Article pubs.acs.org/jpr
Functional Proteome of Macrophage Carried Nanoformulated Antiretroviral Therapy Demonstrates Enhanced Particle Carrying Capacity Andrea L. Martinez-Skinner,† Ram S. Veerubhotla,† Han Liu,† Huangui Xiong,† Fang Yu,‡ JoEllyn M. McMillan,† and Howard E. Gendelman*,†,§ †
Departments of Pharmacology and Experimental Neuroscience, §Internal Medicine, and ‡Biostatistics, University of Nebraska Medical Center, Omaha, Nebraska 68198-5880, United States S Supporting Information *
ABSTRACT: Our laboratory developed long-acting nanoformulations of antiretroviral therapy (nanoART) to improve drug compliance, reduce toxicities, and facilitate access of drug to viral reservoirs. These all function to inevitably improve treatment of human immunodeficiency virus (HIV) infection. Formulations are designed to harness the carrying capacities of mononuclear phagocytes (MP; monocytes and macrophages) and to use these cells as Trojan horses for drug delivery. Such a drug distribution system limits ART metabolism and excretion while facilitating access to viral reservoirs. Our prior works demonstrated a high degree of nanoART sequestration in macrophage recycling endosomes with broad and sustained drug tissue biodistribution and depots with limited untoward systemic toxicities. Despite such benefits, the effects of particle carriage on the cells’ functional capacities remained poorly understood. Thus, we employed pulsed stable isotope labeling of amino acids in cell culture to elucidate the macrophage proteome and assess any alterations in cellular functions that would affect cell−drug carriage and release kinetics. NanoART-MP interactions resulted in the induction of a broad range of activation-related proteins that can enhance phagocytosis, secretory functions, and cell migration. Notably, we now demonstrate that particle−cell interactions serve to enhance drug loading while facilitating drug tissue depots and transportation. KEYWORDS: monocyte-derived macrophages, nanoART, pulsed stable isotope labeling of amino acids in cell culture, proteome, cell function
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INTRODUCTION There is an immediate need to improve antiretroviral therapy (ART) compliance and drug entry into viral reservoirs in the treatment of human immunodeficiency virus (HIV) infection. This is especially noteworthy as improved treatment has the possibility to affect viral eradication of this worldwide epidemic.1−5 To these ends, our laboratory has pioneered long acting nanoformulated ART (nanoART) to improve drug access and enable stable long-lived drug depots.6−10 NanoART may be of particular benefit to patients with limitations in oral drug usage such as malabsorption syndromes or systemic drug toxicities.2,3,11,12 Therefore, a broad range of infected people could benefit from such a therapeutic approach, as nanoART may serve to positively influence drug pharmacokinetics and pharmacodynamics and consequently affect drug entry into HIV reservoirs facilitating the elimination of residual virus.13,14 NanoART is carried in mononuclear phagocytes (MP; monocyte, macrophage); as such, drug-loaded cells “naturally” traffic to lymphoid, gut, brain and other tissue viral reservoirs.15−17 Indeed, nanoART was developed with the goal of optimizing MP storage and release based on particle © 2013 American Chemical Society
size, shape, charge, and coating. This would also best facilitate MP drug depots. Here, the cells serve as Trojan horses for ART entry into the reticuloendothelial system where viral replication is active.18,19 The overarching idea is to build cell-based drug depots and consequently decrease drug metabolism and degradation, while permitting slow drug release at sites of active viral infection for weeks to even months.9,13,14,18 The final effect would lead to monthly drug-dosing intervals while maintaining ART efficacy.9,13,14,18,20 This is facilitated by intracellular nanoART trafficking that parallels virus endocytic sorting.21 We acknowledge that such a laudable goal is not simple. Nonetheless, proof of concept was recently provided by testing nanoART formulations in HIV-1 infected humanized mice. The use of immunodeficient NOD/SCID/IL2r-γnull mice transplanted with human hematopoietic stem cells models chronic HIV infection.13 Our works in this model show that nanoART facilitates drug biodistribution, reduces CD4+ T cell loss, and Received: February 28, 2013 Published: April 1, 2013 2282
dx.doi.org/10.1021/pr400185w | J. Proteome Res. 2013, 12, 2282−2294
Journal of Proteome Research
Article
Zetasizer Nano Series Nano-ZS (Malvern Instruments Inc., Westborough, MA). Once desired characteristics were achieved, samples were centrifuged at 10000× g for 30 min at 4 °C; the resulting pellet was resuspended in surfactant solution containing 9.25% sucrose to adjust tonicity. Final drug concentrations were determined using reverse-phase highperformance liquid chromatography (RP-HPLC).9,10
leads to reductions in viral loads to undetectable levels without demonstrable systemic toxicities. Such findings support nanoART as a viable alternative to oral therapy.13,14 Nonetheless, what remains unrealized is how the drug(s) might reach and eliminate residual virus and what effects the particles themselves might have on the cell carrier. More specifically how the particles affect MP’s functional and migratory capacities needs to be elucidated. To this end, we sought to understand how nanoART cell depots can be established and what long-term toxicities would result. Furthermore, how might this therapeutic approach enhance MP carriage of nanoparticles, bypass lysosomal degradation, and evade host-tissue metabolism? We reasoned that the best means to test such questions is through evaluation of the MP proteome after exposure and carriage of nanoART. This is owed to the fact that proteins participate in virtually every process within the cell governing cellular activities and functions.22 Toward this goal, we used pulsed stable isotope labeling of amino acids in cell culture (pSILAC) to assess changes in de novo protein synthesis in monocyte-derived macrophages (MDMs) following nanoART treatment. This method allows incorporation of isotopically labeled amino acids into newly synthesized proteins that are subsequently identified by mass spectrometry. Previous success with pSILAC was shown in our prior works when macrophage function was assessed during differentiation and viral infection.23,24 pSILAC proved to be an excellent approach when used in tandem with biologic validation to illustrate dynamic nanoART-macrophage interactions. Results show uptake and intracellular distribution of nanoART leads to cellular alterations that serve to facilitate cell trafficking and establishment of macrophage drug depots. These include facilitated phagocytosis, changes in potassium (K+) channel currents, and associated enhanced cell migratory events. The analyses of the dynamic protein differences during particlemacrophage carriage provide new insights into the advantages of combination nanoART (cNanoART) for drug treatments. Perhaps and most importantly, it serves to guide the next stage of investigation toward the translation of long acting ART nanoformulations for treatment of HIV infected people.
