RESUMEN
Desorption electrospray ionization mass spectrometry imaging (DESI-MSI) is one of the least specimen destructive ambient ionization mass spectrometry tissue imaging methods. It enables rapid simultaneous mapping, measurement, and identification of hundreds of molecules from an unmodified tissue sample. Over the years, since its first introduction as an imaging technique in 2005, DESI-MSI has been extensively developed as a tool for separating tissue regions of various histopathologic classes for diagnostic applications. Recently, DESI-MSI has also emerged as a versatile technique that enables drug discovery and can guide the efficient development of drug delivery systems. For example, it has been increasingly employed for uncovering unique patterns of in vivo drug distribution, the discovery of potentially treatable biochemical pathways, revealing novel druggable targets, predicting therapeutic sensitivity of diseased tissues, and identifying early tissue response to pharmacological treatment. These and other recent advances in implementing DESI-MSI as the tool for the development of novel therapies are highlighted in this review.
Asunto(s)
Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masa por Ionización de Electrospray/métodos , Descubrimiento de Drogas , Sistemas de Liberación de Medicamentos , Diagnóstico por ImagenRESUMEN
Efficient delivery of oligonucleotides still remains a challenge in the field of oligonucleotide based therapy. Peptide nucleic acid (PNA), a DNA analogue that is typically synthesized by solid phase peptide chemistry, has been conjugated to a variety of cell penetrating peptides (CPP) as a means of improving its cellular uptake. These CPPs typically deliver their cargoes into cells by an endosomal-dependent mechanism resulting in lower bioavailability of the cargo. Herein, we designed and synthesized PNA-peptide conjugates as splice switching oligonucleotides (SSO) targeting the Mnk2 gene, a therapeutic target in cancer. In humans, the MKNK2 gene, is alternatively spliced, generating isoforms with opposite biological activities: Mnk2a and Mnk2b. It was found that the Mnk2a isoform is down-regulated in breast, lung, brain, and colon tumors and is a tumor suppressor, whereas MnK2b is oncogenic. We have designed and synthesized PNAs that were conjugated to either of the following peptides: a nuclear localization sequence (NLS) or a cytosol localizing internalization peptide (CLIP6). CLIP6-PNA demonstrates effective cellular uptake and exclusively employs a nonendosomal mechanism to cross the cellular membranes of glioblastoma cells (U87). Simple incubation of PNA-peptide conjugates in human glioblastoma cells up-regulates the Mnk2a isoform leading to cancer cell death.
Asunto(s)
Péptidos de Penetración Celular/metabolismo , Portadores de Fármacos/metabolismo , Oligonucleótidos/metabolismo , Ácidos Nucleicos de Péptidos/metabolismo , Secuencia de Bases , Línea Celular Tumoral , Células HeLa , Humanos , Concentración de Iones de Hidrógeno , Oligonucleótidos/genéticaRESUMEN
Efficient delivery of nucleic acids into cells still remains a great challenge. Peptide nucleic acids (PNAs) are DNA analogues with a neutral backbone and are synthesized by solid phase peptide chemistry. This allows a straightforward synthetic route to introduce a linear short peptide (a.k.a. cell-penetrating peptide) to the PNA molecule as a means of facilitating cellular uptake of PNAs. Herein, we have devised a synthetic route in which a cyclic peptide is prepared on a solid support and is extended with the PNA molecule, where all syntheses are accomplished on the solid phase. This allows the conjugation of the cyclic peptide to the PNA molecule with the need of only one purification step after the cyclic peptide-PNA conjugate (C9-PNA) is cleaved from the solid support. The PNA sequence chosen is an antimiR-155 molecule that is complementary to mature miR-155, a well-established oncogenic miRNA. By labeling C9-PNA with fluorescein isothiocyanate, we observe efficient cellular uptake into glioblastoma cells (U87MG) at a low concentration (0.5 µM), as corroborated by fluorescence-activated cell sorting (FACS) analysis and confocal microscopy. FACS analysis also suggests an uptake mechanism that is energy-dependent. Finally, the antimiR activity of C9-PNA was shown by analyzing miR155 levels by quantitative reverse transcription polymerase chain reaction and by observing a reduction in cell viability and proliferation in U87MG cells, as corroborated by XTT and colony formation assays. Given the added biological stability of cyclic versus linear peptides, this synthetic approach may be a useful and straightforward approach to synthesize cyclic peptide-PNA conjugates.