29 research outputs found
Structure and Dynamics of Thermosensitive pDNA Polyplexes Studied by Time-Resolved Fluorescence Spectroscopy
Combining multiple stimuli-responsive functionalities into the polymer design is an attractive approach to improve nucleic acid delivery. However, more in-depth fundamental understanding how the multiple functionalities in the polymer structures are influencing polyplex formation and stability is essential for the rational development of such delivery systems. Therefore, in this study the structure and dynamics of thermosensitive polyplexes were investigated by tracking the behavior of labeled plasmid DNA (pDNA) and polymer with time-resolved fluorescence spectroscopy using fluorescence resonance energy transfer (FRET). The successful synthesis of a heterofunctional poly(ethylene glycol) (PEG) macroinitiator containing both an atom transfer radical polymerization (ATRP) and reversible addition-fragmentation chain-transfer (RAFT) initiator is reported. The use of this novel PEG macroinitiator allows for the controlled polymerization of cationic and thermosensitive linear triblock copolymers and labeling of the chain-end with a fluorescent dye by maleimide-thiol chemistry. The polymers consisted of a thermosensitive poly(N-isopropylacrylamide) (PNIPAM, N), hydrophilic PEG (P), and cationic poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA, D) block, further referred to as NPD. Polymer block D chain-ends were labeled with Cy3, while pDNA was labeled with FITC. The thermosensitive NPD polymers were used to prepare pDNA polyplexes, and the effect of the N/P charge ratio, temperature, and composition of the triblock copolymer on the polyplex properties were investigated, taking nonthermosensitive PD polymers as the control. FRET was observed both at 4 and 37 degrees C, indicating that the introduction of the thermosensitive PNIPAM block did not compromise the polyplex structure even above the polymer's cloud point. Furthermore, FRET results showed that the NPD- and PD-based polyplexes have a less dense core compared to polyplexes based on cationic homopolymers (such as PEI) as reported before. The polyplexes showed to have a dynamic character meaning that the polymer chains can exchange between the polyplex core and shell. Mobility of the polymers allow their uniform redistribution within the polyplex and this feature has been reported to be favorable in the context of pDNA release and subsequent improved transfection efficiency, compared to nondynamic formulations.Peer reviewe
