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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.
Table 1. Physical characteristics of nanoART Table displays potential representative values for the physical characteristics of nanoART formulations manufactured using the indicated surfactants. Values include the mean diameter based upon the intensity of scattered light, the polydispersity index (PDI, an estimate of the distribution of particle size), and the zeta potential values obtained for various nanoformulation samples. 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-2-1,3-benzoxadiazol-4-yl) amino]hexanoyl]-3-trimethylammonium propane.
FIGURE 1. Flow chart summarizing various methods used to manufacture nanoART. Figure 1 summarizes various methods used to manufacture nanoART. The flowchart includes an anticipated time-allotment for each of the critical phases of the respective methods.
FIGURE 2. Representative images of desirable and undesirable NanoART morphology. Scanning electron microscopy analysis (magnification, 15,000X) of RTV nanoART produced by homogenization, wet milling, and sonication on top of a 0.2 μm polycarbonate filtration membrane. Measure bar equals 2.0 μm in all frames. Desirable nanoART consist, on average, of small (≤ 2 μm) self-contained particles with smooth edges that tend to have the same or similar shape. Undesirable nanoART vary greatly in both size and shape and can fuse and/or stick to one another.
FIGURE 3. Images of desirable nanoART solution and of MDM taking up nanoART. Bright field microscopy images (all acquired using a 20x objective) of homogenized RTV-NP and MDM taking up nanoART. After combining 10 μL of RTV-NP solution with 100 μL of PBS on top of a glass cover slip, the particles are visible and resemble sand with only a few of the larger particles being individually identifiable (A). Image of fully differentiated and spindle shaped MDM before nanoART treatment (B). After the cells have taken up nanoART, they become darker and their nuclei become more apparent due to perinuclear distribution of nanoART; however, they still maintain their spindle shaped cell body (C). Once the cells become overloaded with nanoART, the nuclei become obscured; and the cells become rounded, losing their spindle shaped structure and potentially detaching from the bottom of the well (D).
FIGURE 4. Confirmation of cellular incorporation of nanoART into MDM. Transmission electron microscopy (magnification, 15,000x) demonstrates uptake of nanoART into MDM exposed to RTV-NP from homogenization (A), RTV-NP from wet milling (B), RTV-NP from sonication (C), and untreated cells (D). Within the cells, the nanoART should be readily identifiable by their geometric shape. Note the lack of any obvious geometric structures in the control cell (D). An example of each particle has been outlined: red for homogenization (A), blue for wet milling (B), green for sonication (C). Particle structure should resemble that which was seen using SEM. Measure bar equals 5.0 μm in all frames.
FIGURE 5. Diagram of method for testing antiretroviral efficacy of nanoART. In order to test the ability of nanoART to inhibit viral replication MDM must be first treated with nanoART and then exposed to HIV-1ADA. Similar to the ART release studies, MDM are loaded with nanoART and then washed to remove any particles that have not been internalized. NanoART laden MDM are then cultured up to 15 days with one half medium exchange every other day. During this time (generally on days 1, 5, 10, and 15) MDM are challenged with HIV-1ADA. After each viral exposure cells are cultured for another 10 days in order to allow the infection to progress. On day 10 post-infection media samples are collected for RT analysis and cells fixed and stained for p24 antigen. Non-nanoART treated MDM, both infected and uninfected, are also cultured in parallel and tested for presence of p24 antigen and RT activity.
FIGURE 6. Testing of nanoART efficacy in HIV-1 infected MDM by p24 staining. Bright field images (20x objective) of RTV-NP treated MDM 10 days after being challenged with HIV-1ADA. No infection was present when cells were challenged with virus1 day post-nanoART treatment (A); note the presence of NP within the cytoplasm of most cells (A inset indicated by arrows). Infection was present when cells were exposed to virus 10 days after nanoART treatment (B), although much less so than cells not treated with nanoART (D); note the maintained presence of NP in some cells (B inset indicated by arrows), but fewer than at day 1 post-nanoART treatment (A inset). Image of cells that were neither treated with nanoART nor infected (C); note lack of NP within cell cytoplasm (C inset). Image of cells that were not treated with nanoART but exposed to HIV-1ADA (D).
VIDEO 1. Live cell confocal microscopy of poor RTV-NP uptake by MDM. Fluorescent microscopy of MDM labeled green with Vybrant DiO cell-labeling solution and treated with red-labeled RTV-NP. One image was taken every 30 seconds for 4 hour at 60x magnification. Click here to watch video
VIDEO 2. Live cell confocal microscopy of successful RTV-NP uptake by MDM. Fluorescent microscopy of MDM labeled green with Vybrant DiO cell-labeling solution and treated with red-labeled RTV-NP. One image was taken every 30 seconds for 4 hour at 60x magnification. Click here to watch video
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 ≤ 200 nm or as large as ≥ 3 μm. 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 μm 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.
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.