Synthesis and cytotoxic activities of selenium nanoparticles incorporated nano-chitosan

Chitosan (low molecular weight; < 100 KDa, Bioscience company). Sodium tripolyphosphate, 5-Fluorouracil, Selenous acid and Ascorbic acid were delivered from Aldrich. Acetic acid was analytical grade supplied from Adweck.

Selenium nanoparticles (SeNPs) were prepared by chemical reduction of Selenous acid using ascorbic acid. A weight of 0.25 g Selenous acid was dissolved in 50 ml distilled water under stirring and the temperature of this solution was raised to 60 °C. Ascorbic acid (0.025 g) was dissolved in 20 ml distilled water and added to the warm solution of selenous acid under stirring for about 10 min. The solution color was turned into red color indicating the formation of selenium nanoparticles.

Chitosan nanoparticles were prepared by ionic gelation method using sodium tripolyphosphate (TPP) as described in literature [25, 26]. Briefly; dilute solution of the low molecular weight chitosan (0.5 g) was dissolved in 2% aqueous acetic acid (100 ml) under stirring at room temperature till complete dissolution. 0.25 g of TPP was dissolved in distilled water under stirring. The TPP solution was added dropwise to chitosan solution under continuous stirring and after complete addition the solution mixture left under stirring for about additional one hour. Turbid solution of chitosan nanoparticles was obtained. For encapsulation of selenium nanoparticles, the same procedure was used instead 10 or 20% (respect to chitosan weight) of selenous acid was added to the dissolved chitosan solution under heat at 60 ^oC then ascorbic acid was added dropwise and finally gelation with TPP was performed. For encapsulation 5-Fluorouracil drug, 10 mg or 20 mg that dissolved in 10 ml water for each was added to chitosan solution and apply the same procedure.

The drug release studies from the synthesized nanocomposite were performed in 0.1 M phosphate buffer solution (PBS) with two different pH (pH: 4 and 7.4) simulating the acidic and blood pH, respectively. 50 mg from the nanocomposite (Ch-Se10-Flu20) with relatively high initial drug concentration was suspended in 2 ml buffer solution and transfer onto dialysis bag with molecular weight cut off (12–14 KDa), then the dialysis bag was soaked in 50 ml PBS reservoir with mild agitation (100 rpm). One ml of the phosphate buffer reservoir was taken out at different time interval and fresh buffer was added to stimulate drug release from the nanocomposite. The amount of the released drug was determined using UV spectrophotometer at wavelength 266 nm [27].

Chitosan nanoparticles with their encapsulated particles were characterized using FTIR, TEM, DLS, DSC and X-ray as following:

Three cancer cell lines were used to evaluate the anticancer activity of Ch/Se nanocomposite. Human colorectal carcinoma (HCT-116), human liver carcinoma (HepG-2), and human breast adenocarcinoma (MCF-7) in addition to the normal human skin fibroblast (BJ-1) cell line were purchased from the American Type Culture Collection (Rockville, MD, USA) and maintained in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U mL⁻¹ penicillin, and 100 U mL⁻¹ streptomycin. The cells were grown at 37 °C in a humidified atmosphere of 5% CO₂.

The antiproliferative activities on the HepG-2, HCT-116, MCF-7 and BJ-1 were estimated by the 3-[4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. This test is based on MTT cleavage by mitochondrial dehydrogenases form viable cells [28‐30]. Cells were placed in a 96 well sterile microplate (5 × 10⁴ cells well⁻¹) and incubated at 37 °C in serum-free media containing dimethyl sulfoxide (DMSO) and either a series of various concentrations of each compound or doxorubicin (positive control) for 48 h before the MTT assay. After incubation, the media were removed and 40 µL MTT (2.5 mg mL⁻¹) was added to each well. Incubation was resumed for an additional 4 h. The purple formazan dye crystals were solubilized with 200 µL DMSO. Absorbance was measured at 590 nm in a Spectra Max Paradigm Multi-Mode microplate reader (Molecular Devices, LLC, San Jose, CA, USA). Relative cell viability was expressed as the mean percentage of viable cells compared to the untreated control cells. All experiments were conducted in technical triplicate and three biological replicates. All values were reported as mean ± SD. IC₅₀ were determined by SPSS Inc probit analysis (IBM Corp., Armonk, NY, USA).

