Discrete coated nanoparticles

专利摘要:
This invention relates generally to the field of nanoparticles. More particularly, the invention relates to a process for silanizing a magnetic nanoparticle so that the nanoparticle remains discrete, i.e. non-agglomerated during the silanization process. The invention also relates to such a discrete nanoparticle and a composition comprising discrete nanoparticles and various uses of the particle or composition. Figure to be published with the abstract: FIG. 1
公开号:SE1230153A1
申请号:SE1230153
申请日:2011-05-26
公开日:2013-02-11
发明作者:Maria Kempe；Henrik Kempe
申请人:
IPC主号:

专利说明:

Glues the nanoparticles together permanently, resulting in a larger apparent particle size of the preparation.
For in vivo medical applications involving the transport of nanoparticles in the vascular system, aggregates of nanoparticles can obstruct the blood vessels. For ex vivo and in vitro applications, nanoparticle aggregates can block micro-fate system channels, tubing, nozzles, and other types of small-scale devices.
Thus, there is a need for a new method for producing silanized magnetic nanoparticles in which the nanoparticles have not been silanized as aggregates.
Summary of the Invention The present invention preferably relates to alleviating, alleviating or eliminating one or more of the above-identified shortcomings in the art, alone or in any combination, and solving at least the above-mentioned problems by providing a process for producing discrete silanized magnetic nanoparticles according to the attached claims.
The general solution according to the invention is to expose nanoparticles to one or more specific compounds before silanization. This gives a colloidal solution of nanoparticles, which, when the nanoparticles are subsequently silanized, results in discrete silanized nanoparticles.
According to a first aspect of the invention, there is provided a method of coating a magnetic nanoparticle having hydroxyl groups on its surface by forming a layer thereon. The method comprises the steps of subjecting the nanoparticle to a first solution including a compound of formula (I): HO OH Wow HI wherein "n" is an integer ranging from 0 (zero) to 7000. The method also comprises a step of subjecting the nanoparticle to a second solution. solution including a silanizing agent, and a step of allowing the formation of a silanized layer on the magnetic nanoparticle.
According to a second aspect of the invention there is provided a composition obtainable by the process of the first aspect.
According to a third aspect of the invention there is provided a composition comprising substantially discrete, silane coated nanoparticles.
Further embodiments of the invention are defined in the appended claims, as well as in the description.
The present invention has the advantage over the prior art that it results in discrete silanized nanoparticles, i.e. nanoparticles produced by the formation of silanized layers around non-aggregated, singular particles. Thus, a composition comprising said nanoparticles will comprise substantially discrete nanoparticles with a silanized layer on each nanoparticle.
Summary of the Drawings These and other aspects, features and advantages to which the invention is capable will become apparent and clarified from the following description of the embodiments of the present invention, taken in conjunction with the accompanying drawings, in which FIG. 1 is a schematic illustration of a cross section of a nanoparticle according to an embodiment of the invention; FIG. 2 is a graph showing FT-IR spectra of nanoparticles; FIG. 3 is a transmission electron microscopy (TEM) image of nanoparticles; FIG. 4 is an overview of immobilization of tPA according to one embodiment; FIG. Fig. 5A is a schematic instrumental set-up of magnetic targeting of coated nanoparticles in vitro, and Figs. 5B to F are diagrams showing the effect of the capture speed on the capture efficiency of the nanoparticles; FIG. 6 is a photographic representation of a segment of a capillary tube with inserted spirally wound wire; FIG. 7 shows magnetic hysteresis curves of (A) bare magnetite nanoparticles from Example 1, (B) silanized nanoparticles from Example 5, (C) silanized nanoparticles from Example 6, and (D) tPA nanoparticle conjugates from Example 29; and FIG. 8 is a graph showing the residual enzyme activity of the tPA nanoparticle conjugates of Example 28 (gray bars) and Example 29 (black bars) after (A) sonication for 1 hour; or incubation at 4 ° C for (B) 24 hours, (C) 48 hours, (d) 10 days, (E) 21 days and (F) 40 days.
Detailed Description of the Invention Several embodiments of the present invention will be described in more detail below with reference to the accompanying drawings in order that those skilled in the art will be able to practice the invention. However, the invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Furthermore, the terminology used in the detailed description of the particular embodiments illustrated in the accompanying drawings is not intended to be limiting of the invention.
In FIG. 1 is a schematic cross-section of a nanoparticle according to an embodiment shown. The layer (A) of the nanoparticle is an inner core. The layer (B) is a silanized layer or a coating of silica or a silica derivative, applied around the singular nanoparticle so that a discrete silanized nanoparticle is formed. Thus, the nanoparticle is silanized as a non-aggregated, singular particle.
Layer (C) is an optional additional coating, conjugated to layer (B).
Since the nanoparticle is silanized as a non-aggregated singular particle, according to one embodiment, a composition is provided comprising substantially discrete, silanized nanoparticles, such as over 50%, 60%, 70%, 80% or 90% discrete nanoparticles with a silanized layer on each nanoparticles.
According to one embodiment, said nanoparticle is produced by a method which forms a layer on, or coats, a nanoparticle. The nanoparticle can be any type of nanoparticle as long as it has hydroxyl groups on its surface. The process further comprises a step of subjecting the nanoparticle to a first solution including a compound of formula (1): HO OH WOW f) II formula (I) is "n" an integer in the range 0 (zero) to 7000, in the range 0 ( zero) to 2300 or in the range 2 to 800.
In one embodiment, the nanoparticle is a magnetic nanoparticle.
Examples of compounds of formula (I) include, but are not limited to, ethylene glycol, diethylene glycol (DEG), triethylene glycol (TREG), tetraethylene glycol, pentaethylene glycol, hexaethylene glycol, heptaethylene glycol, octaethylene glycol and other oligoethylene glycol polyethylene glycol up to 300,000, such as PEG 400, PEG 2000, PEG 3400, PEG 8000, PEG 20000, PEG 35000, PEG 100000, PEG 200000, and PEG 300000, or a combination thereof.
The nanoparticles are exposed to the above solution by contacting the nanoparticles with the solution while stirring, mixing, shaking, tumbling, and / or sonicating, typically for a period of time between 1 minute and 24 hours, between 5 minutes and 3 hours or between 30 minutes and 1.5 hours to prepare a colloidal solution.
In one embodiment, the solvent of the first solution is selected from the group consisting of: water, methanol, ethanol, n-propanol, isopropanol, N, N-dimethylformamide (DMF), dimethylsulfoxide (DMSO), acetone and acetonitrile, or a combination thereof. .
However, the first solution may also consist of a compound of formula (I), if compound I is itself in a liquid form, such as TREG.
The first solution may also consist of typer your types of compounds of formula (I), all of which are in fl liquid form. In one embodiment, a compound of formula (I) is a liquid and acts as a solvent in the first solution.
In one embodiment, the first solution comprises your types of compounds of formula (I) and a solvent as selected from the group consisting of: water, methanol, ethanol, n-propanol, isopropanol, N, N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), acetone and acetonitrile, or a combination thereof.
In one embodiment, the first solution further comprises at least one base and / or at least one second solvent.
The base may be selected from the group consisting of: ammonia, sodium hydroxide, potassium hydroxide, triethylamine, trimethylamine, dimethylamine, diethylamine, ethylamine, propylamine, N, N-diisopropylethylamine, N-methylmorpholine, N-methylpyrrolidone, oleylamine, ethanolamine, pyridine , methylamine, and piperidine, or a combination thereof.
The second solvent may be selected from the group consisting of: water, methanol, ethanol, n-propanol, isopropanol, N, N-dimethylformamide (DMF), dimethylsulfoxide (DMSO), acetone and acetonitrile, or a combination thereof.
The method further comprises a step of treating the nanoparticle with a second solution comprising a silanizing agent, so as to form a silanized layer, or coating, on the (magnetic) nanoparticle.
The silanizing agent may be a silane.
In one embodiment, the silane is an alkoxysilane, as selected from the group consisting of tetramethoxysilane, tetraethoxysilane, tetra-n-propoxysilane, tetra-isopropoxysilane, tetra-n-butoxysilane, tetra-t-butoxysilane, trimethoxysilane, triethoxysilane -propoxysilane, tri-iso-propoxysilane, tri-n-butoxysilane, tri-t-butoxysilane, trimethoxychlorosilane, triethoxychlorosilane, tri-n-propoxychlorosilane, tri-isopropoxyclorosilane, tri-n-butoxyclorsilanes, tri-n-butoxyclorosilane, tri- , benzyltriethoxysilane, dimethyldimethoxysilane, dimethyldiethoxysilane, and mixtures thereof.
In one embodiment, the silane is a halosilane, as selected from the group consisting of tetrachlorosilane, trichlorosilane, tetrachlorosilane, trifluorosilane, and mixtures thereof.
In one embodiment, the silane is an aminosilane, as selected from the group consisting of 3-aminopropyltrimethoxysilane, 3-aminopropylmethyldimethoxysilane, 3-aminopropyldimethylmethoxysilane, N- (2-aminoethyl) -3-aminopropylmethyldimethoxysilane, N- (Z-aminopropylethyl) trimethoxysilane, 4-aminobutyldimethylmethoxysilane, 4-aminobutyltrimethoxysilane, aminoethylaminomethylphenethyltrimethoxysilane, N- (2-aminoethyl) -3-aminoisobutylmethyldimethoxysilane, N- (6-aminohexyDaminopropyl-trimethoxysiloxynamoxyphenomethoxyphenylmethoxyphenylmethoxyphenylmethoxyphenylmethoxyphenylamino triethoxysilane, 3-aminopropylmethyldiethoxysilane, 3-aminopropyldimethylethoxysilane, N- (2-aminoethyl) -3-aminopropylmethyldiethoxysilane, N- (Z-aminoethyl-S-aminopropyl) triethoxysilane, 4-aminobutyldimethylethoxysilylethylene, (2-aminoethyl) -3-aminoisobutylmethyldiethoxysilane, N- (6-aminohexyl) aminopropyltriethoxysilane, 3- (m-aminophenoxy) propyltriethoxysilane, aminophenyltriethoxysilane an, and mixtures thereof.
In one embodiment, the silane is an olefin-containing silane, such as selected from the group consisting of 3- (trimethoxysilyl) propyl methacrylate, 3- (triethoxysilyl) propyl methacrylate, methacryloxymethyltrimethoxysilane and methacryloxymethyltriethoxysilane, vinyltrimethylthyranethylethylethylene vinyltrimethoxyethyl .
In one embodiment, the silane is a fluorescent silane.
In one embodiment, the silane is a radiopaque silane.
The silanization step can be repeated with the same or another silanizing agent.
The advantage of this is that fl your silanization layers can be obtained.
Silanization is typically carried out at temperatures between 0 ° C and 200 ° C by placing fl ascomas or flasks in a cold room, at room temperature, in a water bath, in an oil bath, in a heating block, in a heating jacket, in a microwave oven, in a microwave oven. accelerated reaction system or in an oven.
In one embodiment, silanization is performed by placement in a microwave oven or in a so-called microwave-accelerated reaction system. This is advantageous because the silanization proceeds rapidly and efficiently.
The mixtures are mixed, stirred, shaken, tumbled, and / or sonicated optionally. Stirring can be performed with an overhead stirrer, a magnetic stirrer, or a homogenizer at 50 rpm to 30,000 rpm, preferably at 200 rpm to 3,000 rpm. The silanization is allowed to proceed between 10 minutes and 72 hours.
