WO2003005029A2 - Nanoparticules magnetiques pour bioseparations et leurs procedes de production - Google Patents

Nanoparticules magnetiques pour bioseparations et leurs procedes de production Download PDF

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Publication number
WO2003005029A2
WO2003005029A2 PCT/CA2002/001003 CA0201003W WO03005029A2 WO 2003005029 A2 WO2003005029 A2 WO 2003005029A2 CA 0201003 W CA0201003 W CA 0201003W WO 03005029 A2 WO03005029 A2 WO 03005029A2
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particles
nanoparticles
magnetite
dextran
solution
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PCT/CA2002/001003
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WO2003005029A3 (fr
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Zhenghe Xu
Yunli Gu
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Governors Of The University Of Alberta
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Priority to AU2002317095A priority Critical patent/AU2002317095A1/en
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Publication of WO2003005029A3 publication Critical patent/WO2003005029A3/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles

Definitions

  • This invention relates to the field of magnetic, preferably paramagnetic or superparamagnetic, and methods and uses thereof, including for bioseparation applications.
  • Particles in the 1-200 nm diameter size range, considered as nanoparticles often exhibit unique optical, electrical, chemical, structural and/or magnetic properties.
  • magnetite particles greater than 1 ⁇ m in diameter are ferromagnetic, exhibiting a large magnetization hysteresis and a strong remnant magnetic induction (M r ) whereas magnetite particles less than 200 nm are superparamagnetic with little magnetization hysteresis while maintaining a strong saturation magnetization (M s ) (Leslie-Pelecky and Rieke, 1996).
  • This class of paramagnetic nanoparticles have a wide range of potential applications, including information storage, color imaging, bioprocessing, diagnostic microbiology, biosensors, drug delivery, magnetic refrigeration, ferrofluids and magnetic switchers. Applications using magnetic nanoparticles as magnetic carriers or magnetic tags have also been described (Williams, 1992).
  • the objective of magnetic carrier technology is to confer magnetic properties to a naturally non-magnetic target so that the target can be separated from the non-target entities using magnetic separators. This is particularly useful for separations in multiphase systems of a complex nature.
  • Figure 1 summarises schematically the processes involved in a typical magnetic carrier technology application. In order to make a target magnetic, the target has to be recognised by and attached to a magnetic carrier.
  • paramagnetic particles to be functionalized, usually by derivatizing a surface or putting a specific ligand on the particle (step I), or by in situ formation of a nanosize magnetic core in a structured template, e.g. micells, vesicles, porous silica or polymer matrix (step II).
  • a structured template e.g. micells, vesicles, porous silica or polymer matrix
  • the particles prepared as such termed as magnetic carrier (b) are added to a biological system containing unwanted targets, such as infected blood cells (white circles in c).
  • unwanted targets such as infected blood cells (white circles in c).
  • the selective attachment of infected cells to the magnetic carriers is accomplished through biomolecular recognition, conferring the magnetic property to the infected cells only (step III).
  • the labelled cells are directed to a desired location (the top of the system in this case), where they can be removed readily without disturbing the system (step IV).
  • a principal attraction of using magnetic carrier technology in clinical applications is its minimised side effect, compared with traditional treatments, such as chemotherapy which destroys healthy cells as well. (Bacri et al., 1997; Mitsumori et al., 1996).
  • an alkali solution is added to a solution of a ferrous and ferric salt.
  • the particles formed are then separated by centrifugation and gel filtration.
  • the particles in the invention have a diameter of about 100-700 Angstrom, more particularly about 300-400 Angstrom.
  • the magnetic-dextran particles and processes for producing same all have deficiencies.
  • biological cell separation using magnetic carrier technology Kerck and Gilpin, 1986
  • the particles must be sufficiently small (in the nano size range) that thermal energy and surface forces prevail.
  • magnetic flocculation a phenomenon where magnetic particles agglomerate by magnetic forces. With particles less than 200 nm, the remnant magnetic induction is negligible so that the permanent magnetization is avoided and magnetic flocculation minimized.
  • the present invention provides an improved method for synthesizing superparamagnetic nanoparticles.
  • These nanoparticles can be used in a variety of applications, for instance environmental, biological, chemical applications. In one embodiment, these nanoparticles are particularly useful in biological applications. In another embodiment, the nanoparticles are useful in tagging, monitoring chemical or biochemical reactions, monitoring the presence, production, synthesis, break down of certain substances, isolation and/or removal of substances from a sample, including purification, detoxifciation, diagnostic, treatment applications.
  • the invention also provides the resultant nanoparticles and methods and uses thereof.
  • the present invention provides a method of synthesizing paramagnetic, preferably superparamagnetic magnetite particles.
