Nanoparticle conjugates

Abstract

Conjugates comprising a drug, cell or biological molecule bound to a photoluminescent polymer nanoparticle, in particular a cross-linked polyfluorene nanoparticle, are described herein, as well as their methods of manufacture and their uses in biological imaging and sensing applications.

Claims

1. A conjugate comprising a drug molecule, biological molecule or cell bound to a photoluminescent polymer nanoparticle, wherein the drug molecule, biological molecule or cell is bound to the photoluminescent polymer nanoparticle by a covalent bond formed by the reaction of functional groups present on the photoluminescent polymer nanoparticle with functional groups present on the drug molecule, biological molecule or cell; and wherein the photoluminescent polymer nanoparticle is formed from a π-conjugated cross-linked polymer or a salt thereof, the π-conjugated cross-linked polymer comprising a) 80-99.9 mol. % of π-conjugated monomers, and b) 0.1-10 mol. % of a cross-linker having the formula III shown below: ##STR00068## and c) one or more functional groups capable of reacting with functional groups on the drug molecule, biological molecule or cell to form a covalent bond linking the nanoparticle to the drug molecule, biological molecule or cell or a first moiety capable of affinity pairing with a second moiety present on the drug molecule, biological molecule or cell; wherein the π-conjugated monomers each independently have a structure according to formula IV shown below: ##STR00069## wherein R.sub.1 and R.sub.2 are each independently hydrogen or a group:
—X-Q wherein X is absent or selected from the group consisting of (1-30C)alkylene, —O-(1-30C)alkylene, —S-(1-30C)alkylene, —NH-(1-30C)alkylene, (2-30C)alkenylene, (2-30C)alkynylene, —[(CH.sub.2).sub.2—O].sub.n—, —[O—(CH.sub.2).sub.2].sub.n—, —[O—CH.sub.2MeCH.sub.2].sub.n—, —[CH.sub.2MeCH.sub.2—O].sub.n— and —[O—Si(R.sub.z).sub.2].sub.n, wherein R.sub.z is (1-4C)alkyl and n is 1 to 30, —[(CH.sub.2).sub.n′—(CF.sub.2).sub.m′]—, wherein n′ is 0-20 and m′ is 1 to 30; and Q is a terminal group selected from the following substituents, or a salt thereof: hydrogen, halogen, methyl, hydroxyl, carboxy, (1-4C)alkoxycarbonyl, amino, —CH═CH.sub.2, —C≡CH, —SH, biotin, streptavidin, antibody, —CF.sub.3, —OSO.sub.3H, —SO.sub.3H, —OPO.sub.2OH and zwitterions and a polymerisable group selected from silane, siloxane, acrylate, epoxy, styrene; or R.sub.1 and R.sub.2 are aryl or heteroaryl groups optionally substituted with a substituent group; or R.sub.1 and R.sub.2 are linked so that, together with the carbon atom to which they are attached, they form ring system optionally substituted with a substituent group; and with the proviso that, in at least one monomer of formula IV, Q is or comprises a group selected from the following substituents, or a salt thereof: amino, carboxy, hydroxyl, halogen, vinyl, alkenyl, alkynyl, carbonyl, thiol, biotin, streptavidin, an antibody or a polymerisable group selected from acrylate, epoxy, and styrene.

2. The conjugate of claim 1, wherein the covalent bond is selected from an amide, disulphide, ether, thioether, amine, imine, enamine or ester linkage.

3. The conjugate of claim 1, wherein the photoluminescent polymer nanoparticle comprises: (i) one or more hydroxyl groups that are capable of reacting with carboxy groups present on the drug molecule, biological molecule or cell to form ester bonds that couple the polymeric nanoparticle to the drug molecule, biological molecule or cell; (ii) one or more amino groups that can react with carboxy groups present on the drug molecule, biological molecule or cell to form amide bonds that couple the polymeric nanoparticle to the drug molecule, biological molecule or cell; (iii) one or more carboxy groups that can react with amino groups present on the drug molecule, biological molecule or cell to form amide bonds that couple the polymeric nanoparticle to the drug molecule, biological molecule or cell; (iv) one or more thiol groups that can react with thiol groups present on the drug molecule, biological molecule or cell to form disulphide bonds that couple the polymeric nanoparticle to the drug molecule, biological molecule or cell; (v) one or more vinyl groups that can react with thiol groups present on the drug molecule, biological molecule or cell to form sulphide bonds that couple the polymeric nanoparticle to the drug molecule, biological molecule or cell; and/or (vi) one or more carbonyl groups that can react with amine groups present on the drug molecule, biological molecule or cell to form imine or enamine bonds that couple the polymeric nanoparticle to the drug molecule, biological molecule or cell.

4. The conjugate of claim 1, wherein the π-conjugated monomers each independently have a structure according to formula V or VI shown below: ##STR00070## wherein R.sub.1 and R.sub.2 are as defined in claim 1; and
—X-Q A.sub.1 and A.sub.2 are independently absent or selected from any one of the following moieties: ##STR00071## ##STR00072## ##STR00073## ##STR00074## ##STR00075## and wherein R.sub.3 and R.sub.4 are each groups R.sub.1 and R.sub.2 as defined in claim 1, or are each independently hydrogen or a group:
—X.sup.1-Q.sup.1 wherein X.sup.1 is selected from (1-30C)alkylene, (2-30C)alkenylene, (2-30C)alkynylene, —[(CH.sub.2).sub.2—O].sub.n—, —[O—(CH.sub.2).sub.2].sub.n—, —[O—CH.sub.2MeCH.sub.2].sub.n, —[CH.sub.2MeCH.sub.2—O].sub.n—, [O—Si(R.sub.z).sub.2].sub.n— wherein R.sub.z is (1-4C)alkyl and n is 1 to 30, and —[(CH.sub.2).sub.n′—(CF.sub.2).sub.m′]— wherein n′ is 0-20 and m′ is 1 to 30; Q.sup.1 is a terminal group selected from the following substituents, or a salt thereof: hydrogen, halogen, methyl, hydroxyl, carboxy, (1-4C)alkoxycarbonyl, amino, —CH═CH.sub.2, —C≡CH, —SH, biotin, streptavidin, —CF.sub.3, —OSO.sub.3H, —SO.sub.3H, —OPO.sub.2OH, zwitterions, and a polymerisable group selected from silane, siloxane, acrylate, epoxy, styrene; M is a metal selected from Jr, Pt, Rh, Re, Ru, Os, Cr, Cu, Pd and Au; L is a ligand independently selected from halo, (1-30C)hydrocarbyl optionally comprising one or more heteroatoms selected from N, O, S, Si, Ge, As or P, or an aryl or heteroaryl group optionally substituted with one or more substituents selected from (1-4C)alkyl, halo, aryl or heteroaryl; X is a heteroatom selected from N, O, P, S, Si, Ge, As or Se; p is 1 to 4; and with the proviso that, in at least one monomer of formula V or VI, Q.sup.1 is or comprises a group selected from amino, carboxy, hydroxyl, halogen, vinyl, alkenyl, alkynyl, carbonyl, thiol, -biotin or streptavidin, —CF.sub.3, —OSO.sub.3H, —SO.sub.3H, —OPO.sub.2OH, and zwitterions.

5. The conjugate of claim 4, wherein A.sub.1 and A.sub.2 are independently absent or selected from any one of the following moieties: ##STR00076## wherein R.sub.3, R.sub.4, M, L and p are as defined in claim 4.

6. The conjugate of claim 4, wherein A.sub.1 or A.sub.2 is absent; or wherein both A.sub.1 and A.sub.2 are absent.

7. The conjugate of claim 4, wherein when present X and X.sup.1 are independently selected from the group consisting of (1-30C)alkylene, (2-30C)alkenylene, (2-30C)alkynylene, —[(CH.sub.2).sub.m(CF.sub.2).sub.n]—, —[(CH.sub.2).sub.2—O].sub.n—, —[O—(CH.sub.2).sub.2].sub.n—, —[O—CHMeCH.sub.2].sub.n—, —[CHMeCH.sub.2—O].sub.n—, and —[O—Si(R.sub.z).sub.2].sub.n—, wherein R.sub.z is methyl, n is 1 to 30, and m is 0 to 30; Q and Q.sup.1 are independently a terminal group selected from the following substituents, or a salt thereof: hydrogen, halogen, methyl, hydroxyl, carboxy, (1-4C)alkoxycarbonyl, amino, —CH═CH.sub.2, —C≡CH, —SH, biotin, streptavidin, —CF.sub.3, OSO.sub.3H, —SO.sub.3H, —OPO.sub.2OH, zwitterions, and a polymerisable group selected from acrylates, epoxy and styrene; M is a metal selected from Ir, Pt, Rh, Re, Ru, Os, Cr, Cu, Pd and Au; L is a ligand independently selected from the group consisting of halo, (1-30C)hydrocarbyl optionally comprising one or more heteroatoms selected from N, O, S, Si or P, or an aryl or heteroaryl group optionally substituted with one or more substituents selected from (1-4C)alkyl, halo, aryl or heteroaryl; and p is 1 to 4; and with the proviso that at least one group Q and/or Q.sup.1 is or comprises a group selected from amino, carboxy, hydroxyl, halogen, vinyl, alkenyl, alkynyl, carbonyl, thiol, biotin, and streptavidin.

