Nanoformulated Antiretroviral Drug Combinations Extend Drug Release and Antiretroviral Responses in HIV-1-Infected Macrophages: Implications for NeuroAIDS Therapeutics
We posit that improvements in pharmacokinetics and biodistributions of antiretroviral therapies (ART) for human immunodeficiency virus type one-infected people can be achieved through nanoformulationed drug delivery systems. To this end, we manufactured nanoparticles of atazanavir, efavirenz, and ritonavir (termed nanoART) and treated human monocyte-derived macrophages (MDM) in combination therapies to assess antiretroviral responses. This resulted in improved drug uptake, release, and antiretroviral efficacy over monotherapy. MDM rapidly, within minutes, ingested nanoART combinations, at equal or similar rates, as individual formulations. Combination nanoART ingested by MDM facilitated individual drug release from 15 to >20 days. These findings are noteworthy as a nanoART cell-mediated drug delivery provides a means to deliver therapeutics to viral sanctuaries, such as the central nervous system during progressive human immunodeficiency virus type one infection. The work brings us yet another step closer to realizing the utility of nanoART for virus-infected people.
Keywordsantiretroviral therapy nanoparticles macrophages drug delivery HIV nanomedicine
Antiretroviral medications are effective at inhibiting viral replication by interrupting the viral life cycle. As a result, antiretroviral therapy (ART) has reduced morbidity and mortality in human immunodeficiency virus type one (HIV-1)-infected people. However, a major limitation of ART is the need for lifelong daily treatment (Garvie et al. 2009; Royal et al. 2009). Suboptimal adherence to daily therapy increases the risk of treatment failure and development of viral resistance and commonly results in accelerated progression of disease (Paterson et al. 2000; Mascolini et al. 2008; Danel et al. 2009; Metzner et al. 2009). Factors such as concomitant drug abuse, psychiatric and mental disorders, and drug side effects often lead to sporadic adherence (Baum et al. 2009; Meade et al. 2009). In this regard, providers are commonly reluctant to prescribe ART to those patients who are poorly compliant because of concerns about the promotion of viral resistance (Bruce et al. 2006a; Bruce et al. 2006b). Complicating matters further are the common cognitive and motor disorders that result as a consequence of HIV-1 infection (Epstein and Gendelman 1993; Kraft-Terry et al. 2009). These risk factors, taken together, often result in poor treatment outcomes (Murri et al. 2006). Means to overcome such concerns are being developed in our laboratories. Indeed, slow release nanoformulated ART, termed nanoART, could travel to sites of infection and slowly release drug(s) with limited tissue toxicities (Kabanov and Gendelman 2007; Nowacek and Gendelman 2009). Such a drug delivery system, if realized, could revolutionize ART and improve outcomes, particularly those within the central nervous system (CNS).
To this end, we synthesized nanoART that are carried within circulating monocyte–macrophages, delivered to virus-target tissues, and released in time periods measured in weeks (Dou et al. 2006, 2007, 2009; Nowacek et al. 2009b). Unfortunately, there are a number of obstacles that need to be overcome before nanoART can be moved from the bench to the bedside. These include optimizing cellular uptake, preventing drug metabolism and degradation, and reducing untoward side effects. In an effort to overcome these obstacles, we used homogenization and sonication techniques to process poorly water-soluble drugs for nanoART manufacture. Previously, we demonstrated that cellular handling of antiretroviral drugs could be accomplished by altering the physical properties of nanoparticles (NP) (Nowacek et al. 2009b). NanoART-treated monocyte-derived macrophages (MDM) could protect against HIV-1 infection for up to 15 days (Nowacek et al. 2009b). However, these studies were performed using single drug formulations. We posit that by using nanoART combination therapy, we could extend the drug release and improve viral inhibition. To accomplish this, we tested nanoformulations of atazanavir (ATV), ritonavir (RTV), and efavirenz (EFV) in combination in MDM cultures. Our results show that MDM can simultaneously uptake three nanoART formulations without a reduction in individual drug levels. In addition, these additions extended the time of drug release and improved antiretroviral efficacy.
