150 research outputs found
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
A new FRAP/FRAPa method for three-dimensional diffusion measurements based on multiphoton excitation microscopy
We present a new convenient method for quantitative three-dimensionally resolved diffusion measurements based on the photobleaching (FRAP) or photoactivation (FRAPa) of a disk-shaped area by the scanning laser beam of a multiphoton microscope. Contrary to previously reported spot-photobleaching protocols, this method has the advantage of full scalability of the size of the photobleached area and thus the range of diffusion coefficients, which can be measured conveniently. The method is compatible with low as well as high numerical aperture objective lenses, allowing us to perform quantitative diffusion measurements in three-dimensional extended samples as well as in very small volumes, such as cell nuclei. Furthermore, by photobleaching/ photoactivating a large area, diffusion along the optical axis can be measured separately, which is convenient when studying anisotropic diffusion. First, we show the rigorous mathematical derivation of the model, leading to a closed-form formula describing the fluorescence recovery/redistribution phase. Next, the ability of the multiphoton FRAP method to correctly measure absolute diffusioncoefficients is tested thoroughly onmanytest solutions of FITC-dextrans covering a wide range of diffusion coefficients. The same is done for the FRAPa method on a series of photoactivatable green fluorescent protein solutions with different viscosities. Finally, we apply the method to photoactivatable green fluorescent protein diffusing freely in the nucleus of living NIH-3T3 mouse embryo fibroblasts. © 2008 by the Biophysical Society
Fluorescence Single Particle Tracking for the Characterization of Submicron Protein Aggregates in Biological Fluids and Complex Formulations
To evaluate the potential of fluorescence single particle tracking (fSPT) for the characterization of submicron protein aggregates in human serum, plasma and formulations containing human serum albumin (HSA).
A monoclonal IgG was covalently labeled with a fluorescent dye and cross-linked with glutaraldehyde. IgG aggregates and fluorescent beads of 0.1 mu m (control) were diluted in buffer, serum and plasma, and their size distributions were analyzed by fSPT and nanoparticle tracking analysis (NTA). In a separate experiment, IgG and HSA, fluorescently labeled with different dyes, were mixed and subjected to heat stress. The stressed sample was analyzed by fSPT using a dual color mode and by NTA.
The accuracy and precision of fSPT proved to be comparable to NTA. fSPT was able to successfully measure all the samples in buffer, serum and plasma. The average size of the cross-linked protein aggregates showed a slight increase in biological fluids. Moreover, fSPT analysis showed that a significant proportion of the aggregates formed by subjecting an IgG/HSA mixture to heat stress were composed of both proteins.
fSPT is a powerful technique for the characterization of submicron protein aggregates in biological fluids and complex formulations
Detection and characterization of subvisible aggregates of monoclonal lgG in serum
To detect and characterize the aggregation of therapeutic monoclonal antibodies in undiluted biological fluids.
Fluorescently labeled subvisible IgG aggregates formed by applying either heat stress or by pH-shift were investigated immediately after addition to human serum, and after 24 h. Unstressed and stressed IgG formulations were analyzed by fluorescence single particle tracking, confocal laser scanning microscopy and flow cytometry.
Unstressed formulations remained free from subvisible aggregates in serum, whereas heat-stressed and pH-shift stressed formulations showed dissimilar aggregation behaviors. The aggregation profile of the heat-stressed formulation diluted in serum remained practically the same as the one diluted in buffer, even after the 24 h incubation period. The pH-shift stressed formulation had strikingly smaller and more numerous subvisible aggregates immediately after dilution in serum compared to buffer. These aggregates became noticeably larger in both diluents after 24 h, but in serum they appeared to be formed by other types of constituents than the labeled protein itself.