After silanization, the magnetic nanoparticles are separated from the solution either by means of a permanent neodymium magnet, by centrifugation, by sedimentation, or by dialysis. The separation of the nanoparticles from the solutions is carried out either directly or after the addition of ethyl acetate or other organic solvent which helps to precipitate the nanoparticles.
The solutions are optionally cooled before separation. The nanoparticles are washed with water and / or methanol and / or other organic solvents. The coated nanoparticles are dried in vacuo at room temperature or in a vacuum oven or used directly for further applications.
The coating process typically results in mass increases between 5 and 100%.
In one embodiment, the method further comprises a step of immobilizing a functional unit on the silanized layer.
The functional unit may be at least one enzyme, protein, antibody, peptide, affinity ligand, oligonucleotide, carbohydrate, lipid, surfactant, aptamer or a pharmaceutically active (drug) molecule to provide derivatized magnetic nanoparticles and combinations thereof.
This is advantageous because the nanoparticle may then be suitable for treatment, diagnostics, separation, purification, or MICR (Magnetic Ink Character Recognition). The functional unit may also be a molecularly imprinted (imprinted) polymer layer, to provide molecularly imprinted (imprinted) magnetic nanoparticles.
The functional unit may further be a polymer containing functional groups to serve as starting points for either stepwise solid phase synthesis or further derivatization by conjugation to an enzyme, protein, antibody, peptide, affinity ligand, oligonucleotide, carbohydrate, lipid, surfactant, aptamer or drug molecule. .
The functional unit may also be a natural or synthetic polymer capable of entrapping or encapsulating drug molecules for later applications in drug administration, said polymer being coated or grafted onto the nanoparticle.
This is advantageous because the nanoparticle may then be suitable for drug administration.
In one embodiment, a composition obtainable by the methods of certain embodiments is described. Said composition comprises essentially discrete nanoparticles with a silanized layer, of silica or silica derivatives, on each nanoparticle, such as over 50%, 60%, 70%, 80% or 90% discrete nanoparticles with a silanized layer on each nanoparticle.
In one embodiment, wherein the nanoparticles are magnetic nanoparticles, the composition comprising substantially discrete nanoparticles having a silanized layer on each nanoparticle can be used as a magnetic ink.
Thus, in one embodiment, a magnetic ink is provided, comprising the composition according to embodiments of the invention.
In one embodiment, wherein the nanoparticles are radiopaque nanoparticles or fluorescent nanoparticles, the composition comprising substantially discrete nanoparticles with a silanized layer on each nanoparticle can be used as a contrast agent or marker.
Examples The following experimental embodiments are provided to make this description thorough and complete and to convey the scope of the invention to those skilled in the art.
The embodiments do not limit the invention, but the invention is limited only by the appended claims.
The methods for synthesizing magnetic nanoparticles of iron oxide can be divided into those performed in aqueous media and those performed in organic media. Synthesis of magnetic nanoparticles of iron oxide by alkaline hydrolysis of iron salts in aqueous media has been described by Massart [Massait, R. IEEE Trans. Magn. 1981, 1 7, 1247-1248]. Massart's method for the synthesis of magnetite begins with a mixture of iron (II) and iron (III) salts in a molar ratio corresponding to the oxidation number of Fe in magnetite (Fe3O4). A number of other publications use variations of this process from mixtures of iron (II) and iron (III) salts to produce magnetite (Fe 3 O 4) or maghemite (y-Fe 2 O 3) [Molday, RS US4452773; Liang et al. J. Radioanal. Nuclear Chem. 2006, 269, 3-7; Horak et al. Bioconjugate Chem. 2007, 18, 635-644; Qaddoura, M .; Hafeli, U. Polym. Preprints 2007, 48, 425-426; Sahoo et al. J. Phys. Chem. B 2005, 109, 3879-3885; Ma, M. et al. Colloids and Surfaces A: Physicochem. Eng Aspects 2003, 212, 219-226; Yamaura, M. et al. J. Magn. Magn.
Mater. 2004, 279, 210-217; Zheng, W. et al. J. Magn. Magn. Mater. 2005, 288, 403-410; Gu, S. et al. J. Colloid Interface Sci. 2005, 289, 419-426]. When the desired product is Fe 3 O 4, the synthesis is sometimes carried out under an inert atmosphere to prevent further oxidation to Fe 2 O 3. Synthesis of magnetic nanoparticles in organic media takes place by thermal decomposition of organometallic compounds, e.g. iron acetyl acetonates or iron acetyl carbonates, in high boiling organic solvents in the presence of surfactants, e.g. fatty acids, oleic acid or hexadecylamine [Burke, N.A.D et al. Chem. Mater. 2002, 14, 4752-4761; Simenoides, K. et al. J. Magn. Magn. Food. 2007, 316, e1-e4]. The resulting nanoparticles after synthesis in organic media are normally covered by hydrophobic molecules which make them soluble only in organic media.
Examples 1 to 4 below relate to the synthesis of naked magnetite nanoparticles (F e 3 O 4) in water.
However, as will be appreciated by one skilled in the art, other synthetic methods are also possible within the scope of the invention.
Example 1 Water was bubbled with a stream of nitrogen for 1 hour and then used to prepare two solutions: the first solution was prepared by dissolving 0.834 g (3 mmol) of FeSO 4 .7H 2 O in 125 ml of water and the second contained 0.842 g (15 mmol). ) KOH and 5.056 g (50 mmol) of KNO 2 in 125 ml of water. The two solutions were sonicated in an ultrasonic bath for 5 minutes and then mixed together in a 250 ml ash fitted with a screw cap to form a green precipitate. The bottle was placed in a preheated (90 ° C) water bath for 2 hours. By the end of the reaction time, a black dense precipitate had formed. The bottle was cooled in cold (8 ° C) water for 15 minutes. The precipitate was separated from the solution by means of a permanent neodymium magnet (N35; 50> <50> <30 mm; 0.48 T at the surface) and washed with water (250 mL> <3) and methanol (MeOH) (250 mL> <3 ). The procedure gave 231 mg of nanoparticles (100% yield). Analysis of the equivalence with ICP-AES (inductively coupled plasma atomic emission spectrometry) and a colorimetric iron analysis indicated 70.5% Fe and 71.5% Fe, respectively. The nanoparticles caused 0.07% hemolysis of diluted blood after 24 hours of incubation and 0.21% and 0.3 0% hemolysis of isolated erythrotrocytes after incubations for 1 hour and 24 hours, respectively.
Fig. 2A shows FT-IR spectra of nanoparticles obtained according to this example.
Example 2 Water was bubbled with a stream of nitrogen for 1 hour and then used to prepare two solutions: the first solution was prepared by dissolving 0.2 g (0.72 mmol) of F eSO 4 ~ 7H 2 O 10 in 30 ml of water and the other contained 0.202 g (3.6 mmol) of KOH and 1,214 g (12 mmol) of KNO; in 30 ml of water. The two solutions were sonicated in an ultrasonic bath for 5 minutes and then mixed to form a green precipitate. The mixture was poured into an HP-500 Plus microwave oven vessel (CEM Corp, Matthews, NC) and subjected to 1200 W microwave treatment with a MARS 5 microwave-accelerated reaction system (CEM Corp, Matthews, NC) with a gradient over 1 minute up to 120 minutes. ° C and then for 15 minutes at constant temperature (120 ° C). The contents of the vessel were then cooled to approx. 65 ° C. A black dense precipitate was separated from the solution by means of a permanent neodymium magnet. The nanoparticles were washed with 25 ml of water. 56 mg of nanoparticles (100% yield) were obtained.
Example 3 Water was bubbled with a stream of nitrogen for 1 hour and then used to prepare two solutions: the first solution was prepared by dissolving 0.2 g (0.72 mmol) of FeSO 4 ° 7H 2 O in 15 ml of water and the second contained 0.202 g (36 mmol ) KOH and 1,214 g (12 mmol) of KNO; in 15 ml of water. The two solutions were sonicated in an ultrasonic bath for 5 minutes. Volumes of 15 ml of triethylene glycol were added to each solution. The solutions were briefly sonicated. The solutions were mixed to form a green precipitate. The mixture was poured into an HP-500 Plus microwave oven vessel (CEM Corp, Matthews, NC) and subjected to 1200 W microwave treatment with a MARS 5 microwave accelerated reaction system (CEM Corp, Matthews, NC) with a gradient over 1 minute up to 120 ° C and then for 15 minutes at constant temperature (120 ° C). The contents of the vessel were cooled to approx. 65 ° C. A black dense precipitate was separated from the solution by means of a permanent neodymium magnet. The nanoparticles were washed with 25 ml of water. An amount of 56 mg of nanoparticles (100% yield) was obtained.
Example 4 One liter of deionized water was heated to 95 ° C in a bottle fitted with a screw cap. An amount of 600 mg FeCl 2 ° 4 H 2 O was added and the ash was placed in a heated (95 ° C) water bath.
The solution was stirred at 8000 rpm with a knife assembly (homogenizer) during the synthesis. A volume of 5 ml of 7 M ammonia solution was added at the start of the synthesis. The reaction was allowed to proceed for 1 hour.
The nanoparticles were separated from the solution with a permanent neodymium magnet and washed by suspension in water 3 times. An amount of 232 mg of nanoparticles (100%) was obtained.
Examples 5 to 27 below relate to the synthesis of silanized magnetite nanoparticles according to various embodiments of the invention. The following description focuses on an embodiment of the present invention applicable to a magnetic nanoparticle and in particular a nanoparticle of magnetite (Fe 3 O 4). However, it will be appreciated that the invention is not limited to this application but can be applied to many other nanoparticles, as long as they have hydroxyl groups on their surface. Examples of such nanoparticles are nanoparticles of maghemite (FegOg), metal iron oxide (MFe2O4 wherein M is Co or Mn), iron (Fe), iron-platinum alloy (FePt), or silica.
In addition to the examples below, the first solution may be any solution according to Table 1.
Table 1. Different compositions of the first solution according to the invention.
No .: Composition: I 2.5 g PEG 8000, 120 ml MeOH, 30 ml ammonia solution (25%) II 5.0 g PEG 400, 240 ml TREG, 60 ml ammonia solution (25%) III 10.0 g PEG 20000, 600 ml MeOH, 150 ml ammonia solution (25%) IV 10.0 g PEG 35000, 600 ml MeOH, 150 ml ammonia solution (25%) V 5.0 g PEG 2000, 600 ml MeOH, 150 ml ammonia solution (25%) VI 2.5 g PEG 8000, 600 ml MeOH, 150 ml ammonia solution (25%) VII 250 ml TREG, 2 ml ammonia solution (25%) VIII 350 ml TREG, 2 ml ammonia solution (25%) IX 150 ml TREG, 1 ml ammonia solution (25%) X 300 ml TREG, 2 ml ammonia solution (25%) XI 150 ml TREG, 0.25 ml ethanolamine XII 10.0 g PEG 3400, 50 ml TREG, 120 ml MeOH, 30 ml ammonia solution (25%) XIII 2.5 g PEG 3400, 120 ml MeOH, 30 ml ammonia solution (25%) XIV 5.0 g PEG 3400, 120 ml MeOH, 30 ml ammonia solution (25%) XV 5.0 g PEG 3400, 240 ml MeOH, 10 ml ammonia solution (25%) XVI 5.0 g PEG 3400, 240 ml TREG, 30 ml ammonia solution (25% ) Example 5 - Silanization with tetraethoxysilane Freshly synthesized magnetite nanoparticles (approx. 231 mg, prepared as described in Example 1) were subjected to, i.e. added to, a solution containing 2.5 g of PEG 8000 in a mixture of 120 ml of MeOH and 30 ml of 25% ammonia solution. The mixture was sonicated for 15 minutes in an ultrasonic bath. The bottles were then placed at room temperature and stirred at 1000 rpm with an overhead stirrer. Silanization of the nanoparticles was started by dropwise addition over 5 minutes of 250 μl of TEOS (tetraethoxysilane, also called tetraethylorthosilicate or orthosilicic tetraethyl ester), dissolved in 3 ml of MeOH. The silanization was continued with continuous stirring for 3 hours at room temperature. After silanization, the nanoparticles were separated directly from the solution using a permanent neodymium magnet. The solutions were decanted and the nanoparticles were washed with MeOH (100 mL> <2), water (100 mL> <4) and finally with MeOH again (100 mL> <4). Before adding any new fresh wash solution, the nanoparticles were separated and held with a magnet while the solutions were decanted. The coated nanoparticles were dried in vacuo at room temperature overnight. The coating procedure resulted in a weight gain of 26%. Composition: 57.0% Fe (according to ICP-AES), 59.6% Fe (according to colorimetric analysis), 3.2% Si (according to ICP-AES), 0.7% C (according to elemental analysis), 0.5% H (according to elemental analysis), 0.3% N (according to elemental analysis).
