# Folic acid-conjugated magnetic triblock copolymer nanoparticles for dual targeted delivery of 5-fluorouracil to colon cancer cells – Cancer Nanotechnology

May 16, 2022

### Synthesis of 5-FU-SPION-PEG-PCL-PEG-FA nanoparticles

We have previously reported the synthesis of 5-FU-SPION-PEG-PCL-PEG-FA nanoparticles by a modified W1/O1/W2 double emulsion solvent evaporation method (Mirzaghavami et al. 2021). These nanoparticles were synthesized in 5 steps in the following order (Fig. 8).

#### Step 1: synthesis of poly (ε-caprolactone) (PCL)

3 ml of ε-caprolactone (27 mmol) was dissolved in 5 ml of dimethylformamide (DMF) in a three-necked round-bottom flask. Then, 5 µl of ethylene glycol (1 mmol) was added under a nitrogen atmosphere. The reaction temperature was gradually increased to 80 °C followed by addition of stannous octoate [Sn (Oct)2] and increasing temperature to 120 °C. After 24 h, the polymer was precipitated in water at 0 °C and then dried at 40 °C. In order to purify polymer, it was dissolved in DMF and precipitated in water. The product was obtained with 82% yield.

#### Step 2: synthesis of adipoyl chloride-functionalized PCL

1g of caprolactone (0.2 mmol) was dissolved in DMF and 146 µl (1 mmol) of adipoyl chloride and catalytic amount of triethylamine was added. The reaction was continued for 24 h at 80 °C. Then functionalized polymer was precipitated in water followed by drying in the vacuum oven at 40 °C. The purified product was obtained by dissolution of crude product in DMF and then precipitated in cold water.

#### Step 3: synthesis of PEG-PCL-PEG

1 g of functionalized PCL was dissolved in 10 mL of dimethyl sulfoxide (DMSO) and afterward 206 mg N,N′-dicyclohexylcarbodiimide (DCC) and 122 mg 4-dimethylaminopyridine (DMAP) was gradually added to solution. After 1 h, 2 g of PEG was added and stirred at room temperature for 24 h. PEG-PCL-PEG copolymer was precipitated in diethyl ether and then dried in vacuum oven at 40 °C. To have more purified product, dissolving in DMSO and precipitating in diethyl ether was repeated.

#### Step 4: synthesis of folic acid-functionalized PEG-PCL-PEG

220 mg of folic acid, 103 mg of DCC and 61 mg of DMAP were dissolved in 10 mL of DMSO and the reaction was continued for 4 h to activate acidic group of folic acid. Then 1 g of PEG-PCL-PEG copolymer was added and the reaction was continued overnight. After 24 h, the product was precipitated in diethyl ether and dried at 40 °C. Dissolving in DMSO and precipitating in diethyl ether was repeated to acquire more purified product (Fig. 8).

#### Step 5: synthesis of 5-FU-loaded magnetite/PEG-PCL-PEG-folic acid

Preparation of the 5-FU-loaded magnetite/PEG-PCL-PEG-folic nanoparticles was performed by using W1/O1/W2 double emulsion solvent evaporation method. For preparation of inner aqueous solution (W1), 10 mg of 5-FU drug was dissolved in 1.5 mL of distilled water containing 10 mg of Tween 60. 30 mg of magnetite nanoparticles was dispersed in 7 mL of dichloromethane (DCM) to prepare an organic phase using an ultrasonic probe. Then 50 mg of the folic acid-conjugated polymer (PEG-PCL-PEG-FA) and 200 mg of Span 60 were added to the oil phase (O1). W2 aqueous solution was made of 100 mg tween 60 dissolved in 8 mL of distilled water and 8 mL of glycerin. The inner aqueous solution (W1) was emulsified in the organic phase (O1) by ultrasonication using the sonicator probe at an output of 50 W for 30 s in an ice-bath to obtain a W1/O1 emulsion. This primary emulsion was emulsified in second aqueous solution (W2) by ultrasonication for 30 s (50 W) in an ice-bath to obtain a W1/O1/W2 double emulsion. The resulting double emulsion was diluted in 30 mL aqueous solution composed of 15 mL distilled water and 15 mL glycerin under mechanical stirring for a period of 3 h at room temperature, and the DCM was removed by solvent evaporation. The obtained magnetic nanoparticles were cleaned by repeating procedure of collecting and re-suspending in distilled water for two times and then were collected with a magnet. Finally, the resulting nanoparticles were dried by freeze-drying and stored at 4 °C. Drug-free nanoparticles were prepared in the same way, except that the inner aqueous solution was prepared with 1.5 mL of distilled water and 10 mg of tween60, but without 5-FU drug.