  • the method comprises: a) crystallizing magnetite to form superparamagnetic particles under controlled crystallization conditions by adding a solution of ferric and ferrous ions to an alkaline solution, thereby limiting the duration of ferric and ferrous ion supersaturation and the extent of magnetite crystal growth; b) controlling aggregation of said magnetite particles to control the size distribution of said particles and; c) coating said particles with a coating substance to generate non-aggregating particles, thereby stabilizing the particle suspension.
  • (b) is performed under conditions that inhibit the oxidation of Fe2+ to Fe3+ iron, such as under redox conditions.
  • redox conditions can be obtained by nitrogen sparging or chemical equivalent thereof, vacuum or other method known in the art, to obtain an environment of reduced or no oxygen.
  • controlling aggregation comprises permitting or reducing or inhibiting aggregation as desired.
  • controlling aggregation means preventing aggregation to the degree desired.
  • preventing or inhibiting aggregation means that aggregation is prevented or inhibited to at least some degree to control crystal growth and particle size, but with regard to a whole sample a certain degree of aggregation may exist.
  • a person skilled in the art upon reading this description would understand the degree of prevention within a particular sample that would be beneficial to produce the desired superparamagnetic nanoparticles of the invention and the amount thereof and factors that can be adjusted to control aggregation.
  • aggregation of particles is controlled by physical means, for instance by sonication, such as ultrasonication. Sometimes an untrasonication prevents aggregation.
  • the solution of ferric and ferrous ions has a pH of about 2.5.
  • the alkaline solution is a sodium hydroxide solution, for example a 1.5M sodium hydroxide solution.
  • the coating substance is a water-soluble substance that is suitable for inhibiting particle aggregation and that can be coupled to a desired target molecule directly or indirectly.
  • the water-soluble substance is a water-soluble polysaccharide for example dextran, dextrin, starch, guar, gelatin, agar, sucrose or dextrose.
  • the coating substance is dextran, such as dextran with a molecular weight of between 4,000 to 2,000,000, preferably, 4,000 - 70,000, more preferably about 35,000 - 45,000, or about 40,000.
  • the coating substance is 0.25M dextran solution.
  • the coating substance is BSA, antibodies, natural or synthetic polymers, protein, polypeptides or nucleic acid polymers.
  • magnetite nanoparticles preferably supermagnetic particles produced by the method of any of the embodiments of the present invention is also intended to be encompassed within the scope of the invention.
  • the invention provides a method of rendering a biological target magnetizable comprising, functionalizing the magnetite nanoparticles produced by the method of the invention to enable them it to couple with the desired target, such as a biological target and then exposing the target to the functionalized magnetite nanoparticles to form a nano particle-target complex.
  • the magnetite nanoparticles of the invention are functionalized by derivatizing the surface of the particles and/or by putting a specific ligand on the particle.
  • it is the coating substance of the particle that is functionalized.
  • the nanoparticles are functionalized by an anti-coating substance antibody, such as a dextran antibody.
  • the biological targets are cells, protein, nucleic acid molecules, such as DNA or RNA.
  • the method includes recovering the nano particle-target complex from a sample by magnetic separation, for instance by exposing the sample to an external magnetic field gradient.
  • the present invention provides an integrated fabrication route for synthesizing superparamagnetic magnetite nanoparticles stabilized by water soluble dextran molecules.
  • the adsorbed dextran molecules on the synthesized magnetite particles exhibit specific binding with anti-dextran antibody complexes which in turn bind with antigen on target cells, making these cells amenable for magnetic separations.
  • Figure 1 is a schematic diagram illustrating the concept of magnetic carrier technology in biological cell separations.
  • Figure 2 is a graph of concentration versus time illustrating the principle for wet chemical synthesis of nanosize solid particles (Hunter, 1993).
  • Figure 3 illustrates the X-ray diffraction patterns of the synthesized solid particles (the vertical lines represent the diffraction peak positions of the standard magnetite with the line height showing the relative intensity of the diffraction peaks.).
  • Figure 4 illustrates the improvement in the signal to noise ratio of the X-ray diffraction patterns of the synthesized solid particles when the redox environment is controlled (with and without nitrogen sparging). The results suggest that the particles formed with redox control have a greater crystalinity.
  • Figure 5 illustrates the magnetization of the synthesized solid particles with (closed circles) and without (open circles) nitrogenation by adding a ferrous/ferric ion solution to an alkaline solution at pH 12.5, showing a strong magnetism with a clear superparamagnetic characteristic for particles prepared with redox control by nitrogenation.
  • Figures 6 is a graph illustrating particle size distribution of the synthesized magnetic particles (adding ferrous/ferric ion solution to an alkaline solution at pH 12.5).
  • Figure 7 is transmission electron micrographs of the synthesized particles showing: A) morphology; and B) electron diffraction pattern.
