A proposed strategy to cure HIV uses latency-reversing agents (LRAs) to reactivate latent proviruses for purging HIV reservoirs. A variety of LRAs have been identified, but none has yet proven effective in reducing the reservoir size in vivo. Nanocarriers could address some major challenges by improving drug solubility and safety, providing sustained drug release, and simultaneously delivering multiple drugs to target tissues and cells. Here, we formulated hybrid nanocarriers that incorporate physicochemically diverse LRAs and target lymphatic CD4 + T cells. We identified one LRA combination that displayed synergistic latency reversal and low cytotoxicity in a cell model of HIV and in CD4 + T cells from virologically suppressed patients. Furthermore, our targeted nanocarriers selectively activated CD4 + T cells in nonhuman primate peripheral blood mononuclear cells as well as in murine lymph nodes, and substantially reduced local toxicity. This nanocarrier platform may enable new solutions for delivering anti-HIV agents for an HIV cure.

Nanocarrier drug delivery systems provide a promising approach for overcoming these three challenges to using LRAs ( 21 – 23 ). Specifically, nanocarrier drug delivery systems allow LRAs to be delivered specifically to target cells such as CD4 + T cells within organs of interest, thus achieving high concentrations at relevant sites while minimizing off-target effects by keeping concentrations low at other sites. However, few studies have reported on the use of nanocarriers for HIV cure, and those studies used single LRAs ( 24 – 27 ). Of these studies, none has investigated CD4-targeting or lymphatic tissue–targeting functions in vivo. Here, we developed lipid-coated poly(lactic-co-glycolic acid) (PLGA) nanoparticles (termed “LCNPs”) to address the current barriers to LRA treatment. Through simple chemical synthesis and bioconjugation processes, we successfully loaded a variety of LRAs into our LCNPs and formulated them to target CD4 + T cells in the LNs. We demonstrate that these LCNPs can co-deliver multiple LRAs with varying release kinetics for synergistic latency reversal, and successfully traffic to LNs after subcutaneous administration. Our CD4-targeted LCNPs incorporate a promising LRA candidate, ingenol-3-angelate (Ing3A), inducing long-acting and specific CD4 + T cell activation in the complex lymphatic environment while substantially reducing local toxicity compared to the free drug. Our approach is unique and potentially transformative because we investigate agents of multiple mechanistic classes and specifically target them to the cell types and tissues sustaining latency.

Strategies to accelerate the decay of the HIV reservoir pool are being explored, but none has yet proven effective in reducing reservoir size in vivo. One strategy, which has been studied extensively, including in clinical studies, is to induce HIV reactivation using latency-reversing agents (LRAs) while continuing suppressive HAART ( 9 ). Upon reversal of the latent state, the reactivated cells can be eliminated by host immune responses or other killing strategies ( 10 ). This “shock and kill” approach is still controversial based on recent safety issues and limited success in reducing reservoir sizes in clinical settings ( 11 ). There are several hypotheses to explain the failures in clinical trials: First, these studies were restricted to individual LRAs from a single class ( 11 , 12 ), but recent evidence shows that combinations of multiple mechanistic classes may be needed to effectively overcome latency in vivo ( 13 – 15 ). Second, some LRAs are associated with high toxicity ( 16 , 17 ) or nonspecific T cell activation that is unnecessary for latency reversal and causes off-target toxicity ( 14 ). Last, LRAs might not achieve sufficient concentrations in the lymphatic tissues including lymph nodes (LNs) and gut-associated lymphoid tissue (GALT), where vast numbers of latently infected CD4 + T cells reside ( 1 , 18 ). Systemic administration of LRAs may work well on circulating CD4 + T cells, but those cells make up less than 2% of the body’s CD4 + T cells ( 19 , 20 ).

Highly active antiretroviral therapy (HAART) has revolutionized the treatment of HIV-1 and transformed it into a chronic disease but does not cure the infection. Long-term HIV infection is maintained by several factors including limited accessibility of antiretroviral drugs (ARVs) to certain anatomical sites where viral replication may occur ( 1 – 3 ), and latent infection of resting cells where integrated provirus is invisible to drug treatment as well as the immune system ( 4 , 5 ). The latent cell pool decays slowly despite suppressive HAART ( 6 , 7 ), requiring patients to take lifelong ARV regimens, associated with short- and long-term side effects ( 8 ).

