RESUMEN
With an ever-increasing burden of vision loss caused by diseases of the posterior ocular segment, there is an unmet clinical need for non-invasive treatment strategies. Topical drug application using eye drops suffers from low to negligible bioavailability to the posterior segment as a result of static and dynamic defensive ocular barriers to penetration, while invasive delivery systems are expensive to administer and suffer potentially severe complications. As the cornea is the main anatomical barrier to uptake of topically applied drugs from the ocular surface, we present an approach to increase corneal permeability of a corticosteroid, dexamethasone sodium-phosphate (DSP), using a novel penetration enhancing agent (PEA). We synthesised a novel polyacetylene (pAc) polymer and compared its activity to two previously described cell penetrating peptide (CPP) based PEAs, TAT and penetratin, with respect to increasing transcorneal permeability of DSP in a rapid ex-vivo porcine corneal assay over 60 min. The transcorneal apparent permeability coefficients (Papp) for diffusion of pAc, and fluorescein isothiocyanate (FITC) conjugated TAT and penetratin were up to 5 times higher (p < 0.001), when compared to controls. When pAc was used in formulation with DSP, an almost 5-fold significant increase was observed in Papp of DSP across the cornea (p = 0.0130), a significant 6-fold increase with TAT (p = 0.0377), and almost 7-fold mean increase with penetratin (p = 0.9540). Furthermore, we investigated whether the PEAs caused any irreversible damage to the barrier integrity of the corneal epithelium by measuring transepithelial electrical resistance (TEER) and immunostaining of tight junction proteins using zonula occludens-1 (ZO-1) and occludin antibodies. There was no damage or structural toxicity, and the barrier integrity was preserved after PEA application. Finally, an in-vitro cytotoxicity assessment of all PEAs in human retinal pigment epithelium cells (ARPE-19) demonstrated that all PEAs were very well-tolerated, with IC50 values of 64.79 mM for pAc and 1335.45 µM and 87.26 µM for TAT and penetratin, respectively. Our results suggest that this drug delivery technology could potentially be used to achieve a significantly higher intraocular therapeutic bioavailability after topical eye drop administration, than currently afforded.
Asunto(s)
Péptidos de Penetración Celular , Córnea , Dexametasona , Sistemas de Liberación de Medicamentos , Permeabilidad , Animales , Dexametasona/administración & dosificación , Dexametasona/farmacocinética , Dexametasona/análogos & derivados , Porcinos , Córnea/metabolismo , Córnea/efectos de los fármacos , Péptidos de Penetración Celular/administración & dosificación , Sistemas de Liberación de Medicamentos/métodos , Humanos , Retina/metabolismo , Retina/efectos de los fármacos , Línea Celular , Productos del Gen tat/administración & dosificación , Productos del Gen tat/química , Administración Oftálmica , Administración Tópica , Soluciones Oftálmicas/administración & dosificación , Proteínas Portadoras/metabolismo , Polímeros/químicaRESUMEN
Murine cytomegalovirus protein M45 contains a RIP homotypic interaction motif (RHIM) that is sufficient to confer protection of infected cells against necroptotic cell death. Mechanistically, the N-terminal region of M45 drives rapid self-assembly into homo-oligomeric amyloid fibrils, and interacts with the endogenous RHIM domains of receptor-interacting serine/threonine protein kinases (RIPK) 1, RIPK3, Z-DNA-binding protein 1, and Toll/interleukin-1 receptor domain-containing adaptor-inducing interferon-ß. Remarkably, all four aforementioned mammalian proteins harbouring such a RHIM domain are key components of inflammatory signalling and regulated cell death (RCD) processes. Immunogenic cell death by regulated necrosis causes extensive tissue damage in a wide range of diseases, including ischaemia reperfusion injury, myocardial infarction, sepsis, stroke, and solid organ transplantation. To harness the cell death suppression properties of M45 protein in a therapeutically usable manner, we developed a synthetic peptide encompassing only the RHIM domain of M45. To trigger delivery of RHIM into target cells, we fused the transactivator protein transduction domain of human immunodeficiency virus 1 to the N-terminus of the peptide. The fused peptide could efficiently penetrate eukaryotic cells, but unexpectedly it eradicated or destroyed all tested cancer cell lines and primary cells irrespective of species without further stimulus through a necrosis-like cell death. Typical inhibitors of different forms of RCD cannot impede this process, which appears to involve a direct disruption of biomembranes. Nevertheless, our finding has potential clinical relevance; reliable induction of a necrotic form of cell death distinct from all known forms of RCD may offer a novel therapeutic approach to combat resistant tumour cells.