### Proton nuclear magnetic resonance analysis

In order to investigate synthesis procedure and characterize the structure of the synthesized products, the 1H nuclear magnetic resonance (NMR) analysis was performed on poly (ε-caprolactone) (PCL), adipoyl chloride-functionalized PCL, PEG-PCL-PEG and folic acid-functionalized PEG-PCL-PEG using a Varian Inova, 500 MHz spectrometer. Deuterated chloroform (CDCl3) was used as solvent of samples to acquire 1H NMR spectra.

### Characterization of size, zeta potential and morphology of nanoparticles

Dynamic light scattering (DLS) analysis was used to characterize the distribution of the hydrodynamic size of drug-free and 5-FU-loaded nanoparticles. The surface charge of nanoparticles was evaluated by Zeta sizer (Nanoflex, Germany).

The morphology of 5-FU-loaded nanoparticles was investigated using a transmission electron microscope (TEM, Zeiss LEO906, Germany). The samples were prepared on 400-mesh carbon-coated copper grid and imaged at accelerating voltage of 100 kV.

### In vitro drug-release profiles

Investigation of in vitro release profile of 5-FU from NPs-FA was carried out based on equilibrium dialysis bag diffusion method at different pH (5.6, 6.8 and 7.4). First, 3 mg of 5-FU loaded NPs-FA was suspended in phosphate buffer saline (PBS) with different pH and transferred to a dialysis bag (MWCO 12,400 Da). The dialysis bag was fully immersed into a tube containing 10 mL PBS and stirred at the shaking speed of 100 rpm at 37 °C. At predetermined time points, 1.5 mL of media was sampled and replaced with an equal volume of PBS. The absorption of released 5-FU was measured using an UV-spectrophotometer at 265 nm.

### Cell culture

The colon cancer cell lines (HCT116, SW480, HT29 and Caco-2) and HUVEC cells were purchased from Stem Cell Technology Research Center and, respectively, cultured in complete RPMI and DMEM/Ham’s F-12 medium supplemented with 10% FBS, penicillin (100 U/mL) and streptomycin (100 mg/mL) under humidified incubator with 5% carbon dioxide at 37 °C.

### Allograft mouse colon tumor models

The CT26 mouse colon cancer cells and BALB/c mice (20–30 g) were purchased from Pasteur Institute of Tehran, Iran. Mice were kept under the standard conditions in accordance with the Helsinki Declaration at the Animal Research Center of Iran University of Medical Sciences, Tehran, Iran. For colon tumor modeling, 2 × 106 of CT26 cells in 100 µL of RPMI medium were injected subcutaneously on the right leg of the mice.

### MTT assay

The cellular cytotoxicity of 5-FU and synthesized nanoparticles was investigated using MTT assay. In brief, HUVEC and colon cancer cell lines (HCT116, SW480, HT29 and Caco-2) were cultured in a 96-well plate at a density of 7 × 103 cells per well. After 24 h, the cells were treated with varying concentrations of 5-FU ranging from 0.6 to 80 µM and equivalent concentrations of drug-free and drug-loaded NPs. After 48 h incubation, the cells were washed and treated with MTT (5 mg/mL). Plates were incubated at 37 °C in the dark. After 4 h, the medium containing MTT was removed, and 100 µL of dimethyl sulfoxide (DMSO) was added. The solution was incubated for 15 min to solubilize formazan crystals. The absorbance was measured at 570 nm using a microplate reader (BioTek, Winooski, VT). Finally, cell viability was determined based on Eq. (1):

$$mathrm{Cell viability}(%)=frac{mathrm{absorbance,of,treated,cells}}{mathrm{absorbance,of,control,cells}}times 100.$$

(1)

To compare the cytotoxicity of 5-FU with 5-FU-loaded nanoparticle on the cell lines, the IC50 values of these agents was calculated regarding to cell viability curves and also a therapeutic index was defined as the ratio of IC50 of normal cell to IC50 of cancer cell.

According to MTT results, the most 5-FU-resistant cell line that necessitates using higher dose of drug, was selected for further assessments.

