TREATING WATER INSOLUBLE
NANOPARTICLES WITH HYDROPHILIC ALPHA-
HYDROXYPHOSPHONIC ACID CONJUGATES,
THE SO MODIFIED NANOPARTICLES AND
THEIR USE AS CONTRAST AGENTS
BACKGROUT^D
[0001] The invention relates generally to treating nanoparticies, particularly
those based on transition metal oxides, to render them sufficiently hydrophilic to foim stable aqueous suspensions and therefore be useful in applications requiring hydrophilicity such as contrast agents in diagnostic imaging such as MRI and X-ray, to the hydrophilic nanoparticies resulting from said treatment, to said stable aqueous suspensions and to the use of said hydropliilic nanoparticies as contrast agents in said imaging. Nanoparticies, i.e particles whose diameters are appropriately measured in nanometers, have been considered for a wide variety of end uses. SomjC of tlrese uses require some degree of hydrophilicity but the material upon which some nanoparticies are based may lack this attribute. For instance, nanoparticies with appropriate imaging properties for use as contrast agents for MR and X-ray imaging are typicall}- based on transition metal oxides which lack suitable hydrophilicity. Therefore efforts have been made to modify the surface properties of these nanoparticies to be miore compatible with aqueous media and give these nanoparticies tlie ability to fonn stable aqueous suspensions. Flowever, in some applications such as use as contrast agents it is also desirable that the nanoparticies have a monodisperse particle size distribution and any surface treatment that results in a polydisperse particle size distribution such as non¬uniform aggregation by complexation in a biological miatrix based on carbohydrates with carboxylate groups is problematic. In addition, in some applications such as in vivo use as contrast agents it is desirable that the surface treatm.ent ha^^e a well defined reproducible structure and be amenable to safety testing. Silane based surface

treatments can be problematic because they can undergo self condensation that interferes with these goals.
[0002] In addition, there has been a need for hydrophilic nanoparticles that do
not suffer a degradation of their hydrophilicity as a result of purification and display suspension stability in aqueous mediums containing electrolytes. For instance, in the preparation of contrast agents for in vivo use in human subjects the candidate nanoparticles would typically be subjected to filtration and be expected to show suspension stability in isotonic aqueous media, i.e. media containing about 150 niM NaCl. There have been efforts to use the adhesion of phosphates for transition metal oxides to im^pait this type of hydrophilicity to nanoparticles using phosphate based materials alone, such as polyphosphoric acid, or linked to In'drophilic moieties, such as polyethylene glycol. In this regard, there is a preference for hydrophilic moieties with an essentially neutral zeta potential for in vivo use in human subjects to avoid undesirable interactions with human tissue. However, such efforts have not yielded so hydrophilically modified nanoparticles that display the desired stability as a colloidal suspension in a 150 mM NaCl aqueous medium after filtration. For instance, such efforts have not yielded suspensions which display an essentially stable (no increase in hydrodynamic diameter (DH)) particle size as measured by dynamic light scattering (DLS) after tangential flow filtration v^'ith a 30 kDa cut off and storage for more than a week in such an aqueous medium.
BRIEF DESCRIPTION
[0003] The present invention invoh'es the disco^'ery of the superior
performance of conjugates of a-hydro.xyphosphonic acid and a hydroph'lic nioiet)' which are linked via the carbon atom caiTying the a-hydroxy group as agents to impro\''e t:ie hydrophilicity of water insoluble nanoparticles, particularly nanopartiiles based on transition metal oxides. The conjugate linkage preserves all three of the a-hydroxyphosphonic acid's hydroxyi groups and this is believed to give the conjugate superior adhesion to nanoparticles. In some embodiments the conjugate has the following Structure I:

0
II . , r ,
P f 1 '^ ^ [ ]
Structure I
wherein S is a spacer, L is a linlcage between S and R and R is a hydrophilic moiety and m and p are 1-5 and n and o are 0-5. In some embodiments, S is a direct bond, an unsubstituted or substituted aliphatic or cycloaliphatic group, an unsubstituted or substituted aryl group, a heteroaliphatic group or a heteroaryl group and in some cases is a straight chain alkyl group of 1 to 10 carbon atoms in length and Lisa direct bond, carbonyl group, ether group, ester group, secondary or tertiary amine, quaternary amine group, amide group, carbamate group or urea group. Suitable nanoparticles are those which are not soluble in water in the classic sense in which the the individual molecules of the solute are uniformly dispersed in the solvent in the manner of sugar or table salt in water. Thus the treatment of nanoparticles which possess some degree of suspensability in water with the alpha-hydroxyphosponic acid conjugates and the resultant nanoparticles with the conjugates adhered are included in the present invention.
[0004] It is of particular interest that the conjugate not include groups or
moieties which could have undesirable reactions with human tissue. Thus, it is convenient that the conjugate display a zeta potential between about -^10 mV and 40 mV, preferably between about -15 mV and 15 mV when adhered to a nanoparticie with it being especially interesting that it display an essentially neutral zeta potential when so adhered. This is conveniently accomplished by utiHzing zv.itterions or non-ionic moieties as the hydrophilic moiety.
[0005] The hydrophilic moieties ma}- be monomeric or polymeric but it is
convenient that they have an essentially neutral net ionic charge. .Ajiiong the polymeric hydrophilic moieties those polyetliers at least partially based on ethylene oxide units such as ethylene oxide/p^'opyiene oxide copolymers and polyethylene glycol are of especial interest. Monomeric hydrophilic moieties viih no net charge,

particularly zwitterions, are convenient for conjugates used to treat nanoparticles to be used in vivo with human subjects because of the greater ease in characterizing them for safety evaluations. Among these those based on 4-piperadinecarboxylic acid are of especial interest.
[0006] It is also convenient for conjugates used to treat nanoparticles to be
used in vivo with human subjects that the linkage between the a-hydroxyphosphonic acid and a hydrophilic moiety be a hydrocarbon, i.e. in Structure I A is a single bond. This minimiizes the probability of any interaction between such treated nanoparticles and human tissue. In this regard, conjugates of the following Structures II and III are of particular interest:
0
OH (H0)2'^ I
OH
Structure II Structure III
[0007] The conjugate is preferably sufficiently hydrophilic that when it is used
to treat nanoparticles at a ratio of about two conjugates per nanoparticle it will render the nanoparticles capable of forming stable colloidal suspensions in aqueous media with a DH determined by DLS of about 500 nm or less. It is particularly convenient that it render so treated nanoparticles hydrophilic enough to display a value of less than one for the log of the distribution coefficient between equal volumes of n-octanol and 0.1 M pH 7.0 3-(N-moipholino) propanesuifonic acid (MOPS) buffer.
[0008] The nanoparticles that are treated with the conjugate to achieve greater
hydrophilicity are preferably based upon transition metals and transition metal compounds such as oxides, carbides, sulfides, nitrides, phosphides, borides, halides,