Π‘ΡΠ°ΡΠ΅Π²Ρ ΠΎΡΠΎΠ±Π»ΠΈΠ²ΠΎΡΡΡ ΠΌΠ΅ΡΠ°Π±ΠΎΠ»ΡΠ·ΠΌΡ ΠΌΡΠΎΠΊΠ°ΡΠ΄Π° Π² Π΄ΠΈΠ½Π°ΠΌΡΡΡ ΡΠΎΠ·Π²ΠΈΡΠΊΡ Π΅ΠΊΡΠΏΠ΅ΡΠΈΠΌΠ΅Π½ΡΠ°Π»ΡΠ½ΠΎΠ³ΠΎ Π³ΡΠΏΠ΅ΡΡΠΈΡΠ΅ΠΎΠ·Ρ
Π ΡΠΊΡΠΏΠ΅ΡΠΈΠΌΠ΅Π½ΡΠ°Ρ
Π½Π° ΠΏΠΎΠ»ΠΎΠ²ΠΎΠ·ΡΠ΅Π»ΡΡ
ΡΠ°ΠΌΡΠ°Ρ
ΠΈ ΡΠ°ΠΌΠΊΠ°Ρ
ΠΊΡΡΡ ΠΈΡΡΠ»Π΅Π΄ΠΎΠ²Π°Π½ΠΎ Π°ΠΊΡΠΈΠ²Π½ΠΎΡΡΡ ΠΏΠ΅ΡΠ΅ΠΊΠΈΡΠ½ΠΎΠ³ΠΎ ΠΎΠΊΠΈΡΠ»Π΅Π½Π½Ρ Π»ΠΈΠΏΠΈΠ΄ΠΎΠ², Π°Π½ΡΠΈΠΎΠΊΡΠΈΠ΄Π°Π½ΡΠ½ΠΎΠΉ Π·Π°ΡΠΈΡΡ ΠΈ ΡΠ½Π΅ΡΠ³ΠΎΠΎΠ±ΡΠ°Π·ΠΎΠ²Π°Π½ΠΈΡ Π² Π΄ΠΈΠ½Π°ΠΌΠΈΠΊΠ΅ ΡΠ°Π·Π²ΠΈΡΠΈΡ ΡΠΈΡΠΎΠΊΡΠΈΠ½ΠΎΠ²ΠΎΠΉ ΠΊΠ°ΡΠ΄ΠΈΠΎΠΌΠΈΠΎΠΏΠ°ΡΠΈΠΈ, ΠΊΠΎΡΠΎΡΡΡ ΠΌΠΎΠ΄Π΅Π»ΠΈΡΠΎΠ²Π°Π»ΠΈ Π²Π²Π΅Π΄Π΅Π½ΠΈΠ΅ΠΌ L-ΡΠΈΡΠΎΠΊΡΠΈΠ½Π° (500 ΠΌΠ³/ΠΊΠ³, Π²Π½ΡΡΡΠΈΠΆΠ΅Π»ΡΠ΄ΠΎΡΠ½ΠΎ, Π΅ΠΆΠ΅Π΄Π½Π΅Π²Π½ΠΎ). Π ΠΌΠΈΠΎΠΊΠ°ΡΠ΄Π΅ ΠΆΠ΅Π»ΡΠ΄ΠΎΡΠΊΠΎΠ² ΡΠ΅ΡΠ΅Π· 5, 10 ΠΈ 15 Π΄Π½Π΅ΠΉ Ρ Π½Π°ΡΠ°Π»Π° ΡΠΊΡΠΏΠ΅ΡΠΈΠΌΠ΅Π½ΡΠ° ΠΎΠΏΡΠ΅Π΄Π΅Π»ΡΠ»ΠΈ ΡΠΎΠ΄Π΅ΡΠΆΠ°Π½ΠΈΠ΅ Π΄ΠΈΠ΅Π½ΠΎΠ²ΡΡ
ΠΈ ΡΡΠΈΠ΅Π½ΠΎΠ²ΡΡ
ΠΊΠΎΠ½ΡΡΠ³Π°Ρ (ΠΠ, Π’Π), Π’ΠΠ-Π°ΠΊΡΠΈΠ²Π½ΡΡ
ΠΏΡΠΎΠ΄ΡΠΊΡΠΎΠ² (Π’ΠΠ-ΠΠ), Π°ΠΊΡΠΈΠ²Π½ΠΎΡΡΡ ΡΡΠΏΠ΅ΡΠΎΠΊΡΠΈΠ΄Π΄ΠΈΡΠΌΡΡΠ°Π·Ρ (Π‘ΠΠ), ΠΊΠ°ΡΠ°Π»Π°Π·Ρ, Π³Π»ΡΡΠ°ΡΠΈΠΎΠ½ΠΏΠ΅ΡΠΎΠΊΡΠΈΠ΄Π°Π·Ρ (ΠΠ) ΠΈ Π³Π»ΡΡΠ°ΡΠΈΠΎΠ½ΡΠ΅Π΄ΡΠΊΡΠ°Π·Ρ (ΠΠ ), ΡΡΠΊΡΠΈΠ½Π°ΡΠ΄Π΅Π³ΠΈΠ΄ΡΠΎΠ³Π΅Π½Π°Π·Ρ (Π‘ΠΠ), ΡΠΈΡΠΎΡ
ΡΠΎΠΌΠΎΠΊΡΠΈΠ΄Π°Π·Ρ (Π¦Π). Π£ΡΡΠ°Π½ΠΎΠ²ΠΈΠ»ΠΈ, ΡΡΠΎ Π³ΠΈΠΏΠ΅ΡΡΠΈΡΠΎΠΊΡΠΈΠ½Π΅ΠΌΠΈΡ Π²ΠΈΠ·ΡΠ²Π°Π»Π° Π½Π°ΠΊΠΎΠΏΠ»Π΅Π½ΠΈΠ΅ Π² ΠΌΠΈΠΎΠΊΠ°ΡΠ΄Π΅ ΠΆΠ΅Π»ΡΠ΄ΠΎΡΠΊΠΎΠ² ΠΊΡΡΡ ΠΠ, Π’Π ΠΈ Π’ΠΠ-ΠΠ, ΡΡΠΎ Π² ΡΠ°ΠΌΠΎΠΊ Π±ΡΠ»ΠΎ Π±ΠΎΠ»Π΅Π΅ ΡΡΡΠ΅ΡΡΠ²Π΅Π½Π½ΡΠΌ, ΡΠ΅ΠΌ Ρ ΡΠ°ΠΌΡΠΎΠ², Π½Π΅ΡΠΌΠΎΡΡΡ Π½Π° Π±ΠΎΠ»ΡΡΡΡ Π°ΠΊΡΠΈΠ²Π½ΠΎΡΡΡ ΠΠ ΡΠ° ΠΠ . ΠΠ΅Π΄ΠΎΡΡΠ°ΡΠΎΡΠ½Π°Ρ ΠΏΡΠΎΡΠ΅ΠΊΡΠΎΡΠ½Π°Ρ ΡΡΡΠ΅ΠΊΡΠΈΠ²Π½ΠΎΡΡΡ ΡΠ΅ΡΠΌΠ΅Π½ΡΠΎΠ² ΡΠΈΡΡΠ΅ΠΌΡ Π³Π»ΡΡΠ°ΡΠΈΠΎΠ½Π° Π±ΡΠ»Π° ΡΠ΅Π·ΡΠ»ΡΡΠ°ΡΠΎΠΌ Π·Π½Π°ΡΠΈΡΠ΅Π»ΡΠ½ΠΎΠ³ΠΎ ΡΠ³Π½Π΅ΡΠ΅Π½ΠΈΡ Π°ΠΊΡΠΈΠ²Π½ΠΎΡΡΠΈ Π‘ΠΠ ΠΈ ΠΊΠ°ΡΠ°Π»Π°Π·Ρ, ΡΡΠΎ ΡΠ²ΠΈΠ΄Π΅ΡΠ΅Π»ΡΡΡΠ²ΠΎΠ²Π°Π»ΠΎ ΠΎ ΡΡΡΠ΅ΡΡΠ²Π΅Π½Π½ΠΎΠΌ Π½Π°ΠΊΠΎΠΏΠ»Π΅Π½ΠΈΠΈ Π°ΠΊΡΠΈΠ²Π½ΡΡ
ΡΠΎΡΠΌ ΠΊΠΈΡΠ»ΠΎΡΠΎΠ΄Π°. ΠΠΊΡΠΈΠ²Π½ΠΎΡΡΡ ΡΠ½Π΅ΡΠ³ΠΎΠΎΠ±ΡΠ°Π·ΠΎΠ²Π°Π½ΠΈΡ Π² ΡΠ°ΠΊΠΈΡ
ΡΡΠ»ΠΎΠ²ΠΈΡΡ
ΡΠΌΠ΅Π½ΡΡΠ°Π»Π°ΡΡ, ΠΎ ΡΠ΅ΠΌ ΡΠ²ΠΈΠ΄Π΅ΡΠ΅Π»ΡΡΡΠ²ΠΎΠ²Π°Π»ΠΎ ΡΠ³Π½Π΅ΡΠ΅Π½ΠΈΠ΅ Π°ΠΊΡΠΈΠ²Π½ΠΎΡΡΠΈ Π‘ΠΠ, ΠΊΠΎΡΠΎΡΠΎΠ΅ Π±ΡΠ»ΠΎ Π°Π½Π°Π»ΠΎΠ³ΠΈΡΠ½ΡΠΌ Π² ΡΠ°ΠΌΡΠΎΠ² ΠΈ ΡΠ°ΠΌΠΎΠΊ, ΠΈ Π¦Π, ΡΡΠΎ Π±ΡΠ»ΠΎ Π±ΠΎΠ»Π΅Π΅ ΡΡΡΠ΅ΡΡΠ²Π΅Π½Π½ΡΠΌ Π² ΡΠ°ΠΌΠΎΠΊ. ΠΠΎΠ»ΡΡΠ΅Π½Π½ΡΠ΅ ΡΠ΅Π·ΡΠ»ΡΡΠ°ΡΡ ΡΠ²ΠΈΠ΄Π΅ΡΠ΅Π»ΡΡΡΠ²ΡΡΡ ΠΎ ΡΠΎΠΌ, ΡΡΠΎ ΡΠ°Π·Π²ΠΈΡΠΈΠ΅ ΡΠΈΡΠΎΠΊΡΠΈΠ½ΠΎΠ²ΠΎΠΉ ΠΊΠ°ΡΠ΄ΠΈΠΎΠΌΠΈΠΎΠΏΠ°ΡΠΈΠΈ Π²ΡΠ·ΡΠ²Π°Π΅Ρ ΡΡΡΠ΅ΡΡΠ²Π΅Π½Π½ΡΠΉ ΠΌΠ΅ΡΠ°Π±ΠΎΠ»ΠΈΡΠ΅ΡΠΊΠΈΠΉ Π΄ΠΈΡΠ±Π°Π»Π°Π½Ρ Π² ΠΌΠΈΠΎΠΊΠ°ΡΠ΄Π΅ ΠΆΠ΅Π»ΡΠ΄ΠΎΡΠΊΠΎΠ² ΡΠ°ΠΌΠΎΠΊ ΠΊΡΡΡ, ΡΡΠΎ ΡΠ°Π·ΡΠ΅ΡΠ°Π΅Ρ ΠΎΠΆΠΈΠ΄Π°ΡΡ Π±ΠΎΠ»Π΅ΠΌ ΠΈΠ½ΡΠ΅Π½ΡΠΈΠ²Π½ΡΠ΅ ΡΡΡΡΠΊΡΡΡΠ½ΡΠ΅ Π½Π°ΡΡΡΠ΅Π½ΠΈΡ.Lipid peroxidation, antioxidant protection and energy production were studied in adult male and female rats with thyroxin