Protocol for Synthesis and Preparation of Metal-Organic Framework (MOF) Nanoparticles (10ml Total Volume)

Dimethylformamide (DMF)* Benzoic Acid

• Zirconium(IV) Chloride Octahydrate (ZrCl4·8H2O)
• Meso-Tetraphenylporphine (TPP, C44H30N4O8)
• Dimethyl Silicone Oil
• Nitrogen Dioxide (NO2)
• STING Agonist (2'3'-cGAMP, Cyclic GMP-AMP Sodium Salt)
• Phosphate-Buffered Saline (PBS)

Dissolve 0.03g of Zirconium(IV) Chloride Octahydrate and 0.22g of Benzoic Acid in 10mL of DMF to create a precursor solution.1. Employ ultrasonic dispersion to ensure complete dissolution and mixing of the solution.

1. Introduce 0.01g of Meso-Tetraphenylporphine (TPP) to the mixture. Again use ultrasonic dispersion for thorough integration.
2. Transfer the mixture to a round-bottom flask equipped with a magnetic stirrer.
3. Introduce Nitrogen Dioxide (NO2) via a dual needle system for gas dispersion.
4. Heat the solution at 90°C for 5h 0m 0s with continuous stirring at 300rpm on a magnetic stirrer. Utilize dimethyl silicone oil as a heating medium.

After synthesis, distribute the MOF suspension evenly into 10 centrifuge tubes (Eppendorf tubes).1. Centrifuge at 12.000rpm to pellet MOF particles.

1. Decant the supernatant and resuspend the pellet in fresh DMF. Repeat this washing step two more times, ensuring the supernatant is clear after the final wash.
2. Resuspend the washed MOF in 1mLof DMF per tube, using ultrasonic dispersion for complete solubilization.
3. Add STING agonist to the MOF suspension at a 1:10 ratio (v/v), e.g., 300µL of STING to 3mLof MOF suspension. Stir the mixture overnight on a magnetic stirrer.
4. Store the unused MOF suspension at 4°C for the next time.

Remove media from cultured cells and wash them with PBS.1. Scrape cells from the culture plate using a cell scraper, simultaneously adding 1mLof PBS to collect the cells in centrifuge tubes.

1. Centrifuge the cell suspension at 400g (approximately 2000rpm) for 0h 5m 0s
2. Decant the supernatant. Resuspend the cell pellet in 500µL of Cell Extraction Reagent (CER) and 50µL of Protease Inhibitor (e.g., PMSF).
3. Incubate the cell suspension on ice for 0h 5m 0s
4. Transfer the cells to a homogenization vessel and physically disrupt the cells (homogenize).
5. Transfer the homogenate back into centrifuge tubes and centrifuge at 800g (approximately 3.000rpm) for 0h 5m 0s.
6. Transfer the supernatant to new tubes. Add cellular fractionation reagents (e.g., P1201 MER) at a 1:10 ratio (v/v), and incubate on ice for 0h 5m 0s

Centrifuge MOF, MOF+STING, and cell membrane preparations at 12000rpm for 0h 30m 0s.1. Decant the supernatant and resuspend the pellet in 200µLof PBS.

Calibrate hand extruders and initially filter the cell membrane suspension through a 0.4µm filter (diameter 19mm).1. Combine the filtrate with MOF and MOF+STING suspensions in separate tubes. Use ultrasonic dispersion for thorough mixing.

1. Filter the combined suspension through a finer 0.2µm filter (diameter 19mm).
2. Adjust final volumes with PBS as necessary.

Now, the nanoparticles are now ready for in vivo or in vitro experimentation. in vivo or in vitro experimentation.