The nanoparticles caused 0.06% hemolysis of diluted blood after 24 hours of incubation and 5.92% and 21.15% respectively of hemolysis of isolated erythrothrocytes after incubations for 1 hour and 24 hours.
FIG. 2B shows an FT-IR spectrum of coated nanoparticles according to this example and FIG. 3B shows a transmission electron microscopy image (TEM) of coated nanoparticles according to this example.
Example 6 - Silanization with tetraethoxysilane Freshly synthesized magnetite nanoparticles (about 231 mg, prepared as described in Example 1) were added to 5 g of PEG 400 in a mixture of 240 ml of triethylene glycol and 60 ml of 25% ammonia solution.
The mixture was sonicated for 1 hour in an ultrasonic bath. The flask was then placed in a preheated (90 ° C) water bath and stirred at 1000 rpm with an overhead stirrer. Silanization of the nanoparticles was started by dropwise addition over 5 minutes of 250 μl of TEOS dissolved in 3 ml of MeOH. The silanization was continued with continuous stirring for 2 hours at 90 ° C. After silanization, the solution was first cooled and then diluted with ethyl acetate (200 ml) to precipitate nanoparticles. The latter step was performed to accelerate the subsequent magnetic separation. The nanoparticles were washed with MeOH (100 mL> <2), water (100 mL> <4) and finally with MeOH again (100 mL> <4). Before adding each new fresh wash solution, the nanoparticles were separated and held with a magnet while the solutions were decanted. The coated nanoparticles were dried in vacuo at room temperature overnight. The coating process resulted in a weight gain of 15%. Composition of the nanoparticles: 60.8% Fe (according to ICP-AES), 64.4% Fe (according to colorimetric iron analysis), 2.5% Si (according to ICP-AES), 0.7% C (according to elemental analysis), 0.4% H (according to elemental analysis), 0.3 % N (according to elemental analysis). The nanoparticles caused no hemolysis of diluted blood after 24 hours of incubation, and 3.94% and 22.3%, respectively, of hemolysis of isolated erythrotrocytes after incubations for 1 hour and 24 hours.
FIG. 2C shows an FT-IR spectrum of coated nanoparticles according to this example, and FIG. 3C shows a transmission electron microscopy image (TEM) of coated nanoparticles according to this example.
Example 7 - Silanization with tetraethoxysilane and 3- (trimethoxysilyl) propyl methacrylate Freshly synthesized magnetite nanoparticles (56 mg) were added to a solution containing 48 ml of triethylene glycol, 1 g of PEG 400 and 12 ml of 25% ammonia solution. The mixture was sonicated for 1 hour in an ultrasonic bath. A volume of 150 μl TEOS was added. The solution was poured into an HP-500 Plus microwave vessel (CEM Corp, Matthews, NC) and subjected to 1200 W microwave treatment with a MARS 5 microwave accelerated reaction system (CEM Corp, Matthews, NC) with a gradient over 1 minute up to 90 ° C and then for 15 minutes at constant temperature (90 ° C). The contents of the vessel 10 were cooled to approx. 60 ° C. A volume of 150 μl of 3- (trimethoxysilyl) propyl methacrylate was added and the solution was mixed. The solution was then again subjected to 1200 W microwave treatment with a gradient for 1 minute up to 60 ° C and then for 15 minutes at a constant temperature (60 ° C). After cooling, ethyl acetate (50 ml) was added. The nanoparticles were separated by a permanent neodymium magnet while the solution was decanted. The nanoparticles were washed with MeOH (50 mL> <3). Before adding each new fresh MeOH wash solution, the nanoparticles were separated and held with a magnet while the solutions were decanted. The silanized nanoparticles were dried in vacuo at room temperature overnight. The coating procedure typically resulted in a weight gain of 20-60 mg (36-107%).
Example 8 - Silanization with N-trimethoxysilylpropyl-N, N, N-trimethylammonium chloride Freshly synthesized magnetite nanoparticles (about 463 mg, prepared as described in Example 1) were added to a solution consisting of 300 ml of triethylene glycol and 2 ml of 25% ammonia solution.
The mixture was sonicated for 10 minutes in an ultrasonic bath. The flask was then placed in a heated (90 ° C) water bath and the solution was stirred at 900 rpm with an overhead stirrer.
Silanization of the nanoparticles was started by adding 15 ml of N-trimethoxysilylpropyl-N, N, N-trimethylammonium chloride (50% in methanol). The silanization was continued with continuous stirring for 2 hours at 90 ° C. After the silanization, the solution was cooled and ethyl acetate (1.2 L) was added to precipitate the nanoparticles. The nanoparticles were separated from the solution by means of a permanent neodymium magnet. The solution was decanted and the nanoparticles were washed with MeOH (200 mL> <2). The coated nanoparticles were dried in vacuo at room temperature overnight.
Example 9 - Silanization with [hydroxy (polyethylene oxo) propyl] -triethoxysilane (8-12 EO) Newly synthesized magnetite nanoparticles (about 231 mg, prepared as described in Example 1) were added to a solution consisting of 150 ml of triethylene glycol and 1 ml of 25% ammonia solution.
The mixture was sonicated for 30 minutes in an ultrasonic bath. The flask was then placed in a heated (95 ° C) water bath and stirred at 900 rpm with an overhead stirrer. Silanization of the nanoparticles was started by adding 1 ml of a 50% solution of [hydroxy (polyethyleneoxo) propyl] -triethoxysilane (8-12 EO) in ethanol. The silanization was continued with continuous stirring for 2 hours at 95 ° C. After silanization, the solution was cooled and ethyl acetate (350 ml) was added to precipitate the nanoparticles. The nanoparticles were separated from the solution by means of a permanent neodymium magnet. The solution was decanted and the nanoparticles were washed with ethyl acetate (100 mL> <2) and MeOH (100 mL> <2). The coated nanoparticles were dried in vacuo at room temperature overnight. Example 10 - Silanization with tetramethoxysilane and [hydroxy (polyethyleneoxo) propyl] triethoxysilane (8-12 EO) Newly synthesized magnetite nanoparticles (about 231 mg, prepared as described in Example 1) were added to a solution consisting of 150 ml triethylene glycol and 1 ml of 25% ammonia solution.
The mixture was shaken to disperse the nanoparticles. The flask was placed in a 95 ° C water bath and the solution was stirred at 900 rpm. Silanization was started by adding a volume of 250 μl of tetramethoxysilane. Stirring was continued at 900 rpm. After 30 minutes, a volume of 1 ml of a 50% solution of [hydroxy (polyethyleneoxo) propyl] triethoxysilane (8-12 EO) in ethanol was added.
Silanization was allowed to proceed with stirring for an additional 1.5 hours at 95 ° C. The solution was cooled and ethyl acetate (about 350 ml) was added. The nanoparticles were separated from the solution by means of a permanent neodymium magnet. The solution was decanted and the nanoparticles were washed with ethyl acetate (100 mL> <2) and MeOH (100 mL> <2). The coated nanoparticles were dried in vacuo at room temperature overnight. The coating resulted in a weight gain of 15 mg (7%).
Example 11 - Silanization with ores-uorescein isothiocyanate-derivatized silane and tetraethoxysilane Fluorescein isothiocyanate (FITC) -derivatized silane was synthesized by reacting 50 mg of 5 mg of 5-ores-uorescein isothiocyanate isomer I with 6 ml of 5-aminanolopropyltri 5 ml. Newly synthesized magnetite nanoparticles (about 231 mg, prepared as described in Example 1) were added to 360 ml of water, 15 g of PEG 2000 and 90 ml of 25% ammonia solution.
The mixture was sonicated for 1 hour in an ultrasonic bath. The flask was then placed in a pre-heated (90 ° C) water bath and stirred at 1000 rpm with an overhead stirrer. Silanization of the nanoparticles was started by adding 1.0 ml of FITC-derivatized silane solution prepared as above. After 15 minutes, 2.25 ml of TEOS was added. The silanization was continued with continuous stirring at 90 ° C for a further 45 minutes and then at room temperature for 13 hours. After silanization, the fluorescent nanoparticles were separated with a permanent neodymium magnet and washed with MeOH (100 mL> <2), water (100 mL> <4), and finally with MeOH again (100 mL> <4). Before adding each new fresh wash solution, the nanoparticles were separated and held with the magnet while the solution was decanted. The fluorescent nanoparticles were dried in vacuo at room temperature overnight. The coating procedure resulted in a weight gain of 72%.
An advantage of these nanoparticles is that they can be used as markers and contrast agents because they are ores uorescent.
Thus, in one embodiment, a contrast agent is provided, comprising the composition according to embodiments of the invention. Example 12 - Silanization with tetraethoxysilane Freshly synthesized magnetite nanoparticles (about 232 mg, prepared as described in Example 1) were added to a solution consisting of 120 ml of water, 5 g of PEG 2000 and 30 ml of 25% ammonia solution.
The mixture was sonicated for 15 minutes in an ultrasonic bath and then stirred at 900 rpm with an overhead stirrer. Silanization of the nanoparticles was started by adding 0.25 ml of TEOS. The silanization was continued with continuous stirring for 40 hours at room temperature. After silanization, the nanoparticles were separated from the solution by means of a permanent neodymium magnet. The solution was decanted off and the nanoparticles were washed with water (100 mL> <2) and MeOH (100 mL> <2). The coated nanoparticles were dried in vacuo at room temperature overnight.
Example 13 - Silanization with tetraethoxysilane An amount of 50 mg of commercial iron (III, III) oxide nanoparticles> 50 nm (Sigma-Aldrich catalog number 637,106) was added to a solution consisting of 48 ml of triethylene glycol, 12 ml of 25% ammonia solution and 1 g of PEG 400 The mixture was sonicated for 1 hour in an ultrasonic bath. A volume of 200 μl of TEOS was added. The solution was kept in an HP-500 Plus microwave oven vessel (CEM Corp, Matthews, NC) and subjected to 1200 W microwave treatment with a MARS 5 microwave accelerated reaction system (CEM Corp, Matthews, NC) with a gradient over 1 minute up to 90 ° C and then for 15 minutes at constant temperature (90 ° C). After cooling, ethyl acetate was added and the nanoparticles were separated from the solution by means of a permanent neodymium magnet.
The solution was decanted and the nanoparticles were washed with MeOH (100 mL> <3). The silanized nanoparticles were dried in vacuo at room temperature overnight. The procedure resulted in a typical weight gain of 25 mg (50%).
Example 14 - Silanization with tetraethoxysilane and 3-aminopropyltriethoxysilane Freshly synthesized magnetite nanoparticles (about 231 mg, prepared as described in Example 1) were added to a solution consisting of 600 ml of MeOH, 2.5 g of PEG 8000, and 150 ml of 25% ammonia solution. The mixture was sonicated for 15 minutes in an ultrasonic bath and then stirred at 900 rpm at room temperature. Silanization of the nanoparticles was started by adding 0.25 ml of TEOS. After 3.5 hours, 0.25 ml of 3-aminopropyltriethoxysilane was added. The silanization was allowed to proceed for an additional 1 hour. The nanoparticles were separated from the solution by means of a permanent neodymium magnet. The solution was decanted and the nanoparticles were washed with MeOH (100 mL> <2), water (100 mL> <2) and MeOH (100 mL> <2). The silanized nanoparticles were dried in vacuo at room temperature overnight. The procedure resulted in a weight gain of 55 mg (24%). Example 15 - Silanization with tetraethoxysilane Freshly synthesized magnetite nanoparticles (about 232 mg, prepared as described in Example 1) were added to a solution consisting of 600 ml of MeOH, 10 g of PEG 20000, and 150 ml of 25% ammonia solution. . The mixture was sonicated for 30 minutes in an sonication bath and then stirred at 1000 rpm at room temperature. Silanization of the nanoparticles was started by adding 0.25 ml of TEOS. After 3 hours, the nanoparticles were separated from the solution by means of a permanent neodymium magnet. The solution was decanted off and the nanoparticles were washed with MeOH (200 mL> <2), water (300 mL> <3) and MeOH (100 mL> <2). The silanized nanoparticles were dried in vacuo at room temperature overnight. The coating procedure resulted in a weight gain of 47 mg (20%).
