+

WO1996011054A9 - Procede de preparation de microbilles et microbilles ainsi preparees - Google Patents

Procede de preparation de microbilles et microbilles ainsi preparees

Info

Publication number
WO1996011054A9
WO1996011054A9 PCT/US1995/012988 US9512988W WO9611054A9 WO 1996011054 A9 WO1996011054 A9 WO 1996011054A9 US 9512988 W US9512988 W US 9512988W WO 9611054 A9 WO9611054 A9 WO 9611054A9
Authority
WO
WIPO (PCT)
Prior art keywords
microspheres
microsphere
process according
silica
ligands
Prior art date
Application number
PCT/US1995/012988
Other languages
English (en)
Other versions
WO1996011054A2 (fr
WO1996011054A3 (fr
Filing date
Publication date
Priority claimed from IL11118694A external-priority patent/IL111186A/xx
Application filed filed Critical
Priority to DE19581787T priority Critical patent/DE19581787T1/de
Priority to AU41934/96A priority patent/AU4193496A/en
Priority to US08/809,957 priority patent/US6103379A/en
Publication of WO1996011054A2 publication Critical patent/WO1996011054A2/fr
Publication of WO1996011054A3 publication Critical patent/WO1996011054A3/fr
Publication of WO1996011054A9 publication Critical patent/WO1996011054A9/fr

