Physico-mechanical and antimicrobial properties of an elastomeric ligature coated with reduced nanographene oxide-nano curcumin subjected to dual-modal photodynamic and photothermal inactivation against Streptococcus mutans biofilms.

BACKGROUND: White spot lesions (WSLs) are a common side effect of fixed orthodontic treatment. Streptococcus mutans is the primary causative agent of WSLs and dental caries on the teeth during treatment. According to the unique features of reduced graphene oxide-nano curcumin (rGO-nCur), this study aimed to investigate the mechanical properties and antimicrobial potency of rGO-nCur coated orthodontic elastomeric ligatures as a novel coating composite following dual-modal photodynamic inactivation (PDI) and photothermal inactivation (PTI) against S. mutans biofilms. METHODS: After confirmation of rGO-nCur synthesis and coating elastomeric ligatures with different concentration levels of 1.25, 2.5, 5, 7.5, and 10 % of rGO-nCur, tensile strength, force decay, extension to tensile strength, and contact angle of the coated elastomeric ligatures were measured using universal testing machine and sessile drop method, respectively. To investigate the mechanism through which irradiated rGO-nCur can inhibit the formation of S. mutans biofilms, intracellular reactive oxygen species (ROS) generation, and increase in temperature of rGO-nCur solutions under the 450 and 980 nm laser irradiation, respectively, were measured. The anti-biofilm activity and inhibition of water-insoluble extracellular polysaccharide (EPS) production ability of irradiated rGO-nCur coated elastomeric ligatures using a 450 nm diode laser (195 J/cm2), a 980 nm diode laser (195 J/cm2), and a combination of both (78 J/cm2 of irradiation from each one) (i.e., PDI, PTI, and dual-modal PDI/PTI, respectively) were determined. Also, the expression of virulence genes involved in biofilm formation (comDE, gtfD, and smuT) was assessed by quantitative real-time polymerase chain reaction (RT-qPCR) following the mentioned treatment. One-way ANOVA test and Tukey post-hoc test at a p-value equal to/or less than 0.05 were used to analyze the obtained data. RESULTS: The synthesis of GO nano-sheets in a layered structure with a thickness of 0.76 nm was confirmed by AFM analysis. FESEM showed that the exfoliated sheet of synthesized GO had several micrometers in lateral size. DLS revealed that the mean particle size and density index of synthesized nCur were 57.47 ± 2.14 nm and 10 % respectively. In DLS analysis, rGO-nCur showed more positive surface charge (24 mV) than the nano-sheets of GO. FESEM confirmed the coating of rGO-nCur on elastomeric ligatures. ANOVA revealed that tensile strength of 1.25, 2.5, and 5 % rGO-nCur coated elastomeric ligatures were not decreased statistically significantly (P > 0.05). Mean tensile strength and recorded force of 7.5 and 10 % rGO-nCur coated elastomeric ligatures decreased significantly after 14 days' immersion in the artificial saliva (P < 0.05). On the 28th day of the study, the mean of the tensile strength of elastomeric ligatures coated with 10 % rGO-nCur (13.03 ± 0.10 N) was recorded as 55.90 % of the initial tensile strength (23.31 ± 0.41 N in uncoated elastomeric ligatures), while the mean tensile strength of elastomeric ligatures coated with 7.5 % rGO-nCur (16.01 ± 0.10 N) was measured as 68.94 % of the initial tensile strength (23.22 ± 0.09 N in uncoated elastomeric ligatures). When comparing the coated elastomeric ligatures at 7.5 % and 10 % to the original uncoated elastomeric ligatures at similar time intervals, statistically significant decreases in extension to tensile strength (0.42 to 0.71 mm or 3.02 to 5.05 %; all P < 0.05) were observed. The largest contact angle was measured in elastomeric ligatures coated by 10 % rGO-nCur followed by 7.5 and 5 % rGO-nCur (128 ± 2.19°, 117 ± 2.23°, and 99 ± 1.83°; respectively). The results revealed a rise of 6.4-fold in intracellular ROS and an 11.2 °C increase in the temperature of rGO-nCur solutions following the 450 nm and 980 nm laser irradiation, respectively. The 5 % rGO-nCur coated elastomeric ligature mediated dual-modal PDI/PTI showed the most inhibition of the biofilm formation of S. mutans by 83.62 % (P = 0.00). Significant reductions in water-insoluble EPS were detected in biofilm cultures of S. mutans on 1.25 % rGO-nCur coated elastomeric ligatures following irradiation with dual waves of the 450 nm and 980 nm diode lasers (i.e., dual-modal PDI/PTI; 96.17 %; P = 0.00). The expression levels of comDE, gtfD, and smuT virulence genes were significantly downregulated (7.52-, 13.92-, and 8.23-fold, respectively) in the biofilm cultures of S. mutans on 1.25 % rGO-nCur coated elastomeric ligatures following dual-modal PDI/PTI in comparison with biofilm cultures on non-coated elastomeric ligatures. CONCLUSION: 5 % rGO-nCur coated elastomeric ligatures following irradiation with dual waves of the 450 and 980 nm diode lasers (dual-modal PDI/PTI), without adverse effects on the physico-mechanical properties of elastomeric ligatures, can be used to inhibit the formation of S. mutans biofilms on the coated elastomeric ligatures around orthodontic brackets.

Specific Targeting of HER2-Positive Head and Neck Squamous Cell Carcinoma Line HN5 by Idarubicin-ZHER2 Affibody Conjugate.

BACKGROUND: Expression of human epidermal growth factor receptor type 2 (HER2) in head and neck squamous cell carcinoma (HNSCC) cell line HN5 can be employed with great opportunities of success for specific targeting of anti-cancer chemotherapeutic agents. OBJECTIVE: In the current study, HER2-specific affibody molecule, ZHER2:342 (an engineered protein with great affinity for HER2 receptors) was selected for conjugation to idarubicin (an anti-neoplastic antibiotic). METHOD: ZHER2:342 affibody gene with one added cysteine code at the its 5' end was synthesized de novo and then inserted into pET302 plasmid and transferred to E. Coli BL21 hosting system. After induction of protein expression, the recombinant ZHER2 affibody molecules were purified using Ni- NTA resin and purity was analyzed through SDS-PAGE. Affinity-purified affibody molecules were conjugated to idarubicin through a heterobifunctional crosslinker, sulfosuccinimidyl 4-(Nmaleimidomethyl) cyclohexane-1-carboxylate (Sulfo-SMCC). Specific toxicity of idarubicin-ZHER2 affibody conjugate against two HER2-positive cells, HN5 and MCF-7 was assessed through MTT assay after an exposure time of 48 hours with different concentrations of conjugate. RESULTS: Idarubicin in the non-conjugated form showed potent toxic effects against both cell lines, while HN5 cells were significantly more sensitive compared to MCF-7 cells. Dimeric ZHER2 affibody showed a mild decreasing effect on growth of both HN5 and MCF-7 cells at optimum concentration. Idarubicin-ZHER2 affibody conjugate at an optimum concentration reduced viability of HN5 cell line more efficiently compared to MCF-7 cell line. CONCLUSION: In conclusion, idarubicin-ZHER2 affibody conjugate in optimum concentrations can be used for specific targeting and killing of HN5 cells.