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For the first time, this study shows the nanoarchitectonic process to obtain an acetogenin-enriched nanosystem (AuNPs-Ac) using an aqueous extract fromAnnona cherimolaMill (ACM) composed of gold nanoparticles embedded in an organic matrix that acts as stabilizing agent and presents anti-inflammatory activity and cytotoxical effect against HepG2 cell line, promoting apoptosis. The synthesis of AuNPs-Ac was confirmed by x-ray diffraction analysis, showing metallic gold as the only phase, and the scanning transmission microscope showed an organic cap covering the AuNPs-Ac. Fourier-transformed infrared suggests that the organic cap comprises a combination of different annonaceous acetogenins, alkaloids, and phenols by the presence of bands corresponding to aromatic rings and hydroxyl groups. High-Performance Liquid Chromatography has demonstrated the presence of annonacin, a potent acetogenin, in the extract of ACM. Anin vitroanti-inflammatory activity of the extract of ACM and the AuNPs-Ac was performed using the albumin denaturation method, showing a nonlinear response, which is better than sodium diclofenac salt in a wide range of concentrations that goes from 200 to 400µg ml-1with both samples. The viability assay was studied using trypan blue, treating IMR90 and HepG2 at different concentrations of AuNPs-Ac. The results defined a median lethal dose of 800µg ml-1against HepG2 through apoptosis according to the ratio of caspase-cleaved 9/alpha-tubulin evaluated. It was also demonstrated that the nanosystem presents a higher cytotoxic effect on the HepG2 cell line than in IMR90, suggesting a targeted mechanism. In addition, the nanosystem performs better than using only the extract of ACM in the anti-inflammatory or antiproliferative test, attributed to their higher surface area.
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Acetogeninas , Anti-Inflamatórios , Apoptose , Ouro , Nanopartículas Metálicas , Extratos Vegetais , Humanos , Acetogeninas/farmacologia , Acetogeninas/química , Células Hep G2 , Apoptose/efeitos dos fármacos , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/química , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Nanopartículas Metálicas/química , Ouro/química , Ouro/farmacologia , Sobrevivência Celular/efeitos dos fármacosRESUMO
ßCDPEG5 and ßCDPEG2 are two derivatives comprising seven PEG linear chains of 5 and 2 kDa, respectively, conjugated to ßCD. As ßCDPEGs display different physicochemical properties than their precursors, they could also trigger distinct cellular responses. To investigate the biological behavior of ßCDPEGs in comparison to their parent compounds, we performed broad toxicological assays on RAW 264.7 macrophages, MC3T3-E1 osteoblasts, and MDCK cells. By analyzing ROS and NO2- overproduction in macrophages, we found that ßCDPEGs induced a moderate stress response without affecting cell viability. Although MC3T3-E1 osteoblasts were more sensitive than MDCK cells to ßCDPEGs and the parent compounds, a similar pattern was observed: the effect of ßCDPEG5 on cell viability and cell cycle progression was larger than that of ßCDPEG2; PEG2 affected cell viability and cell cycle more than ßCDPEG2; cell post-treatment recovery was favorable in all cases, and the compounds had similar behaviors regarding ROS generation. The effect on MDCK cell migration followed a similar pattern. In contrast, for osteoblasts, the interference of ßCDPEG5 with cell migration was smaller than that of ßCDPEG2; likewise, the effect of PEG2 was shorter than its conjugate. Overall, the covalent conjugation of ßCD and PEGs, particularly to yield ßCDPEG2, improved the biocompatibility profile, evidencing that a favorable biological response can be tuned through a thoughtful combination of materials. Moreover, this is the first time that an in vitro evaluation of ßCD and PEG has been presented for MC3T3-E1 and MDCK cells, thus providing valuable knowledge for designing biocompatible nanomaterials constructed from ßCD and PEGs.
