SE1230153A1 - Discrete coated nanoparticles - Google Patents

Discrete coated nanoparticles Download PDF

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Publication number
SE1230153A1
SE1230153A1 SE1230153A SE1230153A SE1230153A1 SE 1230153 A1 SE1230153 A1 SE 1230153A1 SE 1230153 A SE1230153 A SE 1230153A SE 1230153 A SE1230153 A SE 1230153A SE 1230153 A1 SE1230153 A1 SE 1230153A1
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SE
Sweden
Prior art keywords
nanoparticles
solution
nanoparticle
peg
silane
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SE1230153A
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Swedish (sv)
Inventor
Maria Kempe
Henrik Kempe
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Publication of SE1230153A1 publication Critical patent/SE1230153A1/en

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    • A61K49/1818—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles
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    • A61K49/1824—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles
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    • A61K49/0485—Nanoparticles, nanobeads, nanospheres, nanocapsules, i.e. having a size or diameter smaller than 1 micrometer
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    • B82Y15/00—Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y25/00—Nanomagnetism, e.g. magnetoimpedance, anisotropic magnetoresistance, giant magnetoresistance or tunneling magnetoresistance

Abstract

Denna uppfinning hänför sig generellt till området för nanopartiklar.Närmare bestämt hänför sig uppfinningen till ett förfarande för silanisering av en magnetisk nanopartikel så att nanopartikeln förblir diskret, dvs icke-agglomererad under silaniseringsprocessen. Uppfinningen hänför sig också till en sådan diskret nanopartikel och en komposition innefattande diskreta nanopartiklar och olika användningar av partikeln eller kompositionen.Figur som ska offentliggöras med abstraktet: FIG. 1This 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

Description

20 25 30 limmar samman nanopartiklama permanent, vilket resulterar i en större skenbar partikelstorlek hos preparationen. Glues the nanoparticles together permanently, resulting in a larger apparent particle size of the preparation.

För medicinska tillämpningar in vivo, som involverar transport av nanopartiklar i kärlsystemet, kan aggregat av nanopartiklar obstruera blodkärlen. För applikationer ex vivo och in vitro kan aggregat av nanopartiklar hindra mikroflödessystemkanaler, slangar, munstycken, och andra typer av småskaliga anordningar.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.

Således finns det ett behov av en ny metod för framställning av silaniserade magnetiska nanopartiklar i vilka nanopartiklama inte silaniserats som aggregat.Thus, there is a need for a new method for producing silanized magnetic nanoparticles in which the nanoparticles have not been silanized as aggregates.

Sammanfattning av uppfinningen Föreliggande uppfinning avser företrädesvis att mildra, lindra eller eliminera en eller flera av de ovan identifierade bristema inom tekniken, ensamma eller i någon kombination, och löser åtminstone de ovanämnda problemen genom att tillhandahålla ett förfarande för framställning av diskreta silaniserade magnetiska nanopartiklar enligt de bifogade patentkraven.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.

Den generella lösningen enligt uppfinningen är att utsätta nanopartiklar för en eller flera specifika föreningar innan silanisering. Detta ger en kolloidal lösning av nanopartiklar, vilken, när nanopartiklama därefter silaneras, resulterar i diskreta silaniserade nanopartiklar.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.

Enligt en första aspekt av uppfinningen tillhandahålls en metod för beläggning av en magnetisk nanopartikel som har hydroxylgrupper på sin yta genom att bilda ett skikt därpå. Metoden innefattar stegen att utsätta nanopartikeln för en första lösning inkluderande en förening enligt formel (I) : HO OH Wow H I vari "n" är ett heltal i intervallet 0 (noll) till 7000. Metoden innefattar också ett steg att utsätta nanopartikeln för en andra lösning inkluderande ett silaniseringsmedel, och ett steg att tillåta bildning av ett silaniserat skikt på den magnetiska nanopartikeln.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.

Enligt en andra aspekt av uppfinningen tillhandahålls en komposition, som kan erhållas genom förfarandet enligt den första aspekten.According to a second aspect of the invention there is provided a composition obtainable by the process of the first aspect.

Enligt en tredje aspekt av uppfinningen tillhandahålls en komposition, innefattande huvudsakligen diskreta, silanbelagda nanopartiklar.According to a third aspect of the invention there is provided a composition comprising substantially discrete, silane coated nanoparticles.

Ytterligare utföringsforrner av uppfinningen definieras i de underlydande patentkraven, liksom i beskrivningen.Further embodiments of the invention are defined in the appended claims, as well as in the description.

Föreliggande uppfinning har den fördelen jämfört med känd teknik att den resulterar i diskreta silaniserade nanopartiklar, det vill säga nanopartiklar framställda genom bildning av lO 15 20 25 30 35 silaniserade skikt runt icke-aggregerade, singulära partiklar. Således, en komposition omfattande sagda nanopartiklar kommer att omfatta huvudsakligen diskreta nanopartiklar med ett silaniserat skikt på varje nanopartikel.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.

Sammanfattande beskrivning av ritningarna Dessa och andra aspekter, särdrag och fördelar som uppfinningen är kapabel till kommer att framgå och klargöras av följ ande beskrivning av utföringsforrnema av föreliggande uppfinning, varvid hänvisning görs till de bifogade ritningama, i vilka FIG. l är en schematisk illustration av ett tvärsnitt av en nanopartikel enligt en utföringsform av uppfinningen; FIG. 2 är en graf som visar FT-IR-spektra av nanopartiklar; FIG. 3 är en transmissionselektronmikroskopi (TEM)-bild av nanopartiklar; FIG. 4 är en översikt av immobilisering av tPA enligt en utföringsform; FIG. 5A är en schematisk instrumentell uppställning av magnetisk målstyming av belagda nanopartiklar in vitro, och Fig 5B till F är diagram som visar inverkan av flödeshastigheten på infångningseffektiviteten (CE=capture efficiency) av nanopartiklarna; FIG. 6 är fotografisk representation av ett segment av ett kapillärrör med insatt spirallindad tråd; FIG. 7 visar magnetiska hystereskurvor av (A) nakna magnetitnanopartiklar från Exempel l, (B) silaniserade nanopartiklar från Exempel 5, (C) silaniserade nanopartiklar från Exempel 6, och (D) tPA-nanopartikels-konjugat från Exempel 29; och FIG. 8 är en graf som visar den återstående enzymaktiviteten av tPA-nanopartikels- konjugaten från Exempel 28 (grå staplar) och Exempel 29 (svarta staplar) efter (A) ultraljudsbehandling under l h; eller inkubation vid 4 °C under (B) 24 h, (C) 48 h, (d) 10 dagar, (E) 21 dagar och (F) 40 dagar.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.

Detaljerad beskrivning av uppfinningen Flera utföringsforrner av föreliggande uppfinning kommer att beskrivas mer i detalj nedan med hänvisning till bifogade ritningar i syfte att fackmannen inom teknikområdet ska kunna utföra uppfinningen. Uppfinningen kan emellertid utföras i många olika former och skall inte tolkas som begränsad till de utföringsforrner som anges häri. Vidare är den terminologi som används i den detaljerade beskrivningen av de särskilda utföringsfonnema som illustreras i de bifogade ritningama inte avsedda att vara begränsande för uppfinningen.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.

I FIG. l är ett schematiskt tvärsnitt av en nanopartikel enligt en utföringsforrn visad. Skiktet (A) av nanopartikeln är en inre kärna. Skiktet (B) är ett silaniserat skikt eller en beläggning av silika eller ett silikaderivat, applicerat runt den singulära nanopartikeln så att en diskret silaniserad lO 15 20 25 30 nanopartikel bildas. Sålunda, nanopartikeln silaniseras som en icke-aggregerad, singulär partikel.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.

Skiktet (C) är en valfri ytterligare beläggning, konjugerad till skiktet (B).Layer (C) is an optional additional coating, conjugated to layer (B).

Eftersom nanopartikeln silaniseras som en icke-aggregerad, singulär partikel, enligt en utföringsform, tillhandahålls en komposition omfattande huvudsakligen diskreta, silaniserade nanopartiklar, såsom över 50%, 60%, 70%, 80% eller 90% diskreta nanopartiklar med ett silaniserat skikt på Varje nanopartikel.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.

Enligt en utföringsform, framställs nämnda nanopartikel genom en metod som bildar ett skikt på, eller belägger, en nanopartikel. Nanopartikeln kan vara vilken som helst typ av nanopartikel sålänge den har hydroxylgrupper på sin yta. Förfarandet innefattar vidare ett steg att utsätta nanopartikeln för en första lösning inkluderande en förening enligt formel (1): HO OH WOW f) I I formel (I) är “n” ett heltal i intervallet 0 (noll) till 7000, i intervallet 0 (noll) till 2300 eller i intervallet 2 till 800.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.

I en utföringsform är nanopartikeln en magnetisk nanopartikel.In one embodiment, the nanoparticle is a magnetic nanoparticle.

Exempel på föreningar med formel (I) innefattar, men år inte begränsade till etylenglykol, dietylenglykol (DEG), trietylenglykol (TREG), tetraetylenglykol, pentaetylenglykol, hexaetylen- glykol, heptaethylenglykol, oktaetylenglykol och andra oligoethylenglykoler/polyetylenglykoler (även kallade polyetylenoxider) med molekylvikter upp till 300000, såsom PEG 400, PEG 2000, PEG 3400, PEG 8000, PEG 20000, PEG 35000, PEG 100000, PEG 200000, och PEG 300000, eller en kombination därav.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.

Nanopartiklama utsätts för ovanstående lösning genom att låta nanopartiklama komma i kontakt med lösningen under omrörning, blandning, skakning, tumling, och/eller sonikering, typiskt under en tidsperiod mellan 1 minut och 24 h, mellan 5 minuter och 3 h eller mellan 30 minuter och 1,5 h för att framställa en kolloidal lösning.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.

I en utföringsforrn väljs lösningsmedlet hos den första lösningen från gruppen bestående av: vatten, metanol, etanol, n-propanol, iso-propanol, N, N-dimetylformamid (DMF), dimetylsulfoxid (DMSO), aceton och acetonitril, eller en kombination därav.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. .

Emellertid kan den första lösningen även bestå av förening enligt formel (I), om förening I i sig åri flytande form, såsom TREG.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.

Den första lösningen kan även bestå av flera typer av föreningar enligt formel (I), där alla är i flytande form. lO 15 20 25 30 35 I en utföringsforrn är förening enligt formel (I) en vätska och fungerar som ett lösningsmedel i den första lösningen.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.

I en utföringsforrn innefattar den första lösningen flera typer av föreningar enligt forrnel (I) och ett lösningsmedel, såsom vald från gruppen bestående av: vatten, metanol, etanol, n-propanol, iso-propanol, N, N-dimetylforrnamid (DMF), dimetylsulfoxid (DMSO), aceton och acetonitril, eller en kombination därav.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.

I en utföringsforrn innefattar den första lösningen ytterligare åtminstone en bas och/eller åtminstone ett andra lösningsmedel.In one embodiment, the first solution further comprises at least one base and / or at least one second solvent.

Basen kan välj as från gruppen bestående av: ammoniak, natriumhydroxid, kaliumhydroxid, trietylamin, trimetylamin, dimetylamin, dietylamin, etylamin, propylamin, N,N-diisopropyletylamin, N-metylmorfolin, N-metylpyrrolidon, oleylamin, etanolamin, pyridin, 4-dimetylaminopyridin, metylamin, och piperidin, eller en kombination därav.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.

Det andra lösningsmedlet kan väljas från gruppen bestående av: vatten, metanol, etanol, n- propanol, iso-propanol, N,N-dimetylformamid (DMF), dimetylsulfoxid (DMSO), aceton och acetonitril, eller en kombination därav.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.

Förfarandet omfattar vidare ett steg för behandling av nanopartikeln med en andra lösning innefattande ett silaniseringsmedel, så att bildning av ett silaniserat skikt, eller beläggning, på den (magnetiska) nanopartikeln möjliggörs.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.

Silaniseringsmedlet kan vara en silan.The silanizing agent may be a silane.

