RESUMEN
The purpose of this study was to evaluate the efficacy of imiquimod-containing nanovesicles prepared with lipids extracted from the hyperhalophile archaebacterium Halorubrum tebenquichense (nanoARC-IMQ) to induce protection against Trypanosoma cruzi infection. The therapeutic efficacy of archaeolipid nanovesicles was assessed in an experimental murine model of acute infection with T. cruzi. The administration of nanoARQ-IMQ prevented mortality as compared to infected untreated animals, reduced parasitemia levels and diminished myocardial and musculoskeletal lesions in mice infected with a lethal strain of T. cruzi. Our findings suggest that the immunotherapy with nanoARC-IMQ has potential to limit the progression of Chagas disease.
Asunto(s)
Adyuvantes Inmunológicos/uso terapéutico , Enfermedad de Chagas/terapia , Imiquimod/uso terapéutico , Inmunoterapia , Nanopartículas/uso terapéutico , Adyuvantes Inmunológicos/química , Animales , Enfermedad de Chagas/patología , Imiquimod/química , Lípidos/química , Masculino , Ratones , Ratones Endogámicos C3H , Nanopartículas/química , Tamaño de la Partícula , Propiedades de SuperficieRESUMEN
In this work, the in vitro anti-Leishmania activity of photodynamic liposomes made of soybean phosphatidylcholine, sodium cholate, total polar archaeolipids (TPAs) extracted from the hyperhalophile archaea Halorubrum tebenquichense and the photosensitizer zinc phthalocyanine (ZnPcAL) was compared to that of ultradeformable photodynamic liposomes lacking TPAs (ZnPcUDLs). We found that while ZnPcUDLs and ZnPcALs (130 nm mean diameter and -35 mV zeta potential) were innocuous against promastigotes, a low concentration (0.01 µM ZnPc and 7.6 µM phospholipids) of ZnPcALs irradiated at a very low-energy density (0.2 J/cm(2)) eliminated L. braziliensis amastigotes from J774 macrophages, without reducing the viability of the host cells. In such conditions, ZnPcALs were harmless for J774 macrophages, HaCaT keratinocytes, and bone marrow-derived dendritic cells. Therefore, topical photodynamic treatment would not likely affect skin-associated lymphoid tissue. ZnPcALs were extensively captured by macrophages, but ZnPcUDLs were not, leading to 2.5-fold increased intracellular delivery of ZnPc than with ZnPcUDLs. Despite mediating low levels of reactive oxygen species, the higher delivery of ZnPc and the multiple (caveolin- and clathrin-dependent plus phagocytic) intracellular pathway followed by ZnPc would have been the reason for the higher antiamastigote activity of ZnPcALs. The leishmanicidal activity of photodynamic liposomal ZnPc was improved by TPA-containing liposomes.
Asunto(s)
Antiprotozoarios/farmacología , Éteres de Glicerilo/farmacología , Indoles/farmacología , Leishmania/efectos de los fármacos , Leishmania/efectos de la radiación , Liposomas/farmacología , Compuestos Organometálicos/farmacología , Animales , Antiprotozoarios/química , Antiprotozoarios/farmacocinética , Antiprotozoarios/toxicidad , Línea Celular , Supervivencia Celular/efectos de los fármacos , Éteres de Glicerilo/química , Éteres de Glicerilo/farmacocinética , Éteres de Glicerilo/toxicidad , Humanos , Indoles/química , Indoles/farmacocinética , Indoles/toxicidad , Isoindoles , Liposomas/química , Liposomas/farmacocinética , Liposomas/toxicidad , Macrófagos/metabolismo , Ratones , Compuestos Organometálicos/química , Compuestos Organometálicos/farmacocinética , Compuestos Organometálicos/toxicidad , Especies Reactivas de Oxígeno/análisis , Especies Reactivas de Oxígeno/metabolismo , Compuestos de ZincRESUMEN
Archaeosomes (ARC), vesicles made from lipids extracted from Archaea, display strong adjuvant properties. In this study, we evaluated the ability of the highly stable ARC formulated from total polar lipids of a new Halorubrum tebenquichense strain found in Argentinean Patagonia, to act as adjuvant for soluble parasite antigens in developing prophylactic vaccine against the intracellular protozoan T. cruzi, the etiologic agent of Chagas disease. We demonstrated for the first time that C3H/HeN mice subcutaneously immunized with trypanosomal antigens entrapped in these ARC (ARC-TcAg) rapidly developed higher levels of circulating T. cruzi antibodies than those measured in the sera from animals receiving the antigen alone. Enhanced humoral responses elicited by ARC-TcAg presented a dominant IgG2a antibody isotype, usually associated with Th1-type immunity and resistance against T. cruzi. More importantly, ARC-TcAg-vaccinated mice displayed reduced parasitemia during early infection and were protected against an otherwise lethal challenge with the virulent Tulahuén strain of the parasite. Our findings suggest that, as an adjuvant, H. tebenquichense-derived ARC may hold great potential to develop a safe and helpful vaccine against this relevant human pathogen.
