Gelation Mechanism of Erythromycin Ethylsuccinate During Crystallization

Gelation Mechanism of Erythromycin Ethylsuccinate During Crystallization In this paper, the gelation mechanism of erythromycin ethylsuccinate (EES) during crystallization is investigated for the first time. The generated semisolid gel-like phase exhibited a 3D fibrillar network morphology and the typical rheological properties of gels. The fibers inside the gel-like phase were confirmed to be new types of EES solvates using powder X-ray diffraction, thermogravimetric analysis/differential scanning calorimetry, and gas chromatography. The gelation and crystal - lization regions in EES-1-propanol solid–liquid phase diagram were determined. Analyses of solvent parameters showed that moderate solvent polarity may promote EES gelation. Fourier transform infrared spectra, nuclear magnetic resonance spectra, and scanning electron microscopy analyses indicated that through intermolecular hydrogen bonds, EES and solvent molecules assembled into fibers via crystallographic mismatch branching growth. The fibers intertwined into a 3D network microstructure and formed a gel-like phase, completely immobilizing the solution. Keywords Gelation · Solvate · Crystallization · Phase diagram · Molecular self-assembly Introduction propose that gelation allows APIs to be used as molecular gel dosage forms and allows the control of crystal habits of Solution crystallization is widely used in the production APIs. Hence, the study of APIs gelation and the gelation of pharmaceutical crystals. During the crystallization of mechanism is of great importance. active pharmaceutical ingredients (APIs) under various Gelation refers to a phenomenon where, under certain conditions, several studies have reported the occurrence of conditions, the solute molecules in a solution self-assemble gelation phenomenon [1, 2]. Gelation of some APIs, such into a 3D network structure that immobilizes the whole liq- as clopidogrel sulfate [3] and valsartan [4], is rather likely uid phase [5]. At the macroscopic scale, the gelled solu- to form a gel-like phase in their supersaturated state, which tion loses its flowability and turns into a semisolid soft is an unwanted process in industrial crystallization. How- matter. Gelation is driven by the assembly of solute mol- ever, gelation may have some advantages. New perspectives ecules through multiple reversible noncovalent interactions [6–8], including hydrogen bonds, π–π stacking, van der Waals forces, electrostatic, dipole–dipole, and hydrophobic interactions. Electronic supplementary material The online version of this article (https ://doi.org/10.1007/s1220 9-018-0163-5) contains Similar to crystallization, gelation is also a common supplementary material, which is available to authorized users. phase separation phenomenon. During crystallization, mac- roscopic phase separation occurs, leading to a solid–liquid * Ying Bao phase equilibrium, whereas, during gelation, only a semi- yingbao@tju.edu.cn solid phase exists because of a microscopic phase separation. School of Chemical Engineering and Technology, Tianjin Both crystallization and gelation are driven by supersatura- University, Tianjin 300072, China tion. Methods that result in supersaturation or add external Collaborative Innovation Centre of Chemical Science energy to induce the formation of new phases can result in and Engineering (Tianjin), Tianjin 300072, China gelation or crystallization [9–14], such as cooling a hot solu- Department of Chemical and Biochemical Engineering, tion, pH switch, ultrasonic treatment, and optical radiation. Western University, London, ON N6A 5B9, Canada Vol.:(0123456789) 1 3 X. Su et al. Dynamic arrest is regarded as the main mechanism of physi- Water Co., Ltd, China. Deuterated reagents (acetone-d6 and cal gelation [15], which means that a new phase induced D O, 99.9% deuterium content) were purchased from Tianjin by solute molecules is trapped in a kinetically favorable Heowns Biochem Technology Co., Ltd, China. gel-like state rather than a thermodynamic stable state. The metastable state is the outcome of the competition between Preparation of Gel‑Like Phase solute–solute interaction and solvent–solute interaction [16] and occurs in the metastable region in its phase diagram. Method 1: Solventing-out. To achieve a gel-like phase, 1.5 g For APIs, most studies focused on the metastable region of of EES powder was dissolved completely in 10 mL acetone crystallization. Only a few reports involved the metastable and a transparent solution was formed at 20 °C. Under mod- equilibrium phase diagrams that contain the gel-like phase; erate mechanical stirring (300 r/min), 6 mL water was added meanwhile, gelation mechanism remains poorly understood. as an anti-solvent at a constant flowrate (1.00 mL/min) using In this paper, the gelation of a widely used erythromycin a peristaltic pump, and then the whole system turned into a analog antibiotic, erythromycin ethylsuccinate (EES, CAS gel-like state. Gelation also occurred in a 1-propanol–water No. 1264-62-6, Fig.  1) is studied for the first time. First, mixture at different volume ratios. the EES gel-like phase was characterized and the solvate Method 2: Cooling. 1.5 g EES powder was added into a nature of the fibers inside the phase was confirmed. Then, 4 g water–acetone mixture (1 mol/mol) and heated to 51 °C the EES gelation region was developed in the solvent system to make the powder dissolve completely. Then, the hot solu- of 1-propanol. Finally, Fourier transform infrared (FTIR) tion was kept at room temperature to form a gel-like phase. spectra, nuclear magnetic resonance (NMR) spectra, and Gelation also occurred in 1-propanol using a similar method. solvent parameters analyses were conducted to unravel the Method 3: A gel-like phase was also formed when a mod- gelation mechanism at a molecular level. The results of this erate amount of EES powder was added to certain solvents. study provide useful guidelines to understand the gelation In a typical gelation process, 0.37 g EES powder was added mechanism of other APIs. into a 4 g water–acetone mixture (1 mol/mol) at 20 °C under 300 r/min stirring. After 5 min, the solution became turbid and then a gel-like phase emerged. Gelation also occurred in Experimental 1-propanol when the amount of EES powder was changed. Materials Solvent Screening A white crystalline powder of EES (C H NO , MW The gelation of EES was investigated via method 1 in a 43 75 16 862.07) with a mass fraction purity of over 98% was pur- series of solvents including methanol, ethanol, 2-propanol, chased from Wuhan Yuancheng Gongchuang Science and 1, 2-propanediol, DMF, NMP, acetonitrile, formamide, Technology  