cardiomyopathy, which simulated by introduction of L-thyroxine (500 mg / kg, intraperitoneally, daily). In the myocardium of the ventricles after 5, 10 and 15 days from the start of the experiment determined the content of diene and triene conjugate (DC, TC), TBA-active metabolits (TBA-am), activity of superoxide dismutase (SOD), catalase, glutathione peroxidase (GP) and glutathione reductase (GR), succinate dehydrogenase (SDH), cytochrome oxidase (CO). Found that hyperthyroxinemia caused accumulation in myocardium of the ventricles DC, TC and TBA-am mostly in females despite the higher activity of GP and GR. Lack of protective effects of glutathione system enzymes resulted from significant inhibition of SOD and catalase, indicating a significant accumulation of reactive species of oxygen. Activity of the energy production in these conditions decreased. That was proved by the inhibition of SDH in myocardium both sex animals and CO mostly in females. We conclude that the development of thyroxin cardiomyopathy causes metabolic disbalance in myocardium of the ventricles mostly in female rats, which can results in more intense structural damage
Three-dimensional cathodoluminescence imaging and electron backscatter diffraction: tools for studying the genetic nature of diamond inclusions
As a step towards resolving the genesis of inclusions in diamonds, a new technique is presented. This technique combines cathodoluminescence (CL) and electron backscatter diffraction (EBSD) using a focused ion beam-scanning electron microscope (FIB-SEM) instrument with the aim of determining, in detail, the three-dimensional diamond zonation adjacent to a diamond inclusion. EBSD reveals that mineral inclusions in a single diamond have similar crystallographic orientations to the host, within Β±0. 4Β°. The chromite inclusions record a systematic change in Mg# and Cr# from core to the rim of the diamond that corresponds with a ~80Β°C decrease of their formation temperature as established by zinc thermometry. A chromite inclusion, positioned adjacent to a boundary between two major diamond growth zones, is multi-faceted with preferred octahedral and cubic faces. The chromite is surrounded by a volume of non-luminescent diamond (CL halo) that partially obscures any diamond growth structures. The CL halo has apparent crystallographic morphology with symmetrically oriented pointed features. The CL halo is enriched in ~200 ppm Cr and ~80 ppm Fe and is interpreted to have a secondary origin as it overprints