Cyclodextrin mediated delivery of NF-κB and SRF siRNA reduces the invasion potential of prostate cancer cells in vitro.

Prostate cancer is the most common cancer in men of the western world. To date, no effective treatment exists for metastatic prostate cancer and consequently, there is an urgent need to develop new and improved therapeutics. In recent years, the therapeutic potential of RNA interference (RNAi) has been extensively explored in a wide range of diseases including prostate cancer using numerous gene delivery vectors. The aims of this study were to investigate the ability of a non-viral modified cyclodextrin (CD) vector to deliver siRNA to the highly metastatic PC-3 prostate cancer cell line, to quantify the resulting knockdown of the two target genes (RelA and SRF) and to study the effects of the silencing on metastasis. Data from a Matrigel in vitro invasion assay indicated that the silencing of the target genes achieved by the CD vector resulted in significant reductions (P=0.0001) in the metastatic potential of these cells. As the silencing of these target genes was shown not to have a negative impact on cell viability, we hypothesise that the mechanism of invasion inhibition is due, in part, to the significant reduction observed (P⩽0.0001) in the level of pro-inflammatory cytokine, MMP9, which is known to be implicated in the metastasis of prostate cancer.

Early-stage development of novel cyclodextrin-siRNA nanocomplexes allows for successful postnebulization transfection of bronchial epithelial cells.

BACKGROUND: Successful delivery of small interfering RNA (siRNA) to the lungs remains hampered by poor intracellular delivery, vector-mediated cytotoxicity, and an inability to withstand nebulization. Recently, a novel cyclodextrin (CD), SC12CDClickpropylamine, consisting of distinct lipophilic and cationic subunits, has been shown to transfect a number of cell types. However, the suitability of this vector for pulmonary siRNA delivery has not been assessed to date. To address this, a series of high-content analysis (HCA) and postnebulization assays were devised to determine the potential for CD-siRNA delivery to the lungs. METHODS: SC12CDClickpropylamine-siRNA mass ratios (MRs) were examined for size and zeta potential. In-depth analysis of nanocomplex uptake and toxicity in Calu-3 bronchial epithelial cells was examined using IN Cell(®) HCA assays. Nebulized SC12CDClickpropylamine nanocomplexes were assessed for volumetric median diameter (VMD) and fine particle fraction (FPF) and compared with saline controls. Finally, postnebulization stability was determined by comparing luciferase knockdown elicited by SC12CDClickpropylamine nanocomplexes before and after nebulization. RESULTS: SC12CDClickpropylamine-siRNA complexation formed cationic nanocomplexes of ≤200 nm in size depending on the medium and led to significantly higher levels of siRNA associated with Calu-3 cells compared with RNAiFect-siRNA-treated cells at all MRs (p<0.001, n=3×4), with evidence of toxicity only at MRs 50-100. Nebulization of SC12CDClickpropylamine nanocomplexes using the Aeroneb(®) Pro resulted in VMDs of ∼4 µm and FPFs of ∼57% at all MRs. SC12CDClickpropylamine-siRNA-mediated luciferase knockdown was found to be 39.8±3.6% at MR=20 before and 35.6±4.55% after nebulization, comparable to results observed using unnebulized commercial transfection reagent, RNAiFect. CONCLUSIONS: SC12CDClickpropylamine nanocomplexes can be effectively nebulized for pulmonary delivery of siRNA using Aeroneb technology to mediate knockdown in airway cells. To the best of our knowledge, this is the first study examining the suitability of SC12CDClickpropylamine-siRNA nanocomplexes for pulmonary delivery. Furthermore, this work provides an integrated nanomedicine-device combination for future in vitro and in vivo preclinical and clinical studies of inhaled siRNA therapeutics.

Gene silencing of TNF-alpha in a murine model of acute colitis using a modified cyclodextrin delivery system.

Inflammatory bowel disease (IBD) is a chronic relapsing inflammation of the gastrointestinal tract. The cytokine TNF-alpha (TNF-α) plays a pivotal role in mediating this inflammatory response. RNA interference (RNAi) holds great promise for the specific and selective silencing of aberrantly expressed genes, such as TNF-α in IBD. The aim of this study was to investigate the efficacy of an amphiphilic cationic cyclodextrin (CD) vector for effective TNF-α siRNA delivery to macrophage cells and to mice with induced acute-colitis. The stability of CD.siRNA was examined by gel electrophoresis in biorelevant media reflecting colonic fluids. RAW264.7 cells were transfected with CD.TNF-α siRNA, stimulated with lipopolysaccharide (LPS) and TNF-α and IL-6 responses were measured by PCR and ELISA. Female C57BL/6 mice were exposed to dextran sodium sulphate (DSS) and treated by intrarectal administration with either CD.siRNA TNF-α or a control solution. In vitro, siRNA in CD nanocomplexes remained intact and stable in both fed and fasted simulated colonic fluids. RAW264.7 cells transfected with CD.TNF-α siRNA and stimulated with LPS displayed a significant reduction in both gene and protein levels of TNF-α and IL-6. CD.TNF-α siRNA-treated mice revealed a mild amelioration in clinical signs of colitis, but significant reductions in total colon weight and colonic mRNA expression of TNF-α and IL-6 compared to DSS-control mice were detected. This data indicates the clinical potential of a local CD-based TNF-α siRNA delivery system for the treatment of IBD.

