A new function with four adjustable parameters is proposed as a model of complex particle size di... more A new function with four adjustable parameters is proposed as a model of complex particle size distribution curves of particulate materials. A method for determination of its parameters from experimental data obtained by sieve analysis is described and the flexibility of the new function is illustrated on examples of artificial and experimental distributions.
Upconverting nanoparticles are attracting extensive interest as a multimodal imaging tool. In thi... more Upconverting nanoparticles are attracting extensive interest as a multimodal imaging tool. In this work, we report on the synthesis and characterization of gadolinium-enriched upconverting nanoparticles for bimodal magnetic resonance and optical luminescence imaging. NaYF4:Gd3+,Yb3+,Tm3+ core upconverting nanoparticles were obtained by a thermal coprecipitation of lanthanide oleate precursors in the presence of oleic acid as a stabilizer. With the aim of improving the upconversion emission and increasing the amount of Gd3+ ions on the nanoparticle surface, a 2.5 nm NaGdF4 shell was grown by the epitaxial layer-by-layer strategy, resulting in the 26 nm core–shell nanoparticles. Both core and core–shell nanoparticles were coated with poly(ethylene glycol) (PEG)-neridronate (PEG-Ner) to have stable and well-dispersed upconverting nanoparticles in a biological medium. FTIR spectroscopy and thermogravimetric analysis indicated the presence of ∼20 wt % of PEG-Ner on the nanoparticle surface. The addition of inert NaGdF4 shell resulted in a total 26-fold enhancement of the emission under 980 nm excitation and also affected the T1 and T2 relaxation times. Both r1 and r2 relaxivities of PEG-Ner-modified nanoparticles were much higher compared to those of non-PEGylated particles, thus manifesting their potential as a diagnostic tool for magnetic resonance imaging. Together with the enhanced luminescence efficiency, upconverting nanoparticles might represent an efficient probe for bimodal in vitro and in vivo imaging of cells in regenerative medicine, drug delivery, and/or photodynamic therapy.
The protection of phosphonate coatings against the dissolution of upconverting nanoparticles unde... more The protection of phosphonate coatings against the dissolution of upconverting nanoparticles under physiological conditions was significantly improved by increasing the coating-synthesis temperature to 80 °C.
A bioactive sorbent for degradation of high-molecular-weight RNA by RNase A was prepared by immob... more A bioactive sorbent for degradation of high-molecular-weight RNA by RNase A was prepared by immobilization of RNase A on magnetic spherical carriers based on cellulose or poly(HEMA-co-EDMA). Immobilized RNase A was temperature-stable at 30-60 C. The influence of temperature on immobilized enzyme activity was also studied. A comparison of soluble and immobilized RNase A showed a pH shift in maximum of immobilized enzyme activity in dependence on the matrix type. Practical use of RNase A immobilized on magnetic bead cellulose MBC 1 for high-performance size-exclusion chromatography purification of pUC19 plasmid DNA is demonstrated.
Magnetic microspheres (P(HEMA-co-EDMA), P(HEMA-co-GMA), and PGMA with hydrophilic properties were... more Magnetic microspheres (P(HEMA-co-EDMA), P(HEMA-co-GMA), and PGMA with hydrophilic properties were prepared by single-step dispersion polymerisation. Sterically or electrostatically stabilised colloidal magnetite was obtained by precipitation of Fe2+ and Fe3+ salts and encapsulated by dispersion (co)polymerisation of HEMA and GMA. Several enzymes, such as RNase A, DNase I, proteinase K and Salmonella antibodies were immobilised on the microspheres and their applicability in degradation of bacterial RNA, chromosomal and plasmid DNA, magnetic separation of Salmonella cells or degradation of their intracellular inhibitors was demonstrated. Genomic DNA was successfully isolated from cell lysates on weakly acid derivatives of magnetic P(HEMA-co-EDMA) and P(HEMA-co-GMA) microspheres in the presence of PEG 6000 and sodium chloride.
