ROS-Responsive Chitosan Coated Magnetic Iron Oxide Nanoparticles as Potential Vehicles for Targeted Drug Delivery in Cancer Therapy

Background and Objective Cancer cells accumulate high concentrations of reactive oxygen species as a result of their faster and uninhibited metabolic activity. Cancer chemotherapeutic agents release an excess of severe adverse reactions as a result of targeting normal cells. This demands an improvement in targeted drug-delivery systems to selectively discharge anticancer drugs in the vicinity of such highly metabolically and mitotically active cells. Materials and Methods Here, magnetic nanoparticles were synthesized by a traditional co-precipitation technique. Fe3O4@OA-CS-5-FLU-NPs were synthesized by an easy and rapid in situ loading method. The proposed Fe3O4@OA-CS-5-FLU-NPs were productively prepared as well as characterized by various spectroscopic and microscopic studies. Results The targeted drug release profile of the Fe3O4@OA-CS-5-FLU-NPs was studied in the presence of ROS including H2O2 and pH induction. The released product, Fe3O4@OA-CS-5-FLU-NP, exhibited desirable levels of cytotoxicity and demonstrated morphological changes and inhibition of colony formation for A549 and HeLa S3 cancer cells. The IC50 values at 24 hours were 12.9 and 23 μg/mL, respectively. Conclusion In summary, results from the MTT assay, fluorescence staining as well as colony formation assays, revealed that the Fe3O4@OA-CS-5-FLU-NPs were active and safe for anticancer biomedical applications. In summary, the present investigation provides a powerful nanostructured based system for improved cancer theranostics that should be further studied.


Introduction
Successful cancer therapy depends on the outcome of surgical resection of the affected tissue in combination with chemotherapy or radiation therapy. [1][2][3] Although anticancer drugs are efficient at destroying tumor cells, they are potentially harmful as a result of their non-selectivity, which has limited their therapeutic use. [4][5][6] In the last few decades, there have been more and more cases of nanoparticles (NPs), such as Fe 3 O 4 NP, which have played a pivotal role in drug delivery specifically targeting cancer cells. 7 Fe 3 O 4 NP have gained great attention in various biomedical applications such as targeted delivery, localized hyperthermia treatment, as well as contrast agents for magnetic resonance imaging (MRI). [8][9][10] As the Fe 3 O 4 NP are unstable in aqueous media, they cannot be used as drug carriers alone. In order to overcome this drawback, a coating is applied to employ lipids, proteins, carbohydrates, liposomes, synthetic and bio-degradable polymers. [11][12][13][14] Chitosan has been proposed as a potential carrier for drugs and biological macromolecules 15 with potential biomedical and pharmacological applications because of several attractive features such as low cost, safety, biodegradability and biocompatibility. [16][17][18] Chitosan and its derivatives have many desirable biological and biochemical properties which could be attributed to reactive functional groups namely hydroxyl (-OH) and amino (-NH 2 ) groups. On the other hand, chitosan is not suitable for binding to Fe 3 O 4 NP as the resulting magnetic compounds tend to form aggregates of polymer cross-linked complexes and polymer adsorbates. 19,20 Many investigators have attempted to functionalize CS in order to bind onto Fe 3 O 4 NP. Several reports highlight the internalization of some Fe 3 O 4 NP with an enhanced generation of reactive oxygen species (ROS). Enhanced ROS levels have demonstrated synergistic antitumor efficacy by stimulating mitochondria-meditated apoptosis. [21][22][23][24] Fe 3 O 4 NP have been reported to up-regulate ROS including H 2 O 2 which potentially play a key role in biomedical applications especially for targeted drug delivery. 25 The assessment of anticancer drug-loaded nanoparticles and their effect on the induction of cell apoptosis, and the extent of the Fe 3 O 4 NP activated ROS-mediated mitochondrial apoptosis pathway, have not been experimentally investigated. 5-FLU is an anti-metabolite of the pyrimidine class, with a broad spectrum of activity and is quite effective in the chemotherapy of solid malignant tumors, such as that of the abdominal cavity, ovary, liver, brain, breast, etc., either alone or in combination. 26,27 5-FLU affects nucleoside uptake and can be incorporated into RNA as well as DNA, leading to cytotoxicity and cell damage. Oleic acid (OA) is a commonly used surfactant to stabilize magnetic nanoparticles synthesized by traditional co-precipitation methods, and some studies have proved that a strong chemical bond is formed between carboxylic acid and amorphous iron and amorphous iron oxide nanoparticles. OA possesses a non-polar hydrocarbon tail and a polar carboxylic acid head group. Carboxylate anions are known to coordinate with the surface of magnetite, presumably through a coordination of iron atoms with both of the carboxylate oxygens. Oleic acid and chitosan-based magnetic aggregates were investigated here as a controlled drug-delivery system capable of pH alteration and ROS release. 28,29 They also exhibit enhanced levels of biocompatibility as well as desirable drug release characteristics. 30 Herein, we present the synthesis, characterization and evaluation of Fe 3 O 4 @OA-CS-5-FLU-NPs as a combination drug carrier for the pH-responsive release of 5-FLU delivery in A549 and HeLa S3 cell cultures and the resultant synergistic cytotoxicity of 5-FLU and ROS.

