Linda Jensen

Design of an inhalable dry powder formulation of DOTAP-modified PLGA nanoparticles loaded with siRNA

Matrix systems based on biocompatible and biodegradable polymers like the United States Food and Drug Administration (FDA)-approved polymer poly(DL-lactide-co-glycolide acid) (PLGA) are promising for the delivery of small interfering RNA (siRNA) due to favorable safety profiles, sustained release properties and improved colloidal stability, as compared to polyplexes. The purpose of this study was to design a dry powder formulation based on cationic lipid-modified PLGA nanoparticles intended for treatment of severe lung diseases by pulmonary delivery of siRNA. The cationic lipid dioleoyltrimethylammoniumpropane (DOTAP) was incorporated into the PLGA matrix to potentiate the gene silencing efficiency. The gene knock-down level in vitro was positively correlated to the weight ratio of DOTAP in the particles, and 73% silencing was achieved in the presence of 10% (v/v) serum at 25% (w/w) DOTAP. Optimal properties were found for nanoparticles modified with 15% (w/w) DOTAP, which reduced the gene expression with 54%. This formulation was spray-dried with mannitol into nanocomposite microparticles of an aerodynamic size appropriate for lung deposition. The spray-drying process did not affect the physicochemical properties of the readily re-dispersible nanoparticles, and most importantly, the in vitro gene silencing activity was preserved during spray-drying. The siRNA content in the powder was similar to the theoretical loading and the siRNA was intact, suggesting that the siRNA is preserved during the spray-drying process. Finally, X-ray powder diffraction analysis demonstrated that mannitol remained in a crystalline state upon spray-drying with PLGA nanoparticles suggesting that the sugar excipient might exert its stabilizing effect by sterical inhibition of the interactions between adjacent nanoparticles. This study demonstrates that spray-drying is an excellent technique for engineering dry powder formulations of siRNA nanoparticles, which might enable the local delivery of biologically active siRNA directly to the lung tissue.

Elucidating the molecular mechanism of PAMAM-siRNA dendriplex self-assembly: effect of dendrimer charge density.

Dendrimers are attractive vehicles for nucleic acid delivery due to monodispersity and ease of chemical design. The purpose of this study was to elucidate the self-assembly process between small interfering RNA (siRNA) and different generation poly(amidoamine) dendrimers and to characterize the resulting structures. The generation 4 (G4) and G7 displayed equal efficiencies for dendriplex aggregate formation, whereas G1 lacked this ability. Nanoparticle tracking analysis and dynamic light scattering showed reduced average size and increased polydispersity at higher dendrimer concentration. The nanoparticle tracking analysis indicated that electrostatic complexation results in an equilibrium between differently sized complex aggregates, where the centre of mass depends on the siRNA:dendrimer ratio. Isothermal titration calorimetric data suggested a simple binding for G1, whereas a biphasic binding was evident for G4 and G7 with an initial exothermic binding and a secondary endothermic formation of larger dendriplex aggregates, followed by agglomeration. The initial binding became increasingly exothermic as the generation increased, and the values were closely predicted by molecular dynamics simulations, which also demonstrated a generation dependent differences in the entropy of binding. The flexible G1 displayed the highest entropic penalty followed by the rigid G7, making the intermediate G4 the most suitable for dendriplex formation, showing favorable charge density for siRNA binding.

Molecular characterization of the interaction between siRNA and PAMAM G7 dendrimers by SAXS, ITC, and molecular dynamics simulations.

A prerequisite for the use of dendrimers as drug delivery vehicles is the detailed molecular understanding of the drug interaction. The purpose of this study was to characterize the self-assembly process between siRNA and generation 7 poly(amidoamine) dendrimers and the resulting dendriplexes in aqueous solution using structural and calorimetric methods combined with molecular dynamics simulations. Complexes with a length scale of 150 nm showed a decreasing size with increasing amine-to-phosphate ratio by dynamic light scattering. At the molecular level, individual dendrimers studied by small-angle X-ray scattering (SAXS) showed no change in size upon siRNA binding, suggesting a rigid sphere behavior. Isothermal titration calorimetry (ITC) demonstrated exothermic binding with a concentration-dependent collapse of complexes. Both the experimentally determined ΔH(bind) and size were in close accordance with molecular dynamics simulations. This study demonstrates the unique complementarity of SAXS, ITC, and modeling for the detailed description of the molecular interactions between dendrimers and siRNA during dendriplex formation.

