Development and Evaluation of 2-Amino-7-Fluorophenazine 5,10-Dioxide Polymeric Micelles as Antitumoral Agents for 4T1 Breast Cancer

2-Amino-7-fluorophenazine 5,10-dioxide (FNZ) is a bioreducible prodrug, poorly soluble in water, with potential anticancer activity on hypoxic-tumors. This poor solubility limits its potential applications in clinic. Amphiphilic pristine polymeric micelles (PMs) based on triblock copolymers Pluronic® and Tetronic®, glycosylated derivatives and their mixtures with preformed-liposomes (LPS), were analyzed as strategies to improve the bioavailability of FNZ. FNZ encapsulations were performed and the obtaining nanostructures were characterized using UV-visible spectroscopy (UV- VIS), Transmission Electron Microscopy (TEM), Fourier transform infrared analysis and Dynamic Light Scattering (DLS). The most promising nanoformulations were analyzed for their potential toxicity and pharmacologically, at 20 mg/kg FNZ-doses, in a stage-IV murine metastatic-breast tumor model. The results revealed that the solubility of the encapsulated-FNZ increased up to seven times and the analysis (UV-VIS, DLS and TEM) confirmed the interaction between vehicles and FNZ. In all the cases appropriate encapsulation efficiencies (up to 70%), monodisperse nanometric particle sizes (PDI = 0.180–0.335), adequate Z-potentials (-1.59 to -26.4 mV), stabilities and spherical morphologies were obtained. The in vitro profile of FNZ controlled releases corresponded mainly to a kinetic Higuchi model. The in vitro/in vivo biological studies revealed non-toxicity and relevant tumor-weight diminution (up to 61%).Fil: Lecot, Nicole. Universidad de la República; UruguayFil: Dávila Belzunce, María Belén. Universidad de la República; UruguayFil: Sánchez, Carina. Universidad de la República; UruguayFil: Fernández, Marcelo. Universidad de la República; UruguayFil: González, Mercedes. Universidad de la República; UruguayFil: Cabral, Pablo. Universidad de la República; UruguayFil: Cerecetto, Hugo. Universidad de la República; UruguayFil: Glisoni, Romina Julieta. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Nanobiotecnología. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Nanobiotecnología; Argentin

In vitro and in vivo uptake studies of PAMAM G4.5 dendrimers in breast cancer

Background: Breast cancer is the second leading cause of cancer death worldwide. Nanotechnology approaches can overcome the side effects of chemotherapy as well as improve the efficacy of drugs. Dendrimers are nanometric size polymers which are suitable as drug delivery systems. To the best of our knowledge, studies on the application of PAMAM G4.5 (polyamidoamine half generation 4) dendrimers as potential drug delivery systems in breast cancer have not been reported. In this work we developed a PAMAM G4.5 dendrimer containing FITC (fluorescein isothiocyanate) dye to study their uptake by murine breast cancer cells and BALB/c mice breast tumors. Results: We performed a reaction between FITC and PAMAM G4.5 dendrimers which were previously derivatized with piperazine (linker molecule), characterized them by 1H NMR (proton nuclear magnetic resonance) spectroscopy and MALDI-TOF (matrix-assisted laser desorption/ionization- time-of-flight) mass spectrometry. The experimental data indicated that 2 FITC molecules could be bound covalently at the PAMAM G4.5 dendrimer surface, with 17 FITC molecules probably occluded in PAMAM dendrimers cavity. PAMAM-FITC dendrimer (PAMAM G4.5-piperazinyl-FITC dendrimer) size distribution was evaluated by DLS (dynamic light scattering) and TEM (transmission electron microscopy). The nanoparticle hydrodynamic size was 96.3 ± 1.4 nm with a PdI (polydispersion index) of 0.0296 ± 0.0171, and the size distribution measured by TEM was 44.2 ± 9.2 nm. PAMAM-FITC dendrimers were neither cytotoxic in 4T1 cells nor hemolytic up to 24 h of incubation. In addition, they were uptaken in vitro by 4T1 cells and in vivo by BALB/c mice breast tumors. PAMAM G4.5-piperazinyl-FITC dendrimer intracellular distribution was observed through histologic analysis of the tumor by laser confocal microscopy. Conclusion: These results indicate that PAMAM G4.5 dendrimers enter tumor tissue cells, being good candidates to be used as antitumor drug delivery systems for breast cancer treatment and diagnosis

Biorefinery of rice husk to obtain functionalized bioactive compounds

The biomass industrialization valorisation of grains and cereals is considered an opportunity for the countries where agro-industrial activity is one of its main economic activities, rendering new higher-value products with a concomitant solution to waste accumulation issues. To that end, in this work we describe and characterise bioactive compounds generation from rice husk by semisolid fermentation, obtained from 500 g of the material at room temperature and 60% humidity with mixed cultures of Phanerochaete chrysosporium and Gloeophyllum trabeum. The extract was evaluated in different situations: murine mammary tumour cells (4T1), normal cells (NIH 3T3) and in Lactobacillus acidophilus, and Trichoderma harzianum. The results exhibited that the extract inhibited 4T1 cells at concentrations higher than 20μg/mL, but did not inhibit normal cells, and displayed germicide activity after 3 days incubations. We propose that these functionalized compounds have a potential application in industry/agriculture/medicine obtained from rice husk waste

