Characterization of Nanodiamond-based anti-HIV drug Delivery to the Brain

Human Immunodeficiency Virus Type 1 (HIV-1) remains one of the leading causes of death worldwide. Present combination antiretroviral therapy has substantially improved HIV-1 related pathology. However, delivery of therapeutic agents to the HIV reservoir organ like Central nervous system (CNS) remains a major challenge primarily due to the ineffective transmigration of drugs through Blood Brain Barrier (BBB). The recent advent of nanomedicine-based drug delivery has stimulated the development of innovative systems for drug delivery. In this regard, particular focus has been given to nanodiamond due to its natural biocompatibility and non-toxic nature–making it a more efficient drug carrier than other carbon-based materials. Considering its potential and importance, we have characterized unmodified and surface-modified (-COOH and -NH2) nanodiamond for its capacity to load the anti-HIV-1 drug efavirenz and cytotoxicity, in vitro. Overall, our study has established that unmodified nanodiamond conjugated drug formulation has significantly higher drug loading capacity than surface-modified nanodiamond with minimum toxicity. Further, this nanodrug formulation was characterized by its drug dissolution profile, transmigration through the BBB, and its therapeutic efficacy. The present biological characterizations provide a foundation for further study of in-vivo pharmacokinetics and pharmacodynamics of nanodiamond-based anti-HIV drugs.


Characterization of ND, ND-COOH, and ND-NH 2 .
To prepare the ND conjugated nanodrug, well-characterized ND was taken from different batches of ND powder prepared and characterized as per previously published protocol 36 . Figure 1 shows results of transmission electron microscopy (TEM), X-ray diffraction (XRD) and Raman spectroscopy characterization of nanodiamond powders. According to TEM, the average particle size of nanodiamond was 5 nm (3-6 nm as per manufacturer's specifications) ( Fig. 1A-C). The TEM images also showed that the primary diamond particles were partly agglomerated. Measured XRD pattern (Mo K α -radiation) of nanodiamond powder was a characteristic for good quality nanodiamonds. Two broad peaks correspond to diffraction from atomic planes with Miller indices (111) and (220) of the cubic diamond structure. Figure 1E shows Raman spectra of three ND that were used in this study: (a) unmodified, (b) -COOH surface modified and (c) -NH 2 surface modified surface modifications. Raman spectra were collected using Ar + ion laser excitation (λ = 514.5 nm) and show vibrational feature at 1326 cm −1 which were a first-order diamond peak. The peak at 1619 cm −1 was a superposition of sp 2 carbon (G-band) and OH groups on the surface. Both XRD and Raman study showed no graphitization of -COOH, -NH 2 modified ND. Nonetheless, it was important to consider that the overall formulation should be nontoxic to neuronal cells to consider its potential for further study.

ND-COOH induced more ROS compared to ND and ND-NH 2 on SK-N-MC (ROS assay). In order
to determine which formulation would be ideal for anti-HIV-1 drug delivery, it was important to observe whether any of these formulations by itself can induce ROS production to neuronal cells. In this regard, neuroblastoma cells (SK-N-MC) were treated with different concentrations of ND, ND-COOH, and ND-NH 2 , respectively, to observe the effect of these formulations on ROS production. As explained in the (Fig. 2), ND, ND-COOH, and ND-NH 2 had a varied effect on SK-N-MC with respect to ROS production. Compared to antioxidant (catalase) and positive control (H 2 O 2 ), ND and ND-NH 2 formulation treatment did not exhibit any significant changes in ROS production at the different concentrations tested in this study. Whereas, ND-COOH treated cells showed significantly higher ROS release in all tested concentrations on SK-N-MC indicating overall cytotoxicity. Based on ROS assay, it was observed that ND-COOH formulation was substantially inducing more ROS production to SK-N-MC compared to ND and ND-NH 2 .