In vitro penetration properties of solid lipid nanoparticles in intact and barrier-impaired skin.

Treatment of skin diseases implies application of a drug to skin with an impaired epidermal barrier, which is likely to affect the penetration profile of the drug substance as well as the carrier into the skin. To elucidate this, the effect of skin barrier damage on the penetration profile of a corticosteroid applied in solid lipid nanoparticles (SLN) composed of different lipids, varying in polarity, was studied. The studies were carried out in vitro using impaired and intact porcine ear skin, and the SLN were compared with a conventional ointment. It was shown that a significantly higher amount of corticosteroid remained in the skin, intact as well as barrier impaired, when SLN was used as a vehicle. In general, the penetration profile of the drug substance into the skin was affected by the type of lipid used in the formulation and related to lipid polarity and drug substance solubility. When formulated in SLN and applied to intact skin, the permeation of the drug substance across the skin was significantly reduced, as compared to the ointment. Altogether, in both barrier-impaired and intact skin, a higher amount of drug substance remained in the skin during application of SLN for 6, 16, and 24h, as compared to the ointment. These results emphasize the applicability of SLN to create a drug reservoir in skin, with the drug localized distinctively in the stratum corneum.

Circulating clonal cells in multiple myeloma do not express CD34 mRNA, as measured by single‐cell and real‐time RT‐PCR assays

The peripheral blood (PB) mononuclear cells in patients with multiple myeloma (MM) have been reported to include CD34‐expressing cells that are clonally related to the myeloma cells. To determine whether there were elevated levels of CD34 mRNA or whether CD34+ cells in the PB include myeloma‐related cells, we developed a quantitative real‐time and a competitive CD34 RT‐PCR assay working on single flow‐sorted cells. Myeloma‐specific cells were detected with allele‐specific oligonucleotides (ASO) IgH PCR. PBSC products and mononuclear cell fractions in blood from normal donors, untreated and treated myeloma patients were analysed.

Transfer of plasmid RP4 in the spermosphere and rhizosphere of barley seedling

Transfer of plasmid RP4 to indigenous bacteria in bulk soil could only be detected in soil with nutrient amendment. Lack of physiological active donor and recipient cells was apparently one of the limiting factors in un-amended bulk soil. Plasmid transfer was detected both in the spermosphere and rhizosphere of barley seedlings. Transfer occured from seed coated donor bacteria (i) to introduced recipient bacteria and (ii) to indigenous bacteria present in soil. Plasmid transfer was also detected from donor bacteria introduced to the soil to seed coated recipient bacteria. Transfer efficiencies in the rhizosphere were significantly below the transfer efficiencies obtained in the spermosphere. The transfer efficiencies detected in the barley spermosphere were among the highest reported from any natural environment.

MRI-assessed therapeutic effects of locally administered PLGA nanoparticles loaded with anti-inflammatory siRNA in a murine arthritis model

Rheumatoid arthritis is characterized by systemic inflammation of synovial joints leading to erosion and cartilage destruction. Although efficacious anti-tumor necrosis factor α (TNF-α) biologic therapies exist, there is an unmet medical need for safe and more efficient treatment regimens for disease remission. We evaluated the anti-inflammatory effects of poly(dl-lactide-co-glycolide acid) (PLGA) nanoparticles loaded with small interfering RNA (siRNA) directed against TNF-α in vitro and in vivo. The siRNA-loaded PLGA nanoparticles mediated a dose-dependent TNF-α silencing in lipopolysaccharide-activated RAW 264.7 cells in vitro. The severity of collagen antibody-induced arthritis in DBA/1J mice was assessed by paw scoring and compared to the degree of magnetic resonance imaging (MRI)-quantified joint effusion and bone marrow edema. Two intra-articular treatments per joint with nanoparticles loaded with TNF-α siRNA (1 μg) resulted in a reduction in disease activity, evident by a significant decrease of the paw scores and joint effusions, as compared to treatment with PLGA nanoparticles loaded with non-specific control siRNA, whereas the degree of bone marrow edema in the tibial and femoral head remained unchanged. When the siRNA dose was 5 or 10 μg, there was no difference between the specific and the non-specific siRNA treatment groups. These findings suggest that MRI is a promising method for evaluation of early disease progression and treatment in murine arthritis models. In addition, proper siRNA dosing seems to be important for a positive therapeutic outcome in vivo. However, further studies are needed to fully clarify the mechanism(s) underlying the observed anti-inflammatory effects of the siRNA-loaded nanoparticles.

