Methods Development for Blood Borne Macrophage Carriage of Nanoformulated Antiretroviral Drugs

The aim of this procedure is to manufacture and test cellular uptake, secretion, and antiretroviral properties of crystalline antiretroviral nanoparticles, which we refer to as nano art. This is accomplished by first processing the antiretroviral drugs by sonication homogenization, or wet milling. The second step of the procedure is to characterize the physical properties of the nano art particles after primary monocytes are isolated by countercurrent centrifugal ation.

They're cultured for seven days in DMEM containing macrophage colony stimulating factor, or MCSF, which induces their differentiation into monocyte derived macrophages. The third step is to test the uptake, retention and release of nano art in human monocyte derived macrophages. The final step of the procedure is to test the in vitro antiretroviral efficacy of nano art in monocyte derived macrophages.

Ultimately, results can be obtained that show the facilitated uptake and slow release of nano art from cells and robust antiretroviral efficacy. These are determined by measurements of drug levels, by high performance liquid chromatography or HPLC and by testing infected culture supinate materials by measurement of reverse transcriptase activity. Hi, I'm Hanta Aldi and I'm a postdoctoral research fellow in Dr.Howard Gelman's lab at the University of Nebraska Medical Center.

The methods we present can help answer key questions in nanomedicine that include how cell-based drug delivery systems can be used to develop translational research activities. This can be accomplished by using nano formulated antiretroviral therapy with prolonged half wives. Our studies support the potential use of nano art for clinical use.

Hi, my name's Ari Noche and I'm an MD PhD student scholar working in the Carol Schwartz Emerging Neuroscience Laboratory. Under the guidance of Dr.Gendelman, the implication of the techniques that we show you here extend to antiretroviral therapy because nano formulated compounds can simultaneously improve bioavailability and pharmacokinetics while also reducing drug related side effects. By taking clinically available antiretroviral medications and repackaging them into nanoparticles, we may be able to improve compliance and also positively affect disease outcomes.

Hi, I'm Dr.Howard Gendelman. I'm a clinician scientist and professor and chair of the Department of Pharmacology and Experimental Neuroscience here at the University of Nebraska Medical Center. Our aim overall is to simplify antiretroviral drugs to make them easily available and improve the biodistribution and pharmacokinetics.

Now I turn you over to Ari and Sean who will bring you into the research laboratory and show you how the manufacturer and development of these nanoparticles are done. Clinical utility and development here at the University of Nebraska Medical Center To manufacture nano art via wet milling. First weigh the drug crystals and various surfactants that will be used to make the nano formulations and mix them with 10 millimolar heis buffer pH 7.8 Using a T 18 ultra Turx mixer until completely dispersed, the percent of the drug in the formulation should be between one and 2%for continuous milling.

Make sure that the chiller and the compressor are on and that the outlook pressure is around 100 PSI now set up so that the grinding media can be loaded in the tank and add 50 milliliters of beads to the grinding chamber. Using a funnel, make sure that there are no beads in the threads or anywhere outside the grinding chamber to avoid leaks in the mechanical seal. Now, make sure that the O-ring is in its place on the milling chamber.

Then select an appropriate screen mesh size and secure the lids by tightening the nuts. The screen mesh size should be at least half the size of the beads. Use a large screen mesh size to avoid the product from clogging the filter while continuous milling.

After lowering the grinding chamber, add the drug suspension with various amounts of surfactants to the product inlet. The minimum volume of the suspension should be 100 milliliters. This will prevent the milling chamber from overheating and also avoid bead contamination due to less wear on parts.

Next, start the pump. Using the control unit varying the flow rate is required for your formulation. Then turn on the agitator and adjust the speed on the control unit.

The speed can be increased from 600 rotations per minute to 4, 320 rotations per minute. On a micro sur, monitor the temperature using the temperature gauge that is attached on top of the grinding chamber to make sure that there is no overheating. Now mill the sample at various speeds and flow rates and take out small aliquots of about one milliliter of the formulation for size and charge measurements using a diffraction light scattering malvern zeta sizer.