  • Figure 8 illustrates the role of supersaturation level control in synthesis of magnetic particles by adding ferrous/ferric to an alkaline solution at pH 12.5 vs the addition of NaOH solution to ferrous/ferric ion solution to a final pH of 12.5.
  • the process of the present invention is illustrated with open circles and the prior art method is illustrated in closed circles.
  • Figure 9 demonstrates the role of ultrasonication strength in controlling the resultant average particle sizes.
  • Ferrous/ferric solution was added to an alkaline solution at pH 12.5.
  • Figure 10 shows the effect of ultrasonication intensities on the magnetization of the resultant magnetic nanoparticles synthesized by adding ferrous/ferric to an alkaline solution at pH 12.5.
  • Magneticite as used herein is an iron ore that is strongly attracted by a magnet. In one embodiment it has a general formula of Fe 3 O 4 (preferably with a Fe 2+ to Fe 3+ ratio of about 1 :1.5 to about 1 :2.5 , preferably about 1 :2) or chemical equivalents thereof such as (Fe 2+ , M)O Fe 3+ 2 O3 where M can be, but not limited to Zn, Co, Ni, Mn, Cr. These examples are not meant to limit the scope of this invention.
  • the Fe 2+ to Fe 3+ ratio includes any ratio that permits the formation of superparamagnetic nanoparticles in accordance with the method of the present invention.
  • magnet refers to a class of substances that when placed in a magnetic field are magnetized parallel to the line of force in the field and proportional to the intensity of the field.
  • superparamagnetic refers to a class of substances that have a similar magnetism as ferromagnetic materials in the external magnetic field, but does not have a remnant magnetization after removal of the external magnetization field.
  • magnetization hysteresis refers to a lagging effect behind its cause, for instance a magnetic body subject to a varying force. This can result in an internal magnetic field in the material that may show long term drifts.
  • Water soluble as used herein means those substances, (such as polysaccharides, or more specifically dextran,) that will remain in aqueous solution as individual molecules at ambient temperature (e.g. about but not limited to15-25 degrees Celcius.
  • Stablized as used herein in reference to paramagenitc or supermagnetic particles refers to the stability of the resulting particles in solution after the reagents have been mixed. Stabilization means that the particles are stable without aggregation or settling out of solution. Further that there is no change in the chemical composition of the magnetic particles under specified conditions over a particular period of time.
  • particle size distribution as used herein means the particle size range and the proportion of the magnetic nanoparticles at a particular particle size or sizes.
  • redox environment or conditions refers to an environment that has reduced oxygen levels as compared to ambient conditions, preferably that is substantially free of oxygen. It refers to an environment where the net oxidation of iron in the divalent state to the trivalent state is minimized or preferably prevented.
  • an environment can be obtained by nitrogen sparging (e.g. to displace oxygen) or by other means known in the art (vacuum, sparging with other inert gases e.g. argon.
  • nitrogen sparging e.g. to displace oxygen
  • other inert gases e.g. argon.
  • nucleation in reference to crystallization of magnetite as used herein means the formation of a core site of crystalline Fe 3 U4. Each nucleus can spawn a crystal particle.
  • polysaccharide refers to any water soluble polysaccharide, including, but not limited to dextran, dextrin, starch, guar, gelatin, agar, sucrose and dextrose.
  • extract refers to but not limited to neutral or weakly charged dextran with a molecular weight range of about 4,000 to 2,000,000, or 4,000 to 70,000 or, preferably from about 35,000 to 40,000. Examples of such dextran include Dextran T40 from Amersham Pharmacia Biotech.
  • nanoparticle refers to a particle having a size in the order of 10 "9 m range.
  • the nanoparticles of the present invention are in the range from aboutlO to 250nm, 20 to 250nm, 30 to 250nm, 10 to 200nm, preferably about 20 to 200nm, 30 to 200nm, or 50 to 150 nm, with a mean and/or average size of the nanoparticles preferably being 150nm.
  • saturated magnetization also known as maximum magnetization, refers to a point when all of the magnetic moments in a sample are aligned.
  • controlled crystallization means a process wherein the growth or size of crystals can be controlled (increased, decreased or remain the same) during the course of a reaction.
  • Controlled crystallization can be effected through a number of methods in this invention, including but not limited to one or more of the following: sonication intensity, pH of alkaline solution, concentration of iron solution and ratio of ferric to ferrous ions.
  • crystallization is controlled by using sonication at an instrument output of 35%, adding the ferric/ferrous solution to the sodium hydroxide solution with a preferred ratio of two ferric ions to one ferrous ion or functional chemical or obvious equivalents thereof.
  • a number of approaches have been developed to synthesize nanoparticles (Chow and Gonsalves, 1996).
  • the examples include: gas phase atomization (aero sols), evaporation/condensation, bulk wet chemical synthesis with sonochemistry or in templated microreactors such as porous matrix (zeolites), microemulsions, vesicles or micelles.
