RESUMEN

BACKGROUND: Nanomedicine and the pharmaceutical industry demand the investigation of new biomaterials to improve drug therapies. Combinations of lipids, proteins, and polymers represent innovative platforms for drug delivery. However, little is known about the interactions between such compounds and this knowledge is key to prepare successful drug delivery systems. METHODS: Biophysical properties of biohybrid vesicles (BhVs) composed of phospholipids, proteins, and amphiphilic block copolymers, assembled without using organic solvents, were investigated by differential scanning calorimetry and dynamic light scattering. We studied four biohybrid systems; two of them included the effect of incorporating tetracaine. Thermal changes of phospholipids and proteins when interacting with the amphiphilic block copolymers and tetracaine were analyzed. RESULTS: Lysozyme and the copolymers adsorb onto the lipid bilayer modifying the phase transition temperature, enthalpy change, and cooperativity. Dynamic light scattering investigations revealed relevant changes in the size and zeta potential of the BhVs. Interestingly, tetracaine, a membrane-active drug, can fluidize or rigidize BhVs. CONCLUSIONS: We conclude that positively charged regions of lysozyme are necessary to incorporate the block copolymer chains into the lipid membrane, turning the bilayer into a more rigid system. Electrostatic properties and the hydrophilic-lipophilic balance are determinant for the stability of biohybrid membranes. GENERAL SIGNIFICANCE: This investigation provides fundamental information associated with the performance of biohybrid drug delivery systems and can be of practical significance for designing more efficient drug nanocarriers.

Asunto(s)

Liposomas , Polímeros , Liposomas/química , Polímeros/química , Tetracaína , Muramidasa , Membrana Dobles de Lípidos/química , Fosfolípidos/química , Proteínas

RESUMEN

Nowadays many drugs with improved therapeutic efficacy are discovered but cannot be utilized due to their low solubility and insufficient bioavailability. An example of such a drug molecule is a protein kinase C inhibitor that influences an enzyme which plays an important role in several signal transduction cascades. The aim of this study was to formulate a stable nanoparticle dispersion of the PKC inhibitor encapsulated into PLGA nanoparticles (NPs). Encapsulation of the PKC inhibitor into PLGA NPs of 100-200 nm diameter should provide a targeted delivery to the inflammation sites. The NPs were prepared via nanoprecipitation and different surfactants were investigated: Fully and partially hydrolyzed poly(vinyl alcohol) (PVA, Mowiol X-88 and X-98), poloxamers (Pluronic F68 and F127) and polysorbates (Tween 20 and 80). From all surfactants tested, only NPs prepared with partially hydrolyzed PVA (Mowiol X-88) provided the desired stability throughout the downstream processes. These NPs were subsequently analyzed regarding their particle size, polydispersity, encapsulation efficiency and loading capacity. Dynamic light scattering results revealed that monodisperse NPs of 150-220 nm were formed, a size range that favors targeted delivery. The drug encapsulation efficiency varied from 31 to 75% with a drug loading of 1.3-2%. Moreover, the long-term stability was studied and the residual amount of PVA of the NP solutions was quantified via nuclear magnetic resonance (NMR) measurements. The shell-less hen's egg model was used to test toxic effects (hemorrhage, vascular lysis, thrombosis, hemolysis and lethality) of the NPs in a more complex biological system under dynamic flow conditions.

Asunto(s)

Indoles/química , Maleimidas/química , Nanopartículas/química , Polímeros/química , Tensoactivos/química , Animales , Pollos , Estabilidad de Medicamentos , Agregación Eritrocitaria/efectos de los fármacos , Eritrocitos/efectos de los fármacos , Hemólisis/efectos de los fármacos , Indoles/administración & dosificación , Maleimidas/administración & dosificación , Nanopartículas/administración & dosificación , Tamaño de la Partícula , Polímeros/administración & dosificación , Proteína Quinasa C/antagonistas & inhibidores , Ovinos , Tensoactivos/administración & dosificación , Cigoto/efectos de los fármacos

RESUMEN

Simultaneous assessment of excretory liver and kidney function is still an unmet need in experimental stress models as well as in critical care. The aim of the study was to characterize two polymethine-dyes potentially suitable for this purpose in vivo. Plasma disappearance rate and elimination measurements of simultaneously injected fluorescent dyes DY-780 (hepato-biliary elimination) and DY-654(renal elimination) were conducted using catheter techniques and intravital microscopy in animals subjected to different organ injuries, i.e. polymicrobial sepsis by peritoneal contamination and infection, ischemia-reperfusion-injury and glycerol-induced acute kidney-injury. DY-780 and DY-654 showed organ specific and determined elimination routes in both healthy and diseased animals. They can be measured simultaneously using near-infrared imaging and spectrophotometry. Plasma-disappearance rates of DY-780 and DY-654 are superior to conventional biomarkers in indicating hepatic or kidney dysfunction in different animal models. Greatest impact on liver function was found in animals with polymicrobial sepsis whereas glomerular damage due to glycerol-induced kidney-injury had strongest impact on DY-654 elimination. We therefore conclude that hepatic elimination and renal filtration can be assessed in rodents measuring plasma-disappearance rates of both dyes. Further, assessment of organ dysfunction by polymethine dyes correlates with, but outperforms conventional biomarkers regarding sensitivity and the option of spatial resolution if biophotonic strategies are applied. Polymethine-dye clearance thereby allows sensitive point-of-care assessment of both organ functions simultaneously.