Ltd, China. The organic solvents (acetone, cholamine, glycol, DMSO, triethanolamine, isopropyl ether, 1-propanol, etc., analytical reagent grade) were purchased and THF. Water was used as an anti-solvent. from Tianjin Jiangtian Chemical Technology Co., Ltd, China, and were used without further purification. Deionized Characterization water was purchased from Tianjin QingYuanquan Purified Micrographs were acquired by scanning electron microscopy (Hitachi TM3000 tabletop SEM, Japan) and polarized opti- cal microscopy (Olympus BX51, Japan). Powder X-ray Dif- fraction (PXRD) data were collected by a PXRD instrument (Rigaku D/MAX-2500, Japan) under the following conditions: 8°/min scan speed, 0.02° step size, 2°–50° scan range, and Cu Kα radiation (λ = 1.5406 Å). Rheological experiments were conducted on a rheometer (Anton Paar MCR 302, Austria) at 20 °C. Dynamic frequency sweep (DFS) was conducted in an angular frequency range of 100–0.1 rad/s with a strain amplitude of 0.05%, and dynamic strain sweep (DSS) was conducted in a strain range of 0.01–100% at 10 rad/s fre- quency. Thermogravimetric analysis (TGA) and differen- tial scanning calorimetry (DSC) were conducted on a TGA instrument (Mettler Toledo TGA/DSC 1/SF, Switzerland) and Fig. 1 EES molecular structure DSC instrument (Mettler Toledo DSC 1/500, Switzerland), 1 3 Gelation Mechanism of Erythromycin Ethylsuccinate During Crystallization respectively. 5–10 mg of samples was loaded in a crucible and The samples were measured thrice for each Erlenmeyer heated from 25 to 250 °C at 10 °C/min under N atmosphere. flask, and the average value was used to calculate the solute FTIR spectra were determined on a spectrometer (Bruker Ten- solubility. The temperature ranged from 10 to 50 °C over 5 −1 sor 27, Switzerland) with a resolution of 1 cm , and the solid equidistant points. powder was tableted with KBr and scanned in the range of −1 4000–400 cm . NMR spectra were determined on an NMR Gelation Points Measurement spectrometer (Bruker Avance III 400 MHz, Switzerland) in a D O and acetone-d6 mixture (1 mol/mol), which included C- Cooling Method An appropriate amount of EES powder NMR spectra and concentration variation H-NMR spectra, (m ) and 6 g of 1-propanol (m ) were added into a 50-mL 1 2 with a resolution of 0.45 Hz and sensitivity of 250:1. jacketed glass vessel within a thermostat bath. Under a 300 r/min stirring rate, the vessel was heated at 1 °C/min Measurement of the Phase Diagram to dissolve EES completely, and then, it was cooled at the same rate. The temperature was recorded when the solution Solubility Measurement became discernibly turbid. This was assigned as the gela- tion point of EES, after which the sample would completely Dynamic Method 4 g of 1-propanol (designated as m ) was turn into a gel-like phase. The amount of EES powder was poured into a 50-mL jacketed glass vessel, and a thermostat then changed, and the experiments were repeated to obtain bath was used to regulate the experimental temperature. At a series of gelation points at different concentrations. The a stirring rate of 300 r/min, a certain small amount of EES concentration of the solution was calculated by Eq. (1). powder was continuously added to the solvent to approach saturation. The last added amount was not more than 0.01 g. The total amount of EES powder consumed when the slightly Results and Discussion turbid solution did not turn transparent any more was recorded as m . Each measuring point was repeated thrice, and the solu- Gelation Phenomenon and Characterization bility of EES was calculated by Eq. (1): of the EES Gel‑like Phase x = , The EES gel-like phase obtained through three methods (1) showed a white opaque appearance and exhibited no gravi- tational flow under tube inversion test. When heated, the The temperature ranged from 10 to 50 °C, and 5 equidistant points were considered. gel-like phase was separated into solid and liquid phases and eventually turned into a transparent solution. When the Static Method The cloudiness of the slightly turbid solu- tion mentioned above increased with time, suggesting a phase solution was cooled, the gel-like state reappeared, as shown in Fig. 2. The transition temperatures depended on the solu- transition after the EES dissolution. Thus, the static method was used to acquire the solubility data and the equilibrium tion concentration and solvent composition. Long, thin, and flexible fibers were observed in the micro- solid phase was tested. The experiments were carried out in Erlenmeyer flasks, which were shaken in a thermostat shaking scope images of the gel-like phase, and their sizes varied with the preparation methods (Fig. S1–S3). The variation of bath at 300 r/min. Excessive EES powder and 15 g 1-pro- panol were added into the Erlenmeyer flasks, and samples the microscale morphology (Fig. 3) of the gel-like phase was tracked throughout the gelation process by a microscope. On were taken every 2 h. After 8 h, a liquid–solid equilibrium was reached, and then the shaking was stopped for 0.5 h for the initial stage, the emergence of a few single fibers caused the suspension to separate into two phases. A total sample of 5 mL was extracted with a syringe equipped with a 0.22-μm polyvinylidene fluoride filter. The sample was placed in an evaporating dish, and the weight of the dish (X ) and the total weight of the dish and solution (X ) were recorded. The liq- uid samples were evaporated in a vacuum oven at 50 °C and weighed every 12 h until a constant weight (X ) was achieved. The PXRD pattern of the wet solid phase was recorded. The solubility of the equilibrium solid phase was calculated by Eq. (2): X − X 1 0 x = , (2) X − X 2 1 Fig. 2 Reversible thermal transition of EES gel-like phase 1 3 X. Su et al. constant with the increase in strain from 0.01 to 0.1%, which is the linear viscoelastic region (LVR) of the gel-like phase. The low strain limit (0.1%) demonstrated the crystalline nature of the fibers [17]. The DFS was performed within the LVR, as shown in Fig. 4b. In the whole frequency range, G′ was larger than G″, and both were almost independent of frequency, meaning that the sample was unable to flow and remained in a solid-like phase regardless of the bearing force. These rheological properties are similar to those of typical gel substances [18]. Solvatomorphs and Gelation Mechanism of EES Analysis of PXRD Patterns Fig. 3 Microscope images of the EES gel-like phase. a Single fibers, b fiber clusters, c twisted and cross-linked clusters, and d 3D fibrillar network The morphology of the fibers was maintained when the EES gel-like phase was dried in air for 1 h, while the immobi- a slight turbidity of the solution. By keeping the temperature