I en utföringsform, är silanen en alkoxysilan, såsom vald från gruppen bestående av tetrametoxysilan, tetraetoxysilan, tetra-n-propoxysilan, tetra-iso-propoxysilan, tetra-n-butoxysilan, tetra-t-butoxysilan, trimetoxysilan, trietoxysilan, tri-n-propoxysilan, tri-iso-propoxysilan, tri-n- butoxysilan, tri-t-butoxysilan, trimetoxyklorsilan, trietoxyklorsilan, tri-n-propoxyklorsilan, tri-iso- propoxyklorsilan, tri-n-bytoxyklorsilan, tri-t-butoxyklorsilan, bensyltrimetoxysilan, bensyltrietoxysilan, dimetyldimetoxysilan, dimetyldietoxysilan, och blandningar därav.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.

I en utföringsform är silanen en halosilan, såsom vald från gruppen bestående av tetraklorsilan, triklorsilan, tetrafluorsilane, trifluorsilane, och blandningar därav.In one embodiment, the silane is a halosilane, as selected from the group consisting of tetrachlorosilane, trichlorosilane, tetrachlorosilane, trifluorosilane, and mixtures thereof.

I en utföringsform, är silanen en aminosilan, såsom vald från gruppen bestående av 3- aminopropyltrimetoxysilan, 3-aminopropylmetyldimetoxysilan, 3-aminopropyldimetylmetoxysilan, N-(2-aminoetyl)-3-aminopropylmetyldimetoxysilan, N-(Z-aminoetyl-S -aminopropyl)trimetoxysilan, 4-aminobutyldimetylmetoxysilan, 4-aminobutyltrimetoxysilan, aminoetylaminometylfenetyltri- metoxysilan, N-(2-aminoetyl)-3-aminoisobutylmetyldimetoxysilan, N-(6-aminohexyDaminopropyl- trimetoxysilan, 3-(m-aminofenoxy)propyltrimetoxysilan, aminofenyltrimetoxysilan, 3-aminopropyl- trietoxysilan, 3-aminopropylmetyldietoxysilan, 3- aminopropyldimetyletoxysilan, N-(2-aminoetyl)-3- aminopropylmetyldietoxysilan, N-(Z-aminoetyl-S-aminopropyl)trietoxysilan, 4-aminobutyldimetyl- etoxysilan, 4-aminobutyltrietoxysilan, aminoetylaminometylfenetyl trietoxysilan, N-(2-aminoetyl)-3- 10 15 20 25 30 35 aminoisobutylmetyldietoxysilan, N-(6-aminohexyl)aminopropyltrietoxysilan, 3-(m-aminofenoxy)- propyltrietoxysilan, aminofenyltrietoxysilan, och blandningar därav.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.

I en utföringsforrn, är silanen en olefin-innehållande silan, såsom vald från gruppen bestående av 3-(trimetoxysilyl)propylmetakrylat, 3-(trietoxysilyl)propylmetakrylat, metakryloxy- metyltrimetoxysilan, metakryloxymetyltrietoxysilan, vinyltrimetoxysilan, vinyltrietoxysilan, allyltrimetoxysilan, allyltrietoxysilan, vinyltriklorsilan och blandningar därav.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 .

I en utföringsforrn är silanen en fluorescerande silan.In one embodiment, the silane is a fluorescent silane.

I en utföringsform är silanen en radioopak silan.In one embodiment, the silane is a radiopaque silane.

Silaniseringssteget kan upprepas med samma eller annat silaniseringsmedel.The silanization step can be repeated with the same or another silanizing agent.

Fördelen med detta är att flera silaniseringsskikt kan erhållas.The advantage of this is that fl your silanization layers can be obtained.

Silanisering utförs typiskt vid temperaturer mellan 0 °C och 200 °C genom placering av flaskoma eller kolvama i ett kylrum, i rumstemperatur, i ett vattenbad, i ett oljebad, i ett värrneblock, i en värrnemantel, i en mikrovågsugn, i ett mikrovågsugns-accelererat reaktionssystem eller i en ugn.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.

I en utföringsform, utförs silanisering genom placering i en mikrovågsugn eller i ett så kallat mikrovågs-accelererat reaktionssystem. Detta är fördelaktigt, eftersom silaniseringen fortskrider snabbt och effektivt.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.

Blandningama blandas, omrörs, skakas, tumlas, och/eller sonikeras valfritt. Omröming kan genomföras med en överliggande omrörare, en magnetisk omrörare, eller en homogenisator vid 50 varv per minut till 30000 rpm, företrädesvis vid 200 rpm till 3000 rpm. Silaniseringen får fortgå mellan 10 minuter och 72 timmar.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.

Efter silaniseringen separeras de magnetiska nanopartiklama från lösningen antingen med hjälp av en permanent neodymiummagnet, genom centrifugering, genom sedimentering, eller genom dialys. Separationen av nanopartiklama från lösningama utförs antingen direkt eller efter tillsats av etylacetat eller annat organiskt lösningsmedel som hjälper till att precipitera nanopartiklama.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.

Lösningama kyls valfritt före separationen. Nanopartiklama tvättas med vatten och/eller metanol och/eller andra organiska lösningsmedel. De belagda nanopartiklama torkas i vakuum vid rumstemperatur eller i en vakuumugn eller används direkt för ytterligare tillämpningar.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.

Beläggningsförfarandet resulterar typiskt i massökningar mellan 5 och 100%.The coating process typically results in mass increases between 5 and 100%.

I en utföringsforrn innefattar förfarandet vidare ett steg att immobilisera en funktionell enhet på det silaniserade skiktet.In one embodiment, the method further comprises a step of immobilizing a functional unit on the silanized layer.

Den funktionella enheten kan vara minst ett enzym, protein, antikropp, peptid, affinitetsligand, oligonukleotid, kolhydrat, lipid, ytaktivt ämne, aptamer eller en farmaceutiskt aktiv (läkemedels)-molekyl för att tillhandahålla derivatiserade magnetiska nanopartiklar och kombinationer därav.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.

Detta är fördelaktigt eftersom nanopartikeln då kan bli lämpad för behandling, diagnostik, separation, upprening, eller MICR (Magnetic Ink Character Recognition). 10 15 20 25 30 35 Den funktionella enheten kan också vara ett molekylärt avtryckt (imprintat) polymerskikt, för att tillhandahålla molekylärt avtryckta (imprintade) magnetiska nanopartiklar.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.

Den funktionella enheten kan vidare vara en polymer innehållande funktionella grupper att tjäna som utgångspunkter för antingen stegvis fastfas-syntes eller ytterligare derivatisering genom konjugering till ett enzym, protein, antikropp, peptid, affinitetsligand, oligonukleotid, kolhydrat, lipid, ytaktivt ämne, aptamer eller läkemedelsmolekyl.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. .

Den funktionella enheten kan också vara en naturlig eller syntetisk polymer med förmåga att innesluta eller inkapsla läkemedelsmolekyler for senare tillämpningar inom läkemedels- administration, varvid nämnda polymer är belagd eller ympad på nanopartikeln.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.

Detta är fördelaktigt eftersom nanopartikeln sedan kan vara lämplig för läkemedels- administration.This is advantageous because the nanoparticle may then be suitable for drug administration.

I en utföringsforin är en komposition som kan erhållas genom metoderna enligt vissa utföringsformer beskriven. Nämnda komposition innefattar huvudsakligen diskreta nanopartiklar med ett silaniserat skikt, av silika eller silikaderivat, på varje nanopartikel, såsom över 50%, 60%, 70%, 80% eller 90% diskreta nanopartiklar med ett silaniserat skikt på varje nanopartikel.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.

I en utföringsform, vari nanopartiklama är magnetiska nanopartiklar, kan kompositionen innefattande huvudsakligen diskreta nanopartiklar med ett silaniserat skikt på varje nanopartikel användas som ett magnetiskt bläck.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.

Sålunda, i en utföringsforrn, tillhandahålls ett magnetiskt bläck, innefattande kompositionen enligt utföringsforrner av uppfinningen.Thus, in one embodiment, a magnetic ink is provided, comprising the composition according to embodiments of the invention.

I en utföringsforrn, vari nanopartiklama är radioopaka nanopartiklar eller fluorescerande nanopartiklar, kan kompositionen innefattande huvudsakligen diskreta nanopartiklar med ett silaniserat skikt på varje nanopartikel användas som kontrastmedel eller markör.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.

Exempel Följande experimentella utföringsfonner tillhandahålls för att göra denna beskrivning grundlig och fullständig och förmedla omfattningen av uppfinningen för fackmannen inom området.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.

Utföringsforrnema begränsar inte uppfinningen, utan uppfinningen begränsas endast av de bifogade patentkraven.The embodiments do not limit the invention, but the invention is limited only by the appended claims.

Metoderna för syntes av magnetiska nanopartiklar av jämoxid kan delas in i de som utförs i vattenhaltiga medier och de som utförs i organiska medier. Syntes av magnetiska nanopartiklar av jämoxid genom alkalisk hydrolys av j ämsalter i vattenhaltiga medier har beskrivits av Massart [Massait, R. IEEE Trans. Magn. 1981, 1 7, 1247-l248]. Massarts metod för syntes av magnetit börjar med en blandning av jäm(II)- och j ärn(III)-salter i ett molförhållande som motsvarar oxidationstalet hos Fe i magnetit (Fe3O4). Ett antal andra publikationer använder variationer av detta förfarande utgående från blandningar av j ärn(II)- och j ärn(III)-salter för framställning av magnetit (Fe3O4) eller 10 15 20 25 30 35 maghemit (y-Fe2O3) [Molday, R.S. 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. Colloíds and Surfaces A: Physicochem. Eng Aspects 2003, 212, 219-226; Yamaura, M. et al. J. Magn. Magn.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]. När den önskade produkten är Fe3O4, utförs syntesen ibland under en inert atmosfär för att förhindra ytterligare oxidation till Fe2O3. Syntes av magnetiska nanopartiklar i organiska medier sker genom termisk sönderdelning av metallorganiska föreningar, t.ex. j ärnacetylacetonater eller j ärnacetylkarbonater, i högkokande organiska lösningsmedel i närvaro av ytaktiva medel, t.ex. fettsyror, oleinsyra eller hexadecylamin [Burke, N.A.D et al. Chem. Mater. 2002, 14, 4752-4761; Simenoides, K. et al. J. Magn. Magn. Mat. 2007, 316, e1-e4]. De resulterande nanopartiklarna efter syntes i organiska medier täcks normalt av hydrofoba molekyler som gör dem lösliga endast i organiska medier.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.

Exempel 1 till 4 nedan avser syntes av nakna magnetitnanopartiklar (F e3O4) i vatten.Examples 1 to 4 below relate to the synthesis of naked magnetite nanoparticles (F e 3 O 4) in water.

Emellertid, såsom kommer att inses av en fackman inom området, är andra syntesmetoder också möjliga inom ramen för uppfinningen.However, as will be appreciated by one skilled in the art, other synthetic methods are also possible within the scope of the invention.

Exempel 1 Vatten bubblades med en ström av kvävgas under 1 h och användes sedan för framställning av två lösningar: den forsta lösningen framställdes genom att lösa 0,834 g (3 mmol) FeSO4'7H2O i 125 ml vatten och den andra innehöll 0,842 g (15 mmol) KOH och 5,056 g (50 mmol) KNOg i 125 ml vatten. De två lösningama sonikerades i ett ultraljudsbad under 5 minuter och blandades sedan samman i en 250-ml flaska försedd med skruvlock varvid en grön fällning bildades. Flaskan placerades i ett förvärmt (90 °C) vattenbad under 2 timmar. Vid slutet av reaktionstiden hade en svart tät fällning bildats. Flaskan kyldes i kallt (8 °C) vatten under 15 minuter. Fällningen separerades från lösningen genom med hjälp av en permanent neodymiummagnet (N35; 50 >< 50 >< 30 mm; 0.48 T vid ytan) och tvättas med vatten (250 mL >< 3) och metanol (MeOH) (250 mL >< 3). Proceduren gav 231 mg nanopartiklar (100% utbyte). Analys av jämhalten med ICP-AES (induktivt kopplad plasma atomemissionsspektrometri) och en kolorimetrisk järnanalys indikerade 70.5% Fe respektive 71 .5% Fe. Nanopartiklama orsakade 0.07% hemolys av utspätt blod efter 24-timmars inkubering och 0.21% och 0.3 0% hemolys av isolerade erythrotrocyter efter inkubationer underl h respektive 24 h.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. 2 A visar FT-IR-spektra av nanopartiklar som erhållits enligt detta exempel.Fig. 2A shows FT-IR spectra of nanoparticles obtained according to this example.

