Novel chiral metal organic frameworks functionalized composites for facile preparation of optically pure propranolol hydrochlorides

Journal of Pharmaceutical and Biomedical Analysis 172 (2019) 50–57

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Novel chiral metal organic frameworks functionalized composites for facile preparation of optically pure propranolol hydrochlorides Xue Ma a,1 , Wenrui Du a,1 , Yan Li b , Chenfeng Hua c , Ajuan Yu a,∗ , Wuduo Zhao b , Shusheng Zhang b , Fuwei Xie c a College of Chemistry and Molecular Engineering, Key Laboratory of Molecular Sensing and Harmful Substances Detection Technology, Zhengzhou University, Kexue Avenue 100, Zhengzhou, Henan, 450001, PR China b Center of Advanced Analysis and Computational Science, Key Laboratory of Molecular Sensing and Harmful Substances Detection Technology, Zhengzhou University, Kexue Avenue 100, Zhengzhou, Henan, 450001, PR China c Zhengzhou Tobacco Research Institute of CNTC, Fengyang Street 2, Zhengzhou, Henan, 450001, PR China

a r t i c l e

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Article history: Received 20 December 2018 Received in revised form 14 April 2019 Accepted 15 April 2019 Available online 16 April 2019 Keywords: Enantioselective preparation Chiral propranolol hydrochloride (PRO) Metal-organic frameworks Dispersive magnetic nanoparticle solid phase extraction (d-MNSPE) Cytotoxicity

a b s t r a c t Two novel sorbents based on polydopamine (PDA)-coated magnetic graphite oxide-metal organic frameworks nanoparticles [Cu(L-mal)(bpy)]·H2 O (MGO-CuLBH) and [Cu(D-mal)(bpy)]·H2 O (MGO-CuDBH) possessing of both magnetic property and excellent enantioselective ability were prepared and characterized. Solutions of racemic propranolol hydrochloride (Rac-PRO) were chosen to investigate the enantioselective performance of MGO-CuLBH and MGO-CuDBH by dispersive magnetic nanoparticle solid phase extraction (d-MNSPE). The results showed that the nanocomposites have excellent enantioselectivity to PROs with enantiomeric excess (ee) values reaching up to 98%. The entire process with PROs by the d-MNSPE method was fast, convenient and the collected composites could be easily recycled. Multi-stage operations using MGO-CuLBH and MGO-CuDBH were scaled up to obtain milligram quantities of R-propranolol hydrochloride (R-PRO) and S-propranolol hydrochloride (S-PRO). Furthermore, on the basis of the successful preparations, the differences in the cytotoxicity of Rac-PRO, R-PRO and S-PRO on A549 cells in vitro were all evaluated. © 2019 Published by Elsevier B.V.

1. Introduction Chirality has long attracted attention in the drug industry because of great differences in pharmacologic, toxicologic and metabolic properties of the enantiomers [1]. In many situation, opposite enantiomers present unwanted or even toxic side effects [2,3]. The US Food and Drug Administration (FDA) claims that the pharmacologic properties of the single isomers should be clear during the development and data submission of new chiral drugs. Consequently, the enantioseparation has become a growing hotspot for researchers as well as in preparation of optical drugs. These approaches include supercritical fluid chromatography, membrane-based separation, dynamic kinetic resolution and crystallization [4,5] et al., preferential chromatography. Chromatography-based methods, particularly chiral stationary phases, have become an indispensable part of drug preparation.

∗ Corresponding author. E-mail address: [email protected] (A. Yu). 1 These authors contributed equally and should be considered as co-first authors. https://doi.org/10.1016/j.jpba.2019.04.034 0731-7085/© 2019 Published by Elsevier B.V.

