Patent Abstract:
materials and methods for studying and modulating the interaction of carbohydrate - containing moieties with other species are described , in particular , small particles , e . g . clusters of metal or semiconductor atoms , which can be employed as a substrate for immobilising a plurality of ligands comprising carbohydrate groups . these “ nanoparticles ” can then be used to study carbohydrate mediated interactions , e . g . with other carbohydrates or proteins , and as therapeutics and diagnostic reagents .

Detailed Description:
the nanoparticles described herein or their derivatives can be formulated in pharmaceutical compositions , and administered to patients in a variety of forms , in particular to treat conditions ameliorated by the administration of the ligand . by way of example , this may occur as the ligand blocks a carbohydrate mediated interaction that would otherwise tend to lead to a pathology . thus , the nanoparticles may be used as medicament for modulating leukocyte - endothelial cell adhesion , carbohydrate - antibody interactions , carbohydrate - protein bacterial and viral infection , immunological recognition of tumour cells , the inhibition of metastatis and foreign tissue and cell recognition . pharmaceutical compositions for oral administration may be in tablet , capsule , powder or liquid form . a tablet may include a solid carrier such as gelatin or an adjuvant or an inert diluent . liquid pharmaceutical compositions generally include a liquid carrier such as water , petroleum , animal or vegetable oils , mineral oil or synthetic oil . physiological saline solution , or glycols such as ethylene glycol , propylene glycol or polyethylene glycol may be included . such compositions and preparations generally contain at least 0 . 1 wt % of the compound . parenteral administration includes administration by the following routes : intravenous , cutaneous or subcutaneous , nasal , intramuscular , intraocular , transepithelial , intraperitoneal and topical ( including dermal , ocular , rectal , nasal , inhalation and aerosol ), and rectal systemic routes . for intravenous , cutaneous or subcutaneous injection , or injection at the site of affliction , the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen - free and has suitable ph , isotonicity and stability . those of relevant skill in the art are well able to prepare suitable solutions using , for example , solutions of the compounds or a derivative thereof , e . g . in physiological saline , a dispersion prepared with glycerol , liquid polyethylene glycol or oils . in addition to one or more of the compounds , optionally in combination with other active ingredient , the compositions can comprise one or more of a pharmaceutically acceptable excipient , carrier , buffer , stabiliser , isotonicizing agent , preservative or anti - oxidant or other materials well known to those skilled in the art . such materials should be non - toxic and should not interfere with the efficacy of the active ingredient . the precise nature of the carrier or other material may depend on the route of administration , e . g . orally or parenterally . liquid pharmaceutical compositions are typically formulated to have a ph between about 3 . 0 and 9 . 0 , more preferably between about 4 . 5 and 8 . 5 and still more preferably between about 5 . 0 and 8 . 0 . the ph of a composition can be maintained by the use of a buffer such as acetate , citrate , phosphate , succinate , tris or histidine , typically employed in the range from about 1 mm to 50 mm . the ph of compositions can otherwise be adjusted by using physiologically acceptable acids or bases . preservatives are generally included in pharmaceutical compositions to retard microbial growth , extending the shelf life of the compositions and allowing multiple use packaging . examples of preservatives include phenol , meta - cresol , benzyl alcohol , para - hydroxybenzoic acid and its esters , methyl paraben , propyl paraben , benzalconium chloride and benzethonium chloride . preservatives are typically employed in the range of about 0 . 1 to 1 . 0 % ( w / v ). preferably , the pharmaceutically compositions are given to an individual in a “ prophylactically effective amount ” or a “ therapeutically effective amount ” ( as the case may be , although prophylaxis may be considered therapy ), this being sufficient to show benefit to the individual . typically , this will be to cause a therapeutically useful activity providing benefit to the individual . the actual amount of the compounds administered , and rate and time - course of administration , will depend on the nature and severity of the condition being treated . prescription of treatment , e . g . decisions on dosage etc , is within the responsibility of general practitioners and other medical doctors , and typically takes account of the disorder to be treated , the condition of the individual patient , the site of delivery , the method of administration and other factors known to practitioners . examples of the techniques and protocols mentioned above can be found in remington &# 39 ; s pharmaceutical sciences , 16th edition , osol , a . ( ed ), 1980 . by way of example , and the compositions are preferably administered to patients in dosages of between about 0 . 01 and 100 mg of active compound per kg of body weight , and more preferably between about 0 . 