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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
J Control Release. Author manuscript; available in PMC 2012 March 10.
Published in final edited form as:
PMCID: PMC3065529

Analyses of Nanoformulated Antiretroviral Drug Charge, Size, Shape and Content for Uptake, Drug Release and Antiviral Activities in Human Monocyte-Derived Macrophages


Long-term antiretroviral therapy (ART) for human immunodeficiency virus type one (HIV-1) infection shows limitations in pharmacokinetics and biodistribution while inducing metabolic and cytotoxic aberrations. In turn, ART commonly requires complex dosing schedules and leads to the emergence of viral resistance and treatment failures. We posit that the development of nanoformulated ART could preclude such limitations and affect improved clinical outcomes. To this end, we wet-milled 20 nanoparticle formulations of crystalline indinavir, ritonavir, atazanavir, and efavirenz, collectively referred to as “nanoART,” then assessed their performance using a range of physicochemical and biological tests. These tests were based on cell-nanoparticle interactions using monocyte-derived macrophages and their abilities to uptake and release nanoformulated drugs and effect viral replication. We demonstrate that physical characteristics such as particle size, surfactant coating, surface charge, and most importantly shape are predictors of cell uptake and antiretroviral efficacy. These studies bring this line of research a step closer to developing nanoART that can be used in the clinic to affect the course of HIV-1 infection.

Keywords: antiretroviral, nanoparticles, HIV, crystalline, macrophage, monocyte, nanomedicine


The need to improve the bioavailability, pharmacology, cytotoxicities, and interval dosing of antiretroviral medications in the treatment of human immunodeficiency virus (HIV) infection is notable [1-3]. In addition, there is a great need to attenuate viral replication in tissue sanctuaries, specifically the central nervous system (CNS) [4, 5]. One way to achieve such goals is through application of nanoformulated drug delivery approaches [6-9]. Indeed, studies using nanoformulated compounds have served to positively affect the pharmacokinetics and pharmacodynamics of antiretroviral therapy (ART) while simultaneously reducing secondary cellular and tissue toxicities [10-16]. In addition, studies utilizing cell-mediated transport of nanoformulated drugs have shown promise for improving delivery of medications to diseased organs, particularly the central nervous system [13]. The system is based on the capabilities of blood borne macrophages to uptake nanoformulated materials, store them in intracellular compartments, and cross blood vessel walls to deliver drugs to sites of active disease. In addition to their phagocytic, clearance, antigen presentation and secretory functions, macrophages also serve as viral sanctuaries, vehicles for viral transport, and as reservoirs for ongoing HIV-1 replication [17-20]. Recent efforts in our laboratories have focused on developing novel drug delivery systems that utilize monocyte-macrophages for ART delivery for HIV-1 infection [13, 21-23]. Here, nanoformulated drugs are composed of antiretroviral drug crystals and include indinavir (IDV), ritonavir (RTV), atazanavir (ATV), and efavirenz (EFV). For each parental drug, large crystals are fractioned into nanoparticles (NPs) by wet milling in the presence of surfactants. These micro- to nanoformulated antiretroviral drugs are referred to as “nanoART.” Macrophages are then used to uptake nanoART and slowly release them for long periods of time.

In an attempt to improve upon what has already been accomplished, we hypothesized that nanoART could be designed to optimize cell uptake, improve intracellular stability, extend drug release, maintain antiretroviral efficacy, and minimize cellular toxicity within transporting cells. The structure and composition of nanoformulated drugs have important effects on stability, cellular interactions, efficacy and cytotoxicity [24-27]. The current study focused on optimizing monocyte-derived macrophage (MDM) platforms for cell-based delivery of nanoART for therapeutic gains by improving manufacture, characterization and pharmacodynamics. Wet milling was utilized in development because it was previously used to manufacture crystalline nanoparticles of poor water-soluble drugs and can be scaled upwards for clinical use [28, 29].

