# Biodegradable and biocompatible subcutaneous implants consisted of pH-sensitive mebendazole-loaded/folic acid-targeted chitosan nanoparticles for murine triple-negative breast cancer treatment | Journal of Nanobiotechnology

### Material

Medium-molecular weight chitosan (190–310 kDa, MMW), Sodium tripolyphosphate (TPP), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS), folic acid (97%, FA), Tween-80 (a non-ionic surfactant), mebendazole (MBZ), ammonia (25%, NH4OH), acetic acid, dimethyl sulfoxide (DMSO), phosphate-buffered saline powder (pH = 7.4 PBS), methanol and acetone were purchased from Sigma-Aldrich (Germany) and used without further purification unless stated otherwise. RPMI 1640, fetal bovine serum (FBS), MTT (3-(4 5-dimethylthiazol-2-yl)-2 5-diphenyltetrazolium bromide), phosphate buffer saline (PBS), penicillin/streptomycin, ethanol (96%, v/v), trypsin–EDTA (0.25%) were purchased from Merck (Germany).

### Conjugation of folic acid to chitosan (CS-FA preparation)

CS-FA was fabricated as reported in the literature [61]. Briefly, 0.5 g of FA and 0.2 g of EDC were initially dissolved in anhydrous DMSO (20 mL) under constant stirring at room temperature (2 h). Then, the solution was dropped into the CS solution 0.5% (w/v) prepared in acetate buffer (0.1 M, pH 4.7) and at room temperature in dark for 16 h. Thereafter, the pH of the solution was adjusted to 9.0 by the addition of NaOH (1.0 M). The resulting precipitate was collected by centrifugation, then purified by dialysis against phosphate-buffered saline (PBS, pH 7.4) for 2 days and against water for another 4 days. Finally, yellow-colored CS-FA products were collected and freeze-dried.

### Preparation of the CS-FA-MBZ nanoparticles

CS-FA-MBZ nanoparticles were synthesized according to the previously reported method with some modifications [62]. Initially, CS-FA (0.1 g) was dissolved in a solution containing acetic acid (1% v/v, 20 mL), then left under stirring at room temperature in dark for 16 h to prepare a solution of CS-FA (0.5% w/v). The pH of the solution was adjusted to 4.8 by the addition of NaOH (1.0 M). Afterward, 250 µL of Tween-80 was added dropwise and left for 2 h under stirring at 45 °C. In the next step, 0.01 g MBZ was dissolved in 0.5 M methanolic hydrochloride [63] and then added to the former solution and stirred for 30 min. Finally, 10 mL TPP aqueous solution (0.5% w/v) was dropped to the CS-FA solution slowly under magnetic stirring (800 rpm) at room temperature for 1 h then the nanoparticles were collected by centrifugation (12,000 rpm, 30 min). The resulting CS-FA-MBZ nanoparticles were lyophilized and stored.

MBZ was loaded during the formation of CS-FA nanoparticles as reported in the literature [62]. To calculate MBZ encapsulation efficacy (EE%) and loading capacity (LC%), unloaded MBZ content in the supernatant of the last step was determined through a calibration curve of MBZ standard solution by UV–Visible spectroscopy at 234 nm [63]. The MBZ loading ratio of the nanoparticles was calculated by the following Eqs. (1 and 2):

$$\mathrm{MBZ \, encapsulation \, efficacy }\left(\mathrm{EE\%}\right)=\frac{\mathrm{ Mass \, of \, the \,loaded \,MBZ }}{\mathrm{Mass \,of \,the \,initial \,MBZ}} \times 100$$

(1)

$$\mathrm{MBZ \,loading \,capacity }\left(\mathrm{LC\%}\right)=\frac{\mathrm{ Mass \,of \,the \,loaded \,MBZ }}{\mathrm{Mass \,of \,the \,final \,product}} \times 100$$

(2)

### In vitro drug release pattern of the CS-FA-MBZ nanoparticles

To evaluate the release behavior of MBZ from CS-FA-MBZ nanoparticles, 5 mg of the CS-FA-MBZ nanoparticles were immersed in PBS solution containing Tween-80 (0.1% w/v) at pH values of 2.2, 5.5, 6.8, and 7.4 at 37 °C in dark under shaking at 100 rpm. The released MBZ was assessed at 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 1 day, 2 days, 3 days, 5 days, and 7 days time points after immersing the nanoparticles in PBS [64]. At each predetermined time point, the nanoparticles were centrifuged (10,000 rpm for 15 min) and the released medium was collected and replaced with equivalent fresh PBS solution. The cumulative percentage of released MBZ was determined by UV–Visible spectrophotometry at 234 nm.