RESULTS

Development of LCNPs loaded with mechanistically diverse LRAs Combinations of LRAs have recently shown synergistically enhanced potency for latency reversal as compared to single LRAs in multiple ex vivo assays (13). However, the varied physicochemical properties of different LRAs make combined formulation difficult to achieve and administer. In addition, the low potency but high toxicity of some LRAs limits their tolerability when used in monotherapy. To improve LRA potency while minimizing toxicity, we co-formulated and targeted the drugs to CD4+ T cells using lipid-polymer hybrid nanoparticles (termed LCNPs) surface-modified with targeting antibodies and with LRA incorporated into both the lipid bilayer and the polymer core (Fig. 1). Fig. 1 Strategies for loading LRAs into LCNPs. Hydrophobic LRAs were physically encapsulated into the PLGA core. Chol-but, as the prodrug of butyric acid, was inserted into the lipid bilayer. LRAs with hydroxyl or amine groups were conjugated to the PLGA followed by LCNP synthesis. Prs, prostratin; PANO, panobinostat; DIC, N,N′-diisopropylcarbodiimide; DMAP, 4-(dimethylamino)pyridine; RT, room temperature; EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; HoBt, 1-hydroxybenzotriazole; DIPEA, N,N-diisopropylethylamine. For ease of formulation, we first attempted to physically encapsulate all LRAs individually into LCNPs by co-dissolving each compound with PLGA in ethyl acetate, followed by a modified single-emulsion evaporation method as described previously (23). An initial 5 weight % (wt %) (ratio of LRA to PLGA and lipids) input of hydrophobic drugs JQ1, disulfiram (DSF), and Ing3A resulted in drug loadings of 1.70 ± 0.08 wt %, 2.54 ± 0.60 wt %, and 1.00 ± 0.04 wt %, respectively (Table 1). However, prostratin and romidepsin were encapsulated into LCNPs at less than 0.02 wt % (table S1). Physical encapsulation of panobinostat was also impractical because of its poor solubility in ethyl acetate and dichloromethane (DCM). We used DCM as an alternative solvent for romidepsin, which increased its drug loading to 0.2 wt % but failed to improve the loading of either prostratin or panobinostat. We were unable to encapsulate butyric acid, a short fatty acid that has shown potential in HIV latency reversal (28, 29), into the PLGA core. Instead, we inserted its prodrug cholesteryl butyrate (chol-but) into the LCNP lipid bilayer (30), achieving a loading of 2.72 wt % (Fig. 1). Table 1 Physicochemical properties of LCNP-formulated LRAs. BET, bromodomain and extra-terminal proteins; NF-κβ, nuclear factor κ–light chain enhancer of activated B cells; PKC, protein kinase C; HDAC, histone deacetylase; EE, encapsulation efficiency; n.d., not determined. View this table: For Ing3A, prostratin, and panobinostat, we also evaluated chemical conjugation into LCNPs (Fig. 1). The ester or amide bond formed between PLGA and LRA is hydrolyzed to release free LRAs under physiological conditions by host esterases or the esterase-like activity of human serum albumin (31). Chemical conjugation was expected to decrease the release rate of LRAs compared to physical encapsulation. Drugs were conjugated in a 1:1 molar ratio with a low–molecular weight PLGA to increase LRA content. However, we found that the LCNPs using PLGA of molecular weight less than 20 kDa led to poor particle size distribution and low stability (table S2). Therefore, to maximize LCNP drug loading while minimizing polydispersity, we selected a 24- to 38-kDa carboxyl-terminated PLGA. We used proton nuclear magnetic resonance (H-NMR) and high-performance liquid chromatography (HPLC) to verify the conjugation chemistry and efficiency. Both methods supported successful conjugation of each LRA with PLGA (figs. S1 and S2), with drug loadings of 1.62 ± 0.05 wt % (Ing3A), 1.08 ± 0.03 wt % (prostratin), and 0.31 ± 0.03 wt % (panobinostat). All LRA-loaded LCNPs showed well-distributed sizes with diameters of 160 to 190 nm and polydispersity index (PDI) of <0.1. Chol-but–inserted LCNPs (cbLCNPs) showed an increased size of ~220 nm in diameter and significant changes in ζ potential going from positive to negative, as would be expected because of the replacement of the positively charged lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) (Table 1). All LRA-loaded LCNPs were colloidally stable in physiological conditions for more than 10 days in cell culture media supplemented with 10% fetal bovine serum (FBS) (fig. S3).

