WO2010028217A1 - Réactifs de couplage de méthyléther-carbodiimide de poly(éthylèneglycol) pour la fonctionnalisation biologique et chimique de nanoparticules solubles dans l'eau - Google Patents

Réactifs de couplage de méthyléther-carbodiimide de poly(éthylèneglycol) pour la fonctionnalisation biologique et chimique de nanoparticules solubles dans l'eau Download PDF

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WO2010028217A1
WO2010028217A1 PCT/US2009/055992 US2009055992W WO2010028217A1 WO 2010028217 A1 WO2010028217 A1 WO 2010028217A1 US 2009055992 W US2009055992 W US 2009055992W WO 2010028217 A1 WO2010028217 A1 WO 2010028217A1
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poly
ethylene glycol
methyl ether
carbodiimide
nps
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PCT/US2009/055992
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Preston T. Snee
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The Board Of Trustees Of The University Of Illinois
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G18/00—Polymeric products of isocyanates or isothiocyanates
    • C08G18/06—Polymeric products of isocyanates or isothiocyanates with compounds having active hydrogen
    • C08G18/70—Polymeric products of isocyanates or isothiocyanates with compounds having active hydrogen characterised by the isocyanates or isothiocyanates used
    • C08G18/71—Monoisocyanates or monoisothiocyanates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0063—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres
    • A61K49/0065—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the luminescent/fluorescent agent having itself a special physical form, e.g. gold nanoparticle
    • A61K49/0067—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the luminescent/fluorescent agent having itself a special physical form, e.g. gold nanoparticle quantum dots, fluorescent nanocrystals
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G18/00—Polymeric products of isocyanates or isothiocyanates
    • C08G18/06—Polymeric products of isocyanates or isothiocyanates with compounds having active hydrogen
    • C08G18/28—Polymeric products of isocyanates or isothiocyanates with compounds having active hydrogen characterised by the compounds used containing active hydrogen
    • C08G18/40—High-molecular-weight compounds
    • C08G18/48—Polyethers
    • C08G18/50—Polyethers having heteroatoms other than oxygen
    • C08G18/5021—Polyethers having heteroatoms other than oxygen having nitrogen
    • C08G18/5024—Polyethers having heteroatoms other than oxygen having nitrogen containing primary and/or secondary amino groups
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G18/00—Polymeric products of isocyanates or isothiocyanates
    • C08G18/06—Polymeric products of isocyanates or isothiocyanates with compounds having active hydrogen
    • C08G18/70—Polymeric products of isocyanates or isothiocyanates with compounds having active hydrogen characterised by the isocyanates or isothiocyanates used
    • C08G18/72—Polyisocyanates or polyisothiocyanates
    • C08G18/77—Polyisocyanates or polyisothiocyanates having heteroatoms in addition to the isocyanate or isothiocyanate nitrogen and oxygen or sulfur
    • C08G18/78—Nitrogen
    • C08G18/79—Nitrogen characterised by the polyisocyanates used, these having groups formed by oligomerisation of isocyanates or isothiocyanates
    • C08G18/797—Nitrogen characterised by the polyisocyanates used, these having groups formed by oligomerisation of isocyanates or isothiocyanates containing carbodiimide and/or uretone-imine groups

Definitions

  • the invention relates to reagents and methods for the biological and chemical functionalization of water soluble nanoparticles.
  • NPs metal and semiconductor nanoparticles
  • colloidal synthetic methods which render the materials hydrophobic.
  • Such NPs are dispersed in water through surface organic cap exchange or by amphiphilic polymer encapsulation; often, water solubility is achieved via the presence of carboxylic acid functionalities on the solubilizing agents. While this renders the material water soluble, subsequent functionalization of the systems can be very difficult.
  • a method to derivatize carboxylic acid coated NPs is to conjugate chemical and biological moieties containing amine functionality to the NP surface using the water soluble activator l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC).
