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Trichoderma harzianum is a filamentous fungus that can act as a mycoparasite, saprophyte, or a plant symbiotic. It is widely used as a biological control agent against phytopathogenic fungi and can also be used for plant growth promotion and biofortification. Interaction between T. harzianum and phytopathogenic fungi involves mycoparasitism, competition, and antibiosis. Extracellular vesicles (EVs) have been described as presenting a central role in mechanisms of communication and interaction among fungus and their hosts. In this study, we characterized extracellular vesicles of T. harzianum produced during growth in the presence of glucose or S. sclerotiorum mycelia. A set of vesicular proteins was identified using proteomic approach, mainly presenting predicted signal peptides.
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Vesículas Extracelulares , Hypocreales , Trichoderma , Trichoderma/metabolismo , ProteómicaRESUMEN
BACKGROUND: Currently, considerable efforts to standardize methods for accurate assessment of properties and safety aspects of nanomaterials are being made. However, immunomodulation effects upon skin exposure to nanomaterial have not been explored. OBJECTIVES: To investigate the immunotoxicity of single-wall carbon nanotubes, titanium dioxide, and fullerene using the current mechanistic understanding of skin sensitization by applying the concept of adverse outcome pathway (AOP). METHODS: Investigation of the ability of nanomaterials to interact with skin proteins using the micro-direct peptide reactivity assay; the expression of CD86 cell surface marker using the U937 cell activation test (OECD No. 442E/2018); and the effects of nanomaterials on modulating inflammatory response through inflammatory cytokine release by U937 cells. RESULTS: The nanomaterials easily internalized into keratinocytes cells, interacted with skin proteins, and triggered activation of U937 cells by increasing CD86 expression and modulating inflammatory cytokine production. Consequently, these nanomaterials were classified as skin sensitizers in vitro. CONCLUSIONS: Our study suggests the potential immunotoxicity of nanomaterials and highlights the importance of studying the immunotoxicity and skin sensitization potential of nanomaterials to anticipate possible human health risks using standardized mechanistic nonanimal methods with high predictive accuracy. Therefore, it contributes toward the applicability of existing OECD (Organisation for Economic Co-operation and Development) testing guidelines for accurate assessment of nanomaterial skin sensitization potential.
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Rutas de Resultados Adversos , Dermatitis Alérgica por Contacto/etiología , Dermatitis Alérgica por Contacto/inmunología , Fulerenos/efectos adversos , Nanotubos de Carbono/efectos adversos , Titanio/efectos adversos , Antígeno B7-2/metabolismo , Biomarcadores/metabolismo , Citocinas/metabolismo , Dermatitis Alérgica por Contacto/metabolismo , Células HaCaT , Humanos , Inmunomodulación , Queratinocitos/metabolismo , Células U937RESUMEN
Delivery efficiencies of theranostic nanoparticles (NPs) based on passive tumor targeting strongly depend either on their blood circulation time or on appropriate modulations of the tumor microenvironment. Therefore, predicting the NP delivery efficiency before and after a tumor microenvironment modulation is highly desirable. Here, we present a new erythrocyte membrane-camouflaged magnetofluorescent nanocarrier (MMFn) with long blood circulation time (92 h) and high delivery efficiency (10% ID for Ehrlich murine tumor model). MMFns owe their magnetic and fluorescent properties to the incorporation of manganese ferrite nanoparticles (MnFe2O4 NPs) and IR-780 (a lipophilic indocyanine fluorescent dye), respectively, to their erythrocyte membrane-derived camouflage. MMFn composition, morphology, and size, as well as optical absorption, zeta potential, and fluorescent, magnetic, and magnetothermal properties, are thoroughly examined in vitro. We then present an analytical pharmacokinetic (PK) model capable of predicting the delivery efficiency (DE) and the time of peak tumor uptake (tmax), as well as changes in DE and tmax due to modulations of the tumor microenvironment, for potentially any nanocarrier. Experimental PK data sets (blood and tumor amounts of MMFns) are simultaneously fit to the model equations using the PK modeling software Monolix. We then validate our model analytical solutions with the numerical solutions provided by Monolix. We also demonstrate how our a priori nonmechanistic model for passive targeting relates to a previously reported mechanistic model for active targeting. All in vivo PK studies, as well as in vivo and ex vivo biodistribution studies, were conducted using two noninvasive techniques, namely, fluorescence molecular tomography (FMT) and alternating current biosusceptometry (ACB). Finally, histopathology corroborates our PK and biodistribution results.
