Immobilization of Î²-Cyclodextrin-Conjugated Lactoferrin onto

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Immobilization of #-Cyclodextrin Conjugated Lactoferrin onto Polymer Monolith for Enrichment of Ga in Metabolic Residues of Ga-based Anticancer Drugs Haijiao Zheng, Teng-Gao Zhu, Xiqian Li, Guan Wang, and Qiong Jia Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b01003 • Publication Date (Web): 25 Oct 2017 Downloaded from http://pubs.acs.org on October 28, 2017

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Immobilization of β-Cyclodextrin Conjugated Lactoferrin onto Polymer Monolith for Enrichment of Ga in Metabolic Residues of Ga-based Anticancer Drugs

Haijiao Zheng a, Tenggao Zhu a, Xiqian Li b, Guan Wang *,c, and Qiong Jia *,a a

b

c

College of Chemistry, Jilin University, Changchun 130012, China

China-Japan Hospital of Jilin University, Changchun 130033, China

College of Life Sciences, Jilin University, Changchun 130012, China

KEYWORDS: Lactoferrin; β-Cyclodextrin; Monolith; Ga-based anticancer drugs; Metabolic residues

ABSTRACT: Biological materials-functionalized porous monoliths were prepared with lactoferrin (LF) and β-cyclodextrin (β-CD) via a click reaction. With the monolith as an extraction medium, a method combined with ICP-MS was developed for the determination of total gallium originating from metabolic residues of orally bioavailable gallium complexes with tris(8-quinolinolato)gallium (GaQ3) as a representative. The method exhibited favorable adsorption behaviors for gallium with high selectivity, low detection limit (2 ng L‒1), and an enrichment factor of 29-fold with the sample throughput of 30 min−1. The developed approach was validated by the analysis of gallium from GaQ3 metabolic residues in human cell line. Additionally, the practical applicability of this method was evaluated by the determination of gallium in human serum and urine samples from cancer patients. Results illustrated that the

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prepared monolith had potential in Ga-based anticancer drugs analysis in complex biological samples.

INTRODUCTION Chemotherapeutic agents usually contain metals for not only magnetic resonance imaging (gadolinium) and radioisotope imaging (cobalt technetium), but also anticancer drugs, e.g. platinum, rubidium, and gallium. For instance, tris(8-quinolinolato)gallium (GaQ3) has been found to be a therapeutic agent based on tumor-inhibiting metal complexes and used in clinical trials.1 The action modes of GaQ3 have been widely studied including the active form of its release from serum proteins into cytosol and its metabolic transformations by hydrolytic or ligand-exchange mechanisms.2 In order to make its clinical effects more straightforward, cytotoxicity investigations and therapeutic experiments in animals are deemed to be more useful than traditional tests of absorption, distribution, and transport of drugs.3 Consequently, monitoring ultratrace metabolic residuals in cellomics or actual samples from patients suffering from GaQ3 treatment are of great importance. Various analytical methodologies have been employed for the determination of GaQ3 metabolic residues,3–5 among which inductively coupled plasma-mass spectrometry (ICP-MS) is the most powerful one because of its extremely low detection limit, low mass interference, wide linear range, and so on. In recent studies, ICP-MS has been applied for direct quantification of gallium in human urine or serum spiked with Ga-based anticancer drugs.6,7 On the other hand, ICP-MS gains more significant potential as a powerful tool for the assessment of element speciation when combined with a suitable pretreatment technique. It is of great interest to employ biological materials as extraction media in the field of separation and enrichment, such as immobilization of biomolecules on solid supports.8,9 As a