lized solution was completely dried out. The PXRD patterns and solvent content constant, more fibers emerged and gath- of EES fibers are shown in Fig.  5, where the series of dif- ered to form fiber clusters. The clusters twisted and cross- fraction peaks in the PXRD patterns indicate the crystalline linked with each other to generate a 3D network structure. nature of the fibers. The fibers formed in different solvents The mechanical properties of the gel-like phase were showed the same PXRD patterns, indicating identical pack- measured by dynamic rheology characterization. The DSS ing modes of the EES molecules in the crystal structure. curves of the EES gel-like phase are shown in Fig. 4a. Both The obtained Bragg lattice spacing values (d) of 1.99, 0.97, storage modulus (G′) and loss modulus (G″) remained 0.63, 0.55 nm closely followed the ratio of 1:1/2:1/3:1/4, suggesting the existence of an 1D lamellar structure in the nanofibers of the gel-like phase with a layer distance of 1.99 nm [19, 20]. Compared with the PXRD pattern of EES crystals, the PXRD patterns of the fibers showed two new peaks at 4.44° and 13.96°, and the peaks at 8.15°, 11.05°, 15.22°, 17.00°, and 20.68° disappeared, suggesting that the crystalline form of the fibers is different from that of the EES crystals and has not been reported. After drying the samples in a vacuum oven at 50 °C for 24 h, the PXRD patterns of the dried sam- ples (Fig. S4) were different from those of the fibers but Fig. 5 PXRD patterns of EES crystals (black); EES fibers in water– Fig. 4 Rheological curves of the gel-like phase. a DSS and b DFS acetone (red) and 1-propanol (blue) 1 3 Gelation Mechanism of Erythromycin Ethylsuccinate During Crystallization similar to that of the EES crystals. The SEM image of a obtained from the above two sections indicate that the fibers dried sample (Fig. S5) shows a similar morphology to that formed in different solvents were EES solvatomorphs, which of the EES crystals. The changes indicate that the fibers were discovered for the first time. The gelation of the EES transformed to EES crystals. in its solution was due to the formation and cross-linking of the fibrous solvates. Thermal Analysis Conditions for Gelation Figure 6 shows the thermal analysis of EES fibers. The TGA curve of the fibers formed in 1-propanol showed a weight Phase Diagram loss of 2.55 wt%, and the DSC curve had an endothermic peak at the corresponding temperature range, indicating that The solubility data in 1-propanol were measured by both the sample was 1-propanol solvate. For the fibers formed in static and dynamic methods, corresponding to the blue and the water–acetone mixture, a weight loss of 0.90 wt% and red curves in Fig. 7. The equilibrium solid phase in the static an endothermic peak appeared in TGA and DSC curves in method was proved to be EES 1-propanol solvate (Fig. S8), the same temperature range, respectively, proving that the demonstrating the solubility data (blue curve) correspond- sample was also a kind of EES solvate. ing to the solubility of EES 1-propanol solvate. At the same The headspace sampling gas chromatography (GC) analy- temperature, the solubility of the EES solvate is lower than sis verified the existence of acetone and 1-propanol in the that of the EES crystals. Thus, with the addition of the EES fibers formed in water–acetone and 1-propanol, respectively crystals into 1-propanol, the EES crystals transformed into (Figs. S6 and S7). The existence of water in the fibers formed a solvate when the concentration of the EES in solution in the water–acetone mixture was proved by volumetric Karl exceeded the solubility of the solvate. In a series of typical Fischer titration (see Supporting Information). The results experiments, solutions represented by points from L to J 1 1 were prepared. It showed that because few fibrous crystal- line solvates crystallized out under low supersaturation, the solution could not gel in the region between the blue and red curves, which can be regarded as the crystallization region. Gelation would inevitably occur when the concentration is above the red curve, which can be regarded as the gelation region. The lower the supersaturation in the gelation region, the more difficult and time-consuming it is for gelation to occur. Thus, it is indicated that a high degree of supersatura- tion promotes gelation. The gelation method 3 described in Experimental section is hence termed as “trans-crystal gela- tion,” and the solubility of the EES crystals can be regarded as the critical gelation concentration. The black curve in Fig. 7 corresponds to the gelation points of EES measured Fig. 6 DSC and TGA curves of EES fibers. a fibers in 1-propanol and b fibers in water–acetone Fig. 7 Phase diagram of EES in 1-propanol 1 3 X. Su et al. by cooling. Compared with the trans-crystal gelation region, shows the Δδ of the studied gelation solvents, and it ranges 1/2 the solution gels fast in the cooling gelation region, and the from 4.3 to 14 MPa , which is smaller than those of other critical concentration of cooling gelation is higher than that solvents, indicating that the gelation solvents have a better of trans-crystal gelation. dissolving capacity for EES. Combined with the analysis of solubility parameters, it can be concluded that a high solubil- Analysis of Solvent Parameters ity of EES in the solvents is necessary for gelation. A low solubility results in the separation of a small amount of fib- The influence of solvent properties on the EES gelation was ers from the solution, which makes it hard to trap quantities investigated by solvent screening. The solutions of EES in of solvent molecules and ultimately leads to crystallization. acetone, methanol, ethanol, 1-propanol, 2-propanol, DMF, and NMP gelled entirely with added water, while the solution Intermolecular Interaction Analysis in acetonitrile gelled only partially. As shown in Table 1, the solvents that facilitated gelation have something in common: FTIR a dielectric constant range of 18.3–37.5, δ (dipole interac- 1/2 tions) of 6.1–18 MP a in Hansen solubility parameters, and FTIR characterization was performed on the EES crystals π (polarizability parameter) of 0.48–0.88 in Kamlet–Taft −1 solubility parameters [21]. Outside these ranges, it is impos- and fibers. The peaks at 1741 and 1698 cm in Curve a of Fig.  8 represent the C=O stretching vibration of ester sible for gelation to occur in the corresponding solvents. The matching degree of solvent–solute intermiscibility groups and carbonyl of EES, respectively. For fibers, the −1 former peak shifted to 1736 cm , while the latter disap- can be described by the residual of solubility Δδ. A smaller value of Δδ means a better