Exempel 2 Vatten bubblades med en ström av kvävgas under 1 h och användes sedan för framställning av två lösningar: den första lösningen framställdes genom att lösa 0.2 g (0.72 mmol) av F eSO4~7H2O lO 15 20 25 30 35 i 30 ml Vatten och den andra innehöll 0.202 g (3.6 mmol) KOH och 1.214 g (12 mmol) KNO; i 30 ml vatten. De två lösningama sonikerades i ett ultraljudsbad under 5 minuter och blandades sedan varvid en grön fällning bildades. Blandningen hälldes i ett HP-5 00 Plus-mikrovågsugnskärl (CEM Corp, Matthews, NC) och utsattes för 1200 W mikrovågsugnsbehandling med ett MARS 5 mikrovågs- accelererat reaktionssystem (CEM Corp, Matthews, NC) med en gradient över 1 min upp till 120 °C och därefter under 15 minuter vid konstant temperatur (120 °C). Innehållet i kärlet kyldes sedan till ca. 65 °C. En svart tät fällning separerades från lösningen med hjälp av en permanent neodymium- magnet. Nanopartiklarna tvättades med 25 ml vatten. 56 mg nanopartiklar (100% utbyte) erhölls.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.

Exempel 3 Vatten bubblades med en ström av kvävgas under 1 h och användes sedan för framställning av två lösningar: den första lösningen framställdes genom att lösa 0.2 g (0.72 mmol) FeSO4°7H2O i 15 ml vatten och den andra innehöll 0.202 g (36 mmol) KOH och 1.214 g (12 mmol) KNO; i 15 ml vatten. De två lösningama sonikerades i ett ultraljudsbad under 5 minuter. Volymer om 15 ml trietylenglykol sattes till varje lösning. Lösningama sonikerades kortvarigt. Lösningama blandades varvid en grön fällning bildades. Blandningen hälldes i ett HP-500 Plus-mikrovågsugnskärl (CEM Corp, Matthews, NC) och utsattes för 1200 W mikrovågsugnsbehandling med ett MARS 5 mikro- vågsaccelererat reaktionssystem (CEM Corp, Matthews, NC) med en gradient över 1 min upp till 120 °C och därefter under 15 minuter vid konstant temperatur (120 °C). Innehållet i kärlet kyldes till ca. 65 ° C. En svart tät fällning separerades från lösningen med hjälp av en permanent neodymium- magnet. Nanopartiklarna tvättades med 25 ml vatten. En mängd av 56 mg nanopartiklar (100% utbyte) erhölls.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.

Exempel 4 En liter avjoniserat vatten upphettades till 95 °C i en flaska försedd med skruvkork. En mängd av 600 mg FeCl2°4 H20 tillsattes och flaskan placerades i ett upphettat (95 °C) vattenbad.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.

Lösningen omrördes vid 8000 rpm med ett knivaggregat (homogenisator) under syntesen. En volym av 5 ml 7 M ammoniaklösning tillsattes vid starten av syntesen. Reaktionen fick fortgå under 1 h.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.

Nanopartiklama separerades från lösningen med en permanent neodymiummagnet och tvättades genom suspension i vatten 3 gånger. En mängd av 232 mg nanopartiklar (100%) erhölls.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.

Exempel 5 till 27 nedan avser syntes av silaniserade magnetitnanopartiklar enligt olika utföringsformer av uppfinningen. Följande beskrivning fokuserar på en utföringsform av föreliggande uppfinning tillämpbar på en magnetisk nanopartikel och i synnerhet en nanopartikel av magnetit (Fe3O4). Emellertid kommer det att inses att uppfinningen inte är begränsad till denna tillämpning utan kan tillämpas på många andra nanopartiklar, så länge de har hydroxylgrupper på sin yta. 10 15 10 Exempel på sådana nanopartiklar år nanopartiklar av maghemit (FegOg), metall jåmoxid (MFe2O4 vari M är Co eller Mn), järn (Fe), j äm-platina legering (FePt), eller silika.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.

Utöver nedanstående exempel kan den första lösningen vara någon lösning enligt tabell l.In addition to the examples below, the first solution may be any solution according to Table 1.

Tabell 1. Olika sammansåttningar av den första lösningen enligt uppfinningen.Table 1. Different compositions of the first solution according to the invention.

Nr: Sammansättning: I 2.5 g PEG 8000, 120 ml MeOH, 30 ml ammoniaklösning (25%) II 5.0 g PEG 400, 240 ml TREG, 60 ml ammoniaklösning (25%) III 10.0 g PEG 20000, 600 ml MeOH, 150 ml ammoniaklösning (25%) IV 10.0 g PEG 35000, 600 ml MeOH, 150 ml ammoniaklösning (25%) V 5.0 g PEG 2000, 600 ml MeOH, 150 ml ammoniaklösning (25%) VI 2.5 g PEG 8000, 600 ml MeOH, 150 ml ammoniaklösning (25%) VII 250 ml TREG, 2 ml ammoniaklösning (25%) VIII 350 ml TREG, 2 ml ammoniaklösning (25%) IX 150 ml TREG, l ml ammoniaklösning (25%) X 300 ml TREG, 2 ml ammoniaklösning (25%) XI 150 ml TREG, 0.25 ml etanolamin XII 10.0 g PEG 3400, 50 ml TREG, 120 ml MeOH, 30 ml ammoniaklösning (25%) XIII 2.5 g PEG 3400, 120 ml MeOH, 30 ml ammoniaklösning (25%) XIV 5.0 g PEG 3400, 120 ml MeOH, 30 ml ammoniaklösning (25%) XV 5.0 g PEG 3400, 240 ml MeOH, 10 ml ammoniaklösning (25%) XVI 5.0 g PEG 3400, 240 ml TREG, 30 ml ammoniaklösning (25%) Exempel 5 - Silanisering med tetraetoxysilan Nysyntetiserade magnetitnanopartiklar (ca 231 mg, framställda såsom beskrivits i Exempel 1) utsattes för, dvs sattes till, en lösning innehållande 2.5 g PEG 8000 i en blandning av 120 ml MeOH och 30 ml 25% ammoniaklösning. Blandningen ultraljudsbehandlades i 15 minuter i ett ultraljudsbad. Flaskorna placerades sedan i rumstemperatur och omrördes vid 1000 rpm med en överliggande omrörare. Silanisering av nanopartiklama startades genom droppvis tillsats under 5 minuter av 250 ul av TEOS (tetraetoxysilan, även kallad tetraetylortosilikat eller ortosilicictetraetyl- ester), upplöst i 3 ml MeOH. Silaniseringen fortsatte under kontinuerlig omröring under 3 h vid rumstemperatur. Efter silaniseringen, separerades nanopartiklarna direkt från lösningen med hjälp av en permanent neodymiummagnet. Lösningama dekanterades och nanopartiklama tvåttades med MeOH (100 mL >< 2), vatten (100 mL >< 4) och slutligen med MeOH igen (100 mL >< 4). Före tillsats av varje ny farsk tvättlösning separerades och hölls nanopartiklama med en magnet medan lösningama dekanterades. De belagda nanopartiklarna torkades i vakuum vid rumstemperatur över lO 15 20 25 30 35 11 natten. Beläggningsproceduren resulterade i en viktökning med 26%. Sammansättning: 57.0% Fe (enligt ICP-AES), 59.6% Fe (enligt kolorimetrisk j ämanalys), 3.2% Si (enligt ICP-AES), 0.7% C (enligt elementaranalys), 0.5% H (enligt elementaranalys), 0.3% N (enligt elementaranalys).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).

Nanopartiklarna orsakade 0.06% hemolys av utspätt blod efter 24-timmars inkubering och 5.92% respektive 21.15% hemolys av isolerade erytrothrocyter efter inkubationer under 1 h och 24 h.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. 2 B visar ett FT-IR-spektra av belagda nanopartiklar enligt detta exempel och FIG. 3 B visar en transmissionselektronmikroskopibild (TEM) av belagda nanopartiklar enligt detta exempel.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.

Exempel 6 - Silanisering med tetraetoxysilan Nysyntetiserade magnetitnanopartiklar (ca 231 mg, framställda såsom beskrivits i Exempel 1) sattes till 5 g PEG 400 i en blandning av 240 ml trietylenglykol och 60 ml 25% ammoniaklösning.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.

Blandningen sonikerades under 1 timme i ett ultraljudsbad. Flaskan placerades sedan i ett förvärmt (90 °C) vattenbad och omrördes vid 1000 rpm med en överliggande omrörare. Silanisering av nanopartiklama startades genom droppvis tillsats under 5 minuter av 250 ul av TEOS löst i 3 ml MeOH. Silaniseringen fortsatte under kontinuerlig omröming under 2 h vid 90 °C. Efler silaniseringen kyldes lösningen först och späddes sedan med etylacetat (200 ml) för utfällning av nanopartiklar. Det sistnämnda steget utfördes för att påskynda den efterföljande magnetiska separationen. Nanopartiklama tvättades med MeOH (100 mL >< 2), vatten (100 mL >< 4) och slutligen med MeOH igen (100 mL >< 4). Före tillsats av varje ny färsk tvättlösning, separerades och hölls nanopartiklama med en magnet medan lösningama dekanterades. De belagda nanopartiklama torkades i vakuum vid rumstemperatur över natten. Beläggningsförfarandet resulterade i en viktökning med 15%. Sammansättning av nanopartiklarna: 60.8% Fe (enligt ICP-AES), 64.4% Fe (enligt kolorimetrisk järnanalys), 2.5% Si (enligt ICP-AES), 0.7% C (enligt elementaranalys), 0.4% H (enligt elementaranalys), 0.3% N (enligt elementaranalys). Nanopartiklama orsakade ingen hemolys av utspätt blod efter 24-timmars inkubering, och 3.94% respektive 22.3% hemolys av isolerade erytrotrocyter efter inkubationer underl h och 24 h.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 visar ett FT-IR-spektra av belagda nanopartiklar enligt detta exempel och FIG. 3C visar en transmissionselektronmikroskopibild (TEM) av belagda nanopartiklar enligt detta exempel.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.

Exempel 7 - Silanisering med tetraetoxysilan och 3-(trimet0xysilyl)pr0pyl metakrylat Nysyntetiserade magnetitnanopartiklar (56 mg) sattes till en lösning innehållande 48 ml trietylenglykol, 1 g PEG 400 och 12 ml 25% ammoniaklösning. Blandningen sonikerades under 1 hi ett ultraljudsbad. En volym av 150 ul TEOS tillsattes. Lösningen hälldes i ett HP-500 Plus-mikro- vågsugnskärl (CEM Corp, Matthews, NC) och utsattes för 1200 W mikrovågsugnsbehandling med ett MARS 5 mikrovågsaccelererat reaktionssystem (CEM Corp, Matthews, NC) med en gradient över 1 minut upp till 90 °C och därefter under 15 minuter vid konstant temperatur (90 °C). Innehållet i kärlet lO 15 20 25 30 35 12 kyldes till ca. 60 °C. En volym av 150 ul 3-(trimetoxysilyl)propylmetakrylat tillsattes och lösningen blandades. Lösningen fick därefter åter genomgå 1200 W mikrovågsugnsbehandling med en gradient under l min upp till 60 °C och därefter under 15 minuter vid konstant temperatur (60 °C). Efter kylning tillsattes etylacetat (50 ml). Nanopartiklama separerades med hjälp av en permanent neodymiummagnet medan lösningen dekanterades. Nanopartiklama tvättades med MeOH (50 mL >< 3). Före tillsats av varje ny färsk MeOH-tvättlösning, separerades och hölls nanopartiklama med en magnet medan lösningarna dekanterades. De silaniserade nanopartiklama torkades i vakuum vid rumstemperatur över natten. Beläggningsproceduren resulterade typiskt i en viktökning om 20-60 mg (36-107%).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%).

Exempel 8 - Silanisering med N-trimetoxysilylpropyl-N,N,N-trimetylammoniumklorid Nysyntetiserade magnetitnanopartiklar (ca 463 mg, framställda såsom beskrivits i exempel 1) sattes till en lösning bestående av 300 ml trietylenglykol och 2 ml 25% ammoniaklösning.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.

Blandningen ultraljudsbehandlades i 10 minuter i ett ultraljudsbad. Flaskan placerades sedan i ett upphettat (90 °C) vattenbad och lösningen omrördes vid 900 rpm med en överliggande omrörare.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.