The prepared chitosan encapsulated selenium nanoparticles in the presence or absence of 5-fluoruracil were investigated and characterized by different instrumental techniques. Figure 1 indicates the FTIR of the prepared chitosan nanoparticles, and it is clearly observed that the ChNPs showed broad band at 3420 cm⁻¹ corresponding to the stretching of intermolecular bonded OH combined with NH groups. A small sharp band at 3720 cm⁻¹ revealed to free OH groups. The asymmetric and symmetric stretching vibrations were appeared at 2911 and 2858 cm⁻¹ corresponding to the aliphatic CH₂ groups, respectively. However, the peaks at 1631 cm⁻¹ and 1380 cm⁻¹ were attributed to N–H bending and OH bending, respectively. The peak at 1045 cm⁻¹ was corresponding to C–O stretching. The FTIR spectra of Ch-SeNPs showed blue shift for the characteristic peak of O–H group at 3420 cm⁻¹ to (3425.26 cm⁻¹). Moreover, a slight shift in the absorption peaks of chitosan 3434.55 (O H stretch), 2924.13 (C H stretch), 1734.28 (COO stretch), 1070.77 cm⁻¹ (C O stretch) after Se nanoparticles incorporation. The shift of OH band might indicate an interaction between hydroxyl groups of chitosan and surface of SeNPs. The peak at 1631 cm⁻¹ was clearly appeared and shifted to 1644 cm⁻¹, indicating the formation of Se–O bond. Another band at 879 cm⁻¹ was attributed to the Se–O stretching vibrations [22]. The combination mode of chitosan and SeNPs was similar to what was reported previously [8]. A weak peak at 538 cm⁻¹ revealed to C–F group of 5-fluorouracil in Ch–Se–Fu sample.

The particle size and zeta potential measurements were determined using DLS analysis as shown in Fig. 2 and Table 1. The results indicated that the average values of particle size for Ch, Ch-Se10, Ch-Se20, Ch-Se10-Flu10 and Ch-Se10-Flu20 were 207.4, 271.5, 239.5, 251.3 and 358.2 nm, respectively. The total size of nanochitosan was slightly increased from 207.4 to 271.7 nm as clearly observed in Fig. 2 after incorporation of selenium nanoparticle. Such increment might be considered good indication for the nanoparticles stability [8]. Nevertheless, the dramatic increase in the particle size of Ch-Se10-Flu20 might be attributed to the higher of 5Flu concentration. However, for the lower concentration of drug it was 251.3 nm indicating that Ch-Se10-Flu10 was highly recommended candidate for obtaining more disperse nano-capsules.

The Zeta potential of dispersion is measured by applying an electric field across the dispersion. The Zeta potential is the potential difference existing between the surface of a solid particle immersed in a conducting liquid (e.g., water) and the bulk of the liquid; so it is measuring the electrical charges of particles suspended in a liquid. The prepared particles had positive charge ranging from 23.91 to 41.02 mV displaying the positive surface of the nanoparticles which might be resulted from the cationic amine groups on the nanochitosan surface. The value of zeta potential for all prepared nanoparticles was relatively high which indicated that the Ch-SeNPs had better colloidal stability than chitosan nanoparticles alone as well as the stability of the particles in suspension solution without agglomeration or coagulation [24].

The particles size and shapes of the synthesized chitosan nanoparticles and their nanocomposites were investigated and displayed in Fig. 3. It is clearly seen from Fig. 3a that the particles have semispherical shape with an average size about 30–40 nm with some accumulation and interconnected structure. The positively charges of amino groups of chitosan (protonated amines in aqueous acetic acid solution) interacted ionically with negatively charges of sodium tripolyphosphate developing these semi-spherical nanoparticles [31]. Figure 3b–e displays TEM for the composite nanoparticles. Selenium nanoparticle were displayed as spherical dark spots in the chitosan matrix with small particle size ranged 12–20 nm for lower concentration of selenium (Fig. 3b), whereas for Ch-Se20, the size of selenium nanoparticles was ranged of 50–75 nm (Fig. 3c). The morphology of SeNPs was spherical- like with average size of approximately 20.98 nm for Ch-Se10 and 70.74 nm for Ch-Se20. The distribution of selenium nanoparticles (dark spots) was monodispersed and showed a homogeneous structure for the lower and higher concentrations of selenium. At high concentration of selenium nanoparticles the particles might be aggregated forming relatively higher particle size.

Ch-Se-Flu was significantly affected by 5Flu concentration as shown in Fig. 3c, d. However, when the concentration of 5Flu reached 20 mg/mL; SeNPs showed a net-like structure (Fig. 3d) with aggregation of chitosan nanoparticles combined selenium and fluorouracil. The SeNPs were well cross linked and encapsulated with chitosan and 5Flu as appeared in Fig. 3c. It is worth to mention that 5-fluorouracil was well distributed and encapsulated within the Ch-Se nanoparticles with appearance of few aggregates at the outer shell of the samples. These results indicated that 5-Fu could effectively regulate the size of ChSeNPs when used in a specific range of concentrations [32]. In this context, Ch-Se10-Flu10 and Ch-Se10-Flu20 were considered promising system that can be used in drug deliver application.

