Interactions of siRNA loaded dextran nanogel with blood cells

Adsorption and internalization of siRNA loaded dextran nanogels by blood cells were determined using flow cytometry. Positively and negatively charged nanogels with various PEGylation degrees were compared in order to find a formulation showing minimal interactions with blood

The influence of natural pulmonary surfactant on the efficacy of siRNA-loaded dextran nanogels

Aim: Topical administration of siRNA nanocarriers is a promising approach in the treatment of pulmonary disorders. Pulmonary surfactant, covering the entire alveolar surface of mammalian lungs, will be one of the first interfaces that siRNA nanocarriers encounter upon inhalation therapy. Therefore, it is of outstanding importance to evaluate the impact of pulmonary surfactant on the performance of siRNA nanocarriers. Materials & methods: The effect of natural lung-derived surfactants on the siRNA delivery capacity of dextran nanogels (DEX-NGs) was evaluated in vitro using flow cytometry and confocal microscopy. Results: Although the interaction with pulmonary surfactant decreases the cellular internalization of siRNA-loaded DEX-NGs significantly, the gene silencing potential of siRNA-loaded DEX-NGs was maintained. On the other hand, cationic lipid-based siRNA nanocarriers (Lipofectamine (TM) RNAiMAX) were incompatible with pulmonary surfactants. Conclusion: Our data suggest that pulmonary surfactant can enhance the intracellular siRNA delivery by DEX-NGs, thereby possibly providing new therapeutic opportunities

Bio-inspired pulmonary surfactant-modified nanogels : a promising siRNA delivery system

Inhalation therapy with small interfering RNA (siRNA) is a promising approach in the treatment of pulmonary disorders. However, clinical translation is severely limited by the lack of suitable delivery platforms. In this study, we aim to address this limitation by designing a novel bioinspired hybrid nanoparticle with a core-shell nanoarchitecture, consisting of a siRNA-loaded dextran nanogel (siNG) core and a pulmonary surfactant (Curosurf (R)) outer shell. The decoration of siNGs with a surfactant shell enhances the colloidal stability and prevents siRNA release in the presence of competing polyanions, which are abundantly present in biofluids. Additionally, the impact of the surfactant shell on the biological efficacy of the siNGs is determined in lung cancer cells. The presence of the surfactants substantially reduces the cellular uptake of siNGs. Remarkably, the lowered intracellular dose does not impede the gene silencing effect, suggesting a crucial role of the pulmonary surfactant in the intracellular processing of the nanoparticles. In order to surmount the observed reduction in cellular dose, folate is incorporated as a targeting ligand in the pulmonary surfactant shell to incite receptor-mediated endocytosis. The latter substantially enhances both cellular uptake and gene silencing potential, achieving efficient knockdown at siRNA concentrations in the low nanomolar range. (C) 2015 Elsevier B.V. All rights reserved

New physical and chemical approaches for the cytosolic delivery of bio- therapeutics and nanoparticles into cells