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beta-Ciclodextrinas , Macrófagos , Osteoblastos , Polietilenoglicóis/química , Espécies Reativas de Oxigênio/metabolismo , beta-Ciclodextrinas/químicaRESUMO
BACKGROUND: Tamoxifen is the standard endocrine therapy for breast cancers, which require metabolic activation by cytochrome P450 enzymes (CYP). However, the lower and variable concentrations of CYP activity at the tumor remain major bottlenecks for the efficient treatment, causing severe side-effects. Combination nanotherapy has gained much recent attention for cancer treatment as it reduces the drug-associated toxicity without affecting the therapeutic response. RESULTS: Here we show the modular design of P22 bacteriophage virus-like particles for nanoscale integration of virus-driven enzyme prodrug therapy and photodynamic therapy. These virus capsids carrying CYP activity at the core are decorated with photosensitizer and targeting moiety at the surface for effective combinatory treatment. The estradiol-functionalized nanoparticles are recognized and internalized into ER+ breast tumor cells increasing the intracellular CYP activity and showing the ability to produce reactive oxygen species (ROS) upon UV365 nm irradiation. The generated ROS in synergy with enzymatic activity drastically enhanced the tamoxifen sensitivity in vitro, strongly inhibiting tumor cells. CONCLUSIONS: This work clearly demonstrated that the targeted combinatory treatment using multifunctional biocatalytic P22 represents the effective nanotherapeutics for ER+ breast cancer.
Assuntos
Antineoplásicos Hormonais/administração & dosagem , Bacteriófago P22/enzimologia , Neoplasias da Mama/tratamento farmacológico , Sistema Enzimático do Citocromo P-450/administração & dosagem , Fármacos Fotossensibilizantes/administração & dosagem , Tamoxifeno/administração & dosagem , Antineoplásicos Hormonais/farmacologia , Bacteriófago P22/química , Biocatálise , Neoplasias da Mama/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Sistema Enzimático do Citocromo P-450/farmacologia , Portadores de Fármacos/química , Sistemas de Liberação de Medicamentos , Terapia Enzimática , Feminino , Humanos , Células MCF-7 , Modelos Moleculares , Fotoquimioterapia , Fármacos Fotossensibilizantes/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Receptores de Estrogênio/metabolismo , Tamoxifeno/farmacologiaRESUMO
BACKGROUND: The intracellular delivery of enzymes for therapeutic use has a promising future for the treatment of several diseases such as genetic disorders and cancer. Virus-like particles offer an interesting platform for enzymatic delivery to targeted cells because of their great cargo capacity and the enhancement of the biocatalyst stability towards several factors important in the practical application of these nanoparticles. RESULTS: We have designed a nano-bioreactor based on the encapsulation of a cytochrome P450 (CYP) inside the capsid derived from the bacteriophage P22. An enhanced peroxigenase, CYPBM3, was selected as a model enzyme because of its potential in enzyme prodrug therapy. A total of 109 enzymes per capsid were encapsulated with a 70 % retention of activity for cytochromes with the correct incorporation of the heme cofactor. Upon encapsulation, the stability of the enzyme towards protease degradation and acidic pH was increased. Cytochrome P450 activity was delivered into Human cervix carcinoma cells via transfecting P22-CYP nanoparticles with lipofectamine. CONCLUSION: This work provides a clear demonstration of the potential of biocatalytic virus-like particles as medical relevant enzymatic delivery vehicles for clinical applications.
Assuntos
Bacteriófago P22/química , Capsídeo/química , Sistema Enzimático do Citocromo P-450/administração & dosagem , Portadores de Fármacos/química , Proteínas do Capsídeo/química , Linhagem Celular Tumoral , Sistema Enzimático do Citocromo P-450/uso terapêutico , Terapia Enzimática , Feminino , Células HeLa , Humanos , Neoplasias do Colo do Útero/tratamento farmacológico , Neoplasias do Colo do Útero/enzimologiaRESUMO
Fungal plant pathogens are responsible for serious losses in many economically important crop species worldwide. Due to the use of fungicides and the fungi genome plasticity, multi-drug resistant strains are emerging as a new generation of pathogens, causing an expansive range of superficial and systemic plant infections, or new opportunistic fungal pathogens for humans. The group of antagonistic fungi Trichoderma spp. has been widely used to enhance plant growth and for the control of different pathogens affecting crops. Although Neurospora crassa is not a mycoparasitic fungus, its secretion of secondary metabolites with antimicrobial activity has been described. In this work, the effect of crude extract of the monoculture of Trichoderma asperellum T8a or the co-culture with N. crassa as an inhibitory treatment against the fungal pathogens Botrytis cinerea and Fusarium solani was evaluated. The findings demonstrate that the secondary metabolites contained in the T. asperellum crude extract have a clear fungistatic activity against B. cinerea and F. solani. Interestingly, this fungistatic activity highly increases when T. asperellum is co-cultivated with the non-pathogenic fungus N. crassa. Moreover, the co-culture crude extract also showed antifungal activity on post-harvest fruits, and no toxic effects on Murine fibroblast L929 (CCL-1) and murine macrophages RAW 264.7 (TIB-71) were observed. All these results together are solid evidence of the potential of the co-culture crude extract of T. asperellum and N. crassa, as an antifungal agent against phytopathogenic fungi, or post-harvest fruits during the transportation or commercialization time.