After the desired size is obtained, stop the milling using the control unit and with the pump still on, collect the sample into a flask, allowing the drug to drain completely. Remember to switch off the pump when done and remove and clean the beads. Now transfer the mill suspension into 50 milliliter centrifuge tubes and set the centrifuge to 10, 000 rotations per minute for 30 minutes.

Upon completion of the centrifuge cycle, measure the amount of sate and replace with the same amount of fresh surfactant solution. Once Resus suspension is complete, transfer the suspension to the milling chamber and mill for 10 minutes. Take an aliquot of about one milliliter and measure the size and charge of the sample again using the Zeta sizer.

Finally, measure the concentration of the nano art formulations using HBLC to manufacture nano art via homogenization. Using the Avastin Emulex C five, prepare the drug crystals and various surfactants as previously described, and transfer the suspension to the homogenizing vessel and begin recirculation. Make sure that the chiller is on before starting to homogenize the sample.

Increase the pressure gradually until the meter reads 20, 000 plus or minus 2000 PSI and continue to homogenize until the target particle size is achieved. This usually takes between 45 to 60 minutes. Take an Eloqua of about one milliliter periodically and measure the size and charge of the sample using a zeta sizer centrifuge, the homogenized suspension as previously described.

Then after Resus suspension, transfer the suspension to the C five homogenizer. Restart the circulation and return the homogenizing pressure to 20, 000 plus or minus 2000 PSI and homogenize for 30 minutes to manufacture via sonication using the cold Palmer ultrasonic processor. Pour the chloro methane PLGA and drug solution into a 1%PVA surfactant solution and place in an ice bath in the ultrasonic.

Start sonication at 50%amplitude, although the amplitude of the sonicate can be reduced to about 30%amplitude for smaller batches of samples, sonicate the sample for 10 minutes. Take out a small aliquot for particle size analysis. If the size of the particles is greater than 1.5 microns, sonicate the samples at two minute intervals up to a maximum of 16 minutes total.

Observe the samples under the microscope at 20 times magnification and document observations. Use a stir plate to create an adequate vortex for the suspension and continue mixing overnight at room temperature. Collect the suspension after 24 hours and pour into 50 milliliter centrifuge tubes.

Centrifuge the samples at a speed of 8, 000 rotations per minute for 20 minutes at five degrees SIUs decant the supinate and resuspend one half of the sample in 75 milliliters of filtered revers osmosis water and the other half in 75 milliliters of 0.5%Ctal trimethyl ammonium bromide surfactant. Centrifuge the samples again at a speed of 8, 000 rotations per minute for 20 minutes of five degrees Celsius and resuspend each of the pellets with a total of 20 milliliters of mannitol solution. Finally, for particles manufactured via either homogenization or sonication, measure the size of the particles using the zeta sizer and measure the concentration of the samples using HPLC.

To prepare for visualization with microscopy, take the prepared nano art suspensions and sonicate them for 10 seconds. At 20%amplitude with a sonication probe, take a microscope slide cover slip and place it on an inverted microscope. On the cover slip.

Mix 15 microliters of the nano art suspension with 100 microliters of phosphate buffered saline and mix by pipetting up and down several times. Then visualize the nanoparticles using a 20 times objective to visualize morphology. Samples can also be prepared for visualization with scanning electron microscopy.

Here we see images obtained from A-J-E-O-L Jsm 6 3 0 0 F low voltage field emission scanning electron microscope after isolation of bloodborne monocytes by centrifugation at purified monocytes at a concentration of one times 10 to the six cells per milliliter to DMEM supplemented with the following additives. Then plate two times 10, the six cells per well in six well plates within a humidified atmosphere, supplemented with 5%carbon dioxide and a culture for seven days in order to allow monocytes to differentiate into macrophages to analyze cell uptake and collection. Mix nano art with culture media to a final concentration of 100 micromolar and add one milliliter of treatment medium to each well at the desired time point or when cells are fully loaded with nano art.