Click-modified cyclodextrins as nonviral vectors for neuronal siRNA delivery.

RNA interference (RNAi) holds great promise as a strategy to further our understanding of gene function in the central nervous system (CNS) and as a therapeutic approach for neurological and neurodegenerative diseases. However, the potential for its use is hampered by the lack of siRNA delivery vectors which are both safe and highly efficient. Cyclodextrins have been shown to be efficient and low toxicity gene delivery vectors in various cell types in vitro. However, to date, they have not been exploited for delivery of oligonucleotides to neurons. To this end, a modified ß-cyclodextrin (CD) vector was synthesized, which complexed siRNA to form cationic nanoparticles of less than 200 nm in size. Furthermore, it conferred stability in serum to the siRNA cargo. The in vitro performance of the CD in both immortalized hypothalamic neurons and primary hippocampal neurons was evaluated. The CD facilitated high levels of intracellular delivery of labeled siRNA, while maintaining at least 80% cell viability. Significant gene knockdown was achieved, with a reduction in luciferase expression of up to 68% and a reduction in endogenous glyceraldehyde phosphate dehydrogenase (GAPDH) expression of up to 40%. To our knowledge, this is the first time that a modified CD has been used as a safe and efficacious vector for siRNA delivery into neuronal cells.

Characterisation of cationic amphiphilic cyclodextrins for neuronal delivery of siRNA: effect of reversing primary and secondary face modifications.

Significant research is focused on the development of non-viral vectors for delivery of siRNA to neurons and the central nervous system. Cyclodextrins (CDs) have shown great promise as efficient and low toxicity gene delivery vectors in various cell types. Here, we investigate two CDs for siRNA delivery in a neuronal cell model. These CDs were substituted on opposite faces (primary and secondary) with amphiphilic and cationic groups. Physical properties of CD.siRNA complexes, including size, charge and stability were measured. In vitro investigations were carried out in immortalised hypothalamic neurons. Neuronal cell uptake was measured by flow cytometry and cytotoxicity was assessed by MTT assay. Knockdown of a luciferase reporter gene was used as a measure of gene silencing efficiency. Both CDs interacted with siRNA, yielding nanosized cationic complexes which exhibited good stability on storage. A favourable toxicity profile was demonstrated for the CD.siRNA complexes. However, only one of the two CDs mediated high levels of neuronal uptake and efficient gene silencing, equivalent to those achieved with a commercial lipid-based vector. Despite the suitability of both CDs as siRNA delivery vectors in terms of their ability to complex siRNA and the properties of the complexes yielded, only one CD achieved good transfection efficiency. This was likely due to the differences in their chemical structures. The effective CD offers great potential as a novel non-toxic vector for neuronal siRNA delivery.

Opportunities and challenges for oral delivery of hydrophobic versus hydrophilic peptide and protein-like drugs using lipid-based technologies.

Peptide and protein-like drugs are macromolecules currently produced in increasing numbers by the pharmaceutical biotechnology industry. The physicochemical properties of these molecules posebarriers to oral administration. Lipid-based drug-delivery systems have the potential to overcome these barriers and may be utilized to formulate safe, stable and efficacious oral medicines. This review outlines the design of such lipid-based technologies. The mechanisms whereby these formulations enhance the absorption of lipophilic versus hydrophilic peptide and protein-like drugs are discussed. In the case of lipophilic compounds, the advantages of lipid-based drug-delivery systems including increased solubilization, decreased intestinal efflux, decreased intracellular metabolism and possible lymphatic transport are well established as is evident from the success of Neoral and other drug products on the market. In contrast, with respect to hydrophilic compounds, the situation is more complex and, while promising formulation approaches have been studied, issues including reproducibility of response, intersubject variability and duration of response require further optimization before commercially viable products are possible.

Biopharmaceutical challenges associated with drugs with low aqueous solubility--the potential impact of lipid-based formulations.

The percentage of new chemical entities synthesised with low aqueous solubility and high therapeutic efficacy is growing, this presents major challenges for the drug delivery scientists. The role of physicochemical properties in identification of suitable drug candidates for oral lipid-based delivery systems is discussed. A knowledge of the interplay of physicochemical and biopharmaceutical drug properties with the physiological environment of the gastro-intestinal tract (GIT), as a prerequisite to successful formulation design, is reviewed. The importance of excipient selection with an emphasis on bioactive excipients is stressed. The need for more examples of in vitro-in vivo correlations as a means of maximizing the development potential and commercial future for lipid-based formulations, and, promoting confidence within the industry for these delivery systems is highlighted.