8. The conjugate of claim 1, wherein the π-conjugated cross-linked polymer comprises 80-99.9 mol. % of π-conjugated monomers selected from any of the following structures: ##STR00077## wherein R.sub.1, R.sub.2, R.sub.3 and R.sub.4 are each independently selected from: —(CH.sub.2).sub.nR.sub.50 and —(CH.sub.2CH.sub.2O).sub.nR.sub.50; wherein n is 1 to 15 and R.sub.50 is selected from —H, (1-15C)alkyl, —CO.sub.2H, —CO.sub.2(1-6C)alkyl, —CO.sub.2Na, —CH═CH.sub.2, —OC(O)-(biotin) and —OSO.sub.3Na; and X is O.

9. The conjugate of claim 8, wherein R.sub.1 and R.sub.2 are both selected from —(CH.sub.2).sub.7CH.sub.3, —(CH.sub.2).sub.5CO.sub.2Et, —(CH.sub.2).sub.10CO.sub.2H, —(CH.sub.2).sub.4CH═CH.sub.2, —(CH.sub.2).sub.11OSO.sub.3Na, —(CH.sub.2).sub.5CO.sub.2Et, —(CH.sub.2).sub.10CO.sub.2Na, —(CH.sub.2CH.sub.2O).sub.3CH.sub.3, —(CH.sub.2).sub.11OC(O)-(biotin) and —(CH.sub.2CH.sub.2O).sub.12CH.sub.3; and R.sub.3 and R.sub.4 are selected from 2-ethylhexyl, —(CH.sub.2).sub.11OSO.sub.3Na, —(CH.sub.2CH.sub.2O).sub.nCH.sub.3 and —(CH.sub.2).sub.10CO.sub.2Na.

10. A method of forming a conjugate of claim 1, the method comprising the steps of: (i) forming the nanoparticles by emulsion polymerisation, mini-emulsion polymerisation or dispersion polymerisation techniques to provide an aqueous suspension of nanoparticles; (ii) reacting the nanoparticles with the drug molecule, biological molecule or cell so as to form an aqueous suspension of the conjugate.

11. The method of claim 10, wherein the nanoparticles are formed by a cross-coupling polymerisation reaction.

12. The method of claim 10, further comprising the step of purifying the aqueous suspension of nanoparticles.

13. The method of claim 12, wherein the aqueous suspension of nanoparticles is purified by contacting the aqueous suspension of nanoparticles with at least one organic solvent.

14. The method of claim 13, wherein the at least one organic solvent is either selected from the group consisting of polar and non-polar solvents; or is methanol or propanol.

15. The conjugate of claim 1, wherein the π-conjugated monomers comprise monomers having the following structure: ##STR00078##

Description

EXAMPLES

(1) Examples of the invention will now be described, for the purpose of reference and illustration only, with reference to the accompanying figures, in which:

(2) FIG. 1 shows DLS particle size histograms of the cross-linked nanoparticles of Example 1.1 in water (solid line) or THF (broken line).

(3) FIG. 2 shows UV/Vis spectra of the cross-linked nanoparticles of Example 1.1 in water (solid line) or THF (broken line).

(4) FIG. 3 shows PL spectra of the cross-linked nanoparticles of Example 1.1 in water (solid line) or THF (broken line).

(5) FIG. 4 shows DLS particle size histograms of the cross-linked nanoparticles of Example 1.2 in water (solid line) and THF (broken line) dispersants.

(6) FIG. 5 shows UV/Vis (broken line) and PL (solid line) spectra of the cross-linked nanoparticles of Example 1.2.

(7) FIG. 6 shows DLS sizing histograms of cross-linked phosphorescent nanoparticles in water (solid line) or THF (broken line) of the cross-linked nanoparticles of Example 1.3.

(8) FIG. 7 shows UV/Vis spectra of the cross-linked nanoparticles of Example 1.3 in water (solid line) or THF (broken line).

(9) FIG. 8 shows PL spectra of the cross-linked nanoparticles of Example 1.3 in water (solid line) or THF (broken line).

(10) FIG. 9 shows DLS sizing histograms of the cross-linked nanoparticles of Example 1.4 in water (solid line) and THF (broken line).

(11) FIG. 10 shows DLS sizing histograms of the cross-linked nanoparticles of Example 1.5 in water.

(12) FIG. 11 shows DLS sizing histograms of the cross-linked nanoparticles of Example 1.6 in water (broken line) and THF (solid line).

(13) FIG. 12 shows absorption and emission spectra of the cross-linked nanoparticles of Examples 1.4 (FIG. 12a), 1.5 (FIG. 12b) and 1.6 (FIG. 12c).

(14) FIG. 13 shows electrophoresis gel of Nanoparticles conjugated to Streptavidin.

(15) FIG. 14 shows electrophoresis gel of Nanoparticles conjugated to donkey anti-mouse IgG (H+L) secondary antibody.

(16) FIG. 15 shows images obtained by imaging cytometry (example 2.7)

EXAMPLE 1—PREPARATION OF CROSS-LINKED PFO NANOPARTICLES

1.1—Cross-Linked PFO Nanoparticles

(17) Synthesis

(18) Referring to Scheme 1 and Table 1 shown below, sodium dodecyl sulphate (SDS) (50.0 mg) and deionised water (10 mL) were transferred to a Schlenk tube and the resultant solution was degassed by bubbling with argon for 20 minutes. Monomer A (see Table 1), crosslinker B (see Table 1) and monomer C (58.6 mg, 9.12×10.sup.−2 mmol) were dissolved in toluene (1 mL), to which hexadecane (78 μL) was also added, and this solution was degassed for 5 minutes in the same manner. Tetrakis(triphenylphosphine)palladium(0) (2.2 mg, 9.13×10.sup.−3 mmol) was added to the monomer solution, which was then transferred to the reaction vessel. The reaction mixture was emulsified by ultrasonication (Cole Parmer 750W ultasonicator, fitted with microtip, on 22% power) for 2 minutes while cooling with an ice bath. The Schlenk tube was resealed and the miniemulsion was heated to 72° C., followed by addition of 1M aqueous sodium hydroxide solution (365 μL), and the reaction mixture was stirred for 16 hours. After cooling to room temperature, the cap of the reaction vessel was removed and the emulsion was stirred for 5 hours to remove the residual toluene.

(19) ##STR00048##

(20) TABLE-US-00001 TABLE 1 Reaction variables for synthesis of cross-linked PFO nanoparticles Sample Monomer A Crosslinker B Name (mass, moles) (mass, moles) NP-X2.5 45.0 mg 2.9 mg 8.21 × 10.sup.−2 mmol 4.6 × 10.sup.−3 mmol NP-X5 40.0 mg 5.8 mg 7.29 × 10.sup.−2 mmol 9.1 × 10.sup.−3 mmol NP-X10 30.0 mg 11.6 mg 5.47 × 10.sup.−2 mmol 1.82 × 10.sup.−2 mmol
Surfactant Removal and DLS Analysis (Nanoparticles in Water)

(21) A 400 μL aliquot of the crude nanoparticle suspension was diluted with 1.6 mL of deionised water, to which Amberlite XAD-2 resin (20 mg, pre-washed with 2×2 mL of water) was added. The suspension was shaken at room temperature for 15 minutes before decanting off the nanoparticle suspension. This Amberlite XAD-2 purification step was repeated, after which time the suspension no longer foamed upon shaking and was filtered through glass wool prior to dynamic light scattering (DLS) analysis of particle size using a Malvern Zetasizer Nano ZS. Results are shown in Table 2 and FIG. 1.

(22) TABLE-US-00002 TABLE 2 DLS analysis of cross-linked PFO nanoparticles in water Z- Size by St. Sample Average Intensity Dev. Name (d .Math. nm) (d .Math. nm) (nm) Pdl NP-X2.5 128 154 69 0.16 NP-X5 130 151 60 0.14 NP-X10 129 150 56 0.13
DLS Analysis (Nanoparticles in THF)

(23) A 200 μL aliquot of the crude nanoparticle suspension was flocculated through addition of 1.3 mL toluene and the polymer was isolated by centrifugation (14,000 rpm, 1 minute) and decantation of the supernatant. The polymer was dried in air to remove residual methanol before dispersing in tetrahydrofuran (THF, 1 mL). The resultant suspension was measured directly using a Malvern Zetasizer Nano ZS. Results are shown in Table 3 and FIG. 1.

(24) TABLE-US-00003 TABLE 3 DLS analysis of cross-linked PFO nanoparticles in THF Z-Average Size by Intensity St. Dev. Sample name (d. nm) (d. nm) (nm) Pdl NP-X2.5 — — — n/a.sup.[a] NP-X5 174  198 (99.6%)  74 (99.6%) 0.13 4827 (0.4%).sup.[b] 711 (0.4%).sup.[a] NP-X10 147 175 73 0.15 .sup.[a]secondary peak likely to result from a small proportion of aggregated nanoparticles
UV/Vis Analysis (Nanoparticles in Water or THF)

(25) Following surfactant removal via treatment with Amberlite XAD-2, 40 μL of the nanoparticle suspension was diluted with 3 mL of water. UV-Vis absorption spectra of the nanoparticles at this concentration were recorded on a Varian Cary 55 5000UV-Vis-NIR spectrophotometer at room temperature. FIG. 2 shows UV/Vis spectra of the cross-linked PFO nanoparticles.

