RESUMEN
Semiconductor nanocrystals or quantum dots (QDs) have gained significant attention in biomedical research as versatile probes for imaging, sensing, and therapies. However, the interactions between proteins and QDs, which are crucial for their use in biological applications, are not yet fully understood. Asymmetric flow field-flow fractionation (AF4) is a promising method for analyzing the interactions of proteins with QDs. This technique uses a combination of hydrodynamic and centrifugal forces to separate and fractionate particles based on their size and shape. By coupling AF4 with other techniques, such as fluorescence spectroscopy and multi-angle light scattering, it is possible to determine the binding affinity and stoichiometry of protein-QD interactions. Herein, this approach has been utilized to determine the interaction between fetal bovine serum (FBS) and silicon quantum dots (SiQDs). Unlike metal-containing conventional QDs, SiQDs are highly biocompatible and photostable in nature, making them attractive for a wide range of biomedical applications. In this study, AF4 has provided crucial information on the size and shape of the FBS/SiQD complexes, their elution profile, and their interaction with serum components in real time. The differential scanning microcalorimetric technique has also been employed to monitor the thermodynamic behavior of proteins in the presence of SiQDs. We have investigated their binding mechanisms by incubating them at temperatures below and above the protein denaturation. This study yields various significant characteristics such as their hydrodynamic radius, size distribution, and conformational behavior. The compositions of SiQD and FBS influence the size distribution of their bioconjugates; the size increases by intensifying the concentration of FBS, with their hydrodynamic radii ranging between 150 and 300 nm. The results signify that in the alliance of SiQDs to the system, there is an augmentation of the denaturation point of the proteins and hence their thermal stability, providing a more comprehensive understanding of the interactions between FBS and QDs.
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Fraccionamiento de Campo-Flujo , Puntos Cuánticos , Puntos Cuánticos/química , Silicio , Albúmina Sérica Bovina/química , Fraccionamiento de Campo-Flujo/métodos , TemperaturaRESUMEN
The enthalpy and entropy of micellization in water, ΔHmic and ΔSmic, respectively, of three linear amphiphilic BAB block copolymers consisting of either poly(methyl acrylate) (Mn â¼ 1200 and 700 Da) or poly(ethyl acrylate) (Mn â¼ 800 Da) as hydrophobic (B) segments and poly(ethylene oxide) (PEO) as the hydrophilic (A, Mn â¼ 3000 Da) segment were determined by isothermal titration calorimetry (ITC). The ΔHmic and ΔSmic of the cyclic AB block copolymers obtained by cyclization of the linear triblock copolymers were determined under the same conditions. The ΔHmic value of the cyclic copolymers was smaller than that of their linear precursors. The ΔSmic value showed the same trend, but the relative difference between the cyclized and linear copolymers was less pronounced. The hydrodynamic diameter (Dh), critical micelle concentration (CMC), molecular weight (Mw-mic), and second virial coefficient (A2) of the micelles were determined. The Dh value of the cyclic copolymer micelles was smaller than the linear counterpart. On the other hand, the CMC value became larger, whereas the A2 value was comparable or increased by cyclization. Overall, the results suggest that, in the unimer state, the hydrophobic segments of the cyclized copolymers form a tightly coiled structure to minimize contact with water, resulting in the smaller ΔHmic value. Contrary to the linear copolymer micelles, the cyclic copolymer micelles have no "dangling chains", which may explain the topology-driven slight difference in the ΔSmic value.