Links

Definitions

  • the present invention relates to a process for the preparation of microspheres, which may be optionally hollow, and which are consisting of one or more layers of silica shells and may also involve other coatings of different materials, characterized by variety of desirable properties.
  • microspheres of controlled composition are of great importance, because of the potential use of such particles in a wide variety of fields.
  • the complexity of processes for preparing such particles is even greater when the particle to be formed is to be used in delicate biological applications, because then several parameters, which are not easy to handle, should be taken into account, such as types of chemical interactions between the different layers composing the microspheres, as well as the relevant function of the particle within the planned biological application.
  • silica itself consists of particles the diameter of which is about 50 nm, and is obtained, according to sol-gel procedures, by agents such as tetraethoxysilane, [(SiOEt)4], which are converted, via hydrolysis and subsequent condensation, to silica.
  • the microsphere is of diameter of only 0.1 ⁇ m to 0.3 ⁇ m, and is composed only of cationic polystyrene.
  • the coating is made of inorganic material, namely, yttrium basic carbonate.
  • Another important type of coating is one exhibiting magnetic properties.
  • a method for the preparation of ferrite plating of various chemical compositions is known in the art [Abe et al., J. of Applied Physics 57, pp. 3795-3797, (1985)], yet this method is characterized by several disadvantages and limitations.
  • an essential condition for the formation of the ferrite film is the presence of hydroxyl groups on the substrate to be coated, because these groups enhance the adhesion of the film to the surface.
  • the microsphere diameter is smaller than 0.5 ⁇ m, a continued magnetic coating can not be obtained .
  • Abe et al. failed to disclose means for the protection of the ferrite coating, in order to avoid a partial leakage of the ferrite into the solution.
  • the present invention provides processes for the production of microspheres characterized by several desired properties: a) It provides a process for the preparation of silica coating to spherical particles, wherein surfactants of proper nature, adsorbed on the microspheres, serves as a "connective glue" between the microsphere and the silica nanoparticles; b) It provides a process for the preparation of a microsphere coated with a magnetic layer; c) It provides a process for the preparation of hollow silica microspheres; d) It provides a process for the preparation of magnetic microspheres enveloped with silica layers; e) It allows a further modification of the surface of the solid or hollow microspheres, which are coated by magnetic layers and silica layers, to adjust them to several biological applications.
  • microspheres the diameter of which is in the range of about 0.2 ⁇ m up to a few microns, which are made of polymeric materials and contain surfactants of hydrophilic nature on their surfaces.
  • This microspheres are then subjected to further coating stages.
  • the final coated microspheres can then serve in a variety of biological applications.
  • the microspheres can be rendered hollow by removing therefrom the inner polymeric core.
  • the microspheres are obtained by polymerization of monomeric units, such as styrene, chloromethylstyerene, divinylbenzene and methylmethacrylate, in the presence of surfactants of hydrophilic nature and an initiator.
  • monomeric units such as styrene, chloromethylstyerene, divinylbenzene and methylmethacrylate
  • surfactants of hydrophilic nature such as styrene, chloromethylstyerene, divinylbenzene and methylmethacrylate.
  • the choice of the surfactant composition and concentration, as well as other parameters of the reaction, such as initiator type, monomer concentration, governs the distribution of the microspheres diameters.
  • the inventors have found that the quality of the coatings to be crated on the microspheres is also affected by two of these factors, namely, by the surfactant composition and by the initiator. As said, proper surfactants for coating purposes are of hydrophilic nature.
  • the composition of the surfactants may serve as a tool for controlling the coating quality, by choosing proper fractions for the hydrophilic and hydrophobic surfactants.
  • surfactant is meant to indicate a high molecular weight compound having one or more hydrophilic region(s) and one or more hydrophobic region(s).
  • suitable surfactants are polyethylene oxide, polyacrylic acid, copolymers of polyvinyl pyrrolidone- polyvinyl acetate in different ratios.
  • the hydrophilic surfactant is chosen from among polyvinylpyrrolidone, poly aery he acid and polyethyleneoxide.
  • a preferred initiator is benzoylperoxide.
  • the microspheres which comprise surfactants on their surface are treated in an organic solution, i.e. alcoholic solution, which includes alkyi silicates as an agent for the silica production, and a catalyst.
  • alcoholic solution which includes alkyi silicates as an agent for the silica production
  • a catalyst is ammonia.
  • Another purpose of the present invention is to provide a process for the preparation of hollow silica microspheres.
  • these hollow shells are obtained by removing the inner polymeric core of the microspheres by burning or dissolving it.
  • the burning off is performed by subjecting the microspheres to temperatures in the range of 400°C-900°C.
  • the dissolution is carried out by means of appropriate solvents.
  • solvents comprise toluene, dimethylformamide, and the like.
  • the polymeric microspheres, containing proper surfactants on their surface may further be intermediately coated by a magnetic layer.
  • said magnetic layer may be of esO
  • This coating process is carried out in an aqueous solution, in a temperature range of 55°C-90°C, and in a pH range of 8 to 11, preferably between 10-11, since it was found that the magnetic intensity of the coated microspheres is enhanced if more basic conditions are applied.
  • the ferrite formation involves oxidizing part of the Fe+ 2 to Fe +3 > and then interaction between these two spieces to obtain the ferrite..
  • the source for the divalent cation may be, for example, salts such as FeCl 2 -4H 2 ⁇ , and the oxidation reagent may be chosen, for instance, from among NaNO 2 , H 2 O 2 or air.
  • the inventors have found that the magnetic properties of the coated microsphere are determined by the nature of the surfactant adsorbed initially on the particle surface. If hydrophobic surfactants are used, in combination with a hydrophilic one, a significant retardness is observed in the magnetic intensity of these coated microspheres, compared with those obtained with hydrophilic surfactant alone.
