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

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.

Gene Expression in Synovial Membrane Cells After Intraarticular Delivery of Plasmid-Linked Superparamagnetic Iron Oxide Particles—A Preliminary Study in Sheep

Alke Fink, Heinrich Hofmann, Benedikt Steitz

This study evaluated in vivo gene deliver y and subsequent gene expression within cells of the synovium in the presence of static and pulsating magnetic field application following intraar ticular injection of super paramagnetic iron oxide nanopar ticles linked to plasmids containing reporter genes encoding for fluorescent proteins. Plasmids encoding genes for either green fluorescent protein or red fluorescent protein were bound to super paramagnetic nanopar ticles coated with polyethyleneimine. Larger (200–250 nm) and smaller (50 nm) nanopar ticles were compared to evaluate the effects of size on transfection efficiency as well as any associated intraar ticular reaction. Comparisons between groups were evaluated at 24, 72, and 120 h time periods. Inflammator y response was mild to moderate for all injected par ticles, but was present in the majority of synovial membrane samples evaluated. Larger par ticles tended to be associated with more inflammation than smaller ones. Never theless, intraar ticular application of both experimental and control nanoparticles were well tolerated clinically. Gene expression as determined by obser vation of either green or red intracellular fluorescence was difficult to assess by both epifluorescent light, and confocal microscopy. An insufficient concentration of nanopar ticles in relation to joint volume likely resulted in a limited number of samples with positive evidence of iron staining and with suspected positive evidence of cells expressing fluorescent proteins. Our results indicate that intraar ticular administration of functionalized super paramagnetic iron oxide nanopar ticles resulted in a mild to moderate synovitis and there was in conclusive evidence of gene expression. Fur ther research is warranted to determine the best and most effective repor ter assay for assessment of the in vivo gene deliver y into the joints. In addition, the best suited concentration and size of nanopar ticles, which will optimize gene delivery and expression, while minimizing intraarticular inflammation, needs to be determined.

2006

Establishment of recombinant mammalian cell lines

Madiha Derouazi

Recombinant proteins are gaining in importance for therapeutic applications. The proteins are expressed in stable cell lines, within which recombinant DNA has incorporated into the host cells genome. Identification and isolation of extremely high producers from transfected cell populations is a tedious and time-consuming effort. We proposed two different approaches for the establishment of recombinant cell lines: affinity cell sorting and microinjection. Even with the help of single cell sorting and analysis systems (flow cytometry) rarely more than thousand different cell clones, stably expressing a desired gene can be evaluated. We are considering a new approach in which polyclonal cell populations will be established which co-express a protein of interest and an epitope-tagged cell surface receptor. A microbead fixed antibody specific to the epitope tag is hoped to assist in the isolation of rare, but highly productive cells from large suspensions of transfected cells. For this reason we have also developed an efficient method for the bulk transfection of CHO cells. On the side of vector-development for this approach, we have chosen to investigate the utility of a mouse serotonin 5-HT3 receptor as a cell surface marker. This receptor can be expressed at very high levels (up to 1 x 107 receptors per cell) in heterologous systems. The receptor is detectable on the cell surface by means of fluorescent ligands, which allows the determination of transfection efficiencies in suspension cultures. A Flag-tag was fused to the amino terminal end of the receptor without adverse effects on receptor function or ligand binding. Only 5 % of receptor DNA in the transfection cocktail was sufficient to detect positive cells in a population. The specific interaction of the epitope-tagged receptor with the cognate antibody fixed to microbeads was tested in comparison to non-transfected control cells using laser scanning confocal microscopy. A 80% enrichment of cells expressing GFP and the 5-HT3 receptor was achieved in 30 minutes. We have investigated microinjection as a gene transfer method for the establishment of recombinant mammalian cell lines. The conditions for the injection of plasmid DNA into either the nucleus or the cytoplasm of CHO-DG44 cells were established using pMYK-EGFP, an expression vector with the enhanced green fluorescent protein (EGFP) gene under the control of the murine CMV promoter and the puromycin resistance gene for selection. To estimate plasmid copy number during microinjection, it was first necessary to determine the injection volume by fluorescence quantification of injected FITC-dextran. For microinjection into the cytoplasm the volume ranged from 200 to 350 fl and for nuclear microinjection it was about 180 fl. We then investigated the impact of DNA concentration and injection pressure on transient EGFP transient expression following microinjection into the cytoplasm or nucleus. As a general rule, we observed that concentration of the DNA solution injected into the cells was the most important parameter regardless of the pressure or the volume of injection. Recombinant cell lines expressing intracellular EGFP were established following microinjection into the nucleus and the cytoplasm with linear and circular DNA. Selection with puromycin, clonal expansion, and cell banking were completed within three to four weeks post-microinjection. In parallel, recombinant cell lines were established after calcium-phosphate transfection with linear and circular DNA. The number of plasmid copies integrated was determined by Southern blot and the number and localization of integration sites was determined by fluorescence in-situ hybridization (FISH). Growth of the cells and stability of EGFP expression were investigated by cultivation over a two-month period. This approach presents several advantages over other DNA delivery methods such as control of the quantity and localization of DNA delivered into the cell and speed of recovery of recombinant cell lines. This approach may be relevant for the establishment of recombinant cell lines from cells that are not easily transfected by classical methods.

EPFL2005

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