However, expensive apparatus, large solvent and time consuming should be required. Therefore, it is an urgent need to develop new composites with superior enantioselective abilities. Metal-organic frameworks (MOFs), sometimes known as porous sorbent materials, are consisting of metal ions or clusters covalently joined by organic links [6]. In particular, chiral MOFs have great potential for enantioseparation, because of their controllable synthesis, flexibility of the size selectivity, large surface areas, properties which surpass traditional inorganic and organic porous materials [7,8]. The separation of enantiomers by chiral MOFs can be ascribed to the reversible affinity between chiral active sites and optical isomers. In several recent research, it has been reported that enantioseparation of racemic alcohols and sulphoxides with chiral MOFs via chromatography [9], crystallization [10] and membranes [11], but only few showed efficient chiral separation. The chiral MOFs that have been tested represent just the tip of the iceberg and the untested chiral MOFs are proposed for further investigation. As a two-dimensional sheet of carbon network, graphene oxide (GO) is among the most suitable materials for synthesis of MOF composites [12,13]. It is of interest to mix the porous chiral MOF as chiral selectors with graphene nanosheets to capture enantiomers

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efficiently and achieve drug enantioseparation. MOF functionalized composite, evolved from encapsulating GO nanoparticle into MOF, can integrate the advantages of the two constituents and endow the composite with an improved synergystic effect. It is expected that GO can improve chiral MOF formation by increasing the dispersive forces within the MOF, suppressing its aggregation, and controlling the physicochemical properties of MOF such as structure, morphology and size [14,15]. Only with a suitably homochiral environment, one enantiomer can be allowed to pass through, while the other is prevented. While, due to their ultrahigh surface area and the intrinsic topology properties, chiral MOF would significantly ameliorate the dispersibility of graphene in solution. Hauser et al. [16] proposed a new computational simulations of different enantioseparations based on density functional theory using functionalized nanoporous graphene. Recently, our group [17] reported a novel magnetic MOF nanocomposite MGO-ZnCB as a adsorbing material for fast enantioseparation of chiral 1, 1’-bi-2-naphthol and 2, 2’-furoin. This material gave a good enantioselective performance in separation of 1, 1’-bi-2-naphthol and 2, 2’-furoin with enantiomeric excess (ee) values of 74.8% and 57.4%, respectively. To date however, achieving entire separation of the two drug enantiomers such as those of propranolol hydrochloride (PRO) based on MGOMOF by dispersive magnetic nanoparticle solid phase extraction (d-MNSPE) remains a great challenge. However, due to the lack of readily available pure PRO enantiomers, little is known however about the influence of the stereochemical configuration of PRO on their apoptosis-based anticancer activity, for instance, non-small cell lung cancer (NSCLC) [18,19]. Herein, we have prepared and characterized two novel polydopamine (PDA)-coated magnetic graphite oxide-homochiral metal-organic frameworks [Cu(L-mal)(bpy)]·H2 O (MGO-CuLBH) and [Cu(D-mal)(bpy)]·H2 O (MGO-CuDBH) and applied these two nanocomposites as sorbents to the enantioselective capture of PRO enantiomers (Fig. 1). These new composites combine the features of Fe3 O4 , PDA, GO and MOFs, and have demonstrated a number of advantages such as excellent dispersive capabilities, good chiral recognition abilities and rapid magnetic separating performances. A strategy of dispersive magnetic nanoparticle solid phase extraction (d-MNSPE) with rapid extraction and elution processes was optimized for solutions of racemic PRO (Rac-PRO). Both MGOCuLBH and MGO-CuDBH with d-MNSPE procedure exhibited good performance in the enantioselective capture of PROs and the ee values reached up to 98%. The enantioseparation for PROs can be finished in 8 min. After washing with isopropanol, the collected composites are easy recycling and can be reutilized at least six times with no significant performance loss. Multi-stage operations using MGO-CuLBH and MGO-CuDBH were scaled up to obtain milligram quantities of R-propranolol hydrochloride (R-PRO) and S-propranolol hydrochloride (S-PRO). Furthermore, on the basis of the successful preparations, A549 cells, a model cell was chosen to investigate the differences among the cytotoxicity of Rac-PRO and its optically pure enantiomers R-PRO and S-PRO for the first time using a cell counting Kit-8 (CCK-8) assay (Fig. 1). The apoptotic nature of cell death was characterized by flow cytometric analysis with Annexin V-FITC/PI staining and the changes in nuclear morphology with DAPI staining by laser scanning confocal microscopy.