Materials and methods

Preparation and characterization of nanoART

RTV (Shengda Pharmaceutical Co., Zhejiang, China) and EFV (Hetero Labs LTD., Hyderabad, India) were obtained in free base form. The free bases of IDV sulfate (Longshem Co., Shanghai, China) and ATV sulfate (Gyma Laboratories of America Inc., Westbury, NY) were made using a 1N NaOH solution. The surfactants used in this study were: poloxamer-188 (P188; Sigma-Aldrich, Saint Louis, MO), polyvinyl alcohol (PVA) (Sigma-Aldrich, Saint Louis, MO), 1,2-distearoylphosphatidylethanolamine-methyl-polyethyleneglycol conjugate-2000 (mPEG2000DSPE) (Genzyme Pharmaceuticals LLC., Cambridge, MA), sodium dodecyl sulfate (SDS) (Bio-Rad Laboratories, Hercules, CA), and 1,2-dioleoyloxy-3-trimethylammoniumpropane (DOTAP) (Avanti Polar Lipids INC., Alabaster, AL). For preparation of each nanosuspension, surfactants were suspended in 10 mM HEPES buffer solution (pH 7.8) in the following 5 combinations (weight/volume): (1) 0.5% P188 alone; (2) 0.5% PVA and 0.5% SDS; (3) 0.5% P188 and 0.5% SDS; (4) 0.3 P188 and 0.1% mPEG2000DSPE; and (5) 0.5% P188, 0.2% mPEG2000DSPE, and 0.1% DOTAP. Free base drug (either ATV, EFV, IDV or RTV; 0.6% by weight) was then added to surfactant solutions. The suspension was agitated using an Ultraturrax T-18 rotor-stator mixer until a homogeneous dispersion formed. The mixture was then transferred to a NETZSCH MicroSeries Wet Mill (NETZSCH Premier Technologies, LLC, Exton, PA) along with 50 mL of 0.8 mm grinding media (zirconium ceramic beads). The sample was processed for 30 min to 1 hour at speeds ranging from 600 to 4320 rpm until desired particle size was achieved. For determination of particle size, polydispersity, and surface charge, 20 μl of the nanosuspension was diluted 50-fold with distilled/deionized water and analyzed by dynamic light scattering using a Malvern Zetasizer Nano Series Nano-ZS (Malvern Instruments Inc., Westborough, MA). After the desired size was achieved, samples were centrifuged and the resulting pellet resuspended in the respective surfactant solution along with 9.25% sucrose to adjust tonicity. The final drug concentration was determined using high performance liquid chromatography (HPLC).

Human monocyte isolation and cultivation

Human monocytes, obtained by leukapheresis from HIV-1 and hepatitis seronegative donors were purified by counter-current centrifugal elutriation. Monocytes were cultivated in DMEM with 10% heat-inactivated pooled human serum, 1% glutamine, 50 μg/ml gentamicin, 10 μg/ml ciprofloxacin and 1000 U/ml recombinant human macrophage-colony stimulating factor at a concentration of 1×106 cells/ml at 37°C [30].

Electron microscopy

Cell samples were fixed with 3% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) and further fixed with 1% osmium tetroxide in 0.1 M phosphate buffer (pH 7.4) for 1 hour. The samples were then dehydrated in a graduated ethanol series and embedded in Epon 812 (Electron Microscopic Sciences, Fort Washington, PA) for scanning electron microscopy. For transmission electron microscopy, thin sections (80 nm) were stained with uranyl acetate and lead citrate and observed under a Hitachi H7500-I transmission electron microscope (Hitachi High Technologies America Inc., Schaumburg, IL).

