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Catechin adsorption on magnetic hydroxyapatite nanoparticles: A synergistic interaction with calcium ions
Journal Pre-proof Catechin Adsorption on Magnetic Hydroxyapatite Nanoparticles: A Synergistic Interaction with Calcium Ions A.H.M. Yusoff, Midhat Nabil Salimi, Subash C.B. Gopinath, A.M.M. Al Bakri, E.M. Samsudin PII:
S0254-0584(19)31152-6
DOI:
https://doi.org/10.1016/j.matchemphys.2019.122337
Reference:
MAC 122337
To appear in:
Materials Chemistry and Physics
Received Date: 12 May 2019 Revised Date:
9 October 2019
Accepted Date: 16 October 2019
Please cite this article as: A.H.M. Yusoff, Midhat Nabil Salimi, Subash C.B. Gopinath, A.M.M. Al Bakri, E.M. Samsudin, Catechin Adsorption on Magnetic Hydroxyapatite Nanoparticles: A Synergistic Interaction with Calcium Ions, Materials Chemistry and Physics (2019), doi: 10.1016/ j.matchemphys.2019.122337 This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Elsevier B.V. All rights reserved.
Catechin Adsorption on Magnetic Hydroxyapatite Nanoparticles: A Synergistic Interaction with Calcium Ions
A.H.M. Yusoff1, Midhat Nabil Salimi1*, Subash C.B. Gopinath1,2, A.M.M. Al Bakri3, E.M. Samsudin4
1
School of Bioprocess Engineering, Universiti Malaysia Perlis, 02600 Arau, Perlis, Malaysia 2 Institute of Nano Electronic Engineering, Universiti Malaysia Perlis, 01000 Kangar, Perlis, Malaysia 3 Center of Excellence Geopolymer & Green Technology, Universiti Malaysia Perlis, 02600 Arau, Perlis, Malaysia 4 School of Engineering, Taylor’s University, Taylor’s Lakeside Campus, 47500 Subang Jaya, Selangor, Malaysia
A.H.M. Yusoff - [email protected] Midhat Nabil Salimi - [email protected] Subash C.B. Gopinath [email protected] A.M.M. Al Bakri - [email protected] E.M. Samsudin - [email protected]
Correspondence: M.N. Salimi ([email protected]) Tel : 017-6325996; Fax : 04-9798755
Abstract Chemotherapeutic drug in the treatment of cancer is the developing strategy that includes the usage of herbal catechin, is being explored to get more insights. On the other hand, the broad dispersal of flavonoid compounds invariably restricts the therapeutic dosage of the drug at the point of delivery. This study is creating an effective transport mechanism for the delivery of catechin, a core-shell magnetite-hydroxyapatite nano agent (Fe3O4/HA) was used. A batch adsorption portrayed by Vibrating Sample Magnetometer, UV-Vis Spectroscopy, Transmission Electron Microscopy, Field-emission Scanning Electron Microscopy, X-ray powder Diffraction and Fourier-transform Infrared Spectroscopy was employed to investigate Fe3O4/HA on catechin adsorption. Accordingly, it was discovered that the herbal catechin functionalised on the Fe3O4/HA (Fe3O4/HA/Cat) exhibited a dimension of 93 nm and 8.81 nm for the magnetic core. In addition, the Fe3O4/HA/Cat at ambient room temperature was found to consist with properties that were superparamagnetic including acquiring a saturation magnetisation of 9.127 emu/g. Employing the Langmuir model, the data pertaining to catechin adsorption showed that the process was positive in acquiring the capacity and mechanism for higher adsorption (110.97 mg/g). Furthermore, the ability of the catechin to kinetically adsorb on the Fe3O4/HA was well evidenced as observed by applying the pseudo-second-order equation. Accordingly, the findings of this research help to show the chemical compounds and their associated interactions. Also, based on the findings of this study, the ability of Fe3O4/HA to transport catechin electrostatically was possibly attributed to the -OH component of the catechol moiety of catechin and Ca2+ on the HA shell. Keywords: Adsorption Isotherm; Bioactive Antioxidant; Catechin; Magnetic Hydroxyapatite Nanoparticle Introduction Recent developments in finding safer cancer treatment are increasing and it is known that chemotherapeutic drugs attack both cancer and normal cells that can induce highly deleterious and often life-threatening side effects [1]. The drugs for chemotherapy have been found to attack normal cells such as bone marrow cells, digestive tract cells, and hair follicle cells, that ultimately leads to low blood production, hair loss, immunosuppression, and nervous disorders [2]. The current situation urges the search for cancer treatment with a higher safety profile. In the last decades, several longitudinal studies displayed the favourable effects of tea flavonoids including green tea catechin. Catechin is a natural product that is commonly found in tea leaves. It can be subclassified into flavan-3-ols where the carbon
atoms in its structure are assembled in two benzene rings (denoted as (denoted as ring–A and ring–B) and a dihydropyran heterocycle (ring-C) with several hydroxyl groups (Figure 1). It is well known that catechin exhibits several anticancer activities by eliminating carcinogens and inhibiting the growth of cancer cells [3] and selective against cancer cells. Green tea catechin demonstrates cytotoxic activities against bladder cancer [4], colon cancer [5], and skin cancer cells [6], and its cytotoxic activity is comparable to that of chemotherapy drugs such as doxorubicin [7] and 5-fluorouracil [8]. Despite its long preclinical success, delivery of catechin into the cancer site has several problems. Some of the biggest challenges for the treatment using catechin are their poor bioavailability and low targeting at the intended sites. In many cases, only a limited amount of administered catechin dosage reaches the targeted sites, whereas the majority of them are randomly distributed to the rest of the body. A number of delivery systems using the nanoscaled particles, including nanoemulsion [9], liposome [10], and micelle [11] have been tested to overcome these problems. The studies display that catechin chelate with metallic nanoparticle can improve the targeting and induce the apoptosis of the cancer cells. Chen et al. demonstrated that catechin might conjoin with Mg2+ ions to form a chelate complex [12]. Their results indicate that anticancer activity was significantly enhanced due to the local administration of catechin. In another study, Manna et al. used Fe3+ ions to selectively adsorb catechin from natural biosources to develop chemoprotective nanoparticles [13]. However, no single study exists regarding the use of metal Ca2+ ions for catechin chelation. In this study, magnetite–hydroxyapatite nanoparticles (Fe3O4/HA) in the form of a core–shell structure was designed to construct a catechin delivery system. The cooperation of magnetite nanoparticles (Fe3O4) as a core is mostly used for the drug localisation in certain areas using external magnet [14]. Meanwhile, hydroxyapatite (HA) coating layer confers excellent biocompatible and biodegradable properties to the nanoparticles, similar to that of
the inorganic phase of vertebrate’s hard tissues. Here, we used catechin that was extracted from Ficus deltoidea leaves and subsequently used for the production of magnetite–HA– catechin nanocomposites (Fe3O4/HA/Cat). Catechin was conjugated on the Fe3O4/HA surface by utilising Ca2+ ions on the HA structure. Additionally, previous studies provide less information about adsorbate–adsorbent interaction. There is an insufficient scientific understanding of the interaction mechanism between the adsorbate (catechin) and adsorbent (Fe3O4/HA). Therefore, the adsorption process was evaluated through the adsorption isotherms and adsorption kinetics studies using the existing mathematical model. Experimental Section Materials All chemicals and reagents were used without further any purification. Sodium hydroxide (NaOH), iron(II) chloride (FeCl2·4H2O), and iron(III) chloride (FeCl3, 98%) were bought from Acros Organics. Meanwhile, pure ethanol, phosphorus pentoxide, and calcium nitrogen tetrahydrate [Ca(NO3)2·4H2O], were procured from Sigma-Aldrich UK. Dimethyl sulfoxide (DMSO) was purchased from Fisher Scientific. Deionised water was employed in all experiments. Collection of Ficus deltoidea and Extraction of catechin Leaves of Ficus deltoidea were received from a covered greenhouse garden of School of Bioprocess Engineering, Universiti Malaysia Perlis. Distilled water was used to wash the collected leaves and dried at room temperature for two weeks. Then, the dried leaves were chopped and ground. One hundred grams of the grounded leaves were then macerated in methanol and distilled water at a volumetric ratio of 60:40 for 24 h. Whatman filter paper was used to filter the extracts, and the solvent was evaporated from the samples using a rotary evaporator. The filtrate was further extracted to separate catechin. The aqueous crude extract
