RESUMEN
As one of the most devastating forms of trauma, spinal cord injury (SCI) remains a challenging clinical problem. The secondary processes associated with the primary injury, such as overproduction of reactive oxygen species (ROS) and inflammation, lead to concomitant compression of the injured spinal cord and neuronal death. Delivery of copper-zinc superoxide dismutase (SOD1), an efficient ROS scavenger, to the site of injury can mitigate SCI-induced oxidative stress and tissue damage. Towards this goal catalytically active nanoformulations of SOD1 ("nanozymes") are developed as a modality for treatment of SCI. Along with the cross-linked polyion complex of SOD1 with polycation poly(ethylene glycol) (PEG)-polylysine (single-coat (SC) nanozyme), we introduce for the first time the chemically cross-linked multilayer polyion complex in which SOD1 is first incorporated into a polyion complex with polycation, then coated by anionic block copolymer, PEG-polyglutamic acid (double-coat (DC) nanozyme). We developed DC nanozymes with high enzymatic activity and ability to retain and protect SOD1 under physiological conditions. Pharmacokinetic study revealed that DC nanozymes significantly prolonged circulation of active SOD1 in the blood stream compared to free SOD1 or SC nanozymes (half-life was 60 vs 6min). Single intravenous injection of DC nanozymes (5kU of SOD1/kg) improved the recovery of locomotor functions in rats with moderate SCI, along with reduction of swelling, concomitant compression of the spinal cord and formation of post-traumatic cysts. Thus, based on the testing in a rodent model the SOD1 DC nanozymes are promising modality for scavenging ROS, decreasing inflammation and edema, and improving recovery after SCI.
Asunto(s)
Nanopartículas/administración & dosificación , Traumatismos de la Médula Espinal/tratamiento farmacológico , Superóxido Dismutasa-1/administración & dosificación , Enfermedad Aguda , Animales , Femenino , Locomoción/efectos de los fármacos , Masculino , Polímeros/administración & dosificación , Polímeros/farmacocinética , Ratas Sprague-Dawley , Ratas Wistar , Traumatismos de la Médula Espinal/fisiopatología , Superóxido Dismutasa-1/farmacocinéticaRESUMEN
Using computer modeling, virtual screening of high-affinity ligands for immobilization of inulinase - an enzyme that cleaves inulin and fructose-containing polymers to fructose - has been performed. The inulinase molecule from Aspergillus ficuum (pdb: 3SC7) taken from the database of protein structures was used as a protein model and the target for flexible docking. The set of ligands studied included simple sugars (activators, inhibitors, products of enzymatic catalysis), as well as high-molecular weight compounds (polycation and polyanion exchange resins, glycoproteins, phenylalanine-proline peptide, polylactate, and caffeine). Based on the comparative analysis of the values of the total energy and the localization of ligand binding sites, we made several assumptions concerning the mechanisms of interaction of the suggested matrices for the immobilization of enzyme molecules and the structural features of such complexes. It was also assumed that the candidates for immobilization agents meeting the industrial requirements may be glycoproteins, for which we propose an additional incorporation of cysteine residues into their structure, aimed to create disulfide «anchors¼ to the surface.