selenides, tellurides and combinations thereof. Oxides are of particular interest. It is believed that the oxide structure contributes to the adhesion of the a-hydroxyphosphonic acid. Transition metal compounds are useful for preparing contrast agents for MR and X-ray im.aging. The transition metals of the third period of the Periodic Table of Elements are useful for fonning compounds that display paramagnetism and conveniently superparamagnetism and tlierefore are useiiil as MRI contrast agents. Especially convenient are supei-paramagnetic nanoparticles based upon iron oxide and optionally cobalt, copper, manganese, nickel or combinations thereof. Of these, the most convenient are nanoparticles based upon magnetite, maghemite or com.binations that are about 15 nm or less in diameter and display superparamagnetism. These are commonly refen-ed to as superparamagnetic iron oxide (SPIO) particles. Transition metals with atomic numbers greater than 34 and zinc are useftil for preparing compounds useful as X-ra}- contrast agents. Among these hafnium, molybdenum, silver, taiitalum, tungsten, and zirconium are of particular interest with tantalum and particularly tantalum oxide being the most convenient.
[0009] The hydrophilically modified nanoparticles typically have a DH as
deteimined by DLS of 500 mil or less. It is convenient that their DH be 50 nm or less, more preferably 30 mn or less and miOSt preferably that DH be between 3 and 30 mn. If the hydrophilically modified nanoparticles are destined for in vivo use in human subjects as, for instance, MRI or X-ray contrast agents, a particularly convenient Deis about 8 nm or less.
[0010] The hydrophilically modified nanoparticles are conveniently prepared
by reacting them with the conjugate. A convenient approach is to foim a colloidal suspension of the nanoparticles in an organic solvent such as tetrahydrofuran (THF) and then mix it with an organic solution of the conjugate in the same or a different organic solution. The mixture may then be held for an elevated temperature for an extended period until the reaction is essentially complete. Typically temperatures of 50° C or more for 16 hours or more are convenient.

[0011] Stable monodisperse aqueous colloidal suspensions of the
hydrophilically modified nanoparticles are readily obtained. Such suspensions should preferably be stable against filtration such as tangential flow filtration against a 30 kDa cut off and the addition of electrol\1es such as the addition of NaCl to render the aqueous medium isotonic, i.e. about 150 mM of NaCl. Preferably the suspensions are stable for storage periods of one week or greater and more preferably are stable against not only sedimentation but also against growth of the DH as determined by DLS of the suspended nanoparticles. If the suspensions are intended for in vivo use in human subjects it is convenient to render them isotonic by the addition of NaCl, dextrose or combinations thereof
[0012] The stable monodisperse aqueous colloidal suspensions are
conveniently prepared by diluting a colloidal suspension in an organic solvent, A convenient approach is to simply dilute the organic solvent or solvents in which the nanoparticles have been reacted with the conjugate by the addition of water. Another approach is to react a colloidal suspension of the nanoparticles in an organic solvent with the conjugate in water. In either case it is com'enient to remove the unreacted reactants by filtration or organic extraction with a solvent such as hexane or a combination. Any volatiles in the aqueous phase after solvent extraction can be removed by the appHcation of a partial vacuum. Then the hydrophilically modified nanoparticles can be purified by tangential flov/ filtration against a 30kDa filter.
[0013] The hydrophilicalh' modified nanoparticies may be conveniently used
as contrast agents in diagnostic imaging. Common types of such diagnostic imaging are MR and X-ray imaging. In either case, it is con\'enient to use hydrophilically modified nanoparticles which ha\'e a zeta potential between about -15 mV and 15 mV. A coiwenient approach in the in vivo imaging of human subjects is to administer the nanoparticles intraveneously, preferably as a stable isotonic aqueous suspension. If the imaging is to be by MR the nanoparticles should comprise a paramagnetic, preferably supei-paramagnetic species, and most preferably the}- should be iron oxide based such as magnetite or maghemite. If the imaging is to be by X-rav the nanoparticles should comprise a transition metal compound of a metal with an atomic nuiTiber greater than 34 or zinc, preferably gold, hafnium., molybdenum, silver.

tantalum, tungsten or zirconium and most preterably they should be tantalum oxide based. In a particularly interesting embodiment, the hydrophilically modified nanoparticles have a DR of 8 nra or less and clear the body of the subject via the kidney.
DRAWINGS
[0014] These and other features, aspects, and advantages of the present
invention will become better understood when the following detailed description is read with reference to the accompanying drawings in which like characters represent like parts tliroughout the drawings, wherein;
[0015] FIG. 1 is a generic structural fomiula for suitable alpha-hydroxy
phosphonic acids with which to make hydrophilic nanoparticles.
[0016] FIG. 2 is the structural formula for a particularly interesting alpha-
hydroxy phosphonic acid with which to make hydrophilic nanoparticles wherein Me is a methyl group.
[0017] FIG. 3 is a synthetic route to alpha-hydroxy phosphonic acids with
attached hydrophilic moieties R.
[0018] FIG. 4 is a synthetic route to the particularly interesting alpha-hydroxy
phosphonic acid shown in FIG. 2.
[0019] FIG. 5 is a hypothetical schematic representation of the attachment of
an alpha-hydroxy phosphonic acid of the t}'pe whose synthesis is illustrated in FIG. 3 with a hydrophilic moiety R attached to a superparamagnetic iron oxide SPIO nanoparticle.
[0020] FIG. 6A is a Ti weighted image (TE = 4.1 ras) of a tumor in
accordance Example 12 without contrast agent.
[0021] FIG. 6B is a Ti weighted irrage (TIE = 4.1 ms) of a tumor in
accordance Example 12, 30 minutes affer the adininistration of the nanoparticle contrast agent of Examnle 4.