(26) Photoluminescence (PL) Analysis (Nanoparticles in Water or THF)

(27) Following surfactant removal via treatment with amberlite XAD-2, 40 μL of the nanoparticle suspension was diluted with 3 mL of water. PL spectra were recorded on a Varian Cary Eclipse fluorimeter. FIG. 2 shows PL spectra of the cross-linked PFO nanoparticles

(28) Photoluminescence (PL) Analysis (Nanoparticles in Water)

(29) Photoluminescence measurements were obtained using a Fluoromax-4 spectrofluorometer. Measurements were carried out on dilute dispersions of the nanoparticles in water (800 μL, abs>1), using the same volume of water for background measurements. The results are provided in Table 4.

(30) TABLE-US-00004 TABLE 4 Optical properties of PFO nanoparticles in water Sample Name λ.sub.max λ.sub.em.sup.[a] NP-X2.5 390 440 NP-X5 390 438 NP-X10 390 437 .sup.[a]λ.sub.ex = 380 nm

1.2—Ethyl Ester-Functionalised Cross-Linked PFO Nanoparticles

(31) Synthesis

(32) Referring to Scheme 2 shown below, sodium dodecyl sulfate (50.0 mg) and deionised water (10 mL) were transferred to a Schlenk tube and the resultant solution was degassed by bubbling with argon for 20 minutes. Crosslinker A (5.8 mg, 9.12×10.sup.−3 mmol), monomer B (44.4 mg, 7.30×10.sup.−2 mmol) and monomer C (58.6 mg, 9.12×10.sup.−2 mmol) were dissolved in toluene (1 mL), to which hexadecane (78 μL) was also added, and this solution was degassed for 5 minutes in the same manner. Tetrakis(triphenylphosphine)palladium(0) (2.2 mg, 9.13×10.sup.−3 mmol) was added to the monomer solution, which was then transferred to the reaction vessel. The reaction mixture was emulsified by ultrasonication (Cole Parmer 750W ultasonicator, fitted with microtip, on 22% power) for 2 minutes while cooling with an ice bath. The Schlenk tube was resealed and the miniemulsion was heated to 72° C., followed by addition of 1M aqueous sodium hydroxide solution (365 μL), and the reaction mixture was stirred for 16 hours. After cooling to room temperature, the cap of the reaction vessel was removed and the emulsion was stirred for 5 hours to remove the residual toluene.

(33) ##STR00049##
DLS Analysis (Nanoparticles in Water or THF)

(34) Surfactant removal was carried out using the general procedure described in Example 1. Flocculation and resuspension in THF were carried out using the general procedure described in Example 1. DLS analysis was carried out as in Example 1, using either water or THF as the dispersant. The results are provided in Table 5 and FIG. 4.

(35) TABLE-US-00005 TABLE 5 DLS analysis of ethyl ester-functionalised nanoparticles in water or THF Sample Z-Average Size by Intensity St. Dev Name Dispersant (d. nm) (d. nm) (nm) Pdl NP-X5E40 Water 118 139 56 0.14 NP-X5E40 THF 170 204 82 0.16
UV/Vis and PL Analysis (Nanoparticles in Water)

(36) The general UV/Vis and PL analytical_procedures described in Example 1 were used to record the UV/Vis and PL spectra of the nanoparticles in dilute aqueous dispersion. The results are provided in FIG. 5.

(37) PL Analysis (Nanoparticles in Water)

(38) PL measurements were obtained using the general method described in Example 1. The results are provided in Table 6.

(39) TABLE-US-00006 TABLE 6 Optical properties of ethyl ester-functionalised nanoparticles in water Sample Name λ.sub.max λ.sub.em.sup.[a] NP-X5E40 391 432 .sup.[a]λ.sub.ex = 380 nm

1.3—Cross-Linked Phosphorescent Nanoparticles

(40) Method

(41) Referring to Scheme 3 and Table 7 shown below, sodium dodecyl sulfate (50.0 mg) and deionised water (10 mL) were transferred to a Schlenk tube and the resultant solution was degassed by bubbling with argon for 20 minutes. Monomers A (see Table 7), C (20.5 mg, 1.82×10.sup.−2 mmol) and D (58.6 mg, 9.12×10.sup.−2 mmol) and crosslinker B (5.8 mg, 9.12×10.sup.−3 mmol) were dissolved in toluene (1 mL), to which hexadecane (78 μL) was also added, and this solution was degassed for 5 minutes in the same manner. Tetrakis(triphenylphosphine)palladium(0) (2.2 mg, 9.13×10.sup.−3 mmol) was added to the monomer solution, which was then transferred to the reaction vessel. The reaction mixture was emulsified by ultrasonication (Cole Parmer 750W ultasonicator, fitted with microtip, on 22% power) for 2 minutes while cooling with an ice bath. The Schlenk tube was resealed and the miniemulsion was heated to 72° C., followed by addition of 1M aqueous sodium hydroxide solution (365 μL), and the reaction mixture was stirred for 16 hours. After cooling to room temperature, the cap of the reaction vessel was removed and the emulsion was stirred for 5 hours to remove the residual toluene.

(42) ##STR00050##

(43) TABLE-US-00007 TABLE 7 Reaction variables for synthesis of cross-linked phosphorescent nanoparticles Monomer A Monomer A Sample Name Side Chain (R.sup.1) (mass, moles) NP-XIr1 Octyl 30.0 mg 5.47 × 10.sup.−2 mmol NP-XIr2 MeO-PEG3 33.7 mg 5.57 × 10.sup.−2 mmol
DLS Analysis (Nanoparticles in Water or THF)

(44) Surfactant removal was carried out using the general procedure described in Example 1. Flocculation and resuspension in THF were carried out using the general procedure described in Example 1. DLS analysis was carried out as in Example 1, using either water or THF as the dispersant. The results are provided in Table 8 and FIG. 6.

(45) TABLE-US-00008 TABLE 8 DLS analysis of cross-linked phosphorescent nanoparticles in water or THF Z- Size Sample Average by Intensity St. Dev Name Dispersant (d. nm) (d. nm) (nm) Pdl NP-XIr1 Water 131 158  69 0.15 NP-XIr1 THF 167 210 109 0.18 NP-XIr2 Water 126  150 (99.3%)  70 (99.3%) 0.19 4709 (0.7%).sup.[a] 774 (0.7%).sup.[a] NP-XIr2 THF 165 205  98 0.18 .sup.[a]Secondary peak likely to result from a small proportion of aggregated nanoparticles
UV/Vis and PL Analysis (Nanoparticles in Water or THF)

(46) The general UV/Vis and PL analytical_procedures described in Example 1 were used to record the UV/Vis (FIG. 7) and PL (FIG. 8) spectra of the nanoparticles in dilute aqueous dispersion or THF.

(47) PL Analysis (Nanoparticles in Water)

(48) PL measurements were obtained using the general method described in Example 1. The results are provided in Table 9.

(49) TABLE-US-00009 TABLE 9 Optical properties of cross-linked phosphorescent nanoparticles in water Sample Name λ.sub.max λ.sub.em.sup.[a] NP-Ir1 392 609 NP-Ir2 392 609 .sup.[a]λ.sub.ex = 390 nm

1.4—PEG3 Functionalised 10% Cross-Linked PFO Nanoparticles

(50) Synthesis

(51) Referring to Scheme 4 shown below, tetraethylammonium hydroxide solution (40% in water) (0.1567 g, 0.4 mmol), was added to an aqueous solution (50 ml) of non-ionic surfactant, Triton x-102 (2.5 g, 5 wt % in de-ionised water) in a 100 ml three necked round bottom flask. Then contents were then degassed for 30 mins by bubbling nitrogen gas through the stirred solution. Then a separate 10 ml two necked round bottom flask was used to mix together the monomers in the organic solvent prior to addition to the reaction flask. 9,9-dioctylfluorene-2,7-di-boronic acid-bis(1,3-propanediol)ester (0.1151 g, 0.2 mmol), 2,7-dibromo-9,9-bis(2-(2-(2-methoxyethoxy)ethoxy)ethyl)fluorene (0.0967 g, 0.16 mmol) and 2,2′,7,7′-tetrabromo-9,9′-spirobifluorene (0.0126 g, 0.02 mmol) were dissolved in xylene (2 ml). The monomer solution was degassed and then the catalyst IPr*PdTEACl.sub.2 (0.0095 g, 0.008 mmol) was added, followed by further degassing of the resultant solution. A syringe was used to transfer the monomer/catalyst into the stirred surfactant/base solution in the main reaction flask now maintained at 30° C. with stirring and maintaining under nitrogen gas for 24 h.

(52) ##STR00051##
DLS Analysis (Nanoparticles in Water or THF)

(53) 500 μl of sample was transferred to centrifuge vial the 1.5 ml of methanol was added. The sample vial was centrifuged at 14,000 rpm for 5 min then the liquid was decanted. Crude sample was washed with methanol 3 times and re-dispersed in THF in order to measure the size of particles. Neat products without further purification were also investigated. The results are shown in FIG. 9 and Table 10. Concentrations of polymer in water was 23 μg/ml.

(54) TABLE-US-00010 TABLE 10 Particle sizes of CPNs in water and THF at 25° C. Dz Sample Size (nm) (nm) STD (nm) Pdl LM55 Neat 50 44 26.81 0.244 LM55 in THF 108 218 51.80 0.217
Optical Properties

(55) Referring to Table 11 and FIG. 12, LM55 exhibited maxima band at 370 nm but no β-phase was observed.