ND was found to be nontoxic to SK-N-MC cells compared to ND-COOH and ND-NH 2 (MTS assay). ND, ND-COOH, and ND-NH 2 were also evaluated for their effect on the cell viability of SK-N-MC (Fig. 3). The formulations were introduced separately at different concentrations (0.5-1000 µg/ml) to SK-N-MC and incubated for 24 h; MTS assay was then performed as per the manufacturer's instruction (G3582, Promega, Madison, WI, USA). Figure 3 showed that SK-N-MC cell viability did not significantly change with increasing concentration of ND or ND-NH 2 compared to control. Whereas, ND-COOH treatment had a significant effect on reducing cell viability at concentrations higher than 40 µg/ml. Considering the chemical characterization, cytotoxicity, and ROS assay, it was observed that ND and ND-NH 2 were more biocompatible and less toxic to neuronal cells. ND adsorbed comparatively more drug than ND-COOH. Adsorption isotherm of EFV on unmodified ND (milligrams of EFV absorbed per 1 g of ND) (in blue) as a function of C eq (concentration of adsorbate in solution which is equilibrium with ND) is shown in (Fig. 4). The other formulations ND-NH 2 (in red) and  Effect of ND, ND-COOH, & ND-NH 2 on ROS production on SK-N-MC cells: Three formulations were exposed at different concentrations (10-1000 µg/ml) to SK-N-MC cells for 24 h. At the end of incubation, ROS production was measured in treated cells compared to untreated cells. The ROS production was measured in terms of mean ± SE relative fluorescence units (RFU) of eight independent experimental values. The statistical significance between untreated (ND) and ND-COOH treated cells were expressed as p values (**p < 0.001, ***p < 0.0003). There was no statistical significance between untreated (ND) and ND-NH 2 groups. ND-COOH (in black) were also presented in Fig. 4 to demonstrate comparative adsorption of EFV by these formulations. Experimental points were fit by Langmuir adsorption model. This empirical model suggests homogeneous monolayer absorption of the drug on the surface of the adsorbent. Maximum adsorption capacity obtained from Langmuir isotherm is 161 mg EVF/g for unmodified ND. However, this value was not achieved experimentally due to the low solubility of EFV. KL constant which characterizes the bonding strength between the adsorbate (EFV) and the adsorbent (ND) is 23 mL/mg. In this regard, the relatively low value of KL suggested easy desorption of EFV from the ND. Comparative analysis of EFV adsorption on ND, ND-COOH, and ND-NH 2 , respectively, indicated that with respect to time and increasing concentration, there was quite a distinction between the three formulations. In Fig. 4, unmodified ND and ND-NH 2 demonstrated a similar EFV adsorption compared to ND-COOH, which showed relatively low adsorption capacity for EFV at any given concentration and therefore not selected for further characterization in drug adsorption study. On the other hand, ND and ND-NH 2 showed a similar capacity for drug loading and were nontoxic to cells. As ND-NH 2 possessed a similar drug loading capacity and relatively similar biocompatibility as ND, therefore ND was selected for further in vitro characterization.
Sustained release of EFV from ND-EFV (Dissolution study). The sustained drug release profile of the ND-EFV formulation was determined in phosphate buffer saline (PBS) using equilibrium dialysis. The released drugs outside the dialysis bag were sampled at different time intervals (from 30 min up to 14 days) and measured by high-performance liquid chromatography (HPLC). As a positive control, equal amounts of the unformulated EFV (also called free drug or FD) were introduced directly to the in vitro PBS buffer, separately. Results were expressed as ng/ml of drug released from the ND relative to initial drug loading (Fig. 5). Compared to the FD (in blue), ND-EFV (in green) showed significant sustained drug release characteristics indicating improved pharmacokinetics of ND-EFV in vitro. This observation showed probable in vivo drug release pattern where a free drug  is immediately released and get metabolized, whereas the ND-EFV sustained release pattern helps in CNS drug delivery where slow release of the drug is crucial for viral reservoir reduction. The overall chemical characterization of ND-EFV established a potential nanoformulation for anti-HIV drug carrier. Considering drug loading capacity of ND and possible cytotoxicity of ND-EFV at higher concentration, 40 ug/ml ND-EFV was used for further biological characterization.