solubility of a solute in a solvent. peared, which could have been masked by the former. It was speculated that ester groups participated in the intermolecu- Δδ can be calculated from Eq. (3): lar hydrogen bonds as H acceptors, accepting H from the sol- 2 2 2 vent molecules to form the EES solvate fibers. Furthermore, (3) Δ = ( −  ) +  −  +  −  , d d p p h h 1 2 1 2 1 2 the red shift of C–O–C vibration peaks in the ester groups where δ and δ represent the dispersion interactions and and glucosidic bonds also imply the participation of these O d h hydrogen bond interactions, respectively, while subscripts 1 atoms in the supramolecular self-assembly of EES solvate and 2 correspond to EES and solvent, respectively. Table 1 fibers as H acceptors (Table S1). Table 1 Gelation behaviors of * No. Solvent Gelation Dielectric con- Hansen solubility Δδ π [21] EES in different solvents and 1/2 1/2 behavior stant [21] (25 °C)parameters (MPa ) (MPa ) solvent parameters analysis [21, 22] δ δ δ d p h 1 Acetone G 20.7 15.5 10.4 7 4.306 0.62 2 Methanol G 31.2 15.1 12.3 22.3 14.08 0.6 3 Ethanol G 25.7 15.8 8.8 19.4 10.61 0.54 4 Propanol G 22.2 16 6.8 17.4 8.693 0.52 5 2-Propanol G 18.3 15.8 6.1 16.4 7.782 0.48 6 DMF G 36.71 17.4 13.7 11.3 7.778 0.88 7 NMP G 32 18 12.3 7.2 7.077 8 Acetonitrile PG 37.5 15.3 18 6.1 11.15 0.66 9 Formamide P 111 17.2 26.2 19 21.49 0.97 10 Cholamine P 37.72 17.2 15.6 21.3 15.1 11 Glycol P 37.7 0.92 12 DMSO P 48.9 18.4 16.4 10.2 10.36 1 13 Triethanolamine P 29.36 14 Isopropyl ether P 4.49 15 Water P 7.58 16.8 5.7 8 4.2 0.55 16 THF P 78.5 15.6 16 42.3 34.26 1.09 17 EES 13.2 7.5 9.2 – G, PG, and P represent gelation, partial gelation, and precipitate, respectively 1 3 Gelation Mechanism of Erythromycin Ethylsuccinate During Crystallization network structure. Thus, the formation and growth of the EES-solvent supramolecular fibers is in accordance with the mechanism of crystallographic mismatch branching [25]. NMR Concentration variation H NMR spectra are shown in Fig. 10. With the increase in EES concentration, the sys- tem changed from a solution to gel-like state, which led to the weakening of peak intensity and peak splitting, suggesting that the mobility of molecules decreased, and protons could not produce individual sharp signals as they were in the aggregated state in the gel-like phase [26]. The Fig. 8 Solid FTIR absorption curves. a EES crystals, b fibers in water–acetone, and c fibers in 1-propanol H atoms of H O (δ of ~ 4.30 ppm) show downfield chemi- cal shifts with the increasing EES concentration, indicat- ing the participation of H O molecules in the EES–solvent −1 The wide peak of –OH stretching vibration at 3453 cm hydrogen bonds as H donors. of EES crystals indicates that the EES molecules interacted The C NMR spectra (see Fig. S10) were also recorded with each other through hydrogen bonds between hydroxyls. for the EES solution (1.875  mg/mL) and gel-like state For fibers, the –OH stretching vibration peak split into two (3.75 mg/mL). The chemical shift at nearly δ =211.5 ppm peaks, indicating the variation of intermolecular hydrogen (Fig. 11a) corresponds to the C atoms of carbonyl groups. bonds between the hydroxyl groups of the EES molecules As the amount of solvents was much larger, we assigned after fibers formation. Some of the EES–EES intermolecu- the high peak to acetone-d6 and the low peak to the car- lar hydrogen bonds between the hydroxyls were substituted bonyl group on the EES ring. The downfield shifts of these by the new EES–solvent intermolecular hydrogen bonds in peaks indicate the participation of carbonyl groups as H the supramolecular fibers [23, 24], as the small size makes acceptors in the EES–solvent intermolecular hydrogen the solvent molecules bond easily with the EES molecules. bonds. In addition, the peaks of –COOC– in ester groups Moreover, both the out-of-plane bending vibration peak of around 175  ppm (Fig.  11b) showed minute downfield –OH and the bending vibration peak of –OH on the clad- shifts (0.06–0.1 ppm), suggesting the involvement of ester inose ring exhibited a red shift in the fibers, demonstrat- groups in the EES–solvent intermolecular hydrogen bonds. ing the participation of these hydroxyls in intermolecular hydrogen bonds. Because of the presence of many intermolecular hydrogen bonds between the EES and solvent molecules, they assembled together to form 1D lamellar structural units, which assembled into 2D fibers quickly, and many branches were generated around the fibers, as shown in Fig.  9. Finally, the fibers intertwined and cross-linked with each other to construct a 3D self-assembled fibrillar Fig. 9 Images of fibrous crystals in 1-propanol. a optical microscope Fig. 10 Concentration variation H NMR spectra of EES in of crystal and b SEM image of fibers D O:acetone-d6 (v:v = 3:5) 1 3 X. Su et al. 5. Estroff LA, Hamilton AD (2004) Water gelation by small organic molecules. Chem Rev 104:1201–1218 6. Dastidar P (2008) Supramolecular gelling agents: can they be designed? Chem Soc Rev 37:2699–2715 7. Song J, Sun HJ, Sun SJ et al (2013) Synthesis and gel properties of sorbitol derivative gelators. Trans Tianjin Univ 19:319–325 8. 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Gelation Mechanism of Erythromycin Ethylsuccinate During Crystallization

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Abstract

In this paper, the gelation mechanism of erythromycin ethylsuccinate (EES) during crystallization is investigated for the first time. The generated semisolid gel-like phase exhibited a 3D fibrillar network morphology and the typical rheological properties of gels. The fibers inside the gel-like phase were confirmed to be new types of EES solvates using powder X-ray diffraction, thermogravimetric analysis/differential scanning calorimetry, and gas chromatography. The gelation and crystal - lization regions in EES-1-propanol solid–liquid phase diagram were determined. Analyses of solvent parameters showed that moderate solvent polarity may promote EES gelation. Fourier transform infrared spectra, nuclear magnetic resonance spectra, and scanning electron microscopy analyses indicated that through intermolecular hydrogen bonds, EES and solvent molecules assembled into fibers via crystallographic mismatch branching growth. The fibers intertwined into a 3D network microstructure and formed a gel-like phase, completely immobilizing the solution. Keywords Gelation · Solvate · Crystallization · Phase diagram · Molecular self-assembly Introduction propose that gelation allows APIs to be used as molecular gel dosage forms and allows the control of crystal habits of Solution crystallization is widely used in the production APIs. Hence, the study of APIs gelation and the