PILETSKA OLENA (GB)
GUERREIRO ANTONIO (GB)
WHITCOMBE MICHAEL (GB)
POMA ALESSANDRO (GB)
WO2011067563A1 | 2011-06-09 | |||
WO2006129088A1 | 2006-12-07 | |||
WO2010092071A1 | 2010-08-19 | |||
WO2001032760A1 | 2001-05-10 | |||
WO2010081076A2 | 2010-07-15 |
CLAIMS : 1. Ά process for producing nauopartieles of. moleeularly imprinted olyme s comprising: (.1.) i««Tiofo.iiisi,ng a template species on a solid S support to provide a supported template material; (ii) iodating the supported template material, in a reaction vessel having one or more flow inlets,- one or more flow outlets, a cooler/heater: adapted to- affect the temperature of the vessel's contents, and radiation8 source adapted to irradiate the vessel rs: interior (ill) passing a poiymerisafeie composition into the vessel througn. one or more of said flow inlets; (iv) causing the radiation source to irradiate the vessel's interior to effect controlled polymerisation of the polymerisable composition,- while using the cooler/heater to control the tempera nre: thereof; (v) ceasing irradiation at a stage when polyme isation has generated nanoparticles it.h molecular weights in the range 500- 1 , 000 ? 000 Da, at least some of the nanoparticles being bound to the immobilised template; (vi) passing solvent through the vessel via one ox more of said inlets and one or more of said outlets to wash out onreacted components of the polyme isafole composition and unbound polymers; and (vii) passing solvent through the vessel via one or more of said inlets and one or more of said outlets under conditions such that at least some of said bound nanoparticles are separated from the immobilised template and passed out of the vessel to a collection vessel, said conditions comprising one or more of increased t ec <:.:.;:<·.; ! ,,r- : produced by the cooler/heater f and use of a solvent composition that is able to disrupt binding of nanopartielea to template. . A process of cl im 1 wherein step (vii) comprises a first step (a) in which conditions are selected so that nanopartiol.es bound relatively loosely to template are separated and passed oat to a firs said collection 10 vessel; and a second step bid i which conditions are selected so that more tightly bound nanopartioies are separated and passed oat to a second collection vessel, 3< A process of claim 2 wherein the polymerisation step id (iv) is carried out with control of the temperature in the range -20 to 20°; said first step (vii} (a) is carried out with control of the temperature in the range 20 to 40¾; and said second step (vii) (b) is carried out with control of the temperature in the range 60 to 80°, 0 4, A process according to any preceding claim wherein steps {ill; to (vii) are carried out under computer control . 5 5, A process according to claim 4 wherein the polymerisation conditions are controlled to produce particles of mean diameter 40-100 ma. β. A process according to claims 4 or a wherein the polyme isation conditions are ontr lled to produce pa ticles whereof at least 50% ha-ve. diamete s in the range 4u~io0 or-. 7. A process of.' any receding claim wherein tie polymerisation is initerter-basel living radical polycssrisation . 8, A process according to any preceding claim, including a subsequent step of re-binding' nanopartioias: separated in step Cvii) to immobilised1 template and subsequ ntly separating t em therefrom, 9, A process according to any preceding claim including a subsequent step of subjecting the separated nanoparticles to further polymerisation to graft block copolymer segments onto them. 10, A process according to any preceding claim including a subsequent step of using the separated nanoparticles as a drug, as a receptor-specific iigand for analysis or separation, o as a catalyst. 11, A photoreactor for producing nanoparticles of s-iolecuiarly imprinted polymer comprising: a reaction -vessel for holding a solid phase bearing an inci'sobi Ileal template species; a radiation source ortanged to irradiate the interior of ids reaction vessel/ a fl.oidic sysiem in communication wi h tea regctioa vessel for providing an inflow comprising olymer aable monome (s) and a outflow containing synthesiaed nanoparti else; and a computer progra Kied a d arranged lor controlling the product ion. process. ' 12, ¾ photoreaeto according to claim 11 wherein, the reaction vessel contains the solid phase bearing an. iiffiiiob11i seel templa e seecie's , 13. Ά photoreaetor according to claim- 11 or claim 12 including a reservoir containing said polymerisahle corcoov '.' 1 r } coupled to said fluidie system, 14. ¾ photoreaetor according to any of claims 11-13 wherein the reaction vessel is provided with thermostatic control means , 15. A photoreaetor according to any of claims 1.1-14 ale re ha the radiation source is a OV source. 16. A photoreaetor according to any of claims 11-15 adapted to carry out a process according to any of claims l-lOd |
The term "template-directed synt esis'' includes the formation of a ew substance by c emcal modification of a sebstrste, or by the coupling of two or ore i¾o.iecules ; in the presence of a template hich serves as a pattern for new structure formation, a specific example is
"molecular i rinting", based on the polymerisation of vinyl or acrylic monomers in the presence of template or templates (see ret " , 1, 2 } , The traditional approach involves the production of highly cross-linked imprinted polymers, which are insoluble in aqueous and organic solvents * Because of their inherent insolubility, the possibility tc use molecn laxly imprinted polymers (tPIPs) in pharmacology ; and medicine is restricted. Recently, several attempts hare been made to da reicy. protocols fox the preparation of imprinted polymers with relatively ice- co i ecu.; a weights which could exist in soluble or at least colloidal forms . This format will allow polymers to be used as biologically active molecules {drugs, effectors, modulators; inhibitors) in pharmacology and medicine, and as A plastic antibodies'" f replacing:
biological molecules in sensors and in affinity
separation and as catalysts with enzyme--.like properties,
One way to synthesise soluble blips is by controlled:
polymerisation, Controlled polymerisation is defined here as a process which leads to the format ion of
fractions of soluble polymexs with relatively arrow distribution of molecular si es, Typical exaraples of controlled poiyrerisation employ loving polyme isation, 5 as: described below.