was shaken vigorously with chloroform to eliminate caffeine and other impurities from the tea infusion. Afterwards, catechin was extracted from the water layer using ethyl acetate. Catechin was then further concentrated using a rotary evaporator (defined as catechin extract). The catechin extract was preserved at 4 °C. The purity of the catechin extract was checked using a UV–visible (UV–vis) spectrophotometer. A standard curve of catechin extract was obtained prior to the catechin attachment using different concentrations (100–500 µg/mL). The standard curve was plotted based on their respective absorbance (λmax = 280 nm). The information on the standard curve equation and regression value were obtained. Preparation of magnetite-hydroxyapatite nanoparticles (Fe3O4/HA) The synthesis of Fe3O4 nanoparticles was performed via a coprecipitation technique with ferrous chloride and ferric chloride in a 1:2 ratio [15]. The iron precursor solution was first mixed and magnetically stirred until the temperature reached 70 °C. Subsequently, 5 mL of NaOH was dropped into the mixture and the mixture was permitted to stir for another 30 min prior to the collection of Fe3O4 nanoparticles by centrifugation. The precipitate was washed thoroughly with deionised water and then used directly to coat with HA. HA coating was employed by using a rapid sol–gel route reported elsewhere [16]. The stoichiometric amounts of phosphorus pentoxide and calcium nitrate tetrahydrate were dissolved in a separate ethanol solution (molar ratio of 10:6). Fe3O4 nanoparticles (10 mg/mL) were added dropwise into the calcium nitrate tetrahydrate solution under sonication. Next, the mixture was added with phosphorus pentoxide and stirred for 1 h at 500 rpm using an overhead stirrer, which was fitted with a Rushton turbine impeller. After an hour, the speed of the overhead stirrer was adjusted to 200 rpm and continued to stir for another 1 h to allow the gelation process to occur. Then the ageing process was done for 1 h at room
temperature. After that, a rotary evaporator was used to dry the aged gel at 50 mbar in a water bath at 60 °C for 1 h to remove the solvent. The dried samples were further calcined at 800 °C with a heating rate of 5 °C/5 min and then cooled to room temperature before being ground into powder form. Adsorption experiments Batch mode adsorption experiments were performed to determine the loading capacity of catechin on Fe3O4/HA nanocomposites. Preliminary experiments were done to determine the optimal condition for the batch adsorption. For equilibrium adsorption isotherm experiment, 10 mL of catechin extract solution with desired concentrations was mixed with 3 mg of Fe3O4/HA nanocomposites at 25 °C. The suspension was agitated in a shaking bed at 150 rpm for 4 h. The pH of the mixture was adjusted to 5.5 using either NaOH or HCl solutions before and during the adsorption experiments. After equilibrium, the catechin extract loaded nanoparticles and remaining catechin extract was partitioned using centrifugation at 3000 rpm at 10 °C for 30 min. A UV–vis spectrophotometer was used to measure the unloaded catechin concentration at 280 nm by comparing the absorbance of the standard curve. For kinetics study, almost similar experiments were done where a fixed amount of adsorbents was suspended in 300 µg/mL of catechin extracts and samples were collected at various time intervals. The samples were collected to estimate the loaded and unloaded catechin concentrations at the following time intervals: 0.5, 1, 2, 4, 6, 12, and 24 h. The adsorption percentage and adsorption capacity at time t, qt (mg/g) of catechin extract on nanocomposites were calculated using Equations 1.1 and 1.2:
% catechin extract loading =
=
(C − C ) m
C − C X 100 C
(1.1)
(1.2)
where, Ct and C0 represent the catechin extract (mg/mL) concentration at an instant and initial time, t (min), V is the total solution volume (mL), and m is the nanocomposites mass (g). These experiments were performed in triplicates. Adsorption isotherm Adsorption isotherm experiment was carried out to describe the condition of adsorption at equilibrium. Langmuir and Freundlich adsorption isotherms are the common models used to investigate the adsorption of adsorbate on the adsorbent. Thus, both isotherm adsorption models were utilised to fit the experimental data. The Langmuir model is as follows:
=
!"
1 + b"
(1.3)
For fitting the data, the model can be linearized as:
" 1 " = + !
(1.4)
where, qe is the total of adsorbate adsorbed at equilibrium per unit of nanocomposites weight (mg/g), Qo is a constant reflecting a maximum monolayer adsorption (mg/g) , b is the Langmuir constant (mL/mg) that has a correlation to the binding energy of the adsorbate species to the active sites, and Ce is the adsorbate equilibrium concentration (mg/mL). In addition, the equilibrium parameter, RL is utilised to determine the favourability of Langmuir isotherm [17]:
%& =
1 1 + !"
(1.5)
where, C0 is the initial highest concentration of adsorbate (mg/mL) and b is the Langmuir constant (mL/mg).
Meanwhile, Freundlich isotherm can be expressed as:
)/+
= '( "
(1.6)
The linearized form of the equation is given by:
ln ( ) = ln ('( ) + (1/,)ln (" )
(1.7)
where, KF is the Freundlich constant representing the multilayer, n is adsorption capacity of adsorbent (mg/g) and 1/n is indicative the strength of the adsorption reaction to follow the Freundlich isotherm (dimensionless). 1.1.Adsorption kinetics Adsorption kinetics is the vital characteristic elucidating the adsorption efficiency by revealing the solute uptake rate at the solid-solution interface. The generally utilized kinetic models are pseudo-first and pseudo-second orders, as follows: - = /) ( − ) -.
(1.8)
- = /0 ( − )0 -.
(1.9)
where, qe and qt represent the amounts of adsorbate adsorbed (mg/g) per unit of adsorbent at equilibrium and at time t, respectively. k1 is the pseudo-first-order rate constant (1/min) and k2 is the pseudo-second-order rate constant (g/mg.min). The linear forms of pseudo-firstorder and pseudo-second-order models can be represented as follows, by integrating the equations for the boundary conditions t = 0 to t = t and qt = 0 and qt = qt: ln ( − ) = ln ( ) + /) .
(1.10)
. 1 . = + /0 0
(1.11)
In this instance, the values of qe and rate constants k1 and k2 were estimated from the slopes and intercept of the plots. The values of correlation coefficients (R2) were determined from the linear plots and used to evaluate the kinetic models. Magnetite-hydroxyapatite-catechin nanocomposites (Fe3O4/HA/Cat) characterization X-ray diffraction (XRD) analysis was performed on a Shimadzu XRD-6000 diffractometer with Cu Kα1 radiation (λ = 1.5406 Å) recorded over the 2θ range of 20° to 80°. The FTIR spectra of the samples were collected with a Perkin Elmer Spectrum GX spectrophotometer within the range of 4000 to 400 cm−1. The samples were examined in a KBr pellet. Transmission electron microscope (TEM, Philips CM 12) imaging was performed to study the morphology of the samples. The average particles sizes were also determined from the TEM images. The UV–visible spectral study was carried out by using JoscoV-650 spectrophotometer. The magnetic property of the sample was studied utilising a Lakeshore 7404 vibrating sample magnetometer (VSM). The experiment was conducted at room temperature under atmospheric air.