Preparation of Fe 3 O 4 @OA NPs
Fe 3 O 4 magnetic nanoparticles were synthesized by a chemical co-precipitation process. Concisely, 1.0 g of FeCl 3 ·6H 2 O and 0.5 g of FeCl 2 ·4H 2 O were dissolved in 50 mL of double-distilled water through a dynamic stirring process and the solution was allowed to reflux at 80°C. Then, the precipitant of the ammonia solution in 10 mL (NH 4 OH, 25 wt %) was slowly dropped into the above mixture under vigorous stirring for 15 min; 31 the reason for using an ammonia solution was to increase the number and size of the nanoparticles. Ammonia could play a role not only in increasing the reaction rate but also in retarding the growth of the particles when used in different concentration ranges. Finally, the drop-wise addition of oleic acid (3 mL) into the above suspension led to an alteration at the surface of the Fe 3 O 4 NP. 27 This mixture was refluxed at 80°C for 30 min to complete the formation of the Fe 3 O 4 NP. Subsequently, the suspension was cooled down to room temperature and separated by a magnet, cleaned with distilled water and ethanol to remove the reactants and impurities. The final product of Fe 3 O 4 @OA NP (oilbase surface) was desiccated at room temperature under a vacuum at 60°C for 12 h.

Preparation of Fe 3 O 4 @OA-CS-NPs
Chitosan modified Fe 3 O 4 NPs were synthesized by the coprecipitation of Fe 2+ and Fe 3+ salts in the presence of chitosan. 32 Chitosan (0.125 g) was solidified in 30 mL of 1% acetic acid and the pH was tuned to 4.8 by adding 10 M NaOH. The Fe 3 O 4 @OA NPs (1g) were added to 30 mL of a chitosan solution while stirring under an inert atmosphere at 50°C. Then, 30 mL of an aqueous ammonia solution (25%) was gently added to the above mixture to yield small nanoparticles. The resulting mixture was further stirred for 30 min. The colloidal Fe 3 O 4 @OA-CS-NP was continuously washed with distilled water and separated by magnetic decantation repeatedly.

Preparation of Fe 3 O 4 @OA-CS-5-FLU-NPs
Fe 3 O 4 @OA-CS-5-FLU-NPs were prepared based on an in situ filling process as shown in Figure 1. For the loading method, Fe 3 O 4 @OA-CS-NPs (100 mg) were mixed with 5-FLU and stirred at room temperature for 48 h. The negatively charged 5-FLU molecules electrostatically interacted with positively charged CS molecules for the formation of Fe 3 O 4 @OA-CS-5-FLU-NPs. Chitosan is a hydrophilic cationic polymer used to prepare nanoparticles by means of electrostatic interactions. 5-fluorouracil (5-FU) is a hydrophilic drug. Two ionic interactions exist in the reaction process: an intense electrostatic interaction between the positively charged amino group of CS and the negatively charged 5-FLU solution. The suspension was carefully collected using a magnet and washed with an abundant amount of water and ethanol to remove the unreacted particles. Finally, the Fe 3 O 4 @OA-CS-5-FLU-NP powder was obtained after drying under vacuum pressure. 33