(56) TABLE-US-00011 TABLE 11 Summarized optical properties of cross-linked polymer in water Final polymer Size λ.sub.abs λ.sub.em Sample conc. (mg/ml) (nm) (nm) (nm) E.sub.g* LM55 2.5 50 370 420, 441 2.91

1.5—PEG3 Functionalised 5% Cross-Linked PFO Nanoparticles

(57) Synthesis

(58) Referring to Scheme 5 shown below, tetraethylammonium hydroxide solution (40% in water) (0.1567 g, 0.4 mmol), was added to an aqueous solution (50 ml) of non-ionic surfactant, Triton x-102 (2.5 g, 5 wt % in de-ionised water) in a 100 ml three necked round bottom flask. Then contents were then through degassed for 30 mins by bubbling nitrogen gas through the stirred solution. Then a separate 10 ml two necked round bottom flask was used to mix together the monomers in the organic solvent prior to addition to the reaction flask. 9,9-dioctylfluorene-2,7-di-boronic acid-bis(1,3-propanediol)ester (0.1151 g, 0.2 mmol), 2,7-dibromo-9,9-dioctylfluorene (0.0768 g, 0.14 mmol), 2,7-dibromo-9,9-bis(2-(2-(2-methoxyethoxy)ethoxy)ethyl)fluorene (0.0242 g, 0.04 mmol) and 2,2′,7,7′-tetrabromo-9,9′-spirobifluorene (0.0063 g, 0.01 mmol) were dissolved in xylene (2 ml). The monomer solution was degassed and then the catalyst IPr*PdTEACl.sub.2 (0.0095 g, 0.008 mmol) was added, followed by further degassing of the resultant solution. A syringe was used to transfer the monomer/catalyst into the stirred surfactant/base solution in the main reaction flask now maintained at 30° C. with stirring and maintaining under nitrogen gas for 24 h.

(59) ##STR00052##
DLS Analysis (Nanoparticles in Water or THF)

(60) 500 μl of sample was transferred to centrifuge vial the 1.5 ml of methanol was added. The sample vial was centrifuged at 14,000 rpm for 5 min then the liquid was decanted. Crude sample was washed with methanol 3 times and re-dispersed in THF in order to measure the size of particles. Neat products without further purification were also investigated. The results are shown in FIG. 10 and Table 12. Concentrations of polymer in water was 23 μg/ml.

(61) TABLE-US-00012 TABLE 12 Particle sizes of CPNs in water at 25° C. Dz Sample Size (nm) (nm) STD (nm) Pdl LM56 Neat 55 41 26.23 0.381
Optical Properties

(62) Referring to Table 13 and FIG. 12, LM56 showed absorption peak at 378 nm.

(63) TABLE-US-00013 TABLE 13 Summarized optical properties of cross-linked polymer in water Final polymer λ.sub.em Sample conc. (mg/ml) Size (nm) λ.sub.abs (nm) (nm) E.sub.g* LM56 2.5 55 378, 435 421, 436, 453 2.78

1.6—PEG12 Functionalised 10% Cross-Linked PFO Nanoparticles

(64) Synthesis

(65) Referring to Scheme 6 below, tetraethylammonium hydroxide solution (40% in water) (0.1567 g, 0.4 mmol), was added to an aqueous solution (50 ml) of non-ionic surfactant, Triton x-102 (2.5 g, 5 wt % in de-ionised water) in a 100 ml three necked round bottom flask. Then contents were then through degassed for 30 mins by bubbling nitrogen gas through the stirred solution. Then a separate 10 ml two necked round bottom flask was used to mix together the monomers in the organic solvent prior to addition to the reaction flask. 9,9-dioctylfluorene-2,7-di-boronic acid-bis(1,3-propanediol)ester (0.1151 g, 0.2 mmol), 2,7-dibromo-9,9-bis(polyethylene glycol monoether)fluorene (0.2255 g, 0.16 mmol) and 2,2′,7,7′-tetrabromo-9,9′-spirobifluorene (0.0126 g, 0.02 mmol) were dissolved in xylene (2 ml). The monomer solution was degassed and then the catalyst IPr*PdTEACl.sub.2 (0.0095 g, 0.008 mmol) was added, followed by further degassing of the resultant solution. A syringe was used to transfer the monomer/catalyst into the stirred surfactant/base solution in the main reaction flask now maintained at 30° C. with stirring and maintaining under nitrogen gas for 24 h.

(66) ##STR00053##
DLS Analysis (Nanoparticles in Water or THF)

(67) 500 μl of sample was transferred to centrifuge vial the 1.5 ml of methanol was added. The sample vial was centrifuged at 14,000 rpm for 5 min then the liquid was decanted. Crude sample was washed with methanol 3 times and re-dispersed in THF in order to measure the size of particles. Neat products without further purification were also investigated. The results are shown in FIG. 11 and Table 14. Concentrations of polymer in water was 23 μg/ml.

(68) TABLE-US-00014 TABLE 14 Particle sizes of CPNs in water and THF at 25° C. Dz Sample Size (nm) (nm) STD (nm) Pdl LM02-6 Neat 244 13 103.2 0.359 LM02-6 in THF 74 847 10.97 0.489
Optical Properties

(69) Table 15 and FIG. 12 show summarized optical properties for LM02-6 in water.

(70) TABLE-US-00015 TABLE 15 Summarized optical properties of cross-linked polymer in water Final polymer λ.sub.em Sample conc. (mg/ml) Size (nm) λ.sub.abs (nm) (nm) E.sub.g* LM02-6 2.5 244 N/A 419, 441 N/A

1.7-5% 2,1,3-Benzothiadiazole, 35% 9,9-Di(undecanoic acid)fluorene and 5% 9,9′-Spirobifluorene Cross-Linked Polyfluorene Nanoparticles

(71) Synthesis

(72) ##STR00054##

(73) In a Schlenk tube was added water (22.0 mL), sodium dodecyl sulfate (110 mg, 382 μmol) and 1M aqueous sodium hydroxide (1080 μL, 1080 μmol). The solution was purged with argon for 2 hours. In a vial was weighed 9,9-dioctylfluorene-2,7-diboronic acid bis(1,3-propanediol) ester (111.7 mg, 200 μmol), 2,7-dibromo-9,9-di(undecanoic acid)fluorene (96.9 mg, 140 μmol), 2,2′,7,7′-tetrabromo-9,9′-spirobifluorene (12.6 mg, 20 μmol), 4,7-dibromobenzo[c]-1,2,5-thiadiazole (5.9 mg, 20 μmol) tris(dibenzylideneacetone)dipalladium(0) (4.6 mg, 5 μmol), tri(o-tolyl)phosphine (9.1 mg, 30 μmol) and hexadecane (171 μL, 585 μmol). The vial was transferred to an argon filled glovebox, sealed with a rubber septum and removed. Toluene (2.19 mL) was added to the vial and the suspension sonicated until a homogenous solution was achieved. The initial aqueous solution was cooled to 0° C. in an ice bath, the ultrasonic probe inserted and the toluene solution injected rapidly into the water. The solution was ultrasonicated for 1 minute, stirred for 30 seconds and ultrasonicated for 1 further minute. The Schlenk tube was sealed, placed in a preheated oil bath at 50° C. and stirred for 20 hours. The Schlenk was opened and a stream of nitrogen gas passed over the emulsion at 50° C., with stirring. The emulsion was cooled to room temperature, the volume increased to 23.0 mL with deionised water and filtered through a glass wool plug. The emulsion was obtained as a milky dark green solution. DLS (water): Z-average=79.0 nm, Pdl=0.117, D.sub.n=52.4 nm and SD=15.3 nm. UV-Vis Abs. (water): λ.sub.max=380 nm, λ.sub.sec.=450 nm, λ.sub.onset=520 nm. UV-Vis PL (water): λ.sub.max=535 nm, λ.sub.sec.=424 nm.

1.8-40% Di(t-butyl hexanoate)fluorene and 5% 9,9′-Spirobifluorene Cross-Linked Polyfluorene Nanoparticles

(74) Synthesis

(75) ##STR00055##

(76) In a Schlenk tube was added water (22.0 mL), sodium dodecyl sulfate (110 mg, 382 μmol) and 1M aqueous sodium hydroxide (800 μL, 800 μmol). The solution was purged with argon for 2 hours. In a vial was weighed 9,9-dioctyl-9H-fluorene-2,7-diboronic acid bis(pinacol) ester (128.5 mg, 200 μmol), 2,7-dibromo-9,9-di(t-butyl hexanoate)fluorene (106.3 mg, 160 μmol), 2,2′,7,7′-tetrabromo-9,9′-spirobifluorene (12.6 mg, 20 μmol), tetrakis (triphenylphosphine)palladium(0) (5.8 mg, 5 μmol) and hexadecane (171 μL, 585 μmol). The vial was transferred to an argon filled glovebox, sealed with a rubber septum and removed. Toluene (2.19 mL) was added to the vial and the suspension sonicated until a homogenous solution was achieved. The initial aqueous solution was cooled to 0° C. in an ice bath, the ultrasonic probe inserted and the toluene solution injected rapidly into the water. The solution was ultrasonicated for 1 minute, stirred for 30 seconds and ultrasonicated for 1 further minute. The Schlenk tube was sealed, placed in a preheated oil bath at 72° C. and stirred for 20 hours. The Schlenk was opened and a stream of nitrogen gas passed over the emulsion at 50° C., with stirring. The emulsion was cooled to room temperature, the volume increased to 23.0 mL with deionised water and filtered through a glass wool plug. The emulsion was obtained as a milky light green solution. DLS (water): Z-average=129 nm, Pdl=0.226, D.sub.n=64 nm and SD=23.4 nm. UV-Vis Abs. (water): λ.sub.max=384 nm, λ.sub.onset=441 nm. UV-Vis PL (water): λ.sub.max=430 nm, λ.sub.sec.=453 nm, λ.sub.sec.=484 nm.

