Preparation of CSNPs
In this work, chitosan nanoparticles were prepared by ionotropic gelation. This method involves ionic interactions between the positive charge of CS and the negative charge of TPP. The chitosan molecules are gelled when they crosslinked ionically. Ionic crosslinking of chitosan is a noncovalent interaction which can occure in the presence of negatively charged multivalent ions like TPP. Noncovalent or physical crosslinking is more promising for pharmaceutical applications than covalent crosslinking, because it is reversible and may avoid toxicity of the reagents [23].
Previous studies showed that the particle size and surface loadings has been affected by the chitosan concentration, the ratio of CS:TPP, and pH of the solution. The primary aim was to determine the conditions that the nanoparticles can produced with optimal characteristics, such as proper nanoparticle size and minimum polydispersty index. The particle size affected the ability of the drug delivery system to penetrate the tissue and effective drug release [23, 24].
It was found that by increasing the CS concentration the size of the formed particles increase. This also occurs with increasing TPP concentration. In the present study, the suspension of CS-TPP nanoparticles with concentrations of 0.1, 0.2 and 0.3 CS as well as TPP with concentration of 0.45 mg/l was prepared. The effect of different concentrations of these two solutions on particle size and morphological properties was investigated.
Dynamic light scattering (DLS)
As was mentioned before, many factors affected the size and distribution of chitosan nanoparticle including molecular weight of chitosan, CS:TPP ratio, the addition conditions of TPP to chitosan, chitosan and TPP concentration, the pH of chitosan, ambient temperature, and agitation rate. DLS technique was used to measure the hydrodynamic diameter in the nanometer range. Table 1 shows the effect of CS:TPP ratio on the size of chitosan nanoparticle. According to the results the CSNPs sample with size of 99.73 nm and CS:TPP ratio of 1:1 was selected for further analysis.
Table 1
Effect of CS:TPP ratio on the nanoparticle size.
Chitosan conc.
(mg/ml)
|
TPP conc.
(mg/ml)
|
CS:TPP
|
Stirrer velocity
(rpm)
|
Temperature (°C)
|
Nanoparticle diameter (nm)
|
0.1
|
0.45
|
1:1
|
6000
|
25
|
325.84
|
0.2
0.3
|
0.45
0.45
|
1:1
1:2
|
9000
9000
|
25
25
|
680.93
478.97
|
0.2
|
0.45
|
1:1
|
9000
|
25
|
99.73
|
Dynamic light scattering test was also performed after preparing vancomycin loaded chitosan nanoparticles. it was observed in Fig. 3 that the nanoparticles size increased due to drug loading and reached about 100 nm.
The nanoparticles size depends greatly on the ratio of CS:TPP, and with increasing this ratio from 1:1 to 2:1, the nanoparticle size has changed from 305.84 nm to 93.73 nm. The DLS test was also performed after loading vancomycin on the nanoparticles. It was observed that the size of the nanoparticles increased slightly due to the drug loading and the average nanoparticle size reached 100.88 nm (Fig. 3).
Scanning electron microscopy (SEM)
The SEM images of chitosan nanoparticles with and without vancomycin are shown in Fig. 4. As can see in Fig. 4a, the image of unloaded CSNPs revealed a smooth and homogenous surface. surface nanoparticles represent a very uniform and spherical shape morphology with average size of 86 nm. SEM image of vancomycin-loaded chitosan nanoparticles is shown in Fig. 4b. The image also revealed a smooth and homogenous surface without any crystallized vancomycin. On the other hand, the drug is distributed homogenously at molecular level, which corresponds to the results obtained by some researchers [16, 27].
FTIR spectroscopy
Figure 5 shows the FTIR spectra of chitosan, CSNPs, vancomycin, and vancomycin-loaded CSNPs. In the chitosan spectrum (Fig. 5a), a peak at 3360 cm− 1 is observed for O-H group overlapped with N-H group stretching vibrations. The peak appears at 2963 cm− 1 is assigned to the C − H stretching vibration mode. The absorption peaks at 1633 and 1594 cm− 1 are attributed to C = O bending vibration and N-H bending vibration of protonated amino (-NH2) group, respectively. The C-N stretching vibration of the amine group was observed at 1230 cm− 1 and the asymmetric C-O-C stretch was observed at about 1158 cm− 1. A weak peak observed at 1420 cm− 1 is attributed to the bending vibration of O − H [26]. The 1580 cm− 1 peak of –NH bending vibration shifts to 1630 cm− 1 and a new peak appears at position 1530 cm− 1 which is assigned to N-O-P stretching vibration. This indicates that the TPP anions were crosslinked with the ammonium groups of CS to form CS-NPs as illustrated in Fig. 2. Similar results were also obtained in other works [11, 12].
