CN101421035B - Improved preparation of molecular imprinted polymers - Google Patents

Abstract

One aspect is a method for improved preparation of molecular imprinted polymer (MIP) particles, where initial compositions comprising insoluble MIP particles are enriched for those MIP particles that bind a particular target molecule, thus excluding non-binding and weakly binding particles from the final composition. Enrichment is typically accomplished via use of chromatographic methods capable of separating particulate material or by means of agglutination. Another aspect is preparation of improved insoluble MIPs by use of extended micronization of raw MIP particles with a view to expose a large number of binding sites per mass unit of MIP particles. In preferred embodiments the two aspects are combined. The resulting improved MIPs may be used for diagnostic, analytical and therapeutic purposes, notably as orally administered drugs which can bind substances such as cholesterol and bile acids and bile acid salts in the gastrointestinal tract.

Description

The preparation method of the molecularly imprinted polymer improved

Technical field

The present invention relates to the improvement of the preparation of molecularly imprinted polymer (MIP), the invention particularly relates to the binding capacity and specificity that increase MIP, with convenient they in pharmaceutical preparation, be used as the method for trapping agent, described pharmaceutical preparation particularly combines the pharmaceutical preparation of target molecule such as cholesterol and cholic acid in the gastrointestinal tract and cholate.And this improvement prepared by MIP can as a kind of characterizing method of described MIP.

Background technology

The molecular engram of synthetic polymer is under the existence of the target molecule as molecular template, the process of function monomer and crosslinked monomer copolymerizable.Before polymerization, function monomer or form complex via noncovalent interaction and template, or covalent coupling forms the polymerizable derivant of template.After polymerization, the functional group of monomer is remained on appropriate location by highly cross-linked paradigmatic structure.Remove template by the method for solvent extraction and/or chemical cracking subsequently, demonstrate the binding site with target molecule size and shape complementarity.In this way, molecular memory is introduced into polymer (being called now " molecularly imprinted polymer " or " MIP "), and this polymer now can with high specificity again in conjunction with target.

At first, in HPLC particularly in chiral separation MIP be used as immobile phase.Afterwards, their application extension in other analytical technologies, the such as binding analysis of thin layer chromatography, capillary electrophoresis, solid phase extractions and immunoassay type.Binding site has affinity and the selectivity of similar antibody-antigene system usually.To sensing technology, these analogies demonstrate some obvious advantages compared to real antibody.Because their highly cross-linked character, MIP is stable and firm in essence, is conducive to them at extreme environment such as acid, alkali or metal ion, organic solvent, or the application under the existence of High Temperature High Pressure.And MIP low production cost, can long term storage under room temperature, dry state.

Therefore, all in principle MIP can make in order to below method: monomer and target molecule (or template) mixing, self assembly occur, add cross-linking agent and polymerization is initial.After polymerization, polymer is broken into fritter, and target molecule is extracted.If MIP is put into the solution of target molecule, it is (another with reference to Yu Cong that they will be combined with MIP again; Leif Schweitz and Ioana -Surugiu).

General technical step prepared by MIP shows in FIG.

The history of MIP

One of example the earliest prepared by MIP just has description as far back as 1949 (Dickey), and he carrys out Selective recognition dyestuff with a kind of silicon dioxide (waterglass).Had afterwards describe occurred that the self-organizing system of other kinds can set up reticular system, wherein specifically in conjunction with target/analysis thing become possible ( deng, Schweitz etc., and Vlatakis etc.).

the selection of monomer and polymerization

In the seventies and eighties in 20th century (with reference to Shea 1986, Shea 1990 and Wulff 1987), template/target molecule and the direct covalent bond of polymer are described by people for the concept setting up support.Claim directly combine will cause binding site in whole polymer evenly distribution.But this method leaves the problem that polymerization rear pattern plate is removed simultaneously.In order to remove template, needing micro polymer granulation and opening chemical bond.

During preparation MIP; if template is not connected with one of monomer used in polymerization; the MIP that usually can produce; but the experience of document is a large amount of binding site will comprise the binding site of tendency in conjunction with the part of poor specificity in template/analyte molecule, thus product MIP does not have the specificity of expectation.This is extremely important to the MIP for analysis purpose, if when particularly target is the stereoisomer form of isolated molecule, but this is just so unimportant when main target is the situation of the total binding capacity strengthening product MIP.

Analyze situation at some, confirmed that template has to have different characteristics, the MIP namely produced is based upon in template " analogies " instead of uses real template, and object is to not pollute analyzed sample.Be apparent that, find the template analogies that can ensure to analyze specific bond between thing and MIP to be very difficult task.

The diverse method of one of preparation MIP is the mixture by polymerization single polymerization monomer, cross-linking agent and template (or template analogies), and they remain on the form of granule in Emulsion, and such product MIP directly becomes granule (Funke etc.).The size of the MIP granule that this method produces especially depends on concentration and the stir speed (S.S.) (determining the size of drop in Emulsion) of monomer.(in order to particle size be controlled below 1 μm, the rotating speed of agitating solution need be greater than 1000rpm).According to document, the shortcoming of these class methods is preparation time length and productive rate is low.

In a word, prior art describes the difficulty preparing reproducible MIP usually, and wherein capacity and specificity are not taken into account (therefore lower than expectation numerical value).Up to the present, this area does not also find a kind of preparation method (Sellergren 1998) of MIP compositions of reliably feasible tool height binding capacity.

obtain useful particle size

When the method that bulk forms polymerization is used for preparing MIP, micro polymer, before template molecule has extracted, is granulated into very little particle size by highly cross-linked requirement.Micronized method is applied to widely different industrial circle, comprises from the cement industry granule of size (produce mm) to the small grinder (μm sized particles) of thick film ink preparing electronic circuit.Although in fact atomization process is the key component of MIP functional (comprising specificity and capacity); but the practical methods selected all is not described in detail in most of MIP document, only mention and before extract template from MIP, the bulky polymer obtained being pulverized and sieve (sorting granular size).

remove template

In most of document, do not concentrate the removal of description template molecule, although in fact this process is vital for the functional of product MIP.Usually this process is only mentioned by the cleaning method as non-detailed description, and the method uses the solvent of one or more specific named.Especially, when developing MIP and being used for solid phase extractions (SPE), such as, as the instrument analyzing thing pre-concentration, even the residual quantity of template will disturb the availability of MIP.Sorting MIP from substrate for template removal leads to typical filter or centrifugal method completes.

the achievement of known improvement MIP

U.S. Patent number 4,111, describing " a kind of nondistensible three-dimensional polymer; this polymer has the composition of the residue for optically active compounds; this residue can be removed from described polymer chemistry; thus leave the space of the size and dimension of the residue corresponding to described optically active compounds in the physical arrangement of described polymer, and the particular functional group's spatial arrangements in the space of described polymer of chemical constitution corresponding to described optically active compounds residue ... .. " this optically active compounds in 863 is the template that can combine with object MIP subsequently.

At United States Patent (USP) 5,110, in 833, " a kind of method producing synzyme or synthetic antibody; comprise the orientation of monomer around the marking (print) molecule; cross-linking agent adds, the removal of polymer polymerizing and subsequently imprint molecule, thus manufacture corresponding to the hole of described imprint molecule in the polymer " is claimed and is added the specificity of MIP to template molecule.In other words, the U.S. 5,110, the performance mentioned in 833 improves the optimization contacted with monomer unit based on polymerization front template molecule.

At United States Patent (USP) 6,881, in 804, in MIP, porous introducing is described to a kind of method increasing MIP performance by being increased to the path being intended to the interactional space with template.

