Preparation and Analysis of Bacteria Surface-Modified with Polymer Nanoparticles

Cell-based drug carriers present an interesting approach to improve the targeted delivery of therapeutic drugs in vivo. Specifically, bacterial cells possess interesting properties such as motility and sensing that allow some strains to selectively target tumor areas. Immobilization of abiotic materials on the cell surface of such bacterial cells can therefore enable the delivery of therapeutic compounds at the desired location. The work presented in this thesis explores the fabrication and characterization of bacteria-nanoparticle biohybrids as potential carriers for drug delivery applications.Chapter 1 presents a review of the scientific literature that covers the different approaches to immobilize abiotic materials on the surface of bacteria. It highlights the advantages of using cells as carriers for the transport of therapeutics. An effective drug delivery agent should present great motility, viability, biodistribution and biocompatibility with human cells. Examples of various chemical approaches to surface-modify bacteria are presented. Chapter 2 systematically explores the immobilization of polystyrene nanoparticles on the surface of Escherichia coli (E. coli) and Salmonella typhimurium (Salmonella) cells. We compared the nanoparticle immobilization using four different conjugations strategies (electrostatic, covalent and ligand-ligand) and characterized the bio-hybrids systems using flow cytometry and confocal microscopy. Two different microscopy techniques were used: the first was based on 3D-reconstruction of z-stacks of single bacterium to investigate position of nanoparticles on the membrane; the second allowed for a large screening of bacteria followed by image processing determining the area of bacteria covered by nanoparticles. We observed that the efficiency of the surface modification is correlated to the initial nanoparticle/cell ratio and that non-covalent strategies allowed for increased number of biohybrids formed and nanoparticle attached. This study provides an overview of the variety of conjugation strategies (and their conjugation efficiencies) available for fabricating nanoparticle-decorated bacteria and presents us an opportunity to better understand and design bacteria-based target-specific drug delivery systems.Chapter 3 presents the characterization of the previously developed bacteria-nanoparticle biohybrids as potential carriers for nanoparticle transport. The viability of the various conjugates was determined by flow cytometry indicating a decrease in cell viability with increasing numbers of nanoparticles immobilized on the membrane. The motility was studied to investigate the impact of nanoparticle attachment on the bacterial velocity as well as the capacity to transport the cargo. Bacteria were able to transport nanoparticles when prepared with low ratios of nanoparticle/cell.

Covalent and Noncovalent Conjugation of Degradable Polymer Nanoparticles to T Lymphocytes

Harm-Anton Klok, Tanja Thomsen

Cells are attractive as carriers that can help to enhance control over the biodistribution of polymer nanomedicines. One strategy to use cells as carriers is based on the cell surface immobilization of the nanoparticle cargo. While a range of strategies can be used to immobilize nanoparticles on cell surfaces, only limited effort has been made to investigate the effect of these surface modification chemistries on cell viability and functional properties. This study has explored seven different approaches for the immobilization of poly(lactic acid) (PLA) nanoparticles on the surface of two different T lymphocyte cell lines. The cell lines used were human Jurkat T cells and CD4(+) T-EM cells. The latter cells possess blood-brain barrier (BBB) migratory properties and are attractive for the development of cell-based delivery systems to the central nervous system (CNS). PLA nanoparticles were immobilized either via covalent active ester-amine, azide-alkyne cycloaddition, and thiol-maleimide coupling, or via noncovalent approaches that use lectin-carbohydrate, electrostatic, or biotin-NeutrAvidin interactions. The cell surface immobilization of the nanoparticles was monitored with flow cytometry and confocal microscopy. By tuning the initial nanoparticle/cell ratio, T cells can be decorated with up to similar to 185 nanoparticles/cell as determined by confocal microscopy. The functional properties of the nanoparticle-decorated cells were assessed by evaluating their binding to ICAM-1, a key protein involved in the adhesion of CD4(+) TEM cells to the BBB endothelium, as well as in a two-chamber model in vitro BBB migration assay. It was found that the migratory behavior of CD4(+) T-EM cells carrying carboxylic acid-, biotin-, or Wheat germ agglutinin (WGA)-functionalized nanoparticles was not affected by the presence of the nanoparticle payload. In contrast, however, for cells decorated with maleimide-finictionalized nanoparticles, a reduction in the number of migratory cells compared to the nonmodified control cells was observed. Investigating and understanding the impact of nanoparticle-cell surface conjugation chemistries on the viability and properties of cells is important to further improve the design of cell-based nanoparticle delivery systems. The results of this study present a first step in this direction and provide first guidelines for the surface modification of T cells, in particular in view of their possible use for drug delivery to the CNS.