Estimation of Drug Encapsulation Efficiency of Fe 3 O 4 @OA-CS-5-FLU-NPs
The concentration of the Fe 3 O 4 @OA-CS-5-FLU-NP was estimated by UV-Vis spectrophotometry. 10 milligrams of Fe 3 O 4 @OA-CS-5-FLU-NP dissolved in 1 mL of DCM (dichloromethane, to allow for a more extensive and increased drug-loading into the interior of nanoparticle) was taken in a closed glass tube and 10 mL of phosphate buffer (pH 7.4) was added to it (the total volume of the releasing medium was maintained as constant by adding an equal volume of fresh buffer solution). The mixture was stirred for 2h at room temperature. The mixture was centrifuged at 6000 rpm for 10 min after the slow evaporation of DCM. The buffer solution was separated and the amount of 5-FLU in the buffer solution was estimated by spectrophotometry at 270 nm. The following equations were used to determine drug content and encapsulation efficiency: where C t is the concentration of 5-FLU released from Fe 3 O 4 @OA-CS-5-FLU-NP at time "t" and C 0 is the initial concentration of the Fe 3 O 4 @OA-CS-5-FLU-NP.

Statistical Analysis
Tests were completed at least three times each and the data were characterized as the average and standard deviation (mean ± SD) of three independent experiments. One-way ANOVA analysis was used for comparing between groups and P <0.05 was considered a statistically significant difference.

Morphology and FTIR Analyses of Fe 3 O 4 @OA-CS-5-FLU-NPs
The surface morphology of the Fe 3 O 4 @OA-CS-5-FLU-NPs was analyzed through scanning electron micrographs (SEM). As depicted in Figure 2A, the nanoparticles appeared to be spherically-shaped with an average size of 1.5 µm and a smooth outer surface. Under an external magnetic field, Fe 3 O 4 @OA-CS-5-FLU-NP with a diameter of 50 nm can be selectively targeted to affected tissues.
The corresponding energy dispersive X-ray spectrum (EDX) of Fe 3 O 4 @OA-CS-5-FLU-NP is shown in Figure 2B. The characteristic peaks of carbon, nitrogen, oxygen and iron are presented by CS and 5-FLU. Furthermore, the metallic iron peaks show the existence of

Morphology and TEM Analyses of Fe 3 O 4 @OA-CS-5-FLU-NPs
The morphology of the synthesized Fe 3 O 4 @OA-CS-5-FLU-NPs was characterized at 37°C using TEM analysis. The TEM image shown in Figure 4 for Fe 3 O 4 @OA-CS-5-FLU-NPs revealed that the magnetic nanoparticles agglomerated, and the average particle size was about 20-35 nm as measured by DLS. Thus, the polymer on the nanoparticles did not lead to aggregation between the particles.

Magnetic Characteristic Analysis of Fe 3 O 4 @OA-CS-5-FLU-NPs
The magnetic properties of the Fe 3 O 4 @OA NPs and Fe 3 O 4 @OA-CS-5-FLU-NPs were studied by VSM at room temperature and the data is presented in Figure 7A. The higher saturation magnetization value of 73.25 emu/g was obtained  for the Fe 3 O 4 @OA NPs. However, a lower saturation magnetization of 30.21 emu/g was found for the Fe 3 O 4 @OA-CS -5-FLU-NPs. Hence, the gained magnetic targeted drugdelivery for these NPs has potential for broad biomedical applications. The lower value of the saturation magnetization is due to the smaller size of the nanoparticles and the low saturation magnetization of the Fe 3 O 4 @OA-CS-5-FLU-NP composite may be attributed to the presence of a thick polymeric layer of CS and 5-FLU on the Fe 3 O 4 @OA NPs. 39 The Fe 3 O 4 @OA-CS-5-FLU-NPs also retained superior paramagnetic properties at room temperature which is useful in a drug-releasing system. As they do not retain magnetization before and after exposure to an external localized magnetic field, this reduces the probability of particle aggregation due to magnetic dipole attraction. The higher magnetization of the Fe 3 O 4 @OA-CS-5-FLU-NPs is useful for magnetically targeted drug-delivery systems. Hence, the magnetic performances of Fe 3 O 4 @OA-CS-5-FLU-NPs ( Figure 7B) afford good targeted ability for therapeutic applications.