At United States Patent (USP) 6,638, in 498, the monomer of special selection claims the generation for the special MIP of cholic acid, and template molecule is fixed in a kind of support material by U.S. 2004/0157209A1 suggestion before polymerization.The suggestion of all improvement MIP performances relates to the chemical characteristic of monomer or the structure of MIP, and these are all the method steps in MIP preparation or before preparation.

The U.S. 5,994,110 find that the MIP that original position produces forms little polymer/oligomer, comprise the complementary structure with template molecule.These polymer or oligomer form coating around biomolecule or image, and this coating or image are removed by from it, and discrete entity, from deriving here, may be used for such as treating or preventative medicament such as medicine.Due to this kind of production method, the U.S. 5,994,110 do not adopt the micronized step in the preparation of conventional MIP granule.The U.S. 5,994,110 really suggested be separated MIP from azygosperms, but the method proposed all depends on the very little size producing MIP, such as, only have when MIP is solvable entity by chromatography.Demonstrate molecular weight according to the U.S. 5,994, the 110 such as specific MIP indicating tool therapeutic activity and be in one end less in 1-200kDa scope.And the U.S. 5,994,110 1 aspects do not disclose the method for " excellent combination " of the insoluble MIP of any separate out suspended, are openly separated the method for wherein " poor efficiency or azygosperm " on the other hand yet.

Goal of the invention

What an object of the present invention is to provide prepared by MIP improves one's methods, to provide the MIP compositions with enough high binding capacity, thus this compositions is used in medicinal application as the substitute of soluble recepter and antibody.Another object is to provide the MIP compositions of the characteristic with the improvement surmounting prior art MIP compositions.

Summary of the invention

Although trial discussed above is to improve existing MIP technology of preparing, but so far also without any being successfully prepared in Therapeutic Method the feasible substitute that can be used as such as antibody and soluble recepter by the trial of the MIP compositions of Clinical practice, wherein certain target molecules is relevant from the removing of body as the part for the treatment of.

The present inventor ascribes this point to the following fact; although MIP may have very high affinity to particular ligand separately; but the MIP compositions table of such as micronize/grinding reveals the great transmutability to ligand binding affinity; make overall binding capacity cannot be satisfactory like this; and be not suitable for such as clinical application; or in brief; known MIP compositions is usually too low to the binding capacity of target ligands, and a kind of feasible alternative that MIP can not be made to become the upper soluble recepter for the treatment of and antibody is selected.

The present inventor also concentrates on the following fact, little on the research of the impact of the overall binding capacity of MIP compositions about MIP particle size, even if if any.

Therefore the present inventor's instruction is by improving the performance of MIP further in conjunction with the ability sorting MIP granule of template or template analog according to MIP granule, thus the MIP that the target effectively realizing improving concentration combines.By so a kind of functional sorting or purge process, a part can be produced there are space or hole, having being selected the MIP granule of the appropriate combination ability of template or analog, thus the binding capacity of the average affinity that improve between MIP and template and MIP.

And; the present inventor recognizes that the performance of MIP compositions can be improved simply by improvement application micronized step so far; make the average-size of MIP granule less like this, they demonstrate the binding site of raising and the ratio of volume by this method.

Therefore, first aspect of the present invention relates to a kind of method preparing compositions, and this compositions comprises and has target molecule height binding capacity and specific molecularly imprinted polymer (MIP), and described method comprises:

A) obtain the float of the insoluble MIP of binding target molecule, it is prepared as template molecule with the analogies of target molecule or target molecule,

B) by the MIP that suspends through affinity purification method, wherein template molecule or its fragment or its analogies are as trapping agent,

C) be recovered in the MIP in conjunction with trapping agent in affinity purification method, from recovery product, remove trapping agent substantially and not in conjunction with the MIP of trapping agent, and

D) by the MIP that reclaims and optionally carrier, excipient or mixing diluents, to obtain described compositions.

Second aspect, the invention provides a kind of method preparing the MIP had target molecule height binding capacity, described method comprises the former MIP by comprising the template molecule be made up of the analogies of described target molecule or target molecule, through first micronized step so that the size of the MIP granule obtained is enough little, thus can template molecule be removed, remove all template molecules substantially, optionally by the MIP that obtains like this through second micronized step, wherein said first pelletizing step and optionally second pelletizing step make the average diameter of MIP be 50 μm to the maximum.

The third aspect, the invention provides the compositions of a kind of insoluble MIP, has at least one following characteristics:

1) average diameter of MIP is less than 20 μm;

2) average target combines and is at least 1 mass unit target in conjunction with 10 mass unit MIP;

3) all in compositions MIP are substantially in conjunction with same target molecule, and optionally compositions does not comprise all binding sites to target molecule.

4th aspect, the present invention relates to the application of the present composition for the preparation of pharmaceutical preparation, the infection that described pharmaceutical preparation is used for the treatment of, prevents or alleviates cardiovascular disease, hypertension, atherosclerosis, cholelithiasis, cholestatic hepatopathy, hypercholesterolemia, obesity, parasite or microorganism such as antibacterial and fungus cause, or oral toxin cause poisoning.

Finally, 5th aspect, the present invention relates to a kind for the treatment of, alleviation or reduce the method for following disease risks, described disease is selected from the infection caused by cardiovascular disease, hypertension, atherosclerosis, cholelithiasis, cholestatic hepatopathy, hypercholesterolemia, obesity, parasite or microorganism such as antibacterial and fungus, the group of the poisoning composition that oral toxin causes, described method comprises the present composition to using effective dose to its curee in need.

Accompanying drawing explanation

Fig. 1: chart describes a kind of simple MIP preparation method.

Fig. 2: the quick-reading flow sheets of invention process described herein.This process can repeatedly several times.

Fig. 3, part I:

By purification or the sorting of expanded bed adsorption.

MIP in conjunction with the cholesterol sample molecule of template such as coupling bed granule remains, and the MIP do not combined then passes through and is dropped.

Fig. 3, part II:

Functional purification MIP.

MIP in conjunction with the template molecule on bed granule can subsequently by eluting out.The MIP of eluting will have higher specific bond capacity than the rough set of MIP comprising combination and unconjugated MIP.

Fig. 4: the overlapping figure of the MIP granule in embodiment 4.

Overlapping figure comprises two pictures respectively by visible white light and ultraviolet light shooting.White point represents the granule of white light, and stain represents the green fluorescence granule of irradiation under ultraviolet ray.

Detailed Description Of The Invention

definition

Below, in order to ensure the correct understanding to boundary of the present invention and scope, a large amount of terms will be defined.

" molecularly imprinted polymer " (MIP) refers to a kind of polymer comprising hole (or space) corresponding with one or more template molecules at least partly, and wherein template molecule has been incorporated in the monomer host matrix (matrix) comprising cross-linking monomer before polymerization.The polymer obtained after polymerization comprises a large amount of hole corresponding with template molecule shape.Typically MIP sequester (sequester) is granule, be conducive to like this template removal and stay part to target molecule interacts opening hole, its target is identical with template molecule or similar.In this description and claim, term MIP generally refers to the particle form of MIP, means term " MIP " and " MIPs " interchangeably for expressing the plural form of MIP granule and MIP granule respectively.

Will be understood that the MIP used in the present invention is insoluble molecule/entity.Due to they insoluble restriction or stop them to enter body (such as entering circulation) from gastrointestinal tract, these MIP are particularly suited for being applied to gastrointestinal tract as medicament.In other words, when oral medication, will retain substantially for MIP of the present invention and be limited to gastrointestinal tract until they are discharged by feces.

" former MIP " refers to also not through any micronized MIP, in the hole of MIP structure, therefore still comprises template molecule or be at least the derivative vestiges of template molecule.

" micronize " represents that the MIP sequester that still may comprise template is the process of less granule.Any method being applicable to this object all can be applied.