Asunto(s)
Adyuvantes Inmunológicos/administración & dosificación , Enfermedad de Chagas/prevención & control , Halorubrum/química , Liposomas/administración & dosificación , Lípidos de la Membrana/administración & dosificación , Vacunas Antiprotozoos/administración & dosificación , Vacunas Antiprotozoos/inmunología , Adyuvantes Inmunológicos/aislamiento & purificación , Animales , Anticuerpos Antiprotozoarios/sangre , Argentina , Enfermedad de Chagas/inmunología , Modelos Animales de Enfermedad , Femenino , Inmunoglobulina G/sangre , Inyecciones Subcutáneas , Liposomas/aislamiento & purificación , Lípidos de la Membrana/aislamiento & purificación , Ratones Endogámicos C3H , Parasitemia/inmunología , Parasitemia/prevención & control , Análisis de Supervivencia , Células TH1/inmunología , Resultado del TratamientoRESUMEN
Ultradeformable archaeosomes (UDA) are vesicles made of soybean phosphatidylcholine (SPC), sodium cholate (NaChol) and polar lipids from Halorubrum tebenquichense (3:1:3 wt/wt). Although ultradeformable liposomes (UDL, made of SPC and NaChol at 6:1 wt/wt) and UDA were neither captured nor caused cytotoxicity on keratinocytes, UDA was avidly captured by macrophages, their viability being reduced by 0.4-1.6 mg/mL phospholipids by 25 to 60%. Instead, UDL were poorly captured and caused no toxicity. Balb/C mice immunized by the topical route with four doses of ovalbumin (OVA)-loaded UDA, at 75 µg OVA/600 µg phospholipids (125 nm mean size and -42 mV zeta potential), induced IgG titers tenfold to 100-fold higher than those immunized with OVA-loaded UDL at the same dosage. Both matrices penetrate to the same skin depth (nearly 10 µm after 1 hour on excised human skin), being the higher topical adjuvancy and higher phagocytic uptake of UDA related to its glycolipid content. FROM THE CLINICAL EDITOR: This work summarizes key findings related to the development of ultradeformable archaeosomes as vehicles utilized in transdermal delivery systems with improved skin penetration.
Asunto(s)
Lípidos , Liposomas , Fosfatidilcolinas , Colato de Sodio , Administración Tópica , Animales , Línea Celular , Supervivencia Celular/efectos de los fármacos , Sistemas de Liberación de Medicamentos , Femenino , Halorubrum/química , Humanos , Queratinocitos/efectos de los fármacos , Lípidos/administración & dosificación , Lípidos/química , Liposomas/administración & dosificación , Ratones , Fosfatidilcolinas/administración & dosificación , Fosfatidilcolinas/química , Absorción Cutánea/fisiología , Colato de Sodio/administración & dosificación , Colato de Sodio/química , VacunaciónRESUMEN
The archaeolipids (lipids extracted from archaebacterias) are non saponificable molecules that form self sealed mono or bilayers (archaeosomes-ARC). Different to liposomes with bilayers made of conventional glycerophospholipids, the bilayer of ARC posses a higher structural resistance to physico chemical and enzymatic degradation and surface hydrophobicity. In this work we have compared the binding capacity of ARC exclusively made of archaeols containing a minor fraction of sulphoglycophospholipids, with that of liposomes in gel phase on M-like cells in vitro. The biodistribution of the radiopharmaceutical (99m)Tc-DTPA loaded in ARC vs that of liposomes upon oral administration to Wistar rats was also determined. The fluorescence of M-like cells upon 1 and 2h incubation with ARC loaded with the hydrophobic dye Rhodamine-PE (Rh-PE) and the hydrophilic dye pyranine (HPTS) dissolved in the aqueous space, was 4 folds higher than upon incubation with equally labeled liposomes. Besides, 15% of Rh-PE and 13 % of HPTS from ARC and not from liposomes, were found in the bottom wells, a place that is equivalent to the basolateral pocket from M cells. This fact suggested the occurrence of transcytosis of ARC. Finally, 4 h upon oral administration, ARC were responsible for the 22.3 % (3.5 folds higher than liposomes) shuttling of (99m)Tc-DTPA to the blood circulation. This important amount of radioactive marker in blood could be a consequence of an extensive uptake of ARC by M cells in vivo, probably favored by their surface hydrophobicity. Taken together, these results suggested that ARC, proven their adjuvant capacity when administered by parenteral route and high biocompatibility, could be a suitable new type of nanoparticulate material that could be used as adjuvants by the oral route.