Assuntos
Botrytis , Técnicas de Cocultura , Frutas , Fusarium , Trichoderma , Fusarium/efeitos dos fármacos , Fusarium/crescimento & desenvolvimento , Frutas/microbiologia , Frutas/química , Botrytis/efeitos dos fármacos , Botrytis/crescimento & desenvolvimento , Trichoderma/metabolismo , Trichoderma/genética , Animais , Camundongos , Antifúngicos/farmacologia , Antifúngicos/metabolismo , Doenças das Plantas/microbiologia , Doenças das Plantas/prevenção & controle , Neurospora crassa/efeitos dos fármacos , Neurospora crassa/metabolismo , Células RAW 264.7 , Misturas Complexas/farmacologia , Misturas Complexas/químicaRESUMO
This study used a sonochemical synthesis method to prepare (La, Sm)-doped ZnO nanoparticles (NPs). The effect of incorporating these lanthanide elements on the structural, optical, and morphological properties of ZnO-NPs was analyzed. The cytotoxicity and the reactive oxygen species (ROS) generation capacity of ZnO-NPs were evaluated against breast (MCF7) and colon (HT29) cancer cell lines. Their antioxidant activity was analyzed using a DPPH assay, and their toxicity towards Artemia salina nauplii was also evaluated. The results revealed that treatment with NPs resulted in the death of 10.559-42.546% and 18.230-38.643% of MCF7 and HT29 cells, respectively. This effect was attributed to the ability of NPs to downregulate ROS formation within the two cell lines in a dose-dependent manner. In the DPPH assay, treatment with (La, Sm)-doped ZnO-NPs inhibited the generation of free radicals at IC50 values ranging from 3.898 to 126.948 µg/mL. Against A. salina nauplii, the synthesized NPs did not cause death nor induce morphological changes at the tested concentrations. A series of machine learning (ML) models were used to predict the biological performance of (La, Sm)-doped ZnO-NPs. Among the designed ML models, the gradient boosting model resulted in the greatest mean absolute error (MAE) (MAE 9.027, R2 = 0.86). The data generated in this work provide innovative insights into the influence of La and Sm on the structural arrangement and chemical features of ZnO-NPs, together with their cytotoxicity, antioxidant activity, and in vivo toxicity.
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The antioxidant capabilities of nanoparticles are contingent upon various factors, including their shape, size, and chemical composition. Herein, novel Nd-doped CeO2 nanoparticles were synthesized and the neodymium content was varied to investigate the synergistic impact on the antioxidant properties of CeO2 nanoparticles. Incorporating Nd3+ induced changes in lattice parameters and significantly altered the morphology from nanoparticles to nanorods. The biological activity of Nd-doped CeO2 was examined against pathogenic bacterial strains, breast cancer cell lines, and antioxidant models. The antibacterial and anticancer activities of nanoparticles were not observed, which could be associated with the Ce3+/Ce4+ ratio. Notably, the incorporation of neodymium improved the antioxidant capacity of CeO2. Machine learning techniques were employed to forecast the antioxidant activity to enhance understanding and predictive capabilities. Among these models, the random forest model exhibited the highest accuracy at 96.35%, establishing it as a robust computational tool for elucidating the biological behavior of Nd-doped CeO2 nanoparticles. This study presents the first exploration of the influence of Nd3+ on the structural, optical, and biological attributes of CeO2, contributing valuable insights and extending the application of machine learning in predicting the therapeutic efficacy of inorganic nanomaterials.