  • a further object of the present invention is to provide a process for the preparation of solid or hollow microspheres optionally comprising magnetic coatings, and further enveloped by silica layers. These coated microspheres are then subjected to further modification which have extremely important uses.
  • the silica functionalizes in two different levels: it provides a defensive shell to the ferrite coating, and it serves as a source of optional covalent bonds, through which desired ligand may be attached to the system, in order to form the final, desired particle.
  • silica coating of said microspheres (which are optionally magnetic) is performed as hereinbefore described.
  • ligands are added, comprising a functional group at their ⁇ -position.
  • these ligands can form covalent bonds with the silica coating.
  • the ligands may be chosen from among alkylsilane and/or alkylhydroxyl compounds, in particular SiCl3(CH2)nX, Si(OR)3(CH 2 )nX> OH(CH 2 )nX.
  • R is an alkyi substituent
  • n is between 2 to 20 and X is -NH 2 , -CH 3 , -CO 2 R, -CN, etc.
  • the amine group, or other functional groups which can be converted to an amine group because covalent binding of polyaldehyde ligands onto these groups may be further applied.
  • the polyaldehyde derivatized microsphere surface obtained may be now coupled to biomaterials such as proteins.
  • the residual ⁇ -amine groups can be blocked by a proper reagent, for instance, acetic acid N- hydroxysuccin-imide ester.
  • acetic acid N- hydroxysuccin-imide ester the inventors have found that acidic pH conditions improve the content of said aldehyde in the derivatized microsphere.
  • Fig. 6 summarizes various preferred embodiments of the present invention, wherein P is the polymeric microsphere and S is the surfactant adsorbed on its surface. All the above and other characteristics and advantages of the invention will be better understood through the following illustrative and non- limitative description of preferred embodiments thereof.
  • Fig. 1 is a SEM photograph of polystyrene microspheres
  • Fig. 2A is a TEM picture of a polystyrene microsphere of ca. 1.8 ⁇ m coated with three layers of silica nanoparticles of ca. 30 nm average diameter;
  • Figs. 2B and 2C are TEM and SEM pictures, respectively of hollow microspheres obtained by burning off the organic content of these coated microspheres at 800°C for 12 h;
  • Figs. 3 are cross-section pictures obtained by TEM of hollow silica microspheres prepared from polystyrene microspheres of ca. 1.8 ⁇ m average diameter coated with three layers of silica nanoparticles of ca.. 30 nm average diameter;
  • Fig. 4 shows SEM pictures of hollow silica microspheres obtained by burning off the organic core of polystyrene microspheres of ca. 2.3 ⁇ m coated with a single layer of sihca nanoparticles of ca. 30 nm diameter (Fig. 4A), and three layers of similar sihca nanoparticles (Fig. 4B);
  • Fig. 5A illustrates SEM photomicrograph of polystyrene microsphere of ca. 2.3 ⁇ m diameter.
  • Fig. 5B Illustrates the magnetic coating on polystyrene microspheres of ca. 2.3 ⁇ m diameter.
  • Fig. 5C illustrates the first sihca nanoparticles coating on magnetic polystyrene microspheres of ca. 2.3 ⁇ m, prepared according to example 13;
  • Fig. 6 summarizes various preferred embodiments of the present invention, wherein P is the polymeric microsphere and S is the surfactant adsorbed on its surface.
  • Polystyrene microspheres were prepared according to the literature/ C.K. Ober, K.P. Lok and M.L. Hair, J. of Polymer Sci., Polymer Letters Edition 23, 103 (1985)/. Briefly, These microspheres were prepared in a three-neck round- bottom flask equipped with a condenser. The flask was immersed in a constant temperature silicone oil bath at a preset temperature. In a typical reaction, A solution containing PVP (M.W. 360,000, 3.75 g) dissolved in ethanol (156 ml) and methyl cellosove (2-methoxyethanol, 62.5 ml) at room temperature was placed into the reaction flask and mechanically stirred (ca. 200 rpm).
  • the diameter of the formed microspheres were controlled by changing conditions, such as surfactant type, surfactant concentration, initiator type, monomer concentration, reaction time, etc. Thereby, monodispersed and polydispersed microsphere systems in sizes ranging from approximately 0.2 ⁇ m up to several microns were formed.
  • similar polymerization procedure as described above substituting the initiator benzoyl peroxide with azobisisobutironitrile, resulted in the formation of polydispersed microspheres with 4.2 ⁇ m average diameter instead of monodispersed microspheres of 2.3 ⁇ m average diameter obtained when benzoyl peroxide was used.
  • Crosslinked polystyrene microspheres of ca. 0.3 ⁇ m diameter were synthesized by a procedure similar to the procedure described above, substituting styrene with divinylbenzene or substituting styrene with a monomer mixture composed of 45% styrene and 55% divinylbenzene.
  • Microns sized polydispersed crosslinked polystyrene microspheres were prepared by a suspension polymerization process, through a procedure similar to that described in the Q.C. Wang, F. Svec and M.J. Frechet, Polymer Bulletin 28, 569 (1992).
  • Monodispersed polychloromethylstyrene microspheres were prepared by a procedure similar to that described for polystyrene microspheres, substituting the solvent mixture (ethanol + methyl cellosove) with ethanol.
  • 5.0 ml chloromethylstyrene were polymerized in 100 ml ethanol solution containing 1.15 g PVP (M.W. 360,000) and 100 mg azobisisobutironitrile.
  • monodispersed polychloromethylstyrene microspheres of ca. 1.2 ⁇ m diameter (standard deviation of ca. 5%) were formed.
  • Microspheres with a variety of diameters were formed by changing conditions, such as monomer concentration, surfactant concentration, etc.
  • PMMA microspheres Monodispersed polymethylmethacrylate (PMMA) microspheres were prepared by a procedure similar to that described for polystyrene microspheres, substituting the solvent mixture (ethanol + methyl cellosove) with ethanol. In a typical reaction, 23 ml methylmathacrylate were polymerized in 212 ml ethanol solution containing 3.75 g PVP (M.W. 360,000) and 1.5 g bezoylperoxide. Thereby, PMMA microspheres of ca. 2.0 ⁇ m diameter were -In ⁇
  • Microspheres with a variety of diameters were formed. Microspheres with a variety of diameters were formed by changing conditions, such as monomer concentration, surfactant concentration, etc.
