An Alternating Magnetic Field-Controlled Drug Delivery System Based on 4,4′-Azobis (4-cyanovaleric Acid)-Functioned Fe3O4@Chitosan Nanoparticles

Herein, we designed chitosan–coated Fe3O4 nanocomposites for the control release of drugs by an alternating magnetic field (AMF). The chitosan-coated Fe3O4 nanoparticles (Fe3O4@CS) were prepared by a alkaline co-precipitation method, and then, the model drug toluidine blue (TB) was covalently grafted onto the surface of the nanocomposite by a two-step amide reaction with the thermosensitive molecule 4,4′-azobis (4-cyanovaleric acid) (ACVA) as the linker group. The prepared nanocomposites were superparamagnetic and showed high magnetization saturation (about 54.0 emu g−1). In vitro hydrothermal release studies showed that most parts of the TB would be effectively enclosed within the nanocarriers at lower ambient temperatures (23 or 37 °C) due to the molecular bonding of ACVA. The results of kinetic fitting of hydrothermal release data showed that TB released from nanoparticles followed first-order kinetics (R2 > 0.99) and the Korsemeyer–Peppas model (R2 > 0.99, n < 0.5). Most importantly, a single magnetron release experiment demonstrated an approximately linear relationship between the cumulative release of the drug and the duration of action of AMF (R2 = 0.9712). Moreover, the increase in the cumulative release of the drug can be controlled by controlling the switch of the AMF generation device. Therefore, the ACVA-modified Fe3O4@CS nanocarrier designed in this study is a promising model for drug delivery that enables the control of drug release dose by AMF.


Introduction
In recent decades, drug delivery system (DDS) based nanomaterials have gained numerous anticipated achievements, which have increasingly become an essential strategy for diagnosis and disease therapy in the biomedical field [1]. However, the necessity of delivering precise doses to specific sites of diseases at defined times remains a challenge [2]. Stimulus-responsive nanocarriers serve as an intelligent and effective drug delivery platform that can reduce the side effects of the drug and improve the therapeutic efficacy [3]. These stimuli include endogenous (pH, enzymes, and redox) and exogenous (temperature, ultrasound, light, and magnetic fields) [4]. In contrast, the responsiveness of nanocarriers can be designed by the properties of the material itself or by modifying appropriate release triggers [5,6]. Among them, magnetic nanocarrier systems based on external alternating magnetic field (AMF) response have the following particular advantages: (a) no penetration depth restriction by magnetic fields to trigger drug release in the deep animal body without scattering or absorption by tissues, such as light, and (b) less limitation on the type of tissue treated than ultrasound methods strongly attenuated by air and bone structure [7]. Scheme 1. Synthesis schematic of azo-functionalized chitosan-coated Fe3O4 nanoparticles and triggered release under an AMF.

Scheme 2.
The structure schematic of the drug released from Fe3O4@CS nanoparticles under an AMF.

Synthesis of Fe3O4@CS Magnetic Nanoparticle
The Fe3O4@CS nanoparticles were synthesized using modified co-precipitation method [21]. First, a total of 0.01 mol of FeCl3·6H2O (2.203 g) and 0.005 mol of FeSO4·7H2O (1.89 g) were dissolved in 25 mL of deionized water (named A). Subsequently, 0.5 mL of Scheme 2. The structure schematic of the drug released from Fe 3 O 4 @CS nanoparticles under an AMF.

Synthesis of Fe 3 O 4 @CS Magnetic Nanoparticle
The Fe 3 O 4 @CS nanoparticles were synthesized using modified co-precipitation method [21]. First, a total of 0.01 mol of FeCl 3 ·6H 2 O (2.203 g) and 0.005 mol of FeSO 4 ·7H 2 O (1.89 g) were dissolved in 25 mL of deionized water (named A). Subsequently, 0.5 mL of acetic acid and 0.5 g of CS were dissolved in 50 mL of deionized water (named B). Then, in a 250 mL three-neck flask equipped with a mechanical stirring bar, 25 mL of A and 25 mL of B were mixed vigorously stirring at a rate of 150 r/min under the flow of nitrogen for 10 min. When the mixture was heated to 60 • C, 20 mL ammonia was added and stirred for another 1 h under N 2 at 60 • C. The product was washed with deionized water and anhydrous ethanol. The final product was freeze-dried and named as Fe 3 O 4 @CS-1. Fe 3 O 4 @CS-2, Fe 3 O 4 @CS-3, Fe 3 O 4 @CS-4, and Fe 3 O 4 @CS-5 magnetic nanoparticles with CS contents of 2, 3, 4, and 5 wt.% were also prepared, respectively, by altering the CS weight ratio in solution B described above. All the samples obtained were stored at 4 • C for further investigations.