gelation of pharmaceutical crystals. During the crystallization of mechanism is of great importance. active pharmaceutical ingredients (APIs) under various Gelation refers to a phenomenon where, under certain conditions, several studies have reported the occurrence of conditions, the solute molecules in a solution self-assemble gelation phenomenon [1, 2]. Gelation of some APIs, such into a 3D network structure that immobilizes the whole liq- as clopidogrel sulfate [3] and valsartan [4], is rather likely uid phase [5]. At the macroscopic scale, the gelled solu- to form a gel-like phase in their supersaturated state, which tion loses its flowability and turns into a semisolid soft is an unwanted process in industrial crystallization. How- matter. Gelation is driven by the assembly of solute mol- ever, gelation may have some advantages. New perspectives ecules through multiple reversible noncovalent interactions [6–8], including hydrogen bonds, π–π stacking, van der Waals forces, electrostatic, dipole–dipole, and hydrophobic interactions. Electronic supplementary material The online version of this article (https ://doi.org/10.1007/s1220 9-018-0163-5) contains Similar to crystallization, gelation is also a common supplementary material, which is available to authorized users. phase separation phenomenon. During crystallization, mac- roscopic phase separation occurs, leading to a solid–liquid * Ying Bao phase equilibrium, whereas, during gelation, only a semi- yingbao@tju.edu.cn solid phase exists because of a microscopic phase separation. School of Chemical Engineering and Technology, Tianjin Both crystallization and gelation are driven by supersatura- University, Tianjin 300072, China tion. Methods that result in supersaturation or add external Collaborative Innovation Centre of Chemical Science energy to induce the formation of new phases can result in and Engineering (Tianjin), Tianjin 300072, China gelation or crystallization [9–14], such as cooling a hot solu- Department of Chemical and Biochemical Engineering, tion, pH switch, ultrasonic treatment, and optical radiation. Western University, London, ON N6A 5B9, Canada Vol.:(0123456789) 1 3 X. Su et al. Dynamic arrest is regarded as the main mechanism of physi- Water Co., Ltd, China. Deuterated reagents (acetone-d6 and cal gelation [15], which means that a new phase induced D O, 99.9% deuterium content) were purchased from Tianjin by solute molecules is trapped in a kinetically favorable Heowns Biochem Technology Co., Ltd, China. gel-like state rather than a thermodynamic stable state. The metastable state is the outcome of the competition between Preparation of Gel‑Like Phase solute–solute interaction and solvent–solute interaction [16] and occurs in the metastable region in its phase diagram. Method 1: Solventing-out. To achieve a gel-like phase, 1.5 g For APIs, most studies focused on the metastable region of of EES powder was dissolved completely in 10 mL acetone crystallization. Only a few reports involved the metastable and a transparent solution was formed at 20 °C. Under mod- equilibrium phase diagrams that contain the gel-like phase; erate mechanical stirring (300 r/min), 6 mL water was added meanwhile, gelation mechanism remains poorly understood. as an anti-solvent at a constant flowrate (1.00 mL/min) using In this paper, the gelation of a widely used erythromycin a peristaltic pump, and then the whole system turned into a analog antibiotic, erythromycin ethylsuccinate (EES, CAS gel-like state. Gelation also occurred in a 1-propanol–water No. 1264-62-6, Fig.  1) is studied for the first time. First, mixture at different volume ratios. the EES gel-like phase was characterized and the solvate Method 2: Cooling. 1.5 g EES powder was added into a nature of the fibers inside the phase was confirmed. Then, 4 g water–acetone mixture (1 mol/mol) and heated to 51 °C the EES gelation region was developed in the solvent system to make the powder dissolve completely. Then, the hot solu- of 1-propanol. Finally, Fourier transform infrared (FTIR) tion was kept at room temperature to form a gel-like phase. spectra, nuclear magnetic resonance (NMR) spectra, and Gelation also occurred in 1-propanol using a similar method. solvent parameters analyses were conducted to unravel the Method 3: A gel-like phase was also formed when a mod- gelation mechanism at a molecular level. The results of this erate amount of EES powder was added to certain solvents. study provide useful guidelines to understand the gelation In a typical gelation process, 0.37 g EES powder was added mechanism of other APIs. into a 4 g water–acetone mixture (1 mol/mol) at 20 °C under 300 r/min stirring. After 5 min, the solution became turbid and then a gel-like phase emerged. Gelation also occurred in Experimental 1-propanol when the amount of EES powder was changed. Materials Solvent Screening A white crystalline powder of EES (C H NO , MW The gelation of EES was investigated via method 1 in a 43 75 16 862.07) with a mass fraction purity of over 98% was pur- series of solvents including methanol, ethanol, 2-propanol, chased from Wuhan Yuancheng Gongchuang Science and 1, 2-propanediol, DMF, NMP, acetonitrile, formamide, Technology  Ltd, China. The organic solvents (acetone, cholamine, glycol, DMSO, triethanolamine, isopropyl ether, 1-propanol, etc., analytical reagent grade) were purchased and THF. Water was used as an anti-solvent. from Tianjin Jiangtian Chemical Technology Co., Ltd, China, and were used without further purification. Deionized Characterization water was purchased from Tianjin QingYuanquan Purified Micrographs were acquired by scanning electron microscopy (Hitachi TM3000 tabletop SEM, Japan) and polarized opti- cal microscopy (Olympus BX51, Japan). Powder X-ray Dif- fraction (PXRD) data were collected by a PXRD instrument (Rigaku D/MAX-2500, Japan) under the following conditions: 8°/min scan speed, 0.02° step size, 2°–50° scan range, and Cu Kα radiation (λ = 1.5406 Å). Rheological experiments were conducted on a rheometer (Anton Paar MCR 302, Austria) at 20 °C. Dynamic frequency sweep (DFS) was conducted in an angular frequency range of 100–0.1 rad/s with a strain amplitude of 0.05%, and dynamic strain sweep (DSS) was conducted in a strain range of 0.01–100% at 10 rad/s fre- quency. Thermogravimetric analysis (TGA) and differen- tial scanning calorimetry (DSC) were conducted on a TGA instrument (Mettler Toledo TGA/DSC 1/SF, Switzerland) and Fig. 1 EES molecular structure DSC instrument (Mettler Toledo DSC 1/500, Switzerland), 1 3 Gelation Mechanism of Erythromycin Ethylsuccinate During Crystallization respectively. 