Efficacy of LRA-loaded LCNPs in an in vitro model of latent HIV-1 infection LRA action requires release from LCNPs and subsequent binding to intracellular molecular targets. Previously, we and others have shown that physical encapsulation of drugs into LCNPs results in an initial burst release upon mixing with cell culture medium (23). We hypothesized that the physically encapsulated LRAs would show similar burst release, while chemically conjugated LRAs would be released slowly. To test this hypothesis, we measured release kinetics of LRAs from LCNPs in cell culture media (Fig. 2A). As expected, all physically encapsulated LRAs (JQ1, DSF, and Ing3A) showed burst release from LCNPs, resulting in 50% of the drug released between 1 hour and 1.6 days as measured by nonlinear regression (Table 1, Fig. 2A, and table S3). In contrast, chemically conjugated LRAs showed slower release kinetics. For Ing3A and prostratin, which were conjugated by Steglich esterification, it took more than 1 month to reach 50% release. Because Ing3A was both physically encapsulated and chemically conjugated, combining these separate formulations would provide both slow and fast release kinetics. For panobinostat conjugated by amide bond using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) chemistry, 50% of the drug was released over 1.4 days. Fig. 2 In vitro dose-response and time-dependent HIV-1 latency reversal correlates with LRA release kinetics from LCNPs. (A) Release kinetics of physically encapsulated LRAs (red triangle) and chemically conjugated LRAs (blue square) from LCNPs in cell culture media at 37°C. Data were fit by nonlinear regression, detailed in table S3. (B) Dose-response curve for latent HIV reactivation (indicated as a percentage of GFP+ cells) on J-Lat A1 cells incubated with single LRA formulations for 20 hours. Data were fit in the four-parameter log-logistic model, detailed in table S4. (C) Time-dependent curve for latent HIV reactivation of J-Lat A1 cells incubated with single LRA formulations over 5 days. (D) Cell viability of J-Lat A1 cells after incubation with single free LRAs or LCNP-formulated LRAs at different concentrations for 20 hours. Cell viability was measured by monitoring metabolic activity with the CellTiter-Blue Assay. Each experiment was performed once with n = 3 wells of each treatment. Data represent means ± SD. LRA/LCNP, LRA was physically encapsulated into LCNPs (red curve); LRA-LCNP, LRA was chemically conjugated to the PLGA (blue curve). Next, we compared the HIV-1 latency reactivation potency of these LCNP-formulated LRAs with free LRAs on the J-Lat Tat-GFP (A1) cell line model, which expresses green fluorescent protein (GFP) upon reactivation of latent HIV-1 integrated into the cell genome (32, 33). The percentage of GFP+ cells measured by flow cytometry was used to assess the potency of LRAs (Fig. 2B and fig. S4). Physically encapsulated JQ1, DSF, and Ing3A showed similar potency to the free drug used at the same concentrations after 20 hours of treatment. In contrast, all chemically conjugated LRAs (Ing3A, prostratin, and panobinostat) showed lower potency than the free drug, likely due to their slower release from LCNPs (table S3). To investigate whether the slow drug release kinetics could lead to delayed latency activation at later time points, we extended cell incubation with LRA formulations to 5 days and observed that the conjugated LRAs (Ing3A-LCNP, Prs-LCNP, and PANO-LCNP) showed accumulated induction of GFP expression over time (Fig. 2C). In contrast, the free LRAs (DSF, Ing3A, and prostratin) and the physically encapsulated drugs (DSF/LCNP and Ing3A/LCNPs) exhibited constant activity over 5 days. Exceptions are physically encapsulated JQ1 and free panobinostat, which also showed accumulated effects. All the LCNP-formulated LRAs showed equal or lower cytotoxicity compared to the free drug (Fig. 2D), meaning that LCNPs could potentially deliver higher LRA doses required for efficacy while avoiding the high toxicity. cbLCNPs induced only ~5% GFP expression in this cell model at the highest concentration we could achieve (fig. S5A). However, because chol-but reduces nonspecific cell binding (34), we kept it in our formulations for the subsequent targeting and animal studies.