  • Sihca nanoparticles were prepared by the sol-gel technique by polymerization of Si(OEt)4 according to the Stober Method/ W. Stober, A. Fink and E. Bohn, J. Colloid Interface Sci. 26, 62 (1968)/. Briefly, particles of 30 nm average diameter were prepared by adding into a flask according to the listed order the following reagents: ethanol (93.6 ml), distilled water (1.9 ml), ammonium hydroxide (1.3 ml) and Si(OEt)4 (3.2 ml). The resulting solution was then shaken at room temperature for ca. 12 h. The formed nanoparticles were washed by evaporation of the unreacted monomer, ethanol and ammonia.
  • silica nanoparticles of ca. 30 nm average diameter Silica nanoparticles of different sizes were prepared by changing conditions, such as monomer concentration, water concentration, etc.
  • Polyacrolein nanoparticles were synthesized according to the literature/S. Margel, Reactive Polymers 1, 241 (1983)/. Briefly, polyacrolein nanoparticles of ca. 70 nm average diameter were prepared by cobalt irradiation (approx. 1 Mrad) of a deairated solution containing 90 ml distilled water, 1 g sodium dodecyl sulfate and 10 ml acrolein. The formed microspheres were then washed by extensive dialysis against distilled water.
  • Fig. 2A illustrates a TEM phtomicrograph of polystyrene microsphere coated with three layers of sihca monoparticles.
  • Example 1 was repeated, substituting the 3.2 ml Si(OEt)4 with 1.8 ml Si(OEt)4. Similar results were obtained, except that the sihca nanoparticle coating on the polystyrene microspheres decreased from ca. 30 nm average diameter to ca. 20 nm average diameter.
  • Example 3 Preparation of continuous thin coatings of silica nanoparticles on polystyrene microspheres.
  • Dried polystyrene microspheres of ca. 1.8 ⁇ m average diameter containing polyvinlpyrrolidone adsorbed on its surfaces were added into a flask containing ethanol (93.6 ml) and distilled water (1.9 ml). The microspheres were then suspended in the solvent by sonication. Ammonium hydroxide (1.3 ml) and Si(OEt)4 (3.2 ml) were then added to the previous polystyrene suspension. The resulting suspension was then shaken at room temperature for ca. 12 h. A second layer of sihca coating was then prepared by adding to the suspension 0.6 ml distilled water and 3.2 ml Si(OEt)4.
  • Example 1 The reaction was then continued for additional ca. 5h.
  • the formed silica coated polystyrene microspheres were then washed according to the description in Example 1.
  • the percent silica obtained for the first and second coatings of the polystyrene was similar to that obtained in Example 1.
  • a third continuous silica coating on polystyrene microspheres was difficult to prepare because of the difficulties existed in separation of grafted silica nanoparticles from ungrafted silica nanoparticles.
  • Examples 1-3 were repeated substituting the polystyrene microspheres of ca. 1.8 ⁇ m average diameter adsorbed with PVP on its surfaces with similar type microspheres, crosshnked and not crosslinked, with variety of diameters, i.e. ca. 0.3, 2.3, 5.2 and 8.0 ⁇ m average diameter.
  • Cross section photomicrographs demonstrated similar silica coatings as that obtained in Examples 1-3.
  • Examples 1-4 were repeated substituting polystyrene microspheres containing PVP adsorbed on its surfaces with polystyrene microspheres containing other hydrophilic surfactants adsorbed on its surfaces, i.e. polyacrylic acid and polyethyleneoxide.
  • Cross section photomicrographs demonstrated similar silica coatings as that obtained in Examples 1-4.
  • Example 6 Effect of hydrophobic surfactants adsorbed on the polystyrene microspheres on the thin coatings from silica nanoparticles.
  • Examples 1, 3 and 4 were repeated substituting polystyrene microspheres containing PVP adsorbed on its surfaces with polystyrene microspheres containing surfactants with decreased order of hydrophihc/hydrophobic ratio on its surfaces, i.e. PVP, PVP-PVAc (60:40) and PVAc. Elemental analysis measurements showed that the percent silica significantly decreased when the hydrophihc/hydrophobic ratio of the adsorbed surfactants decreased. For example, the percent sihca measured for the first silica coating on polystyrene microspheres prepared with benzoylperoxide was ca. 7.8%, 3.7% and 0.5% for microspheres adsorbed on its surfaces with PVP, PVP-PVAc (60:40) and PVAc, respectively.
  • Example 6 was repeated with polystyrene microspheres prepared with the initiator azobisisobutironitrile instead of benzoylperoxide. Thereby, The hydrophobic character of the formed microspheres increased relative to the microspheres prepared with benzoylperoxide. Thermogravimetric measurements also clearly demonstrated that the percent silica coated on the polystyrene significantly decreased when the hydrophihc/hydrophobic ratio of the adsorbed surfactants decreased. For example, the percent silica measured for the first silica coating on polystyrene microspheres prepared with azobisisobutironitrile was ca.
  • Examples 1-7 were repeated, substituting the polystyrene microspheres with polychloromethylstyrene microspheres and/or polymethylmethacrylate microspheres prepared according to the description in the experimental part.
  • Cross section photomicrographs indicated similar results.
  • Hollow silica microspheres were prepared by burning off (i.e. ca. 400°C or above) the organic core of polystyrene microspheres coated with silica nanoparticles, prepared according to Examples 1-5.
  • Fig. 2A is a TEM picture of a polystyrene microsphere of ca. 1.8 ⁇ m coated with three layers of sihca nanoparticles of ca. 30 nm average diameter.
  • Figures 2B and 2C are TEM and SEM pictures, respectively of hollow microspheres obtained by burning off the organic content of these coated microspheres at 800 °C for 12 h. Infrared measurements indicated the total removal of the organic core from the sihca shell.
  • Fig. 3 are cross-section pictures obtained by TEM of hollow silica microspheres prepared from polystyrene microspheres of ca. 1.8 ⁇ m average diameter coated with three layers of silica nanoparticles of ca.. 30 nm average diameter.
  • Fig. 4 shows SEM pictures of hollow sihca microspheres obtained by burning off the organic core of polystyrene microspheres of ca. 2.3 ⁇ m coated with a single layer of sihca nanoparticles of ca. 30 nm diameter (Fig. 4A) and three layers of similar silica nanoparticles (Fig. 4B).
  • the hollow sihca microspheres composed of three layers of silica nanoparticles are stable while the hollow silica microspheres composed of a single sihca nanoparticles layer are mostly broken. These observation may due to the instability of the coating composed of a single silica layer to stand the vacuum applied for the SEM use.
