**3.5 Thermogravimetric (TGA) analysis**

To evaluate the thermal stability of the chitosan (CS) oxidized chitosan (OCS) and (CS/OCS) lyophilized hydrogel, TGA thermograms were obtained as shown in (**Figure 13**). The TGA of pure chitosan shows two-stage weight loss in the range 40

**15**

**Figure 13.**

biopolymer-based Schiff base is thermally less stable.

The TGA curves of uncoated Fe3O4 and the lyophilized ferrogel (CS-Fe3O4-OCS)

are shown in **Figure 14**. For uncoated Fe3O4 NPs (**Figure 14(a)**), the TGA curve showed that the weight loss over the temperature range 40–750°C was about 2.2%.

*A Novel Drug Delivery System Based on Nanoparticles of Magnetite Fe3O4 Embedded in an Auto…*

to 750°C (**Figure 13**), it is clear that chitosan started to degrade at 40–130°C with 8% weight is due to the loss of water molecules. Initial decomposition around 130°C for pure chitosan can be attributed to the strong water adsorptive nature of chitosan. The second stage of degradation occurred at 340°C and continued up to 460°C [67]. There was 38.43% weight loss occurring in the second stage due to degradation of pure chitosan biopolymer and the temperature at which maximum degradation observed was 274.74°C. At the end of 750°C, the total weight loss of sample was 100% [54, 67]. TGA of oxidized chitosan (OCS) showed two steps of degradation (**Figure 13**), the first stage ranges between 40 and 130°C and shows about 8.2% loss in weight corresponded to water release for the initial step. The second stage decomposition was observed from 300°C and continued up to 460°C, during this time there was 39% weight loss due to the degradation of chitosan. At the end of 700°C, the total weight loss of sample was 90% [10, 54]. For CS/OCS lyophilized hydrogel, the degradation starts at a lower temperature compared to chitosan and oxidized chitosan (**Figure 13**). For the CS/OCS hydrogel, shows two-stage weight loss in three stages. The first stages of degradation takes place from 40 to 160°C with a weight loss of 13% could be due to the loss of both the loosely bound water and tightly bound water [68]. The free water and hydrogen-bonded water are released at a temperature between 40 and 100°C. The hydrogel contains many hydrophilic groups that retain water more tightly in the hydrogel skeleton by polar interaction. As a result, it is harder to lose. Thus, this tightly bound water is released in the temperature region 100–156°C. The second stage of hydrogel degradation started between 160° C and 385° C with a 41% weight loss. The three-stages of degradation biopolymers, at the end of 700°C, the total weight loss of sample was 90%. This phase of the weight loss mainly could be caused by a series of thermal and oxidative decomposition in the process including dehydration of the sugar cycle, degradation, N-deacetylation of the molecular chain of the chitosan cracking unit and vaporization and removal of volatile products. It can be concluded the TGA curve shows at about 222°C. This is probably due to the formation of (-C=N-) and this proves that

*TGA graphs of chitosan (CS), oxidized chitosan (OCS) and (CS/OCS) lyophilized hydrogel.*

*DOI: http://dx.doi.org/10.5772/intechopen.94873*

*A Novel Drug Delivery System Based on Nanoparticles of Magnetite Fe3O4 Embedded in an Auto… DOI: http://dx.doi.org/10.5772/intechopen.94873*