Asunto(s)
Productos del Gen tat/química , Productos del Gen tat/metabolismo , Dominios Proteicos , Proteínas Recombinantes de Fusión/metabolismo , Ribonucleótido Reductasas/química , Ribonucleótido Reductasas/metabolismo , Transducción de Señal/genética , Proteínas Virales/química , Proteínas Virales/metabolismo , Secuencia de Aminoácidos , Amiloide/metabolismo , Animales , Productos del Gen tat/genética , VIH-1/química , Células HT29 , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Células 3T3 NIH , Necroptosis/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Ribonucleótido Reductasas/genética , Células U937 , Proteínas Virales/genéticaRESUMEN
Radiodynamic therapy (RDT) is a recent extension of conventional photodynamic therapy, in which visible/near infrared light irradiation is replaced by a well-tolerated dose of high-energy X-rays. This enables greater tissue penetration to allow non-invasive treatment of large, deep-seated tumors. We report here the design and testing of a drug delivery system for RDT that is intended to enhance intra- or peri-nuclear localization of the photosensitizer, leading to DNA damage and resulting clonogenic cell kill. This comprises a photosensitizer (Verteporfin, VP) incorporated into poly (lactic-co-glycolic acid) nanoparticles (PLGA NPs) that are surface-functionalized with a cell-penetrating HIV trans-activator of transcription (TAT) peptide. In addition to a series of physical and photophysical characterization studies, cytotoxicity tests in pancreatic (PANC-1) cancer cells in vitro under 4 Gy X-ray exposure from a clinical 6 MV linear accelerator (LINAC) showed that TAT targeting of the nanoparticles markedly enhances the effectiveness of RDT treatment, particularly when assessed by a clonogenic, i.e., DNA damage-mediated, cell kill.
Asunto(s)
Composición de Medicamentos , Productos del Gen tat/química , Nanopartículas/química , Copolímero de Ácido Poliláctico-Ácido Poliglicólico/química , Verteporfina/farmacología , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Células Clonales , ADN/metabolismo , Endocitosis/efectos de los fármacos , Humanos , Lípidos de la Membrana/metabolismo , Nanopartículas/ultraestructura , Oxígeno Singlete/metabolismoRESUMEN
OBJECTIVE: Intra-articular drug delivery holds great promise for the treatment of joint diseases such as osteoarthritis. The objective of this study was to evaluate the TAT peptide transduction domain (TAT-PTD) as a potential intra-articular drug delivery technology for synovial joints. DESIGN: Experiments examined the ability of TAT conjugates to associate with primary chondrocytes and alter cellular function both in vitro and in vivo. Further experiments examined the ability of the TAT-PTD to bind to human osteoarthritic cartilage. RESULTS: The results show that the TAT-PTD associates with chondrocytes, is capable of delivering siRNA for chondrocyte gene knockdown, and that the recombinant enzyme TAT-Cre is capable of inducing in vivo genetic recombination within the knee joint in a reporter mouse model. Last, binding studies show that osteoarthritic cartilage preferentially uptakes the TAT-PTD from solution. CONCLUSIONS: The results suggest that the TAT-PTD is a promising delivery strategy for intra-articular therapeutics.
Asunto(s)
Productos del Gen tat , Osteoartritis , Animales , Cartílago/metabolismo , Condrocitos/metabolismo , Productos del Gen tat/química , Productos del Gen tat/metabolismo , Ratones , Osteoartritis/metabolismo , TecnologíaRESUMEN
Nanotechnology is a fast-growing research technology. Nanoparticles have intensive scientific applications in many fields. Depending on the physical and chemical characteristics of a nanoparticle, it can be used either as a treatment agent to fight disease or as a delivery vehicle to transport the therapeutic drug to a specified biological organ, tissue, and cell. Cytotoxicity evaluation of nanoparticles is one of the primary concerns in clinical practices to avoid unpredicted or undesirable interactions that could worsen the case. Iron oxide nanoparticle (IONP) is the most utilized nanoparticle in medical fields for treatment, diagnostic, and imaging. This paper is designated to investigate the cytotoxicity of IONPs that decorated with Trans-Activator of Transcription (TAT) protein. WST-1 assay and flow cytometry were used to assess the cytotoxicity of TAT-IONPs, which showed no significant cytotoxic effect on mammalian breast cancer cells (MCF-7). Nanoparticles accumulation in the cell's cytoplasm was evaluated from TEM images by measuring the size of the endosome. The results indicate that TAT-IONPs can be used as a safe and non-toxic nanoplatform for targeted delivery at 50 µg/ml or less. Also, they present an approach by which the area of intracellular endosome can be assessed from the TEM images of fixed cells. In this study, the endosome size increased in a time-dependent manner.
