Farideh Mahmoodzadeh 1, Marjan Ghorbani 2*, and Behrooz Jannat 1

1 Halal Research Center of IRI, FDA, Tehran, Iran.

2 Stem Cell Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.

Short title: Smart chitosan-based nanogel for cancer treatment

*Corresponding author:

Marjan Ghorbani: [email protected].

Abstract

Incomplete delivery of drugs to the cancerous tissue and the drug resistance mechanisms are limited to the medical applications of anticancer drugs. The colloidal systems offer many advantages due to the non-invasive way of drug administration as well as centralize the delivery of anti-cancer drugs to tumor tissue. Nanogels (Ngs) as a part of these colloidal systems have a good perspective for their ability to incorporate and encapsulate the low-molecular-mass drugs, bio-macromolecules, and proteins. Therefore, we developed pH and redox-responsive Ngs to provide a hopeful prospect to the targeted delivery of anti-cancer drugs in cancer cells. For this purpose, chitosan (CS) was first modified with a chain transfer agent (CTA) and then, the polymerization of 2-Hydroxyethyl methacrylate (HEMA) monomer occurred to create (CTS-g-PHEMA). Hydroxyl groups of HEMA reacted with maleic anhydride molecules for the preparation of CTS-g-PHEMA-maleic acid (MAc). Finally, the double bonds of MAc were used for the grafting of N, N′ bis(acryloyl) cystamine (BAC) as a crosslinker agent to prepare the redox-sensitive Ngs. The biocompatibility, chemical structures, DOX loading capacity, the content of drug released, and in-vitro cytotoxicity effects were also studied. As a result, it is expected that the Ngs can be applied as potential nanomedicine carriers for the treatment of cancer.

Keywords: Chitosan; Doxorubicin; Smart responsive; Nanogels; Drug Delivery; Cancer.

1. Introduction

Cancer is a group of diseases with many possible causes in worldwide and cancer patients is most often treated with radiation or chemotherapy. Chemotherapy is the most common types of treatment, but like other treatments, it often causes side effects [1,2]. Scientists work constantly to develop drugs, drug combinations, and ways of giving treatment with fewer side effects [3]. For example, the nano-sized drug carriers (NDC) have achieved most consideration, in the area of nanomedicine to overcome the problems arising from side effects of chemotherapy via the enhanced penetration effect (EPR) [4,5]. Among the many types of NDC, three-dimensional polymeric networks named nanogels (Ngs) have shown interesting potentials because of robustness and flexibility properties, nanoscale size, and smart responsiveness to external stimuli [6–8]. The Ngs with smart responsiveness to endogenous or exogenous environmental changes (temperature, pH, and redox) can be a candidate for targeted and controlled-delivery of drugs to cancerous cells [9,10]. The advantage of redox-responsive Ngs is due to the stability during their circulation in extracellular compartment and the degradation of Ngs is achieved by the high intracellular glutathione (GSH, a type of strong reducing agent inside the cell) concentration inside cancer cells led to the fast drug release in the nucleus of cells [1,11,12]. Polymeric Ngs based on polysaccharides has developed as the most promising candidate owing to their biocompatibility, biodegradability, functionality, low toxicity, and low cost [6,8,13]. In this regard, chitosan-based Ngs can be prepared via controlled radical polymerization as well as reversible addition of fragmentation chain transfer (RAFT) polymerization technique due to the providing of polymers with narrow dispersity and controlled molecular weight [14,15]. The goal of this study was the development of pH and redox-responsive Ngs based on N-pivaloyl chitosan-graft-poly (hydroxyl ethyl metacrylate)-graft-maleic acid-graft-N,N′ bis(acryloyl) cystamine (BAC) [(CTS-g-PHEMA-g-MAc-g-MBA)] for triggered DOX release in cancer tissue. In the first step, functionalized N-phthaloyl-chitosan with 4-cyano, 4-[(phenylcarbothioyl) sulfanyl] pentanoic acid (RAFT agent) was prepared to provide the macroinitiator. Then, HEMA polymerized via macroinitiator until the hydroxyl groups of HEMA could react with maleic anhydride (MAn) monomers to prepare the CTS-g-PHEMA-g-MAc. In the third step, by using the double bonds of MA, the N,N′ bis(acryloyl) cystamine (BAC) was grafted as a redox-sensitive monomer, and the crosslinker agent to prepare a novel dual-responsive Ngs. The designed Ngs were applied for loading DOX and the improved drug’ anticancer activity was proved by the in vitro assessment such as cellular uptake, cell imaging, DAPI staining, and cell cycle analyses against cancer cells (Scheme 1).

