Palladium Nanoparticles on Chitosan-Coated Superparamagnetic Manganese Ferrite: A Biocompatible Heterogeneous Catalyst for Nitroarene Reduction and Allyl Carbamate Deprotection

Although metallic nanocatalysts such as palladium nanoparticles (Pd NPs) are known to possess higher catalytic activity due to their large surface-to-volume ratio, however, in nanosize greatly reducing their activity due to aggregation. To overcome this challenge, superparamagnetic chitosan-coated manganese ferrite was successfully prepared and used as a support for the immobilization of palladium nanoparticles to overcome the above-mentioned challenge. The Pd-Chit@MnFe2O4 catalyst exhibited high catalytic activity in 4-nitrophenol and 4-nitroaniline reductions, with respective turnover frequencies of 357.1 min−1 and 571.4 min−1, respectively. The catalyst can also be recovered easily by magnetic separation after each reaction. Additionally, the Pd-Chit@MnFe2O4 catalyst performed well in the reductive deprotection of allyl carbamate. Coating the catalyst with chitosan reduced the Pd leaching and its cytotoxicity. Therefore, the catalytic activity of Pd-Chit@MnFe2O4 was proven to be unrestricted in biology conditions.


Introduction
In the field of catalysis, metallic nanocatalysts are known to possess higher catalytic activity due to their large surface-to-volume ratio [1][2][3]. To illustrate, gold nanoparticles NH 2 -MIL-101(Fe) with easier biodegradability, superior catalytic activity, and less toxicity present promising strategies for industrial wastewater treatment [4]. Other researchers, R.T. Ledari et al., provided silver nanoparticles as a catalyst. These nanoparticles stabilized with volcanic pumice and chitosan and were acknowledged as higher-yield biomolecules in industrial applications [5]. However, nanosized-catalysts are usually involved in complicated isolation and recovery processes, as well as activity loss due to their aggregation [6][7][8]. Therefore, the constraints posed by nanosized-catalysts have become a challenge in the application of transition metals as a catalyst including palladium nanoparticles (Pd NPs). Palladium as a primary catalyst has been widely used in numerous applications. Several studies have been carried out to overcome the challenges involved in the use of Pd NPs as a catalyst by immobilizing the nanosized catalyst on insoluble metal supports such as silica [9,10], carbon [11,12], and polymeric materials [13][14][15]. For instance, I. Sargin et al. reported that the preparation of chitosan-carbon nanotube-supported palladium catalyst nanoparticles are efficient in reducing nitroarenes in industrial effluents [16]. Further 4-NA can be used as an ingredient in hair dyes [51], rubber antioxidants [52], aramid textile fibre intermediates, thermoplastics, and cast elastomers [53]. Besides nitroarene reduction, Pd can also play an important catalytic role in the allyl carbamate cleavage [54]. Due to the stability of allyl carbamate in acidic and basic conditions, it is widely used in the protection of the amine group that is crucial in certain organic synthesis reactions [55,56].
In this study, a novel chitosan-coated magnetic Pd NPs catalyst (Pd-Chit@MnFe 2 O 4 ) was successfully designed for nitroarene reduction and allyl carbamate deprotection reactions. The magnetic material MnFe 2 O 4 was coated with chitosan via the ionic gelation method to provide an ideal platform for metal ion immobilization on the support material. Subsequently, the immobilization of Pd NPs on the chitosan-coated MnFe 2 O 4 was performed via the wet impregnation method. The physicochemical data and catalytic properties indicate that the catalyst has high potential for use in both chemical and biological systems.

Preparation of Pd-Chit@MnFe 2 O 4 2.2.1. Preparation of MnFe 2 O 4 Nanoparticles
MnFe 2 O 4 nanoparticles were prepared using the coprecipitation method developed by Pereira et al. [57]. Solution A was prepared by mixing 10 mmol of MnCl 2 ·4H 2 O with 5 mL of 0.1 M HCl (1:4 V/V) in a beaker. Solution B was prepared by dissolving 20 mmol of FeCl 3 ·6H 2 O in 40 mL of DIW. Both solutions were heated to 50 • C and then quickly added to a beaker containing 200 mL of 3.0 M MIPA solution. The reaction mixture was heated to 100 • C and mechanically stirred for 2 h at 1000 rpm. The freshly prepared MnFe 2 O 4 nanoparticles were magnetically isolated and dried under vacuum at room temperature overnight.

