RESUMEN
Ultraviolet C (UV-C) radiation induces apoptosis in mammalian cells via the mitochondrion-mediated pathway. The Bcl-2 family of proteins are the regulators of the mitochondrial pathway of apoptosis and appears responsive to UV-C radiation. It is unknown how the structure and, effectively, the function of these proteins are directly impacted by UV-C exposure. Here, we present the effect of UV-C irradiation on the structure and function of pro-apoptotic Bid-FL and anti-apoptotic Bcl-xlΔC proteins. Using a variety of biophysical tools, we show that, following UV-C irradiation, the structures of Bcl-xlΔC and Bid-FL are irreversibly altered. Bcl-xLΔC is found to be more sensitive to UV stress than Bid-FL Interestingly, UV-C exposure shows dramatic chemical shift perturbations in consequence of dramatic structural perturbations (α-helix to ß-sheet) in the BH3- binding region, a crucial segment of Bcl-xlΔC. Furter it has been shown that UV-exposed Bcl-xlΔC has reduced efficacy of its interactions with pro-apoptotic tBid.
Asunto(s)
Proteínas Reguladoras de la Apoptosis , Apoptosis , Animales , Proteína bcl-X/metabolismo , Proteína Proapoptótica que Interacciona Mediante Dominios BH3/metabolismo , Mitocondrias/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Mamíferos/metabolismoRESUMEN
Apoptosis is a naturally occurring process during the growth and development of multicellular organisms and is increasingly active during times of cellular stress such as in response to intracellular DNA damage when removal of the host cell is paramount to prevent cancer. Unfortunately, once formed, cancer cells become impervious to apoptosis, creating a desperate need to identify an approach to induce apoptosis in these cells. An attractive option is to focus efforts on developing and locating compounds which activate apoptosis using natural compounds. Curcumin is a natural component in turmeric and is well-known for its pharmacological effects in preventing and combating many ailments and has been shown to decrease the rapid proliferation of a wide variety of tumor cells. However, to date, the apoptotic intermediates and interactions through which curcumin exerts its cytotoxic effects are unknown. Motivated by reports linking the intracellular modulation of the concentrations of Bid and Bcl-xL, following curcumin administration to cancer cells, we set out to probe for potential intermolecular interactions of these proteins with curcumin. Using several biophysical techniques, most notably, fluorescence, circular dichroism and nuclear magnetic resonance spectroscopy, we reveal binding interactions of curcumin with both Bcl-xLΔC and full-length Bid (Bid-FL) and prove that this binding is hydrophobically driven and localized to well-known functional regions of each protein. Specifically, our NMR studies show that while Bid-FL interacts with curcumin through its hydrophobic and pore forming helices (α6-α7), Bcl-xLΔC interacts with curcumin via its BH3 binding pocket (α2-α3-α4-α5), a critical region for mediating apoptosis.
Asunto(s)
Proteína Proapoptótica que Interacciona Mediante Dominios BH3/metabolismo , Curcumina/farmacología , Neoplasias/tratamiento farmacológico , Proteína bcl-X/metabolismo , Apoptosis , HumanosAsunto(s)
Enfermedades de los Anexos , Neoplasias Ováricas , Enfermedades de los Anexos/diagnóstico , Enfermedades de los Anexos/cirugía , Diagnóstico Diferencial , Femenino , Humanos , India , Neoplasias Ováricas/diagnóstico , Neoplasias Ováricas/patología , Neoplasias Ováricas/cirugía , Torsión Ovárica , Anomalía Torsional/cirugíaRESUMEN
Paramagnetic relaxation enhancements (PREs) are routinely used to provide long-range distance restraints for the determination of protein structures, to resolve protein dynamics, ligand-protein binding sites, and lowly populated species, using Nuclear Magnetic Resonance Spectroscopy (NMR). Here, we propose a simultaneous 1H-15 N, 1H-13C SESAME based pulse scheme for the rapid acquisition of 1HC/N-R2 relaxation rates for the determination of backbone and sidechain PREs of proteins. The 1HN-R2 rates from the traditional and our approach on Ubiquitin (UBQ) are well correlated (R2 = 0.99), revealing their potential to be used quantitatively. Comparison of the S57C UBQ calculated and experimental PREs provided backbone and side chain Q factors of 0.23 and 0.24, respectively, well-fitted to the UBQ NMR structure, showing that our approach can be used to acquire accurate PRE rates from the functionally important sites of proteins but in at least half the time as traditional methods.
