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

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

The impact of species and cell type on the nanosafety profile of iron oxide nanoparticles in neural cells

Background: While nanotechnology is advancing rapidly, nanosafety tends to lag behind since general mechanistic insights into cell-nanoparticle (NP) interactions remain rare. To tackle this issue, standardization of nanosafety assessment is imperative. In this regard, we believe that the cell type selection should not be overlooked since the applicability of cell lines could be questioned given their altered phenotype. Hence, we evaluated the impact of the cell type on in vitro nanosafety evaluations in a human and murine neuroblastoma cell line, neural progenitor cell line and in neural stem cells. Acute toxicity was evaluated for gold, silver and iron oxide (IO) NPs, and the latter were additionally subjected to a multiparametric analysis to assess sublethal effects. Results: The stem cells and murine neuroblastoma cell line respectively showed most and least acute cytotoxicity. Using high content imaging, we observed cell type-and species-specific responses to the IONPs on the level of reactive oxygen species production, calcium homeostasis, mitochondrial integrity and cell morphology, indicating that cellular homeostasis is impaired in distinct ways. Conclusions: Our data reveal cell type-specific toxicity profiles and demonstrate that a single cell line or toxicity end point will not provide sufficient information on in vitro nanosafety. We propose to identify a set of standard cell lines for screening purposes and to select cell types for detailed nanosafety studies based on the intended application and/or expected exposure

Delivery of mixed-lineage kinase domain-like protein by vapor nanobubble photoporation induces necroptotic-like cell death in tumor cells

Modern molecular medicine demands techniques to efficiently deliver molecules directly into mammalian cells. As proteins are the final mediators of most cellular pathways, efficient intracellular protein delivery techniques are highly desired. In this respect, photoporation is a promising recent technique for the delivery of proteins directly into living cells. Here, we show the possibility to deliver a model saccharide (FD70) and a model protein (FITC-BSA) into murine B16 melanoma cells by using the vapor nanobubble photoporation technique with an efficiency of 62% and 38%, respectively. Next, we delivered the mixed-lineage kinase domain-like (MLKL) protein, the most terminal mediator of necroptosis currently known, and caspase-8 and -3 protein, which are important proteins in the initiation and execution of apoptosis. A significant drop in cell viability with 62%, 71% and 64% cell survival for MLKL, caspase-8 and caspase-3, respectively, was observed. Remarkably, maximal cell death induction was already observed within 1 h after protein delivery. Transduction of purified recombinant MLKL by photoporation resulted in rapid cell death characterized by cell swelling and cell membrane rupture, both hallmarks of necroptosis. As necroptosis has been identified as a type of cell death with immunogenic properties, this is of interest to anti-cancer immunotherapy. On the other hand, transduction of purified recombinant active caspase-3 or -8 into the tumor cells resulted in rapid cell death preceded by membrane blebbing, which is typical for apoptosis. Our results suggest that the type of cell death of tumor cells can be controlled by direct transduction of effector proteins that are involved in the executioner phase of apoptosis or necroptosis