A pinboard by
Tobias Keil

PhD Student, Ludwig-Maximilians-Universität München


Since its discovery in mammalian cells, RNA interference based gene knock down has been exploited to develop efficient and specific nucleic acid based therapeutic strategies. In comparison to systemic delivery, direct localized administration of nucleic acids via the pulmonary route allows higher retention in lung tissues, reduced doses and minimized systemic toxicity and side effects. To harness the full potential of the RNAi strategy, an appropriately designed pulmonary delivery system is required to deliver siRNA to the deep lung areas as well as to increase the siRNA stability and shelf life. We propose novel Nano-In-Microparticles (NIM) that redisperse as nanoparticles (NP) in aqueous environment. The present study characterizes NP consisting of the cationic polymer PEI and bulkDNA (bDNA), mimicking siRNA, embedded in trehalose as bulking agent. NIM are produced by spray drying, which can be easily applied to large scale production. The aim of my thesis is the characterization of produced NIM according to its composition, its ability to deposit in deep lung areas, preservation of transfection efficiencies as well as gene downregulation and its effect on an in vivo rodent model of asthma.


Nonviral siRNA delivery to the lung: investigation of PEG-PEI polyplexes and their in vivo performance.

Abstract: This study describes the physicobiological characterization of PEI- and PEG-PEI polyplexes containing partially 2'-OMe modified 25/27mer dicer substrate siRNAs (DsiRNAs) and their in vivo behavior regarding biodistribution and systemic bioavailability after pulmonary application as well as their ability to knock down gene expression in the lung. Biophysical characterization included circular dichroism of siRNA in polyplexes, condensation efficiency of polymers and in vitro stability. After in vivo application, biodistribution and kinetics of radiolabeled polyplexes were quantified and recorded over time in three-dimensional SPECT images and by end point scintillation counting. The influence on lung tissue and on the humoral and cellular immunosystem was investigated, and finally knockdown of endogenous gene expression in the lung was determined qualitatively. While all of the polymers used in our study were proven to effectively condense siRNA, stability of the complexes depended on the PEG grafting degree. Interestingly, PEI 25 kDa, which showed the least interaction with mucin or surfactant in vitro, performed poorly in vivo. Our nuclear imaging approach enabled us to follow biodistribution of the instilled nanocarriers over time and indicated that PEGylated nanocarriers are more suitable for lung application. While moderate proinflammatory effects were attributed to PEI25k-PEG(2k)(10) nanocarriers, none of the treatments caused histological abnormalities. Our preliminary in vivo knockdown experiment suggests that PEG-PEI/siRNA complexes are promising nanomedicines for pulmonary siRNA delivery. These results encouraged us to further investigate possible adverse effects and to quantify in vivo gene silencing in the lung after intratracheal instillation of PEG-PEI/siRNA complexes.

Pub.: 18 Jul '09, Pinned: 18 Jun '17

Effects of Chemical Conjugation of l-Leucine to Chitosan on Dispersibility and Controlled Release of Drug from a Nanoparticulate Dry Powder Inhaler Formulation

Abstract: This study investigated l-leucine-conjugated chitosan as a drug delivery vehicle in terms of dispersibility and controlled release from a nanoparticulate dry powder inhaler (DPI) formulation for pulmonary delivery using diltiazem hydrochloride (DH) as the model drug. DH-loaded nanoparticles of chitosan and conjugate were prepared by water-in-oil emulsification followed by glutaraldehyde cross-linking. Nanoparticles were characterized by dynamic light scattering for particle size, X-ray photoelectron spectroscopy for surface composition, and twin stage impinger for drug dispersibility. The controlled release of DH was studied in phosphate-buffered saline (pH 7.3 ± 0.2, 37 °C) using UV spectrophotometry. The fine particle fractions of conjugated chitosan with and without drug were higher than those of nonconjugated chitosan nanoparticles. The conjugate nanoparticles were superior to those of unmodified chitosan in drug loading, entrapment efficiency, and controlled release profile. The higher dispersibility was attributed to the amphiphilic environment of the l-leucine conjugate and hydrophobic cross-links, and the release profile reflects the greater swelling. The conjugated chitosan nanoparticles could be useful, after appropriate testing for biodegradability and toxicity, as an alternative carrier for lung drug delivery with enhanced aerosolization and prolonged drug release from nanoparticulate DPI formulations.

Pub.: 21 Mar '16, Pinned: 18 Jul '17

Pulmonary administration of small interfering RNA: The route to go?

