morphology depends on the solute–solvent interaction on various crystal faces [2–4]. Drug crystallization methods include solution crystallization from single or mixed solvents [5–7], supercritical fluids crystallization [8], seeding strategies [9], capillary crystallization [10], polymer-induced heteronucleation [11], heteronucleation on substrates [12], and laser-induced nucleation [13]. Solution crystallization is one of the most popular methods in drug crystallization. Crystallization conditions include solvent/antisolvent species, temperature, additive, supersaturation, agitation etc. In these conditions, solvent/antisolvent species is often considered the most important factor that determines the polymorphs in crystallization. Ionic liquids (ILs), which are also known as room-temperature ionic liquids (RTILs), are salts that are composed only of ions and have a melting point below 100 ◦C[14]. RTILs are used as “green” solvents to replace traditional volatile organic solvents, and it is used in a number of research fields, due to their low melting points, such as synthetic chemistry, catalysis, separation technologies, and bio-enzymatic reactions. In particular, recent research progressed to use them as media for carrying out crystallization. A number of publications emerged for their use in crystal engineering applications for inorganic molecules. [15] For example, polymorphic paracetamol can be successfully crystallized from 1-hexyl-3-methylimidazolium hexafluorophosphate and 1-Butyl-3-methylimidazolium hexafluorophosphate, using cooling crystallization [16]. Among them, the 1-hexyl-3-methylimidazolium bromide (HMImBr) is widely used in the extraction of bioactive natural products because of its advantages, such as selective extraction ability, unique advantages of ideal thermal stability, comprehensive dissolving capacity, negligible vapor pressure, excellent structural designability, etc. However, there was no report on the HMImBr as a medium for drug crystallization [17–20]. Moreover, the anti-solvent crystallization technique using ILs as a solvent is one of the new methods to improve the crystal structure of drugs [21]. In this emerging area, there are relatively few publications that have explored this avenue of research. According to An’s research [22], they used ILs as both solvent and antisolvent for designing crystallization, to modify the crystal morphology and crystal structure of adefovir dipivoxil. However, ILs as antisolvents are not good cleaning agents, so the prepared drug cannot be used clinically. The methods using ILs as solvent and water as antisolvent to prepare new crystal drugs could become greener, cheaper, and easier to scale and industrialize. Paclitaxel (PTX) is a diterpenoid anticancer agent that was discovered in the bark of the yew tree [23]; the structure of PTX is shown in Figure1a. PTX is one of the most e ffective anticancer drugs for treating ovarian cancer, breast cancer, Kaposi’s sarcoma, and non-small-cell lung cancer [24,25]. Its application was also expanded to the treatment of acute rheumatoid arthritis [26,27] and Alzheimer’s disease [28]. However, PTX, which has the brand name Taxol®, is administered in combination with Cremophor EL as an adjuvant, because of the poor solubility of PTX in aqueous media. Unfortunately, Cremophor EL induces hypersensitivity and causes serious side effects in many patients [29]; therefore, finding a new formulation with high water solubility and low cost is necessary. Oral administration is the easiest and most convenient route of drug delivery. However, PTX oral bioavailability is very low mainly because of its poor aqueous solubility. To solve this problem, several approaches were reported, such as solubilization with d-α-tocopherol polyethylene glycol 1000 succinate (TPGS) 400 [30], co-administration with a P-glycoprotein inhibitor [31], microemulsions or nanoemulsions [32], formation of inclusion complexes with cyclodextrins, and entrapment in nanoparticles and polymer micelles [33,34]. However, these methods all have different stability and toxicity problems. Preparation of new PTX crystals powders might be a better method to enhance solubility and oral bioavailability. Designing PTX crystallization to enhance solubility with antisolvent crystallization technique using ILs is yet to be reported. This research aims to prepare PTX crystal powders with high water solubility and bioavailability through an antisolvent crystallization technique using 1-hexyl-3-methylimidazolium bromide (HMImBr) (structure is shown in Figure1b) as solvent and water as antisolvent. Single-factor design was Pharmaceutics 2020, 12, x FOR PEER REVIEW 3 of 16

Pharmaceutics 2020, 12, 1008 3 of 16 state and chemical structure, dissolution rate, and bioavailability of PTX crystal powders were Pharmaceutics 2020, 12, x FOR PEER REVIEW 3 of 16 investigated. utilized to obtain the optimal preparation conditions. Furthermore, the morphology, crystalline state state and chemical structure, dissolution rate, and bioavailability of PTX crystal powders were and chemical structure, dissolution rate, and bioavailability of PTX crystal powders were investigated. investigated.

Figure 1. (a) Chemical structure of the paclitaxel. (b) Chemical structure of the HMImBr.

2. Materials and Methods Figure 1. ((a) Chemical structure of the paclitaxel. ( b)) Chemical Chemical structure of the HMImBr.

2.1.2. Materials Materials and Methods 2. Materials and Methods PTX (99.2% purity) was purchased from Chongqing Taihao Pharmaceutical Co., Ltd. 2.1. Materials (Chongqing,2.1. Materials China). HMImBr was purchased from Shanghai Cheng Jie Chemical Co., Ltd. (Shanghai, China).PTX Analytical-grade (99.2% purity) was ethanol, purchased ethyl from ether, Chongqing and other Taihao reagents Pharmaceutical were obtained Co., Ltd. from (Chongqing, Beijing PTX (99.2% purity) was purchased from Chongqing Taihao Pharmaceutical Co., Ltd. ChemicalChina). HMImBr Reagents was Co. purchased(Beijing, China). from Shanghai Acetonitri Chengle, methanol, Jie Chemical and tert-butyl Co., Ltd. methyl (Shanghai, ether China). were (Chongqing, China). HMImBr was purchased from Shanghai Cheng Jie Chemical Co., Ltd. (Shanghai, HPLCAnalytical-grade grade. Deionized ethanol, ethylwater ether, was andpurified other reagentsthrough werea Milli-Q obtained water from purification Beijing Chemical system Reagents from China). Analytical-grade ethanol, ethyl ether, and other reagents were obtained from Beijing MilliporeCo. (Beijing, (Bedford, China). MA, Acetonitrile, USA). methanol, and tert-butyl methyl ether were HPLC grade. Deionized Chemical Reagents Co. (Beijing, China). Acetonitrile, methanol, and tert-butyl methyl ether were water was puriﬁed through a Milli-Q water puriﬁcation system from Millipore (Bedford, MA, USA). HPLC grade. Deionized water was purified through a Milli-Q water purification system from 2.2. Preparation of PTX Crystal Powders 2.2.Millipore Preparation (Bedford, of PTX MA, Crystal USA). Powders PTX crystal powders were prepared using the antisolvent precipitation technique. HMImBr was used2.2. Preparation asPTX the crystal solvent, of powders PTX and Crystal water were Powderswas prepared used as using the an thetisolvent. antisolvent PTX–HMImBr precipitation solution technique. was HMImBrthen injected was intoused a as beaker the solvent, with andwater, water under was usedstirring, as the at antisolvent. room temperature. PTX–HMImBr The solutionsolid drug was immediately then injected PTX crystal powders were prepared using the antisolvent precipitation technique. HMImBr was precipitatedinto a beaker upon with water,mixing. under After stirring, a certain at time, room the temperature. suspension The was solid centrifuged drug immediately until only precipitated a layer of used as the solvent, and water was used as the antisolvent. PTX–HMImBr solution was then injected precipitationupon mixing. was After left. a certain The suspension time, the suspension was then wasrepeatedly centrifuged washed until with only water a layer for of precipitation8–10 times, into a beaker with water, under stirring, at room temperature. The solid drug immediately throughwas left. repeated The suspension centrifugation, was then and repeatedly the particle washeds were with then water freeze-dried. for 8–10 times,Every throughexperiment repeated was precipitated upon mixing. After a certain time, the suspension was centrifuged until only a layer of repeatedcentrifugation, at least and thrice. the particlesThe schematic were then diagram freeze-dried. of the preparation Every experiment of antisolvent was repeated process at is least shown thrice. in precipitation was left. The suspension was then repeatedly washed with water for 8–10 times, FigureThe schematic 2. diagram of the preparation of antisolvent process is shown in Figure2. through repeated centrifugation, and the particles were then freeze-dried. Every experiment was repeated at least thrice. The schematic diagram of the preparation of antisolvent process is shown in Figure 2.