The living, free-radical polymerisation techniques f such as iniferter polyiaerisat.ion, ni tro ide -emulated radical pel yiperisation , atom-transfer radical polymerisation
!ø {&TRP} a d reversible: ad.ditioe--vtragmentatiorj chain- transfer. ;R¾F?) polymerisation, open new routes for the synthesis of polymers with relatively controlled
molecular weights (see ret. 3-91. dontrollsd/iivbsg polymer!sotion techniques are based on a delicate- balance
IS between dormant and active species that effectively
reduces the concentration of propagating entities in the system and ruinimizes the extent of termina ion- Living polymerisation could be free of side reactions such as termination and chain transfer and thus can generate0 polymers with well defined molecular weight distributions and structures. The same approach can be applied to the formation of copolymers, thus making it possible to produce block copolymers by free radical polymerisation, by proper sequencin of the mon.ois.er additions. LivingS polymerisation has been used Previousl in the production of bulk grafted Miss (see e . 10, ill. Soluble polymer were also produced, by living polymerisation and used later in T production (see refe 12} * Recently, controlled living polymerisation was used for the
0 preparation of ΗΪΡ nanopartici.es (13) . One of the complicat ions in MIP synthesis is the: frequent need to use templates w ic are e pansive and/or
difficult to obtain, such as proteins, sose toxins etc. Such templates are also difficult to recover aft
5 olyme isation and limit the amount of Ml? that can be obtained, Ideally the terspiate should be capable of being re-used to -overcome these limitations. The optima 1 way to achieve thi is b using the template in an
.imm.obilfsed form, immobilised template has been used
10. previously (see US 7393909) , Here the template was
im-tobiiised Onto a silica surface and then polymer was formod in pores around it. By dissolving the silica support and removal of the tem late f HlPs of various morphologies w re obtained. In all of the xam les
:?· disclosed in US 73: : h3909y the surface bearing the
ienobiiised template is lost during the dissolution process and cannot be re-used. In other examples immobilised templates were used fo the production of imprinted surfaces (US 6127154; US 6458599; US 7288415),0 Potentially the template-bearing surfaces disclosed in these reports can be regenerated and used several more times. These approaches can be used for the production of sensors or arrays, but would be difficult to adapt for the production of nanoparnicies or small soluble
5 molecules.
Yet another major problem: associated with hilts is heterogeneity of the binding sites produced, which is generall responsible for high levels o non-specific0 .binding. This problem has been solved by affinity
separation of MIP nanopaxtioies on a column bearing an immobilised target species or template after their product ' ion. (13) . It is clear that in order for affinity separation to e possible, Mlthr should foe in a suitable forso preferably in th foiso of nanoparticfes >
5 h recent invention, addresses t problems relating to the deoelopren.i of high per fosance croe¾-l inked MXF
nanoparticies b disclosing a combination of two
techniques: fi) performing controlled poiymerisatiorp optionall controlled radical poiyperisatiorp in the
50 presence of a surface or surfaces bearing imrrsohi iised
template to forr« imprinted nanoparticiea arid ii;
r t ining the: e no ' parnicies by affinity interaction wi h immobilised template for selection, and purif cation ' purposes. Thus cor earlier application Q 2011/067563 i.S discloses a process for the preparation of a molecoiariy imprinted polymer ( IP '<f in the form of a solution or colloidal suspension of KLIP particles, comprising the steps of:
(a) proYidfng a carrier substance having e template 0 material XT.OCO.;.1 isso on it so as to be exposed, at a
se face;
(b) providing a pcelymerisable eoio osition in contact with said surface;
ic) effec ing controlled polymerisation of said5 poiymerisable composition in contact with said surface, preferably by a living radical polymerisation process, said polymerisation being terminated when HIP particles, capable of forroiea a solution or colloidal suspension have been formed; and
0 {d} separating said HIP particles from, said surface.
This may be followed, by a purification step (e) in which (i) a olot i or; or suspension containing said separated IP particles xa contacted wit a carrier substance
having ' said template material immobi l ised on it so as to be exposed a surf ce . , so that said HIP particles bind to said immobilised template ma '.mom.a.:., iii; unbound
mat eraa is separated from said carrier substance; and subsequently {iii; MIP particles are recovered from the immobilised template material to form a purified solution or colloidal suspension. Background material can be found, in the following
references ,
I, Wulffl G. Makromol.; Chain. Maexdmoi, f . 19 3, 70/71 f Mb ,
2, Vlarakis, G . et 1. Nature, 1993, 361, SA5,
3. oad ; G . ; Hizzardo Ei ,m Solomon; .0 , H - Macromoioculea
1982, 15, 909;
4. Matyjaszemski, K. Xia, 0 " . Chem > Rev. 2001, 101, 2921.
5. Kamu.gaito, M. ; Ando, T . ; Suummoco, M. orem , le- . 2001, 10.1, 3689,
6- Hawker, C, 11 ; Bosman A, > Karth, S, c¾em. Rev,
2001, 1 :1; , 3361.
7. Fischer, H, Ch&m. Rev. 2001, 101, 3581.,
8. Otsu, . Matsumoto, ddv . Polyma Sci , 19 8, .:: 76,
75-13?,
9. bead, G . ; et al. PoLyrn, Inc. 2000, 49, 993-1001..
10. Ruckort, B, Kail, a. J, ; Sellergren B, <J. Mater, Sci,
2002, 12, 2275,
II, Hattori, Kd ; et al. J, r mme . Sci. 2004, 233, 169.
