Polyanionic carbosilane dendrimers as a new adjuvant in combination with latency reversal agents for HIV treatment

Abstract

Background: The major obstacle impeding human immunodeficiency virus-1 (HIV-1) eradication in antiretroviral treatment (ART) treated HIV-1 subjects is the establishment of long-lived latently infected resting CD4⁺ T cells. Due to the fact that no drug has been effective, the search for new drugs and combinations are a priority in the HIV cure. Treatments based on nanotechnology have emerged as an innovative and promising alternative to current and conventional therapies. In this respect, nanotechnology opens up a new door for eliminating latent HIV infection. We studied the role of G1-S4, G2-S16 and G3-S16 polyanionic carbosilane dendrimers in the context of latent HIV-1 persistence. Moreover, we study the efficiency of these dendrimers in combination with latency reversal agents (LRAs) against HIV-1 infection.

Methods: J89GFP lymphocyte and THP89GFP monocyte derived cell lines latently infected with HIV-1 p89GFP were used as an in vitro model of latency for our study. Viability assays by 3-(4-5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) were performed to determine the working concentrations of dendrimers and LRAs. Both cell lines were treated with G1-S4, G2-S16 and G3-S16 either alone or in combination with bryostatin (BRY), romidepsin (RMD) or panobinostat (PNB) for 24 and 48 h. The expression pattern of GFP was measured by flow cytometry and referred as measure of viral reactivation.

Results and discussion: The combination treatment of the dendrimers with the protein kinase C (PKC) agonist did not modify the antilatency activity in J89GFP lymphocyte cell line. Interestingly enough, G3-S16 dendrimer alone and its combination with BRY, RMD or PNB showed a significant increased expression of GFP in the THP89GFP monocyte cell line.

Conclusion: We showed for the first time that nanoparticles, in this case, G3-S16 anionic carbosilan dendrimer may play an important role in new treatments against HIV-1 infection.

Keywords: Dendrimers; HIV-1 latency; Latency reversal agents; Nanomedicine.

Fig. 1

Molecular representation of dendrimers. a G1-S4 with 4 sulfate end groups, b G2-S16 with 16 sulfonate end groups and c G3-S16 with 16 sulfate end groups were represented. The generation of dendrimers is determined by considering that each generation corresponds to the number of repeating layers of silicon atoms

Fig. 2

Mitochondrial activity studies of polyanionic carbosilane dendrimers. MTT assays were performed in a J89GFP lymphocyte and b THP89GFP monocyte cell lines for 24 h and 48 h. Cell lines were treated with increased concentration range (0.2–50 μM) of G1-S4, G2-S16 or G3-S16 dendrimers. Non-treated (NT) cells were used as cell viability control. DMSO was used as cell death control. NT non-treated control, DMSO dimethyl sulfoxide. The mean values (mean ± SD) of two or three independent experiments are shown (*p < 0.05; **p < 0.005; ***p < 0.001)

Fig. 3

Mitochondrial activity studies of latency reversal agents. MTT assays were performed on J89GFP lymphocyte and THP89GFP monocyte cell lines during 24 h. Both cell lines were treated with BRY up to 100 nM, PST up to 20 µM, PNB up to 40 nM, and RMD up to 20 nM. Non-treated (NT) cells were used as cell viability control. DMSO was used as cell death control. NT non-treated control, DMSO dimethyl sulfoxide. The mean values (mean ± SD) of three independent experiments are shown (*p < 0.05; **p < 0.005; ***p < 0.001)

Fig. 4

Membrane integrity studies of carbosilane dendrimers and LRAs combinations. LDH assays were performed on a J89GFP lymphocyte and b THP89GFP monocyte cell lines during 24 h and 48 h. Cell lines were treated with G1-S4 (10 µM), G2-S16 (10 µM) or G3-S16 (1 µM) dendrimers. Both cell lines were treated with BRY (100 nM), PST (20 µM), PNB (40 nM) or RMD (20 nM). Non-treated (NT) cells were used as cell viability control. NT non-treated control, BRY bryostatin, PST prostratin, RMD romidepsin, PNB panobinostat. The mean values (mean ± SD) of three independent experiments are shown (*p < 0.05; **p < 0.005; ***p < 0.001)

Fig. 5

Confocal microscope images of reactivation and LIVE/DEAD cells. J89GFP or THP89GFP cell lines were treated with BRY (100 nM), G2-S16 (10 µM) or G3-S16 (1 µM) for 48 h. After incubation, cells were stained with NUCLEAR-ID^® Blue/Red. Blue: live cells. Green: HIV-1 reactivation. Red: cellular death. Non-treated (NT) cells were used as cell viability control. DMSO was used as cell death control. NT non-treated control; DMSO dimethyl sulfoxide; BRY bryostatin. Representative images of two independent experiments performed in duplicate are shown

Fig. 6

Reactivation profile of BRY, PST, RMD and PNB in combination with G1-S4 (10 µM), G2-S16 (10 µM), G3-S16 (1 µM) dendrimers. J89GFP (a) and THP89GFP (b) cell lines were treated with BRY (100 nM), PST (20 µM), RMD (20 nM) and PNB (40 nm), either alone or in combinations with dendrimers for 24 h and 48 h. The integrated mean fluorescence intensity (iMFI, percentage of GFP expressing cells *MFI) of live cells was used as a measure of HIV-1 reactivation. NT non-treated control, BRY bryostatin, PST prostratin, RMD romidepsin; PNB panobinostat. The mean values (mean ± SD) of two or three independent experiments are shown (*p < 0.05; **p < 0.005; ***p < 0.001)