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. 2021 Feb 15:16:1189-1206.
doi: 10.2147/IJN.S287310. eCollection 2021.

In vitro Study on Synergistic Interactions Between Free and Encapsulated Q-Griffithsin and Antiretrovirals Against HIV-1 Infection

Farnaz Minooei  1 , Joel R Fried  1 , Joshua L Fuqua  2   3   4 , Kenneth E Palmer  3   4   5 , Jill M Steinbach-Rankins  2   3   4   5
Affiliations

In vitro Study on Synergistic Interactions Between Free and Encapsulated Q-Griffithsin and Antiretrovirals Against HIV-1 Infection

Farnaz Minooei et al. Int J Nanomedicine. .

Abstract

Introduction: Human immunodeficiency virus (HIV) remains a persistent global challenge, impacting 38 million people worldwide. Antiretrovirals (ARVs) including tenofovir (TFV), raltegravir (RAL), and dapivirine (DAP) have been developed to prevent and treat HIV-1 via different mechanisms of action. In parallel, a promising biological candidate, griffithsin (GRFT), has demonstrated outstanding preclinical safety and potency against HIV-1. While ARV co-administration has been shown to enhance virus inhibition, synergistic interactions between ARVs and the oxidation-resistant variant of GRFT (Q-GRFT) have not yet been explored. Here, we co-administered Q-GRFT with TFV, RAL, and DAP, in free and encapsulated forms, to identify unique protein-drug synergies.

Methods: Nanoparticles (NPs) were synthesized using a single or double-emulsion technique and release from each formulation was assessed in simulated vaginal fluid. Next, each ARV, in free and encapsulated forms, was co-administered with Q-GRFT or Q-GRFT NPs to evaluate the impact of co-administration in HIV-1 pseudovirus assays, and the combination indices were calculated to identify synergistic interactions. Using the most synergistic formulations, we investigated the effect of agent incorporation in NP-fiber composites on release properties. Finally, NP safety was assessed in vitro using MTT assay.

Results: All active agents were encapsulated in NPs with desirable encapsulation efficiency (15-100%), providing ~20% release over 2 weeks. The co-administration of free Q-GRFT with each free ARV resulted in strong synergistic interactions, relative to each agent alone. Similarly, Q-GRFT NP and ARV NP co-administration resulted in synergy across all formulations, with the most potent interactions between encapsulated Q-GRFT and DAP. Furthermore, the incorporation of Q-GRFT and DAP in NP-fiber composites resulted in burst release of DAP and Q-GRFT with a second phase of Q-GRFT release. Finally, all NP formulations exhibited safety in vitro.

Conclusions: This work suggests that Q-GRFT and ARV co-administration in free or encapsulated forms may improve efficacy in achieving prophylaxis.

Keywords: HIV-1 prevention; antiretrovirals; electrospun fibers; griffithsin; microbicide; nanoparticles; synergy.

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Conflict of interest statement

The authors declare they have no competing interests.

Figures

Figure 1
Figure 1
Scanning electron microscopy images of PLGA nanoparticles loaded with 10% w/w (A) Q-GRFT, (B) TFV, (C) RAL, and (D) DAP. Scale bars represent 200 nm.
Figure 2
Figure 2
The cumulative release of Q-GRFT, TFV, RAL, and DAP from PLGA nanoparticles as a function of (A) total active agent release or (B) the percent of total loading, after exposure to SVF for up to 14 d. Release values are shown as the mean ± standard deviation of three independent NP batches. Please note panel B y-axis is scaled to 20% to more easily visualize differences in release.
Figure 3
Figure 3
The IC50 curves of (A) free and (B) NP-encapsulated active agents after administration to TZM-bl cells 1 hr prior to HIV-1 pseudovirus infection. Infectivity values are normalized to uninfected cells and are shown as the mean ± standard deviation of three NP batches.
Figure 4
Figure 4
The IC50 curves for free ARVs and free Q-GRFT co-administration demonstrate synergistic interactions. (A) Free TFV + free Q-GRFT, (B) free RAL + free Q-GRFT and (C) free DAP + free Q-GRFT. (D) Fold decrease in the IC50 values of Q-GRFT and ARVs after co-administration to TZM-bl cells 1 hr prior to HIV-1 pseudovirus infection. The normalized infectivity values are shown as the mean ± standard deviation of three independent samples. Please note differences in log scale on the x-axis.
Figure 5
Figure 5
The IC50 curves for free drugs and Q-GRFT NP co-administration demonstrate synergistic interactions. (A) Free TFV + Q-GRFT NPs, (B) free RAL + Q-GRFT NPs and (C) free DAP + Q-GRFT NPs. (D) Fold decrease in the IC50 values of Q-GRFT NPs and ARVs after co-administration to TZM-bl cells 1 hr prior to HIV-1 pseudovirus infection. The normalized infectivity values are shown as the mean ± standard deviation of three independent samples. Please note differences in log scale on the x-axis.
Figure 6
Figure 6
The IC50 curves for ARV NP and Q-GRFT NP co-administration. (A) TFV NPs + Q-GRFT NPs, (B) RAL NPs + Q-GRFT NPs and (C) DAP NPs + Q-GRFT NPs. (D) Fold decrease in the IC50 values of Q-GRFT NPs and ARV NPs after co-administration to TZM-bl cells 1 hr prior to HIV-1 pseudovirus infection. The normalized infectivity values are shown as the mean ± standard deviation of three independent samples. Please note differences in log scale on the x-axis.
Figure 7
Figure 7
Combination indices of free Q-GRFT and free ARVs, Q-GRFT NPs and free ARVs, and Q-GRFT NPs and ARV NPs co-administered to TZM-bl cells.
Figure 8
Figure 8
(A) Schematic of NP-fiber composites in which spheres may depict Q-GRFT NPs or DAP NPs. (B) The cumulative release of Q-GRFT and DAP from NP-fiber composites with an initial theoretical loading of 10 µg active agent per mg PEO fiber. Release values are shown as the mean ± standard deviation of three independent NP-fiber batches.
Figure 9
Figure 9
In vitro cytotoxicity of NPs encapsulating different agents administered to (A) VK2/E6E7 (B) End1/E6E7, and (C) Ect1/E6E7 cell lines using MTT assay. Viabilities are shown as the mean ± standard deviation from administration of three independent samples. Statistical significance between experimental groups, as calculated by one-way ANOVA, is represented by **p ≤ 0.01.
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