FUNCTIONAL PROTEOME OF MACROPHAGE CARRIED NANOFORMULATED ANTIRETROVIRAL THERAPY DEMONSTRATES ENHANCED PARTICLE CARRYING CAPACITY

Abstract

Our laboratory has pioneered the development of long-acting nanoformulations of antiretroviral therapy (nanoART). NanoART serves to improve drug compliance, toxicities, and access to viral reservoirs. These all function to 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.

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 is 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 serves 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); and 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 size, shape, charge, and coating. This would also best facilitate MP drug depots while the cells simultaneously serve as Trojan horses for ART entry into the reticuloendothelial system where viral replication is most 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 sorting²¹.

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-γ^null 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 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 drug(s) might reach and eliminate residual virus and what effects the particles themselves might have on the cell carrier. More specifically how the particle affects 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 monocyte-derived macrophage (MDM) 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 functions²². 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 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, induction of potassium channels, and associated enhanced cell migratory events. The analyses of the dynamic protein differences during particle-macrophage 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 towards the translation of long acting ART nanoformulations for treatment of HIV infected people.

Experimental Procedures

NanoART Preparation

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 Labs, Ltd (Hyderabad, India) respectively. The surfactants used for the formulation generation were poloxamer-188 (P188; Sigma-Aldrich, St. Louis, MO) with or without 1,2-distearoyl-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 20,000 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 Zetasizer Nano Series Nano-ZS (Malvern Instruments Inc, Westborough, MA). Once desired characteristics were achieved, samples were centrifuged at 10,000 × g for 30 minutes 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 high-performance liquid chromatography (RP-HPLC)^9,10.

Human Monocyte Isolation and Cultivation

Human monocytes were obtained via leukopheresis from HIV-1, 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, EFV-P188-mPEG and RTV-P188-mPEG individually or in combination), 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 hours then washed with 1X 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]-L-arginine and 149.0 mg/mL [D₄]-L-lysine. All isotopically labeled amino acids were obtained from SIGMA-Isotec, St. Louis MO.

Cell Lysis and Sample Preparation

After 48 hours, cells were washed three times with PBS and collected by the addition of 400 μL lysis buffer (4% sodium dodecyl sulfate, 0.1M dithiothreitol, 0.1M 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 minutes, 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 1cc 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.

Mass Spectrometry

An LTQ Orbitrap XL with Eksigent nano-LC system equipped with two alternating peptide traps and a PicoFrit C18 column-emitter (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 minute 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 60,000 scanning from 300-2000 m/z. Precursor peaks were dynamically excluded after two selections for 60 seconds. 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.

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 < 0.05 and a fold change of at least 1.5 (up/down) were identified as having differential expression.

Bioinformatic Analysis

Ratios and p-values of statistically significant proteins were then entered into Ingenuity Pathway Analysis (IPA) content version 12402621, a web-based program to help model, analyze, and understand the complex data sets. IPA was used to identify molecular and cellular functions, physiological system developments and functions, and network functions and interactions. Analysis settings of the reference set included both genes and endogenous chemicals of both direct and indirect relationships.

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 hours in 6-well plates. Media was then removed and replaced with fresh media without additional nanoART, and 24 hours later cells were treated with IgG-FITC beads for an additional 24 hours. 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, California) 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 amongst 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 ATV-P188, RTV-P188-mPEG, EFV-P188-mPEG, and cNanoART compared to untreated MDM controls.

Cell Migration and Morphology

The ability of MDM to respond to a chemoattractant signal following nanoART treatment was determined using a CytoSelect™ 24-Well Cell Migration Assay provided by Cell Biolabs, Inc., following manufacturers’ protocol. Recombinant human monocyte chemotactic protein 1 (MCP 1; PeproTech, Rocky Hill, NJ) was used as the chemoattractant agent at a concentration of 100 ng/mL following reconstitution. Human monocytes were grown in suspension in Teflon flasks at a density of 2 × 10⁶ cells/mL for 7 days with a half media exchange on day 4. On day 7 cells were centrifuged at 120 × g for 10 min and resuspended in media with or without 100 μM ATV-P188, 100 μM EFV-P188-mPEG, and 100 μM RTV-P188-mPEG individually or in combination. After 8 hours, cells were again centrifuged and resuspended in fresh media. Twelve, 24 and 48 hours later cells were resuspended in serum free media at a density of 2 × 10⁶ cell/mL, and 100 μL of cell suspension was added to the top of the membrane insert; to the bottom of the well, media containing MCP-1 was added. Cells were allowed to migrate across the membrane for 18 hours. Absorbance values were recorded and analyzed against cell numbers; the result was normalized to the control and expressed as a percentage. A one-way ANOVA was performed using the Dunnett’s test for multiple comparisons.