Scheme 1. Cell uptake mechanism for delivery of Doxorubicin (DOX) to the cancerous cell.

2. Experimental

2.1. Materials

HEMA, CS (medium molecular weight, the extent of deacetylation 75–85%), GSH, BAC, MAn, dimethylaminopyridine (DMAP), dicyclohexyl carbodiimide (DCC), WST1 (4-[3-(4-Iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene Disulfonate), phosphate buffered saline (PBS), glutathione (GSH), 4-cyano, 4- [(phenylcarbothioyl) sulfanyl] pentanoic acid (RAFT agent), and phthalic anhydride was purchased from Sigma-Aldrich (Company Ltd., Dorset, UK). The initiator of 2, 2-azobisisobutyronitrile (AIBN; Switzerland) was purchased from Sigma-Aldrich.

2.2. Preparation of CTS-g-PHEMA

The esterification of N-phthaloyl-CS by using RAFT agent was occurred to prepare the CTS macroinitiator [16–18]. In a typical procedure, 0.2 g of CTS, 2.0 mg of AIBN (12 μmol), and 0.5 g of HEMA (3 mmol) were dissolved in 6 ml dried DMF solution in the round-bottom flask. After being purged with argon gas, the flask was placed in an oil bath preset at 75 °C for 8 h. Lastly, the flask was cooled using an ice/water bath to quench the polymerization. To obtain the CTS-g-PHEMA, the product was diluted with 6 mL DMF, and then purred into diethyl ether (300 mL). Finally, the product was filtered and dried in a vacuum oven at 25 °C.

2.3. Preparation of CTS-g-PHEMA-g-MAc

The CTS-g-PHEMA-g-MAc was synthesized according to previously reported approach with minor modification [19]. Briefly, a dry polymerization ampoule was charged with CTS-g-PHEMA macroinitiator (1.00 g), and dried DMF (5 mL). After complete dissolution, 600 mg of MAn (6 mmol) was added to the solution under nitrogen atmosphere. After 24 h, the soluble products were dialyzed and lyophilized for further experiments.

2.4. Preparation of smart Ngs (CTS-g-PHEMA-g-MAc-g-BAC)

Briefly, 40 mg of BAC (0.16 mmol), 2.65 mg of AIBN (0.016 mmol), and 80 mg of CTS-g-PHEMA-g-MAc were dissolved in 10 ml dried DMF solution at the round-bottom flask. After being purged with a nitrogen atmosphere for 30 min, the flask was placed in an oil bath preset at 70 °C for 48. Finally, to remove the unreacted monomer and solvent, the solution was diluted with water (25 mL) and transferred into a dialysis bag (molecular weight cut off 1000 Da) and dialyzed for 3 days to achieve Ngs.

2.5. Characterization

Fourier transform infrared (FTIR) spectroscopy was performed using a Shimadzu 8101M FTIR (Kyoto, Japan) in the pellet form with potassium bromide (KBr) powder at room temperature. Proton nuclear magnetic resonance (1HNMR) spectra were recorded on a Bruker spectrometer (Bruker, Ettlingen, Germany) with an operating frequency of 400 MHz at 25 °C. Transmission electron microscopy (TEM) was conducted on a Philips CM10-TH microscope (Phillips, Eindhoven, Netherlands). By photon correlation spectroscopy, the average diameter was analyzed (Instruments, Malvern, UK).

2.6. Preparations of DOX-Ngs

DOX-Ngs were achieved by using a membrane dialysis method [1] Briefly, 25 mg of smart Ngs was dispersed in 5 mL of DI water to achieve polymer solution. Then, 10 mg of DOX was added to the polymeric solution for 48 h. After this time, the unloaded drug was separated from DOX-Ngs by using an Amicon® filter (molecular weight cutoff 100 KDa kDa, Millipore, UK), and then its concentration was investigated by UV-Vis spectrophotometer at 480 nm. The encapsulation efficiency (EE) and loading capacity (LC) of DOX was calculated by the following equations:

LC(%)=(milligram of drug in nanogels)/(milligram of nanogels)  ×100

EE(%)=(milligram of drug in nanogels)/(milligram of initial added drug)  ×100

2.7. In vitro release study

10 mg of DOX-Ngs were suspended in phosphate buffered saline (30 ml, PBS) and then put into a dialysis bag [20]. To confirm the effective performance of DOX-Ngs, the release behavior of DOX-Ngs were assessed in both simulated tumor tissue (pH 5.3, 40 °C) and physiologic (pH 7.4, 37 °C) conditions. 1 mL of released drug were collected at predetermined time intervals to evaluation the released drug content using UV–vis spectrophotometer at 480 nm. It is essential to note, the aliquots were brought back into the flask to keep the same total solution volume.