Preparation of Chitosan-Coated MnFe 2 O 4
Chitosan-coated MnFe 2 O 4 was prepared based on the method developed by Liew et al. [58]. The mass ratio of MnFe 2 O 4 to chitosan was 1:1.25. The concentration of chitosan solution was 5 mg·mL −1 . The mass and volume ratio of chitosan to TPP used for ionic gelation was 5:1 and 5:2, respectively. The freshly prepared MnFe 2 O 4 (100 mg) was dispersed in a beaker containing 25 mL of the acetic acid solution, followed by ultrasonication for 1 min at room temperature. Chitosan (125 mg) was added to the mixture and stirred at room temperature for 1 h at 1000 rpm. The TPP solution was prepared by dissolving 25 mg of TPP in 10 mL of DIW and then it was added dropwise to the MnFe 2 O 4 /chitosan mixture using a syringe pump at a rate of 1.0 mL·min −1 . The mixture was then stirred at 1000 rpm for 1 h at room temperature. The resulting product, Chit@MnFe 2 O 4 was separated using a magnet, washed with water until pH = 7, and dried under vacuum at room temperature overnight.

Preparation of Magnetic-Supported Pd Catalyst
Magnetic-supported Pd catalyst Pd-Chit@MnFe 2 O 4 was prepared by adding 100 mg of Chit@MnFe 2 O 4 in a beaker containing 5 mL of toluene and 0.05 mmol of Pd(OAc) 2 salt. The reaction mixture was stirred at 80 • C at 40 rpm for 10 min and then further stirred at room temperature for 2 h. Subsequently, the Pd 2+ ion was reduced to Pd NPs by adding 10% hydrazine hydrate solution and stirred for 30 min at room temperature with a stirring speed of 40 rpm. The freshly prepared Pd-Chit@MnFe 2 O 4 was then separated, washed with acetone, and dried under vacuum for 24 h. Superparamagnetic MnFe 2 O 4 was first synthesized through the coprecipitation method by using MIPA as the alkaline agent. Subsequently, the superparamagnetic MnFe 2 O 4 was coated with chitosan via the ionic gelation method using sodium tripolyphosphate (TPP) as a cross-linking agent. The coating approach resulted in tuned surface properties of MnFe 2 O 4 , which are ideal for use as a support for heterogeneous catalysts because chitosan has a high sorption capacity for metals and metal ions [59]. For Pd NPs immobilization, Pd(OAc) 2 was used as a Pd precursor to the introduction of free palladium(II) ions in toluene. The free palladium(II) ions were physically adsorbed on the surface of the Chit@MnFe 2 O 4 via electrostatic interaction. The solid material was then treated with hydrazine hydrate to reduce the palladium(II) ions to metallic palladium nanoparticles (Pd NPs). The loading of Pd on Pd-Chit@MnFe 2 O 4 was further investigated by ICP-MS.

Preparation of Rhodamine 110
Bis-allyloxycarbonyl-protected rhodamine 110 was produced based on the method published by Streu and Meggers [40]. Rhodamine 110 (0.5 mmol) was dissolved in 1.2 mL of dry N,N -dimethylformamide (DMF). The reaction mixture was cooled down to 0 • C under an inert atmosphere. A mixture of allyl chloroformate (1.0 mmol) and pyridine (1.5 mmol) in DMF (0.5 mL) was prepared and then added dropwise to the previous solution containing rhodamine 110. The reaction mixture was warmed to room temperature and stirred for 24 h. The product was extracted using ethyl acetate and concentrated under a vacuum. Column chromatography technique was performed by using hexane: ethyl acetate (2:1) to obtain the pure product. The preparation of non-fluorescence bis-allyloxycarbonyl-protected rhodamine 110 from a fluorescence compound rhodamine 110 by using allyl chloroformate is shown in Scheme 1.

Catalyst Characterization
The structure changes on the synthesized sample were investigated using X-ray diffraction (XRD, Bruker D8-Advanced (Bruker AXS, Bremen, Germany). The instrument

Structural Analysis
The structure changes on the synthesized sample were investigated using X-ray diffraction (XRD, Bruker D8-Advanced (Bruker AXS, Bremen, Germany). The instrument employed Cu-Kα radiation (λ = 0.15406 nm) at 30 kV and 15 mA, and scans were conducted over the 2θ range of 20 • ≤ 2θ ≤ 80 • with a scanning rate of 2 • min −1 . The patterns were matched to the Joint Committee of Powder Diffraction Standard (JCPDS)'s database. The average crystallite size of Pd and MnFe 2 O 4 was calculated by using the Debye-Scherrer formula [60]. The elemental composition and chemical state of the catalyst were determined using X-ray photoelectron spectrometry (XPS) analysis (Kratos Axis Ultra DLD X-ray photoelectron spectrometer). The catalyst sample was prepared in a pellet formed for XPS analysis. The determination of the functional group present on the prepared catalyst was investigated using Fourier transform infrared (FTIR) spectra using Agilent Technologies Cary 630 FTIR spectrometer in the range of 650-4000 cm −1 . Furthermore, the morphology and particle size of the catalyst was examined by transmission electron microscopy (TEM) using Philips CM 12 transmission electron microscope at 100 kV and field emission scanning electron microscopy (FESEM) using SUPRA 55VP Zeiss scanning electron microscope.