Results and discussion Synthesis and characterization of magnetite-hydroxyapatite-catechin nanocomposites (Fe3O4/HA/Cat) Fe3O4/HA was utilised as an adsorbent to adsorb catechin. Fe3O4/HA nanocomposites were prepared by using the rapid sol–gel route and characterised using XRD, FTIR, EDXRF, FESEM, TEM, and VSM analyses. This work focused more on the characterisation of Fe3O4/HA/Cat. The adsorption isotherms and adsorption kinetics were also investigated using the established mathematical models, which permits for a well discussion of the interaction
between the adsorbate and adsorbent, enlightening the understanding of catechin interaction with Ca2+ ions on Fe3O4/HA structure. The XRD patterns of the as-prepared powders are displayed in Figure 2A. Fe3O4/HA sample [Figure 2A (1)] shows its unified phases as it contains the peaks of both magnetite (PDF 19-0629) and HA (PDF 09-0432). There are four characteristic peaks at 2θ = 35.4°, 43.2°, 57.4°, and 62.9° that indicate the presence of Fe3O4 nanoparticles under the HA layer. In addition, typical diffraction peaks of HA at 2θ = 26.1°, 28.9°, 31.8°, 32.9°, 34.1°, 39.7°, 41.9°, 46.7°, 49.5°, and 53.2° reflection planes were also noted in the consolidated samples. These two sets of reflection planes can be assigned to the presence of both structural phase in this sample. The above-described peaks were also observed in the Fe3O4/HA/Cat XRD patterns [Figure 2A (2)]. However, diffraction peaks intensities of Fe3O4/HA/Cat were reduced compared to those of the bare Fe3O4/HA. This reduction in peak intensities is mainly due to the presence of the catechin layer over the adsorbent surfaces. The absence of peaks corresponding to catechin on the Fe3O4/HA/Cat XRD pattern indicates that catechin exists in a highly amorphous form. To assess the possible molecular interactions between adsorbate and adsorbent, the FTIR spectra of the catechin extract, unloaded Fe3O4/HA, and catechin loaded Fe3O4/HA (Fe3O4/HA/Cat) were analysed (Figure 2B). Figure 2B (1) shows that the FTIR for free catechin extract exhibits the characteristics bands of O–H stretching of phenolic groups (3423 cm−1), C–H stretching in aliphatic (2922 cm -1), C=C stretching in aliphatic and aromatic compounds (1657 cm−1), aliphatic CH2 scissor bending (1461 cm−1), aliphatic CH3 symmetric bending (1377 cm−1), C–O stretching in aliphatic and aromatic compounds (1160 cm−1), C–H bending in aromatic (1033 cm−1), and the peaks for the disubstituted aromatic ring (766 and 695 cm−1). The occurrence of these bands is consistent with the FTIR spectra of catechin analytical standard (purity ≥ 99%) reported earlier [12, 18, 19]. In the spectrum of Fe3O4/HA
[Figure 2B (2)], strong absorption bands appeared at around 602 cm−1 that correspond to the bending vibrations of the phosphate group (PO43−, O–P–O). The observed strong bands at 960, 1050, and 1100 cm−1 denote the stretching vibrations of the phosphate group (PO43−, P– O). The presence of magnetite under the HA layer can be identified through the peak at 573 cm−1, which represents the Fe–O stretching vibrations. Compared to Fe3O4/HA, the spectrum of Fe3O4/HA/Cat [Figure 2B (3)] shows a new absorption band at 3419 cm−1, which can be attributed to the O–H stretching of the phenolic group. This observation suggests that catechin is associated with the Fe3O4/HA by –OH of phenolic groups in the catechin structure. The weak absorption bands appeared at 2919 and 1631 cm−1 can be attributed to the C–H stretching of aliphatic and C=C stretching in aliphatic and aromatic rings in catechin moiety, respectively, which further confirms the adsorption of catechin on Fe3O4/HA. The UV–vis spectroscopy analysis was carried out to further confirm the adsorption of catechin on Fe3O4/HA qualitatively and quantitatively. As shown in Figure 3A, the extract showed an intense peak at 280 nm indicating the characteristic absorbance peak of catechin. The solution did not absorb any wavelengths above 300 nm demonstrating that the extract mainly consists of catechin. This observation matches well with Omar’s findings [20] that showed catechin is the most bioactive compound found for flavanols class in Ficus deltoidea leaf. Also, the characteristic wavelength of catechin appeared after the attachment onto Fe3O4/HA, suggesting the success of catechin to bind on Fe3O4/HA. Accordingly, the amount of catechin adsorbed onto the Fe3O4/HA was measured at 280 nm and will be discussed in the next subsection. The size and morphology of Fe3O4/HA were verified using FESEM and TEM micrographs (Figure 3B & C). FESEM micrograph shows a well-dispersed Fe3O4/HA/Cat particles with a uniform size of about 93 nm (Figure 3B). Additionally, a closer view was obtained through the TEM micrograph (Figure 3C) which shows that the Fe3O4/HA/Cat
nanoparticles have a size of more than 100 nm due to particle agglomeration. This result can be attributed to the magnetic dipole-dipole interactions that causes nanoparticles to lose their colloidal stability. However, it is apparent from the micrograph that the dark-coloured particles are associated with Fe3O4 nanoparticles (arrows). The spherical Fe3O4 nanoparticles have an average size of 8.81 nm. A few larger sizes of black particles were also observed which might be attributed to Fe3O4 agglomeration that occurred prior to the HA coating process. On the other hand, the micrograph clearly shows that the HA coating layer encapsulated the Fe3O4 nanoparticles with irregular morphology. Also observed in the micrograph is a layer covering the surfaces of Fe3O4/HA, which is possibly the catechin layer. The effect of the magnetic property before and after catechin adsorption on Fe3O4/HA was investigated using VSM at 300 K. Figure 4A shows that both samples demonstrated a similar hysteresis curve which is typical for superparamagnetic behaviour. The saturation magnetisation (Ms) obtained for the bare Fe3O4/HA was 23.274 emu g-1 [Figure 4A (1)]. Meanwhile, a smaller Ms was obtained after catechin adsorbed on Fe3O4/HA (Fe3O4/HA/Cat), which was 9.127 emu g−1 [Figure 4A (2)]. The lower Ms can be attributed to the diamagnetic catechin layer that can lessen the magnetic domain, resulting in lower net magnetic moments. However, the Ms of Fe3O4/HA/Cat structure found in the current study is appropriate for drug targeting as it was previously reported that 9–25 emu/g is sufficient to drag nanoparticlesdrug complexes to reach their target site in the body by external magnetic means [21-23]. In accordance with this, localization of the nanoparticles in the specific cancer site can be manipulated by the magnetic field. This approach allows for a significant decrease of side effects to the patient’s body.
Adsorption studies Adsorption isotherm The adsorption isotherms were investigated further with the existing mathematical models. Adsorption isotherm describes the adsorption condition between adsorbate and adsorbent as the process reaches an equilibrium state. There are several isotherms, but none of them is observed to fit the experimental data well because of the complex nature of the Fe3O4/HA structure. Thus, a careful evaluation was made, taking into account the significance of the basic hypothesis and the goodness of the fitted data.
In this work, Langmuir and Freundlich models were utilised as they show the best-fit curves. Furthermore, both models are often used for comparison in most literature. The adsorption isotherm plots are displayed in Figure 4B and C and the respective parameters obtained are shown in Table 1. From these data, the RL and 1/n values obtained were 0.15 and 0.42 where both values are in the favourable range for Langmuir (Figure 4B) and Freundlich (Figure 4C), respectively (0–1 for RL and 0.1–1 for 1/n). These findings suggest that the adsorption of catechin was favourable to follow both models. However, the low value of RL indicates the adsorption was more favourable to follow the Langmuir model. The current results are also supported by the higher R2 values obtained from the Langmuir model which is 0.98, while the R2 for the Freundlich model is 0.90. Also, the calculated adsorption capacity values (qe,cal) of the Langmuir model well agree with the adsorption capacities experimental results (qe,exp). These results show that the Langmuir isotherm fitted the data significantly better than the Freundlich isotherm, which implies that the adsorption of catechin on Fe3O4/HA is a monolayer adsorption in nature.