Example 16 - Silanization with tetraethoxysilane Freshly synthesized magnetite nanoparticles (about 231 mg, prepared as described in Example 1) were added to a solution consisting of 600 ml of MeOH, 10 g of PEG 35000, and 150 ml of 25% ammonia solution. The mixture was sonicated for 30 minutes in an sonication bath and then stirred at 1000 rpm at room temperature. Silanization of the nanoparticles was started by adding 0.25 ml of TEOS. After 18 hours, the nanoparticles were separated from the solution by means of a permanent neodymium magnet. The solution was decanted and the nanoparticles were washed with MeOH (200 mL), water (500 mL), and MeOH (200 mL). The silanized nanoparticles were dried in vacuo at room temperature overnight. The coating procedure resulted in a weight gain of 46 mg (20%).
Example 17 - Silanization with tetraethoxysilane Freshly synthesized magnetite nanoparticles (about 231 mg, prepared as described in Example 1) were added to a solution consisting of 600 ml of MeOH, 5 g of PEG 2000 and 150 ml of 25% ammonia solution. The mixture was sonicated for 30 minutes in an sonication bath and then stirred at 1000 rpm at room temperature. Silanization of the nanoparticles was started by adding 0.25 ml of TEOS. After 1 hour, the nanoparticles were separated from the solution by means of a permanent neodymium magnet. The solution was decanted off and the nanoparticles were washed with MeOH (100 mL), water (300 mL> <4) and MeOH (200 mL). The silanized nanoparticles were dried in vacuo at room temperature overnight. The coating resulted in a weight gain of 35 mg (15%).
Example 18 - Silanization with [hydroxy (polyethyleneoxo) propyl] triethoxysilane (8-12 EO) and N-trimethoxysilylpropyl-N, N, N-trimethylammonium chloride Freshly synthesized magnetite nanoparticles (about 231 mg, prepared as described in Example 1) were added to of 150 ml of triethylene glycol. A volume of 0.25 ml of ethanolamine was added. The mixture was sonicated for 10 minutes in an ultrasonic bath. The flask was then placed in a heated (95 ° C) water bath and the solution was stirred at 900 rpm with an overhead stirrer. Silanization of the nanoparticles was started by adding 1 ml of [hydroxy (polyethyleneoxo) propyl] triethoxysilane (8-12 EO) (50% in ethanol) and 4 ml of N-trimethoxysilylpropyl-N, N, N-trimethylammonium chloride (50% in methanol). The silanization was continued with continuous stirring for 2 hours at 95 ° C. The solution was then cooled and ethyl acetate (300 ml) was added to precipitate the nanoparticles.
The nanoparticles were separated from the solution by means of a permanent neodymium magnet.
The solution was decanted and the nanoparticles were washed with ethyl acetate (200 mL> <2) and MeOH (200 mL> <2). The coated nanoparticles were dried in vacuo at room temperature overnight.
Example 19 - Silanization with tetraethoxysilane Freshly synthesized magnetite nanoparticles (about 231 mg, prepared as described in Example 1) were added to a solution consisting of 120 ml of MeOH, 10 g of PEG 3400, and 30 ml of 25% ammonia solution. The mixture was sonicated for 20 minutes in an ultrasonic bath. The solution was stirred at 1000 rpm with an overhead stirrer. Silanization of the nanoparticles was started by adding 1 ml of TEOS. The silanization was continued with continuous stirring for 3 hours at room temperature.
The nanoparticles were separated from the solution by means of a permanent neodymium magnet.
The solution was decanted and the nanoparticles were washed with MeOH (200 mL> <5), water (200 mL> <5) and MeOH (200 mL> <5). The procedure resulted in a weight gain of 253 mg (109%).
Example 20 - Silanization with tetraethoxysilane Freshly synthesized magnetite nanoparticles (about 232 mg, prepared as described in Example 1) were added to a solution consisting of 120 ml of MeOH, 2.5 g of PEG 3400 and 30 ml of 25% ammonia solution. The mixture was sonicated for 20 minutes in an ultrasonic bath. The solution was stirred at 1000 rpm with an overhead stirrer. Silanization of nanoparticles was started by adding 0.25 ml of TEOS. The silanization was continued with continuous stirring for 3 hours at room temperature.
The nanoparticles were separated from the solution by means of a permanent neodymium magnet.
The solution was decanted off and the nanoparticles were washed with MeOH (200 mL X 5), water (200 mL> <5) and MeOH (200 mL> <5). The procedure resulted in a weight gain of 53 mg (23%).
Example 21 - Silanization with tetraethoxysilane Freshly synthesized magnetite nanoparticles (about 232 mg, prepared as described in Example 4) were added to a solution consisting of 120 ml of MeOH, 5 g of PEG 400 and 30 ml of 25% ammonia solution.
The mixture was sonicated for 1 hour in an ultrasonic bath. The solution was stirred at 900 rpm with an overhead stirrer. Silanization of the nanoparticles was started by adding 1 ml of TEOS.
The silanization was continued with continuous stirring for 1 hour at 95 ° C. The nanoparticles were separated from the solution by means of a permanent neodymium magnet. The solution was decanted off and the nanoparticles were washed with MeOH (200 mL> <5), Water (200 mL> <5) and MeOH (200 mL> <5). The procedure resulted in a weight gain of 30 mg (13%).
Example 22 - Silanization with tetraethoxysilane and 3- (trimethoxysilyl) propyl methacrylate Freshly synthesized magnetite nanoparticles (about 278 mg) prepared as described in Example 3 were added to a solution containing 290 ml of triethylene glycol, 6 g of PEG 400, and 70 ml of 25% ammonia solution. The mixture was sonicated for 1 hour in an ultrasonic bath. A volume of 600 μl TEOS was added.
The solution was poured into six HP-500 Plus microwave vessels (CEM Corp, Matthews, NC) and subjected to 1200 W microwave treatment with a MARS 5 microwave accelerated reaction system (CEM Corp, Matthews, NC) with a gradient over 1 minute up to 90 ° C and then for 15 minutes at constant temperature (90 ° C). The contents of the vessels were cooled to approx. 55 ° C and merged. A volume of 600 μl of 3- (trimethoxysilyl) propyl methacrylate was added, the solution was mixed and distributed to six microwave ovens. The solutions were again subjected to 1200 W microwave treatment with a gradient for 1 minute up to 50 ° C and then for 30 minutes at constant temperature (50 ° C).
After cooling, ethyl acetate (400 ml) was added. The nanoparticles were separated and held by a pennant neodymium magnet while the solution was decanted. The nanoparticles were washed with MeOH (100 mL> <3). Before each addition of fresh fresh MeOH wash solution, the nanoparticles were separated and held with a magnet while the solutions were decanted. The silanized nanoparticles were dried in vacuo at room temperature overnight. The coating procedure resulted in a typical weight gain of 171 mg (62%).
Example 23 - Silanization with tetraethoxysilane Freshly synthesized magnetite nanoparticles (56 mg), prepared as in Example 3, were added to a solution consisting of 57 ml of triethylene glycol and 3 ml of 25% ammonia solution. The mixture was sonicated for 1 hour in an ultrasonic bath. A volume of 150 μl TEOS was added. The solution was poured into an HP-500 Plus microwave oven (CEM Corp, Matthews, NC) and subjected to 1200 W microwave treatment with a MARS 5 microwave accelerated reaction system (CEM Corp, Matthews, NC) with a gradient over 1 minute up to 90 ° C and then for 15 minutes at constant temperature (90 ° C). After cooling, ethyl acetate was added and the nanoparticles were separated from the solution by means of a permanent neodymium magnet. The solution was decanted and the nanoparticles were washed with MeOH (50 mL> <3). The silanized nanoparticles were dried in vacuo at room temperature overnight.
The procedure resulted in a weight gain of 31 mg (56%).
Example 24 - Silanization with tetraethoxysilane Freshly synthesized magnetite nanoparticles (56 mg), prepared as in Example 3, were added to a solution consisting of 54 ml of triethylene glycol and 6 ml of 25% ammonia solution. The mixture was sonicated for 1 hour in an ultrasonic bath. A volume of 150 μl TEOS was added. The solution was kept in an HP-500 Plus microwave oven (CEM Corp, Matthews, NC) and subjected to 1200 W microwave treatment with a MARS 5 microwave accelerated reaction system (CEM Corp, Matthews, NC) with a gradient over 1 minute up to 90 ° C and then for 15 minutes at constant temperature (90 ° C). After cooling, ethyl acetate was added and the nanoparticles were separated from the solution using a permanent neodymium magnet. The solution was decanted off and the nanoparticles were washed with MeOH (50 mL> <3). The silanized nanoparticles were dried in vacuo at room temperature overnight.
The procedure resulted in a weight gain of 33 mg (59%).
Example 25 - Silanization with tetraethoxysilane Freshly synthesized magnetite nanoparticles (56 mg), prepared as in Example 3, were added to a solution consisting of 57 ml of triethylene glycol and 3 ml of 40% methylamine solution. The mixture was sonicated for 1 hour in an ultrasonic bath. A volume of 150 μl TEOS was added. The solution was kept in an HP-500 Plus microwave oven dish (CEM Corp, Matthews, NC) and subjected to 1200 W microwave treatment with a MARS 5 microwave accelerated reaction system (CEM Corp, Matthews, NC) with a gradient over 1 minute up to 90 ° C and then for 15 minutes at constant temperature (90 ° C). After cooling, ethyl acetate was added and the nanoparticles were separated from the solution using a permanent neodymium magnet. The solution was decanted off and the nanoparticles were washed with MeOH (50 mL> <3). The silanized nanoparticles were dried in vacuo at room temperature overnight. The procedure resulted in a weight gain of 34 mg (60%).
Example 26 - Silanization with tetraethoxysilane Freshly synthesized magnetite nanoparticles (56 mg), prepared as in Example 3, were added to a solution consisting of 54 ml of triethylene glycol and 6 ml of 40% methylamine solution. The mixture was sonicated for 1 hour in an ultrasonic bath. A volume of 150 μl TEOS was added. The solution was kept in an HP-500 Plus microwave oven (CEM Corp, Matthews, NC) and subjected to 1200 W microwave treatment with a MARS 5 microwave accelerated reaction system (CEM Corp, Matthews, NC) with a gradient over 1 minute up to 90 ° C and then for 15 minutes at constant temperature (90 ° C). After cooling, ethyl acetate was added and the nanoparticles were separated from the solution by means of a permanent neodymium magnet. The solution was decanted off and the nanoparticles were washed with MeOH (50 mL> <3). The silanized nanoparticles were dried in vacuo at room temperature overnight. The procedure resulted in a weight gain of 45 mg (81%).
Example 27 - Silanization with tetraethoxylsilane and iodinated silane Iodized silane was synthesized by reacting 1,266 g (11 mmol) of N-hydroxysuccinimide, dissolved in 50 ml of CH 2 Cl 2, with 4,998 g (10 mmol) of 2,3,5-triiodobenzoic acid, dissolved in 50 ml of CH 2 Cl 2 , and 2,108 g of 10 (11 mmol) EDC (water-soluble carbodiimide), dissolved in 50 ml of CH 2 Cl 2. The reaction was allowed to proceed for 2 days.
