2.12. Quantification of TNF-α Release by Enzyme Linked Immunosorbent Assay (ELISA)

To detect the influence of the developed formulations on the cytokine release (TNF-α) of the macrophage-like dTHP-1 cells after induced inflammation by lipopolysaccharide (LPS), an ELISA was performed [60]. A total of 200,000 THP-1 cells were seeded per well in a 24-well plate and differentiated with PMA to M0 macrophages as described in the section above. Subsequently, the cells were incubated with 500 µL of the different microrod formulations and a concentration of 40, 200 and 400 µg/mL for 2 days at 37 °C in a humidified atmosphere of 5% carbon dioxide. These concentrations represent the same concentrations per cell as used in the cytotoxicity assay. After incubation, the supernatant was discarded and replaced by 500 µL of 200 µg/mL LPS from Salmonella typhimurium for 6 h to induce inflammation of the macrophages leading to an increased release of proinflammatory cytokines such as TNF-α [61]. The resulting supernatants were then centrifuged at 200× g for 10 min to remove cell residues and finally analysed by ELISA for the cytokines. As a positive control (PC), dTHP-1 stimulated with LPS for 6 h without treatment were used. Untreated cells served as a negative control (NC). Further controls included the plain drugs curcumin and siRNA without a carrier system, as well as scrambled siRNA with and without carrier were used to exclude possible unspecific reactions with oligonucleotides.