In vitro Drug Release Studies
Accordingly, these nanoparticles might exhibit cytotoxic activity as carrier molecules consisting of OA along with the drug which could be absorbed by simple diffusion. The in vitro release of 5-FLU from the Fe 3 O 4 @OA-CS-5-FLU-NP aggregates was studied in PBS at different pH values as illustrated in Figure 8A. At the physiological pH of 7.4, 5-FLU uptake (as determined was adsorbed on the Fe 3 O 4 @OA-CS-5-FLU-NPs) was released very slowly and the amount of the released 5-FLU presented as a weight  percentage of the total 5-FLU adsorbed, was only about 62% over a period of 500 min. In contrast, in PBS at pH 5.2, to mimic the intracellular conditions of cancer cells, the release rate of 5-FLU from the Fe 3 O 4 @OA-CS-5-FLU-NP was much faster and the highest release of 5-FLU was over 73% within 500 min. 40 Furthermore, in acidic conditions, the 5-FLU released in a biologically active manner and as an anti-metabolite of the pyrimidine analogue. In acidic medium (pH 5.2), -NH 2 groups in the CS polymer matrix stayed simply protonated into -NH 3 + groups, which can weaken hydrogen bonding interactions between the CS polymer which was dissolved in a buffer solution, and released over the diffusion process. 41 It is well known that chitosan is soluble in water in acidic media (pH = 2-6). At this pH, chitosan swells and its chains undergo deployment due to the electrostatic repulsion of positively charged -NH 3+ groups. These results indicated that the release of 5-FLU from the NPs can be measured by changing the pH, which may facilitate its drug delivery and controlled discharge into cancer cells since the microenvironment of the extracellular tissues of tumors and intracellular lysosomes and endosomes are acidic. [42][43][44] In additional analysis, for the site-specific drug delivery of Fe 3 O 4 @OA-CS-5-FLU-NP in cancer, the 5-FLU release behavior from Fe 3 O 4 @OA-CS-5-FLU-NPs was carried out at pH 5.2 in the presence of H 2 O 2 . The 5-FLU release rate from Fe 3 O 4 @OA-CS-5-FLU-NP at pH 7.4 was very low, while at pH 5.2, there was a high It is assumed that only the CS layer degraded at pH 5.2 which led to the release of 5-FLU, but in the presence of H 2 O 2 , the OA layer also degraded (at the same time breaking of bonds amongst the hydrophilic as well as hydrophobic part of OA) which facilitated a high release of 5-FLU from the Fe 3 O 4 @OA-CS-5-FLU-NPs; OA acts as a modifier in nanoparticle synthesis. OA showed good dispersion ability, and which could be ascribed to the great reduction in high surface energy and dipolar attraction of the nanoparticles. 45 These results clearly exposed that the Fe 3 O 4 @OA-CS -5-FLU-NP had favorable 5-FLU release in the presence of ROS (Figure 9), and that the Fe 3 O 4 @OA-CS-5-FLU-NP are suitable carriers to deliver 5-FLU at targeted cancerous cell sites without worrying about primary release in the circulation. 46 Also, the 5-FLU releasing efficiency from the Fe 3 O 4 @OA-CS-5-FLU-NPs increased with an increase of the mixed concentrations of CS. The increase in polymer concentration led to an increase in both particle size and encapsulation efficiency of the prepared NPs. This was attributed to the increased viscosity that helped to enlarge the size and maximize encapsulation efficiency. 47 The 5-FLU release from the Fe 3 O 4 @OA-CS-5-FLU-NP at pH 5.2 (H 2 O 2 in PBS) demonstrated a high amount of 5-FLU released in a controlled manner, as shown in Figure 10. The release of 5-FLU in cancerous cells was also biologically active under acidic conditions. Due to its Figure 11 Cytotoxic studies of nanoparticles examined by MTT assays against human A549, Hela S3 and MCF-7 cancer cells. The experiments were conducted three times and the data expressed as mean ± SD of individual and statistical data were analyzed using GraphPad Prism 6 software.