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porous support, polymeric monolith has been immobilized with various biomolecules such as polysaccharides or proteins because of its highly cross-linked structure, easy preparation, and diverse surface chemistry.10 Lactoferrin (LF), a kind of glycoprotein consisting of a single polypeptide chain folded into two symmetrical highly homologous lobes (N and C lobes),11 has multiple bioactive properties in physiological functions, such as immune response, antioxidant, regulation of iron absorption in the bowel, anti-carcinogenic and anti-inflammatory properties.12 The above-mentioned properties about LF make it competitive to be immobilized onto solid supports as a biomolecule. For instance, LF-immobilized polylactic acid material exhibited enhanced surface properties mainly concerning biocompatibility, antimicrobial, and antioxidant properties. The material was biodegradable and environmentally friendly, furthermore, the introduced LF significantly improved thermoxidative stability of the polymer support.13 It can be anticipated that LF has favorable potential to be immobilized onto polymeric monolith and utilized in the field of separation and enrichment. However, a literature survey revealed that such work has never been reported so far. Herein, LF was conjugated with β-cyclodextrin (β-CD) via click chemistry and immobilized onto poly(glycidyl methacrylate-ethylene dimethacrylate) (poly(GMA-EDMA)) monolithic support with in-situ polymerization. A polymer monolith microextraction (PMME) method, which was first introduced by Feng’s group in 2006,10,14 was established for selective enrichment of total gallium in GaQ3 metabolic residues combining with ICP-MS. β-CD was introduced because it has a hydrophilic outer surface and lipophilic central cavity and can improve the enrichment capacity because of the host-guest interaction and electron transfer.15 The practical applicability of poly(GMA-EDMA-β-CD-LF) monolith was evaluated by the enrichment of low concentration gallium from GaQ3-incubated cell line and body fluids from cancer patients,


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demonstrating its high selectivity and sensitivity in the analysis of complex biological samples for Ga-based anticancer drug research.

EXPERIMENTAL SECTION Reagents and Instrumentation. GMA, EDMA, LF, γ-methacryloxypropyl trimethoxysilane (γ-MAPS), Ga standard solution (1000 mg mL‒1 in HCl), Traut’s reagent, sodium borate, ethylene diamine tetraacetic acid (EDTA), (NH4)2Fe(SO4)2, and GaCl3 were obtained from Sigma-Aldrich Company (St. Louis, MO, USA). 2,2-Azobis(isobutyronitrile) (AIBN), diethylamine, p-toluenesulfonylchloride (TsCl), Ba(OH)⋅8H2O, BaO, and β-CD were acquired from Aladdin Reagent Company (Shanghai, China). 1,4-Butanediol, 1-propanol, cetyl trimethyl ammonium bromide (CTAB), allyl bromide, potassium acetate, and thiourea were purchased from Tianjin Guangfu Fine Chemical Research Institute (Tianjin, China). Tris(8-quinolinolato) was acquired from Tianjin Huadong Chemical Research Institute (Tianjin, China). GMA and EDMA were distilled under vacuum conditions before use. AIBN was purified by recrystallization from ethanol and dried under vacuum at room temperature prior to use. All the other reagents were used as received. GaQ3 was synthesized by dissolving GaCl3 and 8hydroxyqunoline (1:3, molar ratio) in water. Excess potassium acetate was added into the solution, after which the mixture was heated under reflux for 5 h.15 Ultrapure water was prepared in the laboratory using a Millipore system (Milford, MA) and was used in all the experiments. ICP-MS determinations were performed on a quadrupole ICPMS instrument (Thermo iCAP Qc, Thermo, USA) with a high efficiency quartz concentric nebulizer, of which the optimum operation conditions were summarized in Table S1. Fused silica capillaries (530 µm i.d.) were purchased from Yongnian Optical Fiber Factory (Handan, China).