12, Li, L; Day, M . ; Ding, J. F. Faid, , Macromol ecu! es .
2005, .mm 2620. 13. Guerrei.ro A> R, , Chianeila .! . , Piletska S , ,- Whitcorsbe , J . , Piletsky S. A. (2009), Biosens. B^cel&ctron , , 24, 2740-2743.
14. JaQur-GrodKinski, J v Reactive & Functional Polymers,
2001, 1, 1.
15. Shiia f S PS . et al , MacxomQl®oul<ss . 2003, .36, 7994-8000 ,
16. Y , Q. ; Zeng f . ; Zh . u S . E¾ ;: ¾ .R-K 2005, 34
1612 * Fat nts cited
Fatent numbe Territory Issue Title
date
1 USS, 8S.2 818 US OS-Fsfo- MOLECULA LY IMPRINTED
2005 POLYMERS PRODUCED BY
TEMPLATE POLYMEEIOA ι ION
I US6, 127, 154 US 03-Oct-- METHODS FOR DIRECT SYNTHESIS
2000 OF COMPOUNDS HAVI G
COMPLEMENTARY STRUCTURE O A DESIRED MOLECULAR ENTI Y AND USE THEREOF
WG96 0822 PC? 19-Dec- PREPARATION OF BIOLOGICALLY
1996 ACTIVE MOLECULES BY
MOLECULAR IMPRINTING
US 5,€30, 078 2(,-;0u - PREPARATION OF BlOLOCIOALLY
1997 ACTIVE MOLECULES BY
MOLECULAR IMPRINTING
US7 , 393 ,.909 us 0.1 -Jul- POROUS , OLECULARLY
2008 IMPRINTED POLYMER AND A
PROCESS FOR THE PREPARATION THEREOF
US6, 458, 5:39 j US 01-Oct.- COMPOSITIONS AND METHODS FOR 2002 CAP ORINO ISOLATING
I DETECTING A ALYSI G A D QUAN IFYING NLACRO OLECOLES
DS7 f 288, 415 jus 30~Oct- COMPOSITIO S ' AND METHODS FDR
2007 CAPTURING ISOLATING
DETECTING AN LT KING ND QUANT.1FYTNG ACRQBDLECULES i¾O20ll p675631 ,PCT 9:-June- pREPARATION OF HOLECULA-RLY
2011 IMPRINTED POLYMERS
Disclosure of Invention. The resent invention provides a process for producing nanopartioies of molecaiariy
imprinted polymers corrspri s in :
{1} iimaobilosing template species on a solid
support to provide a supported teraplate material;
(ii) locating the supported template materia! in a reaction vessel having one or more flow inlets, one or
more flow outlets f a cooler/heater adapted to affect the temperature of the vessel's contents, and a radiation
source adapted to irradiate the vessel's interior;
(iii) passing a poiymerisable composition into the vessel through one or rore of said flow inlets; Civ) causing the radiation source to irradiate the vessel's interior to effect controlled polymer isation of the poiyoneri able composit. ion, while using the
cooler/heater to control the temperato.re thereof;
(v) ceasing irradiation at a stage w o a
polyrierisat ion has generated nanopartioies wit molecular weights in the range 500-1,000,000 D , at least some of the nanopart icles being bound to the immobilised template;
(vi) passing solvent through the vessel via one or more of said inlets and one or more of said outlets to wash out unreacted components of the polymer isabie
composition and unbound polymers; and
(vii) passing solvent through the vessel via one or more of said inlets and one or more of said outlets under conditions such that at least some of said bound
nanopartioies are separated from the immobilised template and passed out of the vessel to a col lection vessel, said condi ions comprising one or more of increased
temperature produced by the coole /heater, and use of a solvent composition that is able to disrupt binding of nanopa t icles to template.
it also provides equipment - a photoreactor useful for manufacturing BIB nanopartioies by controlled
photochemical polymerisation. The device consists of a photoreactor with a thermostatically-controlled reaction vessel containing immobilised template t lamps to
irradiate said vessel and a fiuidic system for delivering monomer mixture and collection of the synthesised
nanopartioies , preferably where the operation of the equipment is performed under computer control. e have found that the careful control of reaction
parameters is crucial to the successful and consistent production of useful nanopart icuiate MIPs. Firstly we- have found, that careful contra! of irradiation: ime is ' vit l * Fig.. 1 slces some enpsrimsutai results, showing how radiation i : affects (a) diameter of the produc particles ? and (b) yield, hereas the yield rises fairly constantly wit time,, the diameter increases quite slowly at first . , to a value of about 60 ns at 3.5 minutes, and then rises very rapidly to about 375 na at 4. inu es. Since . have alao found tha the affinity of I.P nanop-srtieies peaks at about 60 nap it is clear that the accurate control of parameters is critical for achieving a practical manufacturing process. in one aspect of the proposed, invention the Hits are produced in the photoreaetor in the presence; of an immobilised template using controlled polymerisation, optionally controlled radical photochemical
polymerisation . The preferred type of the reaction, explored in the present invention is iniferter-foased living radical polymerisation, initiated and controlled by UV i radiation,. The polymerisation reaction is terminated at a stage when the size of the synthesised molecules is relatively sm ll. The product of such a process exists either in soluble or stable colloidal form in solution or suspension. The colloidal suspensions o ' solutions of. IPs according to this invention may be in either aqueous or organic liquids.
The light initiates radical formation and propagation of the polymerisation reaction- The light energy utilized in the present invention is ultraviolet ( "UV ,f 5 light or visible light. However, the liglrt energy can also be hroad : ™bana f delivered by fiber optic cable or by the way of an attenuated total reflectance iiVTR link.