Synthesis of Fe 3 O 4 @CS-ACVA-TB Magnetic Nanoparticles
Fe 3 O 4 @CS-ACVA nanoparticles were synthesized as follows. Firstly, 2.7 mmol of ACVA (756.8 mg) was dissolved in 1% (w/w) NaOH solution, and then, the pH was adjusted to 7.2 through NaOH/HCl (1 M). Secondly, 2.7 mmol of DMTMM (747.1 mg) was added to activate the carboxylic acid of ACVA for 20 min at 4 • C. Thirdly, 0.9 mmol of Fe 3 O 4 @CS-1 was added, and then, the pH was adjusted to 7.2 and stirred for 5 days at 4 • C ice bath. Finally, the product was washed for several times with deionized water and named as Fe 3 O 4 @CS-ACVA.
Subsequently, Fe 3 O 4 @CS-ACVA was dispersed in 100 mL deionized water, and then, 0.9 mmol of DMTMM (249.048 mg) was added to activate the carboxylic acid of Fe 3 O 4 @CS-ACVA for 20 min at 4 • C. After that, 0.9 mmol of TB (336.6 mg) was added to the activated Fe 3 O 4 @CS-ACVA in deionized water, and then, the pH was adjusted to 7.2 through NaOH/HCl (1 M) and stirred for 5 days at 4 • C. Finally, the product was washed several times with deionized water and freeze-dried, named as Fe 3 O 4 @CS-ACVA-TB.

Magneto-thermal Properties Analysis of Fe 3 O 4 @CS Nanoparticles
The measurement of magnetic heating characteristics of the Fe 3 O 4 @CS nanoparticles was performed by using a homemade magnetic hyperthermia device. To evaluate the influence of AMF on the magneto-thermal properties of MNPs, Fe 3 O 4 @CS-1 suspension (3 mg/mL) was exposed to AMF with intensities of 2.64 × 10 8 , 4.23 × 10 8 , 5.82 × 10 8 and 7.40 × 10 8 Am −1 s −1 for 5 min. The cyclic magneto-thermal properties under AMF with intensities of 5.82 × 10 8 Am −1 s −1 for 10 min were also determined at the same time. Additionally, the effect of the concentration of Fe 3 O 4 @CS nanoparticles and the CS content were evaluated. The former was explored by placing the Fe 3 O 4 @CS-1 suspensions with concentrations of 0.5, 1, 3, 6, and 9 mg/mL under AMF with the intensity of 5.82 × 10 8 Am −1 s −1 for 5 min. Yet, the latter was determined by placing 5 mg/mL of Fe 3 O 4 @CS-1, Fe 3 O 4 @CS-2, Fe 3 O 4 @CS-3, Fe 3 O 4 @CS-4, and Fe 3 O 4 @CS-5 suspension in AMF with the intensity of 5.82 × 10 8 Am −1 s −1 for 10 min. The curves of temperature rise were monitored by using an optical fiber thermocouple (IF-C, Inno, Fuzhou, China) in real-time.
where C is the volumetric heat capacity of solution, Vs is the sample volume, m is the mass of Fe 3 O 4 @CS nanoparticles, and dT/dt is the initial slope of the temperature increase curve with time.

Hydrothermal Release of Fe 3 O 4 @CS-ACVA-TB Magnetic Nanoparticles
The Fe 3 O 4 @CS-ACVA-TB suspension (1 mg/mL) was incubated in water bath at 23, 37, 57, and 80 • C in PBS of pH 7.4. Then, 3 mL of supernatant was collected by centrifugal separation (8000 r/min) at the intervals of 0, 1, 3, 6, 9, 12, 24, 36, and 48 h and the absorbance Fe 3 O 4 @CS-ACVA-TB solution (1 mg/mL) was incubated at 23 • C in PBS of pH 7.4. Before being triggered by magnetic heating, 3 mL of supernatant was collected every 1 h by centrifugal separation (8000 r/min) and monitored for the absorbance value at λ = 632 nm with a UV spectrophotometer. After monitoring the release every 1 hour over the course of 4 h, the samples were exposed to an AMF with the intensity of 5.82 × 10 8 Am −1 s −1 for 5, 10, 15, and 20 min followed by recording the absorbance value every 1 h. The samples at 23 • C without AMF treatment performed as the blank control for this experiment.