In vitro Anticancer Studies
To further investigate the cell-toxic properties of the nanoparticles, including 5-FLU and Fe 3 O 4 @OA-CS-NPs and Fe 3 O 4 @OA-CS-5-FLU-NPs, to A549, HeLa S3 and MCF-7 cells, MTT assays using different concentrations (1.5-50 µg/mL) for 24 h at 37º C were conducted. As depicted in Figure 11 and Table 1 51 The Fe 3 O 4 nanoparticle aggregates of different sizes were characterized for their uptake and toxicity in three different cell lines. While the aggregation did not elicit a unique toxic response, the uptake patterns were different between single and aggregated nanoparticles. There was a decrease in uptake of aggregated nanoparticles by HeLa S3 and A549 cells in comparison to single and monodispersed nanoparticles.
In addition, the cytotoxic study revealed cell morphological modifications and nucleus fragmentation using a costaining process with acridine orange (AO) and ethidium bromide (EB) of A549 and HeLa S3 cells. Here, AO was used as a staining reagent of live cells and EB was used as an indicator for dead cells. A549 and HeLa S3 cells were examined in the presence of Fe 3 O 4 @OA-CS-5-FLU-NPs (15 and 40 µg/mL) for 24 h and pictures were captured with the help of AO/EB under fluorescence microscopy. As presented in Figure 12, in the presence of Fe 3 O 4 @OA-CS-5-FLU-NPs, cell death of A549 and HeLa S3 cells gradually increased as specified by the red color which co-stained EB. 52 Moreover, this study further investigated whether the Fe 3 O 4 @OA-CS-5-FLU-NPs had an ability to control A549 and HeLa S3 cell colonization. As shown in Figure 13, complete inhibition of colony development was demonstrated in the presence of Fe 3 O 4 @OA-CS-5-FLU-NPs against A549 and HeLa S3 cells at 15 and 40µg/mL, respectively. However, Fe 3 O 4 @OA-CS-5-FLU-NPs did not affect cell colony formation at a lower concentration of 20µg/mL for the Hela S3 cells. Finally, we conclude here that the Fe 3 O 4 @OA-CS-5-FLU-NPs not only showed cytotoxicity (including morphological changes) but also showed a capability to reduce the colony formation of A549 and HeLa S3 cells. 53

Conclusion
In summary, Fe 3 O 4 @OA-CS-5-FLU-NPs were synthesized by an easy and quick in situ loading method. The proposed Fe 3 O 4 @OA-CS-5-FLU-NPs were successfully prepared as well as characterized by several spectroscopic and microscopic studies. They showed significant ROS reactive drug-releasing properties. Further, the Fe 3 O 4 @OA-CS -5-FLU-NPs showed promising magnetic properties that could be used for magnetic targeted and pH-responsive drug-delivery systems. The pH triggered drug release of 5-FLU from Fe 3 O 4 @OA-CS-5-FLU-NPs includes the ROS-responsive polymeric nanocarriers under marginally acidic conditions of pH 5.2 and pH of 7.4. Meanwhile, the MTT assay, fluorescence staining as well as colony formation assay results, revealed that the Fe 3 O 4 @OA-CS-5-FLU-NPs are active and safe for anticancer biomedical applications, and thus should be further studied.