In several cases, chemisorption leads to monolayer adsorption behaviour rather than multilayer adsorption behaviour. Chemisorption involves electronic interactions between adsorbent’s surface sites and adsorbate molecules, showing a strong ionic bond. It is acknowledged that HA is an amphoteric material where the HA structure can have two interchangeable binding sites, which are the C (Ca2+) and P (PO4−) sites. Both sites can be manipulated by the pH of the medium. By suspending nanocomposites in the solution with a pH value lower than the isoelectric point (pI), the surface of HA becomes positively charged through the Ca2+ ions formation. When the pH value of the solution is greater than the pI value, negatively charged PO4− ions are formed. In our current study, the pI value of the Fe3O4/HA samples was 7.2. As the reaction was carried out at a pH of 5.5 throughout the adsorption experiment, the nanocomposites were left with positively charged Ca2+ ions on their surfaces. The aqueous solution is responsible for protonating the ≡PO− and ≡CaOH groups and forms positively charged ≡CaOH2+ and neutral ≡POH sites on the HA surfaces [24, 25]. Therefore, with respect to the positive surface charge of HA, the interaction between catechin and Fe3O4/HA was determined by electrostatic interaction. Ca2+ ions are known to exhibit a high affinity to several functional groups, such as carboxyl, phosphate, sulphate, hydroxyl, and catechol groups [26, 27]. Meanwhile, catechin is known for its metal-chelating property [12, 28]. In accordance with the FTIR results, we can infer that catechin chelates Ca2+ through the –OH part in B ring. The chemical structure of catechin and schematic illustration of the Fe3O4/HA/Cat formation are shown in Figure 5A. In reviewing the literature, this finding supports earlier studies. Chen et al. fabricated Mg(II)–catechin nanocomposite particles in a one-step approach [12]. They also deduce that catechin chelate metal Mg2+ on –OH part in B ring to form a new complex.
Adsorption kinetics Adsorption kinetics is another important characteristic describing the adsorbate–adsorbent sorption mechanism. The fundamental knowledge of adsorption kinetics is crucial for the design and scale-up of adsorbers, especially for industries. Two kinetic models were analysed to investigate the controlling mechanism of the adsorption process, namely pseudo-first- and pseudo-second-order models. Pseudo-first-order reaction corresponds to a diffusioncontrolled process while the pseudo-second-order reaction describes a chemically rate controlled adsorption mechanism. Using appropriate data, the kinetic data that fit into linear equations can be used to determine the reaction order of an unknown or unclear reaction.
The linearised plots of both models are given in Figure 5B & C. There is a significant difference between the two models. It is clear that the reaction well fitted the pseudo-secondorder kinetic model (Figure 5C), whereas the pseudo-first-order kinetic model (Figure 5B) provided a poor fit for the experimental data. The validity of each model was checked by the R2 value as well as the kinetic parameters (Table 2). The R2 value obtained for the pseudosecond-order kinetic model is closer to unity. Furthermore, the calculated adsorption capacity values (qe2,calc) of the pseudo-second-order kinetic model was in good agreement with the experimental results (qe,exp). Thus, it can be concluded that the pseudo-second-order kinetic model is appropriate to elucidate the adsorption of catechin on Fe3O4/HA. Accordingly, these results suggest that the nature of the adsorption process inclines towards chemisorption. The rate-limiting step in catechin and Ca2+ interaction is chemically rate controlled which involves valence forces through the exchange of electrons between adsorbate and adsorbent.
The pseudo-second-order kinetic model is widely applicable compared to the pseudofirst-order kinetic model. The main advantage of utilising this model is that it is not necessary
to know equilibrium capacity, rate constant, and initial adsorption rate from experiment beforehand [29]. Therefore, it has a small sensitivity to the influence of random experimental errors. Although little is known about the interaction mechanism between catechin and Fe3O4/HA, there are many reports on the interaction mechanism between HA-based and other common therapeutic agents. The Langmuir model and pseudo-second-order kinetic model successfully explained the adsorption of anticancer doxorubicin (DOX) [30], antibiotic oxytetracycline [31], and haemoglobin [32] onto the HA-based adsorbents. For instance, Gu et al. [30] investigated the fast loading of DOX on mesoporous HA. They found that DOX adsorption on HA followed the pseudo-second-order kinetic model and the equilibrium data best fitted the Langmuir model. Similar findings were reported by Bharath et al. in the study of haemoglobin adsorption on Fe3O4/HA nanocomposites [32]. This study aids to make a path to utilise other potential nanomaterials demonstrated in the past [33–38] towards drug delivery purposes. Conclusion In the present work, the adsorption of catechin on core–shell adsorbent consisting of Fe3O4 and HA was studied. The Fe3O4/HA powders were synthesised via rapid sol–gel technique for catechin adsorption from aqueous solution. The as-prepared Fe3O4/HA/Cat nanoparticles were well dispersed and exhibited superparamagnetic property. A clear picture of the interaction between catechin and Fe3O4/HA was inferred from the results of the adsorption isotherms and adsorption kinetics. The output demonstrates that the equilibrium data followed the Langmuir isotherm model compared to the Freundlich model. Moreover, the adsorption kinetics could be best explained by the pseudo-second-order kinetic model than the pseudo-first-order kinetic model, which indicates that the electrostatic interaction proved
to be the dominant mechanism for the adsorption between catechin and Fe3O4/HA nanocomposites. The catechol moieties in the B ring of catechin structure seem to be important for Ca2+ ion chelation. Taken together, this adsorption analysis provides a basic understanding of the adsorption mechanism and useful for the generation of nanoparticlesbased drug delivery system, particularly for phytochemicals. Conflict of interest The authors do not have any conflict of interest to declare.
Data Availability Data is available without any restriction.
Approval All authors approved this submission and not submitted anywhere.
Author Contributions: A.H.M. Yusoff: Conceptualization, Methodology, Data Analysis, Writing-Original draft preparation, Investigation. Midhat Nabil Salimi: Conceptualization, Methodology, Data Analysis, Writing-Original draft preparation Subash C.B. Gopinath: Data Analysis, Reviewing and Editing. A.M.M. Al Bakri: Visualization, Validation, Reviewing and Editing. E.M. Samsudin: Methodology, Reviewing and Editing. References [1]
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Figure Legends Figure 1: Schematic overview of the nanoadsorption on magnetic hydroxyapatite particle towards cancer treatment. Figure 2: (A) XRD patterns for (1) Fe3O4/HA and (2) Fe3O4/HA/Cat. (B) FTIR spectra of (1) catechin extract, (2) Fe3O4/HA and (3) Fe3O4/HA/Cat. Figure 3: (A) UV-Vis spectra of catechin extract, Fe3O4/HA and Fe3O4/HA/Cat. Figure inset is displaying the structure of catechin. Micrographs of the as-prepared Fe3O4/HA, (B) by FESEM and (C) by TEM. The inset is the magnified image showing the Fe3O4 nanoparticles and catechin layer. Figure 4: (A) Magnetization curve at room temperature (300 K) of (1) Fe3O4/HA and (2) Fe3O4/HA/Cat. (B) Langmuir (C) and Freundlich adsorption isotherm plots of catechin onto Fe3O4/HA. Figure 5: (A) Schematic diagram of the fabrication of catechin conjugated on Fe3O4/HA. (B) Adsorption kinetics of catechin adsorbed by Fe3O4/HA using pseudo-first-order and (C) using pseudo-second order.
Table 1: Langmuir and Freundlich isotherm model constant with correlation coefficient Isotherm model
Langmuir
Freundlich
Parameter qe,exp (mg/g) qe,cal (mg/g) Qo (mg/g) b (mL/mg) R2 RL qe,calc (mg/g) KF (mg/g.(mg/mL)-1/n) R2 1/n
Value 110.97 113.88 135.14 0.011 0.98 0.15 124.50 9.28 0.90 0.42
Table 2: Comparison of pseudo-first order and pseudo-second order adsorption rate constant of catechin extract on Fe3O4/HA Kinetic model
Pseudo-first order
Pseudo-second order
Parameter qe,exp (mg/g) qe1,calc (mg/g) k1 × 104 (1/min) R2 qe2,calc (mg/g) k2 × 104 (g/mg.min) R2
Value 82.46 49.82 5.00 0.72 83.33 3.69 0.99
• • • • •
Catechin for treating cancer, we established Fe3O4/HAp, a delivery system Catechin adsorption on Fe3O4/HAp studied by FTIR, XRD, FESEM, TEM, UV-Vis, VSM Functionalized Fe3O4/HAp showed a size of 93 nm and magnetic core size 8.81 nm Saturation magnetization (9.13emu/g) & Langmuir model adsorption (110.97mg/g) shown Kinetic by pseudo-second-order, electrostatic between -OH of catechin and metal Ca2+
S0254-0584(19)31152-6
DOI:
https://doi.org/10.1016/j.matchemphys.2019.122337
Reference:
MAC 122337
To appear in:
Materials Chemistry and Physics
Received Date: 12 May 2019 Revised Date:
9 October 2019
Accepted Date: 16 October 2019
Please cite this article as: A.H.M. Yusoff, Midhat Nabil Salimi, Subash C.B. Gopinath, A.M.M. Al Bakri, E.M. Samsudin, Catechin Adsorption on Magnetic Hydroxyapatite Nanoparticles: A Synergistic Interaction with Calcium Ions, Materials Chemistry and Physics (2019), doi: 10.1016/ j.matchemphys.2019.122337 This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Elsevier B.V. All rights reserved.