**Figure 13.** *TGA graphs of chitosan (CS), oxidized chitosan (OCS) and (CS/OCS) lyophilized hydrogel.*

to 750°C (**Figure 13**), it is clear that chitosan started to degrade at 40–130°C with 8% weight is due to the loss of water molecules. Initial decomposition around 130°C for pure chitosan can be attributed to the strong water adsorptive nature of chitosan. The second stage of degradation occurred at 340°C and continued up to 460°C [67]. There was 38.43% weight loss occurring in the second stage due to degradation of pure chitosan biopolymer and the temperature at which maximum degradation observed was 274.74°C. At the end of 750°C, the total weight loss of sample was 100% [54, 67]. TGA of oxidized chitosan (OCS) showed two steps of degradation (**Figure 13**), the first stage ranges between 40 and 130°C and shows about 8.2% loss in weight corresponded to water release for the initial step. The second stage decomposition was observed from 300°C and continued up to 460°C, during this time there was 39% weight loss due to the degradation of chitosan. At the end of 700°C, the total weight loss of sample was 90% [10, 54]. For CS/OCS lyophilized hydrogel, the degradation starts at a lower temperature compared to chitosan and oxidized chitosan (**Figure 13**). For the CS/OCS hydrogel, shows two-stage weight loss in three stages. The first stages of degradation takes place from 40 to 160°C with a weight loss of 13% could be due to the loss of both the loosely bound water and tightly bound water [68]. The free water and hydrogen-bonded water are released at a temperature between 40 and 100°C. The hydrogel contains many hydrophilic groups that retain water more tightly in the hydrogel skeleton by polar interaction. As a result, it is harder to lose. Thus, this tightly bound water is released in the temperature region 100–156°C. The second stage of hydrogel degradation started between 160° C and 385° C with a 41% weight loss. The three-stages of degradation biopolymers, at the end of 700°C, the total weight loss of sample was 90%. This phase of the weight loss mainly could be caused by a series of thermal and oxidative decomposition in the process including dehydration of the sugar cycle, degradation, N-deacetylation of the molecular chain of the chitosan cracking unit and vaporization and removal of volatile products. It can be concluded the TGA curve shows at about 222°C. This is probably due to the formation of (-C=N-) and this proves that biopolymer-based Schiff base is thermally less stable.

The TGA curves of uncoated Fe3O4 and the lyophilized ferrogel (CS-Fe3O4-OCS) are shown in **Figure 14**. For uncoated Fe3O4 NPs (**Figure 14(a)**), the TGA curve showed that the weight loss over the temperature range 40–750°C was about 2.2%.

*Chitin and Chitosan - Physicochemical Properties and Industrial Applications*

several thousands of micrometers [55]. Moreover, the more crosslinking between amino group and aldehyde group, the diameter of the pores are decreased and the compactness of pores increased. The micrographs of CS/OCS hydrogel it was clearly observed that the hydrogel had a three-dimensional porous structure, which was

*Micrographs of chitosan cross-linked oxidized chitosan hydrogel at low and high magnification.*

*The magnetic hysteresis loops for Fe3O4 NPs and CS@Fe3O4@OCS hydrogel measured by SQUID at room* 

To evaluate the thermal stability of the chitosan (CS) oxidized chitosan (OCS) and (CS/OCS) lyophilized hydrogel, TGA thermograms were obtained as shown in (**Figure 13**). The TGA of pure chitosan shows two-stage weight loss in the range 40

**14**

**Figure 11.**

*temperature.*

**Figure 12.**

beneficial for drug delivery systems.

**3.5 Thermogravimetric (TGA) analysis**

**Figure 14.** *TGA curves for a) uncoated Fe3O4 b) CS-Fe3O4-OCS lyophilized ferrogel.*

Hence, this weight loss is related to removal of the physically adsorbed water and/ or hydroxyl groups on the surface of Fe3O4 nanoparticles. For coated nanoparticles Fe3O4. Similarly, the weight curve of CS/Fe3O4/OCS (**Figure 14(b)**) showed a progressive decrease of 85%. This is due to the degradation of the polymer and hydrogen-bound water in the temperature range of 40–166°C, which forms the polysaccharide structure of OCS and CS. In addition, a uniform and steady decrease in weight loss at 166–630°C is CS/Fe3O4/OCS was observed. This may be partly attributed to the degradation and decomposition of organic skeletal structure, amino groups, and other functional groups. By comparing the curves of CS/Fe3O4/ OCS and Fe3O4, it was observed that Fe3O4 particles are wrapped into CS and OCS can enhance the thermal stability of the whole system. A temperature of 630°C or higher, the remaining material was carbonized completely. The indicated that chitosan and oxidized chitosan coated Fe3O4 successfully and penetrated deeply into in the matrix CS/OCS.