Asunto(s)
Aspergillus/enzimología , Simulación por Computador , Proteínas Fúngicas/química , Glicósido Hidrolasas/química , Simulación del Acoplamiento Molecular , Sitios de UniónRESUMEN
The aim of this study was to create a nanocontainer conjugated with monoclonal antibodies to connexin 43 (Cx43) that is actively expressed at the periphery of C6 glioma and in the astroglia roll zone. Stable vector nanogels with high (up to 35%) cisplatin load were synthesized. The antitumor effects of Cx43-modified cisplatin-loaded nanogels, free cisplatin, and nonspecific drugs were carried out on C6 glioma model. Vector nanogels reduced systemic toxicity of cisplatin, effectively inhibited tumor growth, and significantly prolonged the lifespan of animals with experimental tumors.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Antineoplásicos/farmacología , Neoplasias Encefálicas/terapia , Cisplatino/farmacología , Conexina 43/inmunología , Glioblastoma/terapia , Inmunoconjugados/farmacología , Animales , Anticuerpos Monoclonales/química , Antineoplásicos/química , Neoplasias Encefálicas/inmunología , Neoplasias Encefálicas/mortalidad , Neoplasias Encefálicas/patología , Cisplatino/química , Conexina 43/química , Portadores de Fármacos , Femenino , Geles , Glioblastoma/inmunología , Glioblastoma/mortalidad , Glioblastoma/patología , Inmunoconjugados/química , Ratones , Ratones Endogámicos BALB C , Nanoestructuras/química , Trasplante de Neoplasias , Polietilenglicoles/química , Ácidos Polimetacrílicos/química , Estructura Terciaria de Proteína , Ratas , Ratas Wistar , Técnicas Estereotáxicas , Análisis de Supervivencia , Carga Tumoral/efectos de los fármacosRESUMEN
Methodology for production of calcium-phosphate nanoparticles is developed and its efficacy as a drug carrier system is estimated by example of timolol. Conditions for production of particles with optimal size and resistance are determined, methodology of loading of particles with timolol is developed. Physical parameters of particles (form, size, relief), kinetics of saturation with drug and its release are studied. Packaging of timolol into calcium phosphate nanoparticles was showed to enhance and prolong its hypotensive effect in experiment on healthy rabbits.
Asunto(s)
Fosfatos de Calcio , Sistemas de Liberación de Medicamentos/métodos , Presión Intraocular/efectos de los fármacos , Nanopartículas/química , Timolol/administración & dosificación , Administración Oftálmica , Animales , Portadores de Fármacos/administración & dosificación , Portadores de Fármacos/farmacocinética , Nanotecnología , Conejos , Tecnología Farmacéutica/métodos , Timolol/farmacocinética , Resultado del TratamientoRESUMEN
The aim of this study was to create vector superparamagnetic nanoparticles for tumor cell visualization in vivo by magnetic resonance tomography. A method for obtaining superparamagnetic nanoparticles based on ferric oxide with the magnetic nucleus diameter of 12 ± 3 nm coated with BSA and forming stable water dispersions was developed. The structure and size of the nanoparticles were studied by transmissive electron microscopy, dynamic light scattering, and x-ray phase analysis. Their T2 relaxivity was comparable with that of the available commercial analog. Low cytotoxicity of these nanoparticles was demonstrated by MTT test on primary and immortalized cell cultures. The nanoparticles were vectorized by monoclonal antibodies to connexin 43 (Cx43). Specific binding of vectorized nanoparticles to C6 glioma Cx43-positive cell membranes was demonstrated. Hence, vector biocompatible nanoparticles with high relaxivity, fit for use as MRT contrast for the diagnosis of poorly differentiated gliomas, were created.
Asunto(s)
Medios de Contraste/química , Compuestos Férricos/química , Glioma/patología , Espectroscopía de Resonancia Magnética/métodos , Nanopartículas/química , Anticuerpos Monoclonales/química , Línea Celular Tumoral , HumanosRESUMEN
We developed a method for obtaining iron oxide nanoparticles and their conjugation with monoclonal antibodies to vascular endothelial growth factor. The resultant vector nanoparticles were low-toxic and the antibodies retained their immunochemical activity after conjugation. The study was carried out on rats with intracranial glioma C6 on day 14 after its implantation. The intravenously injected nanoparticles visualized the brain tumor in contrast to nanoparticles conjugated with nonspecific immunoglobulins that did not accumulate in the tumor.