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Human Monocyte Isolation and Cultivation
Human monocytes were obtained via leukopheresis from HIV1, HIV-2, and hepatitis seronegative donors then purified by counter-current centrifugal elutriation. Cells were cultured in Dulbecco’s Modified Eagle’s Media (DMEM) containing 10% heat-inactivated pooled human serum, 1% L-glutamine, 50 μg/ mL gentamicin, 10 μg/mL ciprofloxacin and 2 μg/mL recombinant human macrophage colony stimulating factor (MCSF, a gift from Pfizer Inc. Cambridge, MA).25 Three million cells per well were plated on 6-well plates at a density of 1 × 106 cells/mL for 9 days, with respective half and full medium exchanges on days 5 and 7. Drug Treatment and pSILAC Protein Labeling
Media was removed from half of the MDM plates, and the cells were treated with 100 μM of nanoART (ATV-P188 (nanoATV), EFV-P188-mPEG (nanoEFV) and RTV-P188-mPEG (nanoRTV) individually or in combination (cNanoART)), 100 μM of free drug (ATV, EFV, RTV individually or in combination), and 100 μM of an innocuous drug with the P188-mPEG polymer coating in media without MCSF. Media was removed from the remaining plates that served as controls. Plates were incubated for 8 h then washed with 1× phosphate buffered saline (PBS). pSILAC media containing “heavy” (H) and “medium” (M) labels were added to the treated and control plates, respectively. pSILAC media consists of DMEM phenol red free containing low glucose without leucine, lysine, and arginine substituted with isotopically labeled L-arginine and L-lysine. H media is pSILAC medium containing 87.8 mg/mL [13C6,15N4]-L-arginine and 152.1 mg/mL [13C6,15N2]-L-lysine. M media is pSILAC media containing 86.2 mg/mL [13C6]-Larginine and 149.0 mg/mL [D4]-L-lysine. All isotopically labeled amino acids were obtained from SIGMA-Isotec, St. Louis MO.
EXPERIMENTAL PROCEDURES
Cell Lysis and Sample Preparation
NanoART Preparation
After 48 h, cells were washed three times with PBS and collected by the addition of 400 μL lysis buffer (4% sodium dodecyl sulfate, 0.1 M dithiothreitol, 0.1 M Tris HCl, pH 7.6) to the first well then scraping of cells and sequential transfer to the remaining 5 wells. Cell lysates were heated at 95 °C for 3−5 min, briefly sonicated and stored at −80 °C. Protein concentrations were determined using a Pierce 660 assay (Thermo Fisher Scientific, Rockford, IL). Fifty μg of protein from treated cells was combined with equivalent concentrations from control cells. Proteins from lysates were digested using trypsin (Promega, Madison WI) and resulting peptides were cleaned through an MCX 1 cm3 30 mg extraction cartridge (Waters-Oasis, Milford MA). The peptides were separated into 12 fractions by isoelectric focusing using an Agilent 3100 OFFGEL Fractionator Kit pH 3−10 (Agilent Technologies, Santa Clara, CA) and cleaned using Pierce C-18 PepClean Spin Columns (Thermo Fisher) according to the manufacturers’ instructions as previously described.23,24 Peptide samples were dried using a SpeedVac and resuspended in 6 μL 0.1% formic acid for LC-MS/MS analysis.
