Tailoring Nano-copolymer/CNTs Composite and Its Application in Drug Delivery


 This work aimed to overcome the main drawbacks of some essential anticancer drugs as 5-Fluorouracil (5-FU) by controlled loading with novel drug carriers. By a differential microemulsion technique, nanosized particles derived from a copolymer of poly(methyl methacrylate (MMA) and 2-hydroxyethyl methacrylate (HEMA)) with different monomer ratios have been synthesized and used as a drug carrier. Poly(MMA-co-HEMA)/MWCNT nanocomposite was also synthesized using an in-situ microemulsion polymerization technique and used as a 5-FU carrier. Different techniques have characterized these ground-breaking drug delivery systems such as FT-IR, XRD, TEM, TGA, zeta potential, and a particle size analyzer. The effects of monomer feed composition, 5-FU content, and MWCNTs content on morphological and structural properties, in-vitro 5-FU release, and entrapment efficiency (EE%) have been studied. It was noted that the inclusion of MWCNTs in the 5-FU-loaded polymer increases the thermal stability and raises the entrapment efficiency (EE%) to hit 99% at CNTs:5-FU ratio of 2:1. The anticancer drug release from the co-polymeric nanospheres depends on the HEMA ratio, 5-FU/copolymer ratio, CNT/5-FU ratio, and the pH of the medium. The optimized nanocomposite demonstrated higher anti-tumor activity against the cell lines CaCo-2, MCF-7, and HepG-2 and higher cytotoxicity against HepG-2 relative to CaCo-2 and MCF-7.


Introduction
The development of novel drug delivery systems is imported for improving the pharmacological profiles of several classes of therapeutic molecules and overcoming the drawbacks associated with some essential drugs. As an anticancer drug, 5-FU can achieve effective drug therapy with minimal side effects [1]. This could be achieved by incorporating the drug into a novel carrier which could increase its oral bioavailability and prolong its duration of action.
The microemulsion system is the most efficient drug delivery system, which demonstrates high efficiency of drug trapping, release under a sunken environment, biodegradability, and easy removal from the body [2,3]. The microemulsion technique can produce polymer latexes with particle sizes lower than 50 nm, low viscosity, and wide temperature range stability [4,5]. In situ microemulsion polymerization is considered to be an effective method in drug entrapment during the formation of polymer nanosphere [6][7][8]. In the drug delivery application, CNTs were used due to their hollow nanostructures and fantastic physicochemical features [9][10][11][12]. CNTs are also highly capable of entering biological cells without injury, but their use in pharmacology is limited by the low dispersion of CNTs in aqueous solutions. The modification of CNTs could therefore increase their selective distributions, control the release of the drug, act as an active compartment in increasing the cytotoxicity [13]. MWCNT can be functionalized with polymers to produce MWCNT/polymer nanocomposites with special functions and high biocompatibility [14]. Entezar-Almahdi et al. reported that the MWCNTs functionalization is obligatory to improve their preoccupation and bioavailability [15]. Also, González-Lavado et al. demonstrated that the use of MWCNTs as a drug delivery system improves the efficiency of used chemotherapy drugs [16]. 5-FU is the most commonly used anticancer drug in chemotherapy treatment; however, it inhibits the growth of normal body cells and often induces many side effects like other chemotherapy drugs [17]. The 5-FU treatment rate is less than 15 percent due to its poor bioavailability. Furthermore, 5-FU produces major toxicological damages to the blood and gastrointestinal system [18,19]. So, to realize a stronger treatment with fewer side effects, it is important to establish a hopeful drug delivery system for 5-FU. Entezar-Almahdi et al. summarized the recent Advances in designing 5-FU delivery systems [20]. It was suggested that the loading of the drug into MWCNTs/polymer nanocomposite will be effective in improving its efficiency and reducing the side effects caused by the application of the free drug. However, to the best of our knowledge, there is no detailed report on the implementation of the 5-FU drug delivery utilizing the MWCNT/poly(MMA-co-HEMA) system. This work aimed to synthesize poly(MMA-co-HEMA) with varied monomer ratios and MWCNT/poly(MMA-co-HEMA) nanocomposites and trap them with the 5-FU drug-using differential microemulsion copolymerization method. The polyvinyl pyrrolidone (PVP) was used as a biocompatible emulsifier to produce nanoparticles (NPs) and prevent coagulation. The effects of the MMA/HEMA ratio, 5-FU/copolymer ratio, CNT/5-FU ratio, and the pH of the medium on the colloidal properties and drug delivery performance of the designed carriers have been studied. Zeta potential, thermal stability, drug capture performance, in vitro drug release, and in vitro anti-tumor cytotoxicity against CaCo-2, MCF-7, and HepG-2 cell lines have been also investigated.