Development of fluorescent- and radio-traceable T1307-polymeric micelles as biomedical agents for cancer diagnosis: biodistribution on 4T1 tumor-bearing mice

Abstract In recent years, nanocarriers have been studied as promising pharmaceutical tools for controlled drug-delivery, treatment-efficacy follow-up and disease imaging. Among them, X-shaped amphiphilic polymeric micelles (Tetronic®, poloxamines) display great potential due to their biocompatibility and non-toxic effects, among others. In the present work, polymeric micelles based on the T1307 copolymer were initially decorated with a 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY)-fluorophore in order to determinate its in vivo biodistribution on 4T1 tumor-bearing mice. However, unfavorable results with this probe led to two different strategies. On the one hand, the BODIPY-micelle-loaded, L-T1307-BODIPY, and on the other hand, the 99mTc-micelle-radiolabeled, L-T1307- 99m Tc, were analyzed separately in vivo. The results indicated that T1307 accumulates mainly in the stomach, the kidneys, the lungs and the tumor, reaching the maximum organ-accumulation 2 hours after intravenous injection. Additionally, and according to the results obtained for L-T1307- 99m Tc, the capture of the polymeric micelles in organs could be observed up to 24 hours after injection. The results obtained in this work were promising towards the development of new radiotracer agents for breast cancer based on X-shaped polymeric micelles

Development and evaluation of 2-amino-7-fluorophenazine 5,10-dioxide polymeric micelles as antitumoral agents for 4T1 breast cancer

2-Amino-7-fluorophenazine 5,10-dioxide (FNZ) is a bioreducible prodrug, poorly soluble in water, with potential anticancer activity on hypoxic-tumors. This poor solubility limits its potential applications in clinic. Amphiphilic pristine polymeric micelles (PMs) based on triblock copolymers Pluronic® and Tetronic®, glycosylated derivatives and their mixtures with preformed-liposomes (LPS), were analyzed as strategies to improve the bioavailability of FNZ. FNZ encapsulations were performed and the obtaining nanostructures were characterized using UV-visible spectroscopy (UV-VIS), Transmission Electron Microscopy (TEM) and Dynamic Light Scattering (DLS). The most promising nanoformulations were analyzed for their potential toxicity and pharmacologically, at 20 mg/kg FNZ-doses, in a stage-IV murine metastatic-breast tumor model. The results revealed that the solubility of the encapsulated-FNZ increased up to 14 times and the analysis (UV-VIS, DLS and TEM) confirmed the interaction between vehicles and FNZ. In all the cases appropriate encapsulation efficiencies (greater than 75%), monodisperse nanometric particle sizes (PDI = 0.180–0.335), adequate Z-potentials (−1.59 to −26.4 mV), stabilities and spherical morphologies were obtained. The in vitro profile of FNZ controlled releases corresponded mainly to a kinetic Higuchi model. The in vitro/in vivo biological studies revealed non-toxicity and relevant tumor-weight diminution (up to 61%).ANII: FCE_1_2014_1_104714ANII: POS_FCE_2015_1_100519

Desarrollo de formulaciones nanoestructuradas portadores de fenazinas para su aplicación en oncología