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Portadores de Fármacos/química , Membrana Eritrocítica/química , Compuestos Férricos/química , Colorantes Fluorescentes/química , Nanopartículas Magnéticas de Óxido de Hierro/química , Imanes/química , Compuestos de Manganeso/química , Terapia Fototérmica/métodos , Animales , Carcinoma de Ehrlich/tratamiento farmacológico , Modelos Animales de Enfermedad , Portadores de Fármacos/farmacocinética , Femenino , Compuestos Férricos/farmacocinética , Colorantes Fluorescentes/farmacocinética , Hipertermia Inducida/métodos , Compuestos de Manganeso/farmacocinética , Ratones , Tamaño de la Partícula , Nanomedicina Teranóstica/métodos , Distribución Tisular , Carga Tumoral/efectos de los fármacos , Microambiente Tumoral/efectos de los fármacosRESUMEN
Melanoma is the most aggressive and metastasis-prone form of skin cancer. Conventional therapies include chemotherapeutic agents, either as small molecules or carried by FDA-approved nanostructures. However, systemic toxicity and side effects still remain as major drawbacks. With the advancement of nanomedicine, new delivery strategies emerge at a regular pace, aiming to overcome these challenges. Stimulus-responsive drug delivery systems might considerably reduce systemic toxicity and side-effects by limiting drug release to the affected area. Herein, we report the development of paclitaxel-loaded lipid-coated manganese ferrite magnetic nanoparticles (PTX-LMNP) as magnetosomes synthetic analogs, envisaging the combined chemo-magnetic hyperthermia treatment of melanoma. PTX-LMNP physicochemical properties were verified, including their shape, size, crystallinity, FTIR spectrum, magnetization profile, and temperature profile under magnetic hyperthermia (MHT). Their diffusion in porcine ear skin (a model for human skin) was investigated after intradermal administration via fluorescence microscopy. Cumulative PTX release kinetics under different temperatures, either preceded or not by MHT, were assessed. Intrinsic cytotoxicity against B16F10 cells was determined via neutral red uptake assay after 48 h of incubation (long-term assay), as well as B16F10 cells viability after 1 h of incubation (short-term assay), followed by MHT. PTX-LMNP-mediated MHT triggers PTX release, allowing its thermal-modulated local delivery to diseased sites, within short timeframes. Moreover, half-maximal PTX inhibitory concentration (IC50) could be significantly reduced relatively to free PTX (142,500×) and Taxol® (340×). Therefore, the dual chemo-MHT therapy mediated by intratumorally injected PTX-LMNP stands out as a promising alternative to efficiently deliver PTX to melanoma cells, consequently reducing systemic side effects commonly associated with conventional chemotherapies.
