Silencing E3 Ubiqutin ligase ITCH as a potential therapy to enhance chemotherapy efficacy in p53 mutant neuroblastoma cells

P53 mutations are responsible for drug-resistance of tumour cells which impacts on the efficacy of treatment. Alternative tumour suppressor pathways need to be explored to treat p53- deficient tumours. The E3 ubiquitin ligase, ITCH, negatively regulates the tumour suppressor protein TP73, providing a therapeutic target to enhance the sensitivity of the tumour cells to the treatment. In the present study, two p53-mutant neuroblastoma cell lines were used as in vitro models. Using immunostaining, western blot and qPCR methods, we firstly identified that ITCH was expressed on p53-mutant neuroblastoma cell lines. Transfection of these cell lines with ITCH siRNA could effectively silence the ITCH expression, and result in the stabilization of TP73 protein, which mediated the apoptosis of the neuroblastoma cells upon irradiation treatment. Finally, in vivo delivery of the ITCH siRNA using nanoparticles to the neuroblastoma xenograft mouse model showed around 15–20% ITCH silencing 48 hours after transfection. Our data suggest that ITCH could be silenced both in vitro and in vivo using nanoparticles, and silencing of ITCH sensitizes the tumour cells to irradiation treatment. This strategy could be further explored to combine the chemotherapy/radiotherapy treatment to enhance the therapeutic effects on p53-deficient neuroblastoma

Manufacturing and characterisation of 3D-printed sustained-release Timolol implants for glaucoma treatment

Timolol maleate (TML) is a beta-blocker drug that is commonly used to lower the intraocular pressure in glaucoma. This study focused on using a 3D printing (3DP) method for the manufacturing of an ocular, implantable, sustained-release drug delivery system (DDS). Polycaprolactone (PCL), and PCL with 5 or 10% TML implants were manufactured using a one-step 3DP process. Their physicochemical characteristics were analysed using light microscopy, scanning electronic microscopy (SEM), differential scanning calorimetry (DSC) / thermal gravimetric analysis (TGA), and Fourier-transform infrared spectroscopy (FTIR). The in vitro drug release was evaluated by UV-spectrophotometry. Finally, the effect of the implants on cell viability in human trabecular meshwork cells was assessed. All the implants showed a smooth surface. Thermal analysis demonstrated that the implants remained thermally stable at the temperatures used for the printing, and FTIR studies showed that there were no significant interactions between PCL and TML. Both concentrations (5 & 10%) of TML achieved sustained release from the implants over the 8-week study period. All implants were non-cytotoxic to human trabecular cells. This study shows proof of concept that 3DP can be used to print biocompatible and personalised ocular implantable sustained-release DDSs for the treatment of glaucoma

A method for concentrating lipid peptide DNA and siRNA nanocomplexes that retains their structure and transfection efficiency

Nonviral gene and small interfering RNA (siRNA) delivery formulations are extensively used for biological and therapeutic research in cell culture experiments, but less so in in vivo and clinical research. Difficulties with formulating the nanoparticles for uniformity and stability at concentrations required for in vivo and clinical use are limiting their progression in these areas. Here, we report a simple but effective method of formulating monodisperse nanocomplexes from a ternary formulation of lipids, targeting peptides, and nucleic acids at a low starting concentration of 0.2 mg/mL of DNA, and we then increase their concentration up to 4.5 mg/mL by reverse dialysis against a concentrated polymer solution at room temperature. The nanocomplexes did not aggregate and they had maintained their biophysical properties, but, importantly, they also mediated DNA transfection and siRNA silencing in cultured cells. Moreover, concentrated anionic nanocomplexes administered by convection-enhanced delivery in the striatum showed efficient silencing of the β-secretase gene BACE1. This method of preparing nanocomplexes could probably be used to concentrate other nonviral formulations and may enable more widespread use of nanoparticles in vivo

Liposomal delivery of hydrophobic RAMBAs provides good bioavailability and significant enhancement of retinoic acid signalling in neuroblastoma tumour cells