Catechin Adsorption on Magnetic Hydroxyapatite Nanoparticles: A Synergistic Interaction with Calcium Ions
A.H.M. Yusoff1, Midhat Nabil Salimi1*, Subash C.B. Gopinath1,2, A.M.M. Al Bakri3, E.M. Samsudin4
1
School of Bioprocess Engineering, Universiti Malaysia Perlis, 02600 Arau, Perlis, Malaysia 2 Institute of Nano Electronic Engineering, Universiti Malaysia Perlis, 01000 Kangar, Perlis, Malaysia 3 Center of Excellence Geopolymer & Green Technology, Universiti Malaysia Perlis, 02600 Arau, Perlis, Malaysia 4 School of Engineering, Taylor’s University, Taylor’s Lakeside Campus, 47500 Subang Jaya, Selangor, Malaysia
A.H.M. Yusoff - [email protected] Midhat Nabil Salimi - [email protected] Subash C.B. Gopinath [email protected] A.M.M. Al Bakri - [email protected] E.M. Samsudin - [email protected]
Correspondence: M.N. Salimi ([email protected]) Tel : 017-6325996; Fax : 04-9798755
Abstract Chemotherapeutic drug in the treatment of cancer is the developing strategy that includes the usage of herbal catechin, is being explored to get more insights. On the other hand, the broad dispersal of flavonoid compounds invariably restricts the therapeutic dosage of the drug at the point of delivery. This study is creating an effective transport mechanism for the delivery of catechin, a core-shell magnetite-hydroxyapatite nano agent (Fe3O4/HA) was used. A batch adsorption portrayed by Vibrating Sample Magnetometer, UV-Vis Spectroscopy, Transmission Electron Microscopy, Field-emission Scanning Electron Microscopy, X-ray powder Diffraction and Fourier-transform Infrared Spectroscopy was employed to investigate Fe3O4/HA on catechin adsorption. Accordingly, it was discovered that the herbal catechin functionalised on the Fe3O4/HA (Fe3O4/HA/Cat) exhibited a dimension of 93 nm and 8.81 nm for the magnetic core. In addition, the Fe3O4/HA/Cat at ambient room temperature was found to consist with properties that were superparamagnetic including acquiring a saturation magnetisation of 9.127 emu/g. Employing the Langmuir model, the data pertaining to catechin adsorption showed that the process was positive in acquiring the capacity and mechanism for higher adsorption (110.97 mg/g). Furthermore, the ability of the catechin to kinetically adsorb on the Fe3O4/HA was well evidenced as observed by applying the pseudo-second-order equation. Accordingly, the findings of this research help to show the chemical compounds and their associated interactions. Also, based on the findings of this study, the ability of Fe3O4/HA to transport catechin electrostatically was possibly attributed to the -OH component of the catechol moiety of catechin and Ca2+ on the HA shell. Keywords: Adsorption Isotherm; Bioactive Antioxidant; Catechin; Magnetic Hydroxyapatite Nanoparticle Introduction Recent developments in finding safer cancer treatment are increasing and it is known that chemotherapeutic drugs attack both cancer and normal cells that can induce highly deleterious and often life-threatening side effects [1]. The drugs for chemotherapy have been found to attack normal cells such as bone marrow cells, digestive tract cells, and hair follicle cells, that ultimately leads to low blood production, hair loss, immunosuppression, and nervous disorders [2]. The current situation urges the search for cancer treatment with a higher safety profile. In the last decades, several longitudinal studies displayed the favourable effects of tea flavonoids including green tea catechin. Catechin is a natural product that is commonly found in tea leaves. It can be subclassified into flavan-3-ols where the carbon
atoms in its structure are assembled in two benzene rings (denoted as (denoted as ring–A and ring–B) and a dihydropyran heterocycle (ring-C) with several hydroxyl groups (Figure 1). It is well known that catechin exhibits several anticancer activities by eliminating carcinogens and inhibiting the growth of cancer cells [3] and selective against cancer cells. Green tea catechin demonstrates cytotoxic activities against bladder cancer [4], colon cancer [5], and skin cancer cells [6], and its cytotoxic activity is comparable to that of chemotherapy drugs such as doxorubicin [7] and 5-fluorouracil [8]. Despite its long preclinical success, delivery of catechin into the cancer site has several problems. Some of the biggest challenges for the treatment using catechin are their poor bioavailability and low targeting at the intended sites. In many cases, only a limited amount of administered catechin dosage reaches the targeted sites, whereas the majority of them are randomly distributed to the rest of the body. A number of delivery systems using the nanoscaled particles, including nanoemulsion [9], liposome [10], and micelle [11] have been tested to overcome these problems. The studies display that catechin chelate with metallic nanoparticle can improve the targeting and induce the apoptosis of the cancer cells. Chen et al. demonstrated that catechin might conjoin with Mg2+ ions to form a chelate complex [12]. Their results indicate that anticancer activity was significantly enhanced due to the local administration of catechin. In another study, Manna et al. used Fe3+ ions to selectively adsorb catechin from natural biosources to develop chemoprotective nanoparticles [13]. However, no single study exists regarding the use of metal Ca2+ ions for catechin chelation. In this study, magnetite–hydroxyapatite nanoparticles (Fe3O4/HA) in the form of a core–shell structure was designed to construct a catechin delivery system. The cooperation of magnetite nanoparticles (Fe3O4) as a core is mostly used for the drug localisation in certain areas using external magnet [14]. Meanwhile, hydroxyapatite (HA) coating layer confers excellent biocompatible and biodegradable properties to the nanoparticles, similar to that of
the inorganic phase of vertebrate’s hard tissues. Here, we used catechin that was extracted from Ficus deltoidea leaves and subsequently used for the production of magnetite–HA– catechin nanocomposites (Fe3O4/HA/Cat). Catechin was conjugated on the Fe3O4/HA surface by utilising Ca2+ ions on the HA structure. Additionally, previous studies provide less information about adsorbate–adsorbent interaction. There is an insufficient scientific understanding of the interaction mechanism between the adsorbate (catechin) and adsorbent (Fe3O4/HA). Therefore, the adsorption process was evaluated through the adsorption isotherms and adsorption kinetics studies using the existing mathematical model. Experimental Section Materials All chemicals and reagents were used without further any purification. Sodium hydroxide (NaOH), iron(II) chloride (FeCl2·4H2O), and iron(III) chloride (FeCl3, 98%) were bought from Acros Organics. Meanwhile, pure ethanol, phosphorus pentoxide, and calcium nitrogen tetrahydrate [Ca(NO3)2·4H2O], were procured from Sigma-Aldrich UK. Dimethyl sulfoxide (DMSO) was purchased from Fisher Scientific. Deionised water was employed in all experiments. Collection of Ficus deltoidea and Extraction of catechin Leaves of Ficus deltoidea were received from a covered greenhouse garden of School of Bioprocess Engineering, Universiti Malaysia Perlis. Distilled water was used to wash the collected leaves and dried at room temperature for two weeks. Then, the dried leaves were chopped and ground. One hundred grams of the grounded leaves were then macerated in methanol and distilled water at a volumetric ratio of 60:40 for 24 h. Whatman filter paper was used to filter the extracts, and the solvent was evaporated from the samples using a rotary evaporator. The filtrate was further extracted to separate catechin. The aqueous crude extract