2. Materials and methods 2.1. Chemicals and materials Graphite powder, potassium permanganate, ferric chloride hexahydrate, sodium acetate, poly(ethylene glycol) were purchased from Alfa Aesar (Shanghai, P.R. China). Dopamine hydrochloride, tris(hydroxymethyl)aminomethane, cupric acetate monohydrate

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Silver nitrate, dichlorofluorescein and dextrin were supplied by Sigma-Aldrich (St. Louis, MO, USA). Propranolol hydrochloride, L-()-malic acid, D-(+)-malic acid and 4,4’-bipyridyl were purchased from Energy Chemical (Shanghai, P.R. China). R-PRO and S-PRO standards were purchased from Shanghai Aladdin Bio-Chem Technology Co., LTD. N-hexane, isopropanol, methanol, acetonitrile as chromatographic reagents were purchased from Fisher Scientific (Fair Lawn, NJ, USA). Acetone, ethyl acetate, n-butyl alcohol, Isobutyl alcohol, Isopropyl alcohol, 2-butanol, dichloromethane as analytical grade were purchased from Kermel (Tianjin, P.R. China). Purified water was produced using the Milli-Q system (Millipore, Bedford, MA, USA). RPMI 1640 cell culture medium and fetal bovine serum (FBS) were purchased from Invitrogen (Gibco). The following biological reagents were used: CCK-8 detection kit (Dojindo laboratories, Japan); Annexin V-FITC/PI Apoptosis Detection Kit (Beyotime Institute of Biotechnology (Hangzhou, P.R. China); 4’, 6diamidine-2’-phenylindole dihydrochloride (DAPI, Beijing Leagene Biotech. Co., ltd). 2.2. Instrumentations Scanning electron microscope (SEM) was carried out on an S4300 instrument and Transmission electron microscopic (TEM) characterization was measured on a Tecnai G2 F20 microscope. A JSM-7001 F instrument was applied to characterize the energy dispersive X-ray (EDS) analysis of the nanocomposites. Infrared absorption spectra were recorded on a Nicolet 6700 infrared Fourier transform spectrometer. An X-ray diffractometer was applied to characterize the wide-angle X-ray diffraction (XRD) patterns of prepared materials. A STA 409 PC thermogravimetric analysis (TGA) was used to evaluate the thermal stability of the composites. An Autosorb-1 MP gas adsorption instrument was chosen to determine nitrogen adsorption isotherms of materials. Magnetic properties were analyzed using a Lake Shore VSM 7307 magnetometer operating at a maximum magnetic field of 15 kOe at room temperature. Chromatographic analysis was performed on an Agilent 1200 series system. Melting points were confirmed by a Yuhua X-5 microscopic apparatus and were uncorrected. Optical rotation was determined using a Perkin Elmer 341 spectropolarimeter. High performance liquid chromatography (HPLC) analysis was carried out with an Agilent HPLC 1200 System. The chiral column, Chiralpak IC was purchased from Daicei Chemical Industries, LTD. 2.3. Preparation of Fe3 O4 @PDA-GO@CuLBH (MGO-CuLBH) and Fe3 O4 @PDA-GO@CuDBH (MGO-CuDBH) composites (Fig. S1) Fe3 O4 @PDA. Monodisperse Fe3 O4 microspheres were synthesized by a solvothermal method [20]. Fe3 O4 @PDA was prepared at room temperature. First, 40 mg of dopamine hydrochloride was added to 40 mL of tris buffer (10 mmol L−1 ) and sonicated for 5 min. Then 10 mg of the as-prepared Fe3 O4 was added and sonicated for 30 min. The admixture was mechanically mixed at indoor temperature for 24 h. Finally, the product was separated and gathered with a magnet, cleaned by ultrapure water, ethanol, and desiccated in a vacuum oven. MGO-CuLBH and MGO-CuDBH. CuLBH was obtained by heating a mixture of Cu(OAc)2 ·H2 O, L-malic acid and 4, 4’-bipyridyl in a molar ratio of 1:2:1 in a water-methanol mixture (1/1, v/v) at 100 ◦ C for 24 h according to the method of Zavakhina et al. [21] The MGOCuLBH and MGO-CuDBH composites were synthesized by one-step method. Carboxylic GO [22] was dispersed in 15 mL of a watermethanol solution (1/1, v/v) under ultrasonication for 30 min. Then 0.1 g Fe3 O4 @PDA was added. After sonication for 30 min, a water-methanol (1/1, v/v) solution (5 mL) of Cu(OAc)2 ·H2 O (0.3 g), 4, 4’-bipyridyl (0.23 g) and L-malic acid (0.42 g) or D-malic acid (0.42 g) were added and sonicated. The resulting solution was son-