1.9-2% 9,9-Di(undecanoic acid)fluorene, 5% 2,1,3-Benzothiadiazole, 33% Di(hex-5-en-1-yl)fluorene and 5% 9,9′-Spirobifluorene Cross-Linked Polyfluorene Nanoparticles

(77) Synthesis

(78) ##STR00056##

(79) In a Schlenk tube was added water (22.0 mL), sodium dodecyl sulfate (110 mg, 382 μmol) and 1M aqueous sodium hydroxide (816 μL, 816 μmol). The solution was purged with argon for 2 hours. In a vial was weighed 9,9-dioctylfluorene-2,7-diboronic acid bis(1,3-propanediol) ester (111.7 mg, 200 μmol), 2,7-dibromo-9,9-di(undecanoic acid)fluorene (5.5 mg, 8 μmol), 2,2′,7,7′-tetrabromo-9,9′-spirobifluorene (12.6 mg, 20 μmol), 4,7-dibromobenzo[c]-1,2,5-thiadiazole (5.9 mg, 20 μmol), 2,7-dibromo-9,9-di(hex-5-en-1-yl)fluorene (64.5 mg, 132 μmol), tris(dibenzylideneacetone)dipalladium(0) (4.6 mg, 5 μmol), tri(o-tolyl)phosphine (9.1 mg, 30 μmol) and hexadecane (171 μL, 585 μmol). The vial was transferred to an argon filled glovebox, sealed with a rubber septum and removed. Toluene (2.19 mL) was added to the vial and the suspension sonicated until a homogenous solution was achieved. The initial aqueous solution was cooled to 0° C. in an ice bath, the ultrasonic probe inserted and the toluene solution injected rapidly into the water. The solution was ultrasonicated for 1 minute, stirred for 30 seconds and ultrasonicated for 1 further minute. The Schlenk tube was sealed, placed in a preheated oil bath at 50° C. and stirred for 20 hours. The Schlenk was opened and a stream of nitrogen gas passed over the emulsion at 50° C., with stirring. The emulsion was cooled to room temperature, the volume increased to 23.0 mL with deionised water and filtered through a glass wool plug. The emulsion was obtained as a milky dark green solution. DLS (water): Z-average=101 nm, Pdl=0.166, D.sub.n=55.1 nm and SD=18.1 nm. UV-Vis Abs. (water): λ.sub.max=381 nm, λ.sub.sec.=453 nm, λ.sub.onset=522 nm. UV-Vis PL (water): λ.sub.max=530 nm.

1.10. Synthesis of 5% Benzo[c]-1,2,5-thiadiazole, 45% 9,9-Di(sodium undecanyl sulfate)fluorene and 50% 9,9-Dioctyl(fluorene) Nanoparticles

(80) ##STR00057##

(81) In a Schlenk tube was added water (22.0 mL), sodium dodecyl sulfate (110 mg, 382 μmol) and 1M aqueous sodium hydroxide (1080 μL, 1080 μmol). The solution was purged with argon for 2 hours. In a vial was weighed 9,9-dioctylfluorene-2,7-diboronic acid bis(1,3-propanediol) ester (111.7 mg, 200 μmol), 2,7-dibromo-9,9-di(sodium undecanyl sulfate)fluorene (156.4 mg, 180 μmol), 4,7-dibromobenzo[c]-1,2,5-thiadiazole (5.9 mg, 20 μmol) tris(dibenzylideneacetone)dipalladium(0) (4.6 mg, 5 μmol), tri(o-tolyl)phosphine (9.1 mg, 30 μmol) and hexadecane (171 μL, 585 μmol). The vial was transferred to an argon filled glovebox, sealed with a rubber septum and removed. Toluene (2.19 mL) was added to the vial and the suspension sonicated until a homogenous solution was achieved. The initial aqueous solution was cooled to 0° C. in an ice bath, the ultrasonic probe inserted and the toluene solution injected rapidly into the water. The solution was ultrasonicated for 1 minute, stirred for 30 seconds and ultrasonicated for 1 further minute. The Schlenk tube was sealed, placed in a preheated oil bath at 50° C. and stirred for 16 hours. The Schlenk tube was opened and a stream of nitrogen gas passed over the emulsion at 50° C., with stirring. The emulsion was cooled to room temperature, the volume increased to 23.0 mL with deionised water and filtered through a glass wool plug. The emulsion was obtained as a milky dark green solution.

1.11-5% Benzo[c]-1,2,5-thiadiazole, 35% 9,9-Di(undecanoic acid)fluorene and 5% 9,9′-Spirobifluorene Cross-Linked Polyfluorene Nanoparticles

(82) Synthesis

(83) ##STR00058##

(84) In a Schlenk tube was added water (22.0 mL), sodium dodecyl sulfate (110 mg, 382 μmol) and 1M aqueous sodium hydroxide (1080 μL, 1080 μmol). The solution was purged with argon for 2 hours. In a vial was weighed 9,9-dioctylfluorene-2,7-diboronic acid bis(1,3-propanediol) ester (111.7 mg, 200 μmol), 2,7-dibromo-9,9-di(undecanoic acid)fluorene (96.9 mg, 140 μmol), 2,2′,7,7′-tetrabromo-9,9′-spirobifluorene (12.6 mg, 20 μmol), 4,7-dibromobenzo[c]-1,2,5-thiadiazole (5.9 mg, 20 μmol) tris(dibenzylideneacetone)dipalladium(0) (4.6 mg, 5 μmol), tri(o-tolyl)phosphine (9.1 mg, 30 μmol) and hexadecane (171 μL, 585 μmol). The vial was transferred to an argon filled glovebox, sealed with a rubber septum and removed. Toluene (2.19 mL) was added to the vial and the suspension sonicated until a homogenous solution was achieved. The initial aqueous solution was cooled to 0° C. in an ice bath, the ultrasonic probe inserted and the toluene solution injected rapidly into the water. The solution was ultrasonicated for 1 minute, stirred for 30 seconds and ultrasonicated for 1 further minute. The Schlenk tube was sealed, placed in a preheated oil bath at 50° C. and stirred for 20 hours. The Schlenk tube was opened and a stream of nitrogen gas passed over the emulsion at 50° C., with stirring. The emulsion was cooled to room temperature, the volume increased to 23.0 mL with deionised water and filtered through a glass wool plug. The emulsion was obtained as a milky dark green solution (CPN1). DLS (water): Z-average=79.0 nm, Pdl=0.117, D.sub.n=52.4 nm and SD=15.3 nm. UV-Vis Abs. (water): λ.sub.max=380 nm, λ.sub.sec.=450 nm, λ.sub.onset=520 nm. UV-Vis PL (water): λ.sub.max=535 nm, λ.sub.sec.=424 nm.

1.12-5% 9,9′-Spirobifluorene, 90% Di((4-((2-ethylhexyl)oxy)phenyl)) fluorene Nanoparticles

(85) ##STR00059##

(86) In a 250 mL round bottom flask, fitted with an argon inlet, was added water (100 mL), sodium hydroxide (160 mg, 4.00 mmol) and sodium dodecyl sulfate (551 mg) and the solution purged with argon for 1 hour. Toluene was degassed with argon for 2 hours. In a Schlenk tube was weighed 9,9-di((4-((2-ethylhexyl)oxy)phenyl))fluorene-2,7-diboronic acid bis(pinacol) ester (827 mg, 1.00 mmol), 2,7-dibromo-9,9-di((4-((2-ethylhexyl)oxy)phenyl))fluorene (513 mg, 700 μmol), 2,2′,7,7′-tetrabromo-9,9′-spirobifluorene (63.2 mg, 100 μmol), hexadecane (855 μL), 4,7-dibromobenzo[c]-1,2,5-thiadiazole (29.4 mg, 100 μmol), tris(dibenzylideneacetone) dipalladium(0) (22.4 mg, 25 μmol) and tri(o-tolyl)phosphine (45.6 mg, 150 μmol). The Schlenk tube was subjected to four vacuum-argon cycles. Toluene (10 mL) was added and the suspension sonicated in an ultrasonic bath until a homogenous solution was achieved. The initial aqueous solution was cooled to 0° C. in an ice bath and the toluene solution was added rapidly into the aqueous phase under a stream of argon. A stirrer bar was added and the ultrasonic probe inserted (½″ extender tip) to a depth of 2 cm. The solution was ultrasonicated for 2 minutes at 40% amplitude, stirred for 30 seconds then sonicated for 2 further minutes. The flask was sealed, placed in a preheated oil bath at 70° C. and stirred for 16 hours. The flask was cooled to 50° C. and air passed over the emulsion for 5 hours, with stirring. The emulsion was cooled to room temperature, left to stand for 16 hours and filtered through a glass wool plug. The emulsion was obtained as a bright green cloudy dispersion. DLS: z-Average: 115.9 nm, Pdl: 0.153. UV-Vis Abs. (water): λ.sub.max=391 nm. UV-Vis PL (water): λ.sub.max=422 nm.

