Cell-based carriage utilizes monocytes-macrophages for nanoART uptake, carriage and drug release to sites of active viral replication. Nanoparticles cytoxicities were measured in this report to ensure that this drug delivery system itself does not affect disease. We hypothesized that the physical, biochemical and biological properties of nanoART such as particle shape, size, concentration, surfactant, drug, and manufacturing method could affect monocyte-macrophage cytotoxicity profiles. With this in mind, nanoformulated IDV, RTV, and EFV prepared by homogenization and wet milling were examined for their effects on cellular viability and function. Using primary HBMEC, we assessed the effect of nanoART on barrier function to determine whether these nanoformulations could alter the BBB integrity. The toxicity of the nanoformulations varied between monocytes and macrophages, drug, formulation, and manufacturing methods. The mechanism underlining the differential toxicities is not known, but it is possible that with large size (1600 nm) and poor uptake, H1008 did not aggregate on the cell’s surface. On the contrary, due to their much smaller sizes more H2009 and EFV could be retained on the cell surface affecting the integrity of the cell membrane. In fact, our data demonstrate that the amounts of H1008 in cells or culture media from one to 15 days after nanoART loading was less than 1.6 μg /ml, while the amounts of H2009 from day-1 to day-15 varied from a maximum of 23 to 8.7 μg /ml, and that of P4033 varied from a maximum of 8 to 1.7 μg/ml ().
Particle charge could have contributed to EFV toxicities. EFV was the only formulation with a positive zeta potential (+7.4). The zeta potential is the overall charge a particle acquires in a specific medium and its magnitude provides an indication of the particle stability. It has been shown that particles with low (positive or negative) zeta potentials do not disperse well and are more unstable (Dougherty et al., 2008
; Ghosh et al., 2008
). The low positive zeta potential of P4033 could also have decreased its stability, and increased aggregation could also have contributed to the low cellular uptake. NanoART manufacturing also influenced the outcomes seen. Indeed, wet-milled formulations of both IDV and RTV showed reduced uptake, increased toxicity in both monocytes and MDM and increased TNF-α expression in monocytes, compared to similar formulations prepared by homogenization. The increased toxicity of M1002 versus H1008 could be due to the presence of SDS in the surfactant of the milled nanoparticles in contrast to Tween 80 in the homogenized particles. The surfactant combination of P188 and SDS alone does not appear to be toxic as evidenced by the lack of toxicity of M5004. However, the combination of surfactant and drug could result in differences in toxicological characteristics of the particles.
Wet milling produces small particles by fractionating the surfactant-coated drug crystals down to smaller sizes, and the shape of the particles is determined by the shape of the fractionated crystals. Homogenization produces particles by passing the surfactant-coated crystals through a narrow bore under high pressure. Thus the particles produced by homogenization have smoother edges and are more uniform in shape than those produced by wet milling (). Particle shape affects MDM uptake, retention, and nanoART antiretroviral efficacy (Nowacek et al., in submission); the variation in particle shape could affect the methods and mechanisms of cellular uptake, and particles trafficking inside the cells, resulting in variations in toxicity. Other studies have reported that the biological effects of nanomaterials can be greatly influenced by the mechanisms of internalization, trafficking and sub-cellular storage. (Slowing et al., 2006
; Vallhov et al., 2007
). Studies are currently under way in our laboratories to determine how nanoART are taken up, trafficked, and stored by MDM, and how these may ultimately affect their function. The increased toxicity of milled nanoART over homogenized nanoART suggests that the properties of milled nanoART may adversely affect cell viability and increase the risk of inflammation, compared to homogenized formulations.
Although nanomedicine is a rapidly growing discipline with great potentials in pharmaceutics and pharmacology research where its explicit purpose is to improve drug delivery and pharmacokinetics its perils are also appreciated (Debbage, 2009
; Gendelman et al., 2008
; Khemtong et al., 2009
). Targeted delivery of therapeutically beneficial compounds such as ART serves to combat viral infections even within their hard-to-treat tissue sanctuaries (Muthu and Singh, 2009
). Specifically for HIV, nanoART has a number of distinct advantages over conventional ART. This includes reduction in dosing intervals and improved drug entry into viral tissue reservoirs including those housed within the central nervous system (CNS) (Nowacek and Gendelman, 2009
; Nowacek et al., 2009a
). Our previous reports demonstrated the synthesis, characterization, and tissue distribution of nanoART and showed that such nanoformulations can be loaded in MDM and delivered to viral sanctuaries (Dou et al., 2006
; Dou et al., 2009
; Dou et al., 2007
; Nowacek et al., 2009b
). These studies also showed significant increases in nanoART half-lives and anti-viral effects, compared to non-formulated (free) ART drugs (Dou et al., 2007
; Nowacek et al., 2009b
). Nonetheless, such new applications pose concerns in regards to toxicities of nanoformulated drugs on carrier cells and their effects on tissue viability and function. In the current study, attempts were made to address these concerns. Indeed, we demonstrate formulation and drug concentration dependent nanoART uptake, retention, release and toxicities.