NanoART uptake and release

We used a modified version of a previously published method to study uptake and release of nanoART [23]. After 7 days of differentiation, MDM were treated with 100 M nanoART. Uptake of nanoART was assessed without medium change for 8 h. Adherent MDM were washed with phosphate buffered saline (PBS) and collected by scraping into PBS. Cells were pelleted by centrifugation at 950 × g for 10 min at 4°C. Cell pellets were briefly sonicated in 200 μl of methanol and centrifuged at 20,000 × g for 10 min at 4°C. The methanol extract was stored at −80°C. To study cell retention and release of nanoART, MDM were exposed to 100 μM nanoART for 8 h, washed 3 times with PBS, and fresh nanoART-free media was added. MDM were cultured for 15 days with half medium exchanges every other day. On days 1, 5, 10 and 15 post-nanoART treatment, MDM were collected as described for cell uptake. Both cell extracts and medium were stored at −80° C until HPLC analysis as previously described [22].

Live cell microscopy

MDM were stained using Vybrant DiO cell-labeling solution (Invitrogen Corp., Carlsbad, CA) and viable MDM were identified by green fluorescence. NPs were labeled with lissamine rhodamine B 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt (rDHPE; Invitrogen Corp., Carlsbad, CA) by adding fluorescent phospholipid to the surfactant coating. rDHPE-labeled NPs exhibited a red fluorescence. Based on the amount of tracer added, the number of labeled phospholipid molecules represented a very small fraction of the total coating material and contributed minimally to the thickness of the phospholipid coating. This was confirmed by size measurements that showed no significant differences in the sizes of nanoART formulated with or without rDHPE phospholipid (data not shown). No differences were detected in the uptake or release of drug formulated with the fluorescent phospholipid compared to unlabeled particles (data not shown). Images were captured every 30 sec using a Nikon TE2000-U (Nikon Instruments Inc., Melville, NY) with swept-field confocal microscope, 488 nm (green) and 568 nm (red) laser excitations, and a 60× objective.

NanoART antiretroviral activities

MDM were treated with 100 μM nanoART for 8 h, washed to remove excess drug, and infected with HIV-1ADA at a multiplicity of infection of 0.01 infectious viral particles/cell [30] on days 10 and 15 post-nanoART treatment. Following viral infection, cells were cultured for ten days with half media exchanges every other day. Medium samples were collected on day 10 for measurement of progeny virion production as assayed by reverse transcriptase (RT) activity [31]. Parallel analyses for expression of HIV-1 p24 antigen by infected cells were performed by immunostaining.

Reverse transcriptase activity

Medium samples (10 μl) were mixed with 10 μl of a solution containing 100 mM Tris-HCl (pH 7.9), 300 mM KCl, 10 mM DTT, and 0.1% nonyl phenoxylpolyethoxylethanol-40 (NP-40). The reaction mixture was incubated at 37°C for 15 min. At this time 25 μl of a solution containing 50 mM Tris-HCl (pH 7.9), 150 mM KCl, 5 mM DTT, 15 mM MgCl2, 0.05% NP-40, 10 μg/ml poly(A), 0.250 U/ml oligo d(T)12-18, and 10 μCi/ml 3H-TTP was added to each well and incubated at 37°C for 18 h. Following incubation, 50 μl of ice-cold 10% trichloroacetic acid (TCA) was added to each well, the wells were harvested onto glass fiber filters, and the filters were assessed for 3H-TTP incorporation by β-scintillation spectroscopy [31].


Ten days after HIV-1 infection, cells were fixed with 4% phosphate-buffered paraformaldehyde for 15 min at room temperature (RT). Fixed cells were blocked with 10% BSA w/ 1% Triton X-100 (in PBS) for 30 min at RT and incubated with mouse monoclonal antibodies to HIV-1 p24 (1:100, Dako, Carpinteria, CA) for 3 h. Binding of p24 antibody was detected using a Dako EnVision+ System-HRP labeled polymer anti-mouse secondary antibody and diaminobenzidine staining [22, 23]. Cell nuclei were counterstained with hematoxylin. Images were taken using a Nikon TE300 microscope with a 40× objective.


To determine the effect of nanoART treatment on cell viability, MDM were treated with 100 μM nanoART for 8 h, washed with PBS, and viability assessed using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. No effect on cell viability was observed for any of the formulations at the treatment concentrations used (data not shown).