These data strongly suggest that the effects of nanoART could be enhanced by combination therapy. The data also implies that cell-mediated nanoART delivery can control HIV-1 infection. The use of such technology could both impact the administration of ART and help to eliminate virus from its sanctuaries, such as the CNS, where migration of MDM is a major mechanism for viral dissemination (Kraft-Terry et al. 2009). It may also provide a depot of drug for extended time periods.
Materials and methods
Preparation and characterization of nanoART
NanoART of ATV and RTV were prepared by high-pressure homogenization using an Avestin C-5 homogenizer (Avestin, Inc., Ottawa, Ontario, Canada) as previously described, (Nowacek et al. 2009b). Surfactants used to coat the drug crystals included a block copolymer of ethylene oxide and propylene oxide, Poloxamer 188 (P-188; Spectrum Chemicals, Gardena, CA), 1,2-distearoyl-phosphatidyl-ethanolamine-methyl-poly-ethylene-glycol (DSPE-mPEG2000; Genzyme, Cambridge, MA), and 1-oleoyl-2-[6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl]-3-trimethylammonium propane (DOTAP; Genzyme). To coat the nanosized drug crystals, each surfactant was made up (weight/weight percent) of P-188 (0.5%), mPEG2000-DSPE (0.2%), and DOTAP (0.1%). The nanosuspensions were formulated at a slightly alkaline pH of 7.8 using either 10 mM sodium phosphate or 10 mM HEPES as a buffer. Tonicity was adjusted with glycerin (2.25%) or sucrose (9.25%). Freebase drug was added to the surfactant solution to make a concentration of approximately 2% (weight to volume ratio (percent)). Lissamine rhodamine B 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt (Ex 560 nm/Em 580 nm; Invitrogen, Carlsbad, CA) was used to label ATV-H1045, which appeared as red fluorescence. Vybrant DiO cell-labeling solution (Ex 484 nm/Em 501 nm, Invitrogen) was used to label RTV-H1025, which appeared as green fluorescence. In order to synthesize nanoART, a suspension was prepared by adding crystalline drug to a surfactant solution and mixing for 4–7 min using an Ultra-Turrax T-18 (IKA® Works Inc., Wilmington, NC) rotor-stator mixer to reduce initial particle size. The suspension was homogenized at 20,000 psi for approximately 30 passes or until desired particle size was reached.
For preparation of EFV-P1044, drug freebase was suspended within nanosized droplets of poly(lactic-co-glycolic acid) (PLGA; ratio 50:50 of lactide to glycolide; Sigma-Aldrich, St. Louis, MO) and cetyltrimethyl ammonium bromide (CTAB; Sigma-Aldrich). EFV (1.25 g), PLGA (6.0 g), and CTAB (0.50 g) were dissolved in dichloromethane (50 mL) and added to a 1% poly(vinyl) alcohol solution (500 mL). Particle size was achieved by sonicating at 50% amplitude for 10 min using a 400/600-W sonicator with 3/4-inch high gain probe, stirring overnight to evaporate the dichloromethane-hardened particles. The suspension was then centrifuged, washed with distilled/deionized water, and decanted twice. The particles were suspended in 10% mannitol before being frozen or lyophilized for storage. Vybrant DiD cell-labeling solution (Ex 644 nm/Em 665 nm; Invitrogen) was used to label EFV-P1044, which appeared as far-red fluorescence. For all nanosuspensions, particle size was measured using a HORIBA LA 920 light scattering instrument (HORIBA Instruments Inc., Irvine, CA). Zeta potential was measured by diluting 0.1 ml of the suspension into 9.9 ml of 10 mM HEPES, pH 7.4 on a Malvern Zetasizer Nano series instrument (Malvern Instruments Inc., Westborough, MA). Final drug content of the formulations was determined by reversed phase high-performance liquid chromatography (HPLC; data not shown).
Human monocyte isolation and cultivation
Human monocytes were obtained by leukapheresis from HIV-1 and 2 and hepatitis seronegative donors and were purified by countercurrent centrifugal elutriation (Gendelman et al. 1988). As previously described (Nowacek et al. 2009b), monocytes were cultured at a concentration of 1 × 106 cells/ml in the presence of recombinant human macrophage colony stimulating factor (a generous gift of Wyeth Inc., Cambridge, MA) to induce differentiation to macrophages.