  • EDC water soluble activator l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride
  • NPs metallic and semiconductor nanoparticles
  • Semiconductor NPs may be luminescent and display narrow and size tunable emission spectra, high quantum yields, and exceptional photostability.
  • Water solubilized NPs can be functionalized to produce especially useful systems that have found applications in imaging, tracking, sensing, and labeling in biology. See Mattoussi, H.; Mauro, J. M.; Goldman, E. R.; Anderson, G. P.; Sundar, V. C; Mikulec, F. V.; Bawendi, M. G.
  • Cap exchange involves stripping the native trioctylphosphine/triocylphosphine oxide (TOP/TOPO) surface ligands and replacing them with bifunctional surfactants such as mercapto-acids.
  • TOP/TOPO trioctylphosphine/triocylphosphine oxide
  • Water soluble NPs are often synthesized using systems with carboxylic acid terminated functionality.
  • One approach is to cross link the organic shell with a primary amine containing chemical or biological vector using a carbodiimide activator such as l-ethyl-3-(3- dimethylaminopropyl) carbodiimide hydrochloride (EDC).
  • EDC l-ethyl-3-(3- dimethylaminopropyl) carbodiimide hydrochloride
  • quenching caused by the precipitation of carboxylic acid coated NPs by the use of excess EDC has been observed. See e.g., Mattoussi, H.; Mauro, J. M.; Goldman, E. R.; Anderson, G. P.; Sundar, V. C; Mikulec, F. V.; Bawendi, M. G.
  • the NP aggregation can cause the loss of an entire sample if the material is exposed to excess EDC (on the order of 10 4 per NP by mole), and there are indications that this precipitation is permanent.
  • An embodiment of the invention is the compound poly(ethylene glycol) methyl ether carbodiimide of various masses determined by the length of the poly(ethylene glycol) polymer.
  • An embodiment of the invention is a composition comprising a nanoparticle and poly(ethylene glycol) methyl ether carbodiimide which functionalizes the nanoparticle.
  • An embodiment of the invention is a method for making the compound poly(ethylene glycol) methyl ether carbodiimide comprising exposing amine functional poly(ethylene glycol) methyl ether to ethyl isothiocyanate, followed by exposing to HgO.
  • An embodiment of the invention is a method for making the compound poly(ethylene glycol) methyl ether carbodiimide comprising exposing amine functional poly(ethylene glycol) methyl ether to ethyl isocyanate, followed by exposure to p- toluenesulfonyl chloride in the presence of triethylamine.
  • An embodiment of the invention is a method for making a functional water-soluble nanoparticle comprising exposing amine functional poly(ethylene glycol) methyl ether to ethyl isothiocyanate, followed by exposing to HgO to form a poly(ethylene glycol) methyl ether carbodiimide, and combining the poly(ethylene glycol) methyl ether carbodiimide with a nanoparticle.
  • FIG. 1 is a graph of absorption versus wavelength, and depicts dynamic light scattering characterization of the size of water soluble CdSe/ZnS NPs.
  • FIG. 2 is a graph of absorption versus wavelength, and depicts absorption spectra of water soluble CdSe/ZnS NPs before and after exposure to EDC.
  • FIG. 3 is a graph of % initial emission versus time, and depicts the fluorescence intensity of CdSe/ZnS NPs over time after exposure to the carbodiimide reagents disclosed herein compared to the initial emission.
  • FIG. 4 is graph of absorption versus wavelength, and depicts the absorption spectra of CdSe/ZnS - TAMRA cadaverine dye before and after dialysis using IOOK MW cutoff filters.
  • FIG. 5 is a graph of absorption versus wavelength, and depicts the absorption spectra of CdSe/ZnS and TAMRA cadaverine dye before and after dialysis using IOOK MW cutoff filters.
  • FIG. 6 is a graph of absorption versus wavelength, and depicts the normalized absorption spectra of TAMRA cadaverine dyes bound to CdSe/ZnS NPs after subtraction of the CdSe/ZnS component.