### **3.6 Hydrogel swelling**

To investigate the pH dependent swelling behavior of the CS/OCS hydrogels for drug delivery, PBS with (pH = 1.2; pH = 5.8 and pH = 7.4) at T = 37°C were used to simulate the physiological medium and were used for testing swelling of the hydrogels. The results of the equilibrium-swelling ratio are presented in (**Figure 15**). The CS/OCS hydrogels showed large differences in swelling behavior at different pH values. The pKa value of the D-glucosamine residue in chitosan was approximately 6.2 to 7.0. Therefore, the amino groups in the chitosan were protonated and positively charged in acidic PBS (pH = 1.2 and pH = 5.8), and the electrostatic repulsion between positively charged -NH3+ groups would lead to swelling of the hydrogels. The % equilibrium swelling values were found to be higher at pH = 1.2, than at pH = 7.4. This can be explained by protonation of the unreacted NH2 groups of chitosan at acidic pH, leading to dissociation of the hydrogen bonding involving the amino groups, and thus facilitating the entry of the solvent into the material. The swelling process of hydrogels involves the ionization [69]. Furthermore, the Schiff base bonds

**17**

pH 7.4 medium.

**Figure 15.**

**3.7 In vitro release from the hydrogels**

*A Novel Drug Delivery System Based on Nanoparticles of Magnetite Fe3O4 Embedded in an Auto…*

between -NH2 and -CHO as a cross-linker became weak in PBS (pH = 7.4 at 37°C); the swelling ratio of CS/OCS was 350%, resulting in swelling of the hydrogel. Therefore, the CS/OCS hydrogel in PBS (pH = 1.2 at 37°C) exhibited the largest swelling ratio of 670%; the swelling ratio (pH = 5.8 at 37°C) was 509%. The decreased swelling ratio occurred due to increased crosslinking density in the hydrogels. Meanwhile, the equilibrium swelling ratios of the hydrogels in a pH = 7.4 solution dramatically decreased. This is because hydrogels exhibited pH-responsive swelling behavior, and the hydrogels showed a much higher swelling ratio in an acidic medium than that in a

*% Swelling of CS/OCS hydrogel in buffer solutions of pH = 1,2, pH = 5,8 and pH = 7,4 at T = 37°C.*

The model drugs (5-FU, caffeine and ascorbic acid) was encapsulated in the hydrogel matrix used for the release kinetics in PBS (pH = 7.4 at 37°C) are depicted in (**Figure 16**). The purpose here was to study whether the release of the drug trapped in the hydrogels, as well as by the simple diffusion. In a system, where drug is entrapped in a biodegradable matrix, the release rate depends on three parameters: the size of the drug molecule, the drug solubility (soluble-sparingly solubleinsoluble), the cross-linking density and the degradation rate. The ability of the chitosan-imine-oxidized chitosan hydrogels to act as matrix for controlled release was investigated in vitro by monitoring the release profile of the (5-FU, caffeine and ascorbic acid) three-model drug from system in phosphate buffer of physiological pH (7.4), at the human body temperature of 37°C (**Figure 16**). In order to study the release behavior of 5-FU, caffeine, ascorbic acid incorporated in the chitosan-based hydrogel cross-linked by oxidized chitosan, they were incubated in release media (phosphate buffer pH = 7.4 at 37°C) and evaluated by UV spectrophotometry.