Silanisering av nanopartiklama startades genom tillsats av 15 ml N-trimetoxysilylpropyl-N,N,N- trimetylammoniumklorid (50% i metanol). Silaniseringen fortsatte under kontinuerlig omröming under 2 h vid 90 °C. Efter silaniseringen kyldes lösningen och etylacetat (1 .2 L) tillsattes för att fälla nanopartiklama. Nanopartiklama separerades från lösningen med hjälp av en permanent neodymiummagnet. Lösningen dekanterades och nanopartiklama tvättades med MeOH (200 mL >< 2). De belagda nanopartiklarna torkades i vakuum vid rumstemperatur över natten.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.

Exempel 9 - Silanisering med [hydr0xy(p0lyetylen0x0)pr0pyl]-tríetoxysilan (8-12 EO) Nysyntetiserade magnetitnanopartiklar (ca 231 mg, framställda såsom beskrivits i exempel 1) sattes till en lösning bestående av 150 ml trietylenglykol och 1 ml 25% ammoniaklösning.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.

Blandningen ultraljudsbehandlades i 30 minuter i ett ultraljudsbad. Flaskan placerades sedan i ett upphettat (95 °C) vattenbad och omrördes vid 900 rpm med en överliggande omrörare. Silanisering av nanopartiklama startades genom tillsats av 1 ml av en 50% lösning av [hydroxy(polyetylenoxo) propyl]-trietoxysilan (8-12 EO) i etanol. Silaniseringen fortsatte under kontinuerlig omröring under 2 h vid 95 °C. Efter silanisering kyldes lösningen och etylacetat (350 ml) tillsattes för att fälla nano- partiklama. Nanopartiklama separerades från lösningen med hjälp av en permanent neodymium- magnet. Lösningen dekanterades och nanopartiklama tvättades med etylacetat (100 mL >< 2) och MeOH (100 mL >< 2). De belagda nanopartiklama torkades i vakuum vid rumstemperatur över natten. 10 15 20 25 30 35 13 Exempel 10 - Silanisering med tetrametoxysilan och [hydroxy(polyetylenoxo)propyl] trietoxysilan (8-12 EO) Nysyntetiserade magnetitnanopartiklar (ca 231 mg, framställda såsom beskrivits i Exempel 1) sattes till en lösning bestående av 150 ml trietylenglykol och 1 ml 25% ammoniaklösning.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.

Blandningen skakades för att dispergera nanopartiklama. Flaskan placerades i ett 95 °C vattenbad och lösningen omrördes vid 900 varv per minut. Silanisering startades genom tillsats av en volym av 250 ul tetrametoxysilan. Omröringen fortsattes vid 900 varv per minut. Efter 30 minuter tillsattes en volym av 1 ml av en 50% lösning av [hydroxy(polyetylenoxo)propyl]trietoxysilan (8-12 EO) i etanol.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.

Silanisering fick fortgå under omröring under ytterligare 1.5 h vid 95 °C. Lösningen kyldes och etylacetat (ca 350 ml) tillsattes. Nanopartiklama separerades från lösningen med hjälp av en permanent neodymiummagnet. Lösningen dekanterades och nanopartiklama tvättades med etylacetat (100 mL >< 2) och MeOH (100 mL >< 2). De belagda nanopartiklarna torkades i vakuum vid rumstemperatur över natten. Beläggningen resulterade i en viktökning av 15 mg (7%).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%).

Exempel 11 - Silanisering med fluoresceín isothiocyanate-derivatiserad silan och tetraetoxysilan Fluoresceinisotiocyanat (FITC)-derivatiserad silan syntetiserades genom att reagera en mängd av 50 mg 5-fluoresceinisotiocyanat-isomer I med 6 ml 3-aminopropyltrietoxysilan i 5 ml etanol under 24 timmar. Nysyntetiserade magnetitnanopartiklar (ca 231 mg, framställda såsom beskrivits i Exempel 1) sattes till 360 ml vatten, 15 g PEG 2000 och 90 ml 25% ammoniaklösning.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.

Blandningen sonikerades under 1 h i ett ultraljudsbad. Flaskan placerades sedan i ett förvärrnt (90 °C) vattenbad och ornrördes vid 1000 rpm med en överliggande omrörare. Silanisering av nanopartiklama startades genom tillsats av 1.0 ml FITC-derivatiserad silanlösning framställd enligt ovan. Efter 15 min tillsattes en mängd av 2.25 ml TEOS. Silaniseringen fortsatte under kontinuerlig omröring vid 90 °C under ytterligare 45 minuter och därefter vid rumstemperatur under 13 timmar. Efter silaniseringen separerades de fluorescerande nanopartiklama med en permanent neodymiummagnet och tvättades med MeOH (100 mL >< 2), vatten (100 mL >< 4), och slutligen med MeOH igen ( 100 mL >< 4). Före tillsats av varje ny färsk tvättlösning, separerades och hölls nanopartiklama med magneten medan lösningen dekanterades. De fluorescerande nanopartiklama torkades i vakuum vid rumstemperatur över natten. Beläggningsproceduren resulterade i en viktökning med 72%.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%.

En fördel med dessa nanopartiklar är att de kan användas som markörer och kontrastmedel eftersom de är fluorescerande.An advantage of these nanoparticles is that they can be used as markers and contrast agents because they are ores uorescent.

Sålunda, i en utföringsforrn, tillhandahålls ett kontrastmedel, omfattande kompositionen enligt utföringsformer av uppfinningen. lO 15 20 25 30 35 14 Exempel 12 - Silanisering med tetraetoxysílan Nysyntetiserade magnetitnanopartiklar (ca 232 mg, framställda såsom beskrivits i Exempel 1) sattes till en lösning bestående av 120 ml vatten, 5 g PEG 2000 och 30 ml 25% ammoniaklösning.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.

Blandningen ultraljudsbehandlades i 15 minuter i ett ultraljudsbad och omrördes därefter vid 900 rpm med en överliggande omrörare. Silanisering av nanopartiklama startades genom tillsats av 0.25 ml TEOS. Silaniseringen fortsatte under kontinuerlig omröring under 40 h vid rumstemperatur. Efter silaniseringen separerades nanopartiklama från lösningen med hjälp av en permanent neodymium- magnet. Lösningen dekanterades av och nanopartiklama tvättades med vatten (100 mL >< 2) och MeOH (100 mL >< 2). De belagda nanopartiklama torkades i vakuum vid rumstemperatur över natten.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.

Exempel 13 - Silanisering med tetraetoxysilan En mängd av 50 mg kommersiella jäm(ll, Ill)-oxidnanopartiklar > 50 nm (Sigma-Aldrich katalognummer 637.106) sattes till en lösning bestående av 48 ml trietylenglykol, 12 ml 25% ammoniaklösning och 1 g PEG 400. Blandningen sonikerades under 1 h i ett ultraljudsbad. En volym av 200 ul av TEOS tillsattes. Lösningen hålldes i ett HP-500 Plus-mikrovågsugnskärl (CEM Corp, Matthews, NC) och utsattes för 1200 W mikrovågsugnsbehandling med ett MARS 5 mikrovågs- accelererat reaktionssystem (CEM Corp, Matthews, NC) med en gradient över 1 minut upp till 90 °C och därefter under 15 minuter vid konstant temperatur (90 °C). Efter kylning tillsattes etylacetat och nanopartiklarna separerades från lösningen med hjälp av en permanent neodymiummagnet.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.

Lösningen dekanterades och nanopartiklama tvättades med MeOH (100 mL >< 3). De silaniserade nanopartiklarna torkades i vakuum vid rumstemperatur över natten. Förfarandet resulterade i en typisk viktökning om 25 mg (50%).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%).

Exempel 14 - Silanisering med tetraetoxysílan och 3-aminopropyltrietoxysilan Nysyntetiserade magnetitnanopartiklar (ca 231 mg, framställda såsom beskrivits i Exempel 1) sattes till en lösning bestående av 600 ml MeOH, 2.5 g PEG 8000, och 150 ml 25% ammoniaklösning. Blandningen ultraljudsbehandlades i 15 minuter i ett ultraljudsbad och omrördes därefter vid 900 rpm i rumstemperatur. Silanisering av nanopartiklama startades genom tillsats av 0.25 ml TEOS. Efter 3.5 h tillsattes 0.25 ml 3-aminopropyltrietoxysilan. Silaniseringen fick pågå under ytterligare 1 h. Nanopartiklama separerades från lösningen med hjälp av en permanent neodymiummagnet. Lösningen dekanterades och nanopartiklama tvättades med MeOH (100 mL >< 2), vatten (100 mL >< 2) och MeOH (100 mL >< 2). De silaniserade nanopartiklama torkades i vakuum vid rumstemperatur över natten. Proceduren resulterade i en viktökning av 55 mg (24%). 10 15 20 25 30 35 15 Exempel 15 - Silanisering med tetraetoxysílan Nysyntetiserade magnetitnanopartiklar (ca 232 mg, framställda såsom beskrivits i Exempel 1) sattes till en lösning bestående av 600 ml MeOH, 10 g PEG 20000, och 150 ml 25% ammoniak- lösning. Blandningen ultraljudsbehandlades i 30 minuter i ett ultraljudsbad och omrördes sedan vid 1000 rpm i rumstemperatur. Silanisering av nanopartiklama startades genom tillsats av 0.25 ml TEOS. Efter 3 h separerades nanopartiklarna från lösningen med hjälp av en permanent neodymium- magnet. Lösningen dekanterades av och nanopartiklama tvättades med MeOH (200 mL >< 2), vatten (300 mL >< 3) och MeOH (100 mL >< 2). De silaniserade nanopartiklama torkades i vakuum vid rumstemperatur över natten. Belåggningsproceduren resulterade i en viktökning av 47 mg (20%).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%).

Exempel 16 - Silanisering med tetraetoxysílan Nysyntetiserade magnetitnanopartiklar (ca 231 mg, framställda såsom beskrivits i Exempel 1) sattes till en lösning bestående av 600 ml MeOH, 10 g PEG 35000, och 150 ml 25% ammoniak- lösning. Blandningen ultraljudsbehandlades i 30 minuter i ett ultraljudsbad och omrördes sedan vid 1000 rpm i rumstemperatur. Silanisering av nanopartiklama startades genom tillsats av 0.25 ml av TEOS. Efter 18 h separerades nanopartiklama från lösningen med hjälp av en permanent neodymiummagnet. Lösningen dekanterades och nanopartiklama tvåttades med MeOH (200 ml), vatten (5 00 ml), och MeOH (200 ml). De silaniserade nanopartiklama torkades i vakuum vid rumstemperatur över natten. Belåggningsproceduren resulterade i en viktökning av 46 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%).

Exempel 17 - Silanisering med tetraetoxysílan Nysyntetiserade magnetitnanopartiklar (ca 231 mg, framställda såsom beskrivits i Exempel 1) sattes till en lösning bestående av 600 ml MeOH, 5 g PEG 2000 och 150 ml 25% ammoniak- lösning. Blandningen ultraljudsbehandlades i 30 minuter i ett ultraljudsbad och omrördes sedan vid 1000 rpm i rumstemperatur. Silanisering av nanopartiklama startades genom tillsats av 0.25 ml av TEOS. Efter 1 h separerades nanopartiklama från lösningen med hjälp av en permanent neodymium- magnet. Lösningen dekanterades av och nanopartiklama tvättades med MeOH (100 ml), vatten (300 mL >< 4) och MeOH (200 ml). De silaniserade nanopartiklama torkades i vakuum vid rums- temperatur över natten. Beläggningen resulterade i en viktökning av 35 mg (15%).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%).

Exempel 18 - Silanisering med [hydr0xy(p0lyetylenox0)propyl]trietoxysilan (8-12 EO) och N-trimetoxysilylpr0pyl-N,N,N-trimetylammoniumklorid Nysyntetiserade magnetitnanopartiklar (ca 231 mg, framställda såsom beskrivits i exempel 1) sattes till en lösning bestående av 150 ml trietylenglykol. En volym av 0.25 ml etanolamin tillsattes. Blandningen ultraljudsbehandlades i 10 minuter i ett ultraljudsbad. Flaskan placerades sedan i ett upphettat (95 °C) vattenbad och lösningen omrördes vid 900 rpm med en överliggande 10 15 20 25 30 35 16 omrörare. Silanisering av nanopartiklama startades genom tillsats av 1 ml [hydroxy(polyetyleneoxo) propyl]trietoxysilan (8-12 EO) (5 0% i etanol) och 4 ml N-trimetoxysilylpropyl-N, N, N-trimetyl- ammoniumklorid (50 % i metanol). Silaniseringen fortsatte under kontinuerlig omröring under 2 h vid 95 °C. Lösningen kyldes sedan och etylacetat (300 ml) tillsattes för att falla nanopartiklama.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.

