The work was supported by grants 1P01DA028555-01A1, 2R01 NS034239, 2R37 NS36126, P01 NS31492, P20RR 15635, P01MH64570, and P01 NS43985 (to H.E.G.) from the National Institutes of Health. The authors thank Ms. Robin Taylor for critical reading of the manuscript and outstanding graphic and literary support. We would like to thank Steve Grzelak for his wet-milling expertise. We would also like to thank Dr. Han Chen of the University of Nebraska-Lincoln electron microscopy core facility for supplying the scanning and transmission electron microscopy images. Finally, we would like to thank Megan Marquart for her expertise using live confocal microscopy.

<ol>
	<li>Gendelman, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+isolation+and+propagation+of+human+immunodeficiency+virus+on+recombinant+colony-stimulating+factor+1-treated+monocytes.">Efficient isolation and propagation of human immunodeficiency virus on recombinant colony-stimulating factor 1-treated monocytes.</a> J Exp Med. 167 (4), 1428-1441 (1988).</li><li>Dou, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+macrophage-based+nanoparticle+platform+for+antiretroviral+drug+delivery.">Development of a macrophage-based nanoparticle platform for antiretroviral drug delivery.</a> Blood. 108 (8), 2827-2835 (2006).</li><li>Dou, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+investigations+for+the+morphologic,+pharmacokinetic,+and+anti-retroviral+properties+of+indinavir+nanoparticles+in+human+monocyte-derived+macrophages.">Laboratory investigations for the morphologic, pharmacokinetic, and anti-retroviral properties of indinavir nanoparticles in human monocyte-derived macrophages.</a> Virology. 358, 148-158 (2007).</li><li>Dou, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophage+delivery+of+nanoformulated+antiretroviral+drug+to+the+brain+in+a+murine+model+of+neuroAIDS.">Macrophage delivery of nanoformulated antiretroviral drug to the brain in a murine model of neuroAIDS.</a> J Immunol. 183 (1), 661-669 (2009).</li><li>Nowacek, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoformulated+Antiretroviral+Drug+Combinations+Extend+Drug+Release+and+Antiretroviral+Responses+in+HIV-1-Infected+Macrophages:+Implications+for+NeuroAIDS+Therapeutics.">Nanoformulated Antiretroviral Drug Combinations Extend Drug Release and Antiretroviral Responses in HIV-1-Infected Macrophages: Implications for NeuroAIDS Therapeutics.</a> J Neuroimmune Pharmacol. , (2010).</li><li>Nowacek, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NanoART+synthesis,+characterization,+uptake,+release+and+toxicology+for+human+monocyte-macrophage+drug+delivery.">NanoART synthesis, characterization, uptake, release and toxicology for human monocyte-macrophage drug delivery.</a> Nanomed. 4 (8), 903-917 (2009).</li><li>Kalter, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+HIV+replication+in+macrophage+colony-stimulating+factor-treated+monocytes.">Enhanced HIV replication in macrophage colony-stimulating factor-treated monocytes.</a> J Immunol. 146 (1), 298-306 (1991).</li></ol>

No conflicts of interest declared.

NanoART are designed to improve HIV-1 therapeutics. We have proposed a monocyte-macrophage based drug delivery system as a first step to develop such technologies for clinical use and as a laboratory testing system for nanoformulated drug development. Our past works have shown that the system is viable for such an application5,6.
In the current report, we have illustrated methods for synthesizing NP of commonly used HIV-1 medications (indinavir, IDV; ritonovir, RTV; efavirenz, EFV and atazanavir, ATV). This was achieved by wet milling, homogenization and ultrasonication. Importantly, our approach allows for modifications of nanoART physical characteristics such as size, shape and charge 5,6. By careful selection of surfactants, one can control the charge of the particle from highly negative to neutral to highly positive. By altering the processing time and intensity, one can control the size and render particles as small as &le; 200 nm or as large as &ge; 3 &mu;m. The shape of the particle can also be controlled, but to a lesser extent than the other physical characteristics. For example, spherical particles can be made via sonication with polymers while multi-sided geometric shapes can be produced via wet milling or homogenization. Wet milling and homogenization produce differently shaped particles, but this process is dependent on the fractionation method and cannot be directly controlled for. These properties of the manufacturing methods can enable researches to easily produce nanoART to exact design specifications. This is vitally important since the physicochemical properties of nanoART have a powerful effect upon both their stability and how they interact with cells. For example, we have shown that nanoART that are approximately 1 &mu;m in size, have a strong positive charge, and have a multisided geometric shape are more rapidly taken up by macrophages, more stable once inside the cells, and released over a longer period of time 6.
A method for testing nanoART was developed that allows for simple screening of the particles' ability to be taken up, retained, and released by macrophages, one of the principle target cells of HIV-1, in addition to their capacity to inhibit viral replication. This allows one to easily differentiate nanoART based on performance and identify those that have the best chance of succeeding in an in vivo model, thus, increasing both the efficiency and speed of developing nanoART for human use.
It has been shown that mouse bone marrow macrophages when loaded ex vivo with nanoART and adoptively transferred by intravenous injection into humanized HIV-1 infected mice can travel to sites of active infection, including the central nervous system, and release drugs for up to 2 weeks to inhibit viral replication2-4. In addition, these nanoART loaded cells were also able to effectively reduce the numbers of virus-infected cells in plasma, lymph nodes, spleen, liver and lung as well as protect CD4+ cells2. These studies, although preliminary, demonstrate that a cell-mediated nanoART delivery system can distribute clinically significant amounts of drug to both blood and infected tissues. Because of the encouraging preliminary results from HIV-1 infected mouse studies, we are currently developing a simian immuno deficiency virus infected macaque model to further test the potential of nanoART for clinical use.