was shaken vigorously with chloroform to eliminate caffeine and other impurities from the tea infusion. Afterwards, catechin was extracted from the water layer using ethyl acetate. Catechin was then further concentrated using a rotary evaporator (defined as catechin extract). The catechin extract was preserved at 4 °C. The purity of the catechin extract was checked using a UV–visible (UV–vis) spectrophotometer. A standard curve of catechin extract was obtained prior to the catechin attachment using different concentrations (100–500 µg/mL). The standard curve was plotted based on their respective absorbance (λmax = 280 nm). The information on the standard curve equation and regression value were obtained. Preparation of magnetite-hydroxyapatite nanoparticles (Fe3O4/HA) The synthesis of Fe3O4 nanoparticles was performed via a coprecipitation technique with ferrous chloride and ferric chloride in a 1:2 ratio [15]. The iron precursor solution was first mixed and magnetically stirred until the temperature reached 70 °C. Subsequently, 5 mL of NaOH was dropped into the mixture and the mixture was permitted to stir for another 30 min prior to the collection of Fe3O4 nanoparticles by centrifugation. The precipitate was washed thoroughly with deionised water and then used directly to coat with HA. HA coating was employed by using a rapid sol–gel route reported elsewhere [16]. The stoichiometric amounts of phosphorus pentoxide and calcium nitrate tetrahydrate were dissolved in a separate ethanol solution (molar ratio of 10:6). Fe3O4 nanoparticles (10 mg/mL) were added dropwise into the calcium nitrate tetrahydrate solution under sonication. Next, the mixture was added with phosphorus pentoxide and stirred for 1 h at 500 rpm using an overhead stirrer, which was fitted with a Rushton turbine impeller. After an hour, the speed of the overhead stirrer was adjusted to 200 rpm and continued to stir for another 1 h to allow the gelation process to occur. Then the ageing process was done for 1 h at room
temperature. After that, a rotary evaporator was used to dry the aged gel at 50 mbar in a water bath at 60 °C for 1 h to remove the solvent. The dried samples were further calcined at 800 °C with a heating rate of 5 °C/5 min and then cooled to room temperature before being ground into powder form. Adsorption experiments Batch mode adsorption experiments were performed to determine the loading capacity of catechin on Fe3O4/HA nanocomposites. Preliminary experiments were done to determine the optimal condition for the batch adsorption. For equilibrium adsorption isotherm experiment, 10 mL of catechin extract solution with desired concentrations was mixed with 3 mg of Fe3O4/HA nanocomposites at 25 °C. The suspension was agitated in a shaking bed at 150 rpm for 4 h. The pH of the mixture was adjusted to 5.5 using either NaOH or HCl solutions before and during the adsorption experiments. After equilibrium, the catechin extract loaded nanoparticles and remaining catechin extract was partitioned using centrifugation at 3000 rpm at 10 °C for 30 min. A UV–vis spectrophotometer was used to measure the unloaded catechin concentration at 280 nm by comparing the absorbance of the standard curve. For kinetics study, almost similar experiments were done where a fixed amount of adsorbents was suspended in 300 µg/mL of catechin extracts and samples were collected at various time intervals. The samples were collected to estimate the loaded and unloaded catechin concentrations at the following time intervals: 0.5, 1, 2, 4, 6, 12, and 24 h. The adsorption percentage and adsorption capacity at time t, qt (mg/g) of catechin extract on nanocomposites were calculated using Equations 1.1 and 1.2:
% catechin extract loading =
=
(C − C ) m
C − C X 100 C
(1.1)
(1.2)
where, Ct and C0 represent the catechin extract (mg/mL) concentration at an instant and initial time, t (min), V is the total solution volume (mL), and m is the nanocomposites mass (g). These experiments were performed in triplicates. Adsorption isotherm Adsorption isotherm experiment was carried out to describe the condition of adsorption at equilibrium. Langmuir and Freundlich adsorption isotherms are the common models used to investigate the adsorption of adsorbate on the adsorbent. Thus, both isotherm adsorption models were utilised to fit the experimental data. The Langmuir model is as follows:
=
!"
1 + b"
(1.3)
For fitting the data, the model can be linearized as:
" 1 " = + !
(1.4)
where, qe is the total of adsorbate adsorbed at equilibrium per unit of nanocomposites weight (mg/g), Qo is a constant reflecting a maximum monolayer adsorption (mg/g) , b is the Langmuir constant (mL/mg) that has a correlation to the binding energy of the adsorbate species to the active sites, and Ce is the adsorbate equilibrium concentration (mg/mL). In addition, the equilibrium parameter, RL is utilised to determine the favourability of Langmuir isotherm [17]:
%& =
1 1 + !"
(1.5)
where, C0 is the initial highest concentration of adsorbate (mg/mL) and b is the Langmuir constant (mL/mg).
Meanwhile, Freundlich isotherm can be expressed as:
)/+
= '( "
(1.6)
The linearized form of the equation is given by:
ln ( ) = ln ('( ) + (1/,)ln (" )
(1.7)
where, KF is the Freundlich constant representing the multilayer, n is adsorption capacity of adsorbent (mg/g) and 1/n is indicative the strength of the adsorption reaction to follow the Freundlich isotherm (dimensionless). 1.1.Adsorption kinetics Adsorption kinetics is the vital characteristic elucidating the adsorption efficiency by revealing the solute uptake rate at the solid-solution interface. The generally utilized kinetic models are pseudo-first and pseudo-second orders, as follows: - = /) ( − ) -.
(1.8)
- = /0 ( − )0 -.
(1.9)
where, qe and qt represent the amounts of adsorbate adsorbed (mg/g) per unit of adsorbent at equilibrium and at time t, respectively. k1 is the pseudo-first-order rate constant (1/min) and k2 is the pseudo-second-order rate constant (g/mg.min). The linear forms of pseudo-firstorder and pseudo-second-order models can be represented as follows, by integrating the equations for the boundary conditions t = 0 to t = t and qt = 0 and qt = qt: ln ( − ) = ln ( ) + /) .
(1.10)
. 1 . = + /0 0
(1.11)
In this instance, the values of qe and rate constants k1 and k2 were estimated from the slopes and intercept of the plots. The values of correlation coefficients (R2) were determined from the linear plots and used to evaluate the kinetic models. Magnetite-hydroxyapatite-catechin nanocomposites (Fe3O4/HA/Cat) characterization X-ray diffraction (XRD) analysis was performed on a Shimadzu XRD-6000 diffractometer with Cu Kα1 radiation (λ = 1.5406 Å) recorded over the 2θ range of 20° to 80°. The FTIR spectra of the samples were collected with a Perkin Elmer Spectrum GX spectrophotometer within the range of 4000 to 400 cm−1. The samples were examined in a KBr pellet. Transmission electron microscope (TEM, Philips CM 12) imaging was performed to study the morphology of the samples. The average particles sizes were also determined from the TEM images. The UV–visible spectral study was carried out by using JoscoV-650 spectrophotometer. The magnetic property of the sample was studied utilising a Lakeshore 7404 vibrating sample magnetometer (VSM). The experiment was conducted at room temperature under atmospheric air.