For measures of cytoskeletal protein expression, MDM were cultured for 7-10 days at a density of 1 × 10⁶ cells/mL in 4-well Lab-Tek II cc² chamber slides and treated for 8 hours with respective nanoART. Following 48 hours after drug was removed, the cells were washed three times with PBS and fixed with 4% paraformaldehyde at room temperature for 30 minutes. After fixation, cells were washed an additional three times with PBS and blocked/permeablized with a solution containing 0.1% Triton and 0.5% bovine serum albumin in PBS for 20 minutes. This was then quenched with 50 mM NH₄CL for 10 minutes. Cells were then washed once with 0.1% Triton in PBS and sequentially incubated with primary and secondary antibody. Antibodies used were mouse anti-profilin-1 mAb (Clone 2H11; Synaptic Systems, Goettingen, Germany; 1 μg/mL), FITC-conjugated (goat) anti-mouse IgG mAb (Invitrogen Life Technologies, Carlsbad, CA; 5 μg/mL), and rhodamine-conjugated phalloidin (Invitrogen Life Technologies; 5 units/mL) to detect F-actin with an incubation period of 3 hours and 30 and 40 minutes, respectively, at room temperature. Each treatment was followed by triplicate 5 minute washes with 0.1% saponin in PBS. Nonspecific cross binding of antibodies was tested prior to immunostaining. Cells were mounted using ProLong Gold anti-fading reagent with DAPI (Invitrogen Life Technologies) and imaged using a 63× oil lens on a Zeiss 510 Meta Confocal Laser Scanning Microscope (Jena, Germany). For statistical analysis, a one-way ANOVA was performed using the Dunnett’s test for multiple comparisons.

Measures of Secretory Chemokines and Cytokines

Changes in the concentrations of MDM bioactive factors following nanoART treatment were determined using EMD Millipore’s MILLIPLEX MAP Human Cytokine/Chemokine Magnetic Bead Panel kit (Billerica, MA; HCYTMAG-60K-PX38) analyzed on a Luminex MAGPIX (EMD Millipore Co.). Supernatants were taken from MDM treated with nanoART after 48 hours and stored at −80°C prior to assay determinations. Data were log₂-transformed prior to analyses. To adjust for the correlation between samples and from the same donor, the generalized linear model was used. Cytokine expression was compared between each treatment group versus the control group. The Benjamini-Hochberg method was used to control the false discovery rate. The cytokines with false discovery rate adjusted p-values < 0.05 were identified as statistically significant.

Electrophysiology (K⁺ Channel) Measures

Human monocytes were seeded in 35 mm poly-D-lysine coated dishes and cultured for 7 days at a density of 1 × 10⁶ cells/mL. Prior to the electrophysiology assays, cells were treated with 100 μM ATV-P188 for 8 hours. Whole cell patch-clamp recordings were performed on MDM at 22-23°C using an Axopatch 200B amplifier (Molecular Devices, Sunnyvale, CA) interfaced with a Digidata 1440A digitizer (Molecular Devices) and operated with pClamp Version 8.2 software (Molecular Devices). Whole cell currents were filtered at 1kHz, digitized at 5kHz, and stored on a computer hard disk for off-line analyss. Patch pipettes were fabricated from borosilicate glass capillaries using a Sutter P97 microelectrode puller, with tip resistances of 5.0–8.0 MΩ when filled with pipette solution contained (in mM) 130 K-gluconate, 1 CaCl₂, 1 MgCl₂, 10 EGTA, 10 HEPES, pH 7.3 with KOH (280 mOsm). The bath solution contained (in mM) 135 Na-gluconate, 5 KCl, 1 CaCl₂, 1 MgCl₂, 10 EGTA, 10 HEPES, and pH 7.4 with NaOH (290 mOsm). Tetrodotoxin (1μM) and CdCl₂ (0.2 mM) were added in the bath solution to block sodium and calcium currents, respectively. Whole-cell voltage dependent K⁺ currents were recorded in response to a series of voltage steps from –180 mV to +200 mV in 20 mV increments, starting from a holding potential of −60mV. Membrane capacitance was compensated at 60-70%. The steady-state current magnitudes were measured and current density (pA/pF) was then calculated by dividing the steady-state current magnitudes by the whole cell membrane capacitance. Data were analyzed using Clampfit 8.2 (Molecular Devices) and graphed using OriginLab 8.0 (Northampton, MSA).

Results

Biology of NanoART-MDM Interactions

We optimized the ART nanoformulations by size, charge, polydispersity, and polymer coating. These were reflective of previously published values^15,18,19 and are listed in Figure 1 where MDM were treated with 100 μM of nanoART alone or in combination for 8 hours. Drug content was determined after 8 hours of cell uptake and revealed high concentrations of ATV and RTV and low levels of EFV in cells. MDM treated with cNanoART showed no differences in uptake or retention compared to MDM treated with individual nanoformulations. The amount of nanoART retained within cells after 5 days was highest for ATV while RTV and EFV was minimal. This data is consistent with prior studies^9,18,19. Furthermore, cell viability was assessed by MTT assay and did not reveal any cytotoxicities induced by 100 μM nanoART individually or when used as cNanoART (data not shown).

Figure 1.