FigureFigure 2. 2. SchematicSchematic diagram diagram of the of experimental the experimental proces processesses to prepare to prepare the paclitaxel the paclitaxel (PTX) crystal (PTX) powders.crystal powders.

Figure 2. Schematic diagram of the experimental processes to prepare the paclitaxel (PTX) crystal powders.

Pharmaceutics 2020, 12, 1008 4 of 16

2.3. Optimization of the Antisolvent Precipitation Process Single-factor experiments were performed to determine the optimum conditions for the preparation of PTX crystal powders. Three factors were chosen for analysis in a preliminary experiment—drug concentration, antisolvent-to-solvent volume ratio, and temperature. In this method, only one factor was changed, whereas the others remained constant. When drug concentration was studied, the other factors were set under the following conditions—the antisolvent-to-solvent volume ratio was 5, and the temperature was 25 ◦C. The same method as above was applied to study the other factors. The eﬀects of drug concentrations at 10, 30, 50, and 70 mg/mL were studied. The antisolvent-to-solvent volume ratio was varied at 3:1, 5:1, 7:1, and 9:1. The temperature was set at 25, 50, 75, and 95 ◦C and investigated. Finally, the optimum condition for every factor was determined based on the water solubility of the obtained PTX crystal.

2.4. Characterization of the PTX Crystal Powders

2.4.1. Scanning Electron Microscopy (SEM) and Mean Particle Size (MPS) Detection The morphologies of raw PTX particles and PTX crystal powders were detected using SEM (FEI, Eindhoven, The Netherlands). The samples were prepared through direct deposition onto a carbon tape placed on the surface of an aluminum stub. Before analysis, the samples were coated with gold for 4 min, using a sputter coater (KYKY SBC-12, Beijing, China).

2.4.2. Fourier Transform Infrared Spectroscopy (FT–IR) The chemical components of the raw PTX and PTX crystal powders were analyzed through FT–IR spectroscopy using an IRAﬃnity-1 spectroscope (Shimadzu Corporation, Kyoto, Japan). Every sample was mixed with KBr, using an agate mortar and was compressed into a thin disc. The scanning range 1 1 was 4000–400 cm− with a resolution of 4 cm− .

2.4.3. High-Performance Liquid Chromatography–Mass Spectrometry (HPLC–MS) The raw PTX and PTX crystal powders, each of which was at 1 mg/mL concentration, were dissolved separately in methanol. The sample was diluted as necessary. The HPLC–MS spectra were obtained using the analyst1.4 of API3000 (API, Frederick, MD, USA). The mass spectrometer was operated in the negative ion mode.

2.4.4. X-Ray Diﬀraction (XRD) The crystal forms of the samples were also detected using X-ray (Bruker, Karlsruhe, Germany) with nonmonochromated CuKa radiation. The samples were scanned from 4◦ to 50◦ at rates of 10◦ per minute 0.02◦ per minute. The crystal forms were then measured at a voltage of 40 kV and 40 mA.

2.4.5. Diﬀerential Scanning Calorimetry (DSC) A total of 5 mg raw PTX and PTX crystal powders were investigated using DSC (TA instruments, NewCastle, DE, USA) at an increasing temperature heating rate of 10 ◦C/min from 50 ◦C to 300 ◦C.

2.4.6. Thermogravimetric (TG) Analysis The samples were analyzed using a TG analyzer (Diamond TG/DTA from Perkin–Elmer, Waltham, MA, USA). Approximately 5 mg samples were weighed in open aluminum pans. The test condition was as follows—nitrogen ﬂow rate of 50 mL/min. The percentage weight loss of the samples was monitored from 30 ◦C to 600 ◦C with an increasing heating rate of 10 ◦C/min. Pharmaceutics 2020, 12, 1008 5 of 16

2.4.7. Raman Spectroscopy The Raman spectra of the specimens were collected using a computer-controlled laser Raman microprobe equipped with a Leica DM/LM optical microscope, with a 100 objective (NA, 0.9) and × a charge-coupled device detector attached to a modular research spectrograph (Renishaw InVia, Renishaw PLC, Gloucestershire, UK). The 100 objective was used to increase the precision of the × acquired chemical data, resulting in a laser spot size of 1 µm. A monochromatic, near-infrared ≤ diode laser operating at 633 nm was used to induce the Raman scattering eﬀect. The spectral 1 1 coverage of this model ranged from 100 cm− to 3450 cm− with an average spectral resolution of 1 5 cm− . Before each experiment, a comparison with a silicon standard was performed using the calibration system integrated with the software (WiRE 2.0, Renishaw, Gloucestershire, UK), according to manufacturer’s speciﬁcations to calibrate the wavelength and intensity. The exposure time for each scan was 40 s, and the laser power on the specimen was 8 mW.