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Construction of poly(GMA-EDMA-β-CD-LF) Monolith. A click reaction was used for the synthesis of β-CD-LF with details described in Supporting Information.16 The PMME apparatus was prepared according to the procedure reported in previous studies.10,14 Briefly, it was composed of a 5 mL regular plastic syringe, a plastic pinhead, and the poly(GMA-EDMA-β-CDLF) monolithic column (530 µm, i.d. × 3 cm). The metallic needle of the pinhead was removed and replaced with the monolithic column with adhesive. Then, the other end of the pinhead was coupled to the syringe barrel seamlessly. Characterization. β-CD-LF was characterized by Fourier transformed infrared spectra (FT-IR, Thermo Nicolet 670, USA), for which the determinations were conducted in the frequency range of 4000–400 cm‒1 with a total of 32 scans and the samples were prepared as KBr pellets. The morphology and structure of the as-constructed column were characterized by scanning electron microscopy (SEM, JSM-6360LV, JEOL, Japan). Thermogravimetry (TG) curves were carried out on a Q500 thermal gravimetric analyzer (TA Instruments Inc., USA). An X-ray photoelectron spectrometer (XPS, ESCALAB250, Thermo Electron Corporation, USA) was used to obtain XPS data. Water contact angles were measured with an OCA20 apparatus (Data Physics, Germany) at saturated humidity with the temperature controlled by a super thermostat (Julabo F25, Germany). The surface area and mesopore size distribution of poly(GMA-EDMA-β-CD-LF) monolith were obtained by N2 adsorption-desorption experiments with a JW-BK surface area and pore size analyzer (Beijing, China). The permeability behavior was described by the pressure drops of the monolithic column at different flow rates using MeOH and MeOH-H2O (60:40 v/v) as the mobile phases. Values of the system pressure were measured at each flow rate without and with passing the mobile phases through poly(GMA-EDMA-β-CD-LF) monolith and the difference between the two values was calculated as the pressure drop across the monolith. Then, the permeability of the monolith was calculated based on Darcy's Law:


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B0 =

FηL πr 2 ∆p

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(1)

where F is the linear velocity of the eluent, η is the dynamic viscosity of the mobile phase (0.580 cP), L is the effective length of the column, r is the inner radius of the column, ∆P is the pressure drop across the column, and B0 is permeability (m2). The value of B0 can be used as an index for evaluating the quality of monoliths.17 The poly(GMA-EDMA-β-CD-LF) monolithic column was tested upon their swelling behavior in DMSO, known as an excellent solvent for organic polymers. A measure regarding the swelling tendency of polymer-based packing materials is the swelling propensity (SP) factor,18 which is expressed as:

SP =

p(DMSO) - p(H2O) p(H2O)

(2)

where p is defined as the ratio of back pressure to solvent viscosity (p=P/η). According to Eq. (2), SP=0 implies that the material has a non-swelling property. The higher is the SP factor, the more the material swells.18 Sample Preparation. Blood and urine samples from healthy human and cancer patients were provided by the First Hospital of Jilin University (Changchun, China). Details of the preparation procedures were presented in Supporting Information. Molm-13 cell line was cultured in RPMI 1640 media with 10% fetal bovine serum plus 100 U mL‒1 penicillin and 100 µg mL‒1 streptomycin in a 37 °C humidified atmosphere containing 5% CO2 + 95% air, and then sonicated in an ice-water ultrasonic bath for 5 min (5 s on and 10 s off) to extract proteins. Cell lines were tested for the presence of mycoplasma once a week. The solution was then centrifuged at 20000 rpm for 30 min, and the supernatant was stored at −80 °C before usage. Cytotoxicity Assays and Apoptosis Evaluation. In vitro cytotoxicites of GaQ3 were measured using 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays


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based on Pieters et al’s method.19 Briefly, the cells were treated with variable concentrations of GaQ3 for 20 h. MTT was added to a final concentration of 1 mmol L−1 and incubated for 4 h at 37 °C. The cells were lysed using 10% SDS in 10 mmol L−1 HCl and the optical densities were read by a microplate reader at 590 nm. Molm-13 cells were treated with variable concentrations of GaQ3 for 24 h and subjected to flow