Assuntos
Nanopartículas , Nanoestruturas , Antioxidantes/farmacologia , Antioxidantes/química , Neodímio , Nanopartículas/química , Antibacterianos/farmacologia , Antibacterianos/químicaRESUMO
Ovarian cancer is the leading cause of death among gynecological malignancies. It is detected at late stages when the disease is spread through the abdominal cavity in a condition known as peritoneal carcinomatosis. Thus, there is an urgent need to develop novel therapeutic interventions to target advanced stages of ovarian cancer. Mammary serine protease inhibitor (Maspin) represents an important metastasis suppressor initially identified in breast cancer. Herein we have generated a sequence-specific zinc finger artificial transcription factor (ATF) to up-regulate the Maspin promoter in aggressive ovarian cancer cell lines and to interrogate the therapeutic potential of Maspin in ovarian cancer. We found that although Maspin was expressed in some primary ovarian tumors, the promoter was epigenetically silenced in cell lines derived from ascites. Transduction of the ATF in MOVCAR 5009 cells derived from ascitic cultures of a TgMISIIR-TAg mouse model of ovarian cancer resulted in tumor cell growth inhibition, impaired cell invasion, and severe disruption of actin cytoskeleton. Systemic delivery of lipid-protamine-RNA nanoparticles encapsulating a chemically modified ATF mRNA resulted in inhibition of ovarian cancer cell growth in nude mice accompanied with Maspin re-expression in the treated tumors. Gene expression microarrays of ATF-transduced cells revealed an exceptional specificity for the Maspin promoter. These analyses identified novel targets co-regulated with Maspin in human short-term cultures derived from ascites, such as TSPAN12, that could mediate the anti-metastatic phenotype of the ATF. Our work outlined the first targeted, non-viral delivery of ATFs into tumors with potential clinical applications for metastatic ovarian cancers.
Assuntos
Regulação Neoplásica da Expressão Gênica , Neoplasias Experimentais/metabolismo , Neoplasias Ovarianas/metabolismo , Fatores de Transcrição/biossíntese , Dedos de Zinco , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Animais , Linhagem Celular Tumoral , Feminino , Camundongos , Camundongos Transgênicos , Invasividade Neoplásica , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia , Neoplasias Experimentais/terapia , Análise de Sequência com Séries de Oligonucleotídeos , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Neoplasias Ovarianas/terapia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Mensageiro/farmacologia , Serpinas/biossíntese , Tetraspaninas/genética , Tetraspaninas/metabolismo , Fatores de Transcrição/genética , TranscriptomaRESUMO
Copper oxide nanoparticles (CuO-NPs) were functionalized with specific antibodies to target their antibacterial activity against Gram-positive or Gram-negative bacteria. The CuO-NPs were covalently functionalized to cover their surface with specific antibodies. The differently prepared CuO-NPs were characterized by X-ray diffraction, transmission electron microscopy and dynamic light scattering. The antibacterial activities of the unmodified CuO-NPs and the antibody-functionalized nanoparticles (CuO-NP-AbGram- and CuO-NP-AbGram+ ) were determined for both Gram-negative Escherichia coli and Gram-positive Bacillus subtilis bacteria. The antibody-functionalized NPs showed a differential increase of their antibacterial activity according to the specific antibody. The CuO-NP-AbGram- in E. coli showed reduced half maximal inhibitory concentration (IC50 ) and minimum inhibitory concentration (MIC) values when compared with unfunctionalized CuO-NPs. On the other hand, the CuO-NP-AbGram+ also showed reduced IC50 and MIC values in B. subtilis, when compared with non-functionalized CuO-NPs. Thus, the functionalized CuO nanoparticles with specific antibodies showed enhanced specificity of their antibacterial activity. The advantages of "smart" antibiotic nanoparticles are discussed.
Assuntos
Cobre , Nanopartículas , Escherichia coli , Anticorpos , Antibacterianos/farmacologia , ÓxidosRESUMO
Copper oxide nanoparticles (CuONPs) were synthesized using an eco-friendly method and their antimicrobial and biocompatibility properties were determined. The supernatant and extract of the fungus Ganoderma sessile yielded small, quasi-spherical NPs with an average size of 4.5 ± 1.9 nm and 5.2 ± 2.1 nm, respectively. Nanoparticles were characterized by UV-Vis spectroscopy, transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), dynamic light scattering (DLS), and zeta potential analysis. CuONPs showed antimicrobial activity against Staphylococcus aureus (S. aureus), Escherichia coli (E. coli), and Pseudomonas aeruginosa (P. aeruginosa). The half-maximal inhibitory concentration (IC50) for E. coli was 8.5 µg/mL, for P. aeruginosa was 4.1 µg/mL, and for S. aureus was 10.2 µg/mL. The ultrastructural analysis of bacteria exposed to CuONPs revealed the presence of small CuONPs all through the bacterial cells. Finally, the toxicity of CuONPs was analyzed in three mammalian cell lines: hepatocytes (AML-12), macrophages (RAW 264.7), and kidney (MDCK). Low concentrations (<15 µg/mL) of CuONPs-E were non-toxic to kidney cells and macrophages, and the hepatocytes were the most susceptible to CuONPs-S. The results obtained suggest that the CuONPs synthesized using the extract of the fungus G. sessile could be further evaluated for the treatment of superficial infectious diseases.