  • Hollow silica microspheres were also prepared by dissolving with appropriate solvents (e.g. toluene, dimethylformamide, etc.) the organic core of polystyrene microspheres coated with silica nanoparticles, prepared according to Examples 1-5.
  • solvents e.g. toluene, dimethylformamide, etc.
  • the hollow microspheres obtained in this way usually contained, except silica, also traces of organic polymers which could not be removed by this process.
  • Examples 9-11 were repeated substituting polystyrene microspheres with polychloromethylstyrene microspheres or/and polymethylmethacrylate microspheres coated with sihca nanoparticles prepared according to Example 8. Similar results were obtained.
  • Example 13 Preparation of magnetic thin coatings from Fe ⁇ on polystyrene microspheres.
  • the coating was performed in a six-neck round-bottom flask.
  • One neck in the center was used for mechanical stirring, two other necks were used for purging nitrogen and for the exit of this gas, the other three necks were used for gradual introducing into the flask, during the coating process, deairated aqueous solutions from iron chloride, sodium nitrite and sodium hydroxide, respectively.
  • the flask was immersed in a constant temperature silicone oil bath at a preset temperature.
  • Fig . 5A illustrates SEM photomicrograph of polystyrene microspheres of ca. 2.3 ⁇ m.
  • Fig. 5B illustrates that the iron oxide coating on these polystyrene microspheres is in islands and not continuous. Thermogravimetric measurements shows that the percent iron oxide coating on the microspheres is approximately between 6%-8%.
  • Example 13 was repeated under a variety of conditions: (a) Temperature range between 55°C to 90°C, similar results were obtained; (b) pH range between 8 to 11, the magnetic intensity of the coated microspheres prepared at pH range between 10 to 11 was higher than that prepared at the lower pH range; (c) Mole ratio [NaN ⁇ 2]/[FeCl2 «4H2 ⁇ ] up to 10 times higher than that described in the previous example, similar results were obtained; (d) Addition to the microspheres suspension up to five times more of the different reagents (NaNO2, FeCl2-4H2 ⁇ , and NaOH) causes to the corresponding growth of the iron oxide coating on the microspheres.
  • the different reagents NaNO2, FeCl2-4H2 ⁇ , and NaOH
  • Examples 13-15 were repeated, substituting the polystyrene microspheres of ca. 2.3 ⁇ m containing PVP adsorbed on its surfaces with microspheres of different sizes, i.e. ca. 0.3, 1.8 and 6.0 ⁇ m containing different hydrophilic surfactants adsorbed on its surfaces, i.e. PVP, polyacryhc acid and polyethyleneoxide. Similar results were obtained (percent iron oxide between 6% - 10%.) O 96/11054 PCMJS95/12988
  • Examples 13-15 were repeated, substituting the polystyrene microspheres of ca. 2.3 ⁇ m containing PVP adsorbed on its surfaces with microspheres containing hydrophobic surfactants, such as PVAc.
  • the percent iron oxide of these coated microspheres was significantly lower (i.e. ca. 3%) than that adsorbed with hydrophilic surfactants.
  • Example 13-17 were repeated, substituting polystyrene microspheres with polychloromethylstyrene microspheres and/or polymethylmethacrylate microspheres prepared as described in the experimental section hereof. Similar results were obtained.
  • Example 19 Preparation of thin coatings from silica on magnetic polystyrene microspheres.
  • Examples 1-7 were repeated substituting the polystyrene microspheres with polystyrene microspheres thin coated with Fe3U4, prepared according to the description in Examples 13-17. Similar results were obtained.
  • Fig. 5C illustrates the first silica nanoparticles coating on magnetic polystyrene microspheres of ca. 2.3 ⁇ m, prepared according to example 13. The process described above was also repeated with coatings prepared from sihca nanoparticles of sizes ranging from ca. 20 nm up to ca. 0.1 ⁇ m. The separation of grafted silica nanoparticles from non-grafted was performed with a magnetic field. SEM and TEM photomicrographs demonstrated the complete coating of the microsphere surfaces with silica nanoparticles.
  • Example 8 was repeated substituting the polychloromethylstyrene microspheres and/or polymethylmethacrylate microspheres with similar microspheres thin coated with Fe3U4 prepared according to the description in experiment 18. Similar results were obtained.
  • Example 21 Preparation of magnetic hollow silica microspheres.
  • Examples 9-12 were repeated, substituting the polystyrene microspheres and or polychloromethylstyrene microspheres and/or poltmethylmethacrylate microspheres coated with silica nanoparticles with similar microspheres coated with Fe3 ⁇ 4 and with silica, prepared according to the description in Examples 19 and 20. Similar results were obtained.
  • Silica hollow microspheres and or magnetic silica hollow microspheres prepared by burning off the organic core from the inorganic shell at temperatures above ca. 900 °C were floated on water due to its hydrophobic character.
  • similar hollow microspheres prepared below ca. 600 °C sank in water However, if these dried hollow microspheres are reburned at temperatures above ca. 900 °C they also become hydrophobic and float on water.
  • Example 23 Surface modification of the silica coated microspheres and/or silica hollow microspheres.
  • Derivatization of the previous described solid and hollow sihca microspheres through its hydroxyl groups with ⁇ -functionalized alkylsilane compounds and or ⁇ -functionahzed alkylhydroxyl compounds has been performed by the following general procedure: Dried microspheres were added to the appropriate solvent. The resulting mixture was then sonicated in order to dispersed the particles in the solvent. The derivatization of the suspended microspheres was then accomplished at the desired temperature for the desired period of time by- adding the appropriate ⁇ -functionahzed alkylsilane compound and/or ⁇ - functionalized alkylhydroxyl compound to the microspheres suspension.
  • the derivatized microspheres were then washed from undesired compounds by repeated centrifugation cycles under appropriate conditions (or with a magnetic field if magnetic microspheres were used).
  • the washed derivatized microspheres were then dried with a lyophihzer and/or a vacuum oven. If necessary, in order to prevent nonspecific interactions with the silica (due to its negative charge) blocking of residual sihca with appropriate reagents, such as bovine serum albumin (BSA), was performed by suspending the derivatized microspheres in phosphate buffered sahne (PBS) solution containing 1 % BSA for ca. 2 h at room temperature. The derivatized microspheres were then washed by centrifugation (or with a magnetic field if magnetic microspheres were used) with distilled water and then dried by lyophilization.