Multiple Magnetic Heat Control Releases of Fe 3 O 4 @CS-ACVA-TB Magnetic Nanoparticles
Fe 3 O 4 @CS-ACVA-TB solution (1 mg/mL) was prepared and incubated at 23 • C in PBS of pH 7.4. Before being triggered by magnetic heating, 3 mL of supernatant was collected every 1 h by centrifugal separation (8000 r/min) and monitored for the absorbance value at λ = 632 nm with a UV spectrophotometer. After monitoring the release every 1 h over the course of 4 h, the samples were exposed to an AMF with the intensity of 5.82 × 10 8 Am −1 s −1 for 5 min, followed by monitoring the absorbance value of supernatant every 1 h. The next AMF was not applied until the release of TB leveled off. Three monitoring cycles were performed in total. Similarly, three cycles with AMF times of 10, 15, and 20 min were also performed. The samples at 23 • C without AMF treatment performed as the blank control for this experiment.

Synthesis and Characterization of Magnetic Fe 3 O 4 @CS Nanoparticles
For biomedical applications, chitosan, with rich active amino groups was chosen to wrap Fe 3 O 4 . The advantage of preparing chitosan-coated magnetic composites is that chitosan-conjugated particles can be easily prepared by using a co-precipitation method [23], because the chitosan's amine groups (-NH 2 ) with positive charge can easily interact with negatively charged groups (Fe-OH) on the magnetite nanoparticles (MNPs) through ion exchange reactions [16]. The prepared MNPs (Fe 3 O 4 @CS-NH 2 ) allow for subsequent modification and drug loading. Then, to introduce excellent magneto-thermal response functionality, the azo compound ACVA was chosen as a thermal-sensitive switch, which is gradually being developed as a triggering agent for stimulation-responsive drug delivery systems [24,25]. Here, some of the carboxylic acid of ACVA firstly formed an amide bond with the prepared Fe 3 O 4 @CS-NH 2 nanoparticles, and the product was named as Fe 3 O 4 @CS-ACVA. Subsequently, the remaining carboxylic acid of ACVA formed another amide bond with the amine groups on TB, and the final particles were named Fe 3 O 4 @CS-ACVA-TB.
The products of each step of the grafting process were analyzed by infrared spectroscopy. As shown in Figure 1A, the characteristic peaks at 1626 cm −1 correspond to the N-H stretching vibration in chitosan, 1392 cm −1 corresponds to the C-O bending vibration in chitosan, 2922 cm −1 corresponds to the C-H stretching vibration in chitosan, and 1039 cm −1 corresponds to the C-N vibration in chitosan. A new characteristic absorption peak formed at 1562 cm −1 corresponds to the amide bond, which confirms that ACVA is successfully connected via the amide bond surface of Fe 3 O 4 @CS magnetic nanoparticles. In addition, the C≡N absorption at 2253cm −1 , the absorption peaks at 1719 cm −1 (C=O stretching), 1451 cm −1 (C-H bending) and 2904 cm −1 , 2934 cm −1 and 2981 cm −1 (C-H stretching) also prove that ACVA was successfully grafted on Fe 3 O 4 @CS. Furthermore, the absorption at 1612 cm −1 corresponds to the stretching vibration of C=C of the benzene ring on TB, which indicates that the model drug molecule was successfully grafted onto the surface of Fe 3 O 4 @CS nanoparticles through the ACVA. neering 2022, 9, x FOR PEER REVIEW 7 of 16 magnetization (Ms) of Fe3O4@CS-1 nanoparticles synthesized was 54.0 emu/g at 300 K, which is lower than the saturation magnetization of bare Fe3O4 reported in the previous literature (80-100 emu/g) [26]. The reason for the reduced saturation magnetization of the sample might come from the non-magnetic component chitosan of the nanocomposites. Moreover, the value of Mr / Ms < 0.1 (in Table 1) indicates that the synthesized nanoparticles were superparamagnetic, which is a propitious character of magnetic nanocarriers such as DDS [27].   Figure 2(A) shows the TEM image of Fe3O4@CS-ACVA-TB, which was found to be spherical particles with an average particle size of 9.2 ± 1.3 nm (Figure 2(B)). As Figure  2(A) shows, the prepared particles exhibit a certain degree of aggregation, which may be caused by strong interactions between chitosan molecules. We analyzed the distance between particles in the TEM image, which was performed by measuring 20 pairs of nanoparticles in the adjacent area of the image to estimate the coating thickness of Fe3O4 particles to be about 1.2 nm.