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Thermoresponsive water-soluble polymers, aqueous solutions of which undergo lower critical solution temperature (LCST)-type phase separation, have been investigated in detail for several decades. To develop LCST-type thermoresponsive polymers with new polymer backbone, 4-azido-5-hexynamide (AHA) derivatives were designed as monomers for copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) polymerization. AHA derivatives carrying secondary amide side chains, that is, 4-azido-N-methyl-5-hexynamide (M), 4-azido-N-ethyl-5-hexynamide (E), and 4-azido-N-isopropyl-5-hexynamide (iP), were first synthesized and polymerized by CuAAC to obtain polymers (poly(M), poly(E), and poly(iP)). Contrary to our expectation, poly(M), poly(E), and poly(iP) were insoluble in water and many organic solvents presumably because of the formation of hydrogen bonding between the amide side chains or between the amide side chains and triazole residues in the backbone. Thus, AHA derivatives carrying tertiary amide side chains, that is, 4-azido-N,N-dimethyl-5-hexynamide (MM), 4-azido-N-ethyl-N-methyl-5-hexynamide (ME), 4-azido-N-isopropyl-N-methyl-5-hexynamide (MiP), and 4-azido-N,N-diethyl-5-hexynamide (EE), were also synthesized and polymerized to yield polymers (poly(MM), poly(ME), poly(MiP), and poly(EE)). These polymers were soluble in a number of common organic solvents. It is noteworthy that poly(MM) and poly(ME) were also soluble in water. The phase separation behavior of 1.0 wt % aqueous solutions of poly(MM) and poly(ME) was then investigated by transmittance measurements. These data indicated that poly(ME) was an LCST-type thermoresponsive polymer, whereas poly(MM) was not. A large hysteresis was observed in the transmittance measurements for the poly(ME) aqueous solution because of slow rehydration after phase separation. The phase separation behavior was investigated preliminarily by differential scanning calorimetry and 1H NMR.
RESUMEN
Thermoresponsive self-assembled nanogels were conveniently prepared by cholesterol end-capped poly(N-isopropylacrylamide) (PNIPAM) in water. Both cholesterol end-capped PNIPAMs (telelchelic cholesterol PNIPAM, tCH-PNIPAM) formed flower-like nanogels by the self-assembling of four to five polymer chains with multiple domains of cholesterol in water at 20 °C. Meanwhile, one end-group cholesterol-capped PNIPAM (semitelechelic cholesterol PNIPAM, stCH-PNIPAM) was also formed as a nanogel by the self-assembling of 15-20 polymer chains with 3 to 4 cholesterol domains. The hydrophobic cholesterol domains of tCH-PNIPAM nanogels were maintained above the lower critical solution temperature (LCST) of PNIPAM (>32 °C). Differently, the hydrophobic domains of stCH-PNIPAM were disrupted by cholesterol-free PNIPAM chain ends and formed large mesoglobules above the LCST. These transition controls of hydrophilic end-capped smart polymers may open new methodologies to design thermoresponsive nanosystems.
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Calor , Agua , Resinas Acrílicas , Colesterol/química , Nanogeles , Polímeros/química , TemperaturaRESUMEN
High-sensitivity differential scanning calorimetry (HS-DSC) thermograms of aqueous poly(N-isopropylacrylamide) (PNIPAM) solutions present a sharp unimodal endotherm that signals the heat-induced dehydration/collapse of the PNIPAM chain. Similarly, α,ω-di-n-octadecyl-PNIPAM (C18-PN-C18) aqueous solutions exhibit a unimodal endotherm. In contrast, aqueous solutions of α,ω-hydrophobically modified PNIPAMs with polycyclic terminal groups, such as pyrenylbutyl (Py-PN-Py), adamantylethyl (Ad-PN-Ad), and azopyridine- (C12-PN-AzPy) moieties, exhibit bimodal thermograms. The origin of the two transitions was probed using microcalorimetry measurements, turbidity tests, variable temperature 1H NMR (VT-NMR) spectroscopy, and 2-dimensional NOESY experiments with solutions of polymers of molar mass (M n) from 5 to 20 kDa and polymer concentrations of 0.1 to 3.0 mg/mL. The analysis outcome led us to conclude that the difference of the thermograms reflects the distinct self-assembly structures of the polymers. C18-PN-C18 assembles in water in the form of flower micelles held together by a core of tightly packed n-C18 chains. In contrast, polymers end-tagged with azopyridine, pyrenylbutyl, or adamantylethyl form a loose core that allows chain ends to escape from the micelles, to reinsert in them, or to dangle in surrounding water. The predominant low temperature (T 1) endotherm, which is insensitive to polymer concentration, corresponds to the dehydration/collapse of PNIPAM chains within the micelles, while the higher temperature (T 2) endotherm is attributed to the dehydration of dangling chains and intermicellar bridges. This study of the two phase transitions of telechelic PNIPAM homopolymer highlights the rich variety of morphologies attainable via responsive hydrophobically modified aqueous polymers and may open the way to a variety of practical applications.