Synergistic induction of HIV-1 mRNA levels by Ing3A-LCNP and JQ1/LCNP in CD4+ T cells from virally suppressed individuals Mechanistically distinct LRAs delivered in combination have shown synergistic interactions in ex vivo latent HIV-1 reactivation (13, 14). To identify the best LRA formulations to evaluate for HIV-1 latency reversal in human clinical samples, we first measured viral reactivation and cytotoxicity for every pairwise combination of LCNP-formulated LRA using J-Lat A1 cells. To better compare therapeutic index in future planned nonhuman primate (NHP) studies, our criterion for selecting LRA combinations was that both the free and LCNP-formulated drugs were nontoxic. Hence, we balanced trade-off between toxicity, potency, and synergy for selecting LRA combinations. LRA concentrations were chosen to achieve similar induction of GFP expression (~20% GFP+ cells) when dosed individually, and this concentration was used to measure efficacy of the drug combination (Fig. 3, A and B). Most binary combinations showed higher GFP induction than the corresponding single drugs. We used the Bliss independence model to select LRAs with synergistic effects as measured by Δfa xy > 0 (details in Materials and Methods). JQ1 in combination with any of the other four LRAs, and DSF in combination with Ing3A or prostratin, displayed synergy with Δfa xy above 0.1 (Fig. 3C). Ing3A-LCNP and PANO-LCNP were the most potent, as indicated by the lower dose necessary to achieve equivalent efficacy of ~20% GFP+ cells as well as their median effective dose (ED 50 ) (Figs. 2B and 3A and table S4). However, panobinostat demonstrated high cytotoxicity both individually and in combination with JQ1 (Figs. 2D and 3D). A similar relationship between efficacy and cytotoxicity was observed for DSF. DSF combined with prostratin in LCNPs led to the highest measured synergy and also high cell viability (Fig. 3, C and D). However, this LRA combination required use at 10-fold higher total dose (~18,000 nM) compared to the combination of JQ1/LCNP and Ing3A-LCNP (~1500 nM) (Fig. 3A). The free drug combination of DSF and prostratin also showed low viability (Fig. 3D). Last, the combination of Ing3A and JQ1 was chosen as it showed equivalent and synergistic activity at a lower dose with notably better viability (Fig. 3, A to D). Fig. 3 LCNP-formulated Ing3A and JQ1 enhance latent HIV reactivation, reduce cytotoxicity from J-Lat A1 cells, and synergistically increase HIV-1 mRNA expression in CD4+ T cells from infected individuals on suppressive HAART. (A) Concentrations of single and combination LCNP-formulated LRAs. LRA concentrations were calculated as total LRA in LCNPs. (B) In vitro latent HIV reactivation using single or combination LCNP-formulated LRAs on J-Lat A1 cells for 20 hours. (C) Calculation of synergy for LCNP-formulated LRA combinations using the Bliss independence model. Data are presented as the difference between the observed and predicted percentage of GFP+ cells. fa x or fa y , percentage of GFP expression by drug x or y; fa xy,O , observed percentage of GFP expression by a combination of drug x and y; fa xy,P , predicted percentage of GFP expression by a combination of drug x and y using the equation detailed in Materials and Methods. (D) Cell viability of J-Lat A1 cells after incubation with single or combination LRA formulations for 20 hours. Free or LCNP-formulated LRAs were dosed at the concentrations that achieved similar latent HIV reactivation (JQ1, 1488 nM; DSF, 14,840 nM; Ing3A, 3.5 nM; Prs, 251 nM; and PANO, 13.2 nM). The combination of JQ1 and Ing3A (✫) was chosen for high potency, synergy, and low cytotoxicity. The experiment (A to D) was performed once with n = 3 wells of each treatment. Data represent means ± SD. (E) Intracellular HIV-1 mRNA levels in CD4+ T cells isolated from peripheral blood of infected individuals and treated with free Ing3A, Ing3A-LCNP, free JQ1, JQ1/LCNP, or their binary combinations. Data are presented as fold induction relative