" target molecule " refers to the combinative any molecule of MIP in the context of the present invention.

" template molecule " is usually identical with target molecule, but also can be its analogies (namely have and can be made up of the fragment of target molecule with the three dimensional structure identical at least partly of characteristic matching and the molecule-such as analogies of feature with the three dimensional structure of target molecule).Template is used as in MIP structure, subsequently can " generator " in gap of binding target molecule.

" affinity purification " represents any method utilizing the specific bond of material and binding partners to carry out purifying substance.Much such method utilizes the trapping agent of catching material being incorporated into solid support (such as chromatography substrate).Representative instance as known in the art is, with antibody as the affinity purification of trapping agent being coupled to chromatography pearl, for the antigen of purification binding antibody.It should be understood that according to the affinity purification method of the present invention's application it is that those can catch the method for insoluble, the MIP granule with size discussed here of suspension.Therefore, typical affinity purification method can be to expanded bed adsorption well known by persons skilled in the art (EBA).

" solid phase " refers to any material of the mode grappling trapping agent that can be used to by covalently or non-covalently combining in the context of the present invention.Therefore, the material (plastic polymer, sugar, metal, glass, Silicon stone, rubber etc.) that any routine is prepared for chromatographic material can be used as solid phase.Solid phase material can containing suitable, can coupling trapping agent to the functional group on discussed material.Such derived material is that albumen and other macromole chromatography purification art are known to the skilled.And solid phase can have any physical form, described form allow to catch relatively greatly with insoluble granule such as MIP (compared with single biomolecule such as albumen).Therefore, solid phase with fiber (preferred hollow), the chromatography substrate substrate of EBA (be preferably applicable to), pearl (preferably those can be separated with electromagnetic mode) or any other suitable form, can vide infra.

according to the embodiment of invention purification aspect

As specified above, the present invention relates in its first aspect the method that one prepares the compositions comprising molecularly imprinted polymer (MIP), and described compositions has high binding capacity to target molecule and specificity, and described method comprises:

A) obtain the float of the insoluble MIP of binding target molecule, it is prepared as template molecule with the analogies of target molecule or target molecule,

B) by the MIP that suspends through affinity purification method, wherein template molecule or its fragment or its analogies are as trapping agent,

C) be recovered in the MIP in conjunction with trapping agent in affinity purification method, from recovery product, remove trapping agent substantially and not in conjunction with the MIP of trapping agent, and

D) by the MIP that reclaims and optionally carrier, excipient or mixing diluents, to obtain described compositions (such carrier, excipient and diluent are usually selected from pharmaceutically acceptable and contain the known by the technical staff of the field of medicinal compositions of solid-state small sized particles for preparation).

In other words, this aspect depends on the raising concentration of MIP, this MIP demonstrates the desired sufficiently high affinity to target molecule (or associated clip of its replacer such as target molecule), but also there is the effect of the non-binding fragment removing initial former MIP from MIP compositions-with himself this aspect, the binding capacity of every mass unit MIP particulate composition is promoted significant degree, see embodiment 4 and accompanying drawing 4 like this.Can believe the present inventor be first demonstrate in the insoluble MIP compositions of the prior art prepared by methods known in the art comprise greatly in conjunction with granule, the binding capacity of any this compositions per unit mass can be significantly increased by removing azygosperm.Therefore it will be understood that the preparation of any insoluble MIP can through purification step b), so a first aspect of the present invention can combine the method for the insoluble MIP of any known preparation, particularly those relate to the known method of the mode of the MIP compositions obtaining high power capacity and/or high specific.

Below, the embodiment of the purification schemes of the multiple MIP of being designed for compositions will be discussed in detail.

First group of purification schemes comprises trapping agent and solid phase (such as chromatography substrate) covalently or non-covalently coupling-namely, and this organizes purification schemes and especially comprises typical chromatographic purification methods.Therefore, in chromatography and similarity method, useful any material is all useful, but preferred solid phase is the substrate of cross-linked carbohydrate, synthetic polymer, metallic particles or its combination.

The purification schemes group of second no less important is those groups, and wherein trapping agent is made up of or a part for soluble chemical entity soluble chemical entity (allowing, such as by coagulation method purification, to vide infra).The preferred embodiment of this point comprises some schemes, wherein trapping agent and the some covalent or the non-covalent associations that are selected from dendrimers, the carbohydrate of replacement and the soluble polymer such as polyvinyl alcohol and Polyethylene Glycol of replacement, object is in order to often kind of soluble chemical entity exposes multiple trapping agent.

No matter one or other purification schemes groups whether selected, some embodiment of first aspect present invention comprise make a trapping agent integrating step a) in limit can in conjunction with the part binding site in the MIP of template molecule.In brief, this embodiment ensures that the MIP of the binding specificity or binding affinity only with expectation is retained in purge process, and such as those have MIP that is non-specific or weak binding site and have been removed in purification.

A kind of method removing unspecific binding sites in purge process needs to utilize trapping agent in affinity purification, and wherein said trapping agent is the fragment of template molecule.Have selected this method, energy and the part of other molecule competes in conjunction with the template molecule of MIP can be deleted in trapping agent.When target molecule comprises the binding site of supposition cross reaction, this point is especially practical, and the binding site of cross reaction can produce the MIP in conjunction with uncorrelated target.Pass through example: if technical staff such as wants to prepare the MIP compositions in conjunction with metakentrin, the α subunit removing this molecule in template will be appropriate, because the α subunit of LH, FSH, TSH and hCG is identical.

The alternative method of this method can utilize a kind of setting, wherein trapping agent comprises and adopts any template molecule or its analogies or its fragment that are all attached to the surface of solids or part with specific orientation, to avoid the part of exposure trapping agent substantially to MIP-like this, by with selected degree of functionality coupling trapping agent to its solid support or part, so that the orientation of the trapping agent of coupling becomes unanimous on the whole on all coupling partner, the result realized is the part of trapping agent will be maccessiable in conjunction with MIP, therefore can in conjunction with trapping agent non-can close to part MIP will be out screened in whole purge process.The compositions that therefore product of this purification process will be MIP granule, wherein haply in compositions all MIP granules in conjunction with specific target, but at least one binding site of target is MIP granule not in binding compositions.

Selectively, and restriction trapping agent and MIP between combination be undesirably, there is no obvious advantage or unwanted situation under, preferred purification process of the present invention is that wherein trapping agent comprises the template molecule or its analogies or its fragment that are attached to solid phase surface or part (no matter with any), so that all parts of trapping agent are exposed to the method for MIP substantially on non-specific direction.

When trapping agent and solid support in conjunction with, can from any applicable type, select affinity purification method the purification technique that relies on trapping agent and surface of solids coupling.But preferred purification process is selected from the group be made up of expanded bed adsorption (EBA), paramagnetic beads separation and hollow fiber purification.

Expanded bed adsorption (EBA)

The conventional chromatography method utilizing the bed compressed in pillar, usually due to granule tendency non-specific be trapped within the static space of bed, and can not for separating of granular materials.

The cardinal principle of EBA keeps the chromatography media also referred to as " solid phase " flow, therefore as release, permission granule passes through pillar.The advantage of EBA technology is utilized to be make purification of soluble material from thick raw material or cell culture become possibility, this soluble material is albumen or peptide as a rule, and this purification does not need cleaning step before crossing post such as raw material to be loaded to the filtration before pillar and centrifugal (Van Reis & Zapata; Lihme etc.).Plan is insoluble or granular materials such as cell debris is washed off with being deposited in when target molecule is attached in a solid phase simultaneously.In this way, decrease time and the expense of these processes, make EBA become recommend economically, the valuable technology of the countless molecule of a kind of purification.But if expanded by bed, the volume in the static space of bed increases, granular materials is allowed to pass through also thus correctly can contact with the solid phase of bed, just can in conjunction with getting on if reach required affinity between the granular materials discussed subsequently and bed solid phase.In this case, show EBA and can be used for sorting cells and other granular materialss (Ujam etc.), wherein used from the mononuclear cell of peripheral blood and mix the biotinylated anti-CD 14 antibody being also applied to the EBA system that wherein bed granule has supplemented with Streptavidin subsequently with hemocytoblast and be separated.