*DOI: http://dx.doi.org/10.5772/intechopen.94873*

*A Novel Drug Delivery System Based on Nanoparticles of Magnetite Fe3O4 Embedded in an Auto… DOI: http://dx.doi.org/10.5772/intechopen.94873*

**Figure 15.** *% Swelling of CS/OCS hydrogel in buffer solutions of pH = 1,2, pH = 5,8 and pH = 7,4 at T = 37°C.*

between -NH2 and -CHO as a cross-linker became weak in PBS (pH = 7.4 at 37°C); the swelling ratio of CS/OCS was 350%, resulting in swelling of the hydrogel. Therefore, the CS/OCS hydrogel in PBS (pH = 1.2 at 37°C) exhibited the largest swelling ratio of 670%; the swelling ratio (pH = 5.8 at 37°C) was 509%. The decreased swelling ratio occurred due to increased crosslinking density in the hydrogels. Meanwhile, the equilibrium swelling ratios of the hydrogels in a pH = 7.4 solution dramatically decreased. This is because hydrogels exhibited pH-responsive swelling behavior, and the hydrogels showed a much higher swelling ratio in an acidic medium than that in a pH 7.4 medium.

## **3.7 In vitro release from the hydrogels**

The model drugs (5-FU, caffeine and ascorbic acid) was encapsulated in the hydrogel matrix used for the release kinetics in PBS (pH = 7.4 at 37°C) are depicted in (**Figure 16**). The purpose here was to study whether the release of the drug trapped in the hydrogels, as well as by the simple diffusion. In a system, where drug is entrapped in a biodegradable matrix, the release rate depends on three parameters: the size of the drug molecule, the drug solubility (soluble-sparingly solubleinsoluble), the cross-linking density and the degradation rate. The ability of the chitosan-imine-oxidized chitosan hydrogels to act as matrix for controlled release was investigated in vitro by monitoring the release profile of the (5-FU, caffeine and ascorbic acid) three-model drug from system in phosphate buffer of physiological pH (7.4), at the human body temperature of 37°C (**Figure 16**). In order to study the release behavior of 5-FU, caffeine, ascorbic acid incorporated in the chitosan-based hydrogel cross-linked by oxidized chitosan, they were incubated in release media (phosphate buffer pH = 7.4 at 37°C) and evaluated by UV spectrophotometry.

*Chitin and Chitosan - Physicochemical Properties and Industrial Applications*

Hence, this weight loss is related to removal of the physically adsorbed water and/ or hydroxyl groups on the surface of Fe3O4 nanoparticles. For coated nanoparticles Fe3O4. Similarly, the weight curve of CS/Fe3O4/OCS (**Figure 14(b)**) showed a progressive decrease of 85%. This is due to the degradation of the polymer and hydrogen-bound water in the temperature range of 40–166°C, which forms the polysaccharide structure of OCS and CS. In addition, a uniform and steady decrease in weight loss at 166–630°C is CS/Fe3O4/OCS was observed. This may be partly attributed to the degradation and decomposition of organic skeletal structure, amino groups, and other functional groups. By comparing the curves of CS/Fe3O4/ OCS and Fe3O4, it was observed that Fe3O4 particles are wrapped into CS and OCS can enhance the thermal stability of the whole system. A temperature of 630°C or higher, the remaining material was carbonized completely. The indicated that chitosan and oxidized chitosan coated Fe3O4 successfully and penetrated deeply

*TGA curves for a) uncoated Fe3O4 b) CS-Fe3O4-OCS lyophilized ferrogel.*

To investigate the pH dependent swelling behavior of the CS/OCS hydrogels for drug delivery, PBS with (pH = 1.2; pH = 5.8 and pH = 7.4) at T = 37°C were used to simulate the physiological medium and were used for testing swelling of the hydrogels. The results of the equilibrium-swelling ratio are presented in (**Figure 15**). The CS/OCS hydrogels showed large differences in swelling behavior at different pH values. The pKa value of the D-glucosamine residue in chitosan was approximately 6.2 to 7.0. Therefore, the amino groups in the chitosan were protonated and positively charged in acidic PBS (pH = 1.2 and pH = 5.8), and the electrostatic repulsion between positively charged -NH3+ groups would lead to swelling of the hydrogels. The % equilibrium swelling values were found to be higher at pH = 1.2, than at pH = 7.4. This can be explained by protonation of the unreacted NH2 groups of chitosan at acidic pH, leading to dissociation of the hydrogen bonding involving the amino groups, and thus facilitating the entry of the solvent into the material. The swelling process of hydrogels involves the ionization [69]. Furthermore, the Schiff base bonds

**16**

into in the matrix CS/OCS.