2.4.8. Solid-State Nuclear Magnetic Resonance (SSNMR) All measurements were performed using a broadband 3.2 mm solid-state nuclear magnetic resonance (ssNMR) probe and a 300 MHz Bruker BioSpin GmbH (Bruker, Karlsruhe, Germany) spectrometer. Samples were prepared by packing an adequate amount of each sample, as received, into 3.2 mm silicon nitride (Si3N4) SSNMR rotors. The sample spinning rate was set to 10 kHz. The 13C NMR spectra (100 scans) were recorded using the pulse sequence of cross-polarization magic angle spinning. Prior to each spectrum, the corresponding T1 constant (spin–lattice relaxation) was measured using the pulse sequence of saturation recovery.

2.5. Dissolution Study

2.5.1. Preparation of the Tablets Containing the PTX Crystal The raw PTX and PTX crystal powder complexes were compressed into tablets based on formulations. Microcrystalline cellulose (Avicel PH101, 50 mg/tablet) and Ac-Di-Sol (20 mg/tablet) were added as tablet diluent and disintegrant. The initial 3 mg and 12 mg weight of the PTX samples were converted to 200 mg by adjusting the amount of lactose accordingly. All ingredients of the tablet formulation were mixed for 15 min in a plough shear blender at 400 rev/min. The mixture was blended again for 1 min at 400 rev/min and then compressed into tablets with a total weight of 200 mg, using an Erweka EKO (Rieckermann, Hamburg, Germany) [35].

2.5.2. Kinetic Solubility Test A total of 3 mg tablet of raw PTX and PTX crystal powder were added into 1 L deionized water, and then 12 mg tablet of raw PTX and PTX crystal powder were added into the dissolution medium (artificial gastric juice with 5 mL Tween-80). The dissolution temperature and paddle speed were set at 37 0.5 C and 100 r/min. Consequently, 10 mL aliquots were withdrawn each time at 0.5, 1, 2, 3, 4, 6, 8, ± ◦ 12, 16, 24, 36, and 48 h, and filtered using a 0.22 mm filter. Each 9 mL filtrate of the samples were mixed with 1 mL chloroform, vortexed for 1 min, and centrifuged for 10 min at 5000 rpm. Subsequently, 800 µL of the organic layer was transferred to a clean vial and blow dried at 40 ◦C, under a gentle stream of nitrogen. The dried residue was then reconstituted in 200 µL methanol. After being vortexed for 2 min, the content was centrifuged at 12,000 rpm for 10 min. In addition, 20 µL supernatant was injected for HPLC analysis. Chromatographic analyses were performed on a Waters HPLC system consisting of a pump (model 1525), an autosampler (model 717 plus), and a UV detector (2487 dual λ absorbance detector). The C18(2)column (Diamonsil, 5 µm, 4.6 mm 250 mm, Dikma Technologies, Beijing, China) was used × at 30 ◦C. Methanol, acetonitrile, and water (52.2:22.5:25) was used as the mobile phase. The flow rate was 1.0 mL/min, and the injection volume was 20 µL. The signal was monitored at 227 nm. The linear Pharmaceutics 2020, 12, 1008 6 of 16 regression equations for the reference compound of PTX was Yptx = 3.6 104X 24656 (r2 = 0.9998). × − A good linearity was found for PTX in the range of 0.3–300 µg/mL.

2.6. Bioavailability Study of Paclitaxel Crystal Powders in Rats SD rats (230 10 g) provided by the Harbin Medical University (Harbin, Heilongjiang, China), ± were used in the in vivo experiments. The rats were deprived of food overnight before the experiment. The animal use and care protocol was reviewed and approved by the ethics committee of Harbin Medical University. Two groups of the rats (n = 6) were orally administered with 10 mg/kg of PTX through gavage, using an oral feeding sonde, under ether anesthesia. The PTX dose given to each rat was calculated based on the weight of the rat, at a dose of 10 mg/kg. The blood samples were taken from the eye-ground venous plexus at predetermined time points after 0.5, 1, 1.5, 2, 3, 4, 6, 8, 10, 12, 24, and 48 h. The blood samples were placed on ice (4 ◦C) and centrifuged for 10 min at 3000 rpm, and plasma aliquots were stored at 40 C, until additional extraction and analysis. − ◦ Frozen samples were thawed at room temperature and treated as follows. An aliquot (100 µL) of plasma sample was mixed with 25 µL of the internal standard solution (docetaxel, 4 µg/mL in methanol, previously evaporated). After vortex mixing, the liquid–liquid extraction was accomplished with the addition of 4 mL of tert-butyl methyl ether, following gentle vortex agitation (1 min). The mixture was centrifuged for 10 min at 5000 rpm, and then the organic layer was transferred to a clean vial and blow dried at 40 ◦C, under a gentle stream of nitrogen. Finally, the residue was dissolved in 100 µL reconstitution solution (0.01 M acetonitrile–phosphate buﬀer, pH = 2, 50/50 v/v), transferred to autosampler vials, capped, and placed in the HPLC autosampler. A total of 20 µL aliquot of each sample was injected onto the HPLC column [32].

2.7. Statistical Analysis All experiments were conducted in triplicates and repeated at least three different times. Differences between the groups were assessed by one-way ANOVA, and a p value less than 0.05 was considered to be significant.

3. Results and Discussion

3.1. Optimization of Antisolvent Precipitation Process The factors inﬂuencing the saturation water solubility of PTX crystal powders were optimized using single-factor design. Three factors were chosen for the analysis—drug concentration, antisolvent-to-solvent volume ratio, and temperature. As shown in Figure3a, the saturation solubility in water of the PTX crystal powders increased slowly under 30 mg/mL concentration and increased sharply in the range of 30–50 mg/mL concentration. However, when the concentration reached 50 mg/mL, the saturation solubility in water of PTX crystal powders decreased dramatically. Therefore, 50 mg/mL was considered as the optimal concentration of the PTX solution for the antisolvent precipitation process. As shown in Figure3b, the saturation water solubility of the PTX crystal powders decreased with the increase of temperature. Thus, 25 ◦C was considered as the optimal temperature for the antisolvent precipitation process. Figure3c indicates that the saturation solubility in water of PTX crystal powders initially decreased slowly and then increased sharply when the solvent-to-antisolvent volume ratio increased. However, when the solvent-to-antisolvent volume ratio was 1:7, the water saturation solubility of the PTX crystal powders decreased dramatically. Therefore, 1:7 was considered as the optimal solvent-to-antisolvent volume ratio for the antisolvent precipitation process. Using the single-factor design, the optimum antisolvent precipitation process conditions for PTX were found to be as follows—50 mg/mL concentration of the PTX HMImBr solution, − 7:1 antisolvent-to-solvent volume ratio, and 25 ◦C temperature. Based on the conﬁrmatory water saturation solubility tests, better PTX crystal powders were obtained with water saturation solubility of Pharmaceutics 2020, 12, 1008 7 of 16

6.052 µg/mL. The solubility in water of the PTX raw material and PTX crystal powder was 1.16 µg/mL Pharmaceuticsand 6.05 µg 2020/mL., 12 PTX, x FOR crystal PEER powders REVIEW had an increased solubility of nearly six times in the water.7 Thus, of 16 it feasible to prepare PTX crystal through the antisolvent precipitation process, using HMImBr as a solventPharmaceutics for improving 2020, 12, x FOR the PEER water REVIEW solubility of PTX. 7 of 16

FigureFigure 3. 3. The The effect eeffectﬀect of of each each parameter parameter on on the the water water saturation saturation solubility solubilitysolubility of ofof the thethe PTX PTXPTX crystal crystalcrystal powder. powder.powder. (a(a) ) Drug Drug concentration; concentration; ( b(b)) ) temperature,temperature, temperature, andand and (( cc()c) ) volumevolume volume ratioratio ratio ofof of antisolvent-to-solvent.antisolvent-to-solvent. antisolvent-to-solvent.

