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Abstract Vaginal drug delivery has been shown to be a promising strategy for the prevention of sexually transmitted infections. Therapy delivered at the site of infection has many advantages including improved therapeutic efficacy, reduction in systemic toxicity, and reduced potential for development of drug resistance. We developed a “smart” combination intravaginal ring (IVR) that will (1) provide continuous release of hydroxychloroquine (HCQ) to induce T cell immune quiescence as the first-line of defense and (2) release nanoparticles containing anti-CCR5 siRNA only during sexual intercourse when triggered by the presence of seminal fluid as the second-line of defense. The IVR was capable of releasing HCQ over 25 days with a mean daily release of 31.17 ± 3.06 µg/mL. In the presence of vaginal fluid simulant plus seminal fluid simulant, over 12 × more nanoparticles (5.12 ± 0.9 mg) were released over a 4-h period in comparison to IVR segments that were incubated in the presence of vaginal fluid simulant alone (0.42 ± 0.19 mg). Anti-CCR5 siRNA nanoparticles were able to knockdown 83 ± 5.1% of CCR5 gene expression in vitro in the CD4+ T cell line Sup-T1. The IVR system also demonstrated to be non-cytotoxic to VK2/E6E7 vaginal epithelial cells. Graphical abstract

We designed and engineered novel intravaginal ring (IVR) medical devices via fused deposition modeling (FDM) three-dimensional (3D) printing for controlled delivery of hydroxychloroquine, IgG, gp120 fragment (encompassing the CD4 binding site), and coumarin 6 PLGA-PEG nanoparticles (C6NP). The hydrophilic polyurethanes were utilized to 3D-print reservoir-type IVRs containing a tunable release controlling membrane (RCM) with varying thickness and adaptable micro porous structures (by altering the printing patterns and interior fill densities) for controlled sustained drug delivery over 14 days. FDM 3D printing of IVRs were optimized and implemented using a lab-developed Cartesian 3D printer. The structures were investigated by scanning electron microscopy (SEM) imaging and in vitro release was performed using 5 mL of daily-replenished vaginal fluid simulant (pH 4.2). The release kinetics of the IVR segments were tunable with various RCM (outer diameter to inner diameter ratio ranging from 1.12 to 2.61) produced from FDM 3D printing by controlling the printing perimeter to provide daily zero-order release of HCQ ranging from 23.54 ± 3.54 to 261.09 ± 32.49 µg/mL/day. IgG, gp120 fragment, and C6NP release rates demonstrated pattern and in-fill density-dependent characteristics. The current study demonstrated the utility of FDM 3D printing to rapidly fabricate complex micro-structures for tunable and sustained delivery of a variety of compounds including HCQ, IgG, gp120 fragment, and C6NP from IVRs in a controlled manner. Graphical abstract

RNA interference (RNAi)-mediated gene silencing offers a novel treatment and prevention strategy for human immunodeficiency virus (HIV) infection. HIV was found to infect and replicate in human brain cells and can cause neuroinfections and neurological deterioration. We designed dual-antibody-modified chitosan/small interfering RNA (siRNA) nanoparticles to deliver siRNA across the blood-brain barrier (BBB) targeting HIV-infected brain astrocytes as a strategy for inhibiting HIV replication. We hypothesized that transferrin antibody and bradykinin B2 antibody could specifically bind to the transferrin receptor (TfR) and bradykinin B2 receptor (B2R), respectively, and deliver siRNA across the BBB into astrocytes as potential targeting ligands. In this study, chitosan nanoparticles (CS-NPs) were prepared by a complex coacervation method in the presence of siRNA, and antibody was chemically conjugated to the nanoparticles. The antibody-modified chitosan nanoparticles (Ab-CS-NPs) were spherical in shape, with an average particle size of 235.7 ± 10.2 nm and a zeta potential of 22.88 ± 1.78 mV. The therapeutic potential of the nanoparticles was evaluated based on their cellular uptake and gene silencing efficiency. Cellular accumulation and gene silencing efficiency of Ab-CS-NPs in astrocytes were significantly improved compared to non-modified CS-NPs and single-antibody-modified CS-NPs. These results suggest that the combination of anti-Tf antibody and anti-B2 antibody significantly increased the knockdown effect of siRNA-loaded nanoparticles. Thus, antibody-mediated dual-targeting nanoparticles are an efficient and promising delivery strategy for inhibiting HIV replication in astrocytes. Graphical abstract Graphic representation of dual-antibody-conjugated chitosan nanoparticles for the targeted delivery of siRNA across the blood-brain barrier (BBB) for inhibiting HIV replication in astrocytes. a Nanoparticle delivery to the BBB and penetration. b TfR-mediated transcytosis of nanoparticles across the epithelial cells. c B2R-mediated endocytosis of nanoparticles in astrocytes. d The molecular interactions between HIV-1 Tat protein and Cyclin T1 and Tip110 cellular proteins. e A schematic representation of chitosan nanoparticles with its components. RNAPII RNA polymerase II, TAR transactivation response RNA element, LTR long terminal repeat, Ab antibody, CS chitosan, TPP tripolyphosphate.