Asunto(s)
Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Glioma/patología , Imagen por Resonancia Magnética/métodos , Magnetismo , Nanopartículas/química , Factor A de Crecimiento Endotelial Vascular/inmunología , Animales , Medios de Contraste/química , Femenino , RatasRESUMEN
The blood-brain barrier (BBB) presents a tremendous challenge for the delivery of drugs to the central nervous system (CNS). This includes drugs that target brain receptors for the treatment of obesity and anorexia. Strategic drug delivery to brain (SDDB) is an approach that considers in depth the relations among the BBB, the candidate therapeutic, the CNS target, and the disease state to be treated. Here, we illustrate principles of SDDB with two different approaches to developing drugs based on leptin. In normal body weight humans and in non-obese rodents, leptin is readily transported across the BBB and into the CNS where it inhibits feeding and enhances thermogenesis. However, in obesity, the transport of leptin across the BBB is impaired, resulting in a resistance to leptin. As a result, it is difficult to treat obesity with leptin or its analogs that depend on the leptin transporter for access to the CNS. To treat obesity, we developed a leptin agonist modified by the addition of pluronic block copolymers (P85-leptin). P85-leptin retains biological activity and is capable of crossing the BBB by a mechanism that is not dependent on the leptin transporter. As such, P85-leptin is able to cross the BBB of obese mice at a rate similar to that of native leptin in lean mice. To treat anorexia, we developed a leptin antagonist modified by pegylation (PEG-MLA) that acts primarily by blocking the BBB transporter for endogenous, circulating leptin. This prevents blood-borne, endogenous leptin from entering the CNS, essentially mimicking the leptin resistance seen in obesity, and resulting in a significant increase in adiposity. These examples illustrate two strategies in which an understanding of the interactions among the BBB, CNS targets, and candidate therapeutics under physiologic and diseased conditions can be used to develop drugs effective for the treatment of brain disease.
Asunto(s)
Barrera Hematoencefálica/metabolismo , Sistemas de Liberación de Medicamentos/métodos , Leptina/análogos & derivados , Leptina/administración & dosificación , Animales , Encéfalo/metabolismo , Diseño de Fármacos , RatonesRESUMEN
The results of theoretical studies of the structural and dynamic features of peptides and small proteins have been presented that were carried out by quantum chemical and molecular dynamics methods in high-performance graphic stations, "table supercomputers", using distributed calculations by the CUDA technology.
Asunto(s)
Simulación por Computador , Modelos Moleculares , Proteínas/química , Programas Informáticos , MicrocomputadoresRESUMEN
We propose a method for obtaining superparamagnetic nanoparticles based on iron oxide and their water suspensions. The structure and size of nanoparticles were confirmed by transmission electron microscopy, dynamic light scattering, and X-ray diffraction analysis. The nanoparticles also contained a fluorescent dye Dil C18. Cytotoxicity of obtained aqueous suspension was studied by MTT assay; low toxicity of nanoparticles was demonstrated. High T2-relaxivity of nanoparticles allows using them as a contrast agent for MRI. After incubation of cerebellar sections with nanoparticles vectorized with antibodies to antigen AMVB1, specific visualization of blood vessels was detected.
Asunto(s)
Compuestos Férricos/química , Imagen por Resonancia Magnética/métodos , Nanopartículas/química , Medios de Contraste/química , Microscopía Electrónica de Transmisión , Nanopartículas/ultraestructura , Difracción de Rayos XRESUMEN
AIM: Development a method of treatment of Streptococcus pyogenes bacteria that does not disrupt the integrity of surface structure of cell and provides optimal reproducibility of enzyme preparation activity test results. MATERIALS AND METHODS: Museum cultures of S. pyogenes M29 and S. pyogenes T1 were used, as well as standard strain S.pyogenes T5 (ATCC) and 3 phage-associated lysine PlyC preparations (enzybiotics): 2 isolated from phage C1, third--recombinant enzyme obtained by cloning phage C1 DNA. 3 methods of S. pyogenes cells treatment were used: inactivation by chloroform, antibiotics and heating. RESULTS: Treatment of S. pyogenes cells by rifampicin and gentamicin allows simultaneous turbidimetric determination of enzyme preparations activity and streptococci lysis effectiveness with a good reproducibility of test results. Comparison of kinetic curves of streptococci lysis killed by heating with curves of live culture lysis showed that heat treatment of cells results in a decrease oflysis depth and a reduction of enzyme activity. Pattern and effectiveness of lysis of cells incubated with chloroform approached curve of live streptococci lysis, however this method did not exclude lysis of part of cells and required presence of equipment for work with chemical substances. CONCLUSION. S. pyogenes test culture inactivation method by 2-step treatment of culture with antibiotics that does not disrupt the integrity of surface structure of cells and provides optimal reproducibility of enzyme preparation activity test results was developed.