Drug release of ND-EFV in vitro environment through BBB. ND-EFV was further tested for its drug delivery capacity in crossing the BBB. An in vitro BBB model was set up to mimic the biological barrier that impacts drug delivery to the CNS. ND-EFV (40 ug/ml) was introduced into the upper chamber of the in vitro BBB model and drug content was monitored at the lower chamber as it crossed the other side of the BBB from 30 min up to day two. Unformulated EFV (40 g/ml) was also introduced in a separate setup, which served as a positive control. The BBB drug release study showed that unformulated EFV possesses a very robust drug release in the brain side of the BBB model (Fig. 6). Nonetheless, ND-EFV specifically indicated significantly slower release of EFV compared to unformulated EFV. Thus, we have established that ND-EFV is a potential candidate for anti-HIV-1 drug delivery to the brain due to its ability to cross BBB and extend the retention time of EFV in the CNS.  ND-EFV had no significant effect on neuronal plasticity. In order to observe any deleterious effect of ND-EFV on neuron structural protein, we analyzed the expression of different synaptic plasticity genes in ND-EFV treated SK-N-MC as per published protocol 37,38 . Out of 84 human synaptic plasticity genes, we have observed significant up-regulation of three synaptic plasticity genes (CEBPD, EGR3, and GRIN1) in ND treated neuronal cells compared to control cells. We also observed significant down-regulation of GRM8 (3 fold), HOMER1 (4 fold), and IGF1 (4.8 fold) gene when compared to the untreated control cells (Fig. 7). Nonetheless, Fig. 7(d) gene-gene interaction analysis illustrated that all the genes that are affected by ND-EFV exposure are not directly related to each other with respect to their functional activity. Therefore, there was no connection between up-regulated or down-regulated genes. This observation over all indicated that there is no potential deleterious effect on neuronal plasticity of SK-N-MC in presence of ND-EFV. This finding also opens up a potential for the long-term use of ND-EFV without any side effects on human neurons.
Therapeutic efficacy study of ND-EFV. Anti-HIV-1 efficacy of ND-EFV was tested by introducing the formulation in HIV-1 infected primary human macrophages in vitro condition. Initially, Human peripheral blood mononuclear cells (PBMC) were isolated and incubated to be differentiated to macrophages. Following the differentiation, cells were infected with HIV and FD and ND-EFV were introduced separately to HIV-1 infected macrophages. HIV-1 p24 level at cell supernatant of drug-treated cells indicated a significant difference on viral inhibition by unformulated EFV and ND-EFV. The effect of ND-EFV on HIV-1 replication was observed to be very effective and sustained over a period of seven days. In contrast, the unformulated EFV could control viral replication up to day five, thereafter, p24 returned to untreated levels. Infected and untreated macrophages were kept as positive controls. Overall representation in Fig. 8 confirmed the efficacy of ND-EFV over HIV-1 replication compared to unformulated EFV. This further demonstrated the promise of using ND-EFV for targeted drug delivery towards the CNS. Nonetheless, the present study did not confirm the direct CNS drug delivery of Figure 7. Human synaptic plasticity gene expression in ND-EFV exposed SK-N-MC cells. 84 genes analyzed that is related to the synaptic plasticity of the neurons through PCR array analysis. The only genes that significantly up (in red) /down (in blue) regulated (± ≥3 fold) are shown on the table. (a) 3D-profile of fold change of synaptic plasticity genes in ND exposed SK-N-MC cells. (b) Representative figures for scatter plot analysis of the changes in synaptic plasticity gene expression in ND-EFV exposed SK-N-MC cells: Spots associated with individual human synaptic plasticity gene were collected and converted into log 10 scale. The central line indicates unchanged gene expression. The synaptic plasticity genes with expression levels higher or lower in treated neuronal cells than control cells are expected to produce dots that deviate from the centerline. The dots are allocated to positions that are above or below than the +3 fold or 3-fold line when the differences are greater than three folds. (c) Human synaptic plasticity genes expression in ND-EFV exposed SK-N-MC cells (fold change): Out of 84 genes analyzed, only genes significantly (± ≥ 3 fold) dysregulated were shown in this table (d) Gene-Gene interaction network for human synaptic plasticity genes dysregulated in ND-EFV exposed SK-N-MC cells. ND-EFV and possible consequences. In future, this formulation will be used to conjugate with a specific targeting agent (antibody, ligand, etc.) for direct drug delivery of anti-HIV-1 drugs to the CNS.