  • the approach of the present invention is to control nucleation and particle growth by controlling the crystallization chemistry.
  • this is achieved by adding the iron solution to an alkali solution under conditions that control (e.g. inhibit or minimize) aggregation between magnetic nuclei and/or crystal particles (such as by ultrasonication).
  • This addition method generates a large number of nuclei by creating a local supersaturation level while controlling the level of crystal growth by resultant sparse concentration conditions of the bulk alkaline solution.
  • the particles are then stabilized by coating them with a coating substance that inhibits aggregation between particles and preferably maintains the resultant particle size distribution.
  • the coating substance is preferably a biocompatible substance that can be used in bioseparation, biotagging or biomonitoring applications in vivo and/or in vitro.
  • the coating substance is also preferably selected for its ability to be functionalized to couple with a target substance. This can be done by coupling the coating substance with another substance such as substances that can both couple with the coating substance and a specific desired target molecule.
  • the coating substance can be dextran
  • the other substance can be an anti-dextran antibody that can bind dextran and also has specificity to a desired target such as a particular cell surface receptor, cell type, protein, nucleic acid molecule or antigen so it can form a magnetic nanoparticle - target complex that can be separated from non-magnetic particles by application of a magnetic field, such as an external magnetic field.
  • the coating substance can also be derivatized to couple with the target substance, preferably a biological target.
  • the present invention provides a method to synthesize paramagnetic, preferably superparamagnetic nanoparticles comprising controlled crystallization of magnetite (Fe 3 0 4 ) as well as other chemical equivalent compounds such as (Fe 2+ , M) Fe 3+ 2 O 4 , where M can be, but not limited to Zn, Co, Ni, Mn, Cr, under controlled aggregation conditions and stabilization of the particles by coating with a polysaccharide, preferably a water soluble polysaccharide, such as dextran with a preferable molecular weight between 35,000 to 45,000, but not limited to that range. Other ranges can work, for instance 4,000 to 2,000,000.
  • Other water soluble polysaccharides covered by this invention include but are not limited: dextrin, starch, guar, gelatin, agar, sucrose and dextrose.
  • a solid product in the 30 to 250 nm diameter range was produced by adding a concentrated iron solution to an alkaline solution at pH above 8, preferably at pH 12.5 under ultrasonication to prevent particle aggregation.
  • the particles were stabilized with dextran.
  • the synthesized particles were identified with x-ray diffraction as composed largely of magnetite and to be superparamagnetic with a saturation magnetization of about 62 emu/g as determined with a Quantum Design PPMS magnetometer. Transmission electron micrographs showed that the particles were rounded without noticeably sharp edges. These particles exhibited excellent stability and showed satisfactory blood cell recovery with magnetic separations. This method is an improvement over previous methods.
  • the magnetic nanoparticles of the invention are synthesized under a well controlled redox environment in one step (for instance without requiring subsequent frationation) with controlled particle size and crystallinity.
  • the redox environment controls the oxidation state of iron species and preserves the preferable ratios of iron between two different oxidation states (e.g. ferric to ferrous ratio).
  • the preferred redox environment is one that is of a reduced oxygen or other oxidizing compound, content in aqueous solutions (compared to ambient conditions), preferably free of molecular oxygen, which can be accomplished by sparging with an inert gas such as nitrogen, but may also be done with argon.
  • the particle size distribution of the resultant particles is such that there is no need for separation or fractionation when the reaction finishes because the particles are all in a suitable size range.
  • the suitable size range is between 10 - 250 nm, 10 to 200 nm, 20 to 250 nm, 20 to 200 nm, 30 to 250 nm, 30 to 200 nm, or 50 to 150 nm.
  • the mean and/or average particle size is about 150nm.
  • the present invention in one embodiment provides a method of synthesizing paramagnetic, preferably superparamagnetic magnetite particles comprising:
  • the alkaline solution in one embodiment has a pH of above about 8 and preferably about 12.5.
  • the addition of the iron solution into the alkaline solution limits the duration of ferric and ferrous ion supersaturation and the extent of magnetite crystal growth. As stated it above, it generates a large number of nuclei by creating a local supersaturation level while controlling the level of crystal growth by resultant sparse concentration conditions of the bulk solution.
  • the method at least preferably (a) and preferably (b), is conducted in a controlled redox environment to inhibit or prevent the oxidation of Fe 2+ to Fe 3+ ions.
  • the redox environment is controlled by nitrogen sparging.
  • the coating substance is a polysaccharide, preferably a water soluble polysaccharide, such as dextran, for example a dextran having a molecular weigh of about 40,000.
  • the dextran solution is a .25M dextran solution.
  • the coating substance is Bovine Serum Albumin (BSA) antibodies, lipids, natural or synthetic polymers, protein, polypeptides or nucleic acid polymers.