[0022] FIG 6C is a difference map of the ditTerences be^\'een FIG. 6A and
FIG. 6B.
[0023] FIG. 6D is a To^-weighted image (TE = 18.4 ms) of a tumor in
accordance Example 12 wiihout contrast agent.
[0024] FIG. 6E is a To^-weighted imiage (TE = 18.4 nis) of a tumor in
accordance Example 12 30 minutes after the administration of the nanoparticle contrast agent of Example 4.
[0025] FIG. 6F is an R2* relaxation difference map of the differences between
FIG. 6D and FIG. 6E exhibiting a clear distinction between tumor and muscle tissue.
DETAILED DESCRIPTION
[0026] The conjugates of the present in\'ention are broadly defined to ha\'e a
wide variety of linkages and hydrophilic moieties. The key feature is that the conjugate has the tliree h3'droxyl groups of the a-hydrox}' phosponic acid that are chemically and sterically accessible. While the structure has chiral centers it is expected that all of the individual enantiomers and possible racemic mixtures would be suitable to impart hydrophilicity to water insoluble nanoparlicles.
[0027] These conjugates may have any of the commonly known chemical
linlcages between the a-hydroxy phosponic acid structure and the hydrophilic moiety including those based upon carbon, nitrogen, ox}'gen and sulf-ir. Groups of particular interest are hydrocarbon, carbonyl, ester, ether, secondary or tertiary amine, quaternary amine, urea, carbamate and amide. The intended end use of the nanoparticles to be treated with the conjugate may impact the choice of linkage group. For instance if the nanoparticles are to be used in vivo, particularly in human subjects, it may be desirable to avoid linkages such as quaternary groups which might result in interactions with tissue components such as proteins. The most interesting linkage group from the standpoints of inertness is a hydrocarbon.
[0028] The hydrophilic moiety can be any of those mtoieties known to ha\'e
good compatibility with water including those knov.-n as surfactants. They can be

anionic, cationic or non-ionic. The hydropliilic moiety may be a carbohydrate such as mono, di or oligosaccharide, a non-carbohydrate monomeric polyalcohol, a polyether with ethylene oxide groups, a non-carbohydrate polymer, other than a polyethers with ethylene oxide groups, which has pendant hydroxyl groups such as polyvinyl alcohol, poly(ethylene imine), an aliphatic or cycloaliphatic amine and combinations thereof.
[0029] In some embodiments, the hydrophilic moieties are based on ethylene
oxide with the poly(ethylene oxides) being of specific interest, particularly those with molecular weights equal to or less than about 5000 daltons. especially those with molecular weights equal to or less than about 2000 daltons. The poly(ethylene oxides) with molecular weights of about 350 daltons are of particular interest.
[0030] The hydrophilic moiety can also be a zwitterion having one or more
positively charged moieties, one or more negatively charged moieties and a spacer group in between the charged moieties. For the purposes of this application, combinations of positively charged moieties and negatively charged moieties are considered zwitterions if at physiological pH values they display essentially no net charge. Suitable positively charged moieties include protonated primary am/ines, protonated secondary amines, protonated tertiary alkyl amines, quantemary alkyl amines, protonated amidines, protonated guanidines, protonated pyridines, protonated pyrimidines, protonated pyrazines, protonated purines, protonated imidazoles, protonated pj-^rroles or combinations thereof, suitable negatively charged moieties include deprotonated carboxylic acids, deprotonated sulfonic acids, deprotonated sulfinic acids, deprotonated phosphonic acids, deprotonated phosphoric acids, deprotonated phosphinic acids, or combinations thereof and suitable spacer groups include unsubsituted and substituted aliphatic, cycloaliphatic and aryi groups, heteroaliphatic groups, heteroaryl groups, ethers, amides, esters, carbamates, ureas, or combinations thereof In one embodiment, spacer groups include straight chain alkyl groups of 1 to 10 carbon atoms in length.
[0031] If the intended end use of the nanoparticles is compatible with the
modified h}'drophilic nanoparticles w^ith the adhered alpha-hydroxyphosponic acid conjugates having a net ionic charge, the hydrophilic moiety mav be any of the

positively charged moieties or any of the negatively charged moieties discussed above as suitable for the foimation of zwitterions.
[0032] The intended end use of the nanoparticles to be treated with the
conjugate may impact the choice of hydrophilic moiety. For instance if the nanoparticles are to be used in vivo, particularly in human subjects, it may be desirable to avoid hydrophilic moieties such as ionic groups which might result in interactions with tissue components such as proteins. For in vivo use, hydrophilic moieties with essentially no net charge such as zwitterions and polyethers with ethylene oxide units are of particular interest. For use with human subjects, hydrophilic moieties that are easily and reproducibly characterized for safety evaluation such as monomeric moieties are particularly convenient. Particularly convenient hydrophilic moieiles are those based on 4-piperadinecarboxylic acid which are both monomeric and as zwitterions carry no net charge. For applications in which toxicity is less of a concern such as the in vitro inoculation of cell cultures poly(ethylene)imines may be appropriate hydrophilic moieties.
[0033] Particularly suitable zwitterions are disclosed in U.S. Patent
Application 12/344,604 filed 29 December 2008, incorporated by reference herein.
These include hydrophilic moieties which contain cationic nitrogens and anionic
carboxylic, phosphoric and sulfonic acid based groups such as N,N-dimethyl-3-sulfo-
N-(3-propyl)propan-1 -aminium, 3-(methyl)propyl)amino)propane-1 -sulfonic acid. 3-
(|3ropylamino)propane-1 -sulfonic acid, 2-(ethcxy(hydroxy)phosphor}'lox)')-N,N,N-
trimethylethanam.inium, 2ethyI(hydroxy)phosphorylox_v)-N,N,N-
trimethylethanaminium, N,N,N-tTimethyl-3 -(N-propionylsulfamoyl) propan-1 -aminium, N-((2H-tetrazol-5-yl)methyl)-N,N-dimethyl-propan-1 -aminium, N-(2-carboxyethyl)-N,N-dimethyl-propan-l-aminium. 3-(meth}'lpropyl)am,ino)propanoic acid, 3-(propylamino) propanoic acid, N-(carboxymethyl)-N,N-dimethyl-propan-l-aminium, 2-(methy!amino)acetic acid, 2-(propylamino)acetic acid, 2-(4-propylcarbamoyl) piperazin-l-yl)acetic acid, 3-(4propylcarbaraoyI) piperazin-1-yl)propanoic acid, 2-(m.etliy!(2-propylureido) ethy})amiRo)acetic acid and 2-(2-(propylureido)ethyl)aminoacetic acid.