The solution was extracted with water three times, with saturated sodium chloride solution three times, and finally with water once. The solution was dried over MgSO 4. The solution was then evaporated and the solid product was dried in vacuo overnight. 4,363 g (73% yield) of succinimidyl 2,3,5-triiodobenzoate were obtained. 0.884 g (4 mmol) of 3-aminopropyltriethoxysilane was added to 2,388 g (4 mmol) of succinimidyl-2,3,5-triiodobenzoate dissolved in 8 ml of DMF. The reaction is continued for 2 days to obtain the judged silane.
Newly synthesized magnetite nanoparticles (about 232 mg, prepared as described in Example 4) were added to a solution consisting of 120 ml of MeOH, 5 g of PEG 400 and 30 ml of 25% ammonia solution.
The mixture was sonicated for 1 hour in an ultrasonic bath. The solution was placed in a heated (95 ° C) water bath and stirred at 900 rpm with an overhead stirrer. Silanization of the nanoparticles was started by adding 1 ml of TEOS. The silanization was continued with continuous stirring for 30 minutes at 95 ° C. The iodinated silane reaction mixture, prepared as described above, was then added. The silanization was carried out for a further 3 hours. The nanoparticles were separated from the solution by means of a permanent neodymium magnet. The solution was decanted off and the nanoparticles were washed with MeOH (200 mL> <5), water (200 mL> <5) and MeOH (200 mL> <5).
An advantage of these nanoparticles is that they can be used as X-ray contrast agents or markers, as they are X-ray dense.
Thus, in one embodiment, a contrast agent is provided, comprising the composition according to embodiments of the invention.
Examples 28 to 30 below relate to conjugation of enzyme or peptide to silanized nanoparticles according to embodiments of the invention.
Example 28 - Immobilization of recombinant human tissue plasminonogen activator (tPA) by activating silica-coated nanoparticles with NHSS-EDC Fmoc-Gly-OH (0.595 g, 2 mmol), dissolved in DMF (1 mL), was added to 200 mg of silanized magnetite nanoparticles, synthesized as described in Example 5. The coupling was initiated by the addition of DIPCDI (0.252 g, 2 mmol) in DMF (1 mL) and DMAP (25 mg, 0.2 mmol) in DMF (1 mL).
The reaction was carried out on a rotator for 3 days at room temperature. The nanoparticles were separated from the solution with a permanent magnet and washed with DMF (5 ml> <10). Magnetic separation was performed between the washes. The Fmoc groups were removed by treatment with 5 ml of piperidine-DMF (1: 4) for 5 minutes. After removing the first cleavage solution, 5 ml of fresh piperidine-DMF (1: 4) was added and the mixture was incubated for another 15 minutes. The nanoparticles were washed with DMF (5 mL> <10) and CH 2 Cl 2 (5 mL> <10). Kaiser's qualitative ninhydrin test detected free amino groups in this mode. Succinylation of the free amino groups was performed by the addition of succinic anhydride (0.4 g, 4 mmol) in 6 mL of CH 3 Cl 2 -pyridine (1: 1). The mixture was incubated at room temperature on a rotator for 30 minutes. After magnetic separation and removal of the solution, the nanoparticles were washed with CH 2 Cl 2; (5 mL> <5), DMF (5 mL> <5), and MeOH (5 mL> <5). The Kaiser test was negative, indicating that the succinylation was complete. The nanoparticles were dried in vacuo at room temperature overnight. Elemental analysis: 3% C, 0.6% H, 0.6% N. An amount of 125 mg of the dried succinylated nanoparticles was suspended in 1 ml of water.
The nanoparticles were activated by the addition of NHSS (87 mg, 0.4 mmol) in water (1 mL) and EDC (77 mg, 0.4 mmol) in water (1 mL). The esterification was continued at room temperature on a rotator for 2 hours.
The nanoparticles were separated with a magnet and the reagent solution was removed. The nanoparticles were washed with water (5 ml> <10) and finally suspended in 2.08 ml of water. The enzyme immobilization was performed by adding a solution of recombinant human tissue plasminogen activator, tPA (marketed by Boehringer Ingelheim under the trademark Actilyse; 12.5 mg dissolved in 6.25 ml of water) to the nanoparticle solution. Coupling was continued on an orbital shaker (200 rpm) for 4 hours at 4 ° C. The conjugated tPA nanoparticles were separated with a magnet, the solution was removed and washing was performed with water (5 ml> <5), phosphate buffered saline (PBS), pH 7.4 (5 ml> <3), and water (5 ml> <3). The protein concentration of the washing solutions was determined according to Bradford using bovine serum albumin as a reference. The amount of immobilized tPA was calculated by subtracting the amount of tPA in the wash solutions from the amount of tPA added to the nanoparticles at the start of the immobilization. The immobilization yield was calculated as 100% * (amount of immobilized tPA) / (amount of tPA charged). The immobilization yield was 63%. The TPA charge was calculated as (mass of immobilized tPA) / (mass of nanoparticles). The TPA charge was 63 μg tPA / mg nanoparticles. The enzyme activity of free and immobilized enzyme was determined by monitoring the formation of p-nitroaniline (pNA) spectrophotometrically at 405 nm during the hydrolysis of the substrate H-D-Ile-Pro-Arg-pNA. The assay was performed by mixing 0.25 ml of a solution containing either free tPA or tPA nanoparticle conjugate, 0.25 ml of 100 mM Tris-HCl pH 8.4 containing 100 mM NaCl and 0.25 ml of a 1 mM solution of the substrate in water. The specific enzyme activity was 0.86 U / mg tPA. The enzyme activity yield was calculated as 100% * (total activity of immobilized tPA) / (total activity of charged tPA). The enzyme activity yield was 45%. A reaction scheme is provided in FIG. 4A.
Example 29 - Immobilization of tPA by activating coated nanoparticles with tresyl chloride 280 mg of silanized nanoparticles, synthesized as described in Example 6, first washed with dry acetone (5 ml> <2) and then suspended in 6.7 ml dry acetone and 0.78 ml dry pyridine . Activation with tresyl chloride (0.3 ml) was initiated by dropwise addition to the nanoparticle solution with shaking. The reaction was performed on an orbital shake (1,000 rpm) for 2 hours at 4 ° C. The nanoparticles were then retained with a permanent neodymium magnet and the solution was removed.
The nanoparticles were washed with acetone (5 ml> <3), acetone-water (2: 1) (5 ml> <2), acetone-water (111) (5 ml> <2), acetone-water (132) (5 ml> <2), acetone-water (1: 4) (5 ml> <2) and water (10 ml> <3).
The nanoparticles were then suspended under sonication in 10 ml of water and added dropwise to dialyzed tPA (38 mg) in 80 ml of 0.2 M sodium phosphate buffer pH 8. The coupling of tPA to the tresyl chloride-activated nanoparticles was performed at 4 ° C for 33 hours on an orbital shaker (200 rpm ). The nanoparticles were then separated with a permanent magnet and washed with 0.2 M Tris-HCl pH 8 (50 ml). Blocking of the remaining tresyl groups was performed with 0.2 M Tris-HCl, pH 8 for 23 hours at 4 ° C on an orbital shaker (200 rpm). The TPA nanoparticle conjugates were separated with a magnet and washed with water (10 ml> <2), 50 mM sodium phosphate buffer pH 7 (10 ml), 25 mM sodium phosphate buffer pH 7 (20 ml), and 12.5 mM sodium phosphate buffer pH 7 (20 ml> <2).
Determination of protein concentration, enzyme activity and calculations of immobilization parameters were performed as described in Example 28. The enzyme loading was 71 μg tPA / mg nanoparticles.
The immobilization yield was 52%. The specific enzyme activity was 0.82 U / mg tPA.
The enzyme activity yield was 41%. The TPA nanoparticle conjugates did not cause diluted hemolysis after 24 hours of incubation and 0.15% and 1.03% hemolysis of isolated erythrotrocytes, respectively, after incubations for 1 hour and 24 hours.
FIG. 2D shows FT-IR spectra of coated nanoparticles according to this example and FIG. 3D shows a transmission electron microscopy image (TEM) of coated nanoparticles according to this example.
A reaction scheme is provided in FIG. 4B.
Example 30 - Coupling of an NGR-containing peptide to coated nanoparticles via a PEG spacer Fmoc-Gly-OH (0.298 g, 1 mmol), dissolved in DMF (0.5 ml), was added to 100 mg of silanized magnetite nanoparticles, synthesized as described in Example 5. The coupling was initiated by the addition of DIPCDI (0.126 g, 1 mmol) in DMF (0.5 mL) and DMAP (13 mg, 0.1 mmol) in DMF (0.5 mL).
The reaction was carried out on a rotator for 3 days at room temperature. The nanoparticles were separated from the solution with a permanent magnet and washed with DMF (3 ml> <10). Magnetic separation was performed between the washes. The Fmoc groups were removed by treatment with 3 ml of piperidine-DMF (1: 4) for 5 minutes. After removing the first cleavage solution, 3 ml of fresh piperidine-DMF (1: 4) was added and the mixture was incubated for another 15 minutes. The nanoparticles were washed with DMF (3 mL> <10) and CHQCl; (3 ml X 10). Kaiser's qualitative ninhydrin test detected free amino groups in this mode. Fmoc-NH- (PEG) 2-COOH (48 mg, 0.086 mmol) dissolved 0.4 mL of DMF, DIPCDI (11 mg, 0.086 mmol) in 0.2 mL of DMF and HOBt (12 mg, 0.086 mmol) in 0.2 mL of DMF was added to the nanoparticles . The coupling was performed on a rotator for 24 hours. After the coupling, Kaiser's qualitative ninhydrin test was negative. The fmoc groups were removed by treatment with 30 ml of 22 piperidine-DMF (124) for 5 minutes. After removing the first cleavage solution, 3 ml of fresh piperidine-DMF (114) was added and the mixture was incubated for another 15 minutes.
The nanoparticles were washed with DMF (3 mL> <10) and CH 3 Cl 2 (3 mL> <10). Kaiser's qualitative ninhydrin test detected free amino groups at this point. Ac-Gly-Asn (Trt) -Gly-Arg (Pbf) -Gly- Ahx-Gly-OH (10.8 mg, 9.28 μmol), DIPCDI (5 mg, 40 μmol) and HOAt (5.4 mg, 40 μmol) were dissolved in 0.2 ml of DMF and added to the nanoparticles. The reaction was carried out for 3 days. After the coupling, Kaiser's qualitative ninhydrin test was negative. The nanoparticles were dried in vacuo overnight. The protecting groups were removed by treatment with 0.5 ml of TFA-CH 3 Cl 2 water (90: 5: 5) for 2 hours.
The nanoparticles were washed with 1 mL each of CH 2 Cl 2, DMF, CH 2 Cl 2, MeOH, water, and MeOH.
The nanoparticles were dried in vacuo overnight.
Characterization of Coated Nanoparticles A number of methods have been used to study the coated nanoparticles prepared according to embodiments of the invention.
Transmission electron microscopy The size and morphology of the nanoparticles were studied by transmission electron microscopy (TEM) using a J EOL JEM-1230 (Tokyo, Japan) equipped with a Model 791 Multiscan-Gatan camera (Pleasanton, CA, USA). The samples were placed on Pioloform (polyvinyl butyral) - films and images were taken at 80 kV applied voltage. FIG. Figure 3 shows transmission electron microscopy (TEM) of (A) bare magnetite nanoparticles from Example 1, (B) coated magnetite nanoparticles from Example 5, (C) coated magnetite nanoparticles from Example 6, and (D) tPA nanoparticle conjugates from Example 29.
Dynamic light scattering The hydrodynamic particle size distribution was determined by dynamic light scattering (DLS) using an Ultra Nanotrac particle size analyzer from Microtrac (Montgomeryville, PA, USA). The typical hydrodynamic size in triethylene glycol of naked magnetite particles prepared as in Example 1 was 140 nm. The hydrodynamic size could not be measured in water as the bare nanoparticles aggregated in water. The hydrodynamic size in water of the silanized nanoparticles prepared in Example 5 was 300-365 nm. The hydrodynamic size in water of the silanized nanoparticles prepared in Example 6 was 250-300 nm. 10 15 20 25 30 35 23 Magnetic Characterization Magnetic properties of the nanoparticles embedded in epoxy resin were measured at room temperature with a Princeton Measurement Corp. M2900-2 alternating gradient magnetometer (Princeton, NJ, USA). Magnetic hysteresis loops are shown in FIG. 7.
F T -IR spectroscopy Fourier transform-infrared (FT-IR) spectra of the nanoparticles (in KBr tablets) were measured with a Bruker IFS66 FT-IR spectrometer (Billerica, MA, USA). Spectra are shown in FIG. 2.
Elemental analysis The Fe and Si content of the nanoparticles were analyzed with an Optima 3000 DV ICP-AES instrument (Perkin Elmer, Waltham, MA, USA). Elemental analysis (C, H and N) was performed by Mikrokemi AB (Uppsala, Sweden).