NanoART efficacy

Area under the curve (AUC) was determined over 8 h of MDM drug uptake, 15 days of MDM drug release (cell and medium levels), and 15 days of antiretroviral efficacy (RT activity in supernatant from HIV-infected MDM). Scoring of uptake and release was calculated as a decade-weighted ratio of each formulation as a function of the best AUC for each parental drug/experimental parameter. Scoring of antiretroviral activity was determined from the decade-weighted ratios of the inverse RT activity AUC for each parental drug. The area under the curve (AUC) was determined from the level of each drug or RT activity as a function of time. Within each parental drug, the nanoformulation that yielded the highest AUC for uptake, cell retention, or release into the medium was scored as 10, while the formulation that yielded the lowest RT activity was scored as 10. The remainder of the formulations within each parental drug group was scored as a proportion to the best score of 10 based on the AUC/AUCbest ratio. The scores from each parameter for each drug nanoformulation were averaged to obtain the mean final score for each formulation. The formulations with mean final scores within the top 2 quartiles of each parental drug group were designated for continued testing (GO), while evaluations for those formulations with means within the lower 2 quartiles were discontinued (NOGO).


Manufacture and characterization of nanoART

The 21 nanoART formulations consisted of nanosized drug crystals of free-base antiretroviral drugs coated with a thin layer of phospholipid surfactant. Five different surfactant combinations were used for each drug for a total of 5 formulations per drug. To determine the effect of size on cell uptake and release and on antiretroviral efficacy, an additional RTV formulation of larger particles was made using the surfactants P188/mPEG2000DSPE. All formulations were characterized based on their physical properties including coating, size, charge and shape. The formulations were of similar size and ranged from 233 nm (IDV formulation M1005) to 423 nm (RTV formulation M2005) with an average size of 309 nm (Table. 1). Particle size distributions were not dissimilar to what is known for liposomal or other nanoformulated drug formulations manufactured via wet milling methods [32, 33]. To estimate uniformity in particle size for each formulation, the polydispersity of each formulation was measured. The polydispersity indexes (PDI) ranged from 0.180 (RTV formulation M2004) to 0.301 (ATV formulation M3004), indicating that while most of the particles were close to the calculated average size, there was a spectrum of sizes within each formulation. The additional RTV-P188/mPEG2000DSPE formulation (M2006) at a size of 540 nm was approximately twice the size of M2002 (265 nm). The zeta potential for each formulation was also determined. The most negatively charged formulations were those that contained P188 and SDS as the surfactants (M1004, M2004, M3004 and M4004). Addition of DOTAP imparted a positive charge to formulations M1003, M2003, M3003 and M4003. The remaining surfactant combinations gave the formulations varying degrees of negative charge. Particle morphology varied depending on drug; however, all formulations of the same drug were of similar shape (Fig. 1). IDV and EFV particles were polygonal-shaped with rough edges. ATV formulations resembled long thin rods with smooth edges, while RTV formulations resembled shorter and thicker rods, with smooth edges. Transmission electron microscopy confirmed intracellular inclusion of nanoART and demonstrated that the structural integrity of the nanoART is retained inside the cells.

NanoART morphology and cellular incorporation of nanoART
Table 1
Physicochemical characteristics of nanoART.