These results show that subvisible therapeutic protein aggregates may undergo changes in number, type and size distribution upon contact with human serum. This emphasizes the importance of analytical strategies for monitoring aggregation in undiluted biological fluids
Together is better: mRNA co-encapsulation in lipoplexes is required to obtain ratiometric co-delivery and protein expression on the single cell level
Liposomes can efficiently deliver messenger RNA (mRNA) into cells. When mRNA cocktails encoding different proteins are needed, a considerable challenge is to efficiently deliver all mRNAs into the cytosol of each individual cell. In this work, two methods are explored to co-deliver varying ratiometric doses of mRNA encoding red (R) or green (G) fluorescent proteins and it is found that packaging mRNAs into the same lipoplexes (mingle-lipoplexes) is crucial to efficiently deliver multiple mRNA types into the cytosol of individual cells according to the pre-defined ratio. A mixture of lipoplexes containing only one mRNA type (single-lipoplexes), however, seem to follow the "first come - first serve" principle, resulting in a large variation of R/G uptake and expression levels for individual cells leading to ratiometric dosing only on the population level, but rarely on the single-cell level. These experimental observations are quantitatively explained by a theoretical framework based on the stochasticity of mRNA uptake in cells and endosomal escape of mingle- and single-lipoplexes, respectively. Furthermore, the findings are confirmed in 3D retinal organoids and zebrafish embryos, where mingle-lipoplexes outperformed single-lipoplexes to reliably bring both mRNA types into single cells. This benefits applications that require a strict control of protein expression in individual cells.Drug Delivery Technolog
Dioctadecyldimethylammonium:monoolein nanocarriers for efficient in vitro gene silencing
This study describes a novel liposomal formulation for siRNA delivery, based on the mixture of the neutral lipid monoolein (MO) and cationic lipids of the dioctadecyldimethylammonium (DODA) family. The cationic lipids dioctadecyldimethylammonium bromide (DODAB) and chloride (DODAC) were compared in order to identify which one will most efficiently induce gene silencing. MO has a fluidizing effect on DODAC and DODAB liposomes, although it was more homogeneously distributed in DODAC bilayers. All MO-based liposomal formulations were able to efficiently encapsulate siRNA. Stable lipoplexes of small size (100-160 nm) with a positive surface charge (>+45 mV) were formed. A more uniform MO incorporation in DODAC:MO may explain an increase of the fusogenic potential of these liposomes. The siRNA-lipoplexes were readily internalized by human nonsmall cell lung carcinoma (H1299) cells, in an energy dependent process. DODAB:MO nanocarriers showed a higher internalization efficiency in comparison to DODAC:MO lipoplexes, and were also more efficient in promoting gene silencing. MO had a similar gene silencing ability as the commonly used helper lipid 1,2-dioleyl-3-phosphatidylethanolamine (DOPE), but with much lower cytotoxicity. Taking in consideration all the results presented, DODAB:MO liposomes are the most promising tested formulation for systemic siRNA delivery.This work was supported by FEDER through POFC - COMPETE and by national funds from FCT through the projects PEst-C/BIA/UI4050/2011 (CBM.A), PEst-C/FIS/UI0607/2011 (CFUM), and PTDC/QUI/69795/2006, while Ana Oliveira holds scholarship SFRH/BD/68588/2010. Eloi Feitosa thanks FAPESP (2011/03566-0) and CNPq (303030/2012-7), and Renata D. Adati thanks FAPESP for scholarship (2011/07414-0). K. Raemdonck is a postdoctoral fellow of the Research Foundation - Flanders (FWO-Vlaanderen). We acknowledge NanoDelivery-I&D em Bionanotecnologia, Lda. for access to their equipment
Sustained Oscillations of NF-κB Produce Distinct Genome Scanning and Gene Expression Profiles
NF-κB is a prototypic stress-responsive transcription factor that acts within a complex regulatory network. The signaling dynamics of endogenous NF-κB in single cells remain poorly understood. To examine real time dynamics in living cells, we monitored NF-κB activities at multiple timescales using GFP-p65 knock-in mouse embryonic fibroblasts. Oscillations in NF-κB were sustained in most cells, with several cycles of transient nuclear translocation after TNF-α stimulation. Mathematical modeling suggests that NF-κB oscillations are selected over other non-oscillatory dynamics by fine-tuning the relative strengths of feedback loops like IκBα. The ability of NF-κB to scan and interact with the genome in vivo remained remarkably constant from early to late cycles, as observed by fluorescence recovery after photobleaching (FRAP). Perturbation of long-term NF-κB oscillations interfered with its short-term interaction with chromatin and balanced transcriptional output, as predicted by the mathematical model. We propose that negative feedback loops do not simply terminate signaling, but rather promote oscillations of NF-κB in the nucleus, and these oscillations are functionally advantageous
