RESUMEN
4-hydroxynonenal (HNE), a lipid peroxidation product, is a promising anti-neoplastic drug due to its remarkable anti-cancer activities. However, this possibility has not been explored, because the delivery of HNE is very challenging as a result of its low solubility and its poor stability. This study intentionally designed a new type of lipid nanocapsules specifically for HNE delivery. They consist of a medium chain triglyceride liquid oil core surrounded by a polymer shell. A ß-cyclodextrin-poly(4-acryloylmorpholine) conjugate was selected as the shell component. HNE-loaded nanocapsules were about 350 nm in size with a negative surface charge. They were stable for two years when stored in suspensions at 4 degrees C. In vitro experiments showed that HNE was released from the nanocapsules at a considerable rate. Nanocapsule uptake into cells was evaluated using a fluorescent formulation that revealed rapid internalisation. Cytotoxicity studies demonstrated the safety of the formulation. Enhanced anti-tumoral activity against various cell lines, depending on increased HNE stability, was obtained by using HNE-loaded nanocapsules. In particular, we have demonstrated an increase in anti-proliferative, pro-apoptotic and differentiative activity in several tumour cell lines from different tissues. Moreover, we evaluated the effects of these new nanocapsules on a three-dimensional human reconstructed model of skin melanoma. Interestingly, the encouraging results obtained with topical administration on the epidermal surface could open new perspectives in melanoma treatments.
Asunto(s)
Aldehídos/química , Aldehídos/farmacología , Portadores de Fármacos/química , Lípidos/química , Melanoma/patología , Nanocápsulas/química , Acrilamidas/química , Transporte Biológico , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Ciclodextrinas/química , Estabilidad de Medicamentos , Humanos , Morfolinas/químicaRESUMEN
Micro- and nanobubbles provide a promising non-viral strategy for ultrasound mediated gene delivery. Microbubbles are spherical gas-filled structures with a mean diameter of 1-8 µm, characterised by their core-shell composition and their ability to circulate in the bloodstream following intravenous injection. They undergo volumetric oscillations or acoustic cavitation when insonified by ultrasound and, most importantly, they are able to resonate at diagnostic frequencies. It is due to this behaviour that microbubbles are currently being used as ultrasound contrast agents, but their use in therapeutics is still under investigation. For example, microbubbles could play a role in enhancing gene delivery to cells: when combined with clinical ultrasound exposure, microbubbles are able to favour gene entry into cells by cavitation. Two different delivery strategies have been used to date: DNA can be co-administered with the microbubbles (i.e. the contrast agent) or 'loaded' in purposed-built bubble systems - indeed a number of different technological approaches have been proposed to associate genes within microbubble structures. Nanobubbles, bubbles with sizes in the nanometre order of magnitude, have also been developed with the aim of obtaining more efficient gene delivery systems. Their small sizes allow the possibility of extravasation from blood vessels into the surrounding tissues and ultrasound-targeted site-specific release with minimal invasiveness. In contrast, microbubbles, due to their larger sizes, are unable to extravasate, thus and their targeting capacity is limited to specific antigens present within the vascular lumen. This review provides an overview of the use of microbubbles as gene delivery systems, with a specific focus on recent research into the development of nanosystems. In particular, ultrasound delivery mechanisms, formulation parameters, gene-loading approaches and the advantages of nanometric systems will be described.
