Non-Pigmented Ciliary Epithelium-Derived Extracellular Vesicles Loaded with SMAD7 siRNA Attenuate Wnt Signaling in Trabecular Meshwork Cells In Vitro

Primary open-angle glaucoma is established by the disruption of trabecular meshwork (TM) function. The disruption leads to increased resistance to the aqueous humor (AH), generated by the non-pigmented ciliary epithelium (NPCE). Extracellular vesicles (EVs) participate in the communication between the NPCE and the TM tissue in the ocular drainage system. The potential use of NPCE-derived EVs to deliver siRNA to TM cells has scarcely been explored. NPCE-derived EVs were isolated and loaded with anti-fibrotic (SMAD7) siRNA. EV’s structural integrity and siRNA loading efficiency were estimated via electron microscopy and fluorescence. Engineered EVs were added to pre-cultured TM cells and qRT-PCR was used to verify the transfer of selected siRNA to the cells. Western blot analysis was used to evaluate the qualitative effects on Wnt-TGFβ2 proteins’ expression. EVs loaded with exogenous siRNA achieved a 53% mRNA knockdown of SMAD7 in TM cells, resulting in a significant elevation in the levels of β-Catenin, pGSK3β, N-Cadherin, K-Cadherin, and TGFβ2 proteins in TM cells. NPCE-derived EVs can be used for efficient siRNA molecule delivery into TM cells, which may prove to be beneficial as a therapeutic target to lower intraocular pressure (IOP)

Doxorubicin liposomes cell penetration enhancement and its potential drawbacks for the tumor targeting efficiency

The clinical efficacy of the PEGylated doxorubicin liposomes (PLD) is limited by low tumor accumulation and limited intra-tumoral disposition. Decoration with the cell penetration enhancers (CPEs) can increase the PLD permeability via the biological barriers, however at the expense of enhanced distribution to the non-target organs and tissues, and may interfere with their tumor accumulation and with the resulting anti-cancer effects. We investigated the effect of the surface CPE agent tetraArg-[G-1] -distearoyl glycerol (DAG-Arg(4)) on the systemic and intra-tumoral accumulation of PLD, using a 4 T1-Luc murine orthotopic model of breast cancer, using several analytical approaches. CPE-decorated liposomes undergo efficient in vitro endocytosis, and delivered doxorubicin to the cell nuclei. In vivo, they had lower tumor and spleen accumulation, similar liver accumulation, and higher lung accumulation, as compared to those of the PLD. Despite the lower tumor accumulation, CPE-decorated liposomes induced more prominent in vivo anti-cancer effects, as compared to the PLD, apparently ascribable to the higher intra-tumoral permeability mediated by the CPE surface residues. Overall, liposomes decoration with the CPE residues had mostly beneficial effects on their systemic and intra-tumoral disposition. The mechanisms of the CPE-mediated effects on the liposome disposition should be further assessed with additional experimental models using robust analytical methods with high spatial resolution

Inhibition of Gene Expression and Cancer Cell Migration by CD44v3/6-Targeted Polyion Complexes

In recent years, siRNA technology has emerged as a promising strategy for gene silencing in cancer therapy. We have designed novel CD44-targeted polyion complexes (PICs) composed of poly­(ethylene glycol)-<i>block</i>-polyethylenimine (PEG-<i>b</i>-PEI) and laminin-derived peptides (mA5G27D or mA5G27F) for in vivo siRNA delivery and gene silencing in tumors. The full-length A5G27 peptide (RLVSYNGIIFFLK), from which mA5G27D and mA5G27F are derived, binds to CD44v3 and CD44v6 and inhibits tumor cell migration, invasion, and angiogenesis. Thus, when attached to the surface of PICs, A5G27-based peptides can serve both as targeting ligands to navigate siRNA molecules directly to CD44-overexpressing tumors, and as anti-migratory agents to inhibit tumor progression. The mA5G27D- or mA5G27F-harboring PEG-<i>b</i>-PEI copolymers strongly condensed siRNA molecules into nanosized PICs presenting positive surface charges, low in vitro cytotoxicity, and high serum stability. mA5G27D- or mA5G27F-bearing PICs demonstrated high efficacy and selectivity in delivering siRAC1 into CD44-overexpressing cells, thereby silencing RAC1 mRNA and protein levels in such cells. These PICs presented substantial anti-migratory features in vitro and accumulated significantly in SK-OV-3 tumor-bearing mice, following 3 sequential intraperitoneal (i.p.) injections. Treatment of mice with 8 or 9 sequential parenteral (intravenous, (i.v.) or i.p.) injections of mA5G27F-PEG-<i>b</i>-PEI/siRNA efficiently inhibited tumor growth in two different CD44-overexpressing tumor mouse models (A549 and SK-OV-3), regardless of the type of siRNA (siPLK1 or siLUC) used. The results thus reveal the potential utility of this system for targeted delivery of siRNA molecules into solid tumors to prolong the survival time of mice, while at the same time reducing potential toxicity