  • BSA Bovine Serum Albumin
  • the present invention provides paramagnetic, preferably super paramagnetic nanoparticles that can be functionalized to enable them to be used in bio applications such as bioprocessing, diagnostic microbiology, biosensors, drug delivery, magnetic refrigeration, ferrofluids and magnetic switchers, magnetic carrier technology, and applications in screening and purification or isolation.
  • the nanoparticles are produced by the method of the present invention.
  • the paramagnetic nanoparticles of the invention can be functionalized in many ways known to persons skilled in the art such as by derivatizing the surface of the particles by putting a specific ligand on the particle.
  • the nanoparticles are functionalized by an anti-dextran antibody.
  • the paramagnetic particles of the invention are preferably iron oxides, such as magnetite, in a preferred ferrous to ferric iron ratio of about 1 :2, but not limited to that ratio.
  • Other embodiments include a ratio of ferrous to ferric particles of about 1 :1.5-2.5, doped with additive ions, such as but not limited to Zn, Co, Ni, Mn and Cr.
  • Functionalized paramagnetic or superparamagnetic nanoparticle means a nanoparticle that is stabilized or coated by a substance such as a polysaccharide, such as dextran.
  • the stabilized paramagnetic nanoparticle can be further modified or can directly tag another molecule, preferably a biomolecule (e.g. a biological target) (e.g. a cell, a cell surface receptor, an antibody, a protein, nucleic acid molecules such as DNA or RNA) or other desired molecules in a preferably specific manner, to form a nanoparticle target complex.
  • a biomolecule e.g. a biological target
  • the paramagnetic nanoparticle can confer magnetic properties to said non magnetic target substance that is desired to be tagged, monitored, distinguishable or separated from other molecules or substances.
  • a coating substance that can be coupled to the magnetic nanoparticle of the invention directly to a desired target substance or through an intermediary substance or substances (e.g. a linker) that can both couple with the coating substance and the specific target substance.
  • the dextran is the coating substance, an anti-dextran antibody that also has specificity to the target substance couples to the dextran and then can be used to couple the target substance which can be subsequently removed, separated or detected using a magnetic field.
  • the magnetic nanoparticles can also be functionalized by derivatizing the surface of the particles, putting a specific ligand on the particle through silanation or self- assembly by desired molecules, such as organic silanes, lipids, and surfactant molecules.
  • the invention also encompasses superparamagnetic nanoparticles coated (preferably coupled for instance, through chemical, physiochemical and/or hydrogen binding) with a suitable polysaccharide.
  • suitable polysaccharides include any water soluble polysaccharide, including, but not limited to dextran, dextrin, starch, guar, gelatin, agar, sucrose and dextrose.
  • Preferred polysaccharides are dextran.
  • the polysaccharide is loaded on the superparamagnetic particles at a sutiable loading capacity such as greater than 0.29g dextran/g solids, but the invention is not limited by such a loading capacity.
  • a sutiable loading capacity such as greater than 0.29g dextran/g solids
  • the invention is not limited by such a loading capacity.
  • a person skilled in the art can adjust the loading capacity to a desired level, for instance by adjusting the ratio of magnetite and coating substance, such as dextran used and the coating conditions.
  • the invention provides a paramagnetic, preferably superparamagnetic nanoparticles that have an average particle size of about 150 nm.
  • the size of the nanopartices produced is equally distributed around 150 nm, and preferably within a range of 20 - 250 nm but can be 10 - 250 nm, 30 - 250nm, 10 - 200 nm, 20 - 200 nm, 30 - 200 nm or 50 - 150 nm.
  • the product has a narrow particle size distribution in that the particles are primarily around 150 +/- 20 nm.
  • the invention provides a paramagnetic nanoparticle that can confer magnetic properties to a substance or molecule of interest. It can act as a molecular tag or carrier. The substance or molecule of interest can then be separated from other molecules by application of a magnetic field, such as an external magnetic field gradient and separating the magnetic particles from the non-magnetic particles.
  • a magnetic field such as an external magnetic field gradient
  • the superparamagnetic nanoparticles of the invention can be used in monitoring the presence or amount of a desired substance in an assay, such as a bioassay, (e.g. environmental, diagnostic or other assay).
  • an assay such as a bioassay, (e.g. environmental, diagnostic or other assay).
  • the method can also be used to separate or remove a desired substance from other substances, such as to tag and remove cancerous or other cells or substances from a biological environment be it in vitro or in vivo.
  • the magnetic nanoparticle should be biocompatible, in such that it is not harmful to a subject upon administration.
  • the magnetic nanoparticles of the invention can be formed into a pharmaceutical composition and mixed with suitable pharmaceutically acceptable carriers or excipients, such as disclosed in Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa, USA, 1985.