### Cellular uptake of NPs-FA in vitro

#### Prussian blue staining

The cellular uptake of NPs-FA was visually investigated using the Prussian blue staining assay. The HT 29 and HUVEC cell lines were seeded in 6-well plates. After 24 h, both cell lines were incubated with amount of SPION-PEG-PCL-PEG-FA NPs which has capability of encapsulating IC10 of 5-FU against HT29 within 48 h. After treatment time, the cells were washed with PBS for three times, then fixed with 4% paraformaldehyde solution for 20 min followed by staining with the Prussian blue solution (2% potassium ferrocyanide and 2% hydrochloric acid with 1:1 ratio) for 30 min. Subsequently, cells were washed with PBS, and imaged using an optical microscope at the magnification of 400× (Olympus CK2; Olympus Optical Co., Tokyo, Japan).

#### Inductively coupled plasma optical emission spectrometry (ICP-OES)

In order to quantitatively evaluate the cellular uptake of NPs-FA, both cell lines were cultured in T-25 cell culture flasks at a density of 5 × 105 cells, treated in the same way as described before. After treatment, the cells were washed with PBS, trypsinized, collected and counted for quantification purposes. The cells were digested with 1 mL of concentrated HNO3 at 140 °C for 2 h. The samples were diluted to 5 mL with deionized water and the concentration of iron was measured using an ICP-OES assay (VISTA-PRO, Varian, Australia). Finally, the average iron content per cell was calculated.

#### Cellular uptake of NPs and NPs-FA in vivo

The accumulation of NPs and NPs-FA in colon tumor tissues was assayed by Prussian blue staining. The mice were injected with 20 mg/kg NPs with/without drug (containing 1 mg/kg of free 5-FU) through the tail veins 14 days after tumor implantation. For magnetic drug targeting, the magnets with magnetic intensity of 1.3 T were placed on the mice colon tumor for 1 h. Tumoral mice were killed 1 h after the injection of nanoparticles with or without folic acid and magnetic targeting. For magnetic drug targeting, the magnets were placed on the mice colon tumor for 1 h. After, the tumor tissues were fixed in formalin solution (10%) for 48 h at 25 °C. The samples were dehydrated in different concentrations of ethanol, embedded in paraffin, cryosectioned, and stained with Prussian blue solution. The sections were incubated with a mixture of 10% potassium ferrocyanide and 20% hydrochloric acid for 20 min, then washed and stained with Nuclear Fast Red.

#### In vivo antitumor effects of nanoparticles

Colon tumor model was developed in BALB/c mice (20–30 g) by injecting 2 million CT26 cells. After 12 days, when the tumor size reached about 70 to 100 mm3, the mice were divided into 4 groups of 9:

Group 1: normal saline (control),

Group 2: Free 5-FU solution,

Group 3: SPION-PEG-PCL-PEG-FA (NPs-FA) and magnet for 1 h,

Group 4: 5-FU-loaded NPs-FA and magnet for 1 h.

(Only mice in the treatment groups that received the nanoparticles were exposed to the magnetic field for one hour.) The mice were treated with 5-FU and NPs-FA with/without drug injections through the tail veins every other day (12, 14 and 16 days) of the experiment with 1 mg/kg of free 5-FU and 20 mg/kg of NPs-FA (containing 1 mg/kg of 5-FU). After the treatments, the tumor size was measured daily and the tumor volume was calculated by the following equation:

$$V=left[{mathrm{width}}^{2}/2right]times mathrm{length}.$$

(2)

Furthermore, the 6 mice in each group were monitored for life span and analyzed by the Kaplan–Meier survival curves.

#### Western blotting analysis

Two days after treatment, protein level changes of Bax and Bcl-2 in colon tumor-bearing mice was assessed by Western blotting assay. Tumor tissues were homogenized and total protein extracted with RIPA lysis buffer (Santa Cruz Biotechnology, USA) and quantified using Bradford protein assay. The proteins separated through gel electrophoresis were transferred to nitrocellulose membrane. These membranes were incubated for 24 h at 4 C in the solution containing the primary antibodies: Bax (1:500), Bcl-2 (1:1000) and β-actin (1:500). Subsequently the membranes were washed with mixture of Tris buffered saline and Tween 20 (TBST) and then incubated with a horse radish peroxidase conjugated-secondary antibody. Finally, the protein bands were visualized using enhanced chemiluminescence western Blotting Detection Reagent (ChemiDocXRS; Bio-Rad, South San Francisco, USA). The protein bands were quantified using ImageJ software and then normalized to intensity of β-actin.

### Statistical analysis

The results of all experiments were expressed as mean ± standard deviation and analyzed using one-way analysis of variance (ANOVA) followed by Tukey’s test using Graphpad prism 6. A P-value of 0.05 or lower was considered to be statistically significant.