Protocol for Homogenization of Drug Particles

Homogenization of drug particles is a critical process in pharmaceutical manufacturing. It involves reducing the size of drug particles to enhance the uniformity and efficacy of the final product. This protocol describes the homogenization and zeta sizer protocols for most formulations in the Nanomedicine lab.

• For all formulations, weigh the polymer into a glass vial.
• Disperse the polymer into 15 mL of buffer.
• Mix at medium speed using a magnetic stirrer at room temperature until the polymer is completely dissolved.
• Weigh the drug onto weighted paper.
• Carefully add the weighed drug to the polymer solution.
• Mix overnight using a magnetic stirrer to obtain the complete suspension.
• Turn on homogenizer, compressed air, and pressure gauge.
• Connect the tubing to the homogenizer and homogenizer coil.
• Wash the homogenizer thoroughly. a. Rinse homogenization vessel with 500 ml isopropanol; b. Rinse the homogenization vessel with 500 ml methanol; c. Rinse the homogenization vessel with 500 ml water; d. Fill the homogenization vessel approximately half full with 0.5 M NaOH; apply pressure up to 20,000 psi, alternate pressure on/off; e. Rinse the homogenization vessel with 500 ml water; f. Fill the homogenization vessel with 2% P-407 solution, and apply pressure up to 20,000 psi, alternate pressure on/off; g. Rinse the homogenization vessel with 500 ml water; h. Fill the homogenization vessel approximately half full with water; apply pressure up to 20,000 psi, alternate pressure on/off; i. Use compressed air to blow out any water in the homogenizer/tubing.
• Turn chiller and set to -2°C.
• Remove an aliquot (10 mcL) of suspension and add to 990 mcL fresh HPLC grade water for pre-homogenization particle size, PDI, and zeta potential determined by dynamic light scattering (DLS); mix well.
• Add diluted suspension to low-volume disposable sizing cuvette.
• Place the cuvette in Nano manufacture with the arrow pointing forward, and close the lid.
• Read size and PDI; select size measurement, name sample, change equilibration time to 30 seconds, change cuvette type to low volume disposable sizing cuvette, change number of runs to 3, and start the measurement.
• Once done measuring the size, remove the sizing cuvette and carefully transfer the suspension to a disposable folded capillary cell (zeta cell).
• Place zeta cell and close lid.
• Read zeta potential; select zeta measurement, name sample, change equilibration time to 30 seconds, change the number of runs to 3, and start measurement.
• Once done measuring zeta, highlight the 6 runs (3 sizes, 3 zetas) and record the averaged size, PDI, and zeta.
• Remove and clean the zeta cell with HPLC-grade Methanol and then HPLC-grade water.
• Transfer the suspension to the homogenization vessel, and route the end of the tubing back into the homogenization vessel to create a continuous loop.
• Increase the pressure gradually to 20,000 ± 1,000 psi and homogenize at -4°C, note starting time.
• Regularly (i.e. every hour, every 30 minutes, etc.) monitor the size, PDI, and zeta by taking a 10 mcL aliquot from the homogenization vessel and adding to 990 mcL fresh HPLC grade water; measure size, PDI, and zeta as before.
• Continue to homogenize at 4°C until the target particle size and PDI are achieved.
• Reduce pressure down to 0 and carefully remove nanosuspension from the homogenizing vessel into a clean glass vial; note the finishing time.
• Keep the nanosuspension stirring using a magnetic stirrer to avoid aggregation.
• Clean homogenizer thoroughly.

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• Choose appropriate homogenization tools (e.g., high-shear mixers, bead mills, ultrasonicators) based on the physical characteristics of the drug.
• Determine the duration of homogenization to achieve the target particle size.
• Monitor and control temperatures to avoid degradation or changes in the drug's properties.

Physicochemical Characterization Assays