1.13-5% 9,9′-Spirobifluorene, 10% Di((4-(sodium undecanoyl sulfate)phenyl))fluorene, 80% Di((4-((2-ethylhexyl)oxy)phenyl))fluorene Nanoparticles

(87) ##STR00060##

(88) In a 250 mL round bottom flask, fitted with an argon inlet, was added water (100 mL), sodium hydroxide (160 mg, 4.00 mmol) and 2,7-dibromo-9,9-di((4-(sodium undecanoyl sulfate)phenyl))fluorene (211 mg, 200 μmol) and the solution purged with argon for 2 hours. Toluene was degassed with argon for 2 hours. In a Schlenk tube was weighed 9,9-di((4-((2-ethylhexyl)oxy)phenyl))fluorene-2,7-diboronic acid bis(pinacol) ester (827 mg, 1.00 mmol), 2,7-dibromo-9,9-di((4-((2-ethylhexyl)oxy)phenyl))fluorene (440 mg, 600 μmol), 2,2′,7,7′-tetrabromo-9,9′-spirobifluorene (63.2 mg, 100 μmol), hexadecane (855 μL), tris(dibenzylideneacetone) dipalladium(0) (22.4 mg, 25 μmol) and tri(o-tolyl)phosphine (45.6 mg, 150 μmol). The Schlenk tube was subjected to four vacuum-argon cycles. Toluene (10 mL) was added and the suspension sonicated in an ultrasonic bath until a homogenous solution was achieved. A stirrer bar was added and the ultrasonic probe inserted (½″ extender tip) to a depth of 2 cm. The solution was ultrasonicated for 2 minutes at 40% amplitude, stirred for 30 seconds then sonicated for 2 further minutes. The flask was sealed, placed in a preheated oil bath at 60° C. and stirred for 16 hours. The flask was cooled to 50° C. and air passed over the emulsion for 5 hours, with stirring. The emulsion was cooled to room temperature, left to stand for 16 hours and filtered through a glass wool plug. The emulsion was obtained as a dark green/grey cloudy dispersion. DLS: z-Average: 171.2 nm, Pdl: 0.047. UV-Vis Abs. (water): λ.sub.max=398 nm. UV-Vis PL (water): λ.sub.max=422 nm.

1.14-5% Benzo[c]-1,2,5-thiadiazole, 5% 9,9′-Spirobifluorene, 85% Di((4-((2-ethylhexyl)oxy)phenyl))fluorene Nanoparticles

(89) ##STR00061##

(90) In a 250 mL round bottom flask, fitted with an argon inlet, was added water (100 mL), sodium hydroxide (160 mg, 4.00 mmol) and sodium dodecyl sulfate (551 mg) and the solution purged with argon for 1 hour. Toluene was degassed with argon for 2 hours. In a Schlenk tube was weighed 9,9-di((4-((2-ethylhexyl)oxy)phenyl))fluorene-2,7-diboronic acid bis(pinacol) ester (827 mg, 1.00 mmol), 2,7-dibromo-9,9-di((4-((2-ethylhexyl)oxy)phenyl))fluorene (513 mg, 700 μmol), 2,2′,7,7′-tetrabromo-9,9′-spirobifluorene (63.2 mg, 100 μmol), hexadecane (855 μL), 4,7-dibromobenzo[c]-1,2,5-thiadiazole (29.4 mg, 100 μmol), tris(dibenzylideneacetone) dipalladium(0) (22.4 mg, 25 μmol) and tri(o-tolyl)phosphine (45.6 mg, 150 μmol). The Schlenk tube was subjected to four vacuum-argon cycles. Toluene (10 mL) was added and the suspension sonicated in an ultrasonic bath until a homogenous solution was achieved. The initial aqueous solution was cooled to 0° C. in an ice bath and the toluene solution was added rapidly into the aqueous phase under a stream of argon. A stirrer bar was added and the ultrasonic probe inserted (½″ extender tip) to a depth of 2 cm. The solution was ultrasonicated for 2 minutes at 40% amplitude, stirred for 30 seconds then sonicated for 2 further minutes. The flask was sealed, placed in a preheated oil bath at 70° C. and stirred for 16 hours. The flask was cooled to 50° C. and air passed over the emulsion for 5 hours, with stirring. The emulsion was cooled to room temperature, left to stand for 16 hours and filtered through a glass wool plug. The emulsion was obtained as a bright green cloudy dispersion. DLS: z-Average: 115.1 nm, Pdl: 0.160. UV-Vis Abs. (water): λ.sub.max=387 nm. UV-Vis PL (water): λ.sub.max=526 nm.

1.15-5% Benzo[c]-1,2,5-thiadiazole, 5% 9,9′-Spirobifluorene, 10% Di((4-(sodium undecanoyl sulfate)phenyl))fluorene, 75% Di((4-((2-ethylhexyl)oxy)phenyl))fluorene Nanoparticles

(91) ##STR00062##

(92) In a 250 mL round bottom flask, fitted with an argon inlet, was added water (100 mL), sodium hydroxide (160 mg, 4.00 mmol) and 2,7-dibromo-9,9-di((4-(sodium undecanoyl sulfate)phenyl))fluorene (211 mg, 200 μmol) and the solution purged with argon for 1 hour. Toluene was degassed with argon for 1 hours. In a Schlenk tube was weighed 9,9-di((4-((2-ethylhexyl)oxy)phenyl))fluorene-2,7-diboronic acid bis(pinacol) ester (827 mg, 1.00 mmol), 2,7-dibromo-9,9-di((4-((2-ethylhexyl)oxy)phenyl))fluorene (366 mg, 500 μmol), 2,2′,7,7′-tetrabromo-9,9′-spirobifluorene (63.2 mg, 100 μmol), hexadecane (855 μL), 4,7-dibromobenzo[c]-1,2,5-thiadiazole (29.4 mg, 100 μmol), tris(dibenzylideneacetone) dipalladium(0) (22.4 mg, 25 μmol) and tri(o-tolyl)phosphine (45.6 mg, 150 μmol). The Schlenk tube was subjected to four vacuum-argon cycles. Toluene (10 mL) was added and the suspension sonicated in an ultrasonic bath until a homogenous solution was achieved. The initial aqueous solution was cooled to 0° C. in an ice bath and the toluene solution was added rapidly into the aqueous phase under a stream of argon. A stirrer bar was added and the ultrasonic probe inserted (½″ extender tip) to a depth of 2 cm. The solution was ultrasonicated for 2 minutes at 40% amplitude, stirred for 30 seconds then sonicated for 2 further minutes. The flask was sealed, placed in a preheated oil bath at 70° C. and stirred for 16 hours. The flask was cooled to 50° C. and air passed over the emulsion for 5 hours, with stirring. The emulsion was cooled to room temperature, left to stand for 16 hours and filtered through a glass wool plug. The emulsion was obtained as a bright green cloudy dispersion. DLS: z-Average: 184.5 nm, Pdl: 0.031. UV-Vis Abs. (water): λ.sub.max=393 nm. UV-Vis PL (water): λ.sub.max=527 nm.

1.16-5% Benzo[c]-1,2,5-thiadiazole, 5% 9,9′-Spirobifluorene, 75% 9,9-Dioctyl(fluorene), 10% 9,9-Di(poly(ethylene glycol) monomethyl ether.SUB.(Mn 900).)(fluorene)

(93) ##STR00063##

(94) In a Schlenk tube was added water (20 mL) and sodium hydroxide (32.0 mg, 800 μmol) and the solution purged with argon for 2 hours. Toluene was degassed with argon for 2 hours. In a vial was weighed 9,9-di-n-octylfluorene-2,7-diboronic acid bis(propanediol) ester (111.7 mg, 200 μmol), 9,9-di-n-octyl-2,7-dibromofluorene (54.8 mg, 100 μmol), 2,2′,7,7′-tetrabromo-9,9′-spirobifluorene (12.6 mg, 20 μmol), 2,7-dibromo-9,9-di((poly(ethylene glycol) monomethyl ether.sub.(Mn 900))phenyl))fluorene (86.3 mg, 40 μmol), 4,7-dibromobenzo[c]-1,2,5-thiadiazole (5.9 mg, 20 μmol), hexadecane (150 μL), tris(dibenzylideneacetone) dipalladium(0) (4.6 mg, 5 μmol) and tri(o-tolyl)phosphine (9.1 mg, 30 μmol). The vial was purged with argon for 30 minutes. Toluene (2.00 mL) was added and the suspension sonicated in an ultrasonic bath until a homogenous solution was achieved. The initial aqueous solution and cooled to 0° C. in an ice bath. The toluene solution was added rapidly into the aqueous phase under a stream of argon. A stirrer bar was added and the ultrasonic probe inserted (6 mm microtip) to a depth of 2 cm. The solution was ultrasonicated for 1 minute at 30% amplitude, stirred for 30 seconds and sonicated for 1 further minute. The flask was sealed, placed in a preheated oil bath at 40° C. and stirred for 20 hours. The flask was heated to 50° C. and air passed over the emulsion for 5 hours, with stirring. The emulsion was cooled to room temperature, left to stand for 16 hours and filtered through cotton mesh. The emulsion was obtained as a bright green cloudy dispersion.

