Targeted delivery of a splice-switching oligonucleotide by cationic polyplexes of RGD-oligonucleotide conjugate

Nanoparticle-based delivery has become an important strategy to advance therapeutic oligonucleotides into clinical reality. Delivery by nanocarriers can enhance access of oligonucleotides to their pharmacological targets within cells; preferably, targeting ligands are incorporated into nanoparticles for targeting oligonucleotides to disease sites, often by conjugation to delivery carriers. In this study, a splice-switching oligonucleotide (SSO) was conjugated to a bivalent RGD peptide, and then, the RGD-SSO conjugate was formulated into polyplexes with a cationic polymer polyethylenimine. The resultant polyplexes of RGD-oligonucleotide conjugate demonstrated dramatic increase in the pharmacological response of splicing correction compared to free RGD-SSO conjugate or the polyplexes of unconjugated SSO, through integrin-mediated endocytosis and rapid endosomal release. This study has shown that coupling a targeting ligand to cargo oligonucleotide can maintain the integrin targeting ability after the peptide-oligonucleotide conjugate is complexed with cationic polymer. Preliminary study also revealed that integrin targeting redirects intracellular trafficking of the polyplexes to caveolar pathway and thereby generates greater effectiveness of the oligonucleotide. This study provides a new platform technology to construct multifunctional delivery systems of therapeutic oligonucleotides.     

Improving transfection efficiency of ultrapure oligochitosan/DNA polyplexes by medium acidification

Abstract

Context: Ultrapure oligochitosans (UOCs) have recently been reported as efficient nonviral vectors for corneal and retinal gene delivery. However, the influence of some physicochemical factors on the transfection efficiency, such as the pH, remains unclear. Deeper in vitro research of these factors could provide valuable information for future clinical applications.

Objective: The aim of this study is to determine the influence of the pH decrease on the transfection efficiency of UOC/pDNA polyplexes in HEK293 and ARPE19 cells.

Materials and methods: We elaborated self-assembled UOC/pCMS-EGFP polyplexes. The influence of the most important factors on the particle size and the zeta potential was studied by an orthogonal experimental design. We evaluated, in vitro, the cellular uptake and the transfection efficiency by flow cytometry, and the cytotoxicity of the vectors by CCK-8 assay.

Results and discussion: The pH of the medium strongly influences the physicochemical properties of the polyplexes, and by its modulation we are able to control their superficial charge. A significant increase on the cellular uptake and transfection efficiency of UOCs was obtained when the pH was acidified. Neither of our UOC/pCMS-EGFP polyplexes caused cytotoxicity; however, cells treated with Lipofectamine 2000? showed decreased cell viability.

Conclusion: This kind of UOC vectors could be useful to transfect cells that are in an acidic environment, such as tumor cells. However, additional in vivo studies may be required in order to obtain an effective and safe medicine for nonviral gene therapy purpose.     

Stability and activity of hydroxyethyl starch-coated polyplexes in frozen solutions or lyophilizates

Despite their great potential, gene delivery polyplexes have a number of limitations, including their tendency for aggregation in vivo or upon storage. In previous studies, we could show that hydroxyethyl starch (HES)-decoration of polyplexes reduces aggregation in vitro and in vivo. The current study investigates the ability of HES-decoration to improve the stability of polyplexes upon storage as frozen-liquid or lyophilizate, and uses naked polyplexes or PEGylated ones as controls. For this purpose, freeze-thaw (FT) experiments of the polyplexes were conducted in the presence of standard excipients (glucose, sucrose or trehalose). Dynamic light scattering (DLS) measurements showed that HES-decoration imparted better stability when glucose was used, while both HES and PEG were effective in inhibiting aggregation in the presence of trehalose or sucrose. In contrast, the lyophilized HES-coated polyplexes were more stable than the PEGylated ones as shown by DLS, even after storage for 10 weeks at an elevated temperature. Evaluation of the gene transfer efficiency of the stored samples showed no negative effect of storage, except for the lyophilized naked polyplexes. In general, this study shows that, while both HES- or PEG-coats could prevent aggregation under frozen-liquid storage, the HES-coat resulted in superior protective effect upon lyophilization, with possible advantages for in vivo application. In summary, our developed HES-coats provided effective cryo- and lyoprotection to the DNA polyplexes.     