3.2.3.2. Morphology MorphologyMorphology FigureFigure4 4a,b a,b4a,b show show show the the the SEM SEM SEM of of of raw raw raw PTX PTX PTX particles particles particles and and and PTX PTX PTX crystal crystal crystal powders powders powders that that that were were were made made made using using using the theantisolventthe antisolventantisolvent precipitation precipitationprecipitation process. process.process. SEM analysisSEMSEM analysanalys showedisis showedshowed that raw thatthat PTX rawraw particles PTXPTX particles hadparticles cuboid hadhad structure cuboidcuboid structurecrystalsstructure (Figure crystals crystals4a), (Figure (Figure and most 4a), 4a), of and and the most most crystal of of the powders the cr crystalystal had powders powders uniform had had short uniform uniform rod cuboid short short structurerod rod cuboid cuboid (Figure structure structure4b). The(Figure(Figure particles 4b). 4b). The The of theparticles particles rawPTX of of the the were raw raw big PTX PTX and were were had big big a variedand and had had distribution, a a varied varied distribution, distribution, whereas PTX whereas whereas crystal PTX PTX powders crystal crystal werepowderspowders small were were in size small small and in in hadsize size a and and uniform had had a a distribution. uniform uniform distribution. distribution.

FigureFigure 4. 4. Scanning Scanning electron electron electron micrographs micrographs micrographs of of ofeach each each sample. sample. sample. ( a-1(a-1) ( a-1)Raw Raw) Raw PTX PTX PTX(magnification (magnification (magnification of of 5000); 5000); of 5000); ( a-2(a-2) ) (Rawa-2Raw) RawPTX PTX (magnification PTX(magnification (magnification of of 15000); 15000); of 15,000); ( b-1(b-1) ) (PTX b-1PTX) crystal PTXcrystal crystal powder powder powder (magnification (magnification (magnification of of 10000) 10000) of 10,000) and and ( b-2 and(b-2) ) (PTX b-2PTX) crystalPTXcrystal crystal powder powder powder (magnification (magnification (magnification of of 20000). 20000). of 20,000). 3.3. Chemical Structure 3.3.3.3. Chemical Chemical Structure Structure The FT–IR spectra of raw PTX and PTX crystal powders are compared in Figure5. The spectrum TheThe FT–IR FT–IR spectra spectra of of raw raw PTX PTX and and PTX PTX crystal crystal powders powders are are compared compared in in Figure Figure 5. 5. The The spectrum spectrum showed that absorption peaks of the two have the same shape and frequency position in the showedshowed that that absorption absorption peaks peaks of of the the two two have have the the same same shape shape and and frequency frequency position position in in the the 400–4000 400–4000 cmcm−1− 1range, range, indicating indicating that that the the functional functional group group st structuresructures of of PTX PTX raw raw material material and and crystal crystal powders powders werewere not not significantly significantly different. different.

Pharmaceutics 2020, 12, 1008 8 of 16

1 400–4000 cm− range, indicating that the functional group structures of PTX raw material and PharmaceuticscrystalPharmaceutics powders 2020 2020, ,12 12 were, ,x x FOR FOR not PEER PEER signiﬁcantly REVIEW REVIEW diﬀerent. 88 of of 16 16

1 FigureFigureFigure 5. 5. FT–IRFT–IRFT–IR spectra spectra (Wavenumber:(Wavenumber: 400–4000 400–4000 cm cm−−1−)1) ) ofof of thethe samples. samples. ((a (aa)) ) RawRaw PTX; PTX; and and ( ((bb))) PTX PTXPTX crystalcrystalcrystal powders. powders.powders.

The chemical structures of the two PTX samples were further evaluated using HPLC–MS to TheThe chemicalchemical structuresstructures ofof thethe twotwo PTXPTX sampsamplesles werewere furtherfurther evaluatedevaluated usingusing HPLC–MSHPLC–MS toto determine their molecular weights. As shown in Figure6, the molecular weights were not altered. determinedetermine theirtheir molecularmolecular weights.weights. AsAs shownshown inin FiFiguregure 6,6, thethe molecularmolecular weightsweights werewere notnot altered.altered. Based on the calculation of the mass spectrum, the molecular weight of raw PTX and the PTX crystal BasedBased on on the the calculation calculation of of the the mass mass spectrum, spectrum, the the molecular molecular weight weight of of raw raw PTX PTX and and the the PTX PTX crystal crystal powder was 854.3 and 854.4. Combining the FT–IR and HPLC–MS results, we could determine that powderpowder was was 854.3 854.3 and and 854.4. 854.4. Combining Combining the the FT–IR FT–IR and and HPLC–MS HPLC–MS results, results, we we could could determine determine that that thethethe PTX PTXPTX chemical chemicalchemical structure structurestructure did did not not change change duri duringduringng the the antisolvent antisolvent recrystallizationrecrystallization process.process.

