This method can help answer key questions in the drug delivery field, such as targeted delivery of therapeutics to human cancers and the placenta cystoblast. The main advantage of this technique is that it is a simple and reproducible method to synthesize peptides conjugated in other particles. To begin, add three millimeters of four percent ethanol to a sterile 10 milliliter centrifuge tube.
Then, add 90 micrograms of soybean lecithin stock solution, 210 micrograms of DSPE-PEG-carboxylic acid stock solution, and 750 micrograms of DOX stock solution. Use a one milliliter syringe to pipette two milligrams of PLGA stock solution. Place the centrifuge tube in an ice bath on an ultrasonic processor and sonicate for two minutes.
Then, add PLGA stock solution drop-wise into the centrifuge tube. To purify the DNPs, use a centrifuge filter to wash the solution in 0.1 molar MES buffer. Then, centrifuge the solution at 1000 G for three minutes at four degrees Celsius.
To begin ester activation, add 0.4 milligrams of EDC to one milliliter of DNPs. Then, add 0.24 milligrams of NHS to the reaction. Then, allow the reaction to occur for up to one hour at room temperature in the dark.
After this, mix the reaction components thoroughly and place the reaction on a shaker. To start the amine reaction, use PBS to increase the buffer pH from 7.2 to 7.5. Then, add 0.5 milligrams of pICSA targeting peptide to the reaction solution.
Mix the solution well and place it on a shaker. Then, allow the reaction to proceed at four degrees Celsius overnight in the dark. The next day, fill dialysis bags with the conjugate solution.
Purify the pICSA DNPs with PBS buffer at room temperature for 24 hours in the dark. After culturing cells according to the text protocol, remove the media and add one milliliter of fresh, cold media with five percent FBS and pICSA DNPs or DNPs. Then, incubate the cell and nano-particle mixture for one hour at four degrees Celsius.
After this, remove the media and wash the cells three times with PBS. Then, add one milliliter of fresh media and incubate the cells for 30 minutes at 37 degrees Celsius. After this, remove the media and wash the cells three times with PBS.
Then, add two milliliters of cold, four percent PFA and incubate the mixture at room temperature for 10 minutes. Remove the PFA and wash the cells with two milliliters of PBS. Then, add one milliliter of PBS with DAPI for nuclei staining.
Allow the mixture to incubate at room temperature for 10 minutes. Finally, add mounting medium and image the fluorescence with a fluorescence microscope. Using this protocol, DNPs were successfully prepared and conjugated to pICSA BP.After conjugation, the diameter of the primary DNPs increased to approximately 109 nanometers.
The TEM images of the DNPs and the pICSA DNPs showed that the particles were well dispersed and generally demonstrated spherical morphology. The zeta potential of the nano-particles was negative 20.1 and 29.9 millivolts, indicating that the nano-particles were highly stable. The JEG3 cellular uptake assay indicated the pICSA DNPs bound to the JEG3 cells within 30 minutes.
Therefore, the pICSA BP was efficiently conjugated to the surface. While attempting this procedure, it's important to remember to sonicate the mixture for one to two minutes before adding PLGA. Following this procedure, our methods like HPLC assay can be performed in order to determine the conjugation efficiency more accurately.
After its development, this technique paved the way for researchers in the field of reproductive biology to explore the possibility of placenta-targeting treatment in pregnancy complications.
