Modified cyclodextrins as novel non-viral vectors for neuronal siRNA delivery: focus on Huntington’s disease

Huntington’s Disease (HD) is a rare autosomal dominant neurodegenerative disease caused by the expression of a mutant Huntingtin (muHTT) protein. Therefore, preventing the expression of muHTT by harnessing the specificity of the RNA interference (RNAi) pathway is a key research avenue for developing novel therapies for HD. However, the biggest caveat in the RNAi approach is the delivery of short interfering RNA (siRNAs) to neurons, which are notoriously difficult to transfect. Indeed, despite the great advances in the field of nanotechnology, there remains a great need to develop more effective and less toxic carriers for siRNA delivery to the Central Nervous System (CNS). Thus, the aim of this thesis was to investigate the utility of modified amphiphilic β-cyclodextrins (CDs), oligosaccharide-based molecules, as non-viral vectors for siRNA delivery for HD. Modified CDs were able to bind and complex siRNAs forming nanoparticles capable of delivering siRNAs to ST14A-HTT120Q cells and to human HD fibroblasts, and reducing the expression of the HTT gene in these in vitro models of HD. Moreover, direct administration of CD.siRNA nanoparticles into the R6/2 mouse brain resulted in significant HTT gene expression knockdown and selective alleviation of rotarod motor deficits in this mouse model of HD. In contrast to widely used transfection reagents, CD.siRNA nanoparticles only induced limited cytotoxic and neuroinflammatory responses in multiple brain-derived cell-lines, and also in vivo after single direct injections into the mouse brain. Alternatively, we have also described a PEGylation-based formulation approach to further stabilise CD.siRNA nanoparticles and progress towards a systemic delivery nanosystem. Resulting PEGylated CD.siRNA nanoparticles showed increased stability in physiological saltconditions and, to some extent, reduced protein-induced aggregation. Taken together, the work outlined in this thesis identifies modified CDs as effective, safe and versatile siRNA delivery systems that hold great potential for the treatment of CNS disorders, such as HD.

Aspects of the biology of the parasite Bonamia ostreae with a view to gaining a greater understanding of how to alleviate its impact on the European flat oyster, Ostrea edulis.

The parasite Bonamia ostreae has decimated Ostrea edulis stocks throughout Europe. The complete life cycle and means of transmission of the parasite remains unknown. The methods used to diagnose B. ostreae were examined to determine sensitivity and reproducibility. Two methods, with fixed protocols, should be used for the accurate detection of infection within a sample. A 13-month study of two stocks of O. edulis with varying periods of exposure to B. ostreae, was undertaken to determine if varying lengths of exposure would translate into observations of differing susceptibility. Oyster stocks can maintain themselves over extended periods of time in B. ostreae endemic areas. To identify a well performing spat stock, which could be used to repopulate beds within the region, hatchery bred spat from three stocks found in the North sea were placed on a B. ostreae infected bed and screened for growth, mortality and prevalence of infection. Local environmental factors may influence oyster performance, with local stocks better adapted to these conditions. Sediment and macroinvertebrate species were screened to investigate mechanisms by which B. ostreae may be maintaining itself on oyster beds. Mytilus edulis was positive, indicating that B. ostreae may use incidental carriers as a method of maintaining itself. The ability of oyster larvae to pick up infection from the surrounding environment was investigated by collecting larvae from brooding oysters from different areas. Larvae may acquire the pathogen from the water column during the process of filter feeding by the brooding adult, even when the parents themselves are uninfected. A study was undertaken to elucidate the activity of the parasite during the initial stage of infection, when it cannot be detected within the host. A naïve stock screened negative for infection throughout the trial, using heart imprints and PCR yet B. ostreae was detected by in-situ hybridisation.

Quantum dot-based theranostics.

Luminescent semiconductor nanocrystals, also known as quantum dots (QDs), have advanced the fields of molecular diagnostics and nanotherapeutics. Much of the initial progress for QDs in biology and medicine has focused on developing new biosensing formats to push the limit of detection sensitivity. Nevertheless, QDs can be more than passive bio-probes or labels for biological imaging and cellular studies. The high surface-to-volume ratio of QDs enables the construction of a "smart" multifunctional nanoplatform, where the QDs serve not only as an imaging agent but also a nanoscaffold catering for therapeutic and diagnostic (theranostic) modalities. This mini review highlights the emerging applications of functionalized QDs as fluorescence contrast agents for imaging or as nanoscale vehicles for delivery of therapeutics, with special attention paid to the promise and challenges towards QD-based theranostics.