Nanopartiklarna separerades från lösningen med hjälp av en permanent neodymiummagnet.The nanoparticles were separated from the solution by means of a permanent neodymium magnet.

Lösningen dekanterades och nanopartiklama tvättades med etylacetat (200 mL >< 2) och MeOH (200 mL >< 2). De belagda nanopartiklama torkades i vakuum vid rumstemperatur över natten.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.

Exempel 19 - Silanisering med tetraetoxysilan Nysyntetiserade magnetitnanopartiklar (ca 231 mg, framställda såsom beskrivits i Exempel 1) sattes till en lösning bestående av 120 ml MeOH, 10 g PEG 3400, och 30 ml 25% ammoniak- lösning. Blandningen ultraljudsbehandlades i 20 minuter i ett ultraljudsbad. Lösningen omrördes vid 1000 rpm med en överliggande omrörare. Silanisering av nanopartiklama startades genom tillsats av 1 ml TEOS. Silaniseringen fortsatte under kontinuerlig omröring under 3 h vid rumstemperatur.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.

Nanopartiklarna separerades från lösningen med hjälp av en permanent neodymiummagnet.The nanoparticles were separated from the solution by means of a permanent neodymium magnet.

Lösningen dekanterades och nanopartiklama tvättades med MeOH (200 mL >< 5), vatten (200 mL >< 5) och MeOH (200 mL >< 5). Förfarandet resulterade i en viktökning av 253 mg ( 109%).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%).

Exempel 20 - Silanisering med tetraetoxysílan Nysyntetiserade magnetitnanopartiklar (ca 232 mg, framställda såsom beskrivits i Exempel 1) sattes till en lösning bestående av 120 ml MeOH, 2.5 g PEG 3400 och 30 ml 25% ammoniak- lösning. Blandningen ultraljudsbehandlades i 20 minuter i ett ultraljudsbad. Lösningen omrördes vid 1000 rpm med en överliggande omrörare. Silanisering av nanopartiklar startades genom tillsats av 0.25 ml TEOS. Silaniseringen fortsatte under kontinuerlig omröring under 3 h vid rumstemperatur.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.

Nanopartiklarna separerades från lösningen genom hjälp av en permanent neodymiummagnet.The nanoparticles were separated from the solution by means of a permanent neodymium magnet.

Lösningen dekanterades av och nanopartiklama tvättades med MeOH (200 mL X 5), vatten (200 mL >< 5) och MeOH (200 mL >< 5). Förfarandet resulterade i en viktökning av 53 mg (23%).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%).

Exempel 21 - Silanisering med tetraetoxysílan Nysyntetiserade magnetitnanopartiklar (ca 232 mg, framställda såsom beskrivits i Exempel 4) sattes till en lösning bestående av 120 ml MeOH, 5 g PEG 400 och 30 ml 25% ammoniaklösning.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.

Blandningen sonikerades under 1 h i ett ultraljudsbad. Lösningen omrördes vid 900 rpm med en överliggande ornrörare. Silanisering av nanopartiklama startades genom tillsats av 1 ml TEOS.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.

Silaniseringen fortsatte under kontinuerlig omröring under 1 h vid 95 °C. Nanopartiklama separerades från lösningen med hjälp av en permanent neodymiummagnet. Lösningen dekanterades 10 15 20 25 30 35 17 av och nanopartiklarna tvättades med MeOH (200 mL >< 5), Vatten (200 mL >< 5) och MeOH (200 mL >< 5). Förfarandet resulterade i en viktökning om 30 mg (13%).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%).

Exempel 22 - Silanisering med tetraetoxysilan och 3-(trimet0xysilyl)propylmetakrylat Nysyntetiserade magnetitnanopartiklar (ca 278 mg) framställda såsom beskrivits i Exempel 3 sattes till en lösning innehållande 290 ml trietylenglykol, 6 g PEG 400, och 70 ml 25% ammoniak- lösning. Blandningen sonikerades under 1 h i ett ultraljudsbad. En volym av 600 ul TEOS tillsattes.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.

Lösningen hälldes i sex HP-500 Plus-mikrovågskärl (CEM Corp, Matthews, NC) och utsattes för 1200 W mikrovågsugnsbehandling med ett MARS 5 mikrovågsccelererat reaktionssystem (CEM Corp, Matthews, NC) med en gradient över 1 minut upp till 90 °C och därefter under 15 minuter vid konstant temperatur (90 °C). Innehållet i kärlen kyldes till ca. 55 °C och slogs samman. En volym av 600 ul 3-(trimetoxisilyl)propylmetakrylat tillsattes, lösningen blandades och fördelades på sex mikrovågsugnskärl. Lösningama utsattes återigen för 1200 W mikrovågsugnsbehandling med en gradient under 1 min upp till 50 °C och därefter under 30 minuter vid konstant temperatur (50 °C).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).

Efter kylning tillsattes etylacetat (400 ml). Nanopartiklama separerades och hölls med hjälp av en pennanent neodymiummagnet medan lösningen dekanterades. Nanopartiklama tvättades med MeOH (100 mL >< 3). Före varje tillsats av ny färsk MeOH-tvättlösning, separerades nanopartiklama och hölls med en magnet medan lösningama dekanterades. De silaniserade nanopartiklama torkades i vakuum vid rumstemperatur över natten. Beläggningsproceduren resulterade i en typisk viktökning om 171 mg (62%).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%).

Exempel 23 - Silanisering med tetraetoxysilan Nysyntetiserade magnetitnanopartiklar (56 mg), framställda såsom i Exempel 3, sattes till en lösning bestående av 57 ml trietylenglykol och 3 ml 25% ammoniaklösning. Blandningen sonikerades under 1 hi ett ultraljudsbad. En volym av 150 ul TEOS tillsattes. Lösningen hälldes i ett HP-500 Plus-mikrovågsugnskärl (CEM Corp, Matthews, NC) och utsattes för 1200 W mikrovågsugns- behandling med ett MARS 5 mikrovågsaccelererat reaktionssystem (CEM Corp, Matthews, NC) med en gradient över 1 minut upp till 90 °C och därefter under 15 minuter vid konstant temperatur (90 °C). Efter kylning tillsattes etylacetat och nanopartiklama separerades från lösningen med hjälp av en permanent neodymiummagnet. Lösningen dekanterades och nanopartiklama tvättades med MeOH (50 mL >< 3). De silaniserade nanopartiklama torkades i vakuum vid rumstemperatur över natten.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.

Proceduren resulterade i en viktökning om 31 mg (56%).The procedure resulted in a weight gain of 31 mg (56%).

Exempel 24 - Silanisering med tetraetoxysilan Nysyntetiserade magnetitnanopartiklar (56 mg), framställda såsom i Exempel 3, sattes till en lösning bestående av 54 ml trietylenglykol och 6 ml 25% ammoniaklösning. Blandningen sonikerades 10 15 20 25 30 35 18 under 1 hi ett ultraljudsbad. En volym av 150 ul TEOS tillsattes. Lösningen hålldes i ett HP-500 Plus-mikrovågsugnskärl (CEM Corp, Matthews, NC) och utsattes för 1200 W mikrovågsugns- behandling med ett MARS 5 mikrovågsaccelererat reaktionssystem (CEM Corp, Matthews, NC) med en gradient över 1 minut upp till 90 °C och därefter under 15 minuter vid konstant temperatur (90 °C). Efter kylning tillsattes etylacetat och nanopartiklama separerades från lösningen med hj ålp av en permanent neodymiummagnet. Lösningen dekanterades av och nanopartiklama tvåttades med MeOH (50 mL >< 3). De silaniserade nanopartiklama torkades i vakuum vid rumstemperatur över natten.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.

Förfarandet resulterade i en viktökning om 33 mg (5 9%).The procedure resulted in a weight gain of 33 mg (59%).

Exempel 25 - Silanisering med tetraetoxysilan Nysyntetiserade magnetitnanopartiklar (56 mg), framställda såsom i Exempel 3, sattes till en lösning bestående av 57 ml trietylenglykol och 3 ml 40% metylaminlösning. Blandningen sonikerades under 1 h i ett ultraljudsbad. En volym av 150 ul TEOS tillsattes. Lösningen hålldes i ett HP-5 00 Plus-mikrovågsugnskårl (CEM Corp, Matthews, NC) och utsattes för 1200 W mikrovågs- ugnsbehandling med ett MARS 5 mikrovågsaccelererat reaktionssystem (CEM Corp, Matthews, NC) med en gradient över 1 minut upp till 90 °C och därefter under 15 minuter vid konstant temperatur (90 °C). Efter kylning tillsattes etylacetat och nanopartiklama separerades från lösningen med hj ålp av en permanent neodymiummagnet. Lösningen dekanterades av och nanopartiklama tvåttades med MeOH (50 mL >< 3). De silaniserade nanopartiklama torkades i vakuum vid rumstemperatur över natten. Förfarandet resulterade i en viktökning om 34 mg (60%).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%).

Exempel 26 - Silanisering med tetraetoxysilan Nysyntetiserade magnetitnanopartiklar (56 mg), framställda såsom i Exempel 3, sattes till en lösning bestående av 54 ml trietylenglykol och 6 ml 40% metylaminlösning. Blandningen sonikerades under 1 h i ett ultraljudsbad. En volym av 150 ul TEOS tillsattes. Lösningen hålldes i ett HP-500 Plus-mikrovågsugnskärl (CEM Corp, Matthews, NC) och utsattes för 1200 W mikrovågsugnsbehandling med ett MARS 5 mikrovågsaccelererat reaktionssystem (CEM Corp, Matthews, NC) med en gradient över 1 minut upp till 90 °C och därefter under 15 minuter vid konstant temperatur (90 °C). Efter kylning tillsattes etylacetat och nanopartiklama separerades från lösningen med hjälp av en permanent neodymiummagnet. Lösningen dekanterades av och nanopartiklama tvåttades med MeOH (50 mL >< 3). De silaniserade nanopartiklama torkades i vakuum vid rumstemperatur över natten. Proceduren resulterade i en viktökning om 45 mg (81%).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%).

Exempel 27 - Silanisering med tetraetoxylsilan och joderad silan Joderad silan syntetiserades genom att reagera 1.266 g (11 mmol) N-hydroxisuccinimid, löst i 50 ml CHzClg, med 4.998 g (10 mmol) 2,3,5-trijodbensoesyra, upplösti 50 ml CH2CI2, och 2.108 g lO 15 20 25 30 35 19 (11 mmol) EDC (vattenlöslig karbodiimid), upplöst i 50 ml CHgClg. Reaktionen fick fortgå i 2 dagar.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.

Lösningen extraherades med vatten tre gånger, med mättad natriumkloridlösning tre gånger, och slutligen med vatten en gång. Lösningen torkades över MgSO4. Lösningen indunstades därefter och den fasta produkten torkades i vakuum över natten. 4.363 g (73% utbyte) av succinimidyl 2,3,5- trijodbensoat erhölls. 0.884 g (4 mmol) 3-aminopropyltrietoxysilan sattes till 2.388 g (4 mmol) succinimidyl-2,3,5-trijodbensoat löst i 8 ml DMF . Reaktionen fick pågå under 2 dagar för att erhålla den j oderade silanen.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.

Nysyntetiserade magnetitnanopartiklar (ca 232 mg, framställda såsom beskrivits i Exempel 4) sattes till en lösning bestående av 120 ml MeOH, 5 g PEG 400 och 30 ml 25% ammoniaklösning.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.

Blandningen sonikerades under 1 h i ultraljudsbad. Lösningen placerades i ett upphettat (95 °C) vattenbad och omrördes vid 900 rpm med en överliggande omrörare. Silanisering av nanopartiklama startades genom tillsats av 1 ml TEOS. Silaniseringen fortsatte under kontinuerlig omröring under 30 min vid 95 °C. Den joderade silanreaktionsblandningen, framställd såsom beskrivits ovan, tillsattes därefter. Silaniseringen utfördes under ytterligare 3 h. Nanopartiklama separerades från lösningen med hjälp av en permanent neodymiummagnet. Lösningen dekanterades av och nanopartiklama tvättades med MeOH (200 mL >< 5), vatten (200 mL >< 5) och MeOH (200 mL >< 5).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).