Additionally, through the dynamic light scattering scanner, the average hydrated particle size of Fe3O4@CS and Fe3O4@CS-ACVA-TB (Figure 2(C) and 2(D)) was found to be 80.0 ± 16.6 and 230.6 ± 40.6 nm, respectively. This size is much larger than that TGA was used to analyze the weight of organic magnetic nanoparticles ( Figure 1B). As crystalline water was removed (25~200 • C), the weight loss rate of Fe 3 O 4 @CS, Fe 3 O 4 @CS-ACVA and Fe 3 O 4 @CS-ACVA-TB was 4.5 %,5.5 % and 4.5 %, respectively. The mass of the three samples decreased 17.5 %, 23.5 % and 34.6 %, respectively, after continuing to heat from 200~800 • C under N 2 protection. The results showed that ACVA and TB were present on the nanoparticles' surface in weights of 6.4% and 11.1%, respectively.
Subsequently, Figure 1C shows the XRD patterns of where, D is the crystallite particle size in nm, k refers to grain shape factor taken as unity (based on assumption that shape of particles is spherical), λ is the incident X-ray wavelength of Cu-Kα source of radiation, and θ is the Bragg's angle, while β (in radians) refers to broadening of diffraction line measured full width at half maximum intensity (FWHM). The average crystallite size of Fe 3 O 4 @CS and Fe 3 O 4 @CS-ACVA-TB nanoparticles was 8.97 and 7.94 nm, respectively. The magnetic properties of the Fe 3 O 4 @CS-1 were characterized by a vibrational sample magnetometer. Figure 1D shows the saturation magnetization (M s ) of Fe 3 O 4 @CS-1 nanoparticles synthesized was 54.0 emu/g at 300 K, which is lower than the saturation magnetization of bare Fe 3 O 4 reported in the previous literature (80-100 emu/g) [26]. The reason for the reduced saturation magnetization of the sample might come from the non-magnetic component chitosan of the nanocomposites. Moreover, the value of M r /M s < 0.1 (in Table 1) indicates that the synthesized nanoparticles were superparamagnetic, which is a propitious character of magnetic nanocarriers such as DDS [27].  Figure 2A shows the TEM image of Fe 3 O 4 @CS-ACVA-TB, which was found to be spherical particles with an average particle size of 9.2 ± 1.3 nm ( Figure 2B). As Figure 2A shows, the prepared particles exhibit a certain degree of aggregation, which may be caused by strong interactions between chitosan molecules. We analyzed the distance between particles in the TEM image, which was performed by measuring 20 pairs of nanoparticles in the adjacent area of the image to estimate the coating thickness of Fe 3 O 4 particles to be about 1.2 nm.

The Magneto-Thermal Properties of Fe3O4@CS Magnetic Nanoparticles
To investigate the magneto-thermal conversion capability of Fe3O4@CS nanoparticles exposed to AMF, we changed some possible conditions, including AMF intensity (the safety condition H0 f < 5 × 10 9 Am −1 s −1 ) [28], the concentration of MNPs, and the content of chitosan of MNPs. Due to the Néel-Brown mechanism, the magnetic component in the nanoparticles was heated when exposed to AMF, causing a temperature increase. However, the magneto-thermal response ability of Fe3O4@CS gradually decreased with the in- Additionally, through the dynamic light scattering scanner, the average hydrated particle size of Fe 3 O 4 @CS and Fe 3 O 4 @CS-ACVA-TB ( Figure 2C,D) was found to be 80.0 ± 16.6 and 230.6 ± 40.6 nm, respectively. This size is much larger than that charactered by TEM. On the one hand, the hydrated particle size of nanoparticles would increase with the increase in the surface modifier during the material synthesis. On the other hand, this might be due to the association and aggregation of Fe 3 O 4 nanoparticles in water through the action of van der Waals' forces and/or hydrogen bonds.

