Size-regulated group separation of CoFe2O4 nanoparticles using centrifuge and their magnetic resonance contrast properties

Magnetic nanoparticle (MNP)-based magnetic resonance imaging (MRI) contrast agents (CAs) have been the subject of extensive research over recent decades. The particle size of MNPs varies widely and is known to influence their physicochemical and pharmacokinetic properties. There are two commonly used methods for synthesizing MNPs, organometallic and aqueous solution coprecipitation. The former has the advantage of being able to control the particle size more effectively; however, the resulting particles require a hydrophilic coating in order to be rendered water soluble. The MNPs produced using the latter method are intrinsically water soluble, but they have a relatively wide particle size distribution. Size-controlled water-soluble MNPs have great potential as MRI CAs and in cell sorting and labeling applications. In the present study, we synthesized CoFe₂O₄ MNPs using an aqueous solution coprecipitation method. The MNPs were subsequently separated into four groups depending on size, by the use of centrifugation at different speeds. The crystal shapes and size distributions of the particles in the four groups were measured and confirmed by transmission electron microscopy and dynamic light scattering. Using X-ray diffraction analysis, the MNPs were found to have an inverse spinel structure. Four MNP groups with well-selected semi-Gaussian-like diameter distributions were obtained, with measured T₂ relaxivities (r₂) at 4.7 T and room temperature in the range of 60 to 300 mM⁻¹s⁻¹, depending on the particle size. This size regulation method has great promise for applications that require homogeneous-sized MNPs made by an aqueous solution coprecipitation method. Any group of the CoFe₂O₄ MNPs could be used as initial base cores of MRI T₂ CAs, with almost unique T₂ relaxivity owing to size regulation. The methodology reported here opens up many possibilities for biosensing applications and disease diagnosis.

PACS

75.75.Fk, 78.67.Bf, 61.46.Df     

Multifunctional liposomal drug delivery with dual probes of magnetic resonance and fluorescence imaging

Many liposomal drug carriers have shown great promise in the clinic. To ensure the efficient preclinical development of drug-loaded liposomes, the drug retention and circulation properties of these systems should be characterized. Iron oxide (Fe₃O₄) magnetic nanoparticles (MNPs) are used as T2 contrast agents in magnetic resonance imaging (MRI). Gold nanoclusters (GNCs) contain tens of atoms with subnanometer dimensions; they have very low cytotoxicity and possess superb red emitting fluorescent properties, which prevents in vivo background autofluorescence. The aim of this study was to develop dual imaging, nanocomposite, multifunctional liposome drug carriers (Fe₃O₄-GNCs) comprising MNPs of iron oxide and GNCs. First, MNPs of iron oxide were synthesized by co-precipitation. The MNP surfaces were modified with amine groups using 3-aminopropyltriethoxysilane (APTES). Second, GNCs were synthesized by reducing HAuCl₄·3H₂O with NaBH4 in the presence of lipoic acid (as a stabilizer and nanosynthetic template). The GNCs were grown by adsorption onto particles to control the size and stability of the resultant colloids. Subsequently, dual Fe₃O₄-GNCs imaging probes were fabricated by conjugating the iron oxide MNPs with the GNCs via amide bonds. Finally, liposome nanocarriers were used to enclose the Fe₃O₄-GNCs in an inner phase (liposome@Fe₃O₄-GNCs) by reverse phase evaporation. These nanocarriers were characterized by dynamic light scattering (DLS), fluorescence spectroscopy, X-ray diffraction (XRD), transmission electron microscopy (TEM), Fourier transform infrared (FT-IR) spectrophotometry, superconducting quantum interference device (SQUID), nuclear magnetic resonance (NMR) imaging and in vivo imaging systems (IVIS). These multifunctional liposomal drug delivery systems with dual probes are expected to prove useful in preclinical trials for cancer diagnosis and therapy.     

