Covalent and Noncovalent Conjugation of Degradable Polymer Nanoparticles to T Lymphocytes

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.

Preparation and Analysis of Bacteria Surface-Modified with Polymer Nanoparticles

Lisa Patricia Elisabeth Bader

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.

EPFL2022

Chemistry and Analysis of Nanoparticle-Modified Living Cells

Tanja Thomsen

Conventional therapeutics are often limited by their targeting ability, resulting in harmful and potential fatal side-effects for the patients. Recently, new strategies have been developed to improve target specificity of drugs in order to generate more efficient therapeutics. An innovative concept, examined in this thesis, is the use of cells as therapeutic carriers. This concept termed cell-mediated drug delivery, relies on the intrinsic targeting properties of cells to transport the therapeutic drug to the target location. The conjugation of nanoparticles, loaded with drugs or adjuvants, to the surface of cells enables the transport to the desired location and increases the local drug concentration, reducing the required therapeutic leading to further attenuation of side effects. Chapter 1 introduces the concept of cell-mediated drug delivery. It highlights the advantages of using cells decorated with nanoparticles as carriers for therapeutics as opposed to free (polymer) nanoparticles. Examples of various covalent and non-covalent nanoparticle conjugations strategies are discussed and an overview of all approaches, which have been used previously to attach nanoparticles to the cell surface, is presented. The importance of a thorough in vitro characterization of nanoparticle-modified cells is emphasized. The use of common characterization techniques such as flow cytometry, UV-Vis and fluorescence spectroscopy, fluorescence and electron microscopy, and radiolabeling is discussed in detail and examples of qualitative and quantitative in vitro analysis of nanoparticle- modified cells are reviewed. Chapter 2 contains an extensive in vitro analysis of cell-nanoparticle conjugates using fluorescent- and fluorescent label-free methods. The stability of nanoparticles loaded with cargo attached to cells was investigated by encapsulating three different green fluorescent dyes into nanoparticles and tracking the fluorescence signal over 24 h. Two of the dyes revealed a complete loss of fluorescence of a time period of 24 h, whereas a third entrapped dye and a covalently attached dye did not have a decrease in fluorescence over 24 h. Finally, we developed a fluorescent label-free method as an alternative to characterize nanoparticle-decorated cells without the need for any fluorescent label. Chapter 3 systematically explores conjugation chemistries to immobilize two platforms of functional biodegradable polymer nanoparticles (PEG-PLA (poly(ethylene glycol) - poly(lactide)) nanoparticles and carboxylic PLA nanoparticles) on T cells as potential carrier across the blood-brain barrier (BBB). We compared a series of covalent and non-covalent immobilization strategies by flow cytometry (FACS), confocal microscopy, and impact on cell proliferation and cellular viability. We found that T-cell surface coupling of nanoparticles is dependent on the ratio of nanoparticles to cells and that higher ratios led to more particles attached to cells. Furthermore, the most efficient strategy to attach nanoparticles was via ligand-receptor interactions (lectin - sialic acid and biotin - NeutrAvidin). In addition, nanoparticle-decorated T cells were investigated for their ability to cross an in vitro mouse BBB model under static conditions in a two-chamber assay. Decorating the cells with nanoparticles does not affect their ability to bind ICAM-1 (a protein involved in the transmigration process) or to cross the model biological barrier.

EPFL2020

Biotin-NeutrAvidin Mediated Immobilization of Polymer Micro- and Nanoparticles on T Lymphocytes

Maxime Ayer, Olivier Burri, Romain Guiet, Harm-Anton Klok, Arne Seitz

Cells are powerful carriers that can help to improve the delivery of nanomedicines. One approach to use cells as carriers is to immobilize the nanoparticulate cargo on the cell surface. While a plethora of chemical conjugation strategies are available to bind nanoparticles to cell surfaces, only relatively little is known about the effects of particle size and cell type on the surface immobilization of nanoparticles. This study investigates the biotin-NeutrAvidin mediated immobilization of model polymer nanoparticles with sizes ranging from 40 nm to 1 mu m on two different T cell lines, viz., human Jurkat cells as well as mouse SJL/PLP7 T cells, which are of potential interest for drug delivery across the blood-brain barrier. The nanoparticle cell surface immobilization and the particle surface concentration and distribution were analyzed by flow cytometry and confocal microscopy. The functional properties of nanoparticle-modified SJL/PLP7 T cells were assessed in an ICAM-1 binding assay as well as in a two-chamber setup in which the migration of the particle-modified T cells across an in vitro model of the blood-brain barrier was studied. The results of these experiments highlight the effects of particle size and cell line on the surface immobilization of nanoparticles on living cells.