Colorimetric core analysis The iron content of the nanoparticles was determined by a modification of the method described in Sasikumar PG, Kempe M. Magnetic CLEAR supports for solid-phase synthesis of peptides and small organic molecules. Int J Peptide Res Ther 2007; 13 (1-2): 129-141. Samples of the nanoparticles (1-4 mg) were treated with 0.3 ml of HCl (37%) for 30 minutes. The dissolved samples were quantitatively transferred to 25-ml volumetric flasks and diluted with water. Volumes of 0.5 ml of the diluted solutions were mixed with a hydroxylamine hydrochloride solution (0.25 ml, 0.1 mg / ml), a sodium acetate solution (2.5 ml, 0.1 mg / ml) and a 1,10-phenanthroline monohydrate solution (2.5 ml, 1 mg / ml). The solutions were diluted 2-10 times with water and the absorbance was measured at 508 nm. Standard Fezl solutions for calibration were prepared under identical conditions from FeSO 4 ° 7H 2 O.
Hemolysis test on diluted blood 5 ml of human fresh blood anticoagulated with sodium citrate was diluted with 5 ml of PBS. The blood sample was obtained from a healthy volunteer subject. Volumes of 0.75 ml nanoparticles (0.1 mg / ml) in PBS were incubated in Eppendorf microtubes for 30 minutes at 37 ° C. Negative and positive controls were made by replacing the nanoparticle solution with PBS and water, respectively. Each sample was run in triplicate. Diluted blood (0.25 ml) was added and the samples were incubated on a rotator for 24 hours at 37 ° C. The tubes were then centrifuged (10 min, 5,000 rpm). The absorbance of the supernatants was measured in a spectrophotometer at 545 nm. The hemolysis was calculated as 100% * (Apmv - Anegative contm fi / (Apositive control - Anegative contmn). The bare magnetite nanoparticles from Example 1 caused 0.07% hemolysis. The silanized nanoparticles from Example 5 caused 0.06% hemolysis. The silanized 35 10 15. The 24 nanoparticles from Example 6 and the tPA-conjugated nanoparticles from Example 29 did not give rise to any hemolysis.
Hemolysis test on washed isolated human erythrocytes 2 ml PBS was added to 5 ml human fresh blood anticoagulated with sodium citrate. The mixture was inverted for 2 minutes on a tilting table and then centrifuged at 5,000 rpm for 5 minutes.
The supernatant was removed and the pellet of erythrocytes was washed twice more with 4 ml of PBS by suspension, centrifugation, and decantation. The erythrocytes were finally suspended in 5 ml of PBS. 1 ml volumes of the nanoparticles (0.1 mg / ml) in PBS were incubated at 37 ° C in Eppendorf microtubes. Negative and positive controls were prepared by replacing the nanoparticle solution with PBS and water, respectively. Each sample was run in triplicate. After 30 minutes, 0.1 ml of the erythrocyte suspension was added to each tube. The tubes were incubated on a rotator at 37 ° C for either 1 hour or 24 hours.
The tubes were then centrifuged at 10,000 rpm for 10 minutes. The absorbance of the supernatants was measured in a spectrophotometer at 545 nm. The hemolysis was calculated as described above. The bare magnetite nanoparticles from Example 1 caused 0.2% and 0.30% hemolysis after 1 hour and 24 hours, respectively. The silanized nanoparticles from Example 5 caused 5.92% and 21.15% hemolysis after 1 hour and 24 hours, respectively. The silanized nanoparticles from Example 6 caused 3.94%. and 22.30% hemolysis after 1 hour and 24 hours, respectively. The TPA nanoparticle conjugates from Example 29 caused 0.15% and 1.03% hemolysis after 1 hour and 24 hours, respectively.
Stability studies on the tPA nanoparticle conjugates Enzyme activity analysis was performed on aqueous solutions of tPA nanoparticle conjugates from Example 28 (5.7 mg / ml) and Example 29 (2.4 mg / ml) after sonication in an ultrasonic bath for 1 hour and after incubation at 4 °. C for up to 40 days.
The enzyme activities obtained are shown in FIG. 8.
Examples 31 to 33 relate to target control of magnetic particles.
Example 31 - In vitro target control of magnetic particles into a helical wire in a single-pass-through fl fate experiment A Kantahl D ferromagnetic wire (length 100 mm, Q) 0.13 mm) was wound 15 turns to make a helix (length 40 mm, (Z ) 2 mm). The coil was inserted into a Wiretrol II (Drummond Scientific Company, Broomall, PY, USA) glass capillary tube (length 90 mm, Q) 2.2 mm). The capillary tube was placed between two permanent neodymium magnets (N35; 50> <30> <30 mm; 0.48 T at the surface), at a distance of 3 cm from each magnet. At this distance, the magnetic field was applied to the 0.1 T coil, measured with a Gaussmeter model GM-2 (Alphalab, Saltlake City, UT, USA). Both ends 10 of the capillary tube were connected to silicone tubing (inner 0 2 mm, outer 0 4 mm) as shown in the arrangement in FIG. 5A. Evaluation of the coil capture efficiency of nanoparticles during a passage in a genomic fate experiment was performed by connecting a KDS100 syringe infusion pump (KD Scientific, Holliston, MA, USA). 4.7 ml of a solution of nanoparticles (25 μg / ml water), prepared as described in Example 5, was pumped into the evacuated system at a flow of 0.5-6 ml / min. The death volume was 0.7 ml. The sample (4 ml) was collected and the absorbance was measured at 350 nm in a spectrophotometer.
A standard curve showed a linear relationship between absorbance and the concentration of nanoparticles.
The percentage of nanoparticles retained in the capillary, hereinafter referred to as capture efficiency (CE), was calculated as CE = 100 * (A0 - A) / A0 where A0 is the initial absorbance of the nanoparticle solution and A is the absorbance of the effluent. The experiment was repeated without the coil present in the capillary tube to determine the blank retention. CE is shown in FIG. 5B.
Example 32 - In vitro target guidance of magnetic particles to a helical wire in a recirculation-through fl fate system A Kantahl D ferromagnetic wire (length 100 mm, 0 0.13 mm) was wound 15 turns to make a helix (length 40 mm, 0 2 mm). The coil was inserted into a Wiretrol II (Drummond Scientific Company, Broomall, PY, USA) glass capillary tube (length 90 mm, 2.2 mm). The capillary tube was placed between two permanent neodymium magnets (N35; 50> <30> <30 mm; 0.48 T at the surface), at a distance of 3 cm from each magnet. At this distance, the magnetic field was applied to the 0.1 T coil, measured with a Gaussmeter model GM-2 (Alphalab, Saltlake City, UT, USA). Both ends of the capillary tube were connected to silicone tubing (inner 0 2 mm, outer 0 4 mm) as shown in the arrangement in FIG. 5A. The capture efficiency of recycled nanoparticles was evaluated by connecting a Gilson Minipuls 2 peristaltic pump. The tubing was placed in a closed loop and nanoparticles from Example 5 (4-10 ml, 25 μg nanoparticles / ml water) were recycled at a leaching rate of 1-40 ml / min for 10-60 minutes (except for the evaluation at 1 ml / min, which performed for 90 minutes). At the end of each experiment, the loop was disassembled and a sample of the nanoparticle solution was taken to measure the absorbance and calculate the CE as in Example 30. The CE values are shown in FIG. 5C-F. The coating of nanoparticles on the wire is shown in Figure 6B, for comparison with FIG. 6A, which is the bare wire.
Example 33 - In vivo targeting of magnetic particles and lysis of stent thrombosis (in-stent thrombosis) by tPA nanoparticle conjugate A Kanthal D wire (length 80 mm, 0.13 mm) was woven into a NIR PrimoTM coronary stent (length 16 mm , 0 3 mm, Boston Scienti fi c Scimed, Maple Grove, MN, USA) in a helical configuration. The stent was manually mounted on a MaverickTM coronary balloon (length 30 mm, 0 3 mm, Boston Scienti fi c Scimed). A female domestic pig (40 kg) was pre-sedated, anesthetized, orally intubated with a cuffed endotracheal tube, ventilated with nitrous oxide and oxygen (723), and monitored by electrocardiography (ECG). Radiological procedures were performed in an experimental catheter laboratory (Shimadzu Corp., Kyoto, Japan). Angiogram was obtained by injection of ohexol.
Heparin (5,000 IU) was given intravenously before catheterization. The left femoral artery (artería femoralís sínístra), the left carotid artery (artería carotís communís sínístra) and the right external jugular vein (venajugularís externa dextra) were surgically exposed and 6F introducer sheaths were inserted into the vessels. A stemotomy was performed on the pig. A guide catheter was passed through the introducer into the left carotid artery to the left coronary artery (coronary artery). The catheter was used to place a Doppler fl Fetal Speed Sensor (Jometrics Flowire, Jomed NV), connected to a FloMap monitor (Cardiometrics, Mountain View, CA, USA), and a leader in the interventricular ramus anterior artery coronary artery (left anterior descending artery). . The NIR PrimoTM coronary stent with a Kanthal D-wire was placed in the middle of the LAD, distal to the first diagonal branch, by inflating the balloon to 10 atm for 10 seconds. An intravascular ultrasound probe was passed over the guide wire to image the stent-artery segment at various times. Baseline es fl fate, measured with fl destiny wave, was 20 cm / s after stent insertion. A permanent neodymium magnet (N48, 50 X 15 X 15 mm, 0.48 T at the surface) was applied to the anterior part of the heart, in contact with the stent segment of the LAD. After spontaneous formation of a blood clot in the stent, the baseline minsk decreased to 5 cm / s. A solution of tPA nanoparticle conjugate from Example 29 (40 ml, 0.14 mg / ml) was injected through the guide catheter to the left main coronary artery. The thrombus was lysed by the action of the tPA nanoparticle conjugate Baslinjes fl increased to 15 cm / s.
According to one embodiment, a composition according to embodiments of the invention is provided for use as a medicament.
Specifically, according to one embodiment, there is provided a composition according to embodiments of the invention for the treatment of thrombosis.
Thus, according to one embodiment, there is provided a method of treating thrombosis in a subject, comprising a first step of injecting the composition, comprising coated magnetic nanoparticles according to certain embodiments, into the blood, i.e. the cardiovascular system, of the subject.
Then a magnetic field is applied to the site of the thrombosis, after which the nanoparticles are attracted to the thrombosis by the magnetic field, which dissolves the thrombus.
In one embodiment, a method of treating stent thrombosis in a patient with an implanted magnetizable stent is described, comprising a first step of injecting the composition comprising coated magnetic nanoparticles according to certain embodiments, into the blood, i.e. the cardiovascular system, of the subject. Thereafter, a magnetic field is applied to the site of the stent, after which the nanoparticles are attracted to the stent by the magnetic field, thus dissolving the blood clot. As described above, the magnetic nanoparticles can be conjugated to tPA, i.e., recombinant human tissue plasminogen activator, to further enhance the anti-thrombotic effect.
Although the present invention has been described above with reference to specific embodiments, it is not intended to be limited to the particular form set forth herein. Rather, the invention is limited only by the appended claims and embodiments other than those specified above are equally possible within the scope of these appended claims.
In the claims, the term "comprising" does not exclude the presence of other elements or steps. Furthermore, even if they are individually listed, a plurality of methods, elements or method steps can be implemented by e.g. a device or processor. In addition, although individual features may be included in individual requirements, these may optionally be combined and the inclusion in different requirements does not mean that a combination of features is not possible and / or advantageous. In addition, singular references do not exclude a plurality. The terms "one", "one", "first", "second", etc. do not exclude plurality. Reference numerals in the claims are provided as illustrative examples only and should not be construed as limiting the scope of the claims in any way.