Discussion
EFV is one of the very effective NNRTIs that has been in use in present combined antiretroviral therapy (cART) for the treatment of HIV-1 infection. However, its low bioavailability, poor pharmacokinetics, and subsequent drug resistance have made it necessary to optimize therapeutic delivery agents to make EFV more targeted and effective [39][40][41] . Among the many nanocarbon materials that have been investigated for use as drug carriers, ND, is the most promising due to its many favorable properties, including, chemical inertness, biocompatibility, and easy availability compared to other nanocarbon materials 17 .
According to TEM study ( Fig. 1A-C), nanodiamond particles have a spherical morphology and average particle size in a range of 5-6 nm that is close to vendor's specifications (3-5 nm). As it should be expected, modifications of ND surface did not alter ND particles size, morphology or crystal structure (Fig. 1D). Raman spectra of all samples display the characteristic feature of the diamond phase (peak at 1326 cm −1 ). According to Mochalin et al., (2008) a broad feature around 1612 cm −1 can be assigned to graphitic carbon on ND surface (the G-band) with some contribution from hydroxyl groups that are chemo-or physisorbed onto the surface 42 . Considering the fact that cytotoxicity was one of the deciding factors for screening the right drug formulation for the neuronal cells, ROS and MTS assay was performed with all three formulations. As the neuronal cells are very sensitive to ROS, it was important to screen a formulation that does not induce significant amount of ROS production in the neuronal cells 43 . ROS study explained that ND-COOH can induce more ROS production than ND or ND-NH 2 . Further study on cytotoxicity effect of all three formulations also established that ND-COOH was significantly toxic at a higher concentration above 40ug/ml. Following the surface modification and cell cytotoxicity study, ND, ND-COOH and ND-NH 2 were loaded with EFV as per the protocol mentioned in the method. The EFV adsorption study on ND, ND-COOH, and ND-NH 2 clearly indicated the potential of ND and ND-NH 2 formulation over ND-COOH based on Lingmuir adsorption model. This observation also established the potential of ND and ND-NH 2 over ND-COOH. Moreover, the dynamic stability of surface modified and unmodified ND were characterized in sterile phosphate buffer saline (PBS), DMSO and in combination. Presently, the suspension stability was evaluated by visual observation in this study. Surface modified ND in these experiments had lower stability compared to unmodified ND. Unmodified ND was found to be stable for several hours in PBS suspensions and for several days in DMSO suspension (unpublished data). Experiments on further stability of ND/EFV suspensions is in progress and will be reported elsewhere. In the present study, ND was suspended in 1:9 DMSO-PBS (volume/volume) for chemical characterization. The objective of the study was to find a formulation that is nontoxic to neuronal cells and effective against HIV. In this regard, ND was found to be an ideal formulation because ND-COOH showed cytotoxicity and ND-NH 2 had similar drug loading as ND. Considering all these factors, ND was selected for further characterization in vitro. In the dissolution study, ND-EFV demonstrated an extended release of EFV from drug formulation up to fourteen days indicating much more stable formulation than FD. The possible mechanism of FD release delay could be caused by sustained drug release from the reservoir effect in polymeric vehicles, which may attenuate with time and another retention effect from the dialysis bag itself. It is already reported that the dialysis bag may affect the in vitro drug release 44 . Therefore, even considering this delay impact from dialysis bag, the dissolution profile of ND-EFV was still observed to be adequate for sustained drug release requirement. EFV drug release study in BBB model also explained the potential benefit of ND-EFV over FD with respect to higher drug retention and delivery capacity to the brain which is one of the major impediments of targeted drug delivery. In this regard, it is worth mentioning that in vitro BBB membrane also present a concentration difference of FD between upper and lower chamber of the BBB model that might influence the faster release of FD to the media at the lower chamber. In addition, previous studies have also established the unformulated EFV exposure can disrupt the integrity of BBB and possible toxicity to BBB endothelial cells 40,41 . This might also allow more drug to pass through the BBB in a shorter period of time. The robust release of FD in the brain can also cause neurotoxicity and will get metabolized much faster upon release. Whereas, the sustained release of drug from nanoformulation will provide a constant control on viral replication in the brain with limited toxicity 11 . Since ND-EFV has demonstrated the stability of the biological environment and extended release of EFV crossing BBB, it provided a strong evidence to use this nanoformulation for anti-HIV drug development.