Chemical and magnetic functionalization of graphene oxide as a route to enhance its biocompatibility

The novel approach for deposition of iron oxide nanoparticles with narrow size distribution supported on different sized graphene oxide was reported. Two different samples with different size distributions of graphene oxide (0.5 to 7 μm and 1 to 3 μm) were selectively prepared, and the influence of the flake size distribution on the mitochondrial activity of L929 with WST1 assay in vitro study was also evaluated. Little reduction of mitochondrial activity of the GO-Fe₃O₄ samples with broader size distribution (0.5 to 7 μm) was observed. The pristine GO samples (0.5 to 7 μm) in the highest concentrations reduced the mitochondrial activity significantly. For GO-Fe₃O₄ samples with narrower size distribution, the best biocompatibility was noticed at concentration 12.5 μg/mL. The highest reduction of cell viability was noted at a dose 100 μg/mL for GO (1 to 3 μm). It is worth noting that the chemical functionalization of GO and Fe₃O₄ is a way to enhance the biocompatibility and makes the system independent of the size distribution of graphene oxide.     

Reversible thermoflocculation of magnetic core-shell particles induced by remote magnetic heating

We developed multifunctional magnetic polymer brushes with a tailorable thermoresponsive dispersion behavior that can be activated by AC magnetic fields. Via surface-initiated ATRP, magnetic core-shell nanoparticles are obtained that are composed of nanosized superparamagnetic iron oxide cores and a copolymer shell. The shell consists of oligo(ethylene glycol) methylether methacrylate (OEGMA) and methoxyethyl methacrylate (MEMA) copolymers that show a lower critical solution temperature (LCST) in water.The hybrid structures are easily dispersible in water at room temperature, and show a reversible thermoflocculation at critical temperatures adjustable by the copolymer composition. The phase separation can alternatively be initiated and recorded by magnetic heating caused by magnetic losses in AC fields. This method offers a convenient way for the remote-controlled heating and agglomeration of disperse systems.     

Preparation and evaluation of poly(2-hydroxyethyl aspartamide)-hexadecylamine-iron oxide for MR imaging of lymph nodes

The purpose of this study was to synthesize biocompatible poly(2-hydroxyethyl aspartamide)–C₁₆-iron oxide (PHEA-C₁₆-iron oxide) nanoparticles and to evaluate their efficacy as a contrast agent for magnetic resonance imaging of lymph nodes. The PHEA-C₁₆-iron oxide nanoparticles were synthesized by coprecipitation method. The core size of the PHEA-C₁₆-iron oxide nanoparticles was about 5 to 7 nm, and the overall size of the nanoparticles was around 20, 60, and 150 nm in aqueous solution. The size of the nanoparticles was controlled by the amount of C₁₆. The 3.0-T MRI signal intensity of a rabbit lymph node was effectively reduced after intravenous administration of PHEA-C₁₆-iron oxide with the size of 20 nm. The in vitro and in vivo toxicity tests revealed the high biocompatibility of PHEA-C₁₆-iron oxide nanoparticles. Therefore, PHEA-C₁₆-iron oxide nanoparticles with 20-nm size can be potentially useful as T2-weighted MR imaging contrast agents for the detection of lymph nodes.     

Synthesis and magneto-structural properties of chitosan coated ultrafine cobalt ferrite nanoparticles for magnetic fluid hyperthermia in viscous medium

AC induction heating mediated magnetic fluid hyperthermia of superparamagnetic nanoparticles (MNPs) is being widely explored for localized thermo-therapy of tumours. One of the primary hindrances for rapid adaptation of this technique is the loss of heating efficiency when the MNPs are placed within the viscous tissue medium, which necessitates undesired increase in MNP concentrations or exposure time during practical applications. With an objective to mitigate this, here we report the viscosity independent magnetic hyperthermia properties of biocompatible ultrafine (average size ～ 2.5 nm) chitosan-coated superparamagnetic CoFe₂O₄ MNPs synthesized using a low-cost co-precipitation technique. The presence of the chitosan coating is confirmed from Fourier transform infrared and X-ray photoelectron spectroscopy. The superparamagnetic nature of the synthesized MNPs at 300 K is confirmed from Mössbauer spectroscopy, isothermal and temperature dependent magnetization studies. Experimental findings indicate a higher field-induced heating efficiency for the chitosan-coated MNPs due to superior colloidal stability. The ultrafine size, combined with higher anisotropy energy density, results in viscosity independent Nèel relaxation-dominated magneto-thermal energy conversion for the CoFe₂O₄ MNPs. Experimental results reveal negligible loss of heating efficiency due to partial abrogation of Brownian relaxation when the chitosan-coated MNPs are immobilized in a tissue-equivalent agar medium, which is beneficial for practical applications. The heating efficiency of ～72.1 ± 2.8 W/g_Fe (at 33.1 kA/m and 126 kHz), obtained in the present study for the chitosan-coated MNPs, is higher than the previously documented values for ultrafine CoFe₂O₄ MNPs, which is useful for reducing the exposure time during practical applications. Further, the chitosan coating rendered the ultrafine CoFe₂O₄ MNPs bio-compatible against L929 cell line. The satisfactory magnetic fluid hyperthermia efficiency, negligible room temperature coercivity, retention of the field-induced heating efficiency in tissue-equivalent agar medium due to Nèel-dominated relaxation dynamics and superior biocompatibility, make the chitosan-coated ultrafine CoFe₂O₄ MNPs an attractive candidate for practical MFH applications.     