Elemental Analysis
The elemental composition of the Pd immobilized on the surface of Chit@MnFe 2 O 4 was determined by inductively coupled plasma mass spectrometry (ICP-MS) using a Perkin Elmer ELAN 9000 ICP mass spectrometer (USA). The loading of Pd immobilized on the surface of Chit@MnFe 2 O 4 was determined by dissolving 1.0 mg of Pd-Chit@MnFe 2 O 4 in 3 mL aqua regia. The solution was then topped-up with water to a total volume of 25 mL. The amount of Pd in the solution was determined by ICP-MS.

Magnetic Properties
The magnetic properties of the sample were analysed using vibrating sample magnetometry (VSM) (LakeShore 7404 series) vibration sample magnetometer. To distinguish the metal-oxygen species in the catalyst precursors, UV-Vis measurements were performed using a Shimadzu UV-2450 UV-Vis spectrophotometer over a wavelength range of 200~800 nm.

Catalytic Activity Catalytic Reduction of Nitroarene Compounds and Reusability
The catalytic activity of Pd-Chit@MnFe 2 O 4 was investigated in nitroarene (4-NP and 4-NA) reduction using NaBH 4 as a reducing agent. In a typical reaction, 0.15 mmol of NaBH 4 was added to 3 mL of 0.05 mM 4-NP solution, resulting in a yellow-green solution of 4-nitrophenolate. The Pd-Chit@MnFe 2 O 4 was then added to the solution mixture and the conversion of 4-NP to 4-AP took place immediately. The reduction reaction was monitored using a UV-Vis spectrometer. The optimization of 4-NP reduction was performed with the same procedures by using different masses (0.5 and 1.0 mg) of Pd-Chit@MnFe 2 O 4 . The reduction of 4-NA to 4-PDA was also carried out with the same experimental conditions by using 0.5 mg of Pd-Chit@MnFe 2 O 4 as the catalyst. Control experiments were carried out for both 4-NP and 4-NA reduction respectively by replacing the catalyst with MnFe 2 O 4 , Chit@MnFe 2 O 4 , and without catalyst. The rate of reaction was calculated from the pseudofirst-order kinetic model: where A is the concentration of nitroarene compound at t time, A 0 is the initial concentration of 4-NP or 4-NA and k is the rate of the reaction [28]. For a more accurate comparison, the turnover frequency (TOF) value was calculated according to the equation: where n 0 is the initial amount of 4-NP or 4-NA and n Pd is the number of Pd active sites on the catalyst used. The reusability of Pd-Chit@MnFe 2 O 4 was tested using 20 times scale-up nitroarene reduction in a 50 mL round bottom flask with the same reaction conditions. The catalyst was isolated using a magnet, washed with water, and used in the next cycle of nitroarene reduction. The mass of the isolated catalyst was weighed after all 8 cycles of nitroarene reduction were completed.
Deprotection of Bis-allyloxycarbonyl Rhodamine 110 with and without Thiophenol Deprotection of bis-allyloxycarbonyl rhodamine 110 was performed according to the method developed by Yusop et al. [41]. Deprotection of bis-allyloxycarbonyl rhodamine 110 was carried out in two conditions, with thiophenol, and without thiophenol. A cell density of 5000 cells/well was plated in a 96-well plate and allowed to grow for 24 h. Then, the Pd-Chit@MnFe 2 O 4 was added to the well-incubated HeLa cell and monitored with a fluorescence microscope. After 24 h, the cytotoxicity test result was compared with the untreated cell. The cytotoxicity test was performed in triplicate for both concentrations of Pd-Chit@MnFe 2 O 4 .