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Fig. 1. Schematic diagram of the enantioselective capture process for S-PRO or R-PRO using MGO-CuLBH (or MGO-CuDBH) and an in vitro study of cytotoxicity in A549 cells.

icated for 30 min, and then warmed in an oil-bath at 100 ◦ C for 24 h with mechanically stirred. The percentage of GO was 10% in the final material. After refrigerated to indoor temperature, the outcome was magnetically separated, cleaned by ultrapure water, ethanol, and desiccated in a vacuum oven to obtain corresponding MGO-CuLBH or MGO-CuDBH composites.

procedure for R-PRO using MGO-CuDBH composite was similar to those for the aforementioned S-PRO.

2.5. Analysis of cell viability and apoptosis 2.4. Typical separation procedure for S-PRO (or R-PRO) using MGO-CuLBH (or MGO-CuDBH) composite MGO-CuLBH composite was applied to the enantioseparation of PRO enantiomers. In a single-stage operation, 5 mL of Rac-PRO ethanol solution (0.04 mg mL−1 ) and four various composites (10 mg), inclusive of CuLBH, GO@CuLBH, Fe3 O4 @GO@CuLBH, MGO-CuLBH, were mixed in separate vials and each was sonicated for 5 min. Fe3 O4 @GO@CuLBH-enantiomer and MGO-CuLBHenantiomer complexes were gathered using a magneticiron and the clear liquor was removed. The other enantiomer-complexes were gathered by centrifugation. The gathered enantiomer-complexes were then mixed with 2 × 500 ␮L of isopropanol, rocked and sonicated at indoor temperature for 3 min to obtain the absorbed optical isomers of PRO. The eluents were vaporized under a stream of nitrogen. In the end, the enantiomeric composition of the separated enantiomers were dissolved again in 500 ␮L mobile phase and measured by HPLC with a Chiralpak IC column (4.6 × 150 mm, 5 ␮m), using hexane-isopropanol-triethylamine (83/17/0.2, v/v/v) as the mobile phase with a flow rate of 0.5 mL min−1 at 25 ◦ C. The detection wavelength used for PRO was 254 nm. The ee values were calculated by comparing the enantiomeric ratios. The recovery of R-PRO or S-PRO was determined by the ratio of the peak area of the fraction collected versus the peak area of the corresponding one in the loading buffer. All tests were performed in triplicate. In the multi-stage operation, the MGO-CuLBH composite was separated and recycled, and the desorption solution obtained was preserved and feeded as a loading buffer in the next step. The other procedures are similar to the above. The magnetic separation and enrichment

The human lung adenocarcinoma Cell line (A549) was used in this study. The cells were grown in RPMI-1640 cell medium supplemented with 10% FBS in a humidified atmosphere containing 5% CO2 at 37 ◦ C. All experiments were performed on cells in the logarithmic phase of growth. The Rac-PRO, R-PRO and S-PRO solutions (equal-volumes) with the different concentrations were added to each well of a 96 well microplate containing equivoluminal cells in the culture medium at the same density, and the cells were further incubated in a CO2 incubator at 37 ◦ C. After 24 h, Cell viability was measured using the method based on a WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4nitrophenyl)-5-(2, 4-disulfophenyl)-2H-tetrazolium, monosodium salt) assay with a CCK-8 detection kit according to the manufacturer’s instructions. The cells without treatment was used as a control. Absorbance was estimated by microtitre reader at 450 nm (Molecular Devices Corporation, USA). Cellular apoptosis was determined using the Annexin V-FITC/PI Apoptosis Detection Kit. The cells were treated with 30, 40 or 50 ␮g mL−1 of Rac-PRO, R-PRO and S-PRO, respectively. After 24 h of incubation, the cells were harvested and stained with Annexin V-FITC and PI according to the manufacturer’s instructions and further analyzed by flow cytometry (BD Biosciences, USA). Annexin V-positive, PI-negative cells were scored as apoptotic. Doublestained cells were considered as necrotic/late apoptotic cells. The cells without treatment was used as a control. Besides, the morphology of the cells treated described as above was analyzed by a DAPI staining assay and observed using laser scanning confocal microscopy (Leica TCS SP8, Germany).