基于化学方法制备油酸改性超顺磁性氧化铁纳米粒子

目的 油酸改性超顺磁性氧化铁纳米粒子（O-SPION）被应用于制备高品质的MRI T2阴性造影剂或搭载药物的磁靶向分子探针,不同于传统的物理合成方法,本课题组尝试采过化学方法制备O-SPION,并通过体外实验检测其细胞毒性及细胞透过性。方法 通过共沉淀法制备分散性好、磁响应强的超顺磁性纳米粒子。对油酸的羧基进行活化后,利用缩合反应实现油酸改性SPION的化学合成。采用X-Ray衍射仪、红外光谱仪、激光粒度分布测试仪、透射电镜对产物进行表征。MTT法检测其对人肝癌细胞HepG2的毒性作用,普鲁士蓝染色检测其细胞摄取能力。结果 O-SPION的核心粒径为12?.5 nm的,其具有稳定的化学结构和低表面电势,体外实验证明其低毒或无毒性,且在细胞内的摄取量较原始SPION明显增多。结论 利用化学方法成功合成了O-SPION,为进一步制备高品质MRI造影剂提供实验依据。     

Magnetic Resonance Imaging of Tumor-Infiltrating Lymphocytes by Anti-CD3-Conjugated Iron Oxide Nanoparticles

Immune checkpoint blockade, considered a revolutionary approach in cancer treatment, is only effective in patients with high tumor-infiltrating lymphocytes (TILs). This work aimed to investigate the feasibility of targeted contrast agent (CA) based on dextran-coated superparamagnetic iron oxide nanoparticles (SPIONs-DEX) for TILs detection by magnetic resonance imaging (MRI) studies. To do so, we synthesized an MRI CA by conjugating SPIONs-DEX to an anti-CD3 monoclonal antibody via cyanogen bromide as a cross-linker. In vitro assessments demonstrated the higher labeling efficiency of the developed CA to CD3+ lymphocytes compared to SPIONs-DEX. In vivo MRI of a xenograft model of CD3+ lymphocytes revealed the significant signal loss after the intravenous injection of the bioconjugate by ∼34 % and 21 % in T₂*-weighted and T₂-weighted images, respectively. The histopathological evaluation of xenograft tumors confirmed the labeling of lymphocytes by the targeted CA. This approach could open up a new horizon in the non-invasive assessment of TILs to identify patients eligible for immunotherapy.     

Fabrication and characterization of zinc oxide nanoparticle coated magnetic iron oxide: Effect of S-layers adsorption on surface of oxide

Mixed zinc oxide nanoparticle coated magnetic iron oxide has been prepared by a sol–gel and co-precipitation routes. Magnetic iron oxide nanoparticles were synthesized by co-precipitation of ferric and ferrous ions with ammonia, and then zinc oxide was coated onto the surface of magnetic iron oxide by hydrolysis of zinc precursors. As a result, zinc oxide coated magnetic iron oxide nanoparticles with an average size of 68 nm were obtained. The crystalline bacterial cell surface layer)S-layer (used in this study was isolated from Lactobacillus helveticus ATCC 12046. The S-layer was adsorbed onto the surface of zinc oxide nanoparticle coated magnetic iron oxide. The nanoparticles were analyzed by X-ray powder diffractometry (XRD), infrared spectroscopy (FTIR), transmission electron microscopy (TEM), and field emission scanning electron microscopy (FESEM) were used to characterize the structural and the chemical features of the nanocomposites. The infrared spectra indicate that the S-layer-nanoparticle interaction occurs. This novel nanoparticle showed admirable potential in adsorption of S-layers on the surface of oxides for drug delivery.     