to DMSO control. Statistical analysis was performed using paired one-way analysis of variance (ANOVA) with Bonferroni’s test comparing each group with the DMSO control. *P < 0.05, **P < 0.005. (F) Calculation of synergy for Ing3A and JQ1 combinations using the Bliss independence model. Data are presented as the difference between the observed and predicted fractional effect by the LRAs compared to the PMA/I positive control (see Materials and Methods for details). Statistical analysis was performed using paired Student’s t test. n.s., not significant. (G) Percentage of live cells after treatments, measured by live/dead staining following the fluorescence-activated cell sorting analysis. The experiments (E to G) were performed using peripheral blood from three different individuals, represented as a circle, square, or triangle. Each data point represents the mean fold induction of three replicate LRA treatments of 1 × 106 CD4+ T cells per individual. Error bars represent means ± SD from three individuals. CD4+ T cells isolated from HIV-1–infected individuals under suppressive ART were used to validate the activity of LCNP-formulated Ing3A and JQ1. Cells from three donors were used to test the induction of intracellular HIV-1 mRNA after treatment with Ing3A and JQ1 alone as well as their combination as free drugs and formulated in LCNPs. The single drugs in free or LCNP formulations increased intracellular HIV by one- to fourfold over dimethyl sulfoxide (DMSO) control treatment, but the differences were not statistically significant (Fig. 3E). Combining Ing3A and JQ1 significantly increased intracellular HIV-1 mRNA expression 6.1-fold (free Ing3A + JQ1) and 6.4-fold (LCNP-formulated Ing3A + JQ1), which was similar to the positive control of phorbol 12-myristate 13-acetate plus ionomycin (PMA/I; 8.1-fold induction). Because Ing3A and JQ1 act through different mechanisms, we compared the experimentally observed synergistic effect to the predicted effect by the Bliss independence model. We found that the combined effects from both free and LCNP-formulated Ing3A and JQ1 significantly exceeded the Bliss model prediction, resulting in a calculated Δfa xy values of 0.44 and 0.56 (Fig. 3F), respectively, validating that the combination synergistically induces intracellular HIV-1 mRNA from clinical samples. None of these LRA formulations caused significant cytotoxicity in comparison to the DMSO control (Fig. 3G).

CD4-targeted LCNPs selectively activate CD4+ T cells from pigtail macaque PBMCs We have previously shown that conjugating an anti-CD4 monoclonal antibody (mAb) to our optimized cbLCNPs led to high specificity for CD4+ T cells (34). Here, we conjugated an anti-CD4 mAb to our Ing3A-cbLCNPs (Fig. 4A). We chose conjugated Ing3A as the model drug because our screening showed that it is the most potent LRA and the chemically conjugated formulation provides sustained release, which is especially beneficial for targeting. To facilitate a higher level of passive targeting to the draining LNs (3, 35), we synthesized smaller LCNPs by optimizing the single-emulsion process with a different lipid-to-PLGA ratio (table S5). After conjugation with anti-CD4 mAb, the diameter of LCNPs remained less than 150 nm (Fig. 4A). We confirmed the activity of these smaller Ing3A-LCNPs in J-Lat A1 cells and observed slightly higher latency reversal compared to the larger particle (fig. S6A). To evaluate reactivation of the targeted formulations, we used primary cells obtained from pigtail macaque peripheral blood mononuclear cells (PBMCs) and compared CD69 expression between CD4+ (CD3+CD14−CD8−) and CD8+ (CD3+CD14−CD8+) T cells (fig. S7). CD69 is a marker for T cell activation and positively correlates with HIV-1 latency reversal (25, 36). PBMCs were treated with free drug, bare LCNPs, anti-CD4 LCNPs, and isotype LCNPs (Iso-LCNPs). The targeted Ing3A-CD4-cbLCNPs showed twofold increased CD69 expression in CD4+ T cells compared with CD8+ T cells (Fig. 4B), demonstrating the ability