a major primary diamond growth structure. The diamond zonation adjacent to the chromite is complex and records both syngenetic and protogenetic features based on current inclusion entrapment models. In this specific case, a syngenetic origin is favoured with the complex form of the inclusion and growth layers indicating changes of growth rates at the diamond-chromite interface. Combined EBSD and 3D-CL imaging appears an extremely useful tool in resolving the ongoing discussion about the timing of inclusion growth and the significance of diamond inclusion studies. Β© 2010 The Author(s)
Landbouwgrond met hoge natuurwaarden in Nederland op de kaart
In dit onderzoek is bepaald hoe landbouwgrond met hoge natuurwaarden, ofwel High Nature Value (HNV) landbouwgebieden, in Nederland precies begrensd kan worden, wat de omvang is en wat de belangrijkste kenmerken zijn. In EU-verband is het voorts mogelijk cofinanciering te verkrijgen voor HNV-gebieden binnen POP verband. Gebleken is dat HNV gebieden in Nederland ongeveer tussen de 15% en 20% van het landbouwareaal kunnen beslaan. HNV gebieden liggen vooral in de provincies Drenthe, Friesland en Noord- en Zuid-Holland. Ze overlappen vooral met de nattige maar kleinschalige veenweidegebieden die belangrijk zijn voor broedvogels en overwinteraars, maar ook met de meer open nattere graslanden en akkerbouwgebieden die een belangrijke functie hebben voor overwinterende ganzen en smienten en de meer kleinschalige agrarische landschappen waar specifieke zeldzamere vogel- en vegetatiesoorten voorkomen
PEGylated and targeted extracellular vesicles display enhanced cell specificity and circulation time
Extracellular vesicles (EVs) are increasingly being recognized as candidate drug delivery systems due to their ability to functionally transfer biological cargo between cells. However, the therapeutic applicability of EVs may be limited due to a lack of cell-targeting specificity and rapid clearance of exogenous EVs from the circulation. In order to improve EV characteristics for drug delivery to tumor cells, we have developed a novel method for decorating EVs with targeting ligands conjugated to polyethylene glycol (PEG). Nanobodies specific for the epidermal growth factor receptor (EGFR) were conjugated to phospholipid (DMPE)-PEG derivatives to prepare nanobody-PEG-micelles. When micelles were mixed with EVs derived from Neuro2A cells or platelets, a temperature-dependent transfer of nanobody-PEG-lipids to the EV membranes was observed, indicative of a 