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Carbon quantum dots (CQDs) are a new carbon-based nanomaterial that has attracted tremendous attention due to their excellent fluorescent properties, chemical stability, water solubility, and biocompatibility features. Here, fluorescent CQDs synthesized by a green nanoarchitectonic method using Cinchona Pubescens Vahl extract were evaluated as drug nanocarriers for carboplatin (CBP) delivery. The characterization methods showed CQDs with semispherical shapes and sizes around 5 nm, temperature- and pH-dependent functional groups that interact with the CBP molecule adding specificity to the drug-delivery system. Based on the load efficiency results, it seems that the CQDs can carry almost 100 µg of carboplatin for every 1 mg of CQDs. This is possible due to the self-assembly process that takes place through the interaction between the protonation/deprotonation functional groups of CQDs and the hydrolyzed CBP molecule. Through this process, it is created spherical nanoparticles with an average size of 77.44 nm. The CQDs-CBP nanoparticles release the drug through a diffusion-controlled release mechanism where the acidic media is preferred, and the EPR effect also plays a helpful role. Besides, the viability test shows that the CQDs have almost null cytotoxicity suggesting that they could be used as a promising cancer treatment, improving the efficiency of cell internalization and significantly increasing their drug delivery.
Assuntos
Cinchona , Neoplasias , Pontos Quânticos , Nanoestruturas/química , Cinchona/química , Carbono/química , Neoplasias/terapia , Humanos , Animais , Camundongos , Linhagem Celular , Sobrevivência CelularRESUMO
Two-dimensional (2D) cell culture monolayers are commonly used for toxicological assessments of nanomaterials. Despite their facile handling, they exhibit several constraints due to their structural and complexity differences with three-dimensional (3D) in vitro cell models, such as spheroids. Here, we conducted a comparative nanotoxicological study of fibroblasts (L929) and melanoma (B16-F10) cells, grown in 2D and 3D arrangements. The cytotoxicity, reactive oxygen species (ROS) production, genotoxicity, cell morphology complexity, and uptake of silver nanoparticles (AgNPs) and folic acid-functionalized upconversion nanoparticles (FA-UCNPs) were compared in the two culture arrangements. AgNPs cytotoxicity was higher in spheroids than in monolayer cultures. Furthermore, apoptotic cell percentages and ROS production were higher in 3D than in 2D cell cultures. More importantly, 2D cultures required twice the concentration of AgNPs than the 3D cell models to reach a considerable DNA damage index (c.a. 200). Therefore, spheroids are more sensitive to the genotoxic effects of AgNPs. FA-UCNPs exerted negligible cell toxicity in 2D and 3D cell models. Moreover, AgNPs induced disaggregation and downsizing of spheroids in a facile and concentration-dependent manner. Internalization of FA-UCNPs in spheroids was 20% higher than in the 2D cell arrangements. Collectively, our findings, demonstrated that spheroids are a more sensitive model than monolayers for the assessment of nanoparticle biocompatibility and internalization.