Ln farther aspect of the present invention high affinity particles can be selectively bound to (and late
recovered frost) surfaces bearing immo ilis d template, allowing them to be separated from, lev affinity particles. The surfaces bearing iiamobiiised template can be the s me surfaces endh . are oren in the photoreactor vessel or the can be separate surfaces (e.g. corucaieed within a separate column) with immobilised target, which may be an analogue of, or identica to, the template. The surfaces mentioned here can be corfaces of affinity columns, beads, fibres, wells, icropiates, membranes, filters, pits, nanostructures, vesicles, capsules, samples of tissues, living cells etc. The surface can be solid, serai ---sol id or liquid or fluid (as in the case of micelles or
interfaces). Optionally the particles can be further screened using an additional column or columns, to select a subfraction of the particles which do not possess affinity for a potential interferent compound,
alternatively a similar approach can be employed to select subfraction is) of particles which additionally have affinity to one or more analogues of the template in order to produce "class-selective" binders.
The further aspect of the present invention describes an application of the sunthesised ΜΪΡ naropartides ,
olecules syathssised as described In this invention
; truers , oligomers, polymers, or their mixtures) can be used as drugs in pharmacology and medicine, as receptor- specific- iicands in analytical chemistry (sensors, assays) , tor separation in the biot&ehnelogy,- phaaaaceatical arid ood: industriec and as: eataiysts i synthesis or as replacements for o:u-:.yos a in assays, sensor and othe application.® such as washing powders. iitnli...^^
igu e 1 is a graph showing the rela ionship: between irradiation time and either- particle diaiseter (nm) or polyme yield: (mg) .
figure 2 is a schematic view Of a photoreaetor used for the synthesis of .IP nanopartioi.es -
Figure 3 is an SEM image of MI narioparticies produced : n the phot reactdr. Detailed Description
Ths first aspect of the present invention is a synthetic process used in a photoreaetor. Speci ically, one aspect of the present indention relates to the synthesis of MIPs in the photoreaetor by controlled polymerisation,
optionaiiy controlled, living radical polymerisation (LRP) in the presence of an mmobilized template which can foe a biological receptor, .nucleic acid, cell, spore, virus, oticroorgani sm, tissue s mple, carbohydrate,
oligosaccharide, polysaccharide, peptide, nocleoprotein, mocoprotein, lipoprotein, synthetic protein, glycoprotein glucosaminogiycan, steroid, hormone, immunosuppressant , heparin, antibiotic, vitamin, biomarfcer of a pathological, or disease state, tojiin, pesticide, herbicide, explosive, nerve agent, pollutant., endocrine disrupting compound;, nucleotide, nucleoside, oiifonuciectide, metabolite, secondary met chela e, d ug metabolite,- drug intermedi te or: drug o othe class of tem late known to those skilled in the art. The template can be immobilised onto
p ly eric.,- polysaccharide o glass surfaces:, for exam le i the forts of beads, the surface of waveguides, fibres, ixnaiu.di.ng optical flbr ' es membranes, capillaries or any other serface Portable for the intended application, as know to, those skilled in the art.
Several different forms of controlled polymerisation may foe employed in the present invention. They are all based on the ability to control the addition reaction at leeel snch that predominantly soluble nanepart icies are formed rather than continuous polymer layers or networks. In the eKample of living^ radical polymerisation,
initiator molecules undertake reversible transformations by photochemical stimuli^ reversibly transfor ing dormant species into reactive free-radicals or ions which act as chain propagators. For this condition to apply, the equilibrium constants of the reactions should favour formation of the dormant species and should alloc rapid exchange between the dormant and propagating species. Thus the concentrations of the propagating species «111 be very low and their residence tirne very short,, ehieh reduces the probability of side reactions that lead to termination of the -growing poiys-er chain. Examples of living polymerisation include nitroride-"mebiated
polymerisation (PHP), atom-transfer radical
polymerisation pATRF) and reversible additriors- f.segmentation chain-transfer polymerisation {RAFT) . Flct polymerisation is based on reversible addition- fragmentation, chain, transfer equilibrium, where there is an exchange e ween active and. dormant species. The radicals generated In the iudxration step will propagate by addition. o morro tex until a molecule, ca ble of acting as a chain transfer agent, is en-coun red, and to w ich they lea add in reversible fashion. Generally S living pel yma is tion process allow fo the use of
iniferters. {ioitiatcr t ansfar-aqent ieirminatoc ) which optionally can be made in eonjunction with, conventional initiators to confer a living nature on tbe :
po1 ymeris 11 o .. I■vifer era c n e photo~in1 £erters0 bearing dithiocarbamyi group or thermal-buvifenters
carrying: carbon-carbon or aao groups see .g., ret, 14) ox other types of compound known to those skilled in the art. The preferred kind of inifenters are those that yield different radicals, one carbon radical which is5 reactive and anothe less reactive radical ,· e.g., a
dithioearba yi radical. The carbo radical, which is typically a benzyl radical, can react with, unsaturated monomer to initiate polymerisation. The less reactive radical ,< for example dithioearbamyi radical, can
δ terminate, the polymerisa ion by recombining with a
growing polymer ohain. However the product of
termination can be further dissociated to a new
propagating radical and terminator in response to continued application of the stimulus, e.g. UV
5 irradiation (see e.g., ref, 15) ,
One advantage of living polymerisation in contrast to traditional, radical pel y erisation is that the former proceeds at a low rate and with no observable
0 autoacceleration, while the latter frequentl proceeds with a strong- antoacceleration ( see o . -r . , ref. 16). The present .invention takes 1 advantage of this by performing hi living polymerisation under conditions which f v : o. r the formation of polymers eith relativel low molecular weight. Typically the reaction is stopped at an early stage to : produce polymers with molecular weights of 500- 1,000,000 Da.