[0034] Suitable bydropbilic polyether based hydrophilic moieties are
disclosed in U.S. Patent 5,916,539 issued 29 June 1999, which is incoiporated herein by reference. These include polyethylene glycols (PEG's) of various molecular weights with various terminal groups includ'ng amino and hydroxyl as well as copolymers with polypropylene glycol (PPG).
[0035] The conjugate is preferably sufficiently hydrophilic that it can render
nanoparticles able to form stable aqueous suspensions when it is reacted with the nanoparticles at a ratio of two moles of conjugate to one mole of the metal basis of the nanoparticle. In this regard, the nanoparticle will typically be based on a transition metal compound such as an oxide or a transition metal itself It is convenient to specify the reaction ratio using the moles of elemental metal as this can be readily obtained from an elemental analysis of the starting suspension of nanoparticles in organic solvent. From a knowledge of the chemical make up of the nanoparticles and their average size before treatment, a rough calculation can be made of the amount of conjugate per nanoparticle. It is particularly convenient that the conjugate be hydrophilic enough to give nanoparticles of iron oxide or tantalum oxide of less than 15 nm treated at this ratio sufficient hydrophilicity to display a value of less than one for the log of the distribution coefficient between equal volumes of n-octanol and 0.1 M pH 7.0 MOPS buffer.
[0036] It is of particular interest that the modified hydrophilic nanoparticles
with an adhered alpha-hydroxyphosphonic acid conjugate be sufficiently hydrophilic to foiTU a stable aqueous colloidal suspension that exhibits no substantial change in hydrodynamic diameter (DH) as determined by dynamic light scattering (DLS) in 150 mM NaCl water after tangential flow filtration and storage for one week at room, temperature.
[0037] The nanoparticles that are to be treated with the conjugate can be of
any water insoluble material that can be formed into particles of 500 nm or less to which the a-hydroxy phosphonic acid portion of the conjugate will adhere. It is of interest to use nanoparticles that have utility as contrast agents in MR or X-ray

imaging. However, nanoparticles for other end uses sixh as inir.sion of cell cultures for transfection of genes are also of interest.
[0038] For use as MRI contrast agents the basis cf the nanoparticles should be
a metal or its compounds that are paramagnetic, with those that are superparamagnetic being of particular interest. These metals are conveniently drawn from the transition metals of Period III of the Periodic Table of Elements begimiing with m.anganese and ending with zinc. A pailicularly interesting group of materials are those based upon iron oxide. Especially convenient materials are those known as SPIO's. These materials have the general fonnula [Fen OsJxfFcT 03(^1" 0)1;_x where 1 > x > 0. M" may be a divalent metal ion such as iron, m.anganese, nickel cobalt, magnesium, copper, zinc or a combination thereof. When the metal ion (iVr ) is ferrous ion (Fe" ) and X = 0, the material is magnetite (Fe304), and when x = 1, the material is maghemite (y-Fe203).
[0039] In general, superparamagnetism occurs when crystal-containing
regions of unpaired spins are sufficiently large that they can be regarded as thermodynamically independent, single domain particles called magnetic domains. These magnetic domains display a net magnetic dipole that is larger than the sum of its individual unpaired electrons. In the absence of an applied magnetic field, all the magnetic domains are randomly oriented v^ith no net magnetization. Application of an external magnetic field causes the dipole moments of all magnetic domains to reorient resulting in a net magnetic mom.ent. In some embodiments, these materials demonstrate a spinel crystalline structure as shown by transmission electron microscope (TEM) analysis.
[0040] For use as X-ray contrast agents, the basis of the nanoparticles should
be a metal or its compounds that are substantially more radiopaque than materials typically found in living organism.s. It is con^'enient to use materials with an effective atomic number greater than or equal to 34 when at a concentration of approximately 50 mM. Such materials are likely yield appropriate contrast enhancement of about 30 Hounsfield units (HU) or greater, Vviiich is a minimum enhancement of particular interest. Examples of transition metal elements that may provide this property include

tungsten, tantalum, hafnium, zirconium, molybdenum, silver, and zinc. Tantalum oxide is one particular example of a suitable core composition for use in X-ray imaging applications. Of especial interest are materials that lead to a CT signal in a range from about 100 Hounsfield to about 5000 Hounsfield units.
[0041] The modified hydrophilic nanoparticles to which the alpha-
hydroxyphosphonic acids have been adhered may be used as contrast agents in diagnostic imaging. In such an application, these nanoparticles are administered to a subject, in some embodimients a mammalian subject, and then the subject is subjected to imaging. These nanoparticles have particular utility in MR and X-ray imaging though they may also find utility as contrast agents in ultrasound or radioactive tracer imaging.
[0042] When used in diagnostic imaging, particularly of mammalian subjects
and more particularly of human subjects, the modified hydrophilic nanoparticles to which the alpha-hydroxyphosphonic acids have been adhered are typically taken up in a pharmaceutically acceptable caiTier Avhich m^y or may not comprise one or more excipients. If the administration is to be by injec':ion, particularly parenteral injection, the carrier is typically an aqueous medium tliat has been rendered isotonic by the addition of about 150 mM of NaCl, 5% dextrose or combinations thereof It typically also has the physiological pH of between about 7.3 and 7.4. The administration may be intravascular (IM), subcutaneous (SQ) or most commonly intravenous (IV). However, the administration may also be via implantation of a depot that then slowly releases the nanoparticles to the subject's blood or tissue.
[0043] Alternatively, the administration may be by ingestion for imaging of
the GI tract or by inhalation for imaging of the lungs and airways.
[0044] The administration to human subjects, particularly IV administration,
requires that the modified hydrophilic nanoparticles to which the alpha-hydroxyphosphonic acids have been adhered be non-toxic in the amounts used and free of any infective agents such as bacteria and viruses and also free of any pyrogens.