The present study also investigated the direct effect of ND-EFV on the expression of eighty-four key genes of neurons that are related to synaptic alteration during learning and memories. Alteration of these genes has a direct effect on some of the neuronal events like long-term potentiation (LTP), Long-term depression (LTD) etc. and those could be few potential side effects of ND-EFV. The synaptic plasticity gene array showed that there was no immediate side effect of ND-EFV on neuronal cells. This was also an important observation since unformulated EFV has toxic effects on neurons and brain cells 40,41 . The therapeutic efficacy study also indicated that FD had an immediate effect on HIV replication. However, its efficacy significantly decreased within five days. On the other hand, ND-EFV could control viral replication up to seven days and beyond establishing further the suitability of ND-EFV for long-term anti-HIV therapy.
The investigation of ND towards drug delivery and imaging has only begun very recently. Indeed, to our knowledge, the present study has shown for the first time the application of ND in targeted anti-HIV-1 drug delivery towards the CNS. Our study has established that ND-based nanodrugs are nontoxic to the CNS and specific molecular characterizations of these nanodrugs can cross the BBB to deliver drugs to the CNS. Most importantly, the initial chemical characterization of ND and the following biological characterization of ND-EFV has revealed a promising candidate for nanodrug delivery of anti-HIV-1 drugs to the CNS. However, the present study could not confirm the fate of the ND after EFV release since it was done in in vitro microenvironment. In the future, further in vivo study on ND-EFV will be performed to maximize its potential use as a therapeutic agent for HIV-1 patients and its detailed metabolomics.

Characterization of nanodrug. Unmodified nanodiamond powder (ND) was used as it was for this study.
Amine-functionalized nanodiamond (ND-NH 2 ) was fabricated by the procedure reported 45 . 50 mg ND powder was dispersed in absolute ethanol via sonication for 30 min, and then, an excess solution of APTES was slowly dropped and stirred overnight at refluxing conditions on a water bath. The ND-NH 2 was centrifuged and subsequently washed with ethanol for at least 5 times, which was then dried at 80 °C under vacuum for 12 h. Oxidation (surface modification with carboxyl-groups) was performed at 425 °C in air for 2 h 46 .

Transition electron microscopy (TEM).
The preliminary characterization of the nanodrugs was performed using a Phillips CM-200 200 kV Transmission Electron Microscope (TEM) with Energy Dispersive Spectroscopy (EDS) housed at FIU's Advanced Materials Engineering Research Institute (AMERI). TEM observation was helpful for determining the particle size, morphology and dispersity of the nanodrugs.

X-ray diffraction (XRD).
An X -ray diffraction study of ND was performed using a Bruker GADD/D8 X-Ray diffraction system with Apex Smart CCD and imaging plate detectors and direct-drive rotating anode. The MacSci rotating anode (Molybdenum) operates at 50 kV voltages and 20 mA current. The 2D diffraction patterns obtained were integrated using Fit2D software 47 . This X-ray diffraction method was used for structural characterization of the materials, particle size measurements of the crystalline phases, and for estimating the degree of crystallinity. Kaiser Optical Systems) with volume transmission gratings, a holographic notch filter, and a thermoelectrically cooled charge-coupled device CCD detector (Andor Technology). The Raman system has a spectral resolution of 4 cm −1 . The spectra were usually collected with 10 min exposure.

Raman spectroscopy characterization.