Assuntos
Nanopartículas Metálicas , Esferoides Celulares , Técnicas de Cultura de Células/métodos , Ácido Fólico , Nanopartículas Metálicas/toxicidade , Espécies Reativas de Oxigênio , Prata/toxicidadeRESUMO
Among three-dimensional (3D) scaffold fabrication methods, porous polymers templated using high internal phase emulsions (HIPEs) have emerged as an attractive method due to the facile generation of interconnected porosity through a variety of synthetic routes. These include a bottom-up approach to selectively incorporate nanomaterials onto the inner walls in a nonaqueous environment. In this work, novel nonaqueous HIPEs made of different (meth)acrylate monomers and a deep eutectic solvent (DES) were formulated with nonfunctionalized nanohydroxyapatite (NHA), which also played the role of cosurfactant. Free radical polymerization of HIPEs yielded free-standing nanocomposites with 3D interconnected macroporosity and nonfunctionalized NHA selectively decorating the scaffolds' inner surface. The influence of different polymer functionalities, acrylate or methacrylate, their alkyl tail length, and the presence of NHA on MC3T3-E1 preosteoblast cell proliferation in vitro, reactive oxygen species (ROS) production and alkaline phosphatase (ALP) activity were evaluated. All materials presented promising biocompatibility, non-hemolytic activity, negligible inflammatory response along to remarkably enhanced cell proliferation (e.g., up to 160-fold cell proliferation increase compared with polystyrene plate) in vitro, which open the path for the development of scaffolds in regenerative medicine. It is noteworthy that polyHIPEs studied here were obtained using a green synthetic protocol where nonfunctionalized nanoparticles can be selectively incorporated into a scaffolds' inner walls. This versatile technique allows for the simple construction of 3D bioactive nanocomposite scaffolds with varied compositions for cell culture.
Assuntos
Engenharia Tecidual , Alicerces Teciduais , Proliferação de Células , Durapatita , Emulsões , Porosidade , SolventesRESUMO
Candida albicans (ATCC SC5314) was exposed to biosynthesized copper oxide nanoparticles (CuONPs) to determine their inhibitory capacity. Nanoparticles were polydisperse of small size (5.8 ± 3.5 nm) with irregular shape. The minimum inhibitory concentration (MIC) against C. albicans was 35.5 µg/mL. The production of reactive oxygen species (ROS) of C. albicans was verified when exposed to different concentrations of CuONPs. Ultrastructural analysis of C. albicans revealed a high concentration of CuONPs in the cytoplasm and outside the cell; also, nanoparticles were detected within the cell wall. Cytotoxic analyses using fibroblasts (L929), macrophages (RAW 264.7), and breast (MCF-12) cell lines show good results of cell viability when exposed at the MIC. Additionally, a hemocompatibility analysis was carried out and was found to be below 5%, considered the threshold for biocompatibility. Therefore, it is concluded that the biosynthesized CuONPs have a high potential for developing a topical antifungal treatment.
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Silver nanoparticles (AgNPs) represent an excellent option to solve microbial resistance problems to traditionally used antibiotics. In this work, we report optimized protocols for the production of AgNPs using extracts and supernatants of Trichoderma harzianum and Ganoderma sessile. AgNPs were characterized using UV-Vis spectroscopy and transmission electron microscopy, and the hydrodynamic diameter and Z potential were also determined. The obtained AgNPs were slightly larger using the fungal extract, and in all cases, a quasi-spherical shape was obtained. The mean sizes of AgNPs were 9.6 and 19.1 nm for T. harzianum and 5.4 and 8.9 nm for G. sessile using supernatant and extract, respectively. The AgNPs were evaluated to determine their in vitro antibacterial effect against Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus. The minimum inhibitory concentration (MIC) was determined, and in all cases the AgNPs showed an antimicrobial effect, with a MIC varying from 1.26-5.0 µg/mL, depending on the bacterial strain and type of nanoparticle used. Cytotoxicity analyses of AgNPs were carried out using macrophages and fibroblast cell lines. It was determined that the cell viability of fibroblasts exposed for 24 h to different concentrations of AgNPs was more than 50%, even at concentrations of up to 20 µg/mL of silver. However, macrophages were more susceptible to exposure at higher concentrations of AgNPs as their viability decreased at concentrations of 10 µg/mL. The results presented here demonstrate that small AgNPs are obtained using either supernatants or extracts of both fungal strains. A remarkable result is that very low concentrations of AgNPs were necessary for bacterial inhibition. Furthermore, AgNPs were stable for more than a year, preserving their antibacterial properties. Therefore, the reported optimized protocol using fungal supernatants or extracts may be used as a fast method for synthesizing small AgNPs with high potential to use in the clinic.
