A novel approach based on low-field NMR for the detection of the pathological components of sputum in cystic fibrosis patients.

PURPOSE: Development of a reliable, simple method to monitor lung condition in cystic fibrosis (CF) patients. Lung functionality assessment in CF patients is relevant, as most of them still die of respiratory failure. In lung mucus (sputum) of CF patients, components such as proteins, biopolymers, DNA, bacteria, and mucin are pathologically increased. As lung functionality is related to the amount of the pathological components in the sputum, their determination can help clinicians in monitoring lung condition and planning therapy. METHODS: Low-field NMR was used to evaluate the variation of the relaxation time (T2m ) of the water hydrogens present in CF sputum in relation to the amounts of the pathological components. Low-field NMR was tested in artificial samples (mucin or alginates), then in conditional sputum (saliva from healthy volunteers, added by different amounts of the pathological components), and finally in 12 patients' sputums, in which T2m was correlated to a commonly used lung monitoring test (i.e., forced expiratory volume in the first second). RESULTS: T2m significantly (P < 0.05) differed between samples with and without pathological components and between healthy and CF patients (P < 0.05), in which T2m correlated (r = 0.87) with FEV1 . CONCLUSIONS: The presented method can potentially become a valuable lung-monitoring tool in CF patients. Magn Reson Med 79:2323-2331, 2018. © 2017 International Society for Magnetic Resonance in Medicine.

Polymer-Mediated Delivery of siRNAs to Hepatocellular Carcinoma: Variables Affecting Specificity and Effectiveness.

Despite the advances in anticancer therapies, their effectiveness for many human tumors is still far from being optimal. Significant improvements in treatment efficacy can come from the enhancement of drug specificity. This goal may be achieved by combining the use of therapeutic molecules with tumor specific effects and delivery carriers with tumor targeting ability. In this regard, nucleic acid-based drug (NABD) and particularly small interfering RNAs (siRNAs), are attractive molecules due to the possibility to be engineered to target specific tumor genes. On the other hand, polymeric-based delivery systems are emerging as versatile carriers to generate tumor-targeted delivery systems. Here we will focus on the most recent findings in the selection of siRNA/polymeric targeted delivery systems for hepatocellular carcinoma (HCC), a human tumor for which currently available therapeutic approaches are poorly effective. In addition, we will discuss the most attracting and, in our opinion, promising siRNA-polymer combinations for HCC in relation to the biological features of HCC tissue. Attention will be also put on the mathematical description of the mechanisms ruling siRNA-carrier delivery, this being an important aspect to improve effectiveness reducing the experimental work.

Targeted delivery of siRNAs against hepatocellular carcinoma-related genes by a galactosylated polyaspartamide copolymer.

Given the lack of effective treatments for Hepatocellular carcinoma (HCC), the development of novel therapeutic approaches is very urgent. Here, siRNAs were delivered to HCC cells by a synthetic polymer containing α,ß-poly-(N-2-hydroxyethyl)-D,L-aspartamide-(PHEA) derivatized with diethylene triamine (DETA) and bearing in the side chain galactose (GAL) linked via a polyethylene glycol (PEG) to obtain (PHEA-DETA-PEG-GAL, PDPG). The GAL residue allows the targeting to the asialo-glycoprotein receptor (ASGPR), overexpressed in HCC cells compared to normal hepatocytes. Uptake studies performed using a model siRNA or a siRNA targeted against the enhanced green fluorescence protein, demonstrated the PDPG specific delivery of siRNA to HuH7 cells, a human cellular model of HCC. GAL-free copolymer (PHEA-DETA-PEG-NH2, PDP) or the chemical block of ASGPR, impaired PDPG targeting effectiveness in vitro. The specificity of PDPG delivery was confirmed in vivo in a mouse dorsal skinfold window chamber assay. Functional studies using siRNAs targeting the mRNAs of HCC-related genes (eEF1A1, eEF1A2 and E2F1) delivered by PDPG, significantly decreased HuH7 vitality/number and down regulated the expression of the target genes. Only minor effectiveness was in contrast observed for PDP. In IHH, a human model of normal hepatocytes with reduced ASGPR expression, PDPG barely reduced cell vitality. In a subcutaneous xenograft mouse model of HCC, PDPG-siRNAs reduced HCC tumor growth compared to controls without significant toxic effects. In conclusion, our study demonstrates the valuable potentials of PDPG for the specific delivery of siRNAs targeting HCC-related genes.

In vitro and ex vivo delivery of tailored siRNA-nanoliposomes for E2F1 silencing as a potential therapy for colorectal cancer.