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The equilibrium structures and optical properties of the photolabile caged luciferin, (7-diethylaminocoumarin-4-yl)methyl caged D-luciferin (DEACM-caged D-luciferin), in aqueous solution were investigated via quantum chemical calculations. The probable conformers of DEACM-caged D-luciferin were determined by potential energy curve scans and structural optimizations. We identified 40 possible conformers of DEACM-caged D-luciferin in water by comparing the Gibbs free energy of the optimized structures. Despite the difference in their structures, the conformers were similar in terms of assignments, oscillator strengths and energies of the three low-lying excited states. From the concentrations of the conformers and their oscillator strengths, we obtained a theoretical UV/Vis spectrum of DEACM-caged D-luciferin that has two main bands of shape nearly identical to the experimental UV/Vis spectrum. The absorption bands with maxima ~ 384 and 339 nm were attributed to the electronic excitations of the caged group and the luciferin moiety, respectively, by analysis of the theoretical UV/Vis spectrum. Furthermore, the analysis showed that DEACM-caged D-luciferin is excited in the caged group only by light of wavelength ranging within 400-430 nm, which is in the long-wavelength tail of the 384 nm band. This should be tested to lower damage upon photocleavage.
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Benzotiazoles/química , Cumarinas/química , Modelos Teóricos , Procesos Fotoquímicos , Espectrofotometría UltravioletaRESUMEN
Aqueous dispersions of nanogels that respond to switches in environmental pH and/or temperature by changes in their hydrodynamic radius (Rh ) and/or ζ-potential are prepared by reversible addition-fragmentation chain-transfer (RAFT) polymerization-induced thermal (70 °C) self-assembly (PITSA) of N-isopropylacrylamide (NIPAM) in the presence of a poly(methacrylic acid) (PMAA)-substituted macromolecular chain transfer agent and a cross-linker. Photochromic spiropyran (SP) moieties are coupled to the carboxylic acid groups of the nanogels. Upon UV irradiation, the neutral SP isomerizes to the zwitterionic merocyanine (ME) form. Upon UV light irradiation, microgels formed by assembly of SP nanogels undergo a collective motion toward the UV-light source.
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Acrilamidas/química , Nanogeles/química , Acrilamidas/síntesis química , Hidrodinámica , Concentración de Iones de Hidrógeno , Estructura Molecular , Tamaño de la Partícula , Procesos Fotoquímicos , Polimerizacion , Propiedades de Superficie , Temperatura , Rayos UltravioletaRESUMEN
Poly(2-isopropyl-2-oxazoline)-b-poly(lactide) (PiPOx-b-PLA) diblock copolymers comprise two miscible blocks: the hydrophilic and thermosensitive PiPOx and the hydrophobic PLA, a biocompatible and biodegradable polyester. They self-assemble in water, forming stable dispersions of nanoparticles with hydrodynamic radii (R h) ranging from â¼18 to 60 nm, depending on their molar mass, the relative size of the two blocks, and the configuration of the lactide unit. Evidence from 1H nuclear magnetic resonance spectroscopy, light scattering, small-angle neutron scattering, and cryo-transmission electron microscopy indicates that the nanoparticles do not adopt the typical core-shell morphology. Aqueous nanoparticle dispersions heated from 20 to 80 °C were monitored by turbidimetry and microcalorimetry. Nanoparticles of copolymers containing a poly(dl-lactide) block coagulated irreversibly upon heating to 50 °C, forming particles of various shapes (R h â¼ 200-500 nm). Dispersions of PiPOx-b-poly(l-lactide) coagulated to a lesser extent or remained stable upon heating. From the entire experimental evidence, we conclude that PiPOx-b-PLA nanoparticles consist of a core of PLA/PiPOx chains associated via dipole-dipole interactions of the PLA and PiPOx carbonyl groups. The core is surrounded by tethered PiPOx loops and tails responsible for the colloidal stability of the nanoparticles in water. While the core of all nanoparticles studied contains associated PiPOx and PLA blocks, fine details of the nanoparticles morphology vary predictably with the size and composition of the copolymers, yielding particles of distinctive thermosensitivity in aqueous dispersions.