The rapidly growing interest in biology and medicine is due to ongoing progress in noninvasive in... more The rapidly growing interest in biology and medicine is due to ongoing progress in noninvasive in vitro or in vivo diagnosis and imaging or treatment of various diseases, including monitoring of the survival, migration, and fate of transplanted cells over the long-term. This requires the use of contrast agents, drug delivery vehicles, and separation media often based on magnetic nanoparticles and/or microspheres. This chapter is going to describe approaches to their development at the Institute of Macromolecular Chemistry in Prague, the Czech Republic, during the last twenty-five years.
Magnetic γ-Fe2O3/CeO2 nanoparticles were obtained by precipitation of Ce(NO3)3 with ammonia in th... more Magnetic γ-Fe2O3/CeO2 nanoparticles were obtained by precipitation of Ce(NO3)3 with ammonia in the presence of γ-Fe2O3 seeds. The formation of CeO2 nanoparticles on the seeds was confirmed by transmission electron microscopy linked with selected area electron diffraction, energy-dispersive X-ray spectroscopy, electron energy loss spectroscopy, and dynamic light scattering. The γ-Fe2O3/CeO2 particle surface was functionalized with PEG-neridronate to improve the colloidal stability in PBS and biocompatibility. Chemical and in vitro biological assays proved that the nanoparticles, due to the presence of cerium oxide, effectively scavenged radicals, thus decreasing oxidative stress in the model cell line. PEG functionalization of the nanoparticles diminished their in vitro aggregation and facilitated lysosomal cargo degradation in cancer cells during autophagy, which resulted in concentration-dependent cytotoxicity of the nanoparticles. Finally, the iron oxide core allowed easy magnetic separation of the particles from liquid media and may enable monitoring of nanoparticle biodistribution in organisms using magnetic resonance imaging.
Magnetic hypercrosslinked poly(styrene-co-divinylbenzene) microspheres (mgt.HPS1-NH2 and mgt.HPS2... more Magnetic hypercrosslinked poly(styrene-co-divinylbenzene) microspheres (mgt.HPS1-NH2 and mgt.HPS2-NH2) containing different contents of amino groups were prepared and characterized in this study. The microspheres were used for the capture of uncompacted and compacted bacterial and calf thymus DNAs in the presence of different PEG 6000 and NaCl concentrations. Magnetic macroporous poly(glycidyl methacrylate-co-[2-(methacryloyloxy)ethoxy]acetic acid-co-ethylene dimethacrylate) microspheres containing amino and carboxyl groups [mgt.P(GMA-MOEAA-EDMA)-NH2] and magnetic non-porous carboxyl group-functionalized poly(2-hydroxyethyl methacrylate-co-glycidyl methacrylate) [mgt.P(HEMA-GMA)-COOH] microspheres were used as a control. The size changes of uncompacted and compacted bacterial and calf thymus DNAs were measured in the range of concentrations 0–16% PEG 6000 and 0.5–2.0 M NaCl by dynamic light scattering (DLS). The highest capture of compacted bacterial and calf thymus DNAs was achieved with mgt.HPS1-NH2, mgt.HPS2-NH2 microspheres using 8% PEG 6000 and 2.0 M NaCl and with mgt.P(GMA-MOEAA-EDMA)-NH2 microspheres using 8% and 16% PEG 6000 and 2.0 M NaCl. Developed microspheres were used for DNA isolation from real vegetable (broccoli flowering head) samples. DNA was in quality suitable for PCR.