'post-insertion' mechanism. This process did not affect EV morphology, size distribution, or protein composition. After introduction of PEG-conjugated control nanobodies to EVs, cellular binding was compromised due to the shielding properties of PEG. However, specific binding to EGFR-overexpressing tumor cells was dramatically increased when EGFR-specific nanobodies were employed. Moreover, whereas unmodified EVs were rapidly cleared from the circulation within 10 min after intravenous injection in mice, EVs modified with nanobody-PEG-lipids were still detectable in plasma for longer than 60 min post-injection. In conclusion, we propose post-insertion as a novel technique to confer targeting capacity to isolated EVs, circumventing the requirement to modify EV-secreting cells. Importantly, insertion of ligand-conjugated PEG-derivatized phospholipids in EV membranes equips EVs with improved cell specificity and prolonged circulation times, potentially increasing EV accumulation in targeted tissues and improving cargo delivery
PEGylated and targeted extracellular vesicles display enhanced cell specificity and circulation time
Extracellular vesicles (EVs) are increasingly being recognized as candidate drug delivery systems due to their ability to functionally transfer biological cargo between cells. However, the therapeutic applicability of EVs may be limited due to a lack of cell-targeting specificity and rapid clearance of exogenous EVs from the circulation. In order to improve EV characteristics for drug delivery to tumor cells, we have developed a novel method for decorating EVs with targeting ligands conjugated to polyethylene glycol (PEG). Nanobodies specific for the epidermal growth factor receptor (EGFR) were conjugated to phospholipid (DMPE)-PEG derivatives to prepare nanobody-PEG-micelles. When micelles were mixed with EVs derived from Neuro2A cells or platelets, a temperature-dependent transfer of nanobody-PEG-lipids to the EV membranes was observed, indicative of a 'post-insertion' mechanism. This process did not affect EV morphology, size distribution, or protein composition. After introduction of PEG-conjugated control nanobodies to EVs, cellular binding was compromised due to the shielding properties of PEG. However, specific binding to EGFR-overexpressing tumor cells was dramatically increased when EGFR-specific nanobodies were employed. Moreover, whereas unmodified EVs were rapidly cleared from the circulation within 10 min after intravenous injection in mice, EVs modified