Delivery of bio-therapeutics and nanomaterials into living cells is an important step not only for cell studies but also for therapy and bio-imaging. Clear examples are the intracellular delivery of various classes of nucleic acids (siRNA, µRNA, mRNA, pDNA), peptides and proteins for therapy purposes. As another example, all types of (inorganic/organic) nanoparticles are under investigation as intracellular labels for imaging purposes. Meanwhile it generally accepted that after uptake by cells, nanomaterials typically end up in endo-lysosomal vesicles in which they remain entrapped while they should escape from such compartments and arrive in the cytosolic fluids of the cells. In recent years our team undertook major efforts to understand the biophysics which play a role in (a lack of) escape of nanomaterials from endo-lysosomal vesicles. Vere recently we also discovered new chemical strategies (so named ‘escape adjuvants’) (1) which seems promising to ‘liberate’ nucleic acids (like siRNA) from endo-lysosomal vesicles into the cytosol. Furthermore we explored physical methods (either light (2,3) or ultrasound (4) driven) which directly deliver bio-therapeutics into the cytosol, thereby bypassing the endo-lysosomal routes. This lecture will explain our recent findings in this area, as reported in a serious of recently published papers (1-4). Both pharmaceutical, biological and engineering aspects of our work will be highlighted in the lecture. References 1) Repurposing cationic amphiphilic drugs as adjuvants to induce lysosomal siRNA escape in nanogel transfected cells F. Joris, L. De Backer, T. Van de Vyver, C. Bastiancich, S.C. De Smedt, K. Raemdonck Journal of Controlled Release 2018, in Press 2) Comparison of gold nanoparticle mediated photoporation: vapour nanobubbles outperform direct heating for delivering macromolecules in live cells R.H. Xiong, K. Raemdonck, K. Peynshaert, I. Lentacker, I. De Cock, J. Demeester, S.C. De Smedt, A.G. Skirtach, K. Braeckmans ACS Nano 2014, 8(6): 6288-6296 3) Cytosolic Delivery of Nanolabels Prevents Their Asymmetric Inhentance and Enables Extended Quantitative in Vivo Cell Imaging R.H. Xiong, F. Joris, S.Y. Liang, R. De Rycke, S. Lippens, J. Demeester, A. Skirtach, K. Raemdonck, U. Himmelreich, S.C. De Smedt, K. Braeckmans Nano Letters 2016, 16(10): 5975-5986 4) Sonoprinting and the importance of microbubble loading for the ultrasound mediated cellular delivery of nanoparticles I. De Cock, G.P.R. Lajoinie, M. Versluis, S.C. De Smedt*, I. Lentacker Biomaterials 2016, 83: 294-30

FRAP in pharmaceutical research: practical guidelines and applications in drug delivery

Fluorescence recovery after photobleaching (FRAP) is a fluorescence microscopy technique that has attracted a lot of interest in pharmaceutical research during the last decades. The main purpose of FRAP is to measure diffusion on a micrometer scale in a non-invasive and highly specific way, making it capable of measurements in complicated biomaterials, even in vivo. This has proven to be very useful in the investigation of drug diffusion inside different tissues of the body and in materials for controlled drug delivery. FRAP has even found applications for the improvement of several medical therapies and in the field of diagnostics. In this review, an overview is given of the different applications of FRAP in pharmaceutical research, together with essential guidelines on how to perform and analyse FRAP experiments

Nanocarrier lipid composition modulates the impact of pulmonary surfactant protein B (SP-B) on cellular delivery of siRNA

Two decades since the discovery of the RNA interference (RNAi) pathway, we are now witnessing the approval of the first RNAi-based treatments with small interfering RNA (siRNA) drugs. Nevertheless, the widespread use of siRNA is limited by various extra- and intracellular barriers, requiring its encapsulation in a suitable (nanosized) delivery system. On the intracellular level, the endosomal membrane is a major barrier following endocytosis of siRNA-loaded nanoparticles in target cells and innovative materials to promote cytosolic siRNA delivery are highly sought after. We previously identified the endogenous lung surfactant protein B (SP-B) as siRNA delivery enhancer when reconstituted in (proteo) lipid-coated nanogels. It is known that the surface-active function of SP-B in the lung is influenced by the lipid composition of the lung surfactant. Here, we investigated the role of the lipid component on the siRNA delivery-promoting activity of SP-B proteolipid-coated nanogels in more detail. Our results clearly indicate that SP-B prefers fluid membranes with cholesterol not exceeding physiological levels. In addition, SP-B retains its activity in the presence of different classes of anionic lipids. In contrast, comparable fractions of SP-B did not promote the siRNA delivery potential of DOTAP:DOPE cationic liposomes. Finally, we demonstrate that the beneficial effect of lung surfactant on siRNA delivery is not limited to lung-related cell types, providing broader therapeutic opportunities in other tissues as well