Figure 6. LC–MS spectra of the samples. (a) Raw PTX; and (b) PTX crystal powders. FigureFigure 6. 6. LC–MS LC–MS spectra spectra of of the the samples. samples. ( (aa)) Raw Raw PTX; PTX; and and ( (bb)) PTX PTX crystal crystal powders. powders. 3.4. The Crystal Structure 3.4.3.4. The The Crystal Crystal Structure Structure The raw PTX and PTX crystal powders were subjected to XRD analysis to obtain information The raw PTX and PTX crystal powders were subjected to XRD analysis to obtain information on on crystallineThe raw PTX change and afterPTX crystal the antisolvent powders recrystallization were subjected to process. XRD analysis Figure 7toa obtain and Table information1 show the on crystalline change after the antisolvent recrystallization process. Figure 7a and Table 1 show the XRD crystallineXRD results change for the after raw the PTX. antisolvent As illustrated, recrystalli the twozation samples process. had Figure significantly 7a and di Tablefferent 1 show diffractograms. the XRD resultsresults forfor thethe rawraw PTX.PTX. AsAs illustrated,illustrated, thethe twotwo samplessamples hadhad significantlysignificantly differentdifferent diffractograms.diffractograms. The raw PTX showed several characteristic peaks at 2θ = 5.498◦, 8.866◦, 10.007◦, 11.171◦, 12.368◦, TheThe rawraw PTXPTX showedshowed severalseveral characteristiccharacteristic peakspeaks atat 22θθ == 5.498°,5.498°, 8.866°,8.866°, 10.007°,10.007°, 11.171°,11.171°, 12.368°,12.368°, 13.897◦, 14.506◦, 15.596◦, 17.017◦, 18.727◦, and 21.933◦, whereas the PTX crystal powders (Figure8b) 13.897°,13.897°, 14.506°, 14.506°, 15.596°, 15.596°, 17.017°, 17.017°, 18.727°, 18.727°, andand 21.933°,21.933°, whereas whereas thethe PTXPTX crystalcrystal powderspowders (Figure(Figure 8b)8b) exhibited several characteristic peaks at 5.068◦, 5.923◦, 9.562◦, 10.794◦, 13.491◦, 13.667◦15.225◦, 16.312◦, exhibitedexhibited several several characteristic characteristic peaks peaks at at 5.068°, 5.068°, 5.923°, 5.923°, 9.562°, 9.562°, 10.794°, 10.794°, 13.491°, 13.491°, 13.667°15.225°, 13.667°15.225°, 16.312°, 16.312°, and 20.146◦. This finding suggests that the PTX particles changed their crystal structures after the and 20.146°. This finding suggests that the PTX particles changed their crystal structures after the andantisolvent 20.146°. processing. This finding suggests that the PTX particles changed their crystal structures after the antisolventantisolvent processing. processing.

TableTable 1. 1. Analysis Analysis date date of of XRD. XRD.

Angle(°)Angle(°) RawRaw PTX PTX 5.58°, 5.58°, 6.12°, 6.12°, 8.89°, 8.89°, 9.67°, 9.67°, 10.04°, 10.04°, 11.06°, 11.06°, 12.29°, 12.29°, 13.83°, 13.83°, 15.61°, 15.61°, 17.07°, 17.07°, 18.68°, 18.68°, 19.56°, 19.56°, 21.89° 21.89° CrystalCrystal PTX PTX 5.19°, 5.19°, 6.12°, 6.12°, 9.82°, 9.82°, 11 11.13°,.13°, 12.50°, 12.50°, 13.59°, 13.59°, 16.49°, 16.49°, 20.34° 20.34°

Pharmaceutics 2020, 12, 1008 9 of 16 Pharmaceutics 2020, 12, x FOR PEER REVIEW 9 of 16

Pharmaceutics 2020, 12, x FOR PEER REVIEW 9 of 16

Figure 7. XRD patterns (2θ = 4–50°) of the samples. (a) Raw PTX; and (b) PTX crystal powders. Figure 7. XRD patterns (2θ = 4–50◦) of the samples. (a) Raw PTX; and (b) PTX crystal powders.

Raman spectra (Figure 8) show theTable raw 1. AnalysisPTX in the date range of XRD. of the C–H stretching vibrations of the alkyl (2860–3200 cm−1), the valence vibrations of the carbonyl and amine group (1900–1500 cm−1), and Angle(◦) the C–H stretching vibrations of the benzene ring from 900 cm−1 to 650 cm−1. However, the most Raw PTX 5.58 , 6.12 , 8.89 , 9.67 , 10.04 , 11.06 , 12.29 , 13.83 , 15.61 , 17.07 , 18.68 , 19.56 , 21.89 striking differences between◦ the◦ raw◦ PTX◦ and PTX◦ crystal◦ powders◦ ◦ was that◦ the◦ PTX crystal◦ powders◦ ◦ Crystal PTX 5.19◦, 6.12◦, 9.82◦, 11.13◦, 12.50◦, 13.59◦, 16.49◦, 20.34◦ exhibitedFigure no significant 7. XRD patterns stretching (2θ = 4–50°) and vibration. of the samples. These (a )differences Raw PTX; and in Raman(b) PTX spectracrystal powders. were attributed to the differences of intramolecular hydrogen bonds and intermolecular Van der Waals force; these Raman spectra (Figure 8) show the raw PTX in the range of the C–H stretching vibrations of the differencesRaman could spectra also (Figure indicate8) show the thechange raw PTX of cr inystalline the range structure of the C–H of stretchingPTX after vibrationsthe antisolvent of the alkyl (2860–3200 cm−1),1 the valence vibrations of the carbonyl and amine group (1900–1500 cm−1), and1 recrystallizationalkyl (2860–3200 processing. cm− ), the valence vibrations of the carbonyl and amine group (1900–1500 cm− ), −1 1 −1 1 theand C–H the stretching C–H stretching vibrations vibrations of the ofbenzene the benzene ring from ring 900 from cm 900 to cm650− cmto 650. However, cm− . However,the most strikingthe most differences striking di betweenfferences the between raw PTX the and raw PTX PTX crystal and PTX powders crystal was powders that the was PTX that crystal the PTX powders crystal exhibitedpowders no exhibited significant no significant stretching stretchingand vibration. and These vibration. differences These diinff Ramanerences spectra in Raman were spectra attributed were toattributed the differences to the di offf erencesintramolecular of intramolecular hydrogen hydrogen bonds and bonds intermolecular and intermolecular Van der VanWaals der force; Waals these force; differencesthese differences could couldalso indicate also indicate the change the change of cr ofystalline crystalline structure structure of ofPTX PTX after after the the antisolvent antisolvent recrystallizationrecrystallization processing. processing.

Figure 8. Raman spectra of the samples. (a) Raw PTX, and (b) PTX crystal powders.