Abstract: Ever since the discovery of RNA interference (RNAi), which is a post-transcriptional gene silencing mechanism, researchers have been studying the therapeutic potential of using small interfering RNA (siRNA) to treat diseases that are characterized by excessive gene expression. Excessive gene expression can be particularly harmful if it occurs in a vulnerable organ such as the lungs as they are essential for physiological respiration. Consequently, RNAi could offer an approach to treat such lung diseases. Parenteral administration of siRNA has been shown to be difficult due to degradation by nucleases in the systemic circulation and excretion by the kidneys. To avoid these issues and to achieve local delivery and local effects, pulmonary administration has been proposed as an alternative administration route. Regarding this application, various animal studies have been conducted over the past few years. Therefore, this review presents a critical analysis of publications where pulmonary administration of siRNA in animals has been reported. Such an analysis is necessary to determine the feasibility of this administration route and to define directions for future research. First, we provide background information on lungs, pulmonary administration, and delivery vectors. Thereafter, we present and discuss relevant animal studies. Though nearly all publications reported positive outcomes, several reoccurring challenges were identified. They relate to 1) the necessity, efficacy, and safety of delivery vectors, 2) the biodistribution of siRNA in tissues other than the lungs, 3) the poor correlation between in vitro and in vivo models, and 4) the long-term effects upon (repeated) administration of siRNA. Finally, we present recommendations for future research to define the route to go: towards safer and more effective pulmonary administration of siRNA.

Pub.: 25 May '16, Pinned: 18 Jun '17

Poly(amidoamine) dendrimer nanocarriers and their aerosol formulations for siRNA delivery to the lung epithelium.

Abstract: Small interfering RNA (siRNA)-based therapies have great promise in the treatment of a number of prevalent pulmonary disorders including lung cancer, asthma and cystic fibrosis. However, progress in this area has been hindered due to the lack of carriers that can efficiently deliver siRNA to lung epithelial cells, and also due to challenges in developing oral inhalation (OI) formulations for the regional administration of siRNA and their carriers to the lungs. In this work we report the ability of generation four, amine-terminated poly(amidoamine) (PAMAM) dendrimer (G4NH2)-siRNA complexes (dendriplexes) to silence the enhanced green fluorescent protein (eGFP) gene on A549 lung alveolar epithelial cells stably expressing eGFP. We also report the formulation of the dendriplexes and their aerosol characteristics in propellant-based portable OI devices. The size and gene silencing ability of the dendriplexes was seen not to be a strong function of the N/P ratio. Silencing efficiencies of up to 40% are reported. Stable dispersions of the dendriplexes encapsulated in mannitol and also in a biodegradable and water-soluble co-oligomer were prepared in hydrofluoroalkane (HFA)-based pressurized metered-dose inhalers (pMDIs). Their aerosol characteristics were very favorable, and conducive to deep lung deposition, with respirable fractions of up to 77%. Importantly, siRNA formulated as dendriplexes in pMDIs was shown to keep its integrity after the particle preparation processes, and also after long-term exposures to HFA. The relevance of this study stems from the fact that this is the first work to report the formulation of inhalable siRNA with aerosol properties suitable to deep lung deposition using pMDIs devices that are the least expensive and most widely used portable inhalers. This study is relevant because, also for the first time, it shows that siRNA-G4NH2 dendriplexes can efficiently target lung alveolar epithelial A549 cells and silence genes even after siRNA has been exposed to the propellant environment.

Pub.: 09 May '14, Pinned: 18 Jun '17

Comparison of four different particle sizing methods for siRNA polyplex characterization.

Abstract: The ability to reliably determine the size of siRNA polyplexes is the key for the rational design of particles and their formulation, as well as, their safe application in vivo. At the moment, no standard technique for size measurements is available. Each method has different underlying principles and hence may give different results. Here, four different analytical methods were evaluated for their suitability to analyze the characteristics of homogeneous and heterogeneous siRNA polyplexes: dynamic light scattering (DLS), atomic force microscopy (AFM), nanoparticle trafficking analysis (NTA), and fluorescence correlation spectroscopy (FCS). Three different siRNA polyplex compositions generated with different, precise, and hydrophobically modified oligoaminoamides were used in this study. All of the evaluated methods were suitable for analysis of medium sized, homogeneous siRNA polyplexes (~120 nm). Small particles (<40 nm) could not be tracked with NTA, but with the other three methods. Heterogeneous polyplexes were generally difficult to analyze. Only by visualization with AFM, the heterogeneity of those polyplexes was observable. FCS was the only method suitable for measuring polyplex stability in 90% fetal bovine serum. Physico-chemical characteristics of polyplexes are important quality criterions for successful in vivo application and future formulation development. Therefore, a comprehensive analysis by more than one method is of particular importance.

Pub.: 20 Oct '12, Pinned: 30 Jun '17