We firstly identified 48 kDa molecular form of the unconventional myosin 1c (p48/Myo1C), and isol... more We firstly identified 48 kDa molecular form of the unconventional myosin 1c (p48/Myo1C), and isolated it from blood serum of multiple sclerosis patients. The amount of p48/Myo1C in human blood serum correlated with some autoimmune, hemato‐oncological and neurodegenerative diseases and thus may serve as a potential molecular biomarker. The biological functions of this protein in human blood remain unknown. Previously, we used the monodisperse magnetic poly (glycidyl methacrylate)(mag‐PGMA–NH2) microspheres with immobilized 48/Myo1C and western‐blot analysis, which allowed us to identify IgM and IgG immunoglobulins presenting an affinity to this protein. Here, we used mass spectrometry followed by the western blotting in order to identify other blood serum proteins with affinity to 48/Myo1C. The obtained data demonstrate that 48/Myo1C binds to component 3 of the complement and the antithrombin‐III proteins. A combination of magnetic microparticle‐based affinity chromatography with MAL...
The cytopoxic effect of RL2 lactaptin (the recombinant analog of proteolytic fragment of human ka... more The cytopoxic effect of RL2 lactaptin (the recombinant analog of proteolytic fragment of human kappa‐casein) toward tumor cells in vitro and in vivo presents it as a novel promising antitumor drug. The binding of any drug with serum proteins can affect their activity, distribution, rate of excretion and toxicity in the human body. Here, we studied the ability of RL2 to bind to various blood serum proteins. Using magnetic microparticles bearing by RL2 as an affinity matrix, in combination with mass spectrometry and western blot analysis, we found a number of blood serum proteins possessing affinity for RL2. Among them IgA, IgM and IgG subclasses of immunoglobulins, apolipoprotein A1 and various cortactin isoforms were identified. This data suggests that in the bloodstream RL2 lactaptin takes part in complicate protein–protein interactions, which can affect its activity.
Photodynamic therapy (PDT) has garnered immense attention as a minimally invasive clinical treatm... more Photodynamic therapy (PDT) has garnered immense attention as a minimally invasive clinical treatment modality for malignant cancers. However, its low penetration depth and photodamage of living tissues by UV and visible light, which activate a photosensitizer, limit the application of PDT. In this study, monodisperse NaYF:Yb/Ernanospheres 20 nm in diameter, that serve as near-infrared (NIR)-to-visible light converters and activators of a photosensitizer, were synthesized by high-temperature co-precipitation of lanthanide chlorides in a high-boiling organic solvent (octadec-1-ene). The nanoparticles were coated with a thin shell (≈3 nm) of homogenous silica via the hydrolysis and condensation of tetramethyl orthosilicate. The NaYF:Yb/Er@SiOparticles were further functionalized by methacrylate-terminated groups via 3-(trimethoxysilyl)propyl methacrylate. To introduce a large number of reactive amino groups on the particle surface, methacrylate-terminated NaYF:Yb/Er@SiOnanospheres were...
Oleic acid-stabilized hexagonal NaYF4:Yb(3+)/Er(3+) nanocrystals, emitting green and red luminesc... more Oleic acid-stabilized hexagonal NaYF4:Yb(3+)/Er(3+) nanocrystals, emitting green and red luminescence, were prepared by the high-temperature co-precipitation of lanthanide chlorides. By varying the reaction time and the Ln(3+)/Na(+) ratio, the nanocrystal size can be controlled within the range 16-270 nm. The maximum upconversion quantum yield is achieved under 970 nm excitation. The reverse microemulsion technique using hydrolysis and condensation of tetraethoxysilane is a suitable method to coat the nanocrystal surface with a silica shell to make the particles dispersible and colloidally stable in aqueous media. During the subsequent functionalization, (3-aminopropyl)trimethoxysilane introduced amino groups onto the silica to enable future bioconjugation with the target molecules. All specimens were characterized by TEM microscopy, electron and X-ray diffraction, ATR FT-IR spectroscopy, and upconversion luminescence. Finally, in vitro cytotoxicity and intracellular nanoparticle uptake (using confocal microscopy) were determined with human cervical carcinoma HeLa and mRoGFP HeLa cells, respectively. From the investigated particles, amino-functionalized NaYF4:Yb(3+)/Er(3+) nanocrystals internalized into the cells most efficiently. The nanoparticles proved to be nontoxic at moderate concentrations, which is important when considering their prospective application in biolabeling and luminescence imaging of various cell types.