En fördel med dessa nanopartiklar är att de kan användas som röntgenkontrastmedel eller markörer, eftersom de är röntgentäta.An advantage of these nanoparticles is that they can be used as X-ray contrast agents or markers, as they are X-ray dense.

Sålunda, i en utföringsforrn, tillhandahålls ett kontrastmedel, omfattande kompositionen enligt utföringsforrner av uppfinningen.Thus, in one embodiment, a contrast agent is provided, comprising the composition according to embodiments of the invention.

Exempel 28 till 30 nedan avser konjugering av enzym eller peptid till silaniserade nanopartiklar enligt utföringsforrner av uppfinningen.Examples 28 to 30 below relate to conjugation of enzyme or peptide to silanized nanoparticles according to embodiments of the invention.

Exempel 28 - Immobilisering av rekombinant human vävnadsplasmínonogenaktivator (tPA) genom aktivering av silika-belagda nanopartiklar med NHSS-EDC Fmoc-Gly-OH (0.595 g, 2 mmol), upplöst i DMF (1 ml), sattes till 200 mg silaniserade magnetitnanopartiklar, syntetiserade såsom beskrivits i Exempel 5. Kopplingen initierades genom tillsats av DIPCDI (0.252 g, 2 mmol) i DMF (1 ml) och DMAP (25 mg, 0.2 mmol) i DMF (1 ml).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).

Reaktionen utfördes på en rotator under 3 dagar vid rumstemperatur. Nanopartiklama separerades från lösningen med en perrnanentmagnet och tvättades med DMF (5 ml >< 10). Magnetisk separering utfördes mellan tvättama. Fmoc-gruppema avlägsnades genom behandling med 5 ml piperidin-DMF (1 :4) under 5 min. Efter avlägsnande av den första klyvningslösningen tillsattes 5 ml färsk piperidin- DMF (1 :4) och blandningen inkuberades under ytterligare 15 minuter. Nanopartiklama tvättades med DMF (5 ml >< 10) och CHgClg (5 ml >< 10). Kaisers kvalitativa ninhydrintest detekterade fria aminogrupper i detta läge. Succinylering av de fria aminogruppema utfördes genom tillsats av 10 15 20 25 30 35 20 bärnstensyraanhydrid (0.4 g, 4 mmol) i 6 ml CHgClg-pyridin (1 : 1). Blandningen inkuberades i rumstemperatur på en rotator under 30 min. Efter magnetisk separation och avlägsnande av lösningen, tvättades nanopartiklama med CHQCI; (5 ml >< 5), DMF (5 ml >< 5), och MeOH (5 ml >< 5). Kaiser-testet var negativt, vilket indikerar att succinyleringen var fullständig. Nanopartiklama torkades i vakuum vid rumstemperatur över natten. Elementaranalys: 3% C, 0.6% H, 0.6% N. En mängd av 125 mg av de torkade succinylerade nanopartiklarna suspenderades i 1 ml vatten.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.

Nanopartiklarna aktiverades genom tillsats av NHSS (87 mg, 0.4 mmol) i vatten (1 ml) och EDC (77 mg, 0.4 mmol) i vatten (1 ml). Förestringen fortsatte i rumstemperatur på en rotator under 2 h.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.

Nanopartiklarna separerades med en magnet och reagenslösningen avlägsnades. Nanopartiklarna tvättades med vatten (5 ml >< 10) och suspenderades slutligen i 2.08 ml vatten. Enzym- immobiliseringen genomfördes genom tillsats av en lösning av rekombinant human vävnads- plasminogenaktivator, tPA (saluford av Boehringer Ingelheim under varumärket Actilyse; 12.5 mg upplöst i 6.25 ml vatten) till nanopartikellösningen. Kopplingen fortsatte på en orbital skakapparat (200 rpm) under 4 h vid 4 °C. De konjugerade tPA-nanopartiklama separerades ut med en magnet, lösningen avlägsnades och tvättning utfördes med vatten (5 ml >< 5), fosfat-buffrad saltlösning (PBS), pH 7.4 (5 ml >< 3), och vatten (5 ml >< 3). Proteinkoncentrationen hos tvättlösningama bestämdes enligt Bradford med användning av bovint serumalbumin som referens. Mängden immobiliserat tPA beräknades genom att subtrahera mängden tPA i tvättlösningama från mängden tPA som tillsattes till nanopartiklama vid immobiliseringens start. Immobiliseringsutbytet beräknades som 100% * (mängd immobiliserat tPA)/(mängd satsat tPA). Immobiliseringsutbytet var 63%. TPA-laddningen beräknades som (massan av immobiliserat tPA)/ (massan av nanopartiklama). TPA-laddningen var 63 ug tPA/mg nanopartiklar. Enzymaktiviteten av fritt och immobiliserat enzym bestämdes genom att följa bildningen av p-nitroanilin (pNA) spektrofotometriskt vid 405 nm under hydrolysen av substratet H-D-Ile-Pro-Arg-pNA. Analysen utfördes genom att blanda 0.25 ml av en lösning innehållande antingen fritt tPA eller tPA-nanopartikelskonjugat, 0.25 ml 100 mM Tris-HCl pH 8.4 innehållande 100 mM NaCl och 0.25 ml av en l mM lösning av substratet i vatten. Den specifika enzymaktiviteten var 0.86 U/mg tPA. Enzymaktivitetsutbytet beräknades som 100% * (total aktivitet av immobiliserat tPA)/ (total aktivitet av satsat tPA). Enzymaktivitetsutbytet var 45%. Ett reaktionsschema tillhandahålls i FIG. 4A.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.

Exempel 29 - Immobilisering av tPA genom aktivering av belagda nanopartiklar med tresylklorid 280 mg silaniserade nanopartiklar, syntetiserades såsom beskrivits i Exempel 6, tvättades först med torr aceton (5 ml >< 2) och suspenderades därefter i 6.7 ml torr aceton och 0.78 ml torr pyridin. Aktivering med tresylklorid (0.3 ml) initierades genom droppvis tillsats till nanopartikels- lösningen under skakning. Reaktionen utfördes på en orbitalskak (1 000 rpm) under 2 h vid 4 °C. 10 15 20 25 30 35 21 Nanopartiklarna kvarhölls därefter med en permanent neodymiummagnet och lösningen avlägsnades.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.

Nanopartiklama tvättades med aceton (5 ml >< 3), aceton-vatten (2:1) (5 ml >< 2), aceton-vatten (111) (5 ml >< 2), aceton-vatten (132) (5 ml >< 2), aceton-vatten (1 :4) (5 ml >< 2) och vatten (10 ml >< 3).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).

Nanopartiklama suspenderades sedan under sonikering i 10 ml vatten och sattes droppvis till dialyserat tPA (38 mg) i 80 ml 0.2 M natriumfosfatbuffert pH 8. Kopplingen av tPA till de tresylkloridaktiverade nanopartiklama utfördes vid 4 °C under 33 timmar på en orbital skakapparat (200 rpm). Nanopartiklama separerades därefter med en permanent magnet och tvättades med 0.2 M Tris-HCl pH 8 (50 ml). Blockering av återstående tresylgrupper utfördes med 0.2 M Tris-HCl, pH 8 under 23 h vid 4 °C på en orbital skakapparat (200 rpm). TPA-nanopartikelskonjugaten separerades med en magnet och tvättades med vatten (10 ml >< 2), 50 mM natriumfosfatbuffert pH 7 (10 ml), 25 mM natriumfosfatbuffert pH 7 (20 ml), och 12.5 mM natriumfosfat buffert pH 7 (20 ml >< 2).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).

Bestämning av proteinkoncentration, enzymaktivitet och beräkningar av immobiliseringsparametrar utfördes såsom beskrivits i Exempel 28. Enzymladdningen var 71 ug tPA/mg nanopartiklar.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.

Immobiliseringsutbytet var 52%. Den specifika enzymaktiviteten var 0,82 U/mg tPA.The immobilization yield was 52%. The specific enzyme activity was 0.82 U / mg tPA.

Enzymaktivitetsutbytet var 41%. TPA-nanopartikels-konjugaten orsakade inte någon hemolys av utspätt blod efter 24-timmars inkubering och 0.15% respektive 1.03% hemolys av isolerade erytrotrocyter efter inkubationer under 1 h och 24 h.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 visar FT-IR-spektra av belagda nanopartiklar enligt detta exempel och FIG. 3D visar en transmissionselektronmikroskopibild (TEM) av belagda nanopartiklar enligt detta exempel.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.

Ett reaktionsschema tillhandahålls i FIG. 4B.A reaction scheme is provided in FIG. 4B.

Exempel 30 - Koppling av en NGR-innehållande peptid till belagda nanopartiklar via en PEG-spacer Fmoc-Gly-OH (0.298 g, 1 mmol), upplöst i DMF (0.5 ml), sattes till 100 mg silaniserade magnetitnanopartiklar, syntetiserade såsom beskrivits i Exempel 5. Kopplingen initierades genom tillsats av DIPCDI (0.126 g, 1 mmol) i DMF (0.5 ml) och DMAP (13 mg, 0.1 mmol) i DMF (0.5 ml).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).

Reaktionen utfördes på en rotator under 3 dagar vid rumstemperatur. Nanopartiklama separerades från lösningen med en perrnanentmagnet och tvättades med DMF (3 ml >< 10). Magnetisk separering utfördes mellan tvättningama. Fmoc-gruppema avlägsnades genom behandling med 3 ml piperidin- DMF (1 :4) under 5 min. Efter avlägsnande av den första klyvningslösningen tillsattes 3 ml färsk piperidin-DMF (1 :4) och blandningen inkuberades under ytterligare 15 minuter. Nanopartiklama tvättades med DMF (3 ml >< 10) och CHQCI; (3 ml X 10). Kaisers kvalitativa ninhydrintest detekterade fria aminogrupper i detta läge. Fmoc-NH-(PEG)2-COOH (48 mg, 0.086 mmol) upplösti 0.4 ml DMF, DIPCDI (11 mg, 0.086 mmol) i 0.2 ml DMF och HOBt (12 mg, 0.086 mmol) i 0.2 ml DMF sattes till nanopartiklarna. Kopplingen utfördes på en rotator under 24 timmar. Efter kopplingen var Kaisers kvalitativa ninhydrintest negativt. Fmoc-grupperna avlägsnades genom behandling med 3 lO 15 20 25 30 35 22 ml piperidin-DMF (124) under 5 min. Efter avlägsnande av den första klyvningslösningen tillsattes 3 ml färsk piperidin-DMF (114) och blandningen inkuberades under ytterligare 15 minuter.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.

Nanopartiklarna tvättades med DMF (3 ml >< 10) och CHgClg (3 ml >< 10). Kaisers kvalitativa ninhydrintest detekterade fria aminogrupper vid denna punkt. Ac-Gly-Asn(Trt)-Gly-Arg(Pbf)-Gly- Ahx-Gly-OH (10.8 mg, 9.28 umol), DIPCDI (5 mg, 40 umol) och HOAt (5.4 mg, 40 umol) löstes i 0.2 ml DMF och sattes till nanopartiklama. Reaktionen utfördes under 3 dagar. Efter kopplingen var Kaisers kvalitativa ninhydrintest negativt. Nanopartiklama torkades i vakuum över natten. Skydds- gruppema avlägsnades genom behandling med 0.5 ml TFA-CHgClg-vatten (90:5:5) under 2 h.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.

Nanopartiklarna tvättades med 1 ml vardera av CHgClg, DMF, CHgClg, MeOH, vatten, och MeOH.The nanoparticles were washed with 1 mL each of CH 2 Cl 2, DMF, CH 2 Cl 2, MeOH, water, and MeOH.

Nanopartiklarna torkades i vakuum över natten.The nanoparticles were dried in vacuo overnight.

Karakterisering av belagda nanopartiklar Ett antal metoder användes for att studera de belagda nanopartiklama framställda enligt utföringsformer av uppfinningen.Characterization of Coated Nanoparticles A number of methods have been used to study the coated nanoparticles prepared according to embodiments of the invention.