with nanobody-PEG-lipids were still detectable in plasma for longer than 60 min post-injection. In conclusion, we propose post-insertion as a novel technique to confer targeting capacity to isolated EVs, circumventing the requirement to modify EV-secreting cells. Importantly, insertion of ligand-conjugated PEG-derivatized phospholipids in EV membranes equips EVs with improved cell specificity and prolonged circulation times, potentially increasing EV accumulation in targeted tissues and improving cargo delivery
PEGylated and targeted extracellular vesicles display enhanced cell specificity and circulation time
Extracellular vesicles (EVs) are increasingly being recognized as candidate drug delivery systems due to their ability to functionally transfer biological cargo between cells. However, the therapeutic applicability of EVs may be limited due to a lack of cell-targeting specificity and rapid clearance of exogenous EVs from the circulation. In order to improve EV characteristics for drug delivery to tumor cells, we have developed a novel method for decorating EVs with targeting ligands conjugated to polyethylene glycol (PEG). Nanobodies specific for the epidermal growth factor receptor (EGFR) were conjugated to phospholipid (DMPE)-PEG derivatives to prepare nanobody-PEG-micelles. When micelles were mixed with EVs derived from Neuro2A cells or platelets, a temperature-dependent transfer of nanobody-PEG-lipids to the EV membranes was observed, indicative of a 'post-insertion' mechanism. This process did not affect EV morphology, size distribution, or protein composition. After introduction of PEG-conjugated control nanobodies to EVs, cellular binding was compromised due to the shielding properties of PEG. However, specific binding to EGFR-overexpressing tumor cells was dramatically increased when EGFR-specific nanobodies were employed. Moreover, whereas unmodified EVs were rapidly cleared from the circulation within 10 min after intravenous injection in mice, EVs modified with nanobody-PEG-lipids were still detectable in plasma for longer than 60 min post-injection. In conclusion, we propose post-insertion as a novel technique to confer targeting capacity to isolated EVs, circumventing the requirement to modify EV-secreting cells. Importantly, insertion of ligand-conjugated PEG-derivatized phospholipids in EV membranes equips EVs with improved cell specificity and prolonged circulation times, potentially increasing EV accumulation in targeted tissues and improving cargo delivery