Structural Analysis
The  [61,62]. From the XRD diffractogram of Pd-Chit@MnFe 2 O 4 , three weak diffraction peaks were observed at 2θ = 40.1 • , 46.6 • , and 68.3 • , which were assigned to the (111), (200), and (220) planes of Pd(0), respectively, with a face-centred cubic lattice structure (JCPDS 00-046-1043) [63]. The weak intensity of the diffraction peaks is due to the low Pd content on the catalyst surface. However, the intensity of diffraction peaks related to MnFe 2 O 4 reduced due to chitosan coating and immobilization of Pd NPs [64]. The mean crystallite size of MnFe 2 O 4 was calculated using the Scherrer equation was determined to be 11.9 nm whereas the mean crystallite size of Pd was around 3.4 nm. Noteworthy to mention, the diffraction peaks related to chitosan were not observed in the XRD diffractogram of Chit@MnFe 2 O 4 and Pd-Chit@MnFe 2 O 4 due to the amorphous nature of chitosan or due to the formation of a thin chitosan layer [65]. The presence of diffraction peaks related to MnFe 2 O 4 in the diffractogram of Chit@MnFe 2 O 4 and Pd-Chit@MnFe 2 O 4 indicates that its crystallinity is preserved even in the presence of chitosan and Pd.
The XPS spectrum of Pd 3d is given in Figure 1b-c. The deconvolution of the spectrum resulted in two peaks centred at BE = 334.8 eV and BE = 340.8 eV. The first peak is attributed to the Pd 3d 5/2 whereas the latter is attributed to the Pd 3d 3/2 , which can be ascribed to metallic Pd. The presence of these peaks confirmed that Pd 2+ was mainly reduced to metallic Pd. The XPS result is consistent with the previously reported reports [66]. The minor peaks located at 338.2 eV (Pd 3d 5/2 ) and 343.4 eV (Pd 3d 3/2 ) correspond to Pd(II) species, which have indicated the interaction of metallic Pd with amine groups in chitosan [67,68].   [73], 1060 cm −1 and 1023 cm −1 (-C-O stretching vibration of a secondary alcohol and primary alcohol), respectively [74]. A similar trend was observed on Pd_Chit@MnFe2O4, yet Pd_Chit@MnFe2O4 showed shifting of the stretching vibrations of chitosan's (-C-O-C) from 1023 to 1031 cm −1 . The shifting indicates that the bond strengths of C-O-C in glycosidic bonds increased due to environmental differences after the ionic gelation process [75]. Noted, it also implied that manganese ferrite was successfully coated with chitosan via cross-linked TPP. The case of MnFe2O3 showed the elimination of absorption peaks belonging to -CH2, -CH3, and C-O-C. The morphology and particle size distributions of Chit@MnFe2O4 and of Pd−Chit@MnFe2O4 were characterized by SEM and TEM analyses, respectively. From Figure 2a,c, it can be observed that the MnFe2O4 particles were mostly spherical-shaped with various sizes. The surface morphology of the catalyst was spherically shaped as well (Figure 2e). The average diameter of Pd−Chit@MnFe2O4 determined from the TEM image was about 11 ± 5 nm (Figure 2b) whereas the average diameter of Pd−Chit@MnFe2O4 determined from the SEM image was 10 ± 2 nm (Figure 2f). Figure 2c is the TEM micrograph of Chit@MnFe2O4 showing the average particle size from the cor-  The shifting indicates that the bond strengths of C-O-C in glycosidic bonds increased due to environmental differences after the ionic gelation process [75]. Noted, it also implied that manganese ferrite was successfully coated with chitosan via cross-linked TPP. The case of MnFe 2 O 3 showed the elimination of absorption peaks belonging to -CH 2 , -CH 3, and C-O-C. The morphology and particle size distributions of Chit@MnFe 2 O 4 and of Pd-Chit@MnFe 2 O 4 were characterized by SEM and TEM analyses, respectively. From Figure 2a,c, it can be observed that the MnFe 2 O 4 particles were mostly spherical-shaped with various sizes. The surface morphology of the catalyst was spherically shaped as well (Figure 2e). The average diameter of Pd-Chit@MnFe 2 O 4 determined from the TEM image was about 11 ± 5 nm (Figure 2b) whereas the average diameter of Pd-Chit@MnFe 2 O 4 determined from the SEM image was 10 ± 2 nm (Figure 2f). Figure 2c is the TEM micrograph of Chit@MnFe 2 O 4 showing the average particle size from the corresponding diameter distribution, which is in the range of 6 nm (Figure 2d). Thus, the reported higher size in Pd-Chit@MnFe 2 O 4 is due to the capping of Pd nanoparticles. The catalytic activity of the Pd catalyst is probably due to the following effects: A high absorption capacity was provided by the morphology of Pd nanocomposite which in turn, decreased the induction time. Additionally, the catalytic activity was improved due to the strong synergistic effects of its constituents. (−NO 2 ) group can be reduced to an amine and aniline by the Pd-Chit@MnFe 2 O 4 nanocomposite. Without Pd as catalytic hydrogenation, the catalytic reduction reaction cannot proceed. As the catalyst was added into the system, ions were adsorbed by Pd, due to strong adsorption capacity and reduction reaction removed oxygen and added hydrogen. Then the active species reduced −NO 2 into −NH 2 , which indicated the catalyst's critical role in this reduction. In Figure 2h, EDS analysis indicates that Pd-Chit@MnFe 2 O 4 was mainly composed of C, O, Fe, and Mn, P, and Pd. The P originated from the CS cross-linked with the TPP ion by ionic gelation [58]. The EDS and mapping micrograph proved that the immobilized Pd NPs were well-dispersed on the Chit@MnFe 2 O 4 support (Figure 2g). ions were adsorbed by Pd, due to strong adsorption capacity and reduction reaction removed oxygen and added hydrogen. Then the active species reduced −NO2 into −NH2, which indicated the catalyst's critical role in this reduction. In Figure 2h, EDS analysis indicates that Pd−Chit@MnFe2O4 was mainly composed of C, O, Fe, and Mn, P, and Pd. The P originated from the CS cross-linked with the TPP ion by ionic gelation [58]. The EDS and mapping micrograph proved that the immobilized Pd NPs were well-dispersed on the Chit@MnFe2O4 support ( Figure 2g).