**3.6 Hydrogel swelling**

**Figure 14.**

**Figure 16.**

*In vitro release of 5-FU, caffeine and ascorbic acid embedded in hydrogels (pH = 7,4 at 37°C). Values reported are an average of n = 3 ± standard deviation.*

(**Figure 16**) shows release profiles of 5-FU, caffeine and ascorbic acid up to 26 h of incubation period. As shown in (**Figure 16**), the chitosan hydrogels showed an initial burst release of 5-FU, caffeine and ascorbic acid over a period of 3 h for all incubation media, which was of the order of (5 -FU "52%", caffeine "43%" and ascorbic acid "60%"). This initial rapid release, characterized by a "burst effect», because certain quantities of 5-FU, caffeine and ascorbic acid were localized on the surface of the hydrogels by adsorption which could be released easily by diffusion. After 3h the release percentages of (5-FU "86%", caffeine "89%" and ascorbic acid "91%"), respectively [70]. The remaining part of the drug can be trapped in hydrogels because the amino functions of chitosan can enter into the Schiff reaction with the aldehyde groups of oxidized chitosan. Indeed, the hydrogel contained large pore size, which is beneficial for the diffusion of the drug, this initial bursting effect, a slower sustained and controlled release occurred throughout the incubation period and the amount of release. The release profiles confirmed that the (5-FU, caffeine and ascorbic acid) drugs were encapsulated in hydrogels.

As shown in **Figure 17**, magneto-hydrogel showed an initial burst release of (5-FU, caffeine and ascorbic acid) in a period of 5 h, which was in the range of (37%, 34%, and 42%). The initial burst phase is caused by drug adsorbed on the surface of the nanoparticles Fe3O4 embedded in hydrogel. The release kinetics at pH = 7.4 at 37°C within 45 h clearly indicated that Fe3O4 embedded in hydrogel influenced drugs release. At pH = 7.4 at 37°C, about (95%, 93% and 97%) amounts of 5-FU, caffeine and ascorbic acid were release after 45 h, respectively. Due to the slower swelling rate of nanotransporters in solution, the rate of drug release is also slower. It is well known that there are a large number of amino and hydroxyl groups on the surface of chitosan molecules, which provide functional groups and favorable characteristics for biological molecules. In PBS (pH = 7.4 at 37°C), amino

**19**

**4. Conclusion**

**Figure 17.**

*A Novel Drug Delivery System Based on Nanoparticles of Magnetite Fe3O4 Embedded in an Auto…*

groups of chitosan mainly attach to the surface of the nanoparticles, so as to reduce the surface voids and render the pore blockage, lower penetration, and thus slow down the release rate of the drug [36]. However, due to the magnetic orientation role of MNPs, magnetic nano-drug carriers could be transported by applying an external magnetic field and maintain drug concentration for extended periods. Such rapid transport and slow release of the nanocarriers to the target site may be desirable for many biomedical applications, minimizing drug leakage to undesirable sites and reducing the risk of heart attack due to high dose in a short period of time. These results clearly illustrated that the chitosan hydrogel containing Fe3O4 resulted in a barrier system for the sustained release of 5-Fu, caffeine and ascorbic acid. We speculate that this barrier structure would block the drug loss in the early burst

*Cumulative release profiles of 5-FU-gel, caffeine-gel, ascorbic acid-gel with external magnetic field. Values* 

Many conclusions can be made from the present work, the ferrogels (FG) are cross-linked polymer networks containing magnetic nanoparticles: a) magnetic magnetite (Fe3O4) were synthesized successfully by chemical co-precipitation and has been confirmed using FT-IR, VSM analysis, TGA. The advantage of the co-precipitation method are low cost, rapidity, ease, reproducibility and high-yield synthesis. b) The hydrogel was formulated by cross-linking chitosan (CS) and oxidized chitosan (OCS) via the Schiff-base (-C=N-) reaction. Obviously, these results indicate that this exhibit non-toxic, biodegradable, good injectability, less expensive and respect the environment, quick gelation time, in vitro pH-dependent equilibrated swelling ratios, interconnected porosity. c) Magneto-responsive hydrogels are typically prepared by incorporating magnetic particles into hydrogels

release, which is benefit to reduce the toxic side effects.

