Biophysical and biological contributions of polyamine-coated carbon nanotubes and bidimensional buckypapers in the delivery of miRNAs to human cells

Recent findings in nanomedicine have revealed that carbon nanotubes (CNTs) can be used as potential drug carriers, therapeutic agents and diagnostics tools. Moreover, due to their ability to cross cellular membranes, their nanosize dimension, high surface area and relatively good biocompatibility, CNTs have also been employed as a novel gene delivery vector system. In our previous work, we functionalized CNTs with two polyamine polymers, polyethyleneimine (PEI) and polyamidoamine dendrimer (PAMAM). These compounds have low cytotoxicity, ability to conjugate microRNAs (such as miR-503) and, at the same time, transfect efficiently endothelial cells. The parameters contributing to the good efficiency of transfection that we observed were not investigated in detail. In fact, the diameter and length of CNTs are important parameters to be taken into account when evaluating the effects on drug delivery efficiency. In order to investigate the biophysical and biological contributions of polymer-coated CNTs in delivery of miRNAs to human cells, we decided to investigate three different preparations, characterized by different dimensions and aspect ratios. In particular, we took into account very small CNTs, a suspension of CNTs starting from the commercial product and a 2D material based on CNTs (ie, buckypapers [BPs]) to examine the transfection efficiency of a rigid scaffold. In conclusion, we extensively investigated the biophysical and biological contributions of polyamine-coated CNTs and bidimensional BPs in the delivery of miRNAs to human cells, in order to optimize the transfection efficiency of these compounds to be employed as efficient drug delivery vectors in biomedical applications

Dendrimer-coated carbon nanotubes deliver dsRNA and increase the efficacy of gene knockdown in the red flour beetle Tribolium castaneum

Thanks to Dr Alan S. Bowman at the Institute for Biological and Environmental Sciences, University of Aberdeen for providing facilities and laboratory equipment for insect work and to Kevin S. Mackenzie and staff at the Microscopy and Histology Core Facility at the University of Aberdeen for TEM preparations. Scottish Crucible Project Award 2014 provided financial support for this research. CHE was supported by a Knowledge Transfer Network BBSRC Industrial Case (BB/L502467/1) studentship. CRC was supported by a KTN BBSRC CASE studentship (BB/M503526/1). AM and AC were supported by the Italian Ministry of Health (RF-PE-2011-02347026). EMC was supported by European Union’s Seventh Framework Programme for research, technological development and demonstration under grant agreement no. 613960 (SMARTBEES) and Veterinary Medicines Directorate, Department for Environment Food & Rural Affairs (Project # VM0517).Peer reviewedPublisher PD

Regulation of angiogenesis through the efficient delivery of microRNAs into endothelial cells using polyamine-coated carbon nanotubes

AbstractMicroRNAs (miRNAs) directly regulate gene expression at a post-transcriptional level and represent an attractive therapeutic target for a wide range of diseases. Here, we report a novel strategy for delivering miRNAs to endothelial cells (ECs) to regulate angiogenesis, using polymer functionalized carbon nanotubes (CNTs). CNTs were coated with two different polymers, polyethyleneimine (PEI) or polyamidoamine dendrimer (PAMAM), followed by conjugation of miR-503 oligonucleotides as recognized regulators of angiogenesis. We demonstrated a reduced toxicity for both polymer-coated CNTs, compared with pristine CNTs or polymers alone. Moreover, polymer-coated CNT stabilized miR-503 oligonucleotides and allowed their efficient delivery to ECs. The functionality of PAMAM-CNT-miR-503 complexes was further demonstrated in ECs through regulation of target genes, cell proliferation and angiogenic sprouting and in a mouse model of angiogenesis. This comprehensive series of experiments demonstrates that the use of polyamine-functionalized CNTs to deliver miRNAs is a novel and effective means to regulate angiogenesis

Delivery and imaging of miRNAs by multifunctional carbon nanotubes as innovative therapeutic tool for pediatric pulmonary hypertension