Elemental Analysis
The total concentration of Pd, Mn, and Fe in Pd-Chit@MnFe 2 O 4 detected by ICP-MS analysis is given in Table 1. The loading of Pd on Pd-Chit@MnFe 2 O 4 was determined to be 0.21 mmol·g −1 . The atomic ratio of Fe: Mn was determined to be 2:1, which agrees with the molecular formula of MnFe 2 O 4 . The robustness test indicates that higher Pd leached out from the MnFe 2 O 4 without chitosan coating. The concentration of Pd detected was 26.6 ppb or 0.03 wt%. The Pd concentration was reduced to 10.8 ppb or 0.01 wt% when the MnFe 2 O 4 was coated with chitosan. The observation indicates that the Pd-Chit@MnFe 2 O 4 is safe to be used in the pharmaceutical industry as the leaching level of Pd from the catalyst was lower than the maximum acceptable concentration limits [76,77].

Magnetic Properties
The magnetic properties of Pd-Chit@MnFe 2 O 4 were observed using VSM at room temperature and the magnetization curves are shown in Figure 3 and Table 2 [78]. Of note, the M s value of MnFe 2 O 4 was slightly higher than that of Chit@MnFe 2 O 4 because of the chitosan coating around MnFe 2 O 4 [79], whereas the decrease in the M s value of Pd-Chit@MnFe 2 O 4 indicated that Pd was successfully immobilized on Chit@MnFe 2 O 4 [80]. The coating of chitosan and immobilization of Pd form a magnetically disordered layer around MnFe 2 O 4 , which results in a decrease in the total amount of magnetic phase of the support material and catalyst. The superparamagnetic properties of Pd-Chit@MnFe 2 O 4 allowed the catalyst to be easily separated and recycled by applying an external magnetic field; it was redispersed when the external magnetic field was removed.

Elemental Analysis
The total concentration of Pd, Mn, and Fe in Pd−Chit@MnFe2O4 detected by ICP-MS analysis is given in Table 1. The loading of Pd on Pd−Chit@MnFe2O4 was determined to be 0.21 mmol·g −1 . The atomic ratio of Fe: Mn was determined to be 2:1, which agrees with the molecular formula of MnFe2O4. The robustness test indicates that higher Pd leached out from the MnFe2O4 without chitosan coating. The concentration of Pd detected was 26.6 ppb or 0.03 wt%. The Pd concentration was reduced to 10.8 ppb or 0.01 wt% when the MnFe2O4 was coated with chitosan. The observation indicates that the Pd−Chit@MnFe2O4 is safe to be used in the pharmaceutical industry as the leaching level of Pd from the catalyst was lower than the maximum acceptable concentration limits [76,77].

Magnetic Properties
The magnetic properties of Pd_Chit@MnFe2O4 were observed using VSM at room temperature and the magnetization curves are shown in Figure 3 and Table 2. The MnFe2O4, Chit@MnFe2O4, and Pd_Chit@MnFe2O4 retained high saturation magnetization (Ms) with low coercivity (Hc) and negligible remanent magnetization (Mr). The low squareness ratio (Mr/Ms) of MnFe2O4 (0.03), Chit@MnFe2O4 (0.03), and Pd−Chit@MnFe2O4 (0.05) confirmed their superparamagnetic behaviour [78]. Of note, the Ms value of MnFe2O4 was slightly higher than that of Chit@MnFe2O4 because of the chitosan coating around MnFe2O4 [79], whereas the decrease in the Ms value of Pd−Chit@MnFe2O4 indicated that Pd was successfully immobilized on Chit@MnFe2O4 [80]. The coating of chitosan and immobilization of Pd form a magnetically disordered layer around MnFe2O4, which results in a decrease in the total amount of magnetic phase of the support material and catalyst. The superparamagnetic properties of Pd−Chit@MnFe2O4 allowed the catalyst to be easily separated and recycled by applying an external magnetic field; it was redispersed when the external magnetic field was removed.   The catalytic performance of Pd-Chit@MnFe 2 O 4 was investigated in the reduction of 4-NP and 4-NA using NaBH 4 as a reducing agent. Due to the excess NaBH 4 , the reaction rate depends only on the concentration of the nitroarene compound. Hence, a pseudofirst-order kinetic plot was used to study the rate constant of the nitroarene reduction (Figure 4b,d). The 4-NP reduction profile is shown in Figure 4a. The absorption band at 400 nm belongs to the 4-nitrophenolate ion, which was formed when NaBH 4 was added to the 4-NP solution [81]. The absorption band continuously decreased in the presence of the catalyst. The reduction of the 4-nitrophenolate ion is accompanied by the formation of a new absorption band around 300 nm, which indicates the formation of 4−AP [82].