Nanoformulated drug characteristics of ATV-P188, EFV-P188-mPEG, and RTV-P188-mPEG. A. The physicochemical characteristics of nanoformulated antiretroviral drugs including ^aparticle size as a measure of the z-average diameters, ^bpolydisperisty indices (PDI) as determined by dynamic light scattering, and ^coverall charge of the particle as indicated by the zeta potential in mV. NanoART uptake after 8 hours and retention over 15 days were determined in MDM treated with 100 μM nanoART. ^dDrug levels are expressed in μg/10⁶ cells as mean ± standard deviation. B. The scanning electron microscopy (SEM) images of nanoART at a magnification of 15,000× with a 1 μm measure bar.

pSILAC MDM Protein Profiling Following NanoART Treatment

To better understand the response of MDM to nanoART and predict changes in function and possible cellular toxicities as a result of the drug, polymer coating, or the combination in the form of nanoART we analyzed the global proteomic changes induced using pSILAC. Evaluation of protein content was focused on an 8 hour treatment with 100 μM of nanoART (alone or in combination), free drug (alone or in combination), or polymer coating an innocuous drug (fluconazole). This time point was chosen as prior studies showed consistent cell uptake of nanoART over 8 hours^10,18,19,23. Treatment was followed by a 48 hour incubation in pSILAC media containing H amino acids^10,18,19,23. Untreated controls were incubated in pSILAC media containing M amino acids. After 48 hours, cells were processed for proteomic analyses. Three biological replicates (donors) were analyzed for each treatment and control group. pSILAC ratios were obtained by comparing the levels of newly synthesized proteins in treated MDM to their time-matched untreated controls. For each nanoART treatment (alone or in combination), there were multiple proteins with significantly different ratios, as indicated by p-values ≤ 0.05 for at least 2 of the 3 donors, with changes (up or down regulation) in rates of new protein synthesis. A total of 57 out of 1020 identified proteins were differentially expressed in MDM treated with ATV-P188, 15 of 975 proteins with EFV-P188-mPEG, 89 of 1049 proteins with RTV-P188-mPEG, and 68 of 1031 proteins with cNanoART. Interestingly, very few if any changes in protein expression were seen in MDM treated with the free drug (alone or in combination) or polymer-coated fluconazole vs. their time-matched untreated controls. Zero proteins were differentially expressed in MDM treated with free ATV or EFV out of 1194 and 1181 proteins respectively. Three out of 1093 proteins were differentially expressed with free RTV treatment and 15 of 1090 proteins were differentially expressed in MDM treated with the free drug combination. Only 1 out of 1169 proteins was differentially expressed in MDM treated with polymer-coated fluconazole. These results indicate that neither the free drug nor the polymer coating induce significant changes in MDM. It is a combination of the antiretroviral drug and the polymer coating in the form of nanoART that alters MDM protein production. Furthermore, differentially expressed proteins in MDM treated with nanoART demonstrate the cellular response to treatment, which heralds functional changes. The top differentially expressed proteins for each nanoART treatment can be seen in the supplemental table along with their subcellular location, fold changes, and p-values.

The data show some common proteins amongst all nanoART treatment groups, but more interestingly the data demonstrate the differences in each individual treatment as well as the combinatory effects (cNanoART). IPA was used to help analyze and model the proteomic changes revealing the top biological functions affected, which are compared in Figure 2 for each nanoART treatment. The proportional distribution of proteins for each function demonstrates the similarities and differences in nanoART treatment. All nanoformulations show changes in proteins related to cell death, oxidative stress, and free radical scavenging, which include changes that can be both protective and destructive to the cell. RTV-P188-mPEG shows a large change in proteins related to cellular movement and free radical scavenging, which is also reflected in cNanoART treatment, while ATV-P188 and EFV-P188-mPEG are the only nanoformulations that show significant changes in proteins related to drug metabolism. Not surprisingly, most proteomic effects for individual treatments are also reflected in cNanoART treatment but not all indicating that the effects of one treatment may block the effects of another, but this effect is minimal. For this reason cNanoART treatment was chosen as an all-inclusive representative for further individual protein analysis. A list of proteins for the top biological functions affected by cNanoART treatment in MDM can be found in Table 1. It is clear that the signature of proteins present affects many macrophage functions commonly seen during cell differentiation and activation. Proteins indicative of cellular activation include the upregulation of serum amyloid p component (APCS), S100A8/9, and superoxide dismutase 2 (SOD2)^28-30. APCS has been shown to activate the classical complement pathway for innate immunity and improve the efficiency of phagocytosis and was upregulated over 8-fold in MDM treated with cNanoART. S100A8/9 are calcium-binding proteins that were found upregulated by approximately 10- and 6- fold, respectively. These proteins are highly expressed by activated macrophages. S100A8 is an antioxidant with antimicrobial activity towards bacteria and fungi, while S100A9 promotes phagocytosis and migration ^28,29. In addition SOD2, an oxidative stress protein that increases in response to activation, was also upregulated ³¹. SOD2 is a mitochondrial matrix protein responsible for transforming superoxide into hydrogen peroxide and oxygen. The intracellular killing ability of macrophages has been shown to correlate closely with the capacity of the cell to generate toxic oxygen intermediates ³⁰. Results suggest that nanoART treatment affects cellular functions by inducing proteomic changes indicative of macrophage activation.

Figure 2.

Proportional distribution of proteins in the top functional groups identified by IPA for MDM treated with ATV-P188 (ATV), EFV-P188-mPEG (EFV), RTV-P188-mPEG (RTV), or a combination of all three (cNanoART).

Table 1.

Protein groups affected by MDM cNanoART carriage.