cytometer

analysis

using

AnnexinV-fluorescein

isothiocyanate

(AnnexinV-

FITC)/propidium iodide (PI) Apoptosis Kit (BestBio, China). Briefly, resuspended cells were transferred to clean culture tubes and incubated with 50 µL 1× binding buffer containing AnnexinV-FITC and PI in the dark for 20 min. At the end of incubation, 450 µL 1× binding buffer was added to each tube, vortexed, and analyzed using a BD FACS Calibur Flow Cytometer (Becton, Dickinson and Company, USA). Cells were gated to include the main viable cell population based on forward scatter (FS)/side scatter (SS) characteristics. Apoptotic events from this viable cell gate were recorded as a combination of the AnnexinV+/PI− and AnnexinV+/PI+ events. Analytical Procedures. The PMME process included preconditioning, sorption, washing, and desorption. For preconditioning, 0.2 mL methanol and 0.2 mL phosphate buffer (20 mmol L‒1, pH=6.5) were successively driven to pass through the monolithic capillary tube at a flow rate of 0.1 mL min−1. In the sorption step, 0.8 mL sample solution was prepared at pH of 6.5. After passing through the prepared monolithic capillary at a flow rate of 80 µL min−1, the column was washed with 0.2 mL phosphate buffer (20 mmol L−1, pH=6.5) at a flow rate of 0.1 mL min‒1. The residual sample matrix in the pinhead and the monolithic capillary tube was driven out using a clean syringe. Finally, the retained Ga was eluted with 0.1 mol L−1 HCl and followed by ICP-MS determinations. Blank and standard solutions were subjected to same PMME-ICP-MS procedures. The whole procedure for each sample solution was carried out in triplicate.


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RESULTS AND DISCUSSION Characterization of poly(GMA-EDMA-β-CD-LF) Monolith. FT-IR spectra of β-CD-LF and 2,6-O-allyl-β-CD were shown in Figure S1. In the spectrum of 2,6-O-allyl-β-CD, Figure S1b, the peaks at 3085 and 3020 cm−1 were characteristic for =CH and =CHR, respectively. The peaks at 1645 cm−1 corresponding to C=C and those at 995 and 915 cm−1 attributed to =CH2, indicating that the compound had –CH=CH– functional groups. Compared with Figure S1b, the C–S–C peak of β-CD-LF was found at 585 cm−1 in Figure S1a. In addition, the C=C absorption peak at 1645 cm−1 of 2,6-O-allyl-β-CD became weak, implying that the SH−LF group reacted with 2,6O-allyl-β-CD via a click reaction.20 The monolithic column was synthesized inside a preconditioned fused silica capillary (530 µm i.d. × 30 cm) and the preparation process was shown in Figure 1. The fused silica capillary was vinylized with γ-MAPS (40% in acetone, v/v) to achieve covalent binding between the capillary inner wall and falling materials. Then, the vinylized capillary was filled with a polymerization mixture containing 240 mg GMA, 160 mg EDMA, 1120 mg 1-propanol, 320 mg 1,4-butanediol, 160 mg water, 1% AIBN (with respect to monomers, w/w), and β-CD-LF with different quantities. The mixture was thoroughly mixed by ultrasonication and purged with N2 for 10 min to remove O2. After that, the capillary was sealed at both ends with a rubber septum and immersed in a water bath at 60 °C for 24 h. The capillary was washed with 2 mL potassium phosphate buffer (pH=7.4) for 2 h to remove the unreacted proteins. Finally, it was flushed with 50 mmol L−1 Tris-HCl buffer (pH=6.5) for 1 h and 4 mL MeOH to block the remaining unreacted groups, respectively.21 The capillary column was cut into 3 cm length and stored at 4 °C in 50 mmol L−1 Tris-HCl buffer (pH=6.5) prior to use.


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Figure 1. Construction strategy of poly(GMA-EDMA-β-CD-LF) monolith.