5 and 10 mg / kg of body weight . the nanoparticles may be used as carriers for raising antibody responses against the carbohydrate containing ligands linked to the core particles . these antibodies can be modified using techniques which are standard in the art . antibodies similar to those exemplified for the first time here can also be produced using the teaching herein in conjunction with known methods . these methods of producing antibodies include immunising a mammal ( e . g . mouse , rat , rabbit , horse , goat , sheep or monkey ) with the nanoparticle ( s ). antibodies may be obtained from immunised animals using any of a variety of techniques known in the art , and screened , preferably using binding of antibody to antigen of interest . isolation of antibodies and / or antibody - producing cells from an animal may be accompanied by a step of sacrificing the animal . as an alternative or supplement to immunising a mammal with a nanoparticle , an antibody specific for the ligand and / or nanoparticle may be obtained from a recombinantly produced library of expressed immunoglobulin variable domains , e . g . using lambda bacteriophage or filamentous bacteriophage which display functional immunoglobulin binding domains on their surfaces ; for instance see wo92 / 01047 . the library may be naive , that is constructed from sequences obtained from an organism which has not been immunised with any of the nanoparticles , or may be one constructed using sequences obtained from an organism which has been exposed to the antigen of interest . the term “ monoclonal antibody ” as used herein refers to an antibody obtained from a substantially homogenous population of antibodies , i . e . the individual antibodies comprising the population are identical apart from possible naturally occurring mutations that may be present in minor amounts . monoclonal antibodies can be produced by the method first described by kohler and milstein , nature , 256 : 495 , 1975 or may be made by recombinant methods , see cabilly et al , u . s . pat . no . 4 , 816 , 567 , or mage and lamoyi in monoclonal antibody production techniques and applications , pages 79 - 97 , marcel dekker inc , new york , 1987 . in the hybridoma method , a mouse or other appropriate host animal is immunised with the antigen by subcutaneous , intraperitoneal , or intramuscular routes to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the nanoparticles used for immunisation . alternatively , lymphocytes may be immunised in vitro . lymphocytes then are fused with myeloma cells using a suitable fusing agent , such as polyethylene glycol , to form a hybridoma cell , see goding , monoclonal antibodies : principles and practice , pp . 59 - 103 ( academic press , 1986 ). the hybridoma cells thus prepared can be seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused , parental myeloma cells . for example , if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase ( hgprt or hprt ), the culture medium for the hybridomas typically will include hypoxanthine , aminopterin , and thymidine ( hat medium ), which substances prevent the growth of hgprt - deficient cells . preferred myeloma cells are those that fuse efficiently , support stable high level expression of antibody by the selected antibody producing cells , and are sensitive to a medium such as hat medium . culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the nanoparticles / ligands . preferably , the binding specificity is determined by enzyme - linked immunoabsorbance assay ( elisa ). the monoclonal antibodies of the invention are those that specifically bind to the nanoparticles / ligands . in a preferred embodiment of the invention , the monoclonal antibody will have an affinity which is greater than micromolar or greater affinity ( i . e . an affinity greater than 10 − 6 mol ) as determined , for example , by scatchard analysis , see munson & amp ; pollard , anal . biochem ., 107 : 220 , 1980 . after hybridoma cells are identified that produce neutralising antibodies of the desired specificity and affinity , the clones can be subcloned by limiting dilution procedures and grown by standard methods . suitable culture media for this purpose include dulbecco &# 39 ; s modified eagle &# 39 ; s medium or rpml - 1640 medium . in addition , the hybridoma cells may be grown in vivo as ascites tumours in an animal . the monoclonal antibodies secreted by the subclones are suitably separated from the culture medium , ascites fluid , or serum by conventional immunoglobulin purification procedures such as , for example , protein a - sepharose , hydroxylapatite chromatography , gel electrophoresis , dialysis , or affinity chromatography . nucleic acid encoding the monoclonal antibodies of the invention is readily isolated and sequenced using procedures well known in the art , e . g . by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies . the hybridoma cells of the invention are a preferred source of nucleic acid encoding the antibodies or fragments thereof . once isolated , the nucleic acid is ligated into expression or cloning vectors , which are then transfected into host cells , which can be cultured so that the monoclonal antibodies are produced in the recombinant host cell culture . hybridomas capable of producing antibody with desired binding characteristics are within the scope of the present invention , as are host cells containing nucleic acid encoding antibodies ( including antibody fragments ) and capable of their expression . the invention also provides methods of production of the antibodies including growing a cell capable of producing the antibody under conditions in which the antibody is produced , and preferably secreted . antibodies according to the present invention may