The present inventor reaches a conclusion, if the solid phase of EBA system or bed granule expose similar or be equal to the chemical constitution of the template for the preparation of described MIP, with EBA sorting or can be purified by those granules prepared by MIP technology of similar mode.May be importantly, MIP granule is transported with the flowing of EBA system liquid phase part, and bed granule keeps the expanding volume of relative constancy, in order to be separated by the MIP of bed combination and unconjugated MIP in flow process, the bed granule with relatively high density (> 2g/ml) has superiority by being probably proved to be.The example of this high density pearl provides in Ujam etc.And preferably atresia or show limited porous bed granule, see Chase hereafter.Template such as cholesterol or cholic acid are preferably can guarantee that the orientation of the structure and chemical characteristic that at utmost expose template molecule is coupled to a granule, and the preferred size that has of preparation is the bed that the MIP of 0.2-50 μm (vide infra) is applied to liquefaction, through the suitable response time, preferably with the mobile phase recirculation comprised in conjunction with MIP, unconjugated MIP is washed off such as by improving flowing velocity or just by adding clean dcq buffer liquid in EBA system, making liquid wash out refuse.In conjunction with MIP by add soluble template, by heating, by increase ionic strength or to bed apply physical pressure discharge from bed granule.(see the Chase) atresia discussed before utilization or show limited porous bed granule and be also fine.

As expanding through circulation (flow-through) or the substituting of liquefied bed, bed can by mechanical agitation, mix to the end end of to, rock, method that ultrasound wave and other convection current/mass transportation increase and expanding.The separation being attached to the MIP granule on a granule can have been come by the suitable liquefaction of the bed through the stage of circulating subsequently.Optionally, can be separated according to the difference of density, size, shape, optical characteristics in conjunction with unconjugated MIP, by centrifugal, sedimentation, other modes of filtering, catching or being separated according to size and/or weight, density, shape, color, light emission, light scattering, spreading factor.

So, as described by existing purification technique, granule can by the method sorting of EBA or purification, granule prepared by MIP technology can be conveniently used in, if the chemical constitution that the solid phase of EBA system or bed granule expose is similar to the template for the preparation of described MIP according to this point of the present invention.

By in conjunction with magnetic particle separation

Dynal (Invitrogen) and other companies have developed the technology with paramagnetic beads purification of soluble molecule, typical shla molecule is as albumen, peptide or DNA, but such as combining by specific bond the antibody be fixed in paramagnetic beads carrys out isolated cell and organelle is also suggested.Larger pearl M450 (450 μm) is recommended to be separated granular materials as cell.After carrying capture molecules normally the paramagnetic beads of the antibody that epicyte protein is special and target such as cell or other granular materialss having contacted one suitable period, fixed by the magnetic field applied to shuttle by the paramagnetic particle of antibodies on cell.When paramagnetic particle is fixed, unconjugated cell can be washed off, when being released except paramagnetic particle during demagnetizing field.These pearls can obtain from such as Dynal business, and have different types of activation such as tosyl, epoxy radicals, carboxyl and amino, these groups can be used for coupled antibody or other capture molecules, the template molecule of such as MIP synthesis.

By coupling template such as cholesterol or cholic acid in such magnetic-particle template, reactive MIP will be attached on granule, by applying magnetic field from weak binding or do not separate in conjunction with the MIP of template, the same with the cell isolation method carrying special memebrane protein.

Coagulation is separated

Coagulation is when molecule and granule or cell establish a kind of phenomenon occurred when multivalence interacts; this effect is with the cancellated formation with altered dissolubility or suspension characteristic, and network structure can be detected such as by change or the microscopy of optical characteristics as a result.Coagulation mainly fast on the spot medical care detect in for diagnostic purpose.Promote such as crosslinked between hemocyte solubility element normally bivalence or multivalent antibody (P1a etc.), agglutinin or to the specific antigen of application (Rogers etc.).

Expose multiple template or the template class shla molecule like molecule, the tree (dendrimeric structure) such as provided by such as cholesterol or cholic acid, can be used to collect by such as mechanical agitation, mix to the end end of to, rock, ultrasound wave, the mode such as liquid phase flowing keep MIP that is liquid or that suspend.The MIP of template reaction interacts with the template molecule that exposes on such as dendrimers, and be preferably formed network structure, the tree wherein exposing template will as cross-linking agent.

According to both notable differences in density, size, shape, optical characteristics, by centrifugal, sedimentation, filtration, flow cytometry, to catch or according to other separate modes of size and/or weight, density, shape, color, light emission, light scattering, spreading factor, by the MIP granule do not connected be integrated into agglutinator or cancellated MIP particle separation comes.Subsequently, by applying pressure to agglutinator or network structure, as heating, organic solvent, shake or by adding excess soluble template, extracting template reaction MIP granule is individual particle.

relate to the micronized embodiment of MIP

Preparing in the trial of the special MIP of cholesterol before from prior art, capacity is limited in 17mg cholesterol pr.g MIP, but the non-trace MIP prepared with same method is in conjunction with 13mg cholesterol pr.g MIP (Sellergren 1998).Another trial obtains lower capacity, lower than 1mg cholesterol pr.g MIP (Whitcombe 1995).

The present inventor has found obtaining in gratifying binding capacity, and these prior art problems are because do not have the effective zygote in enrichment MIP particulate composition.

The simplest model of MIP template binding capacity is that pure area is considered.The area that template (such as cholesterol) occupies, as the function of MIP particle surface area, can with making decision particle size and in conjunction with the guidance of the demand of usefulness (percentage ratio that MIP surface area is covered by single template).

Theoretical consideration

The following calculated examples shown in result is below about spherical polymer particles cope match-plate pattern cholesterol.The molecular diameter of cholesterol is set as (1.6nm).

The area A that target (cholesterol) covers can be considered to a circle.This area is set as

A _t=π × r _t ²(r _tthe molecular radius of target)

The area of ball (MIP granule) is set as:

A _mIP=π × d _mIP ²(d _mIPthe diameter of MIP ball)

Needing how many MIP (qualitatively) with the cholesterol in conjunction with q.s to estimate, needing the density of polymer used.The density selected also will be subject to specific restriction.

The binding capacity of MIP is can in conjunction with given quality (m _mIP) the quality (m of target of MIP _t):

$\frac{m_T}{m_{\mathrm{MIP}}}=\frac{n_T\·M_W^T}{V_{\mathrm{Part}}\·{\mathrm{\ρ}}_{\mathrm{MIP}}}=\frac{\frac{A_{\mathrm{MIP}}}{A_T}\·\mathrm{CA}\·M_W^T}{V_{\mathrm{MIP}}\·{\mathrm{\ρ}}_{\mathrm{MIP}}}$

CA represents the area that MIP surface is covered by target.