A new function with four adjustable parameters is proposed as a model of complex particle size di... more A new function with four adjustable parameters is proposed as a model of complex particle size distribution curves of particulate materials. A method for determination of its parameters from experimental data obtained by sieve analysis is described and the flexibility of the new function is illustrated on examples of artificial and experimental distributions.
Upconverting nanoparticles are attracting extensive interest as a multimodal imaging tool. In thi... more Upconverting nanoparticles are attracting extensive interest as a multimodal imaging tool. In this work, we report on the synthesis and characterization of gadolinium-enriched upconverting nanoparticles for bimodal magnetic resonance and optical luminescence imaging. NaYF4:Gd3+,Yb3+,Tm3+ core upconverting nanoparticles were obtained by a thermal coprecipitation of lanthanide oleate precursors in the presence of oleic acid as a stabilizer. With the aim of improving the upconversion emission and increasing the amount of Gd3+ ions on the nanoparticle surface, a 2.5 nm NaGdF4 shell was grown by the epitaxial layer-by-layer strategy, resulting in the 26 nm core–shell nanoparticles. Both core and core–shell nanoparticles were coated with poly(ethylene glycol) (PEG)-neridronate (PEG-Ner) to have stable and well-dispersed upconverting nanoparticles in a biological medium. FTIR spectroscopy and thermogravimetric analysis indicated the presence of ∼20 wt % of PEG-Ner on the nanoparticle surface. The addition of inert NaGdF4 shell resulted in a total 26-fold enhancement of the emission under 980 nm excitation and also affected the T1 and T2 relaxation times. Both r1 and r2 relaxivities of PEG-Ner-modified nanoparticles were much higher compared to those of non-PEGylated particles, thus manifesting their potential as a diagnostic tool for magnetic resonance imaging. Together with the enhanced luminescence efficiency, upconverting nanoparticles might represent an efficient probe for bimodal in vitro and in vivo imaging of cells in regenerative medicine, drug delivery, and/or photodynamic therapy.
The protection of phosphonate coatings against the dissolution of upconverting nanoparticles unde... more The protection of phosphonate coatings against the dissolution of upconverting nanoparticles under physiological conditions was significantly improved by increasing the coating-synthesis temperature to 80 °C.
A bioactive sorbent for degradation of high-molecular-weight RNA by RNase A was prepared by immob... more A bioactive sorbent for degradation of high-molecular-weight RNA by RNase A was prepared by immobilization of RNase A on magnetic spherical carriers based on cellulose or poly(HEMA-co-EDMA). Immobilized RNase A was temperature-stable at 30-60 C. The influence of temperature on immobilized enzyme activity was also studied. A comparison of soluble and immobilized RNase A showed a pH shift in maximum of immobilized enzyme activity in dependence on the matrix type. Practical use of RNase A immobilized on magnetic bead cellulose MBC 1 for high-performance size-exclusion chromatography purification of pUC19 plasmid DNA is demonstrated.
Magnetic microspheres (P(HEMA-co-EDMA), P(HEMA-co-GMA), and PGMA with hydrophilic properties were... more Magnetic microspheres (P(HEMA-co-EDMA), P(HEMA-co-GMA), and PGMA with hydrophilic properties were prepared by single-step dispersion polymerisation. Sterically or electrostatically stabilised colloidal magnetite was obtained by precipitation of Fe2+ and Fe3+ salts and encapsulated by dispersion (co)polymerisation of HEMA and GMA. Several enzymes, such as RNase A, DNase I, proteinase K and Salmonella antibodies were immobilised on the microspheres and their applicability in degradation of bacterial RNA, chromosomal and plasmid DNA, magnetic separation of Salmonella cells or degradation of their intracellular inhibitors was demonstrated. Genomic DNA was successfully isolated from cell lysates on weakly acid derivatives of magnetic P(HEMA-co-EDMA) and P(HEMA-co-GMA) microspheres in the presence of PEG 6000 and sodium chloride.