The Magneto-Thermal Properties of Fe 3 O 4 @CS Magnetic Nanoparticles
To investigate the magneto-thermal conversion capability of Fe 3 O 4 @CS nanoparticles exposed to AMF, we changed some possible conditions, including AMF intensity (the safety condition H 0 f < 5 × 10 9 Am −1 s −1 ) [28], the concentration of MNPs, and the content of chitosan of MNPs. Due to the Néel-Brown mechanism, the magnetic component in the nanoparticles was heated when exposed to AMF, causing a temperature increase. However, the magneto-thermal response ability of Fe 3 O 4 @CS gradually decreased with the increase in non-magnetic components ( Figure 3A) nanoparticles, is almost unchanged when exposed to AMF for the same conditions, which indicates that it possesses good magneto-thermal cycle stability and can be subjected to multiple magnetic heating operations. Nevertheless, the tolerable temperature of normal tissue or cells is about 43 °C [29]. The concentration of 1 mg/mL of Fe3O4@CS nanoparticles and the AMF with the intensity of 5.82 × 10 8 Am −1 s −1 was selected to further experiments to avoid damage to the tissue or cells caused by excessive temperature and to better evaluate the performance of the drug delivery model.

Hydrothermal Release of Fe3O4@CS-ACVA-TB Magnetic Nanoparticles
To investigate the temperature sensitivity performance of ACVA-modified Fe3O4@CS nanocarriers, the TB with high molar absorption coefficient, outstanding thermal stability, and water solubility was used as a model drug molecule. It has been reported that the half-life of the azo compound is 10 h at 57 °C, while the azo chemical bond is nearly broken completely at 80 °C after 48 h [30]. Therefore, the effects of temperature on TB release from nanocarriers were investigated at four different water bath temperatures, 23, 37, 57 and 80 °C, in PBS of pH 7.4, and the cumulative release of TB after 48 h of incubation at 80 °C was used as the total amount of drug. As shown in Figure 4(A), higher temperatures show faster and more drug release. The cumulative release of TB finally reached 17.8 ± 2.5%, 22.3 ± 3.8%, and 49.1 ± 6.1% at 23, 37 and 57 °C after 48 h, respectively. Furthermore, Figure  4 (A) shows the release behavior of Fe3O4@CS-ACVA-TB nanoparticles with two phases, a rapid release within 9 h, and followed from 9 to 48 h, a performance of plateau period  Figure 3B shows the heating situation of the Fe 3 O 4 @CS suspension (3 mg/mL) exposed to different AMF intensities. With the increase in the applied AMF intensity, the sample temperature increased by 14.1, 19.1, 26.1, and 33.8 • C from 26.1 • C within 5 min, indicating that the prepared Fe 3 O 4 @CS nanoparticles have good magneto-thermal properties. Figure 3C depicts the temperature-rising curve within 5 min of concentrations of 0.5, 1, 3, 6, and 9 mg/mL Fe 3 O 4 @CS suspensions exposed to the fixed AMF (5.82 × 10 8 Am −1 s −1 ). Moreover, the temperature increased 8.7, 15.9, 26.1, 43.7 and 56.6 • C from 26.1 • C within 5 min, indicating that the heating rate accelerates with the increase in concentration of Fe 3 O 4 @CS. The results of the three magneto-heat-cooling cycles ( Figure 3D) show that the maximum temperature, which can be reached by the Fe 3 O 4 @CS nanoparticles, is almost unchanged when exposed to AMF for the same conditions, which indicates that it possesses good magneto-thermal cycle stability and can be subjected to multiple magnetic heating operations. Nevertheless, the tolerable temperature of normal tissue or cells is about 43 • C [29]. The concentration of 1 mg/mL of Fe 3 O 4 @CS nanoparticles and the AMF with the intensity of 5.82 × 10 8 Am −1 s −1 was selected to further experiments to avoid damage to the tissue or cells caused by excessive temperature and to better evaluate the performance of the drug delivery model.