  • suitable pharmaceutically acceptable carriers or excipients such as disclosed in Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa, USA, 1985.
  • the nanoparticles can be used in the treatment or diagnosis of certain conditions such as in tagging, detecting and/or removing cancer cells for example from a sample or tissue.
  • the invention can also be extended to detoxification and/or valuable recovery from domestic and industrial wastes.
  • Solution preparation For each test, an iron solution at a ferric to ferrous molar ratio of 2:1 was freshly prepared by dissolving 5.90 g of iron(lll) sulfate tetrahydrate and 3.47 g of iron(ll) sulfate heptahydrate in 100 mL of deionized water in a 250 mL beaker, with a resultant pH of ca. 2.5.
  • An alkaline solution was prepared by adding 6.0 g of sodium hydroxide in 100 mL of deionized water in a 500 mL beaker.
  • To prepare a dextran solution 5.0 g of dextran was added to 45mL of water in a 100 mL beaker.
  • Synthesis of stable magnetite nanoparticle suspensions The prepared iron solution was added to the alkaline solution dropwise within 5 minutes under a strong ultrasonication environment with or without nitrogen sparging to control the redox environment. To avoid oxidation of ferrous to ferric ions during the synthesis, sodium hydroxide aqueous solution was purged for 30 minutes with pure nitrogen through glass pipette tube prior to the addition of ferrous/ferric solutions. The nitrogen purge continued during the addition of the ferric/ferrous ion solutions and the dark precipitates were observed to form immediately during this period.
  • This procedure ensures a local supersaturation level for nucleation, a condition shown in Figure 2 by the solid line, while maintaining a minimum bulk iron concentration to limit the duration of crystal growth represented by the dotted line.
  • the resultant suspension was then transferred to the dextran solution at ambient or room temperature in the range of 15-25°C under agitation for 15 minutes, resulting in a stable dark suspension.
  • the suspension was then centrifuged using a BECKMAN Ultracentrifuge (AllegraTM 64R) at 20,000-g for one hour.
  • the supernatant was decanted and the solids were redispersed in 50 mL deionized water using an ultrasonic bath (FS30, Fisher Scientific).
  • the redispersed particles were centrifuged again to decant the supernatant. This washing procedure was repeated for three times to ensure a complete removal of residual dextran in solution.
  • the washed particles were then redispersed in deionized water.
  • Particle characterization For the purpose of characterization, samples of particles prepared as described were dried in a vacuum oven at room temperature for at least 48 hours. The crystallinity of the particles was characterized using a Philips x-ray powder diffractometer (PW 1730, Philips). Magnetization characteristics of a precisely weighed volume of dry particles was determined at 300 K on a Quantum Design PPMS magnetometer (model 6000).
  • the size distribution of the synthesized particles was measured using a Zetasizer 3000 (Malvern Instrument Inc., Point Roberts, WA).
  • the prepared suspension was diluted with deionized water and the measurement was performed at room temperature.
  • TEM transmission electron microscope
  • the prepared suspension was further diluted with deionized water and a holey carbon TEM grid was dipped into the diluted suspension. By carefully taking the grid out of the suspension, a fine drop of the suspension was retained on the grid. After evaporation of the water under ambient conditions, the specimen was transferred into TEM sample chamber and micrographs were taken in the conventional TEM mode.
  • Example 2 Magnetic Characteristics The magnetization characteristics of the formed particles is shown in
  • a saturation magnetization at 25 emu/g was obtained for the sample prepared without nitrogen sparging as also seen in Figure 5, showing a much weaker magnetization characteristics than the sample prepared with sparging. This is likely due to the higher proportion of maghemite or other iron oxide present in the synthesized particles without nitrogen sparging, as seen in Figure 4.
  • the magnetization loop of the formed solids showed little magnetization hysteresis, confirming that the particles are paramagnetic, a feature highly desired in magnetic carrier technology. It is important to note that the magnetic properties of the resultant particles formed without nitrogenation is highly variable, while with nitrogenation, it is more reproducible, indicating an improved quality control of the synthesis.
  • Particle size is an important characteristic for biological applications.
  • the size distribution of the synthesized particles is shown in Figure 6. It is clear that the particles synthesized as such exhibit a single modal particle size distribution peaking at 130 nm. About 90% of the particles are in the 80 to 230 nm diameter range. The particles in this diameter range are highly stable against gravitational sedimentation and "invisible" to biological cells.
  • the TEM micrograph in Figure 7A shows that the synthesized particles are indeed uniformly distributed around 150 nm diameter range, confirming qualitatively the determined particle size distribution using our Zetasizer.
  • the particles are highly rounded without sharp edges.
  • the electron diffraction pattern in Figure 7B clearly shows that the particles are highly crystalline, consistent with the observations from the XRD patterns.