This also can be reduced to further:

$\frac{m_T}{m_{\mathrm{MIP}}}=\frac{M_W^T\·\mathrm{CA}}{N_A\·r_T^2\·\mathrm{\π}\·r_{\mathrm{MIP}}\·{\mathrm{\ρ}}_{\mathrm{MIP}}}$

This provides the result expected intuitively with mathematical formulae:

● less target size provides larger capacity

● less MIP granule provides larger capacity

● less MIP density provides larger capacity

With reference to the description of the MIP particle surface of the binding characteristic between individual particle

If suppose to add representational 50mM template molecule, be evenly distributed in before polymerization in cumulative volume, the theoretical values as the MIP granule cope match-plate pattern molecule of particle size function can be calculated.The numerical value of template molecule, and in the granule such as pulverized at given volume, possible binding site numerical value can describe by the standard profile with given standard deviation.This does not affect difference between granule in theory, wherein granule s _xstandard deviation between upper two sites is given as:

$s_x^2=\frac{\mathrm{\Σ}{(\mathrm{\Δr}-{\mathrm{\Δ}}_r^{-})}^2}{n-1}$

Wherein Δ r is the actual range between two sites, be the given intermediate distance between two binding sites, determined by the distribution of granule cope match-plate pattern molecule.N represents the binding site numerical value of each granule. reduce with the particle size reduced with n, s _xcan not be changed by the micronize of granule.Only have and be namely less than 10 when granule is minimum ^-8time m (see table 1), occur that every granule is on average less than 1 template molecule, granule will there will be large difference.In fact this species diversity " upwards " " propagation " may be greater than 10 to comprising ^-8the granule of m, but the increase with particle size reduces by difference, can become inessential when having the situation of larger particles of a lot of binding site.

On the other hand, the orientation of template molecule will produce difference.If the longitudinal direction of template molecule and granule are vertical, only have two orientation directions in principle, rotate even if reason is template molecule in upright position around the longitudinal axis of itself, we also suppose that this both direction can not produce different binding sites.But, if the longitudinal direction of template molecule is adjusted to parallel with particle surface, when template molecule rotates around the longitudinal axis of itself, will unlimited many different binding sites be produced.In other words, the numerical value of degree of freedom (position that template molecule may occupy) is unlimited in this case, rotates the often kind of new orientation produced be different from every other binding site by producing in principle around longitudinal axis.In a word, this means that the orientation of template molecule produces the different binding sites of unlimited numerical value.

These binding sites can be characterized by they binding constant Kd to template molecule.Even if some different orientation will produce the binding site of identical Kd undoubtedly, but also can there is the binding site of very different K d, this point below can be clearer.

Result and estimation

Table 1 shows

● on the spherical shell that 0.9nm is thick, as the calculating of the template molecule theoretical values of particle size function.

● expect the calculating of the total value of the binding site found.

● the calculating of the expectation numerical value of high affinity combined sites.

Last two row numerical value be based on the observation delivered (Kempe & Mosbach 1991, deng 1994,1996I & 1996II, Liu, Mosbach 1997 & 1998, Andersson etc. 1995) calculate.The thickness of spherical shell is selected to be assumed to be the radius (Davidson & Hayes, 2002) of the dynamic volume had as the molecule such as cholesterol molecule that length is 1.8nm.Typical MIP every gram containing 20 μm of ol binding sites (Kempe & Mosbach 1991, deng 1994 & 1996I, Liu, Mosbach 1997 & 1998), but consistent with hypothesis above-mentioned, and the change of binding constant (Kd) is very large, and mobility scale is 10 ^-3to 10 ^-9m; Share shared by high affinity combined sites be typically less than total binding site numerical value 1% ( deng 1996II, Andersson etc. 1995).Such as, deng 1996II, describe the MIP of anti-different corticosteroid (such as demonstrating the molecule with cholesterol analog structure), wherein high affinity combined sites (< 10 ^-6m) share shared by is 0.075% and 0.28% respectively.In the calculating of table 1, the numerical value of high affinity combined sites is assumed to 0.5%.In the article of reference here, particle size is 25 μm typically.This size is relatively easily obtained by process polymer in manual mortar (manual mortar).

Table 1

Binding site numerical value on thin layer spherical shell is as the description of particle size function.

In practice, the granule of 10 μm can obtain the maximum of binding site, is 3,700,000 (table 1).If it is after the granule of 1 μm that the granule of 10 μm is pulverized, each granule of 1 μm newly will have 37,000 binding site, namely former 10 μm of granules 1%.The binding site of 10 μm of granules is distributed on 100 new 1 μm of granules like this in principle.Because there is binding site that in a large number may be different to exist, the new 1 μm of granule produced is bound to variant, they each only containing on 10 μm of granules 1% " difference " binding site.If only use the part of high affinity combined sites and 0.5% of total value, obtain same result.But when the numerical value of binding site becomes less, this effect (describing in part 1) can cause binding site little, may facilitate the diversity between individual particle further.

The boundary that " hole " on granule is defined as binding site is controversial, but Kd is greater than 10 ^-5the binding site of M almost has nothing to do in the use for the treatment of use.The Kd value that American market is approved as the monoclonal antibody of medicament is less than 10 ^-7m (Carter 2006).

The distribution of granule (given associated standard deviations) cope match-plate pattern molecule will depend on further, such as during polymerization the temperature of mixture, viscosity, template molecule size, with the parameter such as the interaction of solvent and other monomers, but these parameters are not that to describe the same mode of position and directed assessment as used herein general.With our viewpoint, utilize these procedure parameters by relevant to the difference increased between pulverized particles.

In superincumbent discussion, the numerical value of degree of freedom is only with reference to the position of single template molecule; Suppose directly to reflect distribution that is high and low-affinity binding site with the numerical value of the degree of freedom of template molecule directional association.Because the particulate composition of planned production high specific and capacity, we will adopt suitable high stringency in separation, so that main sorting and select high affine binding site.When correlation values and high affine binding site reach makes this parameter (numerical value) also must be conducive to the size of diversity hardly, but reasonably estimates it may is about 1 μm.

If expect to expose the more multiple binding sites more existing than particle exterior surface, have produce stone, have enough wide so that the method for the granule of the groove not having diffusibility to flow through with limiting for liquid.This method is being called as Poros ^tMuse in the tomographic system (such as Applied Biosystem provides) of substrate, wherein can have fast flow velocity and not affect resolution, because liquid is consistent with circumgranular liquid by the flowing of granule.In more traditional chromatography (namely not comprising HPLC) substrate, flow velocity be limited in enter single matrix granule diffusion velocity on.

If expect the contact utilizing intestinal peristalsis promoting to increase the MIP be combined with cholesterol or cholic acid, trough of belt granule can have superiority.If utilize gel, " exchange " of intestinal juice enters being likely limited in the diffusion rate of gel.Produced MIP gel, main purpose is drug delivery system (release of such as insulin), and delivery system is worked by the opening of the gel when glucose is attached to specific site.Which show and also really likely produce MIP with gel structure.(Wizeman & Kofinas 2001, Seong etc. 2002)

The practical operation of micronize aspect

Therefore, from clearly finding out above, a second aspect of the present invention has derived from a kind of preparation method target molecule to high binding capacity and specific MIP, described method comprises: will comprise the former MIP (namely extract template substantially or do not take the polymerization crosslinking MIP of micronize MIP structure) of the template molecule be made up of described target molecule or its analogies, through first step micronize so as the MIP granule obtained enough little thus can remove/extract template molecule, remove/extract all template molecules substantially, optionally by the MIP that obtains like this through second pelletizing step, wherein said first and optionally second pelletizing step make the average diameter of MIP mostly be 25 μm most.Therefore, through these processes, the MIP obtained has volume and exposes the higher ratio of binding site.Generally for the little MIP particle size obtaining expecting; only with first pelletizing step just enough (the simplest); because permission is removed template molecule by this, but can imagine that wherein having interted two pelletizing step of template removal step will be conducive to the situation that be separated of template with MIP.