The resent invention facilitates the optimisation of the conditions of the reaction in order to generate- particles It ' ll relatively small sice. An important part of the : process is the selection of an appropriate living
initi tor: and o timisation of the condition of the polymerisation reaction,: Alternatively the rate of radical formation add propagation can be controlled by adding in i i o s of the reaction or chain transfer agents such as e cap ' to derivatives.
Living radical polymerisation initiators can be prepared from discrete organic molecules or from riacromoleoul.es. In reality, os compounds that contain a hydrox
carhoxyiic or amino group can foe converted into an initiator, and thus incorporated easily into the polymer. This can either be at the terminus of the polymer in the case of a raono-fonctionai initiator, or in the middle of the polymer in the case of a mo.iti-functional initiator.,
The reaction conditions favouring formation of relatively low molecular weight polymers include, but are not limited, toe {±} using stoichiometric ratio beteeen initiator and monomers; (ii) cooling the reaction or removing the source of 0V or other irradiation, w i h will terminate the formation of new propagating species at an early stage of the reaction; (iii) remo al of the monomers from contact with the growing polymer chain e , g., by filtration or chroma ography; (iv) adding inhibitors to the reaction ) performing polyn¾ I-sa io-ii in very dilute, stolotion ivi adding chain transfer agents, The pre arable option would he removal of the source of irradiation or its interruption.. hlLterhatireiy monomers and other reagents can. e removed from the growing MIP attached to the iroftotdiised, template by elutior ¾s a result of controlled lifing polymerisationy MI? particles can be forraed within the sire range 500-1,000, 000 Da, which could erist ίϊ soluble, or at least colloidal fo s after separation from the iriomobilised template.
In the present invention, monomers which can be used for MIP preparation.,, includingo vinyl monomers,, ally!
monomers,- acetylenes, acryiates, meth&cryi¾t$s,
aorylarrsides, methacryiamides, chloroaerylates, itaconates, trifluoroimsthylaorylateS f derivatives of amino acids, nucleosides, nucleotides, and carbohydrates,
Polymerisat i.on m.a be pe formed in the presence or on the surface of particles containing double bends. Cross- linking monomers are used to fix or stabilise the
structure of the resulting polymer, so that it remains compiexaentary to that of the template. Typical examples of cross-linkers suitable for the synthesis of MIPs include, tout are not limited to, ethylene glycol
disco,hacty1ate, rlinethy.1o1propane trimethacry1.ate f divinylbenrene^ methylene bisacrylarai.de, ethylene
bisacrylamide and Ah h ri -bisacryloylpiperazine , The
function of cross-linking agents can be performed by particles or precursor polymers containing double bonds, or particles or polymers uith .multiple functionalities attaches which cart bind to functional monomers. Those shilled ifs the art could select monomers and cross- linkers suitable tor : a particular system. Al ernatively a variety of combinatorial and computational riethods could e used to assist in this selection.
The synthesise na.no.pa,rt icl es have higher affinity to the inimohil ised template 1 than ;:;;· " cos ;:s , non-apeeific
oligomers and low affinity polymers (e.g. those f ared in the bulk volum-e in the absence of template} . Thus in one aspect of the proposed inyerrtion weakly bound material is reaioved from the hanopa rticles attached to the
immobi 1 ised template by nvshlngl. The separation of high- affinit nanopartioles from immobilised template may be achieved by beaming, which disrupts complex forma ion, by changing solution pH f changing ionic strength,, or through the addition of urea, guanidine, or other substance or substances which interact with the template more strongly than, does the pal peer , h s«pon.d aspect of the present invention relates to the apparatus used for MTJ? manufacture and its functions.
The functio of the reactor is to provide a suitable and controlled environment for the synthesis of Ml
n nops rt to!aa . It suitably includes a fluidic system comprising fluid reservoirs, pumps f valves, tubing, i 1 is and connectors for delivering monomers and solvents, and for ' elation of by-produc s, unreached materials and for collection of the ayntheaised nanopartieles . Optionally the deliver of rea.ge.rnls, washing and elation processes:, as ίβII. as the tempera!are inside the reactor and the irradiation time are controlled by computer. One non- restricting esaraple of .such reactor is presented in
Figure 1 , The reaction vessel 10 can be made from glass, quartz, or plastic, or a combination of two or more of these
m t rials, provided that the ransparency to light at the required wavelength is adequate to initiate
polymerisation within the vessel. The path for light to enter the reacto vessel can be ftorn one direction or from ail directions; alternatively the light source 12 may be located inside the reaction, vessel or in a
transparent compartment within the reaction vessel or light may be channeled into the reaction vessel through one or more optical fibres or w veguides . In oases where the light source 12 is located inside the reaction vessel 10 or in a transparent compartment within the reaction vessel or light may be channeled into the reaction vessel through one or more optical fibres or waveguides, there is no general requirement for the reactor vessel to be transparent, apart from the desirability to be able to visually inspect the reactor contents, so a broader range of materials, including metal, ceramic, fibre-reinforced composites, PTFE or other non-transparent materials may be used.
The polymerisation is initiated by irradiation,
preferably by irradia ion with Up or visible light and normally tabes minutes or hours depending on the
reactivity of the species. The light initiates radical formation and propagates polymerisation reaction. The light energy utilized in the present ii erttion is h? ultraviolet ("UV") light or visible light, However, the ligh energy can also be brQad--band f delivered by fiber optic cable or y the way of an attenuated total,
reflectance (ATR.5 link. In this regard, the photoreactQr in FIG. 1 includes a transparent, window or wave guide for the entry of light energy from a light source such e.g. an ult aviolet lamp. The i,rradiation can also, he
delivered from the inside of the reaction through fiber optic cable or waveguide.