The rapidly growing interest in biology and medicine is due to ongoing progress in noninvasive in... more The rapidly growing interest in biology and medicine is due to ongoing progress in noninvasive in vitro or in vivo diagnosis and imaging or treatment of various diseases, including monitoring of the survival, migration, and fate of transplanted cells over the long-term. This requires the use of contrast agents, drug delivery vehicles, and separation media often based on magnetic nanoparticles and/or microspheres. This chapter is going to describe approaches to their development at the Institute of Macromolecular Chemistry in Prague, the Czech Republic, during the last twenty-five years.
Magnetic γ-Fe2O3/CeO2 nanoparticles were obtained by precipitation of Ce(NO3)3 with ammonia in th... more Magnetic γ-Fe2O3/CeO2 nanoparticles were obtained by precipitation of Ce(NO3)3 with ammonia in the presence of γ-Fe2O3 seeds. The formation of CeO2 nanoparticles on the seeds was confirmed by transmission electron microscopy linked with selected area electron diffraction, energy-dispersive X-ray spectroscopy, electron energy loss spectroscopy, and dynamic light scattering. The γ-Fe2O3/CeO2 particle surface was functionalized with PEG-neridronate to improve the colloidal stability in PBS and biocompatibility. Chemical and in vitro biological assays proved that the nanoparticles, due to the presence of cerium oxide, effectively scavenged radicals, thus decreasing oxidative stress in the model cell line. PEG functionalization of the nanoparticles diminished their in vitro aggregation and facilitated lysosomal cargo degradation in cancer cells during autophagy, which resulted in concentration-dependent cytotoxicity of the nanoparticles. Finally, the iron oxide core allowed easy magnetic separation of the particles from liquid media and may enable monitoring of nanoparticle biodistribution in organisms using magnetic resonance imaging.
Magnetic hypercrosslinked poly(styrene-co-divinylbenzene) microspheres (mgt.HPS1-NH2 and mgt.HPS2... more Magnetic hypercrosslinked poly(styrene-co-divinylbenzene) microspheres (mgt.HPS1-NH2 and mgt.HPS2-NH2) containing different contents of amino groups were prepared and characterized in this study. The microspheres were used for the capture of uncompacted and compacted bacterial and calf thymus DNAs in the presence of different PEG 6000 and NaCl concentrations. Magnetic macroporous poly(glycidyl methacrylate-co-[2-(methacryloyloxy)ethoxy]acetic acid-co-ethylene dimethacrylate) microspheres containing amino and carboxyl groups [mgt.P(GMA-MOEAA-EDMA)-NH2] and magnetic non-porous carboxyl group-functionalized poly(2-hydroxyethyl methacrylate-co-glycidyl methacrylate) [mgt.P(HEMA-GMA)-COOH] microspheres were used as a control. The size changes of uncompacted and compacted bacterial and calf thymus DNAs were measured in the range of concentrations 0–16% PEG 6000 and 0.5–2.0 M NaCl by dynamic light scattering (DLS). The highest capture of compacted bacterial and calf thymus DNAs was achieved with mgt.HPS1-NH2, mgt.HPS2-NH2 microspheres using 8% PEG 6000 and 2.0 M NaCl and with mgt.P(GMA-MOEAA-EDMA)-NH2 microspheres using 8% and 16% PEG 6000 and 2.0 M NaCl. Developed microspheres were used for DNA isolation from real vegetable (broccoli flowering head) samples. DNA was in quality suitable for PCR.