2.8. In vitro cell cytotoxicity

The MDAMB-231 cells were obtained from the Iranian National Cell Bank (Pasteur Institute, Tehran, Iran) and cultivated in RPMI1640. The cells were cultured into flasks before the addition of samples. To study the cytotoxicity effects of developed nanocarriers, the cells were treated with different concentrations of Ngs, DOX, and DOX-Ngs for 48 and 72 h. Finally, the culture medium was aspirated, and 200 µL cultivation medium containing 20 µL of WST1 solution was added to each well. Next, after incubation for 1 h, the optical density was recorded via a micro-plate reader at 450 nm (Elx808, Biotek, USA) [21].

2.9. DAPI staining

MDAMB-231 cells were seeded on glass cover slips in 6-well plates with fresh RPMI1640 containing 10% FBS and 1% penicillin-streptomycin at 37 °C for 24 h [1,22]. Briefly, DOX-Ngs, Ngs and DOX in fresh culture medium were added to substitute the primary media and incubated with cells (5×105) for 1 day. After the treatment, cells were washed three times with cold PBS and fixed in 2 mL 4% paraformaldehyde for 10 min. After that, the cells were stained with DAPI following the manufacturer’s instructions and observed using an inverted fluorescence microscopy (Bh2-RFCA, Olympus, Japan), and the typical photographs were captured.

2.10. Cellular uptake study

Cellular uptake evaluation of MDAMB-231 cells was measured by Flow Cytometry[23]. The cells were seeded in six-well plates at a density of 3 × 105 cells per well and cultured at 37 °C for 24 h. For cellular internalization assay, cells were treated by free DOX or DOX-Ngs at an equivalent DOX concentration of 5 μg/mL. After incubation at 37 °C for 3 h, the original medium was removed and the cells were washed three times with 1 mL of cold PBS solution. Digestion of cells were performed by adding 0.5 mL of trypsin and centrifuged at 1000 rpm for 5 min. The pelletes were washed twice with cold PBS, followed by centrifugation. Finally, the cell were resuspended in 500 μL PBS and the fluorescence of DOX trapped by cells was analyzed using a FACS caliber flow cytometer (Becton Dickinson Immunocytometry Systems, San Jose, CA).

2.11. Cell cycle arrest study

The test is established on the stoichiometric binding of propidium iodide to cumulative amounts of DNA in different cell cycle phases (G0/G1, S, and G2/M) by flow cytometry. The cells (4 × 105 cells/mL) were seeded, cultured, and then treated with DOX, DOX-Ngs, and nano-blank for 24 h. After that, the cells were trypsinized and the pellet of samples was washed twice with PBS, fixed and permeabilized using ethanol (70 %). After 24 h, the incubated cells were centrifuged (300 ×g for 10 min at 4 °C), rinsed in phosphate-buffered saline (PBS), and next treated with RNAase. DNA was then quantitatively stained with propidium iodide for 1 h at 37 °C. Cell population were analyzed using a FACS calibur flow cytometer (Becton Dickinson) and FlowJo software (Treestar, Ashland, OR, USA).

3. Results and discussion

To improve the blood circulation time of anticancer drugs, and to achieve the high accumulation in the tumor regions and consequently high cellular uptake of drugs, the drug delivery nanosystems are considered [24–26]. Engineered DOX nanoparticles were prepared by using BAC as a stabilizing and crosslinker agent. In this research, we provided a novel pH and redox-responsive nanoplatform for delivery of DOX based on chitosan. The brief synthesis protocols of DOX-Ngs are as follows: CS was first modified with a chain transfer agent (CTA) to provide CTS macroinitiator. Then, the grafting polymerization of HEMA monomer was controlled via RAFT polymerization to generate CTS-g-PHEMA. Hydroxyl groups of HEMA applied to react with MAn to prepare of CTS-g-PHEMA-MA. To produce the redox-responsive Ngs, the double bonds of MAn were used for polymerization of BAC as a redox-sensitive crosslinker agent. Last, antitumor drug DOX was loaded via two way: a) by physical entrapment in cavity of Ngs and b) electrostatic interaction with maleic acid groups (Scheme 2).

Author: Farideh Mahmoodzadeh