RESUMEN
A series of azopyridine-terminated poly(N-isopropylacrylamide)s (PNIPAM) (C12-PN-AzPy) (â¼5000 < M w < 20 000 g mol-1, polydispersity index 1.25 or less) were prepared by reversible addition-fragmentation chain-transfer polymerization of NIPAM in the presence of a chain-transfer agent that contains an AzPy group and an n-dodecyl chain. In cold water, the polymers form nanoparticles (5.9 nm < R h < 10.9 nm) that were characterized by light scattering (LS), 1H NMR diffusion experiments, and high-resolution transmission electron microscopy. We monitored the pH-dependent photoisomerization of C12-PN-AzPy nanoparticles by steady-state and time-resolved UV-vis absorption spectroscopy. Azopyridine is known to undergo a very fast cis-to-trans thermal relaxation when the azopyridine nitrogen is quaternized or bound to a hydrogen bond donor. The cis-to-trans thermal relaxation of the AzPy chromophore in an acidic nanoparticle suspension is very fast with a half-life τ = 2.3 ms at pH 3.0. It slows down slightly for nanoparticles in neutral water (τ = 0.96 s, pH 7.0), and it is very slow for AzPy-PNIPAM particles in alkaline medium (τ > 3600 s, pH 10). The pH-dependent dynamics of the cis-to-trans dark relaxation, supported by Fourier transform infrared spectroscopy, 1H NMR spectroscopy, and LS analysis, suggest that in acidic medium, the nanoparticles consist of a core of assembled C12 chains surrounded by a shell of hydrated PNIPAM chains with the AzPy+ end groups preferentially located near the particle/water interface. In neutral medium, the shell surrounding the core contains AzPy groups H-bonded to the amide hydrogen of the PNIPAM chain repeat units. At pH 10.0, the amide hydrogen binds preferentially to the hydroxide anions. The AzPy groups reside preferentially in the vicinity of the C12 core of the nanoparticles. The morphology of the nanoparticles results from the competition between the segregation of the hydrophobic and hydrophilic components and weak attractive interactions, such as H-bonds between the AzPy groups and the amide hydrogen of the PNIPAM repeat units.