Synthesis of magnetite/polyamino-ester dendrimer based on PCL/PEG amphiphilic copolymers via convergent approach for targeted diagnosis and therapy

The aim in this study is to synthesize amphiphilic linear-dendritic-linear block copolymers consisting of a poly ?-caprolactone linear block, poly(amino-ester) dendritic block and m-PEG linear block. G1, G2 and G3 dendrons were produced by sequential acrylation and Micheal addition reactions, using required amounts of acryloyl chloride and diethanolamine respectively to achieve quantitative growth. Amphiphilic dendrons were synthesized from the reaction of hydroxyl group of G1, G2 and G3 with mPEG-adipoyl chloride and their structures were characterized by FT-IR and ¹H NMR spectroscopy. The amphiphilic dendrons can self-assemble and form micelles in water. Their critical micelle concentration (CMC), particle size and zeta potential were determined by fluorescence spectroscopy and dynamic light scattering, respectively. Convergent dendrimers were prepared by self-assembly of the dendrons around oleic acid-stabilized Fe₃O₄ nanoparticles via the ligand exchange method and their morphologies were characterized by transmission electron microscopy (TEM). The in-vitro release behavior of quercetin from dendrimers and hydrolytic degradation of them were investigated at two pHs (7.4 and 5.8).     

Preparation and characterization of fluorescent polymer magnetic particles

Magnetic iron oxide (maghemite, Fe₃O₄) particles were encapsulated with fluorescent polymer phase. The resulting fluorescent magnetic polymer particles were characterized by Fourier transform infrared spectroscopy (FTIR), thermal gravimeter analysis (TGA), reflection optical microscopy, differential scanning calorimeter (DSC), Fritsch particle sizer, scanning electron microscopy (SEM), powder X‐ray diffractometer (XRD), and vibrating sample magnetometer (VSM) measurements. FTIR and XRD confirmed the presence of iron oxide in polymer phase. The TGA and DSC measurements indicated that the magnetic polymer particles have more than 50% iron oxide content and high thermal stability. SEM and reflection optical microscopy under UV light revealed that all maghemite particles were embedded in the polymer spheres and have fluorescent characteristics. The size‐distribution analysis of prepared magnetic particles was shown that the means diameter of the particles slightly increased. According to our magnetometry data, shape of the loops evidences the ferromagnetic character of the material and no evidence of superparamagnetism was seen. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2008     

Preparation of styrene-based, magnetic polymer microspheres by a swelling and penetration process

A swelling and penetration process is proposed to prepare magnetic polymer microspheres. Micron-size polystyrene (PS) particles were swollen in an aqueous solution of N-methyl-2-pyrrolidone (NMP) and then mixed with superparamagnetic iron oxide nanoparticles. The magnetic nanoparticles were able to diffuse into polymer microspheres and were entrapped within the polymer microspheres. The saturation magnetization of resultant magnetic polymer microspheres increased as increasing magnetic nanoparticle concentrations were added to the swelling mixture. A higher ratio of NMP-to-water led to a greater swelling but a larger loss of polymer mass due to polymer chain dissolution in the NMP solution. Sodium dodecyl sulfate (SDS) in the NMP aqueous solution significantly enhanced the swelling and penetration process. The use of SDS could not only shorten the process time but also lower the required NMP concentration. The proposed method also worked well in preparing magnetic polymers microspheres with other styrene-based copolymer beads like poly(styrene–glycidyl methacrylate) (PS–GMA).     

Establishment of a method to determine the magnetic particles in mouse tissues

This work is aimed to evaluate a method to detect the residual magnetic nanoparticles (MNPs) in animal tissues. Ferric ions released from MNPs through acidification with hydrochloric acid can be measured by complexation with potassium thiocyanate. MNPs in saline could be well detected by this chemical colorimetric method, whereas the detected sensitivity decreased significantly when MNPs were mixed with mouse tissue homogenates. In order to check the MNPs in animal tissues accurately, three improvements have been made. Firstly, proteinase K was used to digest the proteins that might bind with iron, and secondly, ferrosoferric oxide (Fe₃O₄) was collected by a magnetic field which could capture MNPs and leave the bio-iron in the supernatant. Finally, the collected MNPs were carbonized in the muffle furnace at 420°C before acidification to ruin the groups that might bind with ferric ions such as porphyrin. Using this method, MNPs in animal tissues could be well measured while avoiding the disturbance of endogenous iron and iron-binding groups.     