Propósito de la investigación: 5,10-Dióxido de 2-amino-7(8)-fluorofenazina(FNZ) es un profármaco biorreducible en condiciones de hipoxia, poco soluble en agua, con potencial actividad anticancerígena sobre tumores hipóxicos. Estapobre solubilidad en medios biológicos, limitaría la aplicación de FNZ en la clínica. Las micelas poliméricas prístinas anfifílicas (MPs) a base decopolímeros tribloque PEO-PPO-PEO, Pluronic® y Tetronic®, sus derivados glicosilados con restos gluconolactonas (Glu) y sus respectivas mezclas con liposomas preformados (LPS), son un grupo novedoso de sistemas nanoestructurados inteligentes y termosensibles que pueden ser utilizados para mejorar la solubilidad, la biodisponibilidad y la permeación de profármacos bioreducibles poco solubles, como FNZ. Métodos: FNZ se encapsuló en MPs y mezclas de MPs con LPS. La caracterización de las nanoestructuras resultantes se realizó mediante espectroscopía UV-visible (UV-VIS), microscopía electrónica de transmisión (TEM), análisis infrarrojo por transformada de Fourier (FTIR), dispersión de luz dinámica (DLS) y microscopía de fuerza atómica (AFM). Asimismo, se seleccionaron las nanoformulaciones más promisorias, en cuanto a las propiedades desplegadas por estas, y se procedió al estudio farmacológico en un modelo de tumor de mama metastásico murino símil fase IV humano (4T1). Estos ensayos fueron realizados a dos dosis del profármaco biorreducible FNZ, de 20 y 200 mg/kg de ratón BALB/c. Finalmente, se procedió al marcado con 99mTc de una de las MPs y del profármaco FNZ para su seguimiento in vivo en el antedicho modelo de cáncer de mama metastásico. Resultados: Los resultados revelaron que la solubilidad del profármaco FNZ encapsulado se incrementó hasta diez veces, respecto a su solubilidad intrínseca en agua. Los resultados analizados por UV-VIS, DLS, MET y AFM confirmaron la interacción de FNZ con MPs, MPs derivatizadas y MPs en mezclas con LPS, y en todos los casos desplegaron apropiadas eficiencias de encapsulación, tamaños de partícula monodispersos, adecuada estabilidad y morfología del tipo esférica. Además, el perfil in vitro de liberación controlada de FNZ, desde todos los sistemas preparados, se ajustó a una cinética que sigue el Modelo de Higuchi, con un mecanismo principal de liberación mediada por la difusión del fármaco y la relajación de las cadenas poliméricas, a medida que el medio de liberación difunde a través del material polimérico. Por último, se logró inhibiciones del tamaño tumoral de hasta un 54%, con mínimos signos de afectación del animal a las concentraciones ensayadas de profármaco. Conclusión: Se diseñaron y desarrollaron nanosistemas portadores de FNZ, un profármaco biorreducible poco soluble en agua, los cuales mostraron desplegarse como potenciales agentes de terapia contra el cáncer de mama.Agencia Nacional de Investigación e InnovaciónUniversidad de la república. Comisión Académica de Posgrad

Development of fluorescent- and radio-traceable T1307-polymeric micelles as biomedical agents for cancer diagnosis: biodistribution on 4T1 tumor-bearing mice

In recent years, nanocarriers have been studied as promising pharmaceutical tools for controlled drug-delivery, treatment-efficacy follow-up and disease imaging. Among them, X-shaped amphiphilic polymeric micelles (Tetronic®, poloxamines) display great potential due to their biocompatibility and non-toxic effects, among others. In the present work, polymeric micelles based on the T1307 copolymer were initially decorated with a 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY)-fluorophore in order to determinate its in vivo biodistribution on 4T1 tumor-bearing mice. However, unfavorable results with this probe led to two different strategies. On the one hand, the BODIPY-micelle-loaded, L-T1307-BODIPY, and on the other hand, the 99mTc-micelle-radiolabeled, L-T1307- 99m Tc, were analyzed separately in vivo. The results indicated that T1307 accumulates mainly in the stomach, the kidneys, the lungs and the tumor, reaching the maximum organ-accumulation 2 hours after intravenous injection. Additionally, and according to the results obtained for L-T1307- 99m Tc, the capture of the polymeric micelles in organs could be observed up to 24 hours after injection. The results obtained in this work were promising towards the development of new radiotracer agents for breast cancer based on X-shaped polymeric micelles

Carboranylanilinoquinazoline EGFR-inhibitors: toward ‘lead-to-candidate’ stage in the drug-development pipeline

Peer reviewe

Glucosylated Polymeric Micelles Actively Target a Breast Cancer Model

This work investigates the potential of glycosylation to actively target nanodrug delivery systems to adult solid tumors overexpressing glucose transporters. The highly hydrophobic fluorescent compound curcumin (CUR) is nanoencapsulated within polymeric micelles of pristine and glucosylated poly(ethylene oxide)-poly(propylene oxide) block copolymers, and their interaction with breast cancer (BC) cells is investigated in vitro and in vivo. The aqueous solubility of CUR is increased more than 50 000-fold and spherical nanoparticles display size in the 40 to 500 nm range, as determined by transmission electron microscopy and by dynamic light scattering, respectively. Uptake studies conducted in the BC cell line 4T1 in vitro demonstrate that glucosylation enhances nanoparticle internalization. Finally, the ability of unmodified and glucosylated polymeric micelles to accumulate in female BALB/c mice bearing 4T1-induced tumors is compared by ex vivo bioimaging with auspicious results.Fil: Lecot, Nicole. Universidad de la República; UruguayFil: Glisoni, Romina Julieta. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Nanobiotecnología. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Nanobiotecnología; ArgentinaFil: Oddone, Natalia. Universidad de la República; UruguayFil: Benech, Juan. Universidad de la República; UruguayFil: Fernández, Marcelo. Universidad de la República; UruguayFil: Gambini, Juan Pablo. Universidad de la República; UruguayFil: Cabral, Pablo Octavio. Universidad de la República; UruguayFil: Sosnik, Alejandro Dario. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Nanobiotecnología. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Nanobiotecnología; Argentin