Hydrothermal Release of Fe 3 O 4 @CS-ACVA-TB Magnetic Nanoparticles
To investigate the temperature sensitivity performance of ACVA-modified Fe 3 O 4 @CS nanocarriers, the TB with high molar absorption coefficient, outstanding thermal stability, and water solubility was used as a model drug molecule. It has been reported that the half-life of the azo compound is 10 h at 57 • C, while the azo chemical bond is nearly broken completely at 80 • C after 48 h [30]. Therefore, the effects of temperature on TB release from nanocarriers were investigated at four different water bath temperatures, 23, 37, 57 and 80 • C, in PBS of pH 7.4, and the cumulative release of TB after 48 h of incubation at 80 • C was used as the total amount of drug. As shown in Figure 4A, higher temperatures show faster and more drug release. The cumulative release of TB finally reached 17.8 ± 2.5%, 22.3 ± 3.8%, and 49.1 ± 6.1% at 23, 37 and 57 • C after 48 h, respectively. Furthermore, Figure 4 (A) shows the release behavior of Fe 3 O 4 @CS-ACVA-TB nanoparticles with two phases, a rapid release within 9 h, and followed from 9 to 48 h, a performance of plateau period (barely released at 23 and 37 • C) or slow release (57 • C). The rapid release during the inaugural 9 h is likely to involve a diffusion mechanism of the dye loaded on the chitosan coating [31]. The plateau period (23 and 37 • C) that occurred from 9 to 48 h proved that most of the dye was immobilized within the Fe 3 O 4 @CS nanocarriers due to the molecular bonding of ACVA. When the temperature continued to increase (57 • C), the azo bond in ACVA became unstable at this time, resulting in the slow release of TB immobilized by ACVA as well. To investigate the long-term stability of the material in the solution, we continued to monitor the concentration of dye in the Fe 3 O 4 @CS-ACVA-TB suspension (1 mg/mL), which had been incubated at 37 • C in PBS of pH 7.4 for 48 h. As shown in Figure 4B, the concentration of TB in the centrifuge tube remained almost unchanged when the time was prolonged to the 9th day. This indicated that the drug delivery model prepared with Fe 3 O 4 @CS as the magnetic core and ACVA as the connecting bridge had good stability, which lays a foundation for future application.  Figure  4(B), the concentration of TB in the centrifuge tube remained almost unchanged when the time was prolonged to the 9th day. This indicated that the drug delivery model prepared with Fe3O4@CS as the magnetic core and ACVA as the connecting bridge had good stability, which lays a foundation for future application.

Kinetic-Analytic Studies of Drug Release
To explain the release behavior of Fe3O4@CS-ACVA-TB at different temperatures, different equations, such as zero-order, first-order, Higuchi and Korsemeyer-Peppas, were used for analysis [32]. Equations (3)-(6), corresponding to each model, are shown below: (i) Zero-order:

Kinetic-Analytic Studies of Drug Release
To explain the release behavior of Fe 3 O 4 @CS-ACVA-TB at different temperatures, different equations, such as zero-order, first-order, Higuchi and Korsemeyer-Peppas, were used for analysis [32]. Equations (3)- (6), corresponding to each model, are shown below: (ii) First-order: (iv) Korsemeyer-Peppas: where M t is the total amount of TB released at time t, M ∞ is the total amount of drug released as time goes to infinity (i.e., complete release), k is a release constant, and n is the release exponent that is used to characterize different release mechanisms. For spherical particles, the value of n indicates Fickian diffusion if n < 0.5, nonFickian or anomalous phenomena if 0.5 < n < 1, and n > 1 implies a lack of time dependence on release kinetics (i.e., zero-order kinetics) [33]. The release constants and correlation coefficients corresponding to each model are shown in the Table 2, which with a high "R 2 " value was considered as the best-fitting model. All values of the R 2 of the first-order release model for nanoparticles were higher than the zero-order model at different temperatures, indicating that the leakage of drugs from nanoparticles follows first-order kinetics. This is because the solubility of polymer chitosan in PBS of pH 7.4 is approximately low, so that the water content in the shell is approximately low, which would induce the low permeability of TB [21]. Therefore, when the thermal decomposition of ACVA occurred continuously in nanoparticles, it would lead to a higher concentration of TB inside the chitosan coating than that in the external solution, thus showing a concentration-dependent diffusion behavior. Figure 5A-D shows the First-order fitted curves at 23, 37, 57 and 80 • C, respectively. All the "n" values of the Korsmeyer-Peppas equation for nanoparticles were less than 0.5, indicating that the drug release followed Fickian diffusion. Moreover, the release constants k in the First-order and Korsmeyer-Peppas models increased with the increase in temperature, indicating that the increase in temperature accelerates the breakage rate of ACVA and the thermal motion of molecules in solution, leading to a rapid increase in drug concentration in the nanoparticles. This is as expected and provides us with the possibility to subsequently investigate the magneto-thermal release behavior of nanoparticles.