Results and discussion Synthesis and characterization of magnetite-hydroxyapatite-catechin nanocomposites (Fe3O4/HA/Cat) Fe3O4/HA was utilised as an adsorbent to adsorb catechin. Fe3O4/HA nanocomposites were prepared by using the rapid sol–gel route and characterised using XRD, FTIR, EDXRF, FESEM, TEM, and VSM analyses. This work focused more on the characterisation of Fe3O4/HA/Cat. The adsorption isotherms and adsorption kinetics were also investigated using the established mathematical models, which permits for a well discussion of the interaction
between the adsorbate and adsorbent, enlightening the understanding of catechin interaction with Ca2+ ions on Fe3O4/HA structure. The XRD patterns of the as-prepared powders are displayed in Figure 2A. Fe3O4/HA sample [Figure 2A (1)] shows its unified phases as it contains the peaks of both magnetite (PDF 19-0629) and HA (PDF 09-0432). There are four characteristic peaks at 2θ = 35.4°, 43.2°, 57.4°, and 62.9° that indicate the presence of Fe3O4 nanoparticles under the HA layer. In addition, typical diffraction peaks of HA at 2θ = 26.1°, 28.9°, 31.8°, 32.9°, 34.1°, 39.7°, 41.9°, 46.7°, 49.5°, and 53.2° reflection planes were also noted in the consolidated samples. These two sets of reflection planes can be assigned to the presence of both structural phase in this sample. The above-described peaks were also observed in the Fe3O4/HA/Cat XRD patterns [Figure 2A (2)]. However, diffraction peaks intensities of Fe3O4/HA/Cat were reduced compared to those of the bare Fe3O4/HA. This reduction in peak intensities is mainly due to the presence of the catechin layer over the adsorbent surfaces. The absence of peaks corresponding to catechin on the Fe3O4/HA/Cat XRD pattern indicates that catechin exists in a highly amorphous form. To assess the possible molecular interactions between adsorbate and adsorbent, the FTIR spectra of the catechin extract, unloaded Fe3O4/HA, and catechin loaded Fe3O4/HA (Fe3O4/HA/Cat) were analysed (Figure 2B). Figure 2B (1) shows that the FTIR for free catechin extract exhibits the characteristics bands of O–H stretching of phenolic groups (3423 cm−1), C–H stretching in aliphatic (2922 cm -1), C=C stretching in aliphatic and aromatic compounds (1657 cm−1), aliphatic CH2 scissor bending (1461 cm−1), aliphatic CH3 symmetric bending (1377 cm−1), C–O stretching in aliphatic and aromatic compounds (1160 cm−1), C–H bending in aromatic (1033 cm−1), and the peaks for the disubstituted aromatic ring (766 and 695 cm−1). The occurrence of these bands is consistent with the FTIR spectra of catechin analytical standard (purity ≥ 99%) reported earlier [12, 18, 19]. In the spectrum of Fe3O4/HA
[Figure 2B (2)], strong absorption bands appeared at around 602 cm−1 that correspond to the bending vibrations of the phosphate group (PO43−, O–P–O). The observed strong bands at 960, 1050, and 1100 cm−1 denote the stretching vibrations of the phosphate group (PO43−, P– O). The presence of magnetite under the HA layer can be identified through the peak at 573 cm−1, which represents the Fe–O stretching vibrations. Compared to Fe3O4/HA, the spectrum of Fe3O4/HA/Cat [Figure 2B (3)] shows a new absorption band at 3419 cm−1, which can be attributed to the O–H stretching of the phenolic group. This observation suggests that catechin is associated with the Fe3O4/HA by –OH of phenolic groups in the catechin structure. The weak absorption bands appeared at 2919 and 1631 cm−1 can be attributed to the C–H stretching of aliphatic and C=C stretching in aliphatic and aromatic rings in catechin moiety, respectively, which further confirms the adsorption of catechin on Fe3O4/HA. The UV–vis spectroscopy analysis was carried out to further confirm the adsorption of catechin on Fe3O4/HA qualitatively and quantitatively. As shown in Figure 3A, the extract showed an intense peak at 280 nm indicating the characteristic absorbance peak of catechin. The solution did not absorb any wavelengths above 300 nm demonstrating that the extract mainly consists of catechin. This observation matches well with Omar’s findings [20] that showed catechin is the most bioactive compound found for flavanols class in Ficus deltoidea leaf. Also, the characteristic wavelength of catechin appeared after the attachment onto Fe3O4/HA, suggesting the success of catechin to bind on Fe3O4/HA. Accordingly, the amount of catechin adsorbed onto the Fe3O4/HA was measured at 280 nm and will be discussed in the next subsection. The size and morphology of Fe3O4/HA were verified using FESEM and TEM micrographs (Figure 3B & C). FESEM micrograph shows a well-dispersed Fe3O4/HA/Cat particles with a uniform size of about 93 nm (Figure 3B). Additionally, a closer view was obtained through the TEM micrograph (Figure 3C) which shows that the Fe3O4/HA/Cat
nanoparticles have a size of more than 100 nm due to particle agglomeration. This result can be attributed to the magnetic dipole-dipole interactions that causes nanoparticles to lose their colloidal stability. However, it is apparent from the micrograph that the dark-coloured particles are associated with Fe3O4 nanoparticles (arrows). The spherical Fe3O4 nanoparticles have an average size of 8.81 nm. A few larger sizes of black particles were also observed which might be attributed to Fe3O4 agglomeration that occurred prior to the HA coating process. On the other hand, the micrograph clearly shows that the HA coating layer encapsulated the Fe3O4 nanoparticles with irregular morphology. Also observed in the micrograph is a layer covering the surfaces of Fe3O4/HA, which is possibly the catechin layer. The effect of the magnetic property before and after catechin adsorption on Fe3O4/HA was investigated using VSM at 300 K. Figure 4A shows that both samples demonstrated a similar hysteresis curve which is typical for superparamagnetic behaviour. The saturation magnetisation (Ms) obtained for the bare Fe3O4/HA was 23.274 emu g-1 [Figure 4A (1)]. Meanwhile, a smaller Ms was obtained after catechin adsorbed on Fe3O4/HA (Fe3O4/HA/Cat), which was 9.127 emu g−1 [Figure 4A (2)]. The lower Ms can be attributed to the diamagnetic catechin layer that can lessen the magnetic domain, resulting in lower net magnetic moments. However, the Ms of Fe3O4/HA/Cat structure found in the current study is appropriate for drug targeting as it was previously reported that 9–25 emu/g is sufficient to drag nanoparticlesdrug complexes to reach their target site in the body by external magnetic means [21-23]. In accordance with this, localization of the nanoparticles in the specific cancer site can be manipulated by the magnetic field. This approach allows for a significant decrease of side effects to the patient’s body.
Adsorption studies Adsorption isotherm The adsorption isotherms were investigated further with the existing mathematical models. Adsorption isotherm describes the adsorption condition between adsorbate and adsorbent as the process reaches an equilibrium state. There are several isotherms, but none of them is observed to fit the experimental data well because of the complex nature of the Fe3O4/HA structure. Thus, a careful evaluation was made, taking into account the significance of the basic hypothesis and the goodness of the fitted data.
In this work, Langmuir and Freundlich models were utilised as they show the best-fit curves. Furthermore, both models are often used for comparison in most literature. The adsorption isotherm plots are displayed in Figure 4B and C and the respective parameters obtained are shown in Table 1. From these data, the RL and 1/n values obtained were 0.15 and 0.42 where both values are in the favourable range for Langmuir (Figure 4B) and Freundlich (Figure 4C), respectively (0–1 for RL and 0.1–1 for 1/n). These findings suggest that the adsorption of catechin was favourable to follow both models. However, the low value of RL indicates the adsorption was more favourable to follow the Langmuir model. The current results are also supported by the higher R2 values obtained from the Langmuir model which is 0.98, while the R2 for the Freundlich model is 0.90. Also, the calculated adsorption capacity values (qe,cal) of the Langmuir model well agree with the adsorption capacities experimental results (qe,exp). These results show that the Langmuir isotherm fitted the data significantly better than the Freundlich isotherm, which implies that the adsorption of catechin on Fe3O4/HA is a monolayer adsorption in nature.