Tailored developed nanoliposomes loaded with a siRNA against the transcription factor E2F1 (siE2F1), were produced and delivered to human colorectal adenocarcinoma cell lines and to intestinal human biopsies. siE2F1 loaded nanoliposomes were produced through a dedicated ultrasound assisted technique producing particles with about 40nm size (Small Unilamellar Vesicles, SUVs) and 100% siRNA encapsulation efficiency. Compared to other production methods, the one proposed here can easily produce particles in the nanometric scale by suitable ultrasonic duty cycle treatments. Furthermore, SUVs have a high degree of size homogeneity, a relevant feature for uniform delivery behaviour. siE2F1-loaded SUVs demonstrated a very low cytotoxicity in cells when compared to a commercial transfection agent. Moreover, SUVs loaded with siE2F1 were effective in the down regulation of the target in cultured colon carcinoma cells and in the consequent reduction of cell growth. Finally, a remarkable uptake and target silencing efficiencies were observed in cultured human biopsy of colonic mucosa. In conclusion, whereas further studies in more complex models are required, the siE2F1-SUVs generated have the potential to contribute to the development of novel effective inflammatory bowel diseases-associated colorectal cancer therapies for a future personalized medicine.

Galactosylated polyaspartamide copolymers for siRNA targeted delivery to hepatocellular carcinoma cells.

The limited efficacy of available treatments for hepatocellular carcinoma (HCC) requires the development of novel therapeutic approaches. We synthesized a novel cationic polymer based on α,ß-poly-(N-2-hydroxyethyl)-d,L-aspartamide (PHEA) for drug delivery to HCC cells. The copolymer was synthesized by subsequent derivatization of PHEA with diethylene triamine (DETA) and with a polyethylene glycol (PEG) derivative bearing galactose (GAL) molecules, obtaining the cationic derivative PHEA-DETA-PEG-GAL. PHEA-DETA-PEG-GAL has suitable chemical-physical characteristics for a potential systemic use and can effectively deliver a siRNA (siE2F1) targeted against the transcription factor E2F1, a gene product involved in HCC. The presence of GAL residues in the polyplexes allows the targeting of HCC cells that express the asialo-glycoprotein receptor (ASGP-R). In these cells, but not in ASGP-R non-expressing cells, PHEA-DETA-PEG-GAL/siE2F1 polyplexes induce the reduction of the mRNA and protein levels of E2F1 and of E2F1-regulated genes, all involved in the promotion of the G1/S phase transition. This results in a decrease of cell proliferation with a G1/G0 phase cells accumulation. Notably, removal of GAL residue almost completely abrogates the targeting capacity of the developed polyplexes. In conclusion, the generated polyplexes demonstrate the potential to effectively contributing to the development of novel anti-HCC therapeutic approaches via a siRNA-targeted delivery.

Dissecting the role of the elongation factor 1A isoforms in hepatocellular carcinoma cells by liposome-mediated delivery of siRNAs.

Eukaryotic elongation factor 1A (eEF1A), a protein involved in protein synthesis, has two major isoforms, eEF1A1 and eEF1A2. Despite the evidences of their involvement in hepatocellular carcinoma (HCC), the quantitative contribution of each of the two isoforms to the disease is unknown. We depleted the two isoforms by means of siRNAs and studied the effects in three different HCC cell lines. Particular care was dedicated to select siRNAs able to target each of the two isoform without affecting the other one. This is not a trivial aspect due to the high sequence homology between eEF1A1 and eEF1A2. The selected siRNAs can specifically deplete either eEF1A1 or eEF1A2. This, in turn, results in an impairment of cell vitality, growth and arrest in the G1/G0 phase of the cell cycle. Notably, these effects are quantitatively superior following eEF1A1 than eEF1A2 depletion. Moreover, functional tests revealed that the G1/G0 block induced by eEF1A1 depletion depends on the down-regulation of the transcription factor E2F1, a known player in HCC. In conclusion, our data indicate that the independent targeting of the two eEF1A isoforms is effective in reducing HCC cell growth and that eEF1A1 depletion may result in a more evident effect.

Recent advances in smart biotechnology: Hydrogels and nanocarriers for tailored bioactive molecules depot.

Over the past ten years, the global biopharmaceutical market has remarkably grown, with ten over the top twenty worldwide high performance medical treatment sales being biologics. Thus, biotech R&D (research and development) sector is becoming a key leading branch, with expanding revenues. Biotechnology offers considerable advantages compared to traditional therapeutic approaches, such as reducing side effects, specific treatments, higher patient compliance and therefore more effective treatments leading to lower healthcare costs. Within this sector, smart nanotechnology and colloidal self-assembling systems represent pivotal tools able to modulate the delivery of therapeutics. A comprehensive understanding of the processes involved in the self-assembly of the colloidal structures discussed therein is essential for the development of relevant biomedical applications. In this review we report the most promising and best performing platforms for specific classes of bioactive molecules and related target, spanning from siRNAs, gene/plasmids, proteins/growth factors, small synthetic therapeutics and bioimaging probes.

Potential Applications of Nanocellulose-Containing Materials in the Biomedical Field.