Materials
MMA and HEMA were purchased from Sigma Aldrich Company(Germany) and used after purification utilizing active Al2O3 and SiO2 gel columns [21]. The purified MMA and HEMA were preserved in dark containers in a refrigerator for use within four weeks. PVP (Bioshop, Canada), (NH4)2S2O8 (APS, Sigma Aldrich, Germany), 5-FU drug (Biobasic Canada Inc.) were used as purchased. MWCNTs were prepared in our NPA Lab [22,23]. C2H6O, HCl, KCl, and NaOH were obtained from El-Nasr Company (Egypt). HNO3 and H2SO4 were obtained from Nen Tech Ltd (UK). KH2PO4 was bought from GenLab (Egypt).

Modification of MWCNTs
MWCNTs were dissolved in HCL (Dil.) with concentration (5%) and stirred with a magnetic stirrer for 30 min (add CNTs gradually not at one time to make sure reaction occurs) there would be effervescence during addition. MWCNTs were filtered using Buchner and collected carefully in a glass bitter dish for drying in an oven at 80° C for 18 hr. Then MWCNTs were dissolved in 1 H2SO4: 3 HNO3 and refluxed for 4 hr at 120° C to get rid of catalyst and open limbs of MWCNTs. Dilution was done by distilled water over the mixture of MWCNTs, H2SO4, and HNO3, then filtration was done and washing with distilled water for 10 times and the product was dried in the dryer at 100°C for 12 hr.

Preparation of the nano-polymeric material
Poly(MMA-co-HEMA) of different monomers ratios were designed through a microemulsion having a 10% monomers mixture, PVP as an emulsifier, and APS as initiator. The copolymerization reaction was performed in a three-necked flask (250 ml) provided with a condenser and two funnels. Mechanical stirring was carried out at 350 rpm in a water bath with a temperature controller. The microemulsion polymerization process included the following steps [24]: The PVP is dissolved in 30 ml of deionized (DI)-H2O in a flask using a mechanical stirrer and then left at room temperature overnight. The APS is then dissolved in 15 mL DI-H2O in a beaker. 30% of this APS solution is added to the PVP solution and left in H2O bath at 65 o C to throw out the dissolved O2. When the temperature reaches the initiator's decomposition temperature (65 o C), the required concentration of the monomers, as well as 60% of the APS solution, dropped wisely added to the aqueous phase via the dropping funnel over 1 h. After the addition of the rest 10% of APS solution, the solution was left for another 2 h to complete the polymerization.

Synthesis of 5-FU-loaded MWCNTs/copolymer nanocomposites
The 5-FU-loaded poly(MMA-co-HEMA)/MWNTs nanocomposites were synthesized using the in-situ microemulsion polymerization technique [25,26]. The weighed amount of the functionalized MWNT was added to the emulsifier's aqueous solution and exposed to ultrasound for 10 min. Then, the 5-FU was added to this aqueous solution in the three-necked flask. The weighted APS was applied to 15 ml DI-H2O and the initiator solution wisely fell through the funnel along with the monomers to begin the polymerization that lasted up to 2 hours until the monomer odor is gone.