Despite the advent of efficacious therapies for pediatric pulmonary hypertension (PH), prevention remains a priority. There are few therapeutic options currently available for children with PH. The advent of nanotechnology provide new opportunity for therapeutic strategy. Carbon nanotubes (CNTs) are emerging as promising systems for transfection of drugs, small molecules and, recently, have been widely studied for the delivery of nucleic acids and small interfering RNAs (siRNAs). In this dissertation, I report the preparation of delivery systems based on carbon nanotubes (CNTs) to deliver microRNAs (miRNAs) in order to regulate the angiogenesis in pulmonary hypertension. These systems have been obtained through the functionalization of CNTs with two polymers: polyethyleneimine (PEI) or polyamidoamine dendrimer (PAMAM), followed by conjugation of microRNA-503 oligonucleotides (a recognized regulator of angiogenesis). The in vitro and in vivo results demonstrated a low toxicity of these compounds and the efficient delivery of miR-503 in endothelial cells. The polymers-CNTs complexed with miR-503 have a functional role in endothelial cells, regulating target genes. Their effects have been demonstrated in a mouse model of angiogenesis (cell proliferation and angiogenic sprouting). In a first method we used an excess of polymer while in a second method we improved the protocol preparation using a reduced amount of polymer. Finally, we developed a multifunctional vector consisting in CNTs functionalized with PAMAM, an oligonucleotide mimicking miR-503 and a near-IR probe (IR-820). CNTs have been injected in the lung of mice, and followed for 24 hours. No relevant inflammation or damage was detected in lung tissue

Aged iPSCs display an uncommon mitochondrial appearance and fail to undergo in vitro neurogenesis

Reprogramming of human fibroblasts into induced pluripotent stem cells (iPSCs) leads to mitochondrial rejuvenation, making iPSCs a candidate model to study the mitochondrial biology during stemness and differentiation. At present, it is generally accepted that iPSCs can be maintained and propagated indefinitely in culture, but no specific studies have addressed this issue. In our study, we investigated features related to the 'biological age' of iPSCs, culturing and analyzing iPSCs kept for prolonged periods in vitro. We have demonstrated that aged iPSCs present an increased number of mitochondria per cell with an altered mitochondrial membrane potential and fail to properly undergo in vitro neurogenesis. In aged iPSCs we have also found an altered expression of genes relevant to mitochondria biogenesis. Overall, our results shed light on the mitochondrial biology of young and aged iPSCs and explore how an altered mitochondrial status may influence neuronal differentiation. Our work suggests to deepen the understanding of the iPSCs biology before considering their use in clinical applications

Biophysical Characterization of Membrane Phase Transition Profiles for the Discrimination of Outer Membrane Vesicles (OMVs) From Escherichia coli Grown at Different Temperatures

Dynamic Light Scattering (DLS), Small Angle X-ray Scattering (SAXS) and Transmission Electron Microscopy (TEM) are physical techniques widely employed to characterize the morphology and the structure of vesicles such as liposomes or human extracellular vesicles (exosomes). Bacterial extracellular vesicles are similar in size to human exosomes, although their function and membrane properties have not been elucidated in such detail as in the case of exosomes. Here, we applied the above cited techniques, in synergy with the thermotropic characterization of the vesicles lipid membrane using a turbidimetric technique to the study of vesicles produced by Gram-negative bacteria (Outer Membrane Vesicles, OMVs) grown at different temperatures. This study demonstrated that our combined approach is useful to discriminate vesicles of different origin or coming from bacteria cultured under different experimental conditions. We envisage that in a near future the techniques employed in our work will be further implemented to discriminate complex mixtures of bacterial vesicles, thus showing great promises for biomedical or diagnostic applications

Keppen-lubinsky syndrome is caused by mutations in the inwardly rectifying K+ channel encoded by KCNJ6

Keppen-Lubinsky syndrome (KPLBS) is a rare disease mainly characterized by severe developmental delay and intellectual disability, microcephaly, large prominent eyes, a narrow nasal bridge, a tented upper lip, a high palate, an open mouth, tightly adherent skin, an aged appearance, and severe generalized lipodystrophy. We sequenced the exomes of three unrelated individuals affected by KPLBS and found de novo heterozygous mutations in KCNJ6 (GIRK2), which encodes an inwardly rectifying potassium channel and maps to the Down syndrome critical region between DIRK1A and DSCR4. In particular, two individuals shared an in-frame heterozygous deletion of three nucleotides (c.455_457del) leading to the loss of one amino acid (p.Thr152del). The third individual was heterozygous for a missense mutation (c.460G>A) which introduces an amino acid change from glycine to serine (p.Gly154Ser). In agreement with animal models, the present data suggest that these mutations severely impair the correct functioning of this potassium channel. Overall, these results establish KPLBS as a channelopathy and suggest that KCNJ6 (GIRK2) could also be a candidate gene for other lipodystrophies. We hope that these results will prompt investigations in this unexplored class of inwardly rectifying K+ channels