Because of its high biocompatibility, bio-degradability, low-cost and easy availability, cellulose finds application in disparate areas of research. Here we focus our attention on the most recent and attractive potential applications of cellulose in the biomedical field. We first describe the chemical/structural composition of cellulose fibers, the cellulose sources/features and cellulose chemical modifications employed to improve its properties. We then move to the description of cellulose potential applications in biomedicine. In this field, cellulose is most considered in recent research in the form of nano-sized particle, i.e., nanofiber cellulose (NFC) or cellulose nanocrystal (CNC). NFC is obtained from cellulose via chemical and mechanical methods. CNC can be obtained from macroscopic or microscopic forms of cellulose following strong acid hydrolysis. NFC and CNC are used for several reasons including the mechanical properties, the extended surface area and the low toxicity. Here we present some potential applications of nano-sized cellulose in the fields of wound healing, bone-cartilage regeneration, dental application and different human diseases including cancer. To witness the close proximity of nano-sized cellulose to the practical biomedical use, examples of recent clinical trials are also reported. Altogether, the described examples strongly support the enormous application potential of nano-sized cellulose in the biomedical field.

Aptamer targeting of the elongation factor 1A impairs hepatocarcinoma cells viability and potentiates bortezomib and idarubicin effects.

The high morbidity and mortality of hepatocellular carcinoma (HCC) is mostly due to the limited efficacy of the available therapeutic approaches. Here we explore the anti-HCC potential of an aptamer targeting the elongation factor 1A (eEF1A), a protein implicated in the promotion of HCC. As delivery methods, we have compared the effectiveness of cationic liposome and cholesterol-mediated approaches. A75 nucleotide long aptamer containing GT repetition (GT75) was tested in three HCC cell lines, HepG2, HuH7 and JHH6. When delivered by liposomes, GT75 was able to effectively reducing HCC cells viability in a dose and time dependent fashion. Particular sensitive were JHH6 where increased apoptosis with no effects on cell cycle were observed. GT75 effect was likely due to the interference with eEF1A activity as neither the mRNA nor the protein levels were significantly affected. Notably, cholesterol-mediated delivery of GT75 abrogated its efficacy due to cellular mis-localization as proven by fluorescence and confocal microscopic analysis. Finally, liposome-mediated delivery of GT75 improved the therapeutic index of the anticancer drugs bortezomib and idarubicin. In conclusion, liposome but not cholesterol-mediated delivery of GT75 resulted in an effective delivery of GT75, causing the impairment of the vitality of a panel of HCC derived cells.

Aptameric GT oligomers need to be complexed to ethoxylated polyethylenimine as pre-paired duplexes to efficiently exert their cytotoxic activity in human lymphoblastic cancer cells.

The aptameric oligonucleotides GT were found to exert a selective, specific and dose-dependent cell growth inhibition effect on a variety of human cancer cells by recognising specific nuclear proteins and among these in particular an isoform of the eukaryotic elongation factor 1A1 (EEF1A1). The potential development of these aptameric oligomers needs that they retain serum and intracellular stabilities. Polycations are safe non-viral carriers of the nucleic acids. We demonstrated that a weakly basic polycation, the ethoxylated polyethylenimine (EPEI), can efficiently deliver cytotoxic GT oligomers when they were complexed as partial pre-paired duplex. In this way, nuclease-resistance of the oligomer was markedly improved and the administration of the duplex complexed with EPEI to lymphoblastic cancer cells caused a specific cytotoxic effect at concentrations lower than that of naked GT. However, the cytotoxic activity of the oligomer-EPEI complex resulted strictly related to the GC content and Tm of the duplex region. The single-stranded GT and the duplex with high GC content and Tm, although complexed with EPEI failed to exert cytotoxicity. Overall results indicated that aptameric oligomers complexed with polycations can be efficiently delivered into the cells and display the desired biological effect designing a balanced partial duplex whose stability can allow oligomer release from the polycation under the physiological cellular conditions.

Novel Lipid and Polymeric Materials as Delivery Systems for Nucleic Acid Based Drugs.

Nucleic acid based drugs (NADBs) are short DNA/RNA molecules that include among others, antisense oligonucleotides, aptamers, small interfering RNAs and micro-interfering RNAs. Despite the different mechanisms of actions, NABDs have the ability to combat the effects of pathological gene expression in many experimental systems. Thus, nowadays, NABDs are considered to have a great therapeutic potential, possibly superior to that of available drugs. Unfortunately, however, the lack of effective delivery systems limits the practical use of NABDs. Due to their hydrophilic nature, NABDs cannot efficiently cross cellular membrane; in addition, they are subjected to fast degradation by cellular and extracellular nucleases. Together these aspects make the delivery of NABDs as naked molecules almost un-effective. To optimize NABD delivery, several solutions have been investigated. From the first attempts described in the beginning of the 1980s, a burst in the number of published papers occurred in the beginning of 1990 s reaching a peak in 2012-13. The extensive amount of work performed so far clearly witnesses the interest of the scientific community in this topic. In the present review, we will concentrate on the description of the most interesting advances in the field. Particular emphasis will be put on polymeric and lipid materials used alone or in combination with a promising delivery strategy based on the use of carbon nanotubes. The data presented suggest that, although further improvements are required, we are not far from the identification of effective delivery systems for NABDs thus making the clinical use of these molecules closer to reality.