Evaluation of cellular uptake and gene transfer efficiency of pegylated poly-L-lysine compacted DNA: implications for cancer gene therapy.

Recent success in phase I/II clinical trials (Konstan, M. W.; Davis, P. B.; Wagener, J. S.; Hilliard, K. A.; Stern, R. C.; Milgram, L. J.; Kowalczyk, T. H.; Hyatt, S. L.; Fink, T. L.; Gedeon, C. R.; Oette, S. M.; Payne, J. M.; Muhammad, O.; Ziady, A. G.; Moen, R. C.; Cooper, M. J. Hum. Gene Ther. 2004, 15 (12), 1255-69) has highlighted pegylated poly-L-lysine (C1K30-PEG) as a nonviral gene delivery agent capable of achieving clinically significant gene transfer levels in vivo. This study investigates the potential of a C1K30-PEG gene delivery system for cancer gene therapy and evaluates its mode of cellular entry with the purpose of developing an optimally formulated prototype for tumor cell transfection. C1K30-PEG complexes have a neutral charge and form rod-like and toroid-like nanoparticles. Comparison of the transfection efficiency achieved by C1K30-PEG with other cationic lipid and polymeric vectors demonstrates that C1K30-PEG transfects cells more efficiently than unpegylated poly-L-lysine and compares well to commercially available vectors. In vivo gene delivery by C1K30-PEG nanoparticles to a growing subcutaneous murine tumor was also demonstrated. To determine potential barriers to C1K30-PEG gene delivery, the entry mechanism and intracellular fate of rhodamine labeled complexes were investigated. Using cellular markers to delineate the pathway taken by the complexes upon cellular entry, only minor colocalization was observed with EEA-1, a marker of early endosomes. No colocalization was observed between the complexes and the transferrin receptor, which is a marker for clathrin-coated pits. In addition, complexes were not observed to enter late endosomes/lysosomes. Cellular entry of the complexes was completely inhibited by the macropinocytosis inhibitor, amiloride, indicating that the complexes enter cells via macropinosomes. Such mechanistic studies are an essential step to support future rational design of pegylated poly-L-lysine vectors to improve the efficiency of gene delivery.

Hypoxia induces neurite outgrowth in PC12 cells that is mediated through adenosine A2A receptors.

Development of the nervous system is a complex process, involving coordinated regulation of diverse cellular processes including proliferation, differentiation and synaptogenesis. Disturbances to brain development such as pre- and perinatal hypoxia have been linked to behavioural and late onset of neurological disorders. This study examines the effect of hypoxia on neurite outgrowth in PC12 cells. Hypoxia not only caused a rapid induction of neurite outgrowth, but also synergistically enhanced nerve growth factor (NGF)-induced neurite outgrowth up to 24 h. Transactivation of TrkA receptors was ruled out since the TrkA inhibitor K252a did not block hypoxia-induced neurite outgrowth. Adenosine deaminase prevented hypoxia-induced neurite outgrowth indicating that the effect is mediated by adenosine. Use of the specific adenosine A2A receptor agonist CGS21680 and antagonist 8-3(chlorostyryl)caffeine demonstrated that activation of this receptor is critical for hypoxia-induced neurite outgrowth. Hypoxia-induced neurite outgrowth was blocked by the adenylate cyclase inhibitor, MDL-12,330A, indicating a role for activation of this enzyme in the pathway. Hypoxia was further shown to cause a decrease in growth-associated protein (GAP)-43 levels and a lack of induction of betaIII tubulin, in contrast to NGF treatment which resulted in increased cellular levels of both of these proteins. These findings suggest that hypoxia induces neurite outgrowth in PC12 cells via a pathway distinct from that activated by NGF. Thus, exposure to hypoxia at critical stages of development may contribute to aberrant neurite outgrowth and could be a factor in the pathogenesis of certain delayed developmental neurological disorders.

A comparison of the permeation enhancement potential of simple bile salt and mixed bile salt:fatty acid micellar systems using the CaCo-2 cell culture model.