Morphology and particle size
A transmission electron microscope (TEM, JEM-1230) operating at 60 kV was used to analyze the morphology of the MWCNTs, copolymers, and nanocomposites. The samples were diluted with DI-H2O not less than 10 times before taking the TEM images. A well-distributed drop of the diluted sample is loaded on a copper grid and dried at room temperature. H3PW12O40 (0.4 %) drop is used as a stain for the dried sample.

Zeta potential characterization
Malvern ZetaSizer (3000-HS) is used to measure the electrophoretic mobilities (μe) of the designed nanostructures.
Here ε and η refer to the medium permittivity and viscosity, respectively. Triplicate measurements were carried out and the average values were recorded.

Drug Entrapment Efficiency (EE)
The EE of the drug for the copolymer NPs and MWCNTs/copolymer nanocomposite was calculated by determining the free drug using an indirect method, as follows [30]: The free drug was separated from polymeric NPs and the nanocomposite by dissolving in methanol where the polymeric NPs are suspended in methanol, and then they separated from the polymeric NPs by refrigerated ultracentrifugation with 50000 rpm for 0.5h. The concentrations of the free drug in the supernatant solutions were quantitatively analyzed by a double beam UV spectrophotometer at the specified wavelength (λmax= 266 nm) after measuring a series of known concentrations of the drugs to draw the calibration curve.
The values of EE were calculated from equation (2) using the actual weight of the drug in the sample and the initial or theoretical weight of the used drug [31,32]. (2)

In-vitro 5-FU release
The in-vitro 5-FU drug release from copolymer NPs and MWCNTs/copolymer nanocomposites were carried out in simulated intestinal solution (pH 7.4) and gastric solution (pH 1.2) via the dialysis bag method [26,33]. with the buffer fluid for a few hours [8]. 500 mg of the 5-FU-loaded copolymeric nanocomposite was added to 5 ml of the buffer fluid, placed in the dialysis bag, and then immersed into a closed vessel having 0.1litre of the buffer @100 rpm stirring and 37°C ± 0.5°C [34]. 5 ml of the sample was withdrawn after regular interval times to carry out the 5-FU release analysis and compensated with an equivalent amount of fresh buffer to preserve the condition of sinking. The samples will be analyzed for drug content using a calibration curve for the drug in each buffer solution by UV spectrophotometer at λmax for 5-FU.
The drug release percent was calculated by equation (3); Total drug (5FU)

Cytotoxicity assay
The cytotoxicity of 5-FU-loaded MWCNTs/ polymer nanocomposite was measured using the technique of tissue culture. HepG-2 (hepatocellular carcinoma), MCF-7 (breast cancer), and CaCo-2 (human colorectal adenocarcinoma) cell lines were delivered from the Pharmacology Unit, National Cancer Institute, Cairo University, Egypt. They were held at 37ºC and 5% CO2 in DMEM media of 10% fetal calf serum, sodium pyruvate, 100 U/ml penicillin, and 100 mg/ml streptomycin until the bioassay of cytotoxicity was performed. The cytotoxicity of the sample was evaluated using Skehan et al. process [35]. Generally, during the night afore applying the prepared samples, we put 100 cells/well on 96-well plates to enable cells to be attached to the walls of the plate. We added diverse concentrations The effect of the MMA/HEMA ratio on the morphology of the formed 5-FU loaded polymeric nanospheres was demonstrated in Figure 1. As the monomer feed ratio increases, the particle size is increased. At MMA/HEMA monomer feed ratio of 90/10, Fig.3A, the diameter of the polymeric nanospheres is ranged from 83 to 97.5 with an average value of 97.5 ± 14 nm. At the feed ratio of 70/30, Figure 3B, the average value of the polymeric nanospheres diameter is increased to 143± 13 nm. From Figure 1C, the average value of the polymeric nanospheres diameter is increased to 187± 14 nm @ 50/50 MMA/HEMA monomer feed ratio. It has been observed from Figure 1 that as the monomer feed ratio increased, the polymer nanospheres could be loaded with high 5-FU drug content to form coreshell (polymer nanosphere/5-FU) morphology as shown in the inset of Figure 1C. The thickness of the shell was 35 ±3 nm.   Figure 4 that the rise of the drug content from 1:20 to 1:6 leading to an increase in the particle size from 143± 13 nm to 177± 10. It is observed from the inset images of Fig.4 that a shell of increased thickness is grown around the polymer nanospheres. As the drug content increased from 1:10 to 1:6, the shell thickness is increased from 23.3± 3.5 nm to 38.9± 6.1 nm. This means that 5-FU could be loaded with high drug content into the polymer nanospheres for a drug to a monomer ratio of 1:6.