Fine-tuning somatostatin receptor signalling by agonist-selective phosphorylation and dephosphorylation: IUPHAR Review 5

The biological actions of somatostatin are mediated by a family of five GPCRs, named sst₁ to sst₅. Somatostatin receptors exhibit equally high-binding affinities to their natural ligand somatostatin-14 and largely overlapping distributions. The overexpression of somatostatin receptors in human tumours is the molecular basis for diagnostic and therapeutic application of the stable somatostatin analogues octreotide, lanreotide and pasireotide. The efficiency of somatostatin receptor signalling is tightly regulated and ultimately limited by the coordinated phosphorylation and dephosphorylation of intracellular carboxyl-terminal serine and threonine residues. Here, we review and discuss recent progress in the generation and application of phosphosite-specific antibodies for human sst₂ and sst₅ receptors. These phosphosite-specific antibodies are unique tools to monitor the spatial and temporal dynamics of receptors phosphorylation and dephosphorylation. Using a combined approach of phosphosite-specific antibodies and siRNA knock-down screening, relevant kinases and phosphatases were identified. Emerging evidence suggests distinct mechanisms of agonist-selective fine-tuning for individual somatostatin receptors. The recently uncovered differences in phosphorylation and dephosphorylation of these receptors may hence be of physiological significance in mediating responses to acute, persistent or repeated stimuli in a variety of target tissues.     

Linear PEI nanoparticles: efficient pDNA/siRNA carriers in vitro and in vivo

Linear polyethylenimine (lPEI, 25 kDa) nanoparticles' (LPN) series was synthesized by varying percentage of cross-linking with 1,4-butanediol diglycidyl ether (BDE) and their size, surface charge, morphology, pDNA protection/release, cytotoxicity and transfection efficiency were evaluated. Synthesized nanoparticles (NPs) were spherical in shape (size: ～109 - 235 nm; zeta potential: +38 to +16 mV). These NPs showed increased buffering capacity with increasing percent cross-linking and also exhibited excellent transfection efficiency (i.e., ～1.3 - 14.7 folds in case of LPN-5) in comparison with lPEI and the commercial transfection agents used in this study. LPN-5 based GFP-specific siRNA delivery resulted in ～86% suppression of targeted gene expression. These particles were relatively nontoxic in vitro (in cell lines) and in vivo (in Drosophila). In vivo gene expression studies using LPN-5 in Balb/c mice through intravenous injection showed maximum expression of the reporter gene in the spleen. These results together demonstrate the potential of these particles as efficient transfection reagents. FROM THE CLINICAL EDITOR: The authors demonstrate a novel method of synthesizing linear PEI nanoparticles to utilize these as transfection agents.     

Pharmaceutical and biological properties of poly(amino acid)/DNA polyplexes

Physicochemical properties of polyplexes formed between pRSVlacZ and poly(amino acid)s were investigated as a paradigm of more complex, synthetic virus-like, DNA delivery systems, that are of interest to many gene delivery laboratories. We observed the interaction between polymer and DNA using ethidium exclusion, and determined the size distributions and the zeta potentials of polyplexes. We correlated these properties with their fundamental interactions with cultured B16 murine melanoma cells, and the resulting efficiency of transfection. A variety of poly(amino acid)s each condensed DNA to produce particles with mean hydrodynamic diameters of approximately 100 nm (a typical span of a population was 80-120nm). Poly(amino acid) polyplexes were unstable in electrolyte solutions such as cell culture media. The apparent particle size increased in electrolyte, depending on the charge ratio, to diameters up to 700 nm. This was thought to be due to aggregation, since neutral particles were most sensitive. When the charge ratio (+/-) exceeded unity polyplexes had positive zeta potentials (which peaked at approximately +30 mV), bound non-specifically to cells, were internalised and in the presence of an endosomolytic agent were able to transfect cells. Though all cationic poly(amino acid)s investigated formed polyplexes with similar physical properties, their biological properties were significantly different. Polyplexes prepared with poly-L-ornithine were the most effective transfection agents, but poly(lys-co-ala, 1: 1) systems appeared to be inactive. This may reflect the differences in uncoupling of DNA and polymer, which is expected to be necessary for passage through the nuclear pore. Uncoupling of polycation and DNA was investigated by exposing the complexes to dextran sulphate. Release of DNA was detected by increased fluorescence at 600 nm in the presence of ethidium. Release of DNA was incomplete from polyplexes formed with high molecular weight polylysine. This may explain the lower levels of transfection observed with high molecular weight polylysine. The significance of these observations for design of advanced non-viral gene delivery systems is discussed.     