Cell Death
Protein ID	Symbol	IPI #a	Subcellular Location	Fold Change	p-Value
Acetyl-CoA Acetyltransferase 1	ACAT1	IPI00414057	Cytoplasm	−2.114	2.50E-02
N-acylsphingosine Amidohydrolase 1	ASAH1	IPI00059685	Cytoplasm	−2.348	4.64E-02
Catalase	CAT	IPI00465436	Cytoplasm	−1.956	4.64E-02
Integrin beta 2	ITGB2	IPI00103356	Plasma Membrane	−3.079	2.21E-02
Nicotinamide Phosphoribosyltransferase	NAMPT	IPI00922378	Extracellular Space	21.699	7.57E-04
Superoxide Dismutase 2	SOD2	IPI00607577	Cytoplasm	9.248	9.65E-04
Serpin Peptidase Inhibitor, clade B, member 2	SERPINB2	IPI00789721	Extracellular Spac	17.306	7.57E-04
Neutrophil Cytosolic Factor 2	NCF2	IPI00910100	Cytoplasm	3.013	1.67E-02
Catechol-O-methyltransferase	COMT	IPI00880186	Cytoplasm	−1.812	3.82E-02
Peroxiredoxin 6	PRDX6	IPI00910553	Cytoplasm	−1.815	4.64E-02
CD74 Molecule, Major Histocompatibility Complex	CD74	IPI00607573	Plasma Membrane	−7.009	4.48E-03
Free Radical Scavenging
Protein ID	Symbol	IPI #	Subcellular Location	Fold Change	p-Value
Aldo-Keto Reductase	AKR1B1	IPI00926520	Cytoplasm	3.466	4.49E-02
Aldehyde Dehydrogenase 2 family	ALDH2	IPI00792207	Cytoplasm	−1.873	4.70E-02
Catalase	CAT	IPI00465436	Cytoplasm	−1.956	4.64E-02
Nicotinamide Phosphoribosyltransferase	NAMPT	IPI00922378	Extracellular Space	21.699	7.57E-04
Neutrophil Cytosolic Factor 2	NCF2	IPI00910100	Cytoplasm	3.013	1.67E-02
Neutrophil Cytosolic Factor 4	NCF4	IPI00878414	Cytoplasm	−2.065	4.64E-02
Peroxiredoxin 6	PRDX6	IPI00910553	Cytoplasm	−1.815	4.64E-02
Superoxide Dismutase 2	SOD2	IPI00607577	Cytoplasm	9.248	9.65E-04
Thioredoxin	TXN	PI00552768	Cytoplasm	8.797	7.57E-04
Immune Cell Trafficking
Protein ID	Symbol	IPI #	Subcellular Location	Fold Change	p-Value
Annexin A5	ANXA5	IPI00329801	Plasma Membrane	1.868	4.64E-02
Dihydropyrimidinase-like 2	DPYSL2	IPI00883655	Cytoplasm	−2.545	1.67E-02
Integrin Alpha X	ITGAX	IPI00641386	Plasma Membrane	−2.505	4.56E-02
Integrin Beta 2	ITGB2	IPI00103356	Plasma Membrane	−3.079	2.21E-02
Peptidylprolyl Isomerase A	PPIA	IPI00939576	Cytoplasm	−1.935	2.58E-02
RAP1A, Member of RAS Oncogene Family	RAP1A	IPI00877120	Cytoplasm	1.914	4.64E-02
S100 Calcium binding protein A8	S100A8	IPI00007047	Cytoplasm	10.053	7.57E-04
S100 Calcium Binding Protein A9	S100A9	IPI00939362	Cytoplasm	5.904	1.15E-03
Stabilin 1	STAB1	IPI00383337	Plasma Membrane	−4.995	3.76E-02
Antigen Presentation
Protein ID	Symbol	IPI #	Subcellular Location	Fold Change	p-Value
CD74 Molecule, Major Histocompatibility Complex	CD74	IPI00607573	Plasma Membrane	−7.009	4.48E-03
Major Histocompatibility Complex class II	HLA-DQA1	IPI00941590	Plasma Membrane	−3.986	4.19E-02
Cathepsin S	CTSS	IPI00910216	Cytoplasm	−2.676	2.20E-02
Endocytosis
Protein ID	Symbol	IPI #	Subcellular Location	Fold Change	p-Value
Actin beta	ACTB	IPI00922693	Cytoplasm	−2.223	1.80E-02
EH-domain containing 1	EHD1	IPI00853038	cytoplasm	3.82	1.39E-02
Integrin beta 2	ITGB2	IPI00103356	Plasma Membrane	−3.079	2.21E-02
RAB5A, Member RAS Oncogene Family	RAB5A	IPI00927674	Cytoplasm	4.167	2.06E-02
S100 Calcium binding protein A8	S100A8	IPI00007047	Cytoplasm	10.053	7.57E-04
Tubulin alpha 1a	TUBA1	IPI00936821	Cytoplasm	2.662	2.35E-02
Oxidative Stress
Protein ID	Symbol	IPI #	Subcellular Location	Fold Change	p-Value
Catalase	CAT	IPI00465436	Cytoplasm	−1.956	4.64E-02
Peroxiredoxin 6	PRDX6	IPI00910553	Cytoplasm	−1.815	4.64E-02
S100 Calcium Binding Protein A9	S100A9	IPI00939362	Cytoplasm	5.904	1.15E-03
Superoxide Dismutase 2	SOD2	IPI00607577	Cytoplasm	9.248	9.65E-04
Cellular Movement
Protein ID	Symbol	IPI #	Subcellular Location	Fold Change	p-Value
S100 Calcium binding protein A8	S100A8	IPI00007047	Cytoplasm	10.053	7.57E-04
Thioredoxin	TXN	IPI00552768	Cytoplasm	8.797	7.57E-04
S100 Calcium Binding Protein A9	S100A9	IPI00939362	Cytoplasm	5.904	1.15E-03
Integrin Alpha X	ITGAX	IPI00641386	Plasma Membrane	−2.505	4.56E-02
Cathepsin S	CTSS	IPI00910216	Cytoplasm	−2.676	2.20E-02
Integrin beta 2	ITGB2	IPI00103356	Plasma Membrane	−3.079	2.21E-02
Stabilin 1	STAB1	IPI00383337	Plasma Membrane	−4.995	3.76E-02
CD74 Molecule, Major Histocompatibility Complex	CD74	IPI00607573	Plasma Membrane	−7.009	4.48E-03
Peptidylprolyl Isomerase A	PPIA	IPI00939576	Cytoplasm	−1.935	2.58E-02
Inflammatory Response
Protein ID	Symbol	IPI #	Subcellular Location	Fold Change	p-Value
Amyloid P Component, Serum	APCS	IPI00022391	Extracellular Space	8.362	3.47E-03
S100 Calcium Binding Protein A9	S100A9	IPI00939362	Cytoplasm	5.904	1.15E-03
Cathepsin S	CTSS	IPI00910216	Cytoplasm	−2.676	2.20E-02
Major Histocompatibility Complex class II	HLA-DQA1	IPI00941590	Plasma Membrane	−3.986	4.19E-02
CD74 Molecule, Major Histocompatibility Complex	CD74	IPI00607573	Plasma Membrane	−7.009	4.48E-03
Phagocytosis
Protein ID	Symbol	IPI #	Subcellular Location	Fold Change	p-Value
Amyloid P Component, Serum	APCS	IPI00022391	Extracellular Space	8.362	3.47E-03
RAB5A, Member RAS Oncogene Family	RAB5A	IPI00927674	Cytoplasm	4.167	2.06E-02
Annexin A5	ANXA5	IPI00329801	Plasma Membrane	1.868	4.64E-02
Lysosomal-Associated Membrane Protein 2	LAMP2	IPI00922445	Plasma Membrane	−1.862	4.76E-02
Integrin beta 2	ITGB2	IPI00103356	Plasma Membrane	−3.079	2.21E-02

