Lois M. Alexander

Multifunctionalized biocompatible microspheres for sensing

We present our achievements in the synthesis and application of multifunctionalized, cross‐linked, polystyrene microspheres, which remarkably and quite generally are taken up by all cell types studied to date. Importantly, the nature of these synthetic beads allows multistep solid‐phase chemistry and the ability to bind essentially any molecule/sensor/nucleic acid to them. These microspheres have a number of advantages over other cellular delivery approaches. First, a diverse range of compounds can be attached to the microspheres, and we have demonstrated that these materials are effectively delivered into cells; second, the cellular cargo can be modulated through modification of bead loading; third, they are large enough to be visualized using standard microscopy techniques; and fourth, their cargos are not diluted within the cell. Populations of cells containing beads can be readily sorted from other cells for subsequent analysis with very high, but controllable, uptake rates, which can be modulated through modulation of the bead size and incubation time. Recent work has focused on the development of these microspheres as sensors for intracellular calcium and pH.

Microsphere-Mediated Protein Delivery into Cells

Delivering the goods: By coupling proteins to varyingly sized polymeric microspheres, it is possible to deliver them to cells in an easy and effective way. For this study a fluorescent protein (EGFP) and a functional enzyme (β‐galactosidase) were coupled to these particles. Evaluation of the cellular uptake after “beadfection” shows that the functionality and activity of these proteins were not adversely affected through coupling to the carrier system; this shows that their functional structure is retained.

Knocking (anti)-sense into cells: the microsphere approach to gene silencing.

200 and 500 nm polymeric microspheres have been conjugated to siRNA targeted against EGFP expressed in human cervical cancer (HeLa) cells and shown to efficiently silence protein expression over 72 h, without detrimental cytotoxicity. Furthermore, with use of an independent Cy5 tracking label on the siRNA-laden microsphere, silencing of EGFP could be assessed by selecting only those cells that contained the delivery vehicle (and thus the siRNA) generating a more accurate picture of microsphere-induced gene silencing.

Microsphere-based tracing and molecular delivery in embryonic stem cells.

Embryonic stem (ES) cells are in vitro cell lines that can differentiate into all lineages of the fetus and the adult. Despite the versatility of genetic manipulation in murine ES cells, these approaches are time-consuming and rely on inefficient transient cellular delivery systems that can only be applied to undifferentiated ES cell cultures. Here we describe a polystyrene microsphere-based system designed to efficiently deliver biological materials into both undifferentiated and differentiating ES cells. Our results demonstrate that these microspheres can be successfully employed for simultaneous cellular labeling and controlled transfer of various cargos such as fluorophores, proteins and nucleic acids into ES cells without any significant toxicity or loss of pluripotency. This versatile delivery system is also effective in other stem cell lines derived from early embryos, trophoblast and neural stem cells.

Synthesis and Characterization of Dual-Functionalized Core-Shell Fluorescent Microspheres for Bioconjugation and Cellular Delivery

The efficient transport of micron-sized beads into cells, via a non-endocytosis mediated mechanism, has only recently been described. As such there is considerable scope for optimization and exploitation of this procedure to enable imaging and sensing applications to be realized. Herein, we report the design, synthesis and characterization of fluorescent microsphere-based cellular delivery agents that can also carry biological cargoes. These core-shell polymer microspheres possess two distinct chemical environments; the core is hydrophobic and can be labeled with fluorescent dye, to permit visual tracking of the microsphere during and after cellular delivery, whilst the outer shell renders the external surfaces of the microspheres hydrophilic, thus facilitating both bioconjugation and cellular compatibility. Cross-linked core particles were prepared in a dispersion polymerization reaction employing styrene, divinylbenzene and a thiol-functionalized co-monomer. These core particles were then shelled in a seeded emulsion polymerization reaction, employing styrene, divinylbenzene and methacrylic acid, to generate orthogonally functionalized core-shell microspheres which were internally labeled via the core thiol moieties through reaction with a thiol reactive dye (DY630-maleimide). Following internal labeling, bioconjugation of green fluorescent protein (GFP) to their carboxyl-functionalized surfaces was successfully accomplished using standard coupling protocols. The resultant dual-labeled microspheres were visualized by both of the fully resolvable fluorescence emissions of their cores (DY630) and shells (GFP). In vitro cellular uptake of these microspheres by HeLa cells was demonstrated conventionally by fluorescence-based flow cytometry, whilst MTT assays demonstrated that 92% of HeLa cells remained viable after uptake. Due to their size and surface functionalities, these far-red-labeled microspheres are ideal candidates for in vitro, cellular delivery of proteins.

Cellular uptake of fluorescent labelled biotin-streptavidin microspheres.

Amino functionalized, cross-linked, polystyrene microspheres were covalently loaded with streptavidin to which was coupled fluorescently labeled biotin and biotinylated-tagged DNA. These biotin–streptavidin microsphere conjugates were then successfully delivered into cells. The application of the streptavidin–biotin technology to these microspheres allows the effective delivery of any biotinylated material into intact mammalian cells, without the need for delicate procedures such as micro-injection.

Multi-modal molecular imaging approaches to detect primary cells in preclinical models

The need to understand cellular trafficking in vivo in situ requires the development and application of novel methodologies for cellular labeling and cell tracking. Here we applied new technologies associated with advances in molecular imaging to demonstrate the feasibility of labeling primary immune cells. We demonstrate the utility of fluorescently tagged polystyrene microspheres, MRI susceptible emulsions and cell entry peptoids. The adaptation of these labeling agents will permit cell specific delivery, diagnostic sensing and the delivery of therapeutic agents to sites of inflammation and infection.