权利要求:
Claims (30)
[1]
A method of forming a layer on a nanoparticle having hydroxyl groups on its surface, comprising the steps of subjecting the nanoparticle to a first solution comprising a compound of formula (1): Ho oH WO / W n I wherein "n" is a integers in the range 0 (zero) to 7000, subjecting the nanoparticle to a second solution comprising a silanizing agent, and enabling the formation of a silanized layer on the nanoparticle.
[2]
The method of claim 1, wherein the nanoparticle is a magnetic nanoparticle.
[3]
A method according to claim 1 or 2, wherein the compound of formula (I) is selected from the group consisting of: ethylene glycol, diethylene glycol (DEG), triethylene glycol (TREG), tetraethylene glycol, pentaethylene glycol, hexaethylene glycol, heptaethylene glycol, octaethylene glycol / other oligoethylene glycol / other polyethylene oxides having molecular weights up to 300000 (such as PEG 400, PEG 2000, PEG 3400, PEG 8000, PEG 20000, PEG 35000, PEG 100000, PEG 200000, and PEG 300000), or a combination thereof.
[4]
A method according to any one of the preceding claims, wherein the silanizing agent is a silane.
[5]
The method of claim 4, wherein the silane is an alkoxysilane.
[6]
A method according to claim 5, wherein the alkoxysilane is selected from the group consisting of tetramethoxysilane, tetraethoxysilane, tetra-n-propoxysilane, tetra-isopropoxysilane, tetra-n-butoxysilane, tetra-t-butoxysilane, trimethoxysilane, triethoxysilane, triethoxysilane -propoxysilane, tri-iso-propoxysilane, tri-n-butoxysilane, tri-t-butoxysilane, trimethoxychlorosilane, triethoxychlorosilane, tri-n-propoxychlorosilane, tri-isopropoxyclorosilane, tri-n-butoxyclorosilanes, tri-n-butoxyclorsilane, tri- , benzyltriethoxysilane, dimethyldimethoxysilane, dimethyldiethoxysilane, and mixtures thereof.
[7]
The method of claim 4, wherein the silane is a halosilane. lO 15 20 25 30 35
[8]
The method of claim 8, wherein the halosilane is selected from the group consisting of tetrachlorosilane, trichlorosilane, tetrachlorosilane, trifluorosilane, and mixtures thereof.
[9]
The method of claim 4, wherein the silane is an amino silane.
[10]
The method of claim 9, wherein the aminosilane is selected from the group consisting of 3-aminopropyltrinethoxysilane, 3-aminopropylnethylldinethoxysilane, 3-aminopropylyldyldiethylnietoxysilane, N- (2-aminoethyl) -3-aminopropylmethyldyrnetoxysilane, N- (Z-ethyl-S-) -N-propyl trimethoxysilane, 4-aniinobutyldinietylnietoxysilan, 4-aminobutyltrinietoxysilan, arninoetylarninometylfenetyltrirnetoxysilan, N- (2-arninoetyl) -3 -aminoisobutylmetyldimetoxysilan, N- (6-an1inohexyl) aniinopropyltrinietoxysilan, 3- (m-aminophenoxy) propyltrirnetoxysilan, arninofenyltrirnetoxysilan, 3-aminopropyltriethoxysilane, 3- aminopropylnethyldiethoxysilane, 3-aminopropylyldinethylethoxysilane, N- (2-aminoethyl) -3-aminopropylnethyldiethoxysilane, N- (2-aminoethyl-3-aminopropyl) triethoxysilane, 4-aminobutyldiniethylethoxysilane, 4-aminoethyleniethylethinoethylethinoethylethylamino -aminoisobutylnethylldietoxysilane, N- (6-aminohexyl) aminopropyltriethoxysilane, 3- (m-aminophenoxy) propyltriethoxysilane, aminophenyl ethoxysilane, and mixtures thereof.
[11]
The method of claim 4, wherein the silane is an olefin-containing olefin.
[12]
The method of claim 11, wherein the olefin-containing silane is selected from the group consisting of 3- (trinethoxysilyl) propyl methacrylate, 3- (triethoxysilyl) propyl methacrylate, methacryloxyynethyltrinethoxysilane, methacryloxynethyltriethoxysilane, vinyltrinylethyloxyethylanyl .
[13]
The method of claim 4, wherein the silane is a fluorescent silane.
[14]
The method of claim 4, wherein the silanizing agent is a radiopaque silane.
[15]
A method according to any one of the preceding claims, wherein the first solution further comprises a base and / or a second solvent.
[16]
The method of claim 15, wherein the base is selected from the group consisting of: ammonia, sodium hydroxide, potassium hydroxide, triethylarnin, trimethylanine, dimethylanine, diethylarnin, ethylarnin, propylanine, N, N-diisopropylethylaniline, N-methylniorfoline, N-oontylpyrrolidine, ethanolamine, pyridine, 4-dinylethylaninopyridine, methylamine, and piperidine, or a combination thereof. lO 15 20 25 30 35
[17]
A method according to claim 15 or 16, wherein the second solvent is selected from the group consisting of: water, methanol, ethanol, n-propanol, isopropanol, N, N-dimethylformamide (DMF), dimethylsulfoxide (DMSO), acetone and acetonitrile, or a combination thereof.
[18]
A method according to any one of the preceding claims, further comprising a step of immobilizing a functional unit on the silanized layer, said functional unit being selected from the group consisting of: enzyme, protein, antibody, peptide, affinity ligand, oligonucleotide, carbohydrate, lipid, surfactant or a pharmaceutically active molecule.
[19]
A composition obtainable by the process according to claims 1 to 18.
[20]
A composition comprising substantially discrete nanoparticles with a silanized layer on each nanoparticle.
[21]
21.
[22]
22.
[23]
23.
[24]
24.
[25]
25.
[26]
26.
[27]
27.
[28]
28.
[29]
A composition according to claim 19 or 20, wherein the nanoparticles are magnetic. A composition according to claims 19 to 21 wherein the nanoparticles are X-ray dense. A composition according to claims 19 to 21 wherein the nanoparticles are non-permeable. A composition according to claims 19 to 21 wherein the nanoparticles are MR active. A composition according to any one of claims 19 to 24 for use as a medicament. A composition according to claim 21 for the treatment of thrombosis. A contrast agent comprising the composition of any one of claims 22 to 24. A magnetic ink comprising the composition of claim 21. A method of treating thrombosis in a subject, comprising the steps of: injecting the composition of claim 21 into the bloodstream of the subject; apply a magnetic field to the site of the thrombosis, and attract nanoparticles to the thrombosis with the magnetic field, thus dissolving the thrombus. lO
[30]
A method of treating stent-tronibose (in-stent-tronibose) in a subject with an implanted niagnetizable stent, comprising the steps of: injecting the composition of claim 21 into the bloodstream of the subject, applying a magnetic field to the site of the stent, and attracting nanoparticles to the stent with the magnetic field, thus dissolving the thrombus.