Asunto(s)
Resonancia Magnética Nuclear Biomolecular , Proteínas/química , Cisteína/genética , Humanos , Proteínas Mutantes/química , Mutación/genética , Reproducibilidad de los Resultados , Ubiquitina/químicaRESUMEN
The ribosomal protein P2 of Plasmodium falciparum, (PfP2), performs certain unique extra-ribosomal functions. During the few hours of cell-division, PfP2 protein moves to the external surface of the infected erythrocytes (IE) as an SDS-resistant oligomer, and at that stage treatment with specific anti- PfP2 antibodies results in an arrest of the parasite cell-division. Amongst the oligomeric forms of PfP2, mainly the homo-tetramer is peripherally anchored on the external surface of the IE. To study the anchoring of PfP2 tetramer on IE-surface, we have explored the binding properties of PfP2 protein. Using NMR and erythrocyte pull-down studies, here we report that the homo-tetrameric PfP2 protein interacted specifically with erythrocytes and not leukocytes. The hydrophobic N-terminal 72 amino acid region is the major interacting domain. The binding of P2 to RBCs was neuraminidase resistant, but trypsin sensitive. The RBC binding was exclusive to the Plasmodium PfP2 protein as even the homologous protein of the closely related Apicomplexan parasite Toxoplasma gondii TgP2 protein did not interact with erythrocytes. Pull down assays, immunoprecipitation and mass spectrometry data showed that erythrocytic Band 3 protein is a possible interactor of Plasmodium PfP2 protein on the erythrocyte surface.
Asunto(s)
Eritrocitos/química , Plasmodium falciparum/química , Proteínas Protozoarias/química , Proteínas Ribosómicas/química , Eritrocitos/metabolismo , Humanos , Plasmodium falciparum/metabolismo , Proteínas Protozoarias/metabolismo , Proteínas Ribosómicas/metabolismoRESUMEN
Protein structure determination using NMR is dependent on experimentally acquired distance restraints. Often, however, an insufficient number of these restraints are available for determining a protein's correct fold, much less its detailed three-dimensional structure. In consideration of this problem, we propose a simple means to acquire supplemental structural restraints from protein surface accessibilities using solvent saturation transfer to proteins (SSTP), based on the principles of paramagnetic chemical-exchange saturation transfer. Here, we demonstrate the utility of SSTP in structure calculations of two proteins, TSG101 and ubiquitin. The observed SSTP was found to be directly proportional to solvent accessibility. Since SSTP does not involve the direct excitation of water, which compromises the analysis of protein protons entangled in the breadth of the water resonance, it has an advantage over conventional water-based magnetization transfers. Inclusion of structural restraints derived from SSTP improved both the precision and accuracy of the final protein structures in comparison to those determined by traditional approaches, when using minimal amounts of additional structural data. Furthermore, we show that SSTP can detect weak protein-protein interactions which are unobservable by chemical shift perturbations.
Asunto(s)
Resonancia Magnética Nuclear Biomolecular/métodos , Proteínas/química , Proteínas de Unión al ADN/química , Complejos de Clasificación Endosomal Requeridos para el Transporte/química , Conformación Proteica , Solventes , Factores de Transcripción/química , Ubiquitina/químicaRESUMEN
Bax is known for its pro-apoptotic role within the mitochondrial pathway of apoptosis. However, the mechanism for transitioning Bax from cytosolic to membrane-bound oligomer remains elusive. Previous nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR) studies defined monomeric Bax as conformationally homogeneous. Yet it has recently been proposed that monomeric Bax exists in equilibrium with a minor state that is distinctly different from its NMR structure. Here, we revisited the structural analysis of Bax using methods uniquely suited for unveiling "invisible" states of proteins, namely, NMR paramagnetic relaxation enhancements and EPR double electron-electron resonance (DEER). Additionally we examined the effect of glycerol, the co-solvent of choice in DEER studies, on the structure of Bax using NMR chemical-shift perturbations and residual dipolar couplings. Based on our combined NMR and EPR results, Bax is a conformationally homogeneous protein prior to its activation.