Magneto-thermal Controlled Release of Fe 3 O 4 @CS-ACVA-TB Nanoparticles
Here, we used two ways of applying AMF to investigate the effect of AMF on the release of TB in Fe 3 O 4 @CS-ACVA-TB: (a) single exposures for a different time and (b) multiple sequential exposures each for the same time.

Single Magneto-thermal Controlled Release of Fe 3 O 4 @CS-ACVA-TB Nanoparticles
To confirm the robustness of the ACVA, Fe 3 O 4 @CS-ACVA-TB suspension (1 mg/mL) was placed at 23 • C before being exposed to AMF. After monitoring the release of dye every 1 h over the course of 4 h with a UV spectrophotometer, the samples were exposed to an AMF (5.82 × 10 8 Am −1 s −1 ) for 5, 10, 15, and 20 min. As shown in Figure 6A-E, about 12.5% release of dye was detected in the first 4 h at 23 • C before exposure to an AMF, showing that the tightness of the ACVA can prevent early leakage of most parts of the TB in nanoparticles. After being exposed to AMF for different times, similar release profiles of TB were observed in Figure 6B-F: (i) after AMF, the burst increase in release for 1 h; (ii) after 1 h, the release efficiency of TB slowed down as the magnetic nanoparticles returned to the original temperature. Moreover, the concentration of TB increased 3.7 ± 0.2%, 4.8 ± 0.2%, 5.5 ± 0.1%, and 6.7 ± 0.2% after 5, 10, 15, and 20 min of AMF trigger time, respectively. The results of the linear fit of the increments of TB concentrations controlled by AMF suggested that the release of the drug was correlated with the AMF trigger time ( Figure 6F).

Multiple Magneto-thermal Controlled Release of Fe3O4@CS-ACVA-TB Nanoparticles
The second type of release study was performed with multiple sequential exposures of AMF to explore whether the material could achieve the effect of multiple controlled releases. To confirm the robustness of the ACVA, Fe3O4@CS-ACVA-TB suspension (1 mg/mL) was placed at 23 °C and was monitored for the release of dye every 1 h. The leakage of dye was less than 15%, observed in the first 4 h at 23 °C before exposure to the first cycle of AMF triggering (5.82 × 10 8 Am −1 s −1 ). A total of three cycles of AMF triggering were performed at room temperature, and the AMF triggering time was 5, 10, 15, and 20 min in every cycle, respectively. The release efficiency of the drug increased with the increase in the AMF time during each magneto-thermal cycle (Figure 7), which is similar with that described above for a single application of AMF. Subsequently, when the applied AMF was withdrawn, the drug release efficiency showed a subsequent increase followed by a plateau. This release pattern reappeared when both the second and third magnetic fields were applied.

Multiple Magneto-thermal Controlled Release of Fe 3 O 4 @CS-ACVA-TB Nanoparticles
The second type of release study was performed with multiple sequential exposures of AMF to explore whether the material could achieve the effect of multiple controlled releases. To confirm the robustness of the ACVA, Fe 3 O 4 @CS-ACVA-TB suspension (1 mg/mL) was placed at 23 • C and was monitored for the release of dye every 1 h. The leakage of dye was less than 15%, observed in the first 4 h at 23 • C before exposure to the first cycle of AMF triggering (5.82 × 10 8 Am −1 s −1 ). A total of three cycles of AMF triggering were performed at room temperature, and the AMF triggering time was 5, 10, 15, and 20 min in every cycle, respectively. The release efficiency of the drug increased with the increase in the AMF time during each magneto-thermal cycle (Figure 7), which is similar with that described above for a single application of AMF. Subsequently, when the applied AMF was withdrawn, the drug release efficiency showed a subsequent increase followed by a plateau. This release pattern reappeared when both the second and third magnetic fields were applied.
As above results showed, it is evident that Fe 3 O 4 @CS nanocomposites have an excellent ability to control drug release by magnetic heating. When AMF is applied, the magnetic nanoparticles would respond rapidly to generate local high temperature, which will break the azo bond in ACVA and then affect the release kinetics of the drug from the nanoparticles, accelerating the release of the drug. With the withdrawal of AMF, the nanoparticles would gradually stabilize, and the release behavior of the drug therein would again follow the release kinetics at the ambient temperature that they are exposed to. Moreover, multiple magneto-thermal releases of Fe 3 O 4 @CS nanocomposites can be achieved by controlling the switch of the AMF generation device as well. As above results showed, it is evident that Fe3O4@CS nanocomposites have an excellent ability to control drug release by magnetic heating. When AMF is applied, the magnetic nanoparticles would respond rapidly to generate local high temperature, which will break the azo bond in ACVA and then affect the release kinetics of the drug from the nanoparticles, accelerating the release of the drug. With the withdrawal of AMF, the nanoparticles would gradually stabilize, and the release behavior of the drug therein would again follow the release kinetics at the ambient temperature that they are exposed to. Moreover, multiple magneto-thermal releases of Fe3O4@CS nanocomposites can be achieved by controlling the switch of the AMF generation device as well.

