Rosario M. Sanchez-Martin

Palladium-mediated intracellular chemistry

Many important intracellular biochemical reactions are modulated by transition metals, typically in the form of metalloproteins. The ability to carry out selective transformations inside a cell would allow researchers to manipulate or interrogate innumerable biological processes. Here, we show that palladium nanoparticles trapped within polystyrene microspheres can enter cells and mediate a variety of Pd(0)-catalysed reactions, such as allylcarbamate cleavage and Suzuki-Miyaura cross-coupling. The work provides the basis for the customization of heterogeneous unnatural catalysts as tools to carry out artificial chemistries within cells. Such in cellulo synthesis has potential for a plethora of applications ranging from cellular labelling to synthesis of modulators or inhibitors of cell function.

The Impact of Combinatorial Methodologies on Medicinal Chemistry

Combinatorial chemistry has had a major impact on the discovery and optimisation of potential lead compounds. This review details some of the fundamental principles behind combinatorial chemistry and describes a variety of methods employed in the search for new therapeutically interesting compounds including the concept of dynamic combinatorial chemistry as a method of selecting active compounds from a mixture. It also outlines methods used to analyse resin bound products and describes how solution phase library generation may be aided by the use of resin bound reagents and scavengers.

The use of solid supports to generate nucleic acid carriers.

Nucleic acids are the foundation stone of all cellular processes. Consequently, the use of DNA or RNA to treat genetic and acquired disorders (so called gene therapy) offers enormous potential benefits. The restitution of defective genes or the suppression of malignant genes could target a range of diseases, including cancers, inherited diseases (cystic fibrosis, muscular dystrophy, etc.), and viral infections. However, this strategy has a major barrier: the size and charge of nucleic acids largely restricts their transit into eukaryotic cells. Potential strategies to solve this problem include the use of a variety of natural and synthetic nucleic acid carriers. Driven by the aim and ambition of translating this promising therapeutic approach into the clinic, researchers have been actively developing advanced delivery systems for nucleic acids for more than 20 years. A decade ago we began our investigations of solid-phase techniques to construct families of novel nucleic acid carriers for transfection. We envisaged that the solid-phase synthesis of polycationic dendrimers and derivatized polyamimes would offer distinct advantages over solution phase techniques. Notably in solid phase synthesis we could take advantage of mass action and streamlined purification procedures, while simplifying the handling of compounds with high polarities and plurality of functional groups. Parallel synthesis methods would also allow rapid access to libraries of compounds with improved purities and yields over comparable solution methodologies and facilitate the development of structure activity relationships. We also twisted the concept of the solid-phase support on its head: we devised miniaturized solid supports that provided an innovative cell delivery vehicle in their own right, carrying covalently conjugated cargos (biomolecules) into cells. In this Account, we summarize the main outcomes of this series of chemically related projects.

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.

Number of Nanoparticles per Cell through a Spectrophotometric Method - A key parameter to Assess Nanoparticle-based Cellular Assays

Engineered nanoparticles (eNPs) for biological and biomedical applications are produced from functionalised nanoparticles (NPs) after undergoing multiple handling steps, giving rise to an inevitable loss of NPs. Herein we present a practical method to quantify nanoparticles (NPs) number per volume in an aqueous suspension using standard spectrophotometers and minute amounts of the suspensions (up to 1 μL). This method allows, for the first time, to analyse cellular uptake by reporting NPs number added per cell, as opposed to current methods which are related to solid content (w/V) of NPs. In analogy to the parameter used in viral infective assays (multiplicity of infection), we propose to name this novel parameter as multiplicity of nanofection.

miRNA in situ hybridization in circulating tumor cells - MishCTC

Circulating tumor cells (CTCs) must be phenotypically and genetically characterized before they can be utilized in clinical applications. Here, we present the first protocol for the detection of miRNAs in CTCs using in situ hybridization (ISH) combined with immunomagnetic selection based on cytokeratin (CK) expression and immunocytochemistry. Locked-Nucleic Acid (LNA) probes associated with an enzyme-labeled fluorescence (ELF) signal amplification approach were used to detect miRNA-21 in CTCs. This protocol was optimized using both epithelial tumor (MDA-MB468) and epithelial non-tumor (MCF-10A) cell lines, and miRNA-21 was selected as the target miRNA because of its known role as an onco-miRNA. Hematopoietic cells do not express miRNA-21; thus, miRNA-21 is an ideal marker for detecting CTCs. Peripheral blood samples were taken from 25 cancer patients and these samples were analyzed using our developed protocol. Of the 25 samples, 11 contained CTCs. For all 11 CTC-positive samples, the isolated CTCs expressed both CK and miRNA-21. Finally, the protocol was applied to monitor miRNA-21 expression in epithelial to mesenchymal transition (EMT)-induced MCF-7 cells, an epithelial tumor cell line. CK expression was lost in these cells, whereas miRNA-21 was still expressed, suggesting that miRNA-21 might be a good marker for detecting CTCs with an EMT phenotype.

Solid-phase synthesis of a lysine-capped bis-dendron with remarkable DNA delivery abilities

Solid-phase synthesis of a generation 3.0 polyamidourea 1-->3 C-branched bis-dendron followed by capping of the peripheral amino groups with L-lysine gave an efficient transfection reagent.

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.

Novel strategy for microsphere-mediated DNA transfection.

A new approach for microsphere-mediated delivery of plasmid DNA has been developed and successfully evaluated. Basic molecular biology techniques were used to linearize and functionalize plasmid DNA by aminomodification, enabling efficient conjugation to carboxy-functionalized microspheres. A T cell hybridoma line was successfully transfected as determined by the efficient expression of a biologically relevant YFP fusion protein. Moreover, our data identified microsphere-mediated delivery of plasmid DNA as a noninvasive, nontoxic, and efficient gene delivery method with the potential to be applied to transfection-resistant, nondividing primary cells, including naïve T cells.

Microsphere-Based Intracellular Sensing of Caspase-3/7 in Apoptotic Living Cells

A novel multifunctional probe to monitor intracellular enzymatic activity in living cells is successfully developed. Their use as accurate intracellular sensors by conjugation of an internal control (that gives an extra feature to both evaluate cellular-uptake efficiency and track probes over time) is reported. In particular, a specific application of these multifunctional microspheres as sensors of caspase-3/7 to monitor apoptosis by flow cytometry is described. The preparation of these devices together with a kinetic study towards caspase-3 and caspase-7 and their evaluation as flow cytometry probe in apoptotic living cells are reported.