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To evaluate the mass transfer property of the monoliths, we investigated the permeability behavior, which is one of most practical factors in designing a novel type of monolithic column. In Table S2, effects of monomer, cross-linker, and porogen ratios on the swelling behavior of the monoliths were listed. It could be observed that the monolith exhibited satisfactory permeability when monomer/porogen and monomer/cross-linker weight ratios were 2/8 and 3/2, respectively. In this study, a ternary porogenic system, 1,4-butanediol/1-propanol/H2O, was employed and ratios of the three components were optimized. It could be seen from Table S2 that when the content of 1,4-butanediol in the porogenic mixture increased from 12.0% to 40.0%, the permeability decreased from 11.21 × 10‒14 to 0.46 × 10‒14 m2, which was calculated based on Darcy's Law. When the content of 1,4-butanediol increased to 44%, it was too hard for the solutions to be pushed through the column. Therefore, it could be concluded that 1,4-butanediol acted as the microprogenic system (good solvent). Compromising the permeability and morphology, column M-1 was selected in the following experiments. In addition, the effect of preparation temperature was investigated. High preparation temperature would not only accelerate the reaction process, but also improve the solubility of porogenic system, thus resulting in a decrease of the permeability (data not shown). Finally, the preparation temperature was kept at 60 °C in water bath. Figure 2 showed SEM images of poly(GMA-EDMA-β-CD-LF) monolith. As could be seen from Figure 2A, the structure of the monoliths exhibited a homogeneous network of macroporous polymer, which was highly crosslinked. To examine the thermal properties and stabilities of poly(GMA-EDMA) and poly(GMA-EDMA-β-CD-LF) monoliths, TG analysis was carried out under N2 atmosphere at a heating rate of 10 ºC min‒1 over the range from room temperature to 800 ºC (Figure 3A). Compared with poly(GMA-EDMA) monolith, poly(GMA-


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EDMA-β-CD-LF) monolith began to decompose at 102.5 °C, indicating that LF was successfully introduced into the monolith.13 The TG curve tended to be constant when the temperature was higher than 450 ºC, illustrating a completed composition of the monolithic material. Results of contact angle tests for poly(GMA-EDMA) and poly(GMA-EDMA-β-CDLF) monoliths were indicated in Figure 3B. The contact angle values of 112.4º and 81.2º were obtained for the unmodified/modified columns, respectively, implying that β-CD-LF enhanced the hydrophilicity of the monolith and could increase the hydrophilic interaction between the monolith and target. In order to prove that β-CD-LF was combined into the monolith, XPS analysis of S was conducted and shown in Figure 3C, showing that S content increased after βCD-LF was introduced into poly(GMA-EDMA) monolith.


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Figure 2. SEM images of poly(GMA-EDMA-β-CD-LF) monolith before (A) and after (B) adsorption and corresponding EDX spectra.


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Figure 3. (A) TG curves, (B) contact angles, and (C) XPS spectra of poly(GMA-EDMA) and poly(GMA-EDMA-β-CD-LF) monoliths, (D) XPS spectrum of poly(GMA-EDMA-β-CD-LF) monolith after adsorption.

The pore size distribution and N2 adsorption-desorption isotherm exhibited typical type-IV hysteresis, indicative of the presence of mesoporous structure of poly(GMA-EDMA) and poly(GMA-EDMA-β-CD-LF) monoliths, respectively (Figures 4A and 4B). The specific surface area of poly(GMA-EDMA-β-CD-LF) monolith was calculated to be 154 m2 g‒1, which was about 5 times that of poly(GMA-EDMA) monolith. This is because both β-CD and LF can increase the specific surface area of the monolith.22 Additionally, the average pore diameters were calculated to be 5.234 µm for poly(GMA-EDMA) and 80.245 nm for poly(GMA-EDMA-βCD-LF) monoliths, respectively. The smaller-sized skeletons of poly(GMA-EDMA-β-CD-LF) monolith provided larger surface area and more reactive sites than the unmodified monolith.


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Figure 4. Pore size distribution curve and N2 adsorption-desorption isotherm: (A) poly(GMAEDMA) and (B) poly(GMA-EDMA-β-CD-LF) monoliths.