  • FIG. 7 is a picture of bottles depicting A) visible light image of CdSe/ZnS NP samples after coupling to amine functional TAMRA dye as a function of the MPEG 350 CD to dye ratio, and B) the emission under UV light excitation of the same samples.
  • FIG. 8 is a graph of normalized emission versus wavelength, and depicts the emission spectra of a CdS/ZnS - fluorescein cadaverine dye conjugate as a function of pH.
  • FIG. 9 is a graph of absorption versus diameter, and depicts the gel permeation chromatography trace of aqueous CdSe/ZnS NPs and amine functional MPEG 750 / NP conjugate.
  • FIG. 10 is a graph of absorption versus diameter, and depicts the gel permeation chromatography trace of aqueous CdSe/ZnS NPs and streptavidin conjugate as a function of the streptavidin to NP ratio used during the synthesis.
  • FIG. 11 is a graph of emission versus wavelength, and depicts the emission of CdSe/ZnS - streptavidin conjugates, wherein the sample was prepared using a 100 ⁇ excess of protein to NP.
  • FIG. 12 is a picture of CdSe/ZnS NPs precipitated by EDC.
  • FIG. 13 is a graph of absorbance versus wavelength, and depicts absorption spectra of blank and tetramethylrhodamine-5-carboxamide cadaverine functionalized Fe 2 O 3 nanoparticles.
  • FIG. 14 is a graph of absorbance versus wavelength, and depicts absorption spectra of blank and streptavidin functionalized CdSe/ZnS nanoparticles, showing residual absorption at 280 nm that is likely due to streptavidin.
  • FIG. 15 is a 13C NMR spectrum of carbodiimide functional poly(ethylene glycol) methyl ether 350, 0 ⁇ 80 ppm.
  • FIG. 16 is a 13C NMR spectrum of carbodiimide functional poly(ethylene glycol) methyl ether 350, 140 ⁇ 150 ppm.
  • FIG. 17 is mass spectrum of amine functional poly(ethylene glycol) methyl ether 750.
  • FIG. 18 is mass spectrum of amine functional poly(ethylene glycol) methyl ether 350.
  • FIG. 19 is the proton NMR spectra of cellulose and biotinylated cellulose. DETAILED DESCRIPTION OF THE INVENTION
  • Embodiments of the invention relate to reagents and methods to eliminate precipitation of nanoparticles (NPs).
  • An embodiment of the invention includes at least one carbodiimide compound that does not cause precipitation of nanoparticles, and which can be used to functionalize an amphiphilic polymer coated NP with a high yield.
  • An embodiment of the invention includes a method of functionalizing a nanoparticle system, including but not limited to a semiconductor nanoparticle system, using a carbodiimide reagent.
  • the carbodiimide reagent does not cause precipitation even at high loading levels and can be used to efficiently functionalize carboxylic acid coated NPs.
  • the NPs were clearly precipitated due to their "snowflake" appearance and loss of emission intensity which was verified in the DLS data shown in the inset of Figure 1.
  • the precipitation of the NPs is evident as the average size of the particulates increases to -1550 nm. There is no peak at 10 nm while the small peak near -100 nm is attributed to small dust particulates.
  • the absorption and emission of the samples before and after EDC precipitation is shown in Fig. 2.
  • Figure 2 depicts absorption spectra of water soluble CdSe/ZnS NPs before and after exposure to EDC.
  • the agglomerization characterized from DLS measurements are reflected by the scattering absorption features in the sample after exposure to EDC. See Inset a of Figure 2.
  • the fluorescence emission spectra of CdSe/ZnS NPs before and after exposure to EDC reveal that the emission intensity of the NPs is reduced by 65% due to the EDC induced precipitation.
  • DLS Figure 1
  • CdSe/ZnS residual amine surfactants, generally used in the synthesis of NPs such as CdSe/ZnS, allowed the NPs to crosslink, which was the source of the observed precipitation.