NanoART uptake

After characterizing their physical properties, the formulations were tested for in vitro PK and cellular handling by MDM. Our previous studies of nanoART uptake in MDM showed that > 95% of absolute uptake occurs by 8 h for most nanoART [12, 21-23]. Therefore, all uptake experiments were performed for 8 h, and the amount of drug contained in the cells at that time was considered the maximum. For all IDV formulations, the rate of uptake was similar, and 85% of maximum uptake occurred by 4 h (Fig. 2A). At 8 h cell drug levels ranged from 10.7 to 16.7 μg/106 cells for M1004 and M1002, respectively. For all RTV formulations, the rate of uptake was also similar (Fig. 2B). By 4 h about 80% of maximum uptake had occurred. Maximum levels of RTV in the cells at 8 h ranged from 18.2 to 27.0 g/106 cells for M2005 and M2004, respectively. In contrast to IDV and RTV, the rate of uptake differed among the ATV formulations and cell levels at 4 h ranged from 65% to 90% of maximum (Fig. 2C). Maximum amount of nanoART uptake occurred for all ATV formulations at 8 h. Maximum cell levels of ATV varied widely among the formulations, ranging from 8.6 to 37.1 g/106 cells for M3005 and M3001, respectively. For all EFV formulations, the rate of uptake was similar; and maximum uptake for most occurred by 1 h (Fig. 2D). There was a narrow range of maximum cell levels for EFV formulations, from 0.5 to 1.5 g/106 cells for M4003 and M4005, respectively. Drug uptake was visualized in real-time using live cell confocal imaging (Supplemental Videos 1 and 2). In these experiments green-labeled MDM were treated with red-labeled M3001 or M3005, and an image was taken every 30 sec for 4 h. The resulting videos support the HPLC measurements of drug uptake. MDM accumulated M3001 particles at a much faster rate and in greater amounts than M3005 particles, as indicated by the number of red NP in the cytoplasm. Supplemental Fig. 1 illustrates the AUC for drug concentrations in MDM over 8 h of incubation. AUCs (total drug concentrations measured in μg/106 cells) were evaluated for all nanoART formulations. These values were used for nanoART formulation scoring of uptake in Table 2.

Timecourse of uptake of IDV, RTV, ATV, and EFV nanoART into MDM
Table 2
In vitro analysis and scoring of nanoART formulations based on drug uptake, release, and anti-retroviral activity.

NanoART cellular retention, and release

After an 8-h loading period with the nanoART, MDM were cultured for another 15 days in drug-free medium to study both cellular retention of nanoART and release of drug into the media. Half-media exchanges occurred every other day over the 15 day period to facilitate release of the drug. For all IDV formulations, the profiles for cellular retention and cell release were similar (Fig. 3). Approximately 90% of what was contained within the cells after loading was released within the first 24 h; however, for all IDV formulations, drug levels were low, but detectable within cells through day 15 (Supplemental Table). IDV concentration within the media followed a steady decline from day 1 to day 10 (Fig. 3). For M1001, low but detectable amounts of drug were found in the medium through day 15; however, for all other IDV formulations, IDV was undetectable in the medium by day 15. For all RTV formulations, the profiles for cellular retention and cell release were also similar (Fig. 3). In contrast to IDV, only 20% of RTV contained within the cells after loading was released within the first 24 h, and drug was still detectable within cells for all RTV formulations on day 15 (Supplemental Table). RTV concentration in the medium declined steadily from day 1 to day 15, with levels still exceeding 6 μg/ml on day 15 for all RTV formulations. As for the IDV and RTV formulations, the profiles for cell retention were similar for all ATV formulations (Fig. 3); however, the absolute amount of drug varied depending on the loading level at 8 h. Within the first 24 h, approximately 4 μg/106 cells of ATV were released for all formulations regardless of the initial cell levels following loading. However, after this initial burst of drug release, the cells retained the drug and only small amounts of were released over time. After 15 days, cells loaded with M3001 and M3004 still retained greater than 50% of the initial amount of drug (Supplemental Table). The profiles of drug levels in media following release of ATV from nanoART-laden cells were again nearly identical for all formulations (Fig. 3). By day 5, the content of ATV within the media was greater than 1.5 μg/ml for all formulations except M3005 and remained between 0.25 and 1.1 μg/ml through day 15. For all EFV formulations, the profiles and amounts for both cellular retention and release were also similar (Fig. 3). As observed for IDV formulations, approximately 90% of EFV that was present within the cells at time zero was released within the first 24 h; however on day 15, drug was still detectable within cells for all EFV formulations (Supplemental Table). EFV concentrations within the medium steadily declined from day 1 to day 15 (Fig. 3), with low levels of detectable drug through day 15 for all EFV formulations (Supplemental Table).