Zeta potential and particle size
The zeta potential was used as a measure of the colloidal stability of the nanoparticle suspension where it was considered as an indicator of the magnitude of electrostatic interaction between colloidal particles. The increase in zeta potential provided good stability against coagulation in the colloid system. The data presented in Table 1 showed low negative zeta potential of the prepared microemulsion lattices and this means that the formed latex is stable. But with increasing the HEMA ratio in the latex, the zeta potential value (ζ) increases from -0.39 mV to -0.6 mV due to the increase in the surface charge with the existence of additional hydroxyl groups in the repeated units [37]. The particle size measurements, Table 1, also show the increase of the particle size from 70 nm to 150 nm as the composition of the MMA/HEMA monomer feed is altered from 90/10 to 50/50. This implies that the particle size is duplicated by increasing the content of HEMA, which is well in line with the particle size found by TEM analysis,  Table 1. The size of the nanoparticle was found to increase as the 5-FU content increased, whereas the particle size increased from 85 to 105 and 120 nm by changing the Drug/ (70/30) copolymer ratio from 1/20 to 1/10 and 1/6, respectively. This indicated that the drug was well loaded into the polymeric NPs as observed in Figure 4. The value of ζ for 5-FU-loaded MWCNTs/(MMA/HEMA50/50) nanocomposite is -1.34 mV. This means that the incorporation of functionalized MWCNTs shifts the zeta potential from -0.6 to -1.34 mV, which results from the existence of the carboxylic groups after the acidic functionalization of the MWCNTs [7,8]. The particle size measurements, Table 1, also show the increase of the particle size from 150 nm to 240 nm by the incorporation of CNTs to form 5-FU loaded-MWCNTs/copolymeric(50/50) nanocomposite with 5-FU/MWCNTs ratio of 1:1. This implies the increase of the particle size by a 1.6 factor, which agrees well with the particle size found by TEM analysis,  Table 2 at the specific temperatures.  MWCNTs/copolymer nanocomposites with CNTs/drug ratios of 1:2 and 2:1. Table 3 presented the TGA values of these samples. The TGA values, Table 3, showed that the 5-FU-loaded MWCNTs/copolymer nanocomposite is more stable than the 5-FU-loaded copolymer (MMA/HEMA50/50). Also, it is noted that the thermal stability is increased with increasing MWCNTs/drug ratio from 1:2 to 2:1 [10,38].      The FTIR spectrum of the drug-loaded copolymer, Figure 7iii, refers to the existence of both 5-FU and the copolymer.
It showed the functional groups of HEMA -OH mode@3384 cm −1 and the -COOH mode of 5-FU @2946 cm −1 with higher intensity than that observed for free 5-FU. The modes@575 and 746 cm −1 are often due to the aromatic bending of C=C, which is observed in the free 5-FU FTIR spectrum but not observed in the free copolymer FTIR spectrum.
The 1719 cm −1 mode corresponds to the C=O of the drug, which suggests the encapsulation of 5-FU into the copolymer. The FTIR of 5-FU-loaded MWCNTs/copolymer nanocomposite is illustrated in Figure 6iv.