SSNMR was used to investigate the chemical structure, because it is a sensitive indicator of hydrogen bonding [36–39], stereochemistry [40], conformation [41], steric forces [42], and electrostatic interactions [43–45]. The PTX crystal structure (Figure 1) contained two molecules per asymmetric unit (i.e., prime=Figure 2 referred 8. Raman to spectra herein of as the molecules samples. ( a1a) Raw and PTX,1b) and and (crystallizesb) PTX crystal in powders.the P21 space group [46]. The twoFigure molecules 8. Raman of PTX spectra were of the10-deacetylbaccatin samples. (a) Raw PTX, III [47] and and(b) PTX phenylisoserine crystal powders. side chain [48]. The SSNMRSSNMR analysis was used of tothese investigate data provided the chemical shift as structure,signments becauseto all molecular it is a sensitive positions indicator (Table 2). of SSNMR was used to investigate the chemical structure, because it is a sensitive indicator of Thehydrogen different bonding chemical [36–39 shifts], stereochemistry could be found [40], at conformation C16 and C17, [41 ],by steric comparing forces [42 the], and chemical electrostatic shift hydrogen bonding [36–39], stereochemistry [40], conformation [41], steric forces [42], and electrostatic principal values (CSPV) of raw PTX with the PTX crystal. This phenomenon was attributed to the interactions [43–45]. The PTX crystal structure (Figure 1) contained two molecules per asymmetric increased bond angle of C16–C17 than raw PTX, in 10-deacetylbaccatin III (Figure 9). The bond angle unit (i.e., prime= 2 referred to herein as molecules 1a and 1b) and crystallizes in the P21 space group [46]. The two molecules of PTX were 10-deacetylbaccatin III [47] and phenylisoserine side chain [48]. The SSNMR analysis of these data provided shift assignments to all molecular positions (Table 2). The different chemical shifts could be found at C16 and C17, by comparing the chemical shift principal values (CSPV) of raw PTX with the PTX crystal. This phenomenon was attributed to the increased bond angle of C16–C17 than raw PTX, in 10-deacetylbaccatin III (Figure 9). The bond angle

Pharmaceutics 2020, 12, 1008 10 of 16 interactions [43–45]. The PTX crystal structure (Figure1) contained two molecules per asymmetric unit (i.e., prime= 2 referred to herein as molecules 1a and 1b) and crystallizes in the P21 space group [46]. The two molecules of PTX were 10-deacetylbaccatin III [47] and phenylisoserine side chain [48]. The SSNMR analysis of these data provided shift assignments to all molecular positions (Table2). PharmaceuticsThe diﬀerent 2020 chemical, 12, x FOR shifts PEER couldREVIEW be found at C16 and C17, by comparing the chemical shift principal 10 of 16 values (CSPV) of raw PTX with the PTX crystal. This phenomenon was attributed to the increased ofbond C16–C17 angle ofchanged C16–C17 because than raw the PTX, space in structure 10-deacetylbaccatin of the unit III cell (Figure was 9loose,). The and bond the angle loose of structure C16–C17 allowedchanged easier because collection the space of structurethe water of molecule the unit cell. Thus, was the loose, PTX and crystal the loose powder structure had a allowed better water easier solubility.collection of the water molecule. Thus, the PTX crystal powder had a better water solubility.

TableTable 2. 2. PartialPartial 13C 13C shift shift assignments assignments in in solid solid paclitaxel. paclitaxel.

Position δ 13C/ppmδ 13 (CrystalC/ppm (Crystal PTX) δ 13C/ppm (Raw PXT) Position δ 13C/ppm (Raw PXT) C18 6.10 PTX) 5.79 C16, C17,C18 C19 16.35 6.10 15.50 5.79 C24,C16, C28 C17, C19 28.99, 31.18 16.35 30.14 15.50 C6C24, C2837.58 28.99, 31.1837.19, 30.1441.29 C8, C3'C6 51.53 37.58 37.19, 51.62 41.29 C8, C3’ 51.53 51.62 C7,C7, C13, C13, C2' C2’ 68.64 68.64 68.93, 68.93, C4,C4, C5 C5 86.75 86.75 88.62 88.62 C22,C22, C25, C25, C26 C26 123.18 123.18 123.10 123.10 C11,C11, C12 C12 134.56 134.56 134.16, 134.16, 136.04 136.04 C21, C23, C4’ 164.97 165.10 C21, C23, C4' 164.97 165.10

FigureFigure 9. 9. TheThe change change of of crystalline crystalline structure structure of of the the PTX. PTX.

TheThe DSC DSC is is a a technique technique used used to to measure measure the the temp temperatureerature and and energy energy variations variations involved involved in in the the phasephase transitions; thesethese variationsvariations are are related related to to the the degree degree of crystallinityof crystallinity and and stability stability of the of solidthe solid state stateof drug. of drug. From From Figure Figure 10, the 10, DSC the thermogram DSC thermogram of the PTX of the revealed PTX revealed one endothermic one endothermic peak at 221.99 peak ◦atC 221.99and one °C exothermic and one peakexothermic at 244.6 ◦peakC, which at 244.6 were attributed°C, which to were the melting attributed and decompositionto the melting of PTX,and decompositionrespectively. While of PTX, the respectively. endothermic While peak and the theendothermic exothermic peak peak and the the PTX exothermic crystal powders peak the were PTX at crystalabout 147.88powders and were 211.7 at◦ C,about respectively. 147.88 and This 211.7 phenomenon °C, respectively. was in This agreement phenomenon with the was Raman in agreement and XRD withanalysis, the Raman which showed and XRD that analysis, the raw which PTX crystal showed structure that the had raw changed, PTX crystal as conﬁrmed structure by had the decreasedchanged, asmelting confirmed point by of the raw decreased PTX. The melting decreased point melting of raw point PTX. indicated The decreased that the melting band energy point alsoindicated decreased, that thewhich band coincided energy also with decreased, the bond anglewhich change coincide ofd C16–C17. with the bond angle change of C16–C17.

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FigureFigureFigure 10. 10. DSCDSC 10. curvesDSC curves curves (Temperature (Temperature (Temperature range: range: range: 50–3000 50–3000 50–3000 °C)◦C) °C)of of the of the thesamples. samples. samples. (a () a (Rawa)) Raw Raw PTX, PTX,PTX, and andand (b (()bb )PTX) PTXPTX crystalcrystalcrystal powders. powders. powders.

AsAs shown shownAs shown by by the theby TG TGthe curve TG curve curve (Figure (Figure (Figure 11), 11 ), 11),when when when th thee thtemperature temperaturee temperature increased increased increased from fromfrom 40 4040 °C ◦°CC to to 240 240 °C, °C,◦C, thethe raw rawthe PTX raw PTX hadPTX had nearlyhad nearly nearly no no weight weightno weight loss, loss, loss, and and andPTX PTX PTX weight weight weight decreased decreased decreased quickly quickly quickly at at ata aatemperature temperaturetemperature of of approximatelyapproximatelyapproximately 240 240 °C. 240◦C. PTX °C. PTX PTXcrystal crystal crystal powders powders powders began began began to to slowly to slowly slowly lose lose lose weight weight weight from from from 40 4040 °C◦ °CC to toto 220 220220 °C ◦°CC and and immediatelyimmediatelyimmediately lost lost weight weightlost weight at at approximately at approximately 220220 ◦ 220°C.C.The °C.The totalThe total total weight weight weight loss loss wasloss was approximatelywas approximately approximately 18.83% 18.83% 18.83% and and22.15% 22.15%and for 22.15% thefor rawthe for PTXraw the and PTXraw PTX PTXand crystal andPTX PTXcrystal powders. crystal powder These powders. results Theses. These showed results results thatshowed showed both kindsthat that both ofboth crystals kinds kinds hadof of crystalsgoodcrystals thermal had good had stability. goodthermal thermal stability. stability.

