Cationic polyaspartamide-based nanocomplexes mediate siRNA entry and down-regulation of the pro-inflammatory mediator high mobility group box 1 in airway epithelial cells

Gioia, Sante Di; Sardo, Carla; Belgiovine, Giuliana; Triolo, Daniela; D’Apolito, Maria; Castellani, Stefano; Carbone, Annalucia; Giardino, Ida; Giammona, Gaetano; Cavallaro, Gennara; Conese, Massimo

doi:10.1016/j.ijpharm.2015.06.017

Cited by 12 publications

(17 citation statements)

References 60 publications

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“…The effect of siRNA on the target mRNA is exerted in the cytosol through the RNA-induced silencing complex (RISC) [ 34 ]. Thus, we studied the uptake and internalisation of siRNA by a cytofluorimetric assay, which allows to distinguish between the extracellular and intracellular siRNA molecules [ 35 ]. Since the target of downregulation studies is the coculture of hAMSCs and CFBE cells, cocultures at 1 : 5 hAMSC : CFBE ratio was used in this assay.…”

Section: Resultsmentioning

confidence: 99%

“…After 24 h of incubation with fluorochrome-conjugated siRNA, cells were treated or not with trypan blue before analysis. Trypan blue quenches extracellular fluorescence allowing to determine intracellular signal only [ 35 ]; therefore, whole cell-associated fluorescence is detected in cells not treated with trypan blue, while internalised fluorescence is revealed in the treated ones. As reported in the Supplementary Figure 1 , trypan blue-treated cells show almost 100% of positivity and this percentage is not different from that of cells evaluated in the absence of trypan blue pretreatment, thereby indicating that internalisation has occurred in all cells.…”

Section: Resultsmentioning

confidence: 99%

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Gap Junctions Are Involved in the Rescue of CFTR-Dependent Chloride Efflux by Amniotic Mesenchymal Stem Cells in Coculture with Cystic Fibrosis CFBE41o- Cells

Carbone

Zefferino

Beccia

et al. 2018

Stem Cells International

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We previously found that human amniotic mesenchymal stem cells (hAMSCs) in coculture with CF immortalised airway epithelial cells (CFBE41o- line, CFBE) on Transwell® filters acquired an epithelial phenotype and led to the expression of a mature and functional CFTR protein. In order to explore the role of gap junction- (GJ-) mediated intercellular communication (GJIC) in this rescue, cocultures (hAMSC : CFBE, 1 : 5 ratio) were studied for the formation of GJIC, before and after silencing connexin 43 (Cx43), a major component of GJs. Functional GJs in cocultures were inhibited when the expression of the Cx43 protein was downregulated. Transfection of cocultures with siRNA against Cx43 resulted in the absence of specific CFTR signal on the apical membrane and reduction in the mature form of CFTR (band C), and in parallel, the CFTR-dependent chloride channel activity was significantly decreased. Cx43 downregulation determined also a decrease in transepithelial resistance and an increase in paracellular permeability as compared with control cocultures, implying that GJIC may regulate CFTR expression and function that in turn modulate airway epithelium tightness. These results indicate that GJIC is involved in the correction of CFTR chloride channel activity upon the acquisition of an epithelial phenotype by hAMSCs in coculture with CF cells.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Gap Junctions Are Involved in the Rescue of CFTR-Dependent Chloride Efflux by Amniotic Mesenchymal Stem Cells in Coculture with Cystic Fibrosis CFBE41o- Cells

Carbone

Zefferino

Beccia

et al. 2018

Stem Cells International

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“…All biological assays were performed by using the airway epithelial cell line H441, obtained from papillary adenocarcinoma of the lung [ 20 ]. Cells were grown in RPMI-1640 medium supplemented with 10% FBS, 1% l -glutamine, and 1% penicillin/streptomycin.…”

Section: Methodsmentioning

confidence: 99%

Nanoparticle delivery of grape seed-derived proanthocyanidins to airway epithelial cells dampens oxidative stress and inflammation