The unique macrophage phenotype reveals many differentially expressed proteins including those related to free radical scavenging, cellular movement, immune cell trafficking, and clathrin mediated endocytosis. For example EHD1, also known as EH-domain containing 1, which acts in early endocytic membrane fusion and membrane trafficking of recycling endosomes, was found to be upregulated by nearly 4-fold. TUBA1A (tubulin alpha 1a) was also upregulated and is the major constituent of microtubules, which serve as a scaffold to determine cell shape and provide a backbone for cell organelles and vesicles to move along. Furthermore, annexin A5, a protein important in membrane-related events along exocytotic and endocytotic pathways, was upregulated.

Surprisingly, major histocompatibility complex class II DQ alpha 1 (HLA-DQA1) was downregulated up to 4-fold. This protein binds peptides derived from antigens that access the endocytic route of antigen presenting cells³². CD74 was also downregulated by approximately 7-fold. HLA-DQA1 forms a complex with HLA-DQB1 and CD74 trimer after which it enters the endosomal/lysosomal system where antigen processing occurs. This seemingly contradicts the hypothesis that the macrophage has an increased ability to mount an immune response. However, lysosomal-associated membrane protein 2 (LAMP2), which increases late endosomal/lysosomal fusion, was decreased by nearly 2-fold. Altogether the data suggest that nanoART is not antigenic and does not enter the endosomal/lysosomal system where it would be degraded but instead may enter early and recycling endosomes where it would be protected from degradation. This is supported by the approximate 4-fold increase in ras-related protein 5A (RAB5A) a rate limiting catalyst for internalization and necessary for plasma membrane/early endosome fusion. Furthermore, downregulation of HLA-DQA1 taken together with the upregulation of S100A8 and SOD2 may indicate that the macrophage is enhanced to mount a cell-mediated response. Indeed, studies have shown that a cell-mediated response to HIV infection is far superior to a humoral response in controlling viral levels^33-37.

