Molecular beacon-coated PMMA nanoparticles for the intracellular detection of tumourassociated mRNA

Introduction Fluorescence imaging coupled to nanotechnology is enabling the development of powerful tools in the biological field for different applications, including cellular imaging, intracellular mRNA monitoring and detection, or cancer diagnosis. In particular, nanomaterials (e.g., nanoparticles, nanowires, or nanotubes) have been explored in many biomedical applications, such as biosensing or molecular imaging, because their properties and functions provide a plethora of advantages for these specific applications: their high volume-to-surface ratio, for example, resulting in a large surface area, enables attachment of a great number of target-specific molecules of interest for ultrasensitive detection [1]. Due to these advantages coupled to the optimum dimensions, generally under 100 nm, the delivery of fluorescent agents or probes to cells and tissues by using nanoparticles or other nanomaterials, is currently receiving a growing interest because such nanodimensional structures can conveniently allow the preparation of small tools to examine cellular mechanisms without interferences. We describe here the use of polymeric nanoparticles made of a core of polymethylmethacrylate (PMMA) surrounded by a shell bearing cationic groups [2] as intracellular delivery tools of molecular beacons (MB), particular fluorescent DNA probes, for the detection and localization of a specific mRNA. MBs are DNA sequences composed of one target-recognition region and two short complementary stem sequences: in absence of the target, the sequence forms a stem–loop structure which brings a quencher and fluorophore, which are located at opposite ends of the MB, into close proximity, resulting in fluorescence quenching. In the presence of a target molecule, hybridization between the target and the loop sequence of the MB induces the opening of the stem resulting in the spatial separation of the fluorophore and quencher with restoration of fluorescence [3]. The MB was specific for the mRNA of survivin, a protein member of the inhibitor of apoptosis (IAP) family, highly expressed in most types of cancer [4]. We report the results of the MB and nanoparticles characterization in vitro. Results PMMA nanoparticles were synthesized at ISOF-CNR by emulsion polymerization and they were constituted by a core of PMMA surrounded by a shell bearing cationic quaternary ammonium and –NH2 groups. The same nanoparticles were also functionalized, during the synthesis, with fluoresceine. The MB was anchored to the surface of PMMA nanoparticles via the commercial sulfhydryl-reactive heterobifunctional crosslinker sulfo-LC-SPDP. Survivin MB was initially characterized in solution in order to establish the working conditions leading to the optimum analytical characteristics, such as sensitivity and selectivity. In particular, the MB (100 nM) was examined after incubation with different target concentrations in two buffers (Tris and DMEM) with an incubation time of 3 h. After modification of the nanoparticles with the sulfo- LC-SPDP crosslinker and the MB, the functionalization of the nanoparticles with the MB was verified by fluorescence measurements (Figure 1) both at 635 (ATTO 647N on the MB) and 488 nm (fluoresceine on the nanoparticles). To further prove the functionality of the MB also after immobilization onto the nanoparticles, the solution, which was proved to contain the nanoparticles bearing the MB, was incubated for 3 h with increasing concentrations of the target (0, 10, 100 and 200 nM) and fluorescence was measured exciting at 635 nm. An increase of fluorescence signal (Figure 2) was observed with the increase of the target concentration, confirming the sensitivity of the MB versus the presence of the target also after its immobilization onto the nanoparticles. The functionality and specificity of the MB was also tested in living cells (A375 cell line) by transfection with a classical lipid agent, Lipofectamine, and imaging by confocal microscopy. A fluorescence increase was observed in the cytoplasm after 1 h from the transfection without fluorescent aggregates or fluorescence in the extracellular environment. On the contrary, no fluorescence was observed in transfected cells not expressing survivin (Bronchial Smooth Muscle Cells and monocytes). Conclusions PMMA nanoparticles have been evaluated as potential intracellular nanodelivery tool of MBs for mRNA sensing. The optical characteristics of the nanoparticles and the functionality of the MB have been evaluated by fluorescence measurements before and after the nanoparticles functionalization. References [1] Parveen, S., Misra, R., Sahoo, S.K., “Nanoparticles: a boon to drug deliv-ery, therapeutics, diagnostics and imaging”, Nanomedicine: Nanotechno-logy, Biology, and Medicine 8, 147–166, (2012). [2] Rimessi, P., et al., “Cationic PMMA Nanoparticles Bind and Deliver Anti-sense Oligoribonucleotides Allowing Restoration of Dystrophin Expression in the mdx Mouse”, Molecular Therapy 17, 820–827 (2009). [3] Wang, K., “Molecular Engineering of DNA: Molecular Beacons”, Angew. Chem. Int. Ed. 47, 2–17 (2008). [4] Sah, N.K., Khan, Z., Khan, G.J., Bisen, P.S., “Structural, functional and therapeutic biology of survivin”, Cancer Letters 244, 164-171 (2006). Contacts Contact information of the corresponding author: Francesco Baldini, Istituto di Fisica Applicata “Nello Carrara” Consiglio Nazionale delle Ricerche Via Madonna del Piano 10, 50019 Sesto Fiorentino, Firenze, Italy Phone: +39 055 5226323 Email: f.baldini@ifac.cnr.it