RESUMEN
The development of tools for the early diagnosis of pancreatic adenocarcinoma is an urgent need in order to increase treatment success rate and reduce patient mortality. Here, we present a modular nanosystem platform integrating soft nanoparticles with a targeting peptide and an active imaging agent for diagnostics. Biocompatible single-chain polymer nanoparticles (SCPNs) based on poly(methacrylic acid) were prepared and functionalized with the somatostatin analogue PTR86 as the targeting moiety, since somatostatin receptors are overexpressed in pancreatic cancer. The gamma emitter 67Ga was incorporated by chelation and allowed in vivo investigation of the pharmacokinetic properties of the nanoparticles using single photon emission computerized tomography (SPECT). The resulting engineered nanosystem was tested in a xenograph mouse model of human pancreatic adenocarcinoma. Imaging results demonstrate that accumulation of targeted SCPNs in the tumor is higher than that observed for nontargeted nanoparticles due to improved retention in this tissue.
Asunto(s)
Adenocarcinoma/genética , Nanopartículas/administración & dosificación , Neoplasias Pancreáticas/genética , Somatostatina/biosíntesis , Adenocarcinoma/diagnóstico , Adenocarcinoma/patología , Animales , Línea Celular Tumoral , Detección Precoz del Cáncer , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Ratones , Nanopartículas/química , Neoplasias Pancreáticas/diagnóstico , Neoplasias Pancreáticas/patología , Polímeros/química , Ácidos Polimetacrílicos/administración & dosificación , Ácidos Polimetacrílicos/química , Somatostatina/química , Ensayos Antitumor por Modelo de Xenoinjerto , Neoplasias PancreáticasRESUMEN
Promutagenic O6-alkylguanine adducts in DNA are repaired in humans by O6-methylguanine-DNA-methyltransferase (MGMT) in an irreversible reaction. Here we describe the synthesis of a phosphoramidite that allows the preparation of oligodeoxyribonucleotides (ODNs) containing a novel tricyclic thio analogue of O6-methylguanine in which the third ring bridges the 6-thio group and C7 of a 7-deazapurine. These ODNs are very poor substrates for MGMT and poorly recognised by the alkyltransferase-like protein, Atl1. Examination of the active sites of both MGMT and Atl1 suggest large steric clashes hindering binding of the analogue. Such analogues, if mutagenic, are likely to be highly toxic.
Asunto(s)
Transferasas Alquil y Aril/química , Guanina/análogos & derivados , O(6)-Metilguanina-ADN Metiltransferasa/química , Oligodesoxirribonucleótidos/química , Compuestos de Sulfhidrilo/química , Transferasas Alquil y Aril/metabolismo , Guanina/química , Guanina/metabolismo , Humanos , Modelos Moleculares , Estructura Molecular , O(6)-Metilguanina-ADN Metiltransferasa/metabolismo , Oligodesoxirribonucleótidos/síntesis química , Oligodesoxirribonucleótidos/metabolismo , Compuestos de Sulfhidrilo/metabolismoRESUMEN
The syntheses of novel tricyclic pyrrolo[2,3-d]pyrimidine analogues of O(6)-methylguanine and S(6)-methylthioguanine are described. The crystal structures and pK(a) values of these analogues are reported. In a standard substrate assay with the human repair protein O(6)-methylguanine-DNA methyltransferase (MGMT) only the oxygen-containing analogue displayed activity.
Asunto(s)
Guanina/análogos & derivados , Cristalografía por Rayos X , Diseño de Fármacos , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Guanina/síntesis química , Guanina/química , Guanina/farmacología , Humanos , Técnicas In Vitro , Cinética , Estructura Molecular , O(6)-Metilguanina-ADN Metiltransferasa/antagonistas & inhibidoresRESUMEN
The need for test systems for nanoparticle biocompatibility, toxicity, and inflammatory or adaptive immunological responses is paramount. Nanoparticles should be free of microbiological and chemical contaminants, and devoid of toxicity. Nevertheless, in the absence of contamination, these particles may still induce undesired immunological effects in vivo, such as enhanced autoimmunity, hypersensitivity reactions, and fibrosis. Here we show that artificial particles of specific sizes affect immune cell recruitment as tested in a dermal air pouch model in mice. In addition, we demonstrate that the composition of nanoparticles may influence immune cell recruitment in vivo. Aside from biophysical characterizations in terms of hydrodynamic diameter, zeta potential, concentration, and atomic concentration of metals, we show that - after first-line in vitro assays - characterization of cellular and molecular effects by dermal air pouch analysis is straightforward and should be included in the quality control of nanoparticles. We demonstrate this for innate immunological effects such as neutrophil recruitment and the production of immune-modulating matrix metalloproteases such as MMP-9; we propose the use of air pouch leukocytosis analysis as a future standard assay.
Asunto(s)
Aire , Bioensayo/métodos , Leucocitosis/inducido químicamente , Ensayo de Materiales/métodos , Nanopartículas/toxicidad , Pruebas de Toxicidad/métodos , Animales , Bioensayo/instrumentación , Ensayo de Materiales/instrumentación , Ratones , Tamaño de la Partícula , Poliestirenos , Pruebas de Toxicidad/instrumentaciónRESUMEN
We show that DNA containing a conformationally-locked anti analogue of O(6)-alkylguanine is a poor substrate for human O(6)-methylguanine-DNA methyltransferase (MGMT) and the alkyltransferase-like protein, Atl1. This highlights the requirement for the syn conformation and rationalises why certain O(6)-alkylguanines are poor MGMT substrates.
Asunto(s)
Transferasas Alquil y Aril/metabolismo , Guanina/metabolismo , O(6)-Metilguanina-ADN Metiltransferasa/metabolismo , Oligodesoxirribonucleótidos/biosíntesis , Cristalografía por Rayos X , Guanina/análogos & derivados , Humanos , Unión Proteica , Estructura Terciaria de ProteínaRESUMEN
The syntheses of novel tricyclic pyrrolo[2,3-d]pyrimidine analogues of S6-methylthioguanine are described. The crystal structures and pKa values of these and related O6-methylguanine analogues are reported. All compounds display higher pKa values than O6-methylguanine with the sulfur-containing analogues being the more basic and exhibiting higher stability in aqueous solution. In a standard substrate assay with the human repair protein O6-methylguanine-DNA methyltransferase (MGMT) only the oxygen-containing analogue displayed activity.