NanoART uptake and release
MDM (2 × 106 cells per well) were cultured with nanoART at a concentration of 100 µM for each nanoformulation alone or when used in combination. Therefore, cells exposed to a combination of the nanoformulations were treated with a total of 300 µM of drug (100 µM of each) within the medium. Uptake of nanoART was assessed without medium change for 24 h with cell collection occurring at indicated times points. Release of drug(s) into surrounding medium from nanoART-treated cells was also evaluated. After an initial 12-h exposure to nanoART, MDM were washed three times with phosphate-buffered saline (PBS), given drug-free medium, and underwent a half media exchange every other day for 20 days. Adherent MDM and corresponding medium samples were collected every other day, stored at −80°C, and processed for HPLC. Prepared samples were assessed by HPLC using triplicate 20-µl injections onto a YMC Octyl C8 column (Waters Inc., Milford, MA) with a C8 guard cartridge. Mobile phase consisting of 48% acetonitrile/52% 25 mM KH2PO4, pH 4.15, was pumped at 0.4 ml/min with UV/Vis detection at 272 nm. For all antiretroviral drugs, quantitations were determined by comparison to a standard curve of each free drug (0.025–100 µg/ml) prepared in methanol.
Samples were fixed by 3% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) and further fixed in 1% osmium tetroxide in 0.1 M phosphate buffer (pH 7.4) for 1 h. Samples were dehydrated in a graduated ethanol series and embedded in Epon 812 (Electron Microscopic Sciences, Fort Washington, PA) for scanning electron microscopy. Thin sections (80 nm) were stained with uranyl acetate and lead citrate and observed under a transmission electron microscope (Hitachi H7500-I) (Hitachi High Technologies America Inc., Schaumburg, IL) for transmission electron microscopy.
NP were labeled with fluorescent phospholipids as described above. Unlabeled MDM were then treated with fluorescent NP for 4 h, washed three times with PBS, and were imaged using a Nikon TE2000-U (Nikon Instruments Inc., Melville, NY) with Swept Field Confocal Microscope, 488- (green), 568- (red), and 633-nm (color) laser excitations, and a 60× objective.
Antiretroviral activities of nanoART
MDM were treated with nanoART for 12 h, washed to remove excess drug, and infected with HIV-1ADA at a multiplicity of infection (MOI) of 0.01 infectious viral particles per cell (Gendelman et al. 1988) on days 5, 10, 15, and 20 after nanoART treatment. Following viral infection, cells were cultured for 10 days with half media exchanges every other day. Culture fluids were collected 10 days after infection for measurements of progeny virion production as assayed by reverse transcriptase (RT) activity (Kalter et al. 1991). Parallel analyses for expression of HIV-1 p24 antigen in infected cells were performed by immunostaining on the same day as culture fluid sampling.
In a 96-well plate, media 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, 0.1% nonyl phenoxylpoly-ethoxylethanol-40 (NP-40), and water. The reaction mixture was incubated at 37°C for 15 min, and 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. Plates were incubated at 37°C for 18 h. Following incubation, 50 µl of cold 10% trichloroacetic acid 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 using a TopCount NXT (PerkinElmer Inc., Waltham, MA; Kalter et al. 1991).
Cells were fixed with 4% phosphate-buffered paraformaldehyde 10 days after HIV-1 infection. Mouse monoclonal antibodies to HIV-1 p24 (1:10, Dako, Carpinteria, CA) were used to visualize p24. Images of cells were acquired using a Nikon TE300 (Nikon, San Diego, CA) and a 40× objective.
To determine any potential toxic effects of NP on cells, MDM were treated with NP for 12 h at the same concentrations as used in all other studies, and cytotoxicity was assessed 24 h later using a mitochondrial 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Mosmann 1983).
All data analyses were carried out using Prism (GraphPad Software, Inc., La Jolla, CA). Significant differences in drug levels in uptake and release studies were determined by two-way analysis of variance (ANOVA) followed by Bonferroni’s multiple comparison test. Significant differences in RT activity were determined by one-way ANOVA followed by Bonferroni’s multiple comparison test. A p value of ≤0.05 was considered significant.