Asunto(s)

Colorantes Fluorescentes , Indoles , Riñón , Hepatopatías , Hígado , Insuficiencia Renal Crónica , Enfermedad Aguda , Lesión Renal Aguda/diagnóstico por imagen , Lesión Renal Aguda/metabolismo , Lesión Renal Aguda/fisiopatología , Animales , Enfermedad Crónica , Colorantes Fluorescentes/farmacocinética , Colorantes Fluorescentes/farmacología , Indoles/farmacocinética , Indoles/farmacología , Riñón/diagnóstico por imagen , Riñón/metabolismo , Riñón/fisiopatología , Pruebas de Función Renal , Hígado/diagnóstico por imagen , Hígado/metabolismo , Hígado/fisiopatología , Hepatopatías/diagnóstico por imagen , Hepatopatías/metabolismo , Hepatopatías/fisiopatología , Ratones , Insuficiencia Renal Crónica/diagnóstico por imagen , Insuficiencia Renal Crónica/metabolismo , Insuficiencia Renal Crónica/fisiopatología

RESUMEN

The self-healing polymer P(LMA-co-MeAMMA) crosslinked with C60-fullerene has been studied by FT-Raman spectroscopy in combination with two-dimensional (2D) correlation analysis and density functional theory calculations. To unveil the molecular changes during the self-healing process mediated by the Diels-Alder equilibrium between 10-methyl-9-anthracenyl groups and C60-fullerene different anthracene-C60-fullerene adducts have been synthesized and characterized by time-, concentration- and temperature-dependent FT-Raman measurements. The self-healing process could be monitored via the C60-fullerene vibrations at 270, 432 and 1469 cm(-1). Furthermore, the detailed analysis of the concentration-dependent FT-Raman spectra point towards the formation of anthracene-C60-fullerene adducts with an unusual high amount of anthracene bound to C60-fullerene in the polymer film, while the 2D correlation analysis of the temperature-dependent Raman spectra suggests a stepwise dissociation of anthracene-C60-fullerene adducts, which are responsible for the self-healing of the polymer.

RESUMEN

Matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI MSI) is a method that allows the investigation of the molecular content of surfaces, in particular, tissues, within its morphological context. The applications of MALDI MSI in the field of large-scale mass spectrometric studies, which are typically denoted by the suffix "omics", are steadily increasing. This is because, on the one hand, technical advances regarding sample collection and preparation, matrix application, instrumentation, and data processing have enhanced the molecular specificity and sensitivity of MALDI MSI; on the other hand, the focus of the "omics" community has moved from establishing an inventory of certain compound classes to exploring their spatial distribution to gain novel insights. Thus, the aim of this mini-review is twofold, to display the state-of-the-art in terms of technical aspects in MALDI MSI and to highlight selected applications in the last two years, which either have significantly advanced a certain "omics" field or have introduced a new one through pioneering efforts.

Asunto(s)

Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Animales , Encéfalo/metabolismo , Encéfalo/patología , Calcinosis/metabolismo , Calcinosis/patología , Ácido Cítrico/análisis , Humanos , Metabolómica , Neoplasias/metabolismo , Neoplasias/patología , Neuropéptidos/análisis , Proteómica

RESUMEN

The thermally healable polymer P(LMA-co-FMA-co-MIMA) has been studied by temperature-dependent FT-Raman spectroscopy, two-dimensional Raman correlation analysis and density functional theory (DFT) calculations. To the best of our knowledge this study reports for the first time on the investigation of a self-healing polymer by means of two-dimensional correlation techniques. The synchronous correlation spectrum reveals that the spectrally overlapping C[double bond, length as m-dash]C stretching vibrations at 1501, 1575, 1585 and 1600 cm(-1) are perfect marker bands to monitor the healing process which is based on a Diels-Alder reaction of furan and maleimide. The comparison between experimental and calculated Raman spectra as well as their correlation spectra showed a good agreement between experiment and theory. The data presented within this study nicely demonstrate that Raman correlation analysis combined with a band assignment based on DFT calculations presents a powerful tool to study the healing process of self-healing polymers.