Remove all treatment medium and wash the cells three times with one milliliter of PBS to remove any particles that have not been taken in by the cells in one milliliter of PBS. Remove adherence cells from the bottom of the well by using a cell lifter and place them into a 1.7 milliliter micro centrifuge tube. Now centrifuge the cells at 1000 RCF at four degrees Celsius for 10 minutes.

Remove the supinate and add 200 microliters of 100%Methanol lies the cells and dissolve nano art by briefly sonic the pellet with a sonic probe at 20%amplitude for two to five seconds. Store the samples in minus 80 degrees Celsius until ready to analyze drug content. The intracellular retention of nano art can also be visualized using transmission electron microscopy after sections are prepared by a microtome as seen here.

See the accompanying text to this article for details to analyze nano arch release treat cells as previously described for analyzing cell uptake and collection. However, instead of collecting cells after washing, replace PBS with two milliliters of cell culture medium without MCSF and without nano art on indicated days or as indicated by the media turning yellow exchange half of the medium at the desired day. Collect one milliliter of the cell medium along with replicate cells and process for drug analysis.

To visualize realtime uptake of nano art bifocal microscopy, first take a DRE such as vibrant and add it to the medium Following the manufacturer's instructions, add the dye containing medium to the mature monocyte derived macrophages, and then rinse the cells three times using one milliliter of culture medium to remove any excess dye. Now mix nano art with culture medium using fluorescently labeled nano art and add treatment medium with labeled nano art. Immediately prior to imaging with a confocal microscope, capture an image every 30 seconds for four hours using a 60 times objective.

Finally, HIV one infection and infectivity assays can be performed. First treat cells as described for visualizing art release. Then on desired days, remove all medium and replace with medium without MCSF containing HIV one a A at a multiplicity of infection of 0.01 viral particles per cell and incubate for 24 hours.

Then remove the viral medium and replace with fresh virus, free media culture cells for 10 days, exchanging half the medium every other day or as necessary to keep the cells alive 10 days post-infection. Collect 10 microliters of medium from each well placed into a 96 well plate and store at minus 80 degrees Celsius until ready to perform reverse transcriptase analysis. Subsequent HIV one P 24 detections can also be performed.

Here we can readily see the morphology of cells exposed to virus at a multiplicative infection of 0.011 day after nano art treatment. In these monocyte derived macrophages, there is no evidence of viral infection and the nanoparticles are readily visible within the cell cytoplasm. Here, cells were exposed to virus 10 days after nano art treatment.

Some of these cells were infected as indicated by P 24 staining and multinucleated giant cell formation, but there was still significant protection as compared to untreated cells. Note that nanoparticles are still visible within the cytoplasm of cells in B, but not as much as was seen in a, these structures are not present in cells that were not treated with nano art. In these live cell confocal videos, we see macrophages labeled in green and nano art labeled in red.

Here we can see that the cells accumulate very few nanoparticles. Here they rapidly accumulate particles as indicated by increased red fluorescence. These differences parallel the findings that are seen when analyzing drug uptake Using HPLC, in addition, we can see the cells actively take up the particles.

The difference between rates of cellular nano art accumulation reflects the effects of physical characteristics such as size, shape, and charge on the abilities of macrophages to uptake and retain nano art. After watching this video, you should have a good understanding of how to manufacture nano art using wet milling homogenization and sonication techniques. These are effective methods to manufacture antiretroviral nanoparticles.

Wet milling and homogenization serve to fractionate larger drug crystal particles into smaller nanoparticles within lipid surfactants, and a sonication technique produces individual nano size drug droplets within a polymer. Here we've also demonstrated how to use human monocyte derived macrophages as a testing platform to assess nano art transport. Our method for testing allows for a very simple screening of a particle's ability to be taken up, retained and released by cells, as well as their capacity to inhibit viral replication.

This allows the researcher to identify nano art that may have the best chance of succeeding in vivo and ultimately for use in infected humans. Complete results of these experiments are published on in our article Online Nano. The article is named Nano Formulated Antiretroviral Drug Combinations Extend Drug Release and Antiretroviral Responses in HIV V one Infected Macrophages, implications for Neuro AIDS Therapeutics in the Journal of Neuro Immune Pharmacology 2010.

Thank you very much.