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>nanoART Manufacture</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Indinavir sulfate</td>
<td>Reagent</td>
<td>Longshem Co., Shanghai, China</td>
<td>Cas # 157810-81-6</td>
<td>&nbsp;</td>
<tr>
<td>Ritonavir</td>
<td>Reagent</td>
<td>China Shengda Pharmaceutical Company, China</td>
<td>Cas # 155213-67-5</td>
<td>&nbsp;</td>
<tr>
<td>Atazanavir sulfate</td>
<td>Reagent</td>
<td>Gyma Laboratories of America INC</td>
<td>Cas # 198904-31-3</td>
<td>&nbsp;</td>
<tr>
<td>Effavirenz</td>
<td>Reagent</td>
<td>Hetero Labs LTD, India</td>
<td>Cas # 154598-52-4</td>
<td>&nbsp;</td>
<tr>
<td>PVA</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>P 8136</td>
<td>&nbsp;</td>
<tr>
<td>SDS</td>
<td>Reagent</td>
<td>Bio-Rad</td>
<td>161-0301</td>
<td>&nbsp;</td>
<tr>
<td>Sucrose</td>
<td>Reagent</td>
<td>Fluka Analytical </td>
<td>84097</td>
<td>&nbsp;</td>
<tr>
<td>P188</td>
<td>Reagent</td>
<td>Sigma Life Science</td>
<td>P 1300</td>
<td>&nbsp;</td>
<tr>
<td>mPEG 2000-DSPE</td>
<td>Reagent</td>
<td>Genzyme </td>
<td>LP-R4-039</td>
<td>&nbsp;</td>
<tr>
<td>Dotap</td>
<td>Reagent</td>
<td>Avanti Polar Lipid, Inc</td>
<td>890890 P</td>
<td>&nbsp;</td>
<tr>
<td>Hepes</td>
<td>Reagent</td>
<td>Sigma Life Science</td>
<td>83264</td>
<td>&nbsp;</td>
<tr>
<td>PLGA 75:25</td>
<td>Reagent</td>
<td>Sigma Life Science </td>
<td>P1941</td>
<td>&nbsp;</td>
<tr>
<td>Dichloromethane</td>
<td>Reagent</td>
<td>Acros</td>
<td>75-09-2</td>
<td>&nbsp;</td>
<tr>
<td>Mannitol</td>
<td>Reagent</td>
<td>Sigma Aldrich</td>
<td>M9546</td>
<td>&nbsp;</td>
<tr>
<td>CTAB</td>
<td>Reagent</td>
<td>Sigma Aldrich</td>
<td>H 9151</td>
<td>&nbsp;</td>
<tr>
<td>Analytical Balance</td>
<td>Tool</td>
<td>Denver Instrument</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>T-18 Mixer</td>
<td>Tool</td>
<td>IKA T 18 basic Ultra-Turrax </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Ultra Sonicator</td>
<td>Tool</td>
<td>Cole Parmer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Wet milling, MicroCer</td>
<td>Tool</td>
<td>Netzsch Fine Particle Technology, LLC</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Homogenizer, EmulsiFlex-C5</td>
<td>Tool</td>
<td>Avestin, Inc.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Zetapotentiometer</td>
<td>Tool</td>
<td>Malvern Instruments</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Gas Manifold System</td>
<td>Tool</td>
<td>Victor 200 Series </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>In Vitro Testing</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>DMEM</td>
<td>Reagent</td>
<td>Mediatech</td>
<td>10-013-CV</td>
<td>&nbsp;</td>
<tr>
<td>Gentamicin</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>15710-064</td>
<td>&nbsp;</td>
<tr>
<td>Ciprofloxin</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>17850</td>
<td>&nbsp;</td>
<tr>
<td>Human MCSF</td>
<td>Reagent</td>
<td>Miltenyi Biotech</td>
<td>130-093-964</td>
<td>&nbsp;</td>
<tr>
<td>Pooled normal human serum</td>
<td>Reagent</td>
<td>Cole-Parmer</td>
<td>WU-88061-68</td>
<td>&nbsp;</td>
<tr>
<td>6-well tissue culture plates</td>
<td>Tool</td>
<td>Corning Life Sciences</td>
<td>3524</td>
<td>&nbsp;</td>
<tr>
<td>Methanol (HPLC Grade)</td>
<td>Reagent</td>
<td>Fisher Scientific</td>
<td>A452SK-4</td>
<td>&nbsp;</td>
<tr>
<td>1.7 ml microcentrifuge tubes</td>
<td>Tool</td>
<td>National Scientific</td>
<td>CN1700GT</td>
<td>&nbsp;</td>
<tr>
<td>Glutaraldehyde solution</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>49632</td>
<td>&nbsp;</td>
<tr>
<td>Osmium tetroxide solution</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>75632</td>
<td>&nbsp;</td>
<tr>
<td>Centrifuge 5417R</td>
<td>Tool</td>
<td>Eppendorf</td>
<td>022621807</td>
<td>&nbsp;</td>
<tr>
<td>F-45-30-11 fixed angle rotor</td>
<td>Tool</td>
<td>Eppendorf</td>
<td>022636006</td>
<td>&nbsp;</td>
<tr>
<td>HIV-1ADA</td>
<td>Reagent</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>HIV-1 p24 mouse monoclonal antibody</td>
<td>Reagent</td>
<td>Dako</td>
<td>M0857</td>
<td>&nbsp;</td>
<tr>
<td>Microscope slide cover slips</td>
<td>Tool</td>
<td>Fisher Scientific</td>
<td>12-548-5P</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>
			<div>
			Abbreviations used in the table: IDV: indinavir; RTV: ritonavir; ATV: atazanavir; EFV: effavirenz; PVA: polyvinylalcohol; SDS sodium dodecyl sulfate; P188: poloxamer 188 (also termed Pluronic F68); mPEG 2000-DSPE: methyl-poly(ethylene-glycol)1,2-distearoyl-phosphatidyl-ethanolamine; DOTAP: (1-oleoyl-2-[6-[(7-nitro-2&#150;1,3-benzoxadiazol-4-yl) amino]hexanoyl]-3-trimethylammonium propane); PLGA: poly(lactic-co-glycolic acid); CTAB: cetyltrimethyl ammonium bromide; DMEM: Dulbecco's Modified Eagle Medium; MCSF: Macrophage Colony-Stimulating Factor; HPLC: high-pressure liquid chromatography; HIV-1: Human Immunodeficiency Virus-1 		</div>