The effects of pravastatin on the normal human placenta: Lessons from <i>ex-vivo</i> models

<div><p>Introduction</p><p>Research in animal models and preliminary clinical studies in humans support the use of pravastatin for the prevention of preeclampsia. However, its use during pregnancy is still controversial due to limited data about its effect on the human placenta and fetus.</p><p>Methods</p><p>In the present study, human placental cotyledons were perfused in the absence or presence of pravastatin in the maternal reservoir (PraM). In addition, placental explants were treated with pravastatin for 5, 24 and 72 h under normoxia and hypoxia. We monitored the secretion of placental growth factor (PlGF), soluble fms-like tyrosine kinase-1 (sFlt-1), soluble endoglin (sEng), endothelial nitric oxide synthase (eNOS) expression and activation and the fetal vasoconstriction response to angiotensin-II.</p><p>Results</p><p>The concentrations of PlGF, sFlt-1 and sEng were not significantly altered by pravastatin in PraM cotyledons and in placental explants compared to control. Under hypoxic conditions, pravastatin decreased sFlt-1 concentrations. eNOS expression was significantly increased in PraM cotyledons but not in pravastatin-treated placental explants cultured under normoxia or hypoxia. eNOS phosphorylation was not significantly affected by pravastatin. The feto-placental vascular tone and the fetal vasoconstriction response to angiotensin-II, did not change following exposure of the maternal circulation to pravastatin.</p><p>Conclusion</p><p>We found that pravastatin does not alter the essential physiological functions of the placenta investigated in the study. The relevance of the study lays in the fact that it expands the current knowledge obtained thus far regarding the effect of the drug on the normal human placenta. This data is reassuring and important for clinicians that consider the treatment of high-risk patients with pravastatin, a treatment that exposes some normal pregnancies to the drug.</p></div

The effect of pravastatin on placental eNOS expression and activation in the human perfused cotyledon.

<p>Isolated cotyledons were perfused for 5 h in the absence (Cntl) or presence of 0.2 micromol/L pravastatin in the maternal (PraM) circulation (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172174#pone.0172174.s002" target="_blank">S1 Fig</a>). A. a representative immunoblot demonstrating eNOS expression and activation, as detected by phosphorylation on serine 1177. B-C. The relative fold of eNOS and p-eNOS^Ser1177 expression is shown by mean ± SEM. D-E. The concentration of NO in collected samples were determined by measurements of nitrite and normalized to gram of perfused cotyledon. Cntl: n = 6; PraM: n = 11.</p

eNOS expression and activation in cultured placental explants exposed to various concentrations of pravastatin.

<p>Placental explants were incubated in the absence or presence of pravastatin (Pra; 1, 10, 200 or 2000 micromol/L) for 24 and 72 h (n = 5) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172174#pone.0172174.s003" target="_blank">S2 Fig</a>). A. a representative immunoblot demonstrating eNOS expression and activation, as detected by phosphorylation on serine 1177. B-C. The relative fold of eNOS and p-eNOS^Ser1177 expression is shown by mean ± SEM. D. The concentration of NO in collected samples were determined by measurements of nitrite and expressed relative to Cntl (untreated cultures).</p

Clinical characteristics of pregnancies.

<p>Clinical characteristics of pregnancies.</p

The effect of pravastatin on placental eNOS expression and activation under reduced O₂ conditions.

<p>A. The effect of pravastatin on sFlt-1 secretion. Incubation for 5 h with pravastatin (0.2 micromol/L) did not affect sFlt-1 secretion from placental explants cultured under reduced O₂ (hypoxia; 1% O₂). Secreted protein levels were normalized against placental explant weight and expressed relative to untreated cultures. B. five immunoblots (corresponding to 5 different normal placentas) demonstrating eNOS expression and activation in untreated explants (C) and explants treated with 0.2 micromol/L pravastatin (P) for 5 h. C-D. The relative fold of eNOS and p-eNOS^Ser1177 expression is shown by mean ± SEM. E. The concentration of NO in the culture medium were determined by measurements of nitrite and expressed relative to Cntl (untreated cultures). n = 5. Pra, pravastatin.</p