Asunto(s)
Proteína X Asociada a bcl-2/química , Glicerol/química , Humanos , Simulación de Dinámica Molecular , Conformación Proteica , Estabilidad ProteicaRESUMEN
There are multiple stages in the life cycle of Plasmodium that invade host cells. Molecular machinery involved is such host-pathogen interactions constitute excellent drug targets and/or vaccine candidates. A screen using a phage display library has previously demonstrated presence of enolase on the surface of the Plasmodium ookinete. Phage-displayed peptides that bound to the ookinete contained a conserved motif (PWWP) in their sequence. Here, direct binding of these peptides with recombinant Plasmodium falciparum enolase (rPfeno) was investigated. These peptides showed specific binding to rPfeno, but failed to bind to other enolases. Plasmodium spp enolases are distinct in having an insert of five amino acids ((104)EWGWS(108)) that is not found in host enolases. The possibility of this insert being the recognition motif for the PWWP containing peptides was examined, (i) by comparing the binding of the peptides with rPfeno and a deletion variant Δ-rPfeno lacking (104)EWGWS(108), (ii) by measuring the changes in proton chemical shifts of PWWP peptides on binding to different enolases and (iii) by inter-molecular docking experiment to locate the peptide binding site. Results from these studies showed that the pentapeptide insert of Pfeno indeed constitutes the binding site for the PWWP domain containing peptide ligands. Search for sequences homologous to phage displayed peptides among peritrophic matrix proteins resulted in identification of perlecan, laminin, peritrophin and spacran. The possibility of these PWWP domain-containing proteins in the peritrophic matrix of insect gut to interact with ookinete cell surface enolase and facilitate the invasion of mosquito midgut epithelium is discussed.
Asunto(s)
Culicidae/parasitología , Proteínas de Insectos/metabolismo , Fosfopiruvato Hidratasa/metabolismo , Plasmodium falciparum/enzimología , Proteínas Protozoarias/metabolismo , Secuencias de Aminoácidos , Animales , Sistema Digestivo/metabolismo , Proteínas de Insectos/genética , Modelos Moleculares , Biblioteca de Péptidos , Péptidos/metabolismo , Fosfopiruvato Hidratasa/química , Fosfopiruvato Hidratasa/genética , Unión Proteica , Conformación Proteica , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Proteínas Recombinantes/metabolismoRESUMEN
Toxoplasma gondii is an apicomplexan parasite, which causes toxoplasmosis. Toxoplasma P2 (TgP2) is a ribosomal protein and exists as supramolecular assembly with other proteins in the ribosome. It is also shown that TgP2 is involved in some extra ribosomal functions. However, till date the protein has evaded structural characterization by any of the known techniques. In this background, we report here a systematic study using a variety of biophysical techniques and NMR, under different conditions of pH and temperature, and deduce that TgP2 consists of only helices and unstructured regions, is a monomer at low pH but forms multimers at higher pH, and has intrinsically a molten globule structure. The C-terminal half is flexible and the helices are concentrated in the N-terminal half of the chain. The dynamism inherent to the molten globule structure may have functional implications for its extra-ribosomal functions. which is contrast to that of human P2.
Asunto(s)
Modelos Moleculares , Fosfoproteínas/química , Proteínas Ribosómicas/química , Toxoplasma/química , Secuencia de Aminoácidos , Dicroismo Circular , Datos de Secuencia MolecularRESUMEN
Intrinsically disordered proteins or such domains in globular proteins are believed to be playing important roles in protein functions by virtue of their ability to adapt themselves to requirements of different binding partners and thereby accord high specificity to the interaction. Eukaryotic ribosomal stalk is made up of a supramolecular assembly of P0, P1 and P2 proteins. In Plasmodium falciparum, homo-oligomers of P2 are also seen which seem to be involved in many non-ribosomal functions of the protein in the parasite, and in all of these the protein interacts with different interactors. Here we show by extensive (15)N NMR relaxation studies that the C-terminal stretch of about 45 residues of the protein always remains as a flexible disordered domain, regardless of the state of association of the protein. The relaxation behaviors and the derived rotational correlation times for this portion of the protein are essentially the same in the presence of different concentrations of urea which produce different mixtures of PfP2 oligomers in rapid exchange, whereas the rest of the protein shows substantial variations with urea concentration in the relaxation behaviors. In other words, the C-terminal domain behaves as if it were an independent intrinsically disordered peptide. This would augment the notion that the C-terminal domain of PfP2 would be acting as a scavenger for different interactors depending upon the different functions of the protein inside the parasite.