Synergism between particle-based multiplexing and microfluidics technologies may bring diagnostics closer to the patient
In the field of medical diagnostics there is a growing need for inexpensive, accurate, and quick high-throughput assays. On the one hand, recent progress in microfluidics technologies is expected to strongly support the development of miniaturized analytical devices, which will speed up (bio)analytical assays. On the other hand, a higher throughput can be obtained by the simultaneous screening of one sample for multiple targets (multiplexing) by means of encoded particle-based assays. Multiplexing at the macro level is now common in research labs and is expected to become part of clinical diagnostics. This review aims to debate on the “added value” we can expect from (bio)analysis with particles in microfluidic devices. Technologies to (a) decode, (b) analyze, and (c) manipulate the particles are described. Special emphasis is placed on the challenges of integrating currently existing detection platforms for encoded microparticles into microdevices and on promising microtechnologies that could be used to down-scale the detection units in order to obtain compact miniaturized particle-based multiplexing platforms
Protein Diffusion in Mammalian Cell Cytoplasm
We introduce a new method for mesoscopic modeling of protein diffusion in an entire cell. This method is based on the construction of a three-dimensional digital model cell from confocal microscopy data. The model cell is segmented into the cytoplasm, nucleus, plasma membrane, and nuclear envelope, in which environment protein motion is modeled by fully numerical mesoscopic methods. Finer cellular structures that cannot be resolved with the imaging technique, which significantly affect protein motion, are accounted for in this method by assigning an effective, position-dependent porosity to the cell. This porosity can also be determined by confocal microscopy using the equilibrium distribution of a non-binding fluorescent protein. Distinction can now be made within this method between diffusion in the liquid phase of the cell (cytosol/nucleosol) and the cytoplasm/nucleoplasm. Here we applied the method to analyze fluorescence recovery after photobleach (FRAP) experiments in which the diffusion coefficient of a freely-diffusing model protein was determined for two different cell lines, and to explain the clear difference typically observed between conventional FRAP results and those of fluorescence correlation spectroscopy (FCS). A large difference was found in the FRAP experiments between diffusion in the cytoplasm/nucleoplasm and in the cytosol/nucleosol, for all of which the diffusion coefficients were determined. The cytosol results were found to be in very good agreement with those by FCS
In Vivo Monitoring of mRNA Movement in Drosophila Body Wall Muscle Cells Reveals the Presence of Myofiber Domains
Background: In skeletal muscle each muscle cell, commonly called myofiber, is actually a large syncytium containing numerous nuclei. Experiments in fixed myofibers show that mRNAs remain localized around the nuclei in which they are produced. Methodology/Principal Findings: In this study we generated transgenic flies that allowed us to investigate the movement of mRNAs in body wall myofibers of living Drosophila embryos. We determined the dynamic properties of GFP-tagged mRNAs using in vivo confocal imaging and photobleaching techniques and found that the GFP-tagged mRNAs are not free to move throughout myofibers. The restricted movement indicated that body wall myofibers consist of three domains. The exchange of mRNAs between the domains is relatively slow, but the GFP-tagged mRNAs move rapidly within these domains. One domain is located at the centre of the cell and is surrounded by nuclei while the other two domains are located at either end of the fiber. To move between these domains mRNAs have to travel past centrally located nuclei. Conclusions/Significance: These data suggest that the domains made visible in our experiments result from prolonged interactions with as yet undefined structures close to the nuclei that prevent GFP-tagged mRNAs from rapidly moving between the domains. This could be of significant importance for the treatment of myopathies using regenerative cellbase
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