Time course of cell retention and release of IDV, RTV, ATV, and EFV nanoART

Antiretroviral efficacy

To determine the effectiveness of nanoART at inhibiting HIV replication, we challenged MDM with HIV-1ADA at 1, 5, 10 and 15 days post-nanoART treatment. After HIV challenge, MDM continued to be cultured and media samples were collected 10 days later for RT analysis. All IDV formulations provided low, but similar antiretroviral efficacy. HIV replication was reduced by approximately 20% when viral challenge occurred on day 15 post-nanoART treatments (Fig. 4). In contrast, all EFV formulations provided nearly full protection against HIV infection through challenge day 15 post-nanoART treatments despite the relatively small amount of drug that remained within the cells. RTV and ATV formulations demonstrated wide spectrums of HIV inhibition. At viral challenge day 15, inhibition ranged from 25% to 60% for the RTV formulations (M2002 and M2004, respectively) and from 20% to 80% for the ATV formulations (M3005 and M3001, respectively). Of interest, RT activity directly correlated with amount of drug retained in the cells for ATV and EFV formulations, with a correlation coefficient of 0.92 for each drug group (data not shown).

Antiretroviral efficacy of nanoART

Expression of HIV-1 p24 antigen was used to verify RT activity and HIV proliferation. For each antiretroviral drug species, the best and worst performing formulations, as determined by uptake, cell retention, drug release and RT activity, were tested for comparison purposes. These formulations were M1004 and M1002 (IDV), M2004 and M2002 (RTV), M3001 and M3005 (ATV), and M4005 and M4003 (EFV). MDM loaded with nanoART were challenged with HIV-1ADA on 1, 5, 10, and 15 days post-nanoART treatment and then tested for the presence of p24 antigen at 10 days post-infection. Empirical evaluation of p24 antigen expression demonstrated a gradual increase of HIV infection over time (indicated by increased brown staining) for all nanoART. However, the best performing formulation of each drug, i.e. M1004, M2004, M3001 and M4005, suppressed the increase in p24 expression to a greater extent than did the worst performing formulation, i.e. M1002, M2002, M3005 and M4003. (Supplemental Fig. 2). Of particular interest, all EFV formulations suppressed viral infection out through challenge day 15. Expression of p24 for all nanoART formulations reflected the level of RT activity.

Scoring system for nanoART

All nanoformulations were evaluated for uptake into and release from MDM, as well as for their anti-retroviral activity in HIV-1-infected MDM. Nanoformulations within each experimental parameter were scored and ranked based on the best performing formulation within each parental drug group (Table 2). Data were ranked based on accumulated scores (Total) and mean final scores. A “Go” decision was given to formulations scoring within the top 2 median quartiles (shaded in green), while a “No Go” designation was given to those scoring in the bottom 2 median quartiles (shaded in red). IDV formulations M1002 and M1005 had the highest mean final scores and thus were given a “Go” decision. For the RTV formulations, the shared mean scores by M2003 and M2005 (7.3) were also the median; thus, only two formulations (M2004 and M2006) were given a “Go” designation. For ATV formulations, M3001 and M3002 were designated “Go.” A clear separation in score, 7.5 vs. 5.1, was observed between the “Go/No Go” ATV formulations. One EFV formulation, M4005, scored the highest for each parameter tested and had a final mean score of 10. The next highest final score for EFV formulations (M4002) was nearly half at 5.1 (M4005). Although the difference in mean final score was substantial for these two formulations, both were given the “Go” decision.


We manufactured, characterized and tested 21 nanoART formulations of 4 antiretroviral drugs to assess nanoART in an MDM in vitro testing system. Drug type, surfactant coating, and shape demonstrated substantive effects on particle uptake, drug release, and antiretroviral responses while those that exerted minor effects were particle charge and size. Surfactant coating varied substantively between drug types. For IDV, RTV and EFV four of the five formulations were tested similarly. The surfactant combination P188/mPEG2000DSPE was designated as a “GO” formulation for all drugs tested with the exception of ATV.