T ransmíssíonselektronmíkroskopí Storlek och morfologi hos nanopartiklama studerades med transmissionselektronmikroskopi (TEM) med användning av en J EOL JEM-1230 (Tokyo, Japan) utrustad med en Multiscan-Gatan kamera modell 791 (Pleasanton, CA, USA). Provema placerades på Pioloform (polyvinylbutyral)- filmer och bilder togs vid 80 kV pålagd spänning. FIG. 3 visar transmissionselektronmikroskopi (TEM) av (A) nakna magnetitnanopartiklar från Exempel 1, (B) ytbelagda magnetitnanopartiklar från Exempel 5, (C) ytbelagda magnetitnanopartiklar från Exempel 6, och (D) tPA-nanopartikels-konjugat från Exempel 29.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.

Dynamisk ljussprídníng Den hydrodynamiska partikelstorleksfördelningen bestämdes genom dynamisk ljusspridning (DLS) med användning av en Ultra Nanotrac partikelstorleksanalysator från Microtrac (Montgomeryville, PA, USA). Den typiska hydrodynamiska storleken i trietylenglykol av nakna magnetitpartiklar framställda som i Exempel 1 var 140 nm. Den hydrodynamiska storleken kunde inte mätas i vatten då de nakna nanopartiklama aggregerade i vatten. Den hydrodynamiska storleken i vatten av de silaniserade nanopartiklama framställda i Exempel 5 var 300-365 nm. Den hydrodynamiska storleken i vatten av de silaniserade nanopartiklama framställda i Exempel 6 var 250-300 nm. 10 15 20 25 30 35 23 Ma gnetísk karakteríserín g Magnetiska egenskaper av nanopartiklarna inbäddade i epoxiharts mättes vid rums- temperatur med en Princeton Measurement Corp. M2900-2 alternerande gradientmagnetometer (Princeton, NJ, USA). Magnetiska hysteresloopar visas i FIG. 7.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 spektroskopí Fourier-transform-infrared (FT-IR) spektra av nanopartiklarna (i KBr-tabletter) mättes med en Bruker IFS66 FT-IR-spektrometer (Billerica, MA, USA). Spektra finns i FIG. 2.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.

Elementaranalys Fe- och Si-innehållet i nanopartiklarna analyserades med ett Optima 3000 DV ICP-AES- instrument (Perkin Elmer, Waltham, MA, USA). Elementaranalys (C, H och N) utfördes av Mikrokemi AB (Uppsala, Sverige).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).

Kolorímetrískjärnanalys J ärnhalten hos nanopartiklarna bestämdes genom en modifiering av metoden beskriven i 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. Prover av nanopartiklama (1 -4 mg) behandlades med 0.3 ml HCl (37%) under 30 min. De upplösta provema överfördes kvantitativt till 25-ml mätkolvar och späddes med vatten. Volymer om 0.5 ml av de utspädda lösningama blandades med en hydroxylaminhydroklorid lösning (0.25 ml, 0.1 mg/ml), en natriumacetatlösning (2.5 ml, 0,1 mg/ml) och en 1,10-fenantrolin monohydratlösning (2.5 ml, 1 mg/ml). Lösningama späddes 2-10 gånger med vatten och absorbansen mättes vid 508 nm. Standard Fezl-lösningar för kalibrering bereddes under identiska förhållanden från FeSO4°7H2O.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.

Hemolys-testpå utspätt blod 5 ml humant färskt blod antikoagulerat med natriumcitrat späddes med 5 ml PBS. Blod- provet erhölls från en frisk frivillig försöksperson. Volymer om 0.75 ml nanopartiklar (0,1 mg/ml) i PBS inkuberades i Eppendorf-mikrorör under 30 min vid 37 °C. Negativa och positiva kontroller gjordes genom att byta ut nanopartikelslösningen mot PBS respektive vatten. Varje prov kördes i triplikat. Utspätt blod (0.25 ml) tillsattes och provema inkuberades på en rotator under 24 h vid 37 °C. Rören centrifugerades därefter (10 min, 5 000 varv per minut). Absorbansen hos supematantema mättes i en spektrofotometer vid 545 nm. Hemolysen beräknades som 100% * (Apmv - Anegativ kontmlfi/ (Apositiv kontroll - Anegativ kontmn). De nakna magnetitnanopartiklama från Exempel 1 orsakade 0.07% hemolys. De silaniserade nanopartiklama från Exempel 5 orsakade 0.06% hemolys. De silaniserade lO 15 20 25 30 35 24 nanopartiklama från Exempel 6 och de tPA-konjugerade nanopartiklama från Exempel 29 gav inte upphov till någon hemolys.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.

Hemolys-test på tvättade isolerade humana erytrocyter 2 ml PBS sattes till 5 ml humant färskt blod antikoagulerat med natriumcitrat. Blandningen vändes under 2 minuter på vippbord och centrifugerades sedan vid 5 000 rpm under 5 minuter.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.

Supematanten avlägsnades och pelleten av erytrocyter tvättades ytterligare två gånger med 4 ml PBS genom suspension, centrifugering, och dekantering. Erytrocytema suspenderades slutligen i 5 ml PBS. Volymer om 1 ml av nanopartiklama (0,1 mg/ml) i PBS inkuberades vid 37 °C i Eppendorf- mikrorör. Negativa och positiva kontroller framställdes genom att byta ut nanopartikelslösningen mot PBS respektive vatten. Varje prov kördes i triplikat. Efter 30 minuter tillsattes 0.1 ml av erytrocyt- suspensionen till varje rör. Rören inkuberades på en rotator vid 37 °C under antingen 1 h eller 24 h.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.

Rören centrifugerades sedan vid 10 000 rpm under 10 minuter. Absorbansen hos supematantema mättes i en spektrofotometer vid 545 nm. Hemolysen beräknades såsom beskrivits ovan. De nakna magnetitnanopartiklama från Exempel 1 orsakade 0.2l% och 0.30% hemolys efter 1 h respektive 24 h. De silaniserade nanopartiklama från Exempel 5 orsakade 5.92% och 21.15% hemolys efter 1 h respektive 24 h. De silaniserade nanopartiklama från Exempel 6 orsakade 3.94% och 22.30% hemolys efter 1 h respektive 24 h. TPA-nanopartikels-konjugaten från Exempel 29 orsakade 0.15% och 1.03% hemolys efter 1 h respektive 24 h.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.

Stabilítetsstudíer på tPA-nanopartikels-konjugaten Enzymaktivitetsanalys gjordes på vattenlösningar av tPA-nanopartikels-konjugat från Exempel 28 (5.7 mg/ml) och Exempel 29 (2,4 mg/ml) efter sonikering i ultraljudsbad under 1 h och efter inkubering vid 4 °C i upp till 40 dagar.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.

De erhållna enzymaktiviteterna visas i FIG. 8.The enzyme activities obtained are shown in FIG. 8.

Exampel 31 till 33 avser målstyming av magnetiska partiklar.Examples 31 to 33 relate to target control of magnetic particles.

Exampel 31 - In vitro målstyming av magnetiska partiklar till en spiralformad tråd i ett enkel-passage-genomflödesexperiment En Kantahl D ferromagnetisk tråd (längd 100 mm, Q) 0.13 mm) virades 15 varv för att tillverka en spiral (längd 40 mm, (Z) 2 mm). Spiralen sattes in i ett Wiretrol II (Drummond Scientific Company, Broomall, PY, USA) glaskapillärrör (längd 90 mm, Q) 2.2 mm). Kapillärröret placerades mellan två permanenta neodymiummagneter (N35; 50 >< 30 >< 30 mm; 0.48 T vid ytan), på ett avstånd av 3 cm från varje magnet. På detta avstånd var det magnetiska fältet applicerat på spiralen 0.1 T, uppmätt med en Gaussmeter modell GM-2 (Alphalab, Saltlake City, UT, USA). Båda ändarna lO 15 20 25 30 35 25 av kapillärröret anslöts till silikonslang (inre 0 2 mm, yttre 0 4 mm) såsom Visas i uppställningen i FIG. 5A. Utvärdering av spiralens infångningseffektivitet av nanopartiklar under en passage i ett genomflödesexperiment utfördes genom att ansluta en KDS100 sprut-infusionspump (KD Scientific, Holliston, MA, USA). 4.7 ml av en lösning av nanopartiklar (25 ug/ml vatten), framställda såsom beskrivits i Exempel 5, pumpades in i det evakuerade systemet med flödet 0.5-6 ml/min. Dödvölymen var 0.7 ml. Utflödet (4 ml) uppsamlades och absorbansen mättes vid 350 nm i en spektrofotometer.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.

En standardkurva visade ett linjärt samband mellan absorbans och koncentrationen av nanopartiklar.A standard curve showed a linear relationship between absorbance and the concentration of nanoparticles.

Den procentuella andelen nanopartiklar kvarhållna i kapillären, nedan kallad infångnings- effektiviteten (capture efficiency, CE), beräknades som CE = 100 * (A0 - A)/A0 där A0 är den initiala absorbansen hos nanopartikelslösningen och A är absorbansen hos utflödet. Experimentet upprepades utan spiralen närvarande i kapillärröret för att bestämma blank-retentionen. CE visas i FIG. 5B.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.

Exempel 32 - In vitro målstyrning av magnetiska partiklar till en spiralformad tråd i ett recirkulations-genomflödessystem En Kantahl D ferromagnetisk tråd (längd 100 mm, 0 0.13 mm) virades 15 varv för att tillverka en spiral (längd 40 mm, 0 2 mm). Spiralen sattes in i ett Wiretrol II (Drummond Scientific Company, Broomall, PY, USA) glaskapillärrör (längd 90 mm, 0 2.2 mm). Kapillärröret placerades mellan två permanenta neodymiummagneter (N35; 50 >< 30 >< 30 mm; 0.48 T vid ytan), på ett avstånd av 3 cm frän varje magnet. På detta avstånd var det magnetiska fältet applicerat på spiralen 0,1 T, uppmätt med en Gaussmeter modell GM-2 (Alphalab, Saltlake City, UT, USA). Båda ändarna av kapillärröret anslöts till silikonslang (inre 0 2 mm, yttre 0 4 mm) såsom visas i uppställningen i FIG. 5A. Infångningseffektiviteten av recirkulerade nanopartiklar utvärderades genom att ansluta en Gilson Minipuls 2 peristaltisk pump. Slangama anordnades i ett slutet kretslopp och nanopartiklar från Exempel 5 (4-10 ml, 25 ug nanopartiklar/ml vatten) recirkulerades med flödeshastighet 1-40 ml/min under 10-60 min (med undantag för utvärderingen vid 1 ml/min, vilken utfördes under 90 minuter). Vid slutet av varje experiment, kopplades slingan isär och ett prov av nanopartikels- lösningen togs för mätning av absorbansen och beräkning av CE som i Exempel 30. CE-värdena visas i FIG. 5C-F. Beläggningen av nanopartiklar på tråden visas i figur 6B, för jämförelse med FIG. 6A, som är den nakna tråden.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.

Exempel 33 - In vivo målsökning av magnetiska partiklar och lysering av stenttrombos (in-stent-trombos) genom tPA-nanopartikels-konjugat En Kanthal D tråd (längd 80 mm, 0 0.13 mm) vävdes in i en NIR PrimoTM koronarstent (längd 16 mm, 0 3 mm, Boston Scientific Scimed, Maple Grove, MN, USA) i en spiralforrnad konfiguration. Stenten monterades manuellt på en MaverickTM koronarballong (längd 30 mm, 0 3 mm, Boston Scientific Scimed). En inhemsk gris av honkön (40 kg) för-sederades, sövdes, 10 15 20 25 30 35 26 intuberades oralt med manschettförsedd endotrakealtub, ventilerades med lustgas och syre (723), och övervakades med elektrokardiografi (EKG). Radiologiska förfaranden gjordes i ett experimentellt kateterlaboratorium (Shimadzu Corp, Kyoto, Japan). Angiogram erhölls genom injektion av j ohexol.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 IE) gavs intravenöst fore kateterisering. Den vänstra femoralartären (artería femoralís sínístra), den vänstra halspulsådem (artería carotís communís sínístra) och den högra yttre jugularvenen (venajugularís externa dextra) exponerades kirurgiskt och 6F införingsanordningar (”introducer sheaths”) infördes i kärlen. En stemotomi utfördes på grisen. Genom införaren i den vänstra halspulsådem fördes en guide-kateter fram till den vänstra huvudkransartären (artería coronaría sínístra). Katetem användes för att placera en Doppler flödeshastighetsgivare (Jometrics Flowire, Jomed NV), kopplad till en FloMap monitor (Cardiometrics, Mountain View, CA, USA), och en ledare i ramus ínterventrícularís anterior arteríae coronaríae sínístrae (left anterior descending artery, LAD). NIR PrimoTM koronarstent med en Kanthal D-tråd placerades i mittdelen av LAD, distalt till den första diagonala grenen, genom uppblåsning av ballongen till 10 atm under 10 sekunder. En intravaskulär ultraljudssond fördes fram över guide-vaj em för att avbilda stent-artär- segmentet vid olika tidpunkter. Baslinj esflödet, mätt med flödes-vaj em, var 20 cm/ s efter stentinsättning. En permanent neodymiummagnet (N48, 50 X l5 X l5 mm, 0,48 T vid ytan) applicerades på den främre delen av hjärtat, i kontakt med den stentsegmentet hos LAD. Efter spontan bildning av en blodpropp i stenten, minskade baslinj esflödet till 5 cm/s. En lösning av tPA- nanopartikels-konjugat från Exempel 29 (40 ml, 0,14 mg/ml) injicerades genom guidekatetern till den vänstra huvudkransartären. Tromben lyserades genom verkan av tPA-nanopartikels-konjugaten Baslinjesflödet ökade till 15 cm/s.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.