Catalytic Reduction of Nitroarenes and Reusability
The catalytic performance of Pd−Chit@MnFe2O4 was investigated in the reduction of 4−NP and 4-NA using NaBH4 as a reducing agent. Due to the excess NaBH4, the reaction rate depends only on the concentration of the nitroarene compound. Hence, a pseudofirst-order kinetic plot was used to study the rate constant of the nitroarene reduction (Figure 4b,d). The 4−NP reduction profile is shown in Figure 4a. The absorption band at 400 nm belongs to the 4-nitrophenolate ion, which was formed when NaBH4 was added to the 4−NP solution [81]. The absorption band continuously decreased in the presence of the catalyst. The reduction of the 4-nitrophenolate ion is accompanied by the formation of a new absorption band around 300 nm, which indicates the formation of 4−AP [82].  The effect of catalyst type and the effect of Pd-Chit@MnFe 2 O 4 mass on the 4-NP reduction reaction was also investigated. The pseudo-first-order plot of the 4-NP reduction is shown in Figure 4b, whereas the k and TOF values are summarized in Table 3  The effect of catalyst type and the effect of Pd−Chit@MnFe2O4 mass on the 4-NP reduction reaction was also investigated. The pseudo-first-order plot of the 4-NP reduction is shown in Figure 4b, whereas the k and TOF values are summarized in Table 3  The catalytic activity of Pd−Chit@MnFe2O4 was further evaluated in the reduction of 4-NA. From Figure 4c, the intensity of an absorption band around 380 nm associated with 4-NA decreased as the reaction continued [83]. The reduction in the absorption intensity of 4-NA is accompanied by the rise in an absorption band associated with 4-PDA around 305 nm [40]. For the control experiment, the pseudo-first-order plots of 4-NA reduction are illustrated in Figure 4d, whereas the k and TOF values are shown in Table 3. In the control experiments, the reaction rate was slow when the experiment was conducted with 0.5 mg of Chit@MnFe2O4 and MnFe2O4. The conversion of 4-NA to 4-PDA was negligible without a catalyst. Noteworthy, when 0.5 mg Pd−Chit@MnFe2O4 was added, the reduction of 4-NA was catalysed at a higher reaction rate of 1.42 min −1 and TOF value of 571.4 min −1 . The complete reaction time was reduced to 2.5 min. Overall, the results showed that Pd−Chit@MnFe2O4 has superior catalytic activity in a reduction reaction of 4-NP and 4-NA.
The pseudo-first-order kinetic plot of 8 consecutive cycles of 4-NP and 4-NA reduction reactions is displayed in Figure 5a and The catalytic activity of Pd-Chit@MnFe 2 O 4 was further evaluated in the reduction of 4-NA. From Figure 4c, the intensity of an absorption band around 380 nm associated with 4-NA decreased as the reaction continued [83]. The reduction in the absorption intensity of 4-NA is accompanied by the rise in an absorption band associated with 4-PDA around 305 nm [40]. For the control experiment, the pseudo-first-order plots of 4-NA reduction are illustrated in Figure 4d, whereas the k and TOF values are shown in Table 3. In the control experiments, the reaction rate was slow when the experiment was conducted with 0.5 mg of Chit@MnFe 2 O 4 and MnFe 2 O 4 . The conversion of 4-NA to 4-PDA was negligible without a catalyst. Noteworthy, when 0.5 mg Pd-Chit@MnFe 2 O 4 was added, the reduction of 4-NA was catalysed at a higher reaction rate of 1.42 min −1 and TOF value of 571.4 min −1 . The complete reaction time was reduced to 2.5 min. Overall, the results showed that Pd-Chit@MnFe 2 O 4 has superior catalytic activity in a reduction reaction of 4-NP and 4-NA.
The pseudo-first-order kinetic plot of 8 consecutive cycles of 4-NP and 4-NA reduction reactions is displayed in Figures 5a and 5b, respectively. Results show that the conversion of 4-NP and 4-NA was~100% for 8 consecutive cycles (as shown in Figure 5c), which indicated that Pd-Chit@MnFe 2 O 4 was stable and can be reused up to 8 consecutive cycles.
The minimum loss of catalyst nanoparticles during the recycling process may lead to a slight increase in reaction time upon cycles for both 4-NP and 4-NA reduction. cycles. The minimum loss of catalyst nanoparticles during the recycling process may lead to a slight increase in reaction time upon cycles for both 4-NP and 4-NA reduction. A plausible mechanism was proposed for Pd−Chit@MnFe2O4 catalysed nitroarenes reduction based on the Langmuir-Hinshelwood model and Haber mechanism [84,85]. The proposed mechanism is shown in Figure 6. First, both reactants adsorbed on the surface-active site of Pd−Chit@MnFe2O4 based on the Langmuir-Hinshelwood model. After the adsorption process, electron transfer occurred between the nitroarene compound and hydride ion, followed by the dehydration process to yield the nitroso compound. The nitroso compound undergoes further reduction to form hydroxylamine compound in a very fast step. Next, the hydroxylamine compound was finally converted to an amine compound via a series of electron transfers followed by the dehydration process. At the end of the catalytic cycle, the amine compound as product desorbed from the surface of Pd−Chit@MnFe2O4, and a new catalytic cycle will begin. A plausible mechanism was proposed for Pd-Chit@MnFe 2 O 4 catalysed nitroarenes reduction based on the Langmuir-Hinshelwood model and Haber mechanism [84,85]. The proposed mechanism is shown in Figure 6. First, both reactants adsorbed on the surface-active site of Pd-Chit@MnFe 2 O 4 based on the Langmuir-Hinshelwood model. After the adsorption process, electron transfer occurred between the nitroarene compound and hydride ion, followed by the dehydration process to yield the nitroso compound. The nitroso compound undergoes further reduction to form hydroxylamine compound in a very fast step. Next, the hydroxylamine compound was finally converted to an amine compound via a series of electron transfers followed by the dehydration process. At the end of the catalytic cycle, the amine compound as product desorbed from the surface of Pd-Chit@MnFe 2 O 4 , and a new catalytic cycle will begin.