A genetically engineered thermally responsive sustained release curcumin depot to treat neuroinflammation.

Radiculopathy, a painful neuroinflammation that can accompany intervertebral disc herniation, is associated with locally increased levels of the pro-inflammatory cytokine tumor necrosis factor alpha (TNFα). Systemic administration of TNF antagonists for radiculopathy in the clinic has shown mixed results, and there is growing interest in the local delivery of anti-inflammatory drugs to treat this pathology as well as similar inflammatory events of peripheral nerve injury. Curcumin, a known antagonist of TNFα in multiple cell types and tissues, was chemically modified and conjugated to a thermally responsive elastin-like polypeptide (ELP) to create an injectable depot for sustained, local delivery of curcumin to treat neuroinflammation. ELPs are biopolymers capable of thermally-triggered in situ depot formation that have been successfully employed as drug carriers and biomaterials in several applications. ELP-curcumin conjugates were shown to display high drug loading, rapidly release curcumin in vitro via degradable carbamate bonds, and retain in vitro bioactivity against TNFα-induced cytotoxicity and monocyte activation with IC50 only two-fold higher than curcumin. When injected proximal to the sciatic nerve in mice via intramuscular (i.m.) injection, ELP-curcumin conjugates underwent a thermally triggered soluble-insoluble phase transition, leading to in situ formation of a depot that released curcumin over 4days post-injection and decreased plasma AUC 7-fold.

Injectable laminin-functionalized hydrogel for nucleus pulposus regeneration.

Cell delivery to the pathological intervertebral disc (IVD) has significant therapeutic potential for enhancing IVD regeneration. The development of injectable biomaterials that retain delivered cells, promote cell survival, and maintain or promote an NP cell phenotype in vivo remains a significant challenge. Previous studies have demonstrated NP cell - laminin interactions in the nucleus pulposus (NP) region of the IVD that promote cell attachment and biosynthesis. These findings suggest that incorporating laminin ligands into carriers for cell delivery may be beneficial for promoting NP cell survival and phenotype. Here, an injectable, laminin-111 functionalized poly(ethylene glycol) (PEG-LM111) hydrogel was developed as a biomaterial carrier for cell delivery to the IVD. We evaluated the mechanical properties of the PEG-LM111 hydrogel, and its ability to retain delivered cells in the IVD space. Gelation occurred in approximately 20 min without an initiator, with dynamic shear moduli in the range of 0.9-1.4 kPa. Primary NP cell retention in cultured IVD explants was significantly higher over 14 days when cells were delivered within a PEG-LM111 carrier, as compared to cells in liquid suspension. Together, these results suggest this injectable laminin-functionalized biomaterial may be an easy to use carrier for delivering cells to the IVD.

A functional polymorphism in the 5HTR2C gene associated with stress responses also predicts incident cardiovascular events.

Previously we have shown that a functional nonsynonymous single nucleotide polymorphism (rs6318) of the 5HTR2C gene located on the X-chromosome is associated with hypothalamic-pituitary-adrenal axis response to a stress recall task, and with endophenotypes associated with cardiovascular disease (CVD). These findings suggest that individuals carrying the rs6318 Ser23 C allele will be at higher risk for CVD compared to Cys23 G allele carriers. The present study examined allelic variation in rs6318 as a predictor of coronary artery disease (CAD) severity and a composite endpoint of all-cause mortality or myocardial infarction (MI) among Caucasian participants consecutively recruited through the cardiac catheterization laboratory at Duke University Hospital (Durham, NC) as part of the CATHGEN biorepository. Study population consisted of 6,126 Caucasian participants (4,036 [65.9%] males and 2,090 [34.1%] females). A total of 1,769 events occurred (1,544 deaths and 225 MIs; median follow-up time = 5.3 years, interquartile range = 3.3-8.2). Unadjusted Cox time-to-event regression models showed, compared to Cys23 G carriers, males hemizygous for Ser23 C and females homozygous for Ser23C were at increased risk for the composite endpoint of all-cause death or MI: Hazard Ratio (HR) = 1.47, 95% confidence interval (CI) = 1.17, 1.84, p = .0008. Adjusting for age, rs6318 genotype was not related to body mass index, diabetes, hypertension, dyslipidemia, smoking history, number of diseased coronary arteries, or left ventricular ejection fraction in either males or females. After adjustment for these covariates the estimate for the two Ser23 C groups was modestly attenuated, but remained statistically significant: HR = 1.38, 95% CI = 1.10, 1.73, p = .005. These findings suggest that this functional polymorphism of the 5HTR2C gene is associated with increased risk for CVD mortality and morbidity, but this association is apparently not explained by the association of rs6318 with traditional risk factors or conventional markers of atherosclerotic disease.