Asunto(s)
Supervivencia Celular/efectos de los fármacos , Productos del Gen tat/química , Nanopartículas Magnéticas de Óxido de Hierro , Humanos , Células MCF-7 , Nanopartículas Magnéticas de Óxido de Hierro/química , Nanopartículas Magnéticas de Óxido de Hierro/toxicidad , Sales de TetrazolioRESUMEN
Hyperpigmentation disorders negatively influence an individual's quality of life and may cause emotional distress. Over the years, various melanogenesis inhibitors (mainly tyrosinase inhibitors) have been developed, most of which with low efficacy or high toxicity. Although metabolic engineering by deviation in the flux of substrate is of considerable interest, trials to develop a melanogenesis inhibitor based on L-tyrosine (L-Tyr) restriction are missing. We propose a novel proteinaceous melanogenesis inhibitor called tyrosine ammonia-lyase (TAL), an enzyme that catalyzes the conversion of L-Tyr to p-coumaric acid and ammonia. Since the cell membrane can act as a barrier for intracellular protein delivery, we have covalently conjugated a recombinant TAL enzyme from Rhodobacter sphaeroides (RsTAL) to a trans-activator of transcription (TAT) cell-penetrating peptide (CPP) to afford the intracellular delivery. The heterologously expressed TAT-RsTAL fusion protein was delivered successfully into B16F10 melanocytes as confirmed by the direct fluorescence microscopy with increased intensity from 30 to 180 min. TAT-RsTAL showed sufficient intracellular activity of about 0.83 ± 0.04 and 0.34 ± 0.03 nmolâ¢mg-1 â¢s-1 for the native and inclusion body-extracted conjugates, respectively. The conjugate inhibited melanin biosynthesis in B16F10 cells in a time-dependent manner. Melanin accumulation was inhibited by 12.7 ± 6.2%, 28.2 ± 5.7%, and 33.9 ± 2.9% compared to the nontreated control groups after 24, 48, and 72 hr of incubation, respectively. L-Tyr restriction had no significant effect on the cell viability up to a concentration of 100 µgml-1 even after 72 hr. According to the observed hypopigmentary effect of the conjugate in this study, TAT-RsTAL can be suggested as a melanogenesis inhibitor for further investigations.
Asunto(s)
Amoníaco-Liasas/metabolismo , Péptidos de Penetración Celular/farmacología , Productos del Gen tat/metabolismo , Melaninas/metabolismo , Melanoma Experimental/tratamiento farmacológico , Animales , Supervivencia Celular/efectos de los fármacos , Péptidos de Penetración Celular/química , Sistemas de Liberación de Medicamentos , Productos del Gen tat/química , Melanocitos/efectos de los fármacos , Melanocitos/metabolismo , Melanocitos/patología , Melanoma Experimental/metabolismo , Melanoma Experimental/patología , Ratones , Rhodobacter sphaeroides/enzimología , Tirosina/metabolismoRESUMEN
Immunotherapy methods using potential tumor microenvironment modulators have elicited durable therapeutic responses in cancer treatment. Immune checkpoint molecule programmed cell death-ligand 1 (PD-L1) and oncogenic transcription factor STAT3 (signal transducer and activator of transcription-3) assigned as inhibitory targets of our study and particular delivery system designed to deliver small interfering RNAs (siRNAs) to silence the targeted genes. Generated trimethyl chitosan (TMC) and thiolated chitosan (TC) nanoparticles (NPs) conjugated with HIV-1-derived TAT peptide and HA (hyaluronic acid) exhibited eligible physicochemical characteristics, notable siRNA encapsulation, serum stability, non-toxicity, controlled siRNA release, and extensive cellular uptake by cancer cells. Dual inhibition with STAT3/PD-L1 siRNA-loaded HA-TAT-TMC-TC NPs led to promising results, including significant downregulation of PD-L1 and STAT3 genes, striking suppressive effects on proliferation, migration, and angiogenesis of breast and melanoma cancer cell lines, and restrained tumor growth in vivo. These findings infer the capability of HA-TAT-TMC-TC NPs containing STAT3/PD-L1 siRNAs as a novel tumor-suppressive candidate in cancer treatment.