The plantoreactor vessel 10: is designed to contain a solid phase 1 bearing an i roobi1i zed teraplate. The so i phase 1.4 can be in the form of beads vpaekeob or loosely filled) ! membranes- or fibres. The beads could foe
stationary or stirred for the duration of some or all of the TP manu acturing process. The reactor vessel
contains one or more entry porta 16 f each optionally fitted with a non-return valve and frit for retaining the solid phase within the reactor vessel, for the delivery of monomers and washing solvents and an ea,u port IS, optionally fitted with a non-return valve and frit for retaining the solid, phase within the reactor vessel f for elution of reagents f unreached monomers and synthesized nanoparticies, connected through a switching valve 20 to direct the flow to an appropriate collection reservoir 22, 24, 26 for wast or product collection. The operation and. use of this embodiment of the invention is more particularly described below.
The o o e mixture,, comprising functional monomer or monomers f iniferter, cross.- iinhe.r or cross-linkers an optional chain- transfer agent or chain-transfer agent
IS dissolved, in an appropriate .solvent, said monomer ■ auK ur.fe aving been selected in order to optimise, template- monomer interactions , is injected into the: reactor vessel which contains, immobili red template f as defined above, The polymerisation process is started when: the reaction vessel content's reaches a predetermined temporatore, preferably a low temperature (typically between. ~Z Q . '' C and ; "cj by irradiation of the vessel contents with UV or with Yisible light, The nanoparticles are formed in solution and on th surface bearing the irimo ilired template. The reaction is terminated by ceasing
irradiation. The unreacted monomers, partially • - • formed poty-mers and particles with low affinity to the template are the removed by washing with a wash solvent which can be the same as or different to the one used in the monomer mixture-, The flow of effluent from the column comprising the washings is directed by the switching valve to a suitable waste reservoir (26) . The
temperature of the reaction vessel is increased to between 20 * C to 40 e Ch at which point the low affinity MI? nanoparticles are eluted by continued flow of the wash solvent, Finally the desired high-affinity MIP nanoparticles are col leered by increasing the reaction vessel temperature to between 60 °C to 80 ' "C and elution of the vessel contents with an elation solvent, which can. be the same solvent as the wash solvent, or a different, solvents, optionally containing one or more compounds as additives in order to facilitate elution of the
nanoparticles. The flow of the eiuted nanoparticie- containing solvent is directed by the switching valve to a suitable vessel or vessels 221 24 for collect Ian of the pro-duct or products of reaction. The elution of ar-oparticles pan also be facilitated and/ox controlled by changing the pH of the eiution solvent or through the addition of acidic, basic or surface-active compounds or through the addition of .compounds which otherwi e disrupt binding between the template and MI nan.opartlG.les. A convenient way to monitor the- te perature within the reaction vessel is by the use of one o more thermrr--- couples 23. The heating and cooling of the monomer mixture in the reacto can b achieved using thermostatic con rol, ^optionally with circulating, liquid) 30 or by using Pe111ex e1sments ,
The benefits coming from this approach are .numerous, including.: the possibility to reuse immobilised, template for ImlP synthesis, the possibility to fractionate MlPs with high affinity from low affinity particles and unreached monomers , easy removal of the synthesised Mips from the template , the ability to post-~ unctionaiise MXPs while still attached to the immobilised template, the ability to fully o partially automate the manufacturing process etc. Other benefits of the invention should be evident to those skilled in the art.
In one preferred type of embodiment of the present invent ion, separation and purification of the synthesised MIP nanoparticlss is achieved on the same solid phase bearing inmo ' bilised template as is used for the
imprinting process . The separation relies on affinity interactions between the synthesised MIP hanopart icies and the immobilised template molecules. The particles with low affinity could, be removed from, the solid phase by washing under mild conditions (optionally at a temperature of between -20 S C to -HQ " ' C and in the absence of c mplex-disrupting agents, pH changes etc) , The specific- dl? nanoparticies with strong affinity tc the template can be removed for collection at elevated temperature, by changing pH or by the use of pom ieao-- disrupting agents or s lvents or combin tions of mixtures the eof .
The synthesised high-affinit MIP nanoparticles may be purified additionally by chromatograph filtration and/or electrophore is. The separation of the
synthesised polymer can foe achieved by affinity
chromatograph ,. or selective elation, when binding of the nariopaxt icles to the same or a similar immobilised template or target compoand is used for the purification of a fraction of polymer particles with the highest affinity to the template , and/or by gel permeation chromatography (si£e™e¾ciosion chromatography) which enables polymer fractions of different iae to foe
separated. Fractionationi separation and purification daring affinity separation is achieved, by ashing and elation using buffers with different pH r ionic strength or through the addition of acids, bases-, surface-acaive agents-, urea, gnanidine, or other substance or substances ' which interact with the template mote strongly than does the polymer. Alternativel fractionation of particles with high affinity can be achieved by filtration,
electrophoresis, chromatographic, separation, washing, centr ifugation. or dialysis. Affinity chromatography is in particular a powerful tool, and is particularly preferred, because it allows for the prepa.rat.ieu of Mips ' with a narro distribution of affinities to the empiate-. Af ini y separa ion pan additionally be used to select a subtraction of particles with high-affinity tor the
target or template structure , hut which have low affinity for one or more intsxfetent compounds by the selection of a fraction which ' binds to the forme but not to the
latter.. Additionally subtractions can be selected which have bread ox claps-specific binding properties by
selecting fractions with high-affinity for the target or template: ' structure, but which also ' show high-affinity for one or more structural analogues of the template or
target, comp ising representativ exam les of the class of compounds fox which binders are: required.