Comparison of Polymeric siRNA Nanocarriers in a Murine LPS-Activated Macrophage Cell Line: Gene Silencing, Toxicity and Off-Target Gene Expression

ABSTRACTPurposeTumor necrosis factor α (TNF-α) plays a key role in the progression of rheumatoid arthritis and is an important target for anti-rheumatic therapies. TNF-α expression can be silenced with small interfering RNA (siRNA), but efficacy is dependent on efficient and safe siRNA delivery vehicles. We aimed to identify polymeric nanocarriers for anti-TNF-α siRNA with optimal efficacy and minimal off-target effects in vitro.MethodsTNF-α silencing with polymeric siRNA nanocarriers was compared in lipopolysaccharide-activated RAW 264.7 macrophages by real-time reverse transcription (RT)-PCR. Expression of non-target genes involved in inflammation, apoptosis, and cell cycle progression was determined by RT-PCR, toxicity evaluated by propidium iodide and annexin V staining.ResultsPAMAM dendrimers (G4 and G7) and dextran nanogels mediated remarkably high concentration-dependent gene silencing and low toxicity; dioleoyltrimethylammoniumpropane-modified poly(DL-lactide-co-glycolide acid) nanoparticles, thiolated, trimethylated chitosan and poly[(2-hydroxypropyl)methacrylamide 1-methyl-2-piperidine methanol] polyplexes were less efficient transfectants. There were minor changes in the regulation of off-target genes, mainly dependent on nanocarrier and siRNA concentration.ConclusionsDextran nanogels and PAMAM dendrimers mediated high gene silencing with minor toxicity and off-target transcriptional changes and are therefore expected to be suitable siRNA delivery systems in vivo.

The Clonal Hierachy in Multiple Myeloma

In this report we evaluated the number and phenotype of blood circulating B-cell subsets at different stages of differentiation in 26 patients with newly diagnosed multiple myeloma (MM). In all patients, plasma cells and/or plasma blasts could be identified by flow cytometry with a mean frequency of 1.20% and 0.07%, respectively. In 76.9% of the patients these cells showed aberrant expression mainly of CD56, CD28 and CD117, none of these markers were found on the earlier B-lymphocytes. Clonal B-cells preceding the plasma blast stage were identified by patient specific IgH RT-PCR on sorted B-cell subsets. The clonal cells included the less differentiated CD38+/CD19+ and CD38-/CD19+ subsets, illustrating that the clonal cells are part of an ongoing differentiation process. Further, the presence of CD38-/CD19+

The CD19 compartment in myeloma includes a population of clonal cells persistent after high-dose treatment

Peripheral blood-localized clonal cells in patients with multiple myeloma (MM) have been reported to be insensitive to chemotherapy and it has been suggested that these cells may include the proliferative compartment in myeloma. In this study, circulating clonal CD19 + cells levels were determined in 10 patients with MM by performing patient-specific RT-PCR on single flow-sorted CD19 + cells. A variable proportion of CD + cells being clonal were identified (0.101-6.130%, mean 1.019%). In 10/10 MM patients clonal CD19 + cells were found after high-dose treatment and peripheral blood stem cell transplantation. In conclusion, CD19 + clonal cells include a population of cells persistent after high dose chemotherapy that may be responsible for relapse.