5–10 mg of samples was loaded in a crucible and The samples were measured thrice for each Erlenmeyer heated from 25 to 250 °C at 10 °C/min under N atmosphere. flask, and the average value was used to calculate the solute FTIR spectra were determined on a spectrometer (Bruker Ten- solubility. The temperature ranged from 10 to 50 °C over 5 −1 sor 27, Switzerland) with a resolution of 1 cm , and the solid equidistant points. powder was tableted with KBr and scanned in the range of −1 4000–400 cm . NMR spectra were determined on an NMR Gelation Points Measurement spectrometer (Bruker Avance III 400 MHz, Switzerland) in a D O and acetone-d6 mixture (1 mol/mol), which included C- Cooling Method An appropriate amount of EES powder NMR spectra and concentration variation H-NMR spectra, (m ) and 6 g of 1-propanol (m ) were added into a 50-mL 1 2 with a resolution of 0.45 Hz and sensitivity of 250:1. jacketed glass vessel within a thermostat bath. Under a 300 r/min stirring rate, the vessel was heated at 1 °C/min Measurement of the Phase Diagram to dissolve EES completely, and then, it was cooled at the same rate. The temperature was recorded when the solution Solubility Measurement became discernibly turbid. This was assigned as the gela- tion point of EES, after which the sample would completely Dynamic Method 4 g of 1-propanol (designated as m ) was turn into a gel-like phase. The amount of EES powder was poured into a 50-mL jacketed glass vessel, and a thermostat then changed, and the experiments were repeated to obtain bath was used to regulate the experimental temperature. At a series of gelation points at different concentrations. The a stirring rate of 300 r/min, a certain small amount of EES concentration of the solution was calculated by Eq. (1). powder was continuously added to the solvent to approach saturation. The last added amount was not more than 0.01 g. The total amount of EES powder consumed when the slightly Results and Discussion turbid solution did not turn transparent any more was recorded as m . Each measuring point was repeated thrice, and the solu- Gelation Phenomenon and Characterization bility of EES was calculated by Eq. (1): of the EES Gel‑like Phase x = , The EES gel-like phase obtained through three methods (1) showed a white opaque appearance and exhibited no gravi- tational flow under tube inversion test. When heated, the The temperature ranged from 10 to 50 °C, and 5 equidistant points were considered. gel-like phase was separated into solid and liquid phases and eventually turned into a transparent solution. When the Static Method The cloudiness of the slightly turbid solu- tion mentioned above increased with time, suggesting a phase solution was cooled, the gel-like state reappeared, as shown in Fig. 2. The transition temperatures depended on the solu- transition after the EES dissolution. Thus, the static method was used to acquire the solubility data and the equilibrium tion concentration and solvent composition. Long, thin, and flexible fibers were observed in the micro- solid phase was tested. The experiments were carried out in Erlenmeyer flasks, which were shaken in a thermostat shaking scope images of the gel-like phase, and their sizes varied with the preparation methods (Fig. S1–S3). The variation of bath at 300 r/min. Excessive EES powder and 15 g 1-pro- panol were added into the Erlenmeyer flasks, and samples the microscale morphology (Fig. 3) of the gel-like phase was tracked throughout the gelation process by a microscope. On were taken every 2 h. After 8 h, a liquid–solid equilibrium was reached, and then the shaking was stopped for 0.5 h for the initial stage, the emergence of a few single fibers caused the suspension to separate into two phases. A total sample of 5 mL was extracted with a syringe equipped with a 0.22-μm polyvinylidene fluoride filter. The sample was placed in an evaporating dish, and the weight of the dish (X ) and the total weight of the dish and solution (X ) were recorded. The liq- uid samples were evaporated in a vacuum oven at 50 °C and weighed every 12 h until a constant weight (X ) was achieved. The PXRD pattern of the wet solid phase was recorded. The solubility of the equilibrium solid phase was calculated by Eq. (2): X − X 1 0 x = , (2) X − X 2 1 Fig. 2 Reversible thermal transition of EES gel-like phase 1 3 X. Su et al. constant with the increase in strain from 0.01 to 0.1%, which is the linear viscoelastic region (LVR) of the gel-like phase. The low strain limit (0.1%) demonstrated the crystalline nature of the fibers [17]. The DFS was performed within the LVR, as shown in Fig. 4b. In the whole frequency range, G′ was larger than G″, and both were almost independent of frequency, meaning that the sample was unable to flow and remained in a solid-like phase regardless of the bearing force. These rheological properties are similar to those of typical gel substances [18]. Solvatomorphs and Gelation Mechanism of EES Analysis of PXRD Patterns Fig. 3 Microscope images of the EES gel-like phase. a Single fibers, b fiber clusters, c twisted and cross-linked clusters, and d 3D fibrillar network The morphology of the fibers was maintained when the EES gel-like phase was dried in air for 1 h, while the immobi- a slight turbidity of the solution. By keeping the temperature lized solution was completely dried out. The PXRD patterns and solvent content constant, more fibers emerged and gath- of EES fibers are shown in Fig.  5, where the series of dif- ered to form fiber clusters. The clusters twisted and cross- fraction peaks in the PXRD patterns indicate the crystalline linked with each other to generate a 3D network structure. nature of the fibers. The fibers formed in different solvents The mechanical properties of the gel-like phase were showed the same PXRD patterns, indicating identical pack- measured by dynamic rheology characterization. The DSS ing modes of the EES molecules in the crystal structure. curves of the EES gel-like phase are shown in Fig. 4a. Both The obtained Bragg lattice spacing values (d) of 1.99, 0.97, storage modulus (G′) and loss modulus (G″) remained 0.63, 0.55 nm closely followed the ratio of 1:1/2:1/3:1/4, suggesting the existence of an 1D lamellar structure in the nanofibers of the gel-like phase with a layer distance of 1.99 nm [19, 20]. Compared with the PXRD pattern of EES crystals, the PXRD patterns of the fibers showed two new peaks at 4.44° and 13.96°, and the peaks at 8.15°, 11.05°, 15.22°, 17.00°, and 20.68° disappeared, suggesting that the crystalline form of the fibers is different from that of the EES crystals and has not been reported. After drying the samples in a vacuum oven at 50 °C for 24 h, the PXRD patterns of the dried sam- ples (Fig. S4) were different from those of the fibers but Fig. 5 PXRD patterns of EES crystals (black); EES fibers in water– Fig. 4 Rheological curves of the gel-like phase. a DSS and b DFS acetone (red) and 1-propanol (blue) 1 3 Gelation Mechanism of Erythromycin Ethylsuccinate During Crystallization similar to that of the EES crystals. The SEM image of a obtained from the above two sections indicate that the fibers dried sample (Fig. S5) shows a similar morphology to that formed in different solvents were EES solvatomorphs, which of the EES crystals. The changes indicate that the fibers were discovered for the first time. The gelation of the EES transformed to EES crystals. in its solution was due to the formation and cross-linking of the fibrous solvates. Thermal Analysis Conditions for Gelation Figure 6 shows the thermal analysis of EES fibers. The TGA curve of the fibers formed in 1-propanol showed a weight Phase Diagram loss of 2.55 wt%, and the DSC curve had an endothermic peak at the corresponding temperature range, indicating that The solubility data in 1-propanol were measured by both the sample was 1-propanol solvate. For the fibers formed in static and dynamic methods, corresponding to the blue and the water–acetone mixture, a weight loss of 0.90 wt% and red curves in Fig. 7. The equilibrium solid phase in the static an endothermic peak appeared in TGA and DSC curves in method was proved to be EES 1-propanol solvate (Fig. S8), the same temperature range, respectively, proving that the demonstrating the solubility data (blue curve) correspond- sample was also a kind of EES solvate. ing to the solubility of EES 1-propanol solvate. At the same The headspace sampling gas chromatography (GC) analy- temperature, the solubility of the EES solvate is lower than sis verified the existence of acetone and 1-propanol in the that of the EES crystals. Thus, with the addition of the EES fibers formed in water–acetone and 1-propanol, respectively crystals into 1-propanol, the EES crystals transformed into (Figs. S6 and S7). The existence of water in the fibers formed a solvate when the concentration of the EES in solution in the water–acetone mixture was proved by volumetric Karl exceeded the solubility of the solvate. In a series of typical Fischer titration (see Supporting Information). The results experiments, solutions represented by points from L to J 1 1 were prepared. It showed that because few fibrous crystal- line solvates crystallized out under low supersaturation, the solution could not gel in the region between the blue and red curves, which can be regarded as the crystallization region. Gelation would inevitably occur when the concentration is above the red curve, which can be regarded as the gelation region. The lower the supersaturation in the gelation region, the more difficult and time-consuming it is for gelation to occur. Thus, it is indicated that a high degree of supersatura- tion promotes gelation. The gelation method 3 described in Experimental section is hence termed as “trans-crystal gela- tion,” and the solubility of the EES crystals can be regarded as the critical gelation concentration. The black curve in Fig. 7 corresponds to the gelation points of EES measured Fig. 6 DSC and TGA curves of EES fibers. a fibers in 1-propanol and b fibers in water–acetone Fig. 7 Phase diagram of EES in 1-propanol 1 3 X. Su et al. by cooling. Compared with the trans-crystal gelation region, shows the Δδ of the studied gelation solvents, and it ranges 1/2 the solution gels fast in the cooling gelation region, and the from 4.3 to 14 MPa , which is smaller than those of other critical concentration of cooling gelation is higher than that solvents, indicating that the gelation solvents have a better of trans-crystal gelation. dissolving capacity for EES. Combined with the analysis of solubility parameters, it can be concluded that a high solubil- Analysis of Solvent Parameters ity of EES in the solvents is necessary for gelation. A low solubility results in the separation of a small amount of fib- The influence of solvent properties on the EES gelation was ers from the solution, which makes it hard to trap quantities investigated by solvent screening. The solutions of EES in of solvent molecules and ultimately leads to crystallization. acetone, methanol, ethanol, 1-propanol, 2-propanol, DMF, and NMP gelled entirely with added water, while the solution Intermolecular Interaction Analysis in acetonitrile gelled only partially. As shown in Table 1, the solvents that facilitated gelation have something in common: FTIR a dielectric constant range of 18.3–37.5, δ (dipole interac- 1/2 tions) of 6.1–18 MP a in Hansen solubility parameters, and FTIR characterization was performed on the EES crystals π (polarizability parameter) of 0.48–0.88 in Kamlet–Taft −1 solubility parameters [21]. Outside these ranges, it is impos- and fibers. The peaks at 1741 and 1698 cm in Curve a of Fig.  8 represent the C=O stretching vibration of ester sible for gelation to occur in the corresponding solvents. The matching degree of solvent–solute intermiscibility groups and carbonyl of EES, respectively. For fibers, the −1 former peak shifted to 1736 cm , while the latter disap- can be described by the residual of solubility Δδ. A smaller value of Δδ means a better solubility of a solute in a solvent. peared, which could have been masked by the former. It was speculated that ester groups participated in the intermolecu- Δδ can be calculated from Eq. (3): lar hydrogen bonds as H acceptors, accepting H from the sol- 2 2 2 vent molecules to form the EES solvate fibers. Furthermore, (3) Δ = ( −  ) +  −  +  −  , d d p p h h 1 2 1 2 1 2 the red shift of C–O–C vibration peaks in the ester groups where δ and δ represent the dispersion interactions and and glucosidic bonds also imply the participation of these O d h hydrogen bond interactions, respectively, while subscripts 1 atoms in the supramolecular self-assembly of EES solvate and 2 correspond to EES and solvent, respectively. Table 1 fibers as H acceptors (Table S1). Table 1 Gelation behaviors of * No. Solvent Gelation Dielectric con- Hansen solubility Δδ π [21] EES in different solvents and 1/2 1/2 behavior stant [21] (25 °C)parameters (MPa ) (MPa ) solvent parameters analysis [21, 22] δ δ δ d p h 1 Acetone G 20.7 15.5 10.4 7 4.306 0.62 2 Methanol G 31.2 15.1 12.3 22.3 14.08 0.6 3 Ethanol G 25.7 15.8 8.8 19.4 10.61 0.54 4 Propanol G 22.2 16 6.8 17.4 8.693 0.52 5 2-Propanol G 18.3 15.8 6.1 16.4 7.782 0.48 6 DMF G 36.71 17.4 13.7 11.3 7.778 0.88 7 NMP G 32 18 12.3 7.2 7.077 8 Acetonitrile PG 37.5 15.3 18 6.1 11.15 0.66 9 Formamide P 111 17.2 26.2 19 21.49 0.97 10 Cholamine P 37.72 17.2 15.6 21.3 15.1 11 Glycol P 37.7 0.92 12 DMSO P 48.9 18.4 16.4 10.2 10.36 1 13 Triethanolamine P 29.36 14 Isopropyl ether P 4.49 15 Water