RESUMEN
At room temperature, poly(N-isopropylacrylamide) (PNIPAM) is soluble in water and methanol, but it is not soluble in certain water/methanol mixtures. This phenomenon, known as cononsolvency, has been explored in great detail experimentally and theoretically in an attempt to understand the complex interactions occurring in the ternary PNIPAM/water/co-nonsolvent system. Yet little is known about the effects of the polymer structure on cononsolvency. To address this point, we investigated the temperature-dependent solution properties in water, methanol, and mixtures of the two solvents of poly(2-cyclopropyl-2-oxazoline) (PcyPOx) and two structural isomers of PNIPAM (M n â¼ 11 kg/mol): poly(2-isopropyl-2-oxazoline) (PiPOx) and poly(2-n-propyl-2-oxazoline) (PnPOx). The phase diagram of the ternary water/methanol/poly(2-propyl-2-oxazolines) (PPOx) systems, constructed based on cloud point (T CP) measurements, revealed that PnPOx exhibits cononsolvency in water/methanol mixtures. In contrast, methanol acts as a cosolvent for PiPOx and PcyPOx in water. The enthalpy, ΔH, and temperature, T max, of the coil-to-globule transition of the three polymers in various water/methanol mixtures were measured by high-sensitivity differential scanning calorimetry. T max follows the same trends as T CP, confirming the cononsolvency of PnPOx and the cosolvency of PiPOx and PcyPOx. ΔH decreases linearly as a function of the methanol content for all PPOx systems. Ancillary high-resolution 1H NMR spectroscopy studies of PPOx solutions in D2O and methanol-d 4, coupled with DOSY and NOESY experiments revealed that the n-propyl group of PnPOx rotates freely in D2O, whereas the rotation of the isopropyl and cyclopropyl groups of PiPOx and PcyPOx, respectively, is limited due to steric restriction. This factor appears to play an important role in the case of the PPOxs/water/methanol ternary system.
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The adsorption of hydrophilic or amphiphilic multiblock copolymers provides a powerful means to produce well-defined "smart" surfaces, especially if one or several blocks are sensitive to external stimuli. We focus here on an A-B-A-B-A copolymer, where A is a cationic poly((3-acrylamido-propyl)-trimethylammonium chloride) (PAMPTMA) block containing 15 (end blocks) or 30 (middle block) repeat units and B is a neutral thermosensitive water-soluble poly(2-isopropyl-2-oxazoline) (PIPOZ) block with 50 repeat units. X-ray reflectivity and quartz crystal microbalance with dissipation monitoring were employed to study the adsorption of PAMPTMA15-PIPOZ50-PAMPTMA30-PIPOZ50-PAMPTMA15 on silica surfaces. The latter technique was employed at different temperatures up to 50 °C. Surface forces and friction between the two silica surfaces across aqueous pentablock copolymer solutions at different temperatures were determined with the atomic force microscopy colloidal probe force and friction measurements. The cationic pentablock copolymer was found to have a high affinity to the negatively charged silica surface, leading to a thin (2 nm) and rigid adsorbed layer. A steric force was encountered at a separation of around 3 nm from hard wall contact. A capillary condensation of a polymer-rich phase was observed at the cloud point of the solution. The friction forces were evaluated using Amontons' rule modified with an adhesion term.
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Controlling the propagation of primary tumors is fundamental to avoiding the epithelial to mesenchymal transition process leading to the dissemination and seeding of tumor cells throughout the body. Here we demonstrate that nanoparticles (NPs) limit the propagation of cell aggregates of CT26 murine carcinoma cells used as tumor models. The spreading behavior of these aggregates incubated with NPs is studied on fibronectin-coated substrates. The cells spread with the formation of a cell monolayer, the precursor film, around the aggregate. We study the effect of NPs added either during or after the formation of aggregates. We demonstrate that, in both cases, the spreading of the cell monolayer is slowed down in the presence of NPs and occurs only above a threshold concentration that depends on the size and surface chemistry of the NPs. The density of cells in the precursor films, measured by confocal microscopy, shows that the NPs stick cells together. The mechanism of slowdown is explained by the increase in cell-cell interactions due to the NPs adsorbed on the membrane of the cells. The present results demonstrate that NPs can modulate the collective migration of cells; therefore, they may have important implications for cancer treatment.