Thus, these nanoparticles should be stable to the necessary purification procedures and not suffer a degradation in their hydrophilicity.
[0045] These nanoparticles may be delivered to the site of adrrjnistration as a
stable aqueous colloidal suspension with the proper osmolality and pH, as a concentrated aqueous colloidal suspension suitable for dilution and adjustment or as a powder, such as obtained by lyophilization, suitable for reconstitution.
Example 1
[0046] Synthesis of a PEG-3 50 Conjugate
[0047] Synthesis of PEG-350 mono(methyl ether) acetaldeliyde. To a
solution containing PEG-350 mono(methyl ether) (3.438 g, 9.82 ramol) dissolved in CH2CI2 (98 mL) was added Dess-Martin Periodinane (5.00 g, 11.79 mmol) and the resulting solution was stiiTed at rt for 20 h. During the reaction a fine, white precipitate was foraied and was removed at the end of the reaction via filtration tlirough a celite pad. The solvent was removed from the filtrate in vacuo to leave a white solid suspended in a yellow oil. The solid was triturated with dieth}'l ether, and the solid was removed by filtration thi-ough a celite pad. Removal of the solvent from the filtrate in vacuo left the product PEG-350 mono(methyl ether) acetaldehyde (3.42 g, 100 %) as a yellow oil. 'H NMR (CDCI3) 5 9.73 (t, J = 4 Hz, IH), 4.16 (d, J = 4 Hz, 2H), 3.65 (m., 24H), 3.38 (s, 3H) ppm. IR (neat) 2873, 1732, 1455, 1350, 1109, 1040, 948, 851, 749 cm'^
[0048] Synthesis of diethyl a-hydroxy PEG-350 mono(n]ethyI ether)
phosphonate. To a solution containing PEG-350 niono(methyl ether) acetaldehyde (3.71 g, 10.7 mmol) dissolved in tetrahydrofuran (53 niL) was added diethyl phosphite (1.77 g, 12.8 mmio]). The solutiGU was coc!:d to 0 ''C, and 1,8-diazabicyclo[5.4.0jundec-7-ene (1.94 g. 12.8 mmol). Alter stirring at 0 °C for 10 min, the rxn was wamied to rt and stirred for an additional 24 h. The solvent was remo\'ed in vacuo to leave a dark yello^\" yellow oil vdiich was purified \'ia colunm chromatography (100% CH2CI2 to 15«'o MeOH/ 85% ClhCh) to give 3.30 g (64 %)

of the desired diethyl a-hydroxy PEG-350 mono(raethyl ether) phosphonate product as a yellow oil. ^H NMR (CDCI3) 6 4.19 (m, 6H), 3.65 (ra, 24H), 3.38 (s, 3H), 1.34 (m, 6H) ppm. ^^P NMR (CDCI3) 5 23.1 ppm. IR (neat) 3343, 2872, 1725, 1453, 1248, 1105,965,850,791cm-'.
[0049] Synthesis of a-hydroxy PEG~350 mono(methy5 ether) phosphorJc
acid. To a solution containing diethyl a-hydroxy PEG-350 mono(raethyl ether) phosphonate (3.61 g, 7.43 mniol) dissolved in methylene chloride (74 mL) was added trimethylsiljd bromide (3.41 g, 22.3 mmol) and the resulting solution was stiiTed at rt for 2 h. The solvent was removed in vacuo to leave a brown oil. The resulting oil was dissolved in acetone (74 mL) and water (0.5 mL) and the resulting solution was stirred at rt for 1.5 h. The solvent was then removed in vacuo to leave the desired a-hydroxy PEG-350 mono(methyl ether) phosphonic acid product (2.66 g, 84 %) as a golden oil. 'H NMR (CDCI3) 5 3.65 (m, 24H), 3.38 (s, 3H). ^'P NMR (CDCI3) 5 24.0 ppm. IR(neat) 3460, 2870, 1727, 1456, 1351, 945, 849 cm"'.
Example 2
[0050] Synthesis of a PEG-1900 Conjugate
[0051] Synthesis of PEG-1900 mono(methyI ether) acetaldehyde. To a
solution containing PEG-1900 mono(methyl ether) (16.32 g, 8.60 mmol) dissolved in CH2CI2 (86 mL) was added Dess-Martin Periodinane (4.00 g, 9.44 mmol) and the resulting solution was stirred at rt for 20 h. During the reaction a fine, white precipitate was formed and was removed at the end of the reaction via filtration through a celite pad. The solvent was removed from the filtrate in vacuo to leave a white solid W'hich was recrystallized from TLfF liexanes to give the desired product (11.6 g, 71 %) as a white solid. 'H NMR (CDCL) 5 9.74 (t, J = I Hz. IH), 4.17 (d. J = 1 ITz 2H), 3.83 (m, 2H), 3.65 (m, 170H), 3.39 (s, 3H).
[0052] Synthesis of diethyl a-hydroxy PEG-1900 mono(methyI ether)
phosphonate. To a solution containing PEG-1900 mono(methyI ether) acetaldehyde (10.74 g, 5.66 mmol) dissolved in tetrahydrof'!ran (57 mL) v/as added diethyl

phosphite (0.938 g, 6.79 mmoi) followed by l,8-diazabicyclo[5.4.0]iindec-7-ene (1.03 g, 6.79 mrool). The reaction Vv'as stirred at it for 72 h. The solve:it was removed in vacuo to leave an orange yellow solid vvhich w-as recr} stallized from TKF/hcxanes to give the desired product (11.08 g, 96 %) as an off white solid. 'li NMR (CDCl:) 5 4.18 (m, 4H), 3.64 (m, 172H), 3.38 (s, 311).
[0053] Synthesis of a-hydrosy PEG-350 inoao(methy! ether) phosphcnic
acid. To a solution containing diethyl a-hydroxy PEG-1900 mono(meth\d ether) phosphonate (11.08 g, 5.44 mniol) dissolved in methylene chloride (54 mL) was added trimethylsilyl bromide (2.49 g, 16.3 mmol) and the resulting solution was stirred at rt for 3 h. The solvent was removed in ^'acuo to leave a brown oil. The resulting oil v»^as dissolved in acetone (54 mL) and water (0.5 mL) and the resulting solution was stirred at rt for 16 h. The solvent was then remoA'ed in vacuo to leave an orange solid, which was recrystallized from TrE^'hexanes to give the desired product (10.77 g, 86 %) as an off white solid. 'H NMR (CDCI3) 5 4.12 (m, 2H), 3.65 (m, 170H), 3.38 (s, 3H).
Comparative Example 3
[0054] Synthesis of a hydrophilic phosphate
[0055] Synthesis of diphenyi PEG-350 inonG(methyl ether) phosphate. To
a solution containing PEG-350 mono(methyl ether) (8.54 g, 24.4 mmol) dissolved in CH2CI2 (80 mL) v.'-as added triethyi amiine (3.68 g, 36.6 mmol) followed by 4-A',.V-dimethylaminopyridine (0.298 g, 2.44 mmol). The resulting solution was cooled to 0 °C and diphenyi chlorophosphate (7.87 g, 29.3 mmol) was added dropv/ise and the reaction was stin'ed at 0 °C for 10 min. The reaction was then warmed to rt and stiixed for an additional 16 h. The reaction was quenched with the addition of 10% HCl (80 mL) and the resulting layers were separated. The organic la\'er was washed with water (80 mL) and brine (80 mL) and was dried over anliydrous MgS04. Filtration and removal of the solvent in vacuo left the desired product (14.2 g, 100%) as a golden oil. 'H NMR (CDCI3) 5 7.34 (m, 4H), 7.22 (m, 6H), 4.38 (m, 2H), 3.73 (m, 2H), 3.64 (m, 24H), 3.54 (m, 2H), 3.38 (s, 3H).