Manufacture and characterization of nanoART
Physicochemical characteristics of nanoformulated ART drugs
P188, mPEG2000-DSPE, DOTAP
PLGA, PVA, CTAB
P188, mPEG2000-DSPE, DOTAP
NP uptake and antiretroviral drug release
For release studies, the culture medium was replaced at 12 h with fresh medium without nanoART, and drug release into the medium was determined up to 20 days by HPLC. In both medium and cells, there was a significant (p < 0.05) difference in the release of drug between a nanoART formulation used alone or in combination. Drug retention within MDM was significantly (p < 0.05) greater as early as day 5 post nanoART treatment for both ATV and EFV when used in combination compared to when used alone (Fig. 3). Drug concentrations within MDM were significantly (p < 0.05) greater for all combined ART treatments 15 days after drug removal and remained so for up to 20 days when compared to individual nanoART treatments (Fig. 3). In the case of EFV-P1044, no drug was detectable in MDM 20 days after treatment when cells were loaded with the nanoformulation alone; however, detectable levels of EFV were observed in the cells treated with the nanoART combo (Fig. 3). In the media, drug was still present at day 20 for all treatments (Fig. 3). However, drug levels within the medium were 1.5- to twofold higher when nanoART were used in combination compared to nanoART used individually (Fig. 3).
Antiretroviral efficacy of combination and individual nanoART therapies
Finding ways to effectively control HIV-1 infection remains a singular goal for ART. While current antiretroviral drugs reduce morbidity and mortality associated with viral infection, there are limitations that prevent ART from eradicating the virus in infected people. Indeed, a combination of drug-related biochemical, physiological, as well as human factors, continue to lead to treatment failure in a proportion of patients (Fellay et al. 2001; Hawkins 2006). Notable is the formation of viral sanctuaries, such as the CNS, where ART compounds are unable to access and virus is allowed to replicate albeit at limited levels (Best et al. 2009; Varatharajan and Thomas 2009). First, NanoART provides an important means to overcome this and other limitations for CNS drug delivery. In this report, we show that nanoART can be manufactured and optimized in laboratory models of HIV infection. Second, combinations of nanoART can improve antiretroviral efficacy without cytotoxicity. Viral breakthrough was seen 20 days after monotherapy for each drug while combination therapy still provided total viral inhibition at 20 days. The improvement in antiretroviral activities with combination therapy could potentially be due to the presence of more drugs and to having drugs with different mechanisms of action. Third, the particles were located within the same subcellular compartments. Interestingly, there was no reduction in the absolute amount of each drug taken up when cells were treated with combo nanoART compared to when each was used alone. Fourth, this work serves to bridge animal studies being developed to assess safety and efficacy with the final goal being human clinical trials. The fact that MDM are able to take up all three nanoART formulations simultaneously provides an important step in this direction. Also, that multiple nanoARTs can be loaded simultaneously and that uptake is not competitive among formulations support its utility in an infected human host.
The cell-based delivery system developed in the current report is based on monocyte–macrophage function and viral dissemination in an infected human host. One focus of such infection is the brain. Indeed, HIV infection of the brain can result in both functional and cognitive deficits, a condition known as HIV-1-associated neurocognitive disorder (HAND; Gray et al. 2001). Some form of neurological dysfunction is displayed in nearly half of people infected with HIV; this makes HAND a not infrequent cause of dementia worldwide (Valcour and Paul 2006; Ellis et al. 2007; Varatharajan and Thomas 2009). This is primarily due to the inability of ART medications to effectively cross the blood–brain barrier (BBB) or to persist within the parenchyma upon entering the CNS (reviewed by Nowacek et al. 2009b). Successful delivery of antiretroviral drugs to the brain and purging of this viral sanctuary could effectively eliminate this form of dementia.
Our laboratory has focused its research efforts on developing long-acting parenteral drug formulations that can be maintained inside cells for extended periods and travel specifically to viral sanctuaries where ART compounds can be released. In the past studies (Dou et al. 2006, 2007, 2009; Nowacek et al. 2009b), we demonstrated that antiretroviral compounds could be manufactured into stable formulations of nanometer-sized drug crystals coated in surfactant polymers, that the physical properties of these particles could be modified, and that these changes had an effect upon cellular handling. Most importantly, we demonstrated that MDM pretreated with nanoART could release drug and inhibit HIV-1 replication for up to 15 days after treatment. However, in all prior studies, the effect of only one formulation was explored at a time. In the clinic, ART involves the use of multiple drugs of different classes simultaneously. We hypothesized that we could further extend the time of drug release and improve viral inhibition over our previous findings by exposing MDM to a “nanoART cocktail.” The fact that multiple formulations showed substantial improvements over a single nanoART preparation was an unexpected finding, as we believed that each would affect uptake of the others. This was not the case.
