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. 2017 Aug;23(4):603-614.
doi: 10.1007/s13365-017-0538-8. Epub 2017 Jul 31.

Novel nanoformulation to mitigate co-effects of drugs of abuse and HIV-1 infection: towards the treatment of NeuroAIDS

Rahul Dev Jayant  1 , Venkata S R Atluri  2 , Sneham Tiwari  2 , Sudheesh Pilakka-Kanthikeel  2 , Ajeet Kaushik  2 , Adriana Yndart  2 , Madhavan Nair  3
Affiliations

Novel nanoformulation to mitigate co-effects of drugs of abuse and HIV-1 infection: towards the treatment of NeuroAIDS

Rahul Dev Jayant et al. J Neurovirol. 2017 Aug.

Abstract

Drug abuse (e.g., methamphetamine-Meth or cocaine-Coc) is one of the major risk factors for becoming infected with HIV-1, and studies show that in combination, drug abuse and HIV-1 lead to significantly greater damage to CNS. To overcome these issues, we have developed a novel nanoformulation (NF) for drug-abusing population infected with HIV-1. In this work, a novel approach was developed for the co-encapsulation of Nelfinavir (Nel) and Rimcazole (Rico) using layer-by-layer (LbL) assembled magnetic nanoformulation for the cure of neuroAIDS. Developed NF was evaluated for blood-brain barrier (BBB) transmigration, cell uptake, cytotoxicity and efficacy (p24 assay) in HIV-1 infected primary astrocyte (HA) in presence or absence of Coc and Meth. Developed magnetic nanoformulation (NF) fabricated using the LbL approach exhibited higher amounts of drug loading (Nel and Rico) with 100% release of both the therapeutic agents in a sustained manner for 8 days. NF efficacy studies indicated a dose-dependent decrease in p24 levels in HIV-1-infected HA (~55%) compared to Coc + Meth treated (~50%). The results showed that Rico significantly subdued the effect of drugs of abuse on HIV infectivity. NF successfully transmigrated (38.8 ± 6.5%) across in vitro BBB model on the application of an external magnetic field and showed >90% of cell viability with efficient cell uptake. In conclusion, our proof of concept study revealed that sustained and concurrent release of sigma σ1 antagonist and anti-HIV drug from the developed novel sustained release NF can overcome the exacerbated effects of drugs of abuse in HIV infection and may solve the issue of medication adherence in the drug-abusing HIV-1 infected population.

Keywords: Blood-brain-barrier (BBB); Drugs of abuse; Magnetic nanoparticle; NeuroAIDS; Sustain release formulation.

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Conflict of interest statement

Disclosure statement: The authors declare no competing financial interests.