Asunto(s)
Proteínas Intrínsecamente Desordenadas/química , Malaria Falciparum/parasitología , Plasmodium falciparum/química , Proteínas Protozoarias/química , Proteínas Ribosómicas/química , Urea/química , Secuencia de Aminoácidos , Humanos , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica , Multimerización de Proteína , Estructura Terciaria de ProteínaRESUMEN
The P2 protein in Plasmodium falciparum has a high tendency to oligomerize, which seems to drive many of its non-ribosomal functions. During nuclear division of the parasite inside RBC, P2 translocates to the RBC surface as a tetramer. From a systematic study using variety of biophysical techniques, NMR spectral characteristics and relaxation dispersion measurements under different conditions of pH and/or urea concentrations, we deduce that (i) PfP2, an almost entirely helical protein, forms a molten globule monomer at low pH, (ii) at physiological pH, and at micro-molar concentrations, PfP2 is a stable tetramer wherein two dimmers associate sideways with close packing of helices at the interface, and (iii) the molten globule characteristic of the monomer is preserved in the tetramer. This dynamism in the structure of PfP2 may have functional implications since it is known that different kinds of oligomers are transiently formed in the parasite.
RESUMEN
The P2 protein (equivalent of L7/L12 in prokaryotes), a member of the ribosomal stalk in eukaryotes, is highly conserved, particularly its C-terminal domain. In order to understand the sequence-structure-function relationships in eukaryotic C-terminal stretches, about which nothing is known at the moment, we have investigated here, the structural characteristics of these domains of P2 proteins from three different species, namely, human, Plasmodium falciparum, and Toxoplasma gondii; the sequence homology among these is 70% although sequence identity is only 36%. About 50 amino acids of the C-terminal domains of P2 from the three species were expressed and purified. Gel filtration studies indicated peaks for both monomer and oligomer at milimolar concentrations and also suggested monomer-multimer equilibrium. Circular Dichroism showed that this domain does not have stable secondary structures. (1)H-(15)N HSQC spectra in every case showed one set of requisite number of peaks as per the sequence. This indicated that there is rapid multimer-monomer equilibrium in solution and the observed peaks which originate from the monomer reflect average chemical shifts. The spectral dispersion in all the cases is narrow, although there are noticeable differences in the three proteins. Detailed NMR investigations revealed that this protein domain is intrinsically disordered although there are short segments with preferred secondary structural propensities at similar places along the sequence. This may suggest that the sequence is selected in evolution to impart disorder, and thereby accord conformational adaptability.
Asunto(s)
Proteínas Intrínsecamente Desordenadas/química , Resonancia Magnética Nuclear Biomolecular/métodos , Fosfoproteínas/química , Plasmodium falciparum/metabolismo , Proteínas Protozoarias/química , Proteínas Ribosómicas/química , Toxoplasma/metabolismo , Dicroismo Circular , Simulación por Computador , Humanos , Proteínas Intrínsecamente Desordenadas/genética , Modelos Moleculares , Fosfoproteínas/genética , Filogenia , Plasmodium falciparum/genética , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Protozoarias/genética , Proteínas Ribosómicas/genética , Homología de Secuencia de Aminoácido , Toxoplasma/genéticaRESUMEN
Interaction of small molecule inhibitors with protein aggregates has been studied extensively, but how these inhibitors modulate aggregation kinetic parameters is little understood. In this work, we investigated the ability of two potential aggregation inhibiting drugs, curcumin and kaempferol, to control the kinetic parameters of aggregation reaction. Using thioflavin T fluorescence and static light scattering, the kinetic parameters such as amplitude, elongation rate constant and lag time of guanidine hydrochloride-induced aggregation reactions of hen egg white lysozyme were studied. We observed a contrasting effect of inhibitors on the kinetic parameters when aggregation reactions were measured by these two probes. The interactions of these inhibitors with hen egg white lysozyme were investigated using fluorescence quench titration method and molecular dynamics simulations coupled with binding free energy calculations. We conclude that both the inhibitors prolong nucleation of amyloid aggregation through binding to region of the protein which is known to form the core of the protein fibril, but once the nucleus is formed the rate of elongation is not affected by the inhibitors. This work would provide insight into the mechanism of aggregation inhibition by these potential drug molecules.