The aim of this study was to compare the permeation enhancing potential and toxicity of simple bile salt and bile salt:fatty acid mixed micellar systems using the CaCo-2 cell culture model. The effects of micellar systems of sodium cholate, (NaC), and sodium taurocholate, (NaTC), on the permeability of the hydrophilic markers, mannitol (182) and polyethylene glycols (PEGS) 900 and 4000, were assessed. Simple micelle systems of the unconjugated bile salt, NaC, caused greater enhancement of the hydrophilic markers than the conjugated bile salt, NaTC. In the case of NaC systems the enhancement was coincident with excess membrane disruption and toxicity as indicated by altered TEERs, TEMs, MTT values, and, the lack of recovery following removal of the enhancer. In contrast, the NaTC systems were less toxic, and, in the simple micelle form the likely mechanism of enhancement of the hydrophilic markers is via a transient effect on the tight junctions. Formation of mixed micellar systems with linoleic acid (LA) accentuated the toxic effects of NaC. In comparison, NaTC:LA mixed micelles showed superior permeability enhancement versus simple micelles without increasing membrane toxicity. The mechanism of enhancement of NaTC:LA appears more complex and involves a possible combination effect on both the paracellular and transcellular routes.

The relationship between rat intestinal permeability and hydrophilic probe size.

PURPOSE: The relationship between rat intestinal permeability (Papp) of a range of hydrophilic probe molecules and probe geometry was examined. METHODS: Molecules studies included mannitol, the polyethylene glycols (PEGs) 400, 900, and 4000, the dextran conjugated dye Texas Red (MW 3000) and the polysaccharide inulin (MW 5500). Molecular surface area, volume and cross-sectional diameter for each probe were determined from computer models. The effect of the bile salt sodium cholate, and bile salt: fatty acid mixed micelles on probe intestinal permeability was also studied. RESULTS: Of the size parameters tested, cross-sectional diameter correlated best with log intestinal permeability. The data was fitted to a relationship of the form Papp = Papp zero exp(-Krca) where rca is the molecular cross sectional radius. Papp zero and K are constants. Estimates of equivalent pore radii (R) were also made; the use of rca giving the most reasonable estimate of R. Absorption of all probes was enhanced by both simple and mixed micellar systems. CONCLUSIONS: For large hydrophilic probes and possibly protein drugs, cross sectional diameter is a more important size parameter than volume based values for evaluating size-related retarded absorption. The relationship established may be used as a tool to assess absorption enhancement potential of excipients.

Variations in morphology of the lumbosacral junction on sagittal MRI: correlation with plain radiography.

OBJECTIVE: To identify on sagittal magnetic resonance imaging (MRI) scans of the lumbar spine the features that indicate the presence of a lumbosacral transitional vertebra (LSTV). DESIGN: One hundred consecutive sagittal T1-and T2-weighted MRI scans of the lumbar spine were reviewed and separated into four types depending upon the absence or presence of disc material between what was considered to be the uppermost sacral segment and the remainder of the sacrum, as follows: type 1: no disc material present; type 2: a small residual disc, not extending for the whole anteroposterior (AP) diameter of the sacrum; type 3: a well-formed disc extending for the whole AP diameter of the sacrum; type 4: a well-formed disc extending for the whole AP diameter of the sacrum with, in addition, an abnormal upper sagittal sacral outline. The corresponding plain radiographs of each patient were than reviewed and assessed for the presence of an LSTV. These were classified according to the method of Castellvi et al. PATIENTS: All patients had been referred for MRI of the lumbar spine, usually because of back pain with or without radiculopathy. There were 51 male and 49 female patients with a mean age of 42 years and an age range of 18-85 years. RESULTS AND CONCLUSIONS: With regard to sacral morphology on MRI, 30 patients had type 1, 42 patients type 2, 16 patients type 3 and 12 patients type 4 morphology. Fifteen patients had an LSTV. There was a good correlation between the presence of a fused LSTV and a type 4 MRI appearance, indicating that this type of LSTV can be identified on sagittal MRI scans.

Localization of recurrent medullary thyroid carcinoma with technetium-99m-methoxyisobutylnitrile scintigraphy: a case report.

This case report demonstrates the successful localization of metastatic medullary thyroid carcinoma with 99mTc-labeled methoxyisobutylnitrile (MIBI). Disease recurrence was initially localized using 201Tl and by immunoscintigraphy with 111In-labeled anti-carcinoembryonic antigen (anti-CEA) antibody fragments. Scintigraphy with 99mTc-MIBI yielded higher target-to-background ratios than 201Tl or 111In-anti-CEA. Technetium-99m-MIBI may be a useful agent in the localization of recurrent medullary thyroid carcinoma.

A comparison of the effect of synthetic and naturally occurring surfactants on the solubility and absorption of clofazimine (B 663).

All simple micellar systems investigated, containing both naturally occurring and synthetic surfactants, enhanced the solubility of clofazimine. The incorporation of fatty acid to form mixed micelles, brought about a further enhancement in drug solubility in the case of naturally occurring surfactants, in contrast, with synthetic surfactants this enhancement decreased. The most effective absorption promoter was a synthetic surfactant: fatty acid mixed micellar system. The results indicate an optimum surfactant: fatty acid ratio at which the solubility and membrane effects of the surfactant system are balanced.