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Doxorubicin (DOX) is a chemotherapeutic agent commonly used for the treatment of solid tumors. However, the cardiotoxicity associated with its prolonged use prevents further adherence and therapeutic efficacy. By encapsulating DOX within a PEGylated liposome, Doxil® considerably decreased DOX cardiotoxicity. By using thermally sensitive lysolipids in its bilayer composition, ThermoDox® implemented a heat-induced controlled release of DOX. However, both ThermoDox® and Doxil® rely on their passive retention in tumors, depending on their half-lives in blood. Moreover, ThermoDox® ordinarily depend on invasive radiofrequency-generating metallic probes for local heating. In this study, we prepare, characterize, and evaluate the antitumoral capabilities of DOX-loaded folate-targeted PEGylated magnetoliposomes (DFPML). Unlike ThermoDox®, DOX delivery via DFPML is mediated by the heat released through dynamic hysteresis losses from magnetothermal converting systems composed by MnFe2O4 nanoparticles (NPs) under AC magnetic field excitation-a non-invasive technique designated magnetic hyperthermia (MHT). Moreover, DFPML dismisses the use of thermally sensitive lysolipids, allowing the use of simpler and cheaper alternative lipids. MnFe2O4 NPs and DFPML are fully characterized in terms of their size, morphology, polydispersion, magnetic, and magnetothermal properties. About 50% of the DOX load is released from DFPML after 30 min under MHT conditions. Being folate-targeted, in vitro DFPML antitumoral activity is higher (IC50 ≈ 1 µg/ml) for folate receptor-overexpressing B16F10 murine melanoma cells, compared to MCF7 human breast adenocarcinoma cells (IC50 ≈ 4 µg/ml). Taken together, our results indicate that DFPML are strong candidates for folate-targeted anticancer therapies based on DOX controlled release.
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The development of rational therapies against complex diseases, such as cancer, has increased in the past few years due to the advances of 'omics' technologies. Concomitantly, several efforts have been made to design sophisticated drug delivery systems in order to increase specificity and drug accumulation in tumor sites. The complexity of these drug delivery systems highlights the need for suitable analytical methods to determine encapsulation/conjugation efficiency of drugs and molecules responsible for the targeted delivery. Therefore, this study focuses on the development and validation of a RP-HPLC-DAD methodology for concurrent quantification of paclitaxel (PTX) and cetuximab (CTX) in immunoliposomes. Chromatographic separation was achieved using a wide pore C8 column, and a gradient mobile phase consisting of 0.1% trifluoroacetic acid (TFA) in Milli-Q water/acetonitrile/isopropanol with a flow rate of 1 mL min-1. Drug peaks were fully separated and detected at 280 nm using UV detector. The method was validated according to ICH and FDA guidelines in terms of specificity and forced degradation studies, system suitability, linearity, limit of detection, limit of quantification, repeatability, intermediate precision, accuracy, robustness, and short-term stability. The developed method was linear over the concentration range of 37.5-150 µg mL-1 of PTX and 75-300 µg mL-1 of CTX. All parameters evaluated satisfied the acceptance criteria, according to both FDA and ICH guidelines. The applicability of the analytical method was assessed following the development of PTX-loaded immunoliposomes conjugated with CTX. Thus, the present study shows a novel, simple, stability-indicating and suitable method to quantify simultaneously PTX and CTX in immunoliposomes.
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Cromatografía de Fase Inversa , Paclitaxel , Cetuximab , Cromatografía Líquida de Alta Presión , Límite de Detección , Paclitaxel/análisisRESUMEN
IR-780 iodide is a fluorescent dye with optical properties in the near-infrared region that has applications in tumor detection and photothermal/photodynamic therapy. This multifunctional effect led to the development of theranostic nanoparticles with both IR-780 and chemotherapeutic drugs such as docetaxel, doxorubicin, and lonidamine. In this work, we developed two albumin-based nanoparticles containing near-infrared IR-780 iodide multifunctional dyes, one of them possessing a magnetic core. Molecular docking with AutoDock Vina studies showed that IR-780 binds to bovine serum albumin (BSA) with greater stability at a higher temperature, allowing the protein binding pocket to better fit this dye. The theoretical analysis corroborates the experimental protocols, where an enhancement of IR-780 was found coupled to BSA at 60 °C, even 30 days after preparation, in comparison to 30 °C. In vitro assays monitoring the viability of Ehrlich ascites carcinoma cells revealed the importance of the inorganic magnetic core on the nanocarrier photothermal-cytotoxic effect. Fluorescence molecular tomography measurements of Ehrlich tumor-bearing Swiss mice revealed the biodistribution of the nanocarriers, with marked accumulation in the tumor tissue (≈3% ID). The histopathological analysis demonstrated strong increase in tumoral necrosis areas after 24 and 72 h after treatment, indicating tumor regression. Tumor regression analysis of nonirradiated animals indicate a IR-780 dose-dependent antitumoral effect with survival rates higher than 70% (animals monitored up to 600 days). Furthermore, an in vivo photothermal therapy procedure was performed and tumor regression was also verified. These results show a novel insight for the biomedical application of IR-780-albumin-based nanocarriers, namely cancer therapy, not only by photoinduced therapy but also by a nonirradiation mechanism. Safety studies (acute oral toxicity, cardiovascular evaluation, and histopathological analysis) suggest potential for clinical translation.