  • CdSe/ZnS was synthesized without the use of amines, at any step, according to some original procedures (see Dabbousi, B. O.; RodriguezViejo, J.; Mikulec, F. V.; Heine, J. R.; Mattoussi, H.; Ober, R.; Jensen, K. F.; Bawendi, M. G.
  • MPEG poly(ethylene glycol) methyl ether
  • Figure 3 Shown in Figure 3 are the normalized emission intensities after correction for the dilution that occurs after reagent injection. More specifically, Figure 3 shows the fluorescence intensity of CdSe/ZnS NPs over time after exposure to the carbodiimide reagents discussed herein compared to the initial emission.
  • Carbodiimide functional poly(ethylene glycol) methyl ether 350 (MPEG 350 CD) was used in this study. The amounts of EDC added to the samples were 10% (B), 33% (C) and 50% (D) by mole relative to the amount of MPEG 350 CD (A, 1 xlO 5 per NP).
  • the PEG 350 CD sample (Ix IO 5 molar excess / NP) shows some CdSe/ZnS brightening; however, this effect is slight and is very likely due to an increase in signal simply from fluorescence waveguiding to the detector via dilution.
  • the sample emission intensity remains relatively constant, which indicates that the nanoparticles are stable in solution.
  • injection of a 10% molar equivalent of EDC to MPEG 350 CD into a fresh 3 mL CdSe/ZnS sample of the same concentration causes a similar, slight increase in the CdSe/ZnS signal; however, over time, the emission intensity decreases.
  • CMC critical micelle concentration
  • Figure 4 depicts the absorption spectra of CdSe/ZnS - TAMRA cadaverine dye before (A) and after (B) dialysis using IOOK MW cutoff filters. Compared to the bare NPs (C), there is clearly retention of dye which must be bound to the NP via carbodiimide coupling. This example represents the highest yield that was achieved with the MPEG 750 CD reagent (23%).
  • Figure 5 depicts the absorption spectra of CdSe/ZnS and TAMRA dye before (A) and after (B) dialysis using IOOK MW cutoff filters. This example represents the highest yield that was achieved with the MPEG 350 CD reagent (93%), which was a significant improvement compared to the best results using MPEG 750 CD ( Figure 4).
  • the first reagent studied was the larger MPEG 750 CD. Initially, it was found that a significant excess of this carbodiimide to dye was necessary to observe an appreciable yield. Generally, using a 100 fold excess and stirring overnight gave -7% yield; the use of more reagent up to 300 ⁇ did not seem to affect the results. Similar efficiency was observed when coupling TAMRA cadaverine to Fe 2 O 3 NPs as shown in Figure 13. Next, the efficiency of coupling the dye to CdSe/ZnS with a 100 fold excess of MPEG 750 CD as a function of time was studied, and it was found that stirring for three days gave the highest observed yield (23%). The data from this sample is shown in Figure 4.
  • Figure 6 depicts the normalized absorption spectra of TAMRA dyes bound to CdSe/ZnS NPs after subtraction of the CdSe/ZnS component. Data are taken from a sample with -33 dyes / NP (B) and -130 dyes / NP (A). Clearly, the higher loading levels have affected the dye's photophysical properties.
  • the 33:1 conjugate was prepared with MPEG 750 CD and the 130:1 loaded sample was prepared with MPEG 350 CD. It can be postulated that perhaps the dye is being encapsulated within the interior of the polymer in the high loading conditions, which can change the absorptive properties of the dye. See Fernandez- Arguelles, M.