The CSNPs spectrum (Fig. 5b), the peak at 3427 cm− 1 is attributed to O-H group overlapped with N-H group stretching vibrations and indicates that hydrogen bonding is increased. The peak at 1594 cm− 1 for bending vibration of N − H shifts to 1635 cm− 1 and the new peak at 1553 cm− 1 is attributed to N-O-P stretching vibration. which implies the crosslinking of TPP anions with ammonium groups of chitosan to form chitosan nanoparticles [28]. Figures 5c and 5d show the FTIR spectrum of vancomycin drug and drug-loaded CSNPs. FTIR spectra of vancomycin show its characteristic broader peak attributed to OH stretching vibration of R-CH2-CH3, COOH, R-NH-R around 3240 cm− 1, carbonyl stretching vibration of amide group at 1655 cm− 1, alkyl chain at 1492 cm− 1-1122 cm− 1 band, R-O-R stretching vibration at 1010 cm− 1 and aromatic peak at 1552 cm− 1.
The spectra of drug-loaded CSNPs in Fig. 5d show the overlapped of N-H stretching vibration peak at 3431 cm− 1 and shifting of carbonyl stretching to 1540 cm− 1-1395 cm− 1, R-O-R stretching similar peaks observed at 1010 cm− 1 and aromatic peak at 1637 cm− 1. Thus, the characteristic spectrum in Fig. 5d show shifting and overlapping of absorption peaks assigned the successful loading of vancomycin on CSNPs.
X-ray diffraction analysis (XRD)
The X-ray patterns of chitosan, chitosan nanoparticles, and vancomycin-loaded CSNPs are shown in Fig. 6. While chitosan has a strong peak at 2θ = 22° (Fig. 6a), CSNPs has a weak and broad peak (Fig. 6b). This is due the decreasing of intensity of the characteristic peak for chitosan nanoparticles which is attributed to the crosslinking. This pattern change can be assigned to the modification of molecules arrangement in the crystal lattice. This intensity reduction is more obvious in the vancomycin-loaded CSNPs pattern. It is clear in Fig. 6c that the pattern shows a stronger peak than that of CSPNs peak at 2θ = 25°.
Thermogravimetric analysis (TGA)
The TGA thermogram of chitosan, chitosan nanoparticles, vancomycin, and vancomycin-loaded chitosan nanoparticles are shown in Fig. 7. It is clear that for chitosan, the first weight loss about 3.5% is occurred at 50°C to 100°C which is attributed to the loss of absorbed or bound water severe weight loss and related to the hydrophilic nature of chitosan [29]. The decomposition is initiated at 200°C and obvious weight loss occurred from 200°C to 450°C which probably corresponds to the dehydration of the anhydro glucosidic ring. The residue remained at the end of the experiment (at 800°C) is about 22%. As can be seen for chitosan nanoparticle, the thermal behavior is changed. The water elimination is occurred within 50°C to 100°C with 7.5% weight loss. It seems that more weight loss at the dehydration stage of CSNPs than CS due to its higher hydrophilicity that resulted in more bound water. CSNPs is decomposed from 150°C to 250°C with 60% remaining residue.
Vancomycin is decomposed at 155°C to 560°C with 20% final remaining residue. The thermal behaviour pattern of drug-loaded CSNPs is similar to CSNPs which the decomposition of CSNPs is occurred from 152°C to 280°C. In general, the results confirm that thermal stability of CSNPs is higher than CS, which can be corresponded to the interactions due to the crosslinking of CS molecules with TPP. Also, the crosslinker reduces the decomposition temperature. These results are consistent with the results of other researchers [28, 29].
Differential scanning calorimetry (DSC)
The DSC results of chitosan, chitosan nanoparticles, vancomycin, and vancomycin-loaded chitosan nanoparticles are shown in Fig. 8. It is obvious that vancomycin has melting point (Tm) of 240°C and a glass transition temperature (Tg) of 72 ℃. The melting point and glass transition temperature for chitosan are 230°C and 67.5 ℃ and for and chitosan nanoparticles 240°C and 75.7 ℃. These temperatures are 248°C and 70 ℃, respectively for vancomycin-loaded chitosan nanoparticles. ve had wider characteristics at melting temperature of 67.5 and glass ℃ 230. Thermography of chitosan nanoparticles The melting temperature of glass and its nanoparticles reached 75.7, 240 degrees Celsius, and the chitosan nanoparticles with drug showed marked signs of vancomycin at 70 degrees Celsius, 248 degrees Celsius.
As can be seen, teicoplanin thermogram shows a melting point (Tm) of 75.3 ℃ and a glass transition temperature (Tg) of 250.6 ℃, while for chitosan these temperatures are 67.5 ℃ and 230 ℃, respectively. Due to the nanoscale of chitosan nanoparticles, their Tm and Tg have risen to 75.5 ℃ and 240 ℃, respectively. The thermogram of teicoplanin loaded chitosan nanoparticles exhibited all characteristic peaks of teicoplanin at 72°C and 250°C, therefore there is no interaction between teicoplanin and chitosan.
Drug release
The vancomycin release from chitosan nanoparticles was evaluated for 100 h. A burst release of vancomycin from chitosan nanoparticles was observed at the initial stage. 40% of vancomycin was released in the first 9 h. Then the release is continued gradually and finally 90 % of vancomycin was released in 100 h. It seems that chitosan nanoparticles reduce the drug release rate and vancomycin-loaded CSPNs are suitable for sustained drug release.