Asunto(s)
Antibacterianos/farmacología , Bacteriólisis/efectos de los fármacos , Nefelometría y Turbidimetría/métodos , Fagos de Streptococcus/enzimología , Streptococcus pyogenes/enzimología , Cloroformo , Gentamicinas/farmacología , Calor , Cinética , Reproducibilidad de los Resultados , Rifampin/farmacología , Fagos de Streptococcus/genética , Streptococcus pyogenes/química , Streptococcus pyogenes/efectos de los fármacos , Streptococcus pyogenes/crecimiento & desarrollo , Streptococcus pyogenes/aislamiento & purificaciónRESUMEN
With the aim to study solvation effects in peptide structure organization, the energy of different types of hydration in simple amines and amides has been analyzed. It was shown based on the quantum-chemical DFT and PM3 calculations of amino derivatives CH3-(CH2)3-NH2, (CH3)2-NH, CH3-NH2, NH3, CH2=CH-NH2, H-CC-NH2, O=C(CH)3-N(CH3)2, O=C(CH3)-NH(CH3), O=C(CH3)-NH2, O=CH-N(CH3)H, and O=CH-NH2 that: (1) in the given set of molecules, the proton acceptor N...H-O variant of hydrogen bonding of NH2-group with a water molecule is dominating only for the simplest amines. Being primordially weaker, the proton donor N-H...OH variant of water H-bonding gradually increases in energy in the given set as the basicity of the compound decreases, and for the case of amides of carbon acids it becomes already a significant channel of the hydration; (2) the intermolecular N-H...O=C bonding of trans-N-methylacetamides, which models the peptide hydrogen bonds in proteins, induces a "planarization" of its initially nonplanar O=C-NH fragments. However, the addition of water molecules to the complex through the proton acceptor N...H-O variant of binding of N atom not only "restores", but even strengthens the "pyramidalization" of valent bonds of peptide groups in this place.
Asunto(s)
Amidas/química , Nitrógeno/química , Péptidos/química , Agua/química , Modelos QuímicosRESUMEN
Intense efforts in the last decades have resulted in numerous inventions to improve drug delivery to CNS. Some of them have significant potential for clinical applications. At the same time, the variety of these strategies is in itself indicative of great difficulties inherent in the transfer of therapeutic and imaging agents across the blood-brain barrier. Combination of several approaches, such as encapsulation of drugs in nanocontainers, incorporation in micellar nanostructures, the use of Pluronic "unimers" for inhibition of drug efflux in endothelial cells of brain capillaries, appears to be the most promising modality. The same refers to the methods of intracellular transport of drug suspensions developed recently. All these achievements provide a solid basis for the further improvement of drug pharmacokinetic properties, reduction of systemic toxicity, increase of therapeutic efficacy, and earlier diagnosis of CNS pathology.
Asunto(s)
Encefalopatías/tratamiento farmacológico , Sistemas de Liberación de Medicamentos/instrumentación , Diseño de Fármacos , Nanopartículas/administración & dosificación , Nanotecnología/métodos , Animales , HumanosRESUMEN
Based on the results of quantum-chemical PM3 calculations, some common structural and thermodynamic properties of low-energy monopeptide conformers have been revealed that may be responsible for the initiation of alpha- or beta-like forms in polypeptide structures.
Asunto(s)
Aminoácidos/química , Péptidos/química , Pliegue de Proteína , Amidas/química , Metano/química , Estructura Secundaria de Proteína , Teoría Cuántica , TermodinámicaRESUMEN
On the basis of ab initio MP2 and semi-empirical PM3 quantum-chemical calculations the bistability of the nonplanar O=C-N-H fragment in the structure of simple amides and dipeptides is discussed. The influence of the nature of amino acids on the structural polymorphism of the peptide group in dipeptides is shown.