FigureFigure 11. TG11. TG curve curve (Temperature (Temperature range: range: 30–600 °C)◦C) of of the the samples. samples. (a) Raw (a) RawPTX; PTX;and (b and) PTX (b crystal) PTX Figurecrystalpowders. 11. powders. TG curve (Temperature range: 30–600 °C) of the samples. (a) Raw PTX; and (b) PTX crystal powders. 3.5. Dissolution3.5. Dissolution Results Results 3.5. DissolutionHPLCHPLC analysis Results analysis results results showed showed that that at concentrations at concentrations of 1.16 of 1.16µg/mL μg/mL and 6.05and µ6.05g/mL, μg/mL, the rawthe PTXraw and PTX crystal powder were soluble in pure water. Moreover, at a concentration of 10.42 µg/mL and HPLCPTX and analysis PTX crystal results powder showed were that soluble at concentrations in pure water. ofMoreover, 1.16 μg/mL at a concentration and 6.05 μg/mL, of 10.42 the μ g/mLraw 24.65 µg/mL, the raw PTX and PTX crystal powder were soluble in artificial gastric juice. The solubility PTX andand PTX 24.65 crystal μg/mL, powder the raw were PTX soluble and PTX in pure crystal water. powder Moreover, were soluble at a concentration in artificial gastricof 10.42 juice. μg/mL The of PTXsolubility crystal of powders PTX crystal was increasedpowders was nearly increased six times near inly water six times and in 2.4 water times and in artificial 2.4 times gastric in artificial juice. and 24.65 μg/mL, the raw PTX and PTX crystal powder were soluble in artificial gastric juice. The Thus,gastric it is feasible juice. Thus, to prepare it is feasible PTX to crystal prepare powders PTX crystal through powders the antisolventthrough the precipitationantisolvent precipitation process to solubility of PTX crystal powders was increased nearly six times in water and 2.4 times in artificial improveprocess and to enhance improve its and solubility enhance in its water. solubility in water. gastric juice. Thus, it is feasible to prepare PTX crystal powders through the antisolvent precipitation The dissolutionThe dissolution profiles profiles of the of rawthe PTXraw PTX and PTXand PTX crystal crystal powders powders in water in water are shownare shown in Figure in Figure 12a. process to improve and enhance its solubility in water. The dissolution12a. The dissolution rate of the rate PTX of the crystal PTX powdercrystal powder increased increased rapidly rapidly from from 25.09% 25.09% to 50.67% to 50.67% within within 8 h, The dissolution profiles of the raw PTX and PTX crystal powders in water are shown in Figure and then8 h, and its dissolution then its dissolution ceased as ceased the time as increased.the time increased. By contrast, By onlycontrast, a small only amount a small of amount the drug of wasthe 12a. The dissolution rate of the PTX crystal powder increased rapidly from 25.09% to 50.67% within dissolveddrug was from dissolved the raw PTXfrom atthe a steadyraw PTX rate at a within steady 6 rate h, and within the 6 maximum h, and the dissolutionmaximum dissolution rate of the rate raw 8 h, and then its dissolution ceased as the time increased. By contrast, only a small amount of the PTX wasof the 15.80%. raw PTX The was solubility 15.80%. test The in solubility artificial gastrictest in juiceartificial for eachgastric PTX juice sample for each was PTX performed sample overwas drug was dissolved from the raw PTX at a steady rate within 6 h, and the maximum dissolution rate of the raw PTX was 15.80%. The solubility test in artificial gastric juice for each PTX sample was

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performed over 48 h. As shown in Figure 12b, the dissolution rate of the PTX crystal powders 48 h.performed As shown over in Figure 48 h. 12Asb, shown the dissolution in Figure rate12b, of the the dissolution PTX crystal rate powders of the increasedPTX crystal slowly powders from increased slowly from 5.56% to 32.50% and that of the raw PTX increased from 2.36% to 25.56% within 5.56%increased to 32.50% slowly and from that 5.56% of the to raw 32.50% PTX and increased that of the from raw 2.36% PTX increased to 25.56% from within 2.36% 36 to h. 25.56% Consequently, within 36 h. Consequently, both of them no longer exhibited dissolution as the time increased. The both36 of h. them Consequently, no longer exhibited both of dissolutionthem no longer as the ex timehibited increased. dissolution The dissolution as the time results increased. proved The that dissolutiondissolution results results proved proved that that the the bond bond angle angle of of C16–C17 C16–C17 changed changed and and that that the the crystal crystal structure structure the bond angle of C16–C17 changed and that the crystal structure increased the dissolution rate. increasedincreased the the dissolution dissolution rate. rate.