Asunto(s)
Técnicas de Transferencia de Gen , Terapia Genética/métodos , Microburbujas , Nanotecnología/métodos , Animales , Técnicas de Transferencia de Gen/tendencias , Terapia Genética/tendencias , Humanos , Microburbujas/uso terapéutico , Nanotecnología/tendenciasRESUMEN
4-Hydroxynonenal (HNE) is the most studied end product of the lipoperoxidation process, by virtue of its relevant biological activity. The antiproliferative and proapoptotic effects of HNE have been widely demonstrated in a great variety of tumor cell types in vitro. Thus, it might represent a promising new molecule in anticancer therapy strategies. However, the extreme reactivity of this aldehyde, as well as its insolubility in water, a limiting factor for drug bioavailability, and its rapid degradation by specific enzymes represent major obstacles to its possible in vivo application. Various strategies can used to overcome these problems. One of the most attractive strategies is the use of nanovehicles, because loading drugs into nanosized structures enhances their stability and solubility, thus improving their bioavailability and their antitumoral effectiveness. Several natural or synthetic polymers have been used to synthesize nanosized structures and, among them, ß-cyclodextrin (ßCD) polymers are playing a very important role in drug formulation by virtue of the ability of ßCD to form inclusion compounds with a wide range of solid and liquid molecules by molecular complexation. Moreover, several ßCD derivatives have been designed to improve their physicochemical properties and inclusion capacities. Here we report that the inclusion complex of HNE with a derivative of ßCD, the ßCD-poly(4-acryloylmorpholine) conjugate (PACM-ßCD), enhances the aldehyde stability. Moreover, the inclusion of HNE in PACM-ßCD potentiates its antitumor effects in several tumor cell lines and in a more complex system, such as a human reconstructed skin carrying melanoma tumor cells.
Asunto(s)
Aldehídos/farmacología , Antineoplásicos/farmacología , Melanoma/tratamiento farmacológico , beta-Ciclodextrinas/farmacología , Técnicas de Cultivo de Célula , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Portadores de Fármacos/farmacología , Ensayos de Selección de Medicamentos Antitumorales , Estabilidad de Medicamentos , Humanos , Concentración 50 InhibidoraRESUMEN
BACKGROUND: The development of nonviral gene delivery systems is one of the most intriguing topics in nanomedicine. However, despite the advances made in recent years, several key issues remain unsettled. One of the main problems relates to the difficulty in designing nanodevices for targeted delivery of genes and other drugs to specific anatomic sites. In this study, we describe the development of a novel chitosan nanobubble-based gene delivery system for ultrasound-triggered release. METHODS AND RESULTS: Chitosan was selected for the nanobubble shell because of its low toxicity, low immunogenicity, and excellent biocompatibility, while the core consisted of perfluoropentane. DNA-loaded chitosan nanobubbles were formed with a mean diameter of less than 300 nm and a positive surface charge. Transmission electron microscopic analysis confirmed composition of the core-shell structure. The ability of the chitosan nanobubbles to complex with and protect DNA was confirmed by agarose gel assay. Chitosan nanobubbles were found to be stable following insonation (2.5 MHz) for up to 3 minutes at 37°C. DNA release was evaluated in vitro in both the presence and absence of ultrasound. The release of chitosan nanobubble-bound plasmid DNA occurred after just one minute of insonation. In vitro transfection experiments were performed by exposing adherent COS7 cells to ultrasound in the presence of different concentrations of plasmid DNA-loaded nanobubbles. In the absence of ultrasound, nanobubbles failed to trigger transfection at all concentrations tested. In contrast, 30 seconds of ultrasound promoted a moderate degree of transfection. Cell viability experiments demonstrated that neither ultrasound nor the nanobubbles affected cell viability under these experimental conditions. CONCLUSION: Based on these results, chitosan nanobubbles have the potential to be promising tools for ultrasound-mediated DNA delivery.
Asunto(s)
Quitosano/química , Microburbujas , Transfección/métodos , Ultrasonografía/métodos , Animales , Células COS , Rastreo Diferencial de Calorimetría , Supervivencia Celular/efectos de los fármacos , Quitosano/farmacología , Chlorocebus aethiops , ADN/administración & dosificación , ADN/química , ADN/farmacocinética , Vectores Genéticos/química , Vectores Genéticos/genética , Hemólisis/efectos de los fármacos , Humanos , Nanotecnología , Tamaño de la Partícula , Plásmidos/química , Plásmidos/farmacocinéticaRESUMEN
The activity of antivirals can be enhanced by their incorporation in nanoparticulate delivery systems. Peculiar polymeric nanoparticles, based on a ß-cyclodextrin-poly(4-acryloylmorpholine) monoconjugate (ß-CD-PACM), are proposed as acyclovir carriers. The experimental procedure necessary to obtain the acyclovir-loaded nanoparticles using the solvent displacement preparation method will be described in this chapter. Fluorescent labeled nanoparticles are prepared using the same method for cellular trafficking studies. The biocompatibility assays necessary to obtain safe nanoparticles are reported. Section 4 of this chapter describes the assessment of the antiviral activity of the acyclovir-loaded nanoparticles.