be modified in a number of ways . indeed the term “ antibody ” should be construed as covering any binding substance having a binding domain with the required specificity . thus , the invention covers antibody fragments , derivatives , functional equivalents and homologues of antibodies , including synthetic molecules and molecules whose shape mimics that of an antibody enabling it to bind an antigen or epitope , here a carbohydrate ligand as defined herein . examples of antibody fragments , capable of binding an antigen or other binding partner , are the fab fragment consisting of the vl , vh , cl and chl domains ; the fd fragment consisting of the vh and ch1 domains ; the fv fragment consisting of the vl and vh domains of a single arm of an antibody ; the dab fragment which consists of a vh domain ; isolated cdr regions and f ( ab ′) 2 fragments , a bivalent fragment including two fab fragments linked by a disulphide bridge at the hinge region . single chain fv fragments are also included . a hybridoma producing a monoclonal antibody according to the present invention may be subject to genetic mutation or other changes . it will further be understood by those skilled in the art that a monoclonal antibody can be subjected to the techniques of recombinant dna technology to produce other antibodies , humanised antibodies or chimeric molecules which retain the specificity of the original antibody . such techniques may involve introducing dna encoding the immunoglobulin variable region , or the complementarity determining regions ( cdrs ), of an antibody to the constant regions , or constant regions plus framework regions , of a different immunoglobulin . see , for instance , ep 0 184 187 a , gb 2 188 638 a or ep 0 239 400 a . cloning and expression of chimeric antibodies are described in ep 0 120 694 a and ep 0 125 023 a . as strategy for tailoring polyvalent carbohydrate surfaces with globular shapes to investigate in solution carbohydrate - to - carbohydrate recognition , an approach was devised by which carbohydrates are linked to gold nanoparticles [ 7 ] . by way of example , the preparation , characterisation and preliminary interaction studies of sugar functionalized monolayer and water soluble gold nanoclusters is disclosed below . thiol derivatised neoglycoconjugates of two biological significant oligosaccharides , the lactose disaccharide ( galβ ( 1 - 4 ) glcβl - or ) 1 and 2 and of the trisaccharide le x antigen : have been prepared to attach them to gold nanoparticles . the trisaccharide le x and the disaccharide lactose build up the glycosphingolipid ( gsl ) le x antigen : which have been proposed to mediate morula compaction and metastasis in healthy and carcinoma mouse cells respectively , via a homotypic carbohydrate - to - carbohydrate interaction . [ 8 ] the lacto and le x protected glyconanoparticles provide a glycocalyx - like surface with chemically well defined synthetic matrix and globular shape . moreover , this approach opens the way to tailor glyconanoparticles containing a variety of carbohydrate ligands as well as different surface density providing an under - control model for structure - function studies and for investigating carbohydrate clustering ) and orientation effects at a surface . [ 10 ] the lacto - and le x functionalised nanoparticles will be our polyvalent model system to mimic gsl clustering in plasma membrane [ 11 ] and to investigate in solution the attractive and repulsive forces involved in cell aggregation via carbohydrate - to - carbohydrate interactions . previous studies using synthetic receptors provided first solid evidence that stabilising interactions between lipophilic carbohydrate surfaces exist in water . [ 12 ] the synthesis of the disulfides 1 , 2 and 3 was carried out by glycosidation of the conveniently protected lactose and le x derivative with 11 - thioacetate - 3 , 6 , 9 - trioxa - undecanol ( for 1 ) and 11 - thioacetate undecanol ( for 2 and 3 ) using the trichloroacetimidate method , see fig1 . [ 13 ] compounds 1 , 2 and 3 were isolated as disulfide forms , and in this form used for the formation of the gold protected glyconanoparticles . the water soluble glyconanoparticles 1 - au , 2 - au and 3 - au were obtained in methanol following the procedure of brust et al for the synthesis of monolayer protected gold nanoclusters . [ 7 a ] a series of gold protected nanoparticles , all of them soluble in organic solvents , have recently been prepared for different purposes . [ 14 ] the lacto - au and le x - au glyconanoparticles are water soluble , stable and can be manipulated as a water soluble biological macromolecules . they have been purified by dialysis and characterised by 1 h - nmr , uv and transmission electron microscopy ( tem ). synthesis of glyconanoparticles : a solution of disulphide 1 , 2 , or 3 ( 0 . 012m , 5 . 5 eq ) in meoh was added to a solution of tetrachloroauric acid ( 0 . 025m , 1 eq ) in water . nabh 4 ( 1m , 22 eq ) in water was added in small portions with rapid stirring . the black suspension that was formed was stirred for additional 2 hours and the solvent was then removed under vacuum . the crude of the reaction was washed with meoh and was centrifuged for 10 minutes . the methanol was removed and the process was repeated several times until the starting material was not detected by tlc . the glyco - nanoparticles are completely insoluble in meoh but quite soluble in water . they were purified by dialysis : 50 mg of crude product was dissolved in 10 ml of water ( nanopure ). this solution was loaded into 10 cm segments of cellulose ester dialysis membrane ( sigma , mwco = 12400 ) and placed in 4 l of water ( nanopure ). the dark glyconanoparticles solution was collected from the dialysis segments and lyophilized . the products obtained were free of salts and starting material ( absence of signals due to disulphide and na + in nmr ). transmission electron microscopy ( tem ) examination of the samples was carried out with a philips cm200 microscope working at 200 kv . a single drop of a 0 . 