Multifunctional Nanotherapeutics for the Treatment of neuroAIDS in Drug Abusers

HIV and substance abuse plays an important role in infection and disease progression. Further, the presence of persistent viral CNS reservoirs makes the complete eradication difficult. Thus, neutralizing the drug of abuse effect on HIV-1 infectivity and elimination of latently infected cells is a priority. The development of a multi-component [antiretroviral drugs (ARV), latency reactivating agents (LRA) and drug abuse antagonist (AT)] sustained release nanoformulation targeting the CNS can overcome the issues of HIV-1 cure and will help in improving the drug adherence. The novel magneto-liposomal nanoformulation (NF) was developed to load different types of drugs (LRAs, ARVs, and Meth AT) and evaluated for in-vitro and in-vivo BBB transmigration and antiviral efficacy in primary CNS cells. We established the HIV-1 latency model using human astrocyte cells (HA) and optimized the dose of LRA for latency reversal, Meth AT in in-vitro cell culture system. Further, PEGylated magneto-liposomal NF was developed, characterized for size, shape, drug loading and BBB transport in-vitro. Results showed that drug released in a sustained manner up to 10 days and able to reduce the HIV-1 infectivity up to ~40–50% (>200 pg/mL to <100 pg/mL) continuously using single NF treatment ± Meth treatment in-vitro. The magnetic treatment (0.8 T) was able to transport (15.8% ± 5.5%) NF effectively without inducing any toxic effects due to NF presence in the brain. Thus, our approach and result showed a way to eradicate HIV-1 reservoirs from the CNS and possibility to improve the therapeutic adherence to drugs in drug abusing (Meth) population. In conclusion, the developed NF can provide a better approach for the HIV-1 cure and a foundation for future HIV-1 purging strategies from the CNS using nanotechnology platform.

corning life sciences, Mexico). The upper chamber of this plate is separated from the lower one by a 10µm thick polycarbonate membrane possessing 3.0µm pores. In a sterile 24-well cell culture plate with the pore density of 2x10 6 pores/cm 2 and cell growth area of 0.33 cm 2 , 2x10 5 HBMEC and HA were grown to confluency on the upper chamber and underside of lower chamber respectively. To assess the effect of NF on the integrity of the in-vitro BBB model, after transmigration assay, paracellular transport of FITC-dextran was measured as previously described by us 6 . Also, intactness of in-vitro BBB was determined by measuring the transendothelial electrical resistance (TEER) using Millicell ERS microelectrodes (Millipore). Magneto-liposomal NF was collected from lower chambers after 3 hr of magnetic treatment and % transmigration was analyzed at different time points using ammonium thiocyanate-based photometric assay 6, 24 .
Intracellular uptake analysis and quantitative analysis: The HA were treated with different concentrations of FITC tagged MLs nanoformulation (50, 100 and 200 µg/mL). The cells were then harvested at 24 hr after treatment, washed and counted; equal amounts of cells (1×10 6 ) were aliquoted in 1.5 ml Eppendorf centrifuge tubes in PBS. Cell acquisition was performed using ImageStreamX Imaging Flow Cytometer (Amnis Corporation, Seattle, WA) equipped with INSPIRE software. A 60x magnification was used for all samples. A minimum of 10,000 cells was analyzed for each sample. Data analysis was performed using the IDEAS software (Amnis Corporation). FITC and DAPI were excited with a 100 mW of 488 nm argon laser. FITC and DAPI fluorescence was collected on channel two (505-560 nm) and channel seven (560-595 nm), respectively. Intensity adjusted bright field images were collected on channel one. Bright field area and total fluorescence intensity were calculated using IDEAS software. Data analysis was performed using the IDEAS software (Amnis Corporation). Data were compensated using a compensation matrix generated using singly stained samples. The compensated data was then gated using the following pattern. First, a focus gate was determined to eliminate cells that were not in the field of focus; second, the focused cells were gated to eliminate doublets and debris.
Gated data was used to generate histograms measuring fluorescence intensity (sum of all pixels in an image), median pixel intensity (pixel intensity value separating the brighter half from the less bright half), and max pixel intensity (intensity of the brightest pixels in an image) for each sample.
The IDEAS software contains wizards to measure internalization and count spots. For the spot counting wizard, subpopulations of cells with low and high NF numbers were manually identified as truth sets, and the software used these data sets to determine the number of FITC-NF spots per cell. The data were reported as a histogram of spots for each sample, and the median spot number was reported. Percent uptake was calculated by multiplying the number of spots per cell by the total cell number. This value (total number of NF taken up by cells) was then divided by the amount of NF added to the cells (which was based on the concentration determination from the ImageStreamX).

MRI and Image analysis:
During imaging sessions, mice were anesthetized under 1.5% isoflurane gas, placed in a body tube cradle and setup in a surface transmit/receive radio frequency coil system used for high-resolution imaging on a Magnex Scientific 4.7 Tesla MR scanner. T2 and T1 relaxometry pulse sequences were run on a Varian VnmrJ 3.1 console. Respiratory rates and core body temperature were monitored continuously throughout the experiments. Mice were first scanned without MNP treatment to establish a baseline and were then scanned following intravenous administration of the Magnetic NF. Scanning lasted 6 h, with a scan collected every 4 intervals (20 TE's total), repetition time (TR) = 3000 ms, field of view 24 mm 2 along the read, phase directions and 1 mm along the slice direction, and data matrix of 128 x 128 x 10 slices. We also assessed T1 using a saturation recovery sequence with TR (in ms) = 50, 500, 950, 1400, 1850, 2300, 2750, 3200, 3650, 4100, 4550, 5000. Signal averaging was used to increase the signal to noise. Images were imported into NIH Image J (rsbweb.nih.gov/ij) and the QuickVol plugin (Schmidt, et al., 2004) (http://www.quickvol.com) for processing of T2 maps. T2 maps were reconstructed from a non-linear regression of the exponential decay signal using the multi-TE value datasets. T1 maps were also reconstructed with QuickVol using a non-linear regression of the saturation recovery signal. Regions of interest (ROI's) were manually delineated using ITK-SNAP program (Yushkevich, et al., 2006). We selected areas near ventricles (dorsomedial striatum near lateral ventricles, hypothalamus near the 3 rd ventricle), and on 3 different ventricular areas (dorsal 3 rd ventricle, lateral ventricles, and the cerebral aqueduct). These were clearly visualized on T2 scans. T2 and T1 relaxation rates were assessed separately using MRI on a series of phantom tubes filled with a mixture of Agarose with varying concentrations of magnetic nanoparticle (0-100µM). Parameters similar to those indicated above were used for T2 mapping.

Figure-S3
In-vitro cytotoxicity evaluation of different LRA's