[0056] Synthesis of PEG-350 mono(iTietayi ether) phosphoric acid. To a
solution containing diphenyl PEG-350 n:ono(methy] ether) pliosphate (14.2 g. 24.4 mmol) dissolved in acetic acid (108 raL) was added platiniiira(IV) oxide hydrate (200 mg) and the resulting suspension was heated to 50 °C and placed under an atmosphere of H? until hydrogen uptake ceased. The reaction was filtered tlrough a celite pad to remove catalyst and the solvent v,'as removed in vacuo to leave tire desired product (10.49 g, 100%) as a clear, yellow oil. 'H NMR (CDCI3) 5 4.20 (m, 2H), 3.67 (m, 24 H), 3.56 (m,2H), 3.39 (s,3H).
Example 3
[0057] Synthesis of superparamagnetic iron oxide (SPIO) nanoparticles.
A 100 mL tlrree-necked round bottom flask was charged v'ith Fe(acac)3 (0.706 g, 2.0 mmol) and anhydrous benzyl alcohol (20 mL). The resulting solulion •was sparged with nitrogen and heated to 165°C for 4 hours under a nitrogen atmosphere. The resulting colloidal suspension of 5 mr^ iron oxide particles (As determined by DLS) was then cooled to, and stored, at room temperature.
Exam.ple 4
[0058] Syntiiesis of a-hydroxy PEG-350 mono(methyI ether) phosphonate
coated superparamagnetic iron oxide aanoparticles. To a colloidal suspension of superparamagnetic iron oxide nanoparticles of E::ample 3 in THF at 1 mg Fe/mL was added the a-hydroxy phosphonic acid conjugate of Example 1 (At a ratio of 1. mol of conjugate per mol of Fe) and the resulting suspension v/as heated at 50 °C for 16 h. The reaction was then cooled to rt, diluted with water, and the brown aqueous solution was v/ashed three times with hexanes. Any remaining volatiles in the aqueous layer were removed in vacuo and the resulting nanoparticles were purified by washing with H2O against a 30 kDa molecular cutoff filter using tangential flovv filtration.
Example 5

[0059] Synthesis oi" a-hydroxy PEG-1900 mono(methyl ether)
phosphonate coated superparamagnetic iron oxide nanoparticles. Example 4 was repeated using the conjugate of Example 2 in place of the conjugate of Example 1.
Comparative Example 2
[0060] Synthesis of a-hydroxy PEG-350 mono(methyI ether) phosphate
coated superparamagnetic iron oxide nanoparticles. Example 4 was repeated using the conjugate of Comparative Example 1.
Example 6
[0061] Synthesis of 5-bromo 1-pentanaI. Oxalyl chloride (2.42 niL, 0.022
mol) was mixed with anhydrous dichloromethane (40 mL) in a 250 mL round bottom flask. The flask was blanketed with nitrogen and the solution was cooled to -78 °C in a dry ice/acetone bath. The reaction mixture was stirred and arihydrous dimethylsulfoxide (3.4 mL, 0.044 mol) was slov^'ly added to the flask followed by 5-bromo-l-pentanol (3.34 g, 0.020 mol) and the reaction mixture was stirred for 15 minutes at -78 °C. Triethylamine (14.0 mL, 0.1 mol) was slo\^!y added to the reaction mixture. When the addition of triethylamine was complete, the reaction was stin-ed for 5 minutes at -78 "C. The reaction was removed from the dry ice acetone bath, wanned to room temperature, and stiiTed for 18 hours at room temperature.
[0062] Vv'ater (100 mL) was added to the reaction mixture. The two-phase
mixture was shaken vigorously in a 500 mL scparatory funnel. The aqueous layer was removed and extracted v/ith dichloromethane (100 mL). This dichloromethane was combined with the dichloromethane f-om tlie reaction mixture. The combined dichloromethane solution was successively Vv-ashed with 100 mL each of l';o HCl(aq), water, saturated NaHC03(aq) and saturated NaCl(aq). The dichloromethane solution was dried with magnesium sulfate and the dichlormethane solution was recovered by filtration. Solvent was removed under \'acuum ]ca\''ir;g a yellov,- Liquid (1.80 g). The major product was confirmed to be 5-bronio l-pcnta:ia! by 'H >sMR. 'LINMR (400 MHz, CDC13) 5 9.81 (m, IH), 3.43 (m, 3H), 2.50(^n, 2H), 2.0-1.4(m, 8K). The reaction product was can'ied on tc Examp'; 7 without ParTier purification.

Example 7
[0063] Synthesis of diethyl (5-bromo-l-hydroxy-pcnt>i)phosphonate. 5-
bromopentanal (1.64g, 0.010 mol) was dissolved in diethylether (15 mL) in a 250 ml round bottom flask. The reaction was blanketed with nitrogen. Lithium perchlorate (7.92 g, 0.075 mol) was added to the reaction and the reaction solution was cooled to 0 °C in an ice bath. Chlorotrimethylsilane (0.631 mL, 0.010 moles) was added to the flask followed by trimethylphosphite (2.1 mL, 0.012 mol). The reaction mixture was stirred for 18 hours at room temperature.
[0064] After 18 hours at room temperature water (40 mL) was added to the
■ reaction followed by dichloromethane (40 mL). The organic phase was transferred to a separatory funnel and washed successively with water (40 mX) and brine (40 mL). The methylene chloride solution was dried with magnesium sulfate and filtered to recover the methylene chloride solution. Solvent was removed under vacuum leaving a yellow oil (3.01 g). The oil was characterized by ^H NMR and "^P NMR and the major product was confirmed to be diethyl (5-bromo-l-hydroxy-pentyl)phosphonate. 'H NMR (400 MHz, CDC13) 5 4.25-4.00 {m, 4H). 3.00-3.43 (m, 2H), 1.78-1.95 (m, 2H), 1.78-1.61 (m, 3H), 1.61-1.41 (m, 2H), 1.40-1.25 (m, 6H). ^'P NMR (600 MHz, CDC13) 5 26.5 (s, IP), 24.2-24.7 (m, 0.3P). The reaction product was carried on to Example 8 without further purification.
Example 8
[0065] Synthesis of diethyl 5-(4-(ethoxycarboRyi)piperidiii-l-yl)-l-
hydroxypent}iphosphonate. Diethyl (5-bromo-l-hydroxy-pentyl)phosphonate (3.02 g, 0.0099 mol) vvas dissolved in anliydrous toluene (100 mL) in a 300 mL round . bottom flask. Triethylamine (2.08 mL, 0.015 mol) was added to the reaction mixture followed by ethylisonipecotate (1.84 mL, 0.012 mol). The mixture was heated to reflux for 18 hours. Solvent WT^S removed under vacuum leaving an orange gum. The gum was dissolved in dichloromethane (100 mL) and Vv-ashed successi\'ely with saturated aqueous NaHC03 (100 mL) and brine (100 niL), The methylene chloride