Polymorphisms in the kinesin-like factor 1 B gene and risk of epithelial ovarian cancer in Eastern Chinese women.

The kinesin-like factor 1 B (KIF1B) gene plays an important role in the process of apoptosis and the transformation and progression of malignant cells. Genetic variations in KIF1B may contribute to risk of epithelial ovarian cancer (EOC). In this study of 1,324 EOC patients and 1,386 cancer-free female controls, we investigated associations between two potentially functional single nucleotide polymorphisms in KIF1B and EOC risk by the conditional logistic regression analysis. General linear regression model was used to evaluate the correlation between the number of variant alleles and KIF1B mRNA expression levels. We found that the rs17401966 variant AG/GG genotypes were significantly associated with a decreased risk of EOC (adjusted odds ratio (OR) = 0.81, 95 % confidence interval (CI) = 0.68-0.97), compared with the AA genotype, but no associations were observed for rs1002076. Women who carried both rs17401966 AG/GG and rs1002076 AG/AA genotypes of KIF1B had a 0.82-fold decreased risk (adjusted 95 % CI = 0.69-0.97), compared with others. Additionally, there was no evidence of possible interactions between about-mentioned co-variants. Further genotype-phenotype correlation analysis indicated that the number of rs17401966 variant G allele was significantly associated with KIF1B mRNA expression levels (P for GLM = 0.003 and 0.001 in all and Chinese subjects, respectively), with GG carriers having the lowest level of KIF1B mRNA expression. Taken together, the rs17401966 polymorphism likely regulates KIF1B mRNA expression and thus may be associated with EOC risk in Eastern Chinese women. Larger, independent studies are warranted to validate our findings.

Genetic Variants of the MDM2 Gene Are Predictive of Treatment-Related Toxicities and Overall Survival in Patients With Advanced NSCLC.

INTRODUCTION: Platinum agents can cause the formation of DNA adducts and induce apoptosis to eliminate tumor cells. The aim of the present study was to investigate the influence of genetic variants of MDM2 on chemotherapy-related toxicities and clinical outcomes in patients with advanced non-small-cell lung cancer (NSCLC). MATERIALS AND METHODS: We recruited 663 patients with advanced NSCLC who had been treated with first-line platinum-based chemotherapy. Five tagging single nucleotide polymorphisms (SNPs) in MDM2 were genotyped in these patients. The associations of these SNPs with clinical toxicities and outcomes were evaluated using logistic regression and Cox regression analyses. RESULTS: Two SNPs (rs1470383 and rs1690924) showed significant associations with chemotherapy-related toxicities (ie, overall, hematologic, and gastrointestinal toxicity). Compared with the wild genotype AA carriers, patients with the GG genotype of rs1470383 had an increased risk of overall toxicity (odds ratio [OR], 3.28; 95% confidence interval [CI], 1.34-8.02; P = .009) and hematologic toxicity (OR, 4.10; 95% CI, 1.73-9.71; P = .001). Likewise, patients with the AG genotype of rs1690924 showed more sensitivity to gastrointestinal toxicity than did those with the wild-type homozygote GG (OR, 2.32; 95% CI, 1.30-4.14; P = .004). Stratified survival analysis revealed significant associations between rs1470383 genotypes and overall survival in patients without overall or hematologic toxicity (P = .007 and P = .0009, respectively). CONCLUSION: The results of our study suggest that SNPs in MDM2 might be used to predict the toxicities of platinum-based chemotherapy and overall survival in patients with advanced NSCLC. Additional validations of the association are warranted.