### Nanoparticle’s characterization and implants fabrication

To assess the structure and interaction of CS, CS-FA, and CS-FA-MBZ, Fourier Transform Infrared Spectroscopy (FTIR) was used by a Bruker Equinox 55 spectrometer with the KBr pellets method. To evaluate the size and morphology of nanoparticles, scanning electron microscopy (SEM; FEI ESEM QUANTA 200, MIRAII and MIRAIII Tescan) and transmission electron microscopy (TEM; Philips RM-208, operating voltage: 100 kV) images were acquired. The average size and size distribution of particles were determined by measuring the diameter of 100 particles of SEM images using ImageJ software. The ultraviolet–visible (UV–vis) spectra were recorded by a PerkinElmer Lambda 950 spectrophotometer (wavelength range: 200–800 nm). 1H-NMR experiment was recorded on Avance III ultrasheild spectrometer manufactured by Bruker at a field strength of 11.7 T (500 MHz) and the corresponding data was collected using MestReNova software. The zeta potential, hydrodynamic size distribution, and polydispersity of the prepared nanoparticles were measured by Dynamic Light Scattering (DLS) (Malvern Instruments). A well-dispersed aqueous suspension of the prepared nanoparticles was applied and Zeta measurements was performed at pH value of 7.5. Each experiment was carried out in triplicate and data were presented as means ± standard deviations. For fabricating an implant, the adequate mass of the synthesized CS-FA-MBZ nanoparticles (according to the mouse body weight and its needed dosage of MBZ) were pressed in a steel die at 1500 psi to form cylindric implants (usually 6 mm diameters and 3 mm height).

### Cell viability assay

MTT assay was employed for the cell viability evaluation according to our previous studies [56, 65]. The 4T1 and L929 cells were separately seeded into 96-well culture plates at 5 × 103 cells/well density. After 24 h incubation, different concentrations (0, 0.5, 1, and 1.5 µM) of MBZ, CS-FA, and CS-FA-MBZ were dissolved in culture medium and added to the wells. The cells were incubated for 48 h and then, the culture media was replaced with RPMI culture medium containing 0.005% MTT solution. After 4 h incubation in the standard cell culture incubator, the medium was discarded and the precipitated formazan crystals were dissolved in dimethyl sulfoxide (DMSO). At last, an absorbance Microplate Reader (BioTek-ELX800, USA) was used to measure the absorbance of the wells at 570 nm wavelength. Subsequently, the below-mentioned Eq. (3) was used to calculate the cell viability percentage of the treated wells in comparison with the control wells (0 µM). The experiment was repeated three times and at least six wells were used for each concentration.

$$Cell\,viablity \left(\%\right)=\frac{\left(OD\,Sample-OD\,Blank\right)}{(OD\,Control-OD\,Blank)}\times 100.$$

(3)

### Animal ethics, care, and handling

All animal experiments complied with the ARRIVE guidelines and were conducted according to the guidelines of the European Communities Council Directive (2010/63/UE) and the Isfahan University of Medical Sciences for the care and use of laboratory animals. Likewise, all the procedures, protocols, and steps were approved by the ethics committee of the Isfahan University of Medical Sciences (IR.MUI.RESEARCH.REC.1399.125). Female BALB/c mice (weight: 25 ± 2 g) were purchased from the Pasteur Institute of Tehran, Iran. The mice were acclimatized to the laboratory environment (24 ± 2 °C temperature, 50 ± 10% relative humidity, and 12 h light/12 h dark cycles) for 14 days before involving in the experiments. All mice were fed sterilized standard mouse chow and water ad libitum. Overdose of Ketamine-Xylazine (KX) solution through intraperitoneal injection was used for the mice sacrifice.

### Tumor implantation

4T1 cancer cells (murine mammary carcinoma) were purchased from the Pastor Institute of Tehran, Iran. The cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS). The cells were incubated at 37 °C in a humidified incubator in 5% CO2 atmosphere. When the cells reached adequate numbers, they were harvested from culture flasks by trypsin and washed three times with PBS. The mice were injected with 2 × 106 cells suspended in 50 µL of FBS-free DMEM-F12, subcutaneously (s.c.) into the left 4th abdominal mammary fat pad.