Asunto(s)
Curcumina/farmacología , Quempferoles/farmacología , Muramidasa/metabolismo , Dicroismo Circular , Fluorescencia , Guanidina/química , Cinética , Microscopía Electrónica de Rastreo , Simulación de Dinámica Molecular , Espectrofotometría UltravioletaRESUMEN
Plasmodium falciparum P2 (PfP2) is a ribosomal stalk protein. It also performs extra ribosomal novel functions that seem to be associated with homo oligomerization . Previous in vitro studies have demonstrated that the protein has a high tendency to self-associate predominantly into an 8-mer. In vitro Heteronuclear Single Quantum Coherence (HSQC) of the pure recombinant protein (rPfP2) and its in-cell (Escherichia coli) HSQC spectrum has very similar features, indicating that the protein intrinsically, both inside the cell and under in vitro conditions, has similar aggregation tendencies. In view of this, we have characterized here the folding and concomitant self-association of rPfP2, using an in vitro dissociation-association strategy. We observed that the residue stretch, (Met31-Leu44) of the rPfP2, mapping to Met1-Leu14 of PfP2 protein acts as a nucleation site for helix formation and subsequent self-association. Further association appears to be driven by hydrophobic and complimentary electrostatic charge interactions on the surfaces formed. One stretch of rPfP2, (Ile97-Ala116), always remains floppy, and this may serve as "hinge" for protein segmental motions. Based on these, we have proposed a possible model for rPfP2 self-association into an 8-mer.
Asunto(s)
Plasmodium falciparum/metabolismo , Proteínas Protozoarias/química , Modelos Moleculares , Unión Proteica/efectos de los fármacos , Conformación Proteica/efectos de los fármacos , Pliegue de Proteína/efectos de los fármacos , Multimerización de Proteína , Proteínas Protozoarias/metabolismo , Urea/farmacologíaRESUMEN
The eukaryotic 60S-ribosomal stalk is composed of acidic ribosomal proteins (P1 and P2) and neutral protein P0, which are thought to be associated as a pentameric structure, [2P1, 2P2, P0]. Plasmodium falciparum P2 (PfP2) appears to play additional non-ribosomal functions associated with its tendency for homo-oligomerization. Recombinant bacterially expressed PfP2 protein also undergoes self-association, as shown by SDS-PAGE analysis and light scattering studies. Secondary structure prediction algorithms predict the native PfP2 protein to be largely helical and this is corroborated by circular dichroism investigation. The (1)H-(15)N HSQC spectrum of native P2 showed only 43 cross peaks compared to the expected 138. The observed peaks were found to belong to the C-terminal region, suggesting that this segment is flexible and solvent exposed. In 9 M urea denaturing conditions the chain exhibited mostly non-native ß structural propensity. (15)N Relaxation data for the denatured state indicated substantial variation in ms-µs time scale motion along the chain. Average area buried upon folding (AABUF) calculations on the monomer enabled identification of hydrophobic patches along the sequence. Interestingly, the segments of slower motion in the denatured state coincided with these hydrophobic patches, suggesting that in the denatured state the monomeric chain undergoes transient hydrophobic collapse. The implications of these results for the folding mechanism and self-association of PfP2 are discussed.