  • the particular CdSe/ZnS NP and dye were chosen such that they may undergo Fluorescent Resonant Energy Transfer (FRET) ⁇ see F ⁇ rster, T. insectskulare Energywa substantial und Fluoreszenz. Ann. Physik 1948, 437, 55-75) from the NP donor to the TAMRA dye acceptor. While the emission spectra show suppressed NP emission with concomitant enhanced dye emission, it was discovered that energy transfer is clearly visible to the eye as shown in Figure 7. Not only can the presence of the dye be observed from the increase of the pink color as more MPEG 350 CD was used in the synthesis of the coupled construct, the emission from the TAMRA dye becomes dominant as the loading levels of dye were increased as confirmed with fluorometry.
  • FRET Fluorescent Resonant Energy Transfer
  • Figure 7 depicts A) visible light images of CdSe/ZnS NP samples after coupling to amine functional TAMRA dye as a function of the MPEG 350 CD to dye ratio (the color becomes a darker shade of pink in the samples from left to right). B) depicts the emission under UV light excitation of the same samples (the control is green, 1 :1 is light green, 10:1 is yellow, and 50:1 is orange). A significant amount of dye is coupled using -50:1 ratio of carbodiimide reagent to dye as evident from energy transfer from the NP to the emissive dye.
  • MPEG CD is a versatile reagent and can be used to derivatize several NP systems to create a variety of functional materials. It was found that using a bi-functional carbodiimide PEG reagent caused NP precipitation, however this was likely due to the activation of carboxylic acid functionalities on separate NPs with the same reagent. Addition of excess base resolubilized the NPs.
  • Figure 8 depicts the emission spectra of a CdS/ZnS - fluorescein cadaverine dye conjugate as a function of pH.
  • the inset shows a scheme of the coupled construct. As the pH is increased from 6.0 to 7.0 and then 8.0, the emission peak located at about 490 nm decreased, while the emission peak located at about 535 nm increased.
  • Figure 9 depicts gel permeation chromatography trace of aqueous CdSe/ZnS NPs and amine functional MPEG 750 / NP conjugate.
  • the increase in size is indicative of the coating of the NPs with the poly(ethylene glycol) methyl ether.
  • the PEGylated NPs were found to have an increase in diameter from 13.7 nm for the unfunctionalized blank NPs to 15.2 nm for the PEGylated NPs. Further, the PEGylated NPs were found to be more resistant to precipitation by salts such as calcium chloride. Consequently, the MPEG 350 CD reagent is effective for the coating of NPs with more than just amine functional dyes.
  • Figure 10 depicts gel permeation chromatography traces of aqueous CdSe/ZnS NPs and streptavidin conjugate as a function of the streptavidin to NP ratio used during the synthesis. The data are normalized to the unaggregated peak near 20 nm diameter. Clearly, a large excess of protein is needed to prevent agglomeration as evident by the increase in the dead volume peak as the protein to NP ratio is lowered. [00066] After a method of preventing the precipitation of the NPs during the coupling of the protein was developed, it was examined how to verify the activity of streptavidin on the surface of the nanoparticles via their interaction with biotin.
  • Figure 11 depicts emission of CdSe/ZnS - streptavidin conjugates: this sample was prepared using a 100 ⁇ excess of protein to NP. The control sample was made by diluting the NP - protein constructs to the same volume as the effluent collected. Less emission is observed from NP - protein conjugates after running through a cellulose column, most likely due to nonspecific adsorption; however, no emission is observed if the column contains biotin.
  • NP water solubilizing polymers see Fernandez- Arguelles, M. T.; Yakovlev, A.; Sperling, R. A.; Luccardini, C; Gaillard, S.; Medel, A. S.; Mallet, J. M.; Brochon, J. C; Feltz, A.; Oheim, M.; Parak, W. J. Synthesis and Characterization of Polymer- coated Quantum Dots with Integrated Acceptor Dyes as FRET-based Nanoprobes. Nano Lett.
  • This reagent can create a variety of interesting functional systems, including magnetic and emissive NPs, fluorescent chemical sensors, and biologically tailored CdSe/ZnS nanoparticles, in high yield. Further, the reagent can be synthesized in two steps using commercially available amine functional poly(ethylene glycol) methyl ether.