  • the average particle size with the conventional procedures is much greater and highly dependent on the initial total iron concentration.
  • the average particle size is hardly dependent on the total iron concentration over the concentration range covered, providing an easy control of the synthesis.
  • Example 5 Cell Separation
  • the magnetic nanoparticle suspensions prepared as such were tested for biological cell separations in a commercial laboratory.
  • the amount of dextran adsorbed on magnetite was determined to be as high as 0.29 g- dextran/g-solids.
  • the presence of the dextran on the nanoparticles not only acted as a particle stabilizer, but also provided a means for coupling with light density human peripheral blood cells through tetrameric antibody complex (TAC) recognition using well established procedures (Thomas, et al., 1992).
  • TAC tetrameric antibody complex
  • nucleated cell suspension at a concentration of 1 x 10 8 cells/mL in buffered cell culture medium (e.g. PBS + 2% Fetal Bovine Serum +1 mM EDTA). Place cells in a 12 x 75 mm tube.
  • buffered cell culture medium e.g. PBS + 2% Fetal Bovine Serum +1 mM EDTA.
  • the CD19 antigen is associated with B cells and the CD14 antigen is associated with monocytes.
  • B cell separations resulted in a purity of 96% and a recovery of 37% of the CD 19+ cells initially present.
  • the monocyte separations resulted in a purity of 97% with a recovery of 66% of the cells initially present.
  • the cell separation was effected by a magnetic field gradient generated without the magnetizable medium used in high gradient magnetic separation. This was possible due to the stronger magnetization of the prepared particles relative to other available particles such as Molday (1984).
  • the particle diameter range of 30 to 250 nm allows for diffusible particles which results in effective targeting to the cells.
  • the superparamegnetic nature of the particles ensures a high stability of the fluids against magnetic flocculation.
  • the particles in this size range are in a sense invisible to biological systems, the particles can be used for in vivo clinical applications such as vehicles for drug delivery and for nondestructive clinical diagnosis.

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  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Medicinal Preparation (AREA)

Abstract

L'invention porte sur la cristallisation dirigée de la magnétite pour former des nanoparticules superparamagnétiques. Lesdites particules, d'un diamètre compris entre dans des plages de 10 à 250 nm, 10 à 200, 20 à 200 et de préférence de 50 à 150 nm sont fortement cristallines et leurs caractéristiques superparamagnétiques correspondent à une saturation de magnétisation de 62 emu/g. Stabilisées par un enrobage, par exemple de dextrane, elles ont des applications potentielles dans la séparation de cellules en biologie, l'administration de médicaments et les diagnostiques cliniques non destructifs.
PCT/CA2002/001003 2001-07-03 2002-07-03 Nanoparticules magnetiques pour bioseparations et leurs procedes de production WO2003005029A2 (fr)

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WO2004102196A1 (fr) * 2003-04-30 2004-11-25 Chengdu Kuachang Medical Industrial Limited Dispositif comprenant des nanostructures destine a une separation ou une analyse, et preparation et mise en oeuvre de ce dispositif
WO2005070471A2 (fr) * 2004-01-20 2005-08-04 Alnis Biosciences, Inc. Articles comprenant un materiau magnetique et des agents bioactifs
CN100365012C (zh) * 2006-07-04 2008-01-30 湖南大学 采用超顺磁性硅壳纳米颗粒静电吸附分离蛋白质的方法
CN100410664C (zh) * 2003-04-30 2008-08-13 成都夸常医学工业有限公司 包含纳米结构的分析或分离用装置、制备方法及其应用
WO2009127045A1 (fr) * 2008-04-16 2009-10-22 Stemcell Technologies Inc. Particules magnétiques
EP2277548A2 (fr) 2003-06-09 2011-01-26 Consejo Superior De Investigaciones Cientificas Nanoparticules magnétiques liées à un ligand
WO2012001546A1 (fr) * 2010-07-02 2012-01-05 Koninklijke Philips Electronics N.V. Détection de groupements actionnés par dispersion
WO2012045902A1 (fr) 2010-10-07 2012-04-12 Consejo Superior De Investigaciones Científicas (Csic) Procédé permettant l'enrobage et la fonctionnalisation de nanoparticules par réaction de michael
US20120154082A1 (en) * 2008-12-03 2012-06-21 Tatung University One dimension nano magnetic wires
US8227262B2 (en) 2005-01-17 2012-07-24 Invitrogen Dynal As Process for preparation of coated polymer particles containing superparamagnetic crystals