In the clinic, ART therapy commonly consists of a drug “cocktail”; this improves pharmacokinetics (PK), more effectively reduces viral loads, and decreases the risk of viral resistance (Zolopa 2009). For example, RTV (a protease inhibitor (PI)) can be used to “boost” serum levels of other PIs, such as ATV (Horberg et al. 2008). In fact, ATV is commonly prescribed in conjunction with a low dose of RTV in order to increase its serum levels (Johnson et al. 2005; Gisslen et al. 2006). A possible addition to ATV–RTV therapy is EFV, a nonnucleoside reverse transcriptase inhibitor. In 2008, the FDA recommended a combination of ATZ with RTV and EFV once daily as a possible treatment in ART-naive patients (Horberg et al. 2008).
In addition, increasing the overall drugs taken up by MDM also extended the time of drug release. A potential explanation for this finding involves intracellular drug metabolism. RTV is a very potent inhibitor of cytochrome P450 enzymes, such as CYP2B6, CYP2D6, CYP3A4/5, and others (Kumar et al. 1999; Hesse et al. 2001; Zeldin and Petruschke 2004; Xu and Desai 2009). In the clinic, RTV is used in ART drug combinations to both increase bioavailability and reduce hepatic clearance of other antiretroviral compounds (Zeldin and Petruschke 2004; Feldt et al. 2005; Gianotti et al. 2007; Xu and Desai 2009). This allows for improved dosing schedules and better viral inhibition (Zeldin and Petruschke 2004; Xu and Desai 2009). The expression of CYP2B6, CYP2D6, and CYP3A4/5 mRNAs have been identified in alveolar macrophages suggesting that macrophages are capable of metabolizing antiretroviral medications (Anttila et al. 1997; Hukkanen et al. 1997, 2002; Raunio et al. 1999; Piipari et al. 2000). Therefore, after nanoART are taken up by MDM, a portion of the drugs may be metabolized by the cells. By including RTV in the nanoART combination, we may be inhibiting nanoART metabolism and improving intracellular stability, thereby extending the time of drug release.
A major challenge for medicine is to improve clinical outcomes for CNS disorders related to aging, infection, and degeneration. These conditions include, but are not limited to, stroke, multiple sclerosis, Parkinson’s disease, Alzheimer’s disease, and HAND. In all cases, disease progression could be slowed, halted, or potentially reversed with targeted drug delivery. Potentially useful medications exist for these diseases; unfortunately, like ART compounds, many are unable to effectively cross the BBB, enter the CNS, and potentiate a positive change (Kabanov and Gendelman 2007; Nowacek et al. 2009a). Many neurologic diseases have an inflammatory component (Perry et al. 1995), thereby allowing immunocytes, such as macrophages, to migrate into sites of inflammation via diapedesis and chemotaxis (Kumagai et al. 1987). Therefore, we anticipate that our model of cell-mediated drug delivery of crystalline NP may be applicable to other major CNS disorders besides HAND.
This work brings us one step closer to realizing the use of nanoART in humans, but much work still needs to be done. In the future, we plan to elucidate mechanisms of nanoART uptake, study NP trafficking, and identify subcellular compartments in which nanoART are stored within MDM. In addition, we plan to investigate in vivo PK, biodistribution, and efficacy upon a single parenteral administration.
The authors thank Janice A. Taylor and James R. Talaska of the Confocal Laser Scanning Microscope Core Facility at the University of Nebraska Medical Center for providing assistance with confocal microscopy, the Nebraska Research Initiative, and the Eppley Cancer Center for their support of the Core Facility. We also thank Ms. Robin Taylor for the critical reading of the manuscript and outstanding graphic and literary support.
Financial and competing interests disclosures
The work was supported by the National Institutes of Health grants 2R01 NS034239, 2R37 NS36126, P01 NS31492, P20RR 15635, P01MH64570, and P01 NS43985 (to H.E.G.) and from a research grant from Baxter Healthcare.
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