Atazanavir (ATV)-sulfate was purchased from Gyma Laboratories of America Inc. (Westbury, NY) and the free base form made with a 1N NaOH solution. The free base form of ritonavir (RTV) and efavirenz (EFV) were obtained from Shengda Pharmaceutical Co (Zhejiang, China) and Hetero Laboratories, Ltd. (Hyderabad, India), respectively. The surfactants used for formulation generation were poloxamer188 (P188; Sigma-Aldrich, St. Louis, MO) with or without 1,2distearoyl-phosphatidyl-ethanolamine-methyl-polyethyleneglycol conjugate-2000 (mPEG; Genzyme Pharmaceuticals LLC, Cambridge, MA). Free base drug was suspended (0.1% by weight) in 10 mM HEPES, pH 7.8. Surfactant was added by weight at 0.5% P188 to ATV or at 0.3% P188 and 0.1% mPEG to both RTV and EFV nanosuspensions. Homogeneous suspensions were achieved using an Avestin C3 high-pressure homogenizer (Avestin Inc., Ottawa, ON) and extruded at 20000 psi until the desired particle size was attained. Polydispersity, particle size and surface charge (zeta potential) were analyzed by dynamic light scattering using a Malvern 2283
dx.doi.org/10.1021/pr400185w | J. Proteome Res. 2013, 12, 2282−2294
Journal of Proteome Research
Article
Mass Spectrometry
Analysis settings of the reference set included both genes and endogenous chemicals of both direct and indirect relationships.
An LTQ Orbitrap XL with Eksigent nano-LC system equipped with two alternating peptide traps and a PicoFrit C18 columnemitter (New Objective, Woburn MA) was used. Samples were loaded onto the peptide trap with 98:2 HPLC water with 1% formic acid: acetonitrile with 1% formic acid and eluted using a 60 min linear gradient of 0−60% acetonitrile with 1% formic acid. A direct infusion of angiotensin was used to tune the instrument, which was calibrated every 2−3 days using standards provided by the manufacturer with Lock Mass. The acquisition method was created in data-dependent mode with one precursor scan in the Orbitrap, followed by fragmentation of the 5 most abundant peaks in the collision induced dissociation (CID), detected in the LTQ. Resolution of the precursor scan was set to 60000 scanning from 300 to 2000 m/ z. Precursor peaks were dynamically excluded after two selections for 60 s. No charge state rejection was used, but previously detected background peaks were included in a mass rejection list. Collision energy was set to 35 using an isolation width of 2 and an activation Q of 0.250.
Biological Validation
The biologic validation assays served to link changes in the MDM proteome indicative of macrophage function. These consisted of changes in particle ingestion, cell migration, secretion, and ion channels. Phagocytosis and Flow Cytometry
The ability of MDM to engulf solid particles (phagocytosis) following nanoART treatment was determined using a phagocytosis assay kit (IgG FITC) obtained from Cayman Chemical Company (Ann Arbor, MI) according to the manufacturer’s instructions. Cells were treated with nanoART or control media for 8 h in 6-well plates. Media was then removed and replaced with fresh media without additional nanoART, and 24 h later cells were treated with IgG-FITC beads for an additional 24 h. Excess beads were removed by washing with PBS, and cells were scraped in 1 mL of assay buffer into a polypropylene tube then analyzed by flow cytometry on a BD FACSCalibur (San Jose, CA) using CellQuest Pro software. A mixed effects model was used to include fixed effects from MDM biological conditions and random donor effects in order to adjust for correlations among the same and divergent donor samples. The Tukey-Kramer method was used to correct for multiple comparisons and identify significant differences between the natural log (ln) median fluorescence intensity values of nanoATV, -RTV, and -EFV , and cNanoART compared to untreated MDM controls.
Quantification and Protein Identification
Resulting data files obtained from LTQ-Orbitrap were submitted to MaxQuant (version 1.2.2.2) for peak list generation. Andromeda26 was used to search peak list files against the International Protein Index (IPI) human database. Search parameters were as follows: 2 maximum missed cleavages; carbamidomethylation of cysteine as fixed modification; N-acetylation of proteins and oxidation of methionine as variable modifications; top 6 MS/MS peaks per 100 Da; and MS/MS mass tolerance of 0.5 Da. Requirements included two unique peptides with a minimum length of 6 amino acids. A 0.01 false discovery rate was applied for both protein and peptide identification. Ratios were obtained for nanoART treated (H) MDM to untreated (M) MDM comparisons. All proteins identified by MaxQuant as contaminants, only identified by site, and those identified by reverse peptide hits were eliminated. Also eliminated were proteins identified by 1 peptide, proteins only present in 1 donor, and proteins that did not trend the same way. Following elimination of proteins the significant expression of the remaining proteins was determined as follows. The log2 ratio of the treatment group to the control group for 3 donors was calculated. The LIMMA method27 was applied to the log2 ratio for all donors and all proteins to evaluate whether the proteins have differential expression between the treatment and the control samples. The LIMMA method fit the log2 ratio data with a linear model to analyze experiments involving multiple comparisons and the empirical Bayes approach was used to borrow information across proteins to estimate the variance of log2 protein abundance ratios. The Benjamini-Hochberg method was used to control the false discovery rate to be less than 0.05. The proteins with the Benjamini-Hochberg adjusted p-value