Preferably, the average diameter through the MIP of pelletizing step is less than 20 μm, such as, be less than 15 μm, is less than 10 μm; be less than 5 μm, be less than 1 μm, be less than 900nm, be less than 800nm; be less than 700nm, be less than 600nm, be even less than 500nm, 400nm, 300nm and 200nm.

Preferably, no matter any ratio, the MIP in the given compositions after micronize does not comprise the granule that diameter is greater than 50 μm (such as diameter is greater than 40,30,20,10 or 1 μm) substantially.

That is, micronize such as can be pulverized, grind, be exploded by any suitable method minimizing MIP size, hammering, ball milling, cryogenic grinding and the method for collision homogenisation and the combination of these methods any obtain.

From clearly drawing above; the present invention's very important embodiment needs the combination of the present invention first and second aspect, namely needs the in question affinity purification method according to the first preparation of the micronized MIP to little MIP particle size of the present invention and first aspect present invention subsequently.

At most of conditions, MIP in tomographic system for analysis purpose or in sensor (sensor) as the substitute of albumen.The size of MIP granule is usually the scope of 25 to 100 μm in such applications.Just as detailed above, such MIP size is too large for the binding capacity obtaining when being used for such as oral MIP compositions expecting, and these large MIP do not select/purification by traditional cell purification method.

So in order to obtain suitable capacity, the size of MIP granule is very crucial.According to above, the method the most simply increasing the capacity of given mass M IP is, by increasing area to the ratio of volume namely, makes MIP granule less and/or increase " activity " area (can in conjunction with the area of MIP of expectation analysis thing).Area increases with the inverse increase of diameter the ratio of volume, and when namely diameter reduces by half, the ratio of area to volume doubles.This means that the binding capacity then obtained can increase by 64 times by the MIP particle diameter of every mass unit is reduced to 0.4 μm from 25 μm.

Table 2: area is to the function of the increment of the ratio of volume as particle diameter.The particle diameter of 25 μm is as the index of increment.

Can draw according to the present invention, by the EBA system of the template with coupling bed granule functionally selected/purification, diameter can be optimized further at the capacity (bound fraction area) of the MIP of 0.5 μm of scope, see the discussion of first aspect present invention above.These MIP granules and the known cell that can be separated by surface character in EBA system are in same size scope.In order to reach the little particle size of expectation, it is necessary for pulverizing by multiple method/grind, because diverse ways optimum is applicable to, between different size field, vide infra.

In order to obtain the particle size expected from bulky polymer, utilization is often needed to exceed a kind of Ginding process.Technology existing at present can not be common to by all polymer from mm size be ground to lower than μm size.

By inserting with the water-swelling material of polymer-compatible and utilizing method (the such as Cryo-Grind becoming humidity freeze ^tMtechnology) size reduce effectively to be broken into by polymer the more granule of about 200 μm.In order to easily change this process, the loose structure of polymer can produce by the method adding fiber (such as cellulose, cellulose acetate) in the polymer.

Can produce little range of polymers by high template concentrations, described little range of polymers can provide high binding capacity and be easy to the polymer of the fragile structure reducing size.

Applying nano grinding/pulverize (such as NETZSCH ball mill), a kind of can by top reduction size (down) method of the particulate abrasive of 20 μm to 40-200nm size, can obtain reduce further size extremely lower than μm granule.(granule of these types is generally used for drug products).

When reducing size, spray by air, the liquid jet or similar mode accelerate MIP, by this MIP at a high speed and carrier current impact solid target, MIP being broken into less granule with force of explosion also will be the possible method reducing MIP size.

Finally, as example display, such as use simple mortar with the physical grinding of MIP, prove MIP effective.

belong to of the present invention to further consider

For the selection of the polymer of MIP

In order to obtain better cohesive between two kinds of materials, wetting tension must be very low, namely must not act on the repulsion of part absorption on polymer surfaces.In order to select the suitable material of MIP, Hansen Solubility Parameter (HSP) (see Hansen CM) will be considered when selecting the monomer being applicable to polymer.

In the case of cholesterol, the HSP of deterministic compound, can find by Study Polymer Melts table the good selection that very hydrophobic polymer will be the MIP preparing energy combined cholesterol.HSP is cholesterol (delta D, the delta P, delta H, R) = (20.4, 2.8, 20.4, 20.4), polymer and cholesterol for MIP HSP ball overlapping list is high density polyethylene (HDPE) > of polyvinyl chloride (PVC) > polyacrylonitrile (PAN) polypropylene (PP) > > polytetrafluoroethylene (PTFE) > poly vinyl acetate (PVAc) > polystyrene (PS) > poly (butyl methacrylate (PBMA) > polycarbonate (PC) > polystyrene - methyl acrylic acid copolymer (PS/PMAA) > polyethylene terephthalate oxalic ester (PETP) > polyurethane (PUR) > polystyrene acrylonitrile (SAN) > polymethyl methacrylate (PMMA) > polyamide such as nylon 66 > poly (vinylidene fluoride (polyvinlydiflurid) (PVDF) > polyvinyl alcohol (PVA).These not all polymer are all easy to cross-linking agent together with matrix polymerization, but list is above only select monomer and the useful starting point of cross-linking agent, thus provide optimal polymer.In order to mate the HSP of any given target ligands, can make copolymer, block polymer and block copolymer (etc.).

Template extraction

From MIP remove template must reduction dimension process after, complete before purification step.If need cooling in reduction dimension process, the solvent fully dissolving template will be the fabulous selection of cooling medium.

Template is removed and by utilizing solvent and solvent mixture separately, or can be combined heating, increase or reduce ionic strength.Conventional method is a soxhlet extraction, the wherein MIP solvent of fresh distillation (if or azeotropic mixture be mixture) semi continuous cleaning.If product (with crosslinked) MIP is resistant to elevated temperatures (template molecule is not), pyrolysismethod destruction template can be applied before extraction and make it to be easy to remove, such as, by using the solvent being seldom used for dissolving template.

the present invention prepares the comprehensive discovery of aspect

The present inventor recognizes, in order to prepare the MIP of high power capacity, need to adopt careful select MIP preparation, micronize, template removal and useful MIP the combination of selection, and the special concern to micronize and selection technique.

Therefore, if by MIP preparation and the traditional method of purification and cell and/or the known selection of albumen career field and purification process combination, first-class result just can be expected.

An example is: in MIP technology, and " selection of monomer ", micronize, extraction and screening are by combined function purification such as EBA.According to the ability on the functionalization of matrices/surface in conjunction with expanded bed of MIP granule come sorting they, traditional " upstream " MIP produces and selects and purification step complementation with " downstream ".

The simplest flow journey of above-described anabolic process is shown in Fig. 2.

The representative example of the useful target molecule (or template molecule) of MIP compositions of the present invention is the molecule found in such as human gi-tract in the gastrointestinal tract.Especially preferred target molecule is the molecule that pathology is relevant.Especially preferred target molecule is cholesterol through selecting or cholic acid or cholate, but toxicant, toxin (comprising antibacterial, virus, fungus and parasite toxin) and the antigen found in pathogen such as antibacterial, virus, fungus and parasite and receptor are also the interested target/templates of MIP compositions prepared in accordance with the present invention.

according to compositions of the present invention and medicinal application

Can believe, at least some the MIP compositions obtained by the inventive method is novel combination material.Therefore, the compositions of the MIP that the present invention relates at least has the one of following characteristics:

1) average diameter of MIP is less than 20 μm;

2) average target combines and is at least 1 mass unit target in conjunction with 10 mass unit MIP;

3) all in compositions MIP are substantially in conjunction with same target molecule, but compositions does not comprise all binding sites to target molecule.