Figures

Figure. 1
Figure. 1
Dose optimization studies in HA: (A & B) Different concentrations of Meth (10–250 µM) were tested to study the synergistic effect on HIV infection and its concentration dependent cytotoxicity profile; (C & D). Different concentrations of Coc (0.1–10 µM) were tested to study the synergistic effect on HIV infection and its concentration dependent cytotoxicity profile; (E & F) Rimcazole (Rico) dose optimization in presence of Coc and Meth for attenuating the synergistic effect on HIV infection. (G) Rimcazole concentration dependent cytotoxicity profile. HA (1×105 cells) were grown in 6 well culture plates and were infected with 20ng of HIV-1 clade B. After overnight infection, unbound virus was washed out with PBS and cells were infected for total of 5 days. After 5 DPI (day of post infection) Meth (10, 25, 250, 500, 1000 µM) and Coc (0.1, 1, 2.5, 5, 10 µM) were added and supernatant from each treated well was collected after 48 hour (Total 7 days of HIV infection). HIV infection levels were measured by p24 ELISA. (*, p ≤ 0.05; **, p ≤ 0.01; NS-Not Significant).
Figure 2
Figure 2
(A) In vitro Rico efficacy studies for attenuating Coc and Meth induced HIV infection: HA (1×105 cells) were grown in 6 well culture plates and were infected with 20ng of HIV-1B for overnight. Unbound virus was washed with PBS and cells were infected for 5 days. After 5 day of infection, HIV infected astrocytes were pretreated (1hour) with Rimcazole (2 and 50 µM) and then treated with optimized dose of Meth (25 µM) and Coc (1 µM). To study the Rico attenuation capacity, supernatant from each treated well was collected after 24 hour of treatment and HIV infection levels were measured by p24 ELISA. (*, p ≤ 0.05; **, p ≤ 0.01; NS-Not Significant); (B) Flow-cytometry analyses to assess expression of sigma (σ1) receptors: Primary astrocytes were treated with either cocaine and/or meth for 48 hour. Following treatment, cells were collected and stained with FITC labeled sigma receptor. Accuri (BD Biosciences) was used for fluorescence acquisition, and data were analyzed with FlowJo software (BD Biosciences). Red line indicates the untreated control, and blue line indicates the treated cells (test). Untreated control was gated based on the isotype control, and the treated cells (test) were gated based on the untreated cells.
Figure. 3
Figure. 3
Nanoformulation (NF) design and characterization: (A) Schematic representation of nanoformulation design; (B) Transmission electron microscopy of MNPs (average size range: 10 ±3 nm); (C) Effect of Dextran sulphate (DS) layer coating with respect to Nel and Rico loading on MNPs; (D) Zeta potential analysis for confirmation of drug deposition on MNPs; (E) Comparative cumulative release profile of uncoated Nel and uncoated Rico (F) Comparative cumulative release of dextran sulphate (DS) coating (1 layer [1BL] and two bilayers [2BL]) nanoformulation in phosphate-buffered saline (pH, 7.4) at 37°C.
Figure. 4
Figure. 4
(A) NF transmigration analysis using in-vitro BBB model: NF was added in the upper chamber of the BBB model, after 3 hour of treatment migrated plain MNP and NF in presence or absence of static magnetic (0.8 T) were calculated for iron content in the lower chamber. Results are expressed as mean ± SE of three independent experiments. Statistical significance was determined using unpaired Student’s t-test. (B) BBB integrity using FITC-dextran transport model: FITC-dextran transport was measured in BBB model after 3 hour of magnetic treatment, FITC-dextran was added on the upper chamber of the insert. After 3 hour of incubation, relative fluorescence units (RFUs) from the basal chambers of the inserts were measured. Results are expressed as % FITC-dextran transport with respect to the untreated control cultures (−ve control= no cell and +ve control= with BBB cells) and represented as mean ± SE of independent experiments. (C) Effect of time with respect to Nanoformulation BBB transmigration: NF was added in the upper chamber of the BBB model, after every 1hour till 6 hour of treatment migrated NF in presence of static magnetic (0.8 T) were calculated for iron content in the lower chamber. Results are expressed as mean ± SE of three independent experiments. Statistical significance was determined using unpaired Student’s t-test.
Figure. 5
Figure. 5
(A) NF efficacy in HIV-1 infected HA in presence of drug of abuse (Meth and Coc): HA (1 × 105 cells) were grown in 6 well culture plates and cells were infected with 20 ng of HIV-1 clade B for overnight. Unbound virus was washed with PBS and cell were infected for 5 days. On 6th day of infection, optimized Coc (1 µM) and Meth (25 µM) were added to cells and treated every day for next 8 days (total 14 days of HIV infection). Drug loaded NF (100 µg/ml) was added only once (on 6th day) to the respective wells and reduction in HIV infection levels were measured by using the p24 ELISA. Results were analyzed with respect to HIV-1 infection v/s HIV+(Meth+Coc) and HIV+(Meth+Coc) v/s NF treatment (*, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001; NS-Not Significant); (B) Qualitative analysis of cellular uptake of FITC-tagged LbL-NF in HA by fluorescence microscopy: FITC-tagged LbL-NF (concentration- 100 µg/mL) in HA after 6 hour of treatment. (A) Control cell bright field image; (B) Cell nuclei stained with DAPI (blue); (C) FITC tagged drug loaded NF (green); (D) Composite image show green and blue fluorescence inside the cells, confirms the cellular uptake of the NF. Images taken at 10× magnification using Fluorescence microscopy (Zeiss, Wetzlar, Germany); (C) In-vitro cytotoxicity of NF: Results show the percentage of cells viability after treatment with different NF concentrations (10, 20, 50 and 100 µg/mL). HA (1 × 105 cells) were grown in 6 well culture plates and cells were infected with 20 ng of HIV-1 for overnight. Uninfected virus was washed and cells were infected for 7 days. On 5th day of infection, NF was added at different concentrations to the respective wells and incubated for 24 and 48h respectively. After total 6th and 7th day of post infection (DPI), cell viability was measured by using the MTT assay (*, p ≤ 0.05; NS-Not Significant);
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