Asunto(s)
Éteres de Glicerilo/administración & dosificación , Éteres de Glicerilo/química , Pentetato de Tecnecio Tc 99m/administración & dosificación , Pentetato de Tecnecio Tc 99m/química , Administración Oral , Animales , Archaea/química , Arilsulfonatos/administración & dosificación , Arilsulfonatos/química , Células CACO-2 , Línea Celular Tumoral , Técnicas de Cocultivo , Portadores de Fármacos/administración & dosificación , Portadores de Fármacos/química , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Liposomas/administración & dosificación , Liposomas/química , Nanopartículas/administración & dosificación , Nanopartículas/química , Ratas , Ratas Wistar , Rodaminas/administración & dosificación , Rodaminas/química , Pentetato de Tecnecio Tc 99m/sangre , Distribución Tisular , Transcitosis/fisiologíaRESUMEN
Being independent of artificial power sources, self administered sunlight triggered photodynamic therapy could be a suitable alternative treatment for cutaneous leishmaniasis, that avoids the need for injectables and the toxic side effects of pentavalent antimonials. In this work we have determined the in vitro leishmanicidal activity of sunlight triggered photodynamic ultradeformable liposomes (UDL). ZnPc is a hydrophobic Zn phthalocyanine that showed 20% anti-promastigote activity (APA) and 20% anti-amastigote activity (AA) against Leishmania braziliensis (strain 2903) after 15min sunlight irradiation (15J/cm(2)). However, when loaded in UDL as UDL-ZnPc (1.25µM ZnPc-1mM phospholipids) it elicited 100% APA and 80% AA at the same light dose. In the absence of host cell toxicity, UDL and UDL-ZnPc also showed non-photodynamic leishmanicidal activity. Confocal laser scanning microscopy of cryosectioned human skin mounted in non-occlusive Saarbrücken Penetration Model, showed that upon transcutaneous administration ZnPc penetrated nearly 10 folds deeper as UDL-ZnPc than if loaded in conventional liposomes (L-ZnPc). Quantitative determination of ZnPc confirmed that UDL-ZnPc penetrated homogeneously in the stratum corneum, carrying 7 folds higher amount of ZnPc 8 folds deeper than L-ZnPc. It is envisioned that the multiple leishmanicidal effects of UDL-ZnPc could play a synergistic role in prophylaxis or therapeutic at early stages of the infection.