Cytokines and Chemokines Secreted from NanoART-Treated MDM

In order to determine the MDM phenotype following nanoART treatment and to examine changes in the secretion of bioactive factors, 38 cytokines and chemokines were analyzed in supernatants taken from nanoART-treated and control untreated cells. The fold changes and p-values of each of the cytokines and chemokines tested for each treatment are seen in Table 2. Further, a network generated in IPA for cNanoART treatment demonstrated interactions between the detected cytokines and chemokines (Figure 3). Results show significant increases in EOTAXIN, IL-8, and IL-7 in MDM treated with ATV-P188, RTV-P188-mPEG, or cNanoART. However, MDM treated with EFV-P188-mPEG showed only decreases in macrophage inflammatory protein one alpha (MIP-1α), tumor necrosis factor alpha (TNF-α), interleukin one receptor antagonist (IL-1RA), macrophage-derived chemokine (MDC) and transforming growth factor alpha (TGF-α). These decreases were also observed in MDM treated with cNanoART. Interestingly, the traditional trends in cytokine and chemokine secretions for macrophage activation, indicative of either an M1 or M2 activation state, were not observed in any of the treatment groups. MIP-1α, a cytokine known to increase in classically activated macrophages, decreased in both MDM treated with cNanoART and EFV-P188-mPEG. TNF-α, another classic M1 secretory product, also decreased in MDM treated with EFV-P188-mPEG. Interestingly, IL-1RA, a cytokine known to block the production of the M1 proinflammatory product IL-1β, also decreased in both MDM treated with EFV-P188-mPEG and cNanoART. Thus, there was no specific activation state identified from nanoART treated MDM. What is clear, however, is that there are changes in secretory products that alter the functional characteristics of MDM. This, notably, included IL-8, which is known to enhance phagocytosis, providing a mechanism of action for altered functional macrophage capacities and is supported by the proteomic results.

Table 2.

Cytokines and chemokines induced by nanoART in MDM.

Treatment	Cytokine/Chemokine	Fold Changea	p-Value
ATV	EOTAXIN	2.59	1.37E-04
ATV	IL_7	1.83	1.70E-04
ATV	IL_8	7.48	1.67E-04
EFV	IL_1RA	−1.94	1.44E-03
EFV	MDC	−3.05	4.22E-03
EFV	MIP_1a	−2.36	1.29E-05
EFV	TGF_a	−1.51	6.18E-03
EFV	TNF_a	−1.90	9.96E-04
RTV	EOTAXIN	1.97	2.02E-03
RTV	IL_7	1.55	2.08E-03
RTV	IL_8	3.64	.77E-03
cNanoART	EOTAXIN	3.16	2.42E-05
cNanoART	G_CSF	1.42	2.88E-03
cNanoART	IL_1RA	−2.23	3.24E-04
cNanoART	IL_7	2.13	2.10E-05
cNanoART	IL_8	13.42	1.57E-05
cNanoART	IL_9	1.30	2.13E-02
cNanoART	IL_15	1.26	3.26E-03
cNanoART	IFN_a	1.40	1.05E-02
cNanoART	MCP_3	1.65	1.93E-02
cNanoART	MDC	−3.77	1.26E-03
cNanoART	MIP_1a	−1.87	2.34E-04
cNanoART	TGF_a	−1.58	3.15E-03
cNanoART	VEGF	1.29	1.74E-02

^aStatistically significant changes in cytokines and chemokines in monocyte-derived macrophages 48 hours after nanoART treatment with ATV-P188 (ATV), EFV-P188-mPEG (EFV), RTV-P188-mPEG (RTV), or a combination of all three (cNanoART) as determined by a Luminex assay.

Figure 3.

Cytokine/chemokine interaction network for cNanoART treated MDM. Cytokines/chemokines were submitted to IPA for analyses to evaluate known functional interactions and are indicated with a connecting line. Red coloration indicates an increase in cytokine/chemokine as compared to controls and green indicates a decrease in expression. Color intensity indicates degree of increase or decrease. Lack of color indicates a molecule involved but not identified in the data set. The cytokine/chemokine interaction network was generated using Ingenuity Pathways Analysis (Winter 2012 release; version 14400082) and the Path Explorer tool.

Functional Protein Validations

Phagocytosis

To validate the phenotypic changes supported by both the proteomic and cytokine/chemokine tests in MDM, which followed nanoART treatment, we determined changes in phagocytosis, K⁺ channel activities, cell migration, and cytoskeletal protein expressions. Phagocytosis was assessed using a phagocytosis assay kit where nanoART-treated and control MDM were fed IgG-FITC beads for 24 hours and fluorescence measured using flow cytometry. The results for each treatment are seen in Figure 4A. Analyses of the mean fluorescence for each experimental variable shows that ATV-P188, RTV-P188-mPEG, and cNanoART treated cells are statistically different from untreated controls. These data demonstrate that the treatments increase macrophage phagocytic capacity. RTV-P188-mPEG treated cells exhibited the highest fluorescent intensity indicating that this specific formulation induced the greatest changes in this functional test. EFV-P188-mPEG cells showed no statistical difference in fluorescence from untreated control cells that may reflect more profound alterations in cell functions by the drug formulation. Overall, the data demonstrate that nanoART can enhance MDM phagocytosis and as such facilitate particle carriage.

Figure 4.

Phagocytic activity, migratory capacity, and colocalization of profilin-1 and F-actin in nanoART treated MDM. MDM were treated for 8 hours with ATV-P188 (ATV), EFV-P188-mPEG (EFV), RTV-P188-mPEG (RTV), or a combination of all three (cNanoART). A. Mean fluorescence intensities of MDM treated with nanoART for 8 hours and given IgG-FITC labeled beads for 24 hours later. Bar indicates mean of individual values. *Significant differences (p<0.01) between the ln median fluorescent values was determined by the Tukey-Kramer method. B. Percent migratory capacity as compared to controls 12 hours after treatment of MDM with nanoART. Bar indicates mean of individual values. *Significant differences (p<0.0001) determined by one-way Analysis of Variance followed by Dunnett’s Multiple Comparison Test. C. Profilin-1 and F-actin distribution in control and nanoART treated MDM 48 hours after treatment. Cells were stained for profilin-1 with mouse anti-profilin-1 (mAb) and FITC-conjugated anti-mouse IgG (green), F-actin by rhodamine-conjugated phalloidin (red), and nuclei by To-Pro (blue). Mander’s Overlap Coefficient for profilin-1 and F-actin was determined using Image J software with JACoP Plug-in.