RESUMEN

Polymer-based gene delivery systems have enormous potential in biomedicine, but their efficiency is often limited by poor biocompatibility. Poly(methacrylate)s (PMAs) are an interesting class of polymers which allow to explore structure-activity relationships of polymer functionalities for polyplex formation in oligonucleotide delivery. Here, we synthesized and tested a library of PMA polymers, containing functional groups contributing to the different steps of gene delivery, from oligonucleotide complexation to cellular internalization and endosomal escape. By variation of the molar ratios of the individual building blocks, the physicochemical properties of the polymers and polyplexes were fine-tuned to reduce toxicity as well as to increase activity of the polyplexes. To further enhance transfection efficiency, a cell-penetrating peptide (CPP)-like functionality was introduced on the polymeric backbone. With the ability to synthesize large libraries of polymers in parallel we also developed a workflow for a mid-to-high throughput screening, focusing first on safety parameters that are accessible by high-throughput approaches such as blood compatibility and toxicity towards host cells and only at a later stage on more laborious tests for the ability to deliver oligonucleotides. To arrive at a better understanding of the molecular basis of activity, furthermore, the effect of the presence of heparan sulfates on the surface of host cells was assessed and the mechanism of cell entry and intracellular trafficking investigated for those polymers that showed a suitable pharmacological profile. Following endocytic uptake, rapid endosomal release occurred. Interestingly, the presence of heparan sulfates on the cell surface had a negative impact on the activity of those polyplexes that were sensitive to decomplexation by heparin in solution. In summary, the screening approach identified two polymers, which form polyplexes with high stability and transfection capacity exceeding the one of poly(ethylene imine) also in the presence of serum.

Asunto(s)

Técnicas de Transferencia de Gen , Oligonucleótidos/química , Ácidos Polimetacrílicos/química , Supervivencia Celular/efectos de los fármacos , Endocitosis , Endosomas , Eritrocitos/efectos de los fármacos , Células HeLa , Hemólisis/efectos de los fármacos , Heparitina Sulfato/administración & dosificación , Heparitina Sulfato/química , Humanos , Luciferasas/genética , Oligonucleótidos/administración & dosificación , Ácidos Polimetacrílicos/administración & dosificación , Relación Estructura-Actividad

RESUMEN

Histopathologic differentiation of nodular lesions in cirrhotic liver is difficult even for experienced hepatopathologists especially regarding diagnosis of hepatocellular carcinoma (HCC) in biopsies. For this reason, new tissue markers are needed to reinforce histopathologic decision-making. With advances in molecular techniques, proteomic analysis may help to confirm the diagnosis of HCC. Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) is a powerful technology which allows to determine and to localize proteins directly in tissue sections. Using MALDI IMS proteomic patterns of cryosections with lesions of HCC (n = 15) and non-malignant fibrotic liver tissue (n = 11) were investigated to establish a classification model of HCC, which was validated in an independent set of tissue to distinguish HCC (n = 10) from regenerative nodules (n = 8). By correlating generated mass spectrometric images with the histology of the tissue sections we found that the expression of 4 proteins as indicated by m/z 6274, m/z 6647, m/z 6222 and m/z 6853 was significantly higher in HCC tissue than in non-tumorous liver tissue. The generated classification model based on the most significant 3 differentially expressed proteins allowed a reliable prediction of benign and malignant lesions in fibrotic liver tissue with a sensitivity and specificity of 90 % in the validation set. The identified MALDI IMS proteomic signature can be diagnostically helpful to allow simplifying the diagnostic process and minimize the risks of delays in establishing the objective final diagnosis and initiating treatment of patients with HCC.

Asunto(s)

Biomarcadores de Tumor/análisis , Carcinoma Hepatocelular/química , Cirrosis Hepática/metabolismo , Neoplasias Hepáticas/química , Proteínas de Neoplasias/análisis , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Carcinoma Hepatocelular/patología , Femenino , Humanos , Cirrosis Hepática/patología , Neoplasias Hepáticas/patología , Masculino , Imagen Molecular/métodos , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Distribución Tisular

RESUMEN

In order to achieve a comprehensive description of biological tissue, spectral information about proteins, lipids, nucleic acids, and other biochemical components need to be obtained concurrently. Different analytical techniques may be combined to record complementary information of the same sample. Established techniques, which can be utilized to elucidate the biochemistry of tissue samples are, for instance, MALDI-TOF-MS and Raman microscopic imaging. With this contribution, we combine these two techniques for the first time. The combination of both techniques allows the utilization and interpretation of complementary information (i.e., the information about the protein composition derived from the Raman spectra with data of the lipids analyzed by the MALDI-TOF measurements). Furthermore, we demonstrate how spectral information from MALDI-TOF experiments can be utilized to interpret Raman spectra.

Asunto(s)

Encéfalo/metabolismo , Lípidos/análisis , Proteínas/análisis , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Espectrometría Raman/métodos , Animales , Ratones

RESUMEN

Polymeric materials containing coordinative units have become a field of increasing interest. The combination of inorganic metal-containing units and macromolecules leads to supramolecular structures with new properties. One promising approach to such systems is the application of metallo-supramolecular initiators for living and controlled polymerization methods. The utilization of bi- and terpyridine units and complexes for this purpose will be discussed in this article.