Preferably, the present composition meets 1,2 or 3 of these features, means that compositions may only have feature 1, one in feature 1 and feature 2 or 2, only has feature 2, feature 2 and 3, only has feature 3 or all 3 features 1,2 and 3.

Preferably, the average diameter of MIP is less than 15 μm, such as, be less than 10 μm, is less than 5 μm, is less than 1 μm, is less than 900nm, be less than 800nm, be less than 700nm, be less than 600nm, be even less than 500nm, 400nm, 300nm and 200nm.

Preferably, no matter any ratio, the MIP in the given compositions of the present invention does not comprise the granule that diameter is greater than 50 μm (such as diameter is greater than 40,30,20,10 or 1 μm) substantially.

The preferred compositions of the present invention in conjunction with any one target molecule above-described, namely with reference to " representative instance of useful target molecule " above.

Compositions of the present invention (with compositions prepared according to the methods of the invention) can be used as medicine, and method that can be roughly the same with antibody compositions uses.But because their stability, MIP compositions is suitable for oral medication, wherein unlike antibody and a lot of soluble protein receptoroid, they stablize constant to the proteolytic degradation maintenance in small intestinal.And based on the fact that MIP can be prepared by the material not through gastrointestinal tract epidermis, molecule/medium that they are correlated with at gastrointestinal pathology for target definite limitation is very useful.The target be applicable to is cholesterol, cholic acid and cholate, but the antigen/part in various toxicant or gastrointestinal tract, pathogen found also has probability.

But, if MIP is by be applicable to, prepared by biocompatible and/or Biodegradable polymeric (such as polylactide (PLA), PGA (PLG)), they also can be used as the outer medicament of intestinal, with use such as antibody and compare with soluble recepter, reduce and cause the immunoreactive danger of less desirable drug resistance agent.In such embodiments, the almost any target molecule that can be the target that antibody or soluble recepter are applicable to can become the target of MIP compositions of the present invention.

Therefore, in preferred embodiments, the present invention relates to a kind for the treatment of, alleviation or reduce the method for disease risks, this disease is selected from the infection that cardiovascular disease, hypertension, atherosclerosis, cholelithiasis, cholestatic hepatopathy, hypercholesterolemia, obesity, parasite, virus or microorganism such as antibacterial and fungus cause, it is poisoning that oral toxin causes, and the method comprises uses the present composition of effective dose or compositions prepared in accordance with the present invention to curee in need.This embodiment of the present invention also comprises the preparation of these compositionss for medicament, the infection that described medicament is used for the treatment of, prevents or alleviates cardiovascular disease, hypertension, atherosclerosis, cholelithiasis, cholestatic hepatopathy, hypercholesterolemia, obesity, parasite, virus or microorganism such as antibacterial and fungus cause, or oral toxin cause poisoning.Consider oral medication typically.

The every consumption per day of expectation of the present invention or MIP compositions prepared in accordance with the present invention is at most 40g every day, but due to the high target capacity of MIP compositions, also can consider less every consumption per day, such as every day maximum 30g, 20g, 10g, 5g and 1g.

The standard method that MIP can know according to industry technical staff is prepared, in particular for oral preparation, wherein expect that MIP uses with the form of powder, Emulsion, encapsulation emulsion, pill and tablet, but also can be used as the composition of food and be applied, wherein MIP dispersibles (disperged) in nearly all food or food.

The formula of the MIP in these compositionss, general with reference to Mark Gibson, CRC press, 2001, it is incorporated to herein by reference.

Certainly, can be used for the application of all types according to MIP compositions of the present invention and MIP compositions prepared in accordance with the present invention, wherein MIP is proposed in the prior art as specific binding partners.So even if the present invention concentrates on the medical application of MIP compositions of the present invention, this does not get rid of the application of MIP compositions disclosed herein in known analytical equipment itself and method.Therefore, the present invention also comprises the quantitative or qualitative method determined of sample target in its scope, the method comprises and being contacted with the present invention or compositions prepared in accordance with the present invention by sample, wherein MIP specific bond target molecule in compositions, carries out quantitatively or qualitatively assessing the target molecule in conjunction with described compositions subsequently.In context, the disclosure with reference to the common preparation relating to MIP above and use is all incorporated to herein by reference.

The foreword of embodiment

Make the commonsense method of molecularly imprinted polymer

Follow General reactions method and prepare MIP, functional monomer is mixed with microsphere and cross-linking monomer in the solvent be applicable to.The selection of monomer, according to the ability of it and microsphere coordination, is the technology of those skilled in the art's routine.Polyreaction, from adding applicable concentration initiator, uses such as ultraviolet (for ultraviolet initiator) or heating (for can pyrolysis initiator) disturbance to start subsequently.

Expire micro polymer firmly crisp to (usually) granulation after polymerization the size of hoping, microsphere, removes by extracting in conjunction with monomer, cross-linking agent and initiator scrap, and this extraction is by directly to clean and/or by by one period of preset time of solvent refluxing.

Embodiment 1

The MIP of template is made with fluorescein

In the flask of 100ml, 1.4ml methacrylate (MAA) monomer, 9.5ml ethylene glycol dimethacrylate (EGDMA), 50mg fluorescein and 10ml oxolane (THF) mix 30 minutes in hot bath (counting roughly 40 DEG C).Slowly add 2g 1,1-azo two (cyclohexane extraction-nitrile) (ACHCN).Solution argon (THF is saturated) cleaning 15 minutes after dissolving.Polymerization irradiates initiation in 48 hours by continuous print ultraviolet light (365nm, 9W).The polymer obtained is yellow fragility, and in manual mortar, micronize makes particle size between 10 μm and 25 μm.Powder refluxes 30 minutes in THF, cleaning and filtration several times in ethanol/THF (75: 25).Air-dry white powder.

Embodiment 2

The MIP of template is made with cholic acid

In the flask of 100ml, 1.4ml methacrylate (MAA) monomer, 9.5ml ethylene glycol dimethacrylate (EGDMA), 2g cholic acid and 12ml oxolane (THF) mix 30 minutes in hot bath.Slowly adding 0.2g 2,2 '-azobis isobutyronitrile/2,2 '-azo is two-(2-methyl propionitrile) (AIBN).Solution argon (THF is saturated) cleaning 15 minutes after dissolving.Polymerization irradiates initiation in 24 hours by continuous print ultraviolet light (365nm, 9W) in ice bath.The polymer obtained is yellow rigidity, and in manual mortar, micronize makes particle size between 25 μm and 50 μm.Powder refluxes 30 minutes in THF, cleans and filter 4 times in ethanol/THF (75: 25).Be bordering on the powder air dried overnight of white.

Embodiment 3

The MIP of template is made with cholic acid

In the flask of 100ml, 2.8ml 2-(dimethylamino)-Ethyl-Methyl acrylic acid (DMA-EMAA) monomer, 9.5ml ethylene glycol dimethacrylate (EGDMA), 2g cholic acid and 12ml oxolane (THF) mix 30 minutes in hot bath.Slowly adding 0.8g 2,2 '-azobis isobutyronitrile/2,2 '-azo is two-(2-methyl propionitrile) (AIBN).After dissolving, solution argon cleans 15 minutes.Polymerization irradiates initiation in 24 hours by continuous print ultraviolet light (365nm, 9W) in ice bath.The polymer obtained is bordering on white, fragility, and in manual mortar, micronize makes particle size between 10 μm and 25 μm.Powder refluxes and reaches 30-60 minute 2 times in THF, cleaning and filter 23 in ethanol/THF (75: 25).White powder air dried overnight.

Embodiment 4

The binding capacity of independent MIP granule

In order to assess in powder binding capacity/specific difference between MIP granule, be provided with the Binding experiment of a most simple form.MIP in testing example 1 is to the binding ability of fluorescein.