Figure S2 represented the effect of flow rate on the back pressure of the monolithic columns. It could be seen that the back pressure was linearly proportional to the flow rate, which proved that the monoliths were stable and did not compress even at high flow rate up to over 800 cm h‒1. These results also implied that collapse could be avoided when the monolith was subjected to flow through liquids. A comparison of poly(GMA-EDMA-β-CD-LF) monolith before and after the adsorption of Ga was studied via SEM and XPS determinations. Figure 2B showed SEM images and corresponding EDX spectra of the prepared monolithic columns after the adsorption process of Ga. EDX results in Figure 2B clearly indicated that Ga was adsorbed on poly(GMA-EDMA-βCD-LF) monolith. Furthermore, the XPS spectrum of poly(GMA-EDMA-β-CD-LF) monolith after the adsorption step was investigated. It could be observed that XPS results shown in Figure 3D were in accordance with those mentioned above.


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Optimization of PMME Conditions. To achieve the best extraction efficiency of Ga with poly(GMA-EDMA-β-CD-LF) monolith, we investigated effects of several experimental parameters on the extraction efficiency including sample pH, sample flow rate, sample volume, eluent volume, and eluent flow rate (Figure S3). The effect of sample pH on the extraction efficiency was examined over the range from 2.0 to 12.0. Results revealed the highest extraction efficiency at pH 6.5, which might be explained that Ga was easy to hydrolyze when pH increased.23 As a consequence, pH 6.5 was selected in the present work to avoid possible hydrolysis of Ga at higher pH. An L9(34) orthogonal test was employed to study the effects of other experimental factors. Results were listed in Table S3. Variance and range analysis were also shown in Figure S3. Finally, the optimal extraction parameters were as follows: sample pH of 6.5, sample flow rate of 0.08 mL min‒1, sample volume of 0.8 mL, eluent volume of 0.05 mL, and eluent flow rate of 0.03 mL min‒1. Interference Study. The potential interference from possible coexisting ions prevailing in biological samples was investigated by adding a series amount of foreign ions into 5 mL sample solution containing 2 µg L‒1 Ga with the general procedure above. Results of coexisting ions tolerance limits in human urine and blood samples were listed in Table S4. The recoveries were determined to range from 85% to 115%, verifying that effects of the coexisting ions from human urine and blood matrices could be considered to be negligible. As could be seen, the concentrations of all studied foreign ions were below their tolerance limits in normal human urine and blood samples. It could be confirmed that the present PMME method had high selectivity for Ga and good application potential in the speciation of Ga in complicated biological samples.


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Method Validation. Under the optimized experimental conditions, the method was linear in the range of 0.02‒20 µg mL‒1 for Ga. The inter-day and intra-day repeatabilities for Ga were in the range of 0.65%‒1.34%, respectively. Detection limit, evaluated as the concentration corresponding to 3 times the standard deviation of 11 runs of the blank solution (3σ), was found to be 5 µg L‒1 for Ga. The enrichment factors of Ga, calculated by the ratio of the slopes of calibration curves with and without employing the PMME method, were determined to be 29fold. Such high enrichment capacities may be ascribed to several aspects as followings: Firstly, poly(GMA-EDMA) monolith possessed a large specific area in the porous structure, providing ideal and numerous binding sites for β-CD-LF immobilization; Secondly, the host-guest interaction between β-CD and Ga was favorable for the enrichment;14 Thirdly, the hydrophilic port of β-CD provided a hydrophilic effect on Ga; Fourthly, LF was linked with β-CD steadily and solidly via a click reaction and LF was capable to bind with Ga,24 and the complexation between them enhanced the adsorption capacity of Ga; Finally, the charge interaction and ion interaction also played important roles in the process.14 The reusability of the poly(GMA-EDMA-β-CD-LF) monolithic column was investigated. As shown in Figure S4, the column efficiency still remained at about 95.9% after 10 days. Intrabatch and inter-batch reproducibilities were also investigated under the optimized conditions, illustrating that intra-batch and inter-batch relative standard deviations (RSDs) for Ga were 2.7% and 3.2%, respectively. It could thus be concluded that the poly(GMA-EDMA-β-CD-LF) monolithic column provided a reliable and reproducible performance for Ga analysis. Method Comparison. The Ga concentrations in blood samples were determined using ICPMS with and without employing the PMME method (data not shown). Results indicated that the Ga concentrations were all lower than 0.015 µg L‒1 without PMME and approximately 30-fold