### Tumor-bearing mice grouping and therapeutic approaches

For this experiment 32 tumor-bearing mice were used. When the tumors’ volume reached 50–70 mm3 (3rd day after the cancer cells injection), mice were divided into four groups (n = 8) including (1) Control, (2) MBZ (40 mg/kg, oral administration, twice a week for 2 weeks), (3) CS-FA implants, (4) CS-FA-MBZ implants. The tumor-bearing mice in the 2nd group were treated with oral administration )p.o.( of MBZ (40 mg/kg, twice a week for 2 weeks according to previous studies [66]) from the 3rd day after the cancer cells injection. In the 3rd and 4th groups, the tumor-bearing mice were anesthetized with intraperitoneally injection of Ketamine-Xylazine (KX) solution (Ketamine: 100 mg/kg, Xylazine: 10 mg/kg). The left flank was shaved and scrubbed with betadine. The scrub solution was wiped away from the surgical site with alcohol 70% and covered with a sterile drape. Then, a small (~ 1 cm) incision was made and the implant was embedded under sterile conditions, and the skin was stitched with nylon (4–0). All the operations were done under complete anesthesia. To manage post-surgical pain, ketoprofen (5 mg/kg) was administered subcutaneously until the next 72 h. It should be mentioned that mice in the Control and MBZ group underwent the same surgery at the same day as two other groups and post-operative pain management protocol to prepare the same condition in all groups. The mice were monitored daily for prolonged signs of pain, weight loss, or surgical site infections. If any signs of pain, wounds infection, massive necrosis, and hemorrhage, diffuse metastasis were observed during any steps of the study, the mice were sacrificed by KX overdose. In the Control group, one incision was made at the left flank of the tumor-bearing mice and sutured without implantation of any implants. To determine tumors’ growth progression, the greatest longitudinal diameter (length) and the greatest transverse diameter (width) of the tumors were measured every 3 days until the 18th after cancer cells injection. Then, the tumor’s volume was calculated by the tumor volume Eq. (4). For survival analysis, the tumor-bearing mice were observed for 70 days after treatment administration. The animals’ death was recorded every day. It should be mentioned that standardized humane endpoints based on the current guidelines for endpoints in animal tumor studies were used [67,68,69].

$$Tumor\,volume=\frac{\left(Tumor\,length\right)\times {(Tumor\,width)}^{2}}{2}$$

(4)

### 4T1 breast tumors’ metastasis

For this experiment, 20 tumor-bearing mice were involved (n = 5) and the groups and therapeutic methods were completely the same as the previous section. The mice were sacrificed by overdose of ketamine/xylazine 30 days after cancer cell implantation and their livers were harvested and fixed in 10% neutral buffered formalin solution. An automatic tissue processor (Sakura, Japan) was employed to process the fixed samples. Then, a microtome (Leica Biosystems, Germany) was utilized to cut 4 µm thickness serial sections from the paraffin-embedded blocks. The sections were stained with Hematoxylin & Eosin (H&E) staining protocol according to previous studies [57, 70]. A minimum of 10 random microscopic fields was observed under the 10 × objective lens of a light microscope (Olympus, Japan) to report the mean number of metastatic colonies per microscopic field of the liver. Furthermore, the occupied area by metastatic colonies in each microscopic field of the liver (magnification × 100) was quantified by the Qupath software. The mean percentage of occupied space by liver metastatic colonies in each microscopic field was reported for each sample.

### Histopathology and blood biochemical assays

For evaluating the safety of the subcutaneous CS-FA-MBZ implants, 20 healthy mice were involved and randomly divided into four groups (n = 5) including (1) Control, (2) MBZ, (3) CS-FA, and (4) CS-FA-MBZ implants according to the “Tumor-bearing mice grouping and therapeutic approaches” section. The mice were monitored for general appearance and behavioral parameters for 30 days. They were under close monitored for any signs of toxicity and behavioral changes including weakness, salivation, anorexia, diarrhea, aggressiveness, eyes and ears discharge, noisy breathing, activity, convulsion, cachexia, pain, or any signs of illness in each group for 30 days [71]. On the 30th day, the mice were sacrificed and blood urea nitrogen (BUN), creatinine (Cr), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) levels were measured in the discarded serums [72]. In addition, lungs, kidneys, liver, and spleen were harvested and fixed, processed, and H&E stained. Histological photographs were obtained using a digital light microscope (Olympus, Japan).

### Statistical analysis

The statistical analyzes were performed using one-way analysis of variance (ANOVA) with Tukey’s post-hoc test by JMP 14.0 software (SAS Institute, Japan). The results were statistically significant at P < 0.05 (*: P <  0.05, ns: not significant). All values were expressed as the mean ± standard deviation.