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Agregación Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Nanopartículas/química , Polímeros/química , Polímeros/farmacología , Animales , Línea Celular Tumoral , Ratones , ViscosidadRESUMEN
The sulfobetaine (SB) moiety, which comprises a quaternary ammonium group linked to a negatively charged sulfonate ester, is known to impart nonfouling properties to interfaces coated with polysulfobetaines or grafted with SB-polymeric brushes. Increasingly, evidence emerges that the SB group is, overall, a better antifouling group than the phosphorylcholine (PC) moiety extensively used in the past. We report here the synthesis of a series of SB-modified chitosans (CH-SB) carrying between 20 and 40 mol % SB per monosaccharide unit. Chitosan (CH) itself is a naturally derived copolymer of glucosamine and N-acetyl-glucosamine linked with a ß-1,4 bond. Analysis by quartz crystal microbalance with dissipation (QCM-D) indicates that CH-SB films (thickness â¼ 20 nm) resist adsorption of bovine serum albumin (BSA) with increasing efficiency as the SB content of the polymer augments (surface coverage â¼ 15 µg cm-2 for films of CH with 40 mol % SB). The cell adhesivity of CH-SB films coated on glass was assessed by determining the spreading dynamics of CT26 cell aggregates. When placed on chitosan films, known to be cell-adhesive, the CT26 cell aggregates spread by forming a cell monolayer around them. The spreading of CT26 cell aggregates on zwitterion-modified chitosans films is thwarted remarkably. In the cases of CH-SB30 and CH-SB40 films, only a few isolated cells escape from the aggregates. The extent of aggregate spreading, quantified based on the theory of liquid wetting, provides a simple in vitro assay of the nonfouling properties of substrates toward specific cell lines. This assay can be adopted to test and compare the fouling characteristics of substrates very different from the chemical viewpoint.
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Betaína/análogos & derivados , Quitosano/análogos & derivados , Adsorción/efectos de los fármacos , Animales , Betaína/síntesis química , Betaína/química , Bovinos , Agregación Celular/efectos de los fármacos , Línea Celular Tumoral , Quitosano/síntesis química , Ratones , Albúmina Sérica Bovina/químicaRESUMEN
Polymersomes are attractive nanocarriers for hydrophilic and lipophilic drugs; they are more stable than liposomes, tunable, and relatively easy to prepare. The copolymer composition and molar mass are critical features that determine the physicochemical properties of the polymersomes including the rate of drug release. We used the triblock-copolymer, poly(2-methyl-2-oxazoline)-block-poly-(dimethysiloxane)-block-poly(2-methyl-2-oxazoline) (PMOXA-PDMS-PMOXA), to form amphipathic polymersomes capable of loading proteins and small hydrophobic agents. The selected agents were unstable neurotrophins (nerve growth factor and brain-derived neurotrophic factor), a large protein CD109, and the fluorescent drug curcumin. We prepared, characterized, and tested polymersomes loaded with selected agents in 2D and 3D biological models. Curcumin-loaded and rhodamine-bound PMOXA-PDMS-PMOXA polymersomes were used to visualize them inside cells. N-Methyl-d-aspartate receptor (NMDAR) agonists and antagonists were also covalently attached to the surface of polymersomes for targeting neurons. Labeled and unlabeled polymersomes with or without loaded agents were characterized using dynamic light scattering (DLS), UV-vis fluorescence spectroscopy, and asymmetrical flow field-flow fractionation (AF4). Polymersomes were imaged and tested for biological activity in human and murine fibroblasts, murine macrophages, primary murine dorsal root ganglia, and murine hippocampal cultures. Polymersomes were rapidly internalized and there was a clear intracellular co-localization of the fluorescent drug (curcumin) with the fluorescent rhodamine-labeled polymersomes. Polymersomes containing CD109, a glycosylphosphatidylinositol-anchored protein, promoted cell migration in the model of wound healing. Nerve growth factor-loaded polymersomes effectively enhanced neurite outgrowth in dissociated and explanted dorsal root ganglia. Brain-derived neurotrophic factor increased dendritic spine density in serum-deprived hippocampal slice cultures. NMDAR agonist- and antagonist-functionalized polymersomes targeted selectively neurons over glial cells in mixed cultures. Collectively, the study reveals the successful incorporation into polymersomes of biologically active trophic factors and small hydrophilic agents that retain their biological activity in vitro, as demonstrated in selected central and peripheral tissue models.