Asunto(s)
Resinas Acrílicas/química , Aciclovir/farmacología , Antivirales/farmacología , Herpesvirus Humano 1/efectos de los fármacos , Nanocápsulas/química , beta-Ciclodextrinas/química , Aciclovir/metabolismo , Animales , Antivirales/metabolismo , Supervivencia Celular/efectos de los fármacos , Chlorocebus aethiops , Activación de Complemento/efectos de los fármacos , Hemólisis/efectos de los fármacos , Herpesvirus Humano 1/crecimiento & desarrollo , Humanos , Ensayo de Materiales , Tamaño de la Partícula , Especies Reactivas de Oxígeno/metabolismo , Piel/efectos de los fármacos , Propiedades de Superficie , Técnicas de Cultivo de Tejidos , Células Vero , Carga Viral , Ensayo de Placa ViralRESUMEN
Three types of cyclodextrin nanosponges were synthetized cross-linking α, ß or γ cyclodextrin with carbonyldiimidazole as cross-linker. Nanosponges are solid nanoparticles previously used as drug carriers. In this studies cyclodextrin nanosponges were developed as oxygen delivery system. For this purpose the three types of nanosponges suspended in water were saturated with oxygen and in vitro characterized. The nanosponge safety was tested on Vero cells. Their ability to release oxygen in the presence and in the absence of ultrasound (US) was determined over time. Oxygen permeation through a silicone membrane was obtained using a ß-cyclodextrin nanosponge/hydrogel combination system. Nanosponge formulations might be potential gas delivery systems showing the ability to store and to release oxygen slowly over time.
Asunto(s)
Portadores de Fármacos/química , Nanopartículas , Oxígeno/administración & dosificación , Animales , Chlorocebus aethiops , Reactivos de Enlaces Cruzados/química , Imidazoles , Permeabilidad , Factores de Tiempo , Ultrasonografía/métodos , Células Vero , alfa-Ciclodextrinas/química , beta-Ciclodextrinas/química , gamma-Ciclodextrinas/químicaRESUMEN
In this paper we report on the investigation, as DNA nonviral carriers, of three samples of an amphoteric polyamidoamine bearing 4-aminobutylguanidine deriving units, AGMA5, AGMA10, and AGMA20, characterized by different molecular weights (M(w) 5100, 10100, and 20500, respectively). All samples condensed DNA in spherical, positively charged nanoparticles and protected it against enzymatic degradation. AGMA10 and AGMA20 polyplexes had average diameters lower than 100 nm. AGMA5 polyplexes were larger. All polyplexes showed negligible cytotoxicity and were internalized in cells. AGMA10 and AGMA20 performed differently from AGMA5 as nucleic acid carriers in vitro. AGMA10 and AGMA20 effectively promoted transfection, whereas AGMA5 was ineffective. FITC-labeled AGMA10 was prepared and the intracellular trafficking of its DNA polyplex was studied. DNA/AGMA10 polyplex was largely localized inside the nucleus, while AGMA10 concentrated in the perinuclear region. DNA/AGMA10 polyplex intravenously administered to mice promoted gene expression in liver but not in other organs without detectable toxic side effects.