Castellani

Trapani

Spagnoletta³

et al. 2018

J Transl Med

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BackgroundChronic respiratory diseases, whose one of the hallmarks is oxidative stress, are still incurable and need novel therapeutic tools and pharmaceutical agents. The phenolic compounds contained in grape are endowed with well-recognized anti-oxidant, anti-inflammatory, anti-cancer, and anti-aging activities. Considering that natural anti-oxidants, such as proanthocyanidins, have poor water solubility and oral bioavailability, we have developed a drug delivery system based on solid lipid nanoparticles (SLN), apt to encapsulate grape seed extract (GSE), containing proanthocyanidins.MethodsPlain, 6-coumarin (6-Coum), DiR- and GSE-loaded SLN were produced with the melt-emulsion method. Physicochemical characterization of all prepared SLN was determined by photon correlation spectroscopy and laser Doppler anemometry. MTT assay (spectrophotometry) and propidium iodide (PI) assay (cytofluorimetry) were used to assess cell viability. Flow cytometry coupled with cell imaging was performed for assessing apoptosis and necrosis by Annexin V/7-AAD staining (plain SLE), cell internalization (6-Coum-SLN) and reactive oxygen species (ROS) production (SLN-GSE). NF-κB nuclear translocation was studied by immunofluorescence. In vivo bio-imaging was used to assess lung deposition and persistence of aerosolized DiR-loaded SLN.ResultsPlain SLN were not cytotoxic when incubated with H441 airway epithelial cells, as judged by both PI and MTT assays as well as by apoptosis/necrosis evaluation. 6-Coum-loaded SLN were taken up by H441 cells in a dose-dependent fashion and persisted into cells at detectable levels up to 16 days. SLN were detected in mice lungs up to 6 days. SLN-GSE possessed 243 nm as mean diameter, were negatively charged, and stable in size at 37 °C in Simulated Lung Fluid up to 48 h and at 4 °C in double distilled water up to 2 months. The content of SLN in proanthocyanidins remained unvaried up to 2 months. GSE-loaded SLN determined a significant reduction in ROS production when added 24–72 h before the stimulation with hydrogen peroxide. Interestingly, while at 24 h free GSE determined a higher decrease of ROS production than SLN-GSE, the contrary was seen at 48 and 72 h. Similar results were observed for NF-κB nuclear translocation.ConclusionsSLN are a biocompatible drug delivery system for natural anti-oxidants obtained from grape seed in a model of oxidative stress in airway epithelial cells. They feature stability and long-term persistence inside cells where they release proanthocyanidins. These results could pave the way to novel anti-oxidant and anti-inflammatory therapies for chronic respiratory diseases.Electronic supplementary materialThe online version of this article (10.1186/s12967-018-1509-4) contains supplementary material, which is available to authorized users.

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“…The results obtained in this study are in line with previous studies which report on materials that exhibit a toxicity prole that are independent of the concentration. 53,54 The test material displaying percentage cell viability >70% can be considered as biologically safe and nontoxic to mammalian cells. 55 The biosafety of all ADs can be attributed to selection of non-toxic and biodegradable building blocks used for their synthesis.…”

Section: In Vitro Cytotoxicitymentioning

confidence: 99%

Non-ionic self-assembling amphiphilic polyester dendrimers as new drug delivery excipients

et al. 2017

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Cationic polyaspartamide-based nanocomplexes mediate siRNA entry and down-regulation of the pro-inflammatory mediator high mobility group box 1 in airway epithelial cells

Cited by 12 publications

References 60 publications

Gap Junctions Are Involved in the Rescue of CFTR-Dependent Chloride Efflux by Amniotic Mesenchymal Stem Cells in Coculture with Cystic Fibrosis CFBE41o- Cells

Gap Junctions Are Involved in the Rescue of CFTR-Dependent Chloride Efflux by Amniotic Mesenchymal Stem Cells in Coculture with Cystic Fibrosis CFBE41o- Cells

Nanoparticle delivery of grape seed-derived proanthocyanidins to airway epithelial cells dampens oxidative stress and inflammation

Non-ionic self-assembling amphiphilic polyester dendrimers as new drug delivery excipients

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