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Caged luciferin compounds of firefly luciferins have recently drawn much attention since firefly bioluminescence, in which D-luciferin acts as a substrate, is widely used in noninvasive gene-expression imaging, studies of in vivo cell trafficking, and the detection of enzyme activity. The objectives of this study are the development of new caged luciferins and the quantitative determination of the photophysical parameters of their photo-decomposition. We synthesized 7-(diethylaminocoumarin)-4-(yl)methyl caged D-luciferin (DEACM-caged D-luciferin) and quantitatively characterized its absorption spectrum, bioluminescence, and photoproducts using chiral HPLC chromatography, as a function of light-irradiation time. We observed that 4â¯min of UV irradiation generated maximum D-luciferin concentrations, which corresponds to 16.2% of the original DEACM-caged-D-luciferin concentration. Moreover, we evaluated not only the rate of photocleavage (0.20/min) from DEACM-caged D-luciferin to luciferin but also the rate of caged-luciferin degradation that did not produce luciferin (0.28/min) and the rate of luciferin decomposition (0.20/min) after exposure to irradiation with a 70â¯mW/cm2 high-pressure mercury lamp (254-600â¯nm). The formation rate of L-luciferin via DEACM-caged-D-luciferin photocleavage was smaller by a factor of 1/10 compared with that of D-luciferin. These quantitative measurements and simultaneous evaluations of photocleavage, degradation, and decomposition are the most important and original methodology presented in this study.
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Benzotiazoles/análisis , Cumarinas/síntesis química , Cumarinas/química , Cinética , Mediciones Luminiscentes , Rotación Óptica , Fotólisis , EstereoisomerismoRESUMEN
Mitochondrial targeting and entry, two crucial steps in fighting severe diseases resulting from mitochondria dysfunction, pose important challenges in current nanomedicine. Cell-penetrating peptides or targeting groups, such as Rhodamine-B (Rho), are known to localize in mitochondria, but little is known on how to enhance their effectiveness through structural properties of polymeric carriers. To address this issue, we prepared 8 copolymers of 3-dimethyl(methacryloyloxyethyl)ammonium propane sulfonate and poly(ethyleneglycol) methacrylate, p(DMAPS-ran-PEGMA) (molecular weight, 18.0 < M n < 74.0 kg/mol) with two different endgroups. We labeled them with Rho groups attached along the chain or on one of the two endgroups (α or ω). From studies by flow cytometry and confocal fluorescence microscopy of the copolymers internalization in HeLa cells in the absence and presence of pharmacological inhibitors, we established that the polymers cross the cell membrane foremost by translocation and also by endocytosis, primarily clathrin-dependent endocytosis. The most effective mitochondrial entry was achieved by copolymers of M n < 30.0 kg/mol, lightly grafted with PEG chains (< 5 mol %) labeled with Rho in the ω-position. Our findings may be generalized to the uptake and mitochondrial targeting of prodrugs and imaging agents with a similar polymeric scaffold.
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Metacrilatos/química , Mitocondrias/metabolismo , Polietilenglicoles/química , Polímeros/química , Compuestos de Amonio Cuaternario/química , Rodaminas/química , Transporte Biológico , Técnicas de Química Sintética , Células HeLa , Humanos , Estructura Molecular , Polímeros/síntesis químicaRESUMEN
We study the spreading of cell aggregates deposited on adhesive substrates decorated with microparticles (MPs). A cell monolayer expands around the aggregate. The cells on the periphery of the monolayer take up the MPs, clearing the substrate as they progress and forming an aureole of cells filled with MPs. We study the dynamics of spreading and determine the width of the aureole and the level of MP internalization in cells as a function of MP size, composition, and density. From the radius and width of the aureole, we quantify the volume fraction of MPs within the cell, which leads to an easy, fast, and inexpensive measurement of the cell - particle internalization.