Asunto(s)
Amidas/química , Aminoácidos/química , Dipéptidos/química , Teoría CuánticaRESUMEN
Neurodegenerative and infectious disorders including Alzheimer's and Parkinson's diseases, amyotrophic lateral sclerosis, and stroke are rapidly increasing as population's age. Alzheimer's disease alone currently affects 4.5 million Americans, and more than $100 billion is spent per year on medical and institutional care for affected people. Such numbers will double in the ensuing decades. Currently disease diagnosis for all disorders is made, in large measure, on clinical grounds as laboratory and neuroimaging tests confirm what is seen by more routine examination. Achieving early diagnosis would enable improved disease outcomes. Drugs, vaccines or regenerative proteins present "real" possibilities for positively affecting disease outcomes, but are limited in that their entry into the brain is commonly restricted across the blood-brain barrier. This review highlights how these obstacles can be overcome by polymer science and nanotechnology. Such approaches may improve diagnostic and therapeutic outcomes. New developments in polymer science coupled with cell-based delivery strategies support the notion that diseases that now have limited therapeutic options can show improved outcomes by advances in nanomedicine.
RESUMEN
To elucidate the mechanism of protein thermostabilization, the thermodynamic properties of small monomeric proteins from mesophilic and thermophilic organisms have been analyzed. Molecular dynamics simulations were employed in the study of dynamic features of charged and polar side chains of amino acid residues. The basic conclusion has been made: surface charged and polar side chains with high conformational mobility can form alternative hydrogen bonded (H-bonded) donor-acceptor pairs. The correlation between the quantitative content of alternative H-bonds per residue and the temperature of maximal thermostability of proteins has been found. The proposed mechanism of protein thermostabilization suggests continuous disruption of the primary H-bonds and formation of alternative ones, which maintain constant the enthalpy value in the native state and prevent a rapid increase of the conformational entropy with the rising temperature. The analysis of the results show that the more residues located in the N- and C-terminal regions and in the extended loops that are capable of forming alternative longer-range H-bonded pairs, the higher the protein thermostability.
Asunto(s)
Proteínas/química , Simulación por Computador , Estabilidad de Medicamentos , Enlace de Hidrógeno , Modelos Moleculares , TermodinámicaRESUMEN
On the basis of the idea of the intrinsic polymorphism of Watson-Crick base pairing in DNA structure, the process of accumulation of the large electric dipole moment in model spiral stacks of canonical non-planar AT and GC pairs was analyzed using the quantum-chemistry methods. The dependence of the value and orientation of electrical dipole moment of a double helix on spiral length, geometry of base H-pairing, and the bending of the major axis of the helix were considered.
Asunto(s)
ADN/química , Modelos Moleculares , Animales , Emparejamiento Base , Electroquímica , Humanos , Conformación de Ácido NucleicoRESUMEN
In an effort to improve the efficacy of antisense delivery, we evaluated polyethyleneimine (PEI, 2 kDa) alone or grafted with nonionic amphiphilic block copolymer Pluronic (P85) as a carrier for Ku86 antisense oligonucleotide (ASO) delivery. Ku86 is an abundant nuclear protein that plays an important role in nonhomologous DNA end joining and has implications in tumorigenesis and acquired drug resistance. Transfection of adherent and suspension cell lines with Ku86 ASOs complexed with P85-g-PEI (2 kDa) conjugates was associated with a specific decrease in Ku86 mRNA levels (EC50<75 nM and EC50<250 nM, respectively, n=3). More importantly, no requirement for reduced serum conditions was necessary during transfection. In contrast, whereas Ku86 ASOs complexed with PEI (2 kDa) alone were effective in decreasing Ku86 mRNA levels in adherent cell lines (EC50<75 nM, n=3), the formulation did not produce any detectable decrease in Ku86 mRNA levels in suspension cell lines. Transfection of adherent cell lines with 500 nM Ku86 ASOs formulated with P85-g-PEI (2 kDa) was associated with a specific decrease (<10% remaining of control) in Ku86 protein expression and a two-fold increased cell death after treatment with ionizing radiation (IR). In athymic nude mice bearing subcutaneous human HT29 colon adenocarcinoma xenografts, Ku86 ASO-P85-g-PEI (2 kDa) administration (15 mg/kg, subcutaneously) with a Q1D x 7 treatment schedule, when combined with a single dose of IR (6 Gy), caused a significant inhibition of HT29 tumor growth compared with mismatch- and naked antisense-pretreated control groups (time from 200 to 1000 mm3, 126.9 versus 84.18 and 87.76 days, P<0.005). A potentiation of the antitumor activity was observed in all mice treated with Ku86 ASO-P85-g-PEI (2 kDa) formulation; however, tumor growth inhibition was reversible upon treatment cessation. No morbidity/mortality or changes in histopathology were observed under this treatment regiment. Our results indicate that P85-g-PEI (2 kDa) conjugates may increase the efficacy of Ku86 ASO delivery in management of resistant malignancies, thus providing a rationale for their evaluation in cancer patients in combination with conventional anticancer therapies.