The MIP of 1.9mg to fluorescein is suspended in 380 μ l ethanol (96%), adds 5 μ l luciferin solution (in ethanol 0.05mg/ μ l) and mixes 5 minutes.Centrifuged suspension removes supernatant.Then the ethanol purge granule 3 times of 300 μ l is used.Washed granule is applied on micro-glass plate, carries out volumetric analysis by the granule (whole granule) under visual counting white light and the granule (in conjunction with MIP granule) that shows green fluorescence when being exposed under ultraviolet light (365nm).Owing to using ultraviolet light, launch optical filter to see that green fluorescence does not need to use.

The photo of Fig. 4 display is the overlapping figure of two photos using white light and ultraviolet light shooting.White point is the granule by white light, and stain is the green fluorescence granule with ultra-vioket radiation.As can be seen from Figure 4 granule (white point) number of white light exceedes the green fluorescence granule (stain) when being exposed to ultraviolet light.Numbers of particles difference visual under two kinds of Different Light shows that micronize MIP causes creating a group in conjunction with MIP granule and a group in conjunction with MIP granule (i.e. residue); Therefore the selection in conjunction with MIP granule based on affinity characteristic effectively will increase binding capacity, and binding capacity every unit of weight MIP used weighs the binding ability of used template.

The photo of display can not present the green fluorescence of (intensity) in various degree irrelevant with particle size by our the green fluorescence granule observed of exemplary illustration.This phenomenon conforms in conjunction with the difference of binding ability between MIP granule from different.This intensity (fluorescence) difference again exemplary illustration MIP granule has different binding abilities really; Therefore the increase of the capacity of MIP realizes by selecting the MIP granule (in this embodiment) of maximum intensity fluorescence.

Here conclusion is, the present embodiment shows that the binding capacity of MIP particulate composition can increase in conjunction with granule by removing.Binding capacity has high-affinity by enrichment and/or multiplely can to improve further the MIP granule of binding pocket of exposure of binding target molecule.

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Claims (35)

1. prepare the method comprising and have the high binding capacity of target molecule and the compositions of specific molecularly imprinted polymer (MIP), described method comprises:

A) obtain the float of the insoluble MIP of binding target molecule, it is prepared as template molecule with described target molecule or target molecule analogies,

B) by the MIP that suspends through affinity purification method, wherein said template molecule or its fragment or its analogies are used as trapping agent,

C) be recovered in the MIP in conjunction with described trapping agent in affinity purification method, from described recovery product, remove described trapping agent and not in conjunction with the MIP of described trapping agent, and

D) by the MIP of recovery and optional carrier, excipient or mixing diluents, to obtain described compositions.

2. method according to claim 1, the covalently or non-covalently coupling of wherein said trapping agent and solid phase.

3. method according to claim 2, wherein said solid phase is selected from by cross-linked carbohydrate, synthetic polymer or its substrate composed group of combining.

4. method according to claim 1, wherein said trapping agent is made up of soluble chemical entity or the part of soluble chemical entity.

5. method according to claim 4, wherein said trapping agent and the some covalent or the non-covalent associations that are selected from dendrimers, the carbohydrate of replacement and the soluble polymer of replacement, object is in order to often kind of soluble chemical entity exposes multiple trapping agent.

6. method according to claim 5, the soluble polymer of wherein said replacement is polyvinyl alcohol or Polyethylene Glycol.

7. the method according to aforesaid arbitrary claim, wherein said trapping agent only with step a) in combine in conjunction with the part binding site in the MIP of described template molecule.

8. method according to claim 1, wherein said trapping agent is the fragment of described template molecule.

9. method according to claim 1, wherein said trapping agent comprises adopting and is anyly all attached to the template molecule of solid phase surface or its analogies or its fragment, to avoid the part exposing described trapping agent to described MIP with specific orientation.

10. method according to claim 1, wherein said trapping agent comprises and adopts any template molecule or its analogies or its fragment that are all attached to part with specific orientation, to avoid the part exposing described trapping agent to described MIP.

11. methods according to claim 1, wherein said trapping agent comprises adopting and is anyly all attached to the template molecule of solid phase surface or its analogies or its fragment with unspecific orientation, so that all parts exposing described trapping agent are to described MIP.

12. want the method described in 1 according to right, and wherein said trapping agent comprises and adopts any template molecule or its analogies or its fragment that are all attached to part with unspecific orientation, so that all parts exposing described trapping agent are to described MIP.

13. methods according to claim 1, wherein said affinity purification method is selected from the group be made up of expanded bed adsorption (EBA), paramagnetic beads separation, hollow fiber purification and coagulation.

14. methods according to any one of claim 1-6 and 8-13, wherein said target molecule is the molecule found in gastrointestinal tract.

15. methods according to claim 14, wherein said gastrointestinal tract is human gi-tract.

16. methods according to claim 14, wherein said target molecule is that pathology is correlated with.

17. methods according to any one of claim 1-6,8-13,15 and 16, wherein said target molecule is selected from cholesterol, cholic acid and cholate.

18. 1 kinds of methods preparing the MIP of the high binding capacity had target molecule, described method comprise by comprise the template molecule be made up of described target molecule or its analogies former MIP through first micronized step to obtain enough little MIP particle size, thus can template molecule be removed, remove all template molecules, optionally by the MIP that obtains like this through second pelletizing step, wherein said first pelletizing step and optionally second pelletizing step make the average diameter of MIP be 50 μm to the maximum, subsequently by the MIP that obtains like this through the method process described in any one of claim 1-13.

19. methods according to claim 18, wherein only perform first pelletizing step.

20. methods according to claim 18 or 19, wherein said micronize is realized by the mode of pulverizing, grinding, blast, hammering, ball milling, cryogenic grinding or collision homogenisation.

21. methods according to claim 18 or 19, wherein said target molecule is the molecule found in gastrointestinal tract.

22. methods according to claim 21, wherein said gastrointestinal tract is human gi-tract.

23. methods according to claim 21, wherein said target molecule is that pathology is correlated with.

24. methods according to any one of claim 18,19,22 and 23, wherein said target molecule is selected from cholesterol, cholic acid and cholate.

The compositions of 25. 1 kinds of insoluble MIP, MIP all in wherein said compositions is in conjunction with same target molecule, and optionally described compositions does not comprise all binding sites to target molecule.

26. compositionss according to claim 25, the average diameter of wherein said MIP is less than 20 μm.

27. compositionss according to claim 25 or 26, wherein average target is combined at least 1 mass unit target in conjunction with 10 mass unit MIP.

28. compositionss according to claim 25 or 26, it is method preparation according to any one of claim 1-24.

29. compositionss according to claim 25 or 26, MIP combined cholesterol, cholic acid or cholate in wherein said compositions.

30. compositionss according to claim 25 or 26, it is used as medicine.

31. application of compositions in useful in preparing drug formulations according to any one of claim 25-29, described pharmaceutical preparation is used for the treatment of, prevents or alleviates cardiovascular disease, hypertension, atherosclerosis, cholelithiasis, cholestatic hepatopathy, hypercholesterolemia, obesity, the infection that caused by parasite, virus or microorganism, or oral toxin cause poisoning.

32. application according to claim 31, wherein said microorganism is antibacterial.

33. application according to claim 31, wherein said microorganism is fungus.

34. application according to claim 31, wherein said pharmaceutical preparation is Orally administered.

35. 1 kinds to the quantitative or qualitative method determined of sample target, described method comprises and being contacted with the compositions according to any one of claim 25-30 or the compositions prepared by any one of claim 1-24 by sample, target molecule described in MIP specific bond in wherein said compositions, carries out quantitatively or qualitatively assessing to the target molecule in conjunction with described compositions subsequently.