The overall goal of this procedure is to form polymer nanoparticles of tunable size. This is accomplished by first dissolving polymer into an organic solvent and adding drug or fluorescent encapsulant. The second step is to emulsify the polymer with a water phase containing the emulsifying agent, and then allowing the particles to harden in a larger aqueous volume as the solvent evaporates.
Next, the particles are washed, collected by centrifugation and lyophilized for long-term storage. The final step is to characterize nanoparticle size and surface morphology with scanning electron microscopy or SEM imaging. Ultimately, SEM is used to show that nanoparticle size can be controlled by varying emulsifier concentration.
The implications of this technique extend toward therapy of a variety of human diseases because particles can be customized to encapsulate and deliver a wide range of therapeutic agents for sustained action. In target tissue. Visual demonstration of this method is critical.
The emulsification steps are difficult to learn because subtle aspects of how the emulsion is formed can have a significant impact on the properties of the in particles. To begin nanoparticle preparation place approximately 100 milligrams of polylactic co glycolic acid, also known as PLGA into a 13 by 100 millimeter test tube. Then using a glass serological pipette transfer one milliliter of ethyl acetate to the sample, cover the top of the tube with the aluminum foil, and then securely para film the foil.
Be sure to wrap the param tightly across the top edge of the vial and the bottom edge of the foil to prevent evaporation of the solvent. Mark the level of the solvent on the test tube and allow the polymer to dissolve overnight. The next day, add additional solvent if any evaporation occurs.
Prepare the following equipment and materials in a fume hood close to the ultrasonic. A vortex two small glass pasture pipettes with rubber bulbs and a magnetic stir plate. Additionally, place a large beaker full of ice water on a stand below the ultrasonic.
Next, add 45 milliliters of 0.3%weight per volume, vitamin E-T-P-G-S to a 200 milliliter glass speaker, and then set the stirring speed to 360 RPM. Then add two milliliters of the 0.3%weight per volume, vitamin E-T-P-G-S to a 13 by 100 millimeter glass test tube. In order to mix in hydrophobic agents, add the encapsulant directly to the polymer solution, being careful to avoid the walls of the test tube and vortex the tube until the encapsulant is homogenously dispersed.
For hydrophilic agents, emulsify the encapsulant with the polymer solution. Add up to 50 microliters of drug in buffer to the surface of the polymer solution. Ultrasonic the mixture on ice for approximately 10 seconds or until the solution is opaque and homogenous.
Next, vortex the test tube containing vitamin E-T-P-G-S on high while holding the test tube vertically. Keep the glass posture pipette one to two centimeters above the top of the vortexing tube, and slowly add the polymer solution dropwise onto the surface of the vortexing ul. Be careful that the pipette doesn't touch the sides of the tube after the entire polymer solution has been added.
Continue vortexing the solution. Now an emulsion for 15 seconds. Immediately transfer the emulsified polymer to the ultrasonic ator.
Keep the test tube immersed in ice water and sonicate the emulsion in three ten second bursts. Move the emulsion up and down the probe to ensure even sonication and avoid touching the probe to the sides or bottom of the test tube. Pause for five to 10 seconds between each ten second sonication to allow the solution to cool before proceeding.
Then add one to two milliliters of 0.3%vitamin E-T-P-G-S from the stirring solution to the emulsion. This will thin the emulsified polymer, making it easier to pour, empty the emulsion into the stirring solution. Wash out any remaining solution from the test tube into the stirring solution.
Allow the nanoparticles to harden by stirring for three hours. If the encapsulant is light sensitive, wrap the beaker in aluminum foil and leave the top open to facilitate solvent evaporation to collect the nanoparticles begin by splitting the heart into nanoparticles into two oak ridge centrifuge tubes of 30 milliliter nominal volume and balance within 0.1 grams of each other. Then centrifuge the nanoparticles in a fixed angle rotor for 15 minutes at 17, 000.
RC f. Longer centrifugation times will result in the collection of a higher fraction of smaller nanoparticles. Discard the supernatant taking care not to disturb the nanoparticle pellet.
Add five milliliters of distilled water and use a water bath, ator and vortex to completely suspend the nanoparticles. Combine the contents of the two centrifuge tubes into one and add 20 milliliters of distilled water for a total volume of 30 milliliters. Then repeat the centrifugation and washing steps twice more for a total of three washes.
The fluid volume of the last pellet Resus suspension should be four to five milliliters. Freeze a small aliquot of telos free nanoparticles for SEM imaging to the rest. Add a weight ratio of one to two TLO to polymer as a cryoprotectant for uniform and complete resus suspension of PLGA nanoparticles.
This will prevent ice crystals from forming which may damage the particle surface and induce aggregation. Transfer the nanoparticles to a pre weighed five milliliter centrifuge tube, then freeze at minus 80 degrees Celsius for at least 30 minutes. Next, uncapped the tube and cover the top by securing lab tissue across the top with a rubber band.
Moving quickly so the contents do not melt if any melting occurs. Refreeze before placing in the lyophilizer, lay lyophilize the five milliliter sample for 72 hours. Then replace the cap and wrap the tube with store lyophilized particles at minus 80 degrees Celsius.
On the day of imaging, place a strip of double-sided carbon tape on an SEM stub. Label the metal portion of the stub with permanent marker for later reference. Use a metal spatula to collect a small quantity of lyophilized nanoparticles and gently spread them across the surface of the tape.
Brush the surface of the stub with lab tissue or use compressed air to remove loose nanoparticles. Visualize particles using a working distance of five to 15 millimeters of beam, strength of five to 12 kilovolts, and a spot size of one to three higher beam strengths may result in regional heating of the sample, which will alter the surface morphology of the particles. Microparticles are observed at 100 times magnification and nanoparticles will be distinguishable at 3000 times.
Magnification focus on the top of a sphere to locate particles that can be easily visualized with SEM, collect at least three images per batch in order to obtain a representative sample of particle size and morphology. Using this method, particles can be created by single emotion that appear as individual non fused spheres with a smooth surface morphology and a broad range of sizes distributed throughout the sample. The microparticles shown here have an average diameter of 730 nanometers.
Shown here are nanoparticles created by single emotion that are encapsulating the hydrophobic drug campin. These particles ended up even smaller with an average diameter of 340 nanometers. Higher concentrations of emulsifier produced smaller particles with average diameters decreasing from 2000 to 200 nanometers.
As the emulsifier concentration increased from 0.01 to 0.3%as shown by the SEM images here. However, when the concentration of vitamin E-T-P-G-S was too high, discrete particles did not form when 1%vitamin E-T-P-G-S was used, heavy fusing and sheetlike structures were found present throughout the entire structure.True. Once you have learned how to fabricate PLG nanoparticles by emulsification, other methods such as surface modification can be performed to engineer nanoparticles for interaction with specific cell types or regions of the body.
After watching this Video, you should have a good understanding of how to form particles with reproducible and tunable size by emulsification.