of our targeted LCNPs to specifically activate CD4+ T cells. Fig. 4 CD4-targeted LCNPs selectively activate CD4+ T cells from macaque PBMCs and accumulate in the draining LNs after subcutaneous injection to mouse left flank. (A) Ing3A-LCNP (<150 nm) conjugated with anti-CD4 mAb, resulting in size distribution measured by NanoSight. DSPE, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine; PEG, polyethylene glycol. (B) CD69 median fluorescence intensity (MFI) of CD4+ (CD14−CD3+CD8−) and CD8+ (CD14−CD3+CD8+) cells (left) and their respective MFI ratio (right) from PBMCs isolated from pigtail macaque blood and treated with free Ing3A, bare LCNPs, CD4-targeted LCNPs, and Iso-LCNPs for 20 hours. The experiment was performed once with blood samples from three pigtail macaques (n = 3). (C) Schedule of injection and tissue harvest. C57BL/6J mice were injected with phosphate-buffered saline (PBS), DiR-loaded CD4-cbLCNPs, and Iso-cbLCNPs subcutaneously at the left flank and sacrificed at 20 hours, 3 days, and 7 days for analysis. (D) Representative fluorescent images of inguinal LNs, spleen (top), and other major organs at 20 hours, 3 days, and 7 days. (E) Region of interest quantification of tissue fluorescence normalized by tissue mass. Statistical significance was calculated using paired two-way ANOVA with Bonferroni’s test. **P < 0.005, ***P < 0.005, ****P < 0.0001. Data represent means ± SD; n = 3 mice per group. (Photo credit: Shijie Cao, Department of Bioengineering, University of Washington)

CD4-LCNP biodistribution in mice over 7 days Most of the latent HIV reservoir is harbored in lymphatic tissues such as LNs, making it inaccessible to many anti-HIV-1 agents (2, 3, 21). Nanocarriers have been developed to target ARVs to anatomical HIV reservoirs including LNs, the central nervous system, and GALTs (21, 23). However, none of those nanocarriers have been used to deliver LRAs to lymphatic tissues. We hypothesized that our nanocarriers could traffic to draining LNs after subcutaneous administration and enter the lymphatic system to deliver LRAs to resident CD4+ T cells. To test this hypothesis, we labeled our LCNPs with DiR dye, subcutaneously injected them into the left flank of C57BL/6J mice, and tracked the LCNPs by imaging the draining and nondraining inguinal LNs, as well as other major tissues and organs (spleen, lung, liver, gut, kidney, heart, brain, and plasma). First, we surprisingly found that LCNPs with DOTAP in the lipid bilayer (dtLCNPs) showed little accumulation to LNs after subcutaneous injection (fig. S8A). In contrast, negatively charged cbLCNPs with chol-but in the lipid bilayer led to a marked increase in LN accumulation (fig. S8B). We assumed that the difference was due to nonspecific cell binding of neutrally to positively charged dtLCNPs at the injection site that hindered LN trafficking. Next, we found that the smaller-size optimized cbLCNPs (<150 nm) enhanced LN accumulation compared to the larger particle (fig. S8C). This was consistent with other reported studies that bigger nanoparticles might be more easily trapped at the injection site instead of efficiently draining to the LN (3, 35). Because of their desirable trafficking properties, as well as the increased targeting specificity reported previously (34), we moved forward with smaller CD4-targeted cbLCNPs for all animal studies. We selected 20 hours, 3 days, and 7 days as time points for investigating the biodistribution of CD4-cbLCNPs in mice (Fig. 4, C to E). For all three time points, both targeted and nontargeted cbLCNPs exhibited preferential accumulation in the left draining inguinal LN compared to other organs. The second highest fluorescent signal among all major organs was observed in the right inguinal LN. Comparing the targeted and nontargeted cbLCNPs, CD4-cbLCNPs showed 2.1- and 2.2-fold increased left LN accumulation over the Iso-cbLCNPs at 20 hours and 3 days, respectively, suggesting that CD4 targeting also led to higher LN accumulation and retention.