Drug Entrapment Efficiency
The values of the drug entrapment efficiency (EE%) were estimated at different monomer feed composition, drug content, and MWCNTs content. The obtained values were presented in Table 4. In general, the EE% values are strongly dependent on monomer feed composition, drug content, and MWCNTs content as shown in Table 4. It was noted that drug EE% was influenced by the monomer feed composition. As the HEMA content increased, the EE% value is increased from 78% to 93% at 5% drug content. Also, the drug EE% is improved from 81% to 90% with increasing the drug content from 5% to 16.6%. Such data shows that by the differential microemulsion polymerization process, we can yield a 5-FU-loaded copolymer nanocomposite with high EE%. Moreover, as seen in Table 4, the EE% is significantly enhanced when the drug is loaded on the MWCNTs/(MMA/HEMA50:50) copolymeric nanocomposite. EE% reached about 99% @ high MWCNTs/drug ratio (2:1). Due to the drug's contact with MWCNTs/copolymer or filling in the inner cavities of the MWCNTs, the integration of MWCNTs into the copolymeric nanocomposite enables packing of higher 5-FU content [42,43].

In Vitro Drug Release Studies
As a function of several variables including MMA/HEMA ratio, pH value, 5-FU, and MWCNTs contents, the release behaviors of the 5-FU are measured and presented in Figure 7. In general, the 5-FU drug release from the designed carriers indicates an initial blast effect, followed by a regulated release of the 5-FU as seen in Figure 7.   [44]. A more hydrophilic copolymer would lead to faster ingestion of aqueous solution, resulting in quick rates of 5-FU dissolutions [27,28].

Influence of the pH value
The behavior of the drug release from the copolymer is studied using buffer solutions with pH values of 7.4 and 1.2, which simulated the intestinal and gastric fluids, Figure 7(C). Overall, because of the weakly bounded 5-FU to the wide surface area of the NPs, the release profiles in Figure 7(C) indicate quick starting release [45]. Also, it was noted that the release rates in the simulated intestine fluid, pH7.4, reached higher values compared to the simulated gastric fluid, pH 1.2. The drug release rates reached ~70%@pH 1.2 and 85%@pH 7.4 after 2h using the copolymer (50/50).
The copolymer will then release fewer doses of the 5-FU drug in the stomach than in the intestine. However, the small difference in the copolymer release rates at pH 1.2 and pH 7.4 showed that the 5-FU-loaded copolymer can be applied at a wide variety of pH values.

Effect of drug content and MWCNTs content
The drug release rates were tested at different drug/copolymer ratios (1:20, 1:10, and 1:6) for the copolymeric MMA/HEMA 50/50 nanospheres, Figure 7(D). It was noted that the copolymer with higher drug loading is faster than that with lower drug loading because the higher drug loading in the nanospheres indicated a lower polymer matrix content, which facilitates drug dissolution from the nanospheres [46].
From Figure 7(E), The 5-FU drug release rate from MWCNTs/copolymer nanocomposite is lower than the release rate from the pure copolymer @ pH 7.4. This can be ascribed to the encapsulation of 5-FU into the MWCNTs' internal cavities [47]. The increase of MWCNT relative to the 5-FU drug could improve the controlling of the 5-FU release [43].

In vitro Anti-tumor Cytotoxicity
The cytotoxicity of the sample was evaluated using Skehan et al. process [35].  Also, the incorporation of MWCNTs increases the EE% to a high ratio (99% @ CNTs: drug ratio of 2:1) which permits loading of higher drug content. Also, 5-FU-loaded MWCNTs/copolymer nanocomposite exhibited a significant antiproliferative effect against CaCo-2, MCF-7, and HepG-2 cell lines. Compared with the other two cell lines, it demonstrated stronger cytotoxicity against the HepG-2 cell line. Consequently, the optimized MWCNTs/copolymer nanocomposite would be a hopeful drug delivery system for the 5-FU drug.