Biological Properties of the Aggregated Form of Chitosan Magnetic Nanoparticle

Abstract

Background/aim: Chitosan-coated iron oxide nanoparticles (Chi-NP) have gained attention because of their biocompatibility, biodegradability, low toxicity and targetability under magnetic field. In this study, we investigated various biological properties of Chi-NP.

Materials and methods: Chi-NP was prepared by mixing magnetic NP with chitosan FL-80. Particle size was determined by scanning and transmission electron microscopes, cell viability by MTT assay, cell cycle distribution by cell sorter, synergism with anticancer drugs by combination index, PGE₂ production in human gingival fibroblast was assayed by ELISA.

Results: The synthetic process of Chi-NP from FL-80 and magnetic NP increased the affinity to cells, up to the level attained by nanofibers. Upon contact with the culture medium, Chi-NP instantly formed aggregates and interfered with intracellular uptake. Aggregated Chi-NP did not show cytotoxicity, synergism with anticancer drugs, induce apoptosis (accumulation of subG1 cell population), protect the cells from X-ray-induced damage, nor affected both basal and IL-1β-induced PGE₂ production.

Conclusion: Chi-NP is biologically inert and shows high affinity to cells, further confirming its superiority as a scaffold for drug delivery.

Keywords: Chitosan; X-ray sensitivity; affinity; cytotoxicity; inflammation; magnetic nanoparticle; uptake.

Figure 1. Two methods that explain how to calculate the cell-bound amount of chitosan, NP and Chi-NP. Method 1, Subtraction method: Cell-bound amount (%)=(Input A405 -Output A405)/Input A405. Method 2, Direct method: Cell-bound amount (%)=Recovered A405/Initial A405. A405 represents absorbance at 405 nm

Figure 2. Cytotoxicity of 5 chitosan samples against HSC-2 cells. HSC-2 cells were incubated for 48 h with the indicated concentrations of chitosan nanofiber (n) or chitosan [FL-80 (l), FH-80 (l), 100 (n) or FM-80 (s)], and then viable cell numbers were determined by the MTT method. Each value represents mean±S.D. of triplicate assays.

Figure 3. Properties of chitosan. (A) Solubility of chitosan in isotonic buffer adjusted at pH 3, pH 5 or pH 6. (B) Absorbance intensity of FL-80 [1.25 (○) and 2.5 (l) mg/ml] at different wavelengths. (C) Log scale plot of absorbance intensity of chitosan FL-80 at different concentrations in H2O (l) and 0.5 M NaOH (l) solution. Each value represents the mean value±S.D (n=6). (D) Calculation of cell-bound FL-60 by the subtraction method (Method 1) Absorbance of FL-80 initially added to HSC-2 cells (input) (l) and after it was recovered from the culture supernatant (output) (l). (E) Calculation of cell-bound FL-80 (l), NP (n) and FL-60+NP (l). (F) Calculation of cell-bound nanofiber (l), NP (n) and FL- 60+nanofiber+NP (l). Each value represents mean±S.D. (n=6).

Figure 4. (A) TEM analysis before chitosan addition. SEM analysis of the magnetic nanoparticles encapsulated by chitosan-coating (B, C) and chitosan nanofibers (D).

Figure 5. Effect of chitosan magnetic nanoparticles on the growth of human malignant and non-malignant cells. Human oral squamous cell carcinoma cell lines (Ca9-22, HSC-2, HSC-3, HSC-4) and three human normal oral cells [HGF (gingival fibroblasts), HPLF (periodontal ligament fibroblasts), HPC (pulp cells)] were incubated for 48 h with the following four lots of chitosan magnetic nanoparticles: 1=nanoparticles+5 mg chitosan+0.5% acetic acid with 5 stirring h. 2=nanoparticles+5 mg chitosan+0.5% acetic acid with overnight stirring. 3=nanoparticles+5 mg chitosan+1% acetic acid with 5 stirring h. 4=nanoparticles+5 mg chitosan+1% acetic acid with overnight stirring. Each value represents means of 6 determinations

Figure 6. TEM analysis of the intracellular fine structure of HGFs, HPLFs and HPCs after 3 h contact to Chi-NP (chitosan LH-80-Fe3O4 nanoparticle) (2 and 10 mg/ml). The formation of clusters in the cytoplasm and around the membrane prevents the internalization of Chi-NP

Figure 7. The combination effect of nanoparticles and popular anticancer drugs. a) Effect of 5-FU, abraxane and cisplatin on the viable cell number of HSC-2 cells. Near-confluent HSC-2 cells were incubated for 48 h with the indicated concentrations (0~125 mg/ml) of 5-FU, (0~12.5 mg/ml) of abraxane and (0~12.5 mg/ml) of cisplatine in the presence of 0.08, 0.16, 0.31, 0.63 or 1.25 mg/ml of Chi-NP and, then, the viable cell number was determined by the MTT method. Each value represents the mean of 6 determinations. Upper column in two experiments (Exp. 1 and Exp. 2) shows the dose-response curve of cell viability. Lower column represents the combination index (CI), calculated as described in Materials and Methods

Figure 8. Effect of Chi-NP on cell-cycle analysis in HSC-2 cells. Cells were incubated for 24 h without (control) or with Chi-NP (0.2 mg/ml) or actinomycin D (1 μM), and then subjected to cell cycle analysis

Figure 9. Effect of Chi-NP on IL-1β-stimulated of PGE2 production in HGFs. HGFs (18 PDL) were incubated for 48 h with the indicated concentrations of Chi-NP in the presence or absence of IL-1β (3 ng/ml). The extracellular concentration of PGE2 was determined by ELISA. Each value represents mean±S.D. of 3 determinations

Figure 10. Effect of Chi-NP on the X-ray (900 mGy)-induced cytotoxicity in HSC-2 cells. After changing the medium with fresh DMEM supplemented with 10%FBS and the indicated concentrations of Chi-NP, HSC-2 cells were exposed to X-ray irradiation at 900 mGy (=28.12 mGy×32 times), and then incubated for 48 h to determine the viable cell number (expressed as absorbance at 560 nm) by the MTT method. Each value represents mean±S.D. of 5 determinations