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Gaucher disease is a genetic disorder and the most common lysosomal disease caused by the deficiency of enzyme ß-glucocerebrosidase (GCase). Although enzyme replacement therapy (ERT) is successfully applied using mannose-exposed conjugated glucocerebrosidase, the lower stability of the enzyme in blood demands periodic intravenous administration that adds to the high cost of treatment. In this work, the enzyme ß-glucocerebrosidase was encapsulated inside virus-like nanoparticles (VLPs) from brome mosaic virus (BMV), and their surface was functionalized with mannose groups for targeting to macrophages. The VLP nanoreactors showed significant GCase catalytic activity. Moreover, the Michaelis-Menten constants for the free GCase enzyme (KM =0.29â mM) and the functionalized nanoreactors (KM =0.32â mM) were similar even after chemical modification. Importantly, the stability of enzymes under physiological conditions (pHâ 7.4, 37 °C) was enhanced by ≈11-fold after encapsulation; this is beneficial for obtaining a higher blood circulation half-life, which may decrease the cost of therapy by reducing the requirement of multiple intravenous injections. Finally, the mannose receptor targeted enzymatic nanoreactors showed enhanced internalization into macrophage cells. Thus, the catalytic activity and cell targeting suggest the potential of these nanoreactors in ERT of Gaucher's disease.
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Doença de Gaucher , Terapia de Reposição de Enzimas , Doença de Gaucher/tratamento farmacológico , Doença de Gaucher/genética , Glucosilceramidase/genética , Humanos , Manose , NanotecnologiaRESUMO
Enzymatic nanoreactors were obtained by galactose-1-phosphate uridylyl-transferase (GALT) encapsulation into plant virus capsids by a molecular self-assembly strategy. The aim of this work was to produce virus-like nanoparticles containing GALT for an enzyme-replacement therapy for classic galactosemia. The encapsulation efficiency and the catalytic constants of bio-nanoreactors were determined by using different GALT and virus coat protein ratios. The substrate affinity of nanoreactors was slightly lower than that of the free enzyme; the activity rate was 16 % of the GALT free enzyme. The enzymatic nanoreactors without functionalization were internalized into different cell lines including fibroblast and kidney cells, but especially into hepatocytes. The enzymatic nanoreactors are an innovative enzyme preparation with potential use for the treatment of classic galactosemia.
Assuntos
Bromovirus/metabolismo , Proteínas do Capsídeo/química , Composição de Medicamentos/métodos , UTP-Hexose-1-Fosfato Uridililtransferase/química , Animais , Proteínas do Capsídeo/isolamento & purificação , Linhagem Celular , Endocitose , Fluoresceína-5-Isotiocianato/química , Galactosemias/tratamento farmacológico , Galactosemias/patologia , Humanos , Cinética , Camundongos , Nanotecnologia , UTP-Hexose-1-Fosfato Uridililtransferase/metabolismo , UTP-Hexose-1-Fosfato Uridililtransferase/uso terapêuticoRESUMO
Luminescent lanthanide downconversion nanoparticles (DCNPs) provide a combination of high luminescence intensity, sharp emission peaks with narrow bandwidth and a large Stokes' shift, leading to high-performance biomedical applications mainly for imaging. The purpose of this study is to present a nanotoxicological study of DCNPs Y2 O3 codoped with Eu3+ and functionalized with folic acid (FA). These assessments include cytotoxicity, genotoxicity, hemocompatibility, and in vitro inflammatory studies. We demonstrated by flow cytometry and confocal microscope the internalization of FA-DCNPs in breast cancer and melanoma cells. They were synthesized by sol-gel method and coated with a thin silica shell to make them biocompatible; also they were functionalized with amino groups and FA ligands that bind to the folate receptors (FR) located on the surface of the cancer cells studied. This functionalization enables the DCNPs to be internalized into the cancer cells via endocytosis by the conjugation FA-FR. The DCNPs were characterized with transmission electron microscope, Fourier transform infrared spectroscopy and photoluminescence. The nanotoxicological assessments demonstrated that both nanoparticles (bare and functionalized) are no cytotoxic and no genotoxic at the tested concentrations (0.01-20 µg/mL) in three cell lines (breast, skin cancer, and osteoblasts). Also they are hemocompatible and do not exert nitric oxide production in vitro by macrophages. The FA-DCNPs were clearly localized into the cell cytoplasm with bright red luminescence. Thus, herein we present a complete nanotoxicological study of FA-DCNPs Y2 O3 codoped with Eu3+ and we conclude that these nanoparticles are biocompatible and can be further used for cancer cells bioimaging.