FigureFigureFigure 12. 12. 12.The The The dissolution dissolution dissolution profiles profiles profiles ofof of thethe the samplessamples samples in in water water ( (a(a),a),), and andand in inin artificial artificialartificial gastric gastric juice juice juice ( b(b ().b). ). 3.6. Bioavailability Study 3.6.3.6. Bioavailability Bioavailability Study Study FigureFigureFigure 13 13 shows13 shows shows the the the pharmacokinetic pharmacokinetic pharmacokinetic profilesprofiles profiles ofof of eacheach each sample.sample. sample. The The PTX PTX concentration concentration concentration in in inrat rat rat plasmaplasmaplasma of theof of the PTXthe PTX PTX crystal crystal crystal powders powders powders and and and the the the raw raw raw PTX PTX PTX group group group reached reached reached the the the maximum maximum maximum of of 15.71of 15.71 15.71µg μ /μmLg/mLg/mL and 0.50andandµg /0.50 mL0.50 afterμ μg/mLg/mL 2 hafter after and 2 12 h h ofand and drug 1 1 h h administration.of of drug drug administration. administration. The PTX The crystalThe PTX PTX powders crystal crystal powders dramaticallypowders dramatically dramatically decreased fromdecreaseddecreased 15.71 µg from /mLfrom to15.71 15.71 1.90 μ µμg/mLgg/mL/mL, to to within 1.90 1.90 μ 2μg/mL,g/mL, h to 6 within h,within and 2 slowly2 h h to to 6 increased6 h, h, and and slowly slowly to 8.30 increased increasedµg/mL within to to 8.30 8.30 6 μ hμg/mL tog/mL 10 h. Then,withinwithin the 6 PTX 6 h h to to concentration 10 10 h. h. Then, Then, the the decreased PTX PTX concentration concentration until the decreased concentration decreased until until was the the concentration nearly concentration 0 µg/mL. was was The nearly nearly results 0 0 μ μg/mL. showedg/mL. thatTheThe the results PTXresults crystal showed showed powders that that the the had PTX PTX a secondcrystal crystal powders peak.powders This had had might a a second second be due peak. peak. to the This This liver might might and be intestinebe due due to to circulationthe the liver liver orand gastricand intestine intestine emptying circulation circulation in specific or or gastric gastric intervals, emptying emptying which in in enabledsp specificecific intervals, theintervals, drug which towhich reach enabled enabled the small the the drug intestinedrug to to reach reach twice. Thisthethe resulted small small intestine intestine in two twice. entries twice. This This into resulted resulted the blood, in in two two which entries entries led into tointo the the the appearance blood, blood, which which of led doubleled to to the the peaks. appearance appearance The same of of trendsdoubledouble were peaks. peaks. exhibited The The same same by raw trends trends PTX; were were the exhibited rawexhibited PTX by significantly by raw raw PTX; PTX; the decreasedthe raw raw PTX PTX from significantly significantly 0.50 µg/ mLdecreased decreased to 0.21 fromµ fromg/mL within0.500.50 1μ μg/mL hg/mL to 4 to h,to 0.21 and0.21 μ slowlyμg/mLg/mL within increasedwithin 1 1 h h to to 4 0.38 4 h, h, and µandg/ mLslowly slowly within increased increased 12 h to to to 16 0.38 0.38 h. Then,μ μg/mLg/mL the within within PTX 12 concentration12 h h to to 16 16 h. h. Then, the PTX concentration decreased until the concentration was nearly 0 μg/mL. The decreasedThen, untilthe PTX the concentrationconcentration was decreased nearly 0 µuntilg/mL. the The concentration bioavailability was of thenearly PTX crystal0 μg/mL. increased The bioavailability of the PTX crystal increased 10.88 times than raw PTX. The significant enhancement 10.88bioavailability times than raw of the PTX. PTX The crystal significant increased enhancement 10.88 times of than oral raw bioavailability PTX. The significant was also enhancement in agreement of oral bioavailability was also in agreement with the result of the dissolution test. In addition, withof the oral result bioavailability of the dissolution was also test. in Inagreemen addition,t with previous the result research of the showed dissolution that the test. bioavailability In addition, of previous research showed that the bioavailability of the super-antiresistant PTX micelles by a one- the super-antiresistantprevious research showed PTX micelles that the by bioavailability a one-step method of the super-antiresistant was almost twice thatPTX ofmicelles the raw by PTX a one- [49]. step method was almost twice that of the raw PTX [49]. However, the bioavailability of the PTX However,step method the bioavailability was almost twice of the that PTX of crystal the raw powders PTX [49]. prepared However, in thisthe studybioavailability were approximately of the PTX crystalcrystal powders powders prepared prepared in in this this study study were were approximately approximately 10.88-fold 10.88-fold greater greater than than that that of of raw raw PTX, PTX, 10.88-fold greater than that of raw PTX, which illustrated that the PTX crystal powders obtained in whichwhich illustrated illustrated that that the the PTX PTX crystal crystal powders powders obtained obtained in in this this study study was was remarkably remarkably effective effective in in this study was remarkably effective in improving the extent of absorption of the PTX, and exhibited improvingimproving the the extent extent of of absorption absorption of of the the PTX, PTX, and and exhibited exhibited potential potential as as a a ne neww oral oral drug drug formulation formulation potential as a new oral drug formulation for clinical applications. forfor clinical clinical applications. applications.

Figure 13. Mean ( SD) plasma concentration-time curves after oral administration of raw PTX (a) and ± PTX crystal powders suspension (b) at a dose of 10 mg/kg for PTX (n = 6). Pharmaceutics 2020, 12, 1008 13 of 16

4. Conclusions In this study, PTX crystal powders were prepared through the antisolvent precipitation process, using HMImBr as solvent and water as antisolvent. The factors influencing the water saturation solubility of the PTX crystal powders were optimized through a single-factor design, and the optimum conditions were determined as follows—50 mg/mL concentration of PTX–HMImBr solution, 1:7 solvent-to-antisolvent volume ratio, and 25 ◦C reaction temperature. Under the above conditions, the PTX crystal powder were obtained with the saturation solubility of 6.05 µg/mL, which was six times higher than that of the raw PTX. The PTX crystal powders obtained were characterized using SEM, FT–IR, HPLC–MS, Raman spectroscopy, XRD, DSC, SSNMR, and TG analysis. The SEM showed that PTX crystal powders were small in size and had a uniform distribution and short rods. Furthermore, the FT–IR and LC–MS results showed that the crystal powders had the same chemical structure as the raw drug. Meanwhile, the XRD, SSNMR, Raman spectra, and DSC analyses proved the change of the crystal structure. The XRD results indicated that the crystal powders had different peaks from that of the raw PTX and that CSPV also varied between the raw PTX and PTX crystal powders at C16 and C17 in 10-deacetylbaccatin III, showing that the bond angle of C16–C17 changed. The DSC curve showed that the melting point of the PTX crystal powders was 147.88 ◦C; the smaller melting point proved the lower bond energy, and this conclusion was consistent with the altered bond angle of C16–C17. The TG analysis showed that the raw PTX and PTX crystal powders had similar thermal stability. Furthermore, the maximum solubility of the PTX crystal powder water in water was 5.6 times higher than the raw drug. The dissolution rate and solubility of PTX crystal powders was 3.21 times higher compared to that of the raw PTX in water and was 1.27 times higher than the raw PTX in artificial gastric juice. The oral bioavailability of the PTX crystal increased 10.88 times than raw PTX. This antisolvent precipitation technique is a promising and economical method for the preparation of highly water-soluble drug particles for commercial use. Moreover, this method provides a good theoretical and practical basis for the development of other fat-soluble drugs.

Author Contributions: Methodology, X.Z.; validation, Q.Y., C.Z. and W.W.; formal analysis, Q.Y. and C.Z.; investigation, L.W. (Lingling Wang), L.W. (Li Wang), Y.L. and Y.G.; writing—original draft preparation, C.Z.; writing—review and editing, W.L. and X.Z.; project administration, X.Z.; funding acquisition, X.Z. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the National Natural Science Foundation of China (No. 21473023) and Heilongjiang Touyan Innovation Team Program (Tree Genetics and Breeding Innovation Team). Acknowledgments: The authors are grateful for the precious comments and careful corrections made by anonymous reviewers. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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