Efficient CD44-targeted magnetic resonance imaging (MRI) of breast cancer cells using hyaluronic acid (HA)-modified MnFe2O4 nanocrystals

Targeted molecular imaging with hyaluronic acid (HA) has been highlighted in the diagnosis and treatment of CD44-overexpressing cancer. CD44, a receptor for HA, is closely related to the growth of cancer including proliferation, metastasis, invasion, and angiogenesis. For the efficient detection of CD44, we fabricated a few kinds of HA-modified MnFe₂O₄ nanocrystals (MNCs) to serve as specific magnetic resonance (MR) contrast agents (HA-MRCAs) and compared physicochemical properties, biocompatibility, and the CD44 targeting efficiency. Hydrophobic MNCs were efficiently phase-transferred using aminated polysorbate 80 (P80) synthesized by introducing spermine molecules on the hydroxyl groups of P80. Subsequently, a few kinds of HA-MRCAs were fabricated, conjugating different ratios of HA on the equal amount of phase-transferred MNCs. The optimized conjugation ratio of HA against magnetic content was identified to exhibit not only effective CD44 finding ability but also high cell viability through in vitro experiments. The results of this study demonstrate that the suggested HA-MRCA shows strong potential to be used for accurate tumor diagnosis.     

Effects of hydrothermal process parameters on the physical,magnetic and thermal properties of Zn0.3Fe2.7O4 nanoparticles for magnetic hyperthermia applications

Effects of hydrothermal temperature and time on physical, magnetic and thermal properties of Zn-substituted magnetite nanoparticles (Zn_0.3Fe_2.7O₄) were assessed. The magnetic nanoparticles were synthesized via citric acid-assisted hydrothermal reduction route at temperatures of 150, 175 and 200 °C for duration of 10, 15 and 20 h. The nanoparticles were characterized by X-ray diffraction (XRD), scanning electron microscope (SEM), transmission electron microscope (TEM), vibrating sample magnetometer (VSM) and specific loss power (SLP) measurements. The results showed that temperature and time of the hydrothermal process both had significant effects on nanoparticles composition and properties. It was observed that at 150 °C, heat generation was insufficient to produce activation energy required for nucleation of Zn_0.3Fe_2.7O₄ spinel nanoparticles, even after a long time. At 175 °C, although temperature was low, but the suitable condition for nucleation of nanoparticles was made and spinel nanoparticles with the size of about 13 nm were formed after 15 h. Nonetheless, since crystallinity and SLP of the nanoparticles was low, they showed weak performance for magnetic hyperthermia. At 200 °C, the required activation energy was provided for nanoparticles nucleation; however, the spinel was oxidized to hematite, resulting in a decrease in thermal and magnetic properties. In overall, the nanoparticles synthesized at 200 °C for 15 h possessed the best characteristics of reasonable purity, saturation magnetization of about 35.9 emu/g and SLP of 18.7 W/g.     

Magnetic resonance imaging of glioma with novel APTS-coated superparamagnetic iron oxide nanoparticles

We report in vitro and in vivo magnetic resonance (MR) imaging of C6 glioma cells with a novel acetylated 3-aminopropyltrimethoxysilane (APTS)-coated iron oxide nanoparticles (Fe₃O₄ NPs). In the present study, APTS-coated Fe₃O₄ NPs were formed via a one-step hydrothermal approach and then chemically modified with acetic anhydride to generate surface charge-neutralized NPs. Prussian blue staining and transmission electron microscopy (TEM) data showed that acetylated APTS-coated Fe₃O₄ NPs can be taken up by cells. Combined morphological observation, cell viability, and flow cytometric analysis of the cell cycle indicated that the acetylated APTS-coated Fe₃O₄ NPs did not significantly affect cell morphology, viability, or cell cycle, indicating their good biocompatibility. Finally, the acetylated APTS-coated Fe₃O₄ nanoparticles were used in magnetic resonance imaging of C6 glioma. Our results showed that the developed acetylated APTS-coated Fe₃O₄ NPs can be used as an effective labeling agent to detect C6 glioma cells in vitro and in vivo for MR imaging. The results from the present study indicate that the developed acetylated APTS-coated Fe₃O₄ NPs have a potential application in MR imaging.     