Embodiments of the present invention may also- .include ' post-modification of the syntbeoissd nanoparticies. The syothesised Ml? nanoparticies can be modified with
another polymer or polymers, linear or cross-linked, or be modified with a compound or compounds bearing specific functional group or groups oith the intention of
introducing one or more specific properties to the MIPs in order to modify their solubility characteristics or biocompatibiiity, to facilitate their extraction or other form of separation, to enable their detection . , to
introduce additional recognition or catalytic
functionality, including but not limiting to a. second molecular imprinting step, attachment of cyciodextrin, crcwo- ether, calixarerse, or curcubitur.il or other
supramolecular binding or catalytic groups, to attach dye molecules, optionally fluo es ent dyes, metal -binding iigands pro-drugs,, antibiotics., i munostimulatlng agents, bioligands etc , , preferably by re-a.ctivet ion of surface- bound iniferter groups or by their selective chemical morlifieatipfg preferably while the MXP naneparticl.es . are still bound, tc the immobilised template phase. An example of this c uld be the formation of linear polymers of one or more hydrophobic sonoiiiers, such that
hydrophobic "tails" are formed which would alloc e.g., extraction of the polymer f om: aqueous solu ion by organic solvent or location of the KI nanopartieie at an. agueous™organic interface, it would be possible to introduce specific binding g ou s, s.u,, bio:ttn f which souid allow selective removal of the polymer ' by an affinity adsorbent or to enable binding tc the- surface of a bioaenscrp cuch. as, a surface plasmon resonarice fSF ) sensor device or an acoustic sensor F an. electrode ox any other sensor device know to those ski 1 led in the art.
Those sbi lied in the art will be fami. liar with the wealth of experimental protocols which allow this modification and corresponding separation to be performed. Preferably the modification is achieved in the pholoreactor - essel directly on the surface while ' the polymer is bound to immobilised template or separately or after additional separation and/or purification steps have been pe forced, as described in the precious example.
In one aspect, the present invention relates to
sequential polymerisation when the imprinted polymer is ■ modified with other types of molecules in order to change the properties or functions of the synthesised particles, It has been mentioned already that, the HIP nanoparticles can be mod fied with another polymer ox functional group to facilitate its separation. One important property of living polyimarisat ion. Is the ability to stop a reaction and continue it late by strsply stopping and re-appiying : C ., UV irradiation of the reaction ni at are. The end of the growing polymer chain, contains an. initiator species which can be re-ac ivated to initiate a new round of polymerisation,. Thus the dor ant polymer chain ends could be exposed to nother monomer or aonoiner mixture and the poivalorisation continued, resulting in the
formation of linear or cross-linked polymers or
copolymers grafted to the surface of the cross--dlirhad nanoparticie, additionally two or more aaanoaaer solutions can be introduced and irradiated sequentially id order to graft block copolymer a rohitechnres , The new monomer or monome a can introduce now functionalities into the polymer particles. Thus in addition to the affinity to the first template, praei doc by the first HIP? an
extended polymer could be produced with affinity to a.
second template: introduced into the system or with
catalytic functionality by imprinting a transition state analogue. The extended graft polymer f copolymer or block copolymer could have fluorescent tags attached to end groups which would be useful in diagnostics > Other types of modification are also possible which, would introduce other func localities such as the ability to generate active species with biocidal properties, catalytic groups, isotopie labels, groups useful for immobilisation,,
sensing, imaging .{e.g. contrast agents) etc. These functionalities could also be introduced into the polymer ■ by using the correspondingly functionalised initiator.
The modification can. be a era. sued directly on the surface while the particle is bound to iimaobilised template or separately or after additional separation and/or
parification ste s have been performed a described in the previous example . The present ioventi.o : n alee concerns applications of the synth sized nanopartxcIes These include applications of the synthesised particles as drugs in pharmacology and medicine, as rceeptor-speeitic ligands in analytical chemistry (sensors, assays) f for separations in
technoio¾ , pharmacology and the food industry nd as catalysts. The soluble nature of the synthesised
polymers makea them ideal candidates for use as drags. The selective binding to an en yre^ receptor ei other 1 biological molecule- could be used to affect biological functions of these molecules. Thus MIFs synthesised by living pol merisation could fce used in vivo fox
modulation of biological processes.
The polymers prepared as described in the present
invention resemble effectors (activator inhibitor or substrate) of the template,, and as such can have
biological activity if the template is involved in a physiological process or an effective analogue of such a molecule or structure. Such polymers can be used, for example, as drugs in pharmacolog and codJ. erne ,
When bound to isotopes or fluorescent tags, HIPs could foe need as selective contrast agents or in other torue- of diagnostics. MIPs integrated with ligands capable of producing in certain conditions e.g. , singlet molecular oxygen, could be used as selective biocidai agents. A variety of other modifications could be proposed by those ski 1 lad in the art to introduce antibiotic properties into MTFs prepared by living po ymer sation. The synthenlead MIPs ecuid be used as a substitute for natural antibodies and. receptors In different forms of assays and sensors, Several features Make MIPs prepared by living polymerisation particularly attractive objects for application in sensors. Thus the aynthesised MIP still contain Initlabor which can foe- used to : form
radicals useful for covalent attachment of polymers to the solid surfaces with s,g. double bond containing functionalities. Thus simple UV irradiation .might be sufficient to bind IP.¾ to surfaces covered with double bonds
The ability to u e affinity chromatogra hy to separate HI? preparations into several fractions with different affinities could be advantageous for the preparation of sensors/assays with varying detection ranges. For eom.e applications Blank polymers (those prepared in the same way as : MIPs, but in the absence of any template; prepared oy living polyiaerisat ion conId also be used. It would be necessary however to prepare such polymers using monomers which possess certain affinity or other properties necessary for this particular application. Those skirled in the art know how to select such monomers by using e.g, compotati one 1 or combinatorial approaches, I I * . is
necessary to clarify that using both MIPs and Blank polymers prepared in a fashion as described in tee corresponding embodiments are covered by the scope of the present invent ion . The present invention will now be further described particularly with references to the following non- limiting examples,
2S £¾¾¾FLES
Ex m le X .