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Fig. 2. TEM (a) and SEM (b) images of MGO-CuLBH composite.

2.6. Statistical analysis An unpaired Student’s test was used for between two-group comparison and oneway ANOVA with Fisher’s LSD for multiplegroup analysis. A probability value, P < 0.05 was considered statistically significant. Results were expressed as mean ± standard deviation (SD) unless otherwise indicated. 3. Results and discussion 3.1. Characterization of the MGO-CuLBH Composite The morphology of synthetic nano- material was characterized by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). As showed in Fig. 2a, the core-shell structure was found for Fe3 O4 @PDA magnetic spheres and the PDA shell was approximately 40 nm. The TEM image of MGO-CuLBH displayed that the edges of GO sheets had wrinkled and could spread out over an extent large enough with no aggregation, thus retaining the ultrahigh surface area of GO. Although the distribution of Fe3 O4 @PDA and GO in CuLBH was not well-dispersed (Fig. 2b), the resulting composites clearly indicated the embedded structures with Fe3 O4 @PDA and GO interspersed in CuLBH. For comparison, the SEM and TEM images of Fe3 O4 , Fe3 O4 @PDA and CuLBH were also provided (Fig. S2). In order to validate the successful formation by PDA, GO and CuLBH, infrared adsorption spectra were measured for Fe3 O4 @PDA, CuLBH and MGO-CuLBH (Fig. 3a). In the spectrum of Fe3 O4 @PDA, the absorption bands at 585 cm−1 and 3383 cm−1 were assigned to the vibration of Fe O Fe and the stretching vibration of O H on the surface. The peak at 1607 cm-1 was attributed to the bending vibration of N H groups. Absorption peaks of 1408 cm−1 and 1290 cm-1 were ascribed to the bending vibration from CH2 and the asymmetric stretching vibration of C O bond. In the spectrum of CuLBH, the broad peak around 3345 cm−1 was associated with the O H asymmetric stretching vibration and the absorption bands at 2964 cm-1 were ascribed to the C H asymmetric stretching of Lmalic acid. The peaks at 1600 cm−1 and 1360 cm-1 were attributed to the C O asymmetric stretching vibration, respectively. The peak at 808 cm−1 was the characteristic bands corresponding to 4, 4’bipyridyl ligands. GO has a characteristic absorption peak of O H at 3400 cm−1 . The spectrum of MGO-CuLBH had not only the characteristic absorption peak of Fe3 O4 @PDA and GO, but also the characteristic absorption peak of CuLBH. Therefore, it could be suggested that the magnetic composite was successfully prepared. Besides, the energy dispersive X-ray (EDS) analysis (Fig. 3b) of the composite verified the existence of Fe, N and Cu.