Asunto(s)
Antígeno B7-H1/antagonistas & inhibidores , Neoplasias de la Mama/terapia , Melanoma Experimental/terapia , Nanopartículas/administración & dosificación , Nanopartículas/química , ARN Interferente Pequeño/genética , Factor de Transcripción STAT3/antagonistas & inhibidores , Animales , Apoptosis , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Movimiento Celular , Proliferación Celular , Quitosano/química , Progresión de la Enfermedad , Femenino , Productos del Gen tat/química , Humanos , Ácido Hialurónico/química , Melanoma Experimental/genética , Melanoma Experimental/patología , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Inflammation, mitochondrial dysfunction and oxidative stress are closely associated with neurological diseases. In this study, Mn-TAT PTD-Ngb, a novel artificial recombinant protein, exerted inhibitory effects on the inflammatory response and inflammasome activation. During the lipopolysaccharide (LPS)-induced inflammatory response, Mn-TAT PTD-Ngb suppressed the nuclear translocation of nuclear factor kappa B (NF-κB) and the release of proinflammatory cytokines and attenuated the phosphorylation of mitogen-activated protein kinase (MAPK). Furthermore, the recombinant protein blocked reactive oxygen species (ROS) production, abated mitochondrial dysfunction and significantly suppressed the assembly of the inflammasome, which led to the overproduction of proinflammatory cytokines IL-1ß and IL-18. Mn-TAT PTD-Ngb increased the level of nuclear factor-erythroid 2 -related factor 2 (Nrf2), which protected against oxidative stress and improved pyroptosis. Mn-TAT PTD-Ngb might be a promising drug for curing neurological diseases.
Asunto(s)
Antioxidantes/metabolismo , Mediadores de Inflamación/metabolismo , Mitocondrias/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Estrés Oxidativo/efectos de los fármacos , Proteínas Recombinantes/administración & dosificación , Animales , Línea Celular , Productos del Gen tat/administración & dosificación , Productos del Gen tat/química , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Mediadores de Inflamación/antagonistas & inhibidores , Manganeso/administración & dosificación , Manganeso/química , Ratones , Mitocondrias/efectos de los fármacos , Estrés Oxidativo/fisiología , Proteínas Recombinantes/química , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiologíaRESUMEN
The paper aims to investigate the cytotoxic effect on tumor cells of irradiated AuNPs in green light and subsequently functionalized with HS-PEG-NH2. The toxicity level of gold conjugates after their functionalization with DOX and TAT peptide was also evaluated. The AuNPs were prepared using the modified Turkevich method and exposed to visible light at a wavelength of 520 nm prior their PEGylation. The optical properties were analyzed by UV-vis spectroscopy, the surface modification was investigated using FTIR and XPS spectroscopies and their sizes and morphologies were evaluated by TEM and DLS techniques. DOX and TAT peptide were linked to the surface of PEGylated AuNPs by reacting their amino groups with glycidyloxypropyl of PEGylated DOX or TAT conjugates under mild conditions at room temperature and in the presence of ethanol as catalyst. The conjugates containing DOX or DOX and TAT have been characterized by fluorescence and FTIR techniques. The changes of electrochemical features were observed using cyclic voltammetry, suggesting a better stability of irradiated nanoparticles. By mass spectrometry it was confirmed that the compounds of interest were obtained. The cell viability test showed that irradiated and non-irradiated nanoparticles coated with PEG are not toxic in normal cells. Tumor cell viability analysis showed that the PEGylated nanoparticles modified with DOX and TAT peptide were more effective than pristine DOX, indicating cytotoxicity up to 10% higher than non-irradiated ones.
Asunto(s)
Doxorrubicina/farmacología , Productos del Gen tat/farmacología , Nanopartículas del Metal/química , Osteosarcoma/tratamiento farmacológico , Antineoplásicos/química , Antineoplásicos/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Doxorrubicina/química , Productos del Gen tat/química , Oro/química , Humanos , Osteosarcoma/genética , Osteosarcoma/patología , Péptidos/química , Péptidos/farmacología , Polietilenglicoles/química , Polietilenglicoles/farmacología , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
On account of their excellent capacity to significantly improve the bioavailability and solubility of chemotherapy drugs, amphiphilic block copolymer-based micelles have been widely utilized for chemotherapy drug delivery. In order to further improve the antitumor ability and to also reduce undesired side effects of drugs, cell-penetrating peptides have been used to functionalize the surface of polymer micelles endowed with the ability to target tumor tissues. Herein, we first synthesized functional polyethylene glycol-polylactic acid (PEG-PLA) tethered with maleimide at the PEG section of the block polymer, which was further conjugated with a specific peptide, the transactivating transcriptional activator (TAT), with an approved capacity of aiding translocation across the plasma membrane. Then, TAT-conjugated, paclitaxel-loaded nanoparticles were self-assembled into stable nanoparticles with a favorable size of 20 nm, and displayed a significantly increased cytotoxicity, due to their enhanced accumulation via peptide-mediated cellular association in human breast cancer cells (MCF-7) in vitro. But when further used in vivo, TAT-NP-PTX showed an acceleration of the drug's plasma clearance rate compared with NP-PTX, and therefore weakened its antitumor activities in the mice model, because of its positive charge, its elimination by the endoplasmic reticulum system more quickly, and its targeting effect on normal cells leading towards being more toxic. So further modification of TAT-NP-PTX to shield TAT peptide's positive charges may be a hot topic to overcome the present dilemma.