Asunto(s)
Indoles/farmacología , Leishmania braziliensis/efectos de los fármacos , Leishmaniasis Cutánea/tratamiento farmacológico , Compuestos Organometálicos/farmacología , Fotoquimioterapia , Fármacos Fotosensibilizantes/farmacología , Luz Solar , Administración Cutánea , Animales , Química Farmacéutica , Chlorocebus aethiops , Composición de Medicamentos , Femenino , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Indoles/administración & dosificación , Indoles/química , Indoles/metabolismo , Indoles/toxicidad , Isoindoles , Liposomas , Ratones , Microscopía Confocal , Compuestos Organometálicos/administración & dosificación , Compuestos Organometálicos/química , Compuestos Organometálicos/metabolismo , Compuestos Organometálicos/toxicidad , Tamaño de la Partícula , Fármacos Fotosensibilizantes/administración & dosificación , Fármacos Fotosensibilizantes/química , Fármacos Fotosensibilizantes/metabolismo , Fármacos Fotosensibilizantes/toxicidad , Piel/metabolismo , Absorción Cutánea , Factores de Tiempo , Células Vero , Compuestos de ZincRESUMEN
BACKGROUND: Archaeosomes (ARC), vesicles prepared from total polar lipids (TPL) extracted from selected genera and species from the Archaea domain, elicit both antibody and cell-mediated immunity to the entrapped antigen, as well as efficient cross priming of exogenous antigens, evoking a profound memory response. Screening for unexplored Archaea genus as new sources of adjuvancy, here we report the presence of two new Halorubrum tebenquichense strains isolated from grey crystals (GC) and black mood (BM) strata from a littoral Argentinean Patagonia salt flat. Cytotoxicity, intracellular transit and immune response induced by two subcutaneous (sc) administrations (days 0 and 21) with BSA entrapped in ARC made of TPL either form BM (ARC-BM) and from GC (ARC-GC) at 2% w/w (BSA/lipids), to C3H/HeN mice (25 microg BSA, 1.3 mg of archaeal lipids per mouse) and boosted on day 180 with 25 microg of bare BSA, were determined. RESULTS: DNA G+C content (59.5 and 61.7% mol BM and GC, respectively), 16S rDNA sequentiation, DNA-DNA hybridization, arbitrarily primed fingerprint assay and biochemical data confirmed that BM and GC isolates were two non-previously described strains of H. tebenquichense. Both multilamellar ARC mean size were 564 +/- 22 nm, with -50 mV zeta-potential, and were not cytotoxic on Vero cells up to 1 mg/ml and up to 0.1 mg/ml of lipids on J-774 macrophages (XTT method). ARC inner aqueous content remained inside the phago-lysosomal system of J-774 cells beyond the first incubation hour at 37 degrees C, as revealed by pyranine loaded in ARC. Upon subcutaneous immunization of C3H/HeN mice, BSA entrapped in ARC-BM or ARC-GC elicited a strong and sustained primary antibody response, as well as improved specific humoral immunity after boosting with the bare antigen. Both IgG1 and IgG2a enhanced antibody titers could be demonstrated in long-term (200 days) recall suggesting induction of a mixed Th1/Th2 response. CONCLUSION: We herein report the finding of new H. tebenquichense non alkaliphilic strains in Argentinean Patagonia together with the adjuvant properties of ARC after sc administration in mice. Our results indicate that archaeosomes prepared with TPL from these two strains could be successfully used as vaccine delivery vehicles.
Asunto(s)
Adyuvantes Inmunológicos/química , Halorubrum/química , Lípidos/inmunología , Liposomas/inmunología , Animales , Formación de Anticuerpos , Composición de Base , Chlorocebus aethiops , ADN de Archaea/genética , Femenino , Halorubrum/genética , Halorubrum/inmunología , Halorubrum/aislamiento & purificación , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Lípidos/química , Liposomas/química , Ratones , Ratones Endogámicos C3H , Filogenia , ARN Ribosómico 16S/genética , Células VeroRESUMEN
BACKGROUND: Leishmania amastigotes live inside resident macrophages in different anatomic sites. Their hidden location is responsible for impairing the accession of therapeutic drugs. Drug delivery systems (DDSs) should allow the adverse effects caused by problematic routes of administration to be avoided as well as enhancing the antileishmanial activity and reducing the toxicity of the medication. However, after 30 years of research in the field, and since leishmaniasis is mostly a disease affecting the poorest populations, currently AmBisome is the only DDS used against the visceral form, and most experimental development only relates to parenteral administration. OBJECTIVE: We critically review the main DDSs designed against the different clinical forms of leishmaniasis. METHODS: A literature search was performed on PubMed and through Google. CONCLUSIONS: On reviewing the experimental and clinical therapeutic performance of former and current DDSs and considering the main obstacles to be overcome, we discuss how nanomedicine can contribute to the development of new and more efficient strategies.
Asunto(s)
Antiparasitarios/uso terapéutico , Sistemas de Liberación de Medicamentos , Leishmaniasis/tratamiento farmacológico , Administración Oral , Administración Tópica , Animales , Antiparasitarios/administración & dosificación , Humanos , Leishmania/efectos de los fármacos , Leishmaniasis/parasitología , Liposomas , NanopartículasRESUMEN
We investigated the in vitro action of an hydrosoluble 2-nitroimidazole, Etanidazole (EZL), against Trypanosoma cruzi, the etiologic agent of Chagas disease. EZL displayed lethal activity against isolated trypomastigotes as well as amastigotes of T. cruzi (RA strain) growing in Vero cells or J774 macrophages, without affecting host cell viability. Although not completely equivalent to Benznidazole (BZL), the reference drug for Chagas chemotherapy, EZL takes advantage in exerting its anti-T. cruzi activity for longer periods without serious toxic side effects, as those recorded in BZL-treated patients. Our present results encourage further experiments to study in depth the trypanocidal properties of this drug already licensed for use in human cancers.