Photo-control of the polyplexes formation between DNA and photo-cation generatable water-soluble polymers

Photo-cation generatable water-soluble polymers (Mw: approximately 1x10(5)) were prepared by radical copolymerization of N,N-dimethylacrylamide and vinyl monomer of triphenylmethane leucohydroxide (malachite green), which generate a cation upon ultraviolet light (UV) irradiation at wavelengths of between 290 and 410 nm. The malachite green contents of the copolymers were 3.6 (0.4 mol%), 7.9 (0.7 mol%), and 15.0 (2.7 mol%) per molecule. When an aqueous solution of the photo-cationized copolymers generated by UV irradiation was mixed with a Tris-HCl buffer (pH 7.4) of DNA (pGL3-control plasmid), dynamic light scattering (DLS) measurements showed the formation of polyplexes between the cationic copolymers and anionic DNA by non-specific electrostatic interaction, which was visualized with a confocal laser scanning microscopy (CLMS). Their mean cumulant diameter was about 150 nm with low polydispersity irrespective of the malachite green content in the copolymers. In the copolymer with the lowest malachite green content, almost all of the amount of the polyplexes was maintained by repeated UV irradiation, but the amount gradually decreased in the dark at 37 degrees C due to dissociation of the polyplexes, synchronized with the neutralization of the cation form of malachite green. The photo-cation generatable copolymers designed here can undergo photo-induced formation of the polyplexes with DNA and thermal polyplex dissociation, which may be used as a model for a novel photo-induced gene delivery system into cells.     

公立医院利用信息化手段实现精确管理的探讨

本文依据精确管理的基本原则与理念,提出公立医院实现管理思维创新化、管理流程精确化、管理参数具体化、管理过程透明化、管理手段人性化的基本内涵和实践意义,并阐述了利用信息化手段进行精细化管理、探索较为规范的管理体制和运行机制的实现途径。     

Inhibition by nitroso-chloramphenicol of the proton translocation in mitochondria

We have found recently that, unlike chloramphenicol (CAP), its nitroreduction derivative nitroso-chloramphenicol (NO-CAP) behaved as a potent inhibitor of the energy-conserving mechanism in mitochondria [Abou-Khalil et al. Biochem. Pharmac.29, 2605 (1980)]. Concentrations of 75 and 250 μM NO-CAP were required to inhibit ATP formation with glutamate and succinate, respectively, whereas similar CAP concentrations were without effect. Testing several key reactions associated with the biosynthesis of ATP, inhibitory concentrations of NO-CAP were found to interfere as follows: (a) the transport of an NAD-linked substrate (e.g. glutamate) into mitochondria was only partially inhibited, whereas that of an FAD-linked substrate (e.g. succinate) was not inhibited but was rather slightly activated; (b) the transport of P_i was only inhibited at about 50%; (c) mitochondrial ADP transport was not affected at all; (d) the ATPase activity, measured either by Pi release in the presence of an uncoupler or by H⁺ ejection, was only slightly affected; and (e) under either phosphorylation or no phosphorylation conditions and in the absence of P_i, NO-CAP was found to completely block mitochondrial H⁺ extrusion resulting from the oxidation of either succinate or glutamate; however, under similar conditions the oxidation of the two substrates was not totally inhibited. The possibility of interference by NO-CAP with reactive mitochondrial thiols groups is discussed in the light of previous data and current experiments showing protection by P_i against NO-CAP effects on respiration. Moreover, NO-CAP as compared to conventional inhibitors of oxidative phosphorylation (e.g. rotenone, antimycin A, oligomycin, mersalyl and others) appeared to have a distinct mode of action on that process. The results demonstrate that the inhibitory effect of NO-CAP is primarily located at the respiratory chain level where the proton translocation activity is fully blocked.     