1.17-5% Benzo[c]-1,2,5-thiadiazole, 5% 9,9′-Spirobifluorene, 74% 9,9-Dioctyl(fluorene), 10% 9,9-Di(sodium undecanoyl sulfate)(fluorene), 1% 9,9-Di(sodium undecanoyl carboxylate)(fluorene)

(95) ##STR00064##

(96) In a Schlenk tube was added water (20 mL) and sodium hydroxide (32.0 mg, 800 μmol) and the solution purged with argon for 2 hours. Toluene was degassed with argon for 2 hours. In a vial was weighed 9,9-di-n-octylfluorene-2,7-diboronic acid bis(propanediol) ester (111.7 mg, 200 μmol), 9,9-di-n-octyl-2,7-dibromofluorene (41.7 mg, 76 μmol), 2,2′,7,7′-tetrabromo-9,9′-spirobifluorene (12.6 mg, 20 μmol), 2,7-dibromo-9,9-di(undecanoic acid)fluorene (2.8 mg, 4.0 μmol), 4,7-dibromobenzo[c]-1,2,5-thiadiazole (5.9 mg, 20 μmol), tris(dibenzylideneacetone) dipalladium(0) (4.6 mg, 5 μmol) and tri(o-tolyl)phosphine (9.1 mg, 30 μmol). The vial was purged with argon for 30 minutes. Toluene (2.00 mL) was added and the suspension sonicated in an ultrasonic bath until a homogenous solution was achieved. 9,9-Di(sodium undecanoyl sulfate)2,7-dibromofluorene (34.7 mg, 40 μmol) was added to the initial aqueous solution and cooled to 0° C. in an ice bath. The toluene solution was added rapidly into the aqueous phase under a stream of argon. A stirrer bar was added and the ultrasonic probe inserted (6 mm microtip) to a depth of 2 cm. The solution was ultrasonicated for 2 minutes at 30% amplitude, stirred for 30 seconds and this sequence repeated four further times. The flask was sealed, placed in a preheated oil bath at 40° C. and stirred for 20 hours. The flask was heated to 50° C. and air passed over the emulsion for 7 hours, with stirring. The emulsion was cooled to room temperature and filtered through a glass wool plug. The emulsion was obtained as a bright green cloudy dispersion.

1.18-5% Benzo[c]-1,2,5-thiadiazole, 5% 9,9′-Spirobifluorene, 70% 9,9-Dioctyl(fluorene), 10% 9,9-Di(sodium undecanoyl sulfate)(fluorene), 5% 9,9-Di(sodium undecanoyl carboxylate)(fluorene)

(97) ##STR00065##

(98) In a Schlenk tube was added water (20 mL) and sodium hydroxide (33.6 mg, 840 μmol) and the solution purged with argon for 2 hours. Toluene was degassed with argon for 2 hours. In a vial was weighed 9,9-di-n-octylfluorene-2,7-diboronic acid bis(propanediol) ester (111.7 mg, 200 μmol), 9,9-di-n-octyl-2,7-dibromofluorene (43.9 mg, 80 μmol), 2,7-dibromo-9,9-di(undecanoic acid)fluorene (13.9 mg, 20.0 μmol), 2,2′,7,7′-tetrabromo-9,9′-spirobifluorene (12.6 mg, 20 μmol), 4,7-dibromobenzo[c]-1,2,5-thiadiazole (5.9 mg, 20 μmol), hexadecane (150 μL), tris(dibenzylideneacetone) dipalladium(0) (4.6 mg, 5 μmol) and tri(o-tolyl)phosphine (9.1 mg, 30 μmol). The vial was purged with argon for 30 minutes. Toluene (2.00 mL) was added and the suspension sonicated in an ultrasonic bath until a homogenous solution was achieved. 9,9-Di(sodium undecanoyl sulfate)2,7-dibromofluorene (34.7 mg, 40 μmol) was added to the initial aqueous solution and cooled to 0° C. in an ice bath. The toluene solution was added rapidly into the aqueous phase under a stream of argon. A stirrer bar was added and the ultrasonic probe inserted (6 mm microtip) to a depth of 2 cm. The solution was ultrasonicated for 2 minutes at 30% amplitude, stirred for 30 seconds and this sequence repeated four further times. The flask was sealed, placed in a preheated oil bath at 40° C. and stirred for 20 hours. The flask was heated to 50° C. and air passed over the emulsion for 5 hours, with stirring. The emulsion was cooled to room temperature and filtered through a glass wool plug. The emulsion was obtained as a bright green slightly cloudy dispersion. DLS: z-Average: 77.1 nm, Pdl: 0.211.

1.19-5% Benzo[c]-1,2,5-thiadiazole, 5% 9,9′-Spirobifluorene, 70% 9,9-Dioctyl(fluorene), 10% 9,9-Di(sodium undecanoyl sulfate)(fluorene), 5% 9,9-Di(sodium undecanoyl carboxylate)(fluorene)

(99) ##STR00066##

(100) In a Schlenk tube was added water (20 mL) and sodium hydroxide (33.6 mg, 840 μmol) and the solution purged with argon for 2 hours. Toluene was degassed with argon for 2 hours. In a vial was weighed 9,9-di-n-octylfluorene-2,7-diboronic acid bis(propanediol) ester (111.7 mg, 200 μmol), 9,9-di-n-octyl-2,7-dibromofluorene (43.9 mg, 80 μmol), 2,7-dibromo-9,9-di(undecanoic acid)fluorene (13.9 mg, 20.0 μmol), 2,2′,7,7′-tetrabromo-9,9′-spirobifluorene (12.6 mg, 20 μmol), 4,7-dibromobenzo[c]-1,2,5-thiadiazole (5.9 mg, 20 μmol), tris(dibenzylideneacetone) dipalladium(0) (4.6 mg, 5 μmol) and tri(o-tolyl)phosphine (9.1 mg, 30 μmol). The vial was purged with argon for 30 minutes. Toluene (2.00 mL) was added and the suspension sonicated in an ultrasonic bath until a homogenous solution was achieved. 9,9-Di(sodium undecanoyl sulfate)2,7-dibromofluorene (34.7 mg, 40 μmol) was added to the initial aqueous solution and cooled to 0° C. in an ice bath. The toluene solution was added rapidly into the aqueous phase under a stream of argon. A stirrer bar was added and the ultrasonic probe inserted (6 mm microtip) to a depth of 2 cm. The solution was ultrasonicated for 2 minutes at 30% amplitude, stirred for 30 seconds and this sequence repeated four further times. The flask was sealed, placed in a preheated oil bath at 40° C. and stirred for 20 hours. The flask was heated to 50° C. and air passed over the emulsion for 5 hours, with stirring. The emulsion was cooled to room temperature and filtered through a glass wool plug. The emulsion was obtained as a bright green clear dispersion. DLS: z-Average: 67.6 nm, Pdl: 0.237. UV-Vis Abs. (water): λ.sub.max=375 nm. UV-Vis PL (water): λ.sub.max=536 nm.

EXAMPLE 2—PREPARATION OF CONJUGATES

2.1—Transfer of Nanoparticle Solution CNP1 to MES Buffer (pH 6.1, 50 mM)

(101) To nanoparticle solution CNP1 (Example 1.11) (2.00 mL) was added MES buffer (8.00 mL (pH 6.1, 50 mM)). The solution was transferred to a ‘Vivapore 10/20 static concentrator’ and the volume reduced to 1.0 mL. MES buffer (9.00 mL (pH 6.1, 50 mM)) was added and the solution transferred to a ‘second vivapore 10/20 static concentrator’ and the volume reduced to 1 mL. The volume was increased to 10.0 mL with MES buffer (9.00 mL (pH 6.1, 50 mM)) to give nanoparticle solution CNP2 (1.16 mg mL.sup.−1, 61 μM, 2.0 μmol mL.sup.−1 CO.sub.2H).

2.2—Nanoparticle Activation with N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride and N-hydroxysulfosuccinimide

(102) To nanoparticle solution CPN1 (Example 1.11) (5.00 mL, 1.16 mg mL.sup.−1, 61 μM, 2.0 μmol mL.sup.−1 CO.sub.2H) was added N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (1.9 mg, 10 μmol) and N-hydroxysulfosuccinimide (4.3 mg, 20 μmol). The solution was stirred for 30 mins then MES buffer (5.00 mL (pH 6.1, 50 mM)) was added. The solution was transferred to a ‘Vivapore 10/20 static concentrator’ and the volume reduced to 0.5 mL. MES buffer (9.50 mL (pH 6.1, 50 mM)) was added and transferred to a second ‘vivapore 10/20 static concentrator’ and the volume reduced to 0.5 mL. MES buffer (4.50 mL (pH 6.1, 50 mM)) was added to give the EDC/NHS activated nanoparticles (CPN2) (1.08 mg mL.sup.−1, 57 μM and (1.9 μmol mL.sup.−1 CO.sub.2H)).

2.3—Bioconjugation of Nanoparticles to Streptavidin

(103) FIG. 13 shows the following nanoparticle samples:

(104) Sample a: Nanoparticle solution CPN 1.

(105) Sample b: Nanoparticle solution CPN2, mixed for 18 hours.

(106) Sample c: Nanoparticle solution CPN 2 (400 μL, 22.8 μmol) was mixed for 16 hours, then quenched with glycine (3.8 μL of 200 μM solution, 0.76 μmol) and mixed for a further 2 hours.

(107) Sample d: To nanoparticle solution CPN2 (332 μL, 18.9 μmol) was added streptavidin (10 μL, 189 μmol (18.9 μM solution in PBS buffer pH 7.2, 12 mM)) and mixed for 16 hours at RT. Glycine (3.2 μL of 200 μM solution, 0.63 μmol) was added and the solution mixed for a further 2 hours.
Sample e: Nanoparticle solution CPN1 (310 μL, 18.9 μmol) was mixed with streptavidin (10 μL, 189 μmol (18.9 μM solution in PBS buffer pH 7.2, 12 mM) and mixed for 18 hours.