Preparation of solid su or . Glass beads were activated by fodill g in ISaOH 4 M for 10
Kdfs tes> then washed with doable-distilled water and acetone and dried at 80 ';' C, The beads were then incubated in a 2% v/v -solution o 3-anunopropyl tri:raetho¾ysiia e i toluene overnight, washed with acetone and subsequently incubated in a 7% v/v solution of gl taraldehyde in BS buffer H 7,2 for 2 hours,- after which they were riaaed with double-di stilled water. The surface imyaobiiisation of the template was performed by incubating the beads in a t rag/mi solution of the template in hBS pH 7,2
overnight at ' '. * . Methytpytroi idone {10% v/v) was also added as co-solvent during the inuYiobilisation of melarnine. The glass beads were washed with water and dried under vacuum, then s ored at 4 ';i C until use, Automated synthesis of imprinted particles with
s ecificity owards t laMLt » The synthesiser used
consisted of two pumps 32, 34 delivering up to eight separate feed lines to a temperature controlled glass reaction vessel 10 {reactor, figure 1} packed with the teEpiate-'derivatised solid support 14, One line 36 was used to supply the monomer mixture,- the remaining 38 were used for the delivery of wash and elation solvents. The outlet 18 of said reacto 10 was connected to a multi'-way valve 20 with sin outlets {only three of which are shown in Fig, 1} and used as a fraction collector, one line 40 used for- waste ftaotionSi the renin ining 2 were connected to collection vials 221- 24. A 0V source 12 comprising four -mercury vapour lamps of 8 W pl ced at 20 era from tee reactor was need to Initiate the polymerisation. The pumps, temperature controller 3uy u ' V: lamps and ufti-way valve were connected to a computer interface 50 -and were computer-eonx rolled by programmable software in a
computer 52. The software allows the control of flow rates- selection of feed lines, reactor temperature ? irradiation timing/tirtaCi reactor eiution time and malti- wa value: settings, * For the synthesis of the- imprinted polymer particles the meiamine-eQated beade (23.5 g} were packed into; the temperature controlled glass: reactor. The following steps Were all programmed into the control software and the synthesiser operated: in automatic mode:. The reactor ¾ s filled with solvent iacetonitrilei delivered by a computer-controlled pump 34 at 2 ed./min. The column was cooled down to -4 C and ml of monomer mixture injected onto the column from a different feed line 36. The composition of the monomer irture was (in % w/w) : AcetonJ.trlie 50.5 % r ethylene glycol
dimethacrylate (BGDMA) 15.5 % f trimethyicipropane
trimethactyiate (TRIM) 15, %, methacrylie acid (MAA) 13,8 benzyl diethyldithiooarbamate { IN ' IFERTER) 3,7 %, pentaer thritoi tstrafcis (3--URercaptopropionate } (CTA) 0,86 %, Pumps were switched off and polymerisation was initiated b UV irradiation which lasted 3,5 in..
Afterwards the solvent pump 34 was switched on in order to elate unreacted monomers and particles with poor or no affinity for the template during 30 min at a flow rate of 2 rd./min and 4 °C , The temperature of the column was then increased to 25 :' C and the solvent switched to acatondtrlie ; formic acid 10 mid and run at a flow rate of 2 rtd./min during 11 min. Finally, to elude particles with high affinity for the template, th temperstere ¾as raised to 60 °G and particles w re collected during SO min at a flow re i e of 2 fid/pin.
EXAMPLE 2„ Antoisatad syntJesisi of im inted p icles ith s ec icifc to ards van omycin. For the synthesis of i;xpi iru. ed .polymer particles, glass heads (23 , 5 g) coated with vancoi¾ycin. were prepared generally as
described i Exam le 1, and loaded into the temperature controlled glass reactor. The setup uaed w s as
described in Example X, The following steps were all ogr med into the control software and the synthesiser operated in automa ic mode. The reactor was filled with solvent haoetonitrile) delivered by a co oter---controlled ump . The column was cooled down to 4 °C and 4 ml of monomer mixture injected onto the column iron a different feed line.. The corsposition of the monomer mixture was {In % w/w) ί Acetors itrile 79 %, A r ~.iso . propylacrylajnide (dlshis) 10%; h ? "'tert"'butylacryiamids (TRATU) 3.5 %, bensyi diethyidithiocarbaroate ( IMIfE EP. } 1.62 , ■ Ν,Ν'- methyienebisacryiamide (BIS) 0,5 % , The poly/nerisation was initiated by UV irradiation which lasted 3.5 rain. Afterwards the solvent pump was switched on in order to sluts unreached race roe rs? and particles with poor or no affinity for the template during 30 in at a flow rats of 2 s;l/cin and. 25 °C . The temperature of the column was then increased to 60 ,;' C to elute particles with high affinity for the template which were collected during 20 rain at a flow rate of 2 rfi/mfn.