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higher after introducing the PMME step. Such results were in accordance with the EF values as mentioned above. Meanwhile, Table S5 listed a comparison study between the developed PMME-ICP-MS method with those employed for the detection of Ga in GaQ3 metabolism residues,3,6,14,25-28 indicating that the present strategy provided lower detection limit values. It should be noted that there have been no publications focusing on the determination of Ga from anticancer drugs metabolism residues in cellomics analysis, implying that the proposed method has potential to be applied in the analysis of Ga-based drugs in medical biochemistry and metabolomics. The present method opens a window for rapid and sensitive monitor of trace metal ions in complicated matrices such as human fluids, which may find more applications in biological, environmental, and pharmaceutical analysis. Cytotoxicity and Assessment of apoptosis. In vitro cytotoxicites of GaQ3 were measured using MTT assays. Results were shown in Figure 5 and Figure S5. In detail, the peptide had half-inhibitory concentrations (IC50) on U937 and Molm-13 cells of 15.3 µmol L‒1 and 8.9 µmol L‒1 for 24 h, respectively. To further investigate the inhibitory mechanism of GaQ3 on AML cancer Molm-13 cells, we applied the fixed cells for flow cytometry analysis after the cells were incubated with 10 µmol L‒1 GaQ3 for 24 h and stained with FITC Annexin V and propidiumiodide. The results indicated that GaQ3-treated cells showed increases of the earlyapoptotic stage (Q3) from 3.23% to 41.5% and late-apoptotic stage (Q2) from 1.06% to 48.8% (Figure 5) compared with the control cells. However, in the necrosis stage (Q1), the GaQ3treated cells (0.052%) did not show an obvious change compared with the control cells (0.007%). All these data suggested that GaQ3 induces the death of AML cancer Molm-13 cells via a typical apoptotic pathway.


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Figure 5. Cytotoxicity and assessment of apoptosis.


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Sample Analysis. The concentrations of Ga in GaQ3 metabolic residues of in Molm-13 cells, blood and urine samples from cancer patients were determined (Table S6 and Table S7). In order to verify the accuracy and feasibility of the method, the samples were spiked with the target analyte with two concentration levels, 0.1 µg L‒1 and 1 µg L‒1. Analytical results listed in Table S7 showed that Ga could be detected in the cancer patient fluids and the recovery values were in the range of 84%−110%. The results also demonstrated that the amount of Ga in GaQ3 metabolic residues in the patient fluids increased as time went on. The recoveries data proved that our method was applicable to the real samples with complicated matrices and did not suffer from negative matrix effects. In a word, poly(GMA-EDMA-β-CD-LF) monolith could be used for GaQ3 metabolic residues analysis and had potential in pharmacological study.

CONCLUSIONS In summary, a facile approach was developed for the preparation of β-CD-LF and its immobilization on poly(GMA-EDMA) monolith via a thiol-ene click reaction. Ascribing to the property of β-CD-LF, the resulting poly(GMA-EDMA-β-CD-LF) monolith exhibited favorable high thermostability, strong hydrophilibity, large surface area, and high mechanical stability. When used for the determination of Ga in GaQ3 metabolic residues, the PMME-ICP-MS method presented a detection limit of 2 ng L‒1. In addition, poly(GMA-EDMA-β-CD-LF) monolith was applied to the enrichment of Ga ion in cell line and human body fluids, opening new possibilities for future biological application, chinical diagnostics, and drug discovery.

Supporting Information

Details about sample preparation, preparation of β-CD-LF, Taguchi’s L9(34) orthogonal array


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experiment, and additional Tables and Figures.

Acknowledgments The project was supported by National Natural Science Foundation of China (21575049).

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Graphical Abstract


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Immobilization of Î²-Cyclodextrin-Conjugated Lactoferrin onto

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