  • PBS phosphate buffered sahne
  • the derivatized microspheres were washed by two centrifugation cycles with bicyclohexyl (or toluene) and another two centrifugation cycles with acetone.
  • the derivatized microsphere surfaces were then dried by lyophihzation.
  • the reduction of the ⁇ -nitrile microsphere surfaces to ⁇ -amine derivatized surfaces was accomplished by suspending the derivatized microspheres at 50°C for ca. 18 h in a THF solution containing 1 M diborane. The reduced surfaces were then washed by centrifugation in THF and then in acetone.
  • the primary amino derivatized microsphere surfaces were then dried by lyophihzation. If necessary, albumin blocking of the derivatized microspheres was then performed as described previously.
  • Example 24 Covalent binding of polyaldehyde ligands onto the ⁇ -amine derivatized microsphere surfaces.
  • the ⁇ -amine derivatized microsphere surfaces prepared as described in example 23 were shaken at room temperature for approximately 5 h in an aqueous solution containing ca. 1% of the different polyaldehyde ligands, i.e. glutaraldehyde or polyacrolein nanoparticles of ca. 70 nm diameter prepared as described in the experimental part.
  • the polyaldehyde derivatized microsphere surfaces were then washed by extensive centrifugation with distilled water. If necessary, in order to decrease nonspecific interactions with biomaterials, i.e.
  • Example 25 Covalent binding of acrolein onto the ⁇ -amine derivatized microsphere surfaces.
  • the ⁇ -amine derivatized microsphere surfaces prepared as described in example 23 were shaken at room temperature for approximately 5 h in an aqueous solution, at pH range between 2-9, containing ca. 1% acrolein.
  • the formed polyaldehyde derivatized microsphere surfaces were then washed by extensive centrifugation with distilled water.
  • the polyaldehyde derivatized microspheres were then treated as described in example 24.
  • Our studies demonstrated that the binding of acrolein at acidic pH, i.e. between pH-2 to pH-4 resulted in approximately 3 to 10 times more aldehyde content in the derivatized microspheres.
  • the solid and/or hollow polyaldehyde derivatized microspheres were shaken at room temperature (or other desired temperature) for few hours with the desired protein in PBS (or other physiological solution). Unbound protein was then removed by centrifugation cycles (or by a magnetic separation) in PBS. Residual aldehyde groups were then blocked with amino hgands, such as BSA, hydroxylamine or ethanol amine in pH-7. The protein conjugated microspheres were kept in PBS (or water) at 4°C or kept dried after lyophihzation.
  • microspheres composed of polyaldehyde derivatized sihca coated polystyrene microspheres of ca. 1.8 ⁇ m diameter were shaken at room temperature for 4 h with 1 mg trypsin in 5 ml PBS. Unbound trypsin was then separated by 3 centrifugation cycles in PBS. Residual aldehyde groups on the microspheres were then blocked by shaking the conjugated microspheres at room temperature for 4 h with BSA (1%) in PBS. Unbound BSA was then removed by 2 centrifugation cycles in PBS and then 2 centrifugation cycles in distilled water. The trypsin conjugated microspheres were then dried by lyophihzation.
  • the foUowing proteins were covalently bound to the different polyaldehyde derivatized microspheres: goat anti-rabbit IgG (G ⁇ ⁇ RIgG), , goat anti-mouse Ig (G°cMIg), protein A, trypsin and lysozyme.
  • G ⁇ ⁇ RIgG goat anti-rabbit IgG
  • G°cMIg goat anti-mouse Ig
  • protein A trypsin and lysozyme.
  • the activity of the trypsin conjugated microspheres prepared as described in example 26 was checked with the substrate oc-N-benzoyl-DL-arginine p- nitroanilide (BAPNA).
  • BAPNA substrate oc-N-benzoyl-DL-arginine p- nitroanilide
  • the conjugated trypsin in reaction with BAPNA in Tris buffer (pH-8) liberated p-nitroanihne of which its absorbance value at 400 nm was measured.
  • ⁇ i-antitrypsin in human serum was based on the inhibitory effect of antitrypsin of serum on the hydrolysis of BAPNA by the conjugated trypsin in Tris buffer. The reaction is stopped by adding acetic acid, and the absorbance is then read at 400 nm. At this wavelength the liberated p- nitroanihne has a molar absorptivity of 10,500. Briefly, before the assay, each examined serum was diluted 1000 fold with Tris buffer. 2 ml of the diluted serum were then incubated at 37°C for 30 min with 1 ml suspension containing 10 mg trypsin-conjugated microspheres in Tris buffer.
  • BAPNA solution prepared by dissolving 100 mg BAPNA in 2.3 ml dimethylsulfoxide, and then diluted 1 ml of this stock solution with 100 ml Tris buffer at pH 8.0
  • the reaction was stopped by adding 1 ml glacial acetic acid.
  • the degree of interaction between the conjugated trypsin and BAPNA was then determined by the absorbance value at 400 nm. Control experiments were carried out with 4% HSA by using the same procedure. The precise amount of ⁇ i-antitrypsin was determined by comparison to a standard curve obtained from a known amount of ⁇ j-antitrypsin.
  • Fresh human red blood cells (HRBC) were shaken for 50 min at 4°C with rabbit anti-HRBC (10 6 HRBC with 0.8 ⁇ g rabbit anti-HRBC) antibodies.
  • the sensitized cells were separated and washed 4 times by centrifugation with PBS.
  • the washed sensitized cells were then shaken at 4°C for 1 h with G «
  • RIgG magnetic conjugated microspheres (5 mg) prepared as described in example 26.
  • the labeled cells were then separated from excess microspheres by 3 centrifugations with PBS.
  • the control cells on the other hand, were not labeled at all.
  • a mixture containing 10 6 human RBC and 10 6 turkey RBC was treated with magnetic microspheres by using the labeling procedure described in (2).
  • a small magnet was then fitted on the outside wall of a vial containing 5 ml PBS suspension of the mixture of cells. After ca. 5 min, cells that were not attracted to the wall were isolated. The attracted cells were resuspended in PBS and the magnetic separation repeated twice. Examination in hght microscopy demonstrated separation efficiency of 98%-100% of the human RBC from the turkey RBC.
  • GocMIg conjugated microspheres (5 mg) prepared as described in example 26 were shaken at 4°C for 1 h with purified mouse splenocytes (10 ⁇ ). The labeled cells were then separated from excess microspheres by 3 centrifugation cycles with PBS. Control experiments carried out similarly, substituting the mouse splenocytes with mouse thymocytes. Examination with hght microscopy demonstrated the specific labehng of the mouse splenocytes with the GocMIg conjugated microspheres. The control cells, on the other hand, were not labeled at all.