In several cases, chemisorption leads to monolayer adsorption behaviour rather than multilayer adsorption behaviour. Chemisorption involves electronic interactions between adsorbent’s surface sites and adsorbate molecules, showing a strong ionic bond. It is acknowledged that HA is an amphoteric material where the HA structure can have two interchangeable binding sites, which are the C (Ca2+) and P (PO4−) sites. Both sites can be manipulated by the pH of the medium. By suspending nanocomposites in the solution with a pH value lower than the isoelectric point (pI), the surface of HA becomes positively charged through the Ca2+ ions formation. When the pH value of the solution is greater than the pI value, negatively charged PO4− ions are formed. In our current study, the pI value of the Fe3O4/HA samples was 7.2. As the reaction was carried out at a pH of 5.5 throughout the adsorption experiment, the nanocomposites were left with positively charged Ca2+ ions on their surfaces. The aqueous solution is responsible for protonating the ≡PO− and ≡CaOH groups and forms positively charged ≡CaOH2+ and neutral ≡POH sites on the HA surfaces [24, 25]. Therefore, with respect to the positive surface charge of HA, the interaction between catechin and Fe3O4/HA was determined by electrostatic interaction. Ca2+ ions are known to exhibit a high affinity to several functional groups, such as carboxyl, phosphate, sulphate, hydroxyl, and catechol groups [26, 27]. Meanwhile, catechin is known for its metal-chelating property [12, 28]. In accordance with the FTIR results, we can infer that catechin chelates Ca2+ through the –OH part in B ring. The chemical structure of catechin and schematic illustration of the Fe3O4/HA/Cat formation are shown in Figure 5A. In reviewing the literature, this finding supports earlier studies. Chen et al. fabricated Mg(II)–catechin nanocomposite particles in a one-step approach [12]. They also deduce that catechin chelate metal Mg2+ on –OH part in B ring to form a new complex.
Adsorption kinetics Adsorption kinetics is another important characteristic describing the adsorbate–adsorbent sorption mechanism. The fundamental knowledge of adsorption kinetics is crucial for the design and scale-up of adsorbers, especially for industries. Two kinetic models were analysed to investigate the controlling mechanism of the adsorption process, namely pseudo-first- and pseudo-second-order models. Pseudo-first-order reaction corresponds to a diffusioncontrolled process while the pseudo-second-order reaction describes a chemically rate controlled adsorption mechanism. Using appropriate data, the kinetic data that fit into linear equations can be used to determine the reaction order of an unknown or unclear reaction.
The linearised plots of both models are given in Figure 5B & C. There is a significant difference between the two models. It is clear that the reaction well fitted the pseudo-secondorder kinetic model (Figure 5C), whereas the pseudo-first-order kinetic model (Figure 5B) provided a poor fit for the experimental data. The validity of each model was checked by the R2 value as well as the kinetic parameters (Table 2). The R2 value obtained for the pseudosecond-order kinetic model is closer to unity. Furthermore, the calculated adsorption capacity values (qe2,calc) of the pseudo-second-order kinetic model was in good agreement with the experimental results (qe,exp). Thus, it can be concluded that the pseudo-second-order kinetic model is appropriate to elucidate the adsorption of catechin on Fe3O4/HA. Accordingly, these results suggest that the nature of the adsorption process inclines towards chemisorption. The rate-limiting step in catechin and Ca2+ interaction is chemically rate controlled which involves valence forces through the exchange of electrons between adsorbate and adsorbent.
The pseudo-second-order kinetic model is widely applicable compared to the pseudofirst-order kinetic model. The main advantage of utilising this model is that it is not necessary
to know equilibrium capacity, rate constant, and initial adsorption rate from experiment beforehand [29]. Therefore, it has a small sensitivity to the influence of random experimental errors. Although little is known about the interaction mechanism between catechin and Fe3O4/HA, there are many reports on the interaction mechanism between HA-based and other common therapeutic agents. The Langmuir model and pseudo-second-order kinetic model successfully explained the adsorption of anticancer doxorubicin (DOX) [30], antibiotic oxytetracycline [31], and haemoglobin [32] onto the HA-based adsorbents. For instance, Gu et al. [30] investigated the fast loading of DOX on mesoporous HA. They found that DOX adsorption on HA followed the pseudo-second-order kinetic model and the equilibrium data best fitted the Langmuir model. Similar findings were reported by Bharath et al. in the study of haemoglobin adsorption on Fe3O4/HA nanocomposites [32]. This study aids to make a path to utilise other potential nanomaterials demonstrated in the past [33–38] towards drug delivery purposes. Conclusion In the present work, the adsorption of catechin on core–shell adsorbent consisting of Fe3O4 and HA was studied. The Fe3O4/HA powders were synthesised via rapid sol–gel technique for catechin adsorption from aqueous solution. The as-prepared Fe3O4/HA/Cat nanoparticles were well dispersed and exhibited superparamagnetic property. A clear picture of the interaction between catechin and Fe3O4/HA was inferred from the results of the adsorption isotherms and adsorption kinetics. The output demonstrates that the equilibrium data followed the Langmuir isotherm model compared to the Freundlich model. Moreover, the adsorption kinetics could be best explained by the pseudo-second-order kinetic model than the pseudo-first-order kinetic model, which indicates that the electrostatic interaction proved
to be the dominant mechanism for the adsorption between catechin and Fe3O4/HA nanocomposites. The catechol moieties in the B ring of catechin structure seem to be important for Ca2+ ion chelation. Taken together, this adsorption analysis provides a basic understanding of the adsorption mechanism and useful for the generation of nanoparticlesbased drug delivery system, particularly for phytochemicals. Conflict of interest The authors do not have any conflict of interest to declare.
Data Availability Data is available without any restriction.
Approval All authors approved this submission and not submitted anywhere.
Author Contributions: A.H.M. Yusoff: Conceptualization, Methodology, Data Analysis, Writing-Original draft preparation, Investigation. Midhat Nabil Salimi: Conceptualization, Methodology, Data Analysis, Writing-Original draft preparation Subash C.B. Gopinath: Data Analysis, Reviewing and Editing. A.M.M. Al Bakri: Visualization, Validation, Reviewing and Editing. E.M. Samsudin: Methodology, Reviewing and Editing. References [1]
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Figure Legends Figure 1: Schematic overview of the nanoadsorption on magnetic hydroxyapatite particle towards cancer treatment. Figure 2: (A) XRD patterns for (1) Fe3O4/HA and (2) Fe3O4/HA/Cat. (B) FTIR spectra of (1) catechin extract, (2) Fe3O4/HA and (3) Fe3O4/HA/Cat. Figure 3: (A) UV-Vis spectra of catechin extract, Fe3O4/HA and Fe3O4/HA/Cat. Figure inset is displaying the structure of catechin. Micrographs of the as-prepared Fe3O4/HA, (B) by FESEM and (C) by TEM. The inset is the magnified image showing the Fe3O4 nanoparticles and catechin layer. Figure 4: (A) Magnetization curve at room temperature (300 K) of (1) Fe3O4/HA and (2) Fe3O4/HA/Cat. (B) Langmuir (C) and Freundlich adsorption isotherm plots of catechin onto Fe3O4/HA. Figure 5: (A) Schematic diagram of the fabrication of catechin conjugated on Fe3O4/HA. (B) Adsorption kinetics of catechin adsorbed by Fe3O4/HA using pseudo-first-order and (C) using pseudo-second order.
Table 1: Langmuir and Freundlich isotherm model constant with correlation coefficient Isotherm model
Langmuir
Freundlich
Parameter qe,exp (mg/g) qe,cal (mg/g) Qo (mg/g) b (mL/mg) R2 RL qe,calc (mg/g) KF (mg/g.(mg/mL)-1/n) R2 1/n
Value 110.97 113.88 135.14 0.011 0.98 0.15 124.50 9.28 0.90 0.42
Table 2: Comparison of pseudo-first order and pseudo-second order adsorption rate constant of catechin extract on Fe3O4/HA Kinetic model
Pseudo-first order
Pseudo-second order
Parameter qe,exp (mg/g) qe1,calc (mg/g) k1 × 104 (1/min) R2 qe2,calc (mg/g) k2 × 104 (g/mg.min) R2
Value 82.46 49.82 5.00 0.72 83.33 3.69 0.99
• • • • •
Catechin for treating cancer, we established Fe3O4/HAp, a delivery system Catechin adsorption on Fe3O4/HAp studied by FTIR, XRD, FESEM, TEM, UV-Vis, VSM Functionalized Fe3O4/HAp showed a size of 93 nm and magnetic core size 8.81 nm Saturation magnetization (9.13emu/g) & Langmuir model adsorption (110.97mg/g) shown Kinetic by pseudo-second-order, electrostatic between -OH of catechin and metal Ca2+