AMER CHEMICAL SOC2021

Miniaturized bioanalytics to probe the function of membrane proteins

Pedro Pascoal

G-protein coupled receptors (GPCRs) are the most abundant class of proteins in the cell body. Such receptors are of major interest as potential therapeutic targets. Downscaling and parallelization of bioanalytics opens novel routes to rapidly screen and identify potential drugs with a decrease in regard to the costs, and elucidate novel functions of signaling networks under physiological conditions. Native vesicles are small autonomous biological containers, which are efficiently produced from all cell lines. They are composed of their mother cell plasma membrane and enclose part of their cytoplasm. Membrane receptors are then exposed at their surface and already demonstrate to induce cellular signaling when exposed to receptor ligands. Native vesicles were investigated in this present work, as novel possibilities to downscale receptor investigation in live cells, using neurokinin 1 receptor (NK1R) as a representative model. Here native vesicle production and purification was optimized. The influence of the cell cycle on the production efficiency was demonstrated. Biological proteins were downregulated in order to produce blebbing cells. Native vesicle characterization was achieved. The receptors also demonstrate to be efficiently labeled by agonist and antagonist, allowing to access information about the binding kinetics as well as KD values. The results are in agreement with those obtained in live cells. Native vesicles also demonstrate to internalize agonist after application, demonstrating receptor desensitization and signaling performance similar as live cells. Confocal microscopy shows that cells expressing the NK1R-CFP have two binding affinities for their main agonist, substance P. Similar results could be observed with flow cytometry. The high affinity binding is related to cholesterol content in the cell membrane and was abolished by cholesterol depletion with methyl-β-cyclodextrin. Micro-contact printing (µCP) was used to (bio)functionalize surfaces with proteins, polymers or functionalized nanoparticles. Precise sample positioning by micro-contact printing shows improves nuclear magnetic resonance excitation and detection, when performed with a planar microcoil probe. µCP was used to produce native vesicle arrays by two procedures, and fluorescent binding assay shows the binding of fluorescent ligands to the receptor. Laser tweezers allow manipulating cell membranes without requiring the use of polystyrene beads. From pulled membranes, native vesicles were produced. In addition from pulled membranes, large tethers were produced and artificial connections were established with neighboring cells. Intercellular communication was investigated by whole-cell patch clamp in dissociated primary dorsal root ganglion neurons after optical induced connection, as well as in HEK cells expressing Cx36. The lab-on-chip assay development demonstrates: the high production of native vesicles in microchannels; the efficient purification obtained by negative dielectrophoresis depletion in MEMS chips; perfect trapping and thus immobilization of native vesicles in a new optical multi-tweezer array. Fluorescence labeling was performed with native vesicles trapped in a multi-tweezer array inside microfluidic channel in the presence of two laminar flows. Optical multi-tweezer array setup shows to be the fastest and more efficient technique in order to perform immobilization and labeling of native vesicles in the microfluidic channel. It is presently the only technique to perform fluorescence measurements when maintaining objects trapped.

EPFL2008

Transmembrane Signaling Analysis in Model Membrane Systems

Luigino Grasso

Studying the complex mechanisms of transducing an extracellular signal across a plasma membrane to initiate an intracellular responce is of fundamental importance for a cell’s function. Transmembrane signaling is the key allowing cells to sense and communicate with its environment. Signal transduction is primarily mediated by numerous membrane receptors. In this thesis we investigated the function of G-protein-coupled receptors (GPCR). GPCRs represent the largest and most diverse group of membrane proteins, encoded by more than 800 genes. They allow cells to recognize as diverse extracellular stimuli as photons, organic odorants, nucleotides, nucleosides, peptides, lipids and proteins, conferring to GPCRs a fundamental role in signal transduction. They are broadly distributed across the entire body and thus involved in a wide range of physiological and pathological processes, which makes GPCRs one of the main targets of modern medicines. Activation of GPCRs initiate numerous intracellular signaling pathways, involving a large number of proteins and molecules. They act as a central molecular activators and integrators of a complex network of signaling pathways. While most of the studies have been performed on cells, the complexity of cellular systems has raised the need to develop simpler model systems to assess the role of individual signaling com- ponents. In this thesis we present different model systems to study the functionality of GPCR signal transduction from the ligand binding to the activation of downstream cellular reactions. The first part of the thesis concerns the isolation of single receptors from cultured cells by detergent solubilization followed by purification and the subsequent receptor reconstitution into giant unilamellar vesicles (GUV). Detergent micelle receptors were analyzed by fluorescence correlation spectroscopy (FCS) and surface plasmon resonance (SPR) measurements. We could determine the conditions of functional solubilization of GPCRs by characterizing the amount of obtained receptor-detergent micelles, their homogeneity and the capacity of the receptor to selectively bind ligands. The receptors were then reconstituted into GUVs and ligand binding was monitored by confocal microscopy. In the second part of the thesis, we investigated different aspects properties of native plasma membrane vesicles. These vesicles are derived from live mammalian cells using v cytochalasin B or optical tweezers; they comprise parts of a cell’s plasma membrane and cytosol. After the characterization of their overall composition, we investigated their ability to mediated transmembrane activation of intracellular signaling reactions upon agonist binding to a GPCR. We could successfully monitor the ligand binding to receptor by FCS, the subsequent G-protein activation by intermolecular FRET and the receptor desensitization by monitoring the arrestin recruitment to the plasma membrane. These vesicles thus represent the smallest autonomous container performing cellular signaling reactions thus functioning like a minimized cell. Finally, preliminary experiments demonstrated the potential of native vesicles to serve as novel drug delivery system since they meet most of the requirements for an efficient drug delivery platform.

EPFL2012