Asunto(s)
Resonancia Magnética Nuclear Biomolecular/métodos , Plasmodium falciparum/metabolismo , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismo , Pliegue de ProteínaRESUMEN
We present here an improvisation of HNN (Panchal, Bhavesh et al., 2001) called RD 3D HNCAN for backbone (HN, CA and (15)N) assignment in both folded and unfolded proteins. This is a reduced dimensionality experiment which employs CA chemical shifts to improve dispersion. Distinct positive and negative peak patterns of various triplet segments along the polypeptide chain observed in HNN are retained and these provide start and check points for the sequential walk. Because of co-incrementing of CA and (15)N, peaks along one of the dimensions appear at sums and differences of the CA and (15)N chemical shifts. This changes the backbone assignment protocol slightly and we present this in explicit detail. The performance of the experiment has been demonstrated using Ubiquitin and Plasmodium falciparum P2 proteins. The experiment is particularly valuable when two neighboring amino acid residues have nearly identical backbone (15)N chemical shifts.
Asunto(s)
Resonancia Magnética Nuclear Biomolecular/métodos , Proteínas/química , Algoritmos , Animales , Glicina/química , Humanos , Hidrógeno/química , Nitrógeno/química , Plasmodium falciparum/química , Prolina/química , Conformación Proteica , Pliegue de Proteína , Ubiquitina/químicaRESUMEN
Aims of this investigation were to prepare and characterize cabergoline intranasal microemulsion formulations, determine brain drug delivery through biodistribution using technetium-99m (99mTc) as a tracer, and assess its performance pharmacodynamically in weight control. Cabergoline microemulsions of different compositions were prepared by water titration method and characterized for globule size and zeta potential. Microemulsion with maximum drug solubilization and stability was considered optimal and taken for further studies with or without addition of mucoadhesive agent. Pharmacokinetics of optimized 99mTc-labeled cabergoline formulations and 99mTc-labeled drug solution were studied by estimating radioactivity in brain and blood of albino rats post intranasal, intravenous, and oral administrations. To confirm localization of drug in brain following intranasal, intravenous, and oral administrations, gamma scintigraphy imaging was also performed. To assess weight control performance of formulations, body weight, white adipose tissue mass, serum lipids, leptin, and prolactin were determined before and after 40 days of intranasal administrations of these formulations to Wistar rats. Microemulsions were found to be stable both physically and chemically when stored at various stress conditions. Brain/blood uptake ratios, drug targeting efficiency, and direct drug transport were found to be highest for drug mucoadhesive microemulsion followed by drug microemulsion and drug solution post-intranasal administration compared to intravenous drug microemulsion. Significant (p<0.05) reduction in assessed pharmacodynamic parameters was observed after intranasal administration of mucoadhesive microemulsion against control group. The results of the studies conclusively demonstrate that intranasal microemulsion formulations developed in this investigation are stable and can deliver cabergoline selectively and in higher amounts to the brain compared to both drug administrations as a solution intranasally or microemulsion intravenously. The results also demonstrate reduction in weight, adipose tissue mass, serum lipids, and serum prolactin after intranasal administration of drug microemulsion. Hence, long-term studies in at least two more animal models followed by extensive clinical evaluation can safely result into a product for clinical use.
Asunto(s)
Fármacos Antiobesidad/farmacología , Ergolinas/administración & dosificación , Ergolinas/farmacocinética , Administración Intranasal , Animales , Cabergolina , Difusión , Estabilidad de Medicamentos , Emulsiones , Ergolinas/química , Ergolinas/farmacología , Femenino , Masculino , Prolactina/metabolismo , Ratas , Solubilidad , Distribución TisularRESUMEN
Polyethylene glycols (PEGs) are potential drug carriers for humanizing the therapeutic index of anti-cancer agents. In this paper, we report on the modification of the anticancer drugs, methotrexate (MTX) and melphalan (L-PAM), covalently linked to PEGs for drug delivery. Conjugates of MTX and L-PAM were analyzed through different spectroscopic techniques. Both conjugates were labeled with (99m)Tc by the classical way, using reducing agents at a physiologic pH. Blood kinetic data revealed the biphasic pattern of clearance. Evaluation of the in vitro cytotoxicity of the drug polymer conjugates on the U87MG human glioma cell line revealed that the conjugates showed enhanced dose-dependent cytotoxicity.
