The overall goal of this procedure is to synthesize polymeric nanoparticles using a nano precipitation method. This is accomplished by first performing an E-D-C-N-H-S reaction to generate A-P-L-G-A PEG copolymer. The PLGA PEG copolymer is then used to generate nanoparticles, encapsulating the drug or cargo of interest through nano precipitation.
At this point, basic biophysical characterization, including size, surface charge, and drug loading efficiency can be performed through dynamic light scattering transmission electron microscopy or HPLC. The implications of this technique extend towards the therapy of tumors because the nanoparticles can deliver poorly soluble anti-cancer therapeutics, which may be more effective than current treatments. Though this method can provide insight into cancer treatment.
It can also be used to study other systems such as cellular trafficking. The nanoparticles can be conjugated with a targeting ligand and visualized in vivo or in vi in vitro through use of an imaging agent demonstrating the technique. Today will be two technicians from our laboratory, Rohit Sukumar and Natalie Cummings, To begin synthesis of the PLGA PEG co-polymer dissolve PLGA, carboxylate in Acetonitrile at a concentration of five millimolar with gentle stirring.
Then add enough NHS and EDC to obtain concentrations of 25 millimolar providing a five X storia metric excess as compared to PLGA gently stir the solution for about one hour to allow the PLGA carboxylate to be converted into PGA NHS. After one hour, precipitate out the PGA NHS reaction product by adding the washing solution methanol at approximately 10 times volume, excess of methanol to the solution centrifuge the solution at 2000 times G to pellet, the P-L-G-A-N-H-S following centrifugation discard the SUP natin to remove the traces of EDC and NHS. This procedure of washing with methanol is repeated at least three times.
After wash is complete. Dry the P-L-G-A-N-H-S under a vacuum for 30 minutes to remove any traces of the washing solution. Now red dissolve the P-L-G-A-N-H-S pellet in aceto NI trial at the same concentration that was initially used to dissolve the PLGA.
Once dissolved. Add hetero bifunctional PEG to the PLGA solution. Add a concentration of five millimolar.
Incubate the mixture solution for 24 hours with constant stirring after 24 hours. Precipitate the PLGA PEG block copolymer reaction product by adding methanol in excess. Perform the washing and centrifugation process and additional three times to remove all excess unreactive peg.
As the final step in the synthesis dry the PLGA PEG block copolymer under a vacuum nanoparticle precipitation begins with dissolving the PLGA PEG block copolymer and the drug to be encapsulated in A-P-L-G-A solvent. The choice of solvent is critical as it influences the properties of the nanoparticle. Then add the polymer drug mixture, dropwise to three to 10 volumes of stirring water to obtain a final polymer concentration of around three milligrams per milliliter.Dropwise.
Addition of the organic solution to the aqueous phase is critical for forming nanoparticles of the correct size. Continue the stirring for two hours under reduced pressure to allow the nanoparticles to form by self-assembly and remove traces of the organic solvent. After two hours of stirring.
Concentrate the nanoparticles by centrifugation at 2, 700 times G for 10 minutes using an Amon filter. Then wash the nanoparticles with PBS to remove any un entrapped drug and follow by centrifugation. Finally, reconstitute the nanoparticles in PBS At this point.
Basic biophysical characterization, including size, surface charge, and drug loading efficiency can be performed to better understand the properties of the nanoparticles. The nanoparticles can be stored as described in the written protocol to characterize the PLGA PEG nanoparticles transmission electron microscopy was used to confirm the size, distribution, and structure of the nanoparticles. The particle size is generally in the nanometer range.
Large particle sizes with uneven size distributions could indicate either an error in the conjugation reaction or that the nano precipitation method needs optimization. Shown here is a drug release kinetic study where the Paclitaxel loading efficiency and release was quantified with standard HPLC. Known fixed quantities of the nanoparticles were dialyzed at fixed time intervals.
The content in the dialysis unit was collected and an equal volume of organic solvent was added to dissolve the nanoparticles. HPLC was then performed on these samples. To quantify the Paclitaxel content, Once mastered, this nano precipitation technique can be properly performed in three hours.
Remember when performing this technique to always add the organic phase slowly to the aqueous phase to prevent generation of large particles after its development. This technique paved the way for cancer researchers in the field of nanomedicine to explore the use of poorly soluble drugs that were once deemed too toxic in cancer patients following this procedure. Other methods, such as in vivo efficacy studies or imaging studies can be performed to answer questions like whether poorly soluble drugs are effective, when systemic toxicity is no longer an issue, or do targeting ligands properly target nanoparticles to their destination.