Conclusions
In summary, CS-modified magnetic nanoparticles (Fe3O4@CS), with a high magnetization (54.0 emu/g), were successfully synthesized by the co-precipitation method. Subsequently, the temperature-sensitive molecule ACVA and the amino-based model drug toluidine blue were modified on the surface of Fe3O4@CS and were used to illustrate that the nanocarriers could control drug release. In vitro hydrothermal release studies showed that in the absence of AMF, most parts of the TB would be effectively enclosed within the nanocarriers at lower ambient temperatures (23 or 37 °C) due to the molecular bonding of ACVA, and the ACVA could remain unbroken for a longer period (at least 9 days in 37 °C). However, with the increase in incubation temperature (57 °C), the ACVA would become unstable, resulting in the slow release of TB immobilized by ACVA. Moreover, the results of kinetic fitting of hydrothermal release data showed that TB released from nanoparticles followed first-order kinetics (R 2 > 0.99) and the Korsemeyer-Peppas model (R 2 > 0.99, n < 0.5). In the next experiments, when the Fe3O4@CS-ACVA-TB nanoparticles were exposed to the AMF, the magnetic core would respond rapidly to generate local high temperature, which would break the azo bond in ACVA and then affect the release kinetics of the drug from the nanoparticles. We effectively controlled the release of TB by adjusting the time of applied AMF on the magnetic nanoparticles, and the results showed an approximately linear relationship between the amount of drug release and the time of AMF action (R 2 = 0.9712). Moreover, the drug could be released gradually even after several magneto-thermal cycles. These results indicated that the ACVA-modified Fe3O4@CS nanocarriers as a magneto thermally responsive drug-controlled release model processes a significant potential in controlling the temperature and drug dose.

Conclusions
In summary, CS-modified magnetic nanoparticles (Fe 3 O 4 @CS), with a high magnetization (54.0 emu/g), were successfully synthesized by the co-precipitation method. Subsequently, the temperature-sensitive molecule ACVA and the amino-based model drug toluidine blue were modified on the surface of Fe 3 O 4 @CS and were used to illustrate that the nanocarriers could control drug release. In vitro hydrothermal release studies showed that in the absence of AMF, most parts of the TB would be effectively enclosed within the nanocarriers at lower ambient temperatures (23 or 37 • C) due to the molecular bonding of ACVA, and the ACVA could remain unbroken for a longer period (at least 9 days in 37 • C). However, with the increase in incubation temperature (57 • C), the ACVA would become unstable, resulting in the slow release of TB immobilized by ACVA. Moreover, the results of kinetic fitting of hydrothermal release data showed that TB released from nanoparticles followed first-order kinetics (R 2 > 0.99) and the Korsemeyer-Peppas model (R 2 > 0.99, n < 0.5). In the next experiments, when the Fe 3 O 4 @CS-ACVA-TB nanoparticles were exposed to the AMF, the magnetic core would respond rapidly to generate local high temperature, which would break the azo bond in ACVA and then affect the release kinetics of the drug from the nanoparticles. We effectively controlled the release of TB by adjusting the time of applied AMF on the magnetic nanoparticles, and the results showed an approximately linear relationship between the amount of drug release and the time of AMF action (R 2 = 0.9712). Moreover, the drug could be released gradually even after several magneto-thermal cycles. These results indicated that the ACVA-modified Fe 3 O 4 @CS nanocarriers as a magneto thermally responsive drug-controlled release model processes a significant potential in controlling the temperature and drug dose.