Palladium-Induced Allyl Carbamate Deprotection
From Figure 7 (entry 1), the control experiment showed that no product was formed (no fluorescence detected) in the absence of Pd-Chit@MnFe 2 O 4 , whereas the deprotection of bis-allyloxycarbonyl rhodamine 110 was successfully performed in the presence of Pd-Chit@MnFe 2 O 4 with and without thiophenol (Figure 7: entry 3). Notably, our newly designed catalyst Pd-Chit@MnFe 2 O 4 successfully catalysed allyl carbamate cleavage without thiophenol as a scavenger (Figure 7: entry 2). High levels of thiophenol have proven to be toxic to living organisms and the environment; therefore, this approach without thiophenol is highly beneficial [86]. The characterization of the reaction products formed after the reaction was determined by the purification and analysis of the cell lysate, which quanti-tatively confirmed the chemical identities via liquid chromatography-mass spectrometry (LC-MS) and high-performance liquid chromatography (HPLC). The reaction mixture was centrifuged for 5 min at 13,000 rpm. The supernatant was collected and passed through a DSC-18LT column which had been prewashed with water. The column was washed with water to remove salts and proteins from the cell lysate, and acetonitrile was used to elute the desired product from the column. The residue was dissolved in methanol and analysed by LC-MS in a positive ionization mode. LC-MS suggested the presence of rhodamine 110 with the m/z found at 331.0. In addition, HPLC analysis of the cell lysate showed retention times with a sample standard of around 3.2 min.

Palladium-Induced Allyl Carbamate Deprotection
From Figure 7 (entry 1), the control experiment showed that no product was formed (no fluorescence detected) in the absence of Pd_Chit@MnFe2O4, whereas the deprotection of bis-allyloxycarbonyl rhodamine 110 was successfully performed in the presence of Pd−Chit@MnFe2O4 with and without thiophenol (Figure 7: entry 3). Notably, our newly designed catalyst Pd−Chit@MnFe2O4 successfully catalysed allyl carbamate cleavage without thiophenol as a scavenger (Figure 7: entry 2). High levels of thiophenol have proven to be toxic to living organisms and the environment; therefore, this approach without thiophenol is highly beneficial [86]. The characterization of the reaction products formed after the reaction was determined by the purification and analysis of the cell lysate, which quantitatively confirmed the chemical identities via liquid chromatographymass spectrometry (LC-MS) and high-performance liquid chromatography (HPLC). The reaction mixture was centrifuged for 5 min at 13,000 rpm. The supernatant was collected and passed through a DSC-18LT column which had been prewashed with water. The column was washed with water to remove salts and proteins from the cell lysate, and acetonitrile was used to elute the desired product from the column. The residue was dissolved in methanol and analysed by LC-MS in a positive ionization mode. LC-MS suggested the presence of rhodamine 110 with the m/z found at 331.0. In addition, HPLC analysis of the cell lysate showed retention times with a sample standard of around 3.2 min.