ROLE OF ICAM-1-MEDIATED ENDOCYTOSIS IN ENDOTHELIAL FUNCTION AND IMPLICATIONS FOR CARRIER-ASSISTED DRUG DELIVERY

Intercellular adhesion molecule 1 (ICAM-1) is a transmembrane protein found on the surface of vascular endothelial cells (ECs). Its expression is upregulated at inflammatory sites, allowing for targeted delivery of therapeutics using ICAM-1-binding drug carriers. Engagement of multiple copies of ICAM-1 by these drug carriers induces cell adhesion molecule (CAM)-mediated endocytosis, which results in trafficking of carriers to lysosomes and across ECs. Knowledge about the regulation behind CAM-mediated endocytosis can help improve drug delivery, but questions remain about these regulatory mechanisms. Furthermore, little is known about the natural function of this endocytic pathway. To address these gaps in knowledge, we focused on two natural binding partners of ICAM-1 that potentially elicit CAM-mediated endocytosis: leukocytes (which bind ICAM-1 via β₂ integrins) and fibrin polymers (a main component of blood clots which binds ICAM-1 via the γ3 sequence). First, inspired by properties of these natural binding partners, we varied the size and targeting moiety of model drug carriers to determine how these parameters affect CAM-mediated endocytosis. Increasing ICAM-1-targeted carrier size slowed carrier uptake kinetics, reduced carrier trafficking to lysosomes, and increased carrier transport across ECs. Changing targeting moieties from antibodies to peptides decreased particle binding and uptake, lowered trafficking to lysosomes, and increased transport across ECs. Second, using cell culture models of leukocyte/EC interactions, inhibiting regulatory elements of the CAM-mediated pathway disrupted leukocyte sampling, a process crucial to leukocyte crossing of endothelial layers (transmigration). This inhibition also decreased leukocyte transmigration across ECs, specifically through the transcellular route, which occurs through a single EC without disassembly of cell-cell junctions. Third, fibrin meshes, which mimic blood clot fragments/remnants, bound to ECs at ICAM-1-enriched sites and were internalized by the endothelium. Inhibiting the CAM-mediated pathway disrupted this uptake. Following endocytosis, fibrin meshes trafficked to lysosomes where they were degraded. In mouse models, CAM-mediated endocytosis of fibrin meshes appeared to remove fibrin remnants at the endothelial surface, preventing re-initiation of the coagulation cascade. Overall, these results support a link between CAM-mediated endocytosis and leukocyte transmigration as well as uptake of fibrin materials by ECs. Furthermore, these results will guide the future design of ICAM-1-targeted carrier-assisted therapies.

Improving Photovoltaics with High Luminescence Efficiency Quantum Dot Layers

A solar cell relies on its ability to turn photons into current. Because short wavelength photons are typically absorbed near the top surface of a cell, the generated charge carriers recombine before being collected. But when a layer of quantum dots (nanoscale semiconductor particles) is placed on top of the cell, it absorbs short wavelength photons and emits them into the cell at longer wavelengths, which enables more efficient carrier collection. However, the resulting power conversion efficiency of the system depends critically on the quantum dot luminescence efficiency – the nature of this relationship was previously unknown. Our calculations suggest that a quantum dot layer must have high luminescence efficiency (at least 80%) to improve the current output of existing photovoltaic (PV) cells; otherwise, it may worsen the cell’s efficiency. Our quantum dot layer (using quantum dots with over 85% quantum yield) slightly reduced the efficiency of our PV cells. We observed a decrease in short circuit current of a commercial-grade cell from 0.1977 A to 0.1826 A, a 7.6% drop, suggesting that improved optical coupling from the quantum dot emission into the solar cell is needed. With better optical coupling, we predict current enhancements between ~6% and ~8% for a solar cell that already has an antireflection coating. Such improvements could have important commercial impacts if the coating could be deployed in a scalable fashion.

Bordetella pertussis from functional genomics to intranasal vaccination.

Whooping cough still represents a major health problem, despite the use of effective vaccines for several decades. Being classically a typical childhood disease, whooping cough in young adults is now more common than it used to be, suggesting that protection after vaccination wanes during adolescence. As an alternative to the current vaccines, we wish to develop live attenuated vaccines to be delivered by the nasal route, such as to mimic the natural route of infection and to induce long lasting immunity. Bordetella pertussis, the etiological agent of whooping cough, produces a number of virulence factors, including toxins. Its recently determined genome sequence makes it now possible to apply functional genomics, such as transcriptomics and systematic knock-out mutagenesis. The expression of most known B. pertussis virulence genes is controlled by the two-component system BvgA/S. DNA microarray analyses have led to the identification of novel genes in the BvgA/S regulon, some of which are activated by BvgA/S and others are repressed by BvgA/S. In addition, some genes appear to be differentially modulated by nicotinic acid and MgSO4, both known to modulate the expression of BvgA/S-regulated genes. Among others, the functional genomics approach has uncovered two strongly BvgA/S-activated genes, named hotA and hotB (for 'homolog of toxin'), the products of which show high sequence similarities to pertussis toxin subunits. The identification of the full array of virulence factors, as well as an integrated understanding of the bacterial physiology should allow us to design attenuated B. pertussis strains useful for intranasal vaccination. A first generation of attenuated strains has already shown full protection in mice after a single intranasal administration. Such strains may also serve as vaccine carriers for heterologous antigens, in order to vaccinate against several different pathogens simultaneously.