  • Dyes such as fluorescein-5-carboxamide cadaverine and tetramethylrhodamine-5- carboxamide cadaverine were purchased from Anaspec (San Jose, CA); streptavidin was purchased from the same source.
  • Poly-acrylic acid (1800 MW), octylamine, biotin, cellulose, thionyl chloride, sodium azide, triphenylphosphine and general solvents were purchased from Sigma- Aldrich (St. Louis, MO).
  • the water soluble carbodiimide l-ethyl-3-(3- dimethylaminopropyl) carbodiimide hydrochloride (EDC) was purchased from Advanced Chemtech (Louisville, Kentucky).
  • PAA octylamine modified poly(acrylic acid)
  • the nanoparticles used herein were all initially hydrophobic; as such, an amphiphilic polymer coating was used to render them water soluble.
  • the NPs in growth solution were precipitated with the addition of a few drops of isopropyl alcohol and excess methanol inside of a pre-weighed glass vial. Materials synthesized in 1-octadecene required more isopropyl alcohol. The flocculate was centrifuged and the supernatant was removed. The precipitation was performed again for some samples by redispersing the NPs in hexanes followed by isopropyl and methyl alcohol addition followed by centrifugation and vacuum drying.
  • NPs become soluble in alcohol solution after one precipitation; these samples were dried after the first isolation step.
  • ⁇ 10 mg bare NPs were collected to which 50 mg amphiphilic PAA polymers were added with 5 mL chloroform and two to three drops of methanol. The mixture was sonicated until the polymer solubilized. After being dried under vacuum, the sample was dissolved in basic water (pH 8-10) to form an aqueous solution of NPs, which were often clear.
  • basic water pH 8-10
  • some samples of CdSe/ZnS and all iron oxide NPs require additional cleaning through 0.1 ⁇ m filters. Dialysis was then performed using IOOK molecular weight cutoff filters from Millipore (Amicon Ultra 15, cat. UFC910024).
  • the reaction solution was purged with N 2 and cooled in an ice bath before 3.0 ml (41 mmol) thionyl chloride was added drop wise.
  • the solution was warmed to room temperature and stirred for 2 hours to convert the hydroxide group to chloride.
  • Thionyl chloride was removed under vacuum; the pressure of the vacuum system was monitored to assure removal of the excess reagent.
  • the product was then diluted with 10.0 mL DMF and the solvent was removed again under reduced pressure at room temperature to aid in the removal of residual trace amounts of thionyl chloride. Addition and removal of DMF was repeated three times total which took ⁇ 4 days.
  • 75 mL DMF together with 2.83 g (43.5 mmol) sodium azide was added and stirred overnight at 85 0 C with aluminum foil coating the flask to protect the material from light. (Warning: sodium azide is a highly toxic contact explosive).
  • the DMF was reduced under vacuum before 60 mL dichloromethane (DCM) was added. A solid precipitate was removed with a glass fritted filter or by centrifugation. The DCM was then removed with vacuum to yield the intermediate azide.
  • DCM dichloromethane
  • Two other amine functional reagents were synthesized, including a methyl ether MPEG with a molecular weight of 750 and a bifunctional amine starting from poly(ethylene glycol) with a molecular weight of 400. These materials are stored at 4 0 C in an airtight container. Despite these precautions, hydrolysis of the product is observed after several months of storage which is not unexpected given the reactivity of the carbodiimide functionality.
  • the samples appeared to react slowly with the HgO; in these cases, the samples were filtered and recharged with an additional 1.0 g of HgO in DCM and stirred overnight. This process was repeated until no more dark green byproducts were observed.
  • the MPEG-CD can be dissolved into cold ether which causes the HgS to precipitate. In a few cases, a residual dark green color was observed which was removed by dissolving the product in water and removing a green insoluble material with centrifugation and filtration. The excess water was then removed immediately under vacuum at room temperature to prevent the hydrolysis of the carbodiimide functionality.