Cytotoxic Assay
A HeLa cell was chosen to investigate the cytotoxicity of Pd−Chit@MnFe2O4 because of the cell's ability to grow rapidly and easily [87]. Figure 8 depicts the cytotoxic evaluation of Pd−Chit@MnFe2O4. As can be seen, Pd−Chit@MnFe2O4 caused low necrosis of HeLa cells and high cell viability (>89%) was observed after 24 h. The viability of HeLa cells treated with Pd−Chit@MnFe2O4 at two concentrations was found to have no substantial

Cytotoxic Assay
A HeLa cell was chosen to investigate the cytotoxicity of Pd-Chit@MnFe 2 O 4 because of the cell's ability to grow rapidly and easily [87]. Figure 8 depicts the cytotoxic evaluation of Pd-Chit@MnFe 2 O 4 . As can be seen, Pd-Chit@MnFe 2 O 4 caused low necrosis of HeLa cells and high cell viability (>89%) was observed after 24 h. The viability of HeLa cells treated with Pd-Chit@MnFe 2 O 4 at two concentrations was found to have no substantial cytotoxicity. Previous literature has reported on low cytotoxicity heterogeneous Pd catalyst catalysing Suzuki-Miyaura cross-coupling and allyl carbamate cleavage in vivo [41]. Due to the low cytotoxicity of Pd-Chit@MnFe 2 O 4 , the Pd-Chit@MnFe 2 O 4 catalyst is suggested as a potential candidate for catalysis in living systems.

BET Analysis
The surface area of the pure Pd and Pd−Chit@MnFe2O4 samples was estimated by Brunauer-Emmett-Teller surface area analysis (BET) (Figure 9a and Figure 9b, respectively). According to the results, the surface area of 25.614 and 2.292 m 2 ·g −1 were found for palladium nanoparticles. The higher BET specific surface area of the Pd−Chit@MnFe2O4 nanocomposite compared with pure Pd can be ascribed to the introduction of Chit@MnFe2O4 particles in the composite and is beneficial to improve the catalytic performance.

BET Analysis
The surface area of the pure Pd and Pd-Chit@MnFe 2 O 4 samples was estimated by Brunauer-Emmett-Teller surface area analysis (BET) (Figures 9a and 9b, respectively). According to the results, the surface area of 25.614 and 2.292 m 2 ·g −1 were found for palladium nanoparticles. The higher BET specific surface area of the Pd-Chit@MnFe 2 O 4 nanocomposite compared with pure Pd can be ascribed to the introduction of Chit@MnFe 2 O 4 particles in the composite and is beneficial to improve the catalytic performance.

Conclusions
Chit@MnFe2O4 was successfully prepared and used as supporting material for heterogeneous Pd NP catalysts. This newly prepared Pd−Chit@MnFe2O4 catalyst demonstrated high catalytic activities in nitroarene reduction as well as excellent catalytic performance in the deprotection of allyl carbamate under biological conditions. Moreover, Pd−Chit@MnFe2O4 was stable for reuse and could be recycled with negligible loss of catalytic activity. The recovery process of Pd−Chit@MnFe2O4 was easy and convenient due to its superparamagnetic properties. Furthermore, this magnetically active chitosancoated Pd catalyst is biocompatible and eco-friendly because it exhibited low cytotoxicity and minimal leaching problems. Thus, it could be suitable for a variety of chemical applications. The biocompatible Pd−Chit@MnFe2O4 is of interest for future applications, predominantly in biomedical and clinical research.

Conclusions
Chit@MnFe 2 O 4 was successfully prepared and used as supporting material for heterogeneous Pd NP catalysts. This newly prepared Pd-Chit@MnFe 2 O 4 catalyst demonstrated high catalytic activities in nitroarene reduction as well as excellent catalytic performance in the deprotection of allyl carbamate under biological conditions. Moreover, Pd-Chit@MnFe 2 O 4 was stable for reuse and could be recycled with negligible loss of catalytic activity. The recovery process of Pd-Chit@MnFe 2 O 4 was easy and convenient due to its superparamagnetic properties. Furthermore, this magnetically active chitosan-coated Pd catalyst is biocompatible and eco-friendly because it exhibited low cytotoxicity and minimal leaching problems. Thus, it could be suitable for a variety of chemical applications. The biocompatible Pd-Chit@MnFe 2 O 4 is of interest for future applications, predominantly in biomedical and clinical research.