The crystallinity of the prepared nanoparticles was investigated using XRD analysis as shown in Fig. 4. The XRD pattern of all nanocomposite showed a well characteristic broad peak around 20° which related to the chitosan molecule [33]. Moreover, the XRD pattern of Se NPs displayed several Bragg's reflections. The clear peaks of cubic phases were recorded at θ = 22.4° (100), 32.50° (101), 45.80° confirming the crystalline nature of the prepared Se NPs [17, 34]. These results indicated that SeNP was not pure amorphous and had the crystalline trigonal phase of Se [34‐36]. However, the peaks displayed at 19, 24 and 29^o were attributed to 5-fluorouracil existence in the prepared nano-carrier [27]. These finding results illustrated the successful preparation of chitosan encapsulated selenium nanoparticles and 5flurouracil.

Thermal stability of chitosan selenium nanocomposites was assessed using TGA and displayed in Fig. 5. All nanoparticles showed small weight losses (about 5%) around 100 °C due to the adsorbed moisture content. The decomposition peak at 240 °C was attributed to chitosan, with weight loss reached 36% and appeared as strong single peak. All the curves showed good thermal stability till about 800 °C. The thermal stability of these nanocomposite resulted from the presence of inorganic nanoparticles (selenium nanoparticle).

The in-vitro release profile of 5-fluorouracil drug from chitosan selenium nanocomposite (Ch-Se10-Flu20) was performed in phosphate buffer solution with different pH (4 and 7.4) mimicking the acidic and pH of blood, respectively. It is obviously displayed in Fig. 6 that a rapid initial drug released for the first 8 h for both buffer medium which may be due to the well-known burst effect, resulted from some amounts of drugs were localized on the surface of the nanocomposite that can be easily released by diffusion. The figure indicated also the higher release of 5-flurouracil drug in the acidic medium that at pH 7.4 which may be attributed to the higher swelling performance of chitosan nanoparticles in acidic medium due to the protonated effect of amine groups in acidic medium.

After the initial rapid release, a slower continued release of 5-fluorouracil was attained within the period of 8–24 h and the release reached to about 74% after 24 h for acidic PBS medium and released about 62% in pH 7.4. The nanocomposites were stable and can retain 5-fluorouracil within their structure particularly at pH7.4 and may be need more time to be fully released. These results demonstrated that this system considered a reasonable candidate for drug delivery for cancer treatment applications.

Four nano-composites in addition to 5-fluorouracil and selenium nanoparticles as blank samples were examined in vitro for their activity on HCT-116, HepG-2, MCF-7 human cancer cells and one human healthy cell line (BJ-1) using the MTT assay. The percentages of intact cells were calculated and compared to those of the control. Activities of these nano-composites against the three carcinoma cell lines were compared to the activity of doxorubicin. All nano-composites suppressed the three cancer cells in a dose-dependent manner as shown in (Figs. 7, 8). In case of HCT-116 human colorectal carcinoma cells: both Fig. 7 and Table 2 showed that three nano-composites (Ch-Se10-Flu20, Ch-Se10 and Se-NP, respectively) had comparable potent cytotoxic activities; the rest of the nanocomposites significantly exhibited moderate cytotoxic activity against HCT-116 relative to that of doxorubicin. The highest activity of nanocomposite containing relatively high concentration of 5-fluorouracil (Ch-Se10-Flu20) indicating the high effect of the well-known drug in suppressing the growth of the cancer cells [37]. 5-fluorouracil interferes with nucleic acid synthesis, inhibits DNA synthesis, and eventually stops cell growth. The high activity of composite containing Se-NP with low concentration (Ch-Se10) may be due to the smaller size of Se NP as shown in Fig. 3b compared with that containing high Se NP (Fig. 3c). Selenium nanoparticle with small size can easily penetrate from the nano-capsule into the cell line suppressing their growth [38]. In case of MCF-7 human breast cancer cells: the four nano-composites (Ch-Se10-Flu10, Ch-Se10-Flu20, Ch-Se20 and Ch-Se10%, respectively) had comparable potent cytotoxic activities; the two corresponding blanks (Fluorouracil and SeNPs, respectively) displayed moderate cytotoxic activity against MCF-7 relative to the reference drug (Fig. 8 and Table 2). The high activity of nanocomposite containing both Se-NP and 5-flurouracil attributed to the combined effect of anticancer property of both the drug and Se nanoparticles [39]. In case of HepG2 human liver cancer cells: three nano-composites and their corresponding blanks (SeNP, Ch-Se10-Flu10, Ch-Se20, blank Flu, and Ch-Se10, respectively) showed cytotoxic activity at higher concentration of nanocomposite (100 µM) compared with their effect on the other cancer cell line at (50 µM). (Ch-Se10-Flu20) represented slight less cytotoxic activity against HepG2 relative to that of doxorubicin (Fig. 9 and Table 2). The synthesized nanocomposites have high potent effect on human breast cancer cells MCF-7 followed human colorectal carcinoma cells HCT-116 then human liver cancer cells HepG2. All compounds were tested against non-tumor fibroblast-derived cell line (BJ) and demonstrated very low cytotoxicity. It is worth to mention that the above data demonstrated that SeNPs synthesized without a stabilizer was unstable and easily agglomerate, which confirmed the important role of Ch for further 5-Flu encapsulation.