Asunto(s)
Agmatina/análogos & derivados , ADN/administración & dosificación , Portadores de Fármacos/química , Técnicas de Transferencia de Gen , Plásmidos/administración & dosificación , Poliaminas/química , Agmatina/efectos adversos , Agmatina/síntesis química , Agmatina/química , Supervivencia Celular/efectos de los fármacos , ADN/genética , Desoxirribonucleasas/química , Estabilidad de Medicamentos , Células HeLa , Humanos , Microscopía Electrónica de Transmisión , Peso Molecular , Plásmidos/genética , Poliaminas/efectos adversos , Poliaminas/síntesis química , Propiedades de Superficie , TransfecciónRESUMEN
An amphoteric thiol-functionalized poly(amidoamine) nicknamed ISA23SH(10%) was synthesized. Rhenium complexes 1 and 2, containing 0.5 and 0.8 equiv of rhenium, respectively, were easily obtained by reacting ISA23SH(10%) with [Re(CO)(3)(H(2)O)(3)](CF(3)SO(3)) in aqueous solution at pH 5.5. Both ISA23SH(10%), and its rhenium complexes were soluble in water under physiological conditions. The resultant solutions were stable, even in the presence of cysteine. Rhenium chelation occurred through the S and N atoms of the cysteamine moiety, as demonstrated by (1)H, (13)C, and (15)N NMR spectroscopy. The diffusion coefficients and the hydrodynamic radii of ISA23SH(10%) and complex 1 were determined by pulsed gradient spin echo (PGSE) NMR experiments. The radius of the rhenium complexes 1 and 2 was always slightly larger than that of the parent polymer. TEM analysis showed that both complexes form spherical nanoparticles with narrow size distributions. Consistent results were obtained by dynamic light scattering. The observed sizes were in good agreement with those evaluated by PGSE. Preliminary in vitro and in vivo biological studies have been performed on complexes 1 and 2 as well as on the parent ISA23SH(10%). Neither hemolytic activity of the two rhenium complexes and the parent polymer, up to a concentration of 5 mg/mL, nor cytotoxic effects were observed on Hela cell after 48 h at a concentration of 100 ng/mL. In vivo toxicological tests showed that ISA23SH(10%) is highly biocompatible, with a maximum tolerated dose (MTD) of 500 mg/kg. No toxic side effects were apparent after the intravenous injection in mice of the two rhenium complexes in doses up to 20 mg/kg.
Asunto(s)
Quelantes/química , Nylons/química , Piperazinas/química , Poliaminas/química , Renio/química , Animales , Células HeLa , Humanos , Ratones , Ratones Endogámicos , Nylons/farmacología , Piperazinas/farmacología , Poliaminas/farmacología , Renio/farmacologíaRESUMEN
Ultrasound (US) energy combined with gas-filled microbubbles has been used for several years in medical imaging. This study investigated the ability of oxygen-loaded chitosan bubbles to exchange oxygen in the presence or in the absence of US. Oxygen delivery is enhanced by sonication and both frequency and time duration of US affected the exchange kinetics.
Asunto(s)
Quitosano/química , Microburbujas , Oxígeno/administración & dosificación , Sonicación/métodos , Línea Celular Tumoral , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Factores de Tiempo , UltrasonidoRESUMEN
Chitosan-coated oxygen microbubbles of average diameter 2.5 mum, narrow size distribution and spherical shape were prepared. A core-shell structure was evidenced by fluorescence microscopy using fluorescent microbubbles. Such microbubbles can be a therapeutic device for vehiculating oxygen to hypoxic tissues, provided they show proper permeability and diffusivity properties and are non-toxic. Our study proves that oxygen is efficiently delivered both in 'in vitro' and 'in vivo' preparations, and can be conveniently metabolized reversing the cellular hypoxic response. Moreover, toxic effects were investigated in human blood and in cultured cells and no evidence for them was found.
Asunto(s)
Portadores de Fármacos , Hipoxia/terapia , Microburbujas , Oxígeno/uso terapéutico , Animales , Células Cultivadas , Quitosano/química , Huevos , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/análisisRESUMEN
Thermo-responsive chitosan microbubbles were developed as new therapeutic device for vehiculating gases to tissues concomitantly to hyperthermic treatments. Aiming at applications to non-invasive temperature monitoring, microbubbles were characterized for acoustic attenuation properties in the 1-15 MHz range both by direct methods and by B-mode Ultrasound imaging up to 43 degrees C, which is the temperature used in clinical hyperthermia. The chitosan microbubbles showed a mean diameter of 1 microm at room temperature, which slightly decreases after heating, enhancing gas delivery. Acoustic attenuation monotonically increases with temperature, being the extent of such variation larger than that observed in tissues. Both the physico-chemical and the acoustic profiles showed reversible variations of microbubbles approaching 43 degrees C, which might be of interest for applications in hyperthermic therapies.