We firstly identified 48 kDa molecular form of the unconventional myosin 1c (p48/Myo1C), and isol... more We firstly identified 48 kDa molecular form of the unconventional myosin 1c (p48/Myo1C), and isolated it from blood serum of multiple sclerosis patients. The amount of p48/Myo1C in human blood serum correlated with some autoimmune, hemato‐oncological and neurodegenerative diseases and thus may serve as a potential molecular biomarker. The biological functions of this protein in human blood remain unknown. Previously, we used the monodisperse magnetic poly (glycidyl methacrylate)(mag‐PGMA–NH2) microspheres with immobilized 48/Myo1C and western‐blot analysis, which allowed us to identify IgM and IgG immunoglobulins presenting an affinity to this protein. Here, we used mass spectrometry followed by the western blotting in order to identify other blood serum proteins with affinity to 48/Myo1C. The obtained data demonstrate that 48/Myo1C binds to component 3 of the complement and the antithrombin‐III proteins. A combination of magnetic microparticle‐based affinity chromatography with MAL...
The cytopoxic effect of RL2 lactaptin (the recombinant analog of proteolytic fragment of human ka... more The cytopoxic effect of RL2 lactaptin (the recombinant analog of proteolytic fragment of human kappa‐casein) toward tumor cells in vitro and in vivo presents it as a novel promising antitumor drug. The binding of any drug with serum proteins can affect their activity, distribution, rate of excretion and toxicity in the human body. Here, we studied the ability of RL2 to bind to various blood serum proteins. Using magnetic microparticles bearing by RL2 as an affinity matrix, in combination with mass spectrometry and western blot analysis, we found a number of blood serum proteins possessing affinity for RL2. Among them IgA, IgM and IgG subclasses of immunoglobulins, apolipoprotein A1 and various cortactin isoforms were identified. This data suggests that in the bloodstream RL2 lactaptin takes part in complicate protein–protein interactions, which can affect its activity.
Photodynamic therapy (PDT) has garnered immense attention as a minimally invasive clinical treatm... more Photodynamic therapy (PDT) has garnered immense attention as a minimally invasive clinical treatment modality for malignant cancers. However, its low penetration depth and photodamage of living tissues by UV and visible light, which activate a photosensitizer, limit the application of PDT. In this study, monodisperse NaYF:Yb/Ernanospheres 20 nm in diameter, that serve as near-infrared (NIR)-to-visible light converters and activators of a photosensitizer, were synthesized by high-temperature co-precipitation of lanthanide chlorides in a high-boiling organic solvent (octadec-1-ene). The nanoparticles were coated with a thin shell (≈3 nm) of homogenous silica via the hydrolysis and condensation of tetramethyl orthosilicate. The NaYF:Yb/Er@SiOparticles were further functionalized by methacrylate-terminated groups via 3-(trimethoxysilyl)propyl methacrylate. To introduce a large number of reactive amino groups on the particle surface, methacrylate-terminated NaYF:Yb/Er@SiOnanospheres were...
Oleic acid-stabilized hexagonal NaYF4:Yb(3+)/Er(3+) nanocrystals, emitting green and red luminesc... more Oleic acid-stabilized hexagonal NaYF4:Yb(3+)/Er(3+) nanocrystals, emitting green and red luminescence, were prepared by the high-temperature co-precipitation of lanthanide chlorides. By varying the reaction time and the Ln(3+)/Na(+) ratio, the nanocrystal size can be controlled within the range 16-270 nm. The maximum upconversion quantum yield is achieved under 970 nm excitation. The reverse microemulsion technique using hydrolysis and condensation of tetraethoxysilane is a suitable method to coat the nanocrystal surface with a silica shell to make the particles dispersible and colloidally stable in aqueous media. During the subsequent functionalization, (3-aminopropyl)trimethoxysilane introduced amino groups onto the silica to enable future bioconjugation with the target molecules. All specimens were characterized by TEM microscopy, electron and X-ray diffraction, ATR FT-IR spectroscopy, and upconversion luminescence. Finally, in vitro cytotoxicity and intracellular nanoparticle uptake (using confocal microscopy) were determined with human cervical carcinoma HeLa and mRoGFP HeLa cells, respectively. From the investigated particles, amino-functionalized NaYF4:Yb(3+)/Er(3+) nanocrystals internalized into the cells most efficiently. The nanoparticles proved to be nontoxic at moderate concentrations, which is important when considering their prospective application in biolabeling and luminescence imaging of various cell types.
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