  • the samples may also be purified by flash chromatography over alumina using methanol as the mobile phase.
  • Reaction yields are typically on the order of 90%.
  • the known method of dehydration of a urea intermediate formed from the reaction of poly(ethylene glycol) methyl ether amine with ethyl isocyanate, for example) with an excess of p-toluenesulfonic acid to form the carbodiimide also reported by Sheehan et al. is effective in this regard.
  • Scheme 1 Synthetic method employed to synthesize carbodiimide functional poly(ethylene glycol) methyl ether (MPEG CD).
  • Inset B the structure of the bi- functional PEG carbodiimide.
  • the emission of the NPs was monitored in kinetic mode for ⁇ 90 minutes with a 1 second integration time.
  • the fluorescence data was corrected for the dilution of the NPs after the injection by dividing the signal by the dilution factor.
  • Accurate dilution factors were calculated from the mass of the sample before and after the injection of the carbodiimide solutions.
  • FIG. 12 is a picture of CdSe/ZnS NPs precipitated by EDC. The precipitation is shown by the holiday "snowdome"- like appearance.
  • FIG. 13 is a graph of absorbance versus wavelength, and depicts absorption spectra of blank and tetramethylrhodamine-5-carboxamide cadaverine functionalized Fe 2 O 3 nanoparticles.
  • the inset shows the emission of the sample under irradiation with a UV light source; the sample does not appear very bright under these conditions as the dye has very little absorption at UV wavelengths.
  • FIG. 14 is a graph of absorbance versus wavelength, and depicts absorption spectra of blank and streptavidin functionalized CdSe/ZnS nanoparticles, showing residual absorption at 280 nm that is likely due to streptavidin, the strength of which suggests that there are about 2 proteins per NP.
  • FIG. 15 is a 13 C NMR spectrum of carbodiimide functional poly(ethylene glycol) methyl ether 350, 0 ⁇ 80 ppm. Four distinct species are observed, which corresponds to the unique carbons at both ends of the functional polymer.
  • FIG. 16 is a 13 C NMR spectrum of carbodiimide functional poly(ethylene glycol) methyl ether 350, 140 ⁇ 150 ppm. Based on data for other carbodiimide reagents, the peak a 141.0 ppm is the central carbon in the carbodiimide functionality.
  • FIG. 17 is mass spectrum of amine functional poly(ethylene glycol) methyl ether 750. Masses are for the positive (polymer + H + ) ion fragments. The difference in the peaks is 44.0 amu, corresponding to OCH 2 CH 2 polymer subunits. The variety of peaks is likely the result of the polydispersity in the original sample.
  • FIG. 18 is a mass spectrum of carbodiimide functionalized poly(ethylene glycol) methyl ether 350. Masses are for the positive (polymer + Na + ) ion fragments. One series of the smaller fragments results from the hydro lyzed product (such as mass peak 609.3 amu and ⁇ amu fragments) may be due to the starting material.
  • FIG. 19 is the proton NMR spectra of cellulose and biotinylated cellulose.
  • the resonances near 3.3 ppm can be associated with biotin, however, other biotin around 2.0 ppm to 1.0 ppm resonances are missing. This may be due to line broadening caused by the fact that these biotin protons are near to the ester that conjugates the biotin to the cellulose which does not dissolve well in neat DMSO.

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Abstract

L'invention concerne des réactifs et des procédés pour synthétiser des nanoparticules solubles dans l'eau. Sous un aspect, méthyléther-carbodiimide de poly(éthylèneglycol) est utilisé comme un réactif de couplage pour la fonctionnalisation biologique et chimique de nanoparticules solubles dans l'eau.
PCT/US2009/055992 2008-09-05 2009-09-04 Réactifs de couplage de méthyléther-carbodiimide de poly(éthylèneglycol) pour la fonctionnalisation biologique et chimique de nanoparticules solubles dans l'eau WO2010028217A1 (fr)

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