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同族专利:

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公开号 | 公开日

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WO2012018290A1|2012-02-09|

引用文献:

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公开号 | 申请日 | 公开日 | 申请人 | 专利标题

---

---

US20080057001A1|2006-05-25|2008-03-06|Xiao-Dong Sun|Contrast agents for imaging|

---

US7999025B2|2008-01-28|2011-08-16|University Of Utah Research Foundation|Asymmetrically-functionalized nanoparticles organized on one-dimensional chains|CA2934401C|2009-11-02|2017-01-10|Pulse Therapeutics, Inc.|Magnetomotive stator system and methods for wireless control of magnetic rotors|

---

US9883878B2|2012-05-15|2018-02-06|Pulse Therapeutics, Inc.|Magnetic-based systems and methods for manipulation of magnetic particles|

---

US9119875B2|2013-03-14|2015-09-01|International Business Machines Corporation|Matrix incorporated fluorescent porous and non-porous silica particles for medical imaging|

法律状态:
2013-03-26| RINS| Reinstatement according to par. 72 patents act|Effective date: 20130308 |

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2016-01-05| NAV| Patent application has lapsed|

优先权:

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申请号 | 申请日 | 专利标题

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US34846310P| true| 2010-05-26|2010-05-26|

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PCT/SE2011/000092|WO2012018290A1|2010-05-26|2011-05-26|Discrete coated nanoparticles|

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