In this work, CuLBH was synthesized successfully by a modified procedure, which a rundkolben in an oil-bath with magnetic stirring was needed. Only by this means, relatively uniform composites composed of magnetic particles and homochiral MOFs were easy to form. Powder X-ray diffraction (PXRD) (Fig. S3) showed that the skeleton structure and coordination numbers of the MOF prepared in normal pressure were consistent with that synthesized at high pressure. Based on the successful synthesis of CuLBH, a chiral MOF functionalized MGO-CuLBH was further prepared. PXRD patterns of the MGO-CuLBH, CuLBH and Fe3 O4 @PDA were shown in Fig. 4a. Most diffraction peaks appeared in PXRD pattern with 2␪ from 5◦ to 70◦ for MGO-CuLBH were assigned to CuLBH. The diffraction peaks of the composite with 2␪ at 30.1◦ , 35.5◦ , 43.2◦ , 56.7◦ and 62.7◦ were marked by red asterisks, corresponding to Fe3 O4 @PDA in the nanocomposite. On account of the low concentration of Fe3 O4 @PDA, the intension of Fe3 O4 @PDA was weak. In order to estimate the thermal stability of the nanoparticles, the thermogravimetric analysis was conducted. As shown in Fig. S4, the significant loss of mass of the composite occurred above 200 ◦ C owing to collapse of the framework, indicating good thermal stability below 200 ◦ C. Fig. 4b displays the N2 adsorption-desorption isotherm of MGOCuLBH. The BET surface area is 205.90 m2 g−1 , while the average pore diameter is 3.7 nm, numbers which are satisfactory for enrichment purposes. The magnetic property was measured at indoor temperature as shown in Fig. S5. The low saturation magnetization value (6.1 emu g−1 ) could be ascribed to the incorporation of nonmagnetic components. Nevertheless, it is enough for the composite to achieve fast magnetic separation. 3.2. Enantioselective capture of S-propranolol hydrochloride (S-PRO) Using MGO-CuLBH Composite Propranolol hydrochloride (PRO), a non-cardioselective ␤adrenergic antagonist, is universally prescribed for the therapy of angina and hypertension. It possesses two enantiomers of RPRO and S-PRO, distributed in a racemic mixture. The two PRO enantiomers have been discovered at one time to possess various pharmacodynamics and pharmacokinetics in some situations [23]. For instance, a racemic mixture of PRO has caused side effects such as broncho-constriction in asthmatic patients, while diabetes in hypertensive patients. The side effects are ascribed to the RPRO [24]. On this account, several different strategies have been put forward to get R-PRO and S-PRO in optical pure forms [25,26]. Professor Jiang [27] presented a semipreparative approach of PRO enantiomers on the cellulose tris(3, 5-dimethylphenylcarbamate) coated chiral stationary phase. Professor Yuan [28] reported that

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Fig. 3. (a) FTIR spectra of Fe3 O4 @PDA, MGO-CuLBH, CuLBH. (b) EDS result from MGO-CuLBH.

Fig. 4. (a) PXRD patterns of Fe3 O4 @PDA, CuLBH, MGO-CuLBH, MGO-CuLBH after six uses; (b) N2 adsorption/desorption isotherm curves of MGO-CuLBH (the inset represents the pore size distribution).

homochiral MOFs act as chiral stationary phases for enantioseparation of PRO. Although the study in the chrial separation of PRO have been developed, several defects such as expensive equipment, large solvent consumption and long process are the disadvantages involved with these reported procedures. Here, solution of racemic PRO was applied to assess the enantioselective performance of MGO-CuLBH by dispersive magnetic nanoparticle solid phase extraction (d-MNSPE). In a typital procedure (Fig. 1), the MGO-CuLBH nanoparticles were incubated with racemic PRO solution, and the MGO-CuLBH-enantiomer complexes were gathered by a magneticiron. Enantiomers retained by MGOCuLBH were cleaned and the eluents were measured by HPLC. The result suggested that the stereoselectivity of S-PRO probably resulted in the most suitable steric hindrance and pore size of MGO-CuLBH. The operation arguments of d-MNSPE strategy was systematically optimized, such as the dosage of MGO-CuLBH, extraction and elution conditions (Fig. 5a–f). It was found that diverse extraction and elution solvents have a remarkable impact on ee values. When ethanol as the extracting solvent and isopropanol as eluting solvent, the ee values was much higher than the other cases. The influence of the solvents on chiral separation may occur as a result of the distinct interactions among S-PRO, sorbent and extraction solvent. In the ethanol solvent system, the ␲-␲ and strong hydrogen bonding interactions between the sorbent and S-PRO could be crucial, however, the separation mechanism is complex. As shown in Fig. 5c, the ee value remained almost constant as the amount of MGO-CuLBH increased from 10 mg to 60 mg. Com-

pared with other levels, 10 mg of MGO-CuLBH was enough to obtain a satisfactory result. Then, 10 mg was the optimum dosage of the sorbent. As shown in Fig. 5d, the ee values increased during the first 5 min of extraction time and the first 3 min of elution time. But after that, there was no significant difference, which demonstrated the fast adsorbing-desorbing equilibrium between the composite and PRO enantiomers. Thus, ethanol (extracting solvent, 5 mL), isopropanol (eluting solvent, 1 mL), extracting time (5 min) and elution time (3 min) were chosen as the optimized conditions. Hence, in virtue of the magnetic separation [29,30], the enantioseparation for PROs can be finished in 8 min with ee value reaching up to 98% (Fig. 5e), and was much more efficient than the traditional approaches which consumed too much time. The reusable performance of MGO-CuLBH was also studied and the results were shown in Fig. 5f. After cleaned by isopropanol and reemployed 6 times, the ee value of PRO is 85%, indicating good reusability. However, the recovery of S-PRO was low (50%) after treated by the d-MNSPE procedure, probably due to the inhomogeneity of MGO-CuLBH. 3.3. Comparison of enantioselective performance with other sorbents A comparison of the enantioselective performance among CuLBH, GO@CuLBH, Fe3 O4 @GO@CuLBH, MGO-CuLBH was performed to further verify the excellent enantioseparation efficiency of the composite. Solutions of Rac-PRO were carried out under the optimized d-MNSPE procedure as discussed before. After eluted