Asunto(s)
Antígenos Nucleares/genética , Proteínas de Unión al ADN/genética , Terapia Genética/métodos , Neoplasias/terapia , Oligonucleótidos Antisentido/administración & dosificación , Transfección/métodos , Animales , Línea Celular Tumoral , Femenino , Expresión Génica , Humanos , Autoantígeno Ku , Ratones , Ratones Endogámicos BALB C , Trasplante de Neoplasias , Poloxaleno , Polietileneimina , Trasplante HeterólogoRESUMEN
The blood-brain barrier (BBB) efficiently restricts penetration of therapeutic agents to the brain from the periphery. Therefore, discovery of new modalities allowing for effective delivery of drugs and biomacromolecules to the central nervous system (CNS) is of great need and importance for treatment of neurodegenerative disorders. This manuscript focuses on three relatively new strategies. The first strategy involves inhibition of the drug efflux transporters expressed in BBB by Pluronic block copolymers, which allows for the increased transport of the substrates of these transporters to the brain. The second strategy involves the design of nanoparticles conjugated with specific ligands that can target receptors in the brain microvasculature and carry the drugs to the brain through the receptor mediated transcytosis. The third strategy involves artificial hydrophobization of peptides and proteins that facilitates the delivery of these peptides and proteins across BBB. This review discusses the current state, advantages and limitations of each of the three technologies and outlines their future prospects.
Asunto(s)
Barrera Hematoencefálica/metabolismo , Sistemas de Liberación de Medicamentos/métodos , Preparaciones Farmacéuticas/metabolismo , Tecnología Farmacéutica/métodos , Animales , Barrera Hematoencefálica/efectos de los fármacos , Sistemas de Liberación de Medicamentos/tendencias , Humanos , Unión Proteica/efectos de los fármacos , Unión Proteica/fisiología , Tecnología Farmacéutica/tendenciasRESUMEN
Passive targeting provides a simple strategy based on natural properties of the carriers to deliver DNA molecules to desired compartments. Polyethylenimine (PEI) is a potent non-viral system that has been known to deliver efficiently both plasmids and oligonucleotides (ODNs) in vitro. However, in vivo systemic administration of DNA/PEI complexes has encountered significant difficulties because these complexes are toxic and have low biodistribution in target tissues. This study evaluates PEI grafted with poly(ethylene oxide) (PEO(8K)-g-PEI(2K)) and PEI grafted with non-ionic amphiphilic block copolymer, Pluronic P85 (P85-g-PEI(2K)) as carriers for systemic delivery of ODNs. Following i.v. injection an antisense ODN formulated with PEO(8K)-g-PEI(2K) accumulated mainly in kidneys, while the same ODN formulated with P85-g-PEI(2K) was found almost exclusively in the liver. Furthermore, in the case of the animals injected with the P85-g-PEI(2K)-based complexes most of the ODN was found in hepatocytes, while only a minor portion of ODN was found in the lymphocyte/monocyte populations. The results of this study suggest that formulating ODN with PEO(8K)-g-PEI(2K) and P85-g-PEI(2K) carriers allows targeting of the ODN to the liver or kidneys, respectively. The variation in the tissue distribution of ODN observed with the two carriers is probably due to the different hydrophilic-lipophilic balance of the polyether chains grafted to PEI in these molecules. Therefore, polyether-grafted PEI carriers provide a simple way to enhance ODN accumulation in a desired compartment without the need of a specific targeting moiety.