We observed that the adverse effects of the nanoformulations were more pronounced in monocytes than MDM, with the exception of H2009. These findings are important and will guide subsequent studies to develop injectable nanoART for in vivo
use. In fact, when drugs are administered by the intravenous (IV) route, they come into direct contact with blood monocytes within minutes and higher nanoART toxicities in monocytes suggest that IV injection may not be the appropriate administration route. We suspect that with other administration routes such as intradermal injections, monocyte uptake will be minimal and drug uptake will be mostly by macrophages or immature dendritic cells. Our subsequent studies will determine the optimal-safer method of nanoART administration in vivo
. Drug toxicity is a major issue for HIV-infected individuals on ART; this often causes patients to stop treatment, resulting in ART failure and emergence of drug-resistant viral strains that perpetuates the HIV/AIDS pandemic (Conway, 2007
; Kiertiburanakul and Sungkanuparph, 2009
; Sandelowski et al., 2009
; Waters and Nelson, 2007
). Controls used in this study included free IDV, RTV, and EFV (not mixed to surfactants), individual and combination surfactants without ART drugs, and fluconazole, an anti-fungal drug. The fact that these various controls induced very little or no cell toxicities suggests that formulation cytotoxicities were primarily due to nanoART drug composition, pharmacokinetics, and pharmacodynamics, not to the surfactants alone.
Prior reports demonstrated that nanoformulations of atazanavir, stavudine, delavirdine, and saquinavir increase drug concentration in HBMEC and alter BBB permeability (Chattopadhyay et al., 2008
; Kuo and Su, 2007
). Parallel studies showed that exposure of aluminum oxide NP to brain endothelial cells induced endothelial injury, loss of the tight junction protein claudin-5 and loss of mitochondrial membrane potential, with toxicities observed at 10 mM NP concentration (Chen et al., 2008
). Our study of nanoART effects on the BBB showed that monocytes or MDM carrying four different nanoformulations of IDV and RTV did not induce significant alteration in the expression of tight junction proteins ZO-2 and occludin in HBMEC at 0.1mM and did not substantively alter TEER at both 0.1 and 0.5mM, although media from MDM carrying H2014 induced some decrease in brain TEER. Overall, direct toxicity of nanoART on cells was more pronounced than their effect on TEER and tight junction protein expression, which could be due to the fact that cytotoxicity and TEER measure different parameters. The alamarBlue™ is an oxidation-reduction (REDOX) assay that detects mitochondrial metabolic activity in cells and as such measures cell proliferation and/or cytotoxicity. These results suggest that nanoART toxicity in monocytes and MDM could be related to oxidative stress. In fact, there is evidence that clinically relevant concentrations of free EFV (3.2 to 12.7 μM) inhibit mitochondrial function in a concentration-dependent manner, and this was associated with reduced adenosine triphosphate levels and increased production of reactive oxygen species (Blas-Garcia et al., 2010
). Interestingly, for many formulations, our study showed higher toxicities of free IDV, RTV, and EFV, compared to similar concentrations of nanoformulated IDV, RTV, and EFV. This suggests that nanomedicine-based delivery of ART drugs could diminish drug cytotoxicity.
TEER measures the endothelial barrier function (the resistance of the paracellular pathway between cells of the brain microvasculature), and any alteration in TEER likely occurs downstream, following physical alterations of the HBMEC and long after any change in mitochondrial or metabolic activity had occurred. Decrease in TEER was observed in HBMEC exposed to medium from H2014-loaded MDM. Our subsequent studies will determine whether this decrease in TEER is due to NP, reactive oxygen species or other soluble factors secreted by nanoART-loaded MDM. If the decreases in TEER are due to soluble factors, it is unlikely to be TNF-α or IL-12 because none of the ART nanoformulation induced TNF-α or IL-12p70 expression in MDM. Overall, the adverse toxicities were seen principally with high doses maximized in in vitro systems. Our study underscores the need to optimize individual formulations to correlate cytotoxicities with nanoART pharmacokinetics and pharmacodynamics to help ensure optimal therapeutic benefits. How the data is used to design human trials will be carefully developed as rodent then monkey pharmacokinetic examinations are undertaken.