solution was dried with magnesium sulfate and reco\'crd by filtration. Solvent was removed under vacuum leaving an orange liquid (1.70 g).
[0066] The orange liquid was purified by silica gel column clironiatography.
A silica gel column (40 g) was eiuted with a solvent gradient starting with 100% dichloromethane and changing to 20% methanol by \'olume in dichlorom?thane o\'er 30 minutes. Fractions that contained the product wc:;2 combined and solvent was removed under vacuum leaving a yellow liquid (0.66 g). The yellow liquid Vv-as characterized by 'H NMR and the major product was identified as diethyl 5-(4-(ethoxycarbonyl) piperidin-l-yl)-l-hydroxypentylphosphonate. ^H NMR (400 mHz, CDC13) 5 4.9-4.5 (s, IH), 4.2-4 (m, 5H), 3.8-3.7 (m, IH), 2.9-2.7 (m, 2H), 2.4-2.1 (m., 3H), 2.1-1.9 (m, 2HX 1.9-1.8 (m, 2H), 1.8-1.3 (m, 8I-I), 1.3-1.2 (m, 5H), 1.2-1.1 (im 3H). The reaction product was earned to Exarr.ple 9 without further purification.
Example 9
[0067] Synthesis of 5-(4-(ethoxycarbonyl)piperidin-l-yl)-l-
hydroxypentylphosphonic acid. Diethyl 5-(4-(ethoxycarbonyl)piperidin-l-yl)-l-hydroxypentylphosphonate (0.66g, 0.0017 mol) was dissolved in dichloromethane (25 mL) in a 100 mL flask. Bromotrimethylsilane (0.69 niL, 0.0052 mol) was added to the reaction mixture. The reaction was stin'ed overnight at room temperature. A_fter overnight stirring, solvent was removed under vacuum leaving an orange gum. The gum was dissolved in acetone (20 mL). Water (0.4 mL) was added. A gum precipitated. Solvent was removed under vacuum leaving a red gum (0.6 g). The gum was characterized by ^H NMR and the product determined to be 5-(4-(ethoxycarbonyl)piperidin-l-yl)-l-hydroxypentylphosphonic acid. IH NMR (400 MHz, CD30D) 5 4.3-4.1 (m, 2H), 3.9-3.4 (m, 3H), 3.4-2.5 (m, 7H), 2.5-1.35, (m, IIH), 1.35-1.2 (m,3H).
Example 10
[0068] Synthesis of-(4-(ethn\;ycarbonyI)piperidin-l-yl)-l-
hydroxypentylphosphonate coated tantalum oxide nanoparticles. A solution of anhydrous methanol (17 mL) containing isobutyric acid (0,242 g. 2.75 mmol) and

water (0.08 g, 4.44 mmol) was degassed for 40 minutes by sparging with N2. This was added with Ta2(OEt)5 (1 g, 2.46 mmol) dropwise and the reaction mixture was stiiTed under N2 atmosphere for 5 h to yield a suspension of 3 to 4 nm nanoparticles. A solution of 5-(4-(ethoxycarbonyI)piperidin-1 -yl)-1 -hydroxypentylphosphonic acid (0.088 g, 0.205 mmol) in methanol (0.5 mL) was added dropwise to the tantalum oxide nanoparticle suspension (1 mL) and was heated at 70 °C overnight under N?. After cooling to room temperature, water (~3 mL) was added dropwise to the reaction mixture After removing methanol by evaporation at reduced pressure on a rotary evaporator, 1 M NH4OH (0.33 mL) was added and the reaction was stirred at 50° C overnight. The reaction mixture was dialyzed against DI v/ater (3 x 2 L) for 24 h using a 3500 Da molecular v/eight cut-off regenerated celluose membrane. Size was determined to be 7 run in water by DLS.
Example 11
[0069] Characterization of Colloidai Suspensions c* SPJO Nanoparticles.
The colloidal suspensions obtained as the resuli of the tangential flow filtration in Examples 4 and 5 and Comparative Example 2 were evaluated for stability and zeta potential.
[0070] The hydrodynaraic diameter (Dj;) was measured via dyucimic light
(DLS) scattering using 150 mM NaCI in water as the suspension medium. The purified SPIO suspension from the tangential flow filtration v/as diluted into 150 mM NaCl in water and passed tlii-ough a 100 nm filter to remove dust prior to DLS analysis using a Brookhaven ZetaPALS. The dilution was carded out to j-ield a minimum of 20,000 counts per second during the DLS measurement. The measurements were made both shortly after the modified nanoparticles were made and after two weeks storage at room temperature. A significant increase in the DH after storage was an indication that nanoparticles had aggregated and that therefore the particular colloidal suspension was not stable.
[0071] The Zeta potential was measured using a Brookhaven ZetaPALS after
diluting the purified SPIO suspension froiTi the tangential floAv filtration 14x with 10

