Hydroxychloroquine-conjugated gold nanoparticles for improved siRNA activity.

Current technology of siRNA delivery relies on pharmaceutical dosage forms to route maximal doses of siRNA to the tumor. However, this rationale does not address intracellular bottlenecks governing silencing activity. Here, we tested the impact of hydroxychloroquine conjugation on the intracellular fate and silencing activity of siRNA conjugated PEGylated gold nanoparticles. Addition of hydroxychloroquine improved endosomal escape and increased siRNA guide strand distribution to the RNA induced silencing complex (RISC), both crucial obstacles to the potency of siRNA. This modification significantly improved gene downregulation in cellulo. Altogether, our data suggest the benefit of this modification for the design of improved siRNA delivery systems.

Hypoxia-targeted siRNA delivery.

Altered vasculature and the resultant chaotic tumor blood flow lead to the appearance in fast-growing tumors of regions with gradients of oxygen tension and acute hypoxia (less than 1.4% oxygen). Due to its roles in tumorigenesis and resistance to therapy, hypoxia represents a problem in cancer therapy. Insufficient delivery of therapeutic agents to the hypoxic regions in solid tumors is recognized as one of the causes of resistance to therapy. This led to the development of hypoxia imaging agents, and the use of hypoxia-activated anticancer prodrugs. Here we show the first example of the hypoxia-induced siRNA uptake and silencing using a nanocarrier consisting of polyethyleneglycol 2000, azobenzene, polyethyleneimine (PEI)(1.8 kDa), and 1,2-dioleyl-sn-glycero-3-phosphoethanolamine (DOPE) units (the nanocarrier is referred to as PAPD), where azobenzene imparts hypoxia sensitivity and specificity. We report hypoxia-activated green fluorescent protein (GFP) silencing in vitro and its downregulation in GFP-expressing tumors after intravenous administration. The proposed nanoformulation represents a novel tumor-environment-responsive modality for cancer targeting and siRNA delivery.

Polymeric micelles containing reversibly phospholipid-modified anti-survivin siRNA: a promising strategy to overcome drug resistance in cancer.

The discovery that survivin, a small anti-apoptotic protein, is involved in chemoresistance, opens a new scenario to overcome the drug resistance in cancer. It was shown that siRNA can efficiently inhibit the expression of survivin in cancer cells. However, the clinical use of siRNA is still hampered by an unfavorable pharmacokinetic profile. To address this problem, earlier we developed a novel system to deliver siRNA into cancer cells. Namely, we reversibly modified the survivin siRNA with a phosphothioethanol (PE) portion via a reducible disulfide bond and incorporated the resulting siRNA-S-S-PE conjugate into nanosized polyethyelene glycol 2000-phosphatidyl ethanolamine (PEG2000-PE)-based polymeric micelles (PM), obtaining survivin siRNA PM. The activity of these nanopreparations was evaluated by survivin protein down-regulation, tumor cell growth inhibition, and chemosensitization of the treated tumor cells to paclitaxel (PXL). We found a significant decrease of cell viability and down-regulation of survivin protein levels after treatment with survivin siRNA PM in several cancer cell lines. In addition, the down-regulation of survivin by treating cells with survivin siRNA PM, elicited a significant sensitization of the cells to PXL, in both sensitive and resistant cancer cell lines. Finally, we demonstrated successful co-delivery of PXL and survivin siRNA in the same PM leading to superior therapeutic activity compared to their sequential administration. Our results support the use of this new platform for the treatment of the most aggressive tumors.

Neurosteroids in the mouse brain: behavioral and pharmacological effects of a 3 beta-hydroxysteroid dehydrogenase inhibitor.

Inhibition of the aggressive behavior of castrated male mice toward lactating female intruders by dehydroepiandrosterone (DHEA) is correlated with a decrease of pregnenolone sulfate (PREG S) concentrations in brain. We attempted to establish a cause to effect relationship by preventing the decrease of PREG S with trilostane (TRIL), a competitive inhibitor of delta 5-3 beta-hydroxysteroid dehydrogenase delta 5 --> 4 isomerase enzyme. Indeed, TRIL elicited a large increase of PREG levels in brain. Those of PREG S were, however, unchanged, and TRIL unexpectedly decreased the aggressive behavior of control castrated males and did not counteract the inhibition elicited by DHEA. The neurosedative progesterone (PROG) metabolite, 3 alpha-hydroxy-5 alpha-pregnan-20-one (TH PROG), undetectable in the brain of control mice, reached nanomolar concentration range in TRIL-treated ones. However, injection of appropriate amounts of PROG, producing an even larger increase of brain TH PROG, had no antiaggressive effect. Finally, the latter was attributed to the large (up to 80 nM) TRIL-induced increase of brain 3 alpha,21-dihydroxy-5 alpha-pregnan-20-one, which like TH PROG potentiates inhibitory gamma-aminobutyric acid (GABA)ergic neurotransmission.

Biosynthesis and assay of neurosteroids in rats and mice: functional correlates.

Pregnenolone (PREG), synthesized de novo in rodent brain, is the precursor of PREG sulfate (S) and progesterone (PROG). PROG is further converted to 5 alpha-pregnane 3, 20-dione (DH PROG) and to 3 alpha-hydroxy-5 alpha-pregnan-20-one (TH PROG). PROG, DH PROG and TH PROG have been measured in the brain of male and female rats. Neither PROG nor DH PROG disappeared from brain, contrary to plasma, after combined adrenalectomy (ADX) and gonadectomy (CX). Trilostane decreased PROG and increased PREG in the brain of CX+ADX rats and mice, in accordance with a precursor to product relationship. As previously described in CX male mice, the neurosteroid DHEA and its analog 3 beta-methyl-androst-5-en-17-one (CH3-DHEA) inhibited the aggressive behavior of female mice towards lactating female intruders. The decrease of biting attacks by DHEA was definitely more prominent in females neonatally imprinted with testosterone. The degree of inhibition of aggressive behavior was related to the decrease of PREG S concentrations in brain. The memory-enhancing effects of DHEA S and PREG S in male mice have been previously documented. Infusion of PREG S (12 fmol) into the nucleus basalis magnocellularis (NBM) of the rat after the acquisition trial enhanced memory performance in a two-trial recognition task (TTRT). Conversely, TH PROG (6 fmol), which potentiates GABAergic neurotransmission, disrupted performance when injected before the acquisition trial. Accordingly, we have found a positive correlation between the performances of 2-year-old rats in the TTRT and the concentrations of PREG S in the hippocampus, namely animals which performed best had the highest steroid levels.