Promoted Transfection Efficiency of pDNA Polyplexes-Loaded Biodegradable Microparticles Containing Acid-Labile Segments and Galactose Grafts

Purpose  

Targeting to antigen-presenting cells and efficient intracellular delivery of pDNA are essential for development of microsphere formulations of DNA vaccine.     

Quantification of DOX bioavailability in biological samples of mice by sensitive and precise HPLC assay

Context: Doxorubicin (DOX)-loaded folate-targeted poly(3-hydroxybutyrate-co-3-hydroxyoctanoate) [P(HB-HO)] nanoparticles [DOX/FA-PEG-P(HB-HO) NPs] were prepared by the W₁/O/W₂ solvent extraction/evaporation method for applications in cancer treatment. However, the biodistribution, pharmacokinetics, and targeting of the nanoparticles (NPs) have not yet been studied.

Objective: The biodistribution, pharmacokinetics, and targeting of DOX/FA-PEG-P(HB-HO) NPs were evaluated in female BALB/c nude mice bearing HeLa tumors.

Materials and methods: Three DOX formulations were injected into the tail vein of the mice at a dosage of 5?mg/kg. At each time point, blood and various tissues were collected. All samples were then processed and analyzed by a validated high performance liquid chromatographic (HPLC) method.

Results: The t_1/2 values of DOX/P(HB-HO) NPs and DOX/FA-PEG-P(HB-HO) NPs were 2.7- and 3.5-times higher than that of free DOX. No significant difference (p?>?0.05) was found in C_max between the NPs and free DOX. The T_max values of the two NPs were prolonged from 0.25 to 1?h. The AUC_0–t values were 1.55- and 3.05-folds higher than that of free DOX, and MRT increased to 15.99?h for DOX/P(HB-HO) NPs and 25.14?h for DOX/FA-PEG-P(HB-HO) NPs. For DOX/FA-PEG-P(HB-HO) NPs, the DOX content in the tumors were 10.81- and 3.33-times higher than those for free DOX and DOX/P(HB-HO) NPs at 48?h, respectively.

Discussion and conclusions: DOX/FA-PEG-P(HB-HO) NPs displayed reduced cardiac toxicity and improved bioavailability. Moreover, the NPs exhibited a significant extent of DOX accumulation in the tumors, thus suggesting that folate-targeted NPs could effectively transport into HeLa tumors with satisfying targeting.     

Fine-tuning serotonin2c receptor function in the brain: molecular and functional implications

The serotonin_2C receptor (5-HT_2CR) is a member of the serotonin₂ family of 7-transmembrane-spanning (7-TMS) receptors, which possesses unique molecular and pharmacological properties such as constitutive activity and RNA editing. The 5-HT_2CR is widely expressed within the central nervous system, where is thought to play a major role in the regulation of neuronal network excitability. In keeping with its ability to modulate dopamine (DA) neuron function in the brain, the 5-HT_2CR is currently considered as a major target for improved treatments of neuropsychiatric disorders related to DA neuron dysfunction, such as depression, schizophrenia, Parkinson's disease or drug addiction.The aim of this review is to provide an update of the functional status of the central 5-HT_2CR, covering molecular, cellular, anatomical, biochemical and behavioral aspects to highlight its distinctive regulatory properties, the emerging functional significance of constitutive activity and RNA editing in vivo, and the therapeutic potential of inverse agonism.     