mM NaCl and passing the diluted SPIO solution through a 100 am fiher to remove dust. Tlae zeta potential for all three colloidal suspensions was within the range ±15 mV range commonly accepted as neutral.
[0072] The results are set forth in Table 1
Table 1
Nanoparticle Coating DH post DR 2 weeks post Zeta
s}-nthesjs synthesis Potential
PEG-350 a-hydroxy phosphonate 10 ± 1 nm 9 ± 1 mn -0.5 mV
PEG-350 Phosphate 50±lnm >100nni 7.3 mV
PEG-1900 a-hydroxy phosphonate 20 ± 1 nm 22 ± 1 irni -5.0 mV
5-(4-(ethoxycarbonyl)piperidin-l- 7 ± 1 nm -1.7 mV
yl)-1 -hydroxypentylphosphonate
Example 12
[0073] Imaging of in vivo tumors by Mil?'. All procedures invoh-ing anim.als
were completed under protocols approved by the GE Global Research Institutional Animal Care and Use Committee. Tumors were induced in female Fischer 344 rats (-150 g) by subcutaneous injection of 2x10^ Mat B III cells (ATCC# CRL1666, ATCC, Manassas, VA) in 0.1 mL Hank's balanced saline solution. The injection site was located dorsally between the shoulder blades. The tumors were imaged 9 da}'s after implantation, when the tumors v/ere -1 cm, ir diameter.
[0074] Imaging Vv-'as conducted on a clinical 3 T GE MR750 scanner using a
custom-built, ~6 cm solenoid recei\'e RF coil. To prepare for imiaging, the rats were anesthetized by IP injection of ketamine and diazepam using 75 and 5 mgdcg doses, respectively. Once immobile, a 24 gauge catheter was placed in a lateral tail vein and coimected to a saline-primed, microbore catheter line extensio.n and stop cock. The dead ^-olume of the catheter, line and stop cock v^'as -0.5 m.L. The prepared animal was then placed within the RF coil and positioned within uie bore of the scanner. A

pre-injection image set was acquired, and then, v,-ithout moving the table or the animal, the PEG-350 a-hydroxy phosphonate coated superparamagnetic iron oxide nanoparticles were injected via the stop cock follovvcd by a saline flush (~0.S mL). Immediately following injection (-starting 30 s post-injection), image sets were collected throughout a dynamic acquisition period of -30 minutes resulting in collection of ~16 post-contrast time points. For the injection, SPIO agent was in physiologic saline at a concentration of 10 mg Fe/mL, and was sterile filtered prior to injection and tested for the presence of endotoxin. The agent w^as dosed at 3 mg Fe/kg body weight.
[0075] A 3D fast gradient echo pulse sequence was employed that allowed
collection of images at 10 echo times. The imaging slab was positioned via the graphical prescription interface such that the tumor was centered within tire transaxial slices and the coverage included the majority of the tumor in depth. The pulse sequence parameters were as follows: pulse sequence: 3D ME fGRE; TE: ranged from'4.1 to 68 ms, with 7.1 ms spacing; TR: 75.5 ms; flip angle: 25 degrees; bandwidth: 62.5 MHz; matrix: 256x256; slice thickness: 0.9 ram; field of view: 8 cm^, yielding a voxel size of 0.31x0.31x0.9. The sequence acquisition time was -2 min.
[0076] The imaging data sets were analyzed using a custom software tool
(CineTool v8.0.2, GE Healthcare) built upon the IDE platform (IDE v. 6.3, ITT Coip., Boulder, CO). In brief, the image analysis tool allowed manual drawing of 3D regions of in interest (ROIs) on the pre-injection series with subsequent calculation of the T2* time constant by exponential regression for every voxel within the drawn ROIs at all time points. Representative images and difference maps are given in Figure 5.
[0077] While only certain features of the invention have been illustrated and
described herein, many modificadons and changes will occur to those skilled in the art. It is, therefore, to be understood that the appended claims are intended to cove-all such modifications and changes as fall within the true spirit of the invention.

CLAIMS:
1. A composition comprising:
a water insoluble nanoparticle to which is adhei-ed at least one alpha-hydroxy phosphonate moiety having the formula:
O
II , , , ,
/-P^^ f If 11 (H0)2 f-"S- L- R
HOJ ^ ^n 0 p
L -^m
wherein S is a spacer, L is a linkage between S and R and R is a polymeric hydrophilic moiety and m and p are 1-5 and n and o are 0-5.
2. The composition of claim 1 wherein R is a polymeric hydrophilic moiety based on ethylene oxide units.
3. The composition of claim 1 or 2 wiierein the polymeric hydrophilic moieties are based on ethylene oxide and wherein the poly(ethylene oxides) has a molecular weight equal or less than 5000 daltons.
4. A composition as claimed in any of claims 1 to 3 wherein the nanoparticle is a superpamiagnetic nanoparticle based on iron oxide.
5. A composition as claimed in any of ciaims I to 4 further comprising a pharmaceutically acceptable carrier or excipient,
6. The composition of claim 5 for use as an i\IRI contrast agent suitable for injection into a human subject.
7. A process of utilizing water insoluble nanoparticles to which are adhered alpha-h}'droxy phosphonate moieties in diagnostic imaging comiprising
a. administering said nanoparticles to a ^t:b;ec:: ^md

b. subjecting said subject to diagnostic imaging in wliicli said nanoparticles act as a contrast agent.
8. The process of claim 7 in whicli tlie alplia-li}'droxy pliosphonate moieties have the
fomiula:
O
II , , , ,
?^ [ ] [ I [
(H0)2 f--S- L—R
HO ^ 0 p
L -'m
wherein S is a spacer, L is a linkage between S and R and R is a poiymeric hydrophilic moiety and m and p are 1-5 and n and o are 0-5.
9. The process of claim 7 or 8 wherein R is a polymeric hydrophilic moiety based en ethylene oxide units.
10. The process of claim 9 w^herein the polymeric hydrophilic moieties are based on ethylene oxide and wherein the poly(ethylene oxides) has a molecular w-eight equal or less than 5000 daltons.
11. The process of any of claims 1 to 10 wherehi the nanoparticle is a superparmagnetic nanoparticle based on iron oxide.
12. A process for making water insoluble nanoparticles to which are adhered alpha-hydroxy phosphonate moieties comprising:
a. providing a suspension of nanoparticles in a suspension agent; and
b. contacting said suspension with said alpha-hydroxy phosphonate moieties.
13. The process of claim 12 in which the alpha-hydroxy phosphonate moieties have the
formula:
O
o r 1 1 1 f 1 ^^^^■'2 In J.J. j,-j- J,,
T-'O " ^ P

wherein S is a spacer, L is a linkage between S and R and R is a polymeric hydrophilic moiety, m and p are 1-5 and n and o are 0-5.
14. The process of claim 13 wherein R is a polymeric hydrophilic moiety based on ethylene oxide imits.
15. The process of claim 14 wherein the polymeric hydrophilic moieties are based on ethylene oxide and wherein the poly(ethylene oxides) has a molecular weight equal or less than 5000 daltons.
Dated this on 02/04/2012 ( A /I /^
HRISHIKESHpA^ HAODHURY
OFtREVFRy & SAGAR
ATTORNEY FOR THE APPLjCANT