Cell Migration

To determine the ability of nanoART treated MDM to respond to a chemotactic signal, we used a transmigration assay. MCP-1 was applied to MDM treated with ATV-P188, EFV-P188-mPEG, RTV-P188-mPEG, cNanoART, and untreated controls 12, 24, and 48 hours after treatment. Cells that transmigrated through a 5 μm insert over 18 hours were lysed and absorbance was recorded. Results are illustrated in Figure 4B, which shows that the % migratory capacity increases after nanoART treatment for all treatment groups after 12 hours. This increase in cellular transmigration was not seen at 24 and 48 hours suggesting that nanoART loading positively affecting migration is transient.

Cytoskeletal Proteins

As another test of cellular changes induced by nanoART, specifically phagocytosis and migration, we determined whether nanoART treatment would lead to changes in the cytoskeletal network of MDM. We studied the colocalization of profilin-1 and F-actin in MDM following treatment with ATV-P188, EFV-P188 mPEG, RTV-P188-mPEG, and cNanoART. Actin microfilaments are one of the three major components of the cytoskeleton and are responsible for cellular movement. Profilin-1 is an actin-binding protein that aids in the restructuring of the cytoskeleton. It controls the spatial and temporal growth of actin microfilaments, an essential process in cellular shape and locomotion. Thus, we tested the idea that cNanoART treatment could increase profilin-1 and F-actin colocalization further supporting observed changes in phagocytosis and cell migration. However, the results failed to demonstrate such changes in cytoskeletal protein expressions and localization (Figure 4C) suggesting that the altered cell biological parameters were linked to other physiological mechanisms and perhaps to ion channels.

K⁺ Channels

Electrophysiological studies of K⁺ channels were thus performed in MDM following nanoART treatment to validate the linkages between macrophage activation, altered cellular migration, and changes in phagocytic capacities. MDM were treated with ATV-P188 for 8 hours after which K⁺ currents were measured. The results in Figure 5 show that nanoART enhances outward delayed K⁺ currents indicative of cellular activation and altered migratory and phagocytic capacities. As such, these alterations positively influence nanoART macrophage depots, which would enable drug delivery to virus-targeted tissues.

Figure 5.

Changes in K⁺ channel currents in response to nanoART treatment in MDM. A. Expression of both outward and inward rectifier K⁺ currents in MDM. Left: Representative whole-cell current traces recorded from an MDM before (Control), during (TEA), and after (wash out) bath application of TEA (40mM). Note that bath application of TEA significantly decreased both the outward and inward K⁺ currents as summarized at +100 mV and −180 mV voltage pulse respectively in lower right bar graph (n=6), **<0.01 compared to control, ##<0.01 compared to TEA group. Upper right shows the voltage pulse protocols utilized to generate the whole-cell currents. Voltage step duration illustrated is not proportional to those exhibited in current traces. B. ATV-P188 increased outward K+ currents in MDM. Exemplary current traces recorded from an untreated (control) and ATV-P188-treated MDM are shown on the left. On the right is the I-V curve illustrating that ATV-P188 enhanced the outward K+ currents at voltage steps above +60mV (n=26), indicating that ATV-P188 induces MDM activation. *<0.05 vs. Control, **<0.01 vs. Control.

Discussion

MP are targets for a variety of microbial pathogens that include HIV^38-45. Paradoxically, they also act as the primary sentinel of the innate immune system, the first line of defense against injury and microbial infections. MP participate in clearing debris, tissue homeostasis, and mounting an adaptive immune response through the secretion of bioactive factors and presentation of antigens to T cells. Despite these functions, MP are among the first cells infected with HIV and are actively involved in replicating and trafficking virus, inducing inflammatory responses, and acting as viral sanctuary sites. For these reasons, our research has sought to develop MP as antiretroviral drug traffickers^{6,7,9,10,13,14,16,19}. This has proven quite successful. Indeed, our prior laboratory and animal studies demonstrated improved antiretroviral pharmacokinetics and pharmacodynamics for nanoART when compared to traditional oral therapies. In particular, these prior studies revealed unambiguously that macrophages harbor drug within recycling endosomal depots facilitating drug delivery to sites of infection while maintaining serum drug concentrations for prolonged time periods, measured in weeks^{9,13,14,17-19,21}.