Retinoid treatment is employed during residual disease treatment in neuroblastoma, where the aim is to induce neural differentiation or death in tumour cells. However, although therapeutically effective, retinoids have only modest benefits and suffer from poor pharmacokinetic properties. In vivo, retinoids induce CYP26 enzyme production in the liver, enhancing their own rapid metabolic clearance, while retinoid resistance in tumour cells themselves is considered to be due in part to increased CYP26 production. Retinoic acid metabolism blocking agents (RAMBAs), which inhibit CYP26 enzymes, can improve retinoic acid pharmacokinetics in pre-clinical neuroblastoma models. Here we demonstrate that in cultured neuroblastoma tumour cells, RAMBAs enhance retinoic acid action as seen by morphological differentiation, AKT signalling and suppression of MYCN protein. Although active as retinoid enhancers, these RAMBAs are highly hydrophobic and their effective delivery in humans will be very challenging. Here we demonstrate that such RAMBAs can be loaded efficiently into cationic liposomal particles, where the RAMBAs achieve good bioavailability and activity in cultured tumour cells. This demonstrates the efficacy of RAMBAs in enhancing retinoid signaling in neuroblastoma cells and shows for the first time that liposomal delivery of hydrophobic RAMBAs is a viable approach, providing novel opportunities for their delivery and application in humans

Development of targeted siRNA nanocomplexes to prevent fibrosis in experimental glaucoma filtration surgery

RNA interference induced by double-stranded, small interfering RNA (siRNA) molecules has attracted great attention as a naturally-occurring approach to silence gene expression with high specificity. The Myocardin-Related Transcription Factor/Serum Response Factor (MRTF/SRF) pathway is a master regulator of cytoskeletal gene expression and thus represents a promising target to prevent fibrosis. A major hurdle to implementing siRNA therapies is the method of delivery and we have thus optimised lipid-peptide-siRNA (LPR) nanoparticles containing MRTF-B siRNAs as a targeted approach to prevent conjunctival fibrosis. We tested fifteen LPR nanoparticle formulations with different lipid compositions, surface charges and targeting or non-targeting peptides in human conjunctival fibroblasts. In vitro, the LPR formulation of DOTMA/DOPE lipid with the targeting peptide Y (LYR) was the most efficient in MRTF-B gene silencing and non-cytotoxic compared to the non-targeting formulation. In vivo, subconjunctival administration of LYR nanoparticles containing MRTF-B siRNAs doubled bleb survival in a pre-clinical rabbit model of glaucoma filtration surgery. Furthermore, MRTF-B LYR nanoparticles reduced the MRTF-B mRNA by 29.6% in rabbit conjunctival tissues, which led to significantly decreased conjunctival scarring with no adverse side effects. LYR-mediated delivery of siRNA shows promising results to increase bleb survival and to prevent conjunctival fibrosis after glaucoma filtration surgery

Minicircle DNA provide enhanced and prolonged transgene expression following airway gene transfer

Gene therapy for cystic fibrosis using non-viral, plasmid-based formulations has been the subject of intensive research for over two decades but a clinically viable product has yet to materialise in large part due to inefficient transgene expression. Minicircle DNA give enhanced and more persistent transgene expression compared to plasmid DNA in a number of organ systems but has not been assessed in the lung. In this study we compared minicircle DNA with plasmid DNA in transfections of airway epithelial cells. In vitro, luciferase gene expression from minicircles was 5-10-fold higher than with plasmid DNA. In eGFP transfections in vitro both the mean fluorescence intensity and percentage of cells transfected was 2-4-fold higher with minicircle DNA. Administration of equimolar amounts of DNA to mouse lungs resulted in a reduced inflammatory response and more persistent transgene expression, with luciferase activity persisting for 2 weeks from minicircle DNA compared to plasmid formulations. Transfection of equal mass amounts of DNA in mouse lungs resulted in a 6-fold increase in transgene expression in addition to more persistent transgene expression. Our findings have clear implications for gene therapy of airway disorders where plasmid DNA transfections have so far proven inefficient in clinical trials