US8333993B1 (en) 2006-12-29 2012-12-18 University Of Central Florida Research Foundation, Inc. Synthesis of polymer coated ceria nanoparticles for biomedical applications
WO2013070653A1 (fr) * 2011-11-09 2013-05-16 Board Of Trustees Michigan State University Synthèse de nanoparticules métalliques avec un agent de coiffage à base de glucide
WO2014089464A1 (fr) * 2012-12-07 2014-06-12 The Trustees Of Dartmouth College Nanoparticules magnétiques, composites, suspensions et colloïdes associés à vitesse d'absorption spécifique élevée
US9248204B2 (en) 2004-08-18 2016-02-02 Bracco Suisse S.A. Gas-filled microvesicles composition for contrast imaging
US9364569B2 (en) 2003-02-04 2016-06-14 Bracco Suisse S.A. Ultrasound contrast agents and process for the preparation thereof
US9750821B2 (en) * 2003-12-22 2017-09-05 Bracco Suisse S.A. Gas-filled microvesicle assembly for contrast imaging

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US5062991A (en) * 1990-06-04 1991-11-05 Coulter Corporation In situ use of gelatin in the preparation of uniform ferrite particles
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Cited By (24)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9364569B2 (en) 2003-02-04 2016-06-14 Bracco Suisse S.A. Ultrasound contrast agents and process for the preparation thereof
CN100410664C (zh) * 2003-04-30 2008-08-13 成都夸常医学工业有限公司 包含纳米结构的分析或分离用装置、制备方法及其应用
WO2004102196A1 (fr) * 2003-04-30 2004-11-25 Chengdu Kuachang Medical Industrial Limited Dispositif comprenant des nanostructures destine a une separation ou une analyse, et preparation et mise en oeuvre de ce dispositif
EP2486944A1 (fr) 2003-06-09 2012-08-15 Consejo Superior De Investigaciones Científicas Nanoparticules magnétiques
EP2277548A2 (fr) 2003-06-09 2011-01-26 Consejo Superior De Investigaciones Cientificas Nanoparticules magnétiques liées à un ligand
US9750821B2 (en) * 2003-12-22 2017-09-05 Bracco Suisse S.A. Gas-filled microvesicle assembly for contrast imaging
WO2005070471A2 (fr) * 2004-01-20 2005-08-04 Alnis Biosciences, Inc. Articles comprenant un materiau magnetique et des agents bioactifs
WO2005070471A3 (fr) * 2004-01-20 2006-03-30 Alnis Biosciences Inc Articles comprenant un materiau magnetique et des agents bioactifs
US9248204B2 (en) 2004-08-18 2016-02-02 Bracco Suisse S.A. Gas-filled microvesicles composition for contrast imaging
US10076580B2 (en) 2004-08-18 2018-09-18 Bracco Suisse S.A. Gas-filled microvesicles composition for contrast imaging
US8227262B2 (en) 2005-01-17 2012-07-24 Invitrogen Dynal As Process for preparation of coated polymer particles containing superparamagnetic crystals
CN100365012C (zh) * 2006-07-04 2008-01-30 湖南大学 采用超顺磁性硅壳纳米颗粒静电吸附分离蛋白质的方法
US8333993B1 (en) 2006-12-29 2012-12-18 University Of Central Florida Research Foundation, Inc. Synthesis of polymer coated ceria nanoparticles for biomedical applications
CN102067250A (zh) * 2008-04-16 2011-05-18 干细胞技术公司 磁性粒子
US9701935B2 (en) 2008-04-16 2017-07-11 Stemcell Technologies Inc. Magnetic particles
WO2009127045A1 (fr) * 2008-04-16 2009-10-22 Stemcell Technologies Inc. Particules magnétiques
US20120154082A1 (en) * 2008-12-03 2012-06-21 Tatung University One dimension nano magnetic wires
WO2012001546A1 (fr) * 2010-07-02 2012-01-05 Koninklijke Philips Electronics N.V. Détection de groupements actionnés par dispersion
WO2012045902A1 (fr) 2010-10-07 2012-04-12 Consejo Superior De Investigaciones Científicas (Csic) Procédé permettant l'enrobage et la fonctionnalisation de nanoparticules par réaction de michael
US9581590B2 (en) 2011-11-09 2017-02-28 Board Of Trustees Of Michigan State University Metallic nanoparticle synthesis with carbohydrate capping agent
WO2013070653A1 (fr) * 2011-11-09 2013-05-16 Board Of Trustees Michigan State University Synthèse de nanoparticules métalliques avec un agent de coiffage à base de glucide
US10203325B2 (en) 2011-11-09 2019-02-12 Board Of Trustees Of Michigan State University Metallic nanoparticle synthesis with carbohydrate capping agent
US11221330B2 (en) 2011-11-09 2022-01-11 Board Of Trustees Of Michigan State University Metallic nanoparticle synthesis with carbohydrate capping agent
WO2014089464A1 (fr) * 2012-12-07 2014-06-12 The Trustees Of Dartmouth College Nanoparticules magnétiques, composites, suspensions et colloïdes associés à vitesse d'absorption spécifique élevée

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