3.1. Fabrication and characterization of the CS-FA-MBZ nanoparticles
For fabrication of the CS-FA-MBZ nanoparticles, at first, folic acid was conjugated to chitosan in the presence of EDC as carboxyl activating agent to produce CS-FA (Figure S1). The activated carboxyl moiety of FA was covalently linked to amine groups of CS . In the next step, the cross-linking reaction between CS-FA and TPP lead to the formation of nanoparticles in which the negatively charged TPP was electrostatically adsorbed to positively charged free protonated amine groups of CS-FA and MBZ was encapsulated during the synthesis process (Figure 1). The successful introduction of folate to CS chains was evaluated by FTIR analysis. The FTIR spectra of CS, FA, and CS-FA were shown in Figure S2. The characteristic bands of CS located at 3422 cm-1 assigned to O-H stretching vibration overlapped with N-H stretching mode. The bands observed at 2920 and 2880 cm-1 were attributed to the C-H stretching vibrations of CS. Moreover, the peaks that appeared at 1656 and 1605 cm-1 corresponded to the C-O stretching vibration of amide I and N-H bending vibration of amide II, respectively . FTIR spectrum of FA showed characteristic bands located at 1696, 1607, and 1486 cm-1 related to the C=O amide stretching vibration of the carboxyl group, N-H bending vibration of CONH, and the stretching vibration of C=C of the phenyl ring of FA, respectively . In the CS-FA spectrum, the absorption peaks at 1635 and 1031 cm-1 could be attributed to the vibration of C-N . The amid band at 1656 cm-1 of CS shifts to 1635 cm-1 because of overlap with the newly formed amide bond, and also a new N-H bending vibration located at 1520 cm-1 confirmed the successful coupling of FA to CS . After the formation of the CS-FA-MBZ nanoparticles, FTIR analysis was carried out to find the drug-polymer compatibility (Figure 2). The characteristic absorption peaks of pure MBZ at 3403, 1717, 1647, and 1523 cm-1 were also observed for CS-FA-MBZ. The stretching vibration of amide I of the carbamate group of MBZ (1717 cm-1) was also observed at the CS-FA-MBZ nanoparticles spectrum in the same location . The N-H stretching and CNH vibration were observed at 3403 and 1523 cm-1 for MBZ, 3405, and 1525 cm-1 for CS-FA-MBZ. Hence, no interaction was observed from FTIR detection.
Figures 3a and b show SEM images of the CS-FA-MBZ nanoparticles at different magnifications, in which the nanoparticles were uniform and exhibited a spherical morphology. The corresponding particle size distribution histograms (dry state) were obtained by measuring the size of 100 nanoparticles in SEM images by the “Image J” software (Figure 3c). A narrow size distribution with a mean size of 153.3 ± 18.4 nm was measured. Moreover, the particle size distribution of the CS-FA-MBZ was determined by DLS measurement (wet state) and the mean size (diameter) of the nanoparticles was measured 182 ± 12.1 nm with a low polydispersity index (PDI<0.2), illustrating a narrow size distribution and confirmed the result obtained from SEM as well (Figure 3d). Due to the measuring of hydrodynamic radius, the DLS analysis showed a slightly larger size for the nanoparticles as compared to SEM photographs . The zeta potential values showed that the positive charge of chitosan due to the presence of the amine groups decreased after FA conjugation (Figure 2e). It could be related to the interaction of FA molecules with these amine groups of CS, leading to the neutralization of the potential value . Additionally, the results depicted that the encapsulation of MBZ into the CS-FA nanoparticles could not induce a noticeable zeta value change in the final nanoparticles with the preserved positive value of +27 mV.
3.2. MBZ loading
MBZ, as a hydrophobic drug, was loaded into the CS-FA nanoparticles. The UV-Vis spectrum of MBZ (Figure 4a) showed the characteristic absorption band at 234 nm which also could be observed for CS-MBZ. This band along with the absorption band of FA at 280 nm with a slight shift to longer wavelengths could be observed in the spectrum of CS-FA-MBZ, indicating the appropriate MBZ loading into the CS-FA nanoparticles. The encapsulation efficacy (EE) and loading capacity (LC) of MBZ were calculated by plotting the standard calibration curve with a linear curve fit equation (Figure S3.). The value of EE and LC were achieved 57.7% and 10.5% respectively, demonstrating the valuable capability of the target system for MBZ loading.
3.3. In vitro MBZ release study
The release behavior of MBZ from the CS-FA-MBZ nanoparticles was evaluated at different pH values of 5.5, 6.8, and 7.4 to mimic tumor microenvironment, physiological conditions (pH =7.4 like bloodstream) and endocytic compartments conditions (pH= 5.5) . As Figure 4b illustrates, the CS-FA-MBZ nanoparticles exhibited a continuous and sustained release profile at different pH values started with a burst release within 6 h followed by a gradual release up to 1 week. The initial fast release could be related to the absorbed MBZ on the surface of nanoparticles . Moreover, a pH-responsive behavior of MBZ was observed. After 1 week, the release of MBZ was around 62 %, 49 %, and 38 % at pH values of 5.5, 6.8, and 7.4, respectively. This behavior could be assigned to the high swelling ability of CS which was induced by protonation of the amine groups of the polymer in an acidic environment . Considering the acidic microenvironment of cancerous tissues and intracellular organelles such as endosomes and lysosomes, the pH-sensitive behavior of the CS-FA-MBZ nanoparticles improves their capability for tumor-specific drug delivery and reduces the probable adverse effects to normal tissues.