P 7.58 16.8 5.7 8 4.2 0.55 16 THF P 78.5 15.6 16 42.3 34.26 1.09 17 EES 13.2 7.5 9.2 – G, PG, and P represent gelation, partial gelation, and precipitate, respectively 1 3 Gelation Mechanism of Erythromycin Ethylsuccinate During Crystallization network structure. Thus, the formation and growth of the EES-solvent supramolecular fibers is in accordance with the mechanism of crystallographic mismatch branching [25]. NMR Concentration variation H NMR spectra are shown in Fig. 10. With the increase in EES concentration, the sys- tem changed from a solution to gel-like state, which led to the weakening of peak intensity and peak splitting, suggesting that the mobility of molecules decreased, and protons could not produce individual sharp signals as they were in the aggregated state in the gel-like phase [26]. The Fig. 8 Solid FTIR absorption curves. a EES crystals, b fibers in water–acetone, and c fibers in 1-propanol H atoms of H O (δ of ~ 4.30 ppm) show downfield chemi- cal shifts with the increasing EES concentration, indicat- ing the participation of H O molecules in the EES–solvent −1 The wide peak of –OH stretching vibration at 3453 cm hydrogen bonds as H donors. of EES crystals indicates that the EES molecules interacted The C NMR spectra (see Fig. S10) were also recorded with each other through hydrogen bonds between hydroxyls. for the EES solution (1.875  mg/mL) and gel-like state For fibers, the –OH stretching vibration peak split into two (3.75 mg/mL). The chemical shift at nearly δ =211.5 ppm peaks, indicating the variation of intermolecular hydrogen (Fig. 11a) corresponds to the C atoms of carbonyl groups. bonds between the hydroxyl groups of the EES molecules As the amount of solvents was much larger, we assigned after fibers formation. Some of the EES–EES intermolecu- the high peak to acetone-d6 and the low peak to the car- lar hydrogen bonds between the hydroxyls were substituted bonyl group on the EES ring. The downfield shifts of these by the new EES–solvent intermolecular hydrogen bonds in peaks indicate the participation of carbonyl groups as H the supramolecular fibers [23, 24], as the small size makes acceptors in the EES–solvent intermolecular hydrogen the solvent molecules bond easily with the EES molecules. bonds. In addition, the peaks of –COOC– in ester groups Moreover, both the out-of-plane bending vibration peak of around 175  ppm (Fig.  11b) showed minute downfield –OH and the bending vibration peak of –OH on the clad- shifts (0.06–0.1 ppm), suggesting the involvement of ester inose ring exhibited a red shift in the fibers, demonstrat- groups in the EES–solvent intermolecular hydrogen bonds. ing the participation of these hydroxyls in intermolecular hydrogen bonds. Because of the presence of many intermolecular hydrogen bonds between the EES and solvent molecules, they assembled together to form 1D lamellar structural units, which assembled into 2D fibers quickly, and many branches were generated around the fibers, as shown in Fig.  9. Finally, the fibers intertwined and cross-linked with each other to construct a 3D self-assembled fibrillar Fig. 9 Images of fibrous crystals in 1-propanol. a optical microscope Fig. 10 Concentration variation H NMR spectra of EES in of crystal and b SEM image of fibers D O:acetone-d6 (v:v = 3:5) 1 3 X. Su et al. 5. Estroff LA, Hamilton AD (2004) Water gelation by small organic molecules. Chem Rev 104:1201–1218 6. Dastidar P (2008) Supramolecular gelling agents: can they be designed? Chem Soc Rev 37:2699–2715 7. Song J, Sun HJ, Sun SJ et al (2013) Synthesis and gel properties of sorbitol derivative gelators. Trans Tianjin Univ 19:319–325 8. 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Anderson VJ, Lekkerkerker HNW (2002) Insights into phase tran- and gelation. Solvent parameters analysis indicated that sol- sition kinetics from colloid science. Nature 416:811–815 16. Vintiloiu A, Leroux JC (2008) Organogels and their use in drug vent polarity was also a key factor for gelation. The results delivery—a review. J Controlled Release 125:179–192 showed that gelation can be avoided by controlling the 17. Terech P, Pasquier D, Bordas V et al (2000) Rheological proper- supersaturation and selecting appropriate solvents. FTIR and ties and structural correlations in molecular organogels. Langmuir NMR analyses suggested that the dominant driving force for 16:4485–4494 18. Wang YJ, Tang LM, Yu J (2008) Investigation of spontaneous the formation of EES solvate fibers was the intermolecular transition from low-molecular-weight hydrogel into macroscopic hydrogen bonds between EES and solvent molecules. The crystals. Cryst Growth Des 8:884–889 crystalline fibers intertwined with each other to construct a 19. Mallia VA, Terech P, Weiss RG (2011) Correlations of properties 3D fibrillar network, immobilizing solvent molecules and and structures at different length scales of hydro- and organo-gels based on N-alkyl-(R)-12-hydroxyoctadecylammonium chlorides. leading to the formation of a gel-like phase. J Phys Chem B 115:12401–12414 20. Yu Y, Wang S, Jia L et al (2016) Organogels from different self- Acknowledgements This study was supported by the National Natural assembling novel l -proline dihydrazide derivatives: gelation Science Foundation of China (Nos. 21576187, 21776203). mechanism and morphology investigations. J Sol-Gel Sci Technol 78:218–227 Open Access This article is distributed under the terms of the Crea- 21. Reichardt C, Welton T (2011) Solvents and solvent effects in tive Commons Attribution 4.0 International License (http://creat iveco organic chemistry. Wiley-VCH Verlag, Weinheim, Germany mmons.or g/licenses/b y/4.0/), which permits unrestricted use, distribu- 22. Li X (2016) A study on the thermodynamic and kinetic character- tion, and reproduction in any medium, provided you give appropriate istics of oiling-out crystallization process of erythromycin ethyl- credit to the original author(s) and the source, provide a link to the succinate. Dissertation, Tianjin University, China (in Chinese) Creative Commons license, and indicate if changes were made. 23. Iwasaki H, Sugawara Y, Adachi T et  al (1993) Structure of 6-O-methylerythromycin A (clarithromycin). Acta Crystallogr Sect C 49:1227–1230 24. Brüning J, Trepte TK, Bats JW et  al (2012) Erythromycin A References dimethyl sulfoxide disolvate 1.43-hydrate. Acta Crystallogr Sect E 68:700–701 1. Saboktakin MR, Tabatabaei RM (2015) Supramolecular hydrogels 25. Liu XY (2005) Gelation with small molecules: from formation as drug delivery systems. 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Transactions of Tianjin UniversitySpringer Journals

Published: May 28, 2018

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