Reactive Oxygen Species (ROS) assay. ROS productions in SK-N-MC following exposure to different concentrations of ND, ND-COOH, and ND-NH 2 were detected using dichlorofluorescein diacetate assay (DCF-DA; Molecular Probes, Eugene, OR) as per previously published protocol 48 . Cells were cultured in 96-well plates (100,000 cells well) overnight to allow 70% confluence. The next day, cells were treated with different concentrations of ND, ND-COOH, ND-NH 2 (10,20,40,80,100,200, 400, 800, and 1000 µg/ml) for 24 h, respectively. The following day, cells were washed and pretreated with antioxidant, catalase (0.001 mg) for 2 h. After incubation, the cells were treated with DCF-DA (100uM) for 1 h at 37 °C, and finally, the cells were read in a BioTek Synergy HT microplate reader (excitation 485 nm and emission 528 nm; BioTek, Winooski, VT). Cells treated with H 2 O 2 (50 µM) for 2 h were included as a positive control. Solution, Madison, WI, USA) in complete 100 µl cell media for 1 h at 37 °C. Following incubation, an absorbance at 490 nm was measured using the BioTek plate reader (BioTek, Winooski, VT, USA). Untreated cells incubated with fresh media were used as a negative control. All measurements were taken as a mean of eight independent experimental values. The net absorbance (A) was taken as an index of cell viability. The cell viability was calculated as sample/control ×100%. The nanoformulations that did not cause more than 10% loss in cell viability after at least 24 hr exposure were considered nontoxic.

Drug adsorption study.
In order to make the initial calibration curve, concentrations of 1:9 DMSO-PBS and EFV were made at 5, 40, 50, and 100 µg/ml from 10 mg/ml stock solution of EFV in a total volume of 2 ml for each concentration. The initial calibration curve was created using the absorbance values of the corresponding concentrations (250 nm). The dilutions were thoroughly mixed and the optical density (OD) value of each concentration was measured twice using Hitachi U-2910 Spectrophotometer (UV Spec.) 2 mg of ND was then added to each glass tube. The tubes were then sonicated in an ultrasonic bath for 2 min. The tubes were then shaken at 25 °C and 190 rpm for approximately 24 h. The contents were then transferred to microfuge tubes and mixed using Eppendorf Thermomixer for 1h at 600 rpm. The solutions were then centrifuged at 13,000 rpm for 1 h. The supernatant of the solutions was removed (conservatively), leaving the ND pellet. The OD values of the supernatant for each concentration were measured using Hitachi U-2910. Based on the measured absorbance values before and after the adsorption, the concentrations of drug in the resulting solutions were calculated using the measured OD values.
Drug analysis through HPLC-MS/MS. The EFV concentration in biological samples was quantitated by a previously reported Selected reaction monitoring (SRM) method 49 In brief; the samples were homogenized with deionized H 2 O at a ratio of 1:2 (w/v). 1 mL of ice-cold acetonitrile was added to a 100 µL homogenized samples which spiked with 10 µL IS (2.0 g/mL lopinavir). The sample was then vortexed for 3 min, shaken for 15 min, and centrifuged at 16,000 × g for 10 min. The supernatant was aspirated, dried under vacuum at room temperature, and dissolved in 100 µL 50% methanol aqueous solvent. Before injection, the sample was centrifuged for 10 min at 16,000 g, 8 µL of supernatant was injected into the HPLC-MS/MS system for analysis. For all samples, the final concentration of IS was 200 ng/mL. The linearity of the method (in the range of 0.2-1000 ng/mL) was confirmed by preparing 3 standard curves on 3 different days.