Selección de soportes para la producción de inoculantes para leguminosas

p.209-217

The use of fluorescence microscopy to define polymer localisation to the late endocytic compartments in cells that are targets for drug delivery

Macromolecular therapeutics and nano-sized drug delivery systems often require localisation to specific intracellular compartments. In particular, efficient endosomal escape, retrograde trafficking, or late endocytic/lysosomal activation are often prerequisites for pharmacological activity. The aim of this study was to define a fluorescence microscopy technique able to confirm the localisation of water-soluble polymeric carriers to late endocytic intracellular compartments. Three polymeric carriers of different molecular weight and character were studied: dextrin (Mw~50,000 g/mol), a N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer (Mw approximately 35,000 g/mol) and polyethylene glycol (PEG) (Mw 5000 g/mol). They were labelled with Oregon Green (OG) (0.3-3 wt.%; <3% free OG in respect of total). A panel of relevant target cells were used: THP-1, ARPE-19, and MCF-7 cells, and primary bovine chondrocytes (currently being used to evaluate novel polymer therapeutics) as well as NRK and Vero cells as reference controls. Specific intracellular compartments were marked using either endocytosed physiological standards, Marine Blue (MB) or Texas-red (TxR)-Wheat germ agglutinin (WGA), TxR-Bovine Serum Albumin (BSA), TxR-dextran, ricin holotoxin, C6-7-nitro-2,1,3-benzoxadiazol-4-yl (NBD)-labelled ceramide and TxR-shiga toxin B chain, or post-fixation immuno-staining for early endosomal antigen 1 (EEA1), lysosomal-associated membrane proteins (LAMP-1, Lgp-120 or CD63) or the Golgi marker GM130. Co-localisation with polymer-OG conjugates confirmed transfer to discreet, late endocytic (including lysosomal) compartments in all cells types. The technique described here is a particularly powerful tool as it circumvents fixation artefacts ensuring the retention of water-soluble polymers within the vesicles they occupy.

Polymorphims in the Interferon-y/Interleukin 26 Gene Region Contribute to Sex Bias in Susceptibility to Rheumatoid Arthritis

Objective:

To determine whether polymorphisms in the interferon-? (IFN?)/interleukin-26 (IL-26; formerly, AK155) gene cluster contribute to sex-based differential susceptibility to rheumatoid arthritis (RA).

Methods:

Four microsatellite markers, located in a 118-kb interval that contains both the IFN? and IL-26 genes on chromosome 12q15, were typed in 251 patients with RA and 198 unrelated healthy controls (all of whom lived in Northern Ireland) by means of polymerase chain reaction–based fragment analysis.

Results:

Marker D12S2510, which is located 3 kb 3' from the IL-26 gene, was significantly associated with RA in women (corrected P [Pcorr] = 0.008, 2 degrees of freedom [2 df]) but not in men (P = 0.99, 2 df). A 3-marker haplotype, IFNGCA*13;D12S2510*8;D12S2511*9, was inferred that showed significant underrepresentation in women with RA (odds ratio 0.50, 95% confidence interval 0.32–0.78; P = 0.002, Pcorr = 0.03) but not in men with RA.

Conclusion:

Our results demonstrate that common polymorphisms in the IFN?/IL-26 gene region may contribute to sex bias in susceptibility to RA, by distorting the propensity of female carriers versus male carriers to contract this disease. These results conform to our recent observations of a role for this gene cluster in sex-based differential susceptibility to another Th1-type inflammatory disease, multiple sclerosis.

Broken symmetry and pseudogaps in ropes of carbon nanotubes

Since the discovery of carbon nanotubes, it has been speculated that these materials should behave like nanoscale wires with unusual electronic properties and exceptional strength. Recently, 'ropes' of close-packed single-wall nanotubes have been synthesized in high yield. The tubes in these ropes are mainly of the (10,10) type3, which is predicted to be metallic. Experiments on individual nanotubes and ropes indicate that these systems indeed have transport properties that qualify them to be viewed as nanoscale quantum wires at low temperature. It has been expected that the close-packing of individual nanotubes into ropes does not change their electronic properties significantly. Here, however, we present first-principles calculations which show that a broken symmetry of the (10,10) tube caused by interactions between tubes in a rope induces a pseudogap of about 0.1 eV at the Fermi level. This pseudogap strongly modifies many of the fundamental electronic properties: we predict a semimetal-like temperature dependence of the electrical conductivity and a finite gap in the infrared absorption spectrum. The existence of both electron and hole charge carriers will lead to qualitatively different thermopower and Hall-effect behaviours from those expected for a normal metal.