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Fig. 5. “Enantioselective capture” process for S-propranolol hydrochloride (S-PRO) using MGO-CuLBH. (a) Optimization of the extracting solvent; (b) Optimization of the eluting solvent; (c) Optimization of the amount of MGO-CuLBH; (d) Optimization of the extracting time and elution time; (e) Optimization results for the propranolol hydrochloride (PRO). Condition: Chiralpak IC column (4.6 × 150 mm, 5 ␮m); mobile phase, hexane/isopropanol/TEA (83/17/0.2, v/v/v); flow rate, 0.5 mL min−1 ; detection wavelength, 254 nm; column temperature, 25 ◦ C; (f) Recycling use of MGO-CuLBH composites.

from these four sorbents, 20 ␮L of eluents was measured by HPLC. The data (Fig. S6) showed that the current MGO-CuLBH based method had much better performance compared to the other three sorbents in terms of ee values. The MGO-CuLBH exhibits excellent chiral recognition toward S-PRO, which may be due to diverse interactions (␲-␲, hydrogen-bonding interactions, et al.) and the chiral surrounding of the pores. The d-MNSPE strategy coupled with HPLC provides an alternative approach for a simple and fast separation and preparation of PRO enantiomers. 3.4. Enantioselective capture of R-PRO using Fe3 O4 @PDA-GO@CuDBH (MGO-CuDBH) composite In a similar way, another homochiral MOF functionalized composite, named MCO-CuDBH, was synthesized with D-malic acid as

chiral ligands. It was been characterized in detail (Fig. S7). MGOCuDBH composite was expected to selectively adsorb R-PRO from solution of Rac-PRO. The experimental method and condition were identical as mentioned above. The ee value of the resulting desorption solution was also measured by HPLC. MGO-CuDBH composite exhibited excellent chiral selectivity for R-PRO over S-PRO with a ee value of 98% (Fig. S8), owing to the introduction of D-malic acid. The results proved that efficient and easy enantioseparation of PRO enantiomers can be realized by applying MGO-CuLBH and MGO-CuDBH composites. As a further experiment for the preparation of S-PRO and R-PRO, a small-batch resolution of racemic PRO was performed in a similar procedure using 500 mg MGO-CuLBH, but yielded the product with a relatively lower ee value of 78% probably due to the material inhomogeneity (for the HPLC spectrum see Fig. S9). To address this

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Fig. 6. (a) The cell apoptosis quantified by the flow cytometry at 24 h after different treatments. Bars of apoptosis rate, *p < 0.05, **p < 0.01, ***p < 0.001, compared with control group. (b) The confocal images of A549 cells at 24 h after various treatments with different concentrations of Rac-PRO, R-PRO and S-PRO, respectively. Scale bar: 15 ␮m.

problem, a multi-stage adsorption/desorption operation was conducted to improve the optical purity. At each stage, MGO-CuLBH composite was separated and recycled, and the desorption solution obtained was preserved and feeded as a loading buffer in the next step. Under the same operational conditions, the ee values of the product gradually increased and after four cycles, S-PRO was finally obtained with a high ee value of 98% (Fig. S8) and a similar recovery to R-PRO. Small quantities (10 mg, 98%) of both PRO enantiomers were prepared using MGO-CuLBH and MGO-CuDBH composites in a short time. Characterization for the preparative enantiomers of R-PRO and S-PRO was shown in Fig. S10 and the results were summarized in Table S1. The presence of chloride was confirmed for PRO enantiomers by the energy dispersive X-ray (EDS) analysis, chloride precipitation titration test and Infrared absorption spectra (FTIR) analysis. Obviously, compared to the standard FTIR spectra of propranolol (Fig. S10k), NH2 + stretching vibration of R-propranolol hydrochloride (Fig. S10i) or S-propranolol hydrochloride (Fig. S10j) was observed at 2700–2200 cm−1 . Isolated PRO enantiomers from the enantioselective separation of Rac-PRO were positively identified to be HCl salts of propranolol44 . And the purity of the products was shown to be adequate for cytotoxic studies and pharmacological analysis at the laboratory level.