Asunto(s)
Etanidazol/farmacología , Tripanocidas/farmacología , Trypanosoma cruzi/efectos de los fármacos , Animales , Chlorocebus aethiops , Concentración 50 Inhibidora , Dosificación Letal Mediana , Ratones , Pruebas de Sensibilidad Parasitaria , Células Vero/efectos de los fármacosRESUMEN
We investigated the in vitro action of an hydrosoluble 2-nitroimidazole, Etanidazole (EZL), against Trypanosoma cruzi, the etiologic agent of Chagas disease. EZL displayed lethal activity against isolated trypomastigotes as well as amastigotes of T. cruzi (RA strain) growing in Vero cells or J774 macrophages, without affecting host cell viability. Although not completely equivalent to Benznidazole (BZL), the reference drug for Chagas chemotherapy, EZL takes advantage in exertingits anti-T. cruzi activity for longer periods without serious toxic side effects, as those recorded in BZL-treated patients. Our present results encourage further experiments to study in depth the trypanocidal properties of this drug already licensed for use in human cancers.
Asunto(s)
Animales , Ratones , Etanidazol , Tripanocidas , Trypanosoma cruzi , Chlorocebus aethiops , Concentración 50 Inhibidora , Dosificación Letal Mediana , Células VeroRESUMEN
A diferencia del resto de las moléculas biológicas, los fosfolípidos son capaces de autoensamblarse espontáneamente. Con ellos es relativamente simples generar estructuras selladas extremadamente estables, de tamaño, forma y empaquetamiento controlables, llamadas liposomas. En este artículo revisaremos el uso de liposomas para generar vectores que mejoren los procesos de transfección en células eucarioticas, tanto in vivo como in vitro. Empleando vectores lipídicos, es potencialmente posible enviar selectivamente un segmento de AND a cualquier sitio del cuerpo, forzarlo a ingresar al interior celular y aun controlar el destino intracelular de la carga transportada. La clave del éxito de la transfección por medio de vectores lipídicos radica en que protegen mecánicamente al AND de la degradación plasmática, ofreciendo a la vez la oportunidad de controlar su biodistribuición, independientemente del tamaño del segmento de AND que se quiera expresar. Asimismo, son no carcinogénicos y pobremente inmunogénicos. Los avances en la química de sintésis de lípidos permitirán construir vectores cada vez más eficientes, que capitan con los altos niveles de transfección de los vectores virales, sumado a las ventajas de extrema versatilidad, facilidad de preparación y bioseguridad propias de la moléculas autoensamblables. (AU)
Asunto(s)
Animales , Humanos , Terapia Genética , Vectores Genéticos , Liposomas , Lípidos , Transfección , Lípidos/química , Liposomas/química , Ácidos Nucleicos/química , Oligonucleótidos/química , ADN/químicaRESUMEN
A diferencia del resto de las moléculas biológicas, los fosfolípidos son capaces de autoensamblarse espontáneamente. Con ellos es relativamente simples generar estructuras selladas extremadamente estables, de tamaño, forma y empaquetamiento controlables, llamadas liposomas. En este artículo revisaremos el uso de liposomas para generar vectores que mejoren los procesos de transfección en células eucarioticas, tanto in vivo como in vitro. Empleando vectores lipídicos, es potencialmente posible enviar selectivamente un segmento de AND a cualquier sitio del cuerpo, forzarlo a ingresar al interior celular y aun controlar el destino intracelular de la carga transportada. La clave del éxito de la transfección por medio de vectores lipídicos radica en que protegen mecánicamente al AND de la degradación plasmática, ofreciendo a la vez la oportunidad de controlar su biodistribuición, independientemente del tamaño del segmento de AND que se quiera expresar. Asimismo, son no carcinogénicos y pobremente inmunogénicos. Los avances en la química de sintésis de lípidos permitirán construir vectores cada vez más eficientes, que capitan con los altos niveles de transfección de los vectores virales, sumado a las ventajas de extrema versatilidad, facilidad de preparación y bioseguridad propias de la moléculas autoensamblables.