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Hipertermia Inducida , Animales , Línea Celular Tumoral , Indoles , Ratones , Simulación del Acoplamiento Molecular , Fototerapia , Distribución TisularRESUMEN
Ascorbic acid (AA) is widely used in cosmetic formulations due to its antioxidant property and ability to increase collagen synthesis. Here, we encapsulated AA in vesicles with different lipid compositions. Negative liposome charge favored AA skin retention, with accumulation of 37 ± 12 and 74 ± 23 µg/cm2 in the epidermis and dermis, respectively, after 6 hours. Drug flux was influenced by the formulation composition, and both the presence of cholesterol and the liposomes surface charge were able to increase the amount of AA crossing the skin. The formulation was stable for at least 30 days and promoted a 7-fold increase in flux compared to free AA. Additionally, liposomes were able to interact better with keratinocytes and fibroblasts membranes. In vitro efficacy studies demonstrated that associating AA to these liposomes resulted in increased effectiveness of type I collagen synthesis by fibroblasts and regeneration of UVA-induced damage in keratinocytes. Our results demonstrate the applicability of AA-negatively charged liposomes in promoting AA cutaneous permeation and increasing the retention and flux of this molecule in the skin. This formulation also increased AA stability and effectiveness, opening new perspectives for its application in view of reducing certain skin ageing outcomes.
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Antioxidantes/administración & dosificación , Ácido Ascórbico/administración & dosificación , Colágeno/metabolismo , Fibroblastos/efectos de los fármacos , Absorción Cutánea , Administración Cutánea , Animales , Antioxidantes/farmacocinética , Antioxidantes/farmacología , Ácido Ascórbico/farmacocinética , Ácido Ascórbico/farmacología , Células 3T3 BALB , Línea Celular , Fibroblastos/metabolismo , Humanos , Liposomas/química , Ratones , Piel/efectos de los fármacos , Piel/metabolismoRESUMEN
PURPOSE: The objective of this study was to develop nanocapsules and nanospheres of polylactide-co-glycolide (PLGA) containing magnetic nanoparticles and rapamycin. METHOD: Magnetic nanoparticles (MP) were obtained by the co-precipitation of Fe(ll) and Fe(III) salts by addition of ammonium hydroxide. Nanocapsules (NC) and nanospheres (NS) containing either uncoated magnetic nanoparticles (MP), MP coated with oleic acid monolayer (MPOA) or MP coated with oleic acid bilayer (MPOA-OA) were prepared by the emulsion evaporation method. Rapamycin was also encapsulated into NC and NS. Morphology, size, size distribution, entrapment efficiency, stability and magnetization characteristics were determined. RESULTS: Non-contact AFM images showed that the composite nanoparticles were almost spherical in shape. The resulting polymeric nanocarriers were found to have a mean diameter of approximately 120 nm with a narrow size distribution. The influence of some experimental parameters on the entrapment efficiency and stability was determined. Nanocapsules and nanospheres prepared with uncoated magnetic nanoparticles exhibited higher entrapment efficiency and stability. Superparamagnetic behavior of the magnetic nanocomposite was demonstrated by magnetization data. These findings may contribute to the development of potential controlled release drug targeting devices based on magnetic polymeric nanocarriers.