3.5. Cytotoxicity of racemic propranolol hydrochloride (Rac-PRO), R-PRO and S-PRO on A549 cells

parison with S-PRO and Rac-PRO in both assays mentioned above (Figs. 6 and S12). 4. Conclusion In this work, two novel MOFs functionalized MGO-CuLBH and MGO-CuDBH sorbents were prepared by a simple approach. They can be applied in enantioseparation of PRO enantiomers. The nanocomposites have several advantages including good chiral recognition abilities, excellent dispersing capabilities and rapid magnetic separation performances. The developed d-MNSPE method presented excellent enantioselectivity to PROs with ee values reaching up to 98%. Taking advantage of the magnetic separation, the d-MNSPE procedure was fast and convenient. In spite of being reused six times, the chiral selection performance and stability of the sorbents remained comparatively steady. Based on their reusability, multi-stage operations using MGO-CuLBH and MGOCuDBH were scaled up to obtain milligram quantities of R-PRO and S-PRO. Based on the successful preparations, extensive studies on the cytotoxicity demonstrated that R-PRO and S-PRO present a significant anti-proliferation effect on A549 cells in comparison with Rac-PRO, and R-PRO displayed more effective inhibition than SPRO. It was found that the total apoptosis levels of A549 cells in R-PRO induced was markedly high as compared with S-PRO and Rac-PRO treated. Novelty statement

To characterize the cytotoxic effects induced by the Rac-PRO, R-PRO or S-PRO, cell survival and apoptosis assays were analyzed. When treated with Rac-PRO, R-PRO or S-PRO, the proliferation of cells was significantly inhibited in a dose-dependent manner compared to the control. In particular, both R-PRO and S-PRO showed a remarkably suppressive effect on the growth of cell while the R-PRO displayed more effective inhibition than S-PRO (Fig. S11). At the meantime, apoptosis induction by Rac-PRO, R-PRO or S-PRO on A549 cells can be detected by both flow cytometry and nuclear morphology after staining with DAPI. In the flow cytometry analysis, the apoptosis ration of A549 cells was larger than that in control after treatment with a different concentration of Rac-PRO, R-PRO or S-PRO for 24 h. Moreover, the rate of cell apoptosis induced by RPRO or S-PRO is higher than that by Rac-PRO (Figs. 6a and S12). The results were in accordance with the results from nuclear morphology after staining with DAPI (Fig. 6b). It was worth mentioning that R-PRO showed stronger induced influence to A549 cells in com-

Two novel sorbents based on polydopamine (PDA)-coated magnetic graphite oxide-metal organic frameworks nanoparticles [Cu(L-mal)(bpy)]·H2 O (MGO-CuLBH) and [Cu(D-mal)(bpy)]·H2 O (MGO-CuDBH) possessing of both magnetic property and excellent enantioselective ability were prepared and characterized. Solutions of racemic propranolol hydrochloride (Rac-PRO) were chosen to investigate the enantioselective performance of MGO-CuLBH and MGO-CuDBH by dispersive magnetic nanoparticle solid phase extraction (d-MNSPE). The results showed that the nanocomposites have excellent enantioselectivity to PROs with enantiomeric excess (ee) values reaching up to 98%. The entire process with PROs by the d-MNSPE method was fast, convenient and the collected composites could be easily recycled. Multi-stage operations using MGO-CuLBH and MGO-CuDBH were scaled up to obtain milligram quantities of R-propranolol hydrochloride (R-PRO) and S-propranolol hydrochloride (S-PRO). Furthermore, on the basis of

X. Ma et al. / Journal of Pharmaceutical and Biomedical Analysis 172 (2019) 50–57

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