1 mg / ml aqueous solution of the gold glyconanoparticles was placed onto a copper grid coated with a carbon film . the grid was left to dry in air for several hours at room temperature . particle size distribution of the au clusters were evaluated from several micrographs using an automatic image analyser . the number of particles selected for consideration was around 400 , which resulted in stable size distribution statistic . [ 0085 ] fig2 shows tem images and core size distribution histograms for the 2 - au and 3 - au gold glyconanoparticles . the gold particles stabilized with the lactose show a narrower and more homogeneous particle size distribution than the particles stabilized with the le x conjugate . a mean diameter of 1 . 8 nm was found in both samples for the gold core of the functionalized nanoparticles . such a mean particle size corresponds , according to previous work , [ 15 ] to an average number of gold atoms per particles of ca . 200 and 70 protecting alkanethiolate glycoconjugates . the aqueous solutions of the nanoparticles were stable during months and no agglomeration was detected by tem . the presentation of the carbohydrate molecules at the nanoparticles surface was then investigated . the molecular properties of the neoglycoconjugates 1 , 2 and 3 suffer a differential change after attaching them to the gold surface . for example , the lacto derivative 2 , which is soluble in methanol and insoluble in water , gives glyconanoparticles 2 - au insoluble in methanol but with good solubility in water . the le x derivative 3 is soluble in methanol and water , its nanoparticle 3 - au , however , is insoluble in methanol and very soluble in water . these differences in solubility can be used to purify the glyconanoparticles from the non - reacted disulfides by washing them with methanol . however , the most significant fact in these changes is that they reveal the influence of clustering at the surface on the carbohydrate presentation to the surrounding . the 1 h - nmr spectra of the glyconanoparticles show clearly these differences ( fig3 ). the spectra of the lacto - nanoparticles 1 - au and 2 - au in d 2 o differ strongly from those of the lacto - disulfides 1 and 2 ( fig3 a spectrum of 1 not shown ) showing the line broadening of slowly rotating macromolecules in solution . the signal of the methylenes closest to the thiolate / au interface completely disappears , as it occurs in the alkanethiol monolayer - protected gold nanoclusters . in contrast , these differences are not founded in the case of the 3 - au nanoparticles . the 1 h - nmr spectra in d 2 o of both 3 and 3 - au show similar broadening for all signals ( fig3 b , a , b ), indicating an intramolecular aggregation already present in the le x disulfide 3 . this self - interaction persists even at highly diluted water solution and is abolished by addition to the d 2 o solution of 3 of increasing amounts of cd 3 od . some well - resolved signals appear in cd 3 od / d 2 o ( 1 : 1 ) solution and in 70 % cd 3 od / d 2 o solution all signal are well - resolved in the spectrum ( fig3 b , c ). the tendency of the le x disulfide 3 to self - assemble in water cannot exclusively be attributed to the hydrophobicity of the aliphatic chain , but rather to the specific partaking of the carbohydrate moiety in this aggregation , as point out the lack of aggregation in water observed in the 1 h - nmr of the lacto - disulfides 1 and 2 ( fig3 a ). the self - aggregation ability will have consequences in the organisation and clustering of le x - containing gsls , as claimed by some authors [ 16 ] and contrary to the proposal of others that the carbohydrate head group plays an insignificant role in formation of glycolipid - enriched microdomains in the plasma membrane . [ 17 ] the steric crowding of the carbohydrate moiety at the nanoparticle surfaces is also shown by the different behaviour of 1 and 2 and their corresponding nanoclusters 1 - au and 2 - au with β - glycosidases . the β - galactosidase of e . coli processes 1 and 2 at a level comparable to lactose itself ( 5 - 10 % relative to the specific activity of gonp ), while the hydrolysis by the enzyme under the same conditions of 1 - au and 2 - au nanoparticles was barely detected (& lt ; 3 % relative to the enzymatic activity with the free ligands 1 and 2 ). these experiments demonstrate that it is possible to use nanoparticles to produce tailored globular carbohydrate models mimicking gsl - clusters in plasma membrane , allowing for the first time investigations to be carried out in solution of a novel mechanism of cell adhesion via carbohydrate - to - carbohydrate interactions . the glyconanoparticle approach described herein provides a strategy to prepare , in a simple way , a great variety of globular carbohydrate arrays that can advantageously compete with other spherical ( dendrimers , liposome ) or linear carbohydrate displays . the lacto - and le x - nanoparticles may be considered appropriate models to intervene in cell - cell adhesion and recognition processes . 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