Enligt en utföringsform, tillhandahålls komposition enligt utföringsforrner av uppfinningen för användning som ett läkemedel.According to one embodiment, a composition according to embodiments of the invention is provided for use as a medicament.

Specifikt, enligt en utföringsform, tillhandahålls komposition enligt utföringsforrner av uppfinningen för behandling av trombos.Specifically, according to one embodiment, there is provided a composition according to embodiments of the invention for the treatment of thrombosis.

Således, enligt en utföringsforrn, ges en metod för behandling av trombos i ett subjekt, innefattande ett första steg för att injicera kompositionen, innefattande belagda magnetiska nanopartiklar enligt vissa utföringsforrner, i blodet, dvs det kardiovaskulära systemet, hos subj ektet.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.

Därefter appliceras ett magnetiskt fält till platsen för trombosen, varefter nanopartiklama attraheras till trombosen med magnetfältet, vilket löser tromben.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.

I en utföringsform, är en metod för behandling av stenttrombos i en patient med en implanterad magnetiserbar stent beskriven, innefattande ett första steg för att injicera kompositionen innefattande belagda magnetiska nanopartiklar enligt vissa utföringsfonner, i blodet, dvs det kardiovaskulära systemet, hos subj ektet. Därefter appliceras ett magnetiskt fält till platsen för stenten, varefter nanopartiklarna attraheras till stenten med magnetfältet, sålunda upplösande blodproppen. lO 15 27 Såsom beskrivits ovan kan de magnetiska nanopartiklama konjugeras till tPA, dvs rekombinant human vävnadsplasminogenaktivator, för att ytterligare förbättra den anti-trombotiska effekten.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.

Fastän föreliggande uppfinning har beskrivits ovan med hänvisning till specifika utföringsformer är den inte avsedd att vara begränsad till den särskilda form som anges häri. Snarare är uppfinningen begränsad endast av de bifogade patentkraven och andra utföringsfonner än den specifika ovan är lika möjliga inom ramen för dessa bifogade patentkrav.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.

I patentkraven utesluter uttrycket "innefattar/innefattande" inte närvaron av andra element eller steg. Vidare, även om de är individuellt listade kan en pluralitet av sätt, element eller metodsteg implementeras av t.ex. en enhet eller processor. Dessutom, även om enskilda särdrag kan vara inkluderade i enskilda krav, kan dessa eventuellt med fördel kombineras och inklusionen i olika krav innebär inte att en kombination av särdrag inte är möjlig och/eller fördelaktig. Dessutom utesluter singulära referenser inte en pluralitet. Terrnema "en", "ett", "första", "andra" osv utesluter inte pluralitet. Hänvisningsbeteckningar i patentkraven tillhandahålls endast som klargörande exempel och skall inte tolkas som begränsning av omfattningen av kraven på något sätt.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.

Claims (30)

10 15 20 25 30 PATENTKRAV10 15 20 25 30 PATENT REQUIREMENTS 1. l. En metod för att bilda ett skikt på en nanopartikel med hydroxylgrupper på sin yta, innefattande stegen utsätta nanopartikeln för en första lösning innefattande en förening enligt forrnel (1): Ho oH WO/W n I vari "n" är ett heltal i intervallet 0 (noll) till 7000, utsätta nanopartikeln för en andra lösning innefattande ett silaniseringsmedel, och möjliggöra bildandet av ett silaniserat skikt på nanopartikeln.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. Metod enligt krav 1, vari nanopartikeln är en magnetisk nanopartikel.The method of claim 1, wherein the nanoparticle is a magnetic nanoparticle. 3. Metod enligt krav 1 eller 2, vari föreningen enligt forrneln (I) är vald från gruppen bestående av: etylenglykol, dietylenglykol (DEG), trietylenglykol (TREG), tetraetylenglykol, pentaetylenglykol, hexaetylenglykol, heptaetylenglykol, oktaetylenglykol och andra oligoetylenglykoler/polyetylenglykoler/polyetylenoxider med molekylvikter upp till 300000 (såsom PEG 400, PEG 2000, PEG 3400, PEG 8000, PEG 20000, PEG 35000, PEG 100000, PEG 200000, och PEG 300000), eller en kombination därav.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. Metod enligt något av de föregående kraven, vari silaniseringsmedlet är en silan.A method according to any one of the preceding claims, wherein the silanizing agent is a silane. 5. Metod enligt krav 4, vari silanen är en alkoxysilan.The method of claim 4, wherein the silane is an alkoxysilane. 6. Metod enligt krav 5, vari alkoxysilanen är vald från gruppen bestående av tetrametoxysilan, tetraetoxysilan, tetra-n-propoxysilan, tetra-iso-propoxysilan, tetra-n-butoxysilan, tetra-t-butoxysilan, trimetoxysilan, trietoxysilan, tri-n-propoxysilan, tri-iso-propoxysilan, tri-n- butoxysilan, tri-t-butoxysilan, trimetoxyklorsilan, trietoxyklorsilan, tri-n-propoxyklorsilan, tri-iso- propoxyklorsilan, tri-n-bytoxyklorsilan, tri-t-butoxyklorsilan, bensyltrimetoxysilan, bensyltrietoxysilan, dimetyldimetoxysilan, dimetyldietoxysilan, och blandningar därav.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. Metod enligt krav 4, vari silanen är en halosilan. lO 15 20 25 30 35The method of claim 4, wherein the silane is a halosilane. lO 15 20 25 30 35 8. Metod enligt krav 8, vari halosilanen är vald från gruppen bestående av tetraklorsilan, triklorsilan, tetrafluorsilan, trifluorsilan, och blandningar därav.The method of claim 8, wherein the halosilane is selected from the group consisting of tetrachlorosilane, trichlorosilane, tetrachlorosilane, trifluorosilane, and mixtures thereof. 9. Metod enligt krav 4, vari silanen är en aniinosilan.The method of claim 4, wherein the silane is an amino silane. 10. Metod enligt krav 9, vari aniinosilanen är vald från gruppen bestående av 3- aniinopropyltrinietoxysilan, 3-aminopropylnietyldinietoxysilan, 3-aminopropyldinietylnietoxysilan, N-(2-an1inoetyl)-3-arninopropylmetyldirnetoxysilan, N-(Z-aminoetyl-S-an1inopropyl)trimetoXysilan, 4-aniinobutyldinietylnietoxysilan, 4-aminobutyltrinietoxysilan, arninoetylarninometylfenetyltrirnetoxysilan, N-(2-arninoetyl)-3 -aminoisobutylmetyldimetoxysilan, N- (6-an1inohexyl)aniinopropyltrinietoxysilan, 3-(ni-aminofenoxy)propyltrirnetoxysilan, arninofenyltrirnetoxysilan, 3-aminopropyltrietoxysilan, 3-arninopropylnietyldietoxysilan, 3- aniinopropyldinietyletoxysilan, N-(2-an1inoetyl)-3-aniinopropylnietyldietoxysilan, N-(2-an1inoetyl-3- arninopropyl)trietoxysilan, 4-aminobutyldinietyletoxysilan, 4-aminobutyltrietoxysilan, aminoetylaniinonietylfenetyltrietoxysilan, N-(2-an1inoetyl)-3 -aminoisobutylnietyldietoxysilan, N-(6- aminohexyl)arninopropyltrietoxysilan, 3-(m-aminofenoxy)propyltrietoxysilan, aminofenyltrietoxysilan, och blandningar därav.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. Metod enligt krav 4, vari silanen är en olefin-innehållande olefin.The method of claim 4, wherein the silane is an olefin-containing olefin. 12. Metod enligt krav 11, vari den olefin-innehållande silanen är vald från gruppen bestående av 3-(trinietoxysilyl)propylrnetakrylat, 3-(trietoxisilyl)propylmetakrylat, metakryloxynietyltrinietoxysilan, metakryloxynietyltrietoxysilan, vinyltrinietoxysilan, vinyltrietoxysilan, allyltrinietoxysilan, allyltrietoxysilan, vinyltriklorsilan, och blandningar därav.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. Metod enligt krav 4, vari silanen är en fluorescerande silan.The method of claim 4, wherein the silane is a fluorescent silane. 14. Metod enligt krav 4, vari silaniseringsmedlet är en radioopak silan.The method of claim 4, wherein the silanizing agent is a radiopaque silane. 15. Metod enligt något av de föregående kraven, vari den första lösningen vidare innefattar en bas och/eller ett andra lösningsmedel.A method according to any one of the preceding claims, wherein the first solution further comprises a base and / or a second solvent. 16. Metod enligt krav 15, vari basen är vald från gruppen bestående av: ammoniak, natriumhydroxid, kaliurnhydroxid, trietylarnin, trirnetylaniin, dimetylaniin, dietylarnin, etylarnin, propylaniin, N,N-diisopropyletylan1in, N-nietylniorfolin, N-nietylpyrrolidon, oleylamin, etanolaniin, pyridin, 4-din1etylan1inopyridin, rnetylamin, och piperidin, eller en kombination därav. lO 15 20 25 30 35The 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. Metod enligt krav 15 eller 16, vari det andra lösningsmedlet är valt från gruppen bestående av: vatten, metanol, etanol, n-propanol, iso-propanol, N,N-dimetylforrnamid (DMF), dimetylsulfoxid (DMSO), aceton och acetonitril, eller en kombination därav.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. Förfarande enligt något av föregående krav, vidare innefattande ett steg att immobilisera en funktionell enhet på det silaniserade skiktet, varvid sagda funktionella enhet är vald från gruppen bestående av: enzym, protein, antikropp, peptid, affmitetsligand, oligonukleotid, kolhydrat, lipid, ytaktivt ämne eller en farmaceutiskt aktiv molekyl.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. En komposition som kan erhållas genom förfarandet enligt krav 1 till 18.A composition obtainable by the process according to claims 1 to 18. 20. En komposition innefattande huvudsakligen diskreta nanopartiklar med ett silaniserat skikt på varje nanopartikel.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. Komposition enligt krav 19 eller 20, vari nanopartiklama är magnetiska. Komposition enligt kraven 19 till 21 vari nanopartiklama är röntgentäta. Komposition enligt kraven 19 till 21 vari nanopartiklama är fluoreseerande. Komposition enligt kraven 19 till 21 vari nanopartiklama är MR-aktiva. Komposition enligt något av kraven 19 till 24 för användning som ett läkemedel. Komposition enligt patentkrav 21 för behandling av trombos. Ett kontrastmedel innefattande kompositionen enligt något av kraven 22 till 24. Ett magnetiskt bläck, innefattande kompositionen enligt krav 21. Ett förfarande för behandling av trombos i ett subjekt, innefattande stegen: injicera kompositionen enligt patentkrav 21 i blodomloppet hos subj ektet; applicera ett magnetfält till platsen för trombosen, och attrahera nanopartiklar till trombosen med magnetfältet, sålunda upplösande tromben. lOA 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. Ett förfarande för behandling av stenttronibos (in-stent-tronibos) i ett subjekt med en iniplanterad niagnetiserbar stent, innefattande stegen: injicera kompositionen enligt patentkrav 21 i blodoniloppet hos subj ektet, applicera ett magnetfält till platsen för stenten, och attrahera nanopartiklar till stenten med niagnetfaltet, sålunda upplösande tromben.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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