*DOI: http://dx.doi.org/10.5772/intechopen.94873*

*reported are an average of n = 3 ± standard deviation.*

*A Novel Drug Delivery System Based on Nanoparticles of Magnetite Fe3O4 Embedded in an Auto… DOI: http://dx.doi.org/10.5772/intechopen.94873*

**Figure 17.**

*Chitin and Chitosan - Physicochemical Properties and Industrial Applications*

(**Figure 16**) shows release profiles of 5-FU, caffeine and ascorbic acid up to 26 h of incubation period. As shown in (**Figure 16**), the chitosan hydrogels showed an initial burst release of 5-FU, caffeine and ascorbic acid over a period of 3 h for all incubation media, which was of the order of (5 -FU "52%", caffeine "43%" and ascorbic acid "60%"). This initial rapid release, characterized by a "burst effect», because certain quantities of 5-FU, caffeine and ascorbic acid were localized on the surface of the hydrogels by adsorption which could be released easily by diffusion. After 3h the release percentages of (5-FU "86%", caffeine "89%" and ascorbic acid "91%"), respectively [70]. The remaining part of the drug can be trapped in hydrogels because the amino functions of chitosan can enter into the Schiff reaction with the aldehyde groups of oxidized chitosan. Indeed, the hydrogel contained large pore size, which is beneficial for the diffusion of the drug, this initial bursting effect, a slower sustained and controlled release occurred throughout the incubation period and the amount of release. The release profiles confirmed that the (5-FU, caffeine

*In vitro release of 5-FU, caffeine and ascorbic acid embedded in hydrogels (pH = 7,4 at 37°C). Values reported* 

As shown in **Figure 17**, magneto-hydrogel showed an initial burst release of (5-FU, caffeine and ascorbic acid) in a period of 5 h, which was in the range of (37%, 34%, and 42%). The initial burst phase is caused by drug adsorbed on the surface of the nanoparticles Fe3O4 embedded in hydrogel. The release kinetics at pH = 7.4 at 37°C within 45 h clearly indicated that Fe3O4 embedded in hydrogel influenced drugs release. At pH = 7.4 at 37°C, about (95%, 93% and 97%) amounts of 5-FU, caffeine and ascorbic acid were release after 45 h, respectively. Due to the slower swelling rate of nanotransporters in solution, the rate of drug release is also slower. It is well known that there are a large number of amino and hydroxyl groups on the surface of chitosan molecules, which provide functional groups and favorable characteristics for biological molecules. In PBS (pH = 7.4 at 37°C), amino

and ascorbic acid) drugs were encapsulated in hydrogels.

**18**

**Figure 16.**

*are an average of n = 3 ± standard deviation.*

*Cumulative release profiles of 5-FU-gel, caffeine-gel, ascorbic acid-gel with external magnetic field. Values reported are an average of n = 3 ± standard deviation.*

groups of chitosan mainly attach to the surface of the nanoparticles, so as to reduce the surface voids and render the pore blockage, lower penetration, and thus slow down the release rate of the drug [36]. However, due to the magnetic orientation role of MNPs, magnetic nano-drug carriers could be transported by applying an external magnetic field and maintain drug concentration for extended periods. Such rapid transport and slow release of the nanocarriers to the target site may be desirable for many biomedical applications, minimizing drug leakage to undesirable sites and reducing the risk of heart attack due to high dose in a short period of time. These results clearly illustrated that the chitosan hydrogel containing Fe3O4 resulted in a barrier system for the sustained release of 5-Fu, caffeine and ascorbic acid. We speculate that this barrier structure would block the drug loss in the early burst release, which is benefit to reduce the toxic side effects.