3.2 The CS-FA-MBZ implants effect on the 4T1 breast tumors’ growth progression
As Figure 5a illustrates, the CS-FA and CS-FA-MBZ implants were s.c. implanted in the left flank of the tumor-bearing mice inside a small incision on the 3rd day after the cancer cell injection. The implants were completely palpable even after suturing the incision. The implants completely degraded until the 18th day in both CS-FA and CS-FA-MBZ groups and nothing was palpable at the implantation site. This means that the implants were dissociated and release their composing agents means CS-FA-MBZ nanoparticle.
The therapeutic effect of the CS-FA-MBZ implants on inhibition of 4T1 tumors’ growth progression was evaluated by serial measurement of tumor’s diameters and compared with the Control, MBZ (40 mg/kg, oral administration, twice a week for two weeks), and CS-FA groups (Figure 5b). All treatments were initiated from the 3rd day after cancer cells injection. As Figure 5b illustrates, the CS-FA-MBZ implants could significantly inhibit the breast tumors growth progression in comparison with all other groups. On the last day of tumors’ volume monitoring (18th day after cancer cells injection), the mean tumors’ volume at the CS-FA-MBZ group (658.3 ± 88.1 mm3) was significantly (P < 0.05) lower than the Control (1050.5 ± 120.7 mm3), MBZ (561.7± 70.3 mm3) and CS-FA (658.3 ± 88.1 mm3) groups. The CS-FA-MBZ implants could cause about 73.3%, 46.5%, 37.3% decrease in the mean tumors’ volume in comparison with the Control, MBZ, and CS-FA groups, respectively. Therefore, CS-FA-MBZ implants exhibit high efficacy in inhibiting breast tumors growth.
On the 6th and 18th days (3rd and 15th days after surgery, respectively), some implants-bearing mice were sacrificed to observe what is happening on the CS-FA and CS-FA-MBZ implants’ site (Figure 5c). As illustrated in Figure 5c, the implants were surrounded by a thin transparent membrane on the 6th day. On the 18th day, the implants were completely degraded and disappeared. As Figure 6 illustrates, histopathological evaluations of the implantation site on the 6th day demonstrated that the surrounding membrane consisted of connective tissue. Also, limited mononuclear cells infiltration was observed at the implants’ bed (Figure 6).
3.3 The CS-FA-MBZ implants effect on metastasis and tumor-bearing mice survival time
Metastasis is the main cause of cancer-related deaths. The formation of metastatic colonies at vital organs like the liver disrupts their function and causes organ failure . Therefore, to evaluate the effect of the CS-FA-MBZ implants on metastasis formation, H&E sections were used to count the metastatic colonies at tumor-bearing mice liver after 35 days from cancer cells injection (Figure 7). Histopathological evaluations demonstrated significant inhibition of the liver metastatic colonies formation at the CS-FA-MBZ (8.6 ± 1.9) treated mice in comparison with the Control (24.5 ± 4.1), MBZ (14.1 ± 2.5), and CS-FA (15.7 ± 3.1) groups (Figure 7a and b). Besides, the metastatic colonies occupied significantly (P < 0.05) lower space in the liver sections (per microscopic field) of the CS-FA-MBZ treated group in comparison with the other groups (Figure 7c). In addition, the CS-FA-MBZ group exhibited about 51%, 24%, and 17% more survival time (days) in comparison with the Control, MBZ, and CS-FA groups, respectively (Figure 7d). This increase in the tumor-bearing mice survival time can be attributed to significant inhibition of 4T1 tumors’ growth and metastasis.
Biocompatibility of the subcutaneous CS-FA-MBZ implants
The safety and biocompatibility of implants are very important for clinical application. Therefore, the CS-FA-MBZ implants were subcutaneously implanted in non-tumor-bearing mice. Then, the implant-bearing mice were exactly monitored according to general appearance and behavioral parameters, blood biochemical analyzes, and histopathological evaluation of vital organs. No sign of change in the mice's appearance, behavioral pattern, and food intake were observed during the 30 days (Tables 1S). On the 30th day, the animals were sacrificed and their plasma was collected for biochemical (Figure 8a) analyses and the vital organs were harvested for histopathological exams (Figure 8b). No sign of organ damage was observed in either H&E sections and blood biochemical analyzes. Chitosan nanoparticles are the main component of CS-FA-MBZ implants. Chitosan is a natural biodegradable biopolymer. The enzymatic degradation of chitosan causes its transformation to some components which are completely safe. Many enzymes have the ability to degrade chitosan and the most well-known one is lysozyme as a non-specific protease. This enzyme which presents in all mammalian tissues and fluids, plays a key role in degradation of chitosan-based implants in vivo. It targets the acetylated residues of chitosan polymer and degrades chitosan to non-toxic oligosaccharides which can be excreted or incorporated to glycosaminoglycans and glycoproteins [30, 74-76]. Also, eight human chitinases (in the glycoside hydrolase 18 family) have been identified, three of which have shown enzymatic activity .