2.4—Bioconjugation of Nanoparticles to Donkey Anti-Mouse IgG (H+L) Secondary Antibody

(108) FIG. 14 shows the following nanoparticle samples:

(109) Sample a: Nanoparticle solution CPN1.

(110) Sample b: Nanoparticle solution CPN2, mixed for 18 hours.

(111) Sample c: Nanoparticle solution CPN2 (400 μL, 22.8 μmol) was mixed for 16 hours, then quenched with glycine (3.8 μL of 200 μM solution, 0.76 μmol) and stirred for a further 2 hours.

(112) Sample d: To nanoparticle solution CPN2 (233 μL, 13.3 μmol) was added the secondary antibody (10 μL, 133 μmol (13.3 μM solution in PBS buffer pH 7.4, 12 mM)) and mixed for 16 hours at RT. Glycine (2.2 μL of 200 μM solution, 0.44 μmol) was added and the solution mixed for a further 2 hours.
Sample e: Nanoparticle solution CPN1 (218 μL, 13.3 μmol) was mixed with the secondary antibody (10 μL, 133 μmol (13.3 μM solution in PBS buffer pH 7.4, 12 mM)) and mixed for 18 hours.

2.5—Nanoparticle Activation with N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC)/N-hydroxysulfosuccinimide (NHS) and Subsequent Coupling

(113) Step 1. EDC/NHS Activation of Nanoparticles

(114) TABLE-US-00016 Component Activated-Nanoparticles Control Nanoparticles (7 mg/ml, CO.sub.2H 72  72 1.8 umol/mL, size = 76 nm, Abs = 375 nm, Em = 536 nm, 4 mM Stock EDC 100 — 10 mM Stock NHS 100 — MES buffer (50 mM pH 6.1) 228 428 Total Volume 500 500

(115) The above volumes were mixed and incubated for 15 mins.

(116) The above reactions were then desalted using PD10 columns (desalting Sephadex G-25) pre-equilibrated in HEPES buffer (10 mM HEPES, 150 mM NaCl, pH7.4) and the fluorescent (green) fractions were collected (˜1.0 ml). The flow-through and other fractions were non-fluorescent.

(117) The proteins were desalted in PD10 columns equilibrated in HEPES buffer (10 mM HEPES, 150 mM NaCl, pH7.4):

(118) (i) Streptavidin-Cy5 stock (16 uM) (Cy5-SA) was ×4 diluted in HEPES (100 uL+300 uL HEPES) and also desalted in a PD10 column in HEPES. Final concentration 3 uM.

(119) (ii) Anti-Goat-IgG-HRP (IgG1) 250 uL+250 uL HEPES

(120) (iii) Anti-biotin-IgG-AKP (IgG2) 100 uL+400 uL HEPES

(121) (iii) PerCP in 500 uL (2 mg/mL).

(122) (iv) A stock 5 mg/mL Biotin-Cadaverine (Bt-Cad) 1 mg/200 uL HEPES (11 mM)

(123) Final volumes of fluorescent fractions were collected (˜600 uL).

(124) Step 2. Coupling Reactions with EDC-NHS Activated Nanoparticles

(125) The desalted samples were coupled as follows:

(126) TABLE-US-00017 Stock Coupling Activated Luminspheres ™ Protein/NH.sub.2 Reaction (uL) (uL) Luminspheres ™ with Activated Rxn-Luminspheres ™ Cy5-SA Cy5-SA 200 200 Luminspheres ™ with Activated Rxn-Luminspheres ™ Bt-Cad Bt-Cad 200 200 Luminspheres ™ with Activated Rxn-Luminspheres ™ IgG1 IgG1 200 200 Luminspheres ™ with Activated Rxn-Luminspheres ™ IgG2 IgG2 200 200 PerCP Activated Rxn-Luminspheres ™ PerCP 200 200

(127) Each 400 uL reaction was incubated for 2 hours at room temperature before addition of 10 uL of 1M ethanolamine. The reaction was then stored at 4° C. overnight.

(128) Step 3. EDC-NHS Activated Nanoparticles Coupling Reaction Evaluation.

(129) Streptavidin-HP SpinTrap columns (highly cross-linked Agarose, 6%) were pre-equilibrated in HEPES (10 mM HEPES, 150 mM NaCl, pH7.4)

(130) (i) Diluted ×10 Biotinylated-Nanoparticles in HEPES (50 uL=450 uL HEPES)-loaded 400 uL on to spin column.

(131) (ii) Used a ×60 dilution of Nanoparticles-control (Nanoparticles only 50 uL=450 uL HEPES)-loaded 400 uL on to spin column.

(132) (iii) Incubated at room temperature for 1 hour.

(133) (iv) Measured fluorescence of supernatant after incubation:

(134) Biotinylated-Nanoparticles=100 RFU, Luminspheres™-control=240 RFU. Indicating that more of Biotinylated-Luminspheres™ bound specifically to the streptavidin-conjugated agarose beads.

(135) (v) Washed both spin columns with HEPES (0.5 mL) the flow through and washes were not fluorescent.

2.6—Synthesis of 5% Benzo[c]-1,2,5-thiadiazole, 5% 9,9′-Spirobifluorene, 70% 9,9-Dioctyl(fluorene), 10% 9,9-Di(sodium undecanoyl sulfate)(fluorene), 5% 9,9-Di(biotin-undecanoate)(fluorene)

(136) ##STR00067##

(137) In a Schlenk tube was added water (20 mL) and sodium hydroxide (32.0 mg, 800 μmol) and the solution purged with argon for 2 hours. Toluene was degassed with argon for 2 hours. In a vial was weighed 9,9-di-n-octylfluorene-2,7-diboronic acid bis(propanediol) ester (111.7 mg, 200 μmol), 9,9-di-n-octyl-2,7-dibromofluorene (43.9 mg, 80 μmol), 9,9-di(biotin-undecanoate)dibromofluorene (22.3 mg, 20.0 μmol), 2,2′,7,7′-tetrabromo-9,9′-spirobifluorene (12.6 mg, 20 μmol), 4,7-dibromobenzo[c]-1,2,5-thiadiazole (5.9 mg, 20 μmol), tris(dibenzylideneacetone) dipalladium(0) (4.6 mg, 5.0 μmol) and tri(o-tolyl)phosphine (9.1 mg, 30 μmol). The vial was purged with argon for 30 minutes. Toluene (2.00 mL) was added and the suspension sonicated in an ultrasonic bath until a homogenous solution was achieved. 9,9-Di(sodium undecanoyl sulfate)2,7-dibromofluorene (34.7 mg, 40 μmol) was added to the initial aqueous solution and cooled to 0° C. in an ice bath. The toluene solution was added rapidly into the aqueous phase under a stream of argon and the ultrasonic probe inserted (6 mm microtip) to a depth of 2 cm. The solution was ultrasonicated for 2 minutes at 30% amplitude, stirred for 30 seconds and this sequence repeated four further times. A stirrer bar was added, the flask was sealed, placed in a preheated oil bath at 40° C. and stirred for 20 hours. The flask was heated to 50° C. and air passed over the emulsion for 5 hours, with stirring. The emulsion was cooled to room temperature, centrifuged at 4,000 rpm for 5 minutes and filtered through a glass wool plug to provide a bright green dispersion (measured mass of emulsion: 23.00 g. Expected concentration: 7.18 mg/mL).

2.7—Nanoparticle-Cell Flow and Imaging Cytometry

(138) Cell Culture and Incubation of Nanoparticles

(139) HEK293 cells were plated into a 6 well plate and allowed to grow to full confluency. A log dilution of nanoparticle (7 mg/ml, CO.sub.2H 1.8 umol/mL, size=76 nm, Abs=375 nm, Em=536 nm) suspension was made in cell culture media (supplemented DMEM) from 1:10.sup.2 to 1:10.sup.6. Media was aspirated from the cells and replaced with media containing the nanoparticle suspension. One well had growth media without nanoparticles to act as the control well. Cells were incubated for 55 minutes at 37° C./5% CO.sub.2 to allow potential binding/uptake of the nanoparticles. Nanoparticles were aspirated from the cells and they were washed twice in PBS. Cells were recovered from the plate by incubation with 0.2 mL Trypsin-EDTA for 5 minutes, 0.3 mL PBS+5% BSA was added to the cells and they were detached with gentle pipetting in 0.5 mL final volume.

(140) Flow Cytometry Analysis

(141) Cells were filtered through a 50 um mesh to remove aggregates into a 5 mL polypropylene round bottom tube. To identify dead cells the viability marker TOPRO-3 was added at a dilution of 1-500. Cells were run on a BD Influx cell sorter with 355, 488, 405, 561 and 647 nm laser line excitation using settings determined by running unlabelled cells. Live cells were identifies from cell debris and dead cell, and 20,000 cells were recorded. Following flow cytometry the sample with the optimal positive signal was chosen to analyse on the Amnis ImageStream imaging cytometer.

(142) Imaging Cytometry

(143) 100 ul of cell suspension was placed in a 1.5 mL eppendorff tube and loaded onto the ISX imaging cytometer. Using 488 (100 mW power setting) and 405 nm (20 mW power setting) excitation cells were identified above beads and 1000 cells were recorded in the bright field, side scatter and two fluorescence emission channels selected to be optimal to the nanoparticles emission, with 40× magnification. Data was analysed using IDEAS v6 software (see FIG. 15).

(144) While specific embodiments of the invention have been described herein for the purpose of reference and illustration, various modifications will be apparent to a person skilled in the art without departing from the scope of the invention as defined by the appended claims.