Abstract

L'invention concerne un procédé de préparation d'une microbille comprenant un enrobage constitué d'une ou plusieurs couches de nanoparticules de silice. Ce procédé consiste a) à produire une microbille en matière polymère présentant un ou plusieurs agents tensioactifs adsorbés à sa surface, b) à recouvrir la surface de ladite microbille d'une couche de nanoparticules de silice au moyen de la polymérisation par germination de silicates d'alkyle à la surface de ladite microbille.
PCT/US1995/012988 1994-10-06 1995-10-05 Procede de preparation de microbilles et microbilles ainsi preparees WO1996011054A2 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
DE19581787T DE19581787T1 (de) 1994-10-06 1995-10-05 Verfahren zur Herstellung von Mikrokügelchen und dadurch hergestellte Mikrokügelchen
AU41934/96A AU4193496A (en) 1994-10-06 1995-10-05 Process for the preparation of microspheres and microspheres made thereby
US08/809,957 US6103379A (en) 1994-10-06 1995-10-05 Process for the preparation of microspheres and microspheres made thereby

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
IL11118694A IL111186A (en) 1994-10-06 1994-10-06 Process for the preparation of microspheres and microspheres made thereby
IL111186 1994-10-06

Publications (3)

Publication Number Publication Date
WO1996011054A2 WO1996011054A2 (fr) 1996-04-18
WO1996011054A3 WO1996011054A3 (fr) 1996-05-23
WO1996011054A9 true WO1996011054A9 (fr) 1996-08-15

Family

ID=11066612

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1995/012988 WO1996011054A2 (fr) 1994-10-06 1995-10-05 Procede de preparation de microbilles et microbilles ainsi preparees

Country Status (4)

Country Link
AU (1) AU4193496A (fr)
DE (1) DE19581787T1 (fr)
IL (1) IL111186A (fr)
WO (1) WO1996011054A2 (fr)