Dissolution study of the ND-EFV. ND-EFV was tested for long-term sustained release capacity in physiological buffer media. Briefly, a solution of ND-EFV nanoparticles (0.5 ml, concentration: 2 mg/20 mg ratio drug/ ND) was placed into a dialysis bag (molecular cutoff: 6 kDa), sealed, and put into a tube filled with 30-40 ml dissolution solution (composition: 0.1% Tween 20 PBS with pH 7.4). This concentration was decided based on the initial standardization of drug loading on ND through adsorption study (unpublished data). Dispersion of the ND-EFV in PBS (pH 7.4) was placed in the dialysis bag (D-bag) (Pur-A-Lyzer Maxi Dialysis Kit, SIGMA) and dialyzed against the respective buffer solution at 37 °C at a speed of 150 rpm. At each time (30 min, day-1, day-5 and day-14), 100 µl of the solution was taken out from the tank and the equal amount of fresh buffer was replenished. The free EFV release study was also measured in similar conditions simultaneously. Final drug concentrations of the collected samples were determined by HPLC as mentioned above. Stratagene Mx3000p qRT-PCR thermal cycler. The threshold cycle (Ct) of each gene was determined by using the Stratagene MaxPro software. CT data was uploaded into the data analysis template on the manufacturer's website (http://pcrdataanalysis.sabiosciences.com/pcr/arrayanalysis.php). The relative expression of each gene in each ND-EFV treated group was compared with the expression in control cells and it was calculated on the website using the ∆∆ CT method with five housekeeping genes as controls. Controls are also included on each array for genomic DNA contamination, RNA quality, and general PCR performance. The result was represented in fold change in the expression in each gene of test and compared with control gene incorporating the statistical significance among the tested gene expression in the table form. The data also represented in 3D plot profile, scatter plot and volcano plot along with tabular form of up or down regulated gene expression. A gene-gene interactive analysis was also presented based on the level of expression including up & down regulation, co-expression, chemical modification and other protein related interaction. A direct gene-gene interaction would have indicated by direct joined lines between two or more genes in the plot and no gene-gene interaction was indicated as no interconnecting lines between different genes spots within the plot. This observation presents the valuable information about functional co-relation of different up and down regulated genes with respect to their function related to neuroplasticity.
Drug delivery through BBB. Primary Human Brain Microvascular Endothelial Cells (HBMVEC) and astrocytes were obtained from Science Research Laboratories, Carlsbad, CA. In vitro BBB model was established in the trans-well plate as per published protocol 50,51 . HBMVEC was inoculated at the upper side of 0.4 um pore size PTFE membrane tissue culture inserts (Corning, NY) at an initial concentration of 10 4 cells/well. A confluent layer of human astrocytes was grown on the lower side of the membrane. After incubation, the integrity of the BBB was measured with transendothelial electrical resistance (TEER) using Millicell ERS microelectrodes (Millipore, Billerica, MA, USA). The typical TEER values of untreated BBB were observed to be around ~200 Ω/cm 2 . For the drug delivery study, HBMVEC were allowed to grow up to 70% confluency and then ND-EFV (40 µg/ml) was introduced into the upper chamber of the transwell insert. Following that, media was collected at different time points (30 min, 1 h, Day-1, and Day-2) from the lower chamber and replenished with fresh media. The drug content of the collected media was measured by HPLC as mentioned above. Simultaneously, the unformulated drug was also introduced in a similar setup and monitored for drug release with respect to the time period.
Therapeutic efficacy. ND-EFV was investigated for anti-HIV-1 efficacy in the primary human macrophage as previously published protocol 48 . Human PBMC were obtained from a healthy subject through purchase from a local clinic and differentiated into macrophages as previously published 49 . PBMC were isolated with Ficoll-Hypaque (Pharmacia) gradient and cells were incubated for differentiation for 7 days in the presence of human macrophage colony stimulating factor (MCSF, Sigma) to macrophages. Following incubation, macrophages were infected with HIV-1 1Ba-L (NIH AIDS research and reference reagent program Cat# 510) (100 ng/ml) for 24 h. Following 24 h of infection (day 0), cells were washed to get rid of any unattached virus and fresh media was added. At the same time, 40 µg/ml of ND-EFV and FD was added separately to these cells in two different setups. The HIV-1 infected macrophages served as a positive control. The ND-EFV/ FD-treated cells were monitored for up to 10 days along with infected control cells. The cell supernatant was collected at day 0, 1, 2, 3, 5, and 7 days post-treatment. The viral p24 level in the supernatant was measured with p24 ELISA assay (Cat. # 0801200, Zeptometrix, USA). The p24 level at different time points provided the level of viral inhibition caused by ND-EFV and FD. The p24 level at the culture supernatant will be inversely proportional to the therapeutic efficacy of ND-EFV. Statistical analysis. Experiments were performed in multiple replicates and the data were presented as mean ± SEM. The statistical significance of each experiment was analyzed by two-tailed paired t-test and with one & two-way ANOVA test with GraphPad Prism software (GraphPad Prism Software Inc. San Diego, CA) and a p-value of ≤0.05 was considered as significant.