Particle shape had a profound impact on nanoART performance. IDV and EFV particles were rounded with irregular edges and showed diminished cell uptake. In contrast, the RTV and ATV were rod-like in shape, with smooth with regular edges. RTV rods were shorter with smoother corners, while ATV were longer rods with sharper edges. The most effective particle uptake was seen with M3001, an ATV formulation and suggesting that longer rods are taken up most rapidly. These results are consistent with studies that examined the effect of particle shape on phagocytosis kinetics in macrophages and found that spherical particles were taken up more slowly than short rods and that long rods were taken up more rapidly than short rods [26, 34, 35].

One of the most important factors that affected nanoART performance was the chemical nature of the parental drug. All ATV formulations demonstrated good PK but relatively poor antiretroviral efficacy. Furthermore, despite the low uptake of EFV nanoART, they exhibited the best antiretroviral efficacies. Of interest, the solubility of free-base ATV is over 300 times greater than that for the other free-base drugs (ATV: 4-5 mg/ml versus IDV: 15 μg/ml, EFV: 9 μg/ml, or RTV: 1-2 μg/ml) [36, 37] and uptake and release of the ATV nanoformulations appeared to be most-influenced by surfactant coating. NanoART may consist of up to 99% pure drug crystal and as a result, particular antiretroviral drugs may be better suited for MDM cell-mediated delivery than others. When comparing the antiretroviral activity of all nanoART formulations within a single drug group, a good predictor of efficacy is how much drug is contained within the cells. For EFV and ATV nanoART formulations, a strong correlation (0.92) was established between how much drug was contained within the cells and the degree of protection against HIV infection. Cells that contained more drug were provided a greater level of protection, regardless of how much drug was present in the surrounding medium. At days 5 and 15, the amount of drug present in the medium for all drug formulations exceeded EC50 levels for anti-HIV activity reported for a variety of HIV strains and host cell types (1.7-25 nM, EFV; 35-200 nM, RTV; 5-29 nM IDV; 2-5 nM ATV) [38]. Additionally, day 5 medium levels for all drugs were equivalent to therapeutic human plasma levels (1.8-4.1 μg/ml, EFV; 3.5-9.6 μg/ml RTV; 0.15-8.0 μg/ml IDV and 0.3-2.2 μg/ml, ATV [39, 40]. Together these results suggest that nanoART primarily exert their antiretroviral effects inside the cell.

While the amount of nanoART contained within MDM is an important indicator of the degree of protection against HIV-1 infection, it is not the sole determinant. Some of the nanoART drugs were highly efficacious in very small amounts, while others that were present in cells at larger amounts were less efficacious. For example, on day 15, levels of IDV in nanoART treated cells were undetectable; yet, HIV-1 infection was still reduced by approximately 20%. In contrast, the amount of EFV, contained in cells after nanoART treatment was extremely low for all formulations, however, the cells were almost completely protected from HIV infection. In addition, ATV nanoART-treated cells had drug levels more than 1000 times that of EFV nanoART-treated cells, but were still infected with HIV to varying degrees. A possible explanation for this phenomenon is that not all nanoART traffic through the cell in an identical manner and may be stored in different subcellular compartments. If true, this would suggest that location of nanoART within the cell could be as important as how much drug actually enters the cell. For example, if nanoART is co-localized to the same endosomal compartment in which HIV replication is occurring, it may take only a small amount of drug to totally inhibit viral replication. On the other hand, nanoART stored in a separate compartment from where HIV replication is occurring, may be less efficacious even if present in larger amounts. The importance of internal mechanisms, intracellular trafficking, and sub-cellular storage of nanomaterials on their biologic effects has been previously demonstrated [41-43]. In the current study, the two factors that had relatively lesser effect upon nanoART performance were size and charge. Other studies have shown that nanoparticle size can greatly affect function, however, no obvious differences in nanoART performance could be seen in the current study based upon particle size alone [41, 42, 44]. This lack of size effect could be due to the similarity in sizes of our nanoART, which ranged from 233 nm to 423 nm and did not generally vary more than 100 nm. An exception was the comparison of overall performance of M2006 and M2002; both were coated with the same surfactant combination but they differed in size by approximately 2-fold. M2002, which performed the worst overall of the RTV nanoART formulations, was about half the size of M2006, which performed second best. This implies that larger nanoART particles may perform better than smaller ones and parallels our previous findings that suggested larger nanoART (closer to 1 μm in size) may be taken up more efficiently by MDM with extended drug release [23]. Particle charge also had more limited effects on nanoART performance. Most of the particles had a strong negative charge (< −15.0 mV), a few had relatively weak charges (between −15 mV and 0 mV), and a few had strong positive charges (> 20 mV). Others have shown that strongly charged NPs are taken up better than those with weak or neutral charges [45]. The nanoART formulation that performed the best for each drug tested had a strong negative charge, while those with weak negative charges (≤ −8.2) ranked in the bottom two. Positively charged particles tended to be ranked in the middle of their groups. This result suggested strongly charged nanoART perform better than those with a neutral charge [23].