Families Citing this family (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19520398B4 (de) 1995-06-08 2009-04-16 Roche Diagnostics Gmbh Magnetisches Pigment
KR100463475B1 (ko) 1995-06-08 2005-06-22 로셰 디아그노스틱스 게엠베하 자기성피그먼트
FR2747669B1 (fr) * 1996-04-22 1998-05-22 Rhone Poulenc Chimie Procede de preparation de particules creuses de silice
AU6021798A (en) 1997-01-21 1998-08-07 W.R. Grace & Co.-Conn. Silica adsorbent on magnetic substrate
JP4092070B2 (ja) * 2000-11-27 2008-05-28 独立行政法人科学技術振興機構 糖誘導体を用いる有機無機複合体および金属酸化物の製造方法
GB0107563D0 (en) 2001-03-27 2001-05-16 Amersham Pharm Biotech Uk Ltd NO synthase assay particles and method
US6890703B2 (en) 2002-03-06 2005-05-10 International Business Machines Corporation Preparation of crosslinked particles from polymers having activatible crosslinking groups
US20040197819A1 (en) * 2003-04-03 2004-10-07 Kimberly-Clark Worldwide, Inc. Assay devices that utilize hollow particles
DE10355409A1 (de) 2003-11-25 2005-06-30 Magnamedics Gmbh Sphärische, magnetische Silicagel-Träger mit vergrößerter Oberfläche für die Aufreinigung von Nukleinsäuren
US9278866B2 (en) 2005-08-10 2016-03-08 The Procter & Gamble Company Hollow silica particles, compositions comprising them, and methods for making same
WO2011131644A1 (fr) * 2010-04-20 2011-10-27 Basf Se Capsule comprenant un ingrédient actif
CN104781321B (zh) * 2012-11-16 2018-01-23 昆塔麦特利斯株式会社 编码的聚合物微粒
DE202016008770U1 (de) * 2016-03-18 2019-08-06 Bundesrepublik Deutschland, vertreten durch den Bundesminister für Wirtschaft und Energie, dieser vertreten durch den Präsidenten der Bundesanstalt für Materialforschung und –prüfung (BAM) Hybride Kern-Schale-Mikropartikel umfassend einen Polymerkern und eine Siliziumdioxidschale mit kontrollierter Struktur und Oberfläche
CN110508222B (zh) * 2019-08-02 2022-03-18 复旦大学 具有介孔二氧化硅壳层的单分散核壳微球及其制备方法
CN113578214B (zh) * 2021-08-11 2023-08-11 天津博蕴纯化装备材料科技有限公司 微米级多孔磁性微球及其制备方法和应用
CN115893980B (zh) * 2022-09-30 2023-08-11 安徽华仕新材有限公司 一种利用球化剂微粉制备多孔支撑体陶瓷的工艺

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2564336A1 (fr) * 1984-05-16 1985-11-22 Ohayon Hanania Dispositif de separation du sang et d'obtention de serum
FR2624873B1 (fr) * 1987-12-18 1992-01-10 Rhone Poulenc Chimie Particules composites magnetisables a base d'organopolysiloxane reticule, leur procede de preparation et leur application en biologie
WO1992016838A1 (fr) * 1991-03-20 1992-10-01 Gec-Marconi Limited Technique de separation
GB9211822D0 (en) * 1992-06-04 1992-07-15 Tioxide Group Services Ltd Composite pigmentary material

Similar Documents

Publication Publication Date Title
US6103379A (en) Process for the preparation of microspheres and microspheres made thereby
WO1996011054A9 (fr) Procede de preparation de microbilles et microbilles ainsi preparees
WO1996011054A2 (fr) Procede de preparation de microbilles et microbilles ainsi preparees
EP1088315B1 (fr) Nucleation et croissance de nanoparticules d'oxyde metallique magnetique et utilisation de ces dernieres
CA2410023C (fr) Nanoparticules enrobees
US5814687A (en) Magnetic polymer particle and process for manufacturing the same
CA1137296A (fr) Dispersions a particules magnetiques
Lan et al. Facile synthesis of monodisperse superparamagneticFe3O4/PMMA composite nanospheres with high magnetization
US20040265233A1 (en) Composite particle containing superparamagnetic iron oxide
WO2009081700A1 (fr) Particule inorganique fine à revêtement polymère et son procédé de production
JP2003519063A (ja) 多層コーティングによる結晶のカプセル化
JP2003513093A (ja) 複合ナノスフェアおよびそれらの生体分子との複合体
US8029897B2 (en) Nanometric or mesoscopic dissymetric particles, and method for preparing same
Kang et al. Biomimetic approach to the formation of gold nanoparticle/silica core/shell structures andsubsequent bioconjugation
US5976426A (en) Latex of calibrated monodisperse magnetizable microspheres, process of preparation and use of the said latex in chemistry or in biology
US4438156A (en) Mono-particle magnetic dispersion in organic polymers for magnetic recording
JPH10270233A (ja) 磁性ポリマー粒子およびその製造方法
CN114192079B (zh) 一种磁性中空聚合物微球及其制备方法和应用
JP4426819B2 (ja) 磁性体内包粒子の製造方法
JP4080798B2 (ja) 核酸結合用磁性担体およびその製造方法
Fernández et al. Functionalization of γ-alumina cores by polyvinylpirrolidone: properties of the resulting biocompatible nanoparticles in aqueous suspension
US20080038176A1 (en) Unsymmetrical Inorganic Particles, and Method for Producing the Same
Yoshinaga et al. Immobilization of protein on monodispersed colloidal silica with poly (ethylene glycol) spacer and application of the composites to immunological agglutination tests
JP2005241547A (ja) 磁性体内包粒子及び免疫測定用粒子
JP4148301B2 (ja) 乾燥ポリマービーズ調製物
点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载