HAI-178 antibody-conjugated fluorescent magnetic nanoparticles for targeted imaging and simultaneous therapy of gastric cancer

The successful development of safe and highly effective nanoprobes for targeted imaging and simultaneous therapy of in vivo gastric cancer is a great challenge. Herein we reported for the first time that anti-α-subunit of ATP synthase antibody, HAI-178 monoclonal antibody-conjugated fluorescent magnetic nanoparticles, was successfully used for targeted imaging and simultaneous therapy of in vivo gastric cancer. A total of 172 specimens of gastric cancer tissues were collected, and the expression of α-subunit of ATP synthase in gastric cancer tissues was investigated by immunohistochemistry method. Fluorescent magnetic nanoparticles were prepared and conjugated with HAI-178 monoclonal antibody, and the resultant HAI-178 antibody-conjugated fluorescent magnetic nanoparticles (HAI-178-FMNPs) were co-incubated with gastric cancer MGC803 cells and gastric mucous GES-1 cells. Gastric cancer-bearing nude mice models were established, were injected with prepared HAI-178-FMNPs via tail vein, and were imaged by magnetic resonance imaging and small animal fluorescent imaging system. The results showed that the α-subunit of ATP synthase exhibited high expression in 94.7% of the gastric cancer tissues. The prepared HAI-178-FMNPs could target actively MGC803 cells, realized fluorescent imaging and magnetic resonance imaging of in vivo gastric cancer, and actively inhibited growth of gastric cancer cells. In conclusion, HAI-178 antibody-conjugated fluorescent magnetic nanoparticles have a great potential in applications such as targeted imaging and simultaneous therapy of in vivo early gastric cancer cells in the near future.     

Albumin-Coated Single-Core Iron Oxide Nanoparticles for Enhanced Molecular Magnetic Imaging (MRI/MPI)

Colloidal stability of magnetic iron oxide nanoparticles (MNP) in physiological environments is crucial for their (bio)medical application. MNP are potential contrast agents for different imaging modalities such as magnetic resonance imaging (MRI) and magnetic particle imaging (MPI). Applied as a hybrid method (MRI/MPI), these are valuable tools for molecular imaging. Continuously synthesized and in-situ stabilized single-core MNP were further modified by albumin coating. Synthesizing and coating of MNP were carried out in aqueous media without using any organic solvent in a simple procedure. The additional steric stabilization with the biocompatible protein, namely bovine serum albumin (BSA), led to potential contrast agents suitable for multimodal (MRI/MPI) imaging. The colloidal stability of BSA-coated MNP was investigated in different sodium chloride concentrations (50 to 150 mM) in short- and long-term incubation (from two hours to one week) using physiochemical characterization techniques such as transmission electron microscopy (TEM) for core size and differential centrifugal sedimentation (DCS) for hydrodynamic size. Magnetic characterization such as magnetic particle spectroscopy (MPS) and nuclear magnetic resonance (NMR) measurements confirmed the successful surface modification as well as exceptional colloidal stability of the relatively large single-core MNP. For comparison, two commercially available MNP systems were investigated, MNP-clusters, the former liver contrast agent (Resovist), and single-core MNP (SHP-30) manufactured by thermal decomposition. The tailored core size, colloidal stability in a physiological environment, and magnetic performance of our MNP indicate their ability to be used as molecular magnetic contrast agents for MPI and MRI.     

Oxidative coupling of aromatic thiols to corresponding disulfides using magnetic particle-supported sulfonic acid catalyst and hydrogen peroxide under mild conditions

A green and efficient method for chemoselective oxidation of aromatic thiols to the corresponding disulfides is reported using a recoverable supported iron oxide nanoparticle (γ-Fe₂O₃–SO₃H) catalyst in the presence H₂O₂ as the oxidant and methanol as the solvent at room temperature. This reaction is operationally simple with an easy work-up, utilizes mild reaction conditions, is high-yielding, clean, and exhibits high selectivity toward aromatic disulfides with no side reactions. The supported iron oxide nanoparticles can be easily recovered using an external magnet from the reaction mixture and reused several times. Reaction progress was monitored by potentiometric titration.