methods development for blood borne macrophage carriage of nanoformulated antiretroviral drugs

Nanoformulated drugs can improve pharmacodynamics and bioavailability while serving also to reduce drug toxicities for antiretroviral (ART) medicines. To this end, our laboratory has applied the principles of nanomedicine to simplify ART regimens and as such reduce toxicities while improving compliance and drug pharmacokinetics. Simple and reliable methods for manufacturing nanoformulated ART (nanoART) are shown. Particles of pure drug are encapsulated by a thin layer of surfactant lipid coating and produced by fractionating larger drug crystals into smaller ones by either wet milling or high-pressure homogenization. In an alternative method free drug is suspended in a droplet of a polymer. Herein, drug is dissolved within a polymer then agitated by ultrasonication until individual nanosized droplets are formed. Dynamic light scattering and microscopic examination characterize the physical properties of the particles (particle size, charge and shape). Their biologic properties (cell uptake and retention, cytotoxicity and antiretroviral efficacy) are determined with human monocyte-derived macrophages (MDM). MDM are derived from human peripheral blood monocytes isolated from leukopacks using centrifugal elutriation for purification. Such blood-borne macrophages may be used as cellular transporters for nanoART distribution to human immunodeficiency virus (HIV) infected organs. We posit that the repackaging of clinically available antiretroviral medications into nanoparticles for HIV-1 treatments may improve compliance and positively affect disease outcomes.

Watch this Scientific Journal Video about Methods Development for Blood Borne Macrophage Carriage of Nanoformulated Antiretroviral Drugs at JoVE.com

Methods Development for Blood Borne Macrophage Carriage of Nanoformulated Antiretroviral Drugs

Nanoparticles of indinavir, ritonavir, efavirenz and atazanavir were manufactured using wet milling, homogenization and ultrasonication. These nanoformulations, collectively termed nanoformulated antiretroviral therapy (nanoART), assessed macrophage-based drug delivery. Monocyte-derived macrophage nanoART uptake, retention and sustained release were determined. These preliminary studies suggest the potential of nanoART for clinical use.

Nanoformulated drugs can improve pharmacodynamics and bioavailability while serving also to reduce drug toxicities for antiretroviral (ART) medicines. To ...

methods-development-for-blood-borne-macrophage-carriage

Research

JoVE Journal

Bioengineering

13.1K Views.  University of Nebraska Medical Center. Nanoparticles of indinavir, ritonavir, efavirenz and atazanavir were manufactured using wet milling, homogenization and ultrasonication. These nanoformulations, collectively termed nanoformulated antiretroviral therapy (nanoART), assessed macrophage-based drug delivery. Monocyte-derived macrophage nanoART uptake, retention and sustained release were determined. These preliminary studies suggest the potential of nanoART for clinical use.