However, lacking from all these prior investigations was an explanation of why the macrophage was such an efficient vehicle for nanoART carriage. We reasoned that the macrophage might sense the nanoparticle as foreign and alter its function accordingly. Macrophage function evolves rapidly following exposure to a broad range of environmental, toxic, injurious, metabolic, and microbial exposures with altered innate immune activities. Such changes facilitate host survival and homeostasis and include debris clearance, intracellular destruction of pathogens, and secretion and presentation of bioactive factors that help orchestrate an adaptive immune response. Based on the particle’s size and shape, it would inevitably move into protective subcellular structures such as the recycling endosomes as we previously demonstrated²¹. We also presumed that an activation state would be operative where phagocytosis and endosomal trafficking are facilitated but degradation in lysosome structures is inhibited. Furthermore, given that nanoART is polymer coated and taken up into subcellular organelles of monocyte-macrophages, we hypothesized that nanoART would not adversely affect these carrier cells but instead would enhance their abilities to carry, store, and release particles to sites of infection thus increasing biodistribution. A major step in achieving such goals is to assess how carriage affects their common functional roles.

To investigate these possibilities, we evaluated the chemical, biological, immune, virologic, and toxicological properties of the macrophage after nanoART exposure. pSILAC was chosen to direct these studies based on its sensitivity and specificity in assessing cellular protein changes in a dynamic manner. Such a scientific approach can identify changes in the cell proteome of the macrophage during drug treatment and trafficking and can uncover cellular and subcellular toxicities linked to the drug itself, drug carriage, and drug release ^24,46. We believed that targeting cell proteomic functions could prove successful and could be used to direct subsequent biological and functional assays. Furthermore, examination of de novo protein synthesis would be reflective of particle–host cell dynamics. Here, nanoformulations of ATV, RTV, and EFV were chosen based on clinical utility and demonstrated robust upregulation of free radical scavenging and migratory and secretory functions for the macrophage exposed to nanoART. These results laid an important groundwork for subsequent functional validations demonstrating enhanced phagocytosis, migratory, and K⁺ channel activities. All are signs that point to nanoART inducing a state of activation in MDM leading to the enhancement of its innate functions.

Historically, activated macrophages were defined simply as cells that secreted inflammatory mediators and killed intracellular pathogens; however, the definition of an activated macrophage is far more complicated with heterogeneous populations that have different physiologies and distinct immunological functions. There are two well-established and generally accepted phenotypes; classically (M1, pro-inflammatory) and alternatively (M2, anti-inflammatory) activated macrophages^51,52. A third class of activated macrophages has been reported and is referred to as type II or wound healing macrophages and include characteristics of both M1 and M2 macrophages⁵¹. Indeed, our data suggest an activation phenotype in which the functional parameters of MDM are enhanced without the production of typical anti- or pro-inflammatory cytokines and chemokines indicative of any of the three classes of activated macrophages, thus the specific phenotype that nanoART induces is unique.

MCP-1 and IL-8 both play prominent roles in macrophage adhesion and migration. As per the latter, IL-8 is involved in endothelium macrophage contact while MCP-1 facilitates cellular transmigration^53-55. However, the roles of both chemokines in cell trafficking is commonly overlapping in function. In regard to tissue damage IL-8 does promote adhesion of macrophages to injured tissue. IL-8’s receptor CXCR2 is expressed on activated macrophages and facilitates the cell’s migration potential^56-58. In all adhesion, transmigration and retention of blood-borne macrophages in virus-infected tissues including the CNS is most notably regulated through IL-8 which can be produced by macrophages themselves. The fact that IL-8 is upregulated in nanoART treated macrophages is additional evidence supporting the phenotypic cell change in promoting vascular penetration and trafficking and in this manner serving to enhance cell-carried ART.

For HIV-1 infection, the tempo and extent of CNS disease, parallels numbers of virus infected and immune activated blood borne macrophages in brain^47,48. Cell trafficking is affected by chemokines, proinflammatory cytokines, and viral proteins. Macrophage brain egress is affected by chemokine receptors and adhesion molecules. These are present on brain microvascular endothelial cells as well as in macrophages^49,50. Once inside the CNS perivascular and parenchymal inflammatory macrophages can alter the permeability of the blood brain barrier and elicit substantive damage to neighboring endothelial and glial cells. This can facilitate both neural damage and further entry of cells into injured brain subregions. The process, all together, is governed by active viral replication. Thus, we posit that the abilities to restrict viral infection would limit the cell “pull” of neuroinflammation while the nanoformulations themselves and their effects on the cell proteome would affect “push” or the drug-carrying cells into the CNS and as such limit damage and secondary injuries. Similar inhibitions of “pull” mechanisms would occur in peripheral organs and as such limit damage by the infiltrating or “pushed” nanoformulated drug carrying macrophages.

Conclusions

Analysis of the proteomic data supports the notion that nanoART enhances macrophages’ abilities to act as an immune sentinel and a vesicle for nanoART carriage. This is supported by the dynamic changes in protein content linked to immune cell migration and recruitment, cytokine and chemokine production, lipid metabolism, free radical scavenging, and cell differentiation and development. The data, taken together, with functional validation studies support the efficacy of particle activated macrophages to act as cell-based delivery vehicles and drug depots. The work also demonstrates how particle-cell interactions facilitate optimal drug loadings into cells facilitating their role as drug depots and transporters of antiretroviral responses. What is new from these studies is that nanoART can facilitate the cell’s functional abilities leading to improved pharmaceutical outcomes. This data is especially important as the cell can direct drug to viral reservoirs and as such parallel inter- and intracellular pathways operative in viral growth. Taken together, these data move us one step closer towards the potential utility of this approach for targeted drug delivery in an infected human.