Effect of cell membrane thiols and reduction-triggered disassembly on transfection activity of bioreducible polyplexes

Bioreducible polyplexes are promising vectors for delivery of nucleic acids due to low toxicity and favorable transfection activity. The often improved transfection is usually explained by enhanced intracellular reductive disassembly of the polyplexes. This study evaluated the effect of enhanced reductive disassembly on transfection activity of plasmid DNA and antisense oligonucleotide (AON) polyplexes based on a series of bioreducible poly(amido amine)s (PAA). We found that the presence of disulfide bonds in PAA had no effect on nucleic acid binding, hydrodynamic size and zeta potential of polyplexes. Increasing the disulfide content in PAA increased susceptibility to reduction-triggered DNA and AON release from the polyplexes. Increasing the disulfide content in PAA increased DNA transfection but had no effect on AON transfection. Plasma membrane protein thiols played a key role in the observed enhancement of DNA transfection. The presence of disulfide bonds in PAA had no significant effect on the rate of intracellular DNA clearance, suggesting that enhanced intracellular disassembly of the bioreducible polyplexes is not a major contributing factor to the improved transfection activity.     

The effect of metyrapone on granuloma induced by carrageenan-impregnated sponges in normal and essential fatty acid deficient rats

Treatment of normal rats with metyrapone (20 mg kg^?1 day^?1, s.c.) for 5 days, starting on the day before implantation, inhibited the production of granuloma, induced by carrageenan-impregnated sponges, determined 8 days after implantation. Exudate volume and prostaglandin (PG) production were unaffected. In essential fatty acid deficient (EFAD) rats, metyrapone did not alter the already existing adrenal hyperplasia due to EFAD and did not affect either granuloma formation or exudate production. The results are discussed in relation to earlier work using adrenalectomy and with regard to the effect of EFA deficiency on adrenal corticosteroid production. It is suggested that metyrapone is a more useful tool than adrenalectomy in studying the role of endogenous corticosteroids.     

Fine-tuning of the mucosal barrier and metabolic systems using the diet-microbial metabolite axis

The human intestinal microbiota has profound effects on human physiology, including the development and maintenance of the host immune and metabolic systems. Under physiological conditions, the intestinal microbiota maintains a symbiotic relationship with the host. Abnormalities in the host-microbe relationship, however, have been implicated in multiple disorders such as inflammatory bowel diseases (IBDs), metabolic syndrome, and autoimmune diseases. There is a close correlation between dietary factors and the microbial composition in the gut. Long-term dietary habits influence the composition of the gut microbial community and consequently alter microbial metabolic activity. The diet-microbiota axis plays a vital role in the regulation of the host immune system, at least partly through altering microbial metabolism. In this review, we will describe the current findings regarding how dietary factors and microbial metabolites regulate the host immune system.     

The preparation and characterisation of drug-loaded alginate and chitosan sponges

Sponges composed of sodium alginate and chitosan were prepared via a freeze drying process in order to assess the utility of mixed sponges as potential wound dressings or matrices for tissue engineering. Sponge preparation involved dissolving both polymers (either individually or mixed) in 1% acetic acid and freeze-drying the corresponding solutions. The mechanical properties of the sponges were assessed using texture analysis and the microstructure examined using scanning electron microscopy. The dissolution of a model drug (paracetamol) from the sponges was assessed as a function of polysaccharide composition. It was noted that the sponges had a flexible yet strong texture, as assessed macroscopically. Measurement of the resistance to compression ('hardness') indicated that the chitosan sponges were the 'hardest' while the alginate sponges showed the least resistance to compression, with all sponges showing a high degree of recovery. In contrast, the breaking force (tensile force) of the sponges were greatest for the single component systems, while the elongation prior to breaking was similar for each material. SEM studies indicated that the mixed systems had a less well-defined microstructure than the single component sponges. This was ascribed to the two polysaccharides interacting in aqueous solution via coulombic forces, leading to a more randomly ordered network being formed on freezing. Dissolution studies indicated that systems containing chitosan alone showed the slowest release profile, with the mixed systems showing a relatively rapid dissolution profile. The use of chitosan and alginates together, therefore, appears to allow the formulator to manipulate both the mechanical properties and the drug release properties of the sponges.