Asunto(s)
Péptidos de Penetración Celular/administración & dosificación , Péptidos de Penetración Celular/química , Paclitaxel/administración & dosificación , Poliésteres/química , Polietilenglicoles/química , Animales , Antineoplásicos Fitogénicos/administración & dosificación , Antineoplásicos Fitogénicos/química , Línea Celular Tumoral , Membrana Celular/metabolismo , Portadores de Fármacos/química , Retículo Endoplásmico/metabolismo , Femenino , Productos del Gen tat/química , Humanos , Células MCF-7 , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos ICR , Micelas , Nanopartículas/química , Tamaño de la Partícula , Polímeros/químicaRESUMEN
HIV transactivator of transcription (Tat) protein could interact with amyloid ß (Aß) peptide which cause the growth of Aß plaques in the brain and result in Alzheimer's disease in HIV-infected patients. Herein, we employ high-resolution atomic force microscopy and quantitative nanomechanical mapping to investigate the effects of Tat protein in Aß peptide aggregation. Our results demonstrate that the Tat protein could bind to the Aß fibril surfaces and result in the formation of Tat-Aß multifibrillar structures. The resultant Tat-Aß multifibrillar aggregates represent an increase in stiffness compared with Aß fibrils due to the increase in ß-sheet formation. The identification and characterization of the Tat-Aß intermediate aggregates is important to understanding the interactions between Tat protein and Aß peptide, and the development of novel therapeutic strategy for Alzheimer's disease-like disorder in HIV infected individuals.
Asunto(s)
Enfermedad de Alzheimer/fisiopatología , Péptidos beta-Amiloides/química , Amiloide/química , Productos del Gen tat/química , Microscopía de Fuerza Atómica/métodos , Placa Amiloide/química , Péptidos beta-Amiloides/análisis , Productos del Gen tat/metabolismo , Humanos , Placa Amiloide/metabolismoRESUMEN
Ischemic stroke caused by a thrombus clog and ischemia is one of the most lethal and disabling cerebrovascular diseases. A sequentially targeted delivery system is highly desired to deliver thrombolytics and neuroprotectant to the site of the thrombus and ischemic penumbra, respectively, to pursue a maximized combinational effect. Inspired by the vital roles that platelets play in thrombus formation, herein, we develop a bioengineered "nanoplatelet" (tP-NP-rtPA/ZL006e) for sequentially site-specific delivery of recombinant tissue plasminogen activator (rtPA) and neuroprotectant (ZL006e) for ischemic stroke treatment. The tP-NP-rtPA/ZL006e consists of a ZL006e-loaded dextran derivative polymeric nanoparticle core and platelet membrane shell conjugated with thrombin-cleavable Tat-peptide-coupled rtPA. Mediated by the cloak of the platelet membrane, tP-NP-rtPA/ZL006e targets the thrombus site and rtPA is triggered to release by the upregulated thrombin. Subsequently, the in situ exposed Tat peptide enhanced penetration of the "nanoplatelet" across the blood-brain barrier into ischemic brain for ZL006e site-specific delivery. From the in vitro and in vivo evaluation, tP-NP-rtPA/ZL006e is demonstrated to significantly enhance the anti-ischemic stroke efficacy in the rat model with middle cerebral artery occlusion, showing a 63 and 72% decrease in ischemic area and reactive oxygen species level compared to that with free drug combination, respectively.
Asunto(s)
Plaquetas/química , Isquemia Encefálica/tratamiento farmacológico , Nanopartículas/química , Accidente Cerebrovascular/tratamiento farmacológico , Animales , Barrera Hematoencefálica/efectos de los fármacos , Isquemia Encefálica/patología , Dextranos/química , Dextranos/farmacología , Fibrinolíticos/química , Fibrinolíticos/farmacología , Productos del Gen tat/química , Productos del Gen tat/farmacología , Humanos , Masculino , Nanopartículas/uso terapéutico , Fármacos Neuroprotectores/química , Fármacos Neuroprotectores/farmacología , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacología , Accidente Cerebrovascular/patología , Trombina/química , Trombina/farmacología , Activador de Tejido Plasminógeno/química , Activador de Tejido Plasminógeno/farmacologíaRESUMEN
The visualization of the long noncoding RNA of prostate cancer gene 3 (lncRNA PCA3), a specific biomarker for androgen receptor-positive prostate cancer, in living cells not only directly reflects the gene expression and localization but also offers better insight into its roles in the pathological processes. Here, we loaded an entropy-driven RNA explorer (EDRE) on the TAT peptide-functionalized titanium carbide MXenes (Ti3C2-TAT) for the imaging of nuclear lncRNA PCA3 in live cells. The EDRE was condensed on the Ti3C2-TAT (Ti3C2-TAT@EDRE) by electrostatic interaction. Ti3C2-TAT@EDRE enables the entering of cells and release of TAT peptides and EDRE in the cytoplasm by the glutathione (GSH)-triggered cleavage of the disulfide bonds in Ti3C2-TAT. The released EDRE is delivered into the nucleus by the nucleus-targeted guidance of TAT peptides, and initiated by the target lncRNA PCA3, subsequently leading to the continuous accumulation of fluorescence signals. Consequently, fluorescence analysis of lncRNA PCA3 at low-picomolar concentrations in vitro as well as sensitive live cell imaging of lncRNA PCA3 in the nucleus of androgen receptor-positive LNCaP prostate cancer cells were achieved, providing a versatile strategy for the monitoring of nucleic acid biomarkers in the nucleus of living cells.
Asunto(s)
Antígenos de Neoplasias/genética , Biomarcadores de Tumor/análisis , Imagen Molecular/métodos , Sondas Moleculares/química , Neoplasias de la Próstata/genética , ARN Largo no Codificante/genética , Titanio/química , Antígenos de Neoplasias/análisis , Antígenos de Neoplasias/química , Entropía , Productos del Gen tat/química , Humanos , Masculino , Nanoestructuras/química , Fragmentos de Péptidos/química , Neoplasias de la Próstata/diagnóstico por imagen , Neoplasias de la Próstata/patología , ARN Largo no Codificante/análisis , ARN Largo no Codificante/química , Células Tumorales CultivadasRESUMEN
The present study investigated combined biological effects of peptide and miRNA in a peptide/miRNA nanocomplex. We utilized TatBim peptide as a cell-penetrating peptide-based RNA carrier with apoptotic activity. miRNA with apoptotic activity (miR-34a) was used for complex formation to investigate the additional effects of the combination with TatBim peptide. TatBim peptide and the miRNA formed nanocomplexes (approximately 250 nm in diameter), and these complexes were efficiently internalized by cells. Despite its efficient cell internalization, apoptotic activity of the nanocomplex decreased with increasing RNA content. However, photosensitizer-attachment to TatBim and photoirradiation significantly improved the apoptotic activity of the nanocomplex by facilitating dispersion of the peptide and RNA in the cytoplasm. Combined apoptotic activity of both TatBim peptide and miR-34a in the nanocomplex was demonstrated by substituting TatBim with Lipofectamine and by substituting miR-34a with scrambled siRNA.
Asunto(s)
Apoptosis/efectos de los fármacos , Péptidos de Penetración Celular/química , Péptidos de Penetración Celular/farmacología , MicroARNs/química , MicroARNs/farmacología , Apoptosis/genética , Proteína 11 Similar a Bcl2/química , Línea Celular Tumoral , Portadores de Fármacos/farmacología , Ensayos de Selección de Medicamentos Antitumorales , Productos del Gen tat/química , Técnicas de Transferencia de Gen , Células HeLa , Humanos , Lípidos/química , Lípidos/farmacología , MicroARNs/administración & dosificación , MicroARNs/genética , Nanocompuestos/química , Fármacos Fotosensibilizantes/síntesis química , Fármacos Fotosensibilizantes/química , Fármacos Fotosensibilizantes/farmacología , Interferencia de ARN , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/química , ARN Interferente Pequeño/farmacología , Rayos XRESUMEN
Abrin is a cytotoxic protein isolated from seeds of leguminous plants and has become a potential bioterrorism weapon for its high toxity and difficult detection. In the early stage of poisoning, Arbin can damage cells and induce apoptosis. Raman spectroscopy is a molecular fingerprint that can identify and compare various intracellular substances. In this work, thiolated polyethylene glycol (mPEG-SH) and cell-penetrating peptide (TAT) modified 70-80â¯nm gold nanostars (AuNSs) have been developed as label-free Raman enhancement substrates to realize real-time and in-situ monitoring of toxin-induced adherent cell apoptosis. The changes for the surface-enhanced Raman scattering (SERS) spectra of cells before and after the damage (0â¯h, 2â¯h, 4â¯h, 8â¯h, 12â¯h and 24â¯h) of Abrin can be characterized via SERS spectroscopy. The intracellular substances at different time can be compared by using differential spectrum analysis and the cells in different states can be identified and distinguished by means of principal component analysis (PCA). The abundant spectral features in SERS spectra can also reveal the molecular dynamics during apoptosis. Results show that SERS spectroscopy provides a platform for in-situ monitoring of substance changes in adherent cells except detecting cell apoptosis induced by toxin in real-time, which achieves a more detailed and comprehensive understanding of the pathogenesis of toxins in molecular biology and provides a new idea for toxicology experiments.
Asunto(s)
Abrina/toxicidad , Apoptosis/efectos de los fármacos , Productos del Gen tat/química , Oro/química , Células Hep G2 , Humanos , Nanoestructuras/química , Polietilenglicoles/química , Espectrometría RamanRESUMEN
A prominent hurdle in developing small molecule probes against RNA is the relative scarcity of general screening methods. In this study, we demonstrate the application of a fluorescent peptide displacement assay to screen small molecule probes against four different RNA targets. The designed experimental protocol combined with statistical analysis provides a fast and convenient method to simultaneously evaluate small molecule libraries against different RNA targets and classify them based on affinity and selectivity patterns.
Asunto(s)
Colorantes Fluorescentes/metabolismo , Péptidos/metabolismo , ARN/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Secuencia de Aminoácidos , Secuencia de Bases , Evaluación Preclínica de Medicamentos , Colorantes Fluorescentes/química , Productos del Gen tat/química , Péptidos/química , ARN/genéticaRESUMEN
OBJECTIVE: Botulinum toxin has many applications in the treatment of central diseases, as biological macromolecules, it is difficult to pass through the blood-brain barrier which greatly limits their application. In this paper, we verified whether the botulinum toxin heavy chain HCS has a specific neural guidance function. METHODS: We have constructed a fusion protein with botulinum toxin heavy chain and a membrane penetrating peptide TAT (TAT-EGFP-HCS). Recombinant plasmid of botulinum toxin light chain (LC) and TAT were also constructed. The biological activity of HCS, LC, TAT-EGFP-HCS and TAT-EGFP-LC were measured by its ability to cleave protein SNAP-25. The intracellular expression efficiency was evaluated by detecting the fluorescence intensity of EGFP in the cells by fluorescence microscopy and FACS. In addition, we also determined the effect of the above plasmid expression on the apoptosis of PC12 cells. Finally, the tissue specificity of TAT-EGFP-HCS in vivo experiments was also examined. RESULTS: In the present study, we have constructed a fusion protein with botulinum toxin heavy chain and a membrane penetrating peptide TAT which can lead the entire molecule through the blood-brain barrier and reach the central nervous system. Moreover, we also examined the biological activities of this recombinant biological macromolecule and its physiological effects on nerve cells in vitro and in vivo. CONCLUSION: TAT-EGFP-HSC expressed in vitro has neural guidance function and can carry large proteins across the cell membrane without influencing the biological activity.