Our prior studies demonstrated that nanoformulated IDV can improve biodistribution and antiretroviral efficacy [12, 13, 21, 23]. Since the introduction of ART, incidences of both mortality and co-morbidities associated with HIV-1 infection have decreased dramatically. However, many limitations associated with ART still remain which prevent full suppression of viral replication in HIV-infected individuals. These limitations include poor PK and biodistribution, life-long treatment, and multiple untoward toxic side effects [46-48]. Since antiretroviral medications are quickly eliminated from the body and do not thoroughly penetrate all organs, dosing schedules tend to be complex and involve large amounts of drug. Patients have difficulty properly following therapy guidelines leading to suboptimal adherence and increased risk of developing viral resistance, which can result in treatment failure and accelerated progression of disease [49]. For HIV-infected patients who also experience psychiatric and mental disorders and/or drug abuse, proper adherence to therapy is even more difficult [50, 51]. Repackaging traditional ART medications into nanoART and using macrophages as transporters offers several advantages for treating HIV-1 infection including: (i) prolonged plasma drug concentrations; (ii) slow and steady drug release; (iii) targeted delivery of drug to sites of active infection; and (iv) reduced toxicity. Our previous work, both in vitro and in vivo, has demonstrated that loading macrophages with nanoART greatly improves biodistribution and efficacy of antiretroviral medications, while simultaneously reducing cytotoxicities [12, 13, 21-23]. We previously described the manufacture of nanoART and testing in an in vitro MDM system. Through these studies, we developed an MDM testing paradigm to establish a platform for nanoART development. We envision in vitro screening of nanoART as the bridge between manufacturing and in vivo testing. In fact, in vivo studies using crystalline antiretroviral NPs have shown potential therapeutic benefit and suggested that upon in vivo administration, these types of NPs are likely taken up by macrophages [52, 53]. In this study the task before us was to thoroughly test this MDM platform by screening a large number of nanoART (> 20) that varied greatly in both physical characteristics and chemical properties. In the process, we hoped to find some specific qualities that affected nanoART function and that could be manipulated through manufacturing to optimize their performance. Ultimately, these manufacturing, characterization and testing systems will serve as a guide for the clinical translation of nanoART for use in infected patients.

Supplementary Material





Acknowledgments and Financial disclosures

The work was supported by the National Institutes of Health grants 1P01 DA028555, 2R01 NS034239, 2R37 NS36126, P01 NS31492, P20RR 15635, P01MH64570, and P01 NS43985 (to H.E.G.) and from a research grant from Baxter Healthcare. The authors thank Ms. Robin Taylor for critical reading of the manuscript and outstanding graphic and literary support. The authors also would like to thank Dr. Han Chen and Dr. You Zhou of the University of Nebraska-Lincoln electron microscopy core facility for supplying the scanning and transmission electron microscopy images. The authors would also like to thank Megan Marquardt and LeAnn Tiede for assistance in acquiring the live-cell confocal microscopy videos.


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