Asunto(s)
Neoplasias Encefálicas/tratamiento farmacológico , Glioma/tratamiento farmacológico , Melfalán/administración & dosificación , Metotrexato/administración & dosificación , Polietilenglicoles/metabolismo , Animales , Antineoplásicos/administración & dosificación , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Humanos , Concentración de Iones de Hidrógeno , Cinética , Ratones , Polietilenglicoles/química , Polímeros/química , Distribución TisularRESUMEN
The objective of the present study was to optimize olanzapine nanoemulsion (ONE), for nose-to-brain delivery. The nanoemulsions and olanzapine mucoadhesive nanoemulsions (OMNEs) were prepared using water titration method and characterized for technical and electrokinetic properties. Biodistribution of nanoemulsions and olanzapine solution (OS) in the brain and blood of rats following intranasal (intranasal) and intravenous (intravenous) administrations were examined using optimized technetium-labeled ((99m)Tc-labeled) olanzapine formulations. The brain/blood uptake ratios of 0.45, 0.88, 0.80, and 0.04 of OS (intranasal), ONE (intranasal), OMNE (intranasal), ONE (intravenous), respectively, at 0.5 h are indicative of direct nose-to-brain transport (DTP). Higher % drug targeting efficiency (%DTE) and %DTP for mucoadhesive nanoemulsions indicated effective brain targeting of olanzapine among the prepared nanoemulsions. Gamma scintigraphy imaging of the rat brain conclusively demonstrated rapid and larger extent of transport of olanzapine by OMNE (intranasal), when compared with OS (intranasal), ONE (intranasal), and ONE (intravenous), into the rat brain.
Asunto(s)
Benzodiazepinas/administración & dosificación , Encéfalo/metabolismo , Sistemas de Liberación de Medicamentos , Emulsiones/administración & dosificación , Nanoestructuras/administración & dosificación , Administración Intranasal , Animales , Antipsicóticos/administración & dosificación , Antipsicóticos/sangre , Antipsicóticos/farmacocinética , Benzodiazepinas/sangre , Benzodiazepinas/farmacocinética , Emulsiones/química , Haloperidol , Inyecciones Intravenosas , Masculino , Nanoestructuras/química , Olanzapina , RatasRESUMEN
The objective of the present study was to synthesize core-corona nanoparticles of doxorubicin (DOX) using hyaluronic acid-polyethyleneglycol-polycaprolactone (HA-PEG-PCL) copolymer for tumor targeting. Targeting efficiency of HA-PEG-PCL nanoparticles was compared with non-HA-containing nanoparticles (methoxy poly ethylene glycol (MPEG)-PCL). The copolymers were chemically synthesized and characterized by IR and NMR spectroscopies. The nanoparticles were characterized for shape and morphology by transmission electron microscopy, particle size, percentage of drug entrapment, and in vitro drug release profile. Differential scanning calorimetry and X-ray diffraction studies were also performed to appraise the crystalline or amorphous nature of DOX inside the polymer matrix. Formulations were prepared using different DOX:polymer ratios (1:1-1:3 w/w) and the optimum formulation with the drug:polymer ratio of 1:1 showed the mean particle size of 95 +/- 5 nm and entrapment efficiency of 95.56% in the case of HA-PEG-PCL nanoparticles, while the values were 115 nm and 95.50%, respectively, in the case of MPEG-PCL nanoparticles. The HA-PEG-PCL nanoparticles could release DOX for up to 17 days, whereas the MPEG-PCL nanoparticles could release it for up to 14 days. The hemolytic toxicity and hematological studies confirmed that both DOX-loaded HA-PEG-PCL and MPEG-PCL nanoparticles were safe and suitable for sustained and targeted drug delivery. The tissue distribution study and tumor growth inhibition were performed after intravenous injection of nanoparticles in Ehrlich ascites tumor (EAT)-bearing mice. The nanoparticles of HA-PEG-PCL copolymer accomplishes efficient delivery of DOX in EAT tumor when compared with the MPEG-PCL nanoparticles by the process of receptor-mediated endocytosis, as well as enhanced permeability and retention effect.