Asunto(s)
Barrera Hematoencefálica/metabolismo , Toxinas Botulínicas/química , Descubrimiento de Drogas/métodos , Productos del Gen tat/química , Proteínas Fluorescentes Verdes/química , Proteínas Recombinantes/farmacocinética , Animales , Apoptosis/efectos de los fármacos , Células Cultivadas , Sistema Nervioso Central/metabolismo , Humanos , Masculino , Ratones , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacología , Proteína 25 Asociada a Sinaptosomas/efectos de los fármacosRESUMEN
Targeted delivery of nanomaterials to specific intracellular locations is essential for the development of many nanomaterials-based biological applications. Thus far the targeting performance has been limited due to various intracellular transport barriers, especially intracellular vesicle trapping. Here we report the application of permeation enhancers based on organic solvents in small percentage to enhance the intracellular targeted delivery of nanomaterials. Previously permeation enhancers based on organic solvents and ionic liquids have been used in overcoming biological transport barriers at tissue, organ, and cellular levels, but this strategy has so far rarely been examined for its potential in facilitating transport of nanometer-scale entities across intracellular barriers, particularly intracellular vesicle trapping. Using the cell nucleus as a model intracellular target and Tat peptide-conjugated quantum dots (QDs-Tat) as a model nanomaterial-based probe, we demonstrate that a small percentage (e.g. 1%) of organic solvent greatly enhances nucleus targeting specificity as well as increasing endocytosis-based cellular uptake of QDs. We combine vesicle colocalization (DiO dye staining), vesicle integrity (calcein dye release), and single-particle studies (pair-correlation function microscopy) to investigate the process of organic solvent-enhanced vesicle escape of QDs-Tat. The organic solvent based vesicle escape-enhancing approach is found to be not only very effective but minimally invasive, resulting in high vesicle escape efficiency with no significant disruption to the membrane integrity of either intracellular vesicles or cells. This approach drastically outperforms the commonly used vesicle escape-enhancing agent (i.e., chloroquine, whose enhancement effect is based on disrupting vesicle integrity) in both potency and minimal invasiveness. Finally, we apply organic solvent-based targeting enhancement to improve the intracellular delivery of the anticancer drug doxorubicin (DOX).
Asunto(s)
Portadores de Fármacos/química , Productos del Gen tat/química , Productos del Gen tat/metabolismo , Espacio Intracelular/metabolismo , Compuestos Orgánicos/química , Puntos Cuánticos/química , Solventes/química , Transporte Biológico , Células HeLa , Humanos , Células MCF-7 , PermeabilidadRESUMEN
RNA-protein interactions permeate biology. Transcription, translation, and splicing all hinge on the recognition of structured RNA elements by RNA-binding proteins. Models of RNA-protein interactions are generally limited to short linear motifs and structures because of the vast sequence sampling required to access longer elements. Here, we develop an integrated approach that calculates global pairwise interaction scores from in vitro selection and high-throughput sequencing. We examine four RNA-binding proteins of phage, viral, and human origin. Our approach reveals regulatory motifs, discriminates between regulated and non-regulated RNAs within their native genomic context, and correctly predicts the consequence of mutational events on binding activity. We design binding elements that improve binding activity in cells and infer mutational pathways that reveal permissive versus disruptive evolutionary trajectories between regulated motifs. These coupling landscapes are broadly applicable for the discovery and characterization of protein-RNA recognition at single nucleotide resolution.
Asunto(s)
Productos del Gen tat/química , ARN Nucleotidiltransferasas/química , Proteínas de Unión al ARN/química , ARN/química , Proteínas Reguladoras y Accesorias Virales/química , Secuencia de Aminoácidos , Bacteriófago lambda/química , Sitios de Unión , Clonación Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Productos del Gen tat/genética , Productos del Gen tat/metabolismo , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Virus de la Inmunodeficiencia Bovina/química , Modelos Moleculares , Conformación de Ácido Nucleico , Unión Proteica , Estructura Secundaria de Proteína , ARN/genética , ARN/metabolismo , ARN Nucleotidiltransferasas/genética , ARN Nucleotidiltransferasas/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Análisis de Secuencia de ARN , Proteínas Reguladoras y Accesorias Virales/genética , Proteínas Reguladoras y Accesorias Virales/metabolismoRESUMEN
Precise engineering of nanoparticles with systematically varied properties (size, charge surface properties, targeting ligands, etc.) remains a challenge, limiting the effective optimization of nanoparticles for particular applications. Herein we report a single-step microfluidic combinatorial approach for producing a library of single and dual-ligand liposomes with systematically-varied properties including size, zeta potential, targeting ligand, ligand density, and ligand ratio. A targeting ligand folic acid and a cell penetrating peptide TAT were employed to achieve the optimal synergistic targeting effect. In 2D cell monolayer models, the single-ligand folic acid modified liposome didn't show any enhanced cellular uptake, while the incorporation of TAT peptide "switched on" the function of folic acid, and induced significant elevated cellular uptake compared to the single ligand modified liposomes, showing a strong synergistic targeting effect. The folic acid and TAT peptide dual-ligand liposome also demonstrated enhanced tumor penetration as observed using 3D tumor spheroid models. The in vivo study further confirmed the improved tumor targeting and longer tumor retention (up to 72â¯h) of the dual-ligand liposomes. Our work not only proved the versatility of this microfluidic combinatorial approach in producing libraries of multifunctional liposomes with controlled properties but also revealed the great potential of the optimized liposome formulation for synergistic targeting effects.