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Óxido de Alumínio/toxicidade , Diagnóstico por Imagem/métodos , Európio/química , Ácido Fólico/química , Substâncias Luminescentes/química , Nanopartículas/toxicidade , Nanoestruturas/toxicidade , Neoplasias/patologia , Animais , Materiais Biocompatíveis , Testes de Carcinogenicidade , Linhagem Celular Tumoral , Sobrevivência Celular , Receptor 1 de Folato/metabolismo , Humanos , Macrófagos/efeitos dos fármacos , Camundongos , Testes de Mutagenicidade , Óxido Nítrico/metabolismo , Células RAW 264.7 , Dióxido de Silício/toxicidade , Radioisótopos de Ítrio/toxicidadeRESUMO
The one-pot cascade reaction of naturally occurring enzymes is exciting for highly selective complex reaction and biodegradable approaches. Tamoxifen is the main drug against breast cancer for decades and induces an anticancerous effect upon metabolic activation by cytochrome P450 (CYP450). Herein, bi-enzymatic nanoreactors (NRs) are developed as a multimodality platform for smart action against breast tumors. CYPBM3 of Bacillus magaterium (CYP) is co-confined with glucose oxidase (GOx) where GOx produces H2O2 in the presence of glucose that elicits the CYP-mediated transformation of tamoxifen. The scintillating and mesoporous LaF3:Tb as nanocarrier showed advantages like a wide range of pore size and positive surface charge for efficient loading of enzyme couple, while the smallest pores were available for substrate/product diffusion. The obtained NRs were camouflaged with human serum albumin (HSA) to overcome premature enzyme leaching and provide active stealth properties. The nanocomposite was characterized for physicochemical properties and glucose-mediated sequential catalysis. The in vitro studies demonstrated the cell internalization of NRs in both ER+ and triple-negative breast cancer cell lines and showed significant cytotoxicity. The developed NRs not only improve the outcomes of endocrine therapy in ER+ cells but also synergistically act with oxidation therapy for enhanced therapeutic effect. Importantly, inhibition of triple-negative cells was also achieved. Thus, the development of the new multimodal nanomedicine of the present work should afford new tools towards the theranosis of breast cancer with minimized adverse effects.
Assuntos
Neoplasias da Mama , Neoplasias de Mama Triplo Negativas , Bacillus , Neoplasias da Mama/tratamento farmacológico , Catálise , Sistema Enzimático do Citocromo P-450 , Feminino , Humanos , Peróxido de HidrogênioRESUMO
Skin burns and ulcers are considered hard-to-heal wounds due to their high infection risk. For this reason, designing new options for wound dressings is a growing need. The objective of this work is to investigate the properties of poly (ε-caprolactone)/poly (vinyl-pyrrolidone) (PCL/PVP) microfibers produced via electrospinning along with sorbents loaded with Argovit™ silver nanoparticles (Ag-Si/Al2O3) as constituent components for composite wound dressings. The physicochemical properties of the fibers and sorbents were characterized using scanning electron microscopy (SEM), differential scanning calorimetry (DSC), Fourier transform infrared spectroscopy (FTIR) and inductively coupled plasma optical emission spectroscopy (ICP-OES). The mechanical properties of the fibers were also evaluated. The results of this work showed that the tested fibrous scaffolds have melting temperatures suitable for wound dressings design (58-60 °C). In addition, they demonstrated to be stable even after seven days in physiological solution, showing no macroscopic damage due to PVP release at the microscopic scale. Pelletized sorbents with the higher particle size demonstrated to have the best water uptake capabilities. Both, fibers and sorbents showed antimicrobial activity against Gram-negative bacteria Pseudomona aeruginosa and Escherichia coli, Gram-positive Staphylococcus aureus and the fungus Candida albicans. The best physicochemical properties were obtained with a scaffold produced with a PCL/PVP ratio of 85:15, this polymeric scaffold demonstrated the most antimicrobial activity without affecting the cell viability of human fibroblast. Pelletized Ag/Si-Al2O3-3 sorbent possessed the best water uptake capability and the higher antimicrobial activity, over time between all the sorbents tested. The combination of PCL/PVP 85:15 microfibers with the chosen Ag/Si